Peptides and peptide mimetics that bind ATG8 proteins, heterobifunctional compounds comprising the peptides and peptide mimetics, and uses thereof

Abstract

This disclosure provides engineered polypeptides with Atg8 binding activity. The polypeptides comprise a LC-3 interacting motif comprising one or more mutations relative to a wild type LC3 -interacting motif. Also provided are heterobifunctional compounds, compositions comprising the compounds or engineered polypeptides, and methods of using the compounds or engineered polypeptides.

Description

[0001] PEPTIDES AND PEPTIDE MIMETICS THAT BIND ATG8 PROTEINS, HETEROBIFUNCTIONAL COMPOUNDS COMPRISING THE PEPTIDES AND PEPTIDE MIMETICS, AND USES THEREOF

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 694,656, filed on September 13, 2024. The contents of which are incorporated herein by reference in their entirety.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under grant number GM148407 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0006] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0007] The contents of the electronic sequence listing (166118 01561. xml; Size: 68,884 bytes bytes; and Date of Creation: September 15, 2025) is herein incorporated by reference in its entirety.

[0008] BACKGROUND

[0009] Autophagy is a catabolic mechanism that aids in the degradation of damaged intracellular material and metabolite recycling. This activity helps meet metabolic needs during nutrient deprivation, genotoxic stress, growth factor withdrawal and hypoxia. However, autophagy plays a paradoxical role in tumorigenesis, depending on the stage of tumor development. Early in tumorigenesis, autophagy is a tumor suppressor via degradation of potentially oncogenic molecules. However, in advanced stages, autophagy promotes the survival of tumor cells by ameliorating stress in the microenvironment. Current autophagy inhibitors are nonspecific and affect many other biological pathways. Accordingly, there is a remaining need in the art for selective, potent autophagy inhibitors as well as autophagy protein ligands.

[0010] SUMMARY

[0011] The invention comprises novel compounds that bind autophagy-associated proteins including members of the ATG8 protein family. One aspect of the present disclosure aspect of the present disclosure provides an engineered polypeptide with ATG8 binding activity, wherein the polypeptide comprises a LC3- interacting motif, and wherein the motif comprises one or more mutations relative to a wild type LC3- interacting motif. In some embodiments, the engineered peptide inhibits GABARAP or LC3 proteins. In some embodiments, a method of treating an autophagy-related disease is provided, the method comprising administering an effective amount of an engineered polypeptide described herein.

[0012] In another aspect, the disclosure provides a heterobifunctional compound having a formula: M-L-T, wherein M is a moiety that binds to a ATG8 protein, L is a bond or linker covalently attaching M and T and, T is a moiety that binds to a target protein. In some embodiments, the ATG8 protein is a Gamma-aminobutyric acid receptor-associated protein (GABARAP) or Microtubule-associated protein lA / lB-light chain 3 (LC3) protein. In some embodiments, M comprises a LC3 -interacting region motif, and wherein the motif comprises one or more mutations relative to a wild-type LC3 interacting motif. In some embodiments, L comprises a polyethylene glycol moiety, an alkyl moiety, a glycol moiety, a heterocycle, an alkyne, a spirocycle or combinations thereof and T binds to a target protein to be degraded by autophagy. In some embodiments, the present disclosure provides a method of targeting a protein for degradation by autophagy, the method comprising binding the target protein to T from a heterobifunctional compound described herein.

[0013] BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention.

[0015] FIG. 1 is a drawing illustrating the process of autophagy, which is a cell recycling mechanism where cytosolic material is shuttled to the lysosome for degradation. By limiting the effect of reactive oxygen species (ROS), autophagy prevents DNA damage and maintains genome integrity. Autophagy has a complex relationship with cancer. In early stage cancer, autophagy can play a cytoprotective role. In late stage cancer, autophagy can promote cancer cell survival. Inhibition of autophagy can resensitize cancer cells to chempotherapy. FIG. 2 is a drawing illustrating the involvement of LC3 and GABARAPs in autophagy. Atg8 proteins are important for cargo recruitment.

[0016] FIG. 3. is drawing demonstrating that Atg8 binding partners interact through the LC3 Interacting Region. The binding domain of Atg8s is called the LIR docking site. In this region there are two hydrophobic pockets formed. HP1 is formed between alpha helix 2 and beta strand 2 and HP2 is formed between beta strand 2 and alpha helix 3. Atg8 binding partners interact with this domain using the LC3 interacting motif or LIR motif. This motif typically consists of an aromatic residue, tryptophan, tyrosine, or phenylalanine, an amino acid, another amino acid, and an aromatic residue, commonly isoleucine, leucine, or valine ([W / Y / F]-XI-X2-[I / L / V]). This is considered the core LIR motif. N-terminal acidic residues increase due to the general basic nature of the n-terminus of Atg8s. C-terminal residues / interactions can also increase binding to Atg8s. The space fill model shows GAB ARAP with the core LIR motif of ULK1 binding in pink. Phenylalanine occupies HP1 and Vai occupies HP2. The protein usually binds in an extended conformation, and N-terminal acidic residues stabilize interactions.

[0017] FIG. 4 is a drawing illustrating strategies employed to improve drug-like properties of the disclosed peptides, including peptide stapling and A -methyl at ion.

[0018] FIG. 5. is a drawing illustrating peptide stapling to stabilize the extended conformation. Shown are examples of stapling strategies including thiol alkylation and Glaser stapling. The table shows the effect of sidechain stapling modifications at various position in the parent peptide (wildtype Ulkl).

[0019] FIG. 6 shows the impact of backbone / V-mcthylation to remove the amide. The table shows the effect of A -methylated amide modification in the parent peptide and the effect on binding GAB ARAP.

[0020] FIGS. 7A-7B are drawings illustrating the fluorescence polarization (FP) assay (FIG. 7A) and AlphaScreen assay (FIG. 7B) utilized in the Examples.

[0021] FIG. 8 shows the structure-based design of stapled peptides. Stapled peptides containing cysteine-substituted residues were stapled with either para or meta- methylbenzene. Binding affinities for recombinant GAB ARAP (nM) were measured by FP. Allyl containing peptides were used as a linear control. J denotes norleucine, a structural analog of methionine. Stapling at position 5 and 7 with a para-xylene linker lead to nanomolar affinity for GAB ARAP. FIG. 9 shows the effect of phenylalanine to tryptophan mutation. Replacement of Phe with Trp lead to a 10-fold increase in affinity for both the parent peptide and the stapled peptide.

[0022] FIG. 10 shows the effect of truncation of IM-20. Removal of either of the termini lead to a ~20-fold decrease in affinity. Simultaneous truncation of the termini lead to significant loss in affinity.

[0023] FIG. 11 shows the effect of .V-methylation of core LIR FVJV. A represent A'-m ethyl phenylalanine, £1 represents A -methyl valine, and II represent Af-m ethyl norleucine. IM-3 showed improved affinity after JV-methylation.

[0024] FIG. 12 shows the effect of phenylalanine to tryptophan mutation of IM-3. Similar to the parent peptide and stapled peptide, replacement of Phe with Trp lead to a 10-fold increase in affinity in the A-methyl peptide.

[0025] FIG. 13 shows Ulkl and IM-26 binding modes. A-methyl in IM-27 points to solvent. Ulkl and IM-27 bind almost identically with slight variations in the N- and C-termini of the peptides.

[0026] FIG. 14 shows truncation of IM-27. Removal of the N-terminus lead to a ~13 fold decrease in affinity. Removal of the C-terminus lead to a ~2-fold decrease in affinity. Simultaneous truncation of the termini lead to a ~16-fold loss in affinity.

[0027] FIG. 15 shows the inhibitory concentration (IC50) of A-methyl series. 0 represents indole. Due to the removal of the terminal amine, a Kd could not be determined for IM-33. Increased truncation of the peptides lead to increased IC50 values.

[0028] FIG. 16 shows results from the IM-20 and IM-27 combination peptide. Combination of the staple and A-methyl modification did not yield an additive effect on binding.

[0029] FIG. 17 depicts an exemplary heterobifunctional compound with the formula M-L-T. In this non-limiting example, M comprises an LC3 / GABARAP ligand (IM-3; SEQ ID NO: 5) and T comprises JQ1, which are connected via a linker (L).

[0030] DETAILED DESCRIPTION

[0031] The invention comprises novel compounds that bind the autophagy-associated protein including members of the Atg8 protein family. The compounds described herein are more potent compounds than naturally occurring inhibitors, are more drug-like and are expected to be more cell-penetrant and bioactive. Autophagy is an essential stress survival pathway transferring cytoplasmic material in autophagosomes to lysosomes for degradation, thereby restoring nutrients and molecular building blocks. Autophagy ensures cellular health by selectively removing toxic macromolecules, damaged organelles, or intracellular pathogens. Accordingly, deregulation of autophagy has been implicated in a broad range of diseases, including cancer, neurodegenerative disorders, and infection. Further targeting proteins to autophagy provides a novel path to target specific degradation.

[0032] Autophagy relies on soluble or membrane-bound cargo receptors that recognize cargo and bring about autophagosome formation at the cargo. The cargo-bound receptors interact with lipidated Atg8 family proteins anchored in the membrane at the concave side of the forming autophagosome. The interaction is mediated by 15- to 20-amino-acid-long sequence motifs called LC3 -interacting region (LIR) motifs that bind to the LIR docking site (LDS) of Atg8 proteins. Mammalian Atg8 proteins comprise two subfamilies, namely Microtubule-associated protein lA / lB-light chain 3 proteins (LC3s), including LC3A, LC3B, and LC3C and Gamma- aminobutyric acid receptor-associated proteins (GABARAPs), including GABARAP, GABARAP-L1, and GAB ARAP -L2. Atg8 proteins promote autophagosome formation, elongation, and closure, as well as fusion with lysosomes.

[0033] The canonical LIR motif is a small O0-X1-X2T3 motif, where O represents an aromatic residue (W / F / Y) and T an aliphatic residue (L / V / I) (whose side chains bind to hydrophobic pocket 1 (HP1) and HP2 of the LIR docking site (LDS), respectively) and X represents any amino acid. The N-terminal region directly preceding the core LIR motif often harbors acidic or phosphorylated residues that stabilize Atg8 binding through electrostatic interactions. ULK1 / 2 (Unc-51 like autophagy activating kinase 1 / 2) is an important protein in autophagy and binds to Atg8 proteins at the LIR motif. Compounds provided herein are based on a natural protein sequence derived from the human protein ULK1 (TDDFVMVPA SEQ ID NO: 1), but which have been modified to promote increased binding, be more drug-like, be more cell-penetrant and bioactive.

[0034] Compounds:

[0035] Disclosed herein includes a heterobifunctional compound having a formula: M-L-T wherein M is a moiety that binds to a Atg8 protein, L is a bond or linker covalently attaching M and T and, T is a moiety that binds to a target protein or is a label. M may bind to an LC3 family protein or a GABARAP family protein, including but not limited to LC3A, LC3B, LC3C, GAB ARAP, GABARAP-Ll, and GAB ARAP -L2. A heterobifunctional compound has the ability to bind or otherwise interact with more than one moiety, for example M may bind to an Atg8 protein and T may bind to a target protein. Examples of heterobifunctional compound may include various protein degradation technologies including but not limited to proteolysis targeting chimera (PROTAC), molecular glue, Chaperone-mediated Protein Degradation / Degrader (CHAMP), Lysosome-Targeting Chimaeras (LYTAC), Bispecific Aptamer Chimera (BIAC), Antibody -based PROTAC (AbTAC), GluTAC, Autophagy-Targeting Chimeras (AUTAC or AUTOTAC), and Autophagosome-Tethering Compound (ATTEC). Suitable labeling moieties are known in the art and include, without limitation, a gold nanoparticle, a protein binding ligand, a hapten, an antigen, a fluorescent compound, a dye, a radioactive isotope, and an enzyme. For example, the label may be a fluorescent compound, such as fluorescein which is utilized in the examples.

[0036] In some embodiments, M comprises a LC3 -interacting motif, and wherein the motif comprises one or more mutations relative to a wild-type LC3 interacting motif. The LC3- interacting region (LIR) motif, which ensures the targeting of autophagy receptors to LC3 and / or GABARAP proteins anchored in the phagophore membrane. The wild-type LC3 interacting region motif may comprise the sequence TDDFVMVPA (SEQ ID NO: 1). In some embodiments, M may be a peptide, a peptide mimetic, a small molecule derived from the peptide, or another small molecule.

[0037] The term “mutation” as used herein indicates any modification of a nucleic acid and / or polypeptide which results in an altered nucleic acid or polypeptide (i.e., relative to the wild-type nucleic acid or polypeptide sequence). Mutations include, for example, point mutations, substitutions, deletions, or insertions of single or multiple residues in a polypeptide or polynucleotide (or the encoded polypeptide), which includes alterations arising within a proteinencoding region of a gene as well as alterations in regions outside of a protein-encoding sequence, such as, but not limited to, regulatory or promoter sequences. A genetic alteration may be a mutation of any type. For instance, the mutation may constitute a point mutation, a frame-shift mutation, an insertion, or a deletion of part or all of a gene. In certain embodiments the mutations are the result of genetic or protein engineering. The compounds provided herein comprise one or more mutations to the wild-type LC3 motif. Mutations may comprise any that increase bioavailability, bioactivity, binding or cell penetration. Binding of the mutated compounds may also alter the function of LC3 and or GABARAP proteins. In some embodiments, the mutations may comprise inclusion of an amino acid substitution, a non-natural amino acid, an amino acid analog, a A' -methyl substitution, a covalent linkage, an N-terminal or C-terminal modification, or a combination thereof. In some embodiments, the compound comprises a tryptophan for phenylalanine substitution. The tryptophan for phenylalanine substitution may be located, with reference to SEQ ID NO: 1, at amino acid position 4, or a homologous position thereof. In some embodiments, the A-m ethyl substitution may comprise A -methyl phenylalanine, A -methyl norleucine, or A -methyl valine or combinations thereof. In some embodiments, the A-methyl substitution is located, with reference to SEQ ID NO: 1, at amino acid position 6, or a homologous position thereof. In some embodiments the non-natural amino acids or amino acid analogs may comprise norleucine, tryptophan analogs or combinations thereof. In some embodiments, the covalent linkage may comprise sidechain stapling. As used herein, “sidechain stapling” refers to the crosslinking of the sidechains of two amino acid residues in a peptide molecule. Suitable sidechain stapling linkages include, without limitation, an ortho- dimethylbenzene (o-xylene) linkage, a meta- dimethylbenzene (m-xylene) linkage, a / wcr-dimethylbenzene (p-xylene) linkage, a 2,2’ - bi s(methyl) 1,1 ’-biphenyl linkage, a bis(methyl)naphthalene linkage, or a 2,4-Hexadiyne linkage. In some embodiments, the sidechain stapling comprises a linkage between amino acids located, with reference to SEQ ID NO: 1, at amino acid positions 5 and 7, or a homologous position thereof. The sidechain stapling may comprise a linkage between any two suitable amino acid residues. In some embodiments, the sidechain stapling comprises a linkage between a cysteine residue and a cysteine residue, a cysteine residue and a penicillamine residue, a penicillamine residue and a penicillamine residue, or a propargyl serine residue and a propargylserine residue. In some embodiments, the covalent linkage comprises an allyl modification. In some embodiments, the covalent linkage comprises an allyl modification of the amino acids located, with reference to SEQ ID NO: 1, at amino acid positions 5 and / or 7, or a homologous position thereof.

[0038] Unnatural amino acids include, but are not limited to, azetidinecarboxylic acid, 2- aminoadipic acid, 3-aminoadipic acid, beta-alanine, naphthylalanine (“naph”), aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2- aminoisobutyric acid, 3-aminoisbutyric acid, 2-aminopimelic acid, tertiary-butylglycine (“tBuG”), 2,4-diaminoisobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N- ethylglycine, N-ethylasparagine, homoproline (“hPro” or “homoP”), hydroxylysine, allohydroxylysine, 3-hydroxyproline (“3Hyp”), 4-hydroxyproline (“4Hyp”), isodesmosine, alloisoleucine, N-methylalanine (“MeAla” or “Nime”), N-alkylglycine (“NAG”) including N- methylglycine, N-methylisoleucine, N-alkylpentylglycine (“NAPG”) including N- methylpentylglycine. N-methylvaline, naphthylalanine, norvaline (“Norval”), norleucine (“Norleu”), octylglycine (“OctG”), ornithine (“Om”), pentylglycine (“pG” or “PGly”), pipecolic acid, thioproline (“ThioP” or “tPro”), homoLysine (“hLys”), and homoArginine (“hArg”).

[0039] The term “amino acid analog” refers to a natural or unnatural amino acid where one or more of the C-terminal carboxy group, the N-terminal amino group and side-chain bioactive group has been chemically blocked, reversibly or irreversibly, or otherwise modified to another bioactive group. For example, aspartic acid-(beta-methyl ester) is an amino acid analog of aspartic acid; N- ethylglycine is an amino acid analog of glycine; or alanine carboxamide is an amino acid analog of alanine. Other amino acid analogs include methionine sulfoxide, methionine sulfone, S- (carboxymethyl)-cysteine, S-(carboxymethyl)-cysteine sulfoxide and S-(carboxymethyl)-cysteine sulfone.

[0040] As used herein, a “conservative” amino acid substitution refers to the substitution of an amino acid in a peptide or polypeptide with another amino acid having similar chemical properties, such as size or charge. Naturally occurring residues may be divided into classes based on common side chain properties, for example: polar positive (or basic) (histidine (H), lysine (K), and arginine (R)); polar negative (or acidic) (aspartic acid (D), glutamic acid (E)); polar neutral (serine (S), threonine (T), asparagine (N), glutamine (Q)); non-polar aliphatic (alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M)); non-polar aromatic (phenylalanine (F), tyrosine (Y), tryptophan (W)); proline and glycine; and cysteine. As used herein, a “semi-conservative” amino acid substitution refers to the substitution of an amino acid in a peptide or polypeptide with another amino acid within the same class.

[0041] In some embodiments, unless otherwise specified, a conservative or semi -conservative amino acid substitution may also encompass non-naturally occurring amino acid residues that have similar chemical properties to the natural residue. These non-natural residues are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include, but are not limited to, peptidomimetics and other reversed or inverted forms of amino acid moieties. Non-conservative substitutions may involve the exchange of a member of one class for a member from another class.

[0042] In some embodiments, the mutations to the LC3 -interacting motif of (M) described herein may result in increased binding affinity of the mutated protein as compared to the wild-type LC3. The equilibrium dissociation constant (Kd) is a quantitative measurement of binding affinity, which is the strength of the interaction between a ligand and a target molecule. M may also bind to an Atg8 protein for a longer duration or be more effective at a lower concentration than a protein with a wild-type LC3- interacting domain. In some embodiments, the heterobifunctional compound provided herein M has a dissociation constant (Kd) for a GAB ARAP or a LC3 protein of less than 500nM, less than 400nM, less than 300nM, less than 200nM, or less than lOOnM, or has a Kd within a range bounded by any of the foregoing. .

[0043] In some embodiments, in the heterobifunctional compound provided herein M may comprise any of those disclosed in Table 1 and shown in Example 1. In some embodiments, M comprises or consists of any one of SEQ ID NOs: 2-38, or comprises or consists of a polypeptide having at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 2-38.

[0044] The disclosed heterobifunctional compounds may include a linker (L) that covalently attaches the Atg8 biding moiety (M) and the target protein moiety (T). The linker connecting M and T in the bifunction compound may be used without limitation as long as it has a structure in which both M and T are structurally connected. Linkers used in the development of heterobifunctional compounds include polyethylene glycol (PEG) linkers, Alkyl-Chain linkers, and Alkyl / ether linkers. In some embodiments, L comprises a polyethylene glycol moiety, an alkyl moiety, a glycol moiety, a heterocycle, an alkyne, a spirocycle or combinations thereof.

[0045] Linkers may be covalently attached by any means to structurally connect M and T. Without limitation such means may comprise solid-phase peptide coupling reactions, click chemistry, and carbodiimide crosslinker chemistry. The linker may be cleavable or non-cleavable and may be of any length. As used herein, the term “conjugate” refers to the joining of two entities by covalent bonds. The entities may be covalently bonded directly or through linking groups using standard synthetic coupling procedures. For examples, two polypeptides may be linked together by simultaneous polypeptide expression typically referred to as a fusion or chimeric protein. One or more amino acids may be inserted into polypeptide as a linking group by incorporation of corresponding nucleic acid sequences into the expression vector. Other contemplated linking groups include polyethylene glycols or hydrocarbons terminally substituted with amino or carboxylic acid groups to allow for amide coupling with polypeptides having amino acids side chains with carboxylic acid or amino groups, respectively. Alternatively, the amino and carboxylic acid groups can be substituted with other binding partners such as an azide and an alkyne, which undergo copper catalyzed formation of triazoles.

[0046] In another example of conjugation, polypeptides are expressed to contain naturally or non- naturally occurring amino acids containing a thiol group. The thiol group can be substituted for an amino group in coupling reactions with carboxylic acids, or two thiol groups when exposed to oxidative conditions react to form disulfides. Additionally, in some embodiments, non-naturally occurring amino acids are incorporated into the polypeptide, allowing for site-specific conjugation of the polypeptide to one or more agents.

[0047] The disclosed heterobifunctional compounds may include a moiety that binds to a target protein (T). In some embodiments, T binds to a target protein to be degraded by autophagy. A target protein may be any protein to be degraded by autophagy. Protein degradation is the process by which proteins are naturally destroyed in a cell. Without limitation target proteins may comprise oncoproteins, monomeric or oligomeric species of aggregation-prone proteins, aggregated proteins, differentially expressed proteins, undruggable proteins, and foreign proteins. Without limitation target proteins may comprise androgen receptor (AR), estrogen receptor (ER), Bruton’s tyrosine kinase (BTK), epidermal growth factor receptor (EGFR), tropomyosin receptor kinase (TRK), Focal adhesion kinase (FAK), Bromodomain and extraterminal (BET), Cyclin-dependent kinases (CDKs), spinocerebellar ataxia type 3 protein, mutated huntingtin (mHTT), methionine aminopeptidase-2 (MetAP2), FKBP12, Translocator protein (TSPO), Bcl-2 family of proteins, bromodomain containing 9 (BRD9), JQ1, and Interleukin- 1 receptor-associated kinase 4 (IRAK4).

[0048] Engineered Polypeptides:

[0049] In another aspect, the present invention provides engineered polypeptides with Atg8 binding activity comprising an LC3 -interacting motif, wherein the motif comprises one or more mutation relative to a wild-type motif. As used herein, an engineered polypeptide comprises a chain of amino acids that have been designed or modified for a specific purpose. The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. A protein may comprise different domains, for example, a nucleic acid binding domain a nucleic acid cleavage domain or an dLC-3 interacting domain or motif.

[0050] The engineered polypeptides of the present invention comprise a LC3 -interacting motif that allows the polypeptide to bind to a member of the Atg8 family, including LC3 A, LC3B, LC3C, GAB ARAP, GABARAP-Ll, and GABARAP-L2. The engineered polypeptide may comprise mutations in the LC3 -interacting motif, as described herein, such that the binding, activity, localization or availability is altered. The engineered polypeptide provided herein may also be a Atg8 ligand.

[0051] In some embodiments, the engineered polypeptide may inhibit the activity of an Atg8 family member including the GABARAP and LC3 proteins. Inhibiting the activity of an Atg8 protein family member includes interfering with, preventing, antagonizing, blocking or reducing the physiological action of an Atg8 protein. In some embodiments an engineered polypeptide provided herein may inhibit or reduce the formation of a phagosome or the process of autophagy. In some embodiments, the engineered polypeptide may be selected from any of those included in Table 1. In some embodiments, the engineered polypeptide comprises or consists of any one of SEQ ID NOs: 2-38, or comprises or consists of a polypeptide having at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 2-38. The engineered polypeptide described herein may be conjugated to one or more compounds. The one or more compounds may comprise a label, an additional polypeptide, a protein, a linker, or a linker conjugated to the label, the additional polypeptide, or the protein. The linker may comprise any of those described herein, for example a PEG linker. Suitable labeling moi eties are known in the art and include, without limitation, a gold nanoparticle, a protein binding ligand, a hapten, an antigen, a fluorescent compound, a dye, a radioactive isotope, and an enzyme.

[0052] Pharmaceutical Compositions:

[0053] In some embodiments, a composition comprising the heterobifunctional compound or a pharmaceutically acceptable salt thereof, as disclosed herein, and a buffer or a pharmaceutically acceptable excipient, carrier, or diluent is provided. In some embodiments, a composition comprising an engineered polypeptide, or a pharmaceutically acceptable salt thereof, as disclosed herein, and a buffer or a pharmaceutically acceptable excipient, carrier, or diluent is provided.

[0054] The compounds and / or engineered polypeptides employed in the compositions and methods disclosed herein may be administered as pharmaceutical compositions and, therefore, pharmaceutical compositions incorporating the compounds and / or engineered polypeptides are considered to be embodiments of the compositions disclosed herein. Such compositions may take any physical form that is pharmaceutically acceptable; illustratively, they can be orally administered pharmaceutical compositions. Such pharmaceutical compositions contain an effective amount of a disclosed compound and / or engineered polypeptides, which effective amount is related to the daily dose of the compound to be administered. Each dosage unit may contain the daily dose of a given compound and / or engineered polypeptide or each dosage unit may contain a fraction of the daily dose, such as one-half or one-third of the dose. The amount of each compound and / or engineered polypeptide to be contained in each dosage unit can depend, in part, on the identity of the particular compound chosen for the therapy and other factors, such as the indication for which it is given. The pharmaceutical compositions disclosed herein may be formulated so as to provide quick, sustained, or delayed release of the active ingredient after administration to the patient by employing well-known procedures.

[0055] In some embodiments, the compounds and / or engineered polypeptides disclosed herein may be formulated as pharmaceutical compositions that include: (a) a therapeutically effective amount of one or more compounds as disclosed herein; and (b) one or more pharmaceutically acceptable carriers, excipients, or diluents. The pharmaceutical composition may include the compound and / or engineered polypeptide in a range of about 0.1 to 2000 mg (preferably about 0.5 to 500 mg, and more preferably about 1 to 100 mg). The pharmaceutical composition may be administered to provide the compound and / or engineered polypeptide at a daily dose of about 0.1 to about 1000 mg / kg body weight (preferably about 0.5 to about 500 mg / kg body weight, more preferably about 50 to about 100 mg / kg body weight). In some embodiments, after the pharmaceutical composition is administered to a subject (e.g., after about 1, 2, 3, 4, 5, or 6 hours post-administration), the concentration of the compound and / or engineered polypeptide at the site of action may be within a concentration range bounded by end-points selected from 0.001 nM, 0.005 nM, 0.01 nM, 0.5 nM, 0.1 nM, 1.0 nM, 10 nM, and 100 nM (e.g., 0.1 nM - 1.0 nM).

[0056] It is understood by those skilled in the art that dosage amount will vary with the activity of a particular inhibitor compound and / or engineered polypeptide, disease state, route of administration, duration of treatment, and like factors well-known in the medical and pharmaceutical arts. In general, a suitable dose will be an amount which is the lowest dose effective to produce a therapeutic or prophylactic effect. If desired, an effective dose of such a compound and / or engineered polypeptide, pharmaceutically acceptable salt thereof, or related composition may be administered in two or more sub-doses, administered separately over an appropriate period of time.

[0057] The pharmaceutical composition may include the compound and / or engineered polypeptide in a range of about 0.1 to 2000 mg. In some embodiments, the pharmaceutical composition may include the compound and / or engineered polypeptide in a range of from about 0.5 to 500 mg. In some embodiments, the pharmaceutical composition may include the compound and / or engineered polypeptide in a range of from about 1 to 100 mg. The pharmaceutical composition may be administered to provide the compound and / or engineered polypeptide at a daily dose of about 0.1 to about 1000 mg / kg body weight. In some embodiments, the pharmaceutical composition may be administered to provide the compound and / or engineered polypeptide at a daily dose of about 0.5 to about 500 mg / kg body weight. In some embodiments, the pharmaceutical composition may be administered to provide the compound and / or engineered polypeptide at a daily dose of about 50 to about 100 mg / kg body weight. In some embodiments, after the pharmaceutical composition is administered to a subject e.g., after about 1, 2, 3, 4, 5, or 6 hours post-administration), the concentration of the compound and / or engineered polypeptide at the site of action may be within a concentration range bounded by end-points selected from 0.001 nM, 0.005 nM, 0.01 nM, 0.5 nM, 0.1 nM, 1.0 nM, 10 nM, and 100 nM (e.g., 0.1 nM - 1.0 nM).

[0058] The compounds and / or engineered polypeptides utilized in the methods disclosed herein may be formulated as a pharmaceutical composition that includes a carrier. For example, the carrier may be selected from the group consisting of proteins, carbohydrates, sugar, talc, magnesium stearate, cellulose, calcium carbonate, and starch-gelatin paste.

[0059] The compounds and / or engineered polypeptides utilized in the methods disclosed herein may be formulated as a pharmaceutical composition that includes one or more binding agents, filling agents, lubricating agents, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, and effervescent agents. Filling agents may include lactose monohydrate, lactose anhydrous, and various starches; examples of binding agents are various celluloses and cross-linked polyvinylpyrrolidone, microcrystalline cellulose, such as Avicel® PH101 and Avicel® PH102, microcrystalline cellulose, and silicified microcrystalline cellulose (ProSolv SMCC™). Suitable lubricants, including agents that act on the flowability of the powder to be compressed, may include colloidal silicon dioxide, such as Aerosil®200, talc, stearic acid, magnesium stearate, calcium stearate, and silica gel. Examples of sweeteners may include any natural or artificial sweetener, such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acsulfame. Examples of flavoring agents are Magnasweet® (trademark of MAFCO), bubble gum flavor, and fruit flavors, and the like. Examples of preservatives may include potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxybenzoic acid such as butylparaben, alcohols such as ethyl or benzyl alcohol, phenolic compounds such as phenol, or quaternary compounds such as benzalkonium chloride.

[0060] Suitable diluents may include pharmaceutically acceptable inert fillers, such as microcrystalline cellulose, lactose, dibasic calcium phosphate, saccharides, and mixtures of any of the foregoing. Examples of diluents include microcrystalline cellulose, such as Avicel® PH101 and Avicel® PHI 02; lactose such as lactose monohydrate, lactose anhydrous, and Pharmatose® DCL21; dibasic calcium phosphate such as Emcompress®; mannitol; starch; sorbitol; sucrose; and glucose.

[0061] Suitable disintegrants include lightly crosslinked polyvinyl pyrrolidone, corn starch, potato starch, maize starch, and modified starches, croscarmellose sodium, cross-povidone, sodium starch glycolate, and mixtures thereof. Examples of effervescent agents are effervescent couples, such as an organic acid and a carbonate or bicarbonate. Suitable organic acids include, for example, citric, tartaric, malic, fumaric, adipic, succinic, and alginic acids and anhydrides and acid salts. Suitable carbonates and bicarbonates include, for example, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium glycine carbonate, L-lysine carbonate, and arginine carbonate. Alternatively, only the sodium bicarbonate component of the effervescent couple may be present.

[0062] The compounds and / or engineered polypeptides utilized in the methods disclosed herein may be administered in conventional dosage forms prepared by combining the active ingredient with standard pharmaceutical carriers or diluents according to conventional procedures well known in the art. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation.

[0063] Pharmaceutical compositions comprising the compounds and / or engineered polypeptides may be adapted for administration by any appropriate route, for example, by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route. Such formulations may be prepared by any method known in the art of pharmacy, for example, by bringing into association the active ingredient with the carrier(s) or excipient(s).

[0064] Pharmaceutical compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or nonaqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid emulsions.

[0065] Pharmaceutical compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient may be delivered from the patch by iontophoresis.

[0066] Pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, impregnated dressings, sprays, aerosols or oils and may contain appropriate conventional additives such as preservatives, solvents to assist drug penetration and emollients in ointments and creams. For applications to the eye or other external tissues, for example the mouth and skin, the pharmaceutical compositions are in some embodiments applied as a topical ointment or cream. When formulated in an ointment, the compound and / or engineered polypeptide may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the compound and / or engineered polypeptide may be formulated in a cream with an oil-in-water cream base or a water- in-oil base. Pharmaceutical compositions adapted for topical administration to the eye include eye drops where the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent.

[0067] Pharmaceutical compositions adapted for topical administration in the mouth include lozenges, pastilles and mouth washes. Pharmaceutical compositions adapted for rectal administration may be presented as suppositories or enemas. Pharmaceutical compositions adapted for nasal administration where the carrier is a solid include a coarse powder having a particle size (e.g., in the range 20 to 500 microns) which is administered in the manner in which snuff is taken (i.e. by rapid inhalation through the nasal passage from a container of the powder held close up to the nose). Suitable formulations where the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient. Pharmaceutical compositions adapted for administration by inhalation include fine particle dusts or mists which may be generated by means of various types of metered dose pressurized aerosols, nebulizers or insufflators. Pharmaceutical compositions adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.

[0068] Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

[0069] Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tableting lubricants, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants, for example potato starch; or acceptable wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well-known in normal pharmaceutical practice. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives, such as suspending agents, for example sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and, if desired, conventional flavoring or coloring agents.

[0070] Optionally, the disclosed compounds and / or engineered polypeptides or pharmaceutical compositions comprising the disclosed compounds and / or engineered polypeptides may be administered with additional therapeutic agents, optionally in combination, in order to treat cell proliferative diseases and disorders. In some embodiments of the disclosed methods, one or more additional therapeutic agents are administered with the disclosed compounds and / or engineered polypeptides or with pharmaceutical compositions comprising the disclosed compounds and / or engineered polypeptides, where the additional therapeutic agent is administered prior to, concurrently with, or after administering the disclosed compounds and / or engineered polypeptides or the pharmaceutical compositions comprising the disclosed compounds and / or engineered polypeptides. In some embodiments, the disclosed pharmaceutical composition is formulated to comprise the disclosed compounds and / or engineered polypeptides and further to comprise one or more additional therapeutic agents, for example, one or more additional therapeutic agents for treating cell proliferative diseases and disorders.

[0071] Methods of preparing pharmaceutical formulations or compositions include the step of bringing an inhibitor compound and / or engineered polypeptide into association with a carrier and, optionally, one or more additional adjuvants or ingredients. For example, standard pharmaceutical formulation techniques can be employed, such as those described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.

[0072] Regardless of composition or formulation, those skilled in the art will recognize various avenues for medicament administration, together with corresponding factors and parameters to be considered in rendering such a medicament suitable for administration.

[0073] Methods:

[0074] The disclosed compounds and / or engineered polypeptides and pharmaceutical compositions comprising the disclosed compounds and / or engineered polypeptides, may be administered in various methods. In one aspect, a method of targeting a protein for degradation by autophagy comprising binding the target protein to T in the heterobifunctional compound in a cellular environment is provided. As used herein, a cellular environment may be in-vivo, or in- vitro or ex-vivo, wherein the components necessary for the process of autophagy are provided. Without limitation the components necessary for autophagy may comprise autophagy-related proteins, chaperone proteins, small molecules, membranes, lysosomes and other organelles, motor proteins, and other cytoskeletal components. In some embodiments, the compound and / or engineered polypeptide is provided as a composition disclosed herein.

[0075] Another aspect of the present disclosure provides a method of inhibiting autophagy comprising administering an engineered polypeptide provided herein, wherein the engineered peptides binds to and inhibits the action of an Atg8 protein. Inhibiting the action of an Atg8 protein may comprise inhibiting, reducing or altering it’s binding to another protein, or it’s localization.

[0076] In another aspect, the disclosed compounds or polypeptides and pharmaceutical compositions may be administered in methods for treating a disease or disorder in a subject in need thereof, including but not limited to autophagy-related diseases.

[0077] As used herein, the term "disorder" refers to a condition in which there is a disturbance of normal functioning. A "disease" is any abnormal condition of the body or mind that causes discomfort, dysfunction, or distress to the person affected or those in contact with the person. Sometimes the term is used broadly to include injuries, disabilities, syndromes, symptoms, deviant behaviors, and atypical variations of structure and function, while in other contexts, these may be considered distinguishable categories. It should be noted that the terms "disease", "disorder", "condition" and "illness", are equally used herein. As used herein, a "subject" may be interchangeable with "patient" or "individual" and means an animal, which may be a human or non-human animal, in need of treatment. Although the methods disclosed herein are particularly intended for the treatment of proliferative disorders in humans, other mammals are included. By way of non-limiting examples, mammalian subjects include monkeys, equines, cattle, canines, felines, mice, rats and pigs.

[0078] A “subject in need thereof’ may include a subject in need of treatment for a disease or disorder associated with autophagy. By way of example, and not limitation, diseases or conditions associated with autophagy may comprise, cancer, Alzheimer’s disease, inflammation, Huntington’s disease, liver disease, Parkinson’s disease, Chron’s disease, diabetes, kidney diseases, lung diseases, skeletal muscle disease, hone diseases, vascular disease, metabolic dysfunction, autoimmune diseases, neurodegenerative disease, heart diseases and lysosomal storage diseases.

[0079] The engineered polypeptides, compounds and pharmaceutical compositions disclosed herein may be administered alone or in combination with one or more additional compounds, pharmaceutical compositions, and / or therapies. For example, a heterobifunctional that inhibited autophagy may be administered as a single compound or in combination with another compound or therapeutic.

[0080] Additional definitions

[0081] The present disclosure is not limited to the specific details of construction, arrangement of components, or method steps set forth herein. The compositions and methods disclosed herein are capable of being made, practiced, used, carried out and / or formed in various ways that will be apparent to one of skill in the art in light of the disclosure that follows. The phraseology and terminology used herein is for the purpose of description only and should not be regarded as limiting to the scope of the claims. Ordinal indicators, such as first, second, and third, as used in the description and the claims to refer to various structures or method steps, are not meant to be construed to indicate any specific structures or steps, or any particular order or configuration to such structures or steps.

[0082] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to facilitate the disclosure and does not imply any limitation on the scope of the disclosure unless otherwise claimed. No language in the specification, and no structures shown in the drawings, should be construed as indicating that any non-claimed element is essential to the practice of the disclosed subject matter.

[0083] Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.”

[0084] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus <10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.

[0085] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0086] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

[0087] In those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or£B or “A and B.”

[0088] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and / or description found in the cited references.

[0089] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0090] The following examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims. EXAMPLES

[0091] The following Examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter.

[0092] Example 1: Development of GABARAP-specific stapled and A-methylated peptide modulators of autophagy

[0093] Inhibiting autophagy has been validated as a possible anti-cancer therapy, especially in conjunction with traditional DNA-damaging chemotherapies. However, current autophagy inhibitors are nonspecific and affect many other biological pathways. GABARAP inhibitors should be much more selective autophagy inhibitors and could be tested as combination therapies with DNA-damaging agents such as cisplatin.

[0094] In addition, GABARAP is one of the proteins that sort cellular proteins for degradation by autophagy. There is biological data that implies that tethering a cellular protein, organelle, or aggregate to GABARAP will induce its degradation by autophagy. Thus, GABARAP ligands are also valuable building blocks for building bifunctional molecules that bind both GABARAP and a second protein, thereby targeting that protein for degradation. Such autophagy-targeting chimeras (AUTACs) or autophagosome-tethering chimeras (ATTECs) are highly active areas of current drug development.

[0095] There are very few small-molecule GABARAP ligands / inhibitors, and none have potency below about 0.5 micromolar. Our compounds are derived from peptides but compounds in the series, e.g., IM-3, are small enough to be considered small-molecule-like. We expect these smaller compounds, which still have potency well below 0.5 micromolar, to be particularly useful for applications in cells, tissues, and animals, and as therapeutics.

[0096] Here, we developed novel compounds that bind the autophagy-associated protein GABARAP. These compounds are based on a natural protein sequence derived from the human protein ULK1 (SEQ ID NO: 1). We have altered the sequence, added unnatural amino acids, added / V-m ethyl substitutions, and added covalent “staples” to promote better binding. The result are much more potent compounds that are also more drug-like and are expected to be more cellpenetrant and bioactive.

[0097] Results

[0098] Table 1 contains a table with peptide sequences (IM-0 to IM-41), their crosslink modification, and their Kd or IC50 value. Kd values for recombinant GABARAP and LC3B were obtained in a fluorescence polarization assay with peptides labeled on their N-termini with fluorescein, with two beta-alanine residues between the fluorescein and the N-terminal amino acid as a linker. IC50 values are for non-fluoresceinated compounds (acetylated N-termini) inhibiting the interaction between peptide KI and recombinant GAB ARAP in an AlphaScreen assay. Table 1

[0099]

[0100] Key: J = Norleucine; II = A-methyl norleucine; A = A-methyl phenylalanine; ft = A-methyl valine; Z= propargylserine; X = penicillamine; 0 = indole; C = cysteine which is covalently modified by the specified “staple”; Underlined residues are modified with specified “staple” NB=No Binding

[0101] Stapled Peptide Selection

[0102] Covalent crosslinking or peptide “stapling” is a common strategy used to conformationally constrain peptides. Stapling has been shown to increase proteolytic stability, increase affinity, and increase cell penetration. This strategy has been widely applied to alpha helices, beta hairpins, and beta strands. Using the crystal structure of Ulkl bound to GABARAP, we designed stapled peptides at the z+2 and z+3 positions. Our goal was to “stretch” these peptides by stapling them with a variety of aryl linkers that matched the length between the alpha carbons of the side chains of interested. Anything that was around the same or a slightly larger distance was considered a good fit.

[0103] Using this method, we designed peptides IM-6, IM-8, and IM- 10. IM-6 was stapled at z+3 and IM-8 and IM- 10 were stapled at z+2. Linear controls of all peptides were tested to determine the effect of mutating the native residues to cysteine (FIG. 8). IM-5 had a loss in affinity of about 5-fold in comparison to the parent peptide but still retained micromolar affinity. In contrast, the stapled peptide IM-6 showed poor binding upon stapling. The control peptide IM-7 and its stapled counterpart IM-8 had a significant loss in affinity upon cysteine mutation and stapling, respectively. With wishing to be bound by a particular theory, we attributed this loss in affinity for IM-6 and IM-8 to a disruption in the native conformation upon stapling these peptides. We also removed the negatively charged residue, Asp, and negatively charged residues are known to stabilize interactions with Atg8s. Removing them has been shown to greatly decrease binding. IM- 9 stapled with meta-xylene and showed similar affinity to its linear control, IM-8, indicating that stapling modification at this position was tolerated. We then decided to try the para-xylene linker, which is slightly wider than the meta-xylene linker. Here, we found that this peptide bound with about a 200 nanomolar affinity, which is about two-fold better than the parent peptide.

[0104] The LIR motif is known to contain the three aromatic amino acids, Phe, Trp, and Tyr, which dock into HP1 of the LIR domain. Native Ulkl LIR contains Phe in this position. Previous work has found that replacing Phe or Tyr with Trp increases affinity to Atg8s by about 10-fold. Therefore, we replaced Phe with Trp to see if the binding affinity would increase (FIG. 9). We first applied this modification to the parent peptide. Indeed, replacing Phe with Trp increased affinity by about 10-fold from around 500 nM to about 50 nM. We then applied this modification to the stapled peptide hit to see if this increase in affinity was synergistic with the staple. We saw a similar increase in affinity of about 10-fold in IM20. After making this modification, we decided to retain it in all future stapled peptides.

[0105] Peptides have a high molecular weight and a high number of hydrogen bond donors, making it extremely difficult for them to penetrate cell membranes. Truncation and replacement of hydrogen bond donors are both tools used to remove these elements from the peptide to make it more passively penetrant / more cell penetrant. To achieve this, we further modified IM-20 by truncating it to the core LIR sequence, a tetrapeptide (FIG. 10). Previous work has shown that when a peptide has fewer than 3 hydrogen bond donors, it is more cell permeable. This would include removing the acidic residues at the N-term of the peptide, known to greatly decrease binding upon removal, and removal of the C-term, which has a milder effect on binding after removal. After truncating the C-term, IM-22 took a large hit to affinity, losing 20-fold affinity for GAB ARAP. Truncation of the N-term in IM-24 caused a similar loss in affinity. Full truncation of the peptide in IM-26 resulted in an almost complete loss in affinity to GAB ARAP. While N- or C-terminal truncation was tolerated, full truncation to the tetrapeptide was not.

[0106] N-methyl selection

[0107] We next evaluated the impact of backbone Mm ethylation on the core LIR motif of Ulkl (FIG. 11). We hypothesized that an A-m ethylated peptide in the optimal binding mode should correlate with a Kd like that of the parent peptide. We observed decreased binding in IM-1, IM-2, and IM-4. The amide hydrogens of IM-2 and IM-4 hydrogen bond with GABARAP, allowing Ulkl to form an intermolecular beta sheet with GABARAP. Destabilizing this interaction expectedly disrupted this interface. The decreased binding of IM1 was an interesting result because this amide did not hydrogen bond with GABARAP in the crystal structure; however, it was close to the surface of the protein. Interestingly, A-methylation at position Nle6 in IM3 increased binding to GABARAP, with a Kd of about 100 nM. We initially predicted that there would be little to no difference in binding when modifying this position because the amide is pointing toward solvent. Without wishing to be bound by a particular theory, we hypothesize that the increase in binding may be due to decreasing the energy barrier of the optimal binding mode, leading to an increase in the affinity of this peptide to GABARAP.

[0108] IM-3 was chosen for further modification due to its high binding affinity. We first applied the Phe to Trp mutation to this peptide, termed IM-27, and saw a similar 10-fold increase in binding affinity, similar to that of IM-17 and IM-20 (FIG. 12).

[0109] We next investigated the binding mode of IM-27 when bound to GABARAP. The crystal structure revealed that IM-27 binds in an almost identical mode to the parent peptide (FIG. 13). The core LIR of IM-27 occupies the LIR domain in the same manner as the core LIR of IM-0. The methyl at the amide Nle6 sticks out to solvent, with the amide overlapping with the amide of the parent peptide. Pro8 of IM-27 overlaps with Pro8 of IM-0, while Ala9 of IM-27 deviates from Ala8 of IM-0, occupying a higher position on GABARAP than in the parent peptide. Asp2 and Asp3 of IM-27 both deviate from the parent peptide, occupying a lower position on GABARAP in comparison to the parent peptide.

[0110] Similar to stapled peptide IM-20, we further modified IM-27 by truncating it to a tetrapeptide (FIG. 14). Truncation of the N-terminus in IM-30 resulted in a 10-fold loss in affinity with a Kd of 129 nM. Truncation of the C-terminus in IM-31 showed a 2-fold loss in binding with a Kd of about 22 nM, demonstrating that truncation at the C-terminus does not affect binding as much as truncation at the N-terminus. This also deviates from the truncation of IM-20, where both N- and C-terminal truncation greatly decreased binding to a similar degree. The fully truncated peptide IM-32 had a Kd of about 163 nM, about 15-fold higher than IM-27. While this was a significant loss in binding, it is still promising that the peptide retained nanomolar affinity after losing its termini, particularly the acidic N-terminus. We next investigated the removal of the terminal amine in IM-33 to increase cell permeability using the AlphaScreen competitive inhibition assay (FIG. 15). The amine-truncated peptide had the lowest IC50, about 16 pM, in comparison to the IM-27 and IM-32, which had an IC50 of about 200 nM and 3 pM, respectively. This drop in binding affinity was most likely due to loss of contacts between GAB ARAP and the terminal amine.

[0111] To determine the permeability of the A-m ethylated peptide series, we used the PAMPA assay and the MDCK cell assay (Table 2). The PAMPA assay showed increasing permeability as IM-27 became more truncated. The MDCK assay showed a similar pattern. The peptides have slightly lower logPe values in this assay, and IM-32 had a slightly lower logPe than IM-33. Overall, these data demonstrated that our strategy of truncation increased permeability with each modification.

[0112] Table 2: Permeability Assessment

[0113] Combination Peptide

[0114] After finding that IM-20 and IM-27 both increase affinity to GABARAP, we combined these modifications into IM-28 and assessed the effect on binding (FIG. 16). Combining these modifications yielded a slightly less potent peptide with a Kd of about 70nM rather than a more potent peptide. This was surprising as we did not expect these modifications to interfere with each other. The crystal structure of IM-27 shows that it binds similarly to the native peptide. IM-20 may have a different binding mode than IM-0 and IM-27. The addition of the A-methyl within the staple / cycle may also introduce too much strain, disrupting the optimal binding mode. Thus, we pursued IM-20 and IM-27 separately.

[0115] The structures of the peptides utilized in this study are shown below: IM-0 (used for Kd)

[0116] IM-0 (used for IC5o) IM-1 (used for Kd)

[0117] IM-1 (used for IC50)

[0118] IM-2 (used for Kd)

[0119]

[0120] IM-3 (used for Kd)

[0121] IM-3 (used for IC50)

[0122] IM-4

[0123] IM-7 (used for IC5o)

[0124]

[0125] 10 IM-12

[0126] IM-16

[0127] IM-20

[0128]

[0129] IM-28

[0130] IM-36

[0131]

[0132] IM-40

[0133]

[0134] Materials and Methods

[0135] General peptide synthesis: Peptides were synthesized by standard Fmoc solid phase peptide synthesis using either an automated Tribute peptide synthesizer (Gyros Peptide Technologies) or an automated Prelude peptide synthesizer (Gyros Peptide Technologies). Peptides were synthesized on rink amide resin. Fmoc deprotection was done in 20% piperidine in DMF. 4 equivalents of amino acid, 4 equiv. of coupling reagent (HBTU or HATU and HOBt or HO At), and 8 equiv. of N,N-diisopropylethylamine (DIPEA) were used at each coupling step. After synthesis, the resin was washed with DMF, DCM, and MeOH, then dried over vacuum. All peptides were globally deprotected and cleaved from the resin with a TFA cleavage cocktail (TFA / H2O / EDT / TIPS 95:2:2: 1) for 3 hrs. After cleavage, peptides were precipitated in cold diethyl ether and centrifuged yielding a peptide pellet. The pellet was dried with nitrogen, redissolved in ACN:H2O (50:50), and lyophilized.

[0136] After lyophilization, the peptides were resuspended in ACN:H2O (50:50) for reverse phase HPLC purification. Peptides were purified using either a C8 or C18 column with a gradient of 5- 100% acetonitrile with 0.1% TFA over 20 min. After purification, peptides were analyzed via analytical HPLC for 95% purity. Peptide mass was analyzed using MALDI-TOF mass spectrometry. MALDI matrix used was 20 mg / mL a-Cyano-4-hydroxy cinnamic acid in ACN:H2O (70:30) with 0.1% TFA. Fluorescein-tagged peptides'. Fluorescein-tagged peptides included two P-alanine residues on the N-terminus to serve as a linker. After synthesis, the peptide was deprotected and the first coupling with 3 equiv. 5 / 6-carboxyfluorescein succinimidyl ester (NHS-fluorescein, Thermo Fisher) and 6 equiv. DIPEA in DMF for 1 hr was performed. The resin was washed with DMF and a second coupling was done overnight with the same equivalents of NHS-fluorescein and DIPEA. After the second coupling, the resin was washed, dried, and the peptide was cleaved and lyophilized.

[0137] Acetylated peptides. Acetylated peptides were deprotected after synthesis and a coupling with 50 equiv. acetic anhydride and 100 equiv. DIPEA in DMF for 20 minutes was performed. After coupling, the resin was rinsed, washed, dried, and the peptide was cleaved and lyophilized.

[0138] Protein expression and purification'. Recombinant His-tagged GAB ARAP and LC3B were expressed in BL21 (DE3) E.coli cells. The cells were transformed with pET-15b expression plasmids encoding each protein. Transformed cells were plated on ampicillin agar plates and incubated at 37°C overnight. Individual colonies were picked and grown in 5mL LB culture medium with 1% ampicillin at 37°C shaking overnight. The 5 mL culture was then added to IL of LB medium with 1% ampicillin and incubated with shaking at 37°C until the OD600 measured >0.6. Protein expression was induced with 1 mL of 0.5 mM Isopropyl P-D-l -thiogalactopyranoside (IPTG). Cultures were incubated for 3 hours with shaking at 37°C. Cells were then pelleted and stored at -80°C.

[0139] For protein purification, cells were resuspended in lysis buffer (25 mM HEPES, 150 nM NaCl, 10 mM imidazole, 0.2% lysozyme, 1 protease inhibitor cocktail pellet, and 0.001% universal nuclease). The resuspended cells were sonicated then centrifuged to separate the lysate from the cellular debris. The clarified lysate was purified via batch affinity purification with HisPur Ni- NTA resin (Thermo Fisher Scientific). The resin was incubated with the lysate at 4°C for one hour, then washed with 25 mM HEPES, 500 mM NaCl, and 20 mM imidazole. After, it was washed with lysis buffer to reduce the salt from 500 mM NaCl to 150 mM NaCl. The protein was eluted from the resin with elution buffer (25 mM HEPES, 150 mM NaCl, and 500 mM imidazole). To remove imidazole, buffer exchange was performed via a desalting column into storage buffer (25 mM HEPES, 150 mMNaCl, pH 7.3). Protein purity and mass were assessed using SDS-PAGE. If needed, proteins were further purified by size exclusion chromatography using storage buffer. Protein was aliquoted and flash frozen in liquid N2, then stored at -80°C. Fluorescence polarization assay. Fluorescein dye-labeled peptides were diluted in assay buffer (25 mM HEPES, 150 mM NaCl, ImM EDTA, and 0.1% Tween-20, pH 7.3) to 10 nM and 10 uL was added in triplicate to a black, 384-well flat-bottom polystyrene plate (Thermo Fisher). 10 uL of serially diluted recombinant GAB ARAP or LC3B was added to all wells with peptide. The plate was covered with foil and incubated with shaking for 1 hour at room temperature. After incubation, the plate was read on a Tecan plate reader at ex= 494 nm and ex= 519 nm. Kd values were determined using curve fits using GraphPad Prism software as described (use citation from Hawley’s paper). Average Kd and standard error of the mean was calculated using three independent trials.

[0140] The curve fit equation used: y= ml +(m2-ml)* (Kd+L+x-(Kd+L+x)2-4*L*x)2*L

[0141] Y is measured polarization, ml is polarization at 0% bound, m2 is polarization at 100% bound, x is the concentration of the protein, and L is the concentration of the probe.

[0142] AlphaScreen Assay. Recombinant GAB ARAP was diluted in assay buffer (25 mM HEPES, 150 mM NaCl, 0.1% Tween-20, 1 mg / mL BSA) to 50 nM and 5 uL was added in duplicate to a white, 384-well polystyrene plate (AlphaPlate, Revvity) to wells for inhibitor peptides, acetylated- K1 (Ac-Kl) control row, and control wells. A 25 uM Ac-Kl solution was prepared then serially diluted. 5 uL of serially diluted Ac-Kl was added to Ac-Kl control row. 20 mM or 2 mM acetylated peptide DMSO stocks were used to prepare a 100 uM solution with either 2.5% or 5% DMSO, respectively. 5 uL of peptide was added to inhibitor wells. The plate was covered with foil, centrifuged at 1200 rpm for 3 minutes, and incubated at room temperature for 45 minutes. Biotinylated KI (Bio-Kl) was diluted to 50 nM and 5 uL was added to wells with inhibitor peptide, Ac-Kl control row, and the positive control wells. The plate was covered with foil, centrifuged, and incubated for 45 minutes. AlphaScreen streptavidin donor beads and nickel chelate acceptor beads (Revvity) were diluted to 100 ug / mL. 5 uL of acceptor beads were added to all wells used. In the dark, 5 uL of donor beads were added to all wells used. The plate was covered with foil, centrifuged, and incubated for 1 hour. Plate was read on Tecan Spark (excitation 680 nM and emission 520-620 nM). Data was normalized using GraphPad Prism software using blank control wells as 0% bound and no inhibitor wells as 100% bound. After, the normalized data was fit with IC50 curve. Average IC50 and standard error of the mean was calculated using three independent trials.

Claims

1. CLAIMSWhat is claimed:

1. An engineered polypeptide with Atg8 binding activity, wherein the polypeptide comprises a LC3-interacting motif, and wherein the motif comprises one or more mutations relative to a wild type LC3- interacting motif.

2. The engineered polypeptide of claim 1, wherein the wild-type LC3 -interacting motif comprises SEQ ID NO: 1 (TDDFVMVPA).

3. The engineered polypeptide of claim 2, wherein the one or more mutations comprises inclusion of a non-natural amino acid, an amino acid analog, a A'-m ethyl substitution, a covalent linkage, an N-terminal or C-terminal modification, or a combination thereof.

4. The engineered polypeptide of claim 3, wherein the JV-m ethyl substitution comprises N- methyl phenylalanine, A -methyl norleucine, A'-m ethyl valine, or a combination thereof, optionally wherein the 7V-methyl substitution is located, with reference to SEQ ID NO: 1, at amino acid position 6, or a homologous position thereof.

5. The engineered polypeptide of claim 3, wherein the non-natural amino acid or amino acid analog comprise norleucine, a tryptophan analog, or a combination thereof.

6. The engineered polypeptide of claim 3, wherein the covalent linkage comprises sidechain stapling.

7. The engineered polypeptide of claim 6, wherein the sidechain stapling comprises an ortho- dimethylbenzene linkage, a / weta-dimethylbenzene linkage, a / ?czra-dimethylbenzene linkage, a 2,2’-bis(methyl)l,l’-biphenyl linkage, a bis(methyl)naphthalene linkage, or a 2,4-Hexadiyne linkage.

8. The engineered polypeptide of claim 6 or 7, wherein the sidechain stapling comprises a linkage between amino acids located, with reference to SEQ ID NO: 1, at amino acid positions 5 and 7, or a homologous position thereof.

9. The engineered polypeptide of claim 3, wherein the covalent linkage comprises an allyl modification, optionally wherein the allyl modification of the amino acids is located, with reference to SEQ ID NO: 1, at amino acid positions 5 and / or 7, or a homologous position thereof.

10. The engineered polypeptide of claim 1, wherein the polypeptide comprises a tryptophan for phenylalanine substitution, optionally wherein the tryptophan for phenylalanine substitution is located, with reference to SEQ ID NO: 1, at amino acid position 4, or a homologous position thereof.

11. The engineered polypeptide of any one of claims 1-10, wherein the polypeptide inhibits the activity of Gamma-aminobutyric acid receptor-associated protein (GABARAP) or Microtubule-associated protein lA / lB-light chain 3 (LC3) protein.

12. The engineered polypeptide of claim 1, wherein the polypeptide is selected from SEQ ID NOs: 2-38.

13. The engineered polypeptide of any of claims 1-12, wherein the polypeptide is conjugated to one or more compounds.

14. The engineered polypeptide of claim 13, wherein the one or more compounds comprises a label, an additional polypeptide, a protein, or a linker optionally conjugated to the label, the additional polypeptide, or the protein.

15. The engineered polypeptide of claim 14, wherein the linker comprises polyether.

16. A composition comprising the engineered polypeptide of any one of claims 1-15 and a buffer or a pharmaceutically acceptable carrier.

17. A method of inhibiting autophagy, the method comprising administering the engineered polypeptide of any one of claims 1-16 or the composition of claim 17 to a subject in need thereof, wherein the engineered peptides binds to and inhibits the action of an Atg8 protein.

18. A heterobifunctional compound having a formula: M-L-T, wherein M is a moiety that binds to a Atg8 protein, L is a bond or linker covalently attaching M and T and, T is a moiety that binds to a target protein or is a label.

19. The heterobifunctional compound of claim 18, wherein the Atg8 protein is a Gamma- aminobutyric acid receptor-associated protein (GABARAP) or Microtubule-associated protein lA / lB-light chain 3 (LC3) protein.

20. The heterobifunctional compound of claim 19, wherein the GABARAP or LC3 protein is selected from GABARAP, GABARAPL1 GABARAPL2, LC3A, LC3B and LC3C.

21. The heterobifunctional compound of claim 18, wherein M comprises a LC3 -interacting region motif, and wherein the motif comprises one or more mutations relative to a wild-type LC3 interacting motif.

22. The heterobifunctional compound of claim 21, wherein the wild-type LC3 interacting motif comprises SEQ ID NO: 1 (TDDFVMVPA).

23. The heterobifunctional compound of claim 21 or 22, wherein the one or more mutations comprises inclusion of a non-natural amino acid, an amino acid analog, a N-methyl substitution, a covalent linkage, an A -term in al or C-terminal modification, or a combination thereof.

24. The heterobifunctional compound of claim 23, wherein the V-m ethyl substitution comprises A'-methyl phenylalanine, A-m ethyl norleucine, or A -methyl valine, or combinations thereof, optionally wherein the A -methyl substitution is located, with reference to SEQ ID NO: 1, at amino acid position 6, or a homologous position thereof.

25. The heterobifunctional compound of claim 23, wherein the non-natural amino acids or amino acid analogs comprise norleucine, a tryptophan analog, or a combination thereof.

26. The heterobifunctional compound of claim 23, wherein the covalent linkage comprises sidechain stapling.

27. The heterobifunctional compound of claim 26, wherein the sidechain stapling comprises an ortho- dimethylbenzene linkage, a meta-dimethylbenzene linkage, a / wcr-dimethylbenzene linkage, a 2,2’-bis(methyl)l,l’-biphenyl linkage, a bis(methyl)naphthalene linkage, or a 2,4- Hexadiyne linkage.

28. The heterobifunctional compound of claim 26 or 27, wherein the sidechain stapling comprises a linkage between amino acids located, with reference to SEQ ID NO: 1, at amino acid positions 5 and 7, or a homologous position thereof.

29. The heterobifunctional compound of claim 23, wherein the covalent linkage comprises an allyl modification, optionally wherein the allyl modification of the amino acids is located, with reference to SEQ ID NO: 1, at amino acid positions 5 and / or 7, or a homologous position thereof.

30. The heterobifunctional compound of claim 23, wherein M comprises a tryptophan for phenylalanine substitution, optionally wherein the tryptophan for phenylalanine substitution is located, with reference to SEQ ID NO: 1, at amino acid position 4, or a homologous position thereof.

31. The heterobifunctional compound of claim 30, wherein M comprises the tryptophan for phenylalanine substitution and the .V-methyl substitution comprising N-methy I norleucine.

32. The heterobifunctional compound of any one of claims 18-31, wherein M has a dissociation constant (Kd) for a GAB ARAP or a LC3 protein of less than 500nM.

33. The heterobifunctional compound of any one of the claims 18-32, wherein M comprises any one of SEQ ID NOs: 2-38.

34. The heterobifunctional compound of any one of claims 18-33, wherein L comprises a polyethylene glycol moiety, an alkyl moiety, a glycol moiety, a heterocycle, an alkyne, a spirocycle, or a combination thereof.

35. The heterobifunctional compound of any one of claims 18-34, wherein T binds to a target protein to be degraded by autophagy.

36. The heterobifunctional compound of any one of claims 18-35, wherein T binds to a AR, ER, BTK, EGFR, TRK, FAK, BET, or CDK.

37. A composition comprising the heterobifunctional compound of any one of claims 18-35 and a buffer or a pharmaceutically acceptable carrier.

38. A method of targeting a protein for degradation by autophagy, the method comprising, in a cellular environment, binding the target protein to T from the heterobifunctional compound of any one of claims 18-36, optionally wherein the heterobifunctional compound is presented as the composition of claim 37.

39. A method of treating an autophagy-related disease, the method comprising administering to a subject in need thereof an effective amount of the composition of claim 16 or claim 37.

40. The method of claim 39, wherein the autophagy-related disease is cancer.

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