Lasso peptide integrin inhibitors

Description

LASSO PEPTIDE INTEGRIN INHIBITORS1. CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 612,957, filed December 20, 2023, the entire contents of which is incorporated herein by reference.2. FIELD

[0002] The field of invention covers engineered lasso peptides having potency and specificity for binding to integrin receptors, use of such engineered lasso peptides in the management, prevention or treatment of an integrin-mediated disease, such as cancer, a fibroproliferative disease, or fibrosis, and compositions and methods of producing such engineered lasso peptides.3. BACKGROUND

[0003] Cancer is a complex disease commonly caused by DNA damage, genetic mutations, or epigenetic modifications that support dysfunctional cellular signaling and aberrant cellular behavior leading to uncontrolled cellular growth. Over two hundred different forms of cancer are known, and hundreds of drugs and drug combinations have been approved as treatments for specific cancer indications. Survival rates remain low, and prevalence is increasing for many cancers (American Cancer Society. Cancer Facts & Figures 2022. Atlanta: American Cancer Society; 2022). Thus, there exists a need for new therapeutic approaches with improved performance to combat these serious malignancies.

[0004] Fibrosis and fibroproliferative diseases are characterized by dysregulation, accumulation and change in the quality of the extracellular matrix resulting from an abnormal or uncontrolled tissue repair response (Wynn, T.A., J. Clin. Invest. 2007, 117, 524-529).Both benign and malignant fibroproliferative disorders are known and include, but are not limited to, pulmonary fibrosis including cystic and idiopathic pulmonary fibrosis, hepatic fibrosis and cirrhosis, pancreatic fibrosis and pancreatitis, renal fibrosis, glial fibrosis, retroperitoneal cavity fibrosis, mediastinal fibrosis, cardiovascular disease and heart fibrosis, myelofibrosis, systemic sclerosis, Dupuytren's contracture, Peyronie’s disease, macular degeneration, hypertrophic scars, skin fibrosis and skin keloids, arthrofibrosis, and Duchenne muscular dystrophy-associated skeletal muscle fibrosis. In these diseases, excessiveconnective tissue accumulates, and slow but continuous tissue contraction leads to progressive deterioration in the normal structure and function of the affected organs, ultimately concluding in organ failure (Wynn, J. Pathol. 2008; 214: 199-210). It is estimated that up to 45% of all deaths in the western world can be attributed to some form of tissue or organ fibrosis (Henderson, et aL, Nature, 2020, 587, 556-566). Despite the large societal burden of fibrotic diseases, there are currently few approved therapies and there exists an urgent need for new effective therapeutic approaches with improved performance to treat fibrosis and fibroproliferative diseases.

[0005] Integrins serve as key adhesion receptors that facilitate cell -cell interactions and engagement between cells and the extracellular matrix (ECM) (Dustin, Cell, 2019, 177, 499- 501). In disease states such as cancer, ECM adhesion through integrins has been shown to promote cancer cell migration, invasion, and metastasis (Su et al., Front. Pharmacol., 2020, 11 :579068; Hamidi and Ivaska, Nat Rev Cancer, 2018, 18, 533-548; Sheldrake et al., J. Med. Chem., 2014, 57(15), 6301-6315; and Sokeland and Schumacher, Mol. Cancer, 2019, 18, 12). Integrin binding to latency-associated peptide (LAP) has also been shown to be a primary mechanism for liberating activated transforming growth factor beta (TGF-P) (Brown and Marshall, Cancers, 2019, 11, 1221; and Stuelten and Zhang, Front. Cell Dev. Biol. 2021, 9:764727). TGF-P plays many important roles in immune and stem cell regulation and differentiation, and displays or induces strong pro-inflammatory, pro-tumorigenic, pro- fibrotic, and immunosuppressive properties in its local environment (Derynck et al., Sci.Signal. 2019, 12(570), eaav5183). In fibrosis, fibroblasts and macrophages increase production of latent TGF-P, which, as in cancer, is activated by integrins avpi, avP6, and avP8 (Munger et al., Cell, 1999, 96, 319-328; Henderson, et al., Nat. Med., 2013, 19(12), 1617-1624; Kitamura et al., J. Clin. Invest., 2011, 121(7), 2863-2875; and Worthington et al., Immunity, 2015, 42, 903-915).

[0006] Despite tremendous efforts in the field of cancer medication, cancer mortality rates remain high across the globe. Similarly, the rate of fibrosis and mortality associated with fibroproliferative diseases continues to increase. Thus, there remains an urgent need for the development of effective new cancer and fibrosis medications. The present disclosure involving the advancement of novel integrin inhibitors meets this need and provides related advantages.4. SUMMARY

[0007] Provided herein are engineered lasso peptides that potently and specifically bind to integrin receptors and act as integrin inhibitors, and related compositions and methods for the management, prevention and / or treatment of an integrin-mediated disease, such as cancer and / or fibrosis. Compositions and biosynthetic methods for producing the engineered lasso peptides are also provided.

[0008] Particularly, in a first aspect of the present disclosure, provided herein is an engineered lasso peptide including, when cyclized, an integrin binding motif within a loop or a ring of the engineered lasso peptide, wherein the integrin binding motif includes the amino acid sequence RGDXi (SEQ ID NO: 782), wherein Xi is any naturally or non-naturally occurring amino acid residue except for phenylalanine (F) or the amino acid residue of a parent scaffold peptide at that corresponding position, wherein the engineered lasso peptide specifically binds an integrin, and wherein the integrin is selected from alpha V beta 6 (avP6), alpha V beta 8 (avP8), and alpha V beta 1 (avpi). In some embodiments, the Xi is a leucine (L), isoleucine (I), tryptophan (W), lysine (K), glutamine (Q), or arginine (R). In some embodiments, Xi is a leucine (L).

[0009] In some embodiments, the integrin binding motif within the engineered lasso peptide includes the amino acid sequence RGDX1X2 (SEQ ID NO: 788), wherein Xi is any naturally or non-naturally occurring amino acid residue except for phenylalanine (F) or the amino acid residue of a parent scaffold peptide at that corresponding position, wherein the engineered lasso peptide specifically binds an integrin, and wherein X2 is independently any natural or non-natural amino acid residue except for the amino acid residue of a parent scaffold peptide at that corresponding position. In some embodiments, Xi and X2 are independently hydrophobic, polar, or charged amino acid residues. In some embodiments, Xi is independently a leucine (L), isoleucine (I), tryptophan (W), lysine (K), glutamine (Q), or arginine (R), and wherein X2 is independently leucine (L), isoleucine (I), phenylalanine (F), tryptophan (W), lysine (K), glutamine (Q), or arginine (R). In some embodiments, Xi is a leucine (L). In some embodiments, Xi and X2 are independently residues containing an aromatic group, a heteroaromatic group, an alkyl group, a hydroxy group, a halide group, an amino group, a carboxylic acid group, a carboxamide group, or combinations thereof.

[0010] In some embodiments, the integrin binding motif within the engineered lasso peptide includes the amino acid sequence X3RGDX1X2 (SEQ ID NO: 793), wherein Xi is any naturally or non-naturally occurring amino acid residue except for phenylalanine (F) or the amino acid residue of a parent scaffold peptide at that corresponding position, wherein theengineered lasso peptide specifically binds an integrin, wherein X2 and X3 are independently any natural or non-natural amino acid residue except for the amino acid residue of a parent scaffold peptide at that corresponding position. In some embodiments, Xi and X2 are independently hydrophobic, polar, or charged amino acid residues. In some embodiments, Xi, X2, and X3 are independently hydrophobic, polar, or charged amino acid residues. In some embodiments, Xi is independently a leucine (L), isoleucine (I), tryptophan (W), lysine (K), glutamine (Q), or arginine (R), and wherein X2 and X3 are independently alanine (A), leucine (L), isoleucine (I), phenylalanine (F), tryptophan (W), lysine (K), glutamine (Q), or arginine (R). In some embodiments, Xi is a leucine (L). In some embodiments, Xi, X2, and X3 are independently residues containing an aromatic group, a heteroaromatic group, an alkyl group, a hydroxyl group, a halide group, an amino group, a carboxylic acid group, a carboxamide group, or combinations thereof.

[0011] In some embodiments, the engineered lasso peptide provided herein further includes an arginine (R), histidine (H), leucine (L), threonine (T), glutamine (Q), asparagine (N), glycine (G), glutamic acid (E), arginine (K), phenylalanine (F), tryptophan (W) at position 3 located in the ring of the engineered lasso peptide. In some embodiments, wherein the engineered lasso peptide further includes an arginine (R) at position 3 located in the ring of the engineered lasso peptide.

[0012] In some embodiments, the engineered lasso peptide provided herein further includes one, two, three, or four amino acid alterations relative to a parent scaffold peptide. In some embodiments, the alterations include an amino acid substitution, deletion or insertion. In some embodiments, the parent scaffold peptide includes the amino acid sequence of SEQ ID NO: 1, and wherein the alterations include a deletion of proline (P) at position 16 and / or a glutamine (Q) at position 11 in the loop of the engineered lasso peptide and an arginine (R), a histidine (H), or a threonine (T) at position 3 located in the ring of the engineered lasso peptide. In some embodiments, the alterations further include an alteration at position 5 or position 6 located in the ring of the engineered lasso peptide. In some embodiments, the alteration at position 5 located in the ring of the engineered lasso peptide includes a glutamine (Q) or serine (S) at position 5. In some embodiments, the alteration at position 6 located in the ring of the engineered lasso peptide includes an isoleucine (I) at position 6.

[0013] In some embodiments, the engineered lasso peptide provided herein selectively inhibits avP6 function. In some embodiments, such an engineered lasso peptide binds to avP6 with an IC50 < 500 nM. In some embodiments, such an engineered lasso peptide includes the amino acid sequence of any one of SEQ ID NOS: 4-9, 11, 13-21, 23, 24, 26-31,33, 34, 36, 40-48, 50, 54-56, 59, 60, 62, 68, 71, 73, 81, 85-90, 93, 95-103, 163, 168, 187, 189, 190, 192-194, 196-209, 216, 218, 220, 221, 223, 229, 231-233, 242, 245, 247, 249, 251-255, 258, 261-263, 270, 271, 274-276, 282-297, 299-301, 303-316, 318-322, 328-332, 344, 346, 347, and 835-840.

[0014] In some embodiments, engineered lasso peptide provided herein selectively inhibits avP8 function. In some embodiments, such an engineered lasso peptide binds to avP8 with an ICso < 500 nM. In some embodiments, such an engineered lasso includes the amino acid sequence of any one of SEQ ID NOS: 4-9, 11, 13-15, 17-21, 23, 24, 26-29, 31, 36, 40-48, 50, 54-56, 59, 60, 62, 68, 71, 73, 81, 85-90, 93, 95-103, 163, 168, 187, 189, 190, 192-194, 196- 200, 203-209, 216, 218, 220, 221, 223, 229, 231-233, 242, 245, 247, 249, 251-253, 255, 258, 261-263, 270, 271, 274-276, 282-297, 299-301, 303-316, 318-322, 328-332, 344, 346, 347, and 835-840.

[0015] In some embodiments, the engineered lasso peptide provided herein selectively inhibits avpi function. In some embodiments, such an engineered lasso peptide binds to avpi with an IC50 < 500 nM. In some embodiments, such an engineered lasso peptide includes the amino acid sequence of any one of SEQ ID NOS: 6, 7, 87, 88, 97, 100, 163 and 187.

[0016] In some embodiments, the engineered lasso peptide provided herein binds to avP6, avP8, and / or avpi with an IC50 < 50 nM.

[0017] In some embodiments, the engineered lasso peptide provided herein preferentially binds to avP6, avP8, and / or avpi as compared to avP3, avP5, allbp5, a5pi and / or a8pi. In some embodiments, such an engineered lasso peptide binds to avP6, avP8, and / or avpi with an IC50 < 50 nM and binds to avP3, avP5, allbp5, a5pi and / or a8pi with an IC50 > 500 nM.

[0018] In some embodiments, the engineered lasso peptide provided herein further includes a leader sequence, thereby generating a lasso precursor peptide. In some embodiments, the leader sequence includes the amino acid sequence selected from: a) MIKHIHFDKLSSSKKNNVPHSAKGVIQIKKSASQLTK (SEQ ID NO: 777) when the engineered lasso peptide is derived from a parent scaffold peptide including SEQ ID NO: 1, b) MMQQKKNDMKKVTLKKLNKRASKVTR (SEQ ID NO 778) when the engineered lasso peptide is derived from a parent scaffold peptide including SEQ ID NO: 343, c) MKKQTFVPKKLVKVGKATELTK (SEQ ID NO 779) when the engineered lasso peptide is derived from a parent scaffold peptide including SEQ ID NO: ID NO 353, d) MERNHETPSDLIDLGAASVETK (SEQ ID NO 780) when the engineered lasso peptide is derived from a parent scaffold peptide including SEQ ID NO: 362, and e)MTQVSPSPLRLIRVGRALDLTR (SEQ ID NO 781) when the engineered lasso peptide is derived from a parent scaffold peptide including SEQ ID NO: 366.

[0019] In a second aspect of the present disclosure, provided herein is a composition including an engineered lasso peptide provided herein and a buffer, a culture medium or a cellular component.

[0020] In a third aspect of the present disclosure, provided herein is a pharmaceutical composition including an engineered lasso peptide provided herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further includes a second therapeutic agent for managing, preventing or treating cancer and / or fibrosis. In some embodiments, the second therapeutic agent is a chemotherapy or immunotherapy for cancer. In some embodiments, the immunotherapy is an anti-cancer vaccine or immune checkpoint modulator. In some embodiments, the second therapeutic agent is an anti-fibrotic therapy.

[0021] In some embodiments, a pharmaceutical composition provided herein is for use in managing, preventing, or treating cancer and / or fibrosis.

[0022] In a fourth aspect of the present disclosure, provided herein is a method of managing, preventing, or treating an integrin-mediated disease in a subject, including administering to the subject a prophylactically or therapeutically effective amount of an engineered lasso peptide provided herein or the pharmaceutical composition provided herein. In some embodiments, upon administration, the engineered lasso peptide: a) binds to the integrin; b) inhibits a function of the integrin; c) inhibits an integrin-mediated signaling pathway; d) reduces integrin levels on the surface of neoplastic cells, immune cells, and / or fibroblasts in a tumor microenvironment (TME) due to integrin internalization; e) reduces integrin levels on the surface of cells in fibrotic tissues; f) downregulates integrin expression on the surface of neoplastic cells, immune cells, and / or fibroblast cells in a TME; and / or g) downregulates integrin expression on the surface of cells in fibrotic tissues. In some embodiments, the inhibition of the function of the integrin and / or the inhibition of the integrin-mediated signaling pathway is measured by: a) inhibition of binding of isolated integrins to extracellular matrix (ECM) components, fibrinogen, and / or latency-associated peptide (LAP); b) inhibition of adhesion of integrin-expressing cells to ECM components, fibrinogen, and / or LAP; c) inhibition of migration of integrin-expressing cells in a matrix containing ECM components, fibrinogen, and / or LAP; d) inhibition of TGF-P activation; e) inhibition of the release of free TGF-P from its latency complex with LAP; f) inhibition of growth and / or metastasis of neoplastic cells; g) inhibition of the conversion of fibroblasts to activated myofibroblasts; and / or h) inhibition of accumulation of ECM in and / or growth of fibrotictissue. In some embodiments, upon administration, the engineered lasso peptide: a) inhibits the phosphorylation and activation of mothers against decapentaplegic homolog (Smad) transcription factors; b) increases the release of interleukin-y; c) increases the release of tumor necrosis factor-a; d) decreases the production of a-smooth muscle actin, collagen, and / or fibronectin; e) reduces the release of one or more growth factors; and / or f) reduces the release of one or more cytokines. In some embodiments, the one or more growth factors includes TGF-p. In some embodiments, the Smad transcription factors include Smad2 or Smad3. In some embodiments, the one or more cytokines includes IL- 17, IL-6, IL-11, and / or IL-22.

[0023] In some embodiments, for a method of managing, preventing, or treating an integrin- mediated disease in a subject provided herein, upon administration, the engineered lasso peptide: a) increases the proliferation and / or anti-tumor activity of CD8+ cytotoxic T cells in a TME; b) increases the proliferation and / or anti-tumor activity of NK cells in a TME; and / or c) decreases the proliferation of immunosuppressive CD4+ T cells in a TME. In some embodiments, the immunosuppressive CD4+ T cells include regulatory T cells and T helper cells.

[0024] In some embodiments, for a method of managing, preventing, or treating an integrin- mediated disease in a subject provided herein, the integrin-mediated disease includes neoplastic cells expressing one or more integrins. In some embodiments, the subject has a tumor including immune cells expressing one or more integrins in the TME. In some embodiments, the subject has a tumor including stromal cells expressing one or more integrins in the TME. In some embodiments, the stromal cells expressing one or more integrins are cancer associated fibroblasts. In some embodiments, the one or more integrins are RGD-binding integrins. In some embodiments, the one or more integrins are selected from avP6, avP8, and avpi.

[0025] In some embodiments, for a method of managing, preventing, or treating an integrin- mediated disease in a subject provided herein, the integrin-mediated disease is cancer. In some embodiments, the cancer is breast cancer, pancreatic cancer, hepatocellular cancer, prostate cancer, ovarian cancer, gastric cancer, brain or spinal cancer, melanoma, cancer of the head and neck, colorectal cancer, bladder cancer, vulvar cancer, esophageal squamous cell carcinoma, renal cancer, cervical cancer, salivary gland carcinoma, lung cancer, multiple myeloma, or Kaposi’s sarcoma. In some embodiments, the brain or spinal cancer is a glioma. In some embodiments, wherein the glioma is a glioblastoma. In some embodiments, the cancer is melanoma, breast cancer, or ovarian cancer.

[0026] In some embodiments, for a method of managing, preventing, or treating an integrin- mediated disease in a subject provided herein, the integrin-mediated disease is a fibroproliferative disease or fibrosis. In some embodiments, the fibroproliferative disease or fibrosis is a pulmonary fibrosis. In some embodiments, the fibroproliferative disease or fibrosis is selected from cystic fibrosis, idiopathic pulmonary fibrosis, hepatic fibrosis, cirrhosis, pancreatic fibrosis, pancreatitis, renal fibrosis, glial fibrosis, retroperitoneal cavity fibrosis, mediastinal fibrosis, cardiovascular disease and heart fibrosis, myelofibrosis, systemic sclerosis, Dupuytren’s contracture, Peyronie’s disease, macular degeneration, hypertrophic scars, skin fibrosis and skin keloids, arthrofibrosis, Duchenne muscular dystrophy-associated skeletal muscle fibrosis, uterine leiomyoma and adenomyosis.

[0027] In some embodiments, for a method of managing, preventing, or treating an integrin- mediated disease in a subject provided herein, the inhibition of the function of the integrin or the integrin-mediated signaling pathway is a maximal percent inhibition of at least about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the reduction of integrin levels is a maximal percent reduction of at least about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the downregulation of integrin expression is a maximal percent downregulation of at least about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0028] In some embodiments, the engineered lasso peptide provided herein and used in a method of managing, preventing, or treating an integrin-mediated disease in a subject provided herein, is conjugated to an agent. In some embodiments, the agent is selected from the group consisting of a radioisotope, a metal chelator, an enzyme, a protein, a peptide, an antibody, an antibody fragment, a nanobody, a cytotoxic compound, a fluorescent compound, a bioluminescent compound, and a chemiluminescent compound.

[0029] In some embodiments, for a method of managing, preventing, or treating an integrin- mediated disease in a subject provided herein, the method further includes co-administering to the subject a second therapeutic agent with the engineered lasso peptide. In some embodiments, the second therapeutic agent is conjugated with the engineered lasso peptide. In some embodiments, the second therapeutic agent is an immunotherapy or chemotherapy. In some embodiments, the immunotherapy is an anti-cancer vaccine or an immune checkpoint modulator.

[0030] In a fifth aspect of the present disclosure, provided herein is a recombinant nucleic acid encoding an engineered lasso peptide provided herein.

[0031] In a sixth aspect of the present disclosure, provided herein is a recombinant nucleic acid encoding a lasso precursor peptide including an amino acid sequence of an engineered lasso peptide provided herein. In some embodiments, the recombinant nucleic acid includes a nucleotide sequence selected from SEQ ID NOS: 390-397, 399, 401-409, 411, 412, 414- 419, 421, 422, 424, 428-436, 438, 442-444, 447, 448, 450, 456, 459, 461, 469, 473-478, 481, 483-491, 551, 556, 575, 577, 578, 580-582, 584-597, 604, 606, 608, 609, 611, 617, 619-621, 630, 633, 635, 637, 639-643, 646, 649-651, 656, 658, 659, 662-664, 670-685, 687-689, 691- 704, 706-710, 716-720, 732, 734, and 735.

[0032] In some embodiments, a recombinant nucleic acid provided herein includes a nucleotide sequence encoding the engineered lasso peptide operatively linked to a promoter.

[0033] In a seventh aspect of the present disclosure, provided herein is a vector including a recombinant nucleic acid provided herein.

[0034] In an eighth aspect of the present disclosure, provided herein is a non-naturally occurring microbial organism including a recombinant nucleic acid provided herein or a vector provided herein.

[0035] In a ninth aspect of the present disclosure, provided herein is a method for producing an engineered lasso peptide using a non-naturally occurring microbial organism, wherein the method includes: a) introducing into the microbial organism a first nucleic acid including the recombinant nucleic acid provided herein or a vector provided herein and a second nucleic acid encoding a lasso peptide biosynthesis component; and b) culturing the microbial organism under a condition suitable for lasso formation to produce the engineered lasso peptide.

[0036] In some embodiments, for a method for producing an engineered lasso peptide provided herein, the first nucleic acid encodes the engineered lasso peptide provided herein, and wherein the lasso peptide biosynthesis component includes a lasso peptidase capable of catalyzing removal of the leader sequence. In some embodiments, the lasso peptide biosynthesis component includes a lasso cyclase capable of cyclizing a linear lasso core sequence to a mature lasso peptide. In some embodiments, the lasso peptide biosynthesis component includes a lasso peptidase and a lasso cyclase, and wherein the method includes introducing the second nucleic acid sequence encoding the lasso cyclase and a third nucleic acid sequence encoding the lasso peptidase. In some embodiments, the lasso peptide biosynthesis component includes a lasso cyclase and a post-translationally modified peptide (RiPP) recognition element (RRE). In some embodiments, the lasso peptide biosynthesis component includes a lasso cyclase and a post-translationally modified peptide (RiPP)recognition element (RRE), and wherein the method includes introducing the second nucleic acid sequence encoding the lasso cyclase and a fourth nucleic acid sequence encoding the RRE. In some embodiments, the lasso peptide biosynthesis component includes a lasso peptidase, a lasso cyclase and a post-translationally modified peptide (RiPP) recognition element (RRE). In some embodiments, the lasso peptide biosynthesis component includes a lasso peptidase, a lasso cyclase and a post-translationally modified peptide (RiPP) recognition element (RRE), and wherein the method includes introducing the second nucleic acid sequence encoding the lasso cyclase, a third nucleic acid sequence encoding the lasso peptidase, and a fourth nucleic acid sequence encoding the RRE. In some embodiments, at least two of the first, second, third and fourth nucleic acid sequences are in a same nucleic acid molecule.

[0037] In some embodiments, for a method for producing an engineered lasso peptide provided herein, the microbial organism is E.coli, Vibrio nalriegens, Burkholderia spp., Corynebacterium gliilamicum. Sphingomonas subterranean, Pseudomonas fluorescens, Pseudomonas putida, Saccharomyces cerevisiae, Pichia pasloris, Rhodococcus jostii, Saccharopolyspora erylhraea, Streptomyces lividans, Streptomyces coelicolor, Streptomyces albus, or Streptomyces venezuelae .

[0038] In some embodiments, for a method for producing an engineered lasso peptide provided herein, the culturing is performed under aerobic and / or glucose-limiting conditions. In some embodiments, the method further includes isolating the engineered lasso peptide from the culture medium of the microbial organism.5. BRIEF DESCRIPTION OF THE FIGURES

[0039] The details of one or more embodiments of the subject application are set forth in the accompanying drawings and the description below. Other features, objects, and benefits of the embodiments described herein will be apparent from the description and drawings, and from the claims. All publications, patents and patent applications cited herein are hereby expressly incorporated by reference for all purposes.

[0040] The embodiments of the description described herein are not intended to be exhaustive or to limit the disclosure to the precise forms disclosed in the following drawings or detailed description. Rather, the embodiments are chosen and described so that others skilled in the art can appreciate and understand the principles and practices of the description.

[0041] FIG. 1A is a schematic illustration of a lasso peptide with the characteristic lasso (lariat) topology.

[0042] FIG. IB is a schematic illustration of the lasso peptide biosynthesis pathway, including the genes and gene products involved in the enzymatic reactions resulting in formation of matured lasso peptide having the characteristic lasso (lariat) topology.

[0043] FIG. 2 is a schematic illustration showing the twenty -four different mammalian integrin receptors derived from twenty-six different a and b subunits.

[0044] FIG. 3 is a schematic illustration of a heterodimer integrin with a and P subunits bound to the extracellular matrix (ECM) protein fibronectin.

[0045] FIG. 4 is a schematic illustration showing integrin-mediated TGF-P activation in the tumor microenvironment through LAP binding to (A) avP8 on the surface of regulatory T cells (Tregs), and / or (B) avP6 on the surface of cancer cells, both leading to free TGF-P which binds to TGF-P receptors on immune cells, such as cytotoxic T cells and NK cells, and suppresses the anti-tumor immune response.

[0046] FIG. 5 is a schematic illustration of cell-free and cell-based methods for producing lasso peptides as part of an iterative workflow involving in silico modeling and experimental testing to validate and optimize biological activities and other properties of lasso peptides according to the present disclosure.

[0047] FIG. 6 is a schematic illustration showing the process of engineering a lasso peptide from a parent scaffold peptide by computational RGDXi (SEQ ID NO: 782) motif scanning that includes introducing the motif into different loop, ring, and / or tail positions, synthesizing engineered lasso peptides grafted with the RGDXi (SEQ ID NO: 782) binding motif with the best predicted positions, such as into the loop positions (A) or into the ring positions (B), and selecting the top performing engineered lasso peptides based on the experimental assays described in the Examples provided herein. The balls represent individual amino acids, where Xi is any naturally or non-naturally occurring amino acid residue or the amino acid residue of a parent scaffold peptide at that corresponding position.

[0048] FIG. 7 illustrates the engineering of parent lasso peptide (SEQ ID NO 1), which displays no binding to integrins, into a specific and selective avP6 / 8 inhibitor by first grafting the motif RGDL (SEQ ID NO: 783) into the loop, leading to specific avP6 / 8 binding, followed by lasso peptide evolution through amino acid deletion and / or mutagenesis. Black balls are sites of amino acid mutations vs parent.

[0049] FIG. 8 provides ELISA curves showing inhibition of avP6 by four lasso-based integrin inhibitors (SEQ ID NOS: 87, 97, 100, and 163) along with the pan-integrin inhibitor MK-0429.

[0050] FIG. 9 shows an exemplary tumor growth inhibition graph for SEQ ID NO 163 after dosing for 18 days in Balb / c mice bearing subcutaneous EMT6 breast cancer tumors in their flank.

[0051] FIG. 10 shows an exemplary tumor inhibition graph for individual Balb / c mice bearing EMT6 tumors after dosing with SEQ ID NO 163. Results shows strong tumor growth inhibition in 7 out of 10 mice and three mice had tumors regress.6. DETAILED DESCRIPTION

[0052] The novel features of the subject matter described herein are set forth specifically in the appended claims. A better understanding of the features and benefits of the present disclose will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the claimed embodiments are utilized. To facilitate a full understanding of the disclosure set forth herein, a number of terms are defined below.6.1. General Techniques

[0053] Techniques and procedures described or referenced herein include those that are generally well understood and / or commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies described in Sambrook et cd.. Molecular Cloning: A Laboratory Manual (4th ed. 2012); Current Protocols in Molecular Biology (Ausubel et al. eds., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009); Monoclonal Antibodies: Methods and Protocols (Albitar ed. 2010); Antibody Engineering Vols 1 and 2 (Kontermann and Diibel eds., 2nd ed. 2010);Molecular Biology of the Cell (6th Ed., 2014); March's Advanced Organic Chemistry (6thed. 2007); Lasso Peptides, (Li, Y.; Zirah, S.; Rebuffet, S., Springer; New York, 2015); Natural Products in Medicinal Chemistry, Methods and Principles in Medicinal Chemistry (Hanessian, S., ed., Wiley-VCH; 1st edition, 2014); and Basic Principles of Drug Discovery and Development (Blass, B. Academic Press; 2ndedition, 2021).6.2. Terminology

[0054] Unless described otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. All patents, applications, published applications, and other publications are incorporated by reference in their entirety. In the event that any description of terms set forth conflicts with any document incorporated herein by reference, the description of term set forth below shall control.Conventions and Abbreviations

[0055] The singular terms “a,” “an,” and “the” as used herein include the plural reference unless the context clearly indicates otherwise.

[0056] The terms “about” and “approximately” mean an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain instances, the term “about” or “approximately” means within 1, 2, 3, or 4 standard deviations. In certain instances, the term “about” or “approximately” means within 50%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range.

[0057] The term “alpha V beta 6” or “avP6” or similar term refers to an integrin having an a chain V subunit and an P chain 6 subunit from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkey (cyno)), dogs, and rodents (e.g., mice and rats), unless otherwise indicated. The term avP6 encompasses “full-length,” unprocessed a chain V subunit and P chain 6 subunit, as well as any form of a chain V subunit and P chain 6 subunit or any fragment thereof that results from processing in the cell. The term avP6 also encompasses naturally occurring variants of avP6, such as isoforms, SNP variants, splice variants and allelic variants. The full-length amino acid sequence of human a chain V subunit can be found at GenBank Accession No. NP 002201.1 and is provided below: MAFPPRRRLRLGPRGLPLLLSGLLLPLCRAFNLDVDSPAEYSGPEGSYFGFAVDFFVP SASSRMFLLVGAPKANTTQPGIVEGGQVLKCDWSSTRRCQPIEFDATGNRDYAKDD PLEFKSHQWFGASVRSKQDKILACAPLYHWRTEMKQEREPVGTCFLQDGTKTVEYA PCRSQDIDADGQGFCQGGFSIDFTKADRVLLGGPGSFYWQGQLISDQVAEIVSKYDP NVYSIKYNNQLATRTAQAIFDDSYLGYSVAVGDFNGDGIDDFVSGVPRAARTLGMV YIYDGKNMSSLYNFTGEQMAAYFGFSVAATDINGDDYADVFIGAPLFMDRGSDGKL QEVGQVSVSLQRASGDFQTTKLNGFEVFARFGSAIAPLGDLDQDGFNDIAIAAPYGG EDKKGIVYIFNGRSTGLNAVPSQILEGQWAARSMPPSFGYSMKGATDIDKNGYPDLI VGAFGVDRAILYRARPVITVNAGLEVYPSILNQDNKTCSLPGTALKVSCFNVRFCLK ADGKGVLPRKLNFQVELLLDKLKQKGAIRRALFLYSRSPSHSKNMTISRGGLMQCEE LIAYLRDESEFRDKLTPITIFMEYRLDYRTAADTTGLQPILNQFTPANISRQAHILLDC GEDNVCKPKLEVSVDSDQKKIYIGDDNPLTLIVKAQNQGEGAYEAELIVSIPLQADFI GVVRNNEALARLSCAFKTENQTRQVVCDLGNPMKAGTQLLAGLRFSVHQQSEMDT SVKFDLQIQSSNLFDKVSPVVSHKVDLAVLAAVEIRGVSSPDHVFLPIPNWEHKENPE TEEDVGPVVQHIYELRNNGPSSFSKAMLHLQWPYKYNNNTLLYILHYDIDGPMNCT SDMEINPLRIKISSLQTTEKNDTVAGQGERDHLITKRDLALSEGDIHTLGCGVAQCLKI VCQVGRLDRGKSAILYVKSLLWTETFMNKENQNHSYSLKSSASFNVIEFPYKNLPIE DITNSTLVTTNVTWGIQPAPMPVPVWVIILAVLAGLLLLAVLVFVMYRMGFFKRVRP PQEEQEREQLQPHENGEGNSET (SEQ ID NO: 784).

[0058] The full-length amino acid sequence of human P chain 6 subunit can be found at GenBank Accession No. Pl 8564 and is provided below:MGIELLCLFFLFLGRNDHVQGGCALGGAETCEDCLLIGPQCAWCAQENFTHPSGVGE RCDTPANLLAKGCQLNFIENPVSQVEILKNKPLSVGRQKNSSDIVQIAPQSLILKLRPG GAQTLQVHVRQTEDYPVDLYYLMDLSASMDDDLNTIKELGSRLSKEMSKLTSNFRL GFGSFVEKPVSPFVKTTPEEIANPCSSIPYFCLPTFGFKHILPLTNDAERFNEIVKNQKISANIDTPEGGFDAIMQAAVCKEKIGWRNDSLHLLVFVSDADSHFGMDSKLAGIVIPND GLCHLDSKNEYSMSTVLEYPTIGQLIDKLVQNNVLLIFAVTQEQVHLYENYAKLIPG ATVGLLQKDSGNILQLIISAYEELRSEVELEVLGDTEGLNLSFTAICNNGTLFQHQKK CSHMKVGDTASFSVTVNIPHCERRSRHIIIKPVGLGDALELLVSPECNCDCQKEVEVN SSKCHHGNGSFQCGVCACHPGHMGPRCECGEDMLSTDSCKEAPDHPSCSGRGDCYC GQCICHLSPYGNIYGPYCQCDNFSCVRHKGLLCGGNGDCDCGECVCRSGWTGEYCN CTTSTDSCVSEDGVLCSGRGDCVCGKCVCTNPGASGPTCERCPTCGDPCNSKRSCIE CHLSAAGQAREECVDKCKLAGATISEEEDFSKDGSVSCSLQGENECLITFLITTDNEG KTIIHSINEKDCPKPPNIPMIMLGVSLAILLIGVVLLCIWKLLVSFHDRKEVAKFEAERS KAKWQTGTNPLYRGSTSTFKNVTYKHREKQKVDLSTDC (SEQ ID NO: 785).

[0059] Other related avP6 integrins that are also encompassed by the term avP6 include fragments, derivatives (e.g., substitution, deletion, truncations, and insertion variants), fusion polypeptides, and interspecies homologs that retain avP6 activity and / or are sufficient to generate avP6 signaling. As those skilled in the art will appreciate, an engineered lasso peptide described herein can bind to an avP6 integrin, a fragment of avP6 integrin, and / or an epitope on the avP6 integrin. An epitope may be part of a larger polypeptide, which may be part of a larger avP6 protein, which, in turn, may be part of a larger avP6 complex. An avP6 integrin may exist in a native or denatured form. An avP6 integrin described herein may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods. An avP6 integrin may include a polypeptide having the same amino acid sequence as a corresponding avP6 integrin derived from nature.

[0060] The term “alpha V beta 8” or “avP8” or similar term refers to an integrin having an a chain V subunit and an P chain 8 subunit from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkey (cyno)), dogs, and rodents (e.g., mice and rats), unless otherwise indicated. The term avP8 encompasses “full-length,” unprocessed a chain V subunit and P chain 8 subunit, as well as any form of a chain V subunit and P chain 8 subunit or any fragment thereof that results from processing in the cell. The term avP8 also encompasses naturally occurring variants of avP8, such as isoforms, SNP variants, splice variants and allelic variants. The full-length amino acid sequence of human a chain V subunit is provided above as SEQ ID NO: 784. The full-length amino acid sequence of human p chain 8 subunit can be found at GenBank Accession No. P26012.1 and is provided below:MCGSALAFFTAAF VCLQNDRRGP ASFLWAAWVF SLVLGLGQGEDNRC AS SNAASC ARCLALGPECGWCVQEDFISGGSRSERCDIVSNLISKGCSVDSIEYPSVHVIIPTENEINTQVTPGEVSIQLRPGAEANFMLKVHPLKKYPVDLYYLVDVSASMHNNIEKLNSVGN DLSRKMAFFSRDFRLGFGSYVDKTVSPYISIHPERIHNQCSDYNLDCMPPHGYIHVLS LTENITEFEKAVHRQKISGNIDTPEGGFDAMLQAAVCESHIGWRKEAKRLLLVMTDQ TSHLALDSKLAGIVVPNDGNCHLKNNVYVKSTTMEHPSLGQLSEKLIDNNINVIFAV QGKQFHWYKDLLPLLPGTIAGEIESKAANLNNLVVEAYQKLISEVKVQVENQVQGIY FNITAICPDGSRKPGMEGCRNVTSNDEVLFNVTVTMKKCDVTGGKNYAIIKPIGFNET AKIHIHRNCSCQCEDNRGPKGKCVDETFLDSKCFQCDENKCHFDEDQFSSESCKSHK DQPVCSGRGVCVCGKCSCHKIKLGKVYGKYCEKDDFSCPYHHGNLCAGHGECEAG RCQCFSGWEGDRCQCPSAAAQHCVNSKGQVCSGRGTCVCGRCECTDPRSIGRFCEH CPTCYTACKENWNCMQCLHPHNLSQAILDQCKTSCALMEQQHYVDQTSECFSSPSY LRIFFIIFIVTFLIGLLKVLIIRQVILQWNSNKIKSSSDYRVSASKKDKLILQSVCTRAVT YRREKPEEIKMDISKLNAHETFRCNF (SEQ ID NO: 786).

[0061] Other related avP8 integrins that are also encompassed by the term avP8 include fragments, derivatives (e.g., substitution, deletion, truncations, and insertion variants), fusion polypeptides, and interspecies homologs that retain avP8 activity and / or are sufficient to generate avP8 signaling. As those skilled in the art will appreciate, an engineered lasso peptide described herein can bind to an avP8 integrin, a fragment of avP8 integrin, and / or an epitope on the avP8 integrin. An epitope may be part of a larger polypeptide, which may be part of a larger avP8 protein, which, in turn, may be part of a larger avP8 complex. An avP8 integrin may exist in a native or denatured form. An avP8 integrin described herein may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods. An avP8 integrin may include a polypeptide having the same amino acid sequence as a corresponding avP8 integrin derived from nature.

[0062] The term “alpha V beta 1” or “avpi” or similar term refers to an integrin having an a chain V subunit and an P chain 1 subunit from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkey (cyno)), dogs, and rodents (e.g., mice and rats), unless otherwise indicated. The term avpi encompasses “full-length,” unprocessed a chain V subunit and P chain 1 subunit, as well as any form of a chain V subunit and P chain 1 subunit or any fragment thereof that results from processing in the cell. The term avpi also encompasses naturally occurring variants of avpi, such as isoforms (including any one of the six known isoforms of human P chain 1 subunit), SNP variants, splice variants and allelic variants. The full-length amino acid sequence of a chain V subunit is provided above as SEQ ID NO: 784. The full-length amino acid sequence of human P chain 1 subunit can be found at GenBank Accession No. P05556 and is provided below:MNLQPIFWIGLISSVCCVFAQTDENRCLKANAKSCGECIQAGPNCGWCTNSTFLQEG MPTSARCDDLEALKKKGCPPDDIENPRGSKDIKKNKNVTNRSKGTAEKLKPEDITQI QPQQLVLRLRSGEPQTFTLKFKRAEDYPIDLYYLMDLSYSMKDDLENVKSLGTDLM NEMRRITSDFRIGFGSFVEKTVMPYISTTPAKLRNPCTSEQNCTSPFSYKNVLSLTNKG EVFNELVGKQRISGNLDSPEGGFDAIMQVAVCGSLIGWRNVTRLLVFSTDAGFHFAG DGKLGGIVLPNDGQCHLENNMYTMSHYYDYPSIAHLVQKLSENNIQTIFAVTEEFQP VYKELKNLIPKSAVGTLSANSSNVIQLIIDAYNSLSSEVILENGKLSEGVTISYKSYCK NGVNGTGENGRKCSNISIGDEVQFEISITSNKCPKKDSDSFKIRPLGFTEEVEVILQYIC ECECQSEGIPESPKCHEGNGTFECGACRCNEGRVGRHCECSTDEVNSEDMDAYCRK ENSSEICSNNGECVCGQCVCRKRDNTNEIYSGKFCECDNFNCDRSNGLICGGNGVCK CRVCECNPNYTGSACDCSLDTSTCEASNGQICNGRGICECGVCKCTDPKFQGQTCEM CQTCLGVCAEHKECVQCRAFNKGEKKDTCTQECSYFNITKVESRDKLPQPVQPDPVS HCKEKDVDDCWFYFTYSVNGNNEVMVHVVENPECPTGPDIIPIVAGVVAGIVLIGLA LLLIWKLLMIIHDRREFAKFEKEKMNAKWDTGENPIYKSAVTTVVNPKYEGK (SEQ ID NO: 787).

[0063] Other related avpi integrins that are also encompassed by the term avpi include fragments, derivatives (e.g., substitution, deletion, truncations, and insertion variants), fusion polypeptides, and interspecies homologs that retain avpi activity and / or are sufficient to generate avpi signaling. As those skilled in the art will appreciate, an engineered lasso peptide described herein can bind to an avpi integrin, a fragment of avpi integrin, and / or an epitope on the avpi integrin. An epitope may be part of a larger polypeptide, which may be part of a larger avpi protein, which, in turn, may be part of a larger avpi complex. An avpi integrin may exist in a native or denatured form. An avpi integrin described herein may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods. An avpi integrin may include a polypeptide having the same amino acid sequence as a corresponding avpi integrin derived from nature.

[0064] The terms “administer,” “administration,” or grammatical equivalent thereof refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., an engineered lasso peptide as described herein) into a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and / or any other method of physical delivery described herein or known in the art. When a disease, disorder, condition, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease, disorder, condition, or symptoms thereof. When a disease, disorder, condition, or symptoms thereof, are being prevented, administration of the substancetypically occurs before the onset of the disease, disorder, condition, or symptoms thereof. When two or more substances are being administered, this can be referred as coadministration and refers to the simultaneous or sequential administration of at least two substances according to the present disclosure. For example, an engineered lasso peptide as disclosed herein can be administered with another therapeutic agent simultaneously or sequentially in separate unit dosage forms or together in a single unit dosage form. When substances are administered repeatedly to maintain the initial therapeutic effect (activity) in a continuous mode (e.g., for a period of time such as days, weeks, months, or years), such administration can be referred to as chronic administration, which is contrast to an acute mode. Another type of administration includes intermittent administration, which is treatment that is not consecutively done without interruption, but rather is cyclic in nature.

[0065] The term “aerobic” when used in reference to a culture or growth condition means that free oxygen (O2) is available in the culture or growth condition. This includes when the dissolved oxygen in the liquid medium is more than 50% of saturation.

[0066] The term “alteration” or grammatical equivalents thereof as used herein in reference to any peptide, polypeptide, protein, nucleic acid or polynucleotide described herein refers to a change in structure of an amino acid residue, an amino acid sequence, a nucleic acid base, or an nucleic acid sequence relative to the starting or reference residue, base or sequence. An alteration of an amino acid residue includes, for example, deletions, insertions and substitutions. An alteration of a nucleic acid base includes, for example, changing one naturally occurring base for a different naturally occurring base, such as changing an adenine to a thymine or a guanine to a cytosine or an adenine to a cytosine or a guanine to a thymine. An alteration of a nucleic acid base can result in an alteration of the encoding peptide, polypeptide or protein by changing the encoded amino acid residue or function of the peptide, polypeptide or protein. An alteration of a nucleic acid base may not result in an alteration of the amino acid sequence or function of encoded peptide, polypeptide or protein, also known as a silent mutation.

[0067] The term “amino acid” refers to naturally occurring and non-naturally occurring alpha-amino acids, as well as alpha-amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring alpha-amino acids. Naturally encoded amino acids are the 22 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, pyrrolysine and selenocysteine). Amino acid analogs or derivatives refers to compounds that have the samebasic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and a side chain R group, such as, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (such as, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. The terms “nonnatural amino acid” or “non-proteinogenic amino acid” or “unnatural amino acid” or “non- canonical” refer to alpha-amino acids that contain different side chains (different R groups) relative to those that appear in the twenty -two common or naturally occurring amino acids listed above. In addition, these terms also can refer to amino acids that are described as having D-stereochemistry, rather than L-stereochemistry of natural amino acids, despite the fact that some amino acids do occur in the D-stereochemical form in nature (e.g., D-alanine and D-serine).

[0068] The terms “binding” and “binds” refer to an interaction between molecules (e.g., nucleic acids, oligonucleotides, proteins, polypeptides, or peptides) including, for example, the formation of a complex. Interactions can be, for example, non-covalent interactions including hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Waals interactions. A complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of the total non- covalent interactions between a single target-binding site of a binding molecule (e.g., protein, polypeptide, or peptide) and a single target site of a target molecule is the affinity of the binding molecule for that target site. For example, the ratio of dissociation rate (koir) to association rate (kon) of a binding protein (e.g., an engineered lasso peptide) to a monovalent target site (k0ff / k0n) is the equilibrium dissociation constant KD, which is inversely related to affinity. The KD is commonly used to describe how tightly a ligand binds to a particular protein, and is the inverse of the association constant. The lower the KD value, the higher the affinity of the binding protein. The value of KD varies for different complexes of binding molecules and target molecules and depends on both konand koir. When the binding of a binding protein results in inhibition of the target molecule, such binding can be described by an inhibition constant (Ki), which is the equilibrium dissociation constant for an enzyme inhibitor, and provides an indication of the potency of an inhibitor. In other words, the Ki also represents a KD, but more narrowly for the binding of an inhibitor to a target molecule; abinding protein whose binding reduces the activity of the target molecule. The binding equilibrium described by the Ki value depends on the kinetic mechanism of inhibition. The KD or Ki for a binding protein (e.g., an engineered lasso peptide) provided herein can be determined using any method provided herein or any other method well known to those skilled in the art. In general, the Ki value is used whenever the binding constant is measured through inhibition kinetics, while the KD value is preferred when the binding is measured more directly (e.g., by fluorescence quenching, isothermal titration calorimetry, or surface plasmon resonance). The affinity at one binding site does not always reflect the true strength of the interaction between a binding protein and the target molecule. When complex target molecule containing multiple, repeating target sites, such as a polyvalent target protein, come in contact with lasso peptides containing multiple target binding sites, the interaction of the lasso peptide with the target protein at one site will increase the probability of a reaction at a second site.

[0069] The term “binding affinity” generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., a binding protein such as an engineered lasso peptide) and its binding partner (e.g., a target protein). Binding affinity may be the intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., an engineered lasso peptide and target protein). The affinity of a binding molecule X for its binding partner Y can generally be represented by the KD. Similarly, the affinity of an inhibiting molecule X for its binding partner Y can generally be represented by the Ki. Affinity can be measured by common methods known in the art, including those described herein. Low-affinity lasso peptides generally bind target proteins slowly and tend to dissociate readily, whereas high-affinity lasso peptides generally bind target proteins faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure. Specific illustrative embodiments include the following: the “KD” or “KD value” can be measured by assays known in the art, for example by a binding assay, including in a radioimmunoassay (RIA), a surface plasmon resonance assay as provided by Biacore®, using, for example, a Biacore®TM-2000 or a Biacore®TM-3000, or by biolayer interferometry using, for example, the Octet®QK384 system, performed with the engineered lasso peptide described herein and its target protein (e.g., integrin); an “on-rate” or “rate of association” or “association rate” or “kon” can also be determined with the same surface plasmon resonance or biolayer interferometry techniques described above using, for example, a Biacore®TM- 2000 or a Biacore®TM-3000, or the Octet®QK384 system; and the “Ki” or “Ki value” can bemeasured by assays known in the art, for example, direct estimation of Ki and rate of enzyme inactivation (kinact) from time-dependent IC50 values as described in Krippendorff etal., J. Biomolecular Screening, 2009, 14(8): 913-923, or an endpoint competition assay as described in Miyahisa etal., Angew Chem Int Ed Engl. 2015, 54(47): 14099-14102.

[0070] The term “carrier” as used herein refers to a substrate that is useful in preparing a composition for delivery of an active component of the composition. Generally, a physiologically acceptable carrier is neither biologically nor otherwise undesirable, which include carriers, excipients, or stabilizers that are nontoxic to a cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers, such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (e.g., fewer than about 10 amino acid residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and / or nonionic surfactants, such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™. The term carrier can also refer to a diluent, adjuvant (e.g., Freund’s adjuvant (complete or incomplete)), or vehicle. Such carriers, including pharmaceutical carriers, can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is an exemplary carrier when a composition (e.g., a pharmaceutical composition) is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients (e.g., pharmaceutical excipients) include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. Compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations, and the like. Oral compositions, including formulations, can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington ’s Pharmaceutical Sciences (A.R.Gennaro, 19th ed. 1995, Mack Publishing Company). Compositions, including pharmaceutical compounds, can contain an engineered lasso peptide, for example, in isolated or purified form, together with suitable amounts of carriers.

[0071] The term “chemotherapy” as used herein refers to systemic treatment a subject suffering from or at risk of suffering from cancer with one or more anticancer drugs. Types of chemotherapy include adjuvant chemotherapy (treatment of a patient after the primary tumor has been removed and there is no evidence that cancer remains in the body; given to improve survival), primary chemotherapy (also referred to as neoadjuvant chemotherapy; treatment of a cancer with an anticancer drug as the primary treatment or prior to surgery or radiation), or combination chemotherapy (the use of two or more anticancer drugs to treat a patient). Anticancer drugs that can be used for chemotherapy include alkylating agents (e.g., cyclophosphamide and mustargen), platinum drugs (e.g., cisplatin, carboplatin, and oxaliplatin), antimetabolites (e.g., 5-fluorouracil, tegafur, and uracil), antibiotics (e.g., doxorubicin, daunorubicin, idarubicin, epirubicin, dactinomycin, and bleomycin), topoisomerase inhibitors (e.g., etoposide, teniposide, topotecan, and irinotecan), antimicrotubule agents (e.g., vincristine, vinblastine, vinorelbine, paclitaxel, docetaxel, and estramustine phosphate) and hormones (e.g., tamoxifen, leuprolide acetate, and goserelin).

[0072] The term “condition suitable for lasso formation,” depending on the context, can refer to, for example, a condition suitable for the expression of one or more protein products in a bacterial host (e.g., a lasso precursor peptide, or a processing enzyme). Exemplary suitable conditions include, but are not limited to, a suitable culturing condition of the bacterial host that enable the protein synthesis and transportation in the host cell. Additionally, or alternatively, depending on the context, the term “condition suitable for lasso formation” can refer to, for example, a condition suitable for post-translational modification of a lasso precursor peptide. Exemplary suitable conditions include, but are not limited to, a suitable temperature and / or incubation time for a lasso cyclase and / or lasso peptidase to process the lasso precursor into a matured lasso peptide.

[0073] The term “conjugate” as used herein refers to the joining together of two or more of the same or different molecules by the formation of a covalent bond. The conjugation of two or more molecules can result in a heterologous molecule being formed (e.g., an engineered lasso peptide and a therapeutic agent).

[0074] The terms “downregulate” and “downregulation” as used herein refer to lowering the rate or level a molecule relative to a control. Downregulation of a molecule can be expressedas a percentage (e.g., 1%, 2%, 5%, 10%, 20%, 25%, 50%, 75%, 90%, 95%, 99%) or by a fold change (i.e., 1 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 4 fold, 5 fold, 10 fold or more).

[0075] The term “effective amount” refers to an amount that is sufficient to effect one or more beneficial or desired results, such as beneficial or desired clinical results, or other desired effects. An effective amount may also be an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate the symptoms and / or underlying cause, prevent the occurrence of symptoms and / or their underlying cause, and / or improve or remediate the damage that results from or is associated with a disease, disorder, or condition described herein. An effective amount may also an amount that produces a prophylactic effect, such as an amount that delays, reduces, or eliminates the appearance of a pathological or undesired condition associated with a disease, disorder, or condition described herein. An effective amount can be administered in one or more administrations.

[0076] The term “encoding” or grammatical equivalents thereof as it is used in reference to nucleic acid molecule refers to a nucleic acid molecule in its native state or when manipulated by methods well known to those skilled in the art that can be transcribed to produce mRNA, which is then translated into a polypeptide and / or a fragment thereof. The antisense strand is the complement of such a nucleic acid molecule, and the encoding sequence can be deduced therefrom.

[0077] The terms “engineered” and “variant” as used here in when used in reference to any peptide, polypeptide, protein, nucleic acid or polynucleotide described herein refer to a sequence of amino acids or nucleic acids having at least one alteration (e.g., substitution) at an amino acid residue or nucleic acid base as compared to a parent sequence. The parent sequence of amino acids or nucleic acids can be, for example, a wild-type sequence or a homolog thereof, or a variant of a wild-type sequence or homolog thereof. Such an engineered or variant sequence of amino acids or nucleic acids is not naturally occurring. Accordingly, an engineered lasso peptide or engineered lasso refers to a non-naturally occurring analog, derivative, or variant of a naturally occurring lasso peptide, which analog, derivative or variant is also a lasso peptide itself.

[0078] The term “glucose-limiting” as used herein when used in reference to culturing conditions references to use of media having a minimal about of glucose needed for survival of the host cell. Such minimal glucose media can include media having no more than about 30 mM, no more than about 25 mM, no more than about 20 mM, no more than about 15 mM, no more than about 10 mM, no more than about 5 mM, no more than about 2 mM glucose.

[0079] The term “IC50” refers an amount, concentration, or dosage of a compound that results in 50% inhibition of a maximal response in an assay that measures such response.

[0080] The term “grafting” or a grammatical equivalent thereof as used herein means inserting a sequence (e.g., in case of a linear motif) or multiple sequences (e.g., in case of a conformational motif), such as an integrin binding motif described herein, into the sequence of a different peptide or polypeptide, such that the grafted peptide or polypeptide gains the activity of the grafted motif (e.g., binding capability and specificity). Accordingly, a motif of a peptide or polypeptide can be “grafted” into another peptide or polypeptide, for example, by replacing a segment (e.g., in case of a linear motif) or segments (e.g., in case of a conformation motif) of the original sequence of the peptide or peptide with the sequence or sequences of the motif.

[0081] The terms “inhibition” or “inhibitor” as used herein refers to the act of or a molecule which is capable of inhibiting or reducing (including partially inhibiting or allosteric inhibition) one or more of the normal biological activities of a target molecule (e.g, an integrin). Inhibitors, for example, act by reducing or suppressing the activity of a target molecule and / or reducing or suppressing signal transduction that normally is induced by the binding of a natural ligand to the target protein. In some instances, an engineered lasso peptide described herein can be characterized an integrin inhibitor because it causes substantially complete inhibition of one or more of the 24 human integrins described herein. In particular, an engineered lasso peptide described herein causes substantially complete inhibition of one or more integrins selected from avP6, avP8, and avpi. A molecule may also be referred to as a “partial inhibitor,” which refers to a molecule which can induce a partial response for example, by partially reducing or suppressing the activity of a target molecule and / or partially reducing or suppressing signal transduction. In some instances, a partial inhibitor mimics the spatial arrangement, electronic properties, or some other physicochemical and / or biological property of the inhibitor. In some instances, in the presence of elevated levels of an inhibitor, a partial inhibitor competes with the inhibitor for occupancy of the target molecule and provides a reduction in efficacy, relative to the inhibitor alone. Inhibition may be partial (such as, 1%, 2%, 5%, 10%, 20%, 25%, 50%, 75%, 90%, 95%, 99%) or complete (z.e., 100%) inhibition.

[0082] The term “immunotherapy” as used herein refers to treatment of a subject suffering from or at risk of suffering from a disease by a method that includes inducing, enhancing, suppressing or other modification of an immune response by use of an immunomodulator. Immunomodulators that can be used for immunotherapy include interleukins, cytokines,chemokines, cytosine phosphate-guanosine, oligodeoxynucleotides and glucans, and cells, such as T cells, lymphocytes, macrophages, dendritic cells, natural killer cells, cytotoxic T lymphocytes, immune checkpoint modulators (e.g., immune checkpoint inhibitors or immune checkpoint stimulators), and vaccines (e.g., anti-cancer vaccines). For example, an immune checkpoint inhibitor works by blocking checkpoint proteins from binding with their partner proteins, thereby preventing the “off’ signal from being sent, allowing T cells to kill a target cell (e.g., a cancer cell). As another example, anti-cancer vaccines help a subject’s immune system to recognize and react to antigens that are specific to cancer cells (e.g., tumor- associated antigens). Use of such immunomodulators or cells supplements, enhances, replaces or otherwise modifies the subject's own inadequate or inappropriate immune response. In the context of cancer, immunotherapy can refer to stimulation of the immune system to reject and destroy tumors, for example, with cytokines or cells. Also, in the context of cancer, adoptive immunotherapy involves the administration of cells having anti-tumor activity, including activated or expanded T cells, lymphocytes, macrophages, dendritic cells, natural killer cells and cytotoxic T lymphocytes. Such cells are administered to a subject with the aim that the cells mediate either directly or indirectly specific immunity to tumor cells and / or antigenic components or regression of the tumor.Active immunotherapy involves injection of cells or proteins to generate either new or enhance systemic immune responses to the administered cell or protein.Passive immunotherapy involves the administration of an antibody.

[0083] The terms “isolated,” “isolate,” and “isolating,” or grammatical equivalent thereof, when used in reference to a nucleic acid, protein, polypeptide, peptide, or cell, refer to a nucleic acid, protein, polypeptide, peptide, or cell that is substantially free of at least one component relative to the referenced nucleic acid, protein, polypeptide, peptide, or cell is found in nature or in its current environment. The term includes a nucleic acid, protein, polypeptide, peptide or cell that is removed from some or all components as it is found in its natural environment. Therefore, an isolated nucleic acid, protein, polypeptide, peptide, or cell is partly or completely separated from other substances as it is found in nature or as it is grown, stored or subsisted in non-naturally occurring environments (e.g., laboratories). Specific examples of isolated nucleic acid, protein, polypeptide, peptide, or cell include a partially pure nucleic acid, protein, polypeptide, peptide, or cell, a substantially pure nucleic acid, protein, polypeptide, peptide, or cell, a cell cultured in a medium that is non-naturally occurring, a protein, polypeptide, or peptide purified from other components and substances present their natural environment, including other proteins, polypeptides, or peptides, or anisolated nucleic acid that is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence. As another example, an isolated nucleic acid can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. A substantially pure molecule can include isolated forms of the molecule.

[0084] The terms “lasso core peptide” and “core peptide” refer to the peptide or the peptide segment of the precursor peptide that is processed into or otherwise forms an engineered lasso peptide having the lariat-like topology. As used herein, a core peptide can have the same amino acid sequence as an engineered lasso peptide, but has not matured to have the lariat-like topology of an engineered lasso peptide. Core peptides can have different lengths of amino acid sequences. For example, the core peptide of the engineered lasso peptides described herein are typically 15 to about 24 amino acids long, but other core peptides can be, for example, about 10 amino acids long to as many as about 65 amino acids long.

[0085] The term “lasso cyclase” as used herein refers to the enzyme capable of catalyzing cyclization of the ring portion of a lasso core peptide.

[0086] The term “lasso peptidase” as used herein refers to the enzyme capable of catalyzing the removal of the leader sequence from a lasso precursor peptide to produce a lasso peptide (e.g., an engineered lasso peptide).

[0087] The terms “lasso peptide” and “lasso” are used interchangeably herein, and is used to refer to a class of peptide or polypeptide having the general lariat-like topology as exemplified in FIG. 1A. As shown in the figure, the lariat-like topology can be generally divided into a ring portion, a loop portion, and a tail portion. Particularly, a region on one end of the peptide forms the ring around the tail on the other end of the peptide, the tail is threaded through the ring, and a middle loop portion connects the ring and the tail, together forming the lariat-like topology. Particularly, the amino acid residues that are joined together to form the ring are herein referred to as the “ring-forming amino acids.” Ring-forming amino acids can be located at the N-terminus or C-terminus of the lasso peptide (“terminal ring-forming amino acid”) and in the middle (but not necessarily the center) of a lasso peptide (“internal ring-forming amino acid”). Internal ring-forming amino acids are typically an aspartic acid (D) or glutamic acid (E) residue located in the middle the lasso peptide. An engineered lasso peptide can be referred to here as being “cyclized” when such a lariat-like topology is formed by the engineered lasso peptide. For example, a lasso peptide can be described as being G1-D9 cyclized when referring to a lasso peptide, which means that thelasso peptide has a N-terminal ring-forming amino acid of a glycine residue (Gl) and an internal ring-forming amino acid of an aspartate residue at position 9 (D9), where the amino group of Gl and the carboxyl group of D9 form an isopeptide bond, thus forming the ring portion of the lasso peptide. The fragment of a lasso peptide between and including the two ring-forming amino acid residues is the ring portion. The fragment of a lasso peptide between the internal ring-forming amino acid and where the peptide threaded through the plane of the ring is the loop portion. The remaining fragment of a lasso peptide starting from where the peptide is threaded through the plane of the ring is the tail portion. Determining the position of a given residue within a lasso peptide is based on counting from the N- terminal residue of the linear core peptide as the first amino acid (position 1). The boundary between the ring and the loop portion of lasso peptide is determined by the formation of an isopeptide bond between the ring-forming amino acids. The boundary between the loop and tail of a lasso peptide can vary, but is generally dependent upon the location of the locking residues and is typically determined based on the 3D structure of the lasso peptide. In addition to the lariat-like topology, additional topological features of a lasso peptide can further include intra-peptide disulfide bonding, such as disulfide bond(s) between the tail and the ring, between the ring and the loop, and / or between different locations within the loop or tail. A lasso peptide can include both naturally-existing peptides and engineered peptides that have the lariat-like topology as described herein.

[0088] The term “lasso peptide biosynthesis component” as used herein refers to a protein including one or more of (i) a lasso peptidase, (ii) a lasso cyclase, and (iii) RRE. In some instances, the lasso peptidase and RRE proteins are fused, while in others, they operate as separate proteins. An exemplary process of lasso peptide production using lasso peptide biosynthesis components from a lasso precursor peptide is depicted in FIG. IB. An additional lasso peptide biosynthesis component can be a transporter that is specific for secreting lasso peptides outside the cell.

[0089] The terms “lasso peptide variant” or “engineered lasso peptide” are used herein interchangeably and refer to a derivative of a natural lasso peptide that has been modified or changed relative to its original structure or atomic composition. The engineered lasso peptide can (i) have at least one amino acid alteration (e.g., substitution(s), insertion(s) or deletion(s)) as compared to the sequence of a lasso peptide; (ii) have at least one modification to the amino acids as compared to a reference lasso peptide, wherein such modifications include, but are not limited to, acylation, biotinylation, O-methylation, N-methylation, amidation, glycosylation, pegylation, esterification, halogenation, amination, hydroxylation,dehydrogenation, prenylation, lipidoylation, heterocyclization, phosphorylation; (iii) have at least one unnatural amino acid(s) as compared to the sequence of a lasso peptide; (iv) have at least one different isotope(s) as compared to the lasso peptide molecule; or any combination of (i) to (iv). An engineered lasso peptide can be a conjugate or fusion made of a lasso peptide or an engineered lasso peptide and one or more additional molecule(s). In some instances, the additional molecule can be another peptide or protein, including but not limited a lasso peptide and a cell surface receptor or an antibody or an antibody fragment. In some instances, the additional molecule can be a non-peptidic molecule, such as a drug molecule, a fatty acid or lipid molecule, an isoprenoid molecule, or an oligonucleotide molecule. In some instances, the engineered lasso peptide retain the same general lasso topology as shown in FIG. 1A. In some instances, production of an engineered lasso peptide may occur by introducing a modification into the gene of a lasso precursor or core peptide, followed by transcription and translation and cyclization using cell-free or cell-based methods, as described herein, leading to an engineered lasso peptide containing that modification. In an alternative aspect, production of an engineered lasso peptide may occur by introducing a modification into a lasso precursor or core peptide, followed by cyclization of each using cell-free or cell-based methods, as described herein, leading to an engineered lasso peptide containing that modification. In another aspect, production of an engineered lasso peptide may occur by introducing a modification into a pre-formed lasso peptide, leading to a lasso peptide containing that modification. In another aspect, engineered lasso peptides are designed using structural information and in silico modeling algorithms, including docking and molecular dynamics algorithms, and such engineered lasso peptide may be produced by cell-free or cell-based methods. In another aspect, engineered lasso peptide are designed de novo using computer algorithms, including artificial intelligence, machine learning, deep learning, neural nets, etc., and such engineered lasso peptides may be produced by cell-free or cell-based methods.

[0090] An engineered lasso peptide that specifically or preferentially binds to a target protein (e.g., an integrin, or a specific integrin like avP6, avP8, or avpi) can be identified, for example, by immunoassays (e.g., ELISA, fluorescent immunosorbent assay, chemiluminescence immune assay, radioimmunoassay (RIA), enzyme multiplied immunoassay, solid phase radioimmunoassay (SPRIA), a surface plasmon resonance (SPR) assay (e.g., Biacore®), a fluorescence polarization assay, a fluorescence resonance energy transfer (FRET) assay, Dot-blot assay, fluorescence activated cell sorting (FACS) assay, orother techniques known to those of skill in the art. Typically, a specific or selective reaction will be at least twice background signal or noise and can be more than 10 times background.

[0091] The terms “lasso precursor peptide” or “precursor peptide” as used herein refer to a precursor that is processed into or otherwise forms a lasso peptide. A lasso precursor peptide can include at least one engineered lasso core peptide portion. A lasso precursor peptide can also include one or more amino acid residues or amino acid fragments that do not belong to an engineered lasso core peptide, such as a leader sequence that facilitates recognition of the lasso precursor peptide by one or more lasso processing enzymes.

[0092] The term “leader sequence” as used in reference to a lasso peptide, such as a lasso precursor peptide, refers to an amino acid sequence that facilitates recognition and processing by the lasso peptide processing enzymes described herein to form a cyclized lasso peptide. The leader sequence may determine substrate specificity of the processing enzymes. Accordingly, a lasso core peptide (e.g., an engineered lasso peptide) having a leader sequence can be referred to as a lasso precursor peptide.

[0093] The terms “manage,” “managing,” and “management” refer to the beneficial effects that a subject derives from a therapy (e.g., a prophylactic or therapeutic agent), which does not result in a cure of the disease. In some instances, a subject is administered one or more therapies (e.g., prophylactic or therapeutic agents, such as an engineered lasso peptide provided herein) to “manage” an integrin-mediated proliferative disease (e.g., cancer or fibrosis), one or more symptoms thereof, so as to prevent the progression or worsening of the disease.

[0094] The term “maximal percent downregulation” as used herein refers to the maximal level of downregulation achievable based a dose-response curve in an assay that measures such a response.

[0095] The term “maximal percent inhibition” as used herein refers to the maximal level of inhibition achievable based a dose-response curve in an assay that measures such a response.

[0096] The term “maximal percent reduction” as used herein refers to the maximal level of reduction achievable based a dose-response curve in an assay that measures such a response.

[0097] The terms “microbial,” “microbial organism” and “microorganism” as used herein refer to any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya. Therefore, the terms encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. The terms also include cells of any species that can be cultured for the production of a biochemical (e.g., a lassopeptide, including an engineered lasso peptide). A cell, such as a microbial organism, that produces an engineered lasso peptide of the present disclosure can include a bacterial and archaea host cells into which nucleic acids encoding the lasso peptide component have been introduced. Suitable host cells are disclosed below.

[0098] The terms “microenvironment” of a neoplastic cell or neoplastic cells or “neoplastic microenvironment” or “tumor microenvironment” refer to elements of the neoplasia milieu that creates a structural and / or functional environment for the neoplastic process to survive, expand, and / or spread. As a non-limiting example, a neoplastic microenvironment is constituted by the cells, molecules, extracellular matrix and / or blood and lymphatic vessels that surround and / or feed one or more neoplastic cells, such as a solid tumor. In certain embodiments, the neoplastic disease is a solid tumor. Exemplary cells or tissue within the tumor microenvironment include, but are not limited to, tumor vasculature, tumor infiltrating lymphocytes, fibroblast reticular cells, endothelial progenitor cells (EPC), cancer-associated fibroblasts, pericytes, other stromal cells, components of the extracellular matrix (ECM), dendritic cells, antigen presenting cells, T-cells, regulatory T-cells, macrophages, neutrophils, and other immune cells located proximal to a tumor. Exemplary cellular functions affecting the tumor microenvironment include, but are not limited to, production of growth factors, cytokines and / or chemokines, response to cytokines, antigen processing and presentation of peptide antigen, regulation of leukocyte chemotaxis and migration, regulation of gene expression, complement activation, regulation of signaling pathways, cell-mediated cytotoxicity, cell-mediated immunity, humoral immune responses, and innate immune responses, etc.

[0099] The terms “modulating” and “modulate” as used herein refer to an effect of altering a biological activity (i.e. increasing or decreasing the activity), especially a biological activity associated with a particular biomolecule such as a, enzyme or cell surface receptor. For example, an inhibitor of a particular biomolecule modulates the activity of that biomolecule, e.g., an enzyme, by decreasing the activity of the biomolecule, such as an enzyme. Such activity is typically indicated in terms of an inhibitory concentration (ICso) of the compound for an inhibitor with respect to, for example, an enzyme or a cell surface receptor.

[0100] The terms “motif’ and “binding motif’ are used interchangeably herein to mean the set of distinct amino acid residues on a binding molecule (e.g., a lasso peptide) that allows a binding interaction to occur between the binding molecule and a target molecule (e.g., a target protein). A binding motif can be linear or conformational, based on the structure and interaction with the binding target. A conformational motif is formed by the 3Dconformation adopted by the interaction of discontinuous segments of amino acid residue(s). In contrast, a linear motif is formed by the interaction of contiguous amino acid residues. The ability of a linear motif to bind a target molecule may not be determined solely by the primary structure of the involved amino acids. Residues that flank such amino acid residues, as well as more distant amino acid residues of the binding molecule can affect the ability of the linear motif to adopt the motif s 3D conformation required for activity. For example, other adjacent or proximal residues can be used to enhance the binding interactions of a binding motif. Accordingly, “expanded motifs” can be created by adding amino acids to an originally defined minimal motif like RGD, such as RGDX1X2 (SEQ ID NO: 788), which is an example of an expanded motif of RGD, wherein Xi and X2 are amino acid residues that have been shown to enhance the binding affinity and / or selectivity of molecules to certain target integrins, relative to RGD alone.

[0101] The terms “naturally occurring,” “natural,” and “native” when used in connection with naturally occurring biological materials, such as nucleic acid molecules, oligonucleotides, amino acids, polypeptides, peptides, metabolites, small molecule natural products, host cells, and the like, refer to materials that are found in or isolated directly from nature and are not changed or manipulated by humans.

[0102] The terms “non-naturally occurring,” “non-natural,” “unnatural” and “non-native” as used herein refer to a material, substance, molecule, cell, nucleic acid, oligonucleotide, nucleotide, enzyme, protein, polypeptide, peptide, or amino acid that is not known to exist or is not found in Nature or that has been structurally modified and / or synthesized by humans. Such terms when used in reference to a microbial organism, cell extract, or nucleic acid of the disclosure mean that the microbial organism, cell extract, or nucleic acid has at least one genetic alteration not normally found in a naturally occurring strain or a naturally occurring nucleic acid of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, introduction of expressible oligonucleotides or nucleic acids encoding polypeptides (e.g., an engineer lasso peptide), nucleic acid additions, substitutions, or deletions and / or other functional disruption of the microbial organism’s genetic material. Such alterations include, for example, nucleotide changes, additions, substitutions or deletions in the genomic coding regions and functional fragments thereof, used for heterologous, homologous or both heterologous and homologous expression of polypeptides. Additional alterations include, for example, nucleotide changes, additions, substitutions or deletions in the genomic non-coding and / or regulatory regions in which the modifications alter expression of a gene or operon. Such terms when used in reference to aprotein, polypeptide, or peptide are used to refer to a protein, polypeptide, or peptide having amino acids that are introduced into the amino acid sequence of the protein, polypeptide, or peptide to modify the properties of the polypeptide.

[0103] The terms “oligonucleotide” and “nucleic acid” refer to oligomers of deoxyribonucleotides (e.g., DNA) or ribonucleotides (e.g., RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless specifically limited otherwise, the term also refers to oligonucleotide analogs including PNA (peptidonucleic acid), analogs of DNA used in antisense technology (phosphorothioates, phosphoroamidates, and the like). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including but not limited to, degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer, M.A., et al.. Nucleic Acid Res., 1991, 19, 5081-1585;Ohtsuka, E. et al., J. Biol. Chem., 1985, 260, 2605-2608; and Rossolini, G.M., et al., Mol. Cell. Probes, 1994, 8, 91-98). “Oligonucleotide,” as used herein, refers to short, generally single-stranded, synthetic polynucleotides that are generally, but not necessarily, fewer than about 200 nucleotides in length. The terms “oligonucleotide” and “polynucleotide” are not mutually exclusive. The description above for oligonucleotides is equally and fully applicable to polynucleotides. Unless specified otherwise, the left-hand end of any singlestranded polynucleotide sequence disclosed herein is the 5’ end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5’ direction. The direction of 5’ to 3’ addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 5’ to the 5’ end of the RNA transcript are referred to as “upstream sequences”; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 3’ to the 3’ end of the RNA transcript are referred to as “downstream sequences.”

[0104] The term “operatively linked” as used herein when used in reference to a nucleic acid encoding a protein, polypeptide, or peptide refers to connection of a nucleotide sequence encoding the protein, polypeptide, or peptide to another nucleotide sequence (e.g., apromoter) is such a way as to allow for the connected nucleotide sequences to function e.g., express the protein, polypeptide, or peptide in the host).

[0105] The terms “parent scaffold peptide,” “parent scaffold,” and “scaffold” as used herein are used interchangeably and mean the lasso peptide from which an engineered lasso peptide was designed from as a starting peptide into which the integrin binding motif was introduced to and, in some instances, further engineered as described herein.

[0106] The term “peptide” as used herein refers to a polymer chain containing between two and fifty (2-50) amino acid residues. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non- naturally occurring amino acid, e.g., an amino acid analog or non-natural amino acid.

[0107] The term “pharmaceutically acceptable” as used herein means being approved by a regulatory agency of the Federal or a state government, or listed in United States Pharmacopeia, European Pharmacopeia, or other generally recognized Pharmacopeia for use in animals, and more particularly in humans.

[0108] The term “physiological conditions” as used herein refers to the conditions of the external or internal milieu that occurs in nature for a healthy or normal functioning organism or cell system. Physiological conditions include a number of parameters, including temperature, pressure, pH, glucose concentration, oxygen concentration, gravity, and electromagnetism, all of which will depend upon the healthy or normal physiological conditions of the organism or cell system. For example, the physiological pH of a normal human body ranges between 7.35 to 7.45, with an average of 7.4. As another example, the arterial blood of a healthy human subject has a pH of 7.35 to 7.45, pCCh of 35 to 45 mmHg, pO2 of 75 to 100 mmHg, HCO3- of 22 to 26 mEq / L and an O2 Sat of greater than 95% as measured by arterial blood gas. Physiological conditions for a subject can be a temperature within the range of 20°C to 40°C, an atmospheric pressure of 1, a pH of 6 to 8, a glucose concentration of 1 to 20 mM, an atmospheric oxygen concentration, and earth gravity and electromagnetism. Physiological conditions relevant to the property being assessed (e.g., solubility) can be reproduced in an artificial system, such as those found in an in vitro experiment.

[0109] The terms “polypeptide” and “protein” are used interchangeably herein to refer to a polymer of greater than about fifty (50) amino acid residues. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturally occurring amino acid, e.g., an amino acid analog.

[0110] The terms “post-translationally modified peptide (RiPP) recognition element” or “RRE” refer to a facilitator protein element encoded in some lasso peptide biosynthetic gene clusters that facilitates the recognition of and binding to the leader sequence by the lasso peptidase during biosynthesis of the lasso peptide.

[0111] The terms “preferential binding” and “preferentially binds to” when used in reference to a particular polypeptide or peptide (an engineered lasso peptide) on a particular target molecule (e.g., integrin avP6) with respect to a reference molecule (e.g., integrin avP3) refer to binding of the target molecule that is measurably higher than binding of the reference molecule, while the reference molecule may or may not also bind to the engineered lasso peptide. For example, an engineered lasso peptide described herein can preferentially binds to avP6 over avP3. Preferential binding can be determined, for example, by determining the binding affinity for the target molecule and the reference molecule. For example, an engineered lasso peptide that preferentially binds to a target molecule over a reference molecule can bind to the target molecule with a KD less than the KD exhibited relative to the reference molecule.

[0112] The terms “prevent,” “preventing,” and “prevention” refer to delaying, precluding the onset of, barring a subject from acquiring, or reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition as described herein and / or its attendant symptoms.

[0113] The terms “proliferative,” “proliferation” and “neoplastic” disease or condition refer to any physiological condition in animals that is characterized by uncontrolled, abnormal growth of tissues or cells, the latter of which can be referred to as “neoplastic cells.” Neoplastic cells, as used herein, can be malignant or benign, and includes both solid tumors or hematologic tumors and / or malignancies, as well as stromal fibroblast masses. Nonlimiting examples of malignant proliferative diseases that can be prevented, treated or managed with the methods and compositions described herein include those mediated by integrin receptor activity, such as cancer, including breast cancer, pancreatic cancer (e.g., pancreatic adenocarcinoma), hepatocellular carcinoma, prostate cancer, ovarian cancer, gastric cancer, brain or spinal cancer (e.g., glioma, such as a glioblastoma), melanoma, cancer of the head and neck, colorectal cancer, bladder cancer, vulvar cancer, esophageal squamous cell carcinoma, renal cancer (e.g., clear-cell renal cell carcinoma), cervical cancer, salivary gland carcinoma, lung cancer (e.g., non-small cell lung cancer and small-cell lung cancer), multiple myeloma, or Kaposi’s sarcoma. Non-limiting examples of fibrotic proliferative diseases that can be prevented, treated or managed with the methods and compositionsdescribed herein include those mediated by integrin receptor activity, such as pulmonary fibrosis including cystic and idiopathic pulmonary fibrosis, hepatic fibrosis and cirrhosis, pancreatic fibrosis and pancreatitis, renal fibrosis, glial fibrosis, retroperitoneal cavity fibrosis, mediastinal fibrosis, cardiovascular disease and heart fibrosis, myelofibrosis, systemic sclerosis, Dupuytren's contracture, Peyronie’s disease, macular degeneration, hypertrophic scars, skin fibrosis and skin keloids, arthrofibrosis, and Duchenne muscular dystrophy-associated skeletal muscle fibrosis.

[0114] The term “promoter” as used herein in reference to a nucleic acid encoding a protein, polypeptide or peptide refers to a nucleotide sequence where transcription of a linked open reading frame (e.g., a nucleotide sequence encoding an engineered lasso peptide) by an RNA polymerase begins. A promoter sequence can be located directly upstream or at the 5' end of the transcription initiation site. RNA polymerase and the necessary transcription factors bind to a promoter sequence and initiate transcription. Promoter sequences define the direction of transcription and indicate which DNA strand will be transcribed, i.e. the sense strand.

[0115] The term “prophylactically effective amount” refers to an amount of a pharmaceutical composition that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing a disease, disorder, or condition as described herein, or associated symptom(s). Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of a disease, disorder, or condition, a prophylactically effective amount can be less than a therapeutically effective amount. The prophylactic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a prophylactically effective amount can be administered in one or more administrations.

[0116] The term “recombinant” as used herein with respect to a nucleic acid, such as a nucleic acid having a gene that encodes a protein or polypeptide (e.g., an engineered lasso peptide described herein), refers to: a nucleic acid that has been artificially supplied to a biological system; a nucleic acid that has been modified within a biological system, or a nucleic acid whose expression or regulation has been manipulated within a biological system. The recombinant nucleic acid can be supplied to the biological system, for example, by introduction of the nucleic acid into genetic material of a microbial organism, such as by integration into a microbial organism chromosome, or as non-chromosomal genetic material such as a plasmid. A recombinant nucleic acid that is introduced into or expressed in a microbial organism can be a nucleic acid that comes from a different organism or species from the microbial organism, or can be a synthetic nucleic acid, or can be a nucleic acid thatis also endogenously expressed in the same organism or species as the microbial organism. A recombinant nucleic acid that is also endogenously expressed in the same organism or species as the microbial organism can be considered heterologous if: the sequence of the recombinant nucleic acid is modified relative to the endogenously expressed sequence, the sequence of a regulatory region such as a promoter that controls expression of the nucleic acid is modified relative to the regulatory region of the endogenously expressed sequence, the nucleic acid is expressed in an alternate location in the genome of the microbial organism relative to the endogenously expressed sequence, the nucleic acid is expressed in a different copy number in the microbial organism relative to the endogenously expressed sequence, and / or the nucleic acid is expressed as non-chromosomal genetic material such as a plasmid in the microbial organism.

[0117] The terms “selective inhibition of’ and “selectively inhibits” as used herein with regard to inhibition of a target molecule by an engineered lasso peptide refer to inhibition of the target molecule activity is measurably stronger than inhibition of a reference molecule activity. For example, in some instances, an engineered lasso peptide selectively inhibits integrins avP6 and avP8 over avP3 and avP5. Selective inhibition can be determined, for example, by determining the IC50 value. For example, an engineered lasso peptide that selectively inhibits a target protein can exhibit an IC50 value for the target protein that is less than the IC50 exhibited for a second target protein. In some instances, the engineered lasso peptide selectively inhibits a target protein with an IC50 for the target protein that is less than half of the IC50 exhibited for a second target protein. In some instances, the lasso peptide selectively inhibits a target protein with an IC50 for the target protein that is about 75%, about 50%, about 25%, about 10%, about 5%, about 2.5%, or about 1% of the IC50 exhibited for a second target protein. In some instances, the ratio between the IC50 exhibited by the engineered lasso peptide with respect to the second target protein and the IC50 exhibited with respect to the target protein is at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 100 fold, at least 500 fold, at least 103fold, at least 104fold, or at least 105fold.

[0118] The terms “specific binding,” “specifically binds to,” and “is specific for” when used in reference to a particular molecule (e.g., protein, polypeptide, or peptide, such as an engineered lasso peptide) refer to binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can bedetermined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target. The terms also include binding where a molecule (e.g., protein, polypeptide, or peptide) binds to a particular protein or fragment of a particular protein without substantially binding to any other protein or protein fragment. Accordingly, an engineered lasso peptide described herein can be described as specifically binding to a target protein when it binds to the target protein with higher affinity than to any cross-reactive target molecule as determined using experimental techniques described herein.

[0119] The terms “subject” and “patient” refer to a mammal, such as a non-primate (e.g., cow, pig, horse, cat, dog, rat, mouse, etc.) or a primate (e.g., monkey, ape, and human). In some instances, the subject is a human. In some instances, the subject is a mammal (e.g., a human) having an integrin receptor-mediated proliferative disease (e.g. cancer or fibrosis), disorder, or condition. In some instances, the subject is a mammal (e.g., a human) at risk of developing an integrin receptor-mediated proliferative disease (e.g. cancer or fibrosis), disorder, or condition.

[0120] The term “substantially” means that something takes place, as a function or activity, to provide the expected outcome or result to a large degree and to a great extent, but still not to the fullest extent. For example, if an engineered lasso peptide is substantially purified, the engineered lasso peptide is isolated and purification steps afford the engineered lasso peptide at purity level above 80%, preferably above 90%, and as high as 99.99%.

[0121] The term “substantially all” refers to at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100%.

[0122] The term “therapeutic agent” refers to any agent that can be used in treating or preventing a disease, disorder, or condition, including in the treatment, prevention, or alleviation of one or more symptoms of an integrin receptor-mediated proliferative disease (e.g., cancer or fibrosis), disorder, or condition and / or a symptom related thereto. In some instances, a therapeutic agent refers to an engineered lasso peptide as described herein.

[0123] The term “substitution” when used in reference to a peptide, polypeptide, protein refers to an amino acid residue that has been substituted for a structurally different amino acid residue. Such substitutions can be a conservative substitution, a non-conservative substitution, a substitution to a specific sub-class of amino acids, or a combination thereof as described herein.

[0124] The term “therapeutically effective amount” as used herein refers to the amount of an agent (e.g., an engineered lasso peptide provided herein or any other agent described herein) that is sufficient to manage, prevent, or treat a given disease, disorder, or condition as described herein, and / or a symptom related thereto. A therapeutically effective amount of an agent of the present disclosure (e.g., an engineered lasso peptide) can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the agent to elicit a desired response in the individual. A therapeutically effective amount encompasses an amount in which any toxic or detrimental effects of the agent are outweighed by the therapeutically beneficial effects. A therapeutically effective amount also encompasses an amount of an engineered lasso peptide or other agent (e.g., drug) effective to treat a disease, disorder, or condition described herein, in a subject or mammal.

[0125] The term “therapy” refers to any protocol, method, and / or agent that can be used in the management, prevention or treatment of an integrin receptor-mediated proliferative disease, disorder, or condition (e.g., cancer or fibrosis). In some instances, the terms “therapies” and “therapy” refer to a biological therapy, supportive therapy, and / or other therapies useful in the management, prevention, and / or treatment of an integrin receptor- mediated proliferative disease, disorder, or condition (e.g., cancer or fibrosis) known to one of skill in the art such as medical personnel.

[0126] The terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some instances, treatment can be administered after one or more symptoms have developed. In other instances, treatment can be administered in the absence of symptoms. For example, treatment can be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of genetic or other susceptibility factors). Treatment can also be continued after symptoms have resolved, for example to prevent or delay their recurrence.

[0127] The terms “upregulate” and “upregulation” as used herein refer to increasing the rate or level of expression or production of a molecule relative to a control. Upregulation of a molecule can be expressed as a percentage (e.g., 1%, 2%, 5%, 10%, 20%, 25%, 50%, 75%, 90%, 95%, 99%) or by a fold change (i.e., 1 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 4 fold, 5 fold, 10 fold or more).

[0128] The term “vector” refers to a substance that is used to carry or include a nucleic acid sequence, including, for example, a nucleic acid sequence encoding an engineered lasso peptide, a lasso precursor peptide (e.g., an engineered lasso peptide having a leadersequence), or lasso processing enzymes as described herein, in order to introduce the nucleic acid sequence into a host cell. Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell’s chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art. When two or more nucleic acid molecules are to be co-expressed (e.g., both an engineered lasso core peptide and a lasso cyclase), both nucleic acid molecules can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The introduction of nucleic acid molecules into a host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the nucleic acid molecules are expressed in a sufficient amount to produce a desired product (e.g., an engineered lasso peptide as described herein), and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.

[0129] The terms “wild-type” or “WT” refers to organisms, cells, genes, lasso peptide biosynthetic gene clusters, enzymes, proteins, oligonucleotides, and the like that are found in Nature and are unchanged relative to these components found in Nature (in the wild).6.3. Integrin Receptors

[0130] Integrins are a class of ubiquitous, heterodimeric, transmembrane cell-surface receptors that regulate cell adhesion, migration, proliferation, and survival in mammals (Winograd-Katz et al. Nat. Rev. Mol. Cell. Biol., 2014, 15, 273-288; Humphries et al., J. Cell Sci., 2006, 119, 3901-3903; and Hynes, Cell, 2002, 110, 673-687). In humans, each consists of an a-subunit and a P-subunit, of which there are 18 and 8 variants, respectively, creatingtwenty-four different heterodimeric integrins (Humphries et aL, J. Cell Sci., 2006, 119, 3901- 3903) (FIG. 2). Generally, integrins are classified based on common a or P-subunits or related properties such a common ligand binding behavior. Integrins may also be categorized as collagen receptors, laminin receptors, leukocyte-specific receptors, and receptors that recognize the Arg-Gly-Asp (RGD) peptide motif (FIG. 2). Eight RGD-binding integrins are known to bind to the RGD motif present in their endogenous ligands, which include fibrinogen and the extracellular matrix (ECM) proteins, such as fibronectin and vitronectin (FIG. 3). Integrin heterodimers are transported from the endoplasmic reticulum to Golgi apparatus, where they are further post-translationally modified and transferred to the cell surface in an inactive state (De Franceschi et al., J. Cell Sci., 2015, 128, 839-852). The integrin a and P subunits are both glycosylated, and their C-terminal ends are connected to each other by a non- covalent interactions, thereby forming aP integrin heterodimers (Seguin et al., Trends Cell Biol., 2015 25, 234-240). Some integrin subunits only appear in a single heterodimer (e.g., P5, P6, and P8), whereas other subunits, such as pi and av, appear in 12 and 5 integrin heterodimers, respectively (Kechagia etal., Nat. Rev. Mol. Cell Biol., 2019, 20, 457-473). The a- and P- subunits are bound in a non-covalent complex with the ligand-binding site at the interface.

[0131] Integrins act as adhesion receptors, with the unusual ability to signal in both directions across the plasma membrane (Hynes, Cell, 2002, 110, 673-687). These events are called ‘inside-out’ signaling (Faull and Ginsberg, J. Am. Soc. Nephrol., 1996, 7, 1091-1097) and ‘outside-in’ signaling (Zhu et al., Blood, 2007, 110, 2475-2483), resulting either from binding to extracellular ligands or from interacting with the cytoskeleton via the integrin intracellular domains. Integrins can, therefore, enable human cells to respond to changes in the extracellular environment (via outside-in signaling) and can influence the extracellular environment itself (via inside-out signaling). Information from outside the cell is communicated intracellularly when the ligand binds to the receptor, resulting in changes in cell polarity, cytoskeletal structure, gene expression, cell survival and proliferation (Shattil et al., Nat. Rev. Mol. Cell. BioL, 2010, 11, 288-300). In the opposite direction, intracellular activators, such as talin-1, bind to the cytoplasmic tail of the P- subunit, evoking a conformational change that shifts the integrin into a high-affinity state, which more readily binds to extracellular ligands and thus promotes cell migration and extracellular matrix (ECM) assembly and remodeling (Klapholz et al., J. Cell Sci., 2017, 130, 2435-2446; and Calderwood et al., Nat. Rev. Mol. Cell Biol., 2013, 14, 503-517).

[0132] As a receptor on the cell membrane, integrins mainly interact with ECM components to mediate cell adhesion (Dustin, Cell, 2019, 177, 499-501). Based on the ECM components recognized, integrins have been grouped into four classes: leukocyte receptors, collagen binding receptors, RGD-binding receptors, and laminin-binding receptors. Different types of integrins can recognize and bind the same ligand (Humphries et al., J. Cell Sci., 2006, 119, 3901-3903). For example, all five av integrins (avpi, avP3, avP5, avP6, and avP8), two pi integrins (a5pi and a8pi), and allbp3 are RGD-binding integrins. The RGD-binding family of integrins recognize the amino acid binding motif Arg-Gly-Asp (RGD) in their endogenous ligands, while the related integrins a4p7 and a4pi are therapeutic targets that are expressed on leukocytes, and also recognize short peptide sequences, one of which is Leu-Asp-Val (LDV). Integrins aipi, a2pi, aiopi, and al ipi binding to laminins and collagens (Humphries et al., 2006). Additionally, three pi integrins (a3pi, a6pi, and a7pi) and a6p4 are highly selective laminin receptors (Marsico et al., Trends Cancer 4, 2018, 537-552). Moreover, a4pi, a4p7, a9pi, and aEp7 recognize similar sequences in their ligands. On the other hand, the same integrins can bind to multiple ligands (Kechagia et al., 2019). For instance, avP3 not only recognizes RGD peptide motifs, but also binds to other ligands, including ADAM (a disintegrin and metalloprotease) family members, COMP (cartilage oligomeric matrix protein), connective tissue growth factor, ICAM-4 (intercellular cell adhesion molecule-4), and MMP- 2 (Seguin et al., 2015). Finally, even when they interact with the same ligands, the integrins can recognize them in a different manner. For example, although integrins a5pi and a4pi are known to bind to fibronectin, their recognition sequences in fibronectin are different. Integrin a5pi recognizes the RGD sequence in fibronectin, whereas integrin a4pi recognizes EILDV and REDV sequences.

[0133] A set of eight integrin heterodimers, consisting of avpi, avP3, avP5, avP6, avP8, a5pi, a8pi, and allbp3, recognize the tripeptide RGD motif within ECM proteins such as fibronectin, tenascin-C, and vitronectin or, in the case of allbp3, fibrinogen (Nieberler et al., Cancers, 2017, 9, 116). Expression of five RGD-integrins, namely avP3, avP5, avP6, avP8, a5pi, has been shown to correlate well with metastasis and poor patient prognosis across a variety of cancers, and integrins avP3, avP5, a5pi are among the most prominent and well- studied integrin subtypes in oncology (Seguin, 2015). In addition to binding to RGD motifs in ECM ligands, three integrins (avP6, avP8, and avpi) have been shown to bind to the RGD motif contained within the latency associated peptide (LAP), which is co-expressed with and non-covalently bound to TGF-P, to activate the inactive, latent form of the TGF-P (Kelly et al. ,Adv. Immunol., 2017, 1-97). The complex formed by LAP with TGF-P helps to retain this cytokine in an inactive form. Integrin binding to LAP has been shown to be a primary mechanism for liberating activated TGF-P (Brown and Marshall, Cancers, 2019, 11, 1221; and Stuelten and Zhang, Front. Cell Dev. Biol. 2021, 9:764727). Once activated, free TGF-P is released in the tumor microenvironment (TME) and binds to TGF-P receptors on immune, tumor, and stromal cells (Yang el al., Trends in Immunology, 2010, 31, 220-227) (FIG. 4). TGF-P signaling also has a strong impact on epithelial-to-mesenchymal transition (EMT) during invasive cancer growth, progression, and metastasis. In addition, TGF-P has a strong immunosuppressive effect in the TME, which greatly reduces the anti-tumor immune response of CD8+ T cells and other immune cells. Thus, reducing the activity of TGF-P through inhibition of avP6, avP8, and avpi integrins represents a compelling strategy for the development of new immune-oncology agents for treating a broad range of cancers. Furthermore, expression of integrins avpi and avP6 in fibrotic tissues has been shown to be associated with the pathology of fibrosis and fibroproliferative diseases through the agency of excess activated TGF-P (Reed etal., Sci Transl Med., 2015, 7, 288ra79288ra79; Decaris et al., Respir Res, 2021, 22:265).6.4. Integrins and Transforming Growth Factor Beta (TGF-P)

[0134] TGF-P exists in three isoforms, TGF-pi, TGF-P2 and TGF-P3, and these structurally- related cytokines share many features, including structurally-related receptors and downstream signaling effectors, yet they often play functionally distinct roles in physiology and disease (Derynck et al., Sci. Signal. 2019, 12(570), eaav5183). Correlative analyses indicate that TGF- pi shows higher and more widespread upregulation in the TME than the other two isoforms and is more robustly associated with failure of immune checkpoint inhibitor responses in patients with cancer (Martin et al., Sci. Transl. Med., 2020, 12, eaay8456). In turn, TGF-P3 plays specific roles during wound healing and fibrosis and is highly upregulated in cancer- associated fibroblasts (Lichtman et al, Wound Rep. Reg., 2016, 24 215-222; and Calon et al., Cancer Cell, 2012, 22, 571-584).

[0135] TGF-P binding to its receptor activates signaling cascades, for example, through phosphorylation of Smad transcription factors, that enhance inflammation and facilitate strong suppression of anti-tumor immune cell responses (Derynck etal., Nat. Rev. Clin. Oncol. 2021, 18, 9-34; and Mariathasan et al., Nature, 2018, 554, 544-548). TGF-P facilitates the conversion of numerous types of tumor resident cells, including fibroblasts, into cancer-associated fibroblasts (CAFs), which contribute to the production of tumor promoting products such asmatrix metalloproteinases 2 and 9, tissue inhibitor of metalloproteinases- 1 (TIMP-1), a-smooth muscle actin and ECM proteins such as collagen and fibronectin. TGF-P also induces production of pro-inflammatory cytokines, such as the interleukins IL6, IL 11, IL 17, and IL22. Inflammation and immunosuppression promoted by activated TGF-P thus allows tumors to grow and proliferate. Direct blockade of TGF-P or TGF-P receptor, for example by antibodies or small molecule inhibitors, has been shown to induce severe adverse events and systemic toxicity due to the important varied roles that this pleiotropic cytokine plays throughout the body (van den Bulk et al.. Clinical Science, 2021, 135 35-52; and Huynh el al., Biomolecules, 2019, 9, 743). Accordingly, blocking the activation of TGF-P by inhibiting the binding between integrins and the LAP-TGF-P complex in the TME represents an important novel strategy for treating cancer.

[0136] TGF-P also plays an analogous role in the development of fibrosis (Frangogiannis, J. Exp. Med. 2020, 217(3) e20190103). Upon minor organ or tissue injury, fibroblasts, platelets, macrophages, neutrophils, and other cells coordinate their responses to produce ECM leading to normal wound healing. Acute or repetitive injury, ageing, and chronic inflammation can afford a dysfunctional wound repair response that can lead to the condition of fibrosis (Henderson et al., Nature, 2020, 587, 556-566; and Reed et al., Sci Transl Med., 2015, 7, 288ra79288ra79). Activated TGF-P induces the transformation of fibroblasts into activated myofibroblasts in fibrotic tissue and into cancer-associated fibroblasts in tumors, both of which produce large amounts of ECM proteins. In fibrosis, activated TGF-P has been shown to (i) inhibit the production of tumor necrosis factor-a, IL1, and interferon-y, (ii) induce production of matrix metalloproteinases 2 and 9, tissue inhibitor of metalloproteinases- 1 (TIMP-1), a- smooth muscle actin and ECM proteins such as collagen and fibronectin, and (iii) promote the production of pro-inflammatory cytokines, such as the interleukins IL6, IL11, IL17, and IL22 (Turner et al. , Cytokine, 1990, 2(3), 211-216; Espevik et al. , J. Exp. Med., 1987, 166, 571- 576; Perez et aL, Nature Comm. 2020, 11 :2608; Henderson etal., Nature, 2020, 587, 556-566; Gough etal., Gastroenterology, 2021, 161(2), 434-452. el5; and Kwak etal., Mol Cancer Res, 2006, 4(3), 209-220), which further enhance the fibrotic response, ultimately leading to excessive ECM accumulation and tissue scarring, a hallmark of fibrosis. In cancer, activated TGF-P exerts similar effects in the tumor microenvironment by promoting the transformation of resident fibroblasts and other cells into cancer-associated fibroblasts (Derynck et al. 2021). Integrin activation of TGF-P is critical for the fibrotic cascade and blocking the activation TGF- P by inhibiting the binding between RGD-integrins and the latent LAP-TGF-P complex represents an important strategy for treating fibrosis.

[0137] Structural studies of the LAP protein and analogous peptides in complex with avP6 and avP8 have demonstrated that these integrins bind to LAP via an RGDLXXL (SEQ ID NO: 789) binding motif, where the LXXL (X = any amino acid) section is displayed on a helix (Dong et al., Nat Struct Mol Biol, 2014, 21, 1091-1096; Dong et al., Nature, 2017, 542, 55- 59; and Campbell etal., Cell 2020, 180, 490-501). This high affinity interaction between LAP- TGF-P and avP6, avP8, and avpi integrins, which is mediated by the RGDLXXL (SEQ ID NO: 789) motif, leads to a conformational change in LAP that allows TFG-P to be released as a free activated cytokine. Accordingly, the RGDL (SEQ ID NO: 783) and RGDLXXL (SEQ ID NO: 789) motifs have been incorporated into various peptides and antibodies in an effort to develop inhibitors of TGF-P activation by these integrins.6.5. RGD-integrin Inhibition

[0138] Identification of the RGD motif in ECM proteins combined with the recognition that eight unique integrins bind to this sequence led to investigation of the involvement of RGD- integrins in disease (Pierschbacher and Ruoslahti, Nature, 1984, 309, 30-33). The RGD- integrin receptors are prevalent throughout the human body, and they have been found to play pivotal roles in wide-ranging physiological processes such as in tissue growth and fibrosis, inflammation, cancer, thrombosis and autoimmune diseases (Ley et al., Nat. Rev. Drug Disc., 2016, 15, 173 - 183). To date, only a few RGD-integrin inhibitor agents have reached the market, including the anti-P3 antibody abciximab and the small molecules tirofiban and eptifibatide, which target integrin allbp3 and are prescribed for the treatment of acute coronary syndrome. No agents have received regulatory approval that specifically target the av-RGD sub-family of integrins, despite numerous compounds entering clinical trials. In addition to pharmacological properties, one major challenge for most RGD-integrin inhibitors to date has been the lack of integrin selectivity, especially with many small molecules that potently inhibit a range of different integrin. Such lack of integrin specificity leads to undesirable adverse events and toxicity, as well as poor efficacy, due to off-target binding. For example, MK-0429 is a potent yet unselective pan-RGD integrin inhibitor that has not advanced in the clinic beyond Phase 2 trials (Coleman et al., J. Med. Chem. 2004, 47, 4829-4837; Rosenthal et al., Asia Pac. J. Clin. Oncol. 2010, 6, 42-48). New RGD-integrin inhibitors that display high potency and high selectivity for specific integrins associated disease pathology are needed.

[0139] Integrins avP3, avP5 and a5pi have been shown to participate in mediating tumor angiogenesis by interacting with the VEGF and angiopoietin-Tie signaling pathways (Huang and Rofstad, J Exp Clin Cancer Res., 2018, 37, 92). Although many different drugs targetinga variety of integrins have been developed, none of them have shown sufficient evidence for their clinical use in patients and the inhibition of a single integrin as a therapeutic target is not a promising approach because different integrins are often responsible for angiogenesis and organ-specific metastasis in human malignancies. However, toxicity is a challenging problem with the current drugs that target several integrins simultaneously due to the prevalence and extensive involvement of these integrins in maintaining normal biological and physiological functions.

[0140] The most advanced and well -documented av-RGD inhibitor is the cyclic pentapeptide cilengitide that inhibits avP3 and avP5, which has progressed to late-stage clinical trials for cancer indications (Mas-Moruno et al. , Anti-Cancer Agents in Medicinal Chemistry, 2010, 10, 753-768; and Scaring! et al., Anticancer Res, 2012, 32, 4213-4224). Expression of integrins avP3 and avP5 has been shown to play a role in angiogenesis and tumor growth, invasion, and metastasis for a range of cancers including melanoma, breast, prostate, pancreatic, ovarian, cervical and glioblastoma as well as being linked to disease progression (Desgrosellie and Cheresh, Nat Rev Cancer, 2010, 10, 9-22). Targeting avP3 and avP5 with inhibitors, such as the small molecule cilengitide or antibody abituzumab (a pan av monoclonal antibody), has been shown to be efficacious in human xenograft mouse models, with cilengitide also observed to be synergistic with radioimmunotherapy. Unfortunately, neither therapeutic has afforded the expected clinical benefits and other RGD integrins have emerged as targets for inhibition.

[0141] The RGD and RGDF (SEQ ID NO: 790) integrin binding motif sequences have been reported to be grafted into the loop of MccJ25, a natural 21-amino acid lasso peptide with an 11 -membered loop, and were shown to be potent inhibitors of integrin avP3 (Knappe et al., Angew. Chem. Int. Ed. 2011, 50, 8714 -8717; and Hegemann et. al., J. Med. Chem. 2014, 57, 5829-5834). However, it is integrins avP6, avP8, and avpi that have been shown to be directly involved in the activation of latent TGF-P, which upon liberation promotes immunosuppression in the TME and progression of the fibrotic process (Brown and Marshall, Cancers, 2019, 11, 1221). Directly blocking TGF-P generally leads to severe adverse events and systemic toxicity (van den Bulk, 2021). An alternative approach to blocking TGF-P function is to specifically bind and selectively inhibit the TGF-P-activating integrins avP6, avP8, and / or avP 1. Inhibitors of avP6, avP8, and / or avpi have been described, including small molecules, peptides, and antibody agents, and these inhibitors have been demonstrated to reduce the activation of TFGP in tumors and in fibrotic tissue. Peptides that have been reported include avP6-selective compounds, such as the linear peptide A20FMDV2 (Hung et al., Eur. J. Med. Chem., 2017, 136, 154el64) and the cyclic peptide c(FRGDLAFp(NMe)K (SEQ ID NO: 791) (Maltsev etal., Angew. Chem. Int. Ed. 2016, 55, 1535 -1539), as well as the avP 8 -selective cyclic peptide c(GLRGDLpP) (SEQ ID NO: 792) (Reichart etal., J Med. Chem. 2019, 62, 2024-2037). The antibodies that selectively target avP6 include 264RAD (Eberlein et aL, Oncogene, 2013, 32, 4406-4416) and BG00001 (Horan etal.,Am JRespir Crit Care Med, 2008, 177, 56-65), which was terminated after Phase 2 clinical studies due to safety concerns. Two other antibody drugs called C6D4 (Takasaka et al., JCI Insight 3, 2018), and ADWA-11 (Dodagatta-Marri et al., Cell Reports, 2021, 36, 109309) were found to be potent and selective inhibitors of avP8 integrin, and the later antibody is currently in Phase 1 clinical trials as a treatment for cancer. Several small molecule inhibitors have been reported for TGF-P-activating integrins, including the avP6-selective compound GSK3008348 (Hall et al., Biochem Pharmacol, 2016, 117, 88- 96; Procopiou et al., J. Med. Chem., 2018, 61, 8417-8443), the avpi -selective compound C8 (Reed et al., Sci Transl Med, 2015, 7, 288ra79288ra79), and the dual selective avP6 / avpi inhibitor PLN-74809, which currently is in Phase 2 clinical trials for IPF (Decaris et al. , Respir. Res., 2021, 22, 265).6.6. Parent Scaffold Peptides

[0142] Bacterially-derived lasso peptides are emerging as a class of natural molecular parent scaffold peptides for drug design (Hegemann et al., Acc. Chem. Res., 2015, 48, 1909-1919; Zhao et al., Amino Acids, 2016, 48, 1347-1356; Maksimov et al., Nat. Prod. Rep., 2012, 29, 996-1006). Lasso peptides are members of the larger class of natural ribosomally synthesized and post-translationally modified peptides (RiPPs). Lasso peptides are derived from a precursor peptide, having a leader sequence and core peptide sequence, which is cyclized through formation of an isopeptide bond between the N-terminal amino group of the linear core peptide and the side chain carboxyl groups of glutamate or aspartate residues located at positions 7, 8, 9, or 10 of the linear core peptide. The resulting macrolactam ring is formed around the C-terminal linear tail, which is threaded through the ring leading to the characteristic lasso (also referred to as lariat) topology with the general structure shown in FIG. 1A, which is held in place through sterically bulky side chains below and sometimes above the plane of the ring, and sometimes containing disulfide bonds between the tail and the ring or alternatively only in the tail.

[0143] A lasso peptide biosynthetic gene cluster corresponds to one or more nucleic acid molecule(s) independently or jointly having one or more coding sequences for a precursor lasso peptide and processing machinery capable of maturing a precursor lasso peptide into a biosynthetic end product as described herein. The coding sequences can include multiple openreading frames (ORFs) each independently coding for one component of the precursor and processing machinery. Alternatively, the coding sequences can include an ORF coding for two or more components of the precursor and processing machinery fused together. Lasso peptide biosynthetic gene clusters typically consist of three main genes, one coding for the precursor peptide (referred to as Gene A), and two for the processing enzymes, a lasso peptidase (referred to as Gene B or B2) and a lasso cyclase (referred to as Gene C) that close the macrolactam ring around the tail to form the unique lariat structure. The precursor peptide consists of a leader sequence that binds to and directs the enzymes that carry out the cyclization reaction, and a core peptide sequence which contains the amino acids that together form the nascent lasso peptide upon cyclization. The processing enzymes encoded by the lasso peptide biosynthetic gene cluster convert the lasso precursor peptide into a matured lasso peptide having the lariatlike topology. Particularly, the lasso peptidase removes from the precursor peptide the additional portion that is not the lasso core peptide, and the lasso cyclase cyclizes a terminal portion of the core peptide around a terminal tail portion to form the lariat-like topology. In addition, most lasso peptide biosynthetic gene clusters contain additional genes, such as those that encode for a small facilitator protein called a RIPP recognition element (RRE, also referred to as Gene E or Bl), those that encode for lasso peptide transporters, those that encode for kinases, or those that encode proteins that are believed to play a role in immunity, such as an isopeptidase (Burkhart et al.. Nat. Chem. BioL, 2015, 11, 564-570; Knappe etal., J. Am. Chem. A e., 2008, 130, 11446-11454; Solbiati etal., J. Bacterio!., 1999, 181, 2659-2662; Fage etal., Angew. Chem. Int. Ed., 2016, 55, 12717 -12721; and Zhu et al., J. Biol. Chem., 2016, 291, 13662-13678).

[0144] The ultimate lasso peptide directly derives from a core peptide that typically includes a linear sequence ranging from about 11 to 50 amino acids in length. The macrolactam ring of a lasso peptide may contain 7, 8, 9, or 10 amino acids, while the loop and tail vary in length.

[0145] A lasso peptide biosynthetic gene cluster can be identified and isolated from the genome of an organism. Computer-based analytical tools can be used to mine genomic information and identify biosynthetic gene clusters encoding lasso peptides. A genomic sequence mining algorithm called RODEO, has enabled identification of over 3000 entirely new lasso peptide biosynthetic gene clusters associated with a broad range of different bacterial species in the GenBank database, which is a vast increase over the 38 lasso peptides previously described in the literature (Tietz et al., Nature Chem Bio, 2017, 13, 470-478; and DiCaprio et al., J. Am. Chem. Soc. 2019, 141, 1, 290-297). Alternatively, a lasso peptide biosynthetic gene cluster can be assembled by artificially producing and combining the nucleic acid componentsof the lasso peptide biosynthetic gene cluster, using genetic manipulating methods and technology known in the art.

[0146] Lasso peptides represent a new type of molecular diversity which could serve as a vast source of novel products for use in the pharmaceutical, diagnostic, agricultural, and consumer industries. While the discovery of new activities and functions can occur by exploring the naturally occurring lasso peptides, a large percentage (>95%) of natural lasso peptides remain as prophetic entities predicted on the basis of genome sequence analyses. Natural lasso peptides represent a useful source of parent scaffold peptide diversity, but discovery of new functionality, such as high potency and selectivity for biological receptors of interest, often requires further engineering of naturally occurring parent scaffold peptides. Engineering of a given parent scaffold peptide can be accomplished by grafting known integrin binding motifs into the lasso structure and / or substituting the natural amino acids of a given lasso peptide with other amino acids, including non-natural amino acids. Exemplary parent scaffold peptides are described herein, including the amino acid sequences of parent scaffold peptides disclosed in Table 1. Accordingly, in some embodiments, an engineered lasso peptide provided herein is based on a parent scaffold peptide having the amino acid sequence of any one of SEQ ID NOs: 1, 343, 353, 362, and 366. Integrin binding motifs and amino acid changes that are introduced into a given parent scaffold peptide can be guided by computational modeling, docking, and molecular dynamics simulations of parent scaffold peptides and engineered lasso peptides. These lasso peptide engineering methods were developed and implemented for the design, discovery, and optimization of lasso peptide-based integrin inhibitors described in this application.6.7. Computational Methods for in silico Design of Lasso Peptides

[0147] In an effort to accelerate drug discovery and to optimization its process, the pharmaceutical industry has been striving over the past forty years to develop and use in silico methods and algorithms to analyze structural information and assist in what is called computer- aided drug design (CADD) (Sliwoski et aL, Pharmacol Rev., 2014, 66:334-395). CADD is capable of increasing the hit rate of new drug compounds because it uses a much more targeted search than traditional high throughput screening (HTS) and combinatorial chemistry. It not only aims to explain the molecular basis of therapeutic activity but also to predict possible derivatives that would improve activity. In a drug discovery campaign, CADD is usually used for three major purposes: (1) filter large compound libraries into smaller sets of predicted active compounds that can be tested experimentally; (2) guide the optimization of lead compounds,whether to increase its affinity or optimize drug metabolism and pharmacokinetics (DMPK) properties, including absorption, distribution, metabolism, excretion, and the potential for toxicity (ADMET); and (3) design new compounds, either by “constructing” starting molecules one functional group at a time or by piecing together fragments into new chemotypes.

[0148] CADD can be classified into two general categories: structure-based and ligand-based. Structure-based CADD relies on the knowledge of the target protein structure to calculate interaction energies for all compounds tested, whereas ligand-based CADD exploits the knowledge of known active and inactive molecules through chemical similarity searches or construction of predictive, quantitative structure-activity relation (QSAR) models (Kalyaanamoorthy and Chen, Drug Discovery Today, 2011, 16(17-18), 831-839). Structurebased CADD is generally preferred where high-resolution structural data of the target proteins are available (e.g., for soluble proteins that can be structurally analyzed by X-ray crystallography or NMR spectrometry). Ligand-based CADD is generally preferred when no or little structural information is available, often for membrane protein targets. The central goal of structure-based CADD is to design compounds that bind tightly to the target (e.g., with large reduction in free energy), improved DMPK / ADMET properties, and are target specific (e.g., have reduced off-target effects) (Pinzi and Rastelli, Int. J. Mol. Sci. 2019, 20, 4331). A successful application of these methods will result in a compound that can be validated in vitro and in vivo and its binding location has been confirmed, ideally through a cocrystal structure.

[0149] One of the common uses in CADD is the screening of virtual compound libraries, also known as virtual high-throughput screening (vHTS). This allows experimentalists to focus resources on testing compounds likely to have any activity of interest. In this way, a researcher can identify an equal number of hits while screening significantly less compounds, because compounds predicted to be inactive with high confidence may be skipped. Avoiding a large population of inactive compounds saves money and time, because the size of the experimental HTS is significantly reduced without sacrificing a large degree of hits (Ripphausen et al., J. Med. Chem., 2010, .53(24), 8461-8467). The largest fraction of hits has been obtained thus far is for G-protein-coupled receptors (GPCRs) followed by kinases. vHTS comes in many forms, including chemical similarity searches by fingerprints or topology, selecting compounds by predicted biologic activity through QSAR models or pharmacophore mapping, and virtual docking of compounds into target of interest, known as structure-based docking. These methods allow the ranking of “hits” from the virtual compound library for acquisition. The ranking can reflect a property of interest such as percent similarity to a query compound or predicted biologic activity, or in the case of docking, the lowest energy scoring poses for eachligand bound to the target of interest. Often initial hits are rescored and ranked using higher level computational techniques that are too time consuming to be applied to full-scale vHTS. It is important to note that vHTS does not aim to identify a drug compound that is ready for clinical testing, but rather to find leads with chemotypes that have not previously been associated with a target. This is not unlike a traditional HTS where a compound is generally considered a hit if its activity (e.g., KD, IC50, or EC50) is close to 10 micromolar. Through iterative rounds of compound synthesis and in vitro testing, a potential drug is first developed into a “lead” with higher affinity, some understanding of its structure-activity-relation, and initial tests for DMPK / ADMET properties. Only after further iterative rounds of lead-to-drug optimization and in vivo testing does a compound reach a clinically appropriate potency and acceptable DMPK / ADMET properties.

[0150] The cost benefit of using computational tools in the lead optimization phase of drug development can be substantial. Development of new drugs can cost anywhere in the range of 500 million to 2 billion dollars, with synthesis and testing of lead analogs being a large contributor to that sum (Wouters et al., JAMA. 2020, 323(9), 844-853). Therefore, it is beneficial to apply computational tools in hit-to-lead optimization to cover a wider chemical space while reducing the number of compounds that must be synthesized and tested in vitro. The computational optimization of a hit compound can involve a structure-based analysis of docking poses and energy profiles for hit analogs, ligand-based screening for compounds with similar chemical structure or improved predicted biologic activity, or prediction of favorable DMPK / ADMET properties. The comparably low cost of CADD compared with chemical synthesis and biologic characterization of large libraries of compounds make these methods attractive to focus, reduce, and diversify the chemical space that is explored (Enyedy and Egan, J Comput AidedMol Des., 2008; 22: 161-168). De novo drug design is another tool in CADD methods, but rather than screening libraries of previously synthesized compounds, it involves the design of novel compounds. A structure generator is needed to sample the space of chemicals. Given the size of the total chemical search space of more than IO60molecules, and total available compound library size of over 95 million chemicals (Rifaioglu et al., Briefings in Bioinformatics, 2019, 20(5), 1878-1912), heuristics are used to focus these algorithms on molecules that are predicted to be highly active, readily synthesizable, devoid of undesirable properties, often derived from a starting parent scaffold peptide with demonstrated activity, etc. Additionally, effective sampling strategies are used while dealing with large search spaces such as active-learning, evolutionary algorithms, metropolis search, or simulated annealing (Reker et al., Chem. Sci., 2016, 7, 3919-3927). The construction algorithms are generally defined aseither linking or growing techniques. Linking algorithms involve docking of small fragments or functional groups such as rings, acetyl groups, esters, etc., to particular binding sites followed by linking fragments from adjacent sites. Growing algorithms, on the other hand, begin from a single fragment placed in the binding site to which fragments are added, removed, and changed to improve activity. Similar to vHTS, the role of de novo drug design is not to design the single compound with nanomolar activity and acceptable DMPK / ADMET properties but rather to design a lead compound that can be subsequently improved.

[0151] Advances in the algorithms for in silico modeling have led to the creation of increasingly powerful tools for analyzing, designing, and optimizing potential new drugs. These new methods involve self-learning algorithms that are referred to by terms including artificial intelligence, machine learning, deep learning, adaptive learning, active learning, evolutionary learning, and deep reinforcement learning (Hessler et al., Molecules, 2018, 23, 2520; Rifaioglu et al. , Briefings in Bioinformatics, 2019, 20(5), 1878-1912). These algorithms are enabling virtual screening (Carpenter et al., Current Pharmaceutical Design, 2018, 24, 3347-3358) and de novo design (Schneider and Clark, Angew. Chemie Int. Ed. Engl., 2019, 58(32), 10792-10803) of small molecule drugs based on extensive data that has been collected over decades of research. Importantly, as training datasets improve, learning algorithms are becoming increasingly proficient at predicting new molecular structures to explore. In certain embodiments of the present disclosure, in silico modeling methods can be applied to peptides and proteins, such as a parent scaffold peptide and its target integrin.

[0152] If a 3D structure of a particular biological target is unavailable, but one or more binding molecules are known, ligand-based design provides an alternative strategy. This scenario holds true for G-protein-coupled receptors (GPCRs), which are the most successful drug targets in terms of therapeutic benefit and sales. A ligand-based strategy, in contrast, can either consider the 3D or the topological structure of one or more known ligands. When possible, target integrin structures or homology models are generally better for defining binding sites and predicting ligand binding interactions.

[0153] Lasso peptides offer a uniquely constrained 3D structure that is amenable to designing new biologically active molecules through predictive in silico modeling. Provided herein are methods that utilize computational algorithms to design and optimize the properties of engineered lasso peptides having an integrin-biding motif. In one aspect, engineered lasso peptides are designed using computational algorithms that enable in silico docking of engineered lasso peptide structures into a binding site of a target integrin.

[0154] In some embodiments, engineered lasso peptides are designed, and binding interactions are refined, using computational algorithms that enable in silico docking and conformational dynamic modeling of lasso peptide structures that are scored and ranked on the basis of predicted binding affinity in a lasso-binding site of protein structures. In some embodiments, engineered lasso peptides are designed using computational algorithms that enable in silico docking of lasso peptide structures into a lasso-binding site of protein structures and the lasso peptide-protein interactions are further refined and ranked using artificial intelligence algorithms. In some embodiments, engineered lasso peptides are designed and binding interactions are refined using computational and artificial intelligence algorithms that enable in silico docking and conformational dynamic modeling of lasso peptide structures that are scored and ranked on the basis of predicted binding affinity in a lasso-binding site of protein structures.

[0155] In some embodiments, engineered lasso peptides are designed and binding interactions with target integrins are refined using in silico docking algorithms (see, e.g., Pagadala et al., Biophys Rev, 2017, 9, 91-102) including, but not limited to, CABS-dock (Kurcinski et al., Protein Science, 2020, 29, 211-222), Macromodel (Mohamadi et al., J. Comput. Chem, 1990, 11, 440-467), FlexPepDock (London et al., Nucleic Acids Res. 2010 39, W249-W253), DynaDock (Antes, Proteins, 2010, 78, 1084-1104), Autodock (Morris et al., J Comput. Chem., 2009, 30, 2785-2791), MOE-Dock (Corbell etal., J Comput Aided Mol Des., 2012, 26, 775-786), Surflex-dock (Jain, J Med Chem, 2003, 46, 499- 511), Glide (Friesner etal., J. Med. Chem., 2004, 47, 1739-1749) AutoDock Vina (Trott and Olson, J. Comput. Chem., 2010, 31 (2), 455-461), or ICM (Neves et al., J Comput Aided Mol Des., 2012, 26, 675-686). In some embodiments, engineered lasso peptides are designed and binding interactions with the target integrin are refined using in silico docking algorithms together with conformational molecular dynamics algorithms, including but not limited to the publicly available or commercial programs, GROMACS, AMBER, CHARMM, and Schroedinger’ s MAESTRO.

[0156] In some embodiments, engineered lasso peptides are designed and binding interactions with target integrins are modeled and analyzed by using artificial intelligence, deep learning, or machine learning algorithms for in silico virtual screening or de novo design to define the overall lasso peptide structural topologies encompassing the loop, ring, and tail size, as well as amino acid residues that suitably fit inside and engage the lasso-binding site of a target integrin in order to optimize lasso peptide binding affinity predictions that may be refined further through iterative in silico docking and molecular dynamics simulations approaches. In some embodiments, one or more different artificial intelligence, deep learning, or machine learningalgorithms are used, including but not limited to, support vector machines (Warmuth et aL, J. Chem. Inf. Comput. Set., 2003, 43, 667-673), random forest (Deshmukh et al., Mol. Biosyst., 2017, 13, 1630-1639), ^-nearest neighbors (Luo etal., Mol. Inf, 2016, 35, 36-41), as well as neural networks with or without autoencoders, such as convolutional neural network (Jimenez et al., J. Chem. Inf. Model., 2018, 58, 287-296), recurrent neural network (Sattarov etal.,J. Chem. Inf. Model. 2019, 59, 1182-1196; and Muller etal.,J. Chem. Inf. Model., 2018, 58, 2, 472-479), deep neural network (Ma et al., J. Chem. Inf. Model., 2015, 55, 263-274), generative neural network (Gupta et al., Mol. Inf, 2018, 37, 1700111), and generative adversarial neural network (Prykhodko et al., J Cheminform., 2019, 11:74; doi.org / 10.1186 / sl3321-019-0397).

[0157] In some embodiments, engineered lasso peptides are designed and binding interactions with target integrins are optimized using in silico docking algorithms together with conformational molecular dynamics algorithms and / or artificial intelligence, deep learning, or machine learning algorithms, and engineered lasso peptides are simultaneously optimized for multiple properties, such as solubility, cell permeability, cellular activity, or stability using artificial intelligence algorithms that include, but are not limited to, fuzzy-logic design simulations (Warszawski et al., J. Mol. Biol., 2014, 426, 4125-4138), iterative stochastic elimination (Stern et al., Isr. J. Chem., 2014, 54, 1338-1357), and / or deep reinforcement learning (Popova et al., Sci. Adv. 2018;4).

[0158] In one aspect, computational and / or experimental methods are used for identifying and grafting an integrin binding motif of a natural or synthetic polypeptide ligand into a lasso peptide topological structure (FIG. 6). In some embodiments, lasso peptides containing motif grafted segments are designed using computational algorithms that enable in silico docking of lasso peptide structures into a binding site of a target integrin structure. In some embodiments, engineered lasso peptides containing motif grafted segments are designed by motif scanning whereby the integrin binding motif is computationally placed at different positions within the loop, ring, and / or tail of the lasso peptide and binding interactions are optimized using computational algorithms that enable in silico docking and conformational dynamic modeling of lasso peptide structures that are scored and ranked on the basis of predicted binding affinity in pocket of protein structures. In some embodiments, engineered lasso peptides containing motif grafted segments are designed by motif scanning whereby the integrin binding motif is computationally placed at different positions within the loop, ring, and / or tail of the lasso peptide and binding interactions are optimized using computational algorithms that enable in silico docking and conformational dynamic modeling of lasso peptide structures that are scoredand ranked on the basis of predicted binding affinity in pocket of protein structures. In some embodiments, engineered lasso peptides containing motif grafted segments are designed using computational algorithms that enable in silico docking of lasso peptide structures into a binding site of a target integrin structures and the lasso peptide-protein interactions are further refined and ranked using artificial intelligence algorithms. In some embodiments, engineered lasso peptides containing motif grafted segments are designed and binding interactions are optimized using computational and artificial intelligence algorithms that enable iterative in silico docking and conformational dynamic modeling of lasso peptide structures that are scored and ranked on the basis of predicted binding affinity in a lasso-binding site of protein structures.

[0159] In some embodiments, an integrin binding motif is computationally introduced into a lasso peptide structure. In some embodiments, motif-grafted lasso peptides are used as a basis for in silico docking and virtual screening against protein targets of interest (e.g. , integrins). In some embodiments, a linear or continuous integrin binding motif is computationally introduced into a lasso peptide structure. In some embodiments, a conformational motif is computationally introduced into a lasso peptide structure.

[0160] In some embodiments, a plurality of integrin binding motifs are computationally introduced into a lasso peptide structure e.g., the structure of a parent scaffold peptide) to create a library of engineered lasso peptides. In some embodiments, a plurality of integrin binding motifs are computationally introduced into the loop of a lasso peptide structure to create a library of engineered lasso peptides. In some embodiments, a plurality of integrin binding motifs are computationally introduced into the ring of a lasso peptide structure to create a library of engineered lasso peptides. In some embodiments, a plurality of integrin binding motifs are computationally introduced into the tail of a lasso peptide structure to create a library of engineered lasso peptides. In some embodiments, a plurality of integrin binding motifs are computationally introduced into the loop, ring, and / or tail of a lasso peptide structure to create a library of engineered lasso peptides. In some embodiments, a plurality of conformational motifs are computationally introduced into a lasso peptide structure to create a library of engineered lasso peptides. In some embodiments, a plurality of conformational motifs are computationally introduced into the loop and ring of a lasso peptide structure to create a library of engineered lasso peptides. In some embodiments, a plurality of conformational motifs are computationally introduced into the ring and tail of a lasso peptide structure to create a library of engineered lasso peptides. In some embodiments, a plurality of conformational motifs are computationally introduced into the loop and tail of a lasso peptide structure to create a library of engineered lasso peptides. In some embodiments, a plurality of conformational motifs arecomputationally introduced into the loop, ring, and tail of a lasso peptide structure to create a library of engineered lasso peptides.

[0161] In some embodiments, a plurality of integrin binding motifs are computationally introduced into a plurality of lasso peptide structures to create a library of engineered lasso peptides. In some embodiments, a plurality of 3D binding motifs are computationally introduced into a plurality of lasso peptide structures to create a library of engineered lasso peptides. In some embodiments, a plurality of conformational motifs are computationally introduced into the loop and ring of a plurality of lasso peptide structures to create a library of engineered lasso peptides. In some embodiments, a plurality of conformational motifs are computationally introduced into the ring and tail of a plurality of lasso peptide structures to create a library of engineered lasso peptides. In some embodiments, a plurality of conformational motifs are computationally introduced into the loop and tail of a plurality of lasso peptide structures to create a library of engineered lasso peptides. In some embodiments, a plurality of conformational motifs are computationally introduced into the loop, ring, and tail of a plurality of lasso peptide structures to create a library of engineered lasso peptides. In some embodiments, a plurality of conformational motifs are computationally introduced into the loop and tail of a plurality of lasso peptide structures to create a library of engineered lasso peptides.

[0162] In some embodiments, following the computational introduction of integrin binding motifs, a plurality of integrin binding motifs are grafted into a parent scaffold peptide and a plurality of engineered lasso peptides are synthesized to create a library of engineered lasso peptides. In some embodiments, following the computational introduction of integrin binding motifs, a plurality of conformational motifs are grafted into a parent scaffold peptide and a plurality of engineered lasso peptides are synthesized to create a library of engineered lasso peptides.

[0163] In one aspect, provided herein are computer-based methods for designing a lasso peptide for binding with a target molecule (e.g., integrin). In some embodiments, the method includes the steps of (a) providing one or more 3D model structures of lasso peptides; (b) docking the one or more 3D model structures of lasso peptides onto a 3D model structure of the target molecule (e.g., integrin) at a lasso-binding site of the target molecule (e.g., integrin), thereby obtaining an optimal docked system; (c) selecting the 3D model structure of the lasso peptide forming the optimal docked system; (d) mapping an integrin binding motif onto the selected 3D model structure of the lasso peptide; and (e) computationally introducing theintegrin binding motif onto the mapped locations of the selected lasso peptide, thereby generating an engineered lasso peptide candidate.

[0164] In some embodiments, an integrin binding motif is the amino acid residue or the group of amino acid residues of a lasso peptide that binds to a target molecule (e.g., integrin). An integrin binding motif as disclosed herein can be a linear motif that contains a fragment of continuous amino acids (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 continuous amino acids) of the lasso peptide, or a conformational motif that consists of amino acid residues in two or more non-continuous regions of the lasso peptide. According to the present disclosure, an integrin binding motif can contain surface groupings of binding moi eties ((e.g., functionally active chemical groups on amino acids or sugar side chains known to promote binding with biological molecules) responsible for binding with the target integrin. The binding moieties can locate on the same amino acid residue or different amino acid residues forming the integrin binding motif. In some embodiments, the integrin binding motif of a lasso peptide can have specific 3D structural characteristics where the binding moieties are arranged and displayed. A lasso peptide can have more than one different integrin binding motifs that bind with different target sites of a target integrin, or bind with different target integrins.

[0165] According to the present disclosure, providing 3D model structures of a protein or peptide can be performed by retrieving known structural information of the protein or peptide from a protein structure database, such as the worldwide Protein Data Bank (wwPDB) (website: www.wwpdb.org), the Cambridge Structure Database (CSD) (website: www.ccdc.cam.ac.uk / solutions / csd-core / components / csd), Molecular Model Database (MMDB) of National Center for Biotechnology Information (NCBI) (website: www.ncbi.nlm.nih.gov / Structure / MMDB / docs / mmdb_help.html), and the Biological Magnetic Resonance Data Bank (BMRB) (website: bmrb.io). In some embodiments, the protein structure database contains readily available modeled 3D structure of a peptide or protein. In other embodiments, the protein structure database contains atomic coordinates of a peptide or protein that can be used to create a 3D model structure of the protein or peptide using a computer modeling algorithm.

[0166] Accordingly, in some embodiments, the step of (a) providing one or more 3D model structures of lasso peptides of the present method includes retrieving one or more known 3D structures of lasso peptide from a protein structure database. In particular embodiments, the protein structure database is selected from the Protein Data Bank (wwPDB), the Cambridge Structure Database, the Molecular Model Database of National Center for Biotechnology Information (NCBI), and the Biological Magnetic Resonance Data Bank (BMRB) database.

[0167] In alternative embodiments, the step of (a) providing one or more 3D model structures of lasso peptides of the present method includes computationally modeling the 3D structure of a lasso peptide based on atomic coordinates of the lasso peptide. In particular embodiments, the atomic coordinates of the lasso peptide are obtained from a protein structure database or scientific literature. In yet particular embodiments, the protein structure database is selected from a protein structure database. In particular embodiments, the protein structure database is selected from the Protein Data Bank (wwPDB), the Cambridge Structure Database, the Molecular Model Database of National Center for Biotechnology Information (NCBI), and the Biological Magnetic Resonance Data Bank (BMRB) database.

[0168] In some embodiments, the step of (a) providing one or more 3D model structures of lasso peptides of the present method includes computationally modeling the 3D structure of a lasso peptide based on atomic coordinates of the lasso peptide. In particular embodiments, the atomic coordinates of the lasso peptide are obtained by subjecting the lasso peptide to nuclear magnetic resonance (NMR) analysis, X-ray crystallography, neutron diffraction, or three- dimensional electron microscopy (3D-EM). In specific embodiments, the atomic coordinates of the lasso peptide are obtained by subjecting the lasso peptide to serial femtosecond crystallography. In specific embodiments, the atomic coordinates of the lasso peptide are obtained by subjecting the lasso peptide to cryogenic electron microscopy (cryo-EM).

[0169] In some embodiments, the step of (a) providing one or more 3D model structures of lasso peptides of the present method includes computationally modeling the 3D structure of a lasso peptide based on atomic coordinates of the lasso peptide. In some embodiments, the computational modeling is performed by molecular replacement modeling. In specific embodiments, the step of (a) providing one or more 3D model structures of lasso peptides of the present method includes computationally modeling the 3D structure of the lasso peptide based on X-ray diffraction data and / or nuclear magnetic resonance (NMR) data of the lasso peptide and atomic coordinates of a reference lasso peptide.

[0170] In some embodiments, the X-ray diffraction data of the lasso peptide are obtained by subjecting a crystal of the lasso peptide to X-ray crystallography analysis. In particular embodiments, the step of (a) providing one or more 3D model structures of lasso peptides of the present method includes (i) obtaining atomic coordinates of the lasso peptide based on the X-ray diffraction data; and (ii) refining the atomic coordinates of the lasso peptide based on the atomic coordinates of the reference lasso peptide. In specific embodiments, the step of (ii) refining the atomic coordinates of the lasso peptide is performed using a molecular replacement method.

[0171] In some embodiments, the NMR data of the lasso peptide includes NMR chemical shift, J-coupling constant, and resonance intensity obtained by subjecting a solution of the lasso peptide to NMR analysis. In particular embodiments, the step of (a) providing one or more 3D model structures of lasso peptides of the present method includes (i) creating an ensemble of structural models of the lasso peptide based on the NMR data; (ii) obtaining mean atomic coordinates of the lasso peptide based on the ensemble of structural models; and (iii) refining the atomic coordinates of the lasso peptide based on the atomic coordinates of the reference lasso peptide.

[0172] In some embodiments, the step of (a) providing one or more 3D model structures of lasso peptides of the present method includes computationally modeling the 3D structure of a lasso peptide based on the amino acid sequence of the lasso peptide and atomic coordinates of a reference lasso peptide, and wherein the lasso peptide has at least 50 percent (%) sequence identity to the reference lasso peptide.

[0173] In some embodiments, the step of (a) providing one or more 3D model structures of lasso peptides of the present method includes computationally modeling the 3D structure of a lasso peptide based on the amino acid sequence of the lasso peptide and atomic coordinates of a reference lasso peptide; and wherein the lasso peptide has at least 50 percent (%) amino acid sequence identity to the reference lasso peptide. In some embodiments, computationally modeling the 3D structure of the lasso peptide is performed by a homology modeling algorithm.

[0174] In specific embodiments involving a reference lasso peptide, the lasso peptide has at least about 50 percent (%) amino acid sequence identity to the reference lasso peptide. In specific embodiments involving a reference lasso peptide, the lasso peptide has at least about 55% amino acid sequence identity to the reference lasso peptide. In specific embodiments involving a reference lasso peptide, the lasso peptide has at least about 60% amino acid sequence identity to the reference lasso peptide. In specific embodiments involving a reference lasso peptide, the lasso peptide has at least about 65% amino acid sequence identity to the reference lasso peptide. In specific embodiments involving a reference lasso peptide, the lasso peptide has at least about 70% amino acid sequence identity to the reference lasso peptide. In specific embodiments involving a reference lasso peptide, the lasso peptide has at least about 75% amino acid sequence identity to the reference lasso peptide. In specific embodiments involving a reference lasso peptide, the lasso peptide has at least about 80% amino acid sequence identity to the reference lasso peptide. In specific embodiments involving a reference lasso peptide, the lasso peptide has at least about 85% amino acid sequence identity to the reference lasso peptide. In specific embodiments involving a reference lasso peptide, the lassopeptide has at least about 90% amino acid sequence identity to the reference lasso peptide. In specific embodiments involving a reference lasso peptide, the lasso peptide has at least about 95% amino acid sequence identity to the reference lasso peptide. In specific embodiments involving a reference lasso peptide, the lasso peptide has at least about 97% amino acid sequence identity to the reference lasso peptide.

[0175] According to the present disclosure, in some embodiments, a 3D model structure of a lasso peptide can be a lasso peptide backbone structure. In some embodiments, a lasso peptide backbone structure can be constructed by computationally modeling the 3D structure of a lasso peptide and further computationally modeling the lasso backbone structure by removing side chains of each amino acid residue that is not an internal ring-forming residue from the 3D structure of lasso peptides.

[0176] Accordingly, in some embodiments, the step of (a) providing one or more 3D model structures of lasso peptides of the present method further includes computationally modeling the lasso backbone structures by removing side chains of each amino acid residue that is not an internal ring-forming residue from the modeled 3D structure of lasso peptides before proceeding to step (b) of the present method.

[0177] In some embodiments, step (b) of the method includes creating the 3D model structure of the target molecule (e.g., integrin) before docking the one or more 3D model structure of lasso peptide onto the 3D model structure of the target molecule (e.g., integrin). In some embodiments, the target molecule (e.g., integrin) has one or more lasso-binding site on its surface, and step (b) of the method includes docking the one or more 3D model structure of lasso peptide onto the lasso-binding site of the target molecule.

[0178] In some embodiments, the target molecule (e.g., integrin) is capable of binding to a ligand, and the lasso-binding site of the target molecule can be the ligand-binding site where the ligand binds to the target molecule.

[0179] In some embodiments, the target molecule (e.g., integrin) is a receptor that has an orthosteric site where a ligand molecule binds. Accordingly, in some embodiments, the lasso- peptide binding site of a target molecule can be an orthosteric site of the target molecule. In some embodiments, the target molecule is a receptor that has at least one allosteric site where an allosteric effector molecule binds. Accordingly, in some embodiments, the lasso-peptide binding site of a target molecule can be an allosteric site of the target molecule.

[0180] In some embodiments, an orthosteric site is the primary binding site on a receptor that is recognized by an endogenous or natural ligand or agonist for that receptor and induces a biological response. For example, the orthosteric site in the muscarinic receptor is the sitethat acetylcholine binds to modulate neuron polarization. In some embodiments, an allosteric site is a specific binding site other than the orthosteric site on a receptor molecule that, upon binding of an allosteric effector molecule, influences the function or activity of that receptor, typically by changing the shape or conformation of the orthosteric site.

[0181] In some embodiments, the target molecule (e.g., integrin) is capable of switching between an active conformation (open conformation) and an inactive conformation (closed conformation). Accordingly, in some embodiments, the lasso-peptide binding site of a target molecule (e.g., integrin) can be a binding site that exists in the active or open conformation of the target peptide. In alternative embodiments, the lasso-peptide binding site of a target molecule (e.g., integrin) can be a binding site that exists in the inactive or closed conformation of the target peptide.

[0182] In some embodiments, the step (b) of the present method includes creating the 3D model structure of the target molecule (e.g., integrin) before docking. Particularly, in some embodiments, creating the 3D model structure of the target molecule (e.g., integrin) includes computationally modeling the 3D structure of the target molecule based on atomic coordinates of the target molecule. In some embodiments, the atomic coordinates of the target molecule (e.g., integrin) are known. In some embodiments, the atomic coordinates of the target molecule (e.g., integrin) can be obtained from a protein structure database or scientific literature. In some embodiments, the atomic coordinates of the target molecule can be obtained from protein structure database selected from the Protein Data Bank (wwPDB), the Cambridge Structure Database, the Molecular Model Database of National Center for Biotechnology Information (NCBI), and the Biological Magnetic Resonance Data Bank (BMRB) database.

[0183] In some embodiments, the atomic coordinates of the target molecule (e.g., integrin) are obtained by subjecting the target molecule to nuclear magnetic resonance (NMR) analysis, X-ray crystallography, neutron diffraction analysis, or three-dimensional electron microscopy (3D-EM). In particular embodiments, the atomic coordinates of the target molecule (e.g., integrin) are obtained by subjecting the target molecule to serial femtosecond crystallography. In particular embodiments, the atomic coordinates of the target molecule (e.g., integrin) are obtained by subjecting the target molecule to cryogenic electron microscopy (cryo-EM).

[0184] In some embodiments, step (b) of the present method includes creating the 3D model structure of the target molecule (e.g., integrin) before docking. Particularly, in some embodiments, creating the 3D model structure of the target molecule (e.g, integrin) includes computationally modeling the 3D structure of the target molecule based on X-ray diffractiondata and / or nuclear magnetic resonance data of the target molecule and atomic coordinates of a reference polypeptide.

[0185] In some embodiments, the X-ray diffraction data of the target molecule (e.g., integrin) are obtained by subjecting a crystal of the target molecule to X-ray crystallography analysis. In some embodiments, creating the 3D model structure of the target molecule (e.g., integrin) includes: (i) obtaining atomic coordinates of the target molecule based on the X-ray diffraction data; (ii) refining the atomic coordinates of the target molecule based on the atomic coordinates of the reference polypeptide; and (iii) computationally modeling the 3D structure of the target molecule based on the refined atomic coordinates. In particular embodiments, the refining step is performed using a molecular replacement algorithm. In particular embodiments, the atomic coordinates of the reference polypeptide are known. In particular embodiments, the atomic coordinates of the reference polypeptide are obtained from a protein structure database selected from the Protein Data Bank (wwPDB), the Cambridge Structure Database, the Molecular Model Database of National Center for Biotechnology Information (NCBI), and the Biological Magnetic Resonance Data Bank (BMRB) database. In some embodiments, the atomic coordinates of the reference polypeptide are obtained from scientific literature.

[0186] In some embodiments, the nuclear magnetic resonance (NMR) data of the target molecule (e.g., integrin) includes NMR chemical shift, J-coupling constant, and resonance intensity obtained from subjecting a solution of the target molecule to NMR analysis. In some embodiments, creating the 3D model structure of the target molecule (e.g., integrin) includes(i) creating an ensemble of structural models of the target molecule based on the NMR data;(ii) obtaining mean atomic coordinates of the target molecule based on the ensemble of structural models; (iii) refining the mean atomic coordinates of the target molecule based on the atomic coordinates of the reference polypeptide; and (iv) computationally modeling the 3D structure of the target molecule based on the refined mean atomic coordinates. In particular embodiments, the refining step is performed using a molecular replacement algorithm. In particular embodiments, the atomic coordinates of the reference polypeptide are known. In particular embodiments, the atomic coordinates of the reference polypeptide are obtained from a protein structure database selected from the Protein Data Bank (wwPDB), the Cambridge Structure Database, the Molecular Model Database of National Center for Biotechnology Information (NCBI), and the Biological Magnetic Resonance Data Bank (BMRB) database. In some embodiments, the atomic coordinates of the reference polypeptide are obtained from scientific literature.

[0187] In some embodiments, step (b) of the present method includes creating the 3D model structure of the target molecule (e.g., integrin) before docking. In some embodiments, creating the 3D model structure of the target molecule includes computationally modeling the 3D structure of the target molecule based on the amino acid sequence of the target molecule and atomic coordinates of one or more reference polypeptide. In some embodiments, computationally modeling the 3D structure of the target molecule is performed by homology modeling. In some embodiments, computationally modeling the 3D structure of the target molecule is performed by protein structure prediction. In some embodiments, computationally modeling the 3D structure of the target molecule is performed by a combination of protein structure prediction and homology modeling. In some embodiments, the atomic coordinates of the reference polypeptide are known. In some embodiments, the atomic coordinates of the reference polypeptide are obtained from a protein structure database or scientific literature. In some embodiments, the protein structural database is selected from worldwide Protein Data Bank (wwPDB), Cambridge Structure Database, Molecular Model Database of National Center for Biotechnology Information (NCBI), and Biological Magnetic Resonance Data Bank (BMRB) database.

[0188] In specific embodiments involving a reference polypeptide, the target molecule e.g., integrin) has at least about 50 percent (%) amino acid sequence identity to the reference peptide. In specific embodiments involving a reference polypeptide, the target molecule has at least about 55% amino acid sequence identity to the reference peptide. In specific embodiments involving a reference polypeptide, the target molecule has at least about 60% amino acid sequence identity to the reference peptide. In specific embodiments involving a reference polypeptide, the target molecule has at least about 65% amino acid sequence identity to the reference peptide. In specific embodiments involving a reference polypeptide, the target molecule has at least about 70% amino acid sequence identity to the reference peptide. In specific embodiments involving a reference polypeptide, the target molecule has at least about 75% amino acid sequence identity to the reference peptide. In specific embodiments involving a reference polypeptide, the target molecule has at least about 80% amino acid sequence identity to the reference peptide. In specific embodiments involving a reference polypeptide, the target molecule has at least about 85% amino acid sequence identity to the reference peptide. In specific embodiments involving a reference polypeptide, the target molecule has at least about 90% amino acid sequence identity to the reference peptide. In specific embodiments involving a reference polypeptide, the target molecule has at least about 95% amino acid sequence identity to the reference peptide. In specific embodiments involving areference polypeptide, the target molecule has at least about 97% amino acid sequence identity to the reference peptide.

[0189] In some embodiments, step (b) of the present method includes docking the one or more 3D model structures of lasso peptides onto a 3D model structure of the target molecule (e.g., integrin) at a lasso-binding site of the target molecule, thereby obtaining an optimal docked system. In some embodiments, the docking includes, for each 3D model structure of the lasso peptide: (b-1) positioning a docked portion of the 3D model structure of the lasso peptide relative to the lasso-binding site of the 3D model structure of the target molecule e.g., integrin) in a first docked pose, thereby forming a docked system; (b-2) scoring the docked system based on structural complementarity between the docked portion and the lasso-binding site; (b-3) repositioning the docked portion relative to the lasso-binding site to a second docked pose, and repeating step (b-2); (b-4) repeating step (b-3) for one or more times; and (b-5) selecting the docked system having the highest score as the optimal docked system before proceeding to step (c).

[0190] In some embodiments, the docked portion of the 3D model structure includes at least one amino acid residue from the loop portion of the lasso peptide. In some embodiments, the docked portion of the 3D model structure includes at least one amino acid residue from the ring portion of the lasso peptide. In some embodiments, the docked portion of the 3D model structure includes at least one amino acid residue from the tail portion of the lasso peptide. In some embodiments, the docked portion of the 3D model structure includes at least one amino acid residue from the loop portion of the lasso peptide and at least one amino acid residue from the ring portion of the lasso peptide. In some embodiments, the docked portion of the 3D model structure includes at least one amino acid residue from the loop portion of the lasso peptide and at least one amino acid residue from the tail portion of the lasso peptide. In some embodiments, the docked portion of the 3D model structure includes at least one amino acid residue from the ring portion of the lasso peptide and at least one amino acid residue from the tail portion of the lasso peptide. In some embodiments, the docked portion of the 3D model structure includes at least one amino acid residue from the loop portion of the lasso peptide, at least one amino acid residue from the ring portion of the lasso peptide, and at least one amino acid residue from the tail portion of the lasso peptide.

[0191] In some embodiments, the docked system includes one or more complementary binding pairs including a first binding moiety located on the lasso peptide, and a second binding moiety located on the target molecule (e.g., integrin). In some embodiments, in a docked system, the first and second binding moieties form binding interaction with one another. Insome embodiments, the binding interaction between the first and second binding moieties is non-covalent interaction. In some embodiments, the binding interaction between the first and second binding moieties is selected from hydrogen bonding interactions, ionic or electrostatic bonding interactions, pi stacking interactions, polar interactions, dipolar interactions, induced dipolar interactions, hydrophobic interactions, and van der Waals interactions.

[0192] In some embodiments, in a docked system, the first binding moiety is located on a first amino acid residue of the lasso peptide, and the second binding moiety is on a second amino acid residue located on the target molecule (e.g., integrin). In some embodiments, the distance between any atom of the first amino acid residue and any atom of the second amino acid residue is less than about 5 Angstroms. In some embodiments, the distance between any atom of the first amino acid residue and any atom of the second amino acid residue is less than about 4 Angstroms. In some embodiments, the distance between any atom of the first amino acid residue and any atom of the second amino acid residue is less than about 3 Angstroms.

[0193] In some embodiments, step (b-2) scoring the docked system based on structural complementarity between the docked portion and the lasso-binding site includes calculating a total binding free energy of the docked system using one or more molecular mechanics force field functions, and assigning a score to the docked system based on the total binding free energy. In some embodiments, the assigned score to a docked system negatively relates to the total binding free energy of the docked system. In some embodiments, a relatively higher score is assigned to a docked system having a relatively lower total binding free energy. In some embodiments, a relatively lower score is assigned to a docked system having a relatively higher total binding free energy.

[0194] Various computational modeling platforms that are known in the art can be used for calculating the free energy differences between different docked molecules and / or poses and total binding free energy of a docked system using molecular mechanics force field functions and scoring the docked system. Exemplary computational modeling platforms include but are not limited to the MOE 2020.09 computational modeling platform (Molecular Operating Environment, Chemical Computing Group Inc. Canada). A variety of force fields and force field parameters can be used in connection with the present disclosure, including but not limited to Amber force fields AMBER99, Amber 10EHT, ffl4SB, and ffl9SB, with amino acids- specific side chain and protein backbone parameters (See, e.g., Ponder and Case, Adv. Prot. Chem. 2003, 66, 27-85; Tian et al., J. Chem. Theory Comput., 2019, 16, 528-552; and Maier et al., J. Chem. Theory Comput., 2015, 11, 3696-3713) and the MMFF94x force field (See, e.g., Halgren, Comput. Chem., 1996, 490-519). Accordingly, in some embodiments, in step(b-2), scoring the docked system based on structural complementarity between the docked portion and the lasso-binding site includes calculating a total binding free energy of the docked system using one or more molecular mechanics force field functions selected from Amber force fields AMBER99, Amber 10EHT, ffl4SB, ffl9SB, and the Merck force field MMFF94x, and assigning a score to the docked system based on the lower total binding free energy. In some embodiments, the score assigned to a docked system negatively relates to the total binding free energy. In some embodiments, a relatively higher score is assigned to a docked system having a relatively lower total binding free energy. In some embodiments, a relatively lower score is assigned to a docked system having a relatively higher total binding free energy.

[0195] In some embodiments, step (b-3) repositioning the docked portion relative to the lassobinding site to a second docked pose includes identifying the second docked pose using an energy minimizing function before repositioning the docked portion, wherein the docked system is predicted to have a lower binding free energy in the second docked pose than in the first docked pose based on the energy minimizing function. Various energy minimizing function known in the art can be used in connection with the present disclosure, including but not limited to the steepest decent algorithm (Jaidhan, B.J., et al. Energy minimization and conformational analysis of molecules using steepest descent method, (IJCSIT) Int J Comp Sci Inf Tech, 2014, 5(3), 3525-3528), the conjugate gradient algorithm (Knyazev and Lashuk, SIAM Journal on Matrix Analysis and Applications. 2008, 29(4): 1267-1280), the truncated Newton algorithms (Nash, Comput. AppL Math., 2000, 124(1-2): 45-59), the limited-memory Broyden-Fletcher-Goldfarb-Shanno (L-BFGS) algorithm (Byrd et al., J. Sci. Comput. 1995, 16 (5): 1190-1208), and / or the genetic algorithm (Le Grand et al., J. Global Opt., 1993, 3(1), 49-66).

[0196] Accordingly, in some embodiments, the energy minimizing function is selected from the steepest descent algorithm, conjugate gradients algorithm, L-BFGS (limited-memory Broyden-Fletcher-Goldfarb-Shanno) algorithm, and genetic algorithms.

[0197] In some embodiments, step (d) mapping an integrin binding motif onto the selected 3D model structure of the lasso peptide includes (d-1) in the optimal docked system, identifying one or more lasso-binding moi eties in the lasso-binding site of the target molecule (e.g., integrin); (d-2) selecting one or more amino acid residues including one or more target-binding moieties complementary to the one or more lasso-binding moieties; and (d-3) mapping an optimal set of positions in the amino acid sequence of the selected lasso peptide for grafting the one or more selected amino acid residues; wherein the grafting places the one or more target-binding moieties at suitable spatial locations and orientations for binding with thecomplementary lasso-binding moieties on the target molecule. In some embodiments, the binding interaction between the complementary lasso-binding moiety and the target-binding moiety is selected from covalent bonding interactions or non-covalent interactions including hydrogen bonding interactions, ionic or electrostatic bonding interactions, polar interactions, dipolar interactions, induced dipolar interactions, pi stacking interactions, H-bond-aromatic interactions, hydrophobic interactions, and van der Waals interactions.

[0198] In some embodiments, step (d-3) mapping an optimal set of positions in the amino acid sequence of the selected lasso peptide for grafting the one or more selected amino acid residues includes: (d-3-1) computationally introducing the one or more selected amino acid residues into the amino acid sequence of the selected lasso peptide at a first set of positions; (d-3 -2) calculating a total binding free energy of the optimal docked system using one or more molecular force field functions; (d-3-3) modifying at least one position in the first set of positions thereby obtaining an adjusted set of positions, and computationally introducing the one or more selected amino acid residues into the amino acid sequence of the selected lasso peptide at the adjusted set of positions; (d-3 -4) repeating step (d-3 -2); (d-3 -5) repeating steps (d-3-3) and (d-3 -4) sequentially for one or more times; and (d-3 -6) selecting the adjusted set of positions associated with the lowest total binding free energy as the optimal map of positions.

[0199] In some embodiments, the molecular mechanics force field functions are selected from Amber force fields AMBER99, Amber 10EHT, ffl4SB, ffl9SB, and Merck force field MMFF94x.

[0200] In some embodiments, in step (d-3-5), wherein repeating steps (d-3-3) and (d-3-4) sequentially for one or more times including repeating steps (d-3-3) and (d-3-4) for at least 1 time, at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, at least fifteen times, at least twenty times, or at least twenty-five times.

[0201] In some embodiments, step (d-3-3) includes identifying the adjusted set of positions using an energy minimizing function before modifying the first set of positions to become the adjusted set of positions, wherein the docked system having the one or more selected amino acid residues grafted into the amino acid sequence of the selected lasso peptide at the adjusted set of positions is predicted to have a lower binding free energy than at the first set of positions based on the energy minimizing function. In some embodiments, the energy minimizing function is selected from the steepest descent algorithm, conjugate gradients algorithm, L- BFGS (limited-memory Broyden-Fletcher-Goldfarb-Shanno) algorithm, and genetic algorithms.

[0202] In some embodiments, the target molecule (e.g., integrin) has a naturally-existing ligand to which the target molecule binds. Accordingly, in some embodiments, the integrin binding motif to be mapped onto the 3D model structure of the lasso peptide corresponds to a continuous fragment of the naturally-existing ligand that binds to the target molecule. In some embodiments, the integrin binding motif is a linear motif. In some embodiments, the integrin binding motif has 100% amino acid sequence identity to the corresponding continuous fragment of the naturally-existing ligand of the target molecule. In some embodiments, the amino acid sequence of the integrin binding motif differ from the corresponding continuous fragment of the naturally-existing ligand of the target molecule by 1, 2, 3, 4, 5, or more than 5 amino acid residue.

[0203] Alternatively, in some embodiments, the integrin binding motif to be mapped onto the 3D model structure of the lasso peptide includes multiple discontinuous fragments that respectively correspond to multiple discontinuous fragments of the naturally-existing ligand to which the target molecule (e.g., integrin) binds. In some embodiments, the integrin binding motif is a conformational motif. In some embodiments, the multiple discontinuous fragments of the integrin binding motif has 100% amino acid sequence identity to the corresponding multiple discontinuous fragments of the naturally-existing ligand. In some embodiments, the multiple discontinuous fragments of the integrin binding motif differ from the corresponding multiple discontinuous fragments of the naturally-existing ligand by 1, 2, 3, 4, 5, or more than 5 amino acid residue.

[0204] In some embodiments, the target molecule (e.g., integrin) has a naturally-existing ligand, wherein the integrin binding motif corresponds to a fragment or fragments of a naturally-existing ligand of the target molecule that are capable of binding with the lassobinding site of the target molecule and forming a ligand-target interface. In these embodiments, step (d) of the present method further includes aligning the docked portion of the 3D model structure of the lasso peptide with a 3D model structure of the corresponding fragment or fragments of the naturally-existing ligand in the ligand-target interface. In particular embodiments, the aligning includes adjusting the spatial position, conformation and / or orientation of at least one target-binding moiety in the docked portion of the lasso peptide to mimic the spatial position, conformation and / or orientation of the corresponding binding moiety of the fragment or fragments of the naturally-existing ligand in the ligand-target interface.

[0205] In some embodiments, the present method further includes (f) mutating one or more amino acid residues of the engineered lasso peptide candidate to produce a first set of lassopeptide binder variants; and (g) ranking the first set of lasso peptide binder variants based on a predicted binding affinity for binding with the target molecule (e.g., integrin). In particular embodiments, step (f) further includes adjusting conformations of the first set of lasso peptide binder variants to produce a second set of lasso peptide binder variants. In particular embodiments, step (g) further includes ranking the second set of lasso peptide binder variants based on the predicted binding affinity for binding with the target molecule. In specific embodiments, in step (f), mutating the amino acid residue of the engineered lasso peptide candidate includes replacing the side chain of the amino acid residue of the engineered lasso peptide candidate with the side chain of a second amino acid that is different from the mutated amino acid residue. In specific embodiments, the second amino acid is a naturally-occurring or a non-natural amino acid.

[0206] In some embodiments, in step (f), mutating the amino acid residue of the engineered lasso peptide candidate includes modifying one or more chemical moieties on the side chain of the mutated amino acid residue. In some embodiments, at least one modified chemical moiety is a target-binding moiety.

[0207] In some embodiments, in step (f), mutating the amino acid residue of the engineered lasso peptide candidate includes modifying one or more side chains of the engineered lasso peptide candidate to complement one or more lasso-binding moieties on the target molecule (e.g., integrin); and wherein the modifying is selected from the group consisting of (i) incorporating into the lasso peptide a neutral or basic side chain that is structurally opposite to an acidic lasso-binding moiety; (ii) incorporating into the lasso peptide a neutral or acid side chain that is structurally opposite to a basic lasso-binding moiety; (iii) incorporating into the lasso peptide a neutral or positively charged side chain that is structurally opposite to a negatively charged lasso-binding moiety; (iv) incorporating into the lasso peptide a neutral or negatively charged side chain that is structurally opposite to a positively charged lassobinding moiety; (v) incorporating into the lasso peptide a sterically smaller side chain that is structurally opposite to a sterically larger lasso-binding moiety; (vi) incorporating into the lasso peptide a sterically larger side chain that is structurally opposite to a sterically smaller lassobinding moiety; (vii) incorporating into the lasso peptide a hydrophobic side chain, preferably a similarly hydrophobic side chain, that is structurally opposite to a hydrophobic lasso-binding moiety; (viii) incorporating into the lasso peptide an acidic, positively charged, or aromatic side chain that is structurally opposite to an aromatic lasso-binding moiety; (ix) incorporating into the lasso peptide an electron-rich aromatic side chain that is structurally opposite to an electron-poor aromatic lasso-binding moiety; (x) incorporating into the lasso peptide an acidor electron-poor aromatic side chain that is structurally opposite to an electron-rich aromatic lasso-binding moiety; (xi) incorporating into the lasso peptide an inducible dipole or multipole side chain that is structurally opposite to an inducible dipole or multipole lasso-binding moiety; (xii) incorporating into the lasso peptide a permanent, inducible dipole or multipole side chain that is structurally opposite to an inducible or multipole lasso-binding moiety; (xiii) incorporating into the lasso peptide an H-bond acceptor side chain that is structurally opposite to an H-bond donor lasso-binding moiety, and (xiv) incorporating into the lasso peptide an H- bond donor side chain that is structurally opposite to an H-bond acceptor lasso-binding moiety.

[0208] In some embodiments, the mutated amino acid residue is in the (i) ring portion of the lasso peptide; (ii) loop portion of the lasso peptide; (iii) tail portion of the lasso peptide; or (iv) any combination of (i) to (iii).

[0209] In some embodiments, the method further includes step (h) synthesizing the engineered lasso peptide candidate or one or more lasso peptide binder variants having the highest rankings. In particular embodiments, in step (h), synthesizing the engineered lasso peptide candidate or the one or more lasso peptide binder variants is performed using a cell- free or cell-based synthesis method.

[0210] In some embodiments, the method further includes creating a database of optimized lasso peptide structures or intermediates thereof generated in any one of steps (a) to (h).

[0211] According to the present disclosure, de novo design of lasso peptides represents an alternative computational strategy for discovering new lasso peptides that can bind with high affinity to target integrins. De novo drug design is based on stochastic structure optimization, evolutionary fitness functions, and combinatorial design principles that potentially represent a vast chemical space. In order to manage the large numbers of possible structures, constraints are placed on the de novo design space. In general, receptor-based constrains or ligand-based constraints are implemented (Schneider and Fechner, Nat Rev Drug Discov. 2005, 4, 649-663). All information that is related to the ligand-receptor interaction forms the primary target constraints for candidate compounds. Such constraints can be gathered both from the 3D receptor structure and from the structures of known ligands of the particular target. Receptorbased design strategies starts with the determination of known binding sites for known ligands and potential binding sites for new ligands. As complementarities in molecular shape and physical and chemical properties are important for specific binding, the binding site is then examined to derive shape constraints for a ligand, as well as specific non-covalent ligandreceptor interactions in the form of hypothetical interaction sites created through hydrogen bonding, van der Waals, electrostatic, and hydrophobic interactions. Receptor groups capableof hydrogen-bonding are of special interest owing to the strongly directional nature of the two interaction partners (i.e., hydrogen-bond acceptor and donor) and often form key interaction sites, especially in protein-protein or protein-peptide complexes. These constraints allow the assignment of ligand atom positions with a complementary hydrogen-bond type within a small region of space and a defined orientation. Key interaction sites have a major role in the effort to reduce the vast number of possible structures because they define strong and explicit requirements for successful receptor-ligand binding. For ligand generation, energetically favorable positions and orientations of functional groups in the binding site are determined. Functional groups (e.g., amino acids with side chains) are placed inside the binding pocket and fragments are then minimized simultaneously using a force field. Groups are discarded if the interaction energy between them and the protein is above a certain threshold. A de novo ligand design run yields a set of pre-docked fragments or sets of functional groups that can be further investigated through docking to choose the most promising ones. This placement of chemical groups provides a starting point for the assembly of complete ligands and scoring the ligandreceptor binding.

[0212] In some embodiments, the integrin binding motif is designed de novo by creating a set of amino acids that complements those in a potential lasso peptide binding site of a target integrin. In some embodiments, a linear or 3D arrangement of a set of amino acids is identified as a potential lasso-binding site in a target integrin. In some embodiments, the linear or 3D arrangement consists of 2-10 amino acids. In some embodiments, a complementary set of amino acids are identified for interacting with the amino acids in the target binding site. In other embodiments, the complementary set of amino acids have properties that facilitate attractive or non-repulsive interactions with the amino acids in the target binding site. In other embodiments, the complementary set of amino acids is grafted onto a parent scaffold peptide.

[0213] Particularly, in some embodiments, provided herein are computer-based methods for designing a lasso peptide for binding with a target molecule (e.g., integrin). In some embodiments, the method includes: (a) providing a model 3D structure of the target molecule (e.g., integrin) including one or more lasso-binding moi eties at defined spatial locations; (b) selecting one or more amino acid residues including one or more target-binding moieties complementary to the one or more lasso-binding moieties; (c) generating a model 3D structure of an integrin binding motif including the selected amino acid residues; wherein the model 3D structure of the integrin binding motif places the one or more target-binding moieties at suitable spatial locations and orientations for binding with the complementary lasso-binding moieties on the target molecule; (d) screening a plurality of model 3D structures of lasso peptides forengineered lasso peptide candidates including at least one target-binding site capable of supporting the model 3D structure of the integrin binding motif; (e) grafting the integrin binding motif onto the target-binding site of the engineered lasso peptide candidate thereby providing a lasso peptide binder; (f) mutating one or more amino acid residues of the lasso peptide binder to produce a plurality of engineered lasso peptide candidates; (g) ranking the engineered lasso peptide candidates for optimal binding with the target molecule using an energy minimizing function; and (h) synthesizing one or more engineered lasso peptide candidates having the highest ranking.

[0214] In some embodiments, step (a) providing a model 3D structure of the target molecule (e.g., integrin) including one or more lasso-binding moi eties at defined spatial locations. In specific embodiments, step (a) is performed by computationally modeling the 3D structure of the target molecule based on atomic coordinates of the target molecule. In specific embodiments, the atomic coordinates of the target molecule are obtained from a protein structure database or scientific literature. In specific embodiments, the protein structure database is selected from worldwide Protein Data Bank (wwPDB), Cambridge Structure Database, Molecular Model Database of National Center for Biotechnology Information (NCBI), and Biological Magnetic Resonance Data Bank (BMRB) database. In specific embodiments, the atomic coordinates of the target molecule are obtained by subjecting the target molecule to nuclear magnetic resonance (NMR) analysis, X-ray crystallography, neutron diffraction analysis, or three-dimensional electron microscopy (3D-EM). In specific embodiments, the X-ray crystallography is serial femtosecond crystallography. In specific embodiments, the 3D-EM is cryogenic electron microscopy (cryo-EM).

[0215] In some embodiments, computationally modeling the 3D structure of the target molecule (e.g., integrin) is based on X-ray diffraction data and / or nuclear magnetic resonance (NMR) data of the target molecule and atomic coordinates of a reference polypeptide; wherein the reference polypeptide has at least 50% amino acid sequence identity to the target molecule. In some embodiments, the X-ray diffraction data of the target molecule are obtained by subjecting a crystal of the target molecule to X-ray crystallography analysis.

[0216] In some embodiments, creating the 3D model structure of the target molecule (e.g., integrin) includes: (i) obtaining atomic coordinates of the target molecule based on the X-ray diffraction data; (ii) refining the atomic coordinates of the target molecule based on the atomic coordinates of the reference polypeptide; and (iii) computationally modeling the 3D structure of the target molecule based on the refined atomic coordinates.

[0217] In some embodiments, the nuclear magnetic resonance (NMR) data of the target molecule (e.g., integrin) includes NMR chemical shift, J-coupling constant, and resonance intensity obtained by subjecting a solution of the target molecule to NMR analysis.

[0218] n some embodiments, creating the 3D model structure of the target molecule (e.g., integrin) includes: (i) creating an ensemble of structural models of the target molecule based on the NMR data; (ii) obtaining mean atomic coordinates of the target molecule based on the ensemble of structural models; (iii) refining the mean atomic coordinates of the target molecule based on the atomic coordinates of the reference polypeptide; and (iv) computationally modeling the 3D structure of the target molecule based on the refined mean atomic coordinates.

[0219] In some embodiments, the atomic coordinates of the reference polypeptide are obtained from a protein structure database or scientific literature. In specific embodiments, the protein structure database is selected from worldwide Protein Data Bank (wwPDB), Cambridge Structure Database, Molecular Model Database of National Center for Biotechnology Information (NCBI), and Biological Magnetic Resonance Data Bank (BMRB) database. In specific embodiments, the refining step is performed using a molecular replacement algorithm.

[0220] In some embodiments, step (a) is performed by computationally modeling the 3D structure of the target molecule (e.g., integrin) based on the amino acid sequence of the target molecule and atomic coordinates of one or more reference polypeptide; and wherein the amino acid sequence of the target molecule is at least about 50% identical to the amino acid sequence of the reference polypeptide.

[0221] In some embodiments, the atomic coordinates of the reference polypeptide are obtained from a protein structure database or scientific literature. In some embodiments, the protein structure database is selected from worldwide Protein Data Bank (wwPDB), Cambridge Structure Database, Molecular Model Database of National Center for Biotechnology Information (NCBI), and Biological Magnetic Resonance Data Bank (BMRB) database. In specific embodiments, computationally modeling the 3D structure of the target molecule (e.g., integrin) is performed using homology modeling. In some embodiments, the homology modeling is performed in combination with a protein structure prediction algorithm. In some embodiments, the protein structure prediction algorithm is trRosetta, AlphaFold, or AlphaFold 2.

[0222] In some embodiments, the spatial locations of the lasso-binding moieties and / or the spatial locations of the target-binding moieties are defined as atomic coordinates. In some embodiments, the integrin binding motif is less than or equal to ten (10) amino acids in length. In some embodiments, the selected amino acid residues in the integrin binding motif areadjacent with one another and / or separated by one or more non-binding amino acid residues. In some embodiments, the non-binding amino acid residue is a glycine residue. In some embodiments, the integrin binding motif corresponds to a fragment or fragments of a naturally- existing ligand of the target molecule (e.g., integrin), wherein the fragment is capable of binding to the lasso-binding site of the target molecule, thereby forming a ligand-target interface.

[0223] In some embodiments, step (c) further includes mapping the model 3D structure of an integrin binding motif to the natural ligand-target molecule (e.g., integrin) interface to align configurations, conformations, and orientations with the ligand-target interface. In some embodiments, wherein in step (d), the plurality of model 3D structures of lasso peptides include known or predicted 3D structures of lasso peptides. In some embodiments, wherein in step (d), the plurality of model 3D structures of lasso peptides are stored in a computer readable medium. In some embodiments, the method further includes creating a database of designed lasso peptide structures or intermediates thereof generated in any one of steps (a) to (h).

[0224] In some embodiments, the method further includes (i) measuring at least one functional characteristics of the synthesized engineered lasso peptide candidates. In specific embodiments, the functional characteristics is selected from the group consisting of the KD, ICso, EC50 values associated with binding of the synthesized engineered lasso peptide candidates to the target molecule.

[0225] In some embodiments, the present method further includes (j) repeating steps (f) through (h) to improve one or more functional characteristics of the engineered lasso peptide candidate. In some embodiments, the method further includes (i) measuring an initial binding affinity of the synthesized engineered lasso peptide candidate for binding with the target molecule e.g., integrin); (j) mutating the synthesized engineered lasso peptide candidate, thereby generating a library of mutated engineered lasso peptide candidates; and (k) screening the library to identify mutated engineered lasso peptide candidates having higher binding affinity to the target molecule than the initial binding affinity.

[0226] In some embodiments, the higher binding affinity is at least 10 folds higher than the initial binding affinity, the method further includes (1) measuring at least one functional characteristics of the identified mutated engineered lasso peptide candidates. In some embodiments, the method further includes repeating steps (f) to (1).6.8. Compositions and Methods of Producing Engineered Lasso Peptides

[0227] In silico modeling is aimed at designing molecules that are predicted to specifically bind and selectively inhibit to a target integrin of interest. Once designed, the engineered lasso peptides are synthesized, isolated, and partially or substantially purified to enable experimental testing for validation of biological activity and other properties. For lasso peptides, there are two main methods for producing the engineered lasso peptides, which involve: (i) cell-free biosynthesis technology, and / or (ii) cell-based production methods (FIG. 5). Accordingly, provided herein are compositions and methods for producing the engineered lasso peptides described herein.

[0228] In some embodiments, to produce an engineered lasso peptide, a lasso precursor peptide is modified at the core peptide sequence, while the leader sequence is maintained the same. The modified precursor peptide can then be processed by corresponding lasso peptidase and / or lasso cyclase into a matured lasso peptide with modified amino acid sequence. Accordingly, in some embodiments, an engineered lasso peptide disclosed herein, having a core peptide sequence, can further include a leader sequence as disclosed herein, thereby generating a lasso precursor peptide that itself is engineered.

[0229] In some embodiments, the leader sequence includes an amino acid sequence of MIKHIHFDKLSSSKKNNVPHSAKGVIQIKKSASQLTK (SEQ ID NO: 777) when the engineered lasso peptide is derived from a parent scaffold peptide including SEQ ID NO: 1. In some embodiments, the leader sequence includes an amino acid sequence of MMQQKKNDMKKVTLKKLNKRASKVTR (SEQ ID NO 778) when the engineered lasso peptide is derived from a parent scaffold peptide including SEQ ID NO: 343. In some embodiments, the leader sequence includes an amino acid sequence of MKKQTFVPKKLVKVGKATELTK (SEQ ID NO 779) when the engineered lasso peptide is derived from a parent scaffold peptide including SEQ ID NO: ID NO 353. In some embodiments, the leader sequence includes an amino acid sequence of MERNHETPSDLIDLGAASVETK (SEQ ID NO 780) when the engineered lasso peptide is derived from a parent scaffold peptide including SEQ ID NO: 362. In some embodiments, the leader sequence includes an amino acid sequence of MTQVSPSPLRLIRVGRALDLTR (SEQ ID NO 781) when the engineered lasso peptide is derived from a parent scaffold peptide including SEQ ID NO: 366. Other leader sequences as well as lasso peptide biosynthesis components can be readily identified and used as appropriate to generate an engineered lasso peptide described herein depending upon the parent scaffold peptide. For example, additional lasso peptide biosynthesis components and corresponding leader sequences are known in theart, such as those disclosed in PCT application publication numbers: WO2019 / 191571, which is incorporated herein by reference in its entirety.

[0230] In some aspects, to produce an engineered lasso peptide, provided herein are recombinant nucleic acids encoding any one of the engineered lasso peptide described herein or a precursor peptide thereof. In some embodiments, provided herein is a recombinant nucleic acid encoding an engineered lasso peptide described herein. In some embodiments, provided herein is a recombinant nucleic acid encoding a precursor peptide to an engineered lasso peptide described herein. In some embodiments, the recombinant nucleic acid includes any one of the nucleotide sequences depicted in Table 1. In some embodiments, the recombinant nucleic acid includes a nucleotide sequence selected from SEQ ID NOS: 390-397, 399, 401- 409, 411, 412, 414-419, 421, 422, 424, 428-436, 438, 442-444, 447, 448, 450, 456, 459, 461, 469, 473-478, 481, 483-491, 551, 556, 575, 577, 578, 580-582, 584-597, 604, 606, 608, 609, 611, 617, 619-621, 630, 633, 635, 637, 639-643, 646, 649-651, 656, 658, 659, 662-664, 670- 685, 687-689, 691-704, 706-710, 716-720, 732, 734, and 735.

[0231] In some embodiments, provided herein is a recombinant nucleic acid that hybridizes under highly stringent hybridization conditions to an isolated nucleic acid encoding an engineered lasso peptide described herein. Accordingly, in some embodiments, the recombinant nucleic acid is an isolated nucleic acid that hybridizes under highly stringent hybridization conditions to a nucleic acid that encodes an engineered lasso peptide that is a variant of a scaffold peptide (SEQ ID NOs: 1, 343, 353, 362, or 366), such as an engineered lasso peptide having an integrin binding motif and / or alterations as described in Table 1, and, in some embodiments, a combination of motifs and / or alterations described in Table 1. In some embodiments, the recombinant nucleic acid molecule is an isolated nucleic acid that hybridizes under highly stringent hybridization conditions to a nucleic acid that encodes an engineered lasso peptide having the integrin binding motif and / or alterations described in Table 1. In some embodiments, the recombinant nucleic acid is an isolated nucleic acid that hybridizes under highly stringent hybridization conditions to a nucleic acid that encodes an engineered lasso peptide having a combination of substitutions described in Table 1.

[0232] A recombinant nucleic acid encoding an engineered lasso peptide described herein also includes a nucleic acid that hybridizes to a nucleic acid disclosed herein or a nucleic acid that hybridizes to a nucleic acid that encodes an amino acid sequence disclosed. Hybridization conditions can include highly stringent, moderately stringent, or low stringency hybridization conditions that are well known to one of skill in the art such as those described herein. Similarly, a recombinant nucleic acid that can be used in the compositions and methodsdescribed herein can be described as having a certain percent sequence identity to a nucleic acid disclosed herein or a nucleic acid that hybridizes to a nucleic acid molecule that encodes an amino acid sequence disclosed herein. For example, the nucleic acid can have at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or be identical, to a nucleotide described herein.

[0233] Stringent hybridization refers to conditions under which hybridized polynucleotides are stable. As known to those of skill in the art, the stability of hybridized polynucleotides is reflected in the melting temperature (Tm) of the hybrids. In general, the stability of hybridized polynucleotides is a function of the salt concentration, for example, the sodium ion concentration, and temperature. A hybridization reaction can be performed under conditions of lower stringency, followed by washes of varying, but higher, stringency. Reference to hybridization stringency relates to such washing conditions. Highly stringent hybridization includes conditions that permit hybridization of only those nucleotide sequences that form stable hybridized polynucleotides in 0.018MNaCl at 65°C, for example, if a hybrid is not stable in 0.018MNaCl at 65°C, it will not be stable under high stringency conditions, as contemplated herein. High stringency conditions can be provided, for example, by hybridization in 50% formamide, 5X Denhart's solution, 5X SSPE, 0.2% SDS at 42°C, followed by washing in 0. IX SSPE, and 0.1% SDS at 65°C. Hybridization conditions other than highly stringent hybridization conditions can also be used to describe the nucleotide sequences disclosed herein. For example, the phrase moderately stringent hybridization refers to conditions equivalent to hybridization in 50% formamide, 5X Denhart's solution, 5X SSPE, 0.2% SDS at 42°C, followed by washing in 0.2X SSPE, 0.2% SDS, at 42°C. The phrase low stringency hybridization refers to conditions equivalent to hybridization in 10% formamide, 5X Denhart's solution, 6X SSPE, 0.2% SDS at 22°C, followed by washing in IX SSPE, 0.2% SDS, at 37°C. Denhart's solution contains 1% Ficoll, 1% polyvinylpyrolidone, and 1% bovine serum albumin (BSA). 20X SSPE (sodium chloride, sodium phosphate, ethylene diamine tetraacetic acid (EDTA)) contains 3M sodium chloride, 0.2M sodium phosphate, and 0.025 M (EDTA). Other suitable low, moderate and high stringency hybridization buffers and conditions are well known to those of skill in the art and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999).

[0234] A recombinant nucleic acid encoding an engineered lasso peptide described herein can have at least a certain sequence identity to a nucleotide sequence disclosed herein.Accordingly, in some aspects described herein, a recombinant nucleic acid encoding an engineered lasso peptide has a nucleotide sequence of at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity, or is identical, to a nucleic acid disclosed in Table 1 or a nucleic acid that hybridizes to a nucleic acid that encodes an amino acid sequence disclosed Table 1.

[0235] It is understood that a recombinant nucleic acid described herein or an engineered lasso peptide described here can exclude a wild-type or parent scaffold peptide sequence, for example, a parent scaffold peptide sequence, such as SEQ ID NOs: 1, 343, 353, 362, or 366. One skilled in the art will readily understand the meaning of a parent scaffold peptide sequene or wild-type sequence based on what is well known in the art. It is further understood that such a recombinant nucleic acid described herein can exclude a nucleotide sequence encoding a naturally occurring amino acid sequence as found in nature. Similarly, an engineered lasso peptide described herein can exclude an amino acid sequence as found in nature. Thus, in some embodiments, the recombinant nucleic acid or engineered lasso peptide described herein is as set forth herein, with the proviso that the encoded amino acid sequence is not the wild-type or parental sequence or a naturally occurring amino acid sequence and / or that the nucleotide sequence is not a wild-type or naturally occurring nucleotide sequence. A naturally occurring amino acid or nucleotide sequence is understood by those skilled in the art as relating to a sequence that is found in a naturally occurring organism as found in nature. Thus, a nucleotide or amino acid sequence that is not found in the same state or having the same nucleotide or encoded amino acid sequence as in a naturally occurring organism is included within the meaning of a recombinant nucleotide and / or amino acid sequence described herein. For example, a nucleotide or amino acid sequence that has been altered at one or more nucleotide or amino acid positions from a parent sequence, including alterations as described herein, are included within the meaning of a nucleotide or amino acid sequence described herein that is not naturally occurring. A recombinant nucleic acid described herein excludes a naturally occurring chromosome that contains the nucleotide sequence, and can further exclude other molecules, as found in a naturally occurring cell, such as DNA binding proteins, for example, proteins such as histones that bind to chromosomes within a eukaryotic cell.

[0236] Thus, a recombinant nucleic acid described here has physical and chemical differences compared to a naturally occurring nucleic acid. A recombinant or non-naturally occurring nucleic acid described herein does not contain or does not necessarily have some or all of thechemical bonds, either covalent or non-covalent bonds, of a naturally occurring nucleic acid as found in nature. A recombinant nucleic acid described herein thus differs from a naturally occurring nucleic acid, for example, by having a different chemical structure than a naturally occurring nucleic acid as found in a chromosome. A different chemical structure can occur, for example, by cleavage of phosphodiester bonds that release a recombinant nucleic acid from a naturally occurring chromosome. A recombinant nucleic acid described herein can also differ from a naturally occurring nucleic acid by isolating or separating the nucleic acid from proteins that bind to chromosomal DNA in either prokaryotic or eukaryotic cells, thereby differing from a naturally occurring nucleic acid by different non-covalent bonds. With respect to nucleic acids of prokaryotic origin, a non-naturally occurring nucleic acid described herein does not necessarily have some or all of the naturally occurring chemical bonds of a chromosome, for example, binding to DNA binding proteins such as polymerases or chromosome structural proteins, or is not in a higher order structure such as being supercoiled. With respect to nucleic acids of eukaryotic origin, a non-naturally occurring nucleic acid described herein also does not contain the same internal nucleic acid chemical bonds or chemical bonds with structural proteins as found in chromatin. For example, a non-naturally occurring nucleic acid described herein is not chemically bonded to histones or scaffold proteins and is not contained in a centromere or telomere. Thus, the non-naturally occurring nucleic acids described herein are chemically distinct from a naturally occurring nucleic acid because they either lack or contain different van der Waals interactions, hydrogen bonds, ionic or electrostatic bonds, and / or covalent bonds from a nucleic acid as found in nature. Such differences in bonds can occur either internally within separate regions of the nucleic acid (that is cis) or such difference in bonds can occur in trans, for example, interactions with chromosomal proteins. In the case of a nucleic acid of eukaryotic origin, a cDNA is considered to be a recombinant or non-naturally occurring nucleic acid since the chemical bonds within a cDNA differ from the covalent bonds, that is the sequence, of a gene on chromosomal DNA. Thus, it is understood by those skilled in the art that recombinant or non-naturally occurring nucleic acid is distinct from a naturally occurring nucleic acid.

[0237] In order to drive transcription and then translation of the recombinant nucleic acid, which can be used to synthesize the engineered lasso peptides described herein, in some embodiments, the recombinant nucleic acid includes a nucleotide sequence encoding the engineered lasso peptide that is operatively linked to a promoter. Moreover, for cell-based production methods, a vector having such recombinant nucleic acids is also provided. Accordingly, in some embodiments, provided herein is a vector having any one of therecombinant nucleic acids described herein. Methods for generating such recombinant nucleic acids are well known in the art, any one of which can be used to generate such recombinant nucleic acids, including the methods described in the Examples.6.8.1. Cell-Free Biosynthesis of Lasso Peptides

[0238] In one aspect, provided herein are methods for producing lasso peptides, including designed lassos that are predicted using in silico modeling, including in vitro cell-free biosynthesis (CFB) methods. In some embodiments, a CFB method is an in vitro (outside the cell) biosynthetic process for the production of one or more peptides or proteins. In some embodiments, CFB occurs in a “cell-free biosynthesis reaction mixture” or “CFB reaction mixture,” which provides various components, such as RNA, proteins, enzymes, co-factors, natural products, small molecules, organic molecules, to carry out protein synthesis outside a living cell. In some embodiments, the CFB reaction mixture can include one or more cell extracts or supplemented cell extracts, or commercially available cell-free reaction media (e.g. PURExpress®). Exemplary CFB methods and systems, including those involving the use of CFB methods for lasso peptides, are described in Si et al. , J. Am. Chem. Soc. 2021, 143, 5917-5927, and in PCT Application WO2019191571A1, both of which are incorporated herein by reference. In some embodiments, cell-free biosynthesis involves the use of one or more isolated precursor peptide, core peptide, lasso peptidase, lasso cyclase, and lasso RRE. In some embodiments, cell-free biosynthesis involves the use of one or more isolated precursor peptide, core peptide, lasso peptidase, lasso cyclase, and lasso RRE, wherein an isolated precursor peptide or core peptide are produced either synthetically or biologically.

[0239] CFB methods employ the enzymes and the biosynthetic and metabolic machinery present inside cells, but without using living cells. CFB methods allow rapid expression of natural biosynthetic genes and pathways and facilitate targeted or phenotypic activity screening of natural products, without the need for plasmid-based cloning or in vivo cellular propagation, thus enabling rapid process / product pipelines (e.g., creation of large quantity of lasso peptide in a short time). Features of the CFB methods for lasso peptide production include that oligonucleotides (linear or circular constructs of DNA or RNA) encoding a minimal set of lasso peptide biosynthesis pathway genes (e.g., Genes A-C in a lasso peptide biosynthetic gene cluster) may be added to a cell extract containing in vitro transcription and translation (TX-TL) machinery for transcribing and translating the genes into the functional enzymes and lasso precursor peptides for production of lasso peptides (see, e.g., Gagoski et al., Biotechnol. Bioeng. 2016; 113: 292-300; Culler etal., PCT Appl. No. WO2017 / 031399). Accordingly, theCFB methods can produce in a CFB reaction mixture at least one, two or more of the engineered lasso peptides.

[0240] In some embodiments, in vitro TX-TL is a biosynthetic process outside an intact cell, where genes or oligonucleotides are transcribed into messenger ribonucleic acids (mRNAs), and mRNAs are translated into proteins or peptides. The in vitro TX-TL machinery act in concert to carry out the in vitro TX-TL. For the sole purpose of illustration, and by way of non-exhaustive and non-limiting examples, in some embodiments, an in vitro TX-TL machinery includes enzyme(s) and co-factor(s) that carry out DNA transcription and / or mRNA translation. In some embodiments, an in vitro TX-TL machinery further includes other small organic or inorganic molecules, such as amino acids, tRNAs or ATP, that facilitate the DNA transcription and / or mRNA translation. Various cellular components known to participate in in vivo transcription and translation can form part of the in vitro TX- TL machinery, see for example, Matsubayashi et al. , Curr Opin Chem Biol. 2014 Oct;22: 158-62; Li, et al., PLoS One, 2014 Sep 2; 9 (9):el06232. In some embodiments, different components can be provided individually and combined to assemble the in vitro TX-TL machinery. Exemplary ways of providing the in vitro TX-TL machinery components include recombinant production, synthesis, and isolation from a cell. In some embodiments, the in vitro TX-TL machinery is provided in the form of one or more cell extract, or one or more supplemented cell extract that includes the in vitro TX-TL machinery.

[0241] In some embodiments, the method for producing a lasso peptide includes (a) providing a CFB system including a minimal set of lasso peptide biosynthesis components; and (b) incubating the CFB system under a suitable condition to produce the lasso peptide.

[0242] In some embodiments, the minimal set of lasso peptide biosynthesis components includes one or more components functions to provide a lasso precursor peptide, and one or more components function to process the lasso precursor peptide into the lasso peptide. In some embodiments, the one or more components function to process the lasso precursor peptide into the lasso peptide consist of a lasso peptidase and a lasso cyclase. In some embodiments, the one or more components function to process the lasso precursor peptide into the lasso peptide consists of a lasso peptidase, a lasso cyclase and an RRE.

[0243] In some embodiments, the minimal set of lasso peptide biosynthesis components includes one or more components functions to provide a lasso core peptide, and one or more components function to process the lasso core peptide into the lasso peptide. In some embodiments, the one or more components function to process the lasso core peptide into the lasso peptide includes one or more selected from a lasso peptidase, a lasso cyclase and an RRE.In some embodiments, the one or more components function to process the lasso core into the lasso peptide consist of a lasso cyclase.

[0244] In various embodiments, the one or more components function to provide a peptide or protein (e.g., a lasso precursor peptide, a lasso core peptide, or lasso peptide biosynthetic enzymes and proteins) in a CFB system can be provided in the form of the peptide or protein are provided in the form of the peptide or protein per se.

[0245] In some embodiments, at least some of the peptide or protein components in the CFB system can be natural peptides or polypeptides. In some embodiments, at least some of the peptide or protein components in the CFB system are derivatives of natural peptides or polypeptides. In some embodiments, at least some of the peptide or protein components in the CFB system are non-natural peptides. In some embodiments, the one or more peptide or protein components of the CFB system can be isolated from nature, such as isolated from microorganisms producing the lasso precursor peptides. In some embodiments, the one or more peptide or protein components of the CFB system can be synthetically or recombinantly produced, using methods known in the art. In some embodiments, the one or more peptide or protein components of the CFB system can be synthesized using the CFB system as described herein, followed by purifying the biosynthesized peptide or protein components from the CFB system.

[0246] In some embodiments, the CFB system includes one or more fusion protein, or a polynucleotide encoding the fusion protein such that the CFB system is capable of producing the fusion protein through in vitro TX-TL.

[0247] In some embodiments, the fusion protein included a lasso precursor peptide or a lasso core peptide fused to one or more lasso peptide biosynthesis components. In some embodiments, the one or more lasso peptide biosynthesis components are selected from (i) a lasso peptidase; (ii) a lasso cyclase; (iii) a RRE; or (iv) any combinations of (i) to (iii). In some embodiments, the one or more lasso peptide biosynthesis components are encoded by the same lasso peptide biosynthetic gene cluster. In other embodiments, the one or more lasso peptide biosynthesis components are encoded by different lasso peptide biosynthetic gene cluster.

[0248] In some embodiments, the fusion protein includes an amino acid linker between the lasso peptidase or the lasso cyclase and the one or more additional peptide or polypeptide. In some embodiments, the fusion protein does not include an amino acid linker between the lasso peptidase or the lasso cyclase and the one or more additional peptide or polypeptide.

[0249] In specific embodiments, the fusion protein includes a lasso precursor peptide fused to a lasso peptidase. In specific embodiments, the fusion protein includes a lasso precursorpeptide fused to a lasso cyclase. In specific embodiments, the fusion protein includes a lasso precursor peptide fused to a RRE. In specific embodiments, the fusion protein includes a lasso precursor peptide fused to a lasso peptidase and a lasso cyclase. In specific embodiments, the fusion protein includes a lasso precursor peptide fused to a lasso peptidase and a RRE. In specific embodiments, the fusion protein includes a lasso precursor peptide fused to a lasso cyclase and a RRE. In specific embodiments, the fusion protein includes a lasso precursor peptide fused to a lasso peptidase, a lasso cyclase and RRE. In specific embodiments, the fusion protein includes a lasso core peptide fused to a lasso peptidase. In specific embodiments, the fusion protein includes a lasso core peptide fused to a lasso cyclase. In specific embodiments, the fusion protein includes a lasso core peptide fused to a RRE. In specific embodiments, the fusion protein includes a lasso core peptide fused to a lasso peptidase and a lasso cyclase. In specific embodiments, the fusion protein includes a lasso core peptide fused to a lasso peptidase and a RRE. In specific embodiments, the fusion protein includes a lasso core peptide fused to a lasso cyclase and a RRE. In specific embodiments, the fusion protein includes a lasso core peptide fused to a lasso peptidase, a lasso cyclase and RRE.

[0250] In some embodiments, the fusion protein included a lasso precursor peptide or a lasso core peptide fused to one or more additional peptide or polypeptide. In some embodiments, the one or more additional peptide or polypeptide includes a peptide or polypeptide that facilitates production of the lasso precursor peptide or lasso core peptide or the lasso peptide derived therefrom through cell-free biosynthesis. Examples of peptide or polypeptide that can be fused with a lasso precursor peptide or a lasso core peptide according to the present disclosure include but are not limited to (i) a peptide or polypeptide that increases the level of transcription of the lasso precursor peptide or lasso core peptide in the CFB system; (ii) a peptide or polypeptide that increases the level of translation of the lasso precursor peptide or lasso core peptide in the CFB system; (iii) a peptide or polypeptide that facilitates the processing of the lasso precursor peptide or lasso core peptide into the lasso peptide; (iv) a peptide or polypeptide that improves stability of the lasso precursor peptide or lasso core peptide or the lasso peptide derived therefrom; (v) a peptide or polypeptide that improves solubility of the lasso precursor peptide or lasso core peptide or the lasso peptide derived therefrom; (vi) a peptide or polypeptide that enables or facilitates the detection of the lasso precursor peptide or lasso core peptide or the lasso peptide derived therefrom; (vii) a peptide or polypeptide that enables or facilitates purification of the lasso precursor peptide or lasso core peptide or the lasso peptide derived therefrom; (viii) a peptide or polypeptide that enables or facilitates immobilization of the lassoprecursor peptide or lasso core peptide or the lasso peptide derived therefrom; or (ix) any combination of (i) to (viii).

[0251] In some embodiments, the fusion protein included a lasso precursor peptide or a lasso core peptide fused to one or more additional peptide or polypeptide. In some embodiments, the one or more additional peptide or polypeptide includes a biologically active peptide or polypeptide. Examples of biologically active peptide or polypeptide that can be fused with a lasso precursor peptide or lasso core peptide according to the present disclosure include but are not limited to (i) a peptide or polypeptide capable of binding to a target molecule (e.g., an antibody or an antigen); (ii) a peptide or polypeptide that enhance cell permeability of the fusion protein; (iii) a peptide or polypeptide capable of conjugating the fusion protein to at least one additional copy of the fusion protein; (iv) a peptide or polypeptide capable of linking the fusion protein to one or more peptidic or non-peptidic molecule; (v) a peptide or polypeptide capable of modulating activity of the lasso precursor peptide or lasso core peptide; (vi) a peptide or polypeptide capable of modulating activity of the lasso peptide derived from the lasso precursor peptide or the lasso core peptide; or (vii) any combinations of (i) to (vi).

[0252] In some embodiments, the fusion protein included a lasso peptidase or a lasso cyclase fused to one or more additional peptide or polypeptide. In some embodiments, the one or more additional peptide or polypeptide is fused to the N-terminus of the lasso peptidase or the lasso cyclase. In some embodiments, the one or more additional peptide or polypeptide is fused at the C-terminus of the lasso peptidase or the lasso cyclase. In some embodiments, a polynucleotide encoding the fusion protein includes a nucleic acid sequence encoding the lasso peptidase or the lasso cyclase, wherein the 5’ end of the nucleic acid sequence is linked to a nucleic acid sequence encoding the one or more additional peptide or polypeptide. In some embodiments, a polynucleotide encoding the fusion protein includes a nucleic acid sequence encoding the lasso peptidase or the lasso cyclase, wherein the 3’ end of the nucleic acid sequence is linked to a nucleic acid sequence encoding the one or more additional peptide or polypeptide. In some embodiments, the fusion protein includes an amino acid linker between the lasso peptidase or the lasso cyclase and the one or more additional peptide or polypeptide. In some embodiments, the fusion protein does not include an amino acid linker between the lasso peptidase or the lasso cyclase and the one or more additional peptide or polypeptide.

[0253] In some embodiments, the fusion protein included a lasso peptidase or a lasso cyclase fused to one or more additional peptide or polypeptide. In some embodiments, the more additional peptide or polypeptide includes a peptide or polypeptide encoded by a lasso peptide biosynthetic gene cluster. Examples of peptide or polypeptide that can be fused with a lassoprecursor peptide or a lasso core peptide according to the present disclosure include but are not limited to (i) a lasso precursor peptide; (ii) a lasso core peptide; (iii) a lasso peptidase; (iv) a lasso cyclase, (v) a RRE; or (vi) any combinations of (i) to (vi). In specific embodiments, the fusion protein includes at least one lasso cyclase and at least one lasso peptidase. In specific embodiments, the fusion protein includes at least one lasso cyclase fused to a RRE. In specific embodiments, the fusion protein includes at least one lasso peptidase fused to a RRE.

[0254] In some embodiments, the fusion protein included a lasso peptidase or a lasso cyclase fused to one or more additional peptide or polypeptide. In some embodiments, the one or more additional peptide or polypeptide includes a peptide or polypeptide that facilitates production of the lasso peptidase or lasso cyclase through cell-free biosynthesis. Examples of peptide or polypeptide that can be fused with the lasso peptidase or lasso cyclase according to the present disclosure include but are not limited to (i) a peptide or polypeptide that increases the level of transcription of the lasso peptidase or lasso cyclase in the CFB system; (ii) a peptide or polypeptide that increases the level of translation of the lasso peptidase or lasso cyclase in the CFB system; (iii) a peptide or polypeptide that improves stability of the lasso peptidase or lasso cyclase; (vi) a peptide or polypeptide that improves solubility of the lasso peptidase or lasso cyclase; (v) a peptide or polypeptide that enables or facilitates the detection of the lasso peptidase or lasso cyclase; (vi) a peptide or polypeptide that enables or facilitates purification of the lasso peptidase or lasso cyclase; (vii) a peptide or polypeptide that enables or facilitates immobilization of the lasso peptidase or lasso cyclase; or (viii) any combination of (i) to (vii).

[0255] In some embodiments, the fusion protein included a lasso peptidase or a lasso cyclase fused to one or more additional peptide or polypeptide. In some embodiments, the one or more additional peptide or polypeptide includes a biologically active peptide or polypeptide. Examples of biologically active peptide or polypeptide that can be fused with a lasso peptidase or a lasso cyclase according to the present disclosure include but are not limited to (i) a peptide or polypeptide capable of modulating the reaction catalyzing activity of the lasso peptidase or lasso cyclase; (ii) a peptide or polypeptide capable of modulating target specificity of the lasso peptidase or lasso cyclase; (iii) an enzyme having the same or different enzymatic activity as the lasso peptidase or lasso cyclase; or any combination of (i) to (iii).

[0256] In some embodiments, the fusion protein included a RIPP recognition element (RRE) fused to one or more additional peptide or polypeptide. In some embodiments, the one or more additional peptide or polypeptide is fused to the N-terminus of the RRE. In some embodiments, the one or more additional peptide or polypeptide is fused at the C-terminus of the RRE. In some embodiments, a polynucleotide encoding the fusion protein includes a nucleic acidsequence encoding the RRE, wherein the 5’ end of the nucleic acid sequence is linked to a nucleic acid sequence encoding the one or more additional peptide or polypeptide. In some embodiments, a polynucleotide encoding the fusion protein includes a nucleic acid sequence encoding the RRE, wherein the 3’ end of the nucleic acid sequence is linked to a nucleic acid sequence encoding the one or more additional peptide or polypeptide. In some embodiments, the fusion protein includes an amino acid linker between the RRE and the one or more additional peptide or polypeptide. In some embodiments, the fusion protein does not include an amino acid linker between RRE and the one or more additional peptide or polypeptide.

[0257] In some embodiments, the fusion protein included a RIPP recognition element (RRE) fused to one or more additional peptide or polypeptide. In some embodiments, the more additional peptide or polypeptide includes a peptide or polypeptide encoded by a lasso peptide biosynthetic gene cluster. Examples of peptide or polypeptide that can be fused with a lasso precursor peptide or a lasso core peptide according to the present disclosure include but are not limited to (i) a lasso precursor peptide; (ii) a lasso core peptide; (iii) a lasso peptidase; (iv) a lasso cyclase, (v) a RRE; or (vi) any combinations of (i) to (vi). In specific embodiments, the fusion protein includes at least one lasso precursor peptide fused to a RRE. In specific embodiments, the fusion protein includes at least one lasso core peptide fused to a RRE. In specific embodiments, the fusion protein includes at least one lasso cyclase fused to a RRE. In specific embodiments, the fusion protein includes at least one lasso peptidase fused to a RRE.

[0258] In some embodiments, the fusion protein included a RIPP recognition element (RRE) fused to one or more additional peptide or polypeptide. In some embodiments, the one or more additional peptide or polypeptide includes a peptide or polypeptide that facilitates production of the RRE through cell-free biosynthesis. Examples of peptide or polypeptide that can be fused with the RRE according to the present disclosure include but are not limited to (i) a peptide or polypeptide that increases the level of transcription of the RRE in the CFB system; (ii) a peptide or polypeptide that increases the level of translation of the RRE in the CFB system; (iii) a peptide or polypeptide that improves stability of the RRE; (vi) a peptide or polypeptide that improves solubility of the RRE; (v) a peptide or polypeptide that enables or facilitates the detection of the RRE; (vi) a peptide or polypeptide that enables or facilitates purification of the RRE; (vii) a peptide or polypeptide that enables or facilitates immobilization of the RRE; or (viii) any combination of (i) to (vii).

[0259] In some embodiments, the fusion protein included a RIPP recognition element (RRE) fused to one or more additional peptide or polypeptide. In some embodiments, the one or more additional peptide or polypeptide includes a biologically active peptide or polypeptide.Examples of biologically active peptide or polypeptide that can be fused with a RRE according to the present disclosure include but are not limited to (i) a peptide or polypeptide capable of modulating the reaction catalyzing activity of the lasso peptidase or lasso cyclase; (ii) a peptide or polypeptide capable of modulating target specificity of the lasso peptidase or lasso cyclase; (iii) an enzyme having the same or different enzymatic activity as the lasso peptidase or lasso cyclase; or any combination of (i) to (iii).

[0260] In particular embodiments, the lasso precursor peptide genes are fused at the 5’- terminus of the DNA template strand of the gene to oligonucleotide sequences that encode peptides or proteins, such as sequences encoding maltose-binding protein (MBP) or small ubiquitin-like modifier protein (SUMO), which enhance the stability, solubility, and production of the desired TX-TL products (Marblestone, J.G., et al., Protein Sci, 2006, 15, 182-189). In particular embodiments, the lasso precursor peptides are fused at the C-terminus of the leader sequences to form conjugates with peptides or proteins, such as maltose-binding protein or small ubiquitin-like modifier protein, which enhance the stability, solubility, and production of the fused MBP-lasso or SUMO-lasso precursor peptide.

[0261] In particular embodiments, the lasso precursor peptide genes or lasso core peptide genes are fused at the 3 ’-terminus of the DNA template strand of the gene to oligonucleotide sequences that encode peptides or proteins, such as sequences encoding maltose-binding protein (MBP) or small ubiquitin-like modifier protein (SUMO), which enhance the stability, solubility, and production of the desired TX-TL products. In particular embodiments, the lasso precursor peptides, lasso core peptides, or lasso peptides are fused at the N-terminus to form conjugates with peptides or proteins, such as maltose-binding protein or small ubiquitin-like modifier protein, which enhance the stability, solubility, and production of the fused MBP- lasso or SUMO-lasso precursor peptide.

[0262] In particular embodiments, the lasso precursor peptide genes or lasso core peptide genes are fused at the 5 ’-terminus of the DNA template strand of the gene to oligonucleotide sequences that encode peptides or proteins, with or without a linker, such as sequences encoding peptide tags for affinity purification or immobilization, including his-tags, strep-tags, or FLAG-tags. In some embodiments, the lasso precursor peptides, lasso core peptides, or lasso peptides are fused at the C-terminus of the core peptides to form conjugates with other peptides or proteins, with or without a linker, such as peptide tags for affinity purification or immobilization, including his-tags, strep-tags, or FLAG-tags.

[0263] In particular embodiments, lasso precursor peptides, lasso core peptides, or lasso peptides are fused to molecules that can enhance cell permeability or penetration into cells, forexample through the use of arginine-rich cell-penetrating peptides such as TAT peptide, penetratin, and flock house virus (FHV) coat peptide (Brock, R., Bioconjug. Chem., 2014, 25, 863-868). In particular embodiments, a lasso precursor peptide gene or core peptide gene is fused at the 3 ’ -terminus to oligonucleotide sequences that encode arginine-rich cell-penetrating peptides or proteins, including oligonucleotide sequences that encode penetratin, and flock house virus (FHV) coat peptide or similar peptides that contain guanidinium groups or a combination of lysine and guanidinium groups (Wender, P.A., et al., Adv. Drug Deliv. Rev., 2008, 60, 452-472). In particular embodiments, a lasso precursor peptide, lasso core peptide, or lasso peptide is fused at the C-terminus to peptides that promote cell penetration such as arginine-rich cell-penetrating peptides or proteins, including amino acid sequences that encode TAT peptide, penetratin, and flock house virus (FHV) coat peptide or similar peptides that contain guanidinium groups or a combination of lysine and guanidinium groups.

[0264] In particular embodiments, the lasso precursor peptide genes or lasso core peptide genes are fused at the 5 ’-terminus of the DNA template strand of the gene to oligonucleotide sequences that encode peptides or proteins, with or without a linker, such as sequences encoding natural or unnatural peptide motifs that are known to bind with high affinity to antibodies, cell surface proteins, or cell surface receptors, including cytokine binding motifs, integrin ligand binding motifs, and the like. In particular embodiments, the lasso precursor peptides, lasso core peptides, or lasso peptides are fused at the C-terminus to peptides or proteins, with or without a linker, such as peptide motifs that are known to bind with high affinity to antibodies, cell surface proteins, or cell surface receptors, including cytokine binding motifs, chemokine binding motifs, integrin ligand binding motifs, and the like.

[0265] Additionally or alternatively, the one or more components function to provide a peptide or protein (e.g., a lasso precursor peptide, a lasso core peptide, or lasso peptide biosynthetic enzymes and proteins) in a CFB system can be provided in the form of a nucleic acid encoding the peptide or protein and in vitro TX-TL machinery capable of producing the peptide or protein via in vitro TX-TL of the coding sequences. In various embodiments, the coding nucleic acid can be DNA, RNA or cDNA. In various embodiments, one or more coding nucleic acid sequences can be contained in the same nucleic acid molecule, such as a vector.

[0266] It is understood that when more than one coding nucleic acid sequences are included in a CFB system, such more than one encoding nucleic acid sequences can be introduced on separate nucleic acid molecules, on polycistronic nucleic acid molecules, or a combination thereof. For example, as disclosed herein, a microbial organism or a cell extract can be engineered to express two or more exogenous nucleic acids encoding lasso precursor peptide,lasso core peptide, lasso peptidase, lasso cyclase or RRE. In the case where two exogenous nucleic acids encoding a desired activity are introduced into a host microbial organism or into a cell extract, it is understood that the two exogenous nucleic acids can be introduced as a single nucleic acid, for example, on a single plasmid or as linear strands of DNA, or on separate plasmids, or can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two exogenous nucleic acids. Similarly, it is understood that more than two exogenous nucleic acids can be introduced into a host organism or into a cell extract in any desired combination, for example, on a single plasmid, or on separate plasmids, or as linear strands of DNA, or can be integrated into the host chromosome at a single site or multiple sites.

[0267] In some embodiments, the in vitro TX-TL machinery is purified from a host cell. In some embodiments, the in vitro TX-TL machinery is provided in the form of a cell extract of a host cell. An exemplary procedure for obtaining a cell extract includes the steps of (i) growing cells, (ii) breaking open or lysing the cells by mechanical, biological or chemical means, (iii) removing cell debris and insoluble materials e.g., by filtration or centrifugation, and (iv) optionally treating to remove residual RNA and DNA, but retaining the active enzymes and biosynthetic machinery for transcription and translation, and optionally the metabolic pathways for co-factor recycle, including but not limited to co-factors such as THF, S- adenosylmethionine, ATP, NADH, NAD and NADP and NADPH. In some embodiments, a cell extract may be further supplemented for improved performance in in vitro TX-TL.

[0268] In some embodiments, a cell extract can be further supplemented with some or all of the twenty proteinogenic naturally-occurring amino acids and corresponding transfer ribonucleic acids (tRNAs), and optionally, may be supplemented with additional components, including but not limited to: (1) glucose, xylose, fructose, sucrose, maltose, or starch, (2) adenosine triphosphate (ATP), and / or adenosine diphosphate (ADP), purine and guanidine nucleotides, adenosine triphosphate, guanosine triphosphate, cytosine triphosphate, and / or uridine triphosphate, or combinations thereof, (3) cyclic-adenosine monophosphate (cAMP) and / or 3 -phosphoglyceric acid (3-PGA), (4) nicotinamide adenine dinucleotides NADH and / or NAD, or nicotinamide adenine dinucleotide phosphates, NADPH, and / or NADP, or combinations thereof, (5) amino acid salts such as magnesium glutamate and / or potassium glutamate, (6) buffering agents such as HEPES, TRIS, spermidine, or phosphate salts, (7) inorganic salts, including but not limited to, potassium phosphate, sodium chloride, magnesium phosphate, and magnesium sulfate, (8) cofactors such as folinic acid and co-enzyme A (CoA), L(-)-5-formyl-5,6,7,8-tetrahydrofolic acid (THF), and / or biotin, (8) RNA polymerase, (9) 1,4- dithiothreitol (DTT), (10) magnesium acetate, and / or ammonium acetate, and / or (11) crowdingagents such as PEG 8000, Ficoll 70, or Ficoll 400, or combinations thereof. In some embodiments, the cell extracts or supplemented cell extracts can be used as a reaction mixture to carry out in vitro TX-TL. In some embodiments, supplementations or adjustments can be made to the cell extract to provide a suitable condition for lasso formation.

[0269] In some embodiments, the in vitro TX-TL machinery is provided in the form of a cell extract or supplemented cell extract of a host cell. In some embodiments, the host cell is the cell of the same organism where the coding nucleic acid is derived from. For CFB of lasso peptides and related molecules thereof, the coding nucleic acid sequences can be identified using one or more computer-based genomic mining tools described herein or known in the art. For example, U.S. Provisional Application Nos. 62 / 652,213 and 62 / 651,028 disclose thousands of sequences from lasso peptide biosynthetic gene clusters identified from various organisms, and provide GenBank accession numbers for various sequences for lasso precursor peptides, lasso peptidase, lasso cyclase and / or RRE. Host organisms where the lasso peptide biosynthetic gene clusters originate can be identified based on the GenBank accession numbers, including but not limited to Caulobacteraceae species (e.g., Caulobacter sp. K31, Caulobacter henricii). Streptomyces species (e.g. Streptomyces nodosus. Streptomyces caalingaensis). Burkholderiaceae species (e.g., Burkholderia thailandensis E264), Pseudomallei species, Bacillus species, Burkholderia species (e.g., Burkholderia thailandensis MSMB43, Burkholderia oklahomensis, Burkholderia pseudomallei), Sphingomonadaceae species (e.g., Sphingobium sp. YBL2, Sphingobium chlorophenolicum, Sphingobium yanoikuyae). In other embodiments, the host cell is a microbial organism known to be applicable to fermentation processes. Exemplary bacteria include species selected from Escherichia coli, Klebsiella oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Streptomyces albus, Clostridium acetobutylicum, Vibrio natriegens, Pseudomonas fluorescens, and Pseudomonas putida. Exemplary yeasts or fungi include species selected from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger and Pichia pastoris. E. coli is a particularly useful host organism since it is a well characterized microbial organism suitable for genetic engineering. Other particularly useful host organisms include Vibrio natriegens, and yeast such as Saccharomyces cerevisiae.

[0270] In some embodiments, the CFB system is configured to produce a lasso peptide. In specific embodiments, the CFB system includes one or more components configured to provide(i) a lasso precursor peptide, (ii) a lasso peptidase, (iii) a lasso cyclase. In specific embodiments, the CFB system includes one or more components configured to provide (i) a lasso core peptide, and (ii) a lasso cyclase. In some embodiments, the CFB system further includes one or more components configured to provide (iv) an RRE. In some embodiments, all of (i) to (iv) above are provided in the CFB system as the corresponding peptide or protein. In alternative embodiments, at least one of (i) to (iv) above is provided in the CFB system as a nucleic acid encoding the corresponding protein, and the CFB system further includes in vitro TX-TL machinery for producing the corresponding protein from the coding nucleic acid. In these embodiments, the CFB systems can be incubated under a condition suitable for lasso formation to produce the lasso peptide. The incubation condition can be designed and adjusted based on various factors known to skilled artisan in the art, including for example, condition suitable for maintain stability of components of the CFB system, conditions suitable for the lasso processing enzymes to exert enzymatic activities, and / or conditions suitable for the in vitro TX-TL of the coding sequences present in the CFB system.

[0271] Without being bound by the theory, it is contemplated that different lasso peptidases can process the same lasso precursor peptide into different lasso core peptide by recognizing and cleaving different leader peptide off the lasso precursor. Additionally, different lasso cyclase can process the same lasso core peptide into distinct lasso peptides by cyclizing the core peptide at different ring-forming amino acid residues. Additionally, different RREs can facilitate different processing by the lasso peptidase and / or lasso cyclase, and thus lead to formation of distinct lasso peptides from the same lasso precursor peptide.

[0272] Accordingly, in some embodiments, to produce a natural lasso peptide, the CFB system includes the lasso precursor peptide, lasso peptidase, and lasso cyclase produced from coding sequences of the same lasso peptide biosynthetic gene cluster (such as Genes A, B, and C of the same lasso peptide biosynthetic gene cluster). In some embodiments, to produce a natural lasso peptide, the CFB system includes the lasso precursor peptide, lasso peptidase, lasso cyclase, and RRE produced from coding sequences of the same lasso peptide biosynthetic gene cluster.

[0273] In some embodiments, to produce a natural lasso peptide, the CFB system includes the lasso core peptide, and lasso cyclase produced from coding sequences of the same lasso peptide biosynthetic gene cluster (such as Genes A and C of the same lasso peptide biosynthetic gene cluster). In some embodiments, to produce a natural lasso peptide, the CFB system includes the lasso core peptide, lasso cyclase, and RRE produced from coding sequences of the same lasso peptide biosynthetic gene cluster.

[0274] In alternative embodiments, to produce a derivative of a natural lasso peptide, at least two of the lasso precursor peptide, lasso peptidase and lasso cyclase in the CFB system are produced from coding sequences of different lasso peptide biosynthetic gene clusters (such as Gene A from one, and Genes B and C from another, lasso peptide biosynthetic gene cluster). In alternative embodiments, to produce a derivative of a natural lasso peptide, at least two of the lasso precursor peptide, lasso peptidase, lasso cyclase and RRE in the CFB system are produced from coding sequences of different lasso peptide biosynthetic gene clusters.

[0275] In alternative embodiments, to produce a derivative of a natural lasso peptide, the lasso core peptide and lasso cyclase in the CFB system are produced from coding sequences of different lasso peptide biosynthetic gene clusters (such as Gene A from one, and Gene C from another, lasso peptide biosynthetic gene cluster). In alternative embodiments, to produce a derivative of a natural lasso peptide, at least two of the lasso core peptide, lasso cyclase and RRE in the CFB system are produced from coding sequences of different lasso peptide biosynthetic gene clusters.

[0276] In various embodiments, the contacting step (a) includes adding a first nucleic acid sequence encoding the peptide into the cell-free biosynthesis reaction mixture, and where the cell-free biosynthesis reaction mixture includes in vitro TX-TL machinery and is configured to express the peptide. In some embodiments, the contacting step (a) includes adding a second nucleic acid sequence encoding the lasso peptide biosynthesis component to the cell-free biosynthesis reaction mixture, and where the cell-free biosynthesis reaction mixture includes in vitro TX-TL machinery configured to express the lasso peptide biosynthesis component. In some embodiments, the lasso peptide biosynthesis component includes a lasso peptidase. In some embodiments, the lasso peptide biosynthesis component includes a lasso cyclase. In some embodiments, the lasso peptide biosynthesis component further includes a post-translationally modified peptide (RiPP) recognition element (RRE).

[0277] More particularly, in some of those embodiments where the lasso peptide biosynthesis component includes a lasso peptidase and a lasso cyclase, the contacting step (a) includes adding the second nucleic acid sequence encoding the lasso cyclase and a third nucleic acid sequence encoding the lasso peptidase. In some of those embodiments where the lasso peptide biosynthesis component includes a lasso cyclase and a post-translationally modified peptide (RiPP) recognition element (RRE), the contacting step (a) includes adding the second nucleic acid sequence encoding the lasso cyclase and a fourth nucleic acid sequence encoding the RRE. In some of those embodiments where the lasso peptide biosynthesis component includes a lasso peptidase, a lasso cyclase and a post-translationally modified peptide (RiPP) recognitionelement (RRE), and where the contacting step (a) includes adding the second nucleic acid sequence encoding the lasso cyclase, a third nucleic acid sequence encoding the lasso peptidase and a fourth nucleic acid sequence encoding the RRE. In some embodiments, the cell-free biosynthesis reaction mixture includes cell extract or supplemented cell extract.

[0278] In some embodiments, CFB reactions are conducted with a minimal set of lasso peptide biosynthesis components combined with genes that encode additional peptides, proteins or enzymes, including genes that encode RiPP recognition elements (RREs) or oligonucleotides that encode RREs that are fused to the 5’ or 3’ end of a lasso precursor peptide gene, a lasso core peptide gene, a lasso peptidase gene or a lasso cyclase gene. In other embodiments, CFB reactions are conducted with a minimal set of lasso peptide biosynthesis components, including lasso precursor peptides, lasso peptidases, or lasso cyclase that are fused to RREs at the N-terminus or C-terminus. In other embodiments, CFB reactions are conducted with a minimal set of lasso peptide biosynthesis components combined and contacted with additional isolated proteins or enzymes, including RiPP recognition elements (RREs).

[0279] In some embodiments, CFB reactions are conducted with a minimal set of lasso peptide biosynthesis components combined and contacted with genes that encode additional proteins or enzymes, including genes that encode lasso peptide modifying enzymes such as N- methyltransferases, O-methyltransferases, biotin ligases, glycosyltransferases, esterases, acylases, acyltransferases, aminotransferases, amidases, hydroxylases, dehydrogenases, halogenases, kinases, RiPP heterocyclases, RiPP cyclodehydratases, peptidylarginine deiminase, and prenyltransferases.

[0280] In some embodiments, CFB reactions are conducted with a minimal set of lasso peptide biosynthesis components combined and contacted with additional isolated proteins or enzymes, including lasso peptide modifying enzymes such as N-methyltransferases, O- methyltransferases, biotin ligases, glycosyltransferases, esterases, acylases, acyltransferases, aminotransferases, amidases, hydroxylases, dehydrogenases, halogenases, kinases, RiPP heterocyclases, RiPP cyclodehydratases, peptidylarginine deiminase, and prenyltransferases.

[0281] CFB methods and systems provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, are conducted in a CFB reaction mixture, including one or more cell extracts that are supplemented with all twenty proteinogenic naturally occurring amino acids and corresponding transfer ribonucleic acids (tRNAs). Cell extracts used in the CFB reaction mixture, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components also may be supplementedwith additional components, including but not limited to, glucose, xylose, fructose, sucrose, maltose, starch, adenosine triphosphate (ATP), and / or adenosine diphosphate (ADP), purine and guanidine nucleotides, adenosine triphosphate, guanosine triphosphate, cytosine triphosphate, and uridine triphosphate, cyclic-adenosine monophosphate (cAMP) and / or 3- phosphoglyceric acid (3-PGA), nicotinamide adenine dinucleotides NADH and / or NAD, or nicotinamide adenine dinucleotide phosphates, NADPH, and / or NADP, or combinations thereof, amino acid salts such as magnesium glutamate and / or potassium glutamate, buffering agents such as HEPES, TRIS, spermidine, or phosphate salts, inorganic salts, including but not limited to, potassium phosphate, sodium chloride, magnesium phosphate, and magnesium sulfate, folinic acid and co-enzyme A (CoA), crowding agents such as PEG 8000, Ficoll 70, or Ficoll 400, L(-)-5-formyl-5,6,7,8-tetrahydrofolic acid, RNA polymerase, biotin, 1,4- dithiothreitol (DTT), magnesium acetate, ammonium acetate , or combinations thereof. For a general description of cell-free extract production and preparation, see: Krinsky, N., et al., PLoS ONE, 2016, 11(10): e0165137.

[0282] In alternative embodiments, the preparation CFB reaction mixtures and cell extracts employed for the CFB methods as provided herein, includes characterization of the CFB reaction mixtures and cell extracts using proteomic approaches to assess and quantify the proteome available for the production of lasso peptides and related molecules thereof. In alternative embodiments,13C metabolic flux analysis (MFA) and / or metabolomics studies are conducted on CFB reaction mixtures and cell extracts to create a flux map and characterize the resulting metabolome of the CFB reaction mixture and cell extract or extracts.

[0283] In other embodiments, the CFB method is performed using: one or a combination of two or more cell extracts from various “chassis” organisms, such as E. coli, optionally mixed with one or a combination of two or more cell extracts derived from other species, e.g., a native lasso peptide-producing organism or relative. This can give the advantage of a robust transcription / translation machinery, combined with any unknown components of the native species that might be needed for proper protein folding or activity, or to supply precursors for the lasso peptide pathway. In alternative embodiments, if these factors are known they can be expressed in the chassis organism prior to making the cell extract or these factors can be isolated and purified and added directly to the CFB reaction mixture or cell extract.

[0284] In alternative embodiments, CFB methods and systems provided herein to produce lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, including the use of cell extracts for in vitro TX-TL systems express lasso peptide biosynthetic gene clusters without the regulatory constraints of the cell. Inalternative embodiments, some or all of the lasso peptide pathway biosynthetic genes are refactored to remove native transcriptional and translational regulation. In alternative embodiments, some or all of the lasso peptide pathway biosynthetic genes are refactored and constructed into operons on plasmids.

[0285] In alternative embodiments, CFB methods, systems and processes, including in vitro TX-TL systems, provided herein to produce lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, are cell-free platforms that can use whole cell, cytoplasmic or nuclear extract from a single organism such as E.coli or Saccharomyces cerevisiae (S. cerevisiae) or from an organism of the Actinomyces genus, e.g., a Streptomyces. In alternative embodiments, CFB methods, systems and processes, including in vitro TX-TL systems, provided herein to produce lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, are cell-free platforms that can use mixtures of whole cell, cytoplasmic, and / or nuclear extracts from the same or different organisms. In alternative embodiments, strain engineering approaches as well as modification of the growth conditions are used (on the organism from which at least one extract is derived) towards the creation of cell extracts as provided herein, to generate mixed cell extracts with varying proteomic and metabolic capabilities in the final CFB reaction mixture. In alternative embodiments, both approaches are used to tailor or design a final CFB reaction mixture for the purpose of synthesizing and characterizing lasso peptides, or for the creation of engineered lasso peptides through combinatorial biosynthesis approaches.

[0286] In alternative embodiments, cell extracts used in the CFB methods, provided herein to produce lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, include whole cell, cytoplasmic or nuclear extracts from a bacterial cell or eukaryotic cell, including insect, plant, fungal, yeast, or mammalian cells. In alternative embodiments, cell extracts used in the CFB methods, provided herein to produce lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, include whole cell, cytoplasmic or nuclear extracts from a bacterial cell or eukaryotic cell, including insect, plant, fungal, yeast, or mammalian cells, and are designed, produced and processed in a way to maximize efficacy and yield in the production of desired lasso peptides or related molecules thereof.

[0287] In an alternative embodiment, cell extracts used in the CFB methods, provided herein to produce lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, derive from at least two different bacterial cells, two different fungal cells; two different yeast cells, two different insect cells, two different plantcells or two different mammalian cells, or combinations of cell extracts from different species and genera thereof. In alternative embodiments, cell extracts used in the CFB methods, provided herein to produce lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, includes an extract derived from: an Escherichia or a Escherichia coli (E. coli): a Streptomyces or an Aclinobacleria: an Ascomycota, Basidiomycota, or a Saccharomycelales: a Penicillium or a Trichocomaceae: a Spodoptera, a Spodoptera frugiperda, a Trichoplusia or a Trichoplusia ni; a Poaceae. a Triticum, or a wheat germ; a rabbit reticulocyte or a HeLa cell.

[0288] In alternative embodiments, cell extracts used in the CFB methods, provided herein to produce lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, includes a cell extract from or includes an extract derived from: any prokaryotic and eukaryotic organism including, but not limited to, bacteria, including Archaea, eubacteria, and eukaryotes, including yeast, plant, insect, animal, and mammal, including human cells. In alternative embodiments, at least one of the cell extracts used in the CFB methods provided herein includes an extract from or includes an extract derived from: Escherichia coli, Saccharomyces cerevisiae, Saccharomyces kluyveri, Candida boidinii, Clostridium kluyveri, Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharoperbutylacetonicum, Clostridium perfringens, Clostridium difficile, Clostridium botulinum, Clostridium tyrobutyricum, Clostridium tetanomorphum, Clostridium tetani, Clostridium propionicum, Clostridium aminobutyricum, Clostridium subterminale, Clostridium sticklandii, Ralstonia eutropha, Mycobacterium bovis, Mycobacterium tuberculosis, Porphyromonas gingivalis, Arabidopsis thaliana, Thermus thermophilus, Pseudomonas species, including Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas fluorescens, Homo sapiens, Oryctolagus cuniculus, Rhodobacter spaeroides, Thermo-anaerobacter brockii, Metallosphaera sedula, Leuconostoc mesenteroides, Chloroflexus aurantiacus, Roseiflexus castenholzii, Erythrobacter, Simmondsia chinensis, Acinetobacter species, including Acinetobacter calcoaceticus and Acinetobacter baylyi, Porphyromonas gingivalis, Sulfolobus tokodaii, Sulfolobus solfataricus, Sulfolobus acidocaldarius, Bacillus subtilis, Bacillus cereus, Bacillus megaterium, Bacillus brevis, Bacillus pumilus, Rattus norvegicus, Klebsiella pneumonia, Klebsiella oxytoca, Euglena gracilis, Treponema denticola, Moorella thermoacetica, Thermotoga maritima, Halobacterium salinarum, Geobacillus stearothermophilus, Aeropyrum pernix, Sus scrofa, Caenorhabditis elegans, Corynebacterium glutamicum, Acidaminococcus fermentans, Lactococcus lactis, Lactobacillus plantarum, Streptococcus thermophilus, Enterobacteraerogenes, Candida, Aspergillus terreus, Pedicoccus pentosaceus, Zymomonas mobilus, Acetobacter pasteurians, Kluyveromyces lactis, Eubacterium barkeri, Bacteroides capillosus, Anaerotruncus colihominis, Natranaerobius thermophilusm, Campylobacter jejuni, Haemophilus influenzae, Serratia marcescens, Citrobacter amalonaticus, Myxococcus xanthus, Fusobacterium nuleatum, Penicillium chrysogenum, marine gamma proteobacterium, butyrate-producing bacterium, Nocardia iowensis, Nocardia farcinica, Streptomyces griseus, Schizosaccharomyces pombe, Geobacillus thermoglucosidasius, Salmonella typhimurium, Vibrio cholera, Heliobacter pylori, Nicotiana tabacum, Oryza sativa, Haloferax mediterranei, Agrobacterium tumefaciens, Achromobacter denitrificans, Fusobacterium nucleatum, Streptomyces clavuligenus, Acinetobacter baumanii, Mus musculus, Lachancea kluyveri, Trichomonas vaginalis, Trypanosoma brucei, Pseudomonas stutzeri, Bradyrhizobium japonicum, Mesorhizobium loti, Bos taurus, Nicotiana glutinosa, Vibrio vulnificus, Vibrio natriegens, Selenomonas ruminantium, Vibrio parahaemolyticus, Archaeoglobus fulgidus, Haloarcula marismortui, Pyrobaculum aerophilum, Mycobacterium smegmatis MC2 155, Mycobacterium avium subsp. paratuberculosis K-10, Mycobacterium marinum M, Tsukamurella paurometabola DSM 20162, Cyanobium PCC7001, Dictyostelium discoideum AX4.

[0289] In alternative embodiments, at least one cell, cytoplasmic or nuclear extract used in the CFB methods, provided herein to produce lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, includes a cell extract from or includes an extract derived from: Acinetobacter baumannii Naval-82, Acinetobacter sp. ADP1, Acinetobacter sp. strain M-l, Actinobacillus succinogenes 130Z, Allochromatium vinosum DSM 180, Amycolatopsis methanolica, Arabidopsis thaliana, Atopobium parvulum DSM 20469, Azotobacter vinelandii DJ, Bacillus alcalophilus ATCC 27647, Bacillus azotoformans LMG 9581, Bacillus coagulans 36D1, Bacillus megaterium, Bacillus methanolicus MGA3, Bacillus methanolicus PB1, Bacillus methanolicus PB-1, Bacillus selenitireducens MLS10 , Bacillus smithii, Bacillus subtilis , Burkholderia cenocepacia, Burkholderia cepacia, Burkholderia multivorans, Burkholderia pyrrocinia, Burkholderia stabilis, Burkholderia thailandensis E264, Burkholderiales bacterium Joshi 001, Butyrate- producing bacterium L2-50, Campylobacter jejuni, Candida albicans, Candida boidinii, Candida methylica, Carboxydothermus hydrogenoformans, Carboxydothermus hydrogenof ormans Z-2901, Caulobacter sp. AP07, Chlor oflexus aggregans DSM 9485, Chloroflexus aurantiacus J-lO-fl, Citrobacter freundii, Citrobacter koseri ATCC BAA-895, Citrobacter youngae , Clostridium, Clostridium acetobutylicum, Clostridium acetobutylicumATCC 824, Clostridium acidurici, Clostridium aminobutyricum, Clostridium asparagiforme DSM 15981, Clostridium beijerinckii , Clostridium beijerinckii NCIMB 8052, Clostridium bolteae ATCC BAA-613, Clostridium carboxidivorans P7, Clostridium cellulovorans 743B, Clostridium difficile, Clostridium hiranonis DSM 13275, Clostridium hylemonae DSM 15053, Clostridium kluyveri, Clostridium kluyveri DSM 555, Clostridium ljungdahli, Clostridium ljungdahlii DSM 13528, Clostridium methylpentosum DSM 5476 , Clostridium pasteurianum, Clostridium pasteurianum DSM 525, Clostridium perfringens, Clostridium perfringens ATCC 13124, Clostridium perfringens str. 13, Clostridium phytofermentans ISDg, Clostridium saccharobutylicum, Clostridium saccharoperbutylacetonicum, Clostridium saccharoperbutylacetonicum Nl-4, Clostridium tetani, Corynebacterium glutamicum ATCC 14067, Corynebacterium glutamicum R, Corynebacterium sp. U-96, Corynebacterium variabile, Cupriavidus necator N-l, Cyanobium PCC7001, Desulfatibacillum alkenivorans AK-01, Desulfitobacterium hafniense, Desulfitobacterium metallireducens DSM 15288, Desulfotomaculum reducens MI-1, Desulfovibrio africanus str. Walvis Bay, Desulfovibrio fructosovorans JJ, Desulfovibrio vulgaris str. Hildenborough, Desulfovibrio vulgaris str. Miyazaki F', Dictyostelium discoideum AX4, Escherichia coli, Escherichia coli K-12 , Escherichia coli K-12 MG1655, Eubacterium hallii DSM 3353 , Flavobacterium frigoris, Fusobacterium nucleatum subsp. polymorphum ATCC 10953 , Geobacillus sp. Y4.1MC1, Geobacillus themodenitrificans NG80-2, Geobacter bemidjiensis Bern, Geobacter sulfurreducens, Geobacter sulfurreducens PCA, Geobacillus stearothermophilus DSM 2334, Haemophilus influenzae, Helicobacter pylori, Homo sapiens, Hydrogenobacter thermophilus, Hydrogenobacter thermophilus TK-6, Hyphomicrobium denitrificans ATCC 51888, Hyphomicrobium zavarzinii, Klebsiella pneumoniae, Klebsiella pneumoniae subsp. pneumoniae MGH 78578, Lactobacillus brevis ATCC 367, Leuconostoc mesenteroides, Lysinibacillus fusiformis, Lysinibacillus sphaericus, Mesorhizobium loti MAFF303099, Metallosphaera sedula, Methanosarcina acetivorans, Methanosarcina acetivorans C2A, Methanosarcina barkeri, Methanosarcina mazei TucOl, Methylobacter marinus, Methylobacterium extorquens, Methylobacterium extorquens AMI, Methylococcus capsulatas, Methylomonas aminofaciens, Moorella thermoacetica, Mycobacter sp. strain JC1 DSM 3803, Mycobacterium avium subsp. paratuberculosis K-10, Mycobacterium bovis BCG, Mycobacterium gastri , Mycobacterium marinum M, Mycobacterium smegmatis, Mycobacterium smegmatis MC2 155, Mycobacterium tuberculosis, Nitrosopumilus salaria BD31, Nitrososphaera gar gensis Ga9.2, Nocardia farcinica IFM 10152, Nocardia iowensis (sp. NRRL 5646), Nostoc sp. PCC 7120, Ogataea angusta, Ogataea parapolymorpha DL-1(Hansenula polymorpha DL-1), Paenibacillus peoriae KCTC 3763, Paracoccus denitrificans, Penicillium chrysogenum, Photobacterium profundum 3TCK, Phytofermentans ISDg, Pichia pastor is, Picrophilus torridus DSM9790, Porphyromonas gingivalis, Porphyromonas gingivalis W83, Pseudomonas aeruginosa PA01, Pseudomonas denitrificans, Pseudomonas knackmussii, Pseudomonas putida, Pseudomonas sp, Pseudomonas syringae pv. syringae B728a, Pyrobaculum islandicum DSM 4184, Pyrococcus abyssi, Pyrococcus furiosus, Pyrococcus horikoshii 0T3, Ralstonia eutropha, Ralstonia eutropha H16, Rhodobacter capsulatus, Rhodobacter sphaeroides, Rhodobacter sphaeroides ATCC 17025, Rhodopseudomonas palustris, Rhodopseudomonas palustris CGA009, Rhodopseudomonas palustris DX-1, Rhodospirillum rubrum, Rhodospirillum rubrum ATCC 11170, Ruminococcus obeum ATCC 29174, Saccharomyces cerevisiae, Saccharomyces cerevisiae S288c, Salmonella enterica, Salmonella enterica subsp. enterica serovar Typhimurium str. LT2, Salmonella enterica typhimurium , Salmonella typhimurium, Schizosaccharomyces pombe, Sebaldella termitidis ATCC 33386 , Shewanella oneidensis MR-1, Sinorhizobium meliloti 1021, Streptomyces coelicolor, Streptomyces griseus subsp. griseus NBRC 13350, Sulfolobus acidocalarius, Sulfolobus solfataricus P-2, Synechocystis str. PCC 6803, Syntrophobacter fumaroxidans, Thauera aromatica, Thermoanaerobacter sp. X514, Thermococcus kodakaraensis, Thermococcus litoralis, Thermoplasma acidophilum, Thermoproteus neutrophilus, Thermotoga maritima, Thiocapsa roseopersicina, Tolumonas auensis DSM 9187, Trichomonas vaginalis G3, Trypanosoma brucei, Tsukamurella paurometabola DSM 20162, Vibrio cholera, Vibrio harveyi ATCC BAA-1116, Vibrio natriegens, Xanthobacter autotrophicus Py2, Yersinia intermedia, or Zea mays.

[0290] In alternative embodiments, cell extracts used in the CFB methods and processes, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, e.g., including at least one of the cell, cytoplasmic or nuclear extracts, have added to them, or further include, supplemental ingredients, compositions or compounds, reagents, ions, trace metals, salts, or elements, buffers and / or solutions. In alternative embodiments, the CFB method and system of the present disclosure, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, use or fabricate environmental conditions to optimize the rate of formation or yield of a lasso peptide or related molecules thereof.

[0291] In alternative embodiments, CFB reaction mixtures and cell extracts used in the CFB methods and systems, provided herein for the synthesis of lasso peptides and related moleculesthereof from a minimal set of lasso peptide biosynthetic pathway components, are supplemented with a carbon source and other essential nutrients. The CFB production system, including cell extracts used in the CFB methods and processes, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, can include, for example, any carbohydrate source. Such sources of sugars or carbohydrate substrates include glucose, xylose, maltose, arabinose, galactose, mannose, maltodextrin, fructose, sucrose and starch.

[0292] In alternative embodiments, CFB methods and systems provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, are conducted in a CFB reaction mixture, including cell extracts that are supplemented with all twenty proteinogenic naturally occurring amino acids and corresponding transfer ribonucleic acids (tRNAs). In alternative embodiments, cell extracts used in the CFB reaction mixture, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, are supplemented with adenosine triphosphate (ATP), and / or adenosine diphosphate (ADP). In alternative embodiments, cell extracts used in the CFB reaction mixture, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, are supplemented with glucose, xylose, maltose, arabinose, galactose, mannose, maltodextrin, fructose, sucrose and / or starch. In alternative embodiments, cell extracts used in the CFB reaction mixture, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, are supplemented with purine and guanidine nucleotides, adenosine triphosphate, guanosine triphosphate, cytosine triphosphate, and uridine triphosphate. In alternative embodiments, cell extracts used in the CFB reaction mixture, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, are supplemented with cyclic- adenosine monophosphate (cAMP) and / or 3 -phosphoglyceric acid (3-PGA). In alternative embodiments, cell extracts used in the CFB reaction mixture, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, are supplemented with nicotinamide adenine dinucleotides NADH and / or NAD, or nicotinamide adenine dinucleotide phosphates, NADPH, and / or NADP, or combinations thereof. In alternative embodiments, cell extracts used in the CFB reaction mixture, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, are supplemented with aminoacid salts such as magnesium glutamate and / or potassium glutamate. In alternative embodiments, cell extracts used in the CFB reaction mixture, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, are supplemented with buffering agents such as HEPES, TRIS, spermidine, or phosphate salts. In alternative embodiments, cell extracts used in the CFB reaction mixture, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, are supplemented with salts, including but not limited to, potassium phosphate, sodium chloride, magnesium phosphate, and magnesium sulfate. In alternative embodiments, cell extracts used in the CFB reaction mixture, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, are supplemented with folinic acid and co-enzyme A (CoA). In alternative embodiments, cell extracts used in the CFB reaction mixture, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, are supplemented with crowding agents such as PEG 8000, Ficoll 70, or Ficoll 400, or combinations thereof. For a general description of cell-free extract production and preparation, see: Krinsky, N., et al., PLoS ONE, 2016, 11(10): e0165137.

[0293] In some embodiments, cell-free biosynthesis of lasso peptides or engineered lasso peptides is conducted with isolated peptide and enzyme components in standard buffered media, such as phosphate-buffered saline or tris-buffered saline, in each case containing salts, ATP, and co-factors required for lasso peptidase and lasso cyclase enzymatic activity. In some embodiments, cell-free biosynthesis of lasso peptides is conducted using genes that require transcription (TX) and translation (TL) to afford the lasso precursor peptide and / or lasso peptide biosynthetic enzymes in situ, and such in vitro biosynthesis processes are conducted in cell extracts derived from prokaryotic or eukaryotic cells (See: Gagoski, D., et al., BiotechnoL Bioeng. 2016; 113: 292-300; Culler, S. et al., PCT Appl. No. WO2017 / 031399). In alternative embodiments, a cell-free biosynthesis process for producing engineered lasso peptides is conducted by contacting an isolated, chemically- or biologically-synthesized precursor peptide with one or more isolated lasso peptidase, lasso cyclase, and lasso RRE. In other embodiments, a cell-free biosynthesis process for producing engineered lasso peptides is conducted by contacting an isolated, chemically- or biologically-synthesized core peptide with one or more isolated lasso peptidase, lasso cyclase, and lasso RRE.6.8.2. Cell-based Biosynthesis of Lasso Peptides

[0294] In a related aspect, provided herein are also methods for producing lasso peptides and engineered lasso peptides, including lasso peptides designed using in silico modeling methods, using a non-naturally occurring microbial organism. Certain cell-based production methods involve cultivating or fermenting a microbial organism that is a natural producer of a lasso peptide of interest. Alternative cell-based production methods involve cloning the genes encoding lasso peptide biosynthesis component into an appropriate vector or plasmid, introducing that vector or plasmid into a microorganism, and propagating or cultivating that organism with the necessary nutrients and under conditions for heterologous production of recombinant lasso peptides of interest (Zhang et al., Front. Microbiol., 2018, 9:289).

[0295] Depending on the lasso peptide biosynthetic pathway constituents of a selected host microbial organism, the non-naturally occurring microbial organisms of the invention will include at least one exogenously expressed lasso peptide pathway-encoding nucleic acid and up to all encoding nucleic acids for one or more lasso peptide biosynthetic pathways. For example, lasso peptide biosynthesis can be established in a host deficient in a pathway enzyme or protein through exogenous expression of the corresponding encoding nucleic acid. In a host deficient in all enzymes or proteins of a lasso peptide pathway, exogenous expression of all enzyme or proteins in the pathway can be included, although it is understood that all enzymes or proteins of a pathway can be expressed even if the host contains at least one of the pathway enzymes or proteins. For example, exogenous expression of all enzymes or proteins in a pathway for production of a lasso peptide can be included, such as a lasso peptide precursor, a lasso peptide peptidase, a lasso peptide cyclase, and / or a lasso peptide RiPP recognition element (RRE).

[0296] Given the teachings and guidance provided herein, those skilled in the art will understand that the number of encoding nucleic acids to introduce in an expressible form will, at least, parallel the lasso peptide pathway deficiencies of the selected host microbial organism. Therefore, a non-naturally occurring microbial organism can have one, two, three, four, five, six, seven, eight, nine or ten, up to all nucleic acids encoding the enzymes or proteins constituting a lasso peptide biosynthetic pathway disclosed herein. In some embodiments, the non-naturally occurring microbial organisms also can include other genetic modifications that facilitate or optimize lasso peptide biosynthesis or that confer other useful functions onto the host microbial organism. One such other functionality can include, for example, augmentation of the synthesis of one or more of the lasso peptide pathway precursors, such as amino acids.

[0297] Generally, a host microbial organism is selected such that it produces the lasso precursor peptide, either as a naturally produced molecule or as an engineered product that either provides de novo production of a desired biosynthesis precursor or increased production of a biosynthesis precursor naturally produced by the host microbial organism. For example, amino acids are produced naturally in a host organism such as E. coli. A host organism can be engineered to increase production of one or more amino acids in order to increase production of lasso precursor, as disclosed herein. Alternatively, a host organism can be engineered to produce a non-natural amino acid that is incorporated into the lasso precursor peptide (Piscotta etal., Chem. Commun., 2015, 51, 409-412; and Al-Toma etal., ChemBioChem 2015, 16, 503- 509). In addition, a microbial organism that has been engineered to produce a desirable lasso precursor peptide can be used as a host organism and further engineered to express enzymes or proteins that processes the lasso precursor peptide into matured lasso peptides containing nonnatural amino acids.

[0298] In some embodiments, a non-naturally occurring microbial organism is generated from a host that contains the enzymatic capability to synthesize a lasso peptide or engineered lasso peptide. In this specific embodiment it can be useful to increase the synthesis or accumulation of a lasso peptide pathway intermediate or product to, for example, drive lasso peptide pathway reactions toward lasso peptide production. Increased synthesis or accumulation can be accomplished by, for example, overexpression of nucleic acids encoding one or more of the above-described lasso peptide pathway enzymes or proteins. Over expression the enzyme or enzymes and / or protein or proteins of the lasso peptide pathway can occur, for example, through exogenous expression of the endogenous gene or genes, or through exogenous expression of the heterologous gene or genes. Therefore, naturally occurring organisms can be readily engineered to be non-naturally occurring microbial organisms for producing lasso peptides or engineered lasso peptides, through overexpression of one, two, three, four, five, six, seven, eight, nine, or ten, that is, up to all nucleic acids encoding a lasso peptide biosynthetic pathway enzymes or proteins. In addition, a non-naturally occurring organism can be generated by mutagenesis of an endogenous gene that results in an increase in activity of an enzyme in the lasso peptide biosynthetic pathway.

[0299] In particularly useful embodiments, exogenous expression of the encoding nucleic acids is employed. Exogenous expression confers the ability to custom tailor the expression and / or regulatory elements to the host and application to achieve a desired expression level that is controlled by the user. However, endogenous expression also can be utilized in other embodiments such as by removing a negative regulatory effector or induction of the gene’spromoter when linked to an inducible promoter or other regulatory element. Thus, an endogenous gene having a naturally occurring inducible promoter can be up-regulated by providing the appropriate inducing agent (Daniel-Ivad et cd.. ACS Chem. Biol. 2017, 12, 628-634), or the regulatory region of an endogenous gene can be engineered to incorporate an inducible regulatory element, thereby allowing the regulation of increased expression of an endogenous gene at a desired time. Similarly, an inducible promoter can be included as a regulatory element for an exogenous gene introduced into a non-naturally occurring microbial organism.

[0300] It is understood that any of the one or more exogenous nucleic acids can be introduced into a microbial organism to produce a non-naturally occurring microbial organism of the invention. The nucleic acids can be introduced so as to confer, for example, a lasso peptide biosynthetic pathway onto the microbial organism. Alternatively, encoding nucleic acids can be introduced to produce an intermediate microbial organism having the biosynthetic capability to catalyze some of the required reactions to confer lasso peptide biosynthetic capability. For example, a non-naturally occurring microbial organism having a lasso peptide biosynthetic pathway can include at least one exogenous nucleic acid encoding desired enzymes or proteins, such as the linear lasso precursor peptide, or alternatively a combination of a lasso peptide peptidase and a lasso peptide cyclase. Thus, it is understood that any combination of one or more genes encoding one or more peptides, enzymes, or proteins of a biosynthetic pathway can be included in a non-naturally occurring microbial organism of the invention. Similarly, it is understood that any combination of two or more enzymes or proteins of a biosynthetic pathway can be included in a non-naturally occurring microbial organism of the invention, for example, lasso peptide peptidase and a lasso peptide cyclase, and so forth, as desired, so long as the combination of enzymes and / or proteins of the desired biosynthetic pathway results in production of the corresponding desired product.

[0301] Accordingly, in some aspects, provided herein is a method for producing an engineered lasso peptide described herein using a non-naturally occurring microbial organism, wherein the method includes introducing into the microbial organism a first nucleic acid containing a recombinant nucleic acid described herein or a vector described herein, and a second nucleic acid encoding a lasso peptide biosynthesis component. Such methods can further include culturing the microbial organism under a condition suitable for lasso formation to produce the engineered lasso peptide. In some embodiments, the first nucleic acid encodes an engineered lasso peptide having a leader sequence (e.g., a precursor peptide), and wherein the lasso peptide biosynthesis component includes a lasso peptidase capable of catalyzingremoval of the leader sequence. In some embodiments, the lasso peptide biosynthesis component also includes a lasso cyclase capable of cyclizing a linear lasso core sequence to a mature lasso peptide. In some embodiments, the lasso peptide biosynthesis component includes a lasso peptidase and a lasso cyclase. In such a method, the method can further include introducing the second nucleic acid sequence encoding the lasso cyclase and a third nucleic acid sequence encoding the lasso peptidase. Examples of lasso peptidases and a lasso cyclases for cyclizing an engineered lasso peptide described herein are provided in Table 2. Accordingly, some embodiments, the methods provided herein include introducing a nucleic acid sequence encoding a lasso peptidase or a lasso cyclase described Table 2.

[0302] In addition to the biosynthesis of lasso peptides as described herein, the non-naturally occurring microbial organisms also can be utilized in various combinations with each other and with other microbial organisms and methods well known in the art to achieve product biosynthesis by other routes. For example, one alternative to produce a lasso peptide other than use of the lasso peptide producers is through addition of another microbial organism capable of converting a lasso peptide pathway intermediate into a lasso peptide or engineered lasso peptide. One such procedure includes, for example, the fermentation of a microbial organism that produces a linear lasso precursor peptide. The linear lasso precursor peptide can then be used as a substrate for a second microbial organism that converts the linear lasso precursor peptide to a lasso peptide. The linear lasso precursor peptide can be added directly to an...

Claims

CLAIMSWhat is claimed is:

1. An engineered lasso peptide comprising, when cyclized, an integrin binding motif within a loop or a ring of the engineered lasso peptide, wherein the integrin binding motif comprises the amino acid sequence RGDXi (SEQ ID NO: 782), wherein Xi is any naturally or non-naturally occurring amino acid residue except for phenylalanine (F) or the amino acid residue of a parent scaffold peptide at that corresponding position, wherein the engineered lasso peptide specifically binds an integrin, and wherein the integrin is selected from alpha V beta 6 (avP6), alpha V beta 8 (avP8), and alpha V beta 1 (avpi).

2. The engineered lasso peptide of claim 1, wherein Xi is a leucine (L), isoleucine (I), tryptophan (W), lysine (K), glutamine (Q), or arginine (R).

3. The engineered lasso peptide of claim 2, wherein Xi is a leucine (L).

4. The engineered lasso peptide of claim 1, wherein the integrin binding motif comprises the amino acid sequence RGDX1X2 (SEQ ID NO: 788), wherein X2 is independently any natural or non-natural amino acid residue except for the amino acid residue of a parent scaffold peptide at that corresponding position.

5. The engineered lasso peptide of claim 4, wherein Xi and X2 are independently hydrophobic, polar, or charged amino acid residues.

6. The engineered lasso peptide of claim 4, wherein Xi is independently a leucine (L), isoleucine (I), tryptophan (W), lysine (K), glutamine (Q), or arginine (R), and wherein X2 is independently leucine (L), isoleucine (I), phenylalanine (F), tryptophan (W), lysine (K), glutamine (Q), or arginine (R).

7. The engineered lasso peptide of claim 6, wherein Xi is a leucine (L).

8. The engineered lasso peptide of claim 4, wherein Xi and X2 are independently residues containing an aromatic group, a heteroaromatic group, an alkyl group, a hydroxy group, a halide group, an amino group, a carboxylic acid group, a carboxamide group, or combinations thereof.

9. The engineered lasso peptide of claim 1, wherein the integrin binding motif comprises the amino acid sequence X3RGDX1X2 (SEQ ID NO: 793), wherein X2 and X3 are independently any natural or non-natural amino acid residue except for the amino acid residue of a parent scaffold peptide at that corresponding position.

10. The engineered lasso peptide of claim 9, wherein Xi and X2 are independently hydrophobic, polar, or charged amino acid residues.

11. The engineered lasso peptide of claim 9, wherein Xi, X2, and X3 are independently hydrophobic, polar, or charged amino acid residues.

12. The engineered lasso peptide of claim 9, wherein Xi is independently a leucine (L), isoleucine (I), tryptophan (W), lysine (K), glutamine (Q), or arginine (R), and wherein X2 and X3 are independently alanine (A), leucine (L), isoleucine (I), phenylalanine (F), tryptophan (W), lysine (K), glutamine (Q), or arginine (R).

13. The engineered lasso peptide of claim 12, wherein Xi is a leucine (L).

14. The engineered lasso peptide of claim 9, wherein Xi, X2, and X3 are independently residues containing an aromatic group, a heteroaromatic group, an alkyl group, a hydroxyl group, a halide group, an amino group, a carboxylic acid group, a carboxamide group, or combinations thereof.

15. The engineered lasso peptide of any one of claims 1 to 14, wherein the engineered lasso peptide further comprises an arginine (R), histidine (H), leucine (L), threonine (T), glutamine (Q), asparagine (N), glycine (G), glutamic acid (E), arginine (K), phenylalanine (F), tryptophan (W) at position 3 located in the ring of the engineered lasso peptide.

16. The engineered lasso peptide of any one of claims 1 to 14, wherein the engineered lasso peptide further comprises an arginine (R) at position 3 located in the ring of the engineered lasso peptide.

17. The engineered lasso peptide of any one of claims 1 to 14, wherein the engineered lasso peptide further comprises one, two, three, or four amino acid alterations relative to a parent scaffold peptide.

18. The engineered lasso peptide of claim 17, wherein the alterations comprise an amino acid substitution, deletion or insertion.

19. The engineered lasso peptide of claim 18, wherein the parent scaffold peptide comprises the amino acid sequence of SEQ ID NO: 1, and wherein the alterations comprise a deletion of proline (P) at position 16 and / or a glutamine (Q) at position 11 in the loop of the engineered lasso peptide and an arginine (R), a histidine (H), or a threonine (T) at position 3 located in the ring of the engineered lasso peptide.

20. The engineered lasso peptide of claim 19, wherein the alterations further comprise an alteration at position 5 or position 6 located in the ring of the engineered lasso peptide.

21. The engineered lasso peptide of claim 20, wherein the alteration at position 5 located in the ring of the engineered lasso peptide comprises a glutamine (Q) or serine (S) at position 5.

22. The engineered lasso peptide of claim 20, wherein the alteration at position 6 located in the ring of the engineered lasso peptide comprises an isoleucine (I) at position 6.

23. The engineered lasso peptide of any one of claims 1 to 22, wherein the engineered lasso peptide selectively inhibits avP6 function.

24. The engineered lasso peptide of claim 23, wherein the engineered lasso peptide binds to avP6 with an ICso < 500 nM.

25. The engineered lasso peptide of claim 23 or 24, wherein the engineered lasso peptide comprises the amino acid sequence of any one of SEQ ID NOS: 4-9, 11, 13-21, 23, 24, 26-31, 33, 34, 36, 40-48, 50, 54-56, 59, 60, 62, 68, 71, 73, 81, 85-90, 93, 95-103, 163, 168, 187, 189, 190, 192-194, 196-209, 216, 218, 220, 221, 223, 229, 231-233, 242, 245, 247, 249, 251-255, 258, 261-263, 270, 271, 274-276, 282-297, 299-301, 303-316, 318-322, 328-332, 344, 346, 347, and 835-840.

26. The engineered lasso peptide of any one of claims 1 to 13, wherein engineered lasso peptide selectively inhibits avP8 function.

27. The engineered lasso peptide of claim 26, wherein the engineered lasso peptide binds to avP8 with an ICso < 500 nM.

28. The engineered lasso peptide of claim 26 or 27, wherein the engineered lasso comprises the amino acid sequence of any one of SEQ ID NOS: 4-9, 11, 13-15, 17- 21, 23, 24, 26-29, 31, 36, 40-48, 50, 54-56, 59, 60, 62, 68, 71, 73, 81, 85-90, 93, 95- 103, 163, 168, 187, 189, 190, 192-194, 196-200, 203-209, 216, 218, 220, 221, 223, 229, 231-233, 242, 245, 247, 249, 251-253, 255, 258, 261-263, 270, 271, 274-276, 282-297, 299-301, 303-316, 318-322, 328-332, 344, 346, 347, and 835-840.

29. The engineered lasso peptide of any one of claims 1 to 13, wherein the engineered lasso peptide selectively inhibits avpi function.

30. The engineered lasso peptide of claim 29, wherein the engineered lasso peptide binds to avpi with an ICso < 500 nM.

31. The engineered lasso peptide of claim 29 or 30, wherein the engineered lasso peptide comprises the amino acid sequence of any one of SEQ ID NOS: 6, 7, 87, 88, 97, 100, 163 and 187.

32. The engineered lasso peptide of any one of claims 1 to 22, wherein the engineered lasso peptide binds to avP6, avP8, and / or avpi with an ICso < 50 nM.

33. The engineered lasso peptide of any one of claims 1 to 22, wherein the engineered lasso peptide preferentially binds to avP6, avP8, and / or avpi as compared to avP3, avP5, allbp5, a5pi and / or a8pi.

34. The engineered lasso peptide of claim 33, wherein the engineered lasso peptide binds to avP6, avP8, and / or avpi with an ICso < 50 nM and binds to avP3, avP5, allbp5, a5pi and / or a8pi with an ICso > 500 nM.

35. The engineered lasso peptide of any one of claims 1 to 34, wherein the engineered lasso peptide further comprises a leader sequence, thereby generating a lasso precursor peptide.

36. The engineered lasso peptide of claim 35, wherein the leader sequence comprises the amino acid sequence selected from: a) MIKHIHFDKLSSSKKNNVPHSAKGVIQIKKSASQLTK (SEQ ID NO: 777) when the engineered lasso peptide is derived from a parent scaffold peptide comprising SEQ ID NO: 1, b) MMQQKKNDMKKVTLKKLNKRASKVTR (SEQ ID NO 778) when the engineered lasso peptide is derived from a parent scaffold peptide comprising SEQ ID NO: 343, c) MKKQTFVPKKLVKVGKATELTK (SEQ ID NO 779) when the engineered lasso peptide is derived from a parent scaffold peptide comprising SEQ ID NO: 1D NO 353, d) MERNHETPSDLIDLGAASVETK (SEQ ID NO 780) when the engineered lasso peptide is derived from a parent scaffold peptide comprising SEQ ID NO: 362, and e) MTQVSPSPLRLIRVGRALDLTR (SEQ ID NO 781) when the engineered lasso peptide is derived from a parent scaffold peptide comprising SEQ ID NO: 366.

37. A composition comprising the engineered lasso peptide of any one of claims 1 to 36 and a buffer, a culture medium or a cellular component.

38. A pharmaceutical composition comprising the engineered lasso peptide of any one of claims 1 to 34 and a pharmaceutically acceptable carrier.

39. The pharmaceutical composition of claim 38, wherein the composition further comprises a second therapeutic agent for managing, preventing or treating cancer and / or fibrosis.

40. The pharmaceutical composition of claim 39, wherein the second therapeutic agent is a chemotherapy or immunotherapy for cancer.

41. The pharmaceutical composition of claim 40, wherein the immunotherapy is an anticancer vaccine or immune checkpoint modulator.

42. The pharmaceutical composition of claim 40, wherein the second therapeutic agent is an anti-fibrotic therapy.

43. The pharmaceutical composition of any one of claims 38 to 42 for use in managing, preventing, or treating cancer and / or fibrosis.

44. A method of managing, preventing, or treating an integrin-mediated disease in a subject, comprising administering to the subject a prophylactically or therapeutically effective amount of the engineered lasso peptide of any one of claims 1 to 34 or the pharmaceutical composition of any one of claims 38 to 43.

45. The method of claim 44, wherein upon administration, the engineered lasso peptide: a) binds to the integrin; b) inhibits a function of the integrin; c) inhibits an integrin-mediated signaling pathway; d) reduces integrin levels on the surface of neoplastic cells, immune cells, and / or fibroblasts in a tumor microenvironment (TME) due to integrin internalization; e) reduces integrin levels on the surface of cells in fibrotic tissues; f) downregulates integrin expression on the surface of neoplastic cells, immune cells, and / or fibroblast cells in a TME; and / or g) downregulates integrin expression on the surface of cells in fibrotic tissues.

46. The method of claim 45, wherein the inhibition of the function of the integrin and / or the inhibition of the integrin-mediated signaling pathway is measured by: a) inhibition of binding of isolated integrins to extracellular matrix (ECM) components, fibrinogen, and / or latency-associated peptide (LAP); b) inhibition of adhesion of integrin-expressing cells to ECM components, fibrinogen, and / or LAP; c) inhibition of migration of integrin-expressing cells in a matrix containing ECM components, fibrinogen, and / or LAP; d) inhibition of TGF-P activation; e) inhibition of the release of free TGF-P from its latency complex with LAP; f) inhibition of growth and / or metastasis of neoplastic cells;g) inhibition of the conversion of fibroblasts to activated myofibroblasts; and / or h) inhibition of accumulation of ECM in and / or growth of fibrotic tissue.

47. The method of claim 44, wherein upon administration, the engineered lasso peptide: a) inhibits the phosphorylation and activation of mothers against decapentaplegic homolog (Smad) transcription factors; b) increases the release of interleukin-y; c) increases the release of tumor necrosis factor-a; d) decreases the production of a-smooth muscle actin, collagen, and / or fibronectin; e) reduces the release of one or more growth factors; and / or f) reduces the release of one or more cytokines.

48. The method of claim 47, wherein the one or more growth factors comprises TGF-p.

49. The method of claim 47, wherein the Smad transcription factors comprise Smad2 or Smad3.

50. The method of claim 47, wherein the one or more cytokines comprises IL- 17, IL-6, IL-11, and / or IL-22.

51. The method of claim 44, wherein upon administration, the engineered lasso peptide: a) increases the proliferation and / or anti-tumor activity of CD8+ cytotoxic T cells in a TME; b) increases the proliferation and / or anti-tumor activity of NK cells in a TME; and / or c) decreases the proliferation of immunosuppressive CD4+ T cells in a TME.

52. The method of claim 51, wherein the immunosuppressive CD4+ T cells comprise regulatory T cells and T helper cells.

53. The method of any one of claims 44 to 52, wherein the integrin-mediated disease comprises neoplastic cells expressing one or more integrins.

54. The method of any one of claims 44 to 53, wherein the subject has a tumor comprising immune cells expressing one or more integrins in the TME.

55. The method of any one of claims 44 to 52, wherein the subject has a tumor comprising stromal cells expressing one or more integrins in the TME.

56. The method of claim 55, wherein the stromal cells expressing one or more integrins are cancer associated fibroblasts.

57. The method of any one of claims 53 to 56, wherein the one or more integrins are RGD-binding integrins.

58. The method of any one of claims 53 to 56, wherein the one or more integrins are selected from avP6, avP8, and avpi.

59. The method of any one of claims 44 to 58, wherein the integrin-mediated disease is cancer.

60. The method of claim 59, wherein the cancer is breast cancer, pancreatic cancer, hepatocellular cancer, prostate cancer, ovarian cancer, gastric cancer, brain or spinal cancer, melanoma, cancer of the head and neck, colorectal cancer, bladder cancer, vulvar cancer, esophageal squamous cell carcinoma, renal cancer, cervical cancer, salivary gland carcinoma, lung cancer, multiple myeloma, or Kaposi’s sarcoma.

61. The method of claim 60, wherein the brain or spinal cancer is a glioma.

62. The method of claim 61, wherein the glioma is a glioblastoma.

63. The method of claim 60, wherein the cancer is melanoma, breast cancer, or ovarian cancer.

64. The method of any one of claims 44 to 58, wherein the integrin-mediated disease is a fibroproliferative disease or fibrosis.

65. The method of claim 64, wherein the fibroproliferative disease or fibrosis is a pulmonary fibrosis.

66. The method of claim 64, wherein the fibroproliferative disease or fibrosis is selected from cystic fibrosis, idiopathic pulmonary fibrosis, hepatic fibrosis, cirrhosis, pancreatic fibrosis, pancreatitis, renal fibrosis, glial fibrosis, retroperitoneal cavity fibrosis, mediastinal fibrosis, cardiovascular disease and heart fibrosis, myelofibrosis, systemic sclerosis, Dupuytren’s contracture, Peyronie’s disease, macular degeneration, hypertrophic scars, skin fibrosis and skin keloids, arthrofibrosis, Duchenne muscular dystrophy-associated skeletal muscle fibrosis, uterine leiomyoma and adenomyosis.

67. The method of claim 45, wherein the inhibition of the function of the integrin or the integrin-mediated signaling pathway is a maximal percent inhibition of at least about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

68. The method of claim 45, wherein the reduction of integrin levels is a maximal percent reduction of at least about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

69. The method of claim 45, wherein the downregulation of integrin expression is a maximal percent downregulation of at least about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

70. The method of any one of claims 44 to 69, wherein the engineered lasso peptide is conjugated to an agent.

71. The method of claim 70, wherein the agent is selected from the group consisting of a radioisotope, a metal chelator, an enzyme, a protein, a peptide, an antibody, an antibody fragment, a nanobody, a cytotoxic compound, a fluorescent compound, a bioluminescent compound, and a chemiluminescent compound.

72. The method of any one of claims 44 to 71, further comprising co-administering to the subject a second therapeutic agent with the engineered lasso peptide.

73. The method of claim 72, wherein the second therapeutic agent is conjugated with the engineered lasso peptide.

74. The method of claim 72 or 73, wherein the second therapeutic agent is an immunotherapy or chemotherapy.

75. The method of claim 73, wherein the immunotherapy is an anti-cancer vaccine or an immune checkpoint modulator.

76. A recombinant nucleic acid encoding the engineered lasso peptide of any one of claims 1 to 34.

77. A recombinant nucleic acid encoding a lasso precursor peptide comprising an amino acid sequence of the engineered lasso peptide of claim 35 or 36.

78. The recombinant nucleic acid of claim 77, wherein the recombinant nucleic acid comprises a nucleotide sequence selected from SEQ ID NOS: 390-397, 399, 401-409, 411, 412, 414-419, 421, 422, 424, 428-436, 438, 442-444, 447, 448, 450, 456, 459, 461, 469, 473-478, 481, 483-491, 551, 556, 575, 577, 578, 580-582, 584-597, 604, 606, 608, 609, 611, 617, 619-621, 630, 633, 635, 637, 639-643, 646, 649-651, 656, 658, 659, 662-664, 670-685, 687-689, 691-704, 706-710, 716-720, 732, 734, and 735.

79. The recombinant nucleic acid of any one of claims 76 to 78, wherein the recombinant nucleic acid comprises a nucleotide sequence encoding the engineered lasso peptide operatively linked to a promoter.

80. A vector comprising the recombinant nucleic acid of any one of claims 76 to 79.

81. A non-naturally occurring microbial organism comprising the recombinant nucleic acid of any one of claims 76 to 79 or the vector of claim 80.

82. A method for producing an engineered lasso peptide using a non-naturally occurring microbial organism, wherein the method comprises: a) introducing into the microbial organism a first nucleic acid comprising the recombinant nucleic acid of any one of claims 76 to 79 or the vector of claim 80 and a second nucleic acid encoding a lasso peptide biosynthesis component; and b) culturing the microbial organism under a condition suitable for lasso formation to produce the engineered lasso peptide.

83. The method of claim 82, wherein the first nucleic acid encodes the engineered lasso peptide of claim 35, and wherein the lasso peptide biosynthesis component comprises a lasso peptidase capable of catalyzing removal of the leader sequence.

84. The method of claim 82 or 83, wherein the lasso peptide biosynthesis component comprises a lasso cyclase capable of cyclizing a linear lasso core sequence to a mature lasso peptide.

85. The method of claim 82 or 83, wherein the lasso peptide biosynthesis component comprises a lasso peptidase and a lasso cyclase, and wherein the method comprises introducing the second nucleic acid sequence encoding the lasso cyclase and a third nucleic acid sequence encoding the lasso peptidase.

86. The method of claim 82 or 83, wherein the lasso peptide biosynthesis component comprises a lasso cyclase and a post-translationally modified peptide (RiPP) recognition element (RRE).

87. The method of claim 82 or 83, wherein the lasso peptide biosynthesis component comprises a lasso cyclase and a post-translationally modified peptide (RiPP) recognition element (RRE), and wherein the method comprises introducing the second nucleic acid sequence encoding the lasso cyclase and a fourth nucleic acid sequence encoding the RRE.

88. The method of claim 82 or 83, wherein the lasso peptide biosynthesis component comprises a lasso peptidase, a lasso cyclase and a post-translationally modified peptide (RiPP) recognition element (RRE).

89. The method of claim 82 or 83, wherein the lasso peptide biosynthesis component comprises a lasso peptidase, a lasso cyclase and a post-translationally modified peptide (RiPP) recognition element (RRE), and wherein the method comprises introducing the second nucleic acid sequence encoding the lasso cyclase, a thirdnucleic acid sequence encoding the lasso peptidase, and a fourth nucleic acid sequence encoding the RRE.

90. The method of any one of claims 82 to 89, wherein at least two of the first, second, third and fourth nucleic acid sequences are in a same nucleic acid molecule.

91. The method of any one of claims 82 to 90, wherein the microbial organism is E.coli, Vibrio nalriegens, Burkholderia spp., Corynebacterium gliilamicum. Sphingomonas subterranean, Pseudomonas fluorescens, Pseudomonas putida, Saccharomyces cerevisiae, Pichia pasloris, Rhodococcus jostii, Saccharopolyspora erylhraea, Streptomyces lividans, Streptomyces coelicolor, Streptomyces albus, or Streptomyces venezuelae .

92. The method of any one of claims 82 to 91, wherein the culturing is performed under aerobic and / or glucose-limiting conditions.

93. The method of any one of claims 82 to 92, wherein the method further comprises isolating the engineered lasso peptide from the culture medium of the microbial organism.

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