Membrane translocation domains and uses thereof

Abstract

Peptides are described that include a membrane translocation domain having one or more cell penetrating peptide motifs, where at least one cell penetrating peptide motif is 3-10 amino acid residues in length and has at least three arginine and / or lysine residues, or where at least one cell penetrating peptide motif is 3-10 amino acid residues in length and has at least two arginine and / or lysine residues, and at least one other cell penetrating peptide motif is 2-8 amino acid residues in length and has at least two hydrophobic residues. Compositions are also disclosed that include cargo motifs bound to the disclosed peptides. Also disclosed are methods for delivering a cargo moiety to a cell, the methods comprising contacting a cell with a peptide disclosed herein.

Description

Detailed Description of the Invention

[0001] [Technical field] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 320,978, filed March 17, 2022, which is incorporated by reference herein in its entirety.

[0002] Government Support Statement This invention was made with Government support under Grant No. GM122459 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] Reference to an Electronically Submitted Sequence Listing This application contains a sequence listing submitted electronically via EFS-Web as an ASCII sequence listing with the filename "103361-223WO1_ST26.xml", a creation date of March 17, 2023, and a size of 392417 bytes. The sequence listing submitted via EFS-Web is a part of this specification and is incorporated by reference in its entirety. [Background technology]

[0004] The ability to effectively deliver biomolecules (e.g., peptides, proteins, and nucleic acids) to the interior of cells (e.g., the cytosol and nucleus) would open the door to a new generation of therapeutic approaches capable of treating many currently difficult-to-treat diseases. Over the past decades, researchers have developed cell-penetrating peptides (Muhammad MN,et al.(2021)Cell penetrating peptides:A versatile vector for co-delivery of drug and genes in cancer.J.Contr.Rel.330:1220-1228, Heitz,F.,et al.(2009)Twenty years of cell-penetrating peptides:from molecular mechanisms to therapeutics.Br.J.Pharmacol.157:195-206), bacterial toxins (Pavlik,BJ,et al.(2017)Repurposed bacterial toxins for human therapeutics.Curr.Topics Peptide Protein Res.18:1-15, Shorter SA,et al.(2017)The potential of toxin-based drug delivery systems for enhanced nucleic acid therapeutic delivery.Expert Op.Drug Deliv.14(5):685-696), viruses (Wang,D.,et al.(2019)Adeno-associated virus vector as a platform for gene therapy delivery.Nat.Rev.Drug Discov.18:358-378), polyplexes (Ita,K.(2020)Polyplexes for gene and nucleic acid delivery:Progress and bottlenecks,Eur.J.Pharm.Sci.150:105358), liposomes (Pattni,BS,et al.(2015)New Developments in Liposomal Drug Delivery.Chem.Rev. 115(19):10938-66) and nanoparticles for various biomolecular cargoes (Mitchell, MJ, et al. (2021) Engineering precision nanoparticles for drug delivery. Nat. Rev. Drug Discov. 20:101-124). Although some delivery methods have been clinically successful (e.g., liposomal delivery of mRNA vaccines (Kim, EM, et al. (2021) Liposomes: Biomedical Applications. Chonnam Med. J. 57(1):27-35)), viral delivery of gene therapy (Wang, D., supra) and bacterial toxin-based anticancer drugs (Pavlik BJ, supra), significant limitations remain. For example, virus-, liposome-, and nanoparticle-based delivery systems are often sequestered by the liver and spleen, limiting their biodistribution to other diseased tissues, while non-viral delivery systems also face the major challenge of endosomal entrapment (Patra, JK, et al. (2018) Nano based drug delivery systems: recent developments and future prospects. J. Nanobiotechnol. 16, 71, Pei D., et al. (2019) Overcoming Endosomal Entrapment in Drug Delivery. Bioconjug. Chem. 30(2): 273-283).

[0005] A family of cyclic CPPs has been discovered that enter mammalian cells by endocytosis and efficiently escape from early endosomes into the cytosol by a vesicle budding and collapse mechanism (Qian Z., et al. (2013) Efficient delivery of cyclic peptides into mammalian cells with short sequence motifs. ACS Chem. Biol. 8(2): 423-31, Qian, Z., et al. (2016) Discovery and Mechanism of Highly Efficient Cyclic Cell-Penetrating Peptides. Biochem. 55(18): 2601-2612, Sahni, A., et al. (2020) Cell-Penetrating Peptides Escape the Endosome by Inducing Vesicle Budding and Collapse. ACS Chem. Biol. 15(9): 2485-2492). These cyclic CPPs have proven highly effective for cytosolic delivery of major drug modalities (e.g., small molecules, peptides, proteins, and nucleic acids) in vitro and in vivo. However, cyclic CPPs are less ideal for protein delivery because they contain non-proteinogenic amino acids and must be chemically synthesized and conjugated to the cargo molecule of interest, making site-specific conjugation of proteins to other entities a major challenge in itself. To overcome this limitation, short CPP motifs (e.g., Arg-Arg-Arg-Arg-Trp-Trp-Trp or R4W3) (SEQ ID NO: 133) have been genetically inserted into the surface loops of target proteins to make them cell-permeable (Chen, K., et al. (2020) Engineering Cell-Permeable Proteins through Insertion of Cell-Penetrating Motifs into Surface Loops. ACS Chem. Biol. 15(9):2568-2576).Although this approach avoids the need for post-translational modification of proteins, it still suffers from several drawbacks. First, it generally requires that structural information of the target protein is available to identify the appropriate surface loop(s) for CPP insertion. Second, even if structural information is already available, it may still be necessary to experimentally test several different insertion sites and multiple CPP sequences to find the optimal combination of insertion site and CPP sequence. For some proteins, it may not be possible to generate insertion mutants without compromising their stability and / or activity. Finally, each target protein is unique in structure and properties, requiring different engineering / optimization campaigns. There is a need for a highly effective and versatile protein delivery system that can eliminate some or all of the above drawbacks. The compositions and methods disclosed herein address these and other needs. [Summary of the Invention]

[0006] Disclosed herein are compounds, compositions, and methods for making and using such compounds and compositions. In one aspect, peptides are disclosed that include a membrane translocation domain with one or more cell penetrating peptide motifs, where at least one cell penetrating peptide motif is 3-10 amino acid residues in length and has at least three arginine and / or lysine residues, or where at least one cell penetrating peptide motif is 3-10 amino acid residues in length and has at least two arginine and / or lysine residues, and where at least one other cell penetrating peptide motif is 2-8 amino acid residues in length and has at least two hydrophobic residues. Also disclosed are compositions that include cargo motifs bound to the disclosed peptides. Also disclosed are methods for delivering a cargo moiety to a cell, the methods comprising contacting the cell with a peptide disclosed herein.

[0007] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will become apparent from the description and drawings, and from the claims.

[0008] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several aspects and, together with the description, serve to explain the principles of the invention. [Brief description of the drawings]

[0009] [Figure 1] Structures of WT FN3 and MTD1–10. The FN3 structure was generated from the PDB file 1ttg, and the MTD1–10 structures were predicted by Phyre2. The inserted CPP motifs in MTD1–10 are highlighted with a thick line. [Figure 2A] Expression and purification of MTD4. [Figure 2B] Expression and purification of MTD4. Figure 2A is an FPLC chromatogram showing the elution of MTD4 from a Ni-NTA column (MTD4 elutes as a broad peak). Figure 2B is an SDS-PAGE showing the expression levels and various fractions during purification on a Ni-NTA column. L, molecular weight marker; U, crude lysate of uninduced cells; I, crude lysate of IPTG-induced cells; CL, crude cell lysate after centrifugation; FT, flow-through fraction; 1-10, Ni-NTA column elution fractions (the strong band is MTD4). [Diagram 3] A-I are live cell confocal microscopy images of HeLa cells after 2 h incubation with 5 μM FN3TMR (Figure 3A), MTD2TMR (Figure 3B), MTD4TMR (Figure 3C), MTD4TMR (Figure 3D), MTD6TMR (Figure 3E), MTD7TMR (Figure 3F), MTD8TMR (Figure 3G), MTD9TMR (Figure 3H), or MTD10TMR (Figure 3I). All images were obtained under the same conditions, including laser power and signal amplification. [Figure 4] Total cellular uptake efficiency of TMR-tagged protein domains measured by flow cytometry. Values ​​represent the mean fluorescence intensity of treated cells. [Diagram 5] Western blot analysis of global pY levels in NIH3T3 cells after 4 h treatment with increasing concentrations of MTD4-PTP1B (0–5 mM). Anti-pY antibody 4G10 was used for pY detection. The same membrane was western blotted again with anti-GAPDH antibody to confirm equal loading. L, molecular weight marker. [Figure 6] A-C: Effects of MTD4-RBDV (Fig. 6A), MTD4-NS1 (Fig. 6B), and NS1-MTD4 (Fig. 6C) on the viability of various cancer cell lines. Cells were treated with the indicated concentrations of fusion proteins or vehicle (PBS) for 72 h at 37 °C. Then, cells were incubated with Cell Titer Glo solution for 15 min and luminescence was measured. Viability values ​​reported are relative to the viability of vehicle (PBS)-treated cells. [Figure 7] Inhibition of Ras-Raf interaction in HEK293T cells by MTD4-RBDV and MTD4-NS1. Cells were transfected with pEF-RLUC8-L15-KrasG12V (or G12D) and pEF-CRAFRBD(1-149)-L15-GFP and incubated at 37°C for 24 h. Cells were then treated with the indicated concentrations of fusion proteins for 20–24 h. BRET signals were measured after adding 10 μM coelenterazine 400a as substrate. [Figure 8] A-B are Western blot analyses. Effects of MTD4-RBDV (FIG. 8A) and MTD4-NS1 (FIG. 8B) on phosphorylation of Akt and MEK in MiaPaCa-2 cells. GAPDH was used as a loading control. [Figure 9] Figure 1 shows that MTD4-RBDV and MTD4-NS1 induce apoptosis in lung cancer H358 cells. Approximately 10,000 cells were treated with the indicated concentrations of MTD4-RBDV or MTD4-NS1 in the presence of 10% FBS for 24 hours, and then stained with Alexa Fluor® 488-Annexin V and propidium iodide before flow cytometry analysis. [Figure 10] A–D are live cell confocal microscopy images of HEK293T cells after incubation for 6 h with buffer (Figure 10A), 10 μM GFP11 (Figure 10B), CPP12-GFP11 (Figure 10C), or MTD4-GFP11 (Figure 10D). [Figure 11] Dose-dependent induction of luciferase activity in HEK293T cells transiently expressing LgBit by HiBit or HiBit conjugated to different delivery vehicles. All y-axis values ​​are relative to those of untreated cells (no HiBit) and represent the mean ± SD of three independent experiments. [Figure 12] AC are live-cell confocal microscopy images of HeLa cells after 2 h incubation with buffer (FIG. 12A), 5 μM SEP (FIG. 12B), or 5 μM MTD4-SEP (FIG. 12C). Top panels, nuclear staining with Hoechst; bottom panels, SEP fluorescence. [Figure 13] A-B, Dephosphorylation of pY protein in HEK293T cells by MTD4-PTP1B. Figure 13A is a Western blot analysis of cell lysates after cells were treated with buffer, PTP1B (WT, 5 mM), or increasing concentrations of MTD4-PTP1B (0-5 mM) for 6 hours. Figure 13B is a Coomassie blue staining of the SDS-PAGE gel in Figure 13A, showing that similar amounts of protein samples were loaded in all lanes. [Figure 14] Biodistribution of MENC protein in various organs of transgenic mice. The green and red channels correspond to the signals from EGFP and m-cherry, respectively. Fluorescence in various tissues was monitored for mice euthanized after 3 hours (EGFP) and 48 hours (mCherry). [Figure 15] A-B are SDS-PAGE analyses of MTD4 (FIG. 15A) and FN3 (FIG. 15B) after incubation with human serum for various times (0-24 h). Protein bands in boxes correspond to MTD4 / FN3 and their degradation products. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The present invention may be understood more readily by reference to the following detailed description of the invention and the examples included therein.

[0011] Before the present compounds, compositions, articles, systems, devices, and / or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods, unless otherwise specified, or to specific reagents, unless otherwise specified, and as such 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 be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described herein.

[0012] All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. Further, the publication dates provided herein may be different from the actual publication dates, which may require independent confirmation. definition

[0013] Throughout this specification, the terms "about" and / or "approximately" may be used in conjunction with numerical values ​​and / or ranges. The term "about" is understood to mean not only the stated value, but also a value close to the stated value.

[0014] Numerical ranges are provided for specific quantities throughout this specification. It is to be understood that these ranges include all values ​​and subranges therein. Thus, a range of "50 to 80" includes all possible values ​​within that range (e.g., 50, 51, 52, 53, 54, 55, 56, etc.) and all possible ranges within that range (e.g., 51 to 79, 52 to 78, 53 to 77, 54 to 76, 55 to 75, 60 to 70, etc.). Furthermore, all values ​​within a given range may be the endpoints of the ranges included within that range (e.g., the range 50 to 80 includes the endpoints 55 to 80, 50 to 75, etc.).

[0015] The term "a" or "an" refers to one or more of that entity, for example, "a polypeptide conjugate" refers to one or more polypeptide conjugates or at least one polypeptide conjugate. Thus, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein. Furthermore, reference to "a polypeptide conjugate" by the indefinite article "a" or "an" does not exclude the possibility that there is more than one polypeptide conjugate, unless the context clearly requires that there is only one polypeptide conjugate.

[0016] As used herein, the term "adjacent" refers to two consecutive amino acids that are covalently linked. "Adjacent" is also used interchangeably with "consecutive."

[0017] The term "carrier" refers to a compound, composition, substance, or structure that, when combined with a compound or composition, aids or facilitates the preparation, storage, administration, delivery, efficacy, selectivity, or any other characteristic of the compound or composition for its intended use or purpose. For example, the carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.

[0018] As used herein, "treat," "treating," "treatment," or variations thereof, refers to any administration of a polypeptide conjugate of the present disclosure that partially or completely relieves, ameliorates, prevents, alleviates, inhibits, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms or characteristics of a disease or condition described herein.

[0019] As used herein, "therapeutically effective" refers to an amount of a polypeptide conjugate of the present disclosure or a complex thereof capable of delivering a therapeutic amount of a therapeutic nucleic acid to a patient.

[0020] As used herein, a "cell-penetrating peptide" or "CPP" refers to any peptide that can permeate a cell membrane. As used herein, a "cyclic cell-penetrating peptide" or "cCPP" refers to any cyclic peptide that can permeate a cell membrane.

[0021] As used herein, "linker" or "L" refers to a moiety that covalently bonds two or more components of a polypeptide conjugate disclosed herein (e.g., a linker can covalently bond a CPP and a group (i.e., P) that binds to a nucleic acid sequence through electrostatic interactions). In some embodiments, the linker can be a natural or unnatural amino acid or polypeptide. In other embodiments, the linker is a synthetic compound that includes two or more suitable functional groups suitable for binding, for example, to a CPP and independently to P. In some embodiments, the linker has a linear length of about 3 to about 100 (e.g., about 3 to about 20) atoms (excluding branch atoms or substituents). In some embodiments, the linker provides a distance between the two groups it links of about 1 Å to about 400 Å.

[0022] As used herein, a "polypeptide" is a chain of at least two amino acids linked together by peptide bonds. There is no upper limit to the number of amino acids that can be included in a polypeptide. Furthermore, a polypeptide may include unnatural amino acids, amino acid analogs, or other synthetic molecules that can be incorporated into a polypeptide.

[0023] As used herein, "monomer" refers to an amino acid residue in a polypeptide. In some embodiments, the amino acid monomer is divalent. In other embodiments, the amino acid monomer may be trivalent if the monomer is further substituted. For example, a cysteine ​​monomer can form peptide bonds independently at the N-terminus and C-terminus, and can also form disulfide bonds.

[0024] As used herein, "amino acid analog" or "analog" (e.g., "arginine analog", "lysine analog" or "histidine analog") refers to a variant of an amino acid that retains at least one function of the amino acid, such as the ability to bind to an oligonucleotide through electrostatic interactions. Such variants may have an extended or shorter side chain (e.g., with one or more -CH2- groups that retain the ability to bind to an oligonucleotide through electrostatic interactions), or the modification may improve the ability to bind to an oligonucleotide through electrostatic interactions. For example, an arginine analog may include an additional methylene or ethylene between the backbone and the guanidine / guanidinium group. Other examples include amino acids with one or more additional substituents (e.g., Me, Et, halogen, thiol, methoxy, ethoxy, C1-haloalkyl, C2-haloalkyl, amine, guanidine, etc.). Amino acid analogs may be monovalent, divalent, or trivalent.

[0025] Throughout this specification, peptides and amino acid monomers are shown as charge-neutral species. It is understood that such species can carry a positive or negative charge depending on the conditions. For example, at pH 7, the N-terminus of an amino acid is protonated and carries a positive charge (-NH3 + ) and the C-terminus of the amino acid is deprotonated and negatively charged (-CO2 - ) Similarly, the side chains of certain amino acids can carry a positive or negative charge.

[0026] Each amino acid may be a natural or unnatural amino acid. The term "unnatural amino acid" refers to an organic compound that is a homologue of a natural amino acid in that it has a similar structure to the natural amino acid and thus mimics the structure and reactivity of the natural amino acid. The unnatural amino acid may be a modified amino acid and / or an amino acid analog that is not one of the 20 common naturally occurring amino acids or the rare natural amino acids selenocysteine ​​or pyrrolysine. The unnatural amino acid may be a D-isomer of a natural amino acid. Thus, as used herein, the term "amino acid" refers to natural and unnatural amino acids, as well as their analogs and derivatives. Examples of suitable amino acids include, but are not limited to, alanine, alloisoleucine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, naphthylalanine, phenylalanine, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, valine, derivatives, or combinations thereof. Analogs of amino acids include those that have a similar, but not identical, structure to an amino acid, for example, due to modifications to the side chain or backbone on the amino acid. Such modifications can increase the hydrophobicity of the side chain, including extending the side chain with one or more hydrocarbons, or increasing the solvent accessible surface area (SASA, as described herein) of amino acids with aromatic rings in the side chain, for example, by conjugating a second aromatic ring or increasing the size of the aromatic ring. Derivatives of amino acids include natural and unnatural amino acids that have been modified (e.g., by substitution) to include hydrophobic groups as described herein. For example, derivatives of lysine include lysine with the side chain substituted with alkylcarboxamidyl. These and others are listed in Table 1 along with their abbreviations used herein.

[0027] [Table 1] TIFF2025509645000002.tif43170

[0028] "Alkyl" or "alkyl group" refers to a fully saturated straight or branched hydrocarbon chain radical having from one to twelve carbon atoms and attached to the remainder of the molecule by a single bond. Alkyl containing any number of carbon atoms from one to twelve is included. Alkyl containing up to twelve carbon atoms is C1-C 12 Alkyl, alkyl containing up to 10 carbon atoms is C1-C 10 An alkyl group is one having up to 6 carbon atoms, which is C1-C6 alkyl, and an alkyl group having up to 5 carbon atoms is C1-C5 alkyl. C1-C5 alkyl includes C5 alkyl, C4 alkyl, C3 alkyl, C2 alkyl, and C1 alkyl (i.e., methyl). C1-C6 alkyl includes all of the moieties described above for C1-C5 alkyl, but also includes C6 alkyl. C1-C 10 Alkyl includes all of the moieties described above for C1-C5 alkyl and C1-C6 alkyl, but also includes C7, C8, C9 and C 10 Alkyl is also included. Similarly, C1-C 12 Alkyl includes all of the above moieties, except C 11 and C 12 Alkyl is also included. C1~C 12 Non-limiting examples of alkyl include methyl, ethyl, n-propyl, i-propyl, sec-propyl, n-butyl, i-butyl, sec-butyl, t-butyl, n-pentyl, t-amyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, and n-dodecyl. Unless stated otherwise specifically in the specification, an alkyl group may be optionally substituted.

[0029] "Alkylene" or "alkylene chain" refers to a fully saturated, straight or branched divalent hydrocarbon chain radical having 1 to 40 carbon atoms. 40Non-limiting examples of alkylene include ethylene, propylene, n-butylene, pentylene, etc. Unless stated otherwise specifically in the specification, an alkylene chain may be optionally substituted as described herein.

[0030] "Alkenyl" or "alkenyl group" refers to a straight or branched hydrocarbon chain radical having from 2 to 12 carbon atoms and having one or more carbon-carbon double bonds. Each alkenyl group is attached to the remainder of the molecule by a single bond. Alkenyl groups containing any number of carbon atoms from 2 to 12 are included. Alkenyl groups containing up to 12 carbon atoms are C2-C 12 Alkenyl, containing up to 10 carbon atoms, is C2-C 10 Alkenyl is an alkenyl having up to 6 carbon atoms, which is C2-C6 alkenyl, and an alkenyl having up to 5 carbon atoms, which is C2-C5 alkenyl. C2-C5 alkenyl includes C5 alkenyl, C4 alkenyl, C3 alkenyl, and C2 alkenyl. C2-C6 alkenyl includes all the moieties described above for C2-C5 alkenyl, but also includes C6 alkenyl. C2-C 10 Alkenyl includes all of the moieties described above for C2-C5 alkenyl and C2-C6 alkenyl, but also includes C7, C8, C9 and C 10 Alkenyl is also included. Similarly, C2-C 12 Alkenyl includes all of the above moieties, except that C 11 and C 12 Alkenyl is also included. C2-C 12Non-limiting examples of alkenyl include ethenyl (vinyl), 1-propenyl, 2-propenyl (allyl), iso-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl , 4-nonenyl, 5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, 1-undecenyl, 2-undecenyl, 3-undecenyl, 4-undecenyl, 5-undecenyl, 6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecenyl, 10-undecenyl, 1-dodecenyl, 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5-dodecenyl, 6-dodecenyl, 7-dodecenyl, 8-dodecenyl, 9-dodecenyl, 10-dodecenyl, and 11-dodecenyl. Unless stated otherwise specifically in the specification, an alkenyl group may be optionally substituted.

[0031] "Alkenylene" or "alkenylene chain" refers to a linear or branched divalent hydrocarbon chain radical having 2 to 40 carbon atoms and having one or more carbon-carbon double bonds. 40 Non-limiting examples of alkenylene include ethenylene (-CH=CH-), propenylene, butenylene, etc. Unless stated otherwise specifically in the specification, an alkenylene chain may be optionally substituted.

[0032] "Alkynyl" or "alkynyl group" refers to a straight or branched hydrocarbon chain radical having from 2 to 12 carbon atoms and having one or more carbon-carbon triple bonds. Each alkynyl group is attached to the remainder of the molecule by a single bond. Alkynyl groups containing any number of carbon atoms from 2 to 12 are included. Alkynyl groups containing up to 12 carbon atoms are C2-C 12 Alkynyl, containing up to 10 carbon atoms, is C2-C 10 Alkynyl is an alkynyl having up to 6 carbon atoms, C2-C6 alkynyl, and an alkynyl having up to 5 carbon atoms is C2-C5 alkynyl. C2-C5 alkynyl includes C5 alkynyl, C4 alkynyl, C3 alkynyl, and C2 alkynyl. C2-C6 alkynyl includes all of the moieties described above for C2-C5 alkynyl, but also includes C6 alkynyl. C2-C 10 Alkynyl includes all of the moieties described above for C2-C5 alkynyl and C2-C6 alkynyl, but also includes C7, C8, C9 and C 10 Alkynyl is also included. Similarly, C2-C 12 Alkynyl includes all of the above moieties, except that C 11 and C 12 Alkynyl is also included. C2-C 12 Non-limiting examples of alkynyl include ethynyl, propynyl, butynyl, pentynyl, etc. Unless stated otherwise specifically in the specification, an alkynyl group may be optionally substituted.

[0033] "Alkynylene" or "alkynylene chain" refers to a linear or branched divalent hydrocarbon chain radical having 2 to 40 carbon atoms and having one or more carbon-carbon triple bonds. 40 Non-limiting examples of alkynylene include ethynylene (-C≡C-), propargylene, etc. Unless stated otherwise specifically in the specification, an alkynylene chain may be optionally substituted.

[0034] "Aryl" refers to a hydrocarbon ring system containing hydrogen, 6-40 carbon atoms, and at least one aromatic ring. For purposes of this disclosure, aryl may be a monovalent or divalent radical (excluding substituents), which may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, and may also include fused or bridged ring systems. Aryl groups include, but are not limited to, radicals derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene. In some embodiments, the aryl radical may be divalent when used as or as part of a linker. Unless otherwise stated in the specification, aryl groups may be optionally substituted.

[0035] As used herein, "aromatic" refers to an unsaturated cyclic molecule having 4n+2π electrons, where n is any integer. The term "non-aromatic" refers to any unsaturated cyclic molecule that does not fall within the definition of aromatic.

[0036] "Carbocyclyl", "carbocyclic ring" or "carbocycle" refers to a ring structure in which each of the atoms forming the ring is carbon. A carbocyclic ring can contain 3 to 20 carbon atoms in the ring. Carbocyclic rings include aryl, cycloalkyl, and fully unsaturated, partially unsaturated, and fully saturated rings. In some embodiments, a carbocyclyl may be divalent when used as a linker or part of a linker. Unless otherwise specified in the specification, a carbocyclyl group may be optionally substituted.

[0037] "Cycloalkyl" refers to a stable non-aromatic monocyclic or polycyclic fully saturated hydrocarbon radical having 3 to 40 carbon atoms and at least one ring, where the ring consists solely of carbon and hydrogen atoms, and may include fused or bridged ring systems. For purposes of this disclosure, cycloalkyl can be a monovalent or divalent radical (excluding substituents). Monocyclic cycloalkyl radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic cycloalkyl radicals include, for example, adamantyl, norbornyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like. In some embodiments, the cycloalkyl radical may be divalent when used as a linker or part of a linker. Unless otherwise specifically stated herein, cycloalkyl groups may be optionally substituted.

[0038] "Cycloalkenyl" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical having 3 to 40 carbon atoms, at least one ring, and one or more carbon-carbon double bonds, where the rings consist only of carbon and hydrogen atoms and may include fused or bridged ring systems. For purposes of this disclosure, cycloalkenyl can be a monovalent or divalent radical (excluding substituents). Monocyclic cycloalkenyl groups include, for example, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, and the like. Polycyclic cycloalkenyl groups include, for example, bicyclo[2.2.1]hept-2-enyl, and the like. In some embodiments, the cycloalkenyl radical may be divalent when used as a linker or as part of a linker. Unless otherwise specifically stated herein, cycloalkenyl groups may be optionally substituted.

[0039] "Cycloalkynyl" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical having 3 to 40 carbon atoms, at least one ring, and one or more carbon-carbon triple bonds, where the rings consist only of carbon and hydrogen atoms and may include fused or bridged ring systems. For purposes of this disclosure, cycloalkynyl can be a monovalent or divalent radical (excluding substituents). Monocyclic cycloalkynyl groups include, for example, cycloheptynyl, cyclooctynyl, and the like. In some embodiments, the cycloalkynyl radical may be divalent when used as a linker or part of a linker. Unless otherwise stated specifically in the specification, cycloalkynyl groups may be optionally substituted.

[0040] "Heterocyclyl", "heterocyclic ring" or "heterocycle" refers to a stable 3- to 20-membered aromatic ring radical consisting of 2 to 12 carbon atoms and 1 to 6 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. For purposes of this disclosure, a heterocyclyl group can be a monovalent or divalent radical (excluding substituents). A heterocyclyl or heterocyclic ring includes heteroaryl, as defined below. Unless otherwise stated specifically in the specification, a heterocyclyl radical can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include fused or bridged ring systems, the nitrogen, carbon or sulfur atoms in the heterocyclyl radical can be optionally oxidized, the nitrogen atom can be optionally quaternized, and the heterocyclyl radical can be partially or fully saturated. Examples of such heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. In some embodiments, heterocyclyl radicals may be divalent when used as or as part of a linker. Unless stated otherwise specifically in the specification, a heterocyclyl group may be optionally substituted.

[0041] "Heteroaryl" refers to a 5- to 20-membered ring system radical containing a hydrogen atom, 1 to 14 carbon atoms, 1 to 6 heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur, and at least one aromatic ring. For purposes of this invention, a heteroaryl radical may be a monovalent or divalent radical (excluding substituents) and may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused or bridged ring systems, and in which the nitrogen, carbon, or sulfur atoms in the heteroaryl radical may be optionally oxidized and the nitrogen atom may be optionally quaternized. Examples include azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indophenyl, iso ... Examples include, but are not limited to, dolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl, 1-oxidopyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e., thienyl).In some embodiments, a heteroaryl radical, when used as or as part of a linker, may be divalent. Unless stated otherwise specifically in the specification, a heteroaryl group may be optionally substituted.

[0042] As used herein, the term "ether" refers to a straight-chain or branched divalent radical moiety -[(CH2) m -O-(CH2) n ] z -, where m, n, and z are each independently selected from 1 to 40. Examples include, but are not limited to, polyethylene glycol. Unless otherwise specified specifically in the specification, ethers may be optionally substituted.

[0043] The term "substituted" as used herein means any of the above groups (i.e., alkylene, alkenylene, alkynylene, aryl, carbocyclyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, heteroaryl, and / or ether) in which at least one hydrogen atom is replaced with a bond to a non-hydrogen atom, such as, but not limited to, a halogen atom, such as F, Cl, Br, and I; an oxygen atom in groups such as hydroxyl, alkoxy, and ester groups; a sulfur atom in groups such as thiol, thioalkyl, sulfone, sulfonyl, and sulfoxide groups; a nitrogen atom in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides, and enamines; a silicon atom in groups such as trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, and triarylsilyl groups; as well as other heteroatoms in various other groups. "Substituted" also refers to any of the above groups in which one or more hydrogen atoms have been replaced with a higher bond (e.g., a double bond or a triple bond) to a heteroatom, such as oxygen in oxo, carbonyl, carboxyl, and ester groups; and nitrogen in groups such as imines, oximes, hydrazones, and nitriles. For example, "substituted" includes any group in which one or more hydrogen atoms have been replaced with -NR g R h , -NR g C(=O)R h , -NR g C(=O)NR g R h , -NR g C(=O)OR h , -NR g SO2R h , -OC(=O)NR g R h , -OR g , -SR g , -SOR g , -SO2R g , -OSO2R g , -SO2OR g , =NSO2R g , and -SO2NR g R h"Substituted" also refers to any of the above groups replaced with one or more hydrogen atoms by -C(=O)R. g , -C(=O)OR g , -C(=O)NR g R h , -CH2SO2R g , -CH2SO2NR g R h In the above, R g and R hare the same or different and are independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and / or heteroarylalkyl. "Substituted" further refers to any of the above groups in which one or more hydrogen atoms are replaced with a bond to an amino, cyano, hydroxyl, imino, nitro, oxo, thioxo, halo, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and / or heteroarylalkyl group. Additionally, each of the aforementioned substituents may be optionally substituted with one or more of the substituents described above. Additionally, one of skill in the art will recognize that "substituted" also includes cases where one or more atoms of any of the above groups are replaced with a substituent listed in this paragraph, and the substituent forms a covalent bond with the CPP, P, or L. For example, in certain embodiments, any of the above groups may be substituted at the first position with a carboxylic acid (i.e., -C(=O)OH) that forms an amide bond with a lysine in the CPP, or the group may be substituted at the second position with a thiol group that forms a disulfide bond with a cysteine ​​(or an amino acid analog having a thiol group).

[0044] As used herein, the term "subject" refers to a target of administration, e.g., a test subject. Thus, the subject of the methods disclosed herein can be a vertebrate, such as a mammal, fish, bird, reptile, or amphibian. Alternatively, the subject of the methods disclosed herein can be a human, a non-human primate, a horse, a pig, a rabbit, a dog, a sheep, a goat, a cow, a cat, a guinea pig, a fish, a bird, a rodent, or a fruit fly. The term does not indicate a particular age or sex. Thus, adult and newborn subjects, as well as fetuses (both male and female), are intended to be encompassed. In some examples, the subject is a mammal. A patient refers to a subject suffering from a disease or disorder. The term "patient" includes human and veterinary subjects. In some examples of the disclosed methods, prior to the administering step, the subject is diagnosed with cancer, an autoimmune disease, and / or a need for treatment of inflammation. In some examples of the disclosed methods, prior to the administering step, the subject is diagnosed with cancer. The term "subject" also includes cells, eg, animal cells, such as human cells.

[0045] As used herein, the term "treatment" refers to the medical management of a patient with the goal of curing, ameliorating, or stabilizing a disease, pathological condition, or disorder. The term includes active treatment, i.e., treatment specifically aimed at ameliorating a disease, pathological condition, or disorder, and also includes causal treatment, i.e., treatment aimed at removing the cause of an associated disease, pathological condition, or disorder. In addition, the term includes palliative treatment, i.e., treatment designed to alleviate symptoms rather than cure a disease, pathological condition, or disorder, and supportive treatment, i.e., treatment used to supplement another specific therapy aimed at ameliorating an associated disease, pathological condition, or disorder. In various embodiments, the term encompasses any treatment of a subject, including a mammal (e.g., a human), including (i) preventing the development of a disease in a subject who may be susceptible to the disease but has not yet been diagnosed as suffering from it, (ii) inhibiting the disease, i.e., arresting its onset, or (iii) relieving the disease, i.e., causing regression of the disease.

[0046] As used herein, the terms "prevent" or "preventing" refer to preventing, averting, eliminating, forestalling, hindering, or impeding something from happening, especially by prior action.

[0047] As used herein, the term "diagnosed" means that a person has undergone a physical examination by a person skilled in the art, e.g., a physician, and has been found to have a condition that can be diagnosed or treated by a compound, composition, or method disclosed herein. For example, "diagnosed with cancer" means that a person has undergone a physical examination by a person skilled in the art, e.g., a physician, and has been found to have a condition that can be diagnosed or treated by a compound or composition that can treat or prevent cancer. As yet another example, "diagnosed as needing to treat or prevent cancer" refers to a person having undergone a physical examination by a person skilled in the art, e.g., a physician, and has been found to have a condition that is characterized by cancer or other disease, and that treating or preventing cancer would be beneficial for the subject.

[0048] As used herein, phrases such as "identified as needing treatment for a disorder" refer to selecting a subject based on the need for treatment for the disorder. For example, a subject can be identified as needing treatment for a disorder (e.g., a disorder related to cancer) based on a previous diagnosis by a person skilled in the art, and then be treated for the disorder. In some instances, it is contemplated that the identification can be performed by a person different from the person who performs the diagnosis. In some instances, it is also contemplated that the administration can be performed by the person who subsequently performs the administration.

[0049] As used herein, the terms "administering" and "administration" refer to any method of providing a pharmaceutical agent to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, intranasal administration, topical administration, intravaginal administration, ocular administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injections such as intravenous administration, intraarterial administration, intramuscular administration, and subcutaneous administration. Administration may be continuous or intermittent. In some examples, the preparations may be administered therapeutically, i.e., administered to treat an existing disease or condition. In some examples, the preparations may be administered prophylactically, i.e., administered to prevent a disease or condition.

[0050] The term "contacting" as used herein refers to combining a disclosed compound and a target (e.g., a cell, a target receptor, a transcription factor, or other biological entity) in such a way that the compound can affect the activity of the target either directly (i.e., by interacting with the target itself) or indirectly (by interacting with another molecule, cofactor, factor, or protein on which the activity of the target depends).

[0051] As used herein, the terms "effective amount" and "effective amount" refer to an amount sufficient to achieve a desired outcome or affect an undesired condition. For example, a "therapeutically effective amount" refers to an amount sufficient to achieve a desired therapeutic outcome or affect an undesired symptom, but generally insufficient to cause adverse side effects. The specific therapeutically effective dosage level for any particular patient will depend on a variety of factors, including the disorder being treated and the severity of the disorder, the particular composition used, the patient's age, weight, general health, sex and diet, time of administration, route of administration, excretion rate of the particular compound used, duration of treatment, drugs used in combination or concomitantly with the particular compound used, and similar factors well known in the medical arts. For example, it is well within the skill of the art to begin administering a compound at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. If necessary, an effective daily dose can be divided into multiple doses for administration. Thus, a single-dose composition can contain such amounts or submultiples thereof to make up the daily dose. In the event of any contraindications, the dosage can be adjusted by the individual physician. Dosage may vary and may be administered in one or more doses per day for one or several days. Guidance can be found in the literature for appropriate dosages for a given class of pharmaceutical agent. In some instances, the preparations may be administered in a "prophylactically effective amount," i.e., an amount effective for the prevention of a disease or condition.

[0052] The term "pharmacologically acceptable" refers to a substance that is not biologically or otherwise undesirable, i.e., that does not cause unacceptable levels of undesirable biological effects or interact in a deleterious manner.

[0053] As used herein, the term "pharmaceutical acceptable carrier" refers to sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions immediately prior to use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms can be ensured by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like. The absorption of injectable pharmaceutical forms can be prolonged by including agents such as aluminum monostearate and gelatin that delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters), and poly(anhydrides). The rate of drug release can be controlled depending on the ratio of drug to polymer and the nature of the particular polymer used. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues. Injectable preparations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium immediately before use. Suitable inert carriers can include sugars such as lactose.

[0054] As used herein and in the final claims, a residue of a chemical species refers to a moiety that is a resulting product of a chemical species in a particular reaction scheme or subsequent formulation or chemical product, regardless of whether the moiety is actually derived from the chemical species. Thus, an amino acid residue in a peptide or protein refers to one or more -OC(O)CH(R)NH- units in the peptide or protein.

[0055] As used herein, the symbol "

[0056] [ka] " (which may hereafter be referred to as a "point bond") denotes a bond that is a point of attachment between two chemical entities, one of which is shown as being attached to the point bond and the other is shown as not being attached to the point bond. For example,

[0057] [ka] " indicates that the chemical entity "XY" is attached to another chemical entity via a bond at the point of attachment. Furthermore, a particular point of attachment to a chemical entity not shown can be specified by inference. For example, the compound CH3-R 3 (In the formula, R 3 is H) or "

[0058] [ka] " is R 3 If "XY" is selected, the bond point is R. 3 This indicates that this is the same bond as the bond shown connecting CH3 to CH4.

[0059] Unless otherwise stated, formulas with chemical bonds shown only as solid lines, not wedges or dashed lines, contemplate each possible isomer, e.g., each enantiomer and diastereomer, as well as mixtures of isomers, e.g., racemic or scalemic mixtures. The compounds described herein may contain one or more asymmetric centers, and thus diastereomers and optical isomers may occur. Unless otherwise stated, the compounds and compositions disclosed herein include all such possible diastereomers and their racemic mixtures, their substantially pure resolved enantiomers, all possible geometric isomers, and their pharma- ceutically acceptable salts. Mixtures of stereoisomers and isolated specific stereoisomers are also included. During the course of the synthetic procedures used to prepare such compounds, or when using racemization or epimerization procedures well known to those skilled in the art, the products of such procedures may be mixtures of stereoisomers.

[0060] Many organic compounds exist in optically active forms that have the ability to rotate the plane of plane-polarized light. In describing optically active compounds, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule around the chiral center(s). The prefixes d and 1 or (+) and (-) are used to indicate the sign of rotation of plane-polarized light by the compound, with (-) or 1 meaning that the compound is levorotatory. Compounds with the prefix (+) or d are dextrorotatory. For a given chemical structure, these compounds, called stereoisomers, are identical except that they are non-superimposable mirror images of one another. A particular stereoisomer may also be called an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is called a racemic mixture. Many of the compounds described herein can have one or more chiral centers and therefore can exist in different enantiomeric forms. If necessary, a chiral carbon can be indicated with an asterisk (*). When a bond to a chiral carbon is shown as a straight line in a disclosed formula, it is understood that both the (R) and (S) configurations of the chiral carbon, and therefore both enantiomers and mixtures thereof, are encompassed by the formula. As used in the art, when it is desired to specify the absolute configuration for a chiral carbon, one of the bonds to the chiral carbon can be shown as a wedge (a bond to an atom above the plane) and the other can be shown as a series of short parallel lines or a wedge of short parallel lines (a bond to an atom below the plane). The Cahn-Ingold-Prelog system can be used to assign the (R) or (S) configuration to a chiral carbon.

[0061] The compounds described herein include both natural and non-natural isotopic abundances of atoms. The disclosed compounds may be isotopically labeled or isotopically substituted compounds identical to those described, except that one or more atoms are replaced with atoms having atomic masses or mass numbers different from the atomic masses or mass numbers typically found in nature. Examples of isotopes that can be incorporated into the compounds disclosed herein include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, e.g., 2 H, 3 H, 13 C. 14 C. 15 N, 18 O. 17 O. 35 S, 18 F, and 36 The compounds further include prodrugs thereof, and pharma- ceutically acceptable salts of the compounds or prodrugs that contain the aforementioned isotopes and / or other isotopes of other atoms are within the scope of the present invention. Certain isotopically labeled compounds, e.g. 3 H and 14 Compounds incorporating radioactive isotopes, such as C, are useful in drug and / or substrate tissue distribution assays. Tritium isotopes (i.e., 3 H) and carbon-14 isotopes (i.e., 14 C) is particularly preferred because it is easy to prepare and detect. 2 Substitution with heavier isotopes such as H may be preferred in some circumstances because it may confer certain therapeutic advantages resulting from increased metabolic stability, such as increased in vivo half-life or reduced dosage requirements. Isotopically labeled compounds and prodrugs thereof can generally be prepared by carrying out the following procedures using readily available isotopically labeled reagents in place of non-isotopically labeled reagents.

[0062] Disclosed are the components used to prepare the compositions disclosed herein, as well as the compositions themselves used within the methods disclosed herein. Where these and other substances are disclosed herein, and combinations, subsets, interactions, groups, etc. of these substances are disclosed, it is understood that although specific reference to each of the various individual and collective combinations and permutations of these compounds cannot be expressly disclosed, each is specifically contemplated and described herein. For example, where a particular compound is disclosed and discussed, and several modifications that can be made to a number of molecules including that compound, all combinations and permutations of that compound and possible modifications are specifically contemplated unless specifically indicated to the contrary. Thus, where a class of molecules A, B, and C is disclosed, as well as a class of molecules D, E, and F, and an example of a combination molecule A-D, each is individually and collectively contemplated, even if each is not individually described, i.e., combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered to be disclosed. Similarly, any subset or combination of these is also disclosed. Thus, for example, subgroups A-E, B-F, and C-E are considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the compositions disclosed herein. Thus, where there are various additional steps that may be performed, it is understood that each of these additional steps can be performed with any particular embodiment or combination of embodiments of the methods disclosed herein.

[0063] Reference will now be made in detail to certain embodiments of the disclosed materials, compounds, compositions, articles and methods, examples of which are illustrated in the accompanying examples and drawings.

[0064] composition Disclosed are cell penetrating peptides and compositions comprising them, which can potentially provide a general vehicle for cytosolic delivery of any peptide or protein cargo, as well as other biomolecules. The disclosed compositions may have better cytosolic delivery efficiency and in vivo stability than simple cell penetrating peptides. Furthermore, the disclosed compositions may escape the endosomes of cells more completely, and thus may transport conjugated cargo out of endosomes more efficiently than in the absence of the disclosed compositions. The disclosed compositions may also have a much broader cargo compatibility (essentially any peptide or protein). Also, synthesis may be simpler, i.e., genetically fusing the membrane translocation domain to the N-terminus, C-terminus, or internal location of the cargo protein. Furthermore, the disclosed compositions may be less likely to be immunogenic and may deliver cargo to nearly any eukaryotic cell (e.g., mammals and plants), unlike bacterial toxins, which may be limited to cells expressing the toxin's specific receptor(s). The disclosed compositions may also have a higher delivery capacity than bacterial toxins, since the delivery capacity of bacterial toxins may be limited by the abundance of receptors on the target cell surface. In further examples, the disclosed compositions do not require or include cofactors such as zinc.

[0065] In certain aspects, peptides are disclosed that include a membrane translocation domain with one or more cell penetrating peptide motifs, where at least one cell penetrating peptide motif is 3-10 amino acid residues in length and has at least three arginine and / or lysine residues. Unlike methods in which a CPP motif is inserted into each target protein, the disclosed compositions and methods include engineered membrane translocation domains that can be genetically or synthetically fused to any target cargo of interest. Another strategy disclosed herein involves splitting the CPP motif into two and inserting them into two different regions of the membrane translocation domain, resulting in a significant increase in cytosolic delivery efficiency.

[0066] Membrane translocation domain The membrane translocation domain portion of the disclosed peptides can be any membrane translocation domain modified to include at least one cell-penetrating motif as described herein, i.e., a peptide sequence capable of crossing a lipid bilayer. In a preferred example, there are two or three cell-penetrating motifs in the membrane translocation domain. For example, at least one cell-penetrating peptide motif can be 3-10 amino acid residues in length and have at least three arginine and / or lysine residues, e.g., 4, 5, or 6 arginine and / or lysine residues. Alternatively, at least one cell-penetrating peptide motif can be 3-10 amino acid residues in length and have at least two arginine and / or lysine residues, and at least one other cell-penetrating peptide motif can be 2-8 amino acid residues in length and have at least two hydrophobic residues. If there is more than one cell penetrating peptide motif, there may be more than one arginine and / or lysine residue within a span of 3-10 amino acids, and there may be another cell penetrating peptide motif with more than one hydrophobic residue within a span of 2-8 amino acids. The cell penetrating peptide motif may be anywhere within the membrane translocation domain.

[0067] In some examples, the membrane translocation domain can be a human membrane translocation domain, such as fibronectin type III. In certain examples, the membrane translocation domain has at least 90%, at least 95%, or at least 97% sequence similarity with SEQ ID NO: 118. In other examples, the membrane translocation domain is a human fibronectin type III having BC, DE, CD, and FG loops, and the cell penetrating peptide motif is in one or more of the BC, DE, CD, or FG loops, for example, the cell penetrating peptide motif is in two of the BC, DE, CD, or FG loops, particularly the BC loop and the FG loop. These loops can be defined as having the following sequences: BC=AVTVR (SEQ ID NO: 31), CD=GGNSPVQ (SEQ ID NO: 32), DE=PGSK (SEQ ID NO: 33), FG=GRGDSPAS (SEQ ID NO: 34).

[0068] In other examples, the membrane translocation domain can be any stably folded protein that can be efficiently expressed, preferably in bacteria. Some further examples of membrane translocation domains are nanobody scaffolds, DARPin scaffolds, and CTPR proteins (consensus tetratricopeptide repeats, Acc. Chem. Res. 2021, 54, 4166-4177).

[0069] Cell-penetrating peptide motifs A cell penetrating peptide (CPP) motif can comprise at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, or at least 6 amino acids, more specifically 3-8, 3-6, 4-8, 4-6, or 6-8 amino acids. In most instances, the CPP motif is replaced with a membrane translocation domain, such that the resulting peptide has the same number of amino acids as the naturally occurring membrane translocation domain.

[0070] In some examples, at least 2, 3, 4, 5, 6, or 7 amino acids of the CPP motif are adjacent arginine residues. In preferred examples, there are 3, 4, or 5 adjacent arginine residues in the CPP motif. In other examples, there are no adjacent arginine residues in the CPP motif. Each amino acid in the CPP motif can be independently a natural or non-natural amino acid. When such adjacent arginine or lysine residues are CPP motifs, no additional CPP motifs (e.g., those containing hydrophobic residues) are required, although such hydrophobic CPP motifs may still be used. When the CPP motif contains two arginine residues, it is preferred that there is another CPP motif with at least two hydrophobic residues within 2-8 amino acids.

[0071] In other examples, at least one, at least two, at least three, or more amino acids of the CPP motif are hydrophobic amino acids, ie, have a hydrophobic side chain. In some examples, the amino acids having hydrophobic side chains are independently selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, naphthylalanine, phenylglycine, homophenylalanine, tyrosine, cyclohexylalanine, piperidine-2-carboxylic acid, cyclohexylalanine, norleucine, 3-(3-benzothienyl)-alanine, 3-(2-quinolyl)-alanine, O-benzylserine, 3-(4-(benzyloxy)phenyl)-alanine, S-(4-methylbenzyl)cysteine, N-(naphthalene-2-yl)glutamine, 3-(1,1'-biphenyl-4-yl)-alanine, tert-leucine, or nicotinoyllysine, each of which is optionally substituted with one or more substituents. In certain examples, each amino acid having a hydrophobic side chain is independently an amino acid having an aromatic side chain. In some embodiments, the amino acid having an aromatic side chain is 3-benzothienyl-L-alanine, naphthylalanine, phenylglycine, homophenylalanine, phenylalanine, tryptophan, or tyrosine, each of which is optionally substituted with one or more substituents.Thus, in some examples, the amino acid having a hydrophobic side chain is phenylalanine, naphthylalanine, tryptophan, or an analog or derivative thereof.In other examples, the CPP motif further comprises at least one phenylalanine, phenylglycine, or histidine, or an analog or derivative thereof.

[0072] [ka]

[0073] In some examples, the CPP motif can include any combination of at least three adjacent arginines, at least two of the amino acids having hydrophobic side chains selected from aryl or heteroaryl, where the aryl and heteroaryl are optionally substituted, and the total number of amino acids in the CPP motif ranges from 5 to about 8 amino acids.

[0074] In some examples, the membrane translocation domain is human fibronectin type III having BC, DE, CD, and FG loops, and the CPP is in one or more of the BC, DE, CD, or FG loops. For example, the CPP motif is in two of the BC, DE, CD, or FG loops. In certain examples, the CPP motif is in either the BC, or the DE, CD, or FG loops, preferably in the BC and FG loops.

[0075] When there is more than one CPP motif, one CPP motif may be a 3-10 amino acid segment having at least two arginine and / or lysine residues and another may be a 2-8 amino acid segment having at least two hydrophobic residues. For example, a membrane translocation domain can have two or more CPPs, with at least one of the motifs being 2-8 amino acid residues and having at least two hydrophobic amino acid residues.

[0076] In this example, the membrane translocation domain may be human fibronectin type III having BC, DE, CD, and FG loops, and the CPP motif may be present in the BC loop, have 2-8 amino acid residues, and have at least two hydrophobic amino acid residues, and the CPP motif may be present in the FG loop, have 3-10 amino acid residues, and have at least three adjacent arginine and / or lysine residues. Alternatively, the CPP motif may be present in the FG loop, have 2-8 amino acid residues, and have at least two hydrophobic amino acid residues, and the CPP motif may be present in the BC loop, have 3-10 amino acid residues, and have at least three adjacent arginine and / or lysine residues.

[0077] When the CPP motif comprises 2-8 amino acid residues and has at least two hydrophobic amino acid residues, it can be WW, FF, WF, FW, WWW, FFF, WFW, FWF, WWF, WFF, FWW, FFW, WYW, WWH, YWW, or WYH. It is preferred that the CPP motif is in the BC loop. It is more preferred that the CPP motif is WW, FW, WF, WYW, WWW, WWH, YWW, WYH, or YWH.

[0078] CPP motifs having 3-10 amino acid residues and at least three adjacent arginine and / or lysine residues can include RRR, RRRR (SEQ ID NO: 159), RRRRR (SEQ ID NO: 160), or any combination of arginine and lysine residues. When this CPP motif is in the FG loop, I can be 3-10 residues in length and any combination of Arg and Lys (and possibly other non-acidic residues). The CPP motif (e.g., WWWRRRR) (SEQ ID NO:161) may be selectively split such that some of the Arg / Lys residues are moved from the FG loop to the BC loop (e.g., WWWR...RRR (SEQ ID NO:161), WWWRR...RR (SEQ ID NO:161), WWWRRRR... (SEQ ID NO:161), etc.), and the CPP motif (e.g., WWWRRRR) (SEQ ID NO:161) may be selectively split such that some of the hydrophobic residues are moved from the BC loop to the FG loop (e.g., WW...WRRR, W...WWRRRR, ...WWWRRRR, etc.) (SEQ ID NO:161). The CPP motif (e.g., WWWRRRR) may be selectively split such that either the BC loop or the FG loop contains a combination of hydrophobic and positively charged residues (e.g., WWR...WRRR, WWRR...WRR, WWRR...RRW, RRW...WWRR, etc.) (SEQ ID NO:161).

[0079] In particular examples, the CPP motif comprises SEQ ID NO: 104, 105, 11, 112, 113, 114, 115, 116, or 117.

[0080] In some examples, the CPP motif can be or include any of the sequences listed in Table 2. In some examples, the cell penetrating peptide can be or include the reverse sequence of any of the sequences listed in Table 2.

[0081] [Table 2] TIFF2025509645000008.tif221170TIFF2025509645000009.tif187170

[0082] The chirality of the amino acids can be selected to improve cytosolic uptake efficiency. In some embodiments, at least two of the amino acids have opposite chirality. In some embodiments, at least two amino acids with opposite chirality may be adjacent to each other. In some embodiments, at least three amino acids have alternating stereochemistry with respect to each other. In some embodiments, at least three amino acids with alternating chirality with respect to each other may be adjacent to each other. In some embodiments, at least two of the amino acids have the same chirality. In some embodiments, at least two amino acids with the same chirality may be adjacent to each other. In some embodiments, at least two amino acids have the same chirality and at least two amino acids have opposite chirality. In some embodiments, at least two amino acids with opposite chirality may be adjacent to at least two amino acids with the same chirality. Thus, in some embodiments, adjacent amino acids in a cCPP may have any of the following sequences: DL, LD, DLLD (SEQ ID NO: 153), LDDL (SEQ ID NO: 154), LDLLD (SEQ ID NO: 155), DLDDL (SEQ ID NO: 156), DLLDL (SEQ ID NO: 157), or LDDLD (SEQ ID NO: 158).

[0083] Cargo section Also described are peptides as disclosed herein, but further comprising a cargo moiety linked to the membrane translocation domain. The cargo moiety can be linked to an amino group (e.g., at the N-terminus), a carboxylate group (e.g., at the C-terminus), or to the side chain of one or more amino acids within the membrane translocation domain.

[0084] When a cargo moiety is attached to the side chain of an amino acid in the membrane translocation domain, the membrane translocation domain comprises an amino acid having a side chain with a suitable functional group for forming a covalent bond (conjugation) with the cargo, or a side chain that can be modified (e.g., via conjugation of a linker) to provide a suitable functional group for forming a covalent bond with the cargo. In some embodiments, the amino acid on the membrane translocation domain that has a side chain suitable for conjugation of the cargo is a cysteine ​​residue, a glutamic acid residue, an aspartic acid residue, a lysine residue, or a 2,3-diaminopropionic acid residue. In such embodiments, the cargo may be directly conjugated to the side chain of the amino acid (e.g., by forming a disulfide bond with a cysteine ​​residue, or an amide bond with a glutamic acid residue or a 2,3-diaminopropionic acid residue), or the cargo may be conjugated to the amino acid side chain via a linker (e.g., PEG).

[0085] The cargo moiety can include any cargo of interest, such as a linker moiety, a detectable moiety, a therapeutic moiety, a targeting moiety, etc., or any combination thereof. In some examples, the cargo moiety can include one or more additional amino acids (e.g., K, UK, TRV), a linker (e.g., the bifunctional linker LC-SMCC), coenzyme A, phosphocoumaryl aminopropionic acid (pCAP), 8-amino-3,6-dioxaoctanoic acid (miniPEG), L-2,3-diaminopropionic acid (Dap or J), L-β-naphthylalanine, L-pipecolic acid (Pip), sarcosine, trimesic acid, 7-amino-4-methylcoumarin (Amc), fluorescein isothiocyanate (FITC), L-2-naphthylalanine, norleucine, 2-aminobutyric acid, rhodamine B (Rho), dexamethasone (DEX), or a combination thereof.

[0086] Detectable Part Detectable moiety can include any detectable label. Examples of suitable detectable labels include, but are not limited to, UV-Vis label, near infrared label, luminescent group, phosphorescent group, magnetic spin resonance label, photosensitizer, photocleavable moiety, chelating center, heavy atom, radioisotope, isotopically detectable spin resonance label, paramagnetic moiety, chromophore, or any combination thereof. In some embodiments, the label can be detected without adding additional reagents.

[0087] In some embodiments, the detectable moiety is a biocompatible detectable moiety, such that the compound may be suitable for use in a variety of biological applications. As used herein, "biocompatible" and "biologically compatible" generally refer to a compound, together with any of its metabolic or decomposition products, that is generally non-toxic to cells and tissues and does not have any significant adverse effects on cells and tissues when the cells and tissues are incubated (e.g., cultured) in their presence.

[0088] The detectable moiety may comprise a luminophore, such as a fluorescent label or a near infrared label. Examples of suitable luminophores include, but are not limited to, metalloporphyrins, benzoporphyrins, azabenzoporphyrins, naphthoporphyrins, phthalocyanines, polycyclic aromatic hydrocarbons such as perylene, perylenediimine, pyrene, azo dyes, xanthene dyes, boron dipyrromethenes, aza-boron dipyrromethenes, cyanine dyes, metal-ligand complexes such as bipyridines, bipyridyls, phenanthrolines, coumarins, ruthenium and iridium acetylacetonates, oxazine derivatives such as acridines, benzophenoxazines, aza-annulenes, squaraines, luminescent nanoparticles, nanocrystals such as 8-hydroxyquinolines, polymethines, quantum dots, carbostyrils, terbium complexes, inorganic fluorophores, ionophores such as crown ether related or derivatized dyes, or combinations thereof. Specific examples of suitable luminophores include Pd(II) octaethylporphyrin, Pt(II)-octaethylporphyrin, Pd(II) tetraphenylporphyrin, Pt(II) tetraphenylporphyrin, Pd(II) meso-tetraphenylporphyrin tetrabenzoporphine, Pt(II) meso-tetraphenylmethylbenzoporphyrin, Pd(II) octaethylporphyrin ketone, Pt(II) octaethylporphyrin ketone, Pd(II) meso-tetra(pentafluorophenyl)porphyrin, Pt(II) meso-tetra(pentafluorophenyl)porphyrin, Ru(II) tris(4,7-diphenyl-1,10-phenanthroline) (Ru(dpp)3), Ru(II) tris(1,10-phenanthroline) (Ru(phen)3), tris(2,2'-Bipyridine)ruthenium(II) chloride hexahydrate (Ru(bpy)3), erythrosine B, fluorescein, fluorescein isothiocyanate (FITC), eosin, iridium(III)((N-methyl-benzimidazol-2-yl)-7-(diethylamino)-coumarin)), indium(III)((benzothiazol-2-yl)-7-(diethylamino)-coumarin)-2-(acetylacetonate), lumogen dyes, Macrolex fluorescent red , Macrolex Fluorescent Yellow, Texas Red, Rhodamine B, Rhodamine 6G, Sulfur Rhodamine, m-Cresol, Thymol Blue, Xylenol Blue, Cresol Red, Chlorophenol Blue, Bromocresol Green, Bromocresol Red, Bromothymol Blue, Cy2, Cy3, Cy5, Cy5.5, Cy7, 4-Nitrophenol, Alizarin, Phenolphthalein, o-Cresolphthalein, Chlorophenol Red, Calmagite , Bromo-xylenol, Phenol Red, Neutral Red, Nitrazine, 3,4,5,6-Tetrabromophenolphthalein, Congo Red, Fluorescein, Eosin, 2',7'-Dichlorofluorescein, 5(6)-Carboxy-fluorescein, Carboxynaphthofluorescein, 8-Hydroxypyrene-1,3,6-trisulfonic acid, Semi-naphthorodafluor, Semi-naphthofluorescein, Tris(4,7-diphenyl-1,10-phenanthroline), Examples of suitable fluorescein-activating agents include, but are not limited to, (4,7-diphenyl-1,10-phenanthroline)ruthenium(II) dichloride, (4,7-diphenyl-1,10-phenanthroline)ruthenium(II) tetraphenylborate, platinum(II) octaethylporphyrin, dialkylcarbocyanines, dioctadecylcycloxacarbocyanine, fluorenylmethyloxycarbonyl chloride, 7-amino-4-methylcoumarin (Amc), green fluorescent protein (GFP), and derivatives or combinations thereof.

[0089] In some examples, the detectable moiety can include rhodamine B (Rho), fluorescein isothiocyanate (FITC), 7-amino-4-methylcoumarin (Amc), green fluorescent protein (GFP), naphthofluorescein (NF), or derivatives or combinations thereof.

[0090] The detectable moiety can be attached to the cell-penetrating peptide moiety at an amino group, a carboxylate group, or a side chain of any amino acid in the cell-penetrating peptide moiety (e.g., an amino group, a carboxylate group, or a side chain of any amino acid in a CPP).

[0091] therapeutic part The disclosed compounds can also include a therapeutic moiety. In some examples, the cargo moiety includes a therapeutic moiety. The detectable moiety can be linked to the therapeutic moiety, or the detectable moiety can also function as a therapeutic moiety. Therapeutic moiety refers to a group that, when administered to a subject, relieves one or more symptoms of a disease or disorder.

[0092] Therapeutic moieties can include a variety of agents, including antagonists, e.g., enzyme inhibitors, and agonists, e.g., transcription factors that increase expression of a desired gene product (although as will be appreciated by those of skill in the art, antagonistic transcription factors can also be used). Additionally, therapeutic moieties include agents that can directly exert and / or induce toxicity to healthy and / or unhealthy cells in the body. Therapeutic moieties can also induce and / or prime the immune system against potential pathogens.

[0093] The therapeutic moiety can include, for example, an anti-cancer agent, an anti-viral agent, an anti-bacterial agent, an anti-inflammatory agent, an immunosuppressant agent, an anesthetic agent, or any combination thereof.

[0094] In some examples, the therapeutic moiety may be a tumor suppressor, a small molecule or peptide-based inhibitor, an enzyme for intracellular enzyme replacement therapy, an oligonucleotide.

[0095] The therapeutic moiety can include an anti-cancer drug. Exemplary anti-cancer drugs include 13-cis-retinoic acid, 2-amino-6-mercaptopurine, 2-CdA, 2-chlorodeoxyadenosine, 5-fluorouracil, 6-thioguanine, 6-mercaptopurine, accutane, actinomycin-D, adriamycin, adrsil, agrylin, ara-cort, aldesleukin, alemtuzumab, alitretinoin, Alkaban-AQ, Alkeran, all-trans retinoic acid, alpha interferon, altretamine, amethopterin, amifostine ... Noglutethimide, anagrelide, anandrone, anastrozole, arabinosylcytosine, Aranesp, Aredia, Arimidex, aromasin, arsenic trioxide, asparaginase, ATRA, Avastin, BCG, BCNU, bevacizumab, bexarotene, bicalutamide, BiCNU, blenoxane, bleomycin, bortezomib, busulfan, busulfex, C225, leucovorin calcium, Campas, Camptosar, camptothecin-11, capecitabine, Carac, carboplatin, carmustine, Cal Mustine wafer, Casodex, CCNU, CDDP, CeeNU, Cervidine, Cetuximab, Chlorambucil, Cisplatin, Citrovorum factor, Cladribine, Cortisone, Cosmegen, CPT-11, Cyclophosphamide, Cytadren, Cytarabine, Cytarabine liposome, Cytosar U, Cytoxan, Dacarbazine, Dactinomycin, Darbepoetin alfa, Daunomycin, Daunorubicin, Daunorubicin hydrochloride, Daunorubicin liposome, Daunoxome, Decadron, Delta-Cortef, Deltazone, Deniroy Quindiftitox, Depocyto, Dexamethasone, Dexamethasone acetate, Dexamethasone sodium phosphate, Dexasone, Dexrazoxane, DHAD, DIC, Geodex, Docetaxel, Doxil, Doxorubicin, Doxorubicin liposome, Droxia, DTIC, DTIC-Dome, Duralon, Efudex, Eligard, Elence, Eloxatin, Elspar, Emcyto, Epirubicin, Epoetin alfa, Erbitux, Erwinia L-asparaginase, Estramustine, Ethiol, Etopophos, Etoposide,Etoposide phosphate, Eurexin, Evista, Exemestane, Fareston, Faslodex, Femara, Filgrastim, Floxuridine, Fludara, Fludarabine, Fluoroplex, Fluorouracil, Fluorouracil (cream), Fluoxymesterone, Flutamide, Folinic acid, FUDR, Fulvestrant, G-CSF, Gefitinib, Gemcitabine, Gemtuzumab ozogamicin, Gemzar, Gleevec, Leuprorelin, Leuprorelin Depot, Matulane, Maxidex, Mechlorethamine, -Mechlorethamine hydrochloride, Medralon, Medrol, Megas, Megestrol, Megestrol acetate, Melphalan, Mercaptopurine, Mesna, Mesnex, Methotrexate, Methotrexate sodium, Methylprednisolone, Mirocel, Letrozole, Neosar, Neulasta, Neumega, Neupogen, Nilandrone, Nilutamide, Nitrogen Mustard, Novardex, Novantrone, Octreotide, Octreotide acetate, Oncasper, Oncovin, Ontac, Onxal, Oprevelkin, Olap Orapred, Orasone, Oxaliplatin, Paclitaxel, Pamidronate, Panretin, Paraplatin, Pediapred, PEG-Interferon, Pegaspargase, Pegfilgrastim, PEG-Intron, PEG-L-Asparaginase, Phenylalanine Mustard, Platinol, Platinol-AQ, Prednisolone, Prednisone, Prelon, Procarbazine, PROCRIT, Proleukin, Prola with Carmustine Implant Prolifeprospan 20, Purinethol, Raloxifene, Rheumatrex, Rituxan, Rituximab, Roveron-A (Interferon alpha-2a), Rubex, Rubidomycin hydrochloride, Sandostatin, Sandostatin LAR, Sargramostim, Solcortef, Solumedrol, STI-571, Streptozocin, Tamoxifen, Targretin, Taxol, Taxotere, Temodar, Temozolomide, Teniposide, TESPA, Thalidomide, Talomid,TheraCys, Thioguanine, Thioguanine Tabloid, Thiophosphoamide, Thioplex, Thiotepa, TICE, Toposar, Topotecan, Toremifene, Trastuzumab, Tretinoin, Trexall, Trisenox, TSPA, VCR, Velban, Velcade, Bepcid, Vesanoid, Viadur, Vinblastine, Vinblastine Sulfate, Vincasar Pfs Pfs), vincristine, vinorelbine, vinorelbine tartrate, VLB, VP-16, Vumon, Xeloda, Zanosar, Zevalin, Zincard, Zoladex, zoledronic acid, Zometa, Gliadel wafer, Gleevec, GM-CSF, goserelin, granulocyte colony-stimulating factor, halotestone, Herceptin, hexadrol, hexalen, hexamethylmelamine, HMM, Hycamtin, Hydrea, hydrocort acetate, hydrocortisone, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, hydrocortone phosphate, hydroxyurea, ibritumomab, ibritumomab tiuxetan, idamycin, idarubicin, ifex, IFN-alpha, ifosfamide, IL2, IL -11, imatinib mesylate, imidazole carboxamide, interferon alpha, interferon alpha-2b (PEG conjugate), interleukin 2, interleukin-11, intron A (interferon alpha-2b), leucovorin, leukelan, leukin, leuprolide, leurocristin, leustatin, liposomal Ara-C, liquid pred, lomustine, L-PAM, L-sarcolysin, methycortene, mitomycin, mitomycin-C, mitoxantrone, M-prednisole, MTC, MTX, mustagen, mustine, mutamycin, myleran, Iressa, irinotecan, isotretinoin, quidrolase, lanacort, L-asparaginase, and LCR. Therapeutic moieties can also include biopharmaceuticals, such as antibodies.

[0096] In some examples, the therapeutic moiety can include an antiviral agent, such as ganciclovir, azidothymidine (AZT), or lamivudine (3TC).

[0097] In some instances, the therapeutic moiety is an antibacterial agent, e.g., acedapsone, acetosulfone sodium, alamethicin, alexidine, amdinocillin, amdinocillin pivoxil, amicycline, amifloxacin, amifloxacin mesylate, amikacin, amikacin sulfate, aminosalicylic acid, aminosalicylic acid sodium, amoxicillin, amphomycin, ampicillin, ampicillin sodium, apalcillin sodium, apramycin, aspartocin, astromycin sulfate, avilamycin, avoparcin, azithromycin, azuromy ... phosphorus, azlocillin sodium, bacampicillin hydrochloride, bacitracin, bacitracin methylene disalicylate, bacitracin zinc, bambermycin, benzoylpas calcium, berithromycin, betamycin sulfate, biapenem, vinilamycin, biphenamine hydrochloride, bispyrithione magsulfex, buticacin, butirocin sulfate, capreomycin sulfate, carbadox, carbenicillin disodium, carbenicillin indanyl sodium, carbenicillin phenyl sodium, carbenicillin potassium, carmonam sodium, cephalosporin ... Chlor, Cefadroxil, Cefamandole, Cefamandole Nafat, Cefamandole Sodium, Cefaparol, Cefatrizine, Cefazaflour Sodium, Cefazolin, Cefazolin Sodium, Cefbuperazone, Cefdinir, Cefepime, Cefepime Hydrochloride, Cefetecol, Cefixime, Cefmenoxime Hydrochloride, Cefmetazole, Cefmetazole Sodium, Cefonicid Monosodium, Cefonicid Sodium, Cefoperazone Sodium, Ceforanide, Cefotaxime Sodium, Cefotetan, Cefotetan Dinatri um, cefotiam hydrochloride, cefoxitin, cefoxitin sodium, cefpimizole, cefpimizole sodium, cefpiramide, cefpiramide sodium, cefpirome sulfate, cefpodoxime proxetil, cefprozil, cefroxadine, cefsulodin sodium, ceftazidime, ceftibuten, ceftizoxime sodium, ceftriaxone sodium, cefuroxime, cefuroxime axetil, cefuroxime pivoxetil, cefuroxime sodium, cephacetrile sodium, cephalexin, cephalexin hydrochloride, cephaloglycin,Cephaloridine, cephalothin sodium, cephapirin sodium, cephradine, cetocycline hydrochloride, cetophenicol, chloramphenicol, chloramphenicol palmitate, chloramphenicol pantothenate complex, chloramphenicol sodium succinate, chlorhexidine phosphanilate, chloroxylenol, chlortetracycline bisulfate, chlortetracycline hydrochloride, cinoxacin, ciprofloxacin, ciprofloxacin hydrochloride, ciloremycin, clarithromycin, clinafloxacin hydrochloride, clindamycin, Clindamycin hydrochloride, clindamycin hydrochloride, clindamycin palmitate hydrochloride, clindamycin phosphate, clofazimine, cloxacillin benzathine, cloxacillin sodium, cloxyquin, colistin methanesulfonate sodium, colistin sulfate, coumermycin, coumermycin sodium, cyclacillin, cycloserine, dalfopristin, dapsone, daptomycin, demeclocycline, demeclocycline hydrochloride, demecycline, denofungin, diaveridine, dicloxacillin, dicloxacillin sodium, sulfate Dihydrostreptomycin, Dipyrithione, Dirithromycin, Doxycycline, Doxycycline calcium, Doxycycline phosphatex, Doxycycline hydrate, Droxacin sodium, Enoxacin, Epicillin, Epitetracycline hydrochloride, Erythromycin, Erythromycin acistrate, Erythromycin estolate, Erythromycin ethylsuccinate, Erythromycin gluceptate, Erythromycin lactobionate, Erythromycin propionate, Erythromycin stearate, Ethambutol lecithin hydrochloride, ethionamide, fleroxacin, floxacillin, fludalanine, flumequine, fosfomycin, fosfomycin tromethamine, fumoxicillin, furazolium chloride, furazolium tartrate, sodium fusidate, fusidic acid, gentamicin sulfate, gloximonam, gramicidin, haloprogin, hetacillin, hetacillin potassium, hexedine, ivafloxacin, imipenem, isoconazole, isepamicin, isoniazid, josamycin, kanamycin sulfate, kitasamycin, levofuraltadone, levopropylcillin potassium,Lexithromycin, Lincomycin, Lincomycin hydrochloride, Lomefloxacin, Lomefloxacin hydrochloride, Lomefloxacin mesylate, Loracarbef, Mafenide, Meclocycline, Meclocycline sulfosalicylate, Megalomycin potassium phosphate, Mequidox, Meropenem, Methacycline, Methacycline hydrochloride, Methenamine, Methenamine hippurate, Methenamine mandelate, Methicillin sodium, Methioprim, Metronidazole hydrochloride, Metronidazole phosphate, Mezlocillin, Mezlocillin sodium, Minocycline, Minocycline Phosphate hydrochloride, mirincamycin hydrochloride, monensin, monensin sodium, nafcillin sodium, nalidixic acid sodium, nalidixic acid, natainisin, nebramycin, neomycin palmitate, neomycin sulfate, neomycin undecylenate, netilmicin sulfate, neuthramycin, niflamide, nifuraldeson, nifuratel, nifuratron, nifurdazil, nifurimide, nifiupirinol, nifluquinazole, nifurthiazole, nitrocycline, nitrofurantoin, nitromide, norfloxacin cin, novobiocin sodium, ofloxacin, onnetoprim, oxacillin, oxacillin sodium, oximonam, oximonam sodium, oxolinic acid, oxytetracycline, oxytetracycline calcium, oxytetracycline hydrochloride, paldimycin, parachlorophenol, paulomycin, pefloxacin, pefloxacin mesylate, penamecillin, penicillin G benzathine, penicillin G potassium, penicillin G procaine, penicillin G sodium, penicillin V, penicillin V benzathine, penicillin V hydraba amine, penicillin V potassium, pentizidone sodium, phenyl aminosalicylate, piperacillin sodium, pirbenicillin sodium, pyridicillin sodium, pirrimycin hydrochloride, pivampicillin hydrochloride, pivampicillin pamoate, pivampicillin probenate, polymyxin B sulfate, porfiromycin, propikacin, pyrazinamide, pyrithione zinc, quindecamin acetate, quinupristin, racephenicol, ramoplanin, ranimicin, relomycin, repromycin, rifabutin, rifametan, rifamexyl, rifamide,Rifampin, Rifapentine, Rifaximin, Rolitetracycline, Rolitetracycline Nitrate, Rosaramycin, Rosaramycin Butyrate, Rosaramycin Propionate, Rosaramycin Sodium Phosphate, Rosaramycin Stearate, Rosoxacin, Roxarsone, Roxithromycin, Sancycline, Sanfetrinem Sodium, Salmoxicillin, Salpicillin, Scopafungin, Sisomicin, Sisomicin Sulfate, Sparfloxacin, Spectinomycin Salt acid salt, spiramycin, stalmycin hydrochloride, stefimycin, streptomycin sulfate, streptonicozide, sulfabenz, sulfabenzamide, sulfacetamide, sulfacetamide sodium, sulfacytine, sulfadiazine, sulfadiazine sodium, sulfadoxine, sulfalene, sulfamerazine, sulfameth, sulfamethazine, sulfamethizole, sulfamethoxazole, sulfamonomethoxine, sulfamoxole, sulfamate Phanilate zinc, sulfanitran, sulfasalazine, sulfasomizole, sulfathiazole, sulfazameth, sulfisoxazole, acetylsulfisoxazole, sulfisoxazole diolamine, sulfomixin, sulopenem, sultamicillin, sancillin sodium, talampicillin hydrochloride, teicoplanin, temafloxacin hydrochloride, temocillin, tetracycline, tetracycline hydrochloride, tetracycline phosphate complex, tetroxoprim, thiazolamide ... The following may be included: amphenicol, tiphencillin potassium, ticarcillin clecil sodium, ticarcillin disodium, ticarcillin monosodium, ticlaton, thiodonium chloride, tobramycin, tobramycin sulfate, tosufloxacin, trimethoprim, trimethoprim sulfate, trisulfapyrimidine, troleandomycin, trospectomycin sulfate, tyrothricin, vancomycin, vancomycin hydrochloride, virginiamycin, or zorbamycin.

[0098] In some instances, the therapeutic moiety can include an anti-inflammatory agent.

[0099] In some examples, the therapeutic moiety can include dexamethasone (Dex).

[0100] In other examples, the therapeutic moiety comprises a therapeutic protein. The protein may be genetically fused to the N-terminus or C-terminus of the MTD. Synthetic peptides containing unnatural amino acids may also be chemically conjugated to side chains, e.g., unique cysteines at the C-terminus of the MTD. Disclosed herein is the delivery of enzymes / proteins to human cells by linking such enzymes / proteins to one of the disclosed cell-penetrating peptide motifs or MTDs. The disclosed cell-penetrating peptide motifs have been tested with proteins (e.g., GFP, PTP1B, actin, calmodulin, troponin C) and shown to function.

[0101] targeting part In some examples, the therapeutic moiety comprises a targeting moiety. The targeting moiety can, for example, comprise a series of amino acids that can target one or more enzyme domains. In some examples, the targeting moiety can comprise an inhibitor for an enzyme that can play a role in diseases such as cancer, cystic fibrosis, diabetes, obesity, or a combination thereof. For example, the targeting moiety can comprise any of the sequences listed in Table 3.

[0102] [Table 3] TIFF2025509645000011.tif220170TIFF2025509645000012.tif52170

[0103] The targeting moiety and the cell-penetrating peptide moiety can overlap, i.e., the residues that form the cell-penetrating peptide moiety can also be part of the sequence that forms the targeting moiety, and vice versa.

[0104] The therapeutic moiety can be attached to the cell-penetrating peptide moiety at an amino group, a carboxylate group, or a side chain of any amino acid of the cell-penetrating peptide moiety (e.g., an amino group, a carboxylate group, or a side chain of any amino acid of a CPP). In some examples, the therapeutic moiety can be attached to a detectable moiety.

[0105] In some examples, the therapeutic moiety can include a targeting moiety that can act as an inhibitor against Ras (e.g., K-Ras), PTP1B, Pin1, Grb2 SH2, CAL PDZ, etc., or a combination thereof.

[0106] Ras is a protein that in humans is encoded by the RAS gene. Normal Ras proteins play a critical role in normal tissue signaling, and mutations in the Ras gene are involved in the development of many cancers. Ras can function as a molecular on / off switch, and once turned on, Ras recruits and activates proteins necessary for the propagation of growth factor and other receptor signals. Mutant forms of Ras have been implicated in a variety of cancers, including lung, colon, and pancreatic cancers, and various leukemias.

[0107] Protein tyrosine phosphatase 1B (PTP1B) is a prototypical member of the PTP superfamily and plays various roles in eukaryotic cell signaling. PTP1B is a negative regulator of the insulin signaling pathway and is considered a promising potential therapeutic target, especially for the treatment of type 2 diabetes. PIP1B is also involved in the development of breast cancer.

[0108] Pin1 is an enzyme that binds to a subset of proteins and plays a role as a post-phosphorylation control in regulating protein function. Pin1 activity can regulate the outcome of proline-directed kinase signaling, which in turn can regulate cell proliferation and cell survival. Deregulation of Pin1 can play a role in a variety of diseases. Upregulation of Pin1 may be involved in certain cancers, and downregulation of Pin1 may be involved in Alzheimer's disease. Inhibitors of Pin1 may have therapeutic implications for the treatment of cancer and immune disorders.

[0109] Grb2 is an adaptor protein involved in signal transduction and cell signaling. The Grb2 protein contains one SH2 domain that can bind to tyrosine phosphorylated sequences. Grb2 is widely expressed and is essential for multiple cellular functions. Inhibition of Grb2 function can impair developmental processes and block transformation and proliferation of various cell types.

[0110] Recently, it was reported that the activity of the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride ion channel protein mutated in cystic fibrosis (CF) patients, is negatively regulated by the CFTR-associated ligand (CAL) through its PDZ domain (CAL-PDZ) (Wolde,M et al. J.Biol.Chem.2007,282,8099). Inhibition of the CFTR / CAL-PDZ interaction was shown to improve the activity of ΔPhe508-CFTR, the most common form of CFTR mutation, by reducing its proteasome-mediated degradation (Cushing,PR et al. Angew.Chem.Int.Ed.2010,49,9907) (Cheng,SH et al.Cell 1990,63,827; Kerem,BS et al.Science 1989,245,1073). Thus, disclosed herein is a method for treating a subject suffering from cystic fibrosis by administering an effective amount of a compound or composition disclosed herein.The compound or composition administered to the subject can include a therapeutic moiety, which can include a targeting moiety that can act as an inhibitor against CAL PDZ.

[0111] In some examples, the therapeutic moiety is a nucleic acid. In some embodiments, the nucleic acid is an antisense compound. In some embodiments, the antisense compound is selected from the group consisting of an antisense oligonucleotide, a small interfering RNA (siRNA), a microRNA (miRNA), a ribozyme, an immunostimulatory nucleic acid, an antagomir, an antimir, a microRNA mimic, a supermir, a U1 adaptor, and an aptamer.

[0112] Also disclosed herein are compositions that include the compounds described herein.

[0113] Also disclosed herein are pharma- ceutically acceptable salts and prodrugs of the disclosed compounds. Pharmaceutically acceptable salts include salts of the disclosed compounds prepared with acids or bases, depending on the particular substituents found on the compound. Under conditions where the compounds disclosed herein are sufficiently basic or acidic to form stable non-toxic acid or base salts, it may be appropriate to administer the compounds as salts. Examples of pharma- ceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, or magnesium salts. Examples of physiologically acceptable acid addition salts include hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, carbonic acid, sulfuric acid, and organic acids (acetic acid, propionic acid, benzoic acid, succinic acid, fumaric acid, mandelic acid, oxalic acid, citric acid, tartaric acid, malonic acid, ascorbic acid, α-ketoglutaric acid, α-glycolic acid, maleic acid, tosylic acid, methanesulfonic acid, and the like). Thus, disclosed herein are hydrochloride, nitrate, phosphate, carbonate, bicarbonate, sulfate, acetate, propionate, benzoate, succinate, fumarate, mandelate, oxalate, citrate, tartrate, malonate, ascorbate, α-ketoglutarate, α-glycolic acid, maleate, tosylate, and mesylate salts. Pharmaceutically acceptable salts of compounds can be obtained by reacting a sufficiently basic compound, such as an amine, with a suitable acid that provides a physiologically acceptable anion, using standard procedures well known in the art. Alkali metal (e.g., sodium, potassium, or lithium) or alkaline earth metal (e.g., calcium) salts of carboxylic acids can also be prepared.

[0114] Linker In various embodiments, the linker is covalently attached to an amino acid on the membrane translocation domain. The linker can be any moiety that conjugates two or more of the membrane translocation domains to the cargo moiety. In some embodiments, the linker can be an amino acid. In other embodiments, the precursor of the linker can be any suitable molecule that can form two or more bonds with the amino acids in the membrane translocation domain and the cargo moiety. Thus, in various embodiments, the precursor of the linker has two or more functional groups, each of which can form a covalent bond with the membrane translocation domain and the cargo moiety. For example, the linker can be covalently attached to the N-terminus, C-terminus, or side chain, or a combination thereof, of any amino acid in the membrane translocation domain. In certain embodiments, the linker forms a covalent bond between the membrane translocation domain and the cargo moiety.

[0115] In some embodiments, the linker is selected from the group consisting of at least one amino acid, alkylene, alkenylene, alkynylene, aryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, heteroaryl, ether, each of which may be optionally substituted as defined above. For example, each of these linkers may be 1-500 atoms in length, e.g., 1-100, 1-250, 10-200, 25-300 atoms in length. Non-limiting examples of linkers include polyethylene glycol, optionally conjugated to a lysine residue. In other examples, the linker is a divalent or trivalent C1-C 50It may be a saturated or unsaturated straight or branched chain alkyl, where 1 to 25 methylene groups are optionally and independently replaced by -N(H)-, -N(C1-C4 alkyl)-, -N(cycloalkyl)-, -O-, -C(O)-, -C(O)O-, -S-, -S(O)-, -S(O)2-, -S(O)2N(C1-C4 alkyl)-, -S(O)2N(cycloalkyl)-, -N(H)C(O)-, -N(C1-C4 alkyl)C(O)-, -N(cycloalkyl)C(O)-, -C(O)N(H)-, -C(O)N(C1-C4 alkyl), -C(O)N(cycloalkyl), aryl, heteroaryl, cycloalkyl, or cycloalkenyl.

[0116] In some embodiments, the linker is covalently attached to the N-terminus or C-terminus of an amino acid on the CPP motif, or to the side chain of glutamine, asparagine, or lysine, or to a modified side chain of glutamine or asparagine (e.g., a reduced side chain bearing an amino group). In certain embodiments, the linker forms a bond with the side chain of glutamine on the CPP motif. In other specific embodiments, the linkers described herein have the structure L-1 or L-2,

[0117] [ka] During the ceremony, AA s is the side chain or terminus of an amino acid on a peptide or staple, AA c is the side chain or terminus of an amino acid in a cCPP, p is an integer from 0 to 10; q is an integer from 1 to 50.

[0118] In other embodiments, the linker may be a proteolytically stable peptide sequence, such as (GGS)n, (GGGS)n, (GSS)n, or (PAS)n, where n is 0-100.

[0119] In some embodiments, the linker can release the cargo moiety from the membrane translocation domain after the polypeptide conjugate enters the cytosol of a cell. In some embodiments, the linker comprises or forms a group after attachment to the membrane translocation domain and the cargo moiety, which is cleaved after cytosolic uptake of the polypeptide conjugate, thereby releasing the cargo moiety. Non-limiting examples of physiologically cleavable linking groups include carbonates, thiocarbonates, thioethers, thioesters, disulfides, sulfoxides, hydrazines, protease-cleavable dipeptide linkers, and the like.

[0120] For example, in embodiments, the linker is covalently attached to the membrane translocation domain via a disulfide bond with the side chain of a cysteine ​​or cysteine ​​analogue, for example, located in the membrane translocation domain or cargo moiety. In some embodiments, the disulfide bond is formed between a thiol group on the precursor of the linker and a side chain of a cysteine ​​or amino acid analogue having a thiol group on the peptide, where the bond to the hydrogen on each thiol group is replaced with a bond to a sulfur atom. Non-limiting examples of amino acid analogues having a thiol group that can be used with the polypeptide conjugates disclosed herein are discussed above.

[0121] Manufacturing method The compounds described herein can be prepared by various methods known to those skilled in the art of organic synthesis, or variations thereof as will be understood by those skilled in the art.The compounds described herein can be prepared from readily available starting materials.Optimal reaction conditions may vary depending on the specific reactants or solvents used, but such conditions can be determined by those skilled in the art.

[0122] Modification of the compounds described herein includes the addition, removal, or movement of various moieties described for each compound. Similarly, if there is one or more chiral centers in the molecule, the chirality of the molecule may change. Furthermore, the synthesis of the compounds may include the protection and deprotection of various chemical groups. The use of protection and deprotection, and the selection of suitable protecting groups, can be determined by those skilled in the art. The chemistry of protecting groups can be found, for example, in Wuts and Greene, Protective Groups in Organic Synthesis, 4th Ed., Wiley & Sons, 2006 (incorporated herein in its entirety by reference).

[0123] Starting materials and reagents used in preparing the disclosed compounds and compositions may be purchased from commercial suppliers, such as Aldrich Chemical Co., (Milwaukee, WI), Acros Organics (Morris Plains, NJ), Fisher Scientific (Pittsburgh, PA), Sigma (St. Louis, MO), Pfizer (New York, NY), GlaxoSmithKline (Raleigh, NC), Merck (Whitehouse Station, NJ), Johnson & Johnson (New Brunswick, NJ), Aventis (Bridgewater, NJ), AstraZeneca (Wilmington, DE), Novartis (Basel, Switzerland), Wyeth (Madison, NJ), Bristol-Myers-Squibb (New York, NY), Roche (Basel, Switzerland), Lilly (Indianapolis, IN), Abbott (Abbott Park, IL), Schering, and others. Plough (Kenilworth, NJ), or Boehringer Ingelheim (Ingelheim, Germany), or may be found in Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991), Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989), Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), March's Advanced Organic Chemistry (John Wiley and Sons, 4th Edition), and Larock's Comprehensive Organic Transformations (VCH Publishers Inc.The pharmaceutical compositions are prepared by methods known to those skilled in the art following procedures described in references such as The Pharmaceutical Journal, 1989. Other materials, such as pharmaceutical carriers, disclosed herein may be obtained from commercial sources.

[0124] The reactions to produce the compounds described herein can be carried out in a solvent, which can be selected by one skilled in the art of organic synthesis. The solvent can be substantially non-reactive with the starting materials (reactants), intermediates, or products under the conditions, i.e., temperature and pressure, at which the reaction is carried out. The reaction can be carried out in one solvent or a mixture of multiple solvents. The formation of the product or intermediate can be monitored according to any suitable method known in the art. For example, the formation of the product can be monitored by nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C), by spectroscopic means such as infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry, or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography.

[0125] The disclosed compounds can be prepared by expression and purification like any other protein. See Chen, K., & Pei, D. (2020). Engineering Cell-Permeable Proteins through Insertion of Cell-Penetrating Motifs into Surface Loops. ACS chemical biology, 15(9), 2568-2576 is incorporated by reference in its entirety for teachings on how to prepare proteins. Other methods for preparing the disclosed compositions include solid-phase peptide synthesis in which the amino acid α-N-terminus is protected by an acid-sensitive or base-sensitive protecting group. Such protecting groups must have the properties of being stable to the conditions of peptide bond formation while at the same time being easily removable without disrupting the growing peptide chain or racemizing any of the chiral centers contained therein. Suitable protecting groups include 9-fluorenylmethyloxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl, 2-cyano-t-butyloxycarbonyl, etc. The 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group is particularly preferred for the synthesis of the disclosed compounds.Other preferred side chain protecting groups are 2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc), nitro, p-toluenesulfonyl, 4-methoxybenzene-sulfonyl, Cbz, Boc, and adamantyloxycarbonyl for side chain amino groups such as lysine and arginine, benzyl, o-bromobenzyloxycarbonyl, 2,6-dichlorobenzyl, isopropyl, t-butyl (t-Bu), cyclohexyl, cyclopenyl, and acetyl (Ac) for tyrosine, t-butyl, benzyl, and tetrahydropyranyl for serine, trityl, benzyl, Cbz, p-toluenesulfonyl, and 2,4-dinitrophenyl for histidine, formyl for tryptophan, benzyl and t-butyl for aspartic acid and glutamic acid, and triphenylmethyl (trityl) for cysteine. In solid phase peptide synthesis, the α-C-terminal amino acid is attached to a suitable solid support or resin. Suitable solid supports useful for the above synthesis are those materials that are inert to the reagents and reaction conditions of the stepwise condensation-deprotection reactions and are insoluble in the media used. Solid supports for the synthesis of α-C-terminal carboxypeptides are 4-hydroxymethylphenoxymethyl copoly(styrene-1% divinylbenzene) or 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidoethyl resin available from Applied Biosystems (Foster City, Calif.).The α-C-terminal amino acid is coupled to the resin by mediated coupling using N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), or O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) in the presence or absence of 4-dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBT), benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) or bis(2-oxo-3-oxazolidinyl)phosphine chloride (BOPCl) in a solvent such as dichloromethane or DMF at a temperature of 10 °C to 50 °C for about 1 to about 24 hours. When the solid support is 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin, the Fmoc group is cleaved with a secondary amine, preferably piperidine, before coupling with the α-C-terminal amino acid as described above. One method of coupling to the deprotected 4(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin is O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU, 1 equivalent) and 1-hydroxybenzotriazole (HOBT, 1 equivalent) in DMF. The coupling of successive protected amino acids can be carried out in an automated polypeptide synthesizer. In one example, the α-N-terminus of the amino acid of the growing peptide chain is protected with Fmoc. Removal of the Fmoc protecting group from the α-N-terminal side of the growing peptide is accomplished by treatment with a secondary amine, preferably piperidine. Each protected amino acid is then introduced in about a three-fold molar excess and the coupling is preferably carried out in DMF. The coupling agents can be O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU, 1 equivalent) and 1-hydroxybenzotriazole (HOBT, 1 equivalent). At the end of the solid phase synthesis, the polypeptide is removed from the resin and deprotected, either sequentially or in a single operation.Removal and deprotection of the polypeptide can be accomplished in a single operation by treating the resin-bound polypeptide with a cleavage reagent containing thianisole, water, ethanedithiol, and trifluoroacetic acid. If the α-C-terminus of the polypeptide is an alkylamide, the resin is cleaved by aminolysis with an alkylamine. Alternatively, the peptide can be removed by transesterification, for example with methanol, followed by aminolysis or direct transamidation. The protected peptide can be purified at this point or used directly in the next step. Removal of side chain protecting groups can be accomplished using the cleavage cocktail described above. The fully deprotected peptide can be purified by a series of chromatographic steps using any or all of the following types: ion exchange on a weakly basic resin (acetate form), hydrophobic adsorption chromatography on underivatized polystyrene-divinylbenzene (e.g., Amberlite XAD), silica gel adsorption chromatography, ion exchange chromatography on carboxymethylcellulose, partition chromatography or countercurrent distribution on e.g. Sephadex G-25, LH-20, high performance liquid chromatography (HPLC), especially reversed-phase HPLC on octyl-silica or octadecylsilyl-silica bonded phase column packings.

[0126] How to use Also provided herein are methods of using the compounds or compositions described herein.Also provided herein are methods for treating a disease or condition in a subject in need of treatment, comprising administering to the subject an effective amount of any of the compounds or compositions described herein.

[0127] Also provided herein is a method of treating, preventing, or ameliorating cancer in a subject. The method includes administering to the subject an effective amount of one or more of the compounds or compositions described herein, or pharma- ceutically acceptable salts thereof. The compounds and compositions described herein, or pharma- ceutically acceptable salts thereof, are useful for treating cancer in humans (e.g., pediatric and geriatric populations) and animals (e.g., veterinary applications). The disclosed methods can optionally include identifying a patient who is in need of, or may be in need of, cancer treatment. Examples of types of cancer treatable by the compounds and compositions described herein include bladder cancer, brain cancer, breast cancer, colon cancer, cervical cancer, gastrointestinal cancer, genitourinary cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, skin cancer, and testicular cancer. Further examples include cancers and / or tumors of the anus, bile duct, bone, bone marrow, intestine (including colon and rectum), eye, gallbladder, kidney, mouth, larynx, esophagus, stomach, testes, cervix, mesothelioma, neuroendocrine, penis, skin, spinal cord, thyroid, vagina, vulva, uterus, liver, muscle, blood cells (including lymphocytes and other immune system cells). Further examples of cancers treatable by the compounds and compositions described herein include carcinoma, Karposi's sarcoma, melanoma, mesothelioma, soft tissue sarcoma, pancreatic cancer, lung cancer, leukemia (acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, and others), lymphoma (Hodgkin's lymphoma and non-Hodgkin's lymphoma), and multiple myeloma.

[0128] The methods of treating or preventing cancer described herein can further include treatment with one or more additional agents (e.g., anti-cancer agents or ionizing radiation). The one or more additional agents and the compounds and compositions described herein or pharma- ceutically acceptable salts thereof can be administered in any order, including simultaneous administration and administration spaced apart in time by up to several days. The methods can also include multiple administrations of one or more additional agents and / or the compounds and compositions described herein or pharma- ceutically acceptable salts thereof. The administration of the one or more additional agents and the compounds and compositions described herein or pharma- ceutically acceptable salts thereof can be by the same route or different routes. When treating with one or more additional agents, the compounds and compositions described herein or pharma- ceutically acceptable salts thereof can be combined into a pharmaceutical composition comprising the one or more additional agents.

[0129] For example, the compounds or compositions described herein, or pharma- ceutically acceptable salts thereof, may be used in combination with additional anticancer agents, such as, for example, 13-cis-retinoic acid, 2-amino-6-mercaptopurine, 2-CdA, 2-chlorodeoxyadenosine, 5-fluorouracil, 6-thioguanine, 6-mercaptopurine, accutane, actinomycin-D, adriamycin, adrsil, agrylin, ara-cort, aldesleukin, alemtuzumab, alitretinoin, Alkaban-AQ, Alkeran, all-trans retinoic acid, alpha Interferon, altretamine, amethopterin, amifostine, aminoglutethimide, anagrelide, anandrone, anastrozole, arabinosylcytosine, aranesp, aredia, arimidex, aromasin, arsenic trioxide, asparaginase, ATRA, avastin, BCG, BCNU, bevacizumab, bexarotene, bicalutamide, BiCNU, blenoxane, bleomycin, bortezomib, busulfan, busulfex, C225, leucovorin calcium, campas, camptosar, camptothecin-11, capecitabine, Carac, Carboplatin, Carmustine, Carmustine Wafer, Casodex, CCNU, CDDP, CeeNU, Cervizin, Cetuximab, Chlorambucil, Cisplatin, Citrovorum Factor, Cladribine, Cortisone, Cosmegen, CPT-11, Cyclophosphamide, Cytadren, Cytarabine, Cytarabine Liposomal, Cytosar U, Cytoxan, Dacarbazine, Dactinomycin, Darbepoetin Alfa, Daunomycin, Daunorubicin, Daunorubicin Hydrochloride, Daunorubicin Liposomal, Daunoxome, Deca Doron, Delta-Cortef, Deltasone, Denileukin diftitox, Depocyte, Dexamethasone, Dexamethasone acetate, Dexamethasone sodium phosphate, Dexasone, Dexrazoxane, DHAD, DIC, Geodex, Docetaxel, Doxil, Doxorubicin, Doxorubicin liposome, Droxia, DTIC, DTIC-Dome, Duralon, Efudex, Eligard, Elence, Eloxatin, Elspar, Emcyte, Epirubicin, Epoetin alfa, Erbitux, Erwinia L-asparaginase, Estramustine,Ethiol, etopophos, etoposide, etoposide phosphate, eurexin, evista, exemestane, fareston, faslodex, femara, filgrastim, floxuridine, fludara, fludarabine, fluoroplex, fluorouracil, fluorouracil (cream), fluoxymesterone, flutamide, folinic acid, FUDR, fulvestrant, G-CSF, gefitinib, gemcitabine, gemtuzumab ozogamicin, gemzar, glivec, leuprorelin, leuprorelin depot, matulane, maxidex, mecu Loretamine, -Mechlorethamine hydrochloride, Medralone, Medrol, Megas, Megestrol, Megestrol acetate, Melphalan, Mercaptopurine, Mesna, Mesnex, Methotrexate, Methotrexate sodium, Methylprednisolone, Mirocel, Letrozole, Neosal, Neulasta, Neumega, Neupogen, Nilandrone, Nilutamide, Nitrogen Mustard, Novaldex, Novantrone, Octreotide, Octreotide acetate, Oncasper, Oncovin, Ontac, Onxal, Oplevel Oprevelkin, Orapred, Orasone, Oxaliplatin, Paclitaxel, Pamidronate, Panretin, Paraplatin, Pediapred, PEG-Interferon, Pegaspargase, Pegfilgrastim, PEG-Intron, PEG-L-Asparaginase, Phenylalanine Mustard, Platinol, Platinol-AQ, Prednisolone, Prednisone, Prelon, Procarbazine, PROCRIT, Proleukin , Prolifeprospan 20 with Carmustine Implant, Purinethol, Raloxifene, Rheumatrex, Rituxan, Rituximab, Roveron-A (Interferon alpha-2a), Rubex, Rubidomycin Hydrochloride, Sandostatin, Sandostatin LAR, Sargramostim, Solucortef, Solumedrol, STI-571, Streptozocin, Tamoxifen, Targretin, Taxol, Taxotere, Temodar, Temozolomide, Teniposide,TESPA, Thalidomide, Thalomid, TheraCys, Thioguanine, Thioguanine Tabloid, Thiophosphoamide, Thioplex, Thiotepa, TICE, Toposar, Topotecan, Toremifene, Trastuzumab, Tretinoin, Trexall, Trisenox, TSPA, VCR, Velban, Velcade, Bepcid, Vesanoid, Viadur, Vinblastine, Vinblastine Sulfate, Vincasar Pfs Pfs), vincristine, vinorelbine, vinorelbine tartrate, VLB, VP-16, Vumon, Xeloda, Zanosar, Zevalin, Zincard, Zoladex, zoledronic acid, Zometa, Gliadel wafer, Gleevec, GM-CSF, goserelin, granulocyte colony-stimulating factor, halotestone, Herceptin, hexadrol, hexalen, hexamethylmelamine, HMM, Hycamtin, Hydrea, hydrocort acetate, hydrocortisone, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, hydrocortisone phosphate, hydroxyurea, ibritumomab, ibritumomab tiuxetan, idamycin, idarubicin, ifex, IFN-alpha, ifosfamide, IL2, IL-11, imatin Nib mesylate, imidazole carboxamide, interferon alpha, interferon alpha-2b (PEG conjugate), interleukin 2, interleukin-11, intron A (interferon alpha-2b), leucovorin, leukelan, leukin, leuprolide, leurocristin, leustatin, liposomal Ara-C, liquid pred, lomustine, L-PAM, L-sarcolysin, methycortene, mitomycin, mitomycin-C, mitoxantrone, M-prednisole, MTC, MTX, mustagen, mustine, mutamycin, myleran, Iressa, irinotecan, isotretinoin, quidrolase, lanacort, L-asparaginase, and LCR may be combined into a pharmaceutical composition. Additional anti-cancer agents may also include biopharmaceuticals, such as antibodies.

[0130] In many tumors and cancers, viral genomes are present in tumor or cancer cells. For example, Epstein-Barr virus (EBV) is associated with many mammalian malignancies. The compounds disclosed herein can also be used alone or in combination with anticancer or antiviral agents, such as ganciclovir, azidothymidine (AZT), lamivudine (3TC), to treat patients infected with viruses that can cause cellular transformation and / or to treat patients suffering from tumors or cancers associated with the presence of viral genomes in cells. The compounds disclosed herein can also be used in combination with virus-based treatments of neoplastic diseases.

[0131] Also described herein is a method of killing tumor cells in a subject. The method includes contacting tumor cells with an effective amount of a compound or composition described herein, and optionally irradiating the tumor cells with an effective amount of ionizing radiation. Additionally provided herein is a method of tumor radiotherapy. The method includes contacting tumor cells with an effective amount of a compound or composition described herein, and irradiating the tumor with an effective amount of ionizing radiation. As used herein, the term ionizing radiation refers to radiation that contains particles or photons that have sufficient energy or can generate sufficient energy through nuclear interactions to cause ionization. An example of ionizing radiation is X-rays. An effective amount of ionizing radiation refers to an amount of ionizing radiation that, when administered in combination with a compound described herein, results in increased cell damage or cell death. Ionizing radiation can be delivered according to methods well known in the art, including administration of radiolabeled antibodies and radioisotopes.

[0132] The methods and compounds described herein are useful for both prophylactic and therapeutic treatments. As used herein, the term "treating" or "treatment" includes prevention, delay of onset, reduction, eradication, or delay of progression of signs or symptoms after onset, and prevention of recurrence. For prophylactic use, a therapeutically effective amount of the compounds and compositions described herein or pharma- ceutically acceptable salts thereof is administered to a subject before onset (e.g., before overt signs of cancer appear), early onset (e.g., when early signs and symptoms of cancer appear), or after cancer progression has been established. Prophylactic administration can occur from days to years before symptoms of infection appear. Prophylactic administration can be used, for example, for chemopreventive treatment of subjects exhibiting precancerous lesions, subjects diagnosed with early-stage malignancies, and subgroups with susceptibility (e.g., familial, racial, and / or occupational) to a particular cancer. Therapeutic treatment includes administering a therapeutically effective amount of the compounds and compositions described herein or pharma- ceutically acceptable salts thereof to a subject after cancer is diagnosed.

[0133] In some examples of the methods of treating, preventing, or ameliorating cancer or tumor in a subject, the compound or composition administered to the subject can include a therapeutic moiety that can include a targeting moiety that can act as an inhibitor against Ras (e.g., K-Ras), PTP1B, Pin1, Grb2 SH2, or a combination thereof.

[0134] The disclosed subject matter also relates to a method for treating a subject suffering from a metabolic disorder or condition. In one embodiment, an effective amount of one or more compounds or compositions disclosed herein is administered to a subject suffering from a metabolic disorder and in need of treatment. In some examples, the metabolic disorder can include type 2 diabetes. In some examples of the method for treating, preventing, or improving a metabolic disorder in a subject, the compound or composition administered to the subject can include a therapeutic moiety, which can include a targeting moiety that can act as an inhibitor against PTP1B. In one particular example of this method, the subject is obese, and the method includes treating the obesity of the subject by administering a composition disclosed herein.

[0135] The disclosed subject matter also relates to a method for treating a subject suffering from cystic fibrosis.In one embodiment, an effective amount of one or more compounds or compositions disclosed herein is administered to a subject suffering from cystic fibrosis and in need of treatment.In some examples of the method for treating cystic fibrosis in a subject, the compound or composition administered to the subject can include a therapeutic moiety, which can include a targeting moiety that can act as an inhibitor against CAL PDZ.

[0136] Additionally, methods of using the disclosed compositions to deliver agricultural products to plant cells are disclosed, the methods comprising contacting the cells with a peptide disclosed herein. Compounds that can be delivered to plants include biodefense activators and biostimulants.

[0137] Compositions, Formulations and Methods of Administration Also disclosed herein are compositions that include the compounds described herein.

[0138] Also disclosed herein are pharma- ceutically acceptable salts and prodrugs of the disclosed compounds. Pharmaceutically acceptable salts include salts of the disclosed compounds prepared with acids or bases, depending on the particular substituents found on the compound. Under conditions where the compounds disclosed herein are sufficiently basic or acidic to form stable non-toxic acid or base salts, it may be appropriate to administer the compounds as salts. Examples of pharma- ceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, or magnesium salts. Examples of physiologically acceptable acid addition salts include hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, carbonic acid, sulfuric acid, and organic acids (acetic acid, propionic acid, benzoic acid, succinic acid, fumaric acid, mandelic acid, oxalic acid, citric acid, tartaric acid, malonic acid, ascorbic acid, α-ketoglutaric acid, α-glycolic acid, maleic acid, tosylic acid, methanesulfonic acid, and the like). Thus, disclosed herein are hydrochloride, nitrate, phosphate, carbonate, bicarbonate, sulfate, acetate, propionate, benzoate, succinate, fumarate, mandelate, oxalate, citrate, tartrate, malonate, ascorbate, α-ketoglutarate, α-glycolic acid, maleate, tosylate, and mesylate salts. Pharmaceutically acceptable salts of compounds can be obtained by reacting a sufficiently basic compound, such as an amine, with a suitable acid that provides a physiologically acceptable anion, using standard procedures well known in the art. Alkali metal (e.g., sodium, potassium, or lithium) or alkaline earth metal (e.g., calcium) salts of carboxylic acids can also be prepared.

[0139] In vivo application of the disclosed compounds and compositions containing them can be achieved by any suitable method and technique known to those skilled in the art now or in the future. For example, the disclosed compounds can be formulated in a physiologically or pharma-ceutically acceptable form and can be administered by any suitable route known in the art, including, for example, oral, nasal, rectal, topical, and parenteral routes of administration. As used herein, the term parenteral includes, for example, subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, and intrasternal administration by injection. The administration of the disclosed compounds or compositions can be a single dose or can be administered continuously or at different intervals, as can be easily determined by those skilled in the art.

[0140] The compounds disclosed herein and compositions containing them can also be administered using liposome technology, time-release capsules, implantable pumps, and biodegradable containers.These delivery methods can advantageously provide a uniform dosage over an extended period of time.The compounds can also be administered in their salt derivative form or crystalline form.

[0141] The compounds disclosed herein can be formulated according to known methods for preparing pharma- ceutically acceptable compositions. Formulations are described in detail in many sources, which are well known and readily available to those skilled in the art. For example, Remington's Pharmaceutical Science by EW Martin (1995) describes formulations that can be used in connection with the disclosed methods. In general, the compounds disclosed herein can be formulated so that an effective amount of the compound is combined with a suitable carrier to facilitate effective administration of the compound. The compositions used may be in various forms. These include, for example, solid, semi-solid, and liquid dosage forms such as tablets, pills, powders, liquid solutions or suspensions, suppositories, injectable and infusible solutions, and sprays. The preferred form depends on the intended method of administration and therapeutic application. The compositions also preferably include conventional pharma- ceutically acceptable carriers and diluents well known to those skilled in the art. Examples of carriers or diluents used with the compounds include ethanol, dimethyl sulfoxide, glycerol, alumina, starch, saline, and equivalent carriers and diluents. To administer such dosages for the desired therapeutic treatment, the compositions disclosed herein can advantageously contain from about 0.1% to 100% by total weight of one or more of the subject compounds, based on the weight of the total composition including any carrier or diluent.

[0142] Preparations suitable for administration include aqueous sterile injection solutions, which may contain, for example, antioxidants, buffers, bactericides, and solutes that render the preparation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions, which may contain suspending agents and thickening agents. The preparations may be provided in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a lyophilized (freeze-dried) state, requiring only the condition of a sterile liquid carrier, for example water for injection, prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, tablets, and the like. In addition to the ingredients specifically mentioned above, it should be understood that the compositions disclosed herein may contain other agents conventional in the art, taking into account the type of formulation being discussed.

[0143] The compounds disclosed herein and compositions comprising them can be delivered to cells through direct contact with cells or via carrier means. Carrier means for delivering compounds and compositions to cells are well known in the art and include, for example, encapsulating the composition in liposome moieties. Another means for delivering compounds and compositions disclosed herein to cells includes binding the compounds to proteins or nucleic acids for delivery to target cells. U.S. Patent No. 6,960,648, U.S. Patent Application Publication Nos. 20030032594 and 20020120100 disclose amino acid sequences that can be coupled to another composition and allow the composition to translocate across biological membranes. U.S. Patent Application Publication No. 20020035243 also describes compositions for transporting biological moieties across cell membranes for intracellular delivery. Compounds can also be incorporated into polymers, examples of which include poly(DL-lactide-co-glycolide) polymer for intracranial tumors, poly[bis(p-carboxyphenoxy)propane:sebacic acid] in a molar ratio of 20:80 (used in GLIADEL), chondroitin, chitin, and chitosan.

[0144] For the treatment of neoplastic disease, the compounds disclosed herein can be administered to a patient in need of treatment in combination with other antitumor or anticancer substances, and / or radiation therapy, and / or photodynamic therapy, and / or surgical treatment to remove the tumor. These other substances or treatments can be administered at the same time as the compounds disclosed herein, or at different times. For example, the compounds disclosed herein can be used in combination with mitotic inhibitors such as taxol or vinblastine, alkylating agents such as cyclophosphamide or ifosfamide, antimetabolites such as 5-fluorouracil or hydroxyurea, DNA intercalators such as adriamycin or bleomycin, topoisomerase inhibitors such as etoposide or camptothecin, angiogenesis inhibitors such as angiostatin, antiestrogens such as tamoxifen, and / or other anticancer drugs or antibodies, such as, for example, GLEEVEC (Novartis Pharmaceuticals Corporation) and HERCEPTIN (Genentech, Inc.), respectively, or immunotherapeutics such as ipilimumab and bortezomib.

[0145] In certain examples, the compounds and compositions disclosed herein can be administered locally to one or more anatomical sites, such as at the site of unwanted cell proliferation (e.g., injected or applied topically to a tumor or skin tumor, such as a tumor site or benign skin tumor), optionally in combination with a pharma- ceutically acceptable carrier, such as an inert diluent. The compounds and compositions disclosed herein can be administered systemically, such as intravenously or orally, optionally in combination with a pharma- ceutically acceptable carrier, such as an inert diluent, or an absorbable edible carrier for oral delivery. They can be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or directly incorporated into the food of the patient's diet. For oral therapeutic administration, the active compound can be combined with one or more excipients and can be used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, aerosol sprays, and the like.

[0146] The disclosed compositions are bioavailable and can be delivered orally. Oral compositions can be tablets, troches, pills, capsules, etc., and can contain a binder such as gum tragacanth, acacia, cornstarch, or gelatin, an excipient such as dicalcium phosphate, a disintegrating agent such as cornstarch, potato starch, alginic acid, a lubricant such as magnesium stearate, and a sweetener such as sucrose, fructose, lactose, or aspartame, or a flavoring such as peppermint, wintergreen oil, or cherry flavor. When the unit dosage form is a capsule, it can contain a liquid carrier such as vegetable oil or polyethylene glycol in addition to the above-mentioned types of materials. Various other materials can be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For example, tablets, pills, or capsules can be coated with gelatin, wax, shellac, sugar, etc. Syrup or elixir can contain the active compound, sucrose or fructose as a sweetener, methylparaben or propylparaben as a preservative, a dye, and a flavoring such as cherry or orange flavor.Of course, any material used in preparing any unit dosage form must be pharma-ceutically acceptable and substantially non-toxic in the amounts used.Furthermore, the active compound can be incorporated into sustained-release preparations and devices.

[0147] The compounds and compositions disclosed herein, including their pharma- ceutically acceptable salts or prodrugs, can be administered intravenously, intramuscularly, or intraperitoneally by infusion or injection.A solution of the active agent or its salt can be prepared in water, optionally mixed with a non-toxic surfactant.Dispersions can also be prepared in glycerol, liquid polyethylene glycol, triacetin, and mixtures thereof, as well as in oils.Under normal conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms.

[0148] Pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions or sterile powders containing the active ingredient, which are suitable for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. The final dosage form must be sterile, fluid, and stable under the conditions of manufacture and storage. The liquid carrier or vehicle may be, for example, a solvent or liquid dispersion medium, including water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by maintaining the required particle size in the case of dispersions, or by the use of surfactants. If necessary, the prevention of the action of microorganisms can be brought about by various other antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be desirable to include isotonic agents, such as sugars, buffers, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents which delay absorption, for example, aluminum monostearate and gelatin.

[0149] Sterile injectable solutions are prepared by incorporating the compounds and / or agents disclosed herein in the required amount in a suitable solvent with various other ingredients as listed above, as needed, followed by filtration sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying technology, which results in a powder of the active ingredient and any additional desired ingredients present in the previously sterile-filtered solution.

[0150] For topical administration, the compounds and agents disclosed herein can be applied as liquids or solids. However, it is generally desirable to combine them with a dermatologically acceptable carrier, which may be solid or liquid, and administer them locally to the skin as a composition. The compounds, agents and compositions disclosed herein can be applied locally to the skin of a subject to reduce the size (which may include complete removal) of malignant or benign tumors or to treat an infected site. The compounds and agents disclosed herein can be applied directly to the growth or infected site. Preferably, the compounds and agents are applied to the growth or infected site in a formulation such as an ointment, cream, lotion, solution, tincture, etc.

[0151] Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina, etc. Useful liquid carriers include water, alcohol or glycol, or water and alcohol / glycol brands in which the compound can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given application. The resulting liquid composition can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area, for example, using pump-action or aerosol sprays.

[0152] Thickening agents such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified cellulose, or modified mineral materials may also be used with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like for application directly to the user's skin.

[0153] Useful dosages of the compounds, agents, and pharmaceutical compositions disclosed herein can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are well known in the art.

[0154] The dosage range of the composition administration is sufficient to produce the desired effect of affecting the symptoms or disorder. The dosage should not be so large as to cause adverse side effects such as undesirable cross-reactions, anaphylactic reactions, etc. In general, the dosage varies according to the age, condition, sex, and degree of disease of the patient, which can be determined by those skilled in the art. If there are any contraindications, the dosage can be adjusted by the individual physician. The dosage can vary and can be administered in one or more doses per day for one or several days.

[0155] Also disclosed is a pharmaceutical composition comprising the compound disclosed herein in combination with a pharma- ceutically acceptable carrier. Pharmaceutical compositions suitable for oral, topical or parenteral administration, comprising an amount of the compound, constitute a preferred embodiment. The dosage administered to a patient, particularly a human, should be sufficient to achieve a therapeutic response in the patient over a reasonable time frame without lethal toxicity, and preferably without causing side effects and morbidity to exceed acceptable levels. Those skilled in the art will recognize that the dosage depends on a variety of factors, including the subject's condition (health), the subject's weight, the type of concomitant therapy (if any), frequency of treatment, therapeutic ratio, and the severity and stage of the disease.

[0156] Also disclosed are kits comprising a compound disclosed herein in one or more containers. The disclosed kits may optionally comprise a pharma- ceutically acceptable carrier and / or diluent. In one embodiment, the kit comprises one or more other components, adjuvants, or supplements described herein. In another embodiment, the kit comprises one or more anti-cancer agents, such as agents described herein. In one embodiment, the kit comprises instructions or packaging material that describes how to administer the compound or composition of the kit. The containers of the kit may be of any suitable material, e.g., glass, plastic, metal, and may be of any suitable size, shape, or configuration. In one embodiment, the compound and / or agent disclosed herein is provided in the kit as a solid, such as a tablet, pill, or powder form. In another embodiment, the compound and / or agent disclosed herein is provided in the kit as a liquid or solution. In one embodiment, the kit comprises an ampoule or syringe that comprises the compound and / or agent disclosed herein in liquid or solution form.

[0157] Although several embodiments of the invention have been described, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. EXAMPLES

[0158] The following examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices, and / or methods claimed herein are made and evaluated, are intended to be merely illustrative of the invention, and are not intended to limit the scope of what the inventors regard as their invention. However, those of ordinary skill in the art should, in light of the present disclosure, understand that many changes can be made in the specific embodiments that are disclosed and still obtain like or similar results without departing from the spirit and scope of the invention.

[0159] Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.

[0160] We selected the human fibronectin type III (FN3) domain as a scaffold for the membrane translocation domain (MTD) (Koide, A., et al. (1998) The physicine type III domain as a scaffold for novel binding protein. J. Mol. Biol. 284 (4): 1141-1151). FN3 is a small (90-100 aa) and highly stable protein that has been widely used to develop monobodies that bind to target proteins with high affinity and specificity (Chandler, PG, et al. (2020) Development and Differentiation in Monobodies Based on the Fibronectin Type 3 Domain. Cells 9 (3): 610). Previous studies have demonstrated that some loop regions of FN3 are resistant to mutations (Steven, A., et al. (2012) Design of novel FN3 domains with high stability by a consensus sequence approach, Protein Engineering, Design and Selection, 25(3): 107-117). In addition, FN3 does not contain any cysteine ​​or disulfide bonds and is therefore stable in the intracellular environment. FN3 easily folds into its native form without any physical or chemical assistance and can be produced in high yields in E. coli. Finally, FN3 is derived from an abundant human extracellular protein and is unlikely to provoke an immune response.

[0161] Design, expression, and purification of MTD The BC, DE, and FG loops of FN3 have previously been shown to be highly tolerant to sequence mutations. The GDSPAS sequence in the FG loop was replaced with RRRWWW (SEQ ID NO: 104) to obtain MTD1 (Table 4). When combined with an arginine residue already present in the FG loop, a putative CPP motif (R4W3) is generated without changing the size of the loop. Similarly, the tetrapeptide AVTV in the BC loop was replaced with WWWRRR (SEQ ID NO: 105) to utilize the existing arginine in the loop to form a putative CPP, W3R4 (Table 4). The size of the BC loop in the resulting mutant MTD2 is increased by two residues. To investigate the possibility of grafting a CPP motif to the other end of FN3, the tripeptide NSP in the CD loop was replaced with the CPP motif R4W3 to obtain MTD3. To test the feasibility of grafting the CPP sequence into two different loops, the relatively hydrophobic tripeptide in the BC loop (VTV) was replaced by WYW, and the hydrophilic motif (GDSPAS, SEQ ID NO: 106) in the FG loop was replaced by RRR to obtain MTD4. Finally, MTD5 was generated by switching the WYW and RRR motifs in MTD4. To test the relative importance of the RRRR and WYW motifs, we also generated two mutants, MTD4a and MTD4b, which contain only half of the CPP motif in the BC and FG loops, respectively. The WYW motif is more hydrophilic than WWW and has been previously reported as an "endosomal escape motif" for cell-penetrating antibodies (Kim, J.-S., et al. (2016) Endosomal acidic pH-induced conformational changes of a cytosol-penetrating antibody mediate endosomal escape. J. Control. Rel. 235:165-175). The loop insertion mutants were analyzed with the online program Phyre2 to predict their folded structures based on sequence homology. All mutants maintained an overall fold similar to wild-type FN3, with the CPP motifs displayed on their surface and constrained to a "ring" topology (Figure 1).

[0162] To further improve the properties of MTD4 (e.g., cell entry efficiency, metabolic stability, and expression yield), the BC and FG loops of FN3 were replaced with different combinations of Y, W, A, and R residues to generate MTD6–10 (Table 4). The total cell entry efficiency of MTD6–10 was assessed by labeling the MTDs at their unique C-terminal cysteines with tetramethylrhodamine-5-maleimide (TMR). HeLa cells were treated with TMR-labeled proteins (5 mM) for 2 h and analyzed by live-cell confocal microscopy. MTD7 TMR and MTD9 TMR MTD4 TMR showed similar uptake to MTD6 TMR , MTD8 TMR , and MTD10 TMR showed much less cell entry (Figures 3A-3I). Moreover, the isolation yields of MTD6-10 ranged from 0.6-6.2 mg per L of E. coli cell culture (Table 4). Note that MTD4 and MTD6 only differ slightly in the BC loop sequence ("WYW" vs. "YWW"), but have dramatic differences in isolation yield (9.4 mg / L vs. 0.9 mg / L) and cell entry efficiency. Similarly, swapping the CPP motif between the BC and FG loops of MTD4 yielded a poorly expressed and much less active mutant (MTD5 in Table 4). These results indicate that proper folding / stability and high cell entry efficiency of MTDs requires not only the presence of an amphipathic CPP motif but also its proper presentation on the protein surface.

[0163] [Table 4]

[0164] A DNA sequence encoding WT FN3 was chemically synthesized and ligated into the prokaryotic expression vector pET-15b. To facilitate protein purification and gene fusion with cargo proteins, a 6-histidine tag and a thrombin cleavage site were added to the N-terminus of FN3, while a flexible linker sequence (GGSGGSGGS, SEQ ID NO: 107) was added to its C-terminus, followed by a recognition site for the restriction endonuclease SacI and a cysteine ​​(Table 5). All loop insertion mutants were generated by a one-step polymerase chain reaction (PCR) method (Qi, D., et al. (2008) A one-step PCR-based method for rapid and efficient site-directed fragment deletion, insertion, and substitution mutagenesis. J. Virol. Meth. 149: 85-9020) and expressed in E. coli. Among the mutant proteins, MTD1 failed to produce significant amounts of soluble protein, whereas WT FN3 and MTD2-5 produced soluble proteins in good yields (Table 4). Figures 2A-B show the expression and purification of MTD4 as an example. All proteins were purified to near homogeneity by metal affinity chromatography on a Ni-NTA column.

[0165] Cloning, expression, and purification of MTD All loop insertion mutants were generated by one-step polymerase chain reaction (PCR) method (Qi, D., supra). The peptide sequence of each construct (Table 5) was confirmed by sequencing the entire coding region of the plasmid DNA. Pilot-scale protein expression was performed to confirm the expression level of the mutant proteins, and all mutants were expressed in 5 mL of E. coli BL21(DE3) bacterial culture. Induction was performed at 37°C in the presence of 0.25 mM IPTG. Expression levels were confirmed by comparing pre- and post-induction whole cell lysates on SDS gels (Figures 2A-B).

[0166] [Table 5] TIFF2025509645000016.tif216170TIFF2025509645000017.tif160170

[0167] The large-scale expression conditions were the same as those used for the small-scale expression, and the E. coli cells were centrifuged and stored at -80°C. The cells were lysed using lysis buffer (50 mL of wash buffer, 0.2 mg / mL lysozyme, 2 mM β-mercaptoethanol, 2 mM PMSF, and 2 tablets of Roche complete protease inhibitor cocktail). After the cell pellet was homogenously resuspended in lysis buffer, the cells were sonicated twice (70% amplification). The crude cell lysate was centrifuged (12000 g for 20 min) and the soluble cell lysate was collected. Protein purification was performed using fast protein liquid chromatography (FPLC), and the soluble cell lysate was loaded onto a Ni-NTA column (containing 15 mM imidazole). The column was washed extensively with wash buffer (50 mM Tris, pH 7.4, 300 mM NaCl, 5% glycerol, and 50 mM imidazole). Proteins were eluted with a linear gradient of 50-500 mM imidazole (pH 7.4) in wash buffer over 30 min.

[0168] Cloning, expression, and purification of MTD4-PTP1B, MTD4-NS1, MTD4-RBDV, MTD4-SEP, MTD4-GFP11, and MENC The coding sequence of PTP1B (amino acids 1-321) was amplified by PCR using plasmid DNA as a template and primers containing SacI and BamHI restriction sites at the 5' and 3' ends of the PTP1B coding sequence, respectively. The PCR product was digested with the restriction enzymes SacI and BamHI and ligated into plasmid pET-15b-MTD4 linearized with the same two enzymes. This fused PTP1B to the C-terminus of MTD4. Other pET15b-based plasmids encoding MTD4-RBDV, MTD4-NS1, MTD4-SEP, and MENC fusion proteins were constructed similarly, but with the addition of XhoI restriction sites rather than BamHI at the 3' ends of the RBDV, NS1, SEP, and ENC coding sequences. The GFP11 peptide was inserted into the C-terminus of MTD4 by a one-step PCR reaction (D. Qi, et al., "A one-step PCR-based method for rapid and efficient site-directed fragment deletion, insertion, and substitution mutagenesis", J Virol Methods, 149(1):85-90, 2008). The authenticity of the DNA construct was confirmed by restriction enzyme mapping and sequencing of the entire coding sequence.

[0169] E. coli BL21(DE3) cells transformed with the appropriate plasmids were grown at 37°C in LB medium supplemented with 75 mg / L ampicillin. OD 600When the NA reached 0.6, 0.25 mM IPTG was added and the cells were induced for 4 h at 37 °C. The cells were pelleted by centrifugation. For MTD4-RBDV, the cell pellet was resuspended in 50 mL (per liter of cell culture) of lysis buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 25 mM imidazole, 3 mM β-mercaptoethanol, protease inhibitor cocktail tablet, and 20 mg / ml lysozyme]. The cells were sonicated briefly and centrifuged at 12,000 g for 20 min. The crude lysate was loaded onto a 5 mL Histrap Ni-NTA column attached to an FPLC. The column was washed extensively with wash buffer (50 mM Tris, pH 7.4, 300 mM NaCl, 5% glycerol, and 50 mM imidazole). Protein was eluted with wash buffer containing 500 mM imidazole. MTD4-PTP1B, MTD4-SEP, MENC, MTD4-GFP11, and MTD4-NS1 were similarly purified, except that for MTD4-NS1, the cell pellet was resuspended in 50 mM Tris (pH 8), 500 mM NaCl, 25 mM imidazole, 3 mM β-mercaptoethanol, a protease inhibitor cocktail tablet, and 20 mg / ml lysozyme. MENC protein was expressed in the BL21 Rosetta pLysS cell line, and induction was performed overnight at 18°C. The specific activity of MTD4-PTP1B was measured using p-nitrophenyl phosphate (pNPP) as a substrate and was found to be comparable to that of WT PTP1B.

[0170] Cell entry efficiency of MTD Wild-type FN3 and MTD were fluorescently labeled at their single C-terminal cysteine ​​using tetramethylrhodamine-5-maleimide (TMR). HeLa (human cervical cancer) cells were incubated with TMR-labeled proteins and imaged without fixation by confocal microscopy. Interestingly, WT FN3 showed prominent cellular entry, despite a punctate intracellular fluorescence pattern, indicating that most of the internalized protein was trapped within endosomes / lysosomes (Figure 3A). In contrast, MTD4 TMRIn addition to punctate fluorescence, HeLa cells treated with β-lactamase exhibited diffuse fluorescence that was easily visible throughout the entire cell volume (including the nucleus) (Figure 3B). The presence of diffuse intracellular fluorescence indicates internalized MTD4. TMR This indicates that a significant portion of MTD2 leaves the endosome and successfully reaches the cytosol (and nucleus). TMR The fluorescence pattern of cells treated with MTD5 is between those of cells treated with FN3 and MTD4, and although some diffuse fluorescence is visible, the fluorescence is predominantly punctate (Figure 3B). TMR did not result in significant intracellular fluorescence (data not shown).

[0171] To quantify cell entry efficiency, cells were then analyzed by flow cytometry and the results were compared to those of a previously reported highly efficient cyclic CPP, CPP12. Preliminary data, after adjusting for the degree of dye labeling of the proteins, showed that MTD4 entered HeLa cells more efficiently than FN3 or MTD2, but not as efficiently as CPP12 (Figure 4).

[0172] Intracellular delivery of PTP1B To demonstrate functional delivery of protein cargo to the cytosol of mammalian cells by MTD, protein tyrosine phosphatase 1B (PTP1B) was selected as cargo and genetically fused to the C-terminus of MTD4. Tyrosine phosphorylation is usually restricted to cytosolic and nuclear proteins, or to the cytosolic domains of transmembrane proteins. PTP1B is a broad-specificity phosphatase that catalyzes the dephosphorylation of many intracellular proteins (Selner, NG, et al. (2014) Diverse levels of sequence selectivity and catalytic efficiency of protein-tyrosine phosphatases. Biochem. 53(2):397-412). Cytosolic delivery of PTP1B is expected to reduce phosphotyrosine (pY) levels of intracellular proteins, which can be easily monitored by anti-pY Western blotting. NIH3T3 cells were treated with different concentrations of MTD4-PTP1B for 4 h, washed, and lysed in the presence of protease and phosphatase inhibitors. Cellular proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with anti-pY antibody 4G10. MTD4-PTP1B reduced pY levels in HeLa cells in a dose-dependent manner, with almost complete abolition of pY at 5 mM (Figure 5).

[0173] Intracellular delivery of Ras inhibitors To further demonstrate the utility of MTD4 and generate a cell-permeable protein with therapeutic potential, MTD4 was fused to two previously reported proteins that bind mutant KRas with high affinity and specificity. Mutations in Ras (including K-, H-, and NRas) are found in approximately 30% of all human cancers, making Ras one of the most important targets for cancer therapeutics (prior art, IA, et al. (2012) A comprehensive survey of Ras mutations in cancer. Cancer Res. 72(10):2457-67, Khan, I., et al. (2020) Therapeutic targeting of RAS: New hope for drugging the “undruggable”. Biochim. Biophys. Acta, Mol. Cell Res.1867,118570). Unfortunately, Ras is one of the most challenging (and undruggable) drug targets because it is intracellular and lacks a major binding pocket on its surface for small molecules to bind. To date, several small molecules that covalently modify (and inhibit) G12C mutant KRas have been introduced into the clinic, one of which has been approved by the FDA, demonstrating that Ras is a viable target for treating Ras mutant cancers (Moore,AR,et al.(2020)RAS-targeted therapies:is the undruggable drugged?Nature Rev.Drug Disc.19(8):533-552). A potent noncovalent inhibitor selective for KRas G12D mutant was also recently reported (Wang,X.,et al.(2021)Identification of MRTX1133,a Noncovalent,Potent,and Selective KRASG12D Inhibitor. J. Med. Chem. 10.1021 / acs.jmedchem.1c01688). However, for many other Ras mutants (e.g., G12V, G12S, G13C, and Q61H), no such drugs exist.

[0174] Previous researchers have generated potent protein inhibitors of KRas by phage display screening. Wiechmann et al. screened a phage display library of C-Raf Ras binding domains (RBDs) and identified WT C-Raf RBD (KRas) in the effector binding site of GTP-bound HRas. D They identified several mutants, RBDV, that bind with approximately 20-fold higher affinity than Ras GTPases (approximately 3 nM) (Wiechmann, S., et al. (2020) Conformation-specific inhibitors of activated Ras GTPases reveal limited Ras dependency of patient-derived cancer organoids. J. Biol. Chem. 295(14):4526-4540). Expression of RBDV in Ras-mutated cancer cells inhibited Ras signaling, leading to apoptosis of the cancer cells. Similarly, Koide and coworkers screened a large library of phage-displayed FN3 mutants against HRas and found a strong binder called "NS1" that binds to an allosteric site and prevents dimerization of Ras on the plasma membrane (Spencer-Smith, R., et al. (2017) Inhibition of RAS function through targeting an allosteric regulatory site. Nat. Chem. Biol. 13:62-68). When expressed in cells, NS1 also inhibited Ras signaling and induced apoptotic death of Ras mutant cancer cells (Khan, I., et al. (2019) Targeting the α4-α5 dimerization interface of K-RAS inhibits tumor formation in vivo. Oncogene 38(16):2984-2993). Unlike RBDV, which binds all Ras isoforms (K-, H-, and NRas) in the GTP-bound state, NS1 binds HRas (K-, H-, and NRas) regardless of nucleotide identity. D = 13 nM) and KRAS (K D=65 nM). Unfortunately, RBDV and NS-1 are not suitable as therapeutic agents because they cannot penetrate the cell membrane to reach the target protein.

[0175] One of the RBDVs (RBDV3) or NS1 was genetically fused to the C-terminus of MTD4 to generate MTD4-RBDV and MTD4-NS1, respectively. The proteins were expressed in E. coli and purified to near homogeneity by metal affinity chromatography. The fusion proteins were then tested for their ability to reduce the viability of Ras mutant cancer cells. MTD4-RBDV dose-dependently reduced the viability of non-small cell lung cancer (H358), pancreatic ductal adenocarcinoma (Mia PaCa-2), non-small cell lung cancer (A549), and colon cancer cells (SW480), with IC 50 The IC values ​​were 1.2 ± 0.2, 1.2 ± 0.1, 1.7 ± 0.2, and 2.2 ± 0.4 μM, respectively (Figure 6A). Similarly, MTD4-NS1 reduced the viability of the aforementioned cells in a dose-dependent manner, with IC 50 The values ​​were 1.4 ± 0.2, 1.5 ± 0.1, 1.8 ± 0.1, and 0.9 ± 0.2 μM, respectively (Figure 6B). To verify the selectivity of NS1 for KRas and HRas but not for NRas, we used H1915 non-small cell lung cancer cells (HRAS Q61L ) mutant and H1299 non-small cell lung cancer cells (NRAS Q61K Both fusion proteins in the MTD4-RBDV mutant were tested. MTD4-RBDV was active in both cells, with IC 50 were 4.7 ± 1.1 μM and 2.4 ± 0.3 μM, respectively. Surprisingly, MTD4-NS1 was active in both cell lines, with IC 50 The IC values ​​were 1.5±0.2 μM and 1.6±0.2 μM. NS1 was fused to the N-terminus of MTD4 to generate NS1-MTD4. NS1-MTD4 also dose-dependently reduced the viability of H358 and MiaPaCa-2 cells, and the IC 50 The IC values ​​were 1.1 + 0.1 μM and 1.8 + 0.2 μM, respectively (Figure 6C). NS1-MTD4 also reduced the viability of H1299 cells, with IC 50 was 1.6±0.2 μM.

[0176] To determine whether the loss of cancer cell viability was caused by on-target inhibition of Ras-effector protein interactions and Ras signaling, a bioluminescence resonance energy transfer (BRET) assay developed by Rabbitts and coworkers was used (Bery, N., et al. (2018) BRET-based RAS biosensors that show a novel small molecule is an inhibitor of RAS-effector protein-protein interactions. eLife 7:e37122). Human embryonic kidney (HEK293T) cells, which do not carry Ras mutations and are relatively insensitive to Ras inhibitors, were transfected with plasmid DNA encoding KRas G12V (or G12D) luciferase and c-Raf RBD green fluorescent protein (GFP) fusion. Interaction between KRas mutants and the RBD generates a BRET signal from the luciferase donor to the GFP acceptor upon addition of the membrane-permeable luciferase substrate coelenterazine 400a. Inhibitors that block the Ras-RBD interaction are expected to reduce the BRET signal. As shown in Figure 7, MTD4-RBDV dose-dependently reduced the BRET signal in HEK293T cells transfected with either the KRas G12V or G12D mutant, with an IC 50The values ​​were 5–10 μM. MTD4-NS1 also reduced the BRET signal, but not as potently as MTD4-RBDV, which is in good agreement with their relative Ras-binding affinities. Furthermore, unlike RBDV, NS1 does not bind to effector binding sites and does not directly compete with Raf for binding to HRas or KRas. Instead, NS1 binds to the dimerization site of HRas / KRas and indirectly inhibits Ras-Raf interaction. Note that for MTD4-NS1, the BRET ratio suddenly decreased at 10 μM protein. This could be caused in part by a decrease in HEK293T cell viability at high inhibitor concentrations. The latter again suggests that in addition to on-target inhibition of Ras signaling, MTD4-NS1 may also have off-target effects.

[0177] The on-target activity of the fusion protein was further evaluated by examining the phosphorylation levels of signaling proteins downstream of Ras by Western blot analysis. Ras activates the Raf / MEK / ERK and PI3K / Akt signaling pathways, increasing the phosphorylation of the protein kinases MEK, ERK, and Akt. Inhibition of Ras function should decrease the phosphorylation levels of Akt and MEK. Indeed, treatment of MiaPaCa-2 cells with MTD4-RBDV dose-dependently decreased the phosphorylation of Akt and MEK, indicating that the phosphorylation of Akt and MEK was significantly increased by 100% compared with that of the IC 50 The values ​​ranged from 1 to 3 μM, while total Akt and MEK levels remained relatively constant (Figure 8A). MTD4-NS1 also reduced p-Akt and p-MEK levels, but not as potently as MTD4-RBDV (Figure 8B).

[0178] We tested whether MTD4-RBDV and MTD4-NS1 induce apoptosis in Ras mutant cancer cells. Therefore, H358 lung cancer cells were treated with the fusion proteins for 24 hours and stained with Alexa Fluor™ 488-Annexin V and propidium iodide before flow cytometry analysis. MTD4-RBDV showed a dose-dependent increase in the Annexin V positive cell population, indicating that apoptosis was responsible for the decreased viability observed in the Cell Glo assay (Figure 9). MTD4-NS1 also caused strong apoptosis at concentrations as low as 2.5 μM (Figure 9).

[0179] Cytosolic delivery efficiency of MTD4 Of the 10 MTDs generated, MTD4 has a relatively high expression yield in E. coli and excellent total cell entry efficiency as monitored by confocal microscopy. Therefore, we selected MTD4 for further evaluation and measured its cytosolic delivery efficiency using two different methods. First, we used a green fluorescent protein (GFP) complementation assay, which involves conjugating the 16-aa peptide GFP11 (corresponding to the 11th β-strand of GFP) to the superfolder GFP1–10 to form a functional GFP. GFP11 was genetically fused to the C-terminus of MTD4. As a comparison, GFP11 was also chemically conjugated to CPP12. HEK293T cells were transiently transfected to express GFP1–10 proteins and incubated with 10 mM GFP11, MTD4-GFP11, or CPP12-GFP11 for 6 h and examined by live-cell confocal microscopy. Cells treated with MTD4-GFP11 showed strong and diffuse fluorescence throughout the cell volume (Figures 10A-D). Cells treated with CPP12-GFP11 also showed strong fluorescence, but the signal was more punctate. In contrast, untreated cells and cells treated with unconjugated GFP11 showed almost no fluorescence.

[0180] A previously reported luciferase complementation assay was used by conjugating HiBit, an 11-residue peptide derived from NanoLuc (VSGWRLFKKIS) (SEQ ID NO: 151), to FN3, MTD, and CPP12 via disulfide bonds (SLYTeo, Jet al., "Unravelling cytosolic delivery of cell penetrating peptides with a quantitative endosomal escape assay", Nat Commun, 12(1):3721, 2021). HEK293T cells were transfected with plasmid DNA encoding the 18-kDa subunit of NanoLuc (LgBit) and then incubated with HiBit or HiBit conjugates. Upon successful delivery to the cytosol, the HiBit peptide is released from the conjugate by intracellular thiols (e.g., glutathione) and specifically binds to cytosolic LgBit to form catalytically active luciferase, whose activity is quantified in real time by the addition of the cell-permeable substrate furimazine. CPP12-SS-HiBit, MTD4-SS-HiBit, and MTD2-SS-HiBit, but not FN3-SS-HiBit, increased luciferase activity in HEK293T cells in a dose-dependent manner (Figure 11). Interestingly, MTD4 and CPP12 showed similar cytosolic delivery efficiency at high concentrations (e.g., 5 μM), but at low concentrations (e.g., 0.19 μM and 0.56 μM), MTD4 was 5-10 times more active than CPP12 (Figures 10A-D). Unconjugated HiBit also exhibited some luciferase activity at high concentrations, likely due to the weak intrinsic cell-penetrating activity of HiBit given its amphipathic sequence (MK Schwinn et al., "CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide", ACS Chem Biol, 13(2):467-474, 2018).

[0181] Intracellular delivery of additional protein cargo We first tested MTD4 for its ability to deliver a protein cargo, Superecliptic pHluorin (SEP). SEP is a pH-sensitive variant of GFP (pKa ≈7.2) that is highly fluorescent in the neutral environment of the mammalian cytosol (pH 7.4) but is essentially non-fluorescent within endosomes (pH 5.5–6.5) or lysosomes (pH 4.5–5.5) (S. Sankaranarayanan, et al., "The use of pHluorins for optical measurements of presynaptic activity", Biophys J, 79(4):2199-208, 2000). Thus, intracellular fluorescence primarily reflects the amount of SEP that successfully reached the cytosol. We genetically fused SEP to the C-terminus of MTD4 and purified the MTD4-SEP fusion protein from E. coli. HeLa cells were incubated with 5 mM SEP or MTD4-SEP for 2 h and imaged by live-cell confocal microscopy. Cells treated with MTD4-SEP displayed strong fluorescence throughout the cell volume, whereas no fluorescence was detected in untreated cells or cells treated with SEP (FIGS. 12A-C).

[0182] To demonstrate functional delivery of protein cargo to the cytosol of mammalian cells, protein tyrosine phosphatase 1B (PTP1B) was selected as cargo and genetically fused to the C-terminus of MTD4. Tyrosine phosphorylation is usually restricted to cytosolic and nuclear proteins, or to the cytosolic domains of transmembrane proteins. PTP1B is a broad-specificity phosphatase that catalyzes the dephosphorylation of many intracellular proteins (NG Selner et al., "Diverse levels of sequence selectivity and catalytic efficiency of protein-tyrosine phosphatases", Biochemistry, 53(2):397-412, 2014). Cytosolic delivery of PTP1B is expected to reduce phosphotyrosine (pY) levels of intracellular proteins, which can be easily monitored by anti-pY Western blotting. HEK293T cells were treated with different concentrations of MTD4-PTP1B for 6 h, washed, and lysed in the presence of protease and phosphatase inhibitors. Cellular proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with anti-pY antibody 4G10. MTD4-PTP1B dose-dependently reduced global pY levels in HEK293T cells, and EC 50 Values ​​were below 5 nM (Figures 13A-B). Based on the fact that cells treated with 5 nM MTD4-PTP1B had lower pY levels than cells treated with 5 mM unconjugated PTP1B (WT), we estimate that MTD4 increases cytosolic entry of PTP1B by more than 1000-fold.

[0183] In-vivo biodistribution of MTD4-EGFP-NLS-Cre To study the biodistribution of MTD4 in mice, EGFP-NLS-Cre protein was genetically fused to the C-terminus of MTD4. Cre recombinase is an enzyme derived from P1 bacteriophage that catalyzes site-specific DNA recombination (K. Abremski, et al., "Bacteriophage P1 site-specific recombination. Purification and properties of the Cre recombinase protein", J Biol Chem, 259(3):1509-14, 1984). Successful delivery of MTD4-EGFP-NLS-Cre (MENC) fusion protein into cells of transgenic mice is expected to result in DNA recombination events and activation of red fluorescent protein (mCherry) expression. Treatment of primary cells derived from transgenic mice with 1 μM MENC in vitro resulted in mild expression of mCherry after 48 h (data not shown).

[0184] Encouraged by the in vitro data, MENC (8 mg / kg) was injected into the tail vein of four transgenic mice, two mice were euthanized after 3 h and the other mice were euthanized 48 h after the treatment. Various organs were harvested, fixed, embedded, sliced ​​and visualized under confocal microscopy. To reduce any autofluorescence from the tissues, the samples were treated with true black dye. Strong EGFP fluorescence was observed in most tissues except the brain, indicating that MENC is widely biodistributed in various organs (Figure 14). Meanwhile, mCherry signal was weak in most organs even after 48 h, which is probably due to the fact that the nuclear localization sequence (NLS) is sandwiched between two protein domains, resulting in low nuclear localization efficiency. Nevertheless, the confocal images clearly show the biodistribution of MENC in various mouse organs.

[0185] Serum stability of MTD4 MTD4 was incubated with human serum for various times and analyzed by SDS-PAGE. MTD4 appears to undergo proteolysis at a single site near the C-terminus, as the cleavage product is slightly smaller than MTD4 and can bind to a nickel affinity column via its N-terminal 6xHis tag (Figure 15A). Degradation of MTD4 is t 1 / 2 occurs in about 3 hours and is nearly complete after 24 hours. In comparison, FN3 appears to undergo a similar cleavage, but at 1 / 2 for over 24 hours (FIG. 15B). Mass spectrometry confirmed cleavage of FN3 and MTD4.

[0186] Other suitable MTD scaffolds All current MTDs are derived from the 10th FN3 domain of human fibronectin, but it has been hypothesized that many other protein domains may also serve as suitable scaffolds for engineering additional MTDs. In general, a good scaffold should contain two or more adjacent surface loops (to incorporate two CPP motifs), be disulfide-free but stably folded (to tolerate sequence changes), be expressed in high yields in E. coli or eukaryotic hosts, and have no biological function of its own. Based on these criteria, examples of suitable MTD scaffolds include other FN3 domains of fibronectin (Kornblihtt AR, et al., Primary structure of human fibronectin: differential splicing may generate at least 10 polypeptides from a single gene. EMBO J. 4(7):1755-1759, 1985), nanobodies (Muyldermans S., "Nanobodies: natural single-domain antibodies". Annu Rev Biochem. 82:775-797, 2013), designed ankyrin repeat proteins (DARPins) (Stumpp MT, et al., "DARPins: a true alternative to antibodies". Curr Opin Drug Discov Devel. 10(2):153-159, 2007), and consensus tetratricopeptide repeats (CTPRs) (Uribe KB, et al., "Engineered Repeat Protein Hybrids: The New Horizon for Biologic Medicines and Diagnostic Tools". Acc Chem Res. 54(22):4166-4177, 2021), Anticalin (Rothe C, et al., Anticalin TMProteins as Therapeutic Agents in Human Diseases. BioDrugs. 32(3): 233-243, 2018), nanofitin / affitin (Goux M, et al., "Nanofitin as a New Molecular-Imaging Agent for the Diagnosis of Epidermal Growth Factor Receptor Over-Expressing Tumors". Bioconjug Chem. 28(9): 2361-2371, 2017), affimer (Tiede C, et al., "Affimer proteins are versatile and renewable affinity reagents". Elife. 6: e24903, 2017), affilin (Ebersbach H, et al., "Affilin-novel binding molecules based on human gamma-B-crystallin, an all beta-sheet protein". J Mol Biol. 372(1):172-185, 2007), FHA domain (Durocher D, et al., "The FHA domain". FEBS Lett. 513(1):58-66, 2002), and SH2 domain (Pawson T, et al., "SH2 domains, interaction modules and cellular wiring". Trends Cell Biol. 11(12):504-511, 2001).

[0187] Assay Confocal microscopy HeLa cells were cultured at 5 × 10 in a 35 / 10 mm glass-bottom microwell dish with four compartments. 4Cells were seeded at a density of 1000 cells / mL and cultured overnight in DMEM containing 10% FBS and 1% Abs. Cells were washed twice with DPBS and treated with 5 μM TMR-labeled proteins for 2 h in phenol red-free DMEM containing 1% FBS and 1% Abs. Cells were washed twice with DPBS, replenished with phenol red-free DMEM, and imaged on a Nikon A1R live cell imaging confocal microscope. Image analysis was performed using NIS Elements AR.

[0188] Flow cytometry HeLa cells were cultured in a 24-well plate at 7.5 × 10 4 Cells were seeded at a density of 1000 cells / well. On the day of the experiment, cells in DMEM medium supplemented with 1% FBS and 1% Abs were incubated with 5 μM TMR-labeled protein for 2 h. Cells were washed with cold DPBS and harvested by trypsinization. Detached cells were washed twice with DPBS, resuspended in DPBS, and analyzed by flow cytometry (BD FACS Aria III).

[0189] Western blotting NIH-3T3 cells were cultured at 10 6Cells were seeded at a density of 1000 cells / well at 37°C and 5% CO2. Cells were starved in serum-free medium for 3 h. Cells were treated with different concentrations of MTD4-PTP1B for 4 h and stimulated with EGF (50 ng / mL) for 10 min. Cells were harvested, washed with PBS, and lysed in 100 μL of Pierce RIPA buffer (Thermo) containing protease, phosphatase inhibitors, and sodium pervanadate for 30 min on ice. Lysates were centrifuged at 15,000 rpm for 20 min. Total protein concentration of each sample was measured using a BCA protein assay kit (Thermo). Equal amounts of protein were loaded into each lane of a 10% SDS-PAGE gel (120 V, 2.5 h). Proteins were electrophoretically transferred to a nitrocellulose membrane at 4°C (90 V, 2.5 h). The membrane was blocked with 5% BSA in TBST buffer (20 mM Tris pH 7.5, 150 mM NaCl, 0.1% (v / v) Tween-20) for 1 h at room temperature. Finally, the membrane was incubated with anti-pY antibody 4G10 (1:1000 dilution) overnight at 4 °C. The membrane was washed three times with TBST and incubated with fluorescently labeled secondary antibody (1:10,000 dilution) for 2 h at room temperature. The membrane was washed again three times with TBST and the signal was acquired using a LICOR Odyssey CLx instrument.

[0190] For Western blotting with anti-p-Akt and anti-p-MEK antibodies. MiaPaCa-2 cells were seeded in 12-well plates at a density of 150,000 cells / well in DMEM medium supplemented with 10% FBS and 1% ABS and incubated at 37°C and 5% CO2. The next day, cells were treated with PBS or various concentrations of MTD4-RBDV or MTD4-NS1 for 4 hours and then stimulated with EGF (50 ng / mL) for 10 minutes. Cells were harvested, lysed, and analyzed by Western blotting with anti-p-Akt and anti-p-MEK antibodies as described for MTD4-PTP1B.

[0191] HEK293T cells were cultured in 6-well plates at 30 × 10 5Cells were seeded at a density of 1000 cells / well at 37°C and 5% CO2. Cells were treated with different concentrations of MTD4-PTP1B in serum-free medium for 6 h. Cells were harvested, washed with PBS, and lysed in 100 μL of Pierce RIPA buffer (Thermo) containing protease, phosphatase inhibitors, and sodium pervanadate for 30 min on ice. Lysates were centrifuged at 15,000 rpm for 20 min. Total protein concentration of each sample was measured using a BCA protein assay kit (Thermo). Equal amounts of protein were loaded into each lane of a 10% SDS-PAGE gel (120 V, 2.5 h). Proteins were electrophoretically transferred to nitrocellulose membranes at 4°C (90 V, 2.5 h). The membrane was blocked with 5% BSA in TBST buffer (20 mM Tris pH 7.5, 150 mM NaCl, 0.1% (v / v) Tween-20) for 1 h at room temperature. Finally, the membrane was incubated with anti-pY antibody 4G10 (1:1000 dilution) overnight at 4 °C. The membrane was washed three times with TBST and incubated with fluorescently labeled secondary antibody (1:10,000 dilution) for 2 h at room temperature. The membrane was washed again three times with TBST and the signal was acquired using a LICOR Odyssey CLx instrument.

[0192] Cell viability assay H358, MiaPaCa-2, H1915, or H1299 cells were seeded in white 96-well plates (5000 cells / well). The next day, cells were treated with serially diluted protein solutions or PBS. Cells were incubated at 37°C, 5% CO2 for 72 hours. After adding Cell Titer Glo and incubating for an additional 15 minutes, luminescence was measured on a TECAN instrument. Viability values ​​reported are relative to the viability of control cells treated with PBS.

[0193] BRET assay HEK293T were seeded (650,000 / well) in 6-well plates and incubated at 37°C, 5% CO2. The next day, cells were transfected with pEF-RLUC8-L15-KrasG12D and pEF-CRAFRBD(1-149)-L15-GFP, or pEF-RLUC8-L15-KrasG12V and pEF-CRAFRBD(1-149)-L15-GFP at a 1:2 ratio (50ng KRAS and 100ng CRAFRBD-GFP). After transfection, cells were incubated at 37°C for 24 hours. The next day, cells were seeded (50,000 cells / well) in white 96-well plates and incubated at 37°C for 4 hours, followed by the addition of various concentrations of MTD4-RBDV or MTD4-NS1. After incubation for 20–24 h and addition of 10 μM coelenterazine 400a substrate, the BRET signal was measured on a TECAN instrument.

[0194] Annexin-V / PI staining H358 cells were cultured in 12-well microplates at 10 × 10 in 1 mL of RPMI containing 10% FBS and 1% Abs. 4Cells were seeded at a density of 1000 cells / well and incubated overnight at 37°C, 5% CO2. The next day, the medium was removed, the cells were washed with DPBS, and treated with various concentrations of MTD4-RBDV or MTD4-NS1 in 1 mL of DMEM medium containing 10% FBS for 24 h at 37°C, 5% CO2. To harvest the cells, the medium was collected in a 15 mL Falcon tube. The cells were washed with DPBS, and the wash was combined with the medium in the corresponding Falcon tube. The adherent cells were treated with 250 μL / well of 0.25% trypsin for 3 min at 37°C and transferred back to the respective Falcon tubes. The cells were pelleted by centrifugation at 300g for 5 min at 4°C and washed twice with DPBS to remove residual trypsin. Annexin V staining was then performed according to the Invitrogen protocol. The cell pellet was resuspended in 100 μL of 1X Annexin Binding Buffer. Next, 5 μL of Alexa Fluor® 488 Annexin V and 1 μL of propidium iodide (PI, 100 μg / ml) were added to the cell suspension and incubated for 15 min at room temperature. Finally, 400 μL of annexin binding buffer was added to each tube immediately before analyzing the fluorescence emission at 530 nm and 575 nm on a BD LSR Fortessa flow cytometer.

[0195] Peptide synthesis and conjugation of HiBit peptides All peptides were synthesized manually on rink amide resin using standard Fmoc chemistry. A typical coupling reaction included 5 equivalents of Fmoc amino acid, 5 equivalents of HATU, and 10 equivalents of diisopropylethylamine (DIPEA), and proceeded with mixing at room temperature (RT) for 30 min. In the case of CPP12, which has a single cysteine ​​at the C-terminus, the allyl group on the C-terminal Glu residue was removed after addition of the last (N-terminal) residue by treatment with 0.3 equivalents of Pd(PPh3)4 and 10 equivalents of phenylsilane in anhydrous DCM in the dark (3 × 15 min). The resin was washed twice with sodium dimethyldithiocarbamate (SDDCM, 0.5 M in DMF) and the terminal Fmoc group was removed by treatment with 20% piperidine in NDMF. The resin was washed extensively with DMF and DCM and incubated in 1 M 1-hydroxybenzotriazole (HOBt) in DMF for 20 min. The peptide was cyclized using 10 equivalents of PyBOP, 10 equivalents of HOBT, and 20 equivalents of DIPEA in DMF at room temperature for 1 hour. Cleavage and deprotection of the peptide was performed on the resin at room temperature for 3 hours using 92.5 / 2.5 / 2.5 / 2.5 (v / v) TFA / triisopropylsilane / 1,3-dimethoxybenzene / water. HiBit peptide with an N-terminal cysteine ​​(CVSGWRLFKKIS) (SEQ ID NO: 152) was synthesized as described above. The purified HiBit peptide was activated with 2,2'-dipyridyl disulfide (PyS-HiBit) and isolated. For conjugation, 1 equivalent of PyS-HiBit peptide was incubated with CPP12-Cys in methanol containing 2% acetic acid, and the conjugated peptide was purified by reversed-phase HPLC on a semi-preparative Waters XBridge C18 column. Similarly, 3 equivalents of PyS-HiBit were incubated with MTD without reducing agent for 3 h, and the reaction mixture was passed through a spin desalting column to remove excess HiBit peptide. Conjugation was confirmed by running a non-reducing SDS-PAGE gel and monitoring a shift of approximately 1.3 kDa.

[0196] HiBit delivery assay. HEK293T cells were cultured at 60 × 10 4 Cells were seeded in 6-well plates at a density of 10,000 cells / well. The next day, cells were transfected with 0.5 μg LgBit plasmid DNA for 24 h using Lipofectamine 2000. Cells were re-seeded in 96-well plates overnight at a density of 10,000 cells / well in seeding medium. Cells were treated with various concentrations of conjugated or unconjugated HiBit peptides for 4 h in medium supplemented with 1% FBS and 1% Abs. Cells were washed twice with DPBS before adding 100 μL OPTIMEM and 25 μL NanoLuc reagent per well. Luminescence was measured immediately using a Tecan Infinite M1000 Pro microplate reader. Values ​​were normalized to those of untreated cells and plotted against peptide concentration as mean ± SD using GraphPad Prism software.

[0197] GFP complementation assay. HEK293T cells were cultured at 60 × 10 4 The cells were seeded in 6-well plates at a density of 1 × 10 cells / well. The next day, the cells were transfected with 1 μg of GFP1-10 plasmid DNA for 24 h using Lipofectamine 2000. The cells were then seeded at 5 × 10 cells / well in 35 / 10 mm glass-bottom microwell dishes with four compartments. 4 Cells were seeded at a density of 1000 cells / mL and cultured overnight in DMEM containing 10% FBS and 1% Abs. Cells were washed twice with DPBS and treated with DPBS (untreated control) or 10 μM GFP11, CPP12-GFP11, or MTD4-GFP11 for 6 h in medium supplemented with 1% FBS and 1% Abs. Cells were washed twice with DPBS, supplemented with phenol red-free medium, and imaged on a Nikon A1R live cell imaging confocal microscope. Image analysis was performed using NIS Elements AR.

[0198] Biodistribution studies All animal experiments were performed in accordance with institutional animal care guidelines and in accordance with committee-approved protocols. Endotoxins were removed from concentrated MENC protein using Pierce High Capacity Endotoxin Removal Resin. Endotoxin-free MENC protein (8 mg / kg) was injected intravenously via the tail vein into four floxed mice (loxP-modified transgenic mice). Two mice were sacrificed after 3 hours and the other two after 48 hours. Various organs were harvested, fixed in 4% paraformaldehyde for 24 hours, and transferred to a 70% ethanol solution. Fixed samples were sent to iHisto Inc. for embedding, slicing, and slide preparation. For imaging, slides were processed by washing twice with 100% xylene, 100% ethanol, 95% ethanol, and ddH2O. Slides were stained with True Black dye (Biotium), mounted, and sealed in toluene. Slides were imaged with a Nikon A1R confocal microscope and analyzed using NIS Elements AR software.

[0199] Serum stability assay FN3 or MTD4 (10 μM) was incubated with 25% clarified human serum in a total volume of 100 μL. The mixtures were incubated at 37°C and 10 μL aliquots were taken at different time points. The aliquots were immediately mixed with 10 μL of 2xSDS loading buffer, boiled for 5 min, and stored at -20°C. After up to 24 h of incubation, all aliquots were analyzed on 15% SDS-PAGE gels and the gels were stained with Coomassie blue. The gels were scanned in the 700 nm channel on an Odyssey Clx Imager (LI-COR).

[0200] [Table 6] TIFF2025509645000019.tif215170TIFF2025509645000020.tif217170TIFF2025509645000021.tif217170TIFF2025509645000022.tif218170TIFF2025509645000023.tif215170TIFF2025509645000024.tif24170

Claims

1. A peptide comprising a membrane translocation domain having one or more cell-penetrating peptide motifs, wherein at least one cell-penetrating peptide motif is 3 to 10 amino acid residues in length and has at least three arginine and / or lysine residues, or at least one cell-penetrating peptide motif is 3 to 10 amino acid residues in length and has at least two arginine and / or lysine residues, and at least one other cell-penetrating peptide motif is 2 to 8 amino acid residues in length and has at least two hydrophobic residues.

2. The peptide of claim 1, wherein the membrane translocation domain is a mammalian membrane translocation domain.

3. The peptide according to claim 2, wherein the membrane translocation domain is human fibronectin type III.

4. The peptide of claim 3, wherein the human fibronectin type III has 90% sequence similarity with SEQ ID NO:

118.

5. The peptide of claim 1, wherein the cell-penetrating peptide motif has 3 to 10 adjacent arginine residues.

6. The peptide of claim 3, wherein the membrane translocation domain is human fibronectin type III having BC, DE, CD, and FG loops, and one or more of the BC, DE, CD, or FG loops have a cell-penetrating peptide motif.

7. The peptide of claim 6, wherein the membrane translocation domain is human fibronectin type III having BC, DE, CD, and FG loops, and two of the BC, DE, CD, or FG loops have a cell-penetrating peptide motif.

8. The peptide of claim 6, wherein the membrane translocation domain is human fibronectin type III having BC, DE, CD, and FG loops, and any of the BC, DE, CD, or FG loops has a cell-penetrating peptide motif.

9. The peptide of claim 6, wherein the membrane translocation domain is human fibronectin type III having BC, DE, CD, and FG loops, and the BC and FG loops have cell-penetrating peptide motifs.

10. The peptide of claim 9, wherein the cell-penetrating peptide motif in the BC loop has 2 to 8 amino acid residues and has at least two hydrophobic amino acid residues, and the cell-penetrating peptide motif in the FG loop has 3 to 10 amino acid residues and has at least two adjacent arginine and / or lysine residues.

11. The peptide according to claim 9, wherein the cell-penetrating peptide motif in the FG loop has 2 to 8 amino acid residues and has at least two hydrophobic amino acid residues, and the cell-penetrating peptide motif in the BC loop has 3 to 10 amino acid residues and has at least two adjacent arginine and / or lysine residues.

12. 12. The peptide of any one of claims 1 to 11, wherein a second cell-penetrating peptide motif is present and is WW, FF, WF, FW, WWF, WFF, FWW, FFW, WYW, WWH, YWW, or WYH.

13. 12. The peptide of claim 1, wherein the cell-penetrating peptide motif is RRRWWW (SEQ ID NO: 104) or WWWRRR (SEQ ID NO: 105).

14. 12. The peptide of any one of claims 1 to 11, comprising TGRRRRRWWWSKPI (SEQ ID NO: 111), APWWWRRRRYY (SEQ ID NO: 112), GGRRRRRWWWVQE (SEQ ID NO: 113), APAWYWRYY (SEQ ID NO: 114), TGRRRRSKPI (SEQ ID NO: 115), APARRRRYY (SEQ ID NO: 116), or TGWYWRSKPI (SEQ ID NO: 117).

15. 12. The peptide of any one of claims 1 to 11, comprising SEQ ID NO: 119, 120, 121, 122, 123, 124, or 125.

16. The peptide of claim 1, further comprising a cargo moiety linked to the membrane translocation domain.

17. 17. The peptide of claim 16, wherein the cargo moiety is linked to the membrane translocation domain at the N-terminus or C-terminus of the membrane translocation domain or by a side chain within the membrane translocation domain.

18. 17. The peptide of claim 16, wherein the cargo moiety is selected from the group consisting of a detectable moiety, a targeting moiety, a therapeutic moiety, or any combination thereof.

19. 12. The peptide of any one of claims 1 to 11, comprising SEQ ID NO: 126, 127, or 128.

20. 1. A composition for delivering a cargo moiety to a cell, comprising SEQ ID NO: 122 covalently attached to a cargo, wherein the cargo moiety is selected from the group consisting of a detectable moiety, a targeting moiety, a therapeutic moiety, or any combination thereof.

21. 20. A method for delivering a cargo moiety to a cell, the method comprising contacting the cell with a peptide according to any one of claims 16 to 18.

22. 19. A method for delivering an agricultural product to a plant cell, the method comprising contacting said cell with a peptide according to any one of claims 16 to 18.

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