Synthetic bi-functional degraders of integrins

Abstract

Compositions and methods are provided for selective degradation of integrin, which finds use, for example, in the treatment of fibrosis. Compounds of interest as bifunctional integrin-degrading molecules comprising a TG2 binding moiety linked to an integrin binding moiety.

Description

[0001] SYNTHETIC BI-FUNCTIONAL DEGRADERS OF INTEGRINS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] Pursuant to 35 U.S.C. § 119 (e), this application claims priority to the filing date of United States Provisional Patent Application Serial No.63 / 525,911 filed July 10, 2023, the disclosure of which application is herein incorporated by reference. BACKGROUND

[0002] Integrins, the principal extracellular matrix receptors, are implicated in diverse fibrotic diseases such as renal fibrosis, cardiac fibrosis, hepatic fibrosis, pulmonary fibrosis, cystic fibrosis, and scleroderma fibrosis. There are over 20+ αV integrin inhibitors in preclinical studies and active clinical trials, targeting pulmonary, hepatic, and renal fibrotic diseases, as well as eye diseases. Although no αV integrin inhibitor has been approved by the FDA, several drugs have progressed to phase II clinical trials.

[0003] Transglutaminase 2 (TG2) has been shown to interact with αVβ1, αVβ3, and αVβ5 integrin. Additionally, its enzymatic activity is upregulated in pathological conditions such as renal and pulmonary fibrosis. Three of the five αV integrins (αVβ1, αVβ3, and αVβ5) are expressed in renal and lung fibroblasts, a key cell type that produces extracellular matrix fibers in renal and pulmonary fibrosis. Although αVβ6 was initially thought to be critical in renal and pulmonary fibrosis, it was later found that αVβ6 is expressed in epithelial cells but not in fibroblasts. Independent studies using human primary lung and kidney fibroblasts and rat primary kidney fibroblasts showed that αVβ5 and αVβ3 are expressed at high levels in fibroblasts and are also upregulated upon induction of fibrosis. Overexpression of αV integrins, including αVβ3 and αVβ5, in human fibroblasts promotes latent TGFβ1 activation, a key player in fibrotic progression. Importantly, TG2 activation also promotes latent TGFβ1 activation and has been identified as a promising drug target for diseases such as renal and pulmonary fibrosis.

[0004] Fibrosis and other conditions associated with integrin dysregulation remain a significant medical challenge. The development of therapies is of interest and is addressed herein. SUMMARY

[0005] Bifunctional integrin-degrading molecules are disclosed, comprising a TG2 binding moiety linked to an integrin binding moiety. The integrin binding moiety binds to an integrin of interest, and the TG2 binding moiety is a substrate or inhibitor of TG2. Selective TG2 binding induces receptor mediated endocytosis of the bifunctional molecule along with both the bound TG2 and integrin (FIG.1). This endocytosis occurs via the cell-surface receptor LRP-1. Once inside the endo-lysosomal compartment, the bound TG2 and integrin are released and degraded. These bifunctional molecules may be referred to as LYTACs (lysosomal targeting chimeras).

[0006] In some embodiments the integrin binding moiety selectively binds to an integrin of interest. In some embodiments the integrin binding moiety is selective for a class of integrins. In some embodiments the integrin binding moiety is selective for one or more αV-containing integrins, e.g. αVβ1, αVβ3, αVβ5, αVβ6, αVβ8. Integrins of interest include human integrin proteins. The integrin binding moiety may comprise an RGD motif or RGD mimetics. The integrin binding moiety may comprise an antibody, nanobody, aptamer, etc. or a binding domain therefrom; peptides; cyclic peptides; synthetic molecules; etc. as known in the art.

[0007] In an embodiment, the TG2 binding moiety comprises or consists of a polypeptide comprising the pentapeptide sequence: Pro-X-Z-R I, where: X is selected from amino acids that engage TG2 through the formation of a covalent enzyme-compound intermediate, including without limitation glutamine, α-diazoketones, α- halo-ketones, αβ-unsaturated carbonyl compounds, and αβ-unsaturated sulfones; Z is selected from ptide YP, where P is proline and Y is any amino acid, including unnatural amino acids with easily appended functional groups, including without limitation primary / secondary amines, alcohols, and carboxylic acids; R is a natural or non-natural aromatic amino acid, e.g. tyrosine (Y), phenylalanine (F), and tryptophan (W), naphthylalanine, etc.; and the C-terminus of the pentapeptide can be a carboxylic acid, an ester, or an amide.

[0008] In some embodiments the TG2 binding moiety comprises or consists of the peptide sequence Ac-PQLPF-NH2; or the modified peptide where the reactive glutamine residue is replaced by an electrophilic warhead. In situations where the TG2 binding moiety is an enzyme inhibitor, TG2 inhibition itself can be clinically useful because TG2 is a drug target for renal and pulmonary fibrosis.

[0009] The TG2 binding moiety of formula I is linked to the integrin binding moiety at the C- terminus of the pentapeptide, and / or to Z through a linker, through covalent direct conjugation, through non-covalent high affinity pairing, and the like.

[0010] In an embodiment, the bifunctional molecule has a structure selected from Formulas II, III and IV, as shown below:

[0002] III; or R is an aromatic amino acid, e.g. phenylalanine, tyrosine, tryptophan, etc.; Link is a linker, e.g. small organic linker such as PEGn (n=1-10), PEGn diamine, piperazine, 4-aminopiperidine, 3,9-diazaspiro[5.5]undecane, linkers amenable to click chemistry, hydrocarbon, etc., or a peptide linker such as GGGGS, Gn, (GGS)n, etc.; and Integrin ligand is an integrin binding moiety as disclosed above.

[0011] In certain specific embodiments, a bifunctional integrin-degrading molecule has the structure:

[0003]

[0012] In certain specific embodiments, a bifunctional integrin-degrading molecule has the structure: H2N where n=0-3; T is the TG2 binding moiety as specified by any of Formulas I-IV disclosed above, and is coupled to the integrin binding moiety through an amide linkage.

[0013] In certain specific embodiments, a bifunctional integrin-degrading molecule has the structure: TH where T is the TG2 binding moiety as specified by any of Formulas I-IV disclosed above and is coupled to the integrin binding moiety through an amide linkage.

[0014] In certain specific embodiments, a bifunctional integrin-degrading molecule has the structure: where X = O, NH or CH2; T is the TG2 binding moiety as specified by any of Formulas I-IV disclosed above and is coupled to the integrin binding moiety through an amide linkage.

[0015] In certain specific embodiments, a bifunctional degrader is comprised of an αVβ5- specific integrin binding moiety with the structure: T1 where T1is H or a TG2 binding group, and T2is H or a TG2 binding group as specified by any of Formulas I-IV disclosed above, with the caveat that one of T1 and T2 are a TG2 binding group, usually where only one of T1and T2is a TG2 binding group.

[0016] In some embodiments a therapeutic formulation is provided, comprising a bifunctional integrin-degrading molecule of the disclosure, and a pharmaceutically acceptable excipient. The bifunctional integrin-degrading molecule may selectively bind to and degrade an αV integrin. The αV integrin may be αVβ5 integrin. The formulation may be a unit dose formulation. Alternatively, a unit dose formulation may comprise lyophilized bifunctional integrin-degrading molecule.

[0017] In some embodiments, methods are provided for treatment of fibrosis, the method comprising administering an effective dose of a bifunctional integrin-degrading molecule of the disclosure to an individual in need thereof. The individual may be monitored during treatment to determine that the treatment is effective in reducing or preventing fibrosis, compared to an untreated individual.

[0018] In some embodiments the fibrosis is renal fibrosis. When the fibrosis is renal fibrosis, it may be caused by hypertension, diabetes, obstruction, or infection. In some embodiments the fibrosis is a pulmonary fibrosis, such as idiopathic pulmonary fibrosis, and the like. In some embodiments the fibrosis is an intestinal fibrosis, which is a common complication of inflammatory bowel disease. In some embodiments, the fibrosis is selected from skin fibrosis, primary sclerosing cholangitis, liver cirrhosis, non-alcoholic steatohepatitis (NASH), and non- alcoholic fatty liver disease (NAFLD). In some embodiments the fibrosis is associated with cancer and tumor growth, i.e. tumor related tissue fibrosis, including without limitation lung cancer, liver cancer, skin cancer, sarcoma, osteosarcoma, etc. In other embodiments the fibrosis is associated with chronic inflammation or injury.

[0019] Compositions and kits for practicing the methods of the disclosure are also provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

[0021] FIG.1: Schematic of integrin degradation.

[0022] FIGS.2A-2E: Compound 100 degrades cell surface integrin αvβ5. (A) Dose response to 100. Cell surface integrin decreases with treatments up to about 1 μM; as is common for bifunctional molecules, higher concentrations lead to less efficient endocytosis. (B) Quantification of (A). (C) Time dependence of treatment with 100 (D) Treatment with the integrin ligand, RGDVF, alone does not lead to a corresponding decrease in surface integrin levels (10 μΜ RGDVF, 6 h treatment). (E) RAP treatment rescues some of the membrane integrin levels (10 μΜ 100, 6h treatment).

[0023] FIGS.3A-3B. Compound 101 degrades cell surface integrin αvβ5. (A) Dose response to 101.Cell surface integrin decreases with treatments up to about1 μM;as is common for bifunctional molecules, higher concentrations lead to less efficient endocytosis. (B) Quantification of (A). Note: 0 μM condition is the same for data shown for 100 and 101.

[0024] FIG. 4A-4C. Anti-fibrotic effect of compound 101. (A) Treatment of human lung fibroblasts (IMR90 cells) with 0, 1, 5, and 20 ng / mL of TGFβ1 for two days induced expression of αSMA, which is used as a molecular marker for a fibroblast-to-myofibroblast transition. (B) Treatment of IMR90 cells with 20 ng / mL of TGFβ1 in the presence of 0, 1, 3, 10, or 30 µM of 101 showed a significant decrease in αSMA expression with a maximal effect at 3 µM 101. Fold change represents normalized αSMA intensity relative to the average value in the absence of 101. (C) The integrin ligand alone, RGDVF, did not affect αSMA expression at any tested concentration.

[0025] FIG.5A-5C. Effect of compound 101 on macrophages derived from the RAW264.7 cell line. (A) Expression of integrin β5 is increased when M0 macrophages are differentiated into M2 macrophages in the presence of 20 ng / mL IL-4. Treatment with 1, 3, and 10 µM of 101 led to degradation of integrin β5 as assessed by Western blot. RGDVF alone did not have an equivalent effect.101 treatment also reduced both (B) the total released TGFβ and (C) the active form of TGFβ produced by these macrophages, whereas RGDVF did not show the corresponding effect.

[0026] FIG.6. Effect of 103 on primary human lung fibroblast cells. Expression of integrin β5 by these cells is increased in the presence of 20 ng / mL of TGFβ1 for 2 days. Treatment with 103 for 24 h induced a dose-dependent changes of integrin β5. Degradation of integrin β5 reaches a maximum at 1 µM 103 concentration, approximately to the pre-TGFβ level. At higher concentrations, integrin β5 increases, presumably due to the well-documented hook effect associated with bifunctional molecules.

[0027] FIG. 7. Long-lasting effect of integrin degradation by 103. Time dependence of degradation was assessed by adding 103 to cultures of primary human lung fibroblasts for 1, 2, 4, 8, and 16 h, followed by compound washout and cellular analysis after 16 h. As little as 1-2 h of 103 treatment was sufficient to maintain reduced integrin levels for at least 16 h.

[0028] FIG.8. Effect of 105 on αvβ5 integrin degradation in primary human lung fibroblast cells. Cells were treated with 20 ng / mL of TGFβ1 for 2 days to induce fibroblast-to-myofibroblast transition. Subsequent treatment with compound 5 for 24 h induced degradation of integrin β5. Degradation of integrin β5 reaches a maximum in the presence of 3 µM compound. At higher degrader concentrations of 10 and 100 µM, the β5 degradation effect diminishes. This is due to the well- documented hook effect associated with bifunctional molecules. The experimental details are analogous to those shown in FIG.6. DETAILED DESCRIPTION OF THE EMBODIMENTS Definitions

[0029] It is to be understood that this invention is not limited to the particular methodology, products, apparatus and factors described, as such methods, apparatus and formulations may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by appended claims.

[0030] It must be noted that as used herein and in the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a drug candidate" refers to one or mixtures of such candidates, and reference to "the method" includes reference to equivalent steps and methods known to those skilled in the art, and so forth.

[0031] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing devices, formulations and methodologies which are described in the publication and which might be used in connection with the presently described invention.

[0032] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0033] In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention.

[0034] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the peptide" includes reference to one or more peptides and equivalents thereof, e.g. polypeptides, known to those skilled in the art, and so forth.

[0035] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0036] The terms “individual,” “host,” “subject,” and “patient” are used interchangeably herein, and refer to an animal, including, but not limited to, human and non-human primates, including simians and humans; rodents, including rats and mice; bovines; equines; ovines; felines; canines; and the like. "Mammal" means a member or members of any mammalian species, and includes, by way of example, canines; felines; equines; bovines; ovines; rodentia, etc. and primates, e.g., non-human primates, and humans. Non-human animal models, e.g., mammals, e.g. non-human primates, murines, lagomorpha, etc. may be used for experimental investigations.

[0037] As used herein, the terms “determining,” “measuring,” “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitative determinations.

[0038] The term “diagnosis” is used herein to refer to the identification of a molecular or pathological state, disease or condition in a subject, individual, or patient.

[0039] The term “prognosis” is used herein to refer to the prediction of the likelihood of death or disease progression, including recurrence, spread, and drug resistance, in a subject, individual, or patient. The term “prediction” is used herein to refer to the act of foretelling or estimating, based on observation, experience, or scientific reasoning, the likelihood of a subject, individual, or patient experiencing a particular event or clinical outcome. In one example, a physician may attempt to predict the likelihood that a patient will survive.

[0040] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a mammal being assessed for treatment and / or being treated. In an embodiment, the mammal is a human. The terms "subject," "individual," and "patient" thus encompass individuals having fibrosis, including without limitation, tumor fibrosis, cardiac fibrosis, liver fibrosis, kidney fibrosis, pulmonary fibrosis, dermal scarring and keloids, Alzheimer's disease, etc. Subjects may be human, but also include other mammals, particularly those mammals useful as laboratory models for human disease, e.g. mouse, rat, etc.

[0041] Integrins are a family of cell surface receptors that play a crucial role in mediating cell adhesion to the extracellular matrix. In mammals, integrins have 18 different α subunits and 8 different β subunits, which form 24 distinct αβ heterodimers. The five member αV family of integrins (αVβ1, αVβ3, αVβ5, αVβ6, αVβ8) plays a crucial role in the progression of fibrosis. For example, genetic deletion of αV integrins or pan-αV blockage using small molecule drugs drastically attenuate fibrosis in multiple organs including the liver, lung, and kidney.

[0042] One important mechanism by which integrins interact with their ligands involves the recognition of a specific amino acid sequence known as the Arg-Gly-Asp (RGD) motif. The RGD motif, commonly found in various extracellular matrix proteins such as fibronectin and vitronectin, acts as a binding site for integrins, particularly the αvβ1, αvβ3, αvβ5 and α5β1 integrin subtypes. When an integrin receptor encounters an RGD motif, it undergoes conformational changes that enable it to tightly bind to the motif, resulting in cell adhesion and initiation of signaling cascades that regulate various cellular processes such as migration, proliferation, and survival.

[0043] Adhesion proteins that contain the RGD sequence include fibronectin, vitronectin, osteopontin, fibrinogen, von Willebrand factor, thrombospondin, laminin, entactin, tenascin, milk fat globule-epidermal growth factor 8 (MFEG8), latency-associated peptide of TGFβ1 / 3 (LAP-TGFβ1 / 3), and bone sialoprotein. The RGD sequence displays specificity to about half of the 20 known integrins including the α5β1, α8β1, αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, and αiiibβ3 integrins, and, to a lesser extent, the α2β1, α3β1, α4β1, and α7β1 integrins.

[0044] The compounds of the disclosure comprise an integrin binding moiety. The moiety may comprise, for example, a peptide linked to either or both ends of an RGD sequence, for example of the structure RGDXX, XXRGDXX, etc., where X is any amino acid. The identity of the residues “X” can be varied in that, together, the X residues flanking the binding motif (RGD, RYD, etc.), provide a certain structure that will selectively recognize the ligand. Alternatively, the integrin binding moiety may comprise an antibody, nanobody, aptamer, etc. or a binding domain therefrom; cyclic peptides; synthetic molecules; etc. as known in the art.

[0045] In some embodiments the integrin binding moiety selectively binds to an integrin of interest. In some embodiments the integrin binding moiety is selective for a class of integrins. In some embodiments the integrin binding moiety is selective for one or more αV-containing integrins, e.g. αVβ1, αVβ3, αVβ5, αVβ6, αVβ8. Integrins of interest include human integrin proteins. The integrin binding moiety may comprise an RGD motif. The integrin binding moiety may comprise an antibody, nanobody, aptamer, etc. or a binding domain therefrom; peptides; cyclic peptides; synthetic molecules; etc. as known in the art.

[0046] In some embodiments the integrin binding moiety is selective for binding αVβ5. Binding moieties of interest include RGD containing peptides, for example peptides of the formula RGDX1X2 where X is any amino acid, e.g. RGDVF, RGDNF, RGDNY, etc.; small molecules, e.g. compounds reported by Lippa et al.; cyclic peptides, e.g. c(RGDfV), c(Arg-Gly-Asp-d- Phe-[NMe]Val); antibodies, nanobodies, aptamers, etc. See, for example, Yamada et al. (2022) FASEB J.36(7):e22389; Lippa et al. (2020) Eur. J. Med. Chem.208, 112719; Kapp et al. (2017) Sci Rep. 11;7:39805; Belvisi et al. (2005) Mol Cancer Ther. 4(11):1670-80, Stuiver and Smith (1995) Hybridoma 14(6):545-50, etc., each herein specifically incorporated by reference.

[0047] Examples of pan-αv integrin binding monoclonal antibodies of interest include intetumumab (O’Day et al, 2011), abituzumab (Hussain et al, 2016), Ab-31 (Zhang et al, 2021) and 17-E6 (Mitjans et al, 1998). Examples of αVβ5 integrin-selective antibodies of interest include monoclonal antibodies P1F6 (Abcam #ab177004), P5H9 (BioTechne #MAB2528) and ALULA (BD Biosciences #AB2739376).

[0048] Peptidic integrin binding moieties of interest include peptides of the formula RGDX1X2, e.g. RGDVF, RGDNF, RGDNY, etc. Other linear peptides of interest include, e.g. RWrNK (Zhang et al, 2019) and members of the disintegrin family (Oliveira et al, 2022).

[0049] Cyclic integrin binding peptides of interest include, e.g. c(RGDfV), c(Arg-Gly-Asp-d- Phe-[NMe]Val), etc.

[0050] Small molecules that bind to multiple αV-integrins of interest include, e.g. MK-0429 (Coleman et al, 2004), bexotegrast, CWHM-12 and ST1646.

[0051] Small molecules that selectively bind αVβ5 integrin include compounds described by Lippa, et al (2020).

[0052] Transglutaminase 2 (TG2) is a member of the human transglutaminase family of enzymes, which is abundantly expressed in various tissues and is found in both intra- and extra-cellular locations (Lorand and Graham, 2003). It possesses the catalytic activity of deamidating glutamine sidechains on substrate peptides or proteins or crosslinking them with biogenic small molecule or protein-bound amines. An example of a high-affinity TG2 substrate is SEQ ID NO:1, LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF, a 33-mer gluten peptide that reveals HLA-DQ2 epitopes upon TG2-catalyzed deamidation at the underlined glutamine residues. Upon forming a covalent bond with the active site Cys277 residue of human TG2, some TG2 ligands facilitate efficient receptor-mediated endocytosis in a manner that depends upon activity of low-density lipoprotein receptor-related protein 1 (LRP1) (Loppinet et al., 2023 Cell Chemical Biology 30, 55–68, herein specifically incorporated by reference). Receptor mediated endocytosis leads to transport of TG2 and its bound ligand partner(s) into the lysosome, where these substances are enzymatically degraded (FIG.1).

[0053] In an embodiment, the TG2 binding moiety comprises or consists of a polypeptide comprising the pentapeptide sequence: Pro-X-Z-R I, where X is selected from amino acids that engage TG2 through the formation of a covalent enzyme-compound intermediate, including without limitation glutamine, α-diazoketones, α- halo-ketones, αβ-unsaturated carbonyl compounds, and αβ-unsaturated sulfones; Z is selected from ptide YP, where P is proline and Y is any amino acid, including unnatural amino acids with easily appended functional groups, including without limitation primary / secondary amines, alcohols, and carboxylic acids; R is a natural or non-natural aromatic amino acid, e.g. tyrosine (Y), phenylalanine (F), and tryptophan (W), naphtylalanine, etc.; and the C-terminus of the pentapeptide can be a carboxylic acid, an ester, or an amide. In some embodiments the TG2 binding moiety comprises or consists of the peptide sequence Ac-PQLPF-NH2; or the modified peptide where the reactive glutamine residue is replaced by an electrophilic warhead.

[0054] The TG2 binding moiety of formula I is linked to the integrin binding moiety at the N- or C-terminus of the pentapeptide, and / or to Z through a linker, through covalent direct conjugation, through non-covalent high affinity pairing, and the like. In an embodiment, the bifunctional molecule has a structure selected from R is an aromatic amino acid, e.g. phenylalanine, tyrosine, tryptophan, etc.; Link is a linker, e.g. small organic linker such as PEGn, PEGndiamine, piperazine, 4- aminopiperidine, 3,9-diazaspiro[5.5]undecane, linkers amenable to click chemistry, hydrocarbon, etc., or a peptide linker such as GGGGS, Gn, (GGS)n, etc.;

[0055] Integrin ligand is an integrin binding moiety as disclosed above.

[0056] In certain specific embodiments, a bifunctional integrin-degrading molecule has the structure:

[0057] In certain specific embodiments, a bifunctional integrin-degrading molecule has the structure: H2N where n=0-3; T is the TG2 binding moiety as specified by any of Formulas I-IV disclosed above, and is coupled to the integrin binding moiety through an amide linkage.

[0058] In certain specific embodiments, a bifunctional integrin-degrading molecule has the structure: TH where T is the TG2 binding moiety as specified by any of Formulas I-IV disclosed above and is coupled to the integrin binding moiety through an amide linkage.

[0059] In certain specific embodiments, a bifunctional integrin-degrading molecule has the structure: H N where X = O, NH or CH2; T is the TG2 binding moiety as specified by any of Formulas I-IV disclosed above and is coupled to the integrin binding moiety through an amide linkage.

[0060] In certain specific embodiments, a bifunctional degrader is comprised of an αVβ5- specific integrin binding moiety with the structure: T1 where T1 is H or a TG2 binding group, and T2 is H or a TG2 binding group as specified by any of Formulas I-IV disclosed above, with the caveat that one of T1 and T2 are a TG2 binding group, usually where only one of T1and T2is a TG2 binding group.

[0061] The term “amino acid” includes both naturally occurring and synthetic amino acids and includes both the D and L form of the acids as well as the racemic form. More specifically, amino acids contain up to ten carbon atoms. They may contain an additional carboxyl group, and heteroatoms such as nitrogen and sulfur. Preferably the amino acids are α and β-amino acids. The term α-amino acid refers to amino acids in which the amino group is attached to the carbon directly attached to the carboxyl group, which is the α-carbon. The term β-amino acid refers to amino acids in which the amino group is attached to a carbon one removed from the carboxyl group, which is the β-carbon. The amino acids described here are referred to in standard IUPAC single letter nomenclature, with “X” meaning any amino acid.

[0062] The term “substantial identity” in the context of a peptide indicates that a peptide comprises a sequence with at least 70% sequence identity to a reference sequence, preferably 80%, more preferably 85%, most preferably at least 90% or at least 95% sequence identity to the reference sequence over a specified comparison window. As used herein, “percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.

[0063] The terms “specific binding,” “selective binding”, “specifically binds,” and the like, refer to non-covalent or covalent preferential binding to a molecule relative to other molecules or moieties in a solution or reaction mixture (e.g., an antibody specifically binds to a particular polypeptide or epitope relative to other available polypeptides). In some embodiments, the affinity of one molecule for another molecule to which it specifically binds is characterized by a KD(dissociation constant) of 10-5M or less (e.g., 10-6M or less, 10-7M or less, 10-8M or less, 10-9M or less, 10-10M or less, 10-11M or less, 10-12M or less, 10-13M or less, 10-14M or less, 10-15M or less, or 10-16M or less). "Affinity" refers to the strength of binding, increased binding affinity being correlated with a lower Kd.

[0064] Linker. The TG2 binding moiety and the integrin binding moiety may be separated by a linker, e.g. a polypeptide linker, or a non-peptidic linker, etc. In some embodiments the linker is a rigid linker, in other embodiments the linker is a flexible linker. In some embodiments, the linker moiety is a peptide linker. In some embodiments, a peptide linker comprises 1 to 10 amino acids. In some embodiments, the peptide linker comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids. Exemplary linkers include linear peptides having at least two amino acid residues such as Gly-Gly, Gly-Ala-Gly, Gly-Pro-Ala, Gly-Gly-Gly-Gly-Ser. Suitable linear peptides include poly glycine, polyserine, polyproline, polyalanine and oligopeptides consisting of alanyl and / or serinyl and / or prolinyl and / or glycyl amino acid residues. In one embodiment a linker comprises the amino acid sequence GSTSGSGKSSEGKG, or (GGGGS)n, where n is 1, 2, 3, 4, 5, etc.; however, many such linkers are known and used in the art and may serve this purpose. Examples of linkers used in bifunctional small molecules are provided in Cao et al. (2022). For example, small organic linkers such as PEGn, PEGn diamine, piperazine, 4-aminopiperidine, 3,9-diazaspiro[5.5]undecane, and linkers amenable to click chemistry may be used.

[0065] In some embodiments a linker is a small aliphatic or aromatic group with at least one terminal nitrogen that forms an amide bond with the carboxy terminus of the TG2 binding moiety, e.g. linkers known and used in click chemistry reactions, see Fantoni et al. (2021) Chem. Rev. 2021, 121, 12, 7122–7154, herein incorporated by reference.

[0066] A “cleavable linker” is a linker that has one or more cleavable groups that may be broken by the result of a reaction or condition. The term “cleavable group” refers to a moiety that allows for release of a component of the solid support or oligomer of the invention by cleaving a bond linking the released moiety to the remainder of the conjugate. Exemplary cleavage mechanisms of use both in preparing and using the oligomers and solid supports of the invention are enzymatically or otherwise chemically mediated.

[0067] In addition to enzymatically cleavable groups, it is within the scope of the present invention to include one or more sites that are cleaved by the action of an agent other than an enzyme. Exemplary non-enzymatic cleavage agents include, but are not limited to, acids, bases, light (e.g., nitrobenzyl derivatives, phenacyl groups, ortho-hydroxcinnamate esters, benzoin esters, piperidines, piperazines, 3-azaspiro[5.5]undecanes), and heat. Many cleavable groups are known in the art. See, for example, Jung et al., Biochem. Biophys. Acta, 761: 152-162 (1983); Joshi et al., J. Biol. Chem., 265: 14518-14525 (1990); Zarling et al., J. Immunol., 124: 913-920 (1980); Bouizar et al., Eur. J. Biochem., 155: 141-147 (1986); Park et al., J. Biol. Chem., 261: 205-210 (1986); Browning et al., J. Immunol., 143: 1859-1867 (1989). Moreover a broad range of cleavable, bifunctional (both homo- and hetero- bifunctional) spacer arms are commercially available.

[0068] An exemplary cleavable group is cleavable by a reagent, e.g. sodium hydroxide, ammonia or other amine. In various embodiments the cleavable linker is readily cleaved at room temperature or under heat. An example of linker used is a valine-citrulline dipeptide linker that is cleaved by cathepsin B in the lysosome.

[0069] Chemical groups that find use in linking binding domains include carbamate, amide (amine plus carboxylic acid), ester (alcohol plus carboxylic acid), thioether (haloalkane plus sulfhydryl; maleimide plus sulfhydryl), Schiff's base (amine plus aldehyde), urea (amine plus isocyanate), thiourea (amine plus isothiocyanate), sulfonamide (amine plus sulfonyl chloride), disulfide, hydrazone, lipids, and the like, as known in the art.

[0070] The linkage between binding domains may comprise spacers, e.g. alkyl spacers, which may be linear or branched, usually linear, and may include one or more unsaturated bonds; usually having from one to about 300 carbon atoms; more usually from about one to 25 carbon atoms; and may be from about three to 12 carbon atoms. Spacers of this type may also comprise heteroatoms or functional groups, including amines, ethers, phosphodiesters, and the like. Specific structures of interest include: (CH2CH2O)n where n is from 1 to about 12; (CH2CH2NH)n, where n is from 1 to about 12; [(CH2)n(C=O)NH(CH2)m]z, where n and m are from 1 to about 6, and z is from 1 to about 10; [(CH2)nOPO3(CH2)m]z where n and m are from 1 to about 6, and z is from 1 to about 10. Such linkers may include polyethylene glycol, which may be linear or branched. Examples of more rigidified linkers include piperidines, piperazines and 3-azaspiro[5.5]undecanes.

[0071] The compound may comprise a homo- or heterobifunctional linker having a group at one end capable of forming a stable linkage to cargo. Illustrative entities include: azidobenzoyl hydrazide, N-[4-(p-azidosalicylamino)butyl]-3'-[2'-pyridyldithio]propionamide), bis-sulfosuccinimidyl suberate, dimethyladipimidate, disuccinimidyltartrate, N-γ- maleimidobutyryloxysuccinimide ester, N-hydroxy sulfosuccinimidyl-4-azidobenzoate, N- succinimidyl [4-azidophenyl]-1,3'-dithiopropionate, N-succinimidyl [4- iodoacetyl]aminobenzoate, glutaraldehyde, NHS-PEG-MAL; succinimidyl 4-[N- maleimidomethyl]cyclohexane-1-carboxylate; 3-(2-pyridyldithio)propionic acid N- hydroxysuccinimide ester (SPDP); N, N'-(1,3-phenylene) bismaleimide; N, N'-ethylene-bis- (iodoacetamide); or 4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid N- hydroxysuccinimide ester (SMCC); m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), and succinimide 4-(p-maleimidophenyl)butyrate (SMPB), an extended chain analog of MBS. The succinimidyl group of these cross-linkers reacts with a primary amine, and the thiol- reactive maleimide forms a covalent bond with the thiol of a cysteine residue.

[0072] Other reagents useful for this purpose include: p,p'-difluoro-m,m'- dinitrodiphenylsulfone (which forms irreversible cross-linkages with amino and phenolic groups); dimethyl adipimidate (which is specific for amino groups); phenol-1,4- disulfonylchloride (which reacts principally with amino groups); hexamethylenediisocyanate or diisothiocyanate, or azophenyl-p-diisocyanate (which reacts principally with amino groups); disdiazobenzidine (which reacts primarily with tyrosine and histidine); O- benzotriazolyloxy tetramethuluronium hexafluorophosphate (HATU), dicyclohexyl carbodiimde, bromo-tris (pyrrolidino) phosphonium bromide (PyBroP); N,N-dimethylamino pyridine (DMAP); 4-pyrrolidino pyridine; N-hydroxy benzotriazole; and the like. Homobifunctional cross-linking reagents include bismaleimidohexane ("BMH").

[0073] The term “Alkyl” refers to a C1-C20alkyl that may be linear, branched, or cyclic. “Lower alkyl”, as in “lower alkyl”, or “substituted lower alkyl”, means a C1-C10 alkyl. The term “alkyl”, “lower alkyl” or “cycloalkyl” includes methyl, ethyl, isopropyl, propyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl, hexyl, cyclopropyl, cyclopropylmethyl, cyclobutyl, cyclobutylmethyl, cyclopentyl, cyclopentylmethyl, cyclohexyl, cyclohexylmethyl, C6 to C12 spirocycles, cyclopropylethyl, cyclobutylethyl, decalinyl, Bicyclo-[1.1.1]-pentyl, norboranyl, bicylo-[2.2.2]-octyl, cubyl, adamantanyl and related cage hydrocarbon moieties. In certain embodiments, the alkyl is a C1-C20 alkyl. In certain embodiments the alkyl group is poly deuterated.

[0074] A “substituted alkyl” is an alkyl which is typically mono-, di-, or tri-substituted with heterocycloalkyl, aryl, substituted aryl, heteroaryl, nitro, cyano (also referred to herein as nitrile), azido, halo, −OR, -SR, -SF5, -CHO, −COR, −C(O)OR, -C(O)-NR2, −OC(O)R, - OC(O)NR2, -OC(O)OR, --P(O)(OR)2, -OP(O)(OR)2, −NR2, -N+R3 (wherein a counterion may be present), −CONR2, −NRCOR, -NHC(O)OR, -NHC(O)NR2, -NHC(NH)NR2, SO3- , -SO2OR, -OSO2R, -SO2NR2, or -NRSO2R, where each R is, independently, hydrogen, lower alkyl, R′-substituted lower alkyl, aryl, R′-substituted aryl, heteroaryl, heteroaryl(alkyl), R′-substituted aryl(alkyl), or aryl(alkyl) and each R′ is, independently, hydroxy, halo, alkyloxy, cyano, thio, SF5, nitro, alkyl, halo- alkyl, or amino. Substituted alkyls which are substituted with one to three of the substituents selected from the group consisting of alkynyl, cyano, halo, alkyloxy, thio, nitro, amino, or hydroxy are particularly of interest.

[0075] The term “Aryl” refers to an aromatic ring having (4n+2) pi electrons that may contain 6 to 20 ring carbon atoms, and be composed of a single ring (e.g., phenyl), or two or more condensed rings, such as 2 to 3 condensed rings (e.g., naphthyl), or two or more aromatic rings, such as 2 to 3 aromatic rings, which are linked by a single bond (e.g., biphenylyl). In certain cases, the aryl is C6-C16 or C6 to C14. In certain embodiments the alkyl group has one or more hydrogen atoms replaced with deuterium.

[0076] Heteroaryl means an aromatic ring system containing (4n+2)pi electrons and comprised of 1 to 10 ring carbon atoms and 1 to 5 heteroatoms selected from O, N, S, Se, having a single ring (e.g., thiophene, pyridine, pyrazine, imidazole, oxazole, tetrazole, etc.), or two or more condensed rings, for example 2 to 3 condensed rings (e.g., indole, benzimidazole, quinolone, quinoxaline, phenothiazine, etc.), or two or more aromatic rings, such as 2 to 3 aromatic rings, which are linked by a single bond (e.g., bipyridyl). In some cases, the heteroaryl is C1-C16, and a selection of 1 to 5 heteroatoms consisting of S, Se, N, and O.

[0077] The term “heterocycloalkyl”, “heterocycle”, “heterocyclic group” or “heterocyclyl” refers to a saturated or unsaturated nonaromatic ring system containing 1 to 10 ring carbon atoms and 1 to 5 heteroatoms selected from O, N, S, Se, having a single ring (e.g., tetrahydrofuran, aziridine, azetidine, pyrrolidine, piperidine, tetrathiopyran, hexamethylene oxide, oxazepane, etc.), or two or more condensed rings, such as 2 to 3 condensed rings (e.g., indoline, tetrahydrobenzodiazapines, etc., including fused, bridged and spiro ring systems, having 3- 15 ring atoms, included 1 to 4 heteroatoms. In certain cases, the heterocycloalky is C1-C16, and a selection of 1 to 5 heteroatoms consisting of S, Se, N, and O. In fused ring systems, one or more of the rings can be cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, provided that the point of attachment is through the non-aromatic ring. In certain embodiments, the nitrogen and / or sulfur atom(s) of the heterocyclic group are optionally oxidized to provide for the N- oxide, -S(O)-, or –SO2- moieties.

[0078] Examples of heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, benzimidazole, pyrazole, benzopyrazole, tetrazole, 1,2,3-triazole, benzotriazole, 1,2,4-triazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, benzoisothiazole, phenazine, isoxazole, benzoisooxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7- tetrahydrobenzo[b]thiophene, thiazole, benzothiazole, thiazolidine, furan, benzofuran, thiophene, benzothiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to as thiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, benzotetrahydrofuranyl, and the like.

[0079] Substituted heterocycloalkyl, aryl, heteroaryl are optionally substituted with, hydrogen, 1 to 3 alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkyl(alkyl), aryl, substituted aryl, aryl(alkyl), -SO2NR5R5, -PO3H2, -NR5SO2R6or –NR5C(=O)R6, wherein R5and R6are independently, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkyl(alkyl), aryl, optionally substituted heterocycloalkyl, aryloxy, heteroaryl, heteroaryl(alkyl), or R5and R6together are -(CH2)3-6- or -(CH2)0-3X(CH2)0-3- where X= NR, O, S, SO2, substituted aryl(alkyl), halo(alkyl), SF5, NR53+, azido, cyano (also referred to herein as nitrile), -OR5, -SR5, -NR5R6, halogen, nitro, SCH3, OCF3, SO2CH3, SCF3, SO2CF3, CF3, -SO2OR5, -OSO2R5, CCl3, -C(=O)R5, -C(=O)OR5; -C(=O)NR5R6, -OC(=O)R5.

[0080] By "substituted" as in "substituted alkyl," "substituted aryl," and the like, as alluded to in some of the aforementioned definitions, is meant that in the hydrocarbyl, alkyl, aryl, or other moiety, at least one hydrogen atom bound to a carbon (or other) atom is replaced with one or more non-hydrogen substituents. Examples of such substituents include, without limitation, functional groups, and the hydrocarbyl moieties C1-C24 alkyl (including C1-C18 alkyl, further including C1-C12 alkyl, and further including C1-C6 alkyl), C2-C24 alkenyl (including C2-C18 alkenyl, further including C2-C12 alkenyl, and further including C2-C6 alkenyl), C2-C24 alkynyl (including C2-C18 alkynyl, further including C2-C12 alkynyl, and further including C2- C6 alkynyl), C5-C30 aryl (including C5-C20 aryl, and further including C5-C12 aryl), and C6- C30 aralkyl (including C6-C20 aralkyl, and further including C6-C12 aralkyl). The above- mentioned hydrocarbyl moieties may be further substituted with one or more functional groups or additional hydrocarbyl moieties such as those specifically enumerated. Unless otherwise indicated, any of the groups described herein are to be interpreted as including substituted and / or heteroatom-containing moieties, in addition to unsubstituted groups.

[0081] “Sulfonyl” refers to the group SO2-alkyl, SO2-substituted alkyl, SO2-alkenyl, SO2- substituted alkenyl, SO2-alkynyl, SO2-substituted alkynyl, SO2-cycloalkyl, SO2-substituted cylcoalkyl, SO2-cycloalkenyl, SO2-substituted cylcoalkenyl, SO2-aryl, SO2-substituted aryl, SO2-heteroaryl, SO2-substituted heteroaryl, SO2-heterocyclic, and SO2-substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. Sulfonyl includes, by way of example, methyl-SO2-, phenyl-SO2-, and 4-methylphenyl-SO2-. Sulfonimidoyl refers to S(O)(NH)-bonded as for sulfonyl defined above.

[0082] The term "water-soluble group" refers to a functional group that is well solvated in aqueous environments and that imparts improved water solubility to the compound to which it is attached. Water-soluble groups of interest include, but are not limited to, polyalcohols, straight chain or cyclic saccharides, primary, secondary, tertiary, or quaternary amines and polyamines, sulfate groups, sulfonate groups, sulfinate groups, carboxylate groups, phosphate groups, phosphonate groups, phosphinate groups, ascorbate groups, glycols, including polyethylene glycols (PEG) and modified PEGs, and polyethers. In some instances, water-soluble groups are primary, secondary, tertiary, and quaternary amines, carboxylates, phosphonates, phosphates, sulfonates, sulfates, -N(H)0-1(CH2CH2OH)1-2, - NHCH2CH2N(CH3)2-3, -NHCH2CH2SO3H, -NHCH2CH2PO3H2 and -NHCH2CH2CO2H, -- (CH2CH2O)yyCH2CH2XRyy, --(CH2CH2O)yyCH2CH2X--, --X(CH2CH2O)yyCH2CH2--, glycol, oligoethylene glycol, and polyethylene glycol, wherein yy is selected from 1 to 1000, X is selected from O, S, and NRZZ, and RZZand RYYare independently selected from H and C1-3 alkyl.

[0083] The term “carboxy isostere” refers to standard medicinal bioisosteric replacement groups for carboxylic acids, amides and ester. These include, but are not limited to: acyl cyanamide, tetrazoles, hydroxychromes, 3-hydroxy-1,2,4-triazoles, 1-hydroxy pyrazoles, 2,4- dihydroxy imidazoles, 1-hydroxy imidazole, 1-hydroxy 1,2,3-triazole, alkylsulfonyl carboxamides, hydroxy isoxazoles, 5-hydroxy 1,2,4-oxadiazoles, thiazoles, 1,2,4- oxadiazoles, 1,2,4-oxadiazolones, oxazoles, triazoles, thiazoles, others hydroxamic acids, sulfonimide, acylsulfonamide, sulfonylureas, oxadiazolone, thiazolidinediones, oxadiazole, thiadiazole, isothiazoles, difluorophenols, tetramic acids, tetronic acids, squaric acids, hydroxyquinoline-ones, hydroxyquinoline-2-ones, boronic acids and phosphoric acids.

[0084] By the term “functional groups” is meant chemical groups such as halo, hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C20 aryloxy, acyl (including C2-C24 alkylcarbonyl (-CO-alkyl) and C6-C20 arylcarbonyl (-CO-aryl)), acyloxy (- O-acyl), C2-C24 alkoxycarbonyl (-(CO)-O-alkyl), C6-C20 aryloxycarbonyl (-(CO)-O-aryl), halocarbonyl (-CO)-X where X is halo), C2-C24 alkylcarbonato (-O-(CO)-O-alkyl), C6-C20 arylcarbonato (-O-(CO)-O-aryl), carboxy (-COOH), carboxylato (-COO- ), carbamoyl (-(CO)- NH2), mono-substituted C1-C24 alkylcarbamoyl (-(CO)-NH(C1-C24 alkyl)), di-substituted alkylcarbamoyl (-(CO)-N(C1-C24 alkyl)2), mono-substituted arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH2), carbamido (-NH-(CO)-NH2), cyano (-C≡N), isocyano (-N+≡C-), cyanato (-O-C≡N), isocyanato (-O-N+≡C-), isothiocyanato (-S-C≡N), azido (-N=N+=N-), formyl (-(CO)-H), thioformyl (-(CS)-H), amino (-NH2), mono- and di-(C1-C24 alkyl)-substituted amino, mono- and di-(C5-C20 aryl)-substituted amino, C2-C24 alkylamido (-NH-(CO)-alkyl), C5-C20 arylamido (-NH-(CO)-aryl), imino (-CR=NH where R = hydrogen, C1-C24 alkyl, C5- C20 aryl, C6-C20 alkaryl, C6-C20 aralkyl, etc.), alkylimino (-CR=N(alkyl), where R = hydrogen, alkyl, aryl, alkaryl, etc.), arylimino (-CR=N(aryl), where R = hydrogen, alkyl, aryl, alkaryl, etc.), nitro (-NO2), nitroso (-NO), sulfo (-SO2-OH), sulfonato (-SO2-O-), C1-C24 alkylsulfanyl (-S-alkyl; also termed "alkylthio"), arylsulfanyl (-S-aryl; also termed "arylthio"), C1-C24 alkylsulfinyl (-(SO)-alkyl), C5-C20 arylsulfinyl (-(SO)-aryl), C1-C24 alkylsulfonyl (- SO2-alkyl), C5-C20 arylsulfonyl (-SO2-aryl), phosphono (-P(O)(OH)2), phosphonato (-P(O)(O- )2), phosphinato (-P(O)(O-)), phospho (-PO2), and phosphino (-PH2), mono- and di-(C1-C24 alkyl)-substituted phosphino, mono- and di-(C5-C20 aryl)-substituted phosphine. In addition, the aforementioned functional groups may, if a particular group permits, be further substituted with one or more additional functional groups or with one or more hydrocarbyl moieties such as those specifically enumerated above.

[0085] When the term "substituted" appears prior to a list of possible substituted groups, it is intended that the term apply to every member of that group. For example, the phrase "substituted alkyl and aryl" is to be interpreted as "substituted alkyl and substituted aryl."

[0086] In addition to the disclosure herein, the term “substituted,” when used to modify a specified group or radical, can also mean that one or more hydrogen atoms of the specified group or radical are each, independently of one another, replaced with the same or different substituent groups as defined below.

[0087] In addition to the groups disclosed with respect to the individual terms herein, substituent groups for substituting for one or more hydrogens (any two hydrogens on a single carbon can be replaced with =O, =NR70, =N-OR70, =N2or =S) on saturated carbon atoms in the specified group or radical are, unless otherwise specified, -R60, halo, =O, -OR70, -SR70, -NR80R80, trihalomethyl, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -SO2R70, -SO2O–M+, -SO2OR70, -OSO2R70, -OSO2O–M+, -OSO2OR70, -P(O)(O–)2(M+)2, -P(O)(OR70)O–M+, -P(O)(OR70) 2, -C(O)R70, -C(S)R70, -C(NR70)R70, -C(O)O–M+, -C(O)OR70, -C(S)OR70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(O)R70, -OC(S)R70, -OC(O) O-M+, -OC(O)OR70, -OC(S)OR70, -NR70C(O)R70, -NR70C(S)R70, -NR70CO2–M+, -NR70CO2R70, -NR70C(S)OR70, -NR70C(O)NR80R80, -NR70C(NR70)R70and -NR70C(NR70)NR80R80, where R60is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl, each R70is independently hydrogen or R60; each R80is independently R70or alternatively, two R80’s, taken together with the nitrogen atom to which they are bonded, form a 5-, 6- or 7-membered heterocycloalkyl which may optionally include from 1 to 4 of the same or different additional heteroatoms selected from the group consisting of O, N and S, of which N may have -H or C1-C3 alkyl substitution; and each M+is a counter ion with a net single positive charge. Each M+may independently be, for example, an alkali ion, such as K+, Na+, Li+; an ammonium ion, such as+N(R60)4; or an alkaline earth ion, such as [Ca2+]0.5, [Mg2+]0.5, or [Ba2+]0.5(“subscript 0.5 means that one of the counter ions for such divalent alkali earth ions can be an ionized form of a compound of the invention and the other a typical counter ion such as chloride, or two ionized compounds disclosed herein can serve as counter ions for such divalent alkali earth ions, or a doubly ionized compound of the invention can serve as the counter ion for such divalent alkali earth ions). As specific examples, -NR80R80is meant to include -NH2, -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, 4N- methyl-piperazin-1-yl, N-morpholinyl, -N(H)0-1(CH2CH2OH)1-2, -NHCH2CH2N(CH3)2-3, - NHCH2CH2SO3H, -NHCH2CH2PO3H2and -NHCH2CH2CO2H.

[0088] In addition to the disclosure herein, substituent groups for hydrogens on unsaturated carbon atoms in “substituted” alkene, alkyne, aryl and heteroaryl groups are, unless otherwise specified, -R60, halo, -O-M+, -OR70, -SR70, -S–M+, -NR80R80, trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -SO2R70, -SO3–M+, -SO3R70, -OSO2R70, -OSO3–M+, -OSO3R70, -PO3-2(M+)2, -P(O)(OR70)O–M+, -P(O)(OR70)2, -C(O)R70, -C(S)R70, -C(NR70)R70, -CO2–M+, -CO2R70, -C(S)OR70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(O)R70, -OC(S)R70, -OCO2–M+, -OCO2R70, -OC(S)OR70, -NR70C(O)R70, -NR70C(S)R70, -NR70CO2–M+, -NR70CO2R70, -NR70C(S)OR70, -NR70C(O)NR80R80, -NR70C(NR70)R70and -NR70C(NR70)NR80R80, where R60, R70, R80and M+are as previously defined, provided that in case of substituted alkene or alkyne, the substituents are not -O-M+, -OR70, -SR70, or -S–M+.

[0089] In addition to the groups disclosed with respect to the individual terms herein, substituent groups for hydrogens on nitrogen atoms in “substituted” heteroalkyl and cycloheteroalkyl groups are, unless otherwise specified, -R60, -O-M+, -OR70, -SR70, -S-M+, -NR80R80, trihalomethyl, -CF3, -CN, -NO, -NO2, -S(O)2R70, -S(O)2O-M+, -S(O)2OR70, -OS(O)2R70, -OS(O )2O-M+, -OS(O)2OR70, -P(O)(O-)2(M+)2, -P(O)(OR70)O-M+, -P(O)(OR70)(OR70), -C(O)R70, -C(S )R70, -C(NR70)R70, -C(O)OR70, -C(S)OR70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(O)R70, -OC (S)R70, -OC(O)OR70, -OC(S)OR70, -NR70C(O)R70, -NR70C(S)R70, -NR70C(O)OR70, -NR70C(S) OR70, -NR70C(O)NR80R80, -NR70C(NR70)R70and -NR70C(NR70)NR80R80, where R60, R70, R80and M+are as previously defined.

[0090] In some embodiments the formulas and compounds disclosed herein comprise the peptidomimetic , typically replaces a Y-Pro dipeptide moiety. Such peptidomimetics are deployed to enhance pharmacokinetic characteristics such as oral bioavailability and half-life. Synthesis of this peptidomimetic has been described by Dragovich, et al (2002). The peptidomimetic may be referred to as “HWE”, for Horner- Wadsworth-Emmons, which is the named chemical reaction used to make it.

[0091] As used herein, the term “electrophilic warhead” may refer to, without limitation, moieties such as: e

[0092] Salts include but are not limited to: Na, K, Ca, Mg, ammonium, tetraalkyl ammonium, aryl and alkyl sulfonates, phosphates, carboxylates, sulfates, Cl, Br, and guanidinium.

[0093] Unless otherwise specified, reference to an atom is meant to include isotopes of that atom. For example, reference to H is meant to include1H,2H (i.e., D) and3H (i.e., T), and reference to C is meant to include12C and all isotopes of carbon (such as13C).

[0094] In addition to the disclosure herein, in a certain embodiment, a group that is substituted has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.

[0095] Unless indicated otherwise, the nomenclature of substituents that are not explicitly defined herein are arrived at by naming the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment. For example, the substituent “heterocycloalkyl(alkyl)” refers to the group (heterocycloalkyl)-(alkyl)-.

[0096] As to any of the groups disclosed herein which contain one or more substituents, it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and / or synthetically non-feasible. In addition, the subject compounds include all stereochemical isomers arising from the substitution of these compounds.

[0097] In certain embodiments, a substituent may contribute to optical isomerism and / or stereo isomerism of a compound. Salts, solvates, hydrates, and prodrug forms of a compound are also of interest. Polymorphic, pseudo-polymorphic, amorphous and co-crystal forms of a compound are also of interest. All such forms are embraced by the present disclosure. Thus, the compounds described herein include salts, solvates, hydrates, prodrug and isomer forms thereof, including the pharmaceutically acceptable salts, solvates, hydrates, prodrugs and isomers thereof. In certain embodiments, a compound may be a metabolized into a pharmaceutically active derivative.

[0098] A "prodrug" is a derivative of a compound described herein, the pharmacologic action of which results from the conversion by chemical or metabolic processes in vivo to the active compound, including through release by cleavage of a linker. Prodrugs include compounds wherein an amino acid residue, or a polypeptide chain of two or more (e.g., two, three or four) amino acid residues is covalently joined through an amide or ester bond to a free amino, hydroxyl or carboxylic acid group of the compound. Additional types of prodrugs are also encompassed. For instance, free carboxyl groups can be derivatized as amides or alkyl esters. Prodrug esters as employed herein includes esters and carbonates formed by reacting one or more hydroxyls of compounds of the method of the invention with alkyl, alkoxy, or aryl substituted acylating agents employing procedures known to those skilled in the art to generate acetates, pivalates, methylcarbonates, benzoates and the like.

[0099] As further examples, free hydroxyl groups may be derivatized using groups including but not limited to hemisuccinates, phosphate esters, dimethylaminoacetates, and phosphoryloxymethyloxycarbonyls, as outlined in Advanced Drug Delivery Reviews, 1996, 19, 115. Carbamate prodrugs of hydroxyl and amino groups are also included, as are carbonate prodrugs, sulfonate prodrugs, sulfonate esters and sulfate esters of hydroxyl groups. Free amines can also be derivatized to amides, sulfonamides or phosphonamides. All of the stated prodrug moieties may incorporate groups including but not limited to ether, amine and carboxylic acid functionalities. Moreover, any compound that can be converted in vivo to provide the bioactive agent (e.g., a compound of formula I) is a prodrug within the scope of the invention. Various forms of prodrugs are well known in the art. A comprehensive description of prodrugs and prodrug derivatives are described in: (a) The Practice of Medicinal Chemistry, Camille G. Wermuth et al., (Academic Press, 1996); (b) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985); (c) A Textbook of Drug Design and Development, P. Krogsgaard-Larson and H. Bundgaard, eds., (Harwood Academic Publishers, 1991).

[0100] As used herein, a "therapeutically effective amount" refers to that amount of the therapeutic agent sufficient to treat or manage a disease or disorder. A therapeutically effective amount may refer to the amount of therapeutic agent sufficient to delay or minimize the onset of disease. A therapeutically effective amount may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease. Further, a therapeutically effective amount with respect to a therapeutic agent of the invention means the amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of a disease.

[0101] As used herein, the term “dosing regimen” refers to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).

[0102] The term "diagnosis" is used herein to refer to the identification of a molecular or pathological state, disease or condition, such as the identification of fibrosis. The methods of the invention may further comprise analysis of fibrosis or fibrotic activity following treatment according to the methods as claimed. Analysis of fibrosis may be made by obtaining a biological sample and examining molecular or pathological state, disease or condition, and the like.

[0103] As used herein, the terms "treatment," "treating," and the like, refer to administering an agent, or carrying out a procedure for the purposes of obtaining an effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of effecting a partial or complete cure for a disease and / or symptoms of the disease. "Treatment," as used herein, covers any treatment of fibrosis in a mammal, particularly in a human, and includes: (a) preventing the development of fibrosis; (b) inhibiting ongoing fibrosis, i.e., arresting its development; and (c) relieving fibrosis, i.e., causing regression of fibrosis.

[0104] Treating may refer to any indicia of success in the treatment or amelioration or prevention of fibrosis, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of an examination by a physician. Accordingly, the term "treating" includes the administration of the compounds or agents of the present invention to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with fibrosis. The term "therapeutic effect" refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject.

[0105] “In combination with”, "combination therapy" and "combination products" refer, in certain embodiments, to the concurrent administration to a patient of a first therapeutic (i.e., first therapeutic agent) and the compounds as used herein. When administered in combination, each component can be administered at the same time or sequentially in any order at different points in time. Thus, each component can be administered separately but sufficiently closely in time so as to provide the desired therapeutic effect. First therapeutic agents contemplated for use with the methods of the present invention include any other agent for use in the treatment of fibrosis. Examples of such therapeutic agents include but are not limited anti-fibrotic agents.

[0106] "Concomitant administration" of a known therapeutic agent with a pharmaceutical composition of the present invention means administration of the therapeutic agent and inhibitor agent at such time that both the known therapeutic agent and the composition of the present invention will have a therapeutic effect. Such concomitant administration may involve concurrent (i.e. at the same time), prior, or subsequent administration of the drug with respect to the administration of a compound of the present invention. A person of ordinary skill in the art would have no difficulty determining the appropriate timing, sequence and dosages of administration for particular drugs and compositions of the present invention. Therapeutic agents contemplated for concomitant administration according to the methods of the present invention include any other agent for use in the treatment of fibrosis.

[0107] As used herein, the term "correlates," or "correlates with," and like terms, refers to a statistical association between instances of two events, where events include numbers, data sets, and the like. For example, when the events involve numbers, a positive correlation (also referred to herein as a "direct correlation") means that as one increases, the other increases as well. A negative correlation (also referred to herein as an "inverse correlation") means that as one increases, the other decreases.

[0108] "Dosage unit" refers to physically discrete units suited as unitary dosages for the particular individual to be treated. Each unit can contain a predetermined quantity of active compound(s) calculated to produce the desired therapeutic effect(s) in association with the required pharmaceutical carrier. The specification for the dosage unit forms can be dictated by (a) the unique characteristics of the active compound(s) and the particular therapeutic effect(s) to be achieved, and (b) the limitations inherent in the art of compounding such active compound(s).

[0109] "Pharmaceutically acceptable excipient "means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous. The terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a human without the production of undesirable physiological effects to a degree that would prohibit administration of the composition.

[0110] A "therapeutically effective amount" means the amount that, when administered to a subject for treating a disease, is sufficient to effect treatment for that disease.

[0111] The phrase "determining the treatment efficacy" and variants thereof can include any methods for determining that a treatment is providing a benefit to a subject. The term "treatment efficacy" and variants thereof are generally indicated by alleviation of one or more signs or symptoms associated with the disease and can be readily determined by one skilled in the art. "Treatment efficacy" may also refer to the prevention or amelioration of signs and symptoms of toxicities typically associated with standard or non-standard treatments of a disease. Determination of treatment efficacy is usually indication and disease specific and can include any methods known or available in the art for determining that a treatment is providing a beneficial effect to a patient. For example, evidence of treatment efficacy can include but is not limited to remission of the disease or indication. Further, treatment efficacy can also include general improvements in the overall health of the subject, such as but not limited to enhancement of patient life quality, increase in predicted subject survival rate, decrease in depression or decrease in rate of recurrence of the indication (increase in remission time). (See, e.g., Physicians' Desk Reference (2010).)

[0112] Fibrosis is the formation of excess connective tissue causing stromal hardening and scar formation. Fibroblasts are connective-tissue cells of mesenchymal origin. They are stromal cells which control tissue integrity. Fibroblasts maintain ECM homeostasis through both deposition of ECM and secretion of matrix metalloproteinases (MMPs) to remodel the ECM. Fibroblasts also regulate adjacent epithelial cells directing epithelial proliferation and differentiation.

[0113] Fibroblasts are considered the main effectors of fibrosis in both normal and pathologic settings. During inflammation, fibroblasts become “activated” and are referred to as myofibroblasts which are the main collagen producers in the body. Fibroblasts associated with normal wound healing are phenotypically distinct from fibroblasts associated with cancer; fibroblasts within the TME are referred to as cancer-associated fibroblasts (CAFs) and they have a unique expression profile and function which significantly contributes to cancer-related fibrosis. In contrast to normal fibroblasts, CAFs have increased autocrine signaling ability and proliferation tendencies.

[0114] Exemplary forms of fibrosis include, but are not limited to, tumor fibrosis, cardiac fibrosis, liver fibrosis such as liver cirrhosis, renal and bladder fibrosis, pulmonary fibrosis, intestinal fibrosis related to inflammatory bowel diseases, dermal scarring and keloids, wound healing and adhesions, post-irradiation fibrosis, fibrosis related to chronic graft v host disease (GvHD), and Alzheimer's disease. In still further embodiments, cardiac fibrosis is associated with hypertension, hypertensive heart disease (HHD), myocardial infarction (Ml), cardiac scarring related to ischemia congestive heart failure, cardiomyopathy, post-myocardial infarction defects in heart function, atherosclerosis, and restenosis. Kidney fibrosis may include, but not be limited to, diabetic nephropathy, vesicoureteral reflux, tubulointerstitial renal fibrosis, glomerulonephritis or glomerular nephritis (GN), focal segmental glomerulosclerosis, membranous glomerulonephritis, or mesangiocapillary GN. Liver fibrosis may include, but not be limited to, cirrhosis, and associated conditions such as chronic viral hepatitis, non-alcoholic fatty liver disease (NAFLD), alcoholic steatohepatitis (ASH), non- alcoholic steatohepatitis (NASH), primary biliary cirrhosis (PBC), biliary cirrhosis, autoimmune hepatitis). Pulmonary fibrosis may include idiopathic pulmonary fibrosis (IPF) or cryptogenic fibrosing alveolitis, chronic fibrosing interstitial pneumonia, interstitial lung disease (ILD), and diffuse parenchymal lung disease (DPLD)), lung scarring including without limitation damage from bacterial viral or fungal infection, emphysema, chronic obstructive pulmonary disease (COPD); and chronic asthma may also be prevented, treated, or ameliorated with compositions of described herein. Also included is fibrosis of the eye and lens, for example glaucoma; age-related macular degeneration (wet AMD and dry AMD), fibrosis of the lens, periorbital fibrosis as in lgG4-related disease, hyperthyroidism, etc. Uterine fibroids are also if interest for treatment.

[0115] One example of fibrosis of the lung is idiopathic pulmonary fibrosis (IPF), the most common form of idiopathic interstitial pneumonia, causes progressive pulmonary fibrosis. Symptoms and signs develop over months to years and include exertional dyspnea, cough, and fine (Velcro) crackles. Diagnosis is based on history, physical examination, high- resolution CT, and / or lung biopsy, if necessary. Treatment may include antifibrotic drugs and oxygen therapy. Most patients deteriorate; median survival is about 3 years from diagnosis.

[0116] A variety of drugs have been tried in various fibroses, particularly pulmonary fibrosis, with very little success. Anti-inflammatory drugs including prednisolone and azathioprine have little effect on fibrosis suggesting that inflammation is only the initiator, but not the driver of the disease. The use of non-specific anti-proliferative agents like colchicine and cyclophosphamide will also prevent repair of the fibrotic tissue by impairing e.g. epithelial growth. Treatment with IFN-γ has shown some utility but is limited by severe side effects.

[0117] By the time a typical patient presents with fibrosis-related symptoms (e.g. difficulty breathing for pulmonary fibrosis, cirrhosis for liver fibrosis, etc.), the fibrosis in the target organ is often quite severe, with much of the target organ architecture having been replaced with extracellular matrix. Stopping this ongoing fibrosis can extend lifespan and improve quality of life. Areas of the target organ where the fibrosis is not extensive may be restored to normal architecture with suitable treatment.

[0118] In some embodiments, tumor fibrosis is associated with pancreatic cancer. Pancreatic cancer is characterized by a prominent desmoplastic / stromal reaction. Pancreatic stellate cells (PSCs) are the principal source of fibrosis in the stroma and interact closely with cancer cells to create a tumor facilitatory environment that stimulates local tumor growth and distant metastasis. Pancreatic fibrosis is initiated when PSCs become activated and undergo morphological and functional changes, so that the rate of extracellular matrix (ECM) deposition exceeds the rate of ECM degradation in the gland. It is now well established that pancreatic cancer cells activate PSCs leading to increased fibrosis. There is significant evidence showing that the intense stromal / desmoplastic reaction around tumor elements (a feature of the majority of pancreatic cancers) plays an important role in tumor progression. A key histopathological feature of pancreatic cancer which is associated with its innate clinical and biological aggressiveness is its desmoplastic (stromal) reaction. Stroma production is stimulated by cancer-cell derived growth factors including transforming growth factor-β (ΤGFβ), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), insulin-like growth factor 1 (IGF-1), and epidermal growth factor (EGF). The desmoplastic reaction is composed of extracellular matrix (ECM) proteins, primarily type I and III collagen, fibronectin and proteoglycans; small endothelium lined vessels; and a diverse population of cells including inflammatory cells, fibroblasts and stellate cells. The stroma can form up to 90% of the tumor volume, a property which is unique to pancreatic cancer. The tumor microenvironment in pancreatic cancer plays a role in its chemoresistance.

[0119] Chronic inflammation results in fibrosis and chronic fibrosis also predisposes to cancer initiation. In addition to cancer-induced chronic inflammation as a driver of fibrosis, cancer treatments also play an important role in creating the fibrotic tumor microenvironment (TME). Organ fibrosis, most notably pulmonary fibrosis, is a known toxicity of multiple chemotherapeutic agents including bleomycin, gemcitabine, and methotrexate. In vitro and in vivo studies demonstrate chemotherapy may promote an inflammatory and fibrotic microenvironment likely through tissue injury related to oxidative stress. Tissues exposed to chemotherapy undergo similar stages of wound healing including inflammation with influx of immune cells, followed by fibroblast activation and proliferation and remodeling which involves the accumulation and cross-linking of ECM.

[0120] Cancer develops within a complex microenvironment critical to supporting tumor survival, growth, and metastasis. This tumor microenvironment (TME) is composed of a web of vasculature, extracellular matrix (ECM), stromal cells, immune cells, and soluble signaling molecules which form a dynamic “organ” critical to the pathophysiology of cancer. Within the TME, cancer-associated fibrosis has emerged as a critical regulator of cancer behavior. Indeed, fibrosis is a hallmark of cancer. Up to 20% of cancers are linked to chronic inflammation-related fibrosis (either from infectious or autoimmune etiologies) including hepatocellular, gastric, esophageal, head and neck, colon, pancreatic, cervix, and vulvar cancers.

[0121] Fibrosis has been reported to support cancer growth through a variety of mechanisms including direct cellular interactions, immune modulation, and ECM remodeling. As a stromal progenitor cell, significantly impact the formation of the TME and are important mediators of fibrosis. Fibrosis is important in not only established tumor sites but also in the creation of a premetastatic niche. In vivo models demonstrate increased fibronectin expression in the stroma of future metastatic sites.

[0122] Fibrosis may be monitored, e.g. in diagnosis, during and subsequent to treatment to assess the efficacy of treatment. The presence of fibrosis can be detected by means known in the art, for example by examination of tissue for excess scarring. Prior to fibrosis, an individual may be determined to be susceptible based on undesirable increase in inflammatory mediators that can exacerbate tissue injury, such as IL-1, TNF-α and reactive oxygen and nitrogen species. Profibrotic mediators such as TGF-β1 may be present. Also present are activated myofibroblasts, which may be resistant to induction of apoptosis. Methods of monitoring fibrosis may include, for example, chest X-ray that image the scar tissue typical of pulmonary fibrosis, and it may be useful for monitoring the course of the illness and treatment. Computerized tomography (CT) scan to combine X-ray images taken from many different angles to produce cross-sectional images of internal structures in the body. A high-resolution CT scan can be particularly helpful in determining the extent of lung damage caused by pulmonary fibrosis. An echocardiogram uses sound waves to visualize the heart. Functional tests can be useful, e.g. with pulmonary fibrosis, including pulmonary function tests, e.g. spirometry, pulse oximetry, exercise stress test, arterial blood gas test, etc. Methods of Treatment

[0123] Individuals diagnosed or at risk of developing a fibrotic disease are treated by administering a bifunctional molecule of the disclosure, in a dose effective to degrade integrin and reduce or prevent fibrosis. In some embodiments the fibrosis is renal fibrosis. When the fibrosis is kidney fibrosis, the kidney fibrosis may be caused by hypertension, diabetes, obstruction, or infection. In some embodiments the fibrosis is pulmonary fibrosis, such as idiopathic pulmonary fibrosis. In some embodiments, the fibrosis is skin fibrosis, kidney fibrosis, intestinal fibrosis, liver fibrosis such as liver cirrhosis, non-alcoholic steatohepatitis (NASH), and non-alcoholic fatty liver disease (NAFLD). In some embodiments the fibrosis is associated with cancer and tumor growth, i.e. tumor related tissue fibrosis, including without limitation lung cancer, skin cancer, sarcoma, etc. In other embodiments the fibrosis is associated with chronic inflammation or injury such as irradiation in tissues, including without limitation fibrosis of liver, lung, kidney, uterus, the eye and the lens, lgG4-related disease, chronic GvHD, and the like.

[0124] Methods include administering to a subject in need of treatment a therapeutically effective amount or an effective dose of a bifunctional molecule of the disclosure. In some embodiments, effective doses of the therapeutic entity of the present invention described herein vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the patient is a human but nonhuman mammals including transgenic mammals can also be treated. Treatment dosages need to be titrated to optimize safety and efficacy.

[0125] In some embodiments reduction in fibrosis is monitored during or subsequent to treatment. Monitoring may include, without limitation, detecting a decrease in fibrotic cells, decrease in fatty infiltrating cells, etc.

[0126] The effective dose of a bifunctional molecule of the disclosure can vary with the agent, but will generally range from about 0.01 mg / kg, from about 0.05 mg / kg, from about 0.1 mg / kg, up to about 50 mg / kg, up to about 40 mg / kg, up to about 30 mg / kg, up to about 20 mg / kg, up to about 10 mg / kg, up to about 5 mg / kg; up to about 1 mg / kg, up to about 0.5 mg / kg; where the dose may vary with the specific agent and recipient.

[0127] The agent may be administered one or a plurality of days, and in some embodiments is administered daily, every two days, semi-weekly, weekly, etc. for a period of from about 1, about 2, about 3, about 4, about 5, about 6, about 7 or more weeks, up to a chronic maintenance level of dosing.

[0128] In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patent can be administered a prophylactic regime.

[0129] In still yet some other embodiments, for prophylactic applications, pharmaceutical compositions or medicaments are administered to a patient susceptible to, or otherwise at risk of a disease or condition in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the outset of the disease, including biochemical, histologic and / or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease.

[0130] In other embodiments, for therapeutic applications, therapeutic entities of the present invention are administered to a patient suspected of, or already suffering from such a disease in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease (biochemical, histologic and / or behavioral), including its complications and intermediate pathological phenotypes in development of the disease. An amount adequate to accomplish therapeutic or prophylactic treatment is defined as a therapeutically- or prophylactically- effective dose. In both prophylactic and therapeutic regimes, agents are usually administered in several dosages until a sufficient response has been achieved.

[0131] According to the present invention, compositions can be administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal or intramuscular means. The most typical route of administration is intravenous although other routes can be equally effective.

[0132] For parenteral administration, compositions of the invention can be administered as injectable dosages of a solution or suspension of the substance in a physiologically acceptable diluent with a pharmaceutical carrier that can be a sterile liquid such as water, oils, saline, glycerol, or ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, surfactants, pH buffering substances and the like can be present in compositions. Other components of pharmaceutical compositions are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, and mineral oil. In general, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions. Antibodies and / or polypeptides can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained release of the active ingredient. An exemplary composition comprises a polypeptide at 1 mg / mL, formulated in aqueous buffer consisting of 10 mM Tris, 210 mM sucrose, 51 mM L-arginine, 0.01 % polysorbate 20, adjusted to pH 7.4 with HCI or NaOH.

[0133] Typically, compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997. The agents of this invention can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient.

[0134] Additional formulations suitable for other modes of administration include oral, intranasal, and pulmonary formulations, suppositories, subcutaneous and transdermal applications.

[0135] For suppositories, binders and carriers include, for example, polyalkylene glycols or triglycerides; such suppositories can be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1 %-2%. Oral formulations include excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%-95% of active ingredient, preferably 25%-70%.

[0136] Topical application can result in transdermal or intradermal delivery. Topical administration can be facilitated by co-administration of the agent with cholera toxin or detoxified derivatives or subunits thereof or other similar bacterial toxins. Glenn et al., Nature 391:851, 1998. Co-administration can be achieved by using the components as a mixture or as linked molecules obtained by chemical crosslinking or expression as a fusion protein.

[0137] In some embodiments the compositions are delivered subcutaneously. Subcutaneous (SC) injection is currently the most common route of self-administering biopharmaceuticals such as proteins and peptides. In addition to a simple injection, the formulation may comprise additional agents to enhance SC delivery. Chemical penetration enhancers that can disrupt the skin barrier and provide an adding driving force for transporting therapeutics. Chemical enhancers can insert into the highly ordered lipid bilayer in stratum corneum to disorganize molecular packing or extract lipids to create lipid-packing defects of nanometer dimensions, thus leading higher transport efficiency. Nano / micro vesicles, such as liposomes and nano / microemulsions, are useful as chemical enhancers. They can not only improve skin permeability but also act as the vehicles for drug solubilization and drug transport through the skin.

[0138] In addition to the chemical penetration enhancers, electrical instruments that facilitate transport through the skin can be used to enhance delivery efficiency through the skin by providing additional driving force via electrical interactions or introducing transient perturbation of the stratum corneum via high-voltage electrical pulse. Electroporation has evolved as another technique for electrically assisted transdermal drug delivery. The procedure of electroporation involves using short, high-voltage pulses to induce transient perturbation in the stratum corneum by creating micro-pathways across its lipid bilayers.

[0139] Besides electrical field, mechanical force is another alternative to produce transient channels on the surface of the skin for transdermal drug delivery. Ultrasound and jet injection are two representative mechanical force-triggered methods for drug delivery. Ultrasound can enhance the permeability of drugs across the skin by hyperthermia or cavitation effect. Jet injection applies the high-speed liquid to disrupt the surface of the skin to dispense insulin solution within the skin tissue.

[0140] Microneedle (MN) techniques have provided an alternative method for transdermal protein delivery. The micro-scaled needles are able to painlessly disrupt the stratum corneum and reach the epidermal and dermal layer for drug release. Solid MNs are designed to pierce the skin to improve the drug transport; hollow MNs are used for injection of a fluid drug formulation through the opening on the skin caused by needles, and dissolving or degradable MNs are made from polymers with encapsulated drugs. Dissolving polymers include, for example, hyaluronic acid (HA), carboxymethylcellulose (CMC), chitosan, alginate, polyvinylpyrrolidone (PVP), and polyvinyl alcohol (PVA).

[0141] Alternatively, transdermal delivery can be achieved using a skin patch or using transferosomes. Paul et al., Eur. J. Immunol. 25: 3521-24, 1995; Cevc et al., Biochem. Biophys. Acta 1368: 201-15, 1998. The pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration. Preferably, a therapeutically effective dose will provide therapeutic benefit without causing substantial toxicity.

[0142] In some embodiments, the compounds disclosed herein are formulated for sustained release, e.g. a transdermal patch or implant. Subcutaneous, sustained release formulations include, for example, polymer-based micro / nanocarriers. Colloidal micro / nanoparticle systems with large surface areas significantly enhance epithelial permeability and improve the bioavailability of protein and peptide drugs. Various biodegradable polymers approved by the FDA are commercially available for drug delivery in micro / nanoparticle formulations. By modifying the polymer structure, these polymeric carriers, such as Poly (lactic-co-glycolic acid) (PLGA), Poly (lactic acid) (PLA) or Chitosan (CS), can realize structural stability and sustained release of the protein and peptide drugs. Other polymer-based carriers include poly (hydroxybutyrate-co-hydroxyhexanoate (PHBHHx). Another biodegradable, biocompatible polymeric carrier is microspheres of p(CPP:SA). After the water-soluble degradation products formed, the anhydride bonds of this CPP:SA copolymer are hydrolyzed.

[0143] In situ gelling systems can be used as drug delivery systems, for example temperature- sensitive hydrogels are aqueous polymer solutions at room temperature and transform to a sol-gel form at physiological temperature, which may be comprised of PLGA and varied ratios of benzyl alcohol (BA) and benzyl benzoate (BB). Examples thermogelling triblock copolymers, such as PLGA–PEG–PLGA triblock copolymer, poly (ε-caprolactone-co-glycolic acid)-poly (ethylene glycol)-poly (ε-caprolactone-co-glycolic acid) (PCGA-PEG-PCGA) and PLGA-PEG-PLGA. Poly (ethylene oxide)–poly (propylene oxide)–poly (ethylene oxide) (PEO–PPO–PEO), also named Pluronic, is a nonionic amphiphilic triblock copolymer that made by a propylene glycol initiator and the sequential addition of propylene and ethylene oxides. The PF-127 gel can be used as a controlled delivery system for peptides. Poly (ethylene glycol)-block-poly (alanine-co-phenyl alanine) (PEG-PAF) is another applicable material.

[0144] Liposomes, small lipid spheres that are composed of concentric lipid bilayers surrounding aqueous compartments, are applicable as sustained delivery systems. Multivesicular liposomes (MLVs) consist of nonconcentric multiple lipid layers. As the diameter of MLVs is generally in the range of tens of microns, a high amount of drugs can be loaded to maintain sustained drug release. Vesicular phospholipid gels (VPGs) are semisolid liposomal dispersions containing aqueous compartments in the core of the vesicles or between the vesicles; thus, they can be used to carry and sustain the release of hydrophilic, amphiphilic or lipophilic drugs. Another kind of lipid gel is the the phospholipid-based phase separation gel (PPSG), which convert from the sol state to the solid or semisolid state after subcutaneous injection.

[0145] Inorganic micro / nanoparticles such as mesoporous silicon (PSi) and silica, are applicable as long-lasting drug delivery systems.

[0146] Toxicity of the agents described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic for use in human. The dosage of the proteins described herein lies preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch.1 ).

[0147] Also within the scope of the invention are kits comprising the compositions of the invention and instructions for use. The kit can further contain a least one additional reagent. Kits typically include a label indicating the intended use of the contents of the kit. The term label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit.

[0148] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. It is also understood that the terminology used herein is for the purposes of describing particular embodiments

[0149] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or only and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the appended claims. Experimental Example 1 Targeted lysosomal degradation of αV integrins through the TG2 / LRP-1 pathway

[0150] LYTACs (lysosomal targeting chimeras) are related bifunctional conjugates that bridge endocytic receptors (e.g., cation-independent mannose-6-phosphate receptor, asialoglycoprotein receptor) to a target extracellular or cell-surface protein of interest, thereby enabling internalization and lysosomal degradation of the target protein. The discovery of LYTACs has spawned an intense quest for new receptor-mediated endocytic mechanisms that can be hijacked for lysosomal delivery of pathogenically relevant molecules in the extracellular matrix or on the plasma membranes of malfunctioning cells.

[0151] We recently characterized (Loppinet et al., 2023; FIG.1) a novel receptor-mediated endocytic pathway that enables gluten peptides, which are primary T cell antigens in celiac disease (CeD) pathogenesis, to be simultaneously deamidated and concentrated in the endo- lysosomal system of antigen presenting cells. This pathway depends on both LRP-1, a cell surface receptor involved receptor-mediated endocytosis, as well as an extracellular enzyme, transglutaminase 2 (TG2).

[0152] Given the extraordinary ability of catalytically active extracellular TG2 to deliver low- abundance substrates into the lysosomes of cells expressing the LRP-1 receptor, we sought to hijack the TG2 / LRP-1 pathway for targeted lysosomal delivery and subsequent degradation of molecules on the cell surface or in the extracellular matrix. This example describes the design, synthesis, and characterization of bifunctional molecules harboring a ligand of a target protein family of interest, integrins of the αV subfamily, and a high affinity TG2 substrate or inhibitor.

[0153] Integrins are a family of over 20 heterodimeric transmembrane proteins each comprised of an α and a β subunit. Dysregulated integrin activity has been shown to be important in many cancers, inflammatory diseases, and fibrotic conditions. Accordingly, integrin targeting drugs have been approved for a variety of indications including dry eye disease (lifitegrast), psoriasis (efalizumab), inflammatory bowel disease (vedolizumab), and cardiovascular thrombosis (abciximab, eptifibatide) with many others under development. Importantly, all approved integrin-targeting small molecule and antibody drugs are receptor antagonists that passively block integrin signaling. There remains a need to explore the therapeutic relevance of irreversibly eliminating a target integrin from the cell surface via protein degradation.

[0154] We sought to engineer a prototypical agent for targeted degradation of αvβ5 integrin, a representative member of the αV subfamily, which is inhibited by the pentapeptide sequence RGDVF. To this end we synthesized compound 100 in which Ac-PQLPF-NH2 is linked to RGDVF via a GGGGS linker. TG2 has similar specificity for 100 compared to Ac- PQLPF-NH2(kcat / KM= 0.16 ± 0.04 μM-1min-1). Treatment of NRK cells with increasing concentrations of the 100 in the presence of 100 μg / mL α2-macroglobulin markedly decreased the presence of αVβ5 on the cell surface in a dose dependent manner. Interestingly, a hook effect is observed, with maximal degradation at 1 μΜ HB320 and cell surface integrin increasing after this point (FIG.2A, B). Degradation is also time dependent (FIG. 2C) reaching maximum degradation after 6 h treatment. Finally, the integrin ligand alone, RGDVF, did not lead to a corresponding decrease in surface αVβ5 (FIG.2D). Further confirming the involvement of the LRP-1 pathway, pre-treating the cells with RAP, a known LRP-1 binder, rescues most of the surface αVβ5 (FIG.2E). We also synthesized 101, which harbors HWE in the position of the reactive glutamine residue. Treatment with 101 leads to similar results as 100 (FIG.3).

[0155] To demonstrate anti-fibrotic activity of 101, human lung fibroblasts (IMR90 cells) were treated with 0, 1, 5, and 20 ng / mL of TGFβ1 for two days to induce a fibroblast-to- myofibroblast transition. For this, IMR-90 cells (ATCC, CCL-186) were cultured following the ATCC protocol. As described by Nichols et al. (1977), IMR-90 cells are a human diploid fibroblast strain. Cells were seeded on vitronectin-coated glass bottom 24-well plates, incubated at 37°C with 5% CO₂ for 24 h, and then treated with 20 ng / mL of human transforming growth factor-beta 1 (TGFβ1) (PeproTech#100-21) for 48 h to induce alpha- smooth muscle actin (α-SMA) expression (FIG 4A). Next, cells were treated with different concentrations of 101 or control reagents for 24 h. After treatment, cells underwent immunostaining to detect α-SMA expression using established protocols. Primary antibody staining was performed using a mouse anti-ɑ-SMA antibody (Sigma-Aldrich, 1A4) at a dilution of 1:500 to 1:1000 in blocking buffer, with an overnight incubation at 4°C. After washing three times by PBS, cells were stained with anti-mouse secondary antibodies in blocking buffer for 30-60 minutes at RT. In the presence of 0, 1, 3, 10, or 30 µM of 101, a significant decrease in αSMA expression was observed (FIG.4B) with a maximal effect at 3 µM 101. The integrin ligand alone, RGDVF, did not affect αSMA expression at any tested concentration (FIG.4C).

[0156] Macrophages are another important class of cells that contribute to the progression of fibrosis by secreting profibrotic cytokines, including TGFβ, IL-1β, and PDGF, which promote the fibroblast-to-myofibroblast transition. To demonstrate the anti-fibrotic activity of 101 on macrophages, the RAW 264.7 cell line (ATCC, TIB-71) was used as a model. Cells were cultured as recommended (https: / / www.atcc.org / products / tib-71), following the method of Raschke et al. (1978). To induce differentiation into M2a macrophages, RAW 264.7 cells (referred to as M0 cells) were treated with 20 ng / mL interleukin-4 (IL-4) for 48 h. Following differentiation, cells were treated with either 101 or RGDVF peptides in DMEM for an additional 24 h. To monitor TGFβ1 expression, a sensitive bioassay was utilized as described by Tesseur et al. (2006). This assay involved the use of MFB-F11 cells, which are mouse embryonic fibroblasts derived from TGFβ1 deficient (Tgfb1- / -) mice. These cells were stably transfected with a reporter plasmid containing TGF-β responsive Smad-binding elements (SBE) linked to a secreted alkaline phosphatase (SEAP) reporter gene. The protocol followed involved culturing MFB-F11 cells and treating them with conditioned media from the RAW 264.7 cell treatments. The SEAP activity, indicative of TGFβ1 expression, was measured by Great EscAPe™ SEAP Chemiluminescence Kit 2.0 (Takara # 631738) to the manufacturer’s instructions.

[0157] Expression of integrin β5 is increased when M0 macrophages are differentiated into M2 macrophages through exposure to 20 ng / mL IL-4 (FIG 5A). Treatment with 1, 3, and 10 µM of 101 led to degradation of integrin β5 as assessed by Western blot (FIG 5A). RGDVF alone did not have an equivalent effect.101 treatment also reduced both the total released TGFβ and the active form of TGFβ produced by these macrophages, whereas RGDVF did not show the corresponding effect (FIG 5B-C).

[0158] Compound 102 is another useful example of a peptide integrin degrader: H2N 102NHTwhere T is AcP(HWE)LPFGGGGS-. Synthesis of 102 follows methods used to synthesize the commercial drug cilengitide, except that the Val residue of cilengitide is replaced with a suitably protected Lys residue. Upon deprotection, the primary amine of the cyclic peptide is coupled to the carboxy terminus of the TG2 binding moiety through the GGGGS linker. Example 2 Bi-functional degraders of αV integrins harboring a non-peptidic integrin binding moiety

[0159] This example describes bifunctional molecules that induce degradation of cell-surface integrins by engaging integrin through non-peptidic moieties. Such molecules have the anticipated benefit of improved DMPK properties over the peptidic compounds described in the previous example. The synthesis of compounds in this example follows standard methods described in the art, for example those found in the Reaxys database (www.reaxys.com).

[0160] The synthesis of compound 103 (which is based on the non-peptidic pan-αV ligand, MK-0429) is outlined below. In this scheme the aza-Michael reaction between the allylimidazolidinone and the methoxypyridinyl acrylate ester yields a racemic form the integrin engaging moiety, although enantioselective synthesis of the desired stereoisomer can be readily achieved by other established methods. The identity of 103 was confirmed by mass spectrometry and by verifying that human TG2 had comparable specificity for this compound as it did for Ac-PQLPF-NH2. OMe N O OOO NaHN+OP O THF O CO2tBu OMe N OMe (a) NaHMDS N O O Br O O HN N MeCOtBuN NH2N (b) NaOH, MeOH KOtBu N CO2tBuMeO MeO NNH2H2NN 9-BBN, SPhos, Pd G1, K3PO4DIPEA O O N2tCO2tNBuDMSO, 120 °C, 12 hCO BuN N FN ClF N(±)H2NNN H O MeO O N HNNH DIPEA N+ ONCO2tBuHATUO CH2Cl :DMF NO2O 2N H ONH= 2:1 N H O OH OH2 h N H2NNN ONH N N OH H O H N O H O O O MeO HNNH N N TFA O NOOO 2NCO2tBuH ONHOHCH2Cl2N H O O N H N N OHN H N N O H N N N O H O H O O MeO HNHNN N O NOOO CO2H O2 NHNHN H O OH N N H O H ONH N N N O H N N N O H O H 103

[0161] The αvβ5 degrader activity of 103 was verified in primary human lung fibroblast cells obtained from ATCC. Similar to IMR90 fibroblasts, expression of integrin β5 by these cells is increased in the presence of 20 ng / mL of TGFβ1 for 2 days. Treatment with 103 for 24 h induced a dose-dependent changes of integrin β5 (FIG. 6). Degradation of integrin β5 reaches a maximum at a 1 µM concentration of 103, approximately to the pre-TGFβ level. At higher concentrations of 103, integrin β5 increase, presumably due to the well-documented hook effect associated with bifunctional molecules. The time dependence of degradation was assessed by adding 103 to cultures of primary human lung fibroblasts for 1, 2, 4, 8, and 16 h, followed by compound washout and cellular analysis after 16 h. As little as 1-2 h of treatment with 103 was sufficient to maintain reduced integrin levels for at least 16 h (FIG. 7), emphasizing the sustained effect of an integrin degrader after washout. In contrast, integrin activity recovers rapidly after drug washout when conventional antagonists are used to block this class of receptors.

[0162] Compound 104 is another example of an integrin degrader based on the non-peptidic pan-αV ligand, MK-0429.

[0163] Compound 105, an integrin degrader based on a non-peptidic αVβ5-specific ligand (Lippa et al, 2019), was synthesized as outlined below. Its identity was confirmed by mass spectrometry and by verifying that human TG2 had comparable specificity for 105 as it did for Ac-PQLPF-NH2.

[0164] The αvβ5 degrader activity of 105 was also verified in primary human lung fibroblast cells (FIG.8). Cells were treated with 20 ng / mL of TGFβ1 for 2 days to induce fibroblast-to-myofibroblast transition. Subsequent treatment with 105 for 24 h induced degradation of integrin β5. Degradation of integrin β5 reaches a maximum in the presence of 3 µM compound.

[0165] The presence of a bromophenyl substituent in the parent non-peptidic αVβ5-specific ligand reported by Lippa et al (2019) enables an alternate strategy for attachment of the TG2 engager moiety, as shown in the bifunctional integrin degrader 106. References

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Claims

WHAT IS CLAIMED IS:

1. A bifunctional integrin-degrading molecule, comprising: a TG2 binding moiety linked to an integrin binding moiety.

2. The bifunctional molecule of claim 1, wherein the TG2 binding moiety comprises or consists of a polypeptide comprising the pentapeptide sequence Pro-X-Z-R (formula I); wherein X is selected from amino acids that engage TG2 through the formation of a covalent enzyme-compound intermediate, selected from glutamine, α-diazoketones, α-halo-ketones, αβ-unsaturated carbonyl compounds, and αβ-unsaturated sulfones; Z is selected fromptide YP, where P is proline and Y is any amino acid or unnatural amino acid; R is a natural or non-natural aromatic amino acid; and the C-terminus of the pentapeptide is a carboxylic acid, an ester, or an amide.

3. The bifunctional molecule of claim 1 or claim 2, wherein the TG2 binding moiety comprises or consists of the peptide sequence Ac-PQLPF-NH2; or a modified peptide where the reactive glutamine residue is replaced by an electrophilic warhead.

4. The bifunctional molecule of claim 1 or claim 2, having a structure selected from:III; orIVwhereinR is an aromatic amino acid; Link is a linker.

5. The bifunctional molecule of claim 4, wherein the linker is selected from PEGn, PEGndiamine, piperazine, 4-aminopiperidine, 3,9-diazaspiro[5.5]undecane, linkers amenable to click chemistry, a hydrocarbon, and a peptide linker.

6. The bifunctional molecule of claim 5, where the linker is a small aliphatic or aromatic group with at least one terminal nitrogen that forms an amide bond with the carboxy terminus of the TG2 binding moiety.

7. The bifunctional molecule of any of claims 1-2 and 4-6, of either structure below:

8. The bifunctional molecule of any of claims 1-7, wherein the integrin binding moiety selectively binds to an integrin or class of integrins of interest.

9. The bifunctional molecule of any of claims 1-7, wherein the integrin binding moiety is selective for one or more of αVβ1, αVβ3, αVβ5, αVβ6, αVβ8.

10. The bifunctional molecule of any of claims 8-9, wherein the integrin binding moiety is selective for binding human αVβ5 integrin.

11. The bifunctional molecule of any of claims 1-10, wherein the integrin binding moiety is a peptide comprising an RGD sequence.

12. The bifunctional molecule of claim 11, wherein the peptide is of the formula RGDX1X2.

13. The bifunctional molecule of claim 12, wherein the peptide has the sequence RGDVF, RGDNF, or RGDNY.

14. The bifunctional molecule of any of claims 1-10, wherein the integrin binding moiety is selected from a peptide, cyclic peptide, small molecule, antibody or antibody binding fragment, nanobody, aptamer.

15. The bifunctional molecule of any of claims 1-6, wherein the cyclic peptide has the structure: H2Nwhere T is the TG2 binding moiety, coupled to the cyclic peptide through an amide linkage; and n=0-3.

16. The bifunctional molecule of any of claims 1-6, wherein the compound has the structure: Twhere T is the TG2 binding moiety, coupled to the integrin binding moiety through an amide linkage.

17. The bifunctional molecule of any of claims 1-6, wherein the compound has the structure:where T is the TG2 binding moiety, coupled to the integrin binding moiety through an amide linkage, X is O, NH or CH2.

18. The bifunctional molecule of any of claims 1-6, wherein the compound has the structure: T1wherein T1is H or the TG2 binding moiety, and T2is H or the TG2 binding moiety as specified by any of Formulas I-IV disclosed above, with the caveat that one of T1 and T2 are a TG2 binding moiety, usually where only one of T1and T2is a TG2 binding moiety.

19. A pharmaceutical composition comprising a bifunctional molecule of any of claims 1-18, and a pharmaceutically acceptable excipient.

20. A unit dose of a pharmaceutical composition of claim 19.

21. A method for prevention or treatment of fibrosis in a mammalian patient, the method comprising: administering a bifunctional molecule of any of claims 1-18 to the mammalian patient in a dose effective to prevent or reduce fibrosis.

22. The method of claim 21, wherein the dose is effective to achieve pharmacologic blocking of transglutaminase 2 and degradation of integrin.

23. The method of claim 21 or 22, wherein the fibrosis is selected from pulmonary fibrosis, skin fibrosis, liver cirrhosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), cancer-associated fibrosis, intestinal fibrosis and kidney fibrosis.

24. The method of any of claims 21-23, wherein the bifunctional molecule is administered locally at the site of cancer or fibrotic tissue.

25. The method of any of claims 21-23, wherein the bifunctional molecule is administered systemically.

26. The method of any of claims 21-25, wherein the individual has been analyzed after prevention or treatment by the method comprising: determining the extent of fibrosis in the patient or in a patient biological sample.

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