SYNTHESIS OF COVALENT PROTEIN DIMERS THAT CAN INHIBIT MYC-DRIVEN TRANSCRIPTION

The disclosure relates to covalent protein dimers of MYC, MAX, and Omomyc; pharmaceutical compositions comprising the covalent protein dimers; methods of making the covalent protein dimers; and methods of treating disorders associated with MYC dysregulation (e.g., cancer) with the covalent protein dimers.

SEQUENCE LISTING

The instant application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII file, created on Jul. 20, 2022, is named 20221025-021203-3.txt and is 3,901 bytes in size.

BACKGROUND

The transcription factor protein MYC forms a heterodimer with MAX in order to bind to the E-Box DNA sequence (CACGTG). The MYC/MAX protein complex is part of the basic-helix-loop-helix/leucine-zipper (bHLH/Lz) transcription factor family and initiates several cellular processes, including cell proliferation and survival. MAX, alternatively, can homodimerize, compete for the E-Box DNA binding site, and inhibit MYC/MAX-driven transcription. MYC/MAX and MAX/MAX, thus, have opposite activities, and MYC overexpression is observed in >50% of human cancers.

Promising strategies to inhibit the oncogenic MYC activity rely on stabilizing the natural MAX/MAX dimer or delivering protein analogs with a similar mechanism of action. The targeting of MYC with small molecules has largely remained elusive, mainly because the structure of MYC presents no binding pockets for small molecule ligands. Recent attempts to overcome the challenge of drugging MYC include a small molecule stabilizer of the MAX/MAX complex that inhibits the proliferation of several cancer cell lines and reduces tumor burden in murine cancer models. An alternate approach involves the artificial miniprotein Omomyc, a dominant-negative form of MYC that can compete for E-Box DNA binding and inhibit MYC/MAX dependent transcription, ultimately resulting in tumor growth inhibition in various mouse models of cancer.

Omomyc, like MYC and MAX, has to form dimeric complexes to be functional and bioactive. MYC, MAX, and Omomyc can interact with each other in different combinations. Upon delivery of a monomer to the cell, the dominating complex formed depends on the other proteins' cellular concentrations and is difficult to predict. The direct administration of defined and stable dimeric complexes would offer a superior degree of control over the concentration and composition of the bioactive dimer inhibitor, in addition to a potentially higher structural stability.

Preparing homogeneous, stable, well-defined protein-protein conjugates can be a challenge. Chemical synthesis approaches to generate covalently linked multimeric proteins have been mainly focused on preparing ubiquitinylated or sumoylated proteins. These strategies relied on chemical ligation or chemoenzymatic workflows, requiring the incorporation of unnatural amino acids or engineered recognition sequences, respectively. In addition, ligation based strategies to prepare covalently linked HIV protease heterodimers have been reported with the aim to study asymmetric mutations of this enzyme dimer. Previous dimerization strategies of MYC/MAX analogs relied on either disulfide formation at the C-terminus of the leucine zipper region of the transcription factor analogs or the formation of oxime and thioester linkages between MYC and MAX or between MAX and MAX. While these defined dimers enabled DNA-binding studies, the reported strategies relied on dovetails with low chemical stability in a biological milieu, making them unsuitable for bioactivity studies in vivo. Accordingly, there is a need for biologically stable dimers of MYC, MAX, and Omomyc.

SUMMARY

The present disclosure provides, inter alia, a covalent protein dimer, or a pharmaceutically acceptable salt thereof, comprising: a first polypeptide comprising a C-terminus and an N-terminus, wherein the first polypeptide comprises a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3; a second polypeptide comprising a C-terminus and an N-terminus, wherein the second polypeptide comprises a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3; and a linker covalently linking the C-terminus of the first polypeptide to the C-terminus of the second polypeptide.

In another aspect, the disclosure provides a covalent protein dimer, or a pharmaceutically acceptable salt thereof, having a structure according to Formula (I):

wherein:Y1is a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3;Y2is a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3;Z1is —O—, —NH—, or —S—;Z2is —O—, —NH—, or —S—;R1is absent, C1-10alkyl, or C1-10heteroalkyl;R2is absent, C1-10alkyl, or C1-10heteroalkyl;W is C1-10alkyl, C1-10heteroalkyl, C6-10aryl, or 5- to 10-membered heteroaryl;L is absent or a linker;R is H, a nitrogen protecting group, biotin, a fluorescent dye, a nuclear-targeting moiety, or a cell-penetrating moiety; andn is 0 or 1.

In an embodiment, the covalent protein dimer has a structure according to Formula (Ib):

wherein:Y1is a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3;Y2is a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3;L is absent or a linker;R is H, a nitrogen protecting group, biotin, a fluorescent dye, a nuclear-targeting moiety, or a cell-penetrating moiety; andn is 0 or 1.

In another aspect, the disclosure provides a covalent protein dimer, or a pharmaceutically acceptable salt thereof, having a structure according to Formula (II):

wherein:Y1is a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3;Y2is a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3;Z1independently is —O—, —NH—, or —S—;Z2independently is —O—, —NH—, or —S—;R1independently is C1-10alkyl or C1-10heteroalkyl;A is C6-10aryl or 5- to 10-membered heteroaryl;L independently is absent or a linker;R independently is H, a nitrogen protecting group, biotin, a fluorescent dye, a nuclear-targeting moiety, or a cell-penetrating moiety; andn independently is 0 or 1.

In some embodiments, the covalent protein dimer has a structure according to Formula (IIb):

wherein:Y1is a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3; andY2is a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3.

In another aspect, the disclosure provides a pharmaceutical composition comprising a covalent protein dimer of the disclosure and a pharmaceutically acceptable carrier. In another aspect, the disclosure provides a method of treating a disease or disorder characterized by MYC dysregulation in a subject in need thereof, the method comprising administering to the subject a covalent protein dimer of the disclosure. In an embodiment, the disease or disorder is cancer. In another aspect, the disclosure provides a method of making a covalent protein dimer of the disclosure.

DETAILED DESCRIPTION

Definitions

Listed below are definitions of various terms used to describe the compounds and compositions disclosed herein. These definitions apply to the terms as they are used throughout this specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group.

Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and peptide chemistry are those well-known and commonly employed in the art.

As used herein, the articles “a” and “an” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.

As used herein, the term “about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. As used herein when referring to a measurable value such as an amount, a temporal duration, and the like, the term “about” is meant to encompass variations of ±20% or ±10%, including ±5%, ±1%, and ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

The term “administration” or the like as used herein refers to the providing a therapeutic agent to a subject. Multiple techniques of administering a therapeutic agent exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary, and topical administration.

The term “treat,” “treated,” “treating,” or “treatment” includes the diminishment or alleviation of at least one symptom associated or caused by the state, disorder or disease being treated. In certain embodiments, the treatment comprises bringing into contact with a subject an effective amount of a covalent protein dimer of the disclosure for conditions related to cancer.

As used herein, the term “prevent” or “prevention” means no disorder or disease development if none had occurred, or no further disorder or disease development if there had already been development of the disorder or disease. Also considered is the ability of one to prevent some or all of the symptoms associated with the disorder or disease.

As used herein, the term “patient,” “individual,” or “subject” refers to a human or a non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and marine mammals. Preferably, the patient, subject, or individual is human.

As used herein, the terms “effective amount,” “pharmaceutically effective amount,” and “therapeutically effective amount” refer to a nontoxic but sufficient amount of an agent to provide the desired biological result. That result may be reduction or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

As used herein, “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the present disclosure, and are physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the compositions. The “pharmaceutically acceptable carrier” may further include a pharmaceutically acceptable salt of the compound disclosed herein. Other additional ingredients that may be included in the pharmaceutical compositions are known in the art and described, for example, in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

As used herein, the term “MYC” refers to the protein MYC proto-oncogene encoded by the MYC gene, which is a member of the myc family of transcription factors, and has the following sequence:

As used herein, the term “MAX” refers to the transcription factor myc-associated factor X, which is encoded by the MAX gene and has the following sequence:

As used herein, the term “Omomyc” refers the artificial mini-protein that functions as a dominant-negative form of MYC and has the following sequence:

As used herein the nomenclature “protein-protein” (e.g., MAX-MAX or MYC-MAX) indicates a covalent dimer whereas the nomenclature “protein/protein” (e.g., MAX/MAX or MYC/MAX) indicates a non-covalent dimer.

As used herein, the term “alkyl,” by itself or as part of another substituent means, unless otherwise stated, a straight or branched chain hydrocarbon having the number of carbon atoms designated (i.e., C1-C6alkyl means an alkyl having one to six carbon atoms) and includes straight and branched chains. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert butyl, pentyl, neopentyl, and hexyl. Other examples of C1-C6alkyl include ethyl, methyl, isopropyl, isobutyl, n-pentyl, and n-hexyl.

As used herein “heteroalkyl” refers to an alkyl group wherein one or more carbon atoms has been replaced with a heteroatom selected from O, S, or N, wherein alkyl is as defined herein.

As used herein, the term “alkoxy” refers to the group —O-alkyl, wherein alkyl is as defined herein. Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, t-butoxy and the like.

As used herein, the term “alkenyl” refers to a monovalent group derived from a hydrocarbon moiety containing, in certain embodiments, from two to six, or two to eight carbon atoms having at least one carbon-carbon double bond. The alkenyl group may or may not be the point of attachment to another group. The term “alkenyl” includes, but is not limited to, ethenyl, 1-propenyl, 1-butenyl, heptenyl, octenyl and the like.

As used herein, the term “halo” or “halogen” alone or as part of another substituent means, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom, preferably, fluorine, chlorine, or bromine, more preferably, fluorine or chlorine.

As used herein, the term “cycloalkyl” means a non-aromatic carbocyclic system that is fully or partially saturated having 1, 2 or 3 rings wherein such rings may be fused. The term “fused” means that a second ring is present (i.e., attached or formed) by having two adjacent atoms in common (i.e., shared) with the first ring. Cycloalkyl also includes bicyclic structures that may be bridged or spirocyclic in nature with each individual ring within the bicycle varying from 3-8 atoms. The term “cycloalkyl” includes, but is not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo [3.1.0] hexyl, spiro [3.3] heptanyl, and bicyclo [1.1.1] pentyl.

As used herein, the term “aromatic” refers to a carbocycle or heterocycle with one or more polyunsaturated rings and having aromatic character, i.e., having (4n+2) delocalized π (pi) electrons, where n is an integer.

As used herein, the term “aryl” means an aromatic carbocyclic system containing 1, 2 or 3 rings, wherein such rings may be fused, wherein fused is defined above. If the rings are fused, one of the rings must be fully unsaturated and the fused ring(s) may be fully saturated, partially unsaturated or fully unsaturated. The term “aryl” includes, but is not limited to, phenyl, naphthyl, indanyl, and 1,2,3,4-tetrahydronaphthalenyl. In some embodiments, aryl groups have 6 carbon atoms. In some embodiments, aryl groups have from six to ten carbon atoms. In some embodiments, aryl groups have from six to sixteen carbon atoms.

It is to be understood that if an aryl, heteroaryl, cycloalkyl, or heterocyclyl moiety may be bonded or otherwise attached to a designated moiety through differing ring atoms (i.e., shown or described without denotation of a specific point of attachment), then all possible points are intended, whether through a carbon atom or, for example, a trivalent nitrogen atom. For example, the term “pyridinyl” means 2-, 3- or 4-pyridinyl, the term “thienyl” means 2- or 3-thienyl, and so forth.

As used herein, the phrase “protecting group” refers to a functional group introduced into a molecule by chemical modification of an oxygen atom, a nitrogen atom, or a sulfur atom to obtain chemoselectivity in a subsequent chemical reaction. Examples of hydroxyl protecting groups include, but are not limited to methoxymethyl (MOM), tetrahydropyranyl (THP), allyl, benzyl (Bn), tert-butyldimethylsilyl (TBDMS), pivaloyl (Piv), and benzoyl (Bz). Examples of nitrogen protecting groups include, but are not limited to, allyloxycarbonyl (Alloc), carbobenzyloxy (Cbz), tert-butyloxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc), acetyl (Ac), benzoyl (Bz), tosyl (Ts), and benzyl (Bn). Examples of sulfur protecting groups include, but are not limited to methoxymethyl (MOM), allyl, trityl (Trt), trichloroacetyl, pivaloyl (Piv), and benzoyl (Bz).

As used herein, the term “optionally substituted” means that the referenced group may be substituted or unsubstituted. As used herein, the term “substituted” means that an atom or group of atoms has replaced hydrogen as the substituent attached to another group.

Covalent Protein Dimers

Provided herein are covalent protein dimers that inhibit the activity of the MYC/MAX complex, which are useful in the treatment of MYC-related disorders, including cancer and other proliferation diseases.

In an aspect, provided herein is a covalent protein dimer, or a pharmaceutically acceptable salt thereof, comprising:a first polypeptide comprising a C-terminus and an N-terminus, wherein the first polypeptide comprises a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3;a second polypeptide comprising a C-terminus and an N-terminus, wherein the second polypeptide comprises a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3; anda linker covalently linking the C-terminus of the first polypeptide to the C-terminus of the second polypeptide.

In some embodiments, the first polypeptide comprises a degree of identity of at least 90% with respect to SEQ ID NO: 1, 2, or 3. In some embodiments, the first polypeptide comprises a degree of identity of at least 95% with respect to SEQ ID NO: 1, 2, or 3. In some embodiments, the first polypeptide comprises a sequence represented by SEQ ID NO: 1, 2, or 3.

In some embodiments, the second polypeptide comprises a degree of identity of at least 90% with respect to SEQ ID NO: 1, 2, or 3. In some embodiments, the second polypeptide comprises a degree of identity of at least 95% with respect to SEQ ID NO: 1, 2, or 3. In some embodiments, the second polypeptide comprises a sequence represented by SEQ ID NO: 1, 2, or 3.

In some embodiments, the first polypeptide is at least 85% identical to SEQ ID NO: 2, and the second polypeptide is at least 85% identical to SEQ ID NO: 2; the first polypeptide is at least 85% identical to SEQ ID NO: 3, and the second polypeptide is at least 85% identical to SEQ ID NO: 3; the first polypeptide is at least 85% identical to SEQ ID NO: 1, and the second polypeptide is at least 85% identical to SEQ ID NO: 2; or the first polypeptide is at least 85% identical to SEQ ID NO: 3, and the second polypeptide is at least 85% identical to SEQ ID NO: 2.

In some embodiments, the first polypeptide is at least 85% identical to SEQ ID NO: 2, and the second polypeptide is at least 85% identical to SEQ ID NO: 2. In some embodiments, the first polypeptide is at least 90% identical to SEQ ID NO: 2, and the second polypeptide is at least 90% identical to SEQ ID NO: 2. In some embodiments, the first polypeptide is at least 95% identical to SEQ ID NO: 2, and the second polypeptide is at least 95% identical to SEQ ID NO: 2. In some embodiments, the first polypeptide comprises a sequence represented by SEQ ID NO: 2, and the second polypeptide comprises a sequence represented by SEQ ID NO: 2.

In some embodiments, the first polypeptide is at least 85% identical to SEQ ID NO: 3, and the second polypeptide is at least 85% identical to SEQ ID NO: 3. In some embodiments, the first polypeptide is at least 90% identical to SEQ ID NO: 3, and the second polypeptide is at least 90% identical to SEQ ID NO: 3. In some embodiments, the first polypeptide is at least 95% identical to SEQ ID NO: 3, and the second polypeptide is at least 95% identical to SEQ ID NO: 3. In some embodiments, the first polypeptide comprises a sequence represented by SEQ ID NO: 3, and the second polypeptide comprises a sequence represented by SEQ ID NO: 3.

In some embodiments, the first polypeptide is at least 85% identical to SEQ ID NO: 1, and the second polypeptide is at least 85% identical to SEQ ID NO: 2. In some embodiments, the first polypeptide is at least 90% identical to SEQ ID NO: 1, and the second polypeptide is at least 90% identical to SEQ ID NO: 2. In some embodiments, the first polypeptide is at least 95% identical to SEQ ID NO: 1, and the second polypeptide is at least 95% identical to SEQ ID NO: 2. In some embodiments, the first polypeptide comprises a sequence represented by SEQ ID NO: 1, and the second polypeptide comprises a sequence represented by SEQ ID NO: 2.

In some embodiments, the first polypeptide is at least 85% identical to SEQ ID NO: 3, and the second polypeptide is at least 85% identical to SEQ ID NO: 2. In some embodiments, the first polypeptide is at least 90% identical to SEQ ID NO: 3, and the second polypeptide is at least 90% identical to SEQ ID NO: 2. In some embodiments, the first polypeptide is at least 95% identical to SEQ ID NO: 3, and the second polypeptide is at least 95% identical to SEQ ID NO: 2. In some embodiments, the first polypeptide comprises a sequence represented by SEQ ID NO: 3, and the second polypeptide comprises a sequence represented by SEQ ID NO: 2.

In general, the linker is a chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches the C-terminus of the first polypeptide to the C-terminus of the second polypeptide. Exemplary linkers may comprise at least one optionally substituted; saturated or unsaturated; linear, branched or cyclic alkyl group or an optionally substituted aryl group. The linker may also be a polypeptide (e.g., from about 1 to about 50 amino acids or more, or from about 1 to about 5 amino acids). In some embodiments, the linker is biologically stable and is not readily cleavable under physiological environments or conditions.

In another aspect, provided herein is a covalent protein dimer, or a pharmaceutically acceptable salt thereof, having a structure according to Formula (I):

wherein:Y1is a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3;Y2is a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3;Z1is —O—, —NH—, or —S—;Z2is —O—, —NH—, or —S—;R1is absent, C1-10alkyl, or C1-10heteroalkyl;R2is absent, C1-10alkyl, or C1-10heteroalkyl;W is C1-10alkyl, C1-10heteroalkyl, C6-10aryl, or 5- to 10-membered heteroaryl;L is absent or a linker;R is H, a nitrogen protecting group, biotin, a fluorescent dye, a nuclear-targeting moiety, or a cell-penetrating moiety; andn is 0 or 1.

In some embodiments, the covalent protein dimer of Formula (I) has a structure according to Formula (Ia):

wherein:Y1is a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3;Y2is a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3;Z1is —O—, —NH—, or —S—;Z2is —O—, —NH—, or —S—;W is C1-10alkyl or C1-10heteroalkyl;L is absent or a linker;R is H, a protecting group, a fluorescent dye, biotin, a nuclear-targeting moiety, or a cell-penetrating moiety; andn is 0 or 1.

In some embodiments, the covalent protein dimer of Formula (I) has a structure according to Formula (Ib):

wherein:Y1is a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3;Y2is a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3;L is absent or a linker;R is H, a nitrogen protecting group, biotin, a fluorescent dye, a nuclear-targeting moiety, or a cell-penetrating moiety; andn is 0 or 1.

In some embodiments, Y1comprises a C-terminus and an N-terminus, wherein the C-terminus forms a bond with Z1or —NH—.

In some embodiments, Y2comprises a C-terminus and an N-terminus, wherein the C-terminus forms a bond with Z2or —NH—.

In some embodiments, if one of Y1or Y2is at least 85% identical to SEQ ID NO: 1, then the other is at least 85% identical to SEQ ID NO: 2.

In some embodiments, Y1is at least 90% identical to SEQ ID NO: 1, 2, or 3. In some embodiments, Y1is at least 95% identical to SEQ ID NO: 1, 2, or 3. In some embodiments, Y1comprises a sequence represented by SEQ ID NO: 1, 2, or 3.

In some embodiments, Y2is at least 90% identical to SEQ ID NO: 1, 2, or 3. In some embodiments, Y2is at least 95% identical to SEQ ID NO: 1, 2, or 3. In some embodiments, Y2comprises a sequence represented by SEQ ID NO: 1, 2, or 3.

In some embodiments, Y1is at least 85% identical to SEQ ID NO: 2, and Y2is at least 85% identical to SEQ ID NO: 2;Y1is at least 85% identical to SEQ ID NO: 3, and Y2is at least 85% identical to SEQ ID NO: 3; Y1is at least 85% identical to SEQ ID NO: 1, and Y2is at least 85% identical to SEQ ID NO: 2; or Y1is at least 85% identical to SEQ ID NO: 3, and Y2is at least 85% identical to SEQ ID NO: 2.

In some embodiments, Y1is at least 85% identical to SEQ ID NO: 2, and Y2is at least 85% identical to SEQ ID NO: 2. In some embodiments, Y1is at least 90% identical to SEQ ID NO: 2,and Y2is at least 90% identical to SEQ ID NO: 2. In some embodiments, Y1is at least 95% identical to SEQ ID NO: 2, and Y2is at least 95% identical to SEQ ID NO: 2. In some embodiments, Y1comprises a sequence represented by SEQ ID NO: 2, and Y2comprises a sequence represented by SEQ ID NO: 2.

In some embodiments, Y1is at least 85% identical to SEQ ID NO: 3, and Y2is at least 85% identical to SEQ ID NO: 3. In some embodiments, Y1is at least 90% identical to SEQ ID NO: 3, and Y2is at least 90% identical to SEQ ID NO: 3. In some embodiments, Y1is at least 95% identical to SEQ ID NO: 3, and Y2is at least 95% identical to SEQ ID NO: 3. In some embodiments, Y1comprises a sequence represented by SEQ ID NO: 3, and Y2comprises a sequence represented by SEQ ID NO: 3.

In some embodiments, Y1is at least 85% identical to SEQ ID NO: 1, and Y2is at least 85% identical to SEQ ID NO: 2. In some embodiments, Y1is at least 90% identical to SEQ ID NO: 1, and Y2is at least 90% identical to SEQ ID NO: 2. In some embodiments, Y1is at least 95% identical to SEQ ID NO: 1,and Y2is at least 95% identical to SEQ ID NO: 2. In some embodiments, Y1comprises a sequence represented by SEQ ID NO: 1, and Y2comprises a sequence represented by SEQ ID NO: 2.

In some embodiments, Y1is at least 85% identical to SEQ ID NO: 3, and Y2 is at least 85% identical to SEQ ID NO:2. In some embodiments, Y1is at least 90% identical to SEQ ID NO: 3, and Y2is at least 90% identical to SEQ ID NO: 2. In some embodiments, Y1is at least 95% identical to SEQ ID NO: 3, and Y2is at least 95% identical to SEQ ID NO: 2. In some embodiments, Y1comprises a sequence represented by SEQ ID NO: 3, and Y2comprises a sequence represented by SEQ ID NO: 2.

In some embodiments, R1is absent or C1-10alkyl. In some embodiments, R2is absent or C1-10alkyl.

In some embodiments, W is C1-10alkyl or C1-10heteroalkyl. In some embodiments, W is C1-5alkyl or C1-5heteroalkyl.

In some embodiments, L is absent.

In some embodiments, L is a linker. Exemplary linkers may comprise at least one optionally substituted; saturated or unsaturated; linear, branched or cyclic alkyl group or an optionally substituted aryl group. In some embodiments, the linker is a polypeptide. In some embodiments, the linker comprises one to fifty amino acids. In some embodiments, the linker comprises one to twenty-five amino acids. In some embodiments, the linker comprises one to ten amino acids. In some embodiments, the linker comprises one to five amino acids. In some embodiments, L is β-alanine.

In some embodiments, R is H.

In some embodiments, R is a nitrogen protecting group that is not 9-fluorenylmethyloxycarbonyl (Fmoc). In some embodiments, R is a nitrogen protecting group selected from the group consisting of allyloxycarbonyl (Alloc), carbobenzyloxy (Cbz), tert-butyloxycarbonyl (Boc), acetyl (Ac), benzoyl (Bz), tosyl (Ts), and benzyl (Bn). In some embodiments, R is Alloc or Boc.

In some embodiments, R is a fluorescent dye. Fluorescent dyes suitable for the covalent protein dimers include any fluorescent dye known in the art that may be covalently linked to dimer by way of the nitrogen atom adjacent variable R. Non-limiting examples of fluorescent dyes include Alexa Fluor fluorescent dyes, DyLight Fluor fluorescent dyes, rhodamine dyes, blue fluorescent protein (BFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), Cascade Blue™, Marina Blue™, Pacific Orange™, Oregon Green™, Cascade Yellow™, BODIPY, coumarin, methoxycoumarin, aminomethylcoumarin (AMCA), dansyl, 5-TAMRA, fluorescein, mBanana, mOrange, mHoneydew, mTangerine, mCherry, and mPlum. In some embodiments, the fluorescent dye is 5-TAMRA.

In some embodiments, R is a nuclear targeting moiety. In some embodiments, R is Mach3 having the sequence:

In some embodiments, n is 0. In some embodiments, n is 1.

In another aspect, provided herein is a covalent protein dimer, or a pharmaceutically acceptable salt thereof, having a structure according to Formula (II):

wherein:Y1is a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3;Y2is a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3;Z1independently is —O—, —NH—, or —S—;Z2independently is —O—, —NH—, or —S—;R1independently is C1-10alkyl or C1-10heteroalkyl;A is C6-10aryl or 5- to 10-membered heteroaryl;L independently is absent or a linker;R independently is H, a nitrogen protecting group, biotin, a fluorescent dye, a nuclear-targeting moiety, or a cell-penetrating moiety; andn independently is 0 or 1.

In some embodiments, the covalent protein dimer of Formula (II) has a structure according to Formula (IIa):

wherein:Y1is a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3;Y2is a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3;L independently is absent or a linker;R independently is H, a nitrogen protecting group, biotin, a fluorescent dye, a nuclear-targeting moiety, or a cell-penetrating moiety; andn independently is 0 or 1.

In some embodiments, the covalent protein dimer of Formula (II) has a structure according to Formula (IIb):

wherein:Y1is a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3; andY2is a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3.

In some embodiments, Y1comprises a C-terminus and an N-terminus, wherein the C-terminus forms a bond with Z2or —NH—.

In some embodiments, Y2comprises a C-terminus and an N-terminus, wherein the C-terminus forms a bond with Z2or —NH—.

In some embodiments, Y1is at least 90% identical to SEQ ID NO: 1, 2, or 3. In some embodiments, Y1is at least 95% identical to SEQ ID NO: 1, 2, or 3. In some embodiments, Y1comprises a sequence represented by SEQ ID NO: 1, 2, or 3.

In some embodiments, Y2is at least 90% identical to SEQ ID NO: 1, 2, or 3. In some embodiments, Y2is at least 95% identical to SEQ ID NO: 1, 2, or 3. In some embodiments, Y2comprises a sequence represented by SEQ ID NO: 1, 2, or 3.

In some embodiments, Y1is at least 85% identical to SEQ ID NO: 2, and Y2is at least 85% identical to SEQ ID NO: 2; Y1is at least 85% identical to SEQ ID NO: 3, and Y2is at least 85% identical to SEQ ID NO: 3; Y1is at least 85% identical to SEQ ID NO: 1, and Y2is at least 85% identical to SEQ ID NO: 1; Y1is at least 85% identical to SEQ ID NO: 1, and Y2is at least 85% identical to SEQ ID NO: 2; Y1is at least 85% identical to SEQ ID NO: 3, and Y2is at least 85% identical to SEQ ID NO: 2; or Y1is at least 85% identical to SEQ ID NO: 3, and Y2is at least 85% identical to SEQ ID NO: 1;.

In some embodiments, Y1is at least 85% identical to SEQ ID NO: 2, and Y2is at least 85% identical to SEQ ID NO: 2. In some embodiments, Y1is at least 90% identical to SEQ ID NO: 2, and Y2is at least 90% identical to SEQ ID NO: 2. In some embodiments, Y1is at least 95% identical to SEQ ID NO: 2, and Y2is at least 95% identical to SEQ ID NO: 2. In some embodiments, Y1comprises a sequence represented by SEQ ID NO: 2, and Y2comprises a sequence represented by SEQ ID NO: 2.

In some embodiments, Y1is at least 85% identical to SEQ ID NO: 3, and Y2is at least 85% identical to SEQ ID NO: 3. In some embodiments, Y1is at least 90% identical to SEQ ID NO: 3, and Y2is at least 90% identical to SEQ ID NO: 3. In some embodiments, Y1is at least 95% identical to SEQ ID NO: 3, and Y2is at least 95% identical to SEQ ID NO: 3. In some embodiments, Y1comprises a sequence represented by SEQ ID NO: 3, and Y2comprises a sequence represented by SEQ ID NO: 3.

In some embodiments, Y1is at least 85% identical to SEQ ID NO: 1, and Y2is at least 85% identical to SEQ ID NO: 1. In some embodiments, Y1is at least 90% identical to SEQ ID NO: 1, and Y2is at least 90% identical to SEQ ID NO: 1. In some embodiments, Y1is at least 95% identical to SEQ ID NO: 1, and Y2is at least 95% identical to SEQ ID NO: 1. In some embodiments, Y1comprises a sequence represented by SEQ ID NO: 1, and Y2comprises a sequence represented by SEQ ID NO: 1.

In some embodiments, Y1is at least 85% identical to SEQ ID NO: 1, and Y2is at least 85% identical to SEQ ID NO: 2. In some embodiments, Y1is at least 90% identical to SEQ ID NO: 1, and Y2is at least 90% identical to SEQ ID NO: 2. In some embodiments, Y1is at least 95% identical to SEQ ID NO: 1, and Y2is at least 95% identical to SEQ ID NO: 2. In some embodiments, Y1comprises a sequence represented by SEQ ID NO: 1, and Y2comprises a sequence represented by SEQ ID NO: 2.

In some embodiments, Y1is at least 85% identical to SEQ ID NO: 3, and Y2is at least 85% identical to SEQ ID NO: 2. In some embodiments, Y1is at least 90% identical to SEQ ID NO: 3, and Y2is at least 90% identical to SEQ ID NO: 2. In some embodiments, Y1is at least 95% identical to SEQ ID NO: 3, and Y2is at least 95% identical to SEQ ID NO: 2. In some embodiments, Y1comprises a sequence represented by SEQ ID NO: 3, and Y2comprises a sequence represented by SEQ ID NO: 2.

In some embodiments, Y1is at least 85% identical to SEQ ID NO: 3, and Y2is at least 85% identical to SEQ ID NO: 1. In some embodiments, Y1is at least 90% identical to SEQ ID NO: 3, and Y2is at least 90% identical to SEQ ID NO: 1. In some embodiments, Y1is at least 95% identical to SEQ ID NO: 3, and Y2is at least 95% identical to SEQ ID NO: 1. In some embodiments, Y1comprises a sequence represented by SEQ ID NO: 3, and Y2comprises a sequence represented by SEQ ID NO: 1.

In some embodiments, A is C6-10aryl. In some embodiments, A is 5- to 10-membered heteroaryl. In some embodiments, A is phenyl or 5- to 6-membered heteroaryl. In some embodiments, A is phenyl.

In some embodiments, L is absent.

In some embodiments, L is a linker. Exemplary linkers may comprise at least one optionally substituted; saturated or unsaturated; linear, branched or cyclic alkyl group or an optionally substituted aryl group. In some embodiments, the linker is a polypeptide. In some embodiments, the linker comprises one to fifty amino acids. In some embodiments, the linker comprises one to twenty-five amino acids. In some embodiments, the linker comprises one to ten amino acids. In some embodiments, the linker comprises one to five amino acids. In some embodiments, L is β-alanine.

In some embodiments, R is a nitrogen protecting group that is not 9-fluorenylmethyloxycarbonyl (Fmoc). In some embodiments, R is a nitrogen protecting group selected from the group consisting of allyloxycarbonyl (Alloc), carbobenzyloxy (Cbz), tert-butyloxycarbonyl (Boc), acetyl (Ac), benzoyl (Bz), tosyl (Ts), and benzyl (Bn). In some embodiments, R is Alloc or Boc.

In some embodiments, R is a fluorescent dye. Fluorescent dyes suitable for the covalent protein dimers include any fluorescent dye known in the art that may be covalently linked to dimer by way of the nitrogen atom adjacent variable R. Non-limiting examples of fluorescent dyes include Alexa Fluor fluorescent dyes, DyLight Fluor fluorescent dyes, rhodamine dyes, blue fluorescent protein (BFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), Cascade Blue™, Marina Blue™, Pacific Orange™, Oregon Green™, Cascade Yellow™, BODIPY, coumarin, methoxycoumarin, aminomethylcoumarin (AMCA), dansyl, 5-TAMRA, fluorescein, mBanana, mOrange, mHoneydew, mTangerine, mCherry, and mPlum. In some embodiments, the fluorescent dye is 5-TAMRA.

In some embodiments, R is a nuclear targeting moiety. In some embodiments, R is Mach3 having the sequence:

In some embodiments, n is 0. In some embodiments, n is 1.

The covalent protein dimers disclosed herein may exist as tautomers and optical isomers (e.g., enantiomers, diastereomers, diastereomeric mixtures, racemic mixtures, and the like).

In an aspect, provided herein is a pharmaceutical composition comprising a covalent protein dimer disclosed herein and a pharmaceutically acceptable carrier.

In an embodiment, the pharmaceutical compositions described herein include a therapeutically or prophylactically effective amount of a compound described herein. The pharmaceutical composition may be useful for treating a proliferative disease in a subject in need thereof, preventing a proliferative disease in a subject in need thereof, or inhibiting the activity of MYC in a subject, biological sample, tissue, or cell. In some embodiments, the proliferative disease is cancer.

Synthesis of Covalent Protein Dimers

Also provided herein are methods of making the covalent protein dimers disclosed herein. Accordingly, in an aspect, the disclosure provides a method of making a covalent protein dimer, or a pharmaceutically acceptable salt thereof, having a structure according to Formula (I):

wherein:Y1is a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3;Y2is a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3;Z1is —O—, —NH—, or —S—;Z2is —O—, —NH—, or —S—;R1is absent, C1-10alkyl, or C1-10heteroalkyl;R2is absent, C1-10alkyl, or C1-10heteroalkyl;W is C1-10alkyl, C1-10heteroalkyl, C6-10aryl, or 5- to 10-membered heteroaryl;L is absent or a linker;R is H, a nitrogen protecting group, biotin, a fluorescent dye, a nuclear-targeting moiety, or a cell-penetrating moiety; andn is 0 or 1;the method comprising:(a) reacting a first resin-bound, side-chain-protected peptide having a structure according to Formula (III):

with one or more amino acids corresponding to the amino acids of the polypeptide represented by Y2to provide a second resin-bound, side-chain-protected peptide having a structure according to Formula (IV):

wherein PG1and PG2are non-identical protecting groups, and wherein neither PG1nor PG2are Fmoc;(b) removing PG2from the second resin-bound, side-chain-protected peptide to provide a third resin-bound, side-chain-protected peptide having a structure according to Formula (V):

(c) reacting the third resin-bound, side-chain-protected peptide with one or more amino acids corresponding to the amino acids of the polypeptide represented by Y1to provide a fourth resin-bound, side-chain protected peptide having a structure according to Formula (VI):

and(d) cleaving the fourth resin-bound, side-chain-protected peptide from the resin to provide the covalent protein dimer.

In some embodiments, the covalent protein dimer of Formula (I) has a structure according to Formula (Ia):

wherein:Y1is a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3;Y2is a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3;Z1is —O—, —NH—, or —S—;Z2is —O—, —NH—, or —S—;W is C1-10alkyl or C1-10heteroalkyl;L is absent or a linker;R is H, a protecting group, a fluorescent dye, biotin, a nuclear-targeting moiety, or a cell-penetrating moiety; andn is 0 or 1;the method comprising:(a) reacting a first resin-bound, side-chain-protected peptide having a structure according to Formula (IIIa):

with one or more amino acids corresponding to the amino acids of the polypeptide represented by Y2to provide a second resin-bound, side-chain-protected peptide having a structure according to Formula (IVa):

wherein PG1and PG2are non-identical protecting groups, and wherein neither PG1nor PG2are Fmoc;(b) removing PG2from the second resin-bound, side-chain-protected peptide to provide a third resin-bound, side-chain-protected peptide having a structure according to Formula (Va):

(c) reacting the third resin-bound, side-chain-protected peptide with one or more amino acids corresponding to the amino acids of the polypeptide represented by Y1to provide a fourth resin-bound, side-chain protected peptide having a structure according to Formula (VIa):

and(d) cleaving the fourth resin-bound, side-chain-protected peptide from the resin to provide the covalent protein dimer.

In some embodiments, the covalent protein dimer of Formula (I) has a structure according to Formula (Ib):

wherein:Y1is a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3;Y2is a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3;L is absent or a linker;R is H, a nitrogen protecting group, biotin, a fluorescent dye, a nuclear-targeting moiety, or a cell-penetrating moiety;n is 0 or 1;the method comprising:(a) reacting a first resin-bound, side-chain-protected peptide having a structure according to Formula (IIIb):

with one or more amino acids corresponding to the amino acids of the polypeptide represented by Y2to provide a second resin-bound, side-chain-protected peptide having a structure according to Formula (IVb):

wherein PG1and PG2are non-identical nitrogen protecting groups, and wherein neither PG1nor PG2are Fmoc;(b) removing PG2from the second resin-bound, side-chain-protected peptide to provide a third resin-bound, side-chain-protected peptide having a structure according to Formula (Vb):

(c) reacting the third resin-bound, side-chain-protected peptide with one or more amino acids corresponding to the amino acids of the polypeptide represented by Y1to provide a fourth resin-bound, side-chain protected peptide having a structure according to Formula (VIb):

and(d) cleaving the fourth resin-bound, side-chain-protected peptide from the resin to provide the covalent protein dimer.

In some embodiments, prior to step (d), the method comprises removing PG1to provide a deprotected nitrogen atom therein, and covalently attaching biotin, a fluorescent dye, a nuclear-targeting moiety, or a cell-penetrating moiety to the deprotected nitrogen atom.

In some embodiments, each of the one or more amino acids of steps (a) and (c) comprises an Fmoc-protected backbone amino group, wherein the corresponding Fmoc group is deprotected after each amino acid is attached to the resin-bound, side-chain-protected peptide.

In some embodiments, each one of steps (a) and (c) is performed in the presence of a coupling agent. Coupling agents suitable for the methods disclosed herein include those known in the art to facilitate peptide bond formation. Exemplary non-limiting coupling agents include (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), hexafluorophosphate azabenzotriazole tetramethyl uronium (HATU), hexafluorophosphate benzotriazole tetramethyl uronium (HBTU), 2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU), and hydroxybenzotriazole (HOBt).

In some embodiments, each one of steps (a) and (c) comprises the addition of N,N-diisopropylethylamine (DIEA).

In another aspect, the disclosure provides a method of making a covalent protein dimer, or a pharmaceutically acceptable salt thereof, having a structure according to Formula (I):

wherein:Y1and Y2are identical and each represents a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3;Z1is —O—, —NH—, or —S—;Z2is —O—, —NH—, or —S—;R1is absent, C1-10alkyl, or C1-10heteroalkyl;R2is absent, C1-10alkyl, or C1-10heteroalkyl;W is C1-10alkyl, C1-10heteroalkyl, C6-10aryl, or 5- to 10-membered heteroaryl;L is absent or a linker;R is H, a protecting group, biotin, a fluorescent dye, a nuclear-targeting moiety, or a cell-penetrating moiety; andn is 0 or 1;the method comprising:(a) reacting a first resin-bound, side-chain-protected peptide having a structure according to Formula (VII):

with one or more amino acids corresponding to the amino acids of the polypeptide represented by Y1to provide a second resin-bound, side-chain-protected peptide having a structure according to Formula (VI):

wherein PG1is a nitrogen protecting group that is not Fmoc; and(b) cleaving the second resin-bound, side-chain-protected peptide from the resin to provide the covalent protein dimer.

In some embodiments, the covalent protein dimer of Formula (I) has a structure according to Formula (Ia):

wherein:Y1and Y2are identical and each represents a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3;Z1is —O—, —NH—, or —S—;Z2is —O—, —NH—, or —S—;W is C1-10alkyl or C1-10heteroalkyl;L is absent or a linker;R is H, a protecting group, a fluorescent dye, biotin, a nuclear-targeting moiety, or a cell-penetrating moiety; andn is 0 or 1;the method comprising:(a) reacting a first resin-bound, side-chain-protected peptide having a structure according to Formula (VIIa):

with one or more amino acids corresponding to the amino acids of the polypeptide represented by Y1to provide a second resin-bound, side-chain-protected peptide having a structure according to Formula (VIa):

wherein PG1is a nitrogen protecting group that is not Fmoc; and(b) cleaving the second resin-bound, side-chain-protected peptide from the resin to provide the covalent protein dimer.

In some embodiments, the covalent protein dimer of Formula (I) has a structure according to Formula (Ib):

wherein:Y1and Y2are identical and each represents a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3;L is absent or a linker;R is H, a nitrogen protecting group, biotin, a fluorescent dye, a nuclear-targeting moiety, or a cell-penetrating moiety;n is 0 or 1;the method comprising:(a) reacting a first resin-bound, side-chain-protected peptide having a structure according to Formula (VIIb):

with one or more amino acids corresponding to the amino acids of the polypeptide represented by Y1to provide a second resin-bound, side-chain-protected peptide having a structure according to Formula (VIb):

wherein PG1is a nitrogen protecting group that is not Fmoc; and(b) cleaving the second resin-bound, side-chain-protected peptide from the resin to provide the covalent protein dimer.

In some embodiments, prior to step (b), the method comprises removing PG1to provide a deprotected nitrogen atom therein, and covalently attaching biotin, a fluorescent dye, a nuclear-targeting moiety, or a cell-penetrating moiety to the deprotected nitrogen atom.

In some embodiments, each of the one or more amino acids of step (a) comprise an Fmoc-protected backbone amino group, and wherein the corresponding Fmoc group is deprotected after each amino acid is attached to the resin-bound, side-chain-protected peptide.

In some embodiments, step (a) comprises the addition of N,N-Diisopropylethylamine (DIEA).

In another aspect, the disclosure provides a method of making a covalent protein dimer, or a pharmaceutically acceptable salt thereof, having a structure according to Formula (II):

wherein:Y1is a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3;Y2is a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3;Z1independently is —O—, —NH—, or —S—;Z2independently is —O—, —NH—, or —S—;R1independently is C1-10alkyl or C1-10heteroalkyl;A is C6-10aryl or 5- to 10-membered heteroaryl;L independently is absent or a linker;R independently is H, a nitrogen protecting group, biotin, a fluorescent dye, a nuclear-targeting moiety, or a cell-penetrating moiety; andn independently is 0 or 1;the method comprising:(a) reacting a polypeptide having a structure according to Formula (VIII):

with a compound of Formula (IX):

to provide a polypeptide having a structure according to Formula (X):

wherein:X and X′ are each, independently, F, Cl, Br, I, or OTf, andLig is a phosphine ligand; and(b) reacting the polypeptide of Formula (X) with a polypeptide having a structure according to Formula (XI):

to provide the covalent protein dimer.

In some embodiments, the covalent protein dimer of Formula (II) has a structure according to Formula (IIa):

wherein:Y1is a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3;Y2is a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3;L independently is absent or a linker,R independently is H, a nitrogen protecting group, biotin, a fluorescent dye, a nuclear-targeting moiety, or a cell-penetrating moiety; andn independently is 0 or 1;the method comprising:(a) reacting a polypeptide having a structure according to Formula (VIIIa):

with a compound of Formula (IX):

to provide a polypeptide having a structure according to Formula (Xa):

wherein:X and X′ are each, independently, F, Cl, Br, I, or OTf; andLig is a phosphine ligand; and(b) reacting the polypeptide of Formula (Xa) with a polypeptide having a structure according to Formula (XIa):

to provide the covalent protein dimer.

In some embodiments, the covalent protein dimer of Formula (II) has a structure according to Formula (IIb):

wherein:Y1is a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3; andY2is a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3;the method comprising:(a) reacting a polypeptide having a structure according to Formula (VIIIb):

with a compound of Formula (IX):

to provide a polypeptide having a structure according to Formula (Xb):

wherein:X and X′ are each, independently, F, Cl, Br, I, or OTf; andLig is a phosphine ligand; and(b) reacting the polypeptide of Formula (Xb) with a polypeptide having a structure according to Formula (XIb):

to provide the covalent protein dimer.

In some embodiments, the compound of Formula (IX), (IXa), or (IXb) is provided in molar excess with respect to the polypeptide of Formula (VIII), (VIIIa), or (VIIIb). In some embodiments, the compound of Formula (IX), (IXa), or (IXb) and the polypeptide of Formula (VIII), (VIIIa), or (VIIIb) are provided in a molar ratio from about 10:1 to about 2:1. In some embodiments the compound of Formula (IX), (IXa), or (IXb) and the polypeptide of Formula (VIII), (VIIIa), or (VIIIb) are provided in a molar ratio of about 5:1.

In some embodiments, X and X′ are I.Lig may be any phosphine ligand known in the art to be useful in cross-coupling reactions. By non-limiting example, Lig may be JohnPhos, DavePhos, XPhos, SPhos, MePhos, RuPhos, BrettPhos, PhDavePhos, tBuXPhos, tBuMePhos, tBuBrettPhos, tBuDavePhos, or JackiePhos. In some embodiments, Lig has a structure according to Formula (XII):

In some embodiments, Lig is

In another aspect, the disclosure provides a method of making a covalent protein dimer having a structure according to Formula (II):

wherein:Y1and Y2are identical and each represents a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3;Z1independently is —O—, —NH—, or —S—;Z2independently is —O—, —NH—, or —S—;R1independently is C1-10alkyl or C1-10heteroalkyl;A is C6-10aryl or 5- to 10-membered heteroaryl;L independently is absent or a linker;R independently is H, a nitrogen protecting group, biotin, a fluorescent dye, a nuclear-targeting moiety, or a cell-penetrating moiety; andn independently is 0 or 1;the method comprising reacting a polypeptide having a structure according to Formula (VIII):

with a compound of Formula (IX):

In some embodiments, the covalent protein dimer of Formula (II) has a structure according to Formula (IIa):

wherein:Y1is a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3;Y2is a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3;L independently is absent or a linker;R independently is H, a nitrogen protecting group, biotin, a fluorescent dye, a nuclear-targeting moiety, or a cell-penetrating moiety; andn independently is 0 or 1;the method comprising reacting a polypeptide having a structure according to Formula (VIIIa):

with a compound of Formula (IX):

In some embodiments, the covalent protein dimer of Formula (II) has a structure according to Formula (IIb):

wherein:Y1and Y2are identical and each represents a polypeptide comprising a degree of identity of at least 85% with respect to SEQ ID NO: 1, 2, or 3;the method comprising reacting a polypeptide having a structure according to Formula (VIIIb):

with a compound of Formula (IX):

to provide the covalent protein dimer,wherein:X and X′ are each, independently, F, Cl, Br, I, or OTf; andLig is a phosphine ligand.In some embodiments, Z1is —S—. In some embodiments, Z2is —NH—.In some embodiments, X and X′ are I.Lig may be any phosphine ligand known in the art to be useful in cross-coupling reactions. By non-limiting example, Lig may be JohnPhos, DavePhos, XPhos, SPhos, MePhos, RuPhos, BrettPhos, PhDavePhos, tBuXPhos, tBuMePhos, tBuBrettPhos, tBuDavePhos, or JackiePhos. In some embodiments, Lig has a structure according to Formula (XII):

In some embodiments, Lig is

Methods of Treatment

In an aspect, provided herein is a method of treating a disease or disorder characterized by MYC dysregulation in a subject in need thereof, the method comprising administering to the subject a covalent protein dimer of the present disclosure. In some embodiments, the disease or disorder characterized by MYC dysregulation is an immune disorder, such as myasthenia gravis, psoriasis, pemphigus vulgaris, and atherosclerosis. In some embodiments, the disease or disorder is cancer. In certain embodiments, the cancer is selected from the group consisting of pancreatic cancer, lung cancer, prostate cancer, breast cancer, ovarian cancer, kidney cancer, liver cancer, brain cancer, neuroblastoma, colorectal cancer, and hematological malignancies.

Also described are methods for contacting a cell or a biological sample with an effective amount of a covalent protein dimer of the disclosure.

In yet another aspect, provided herein is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a covalent protein dimer of the present disclosure.

The term “cancer” refers to any cancer caused by the proliferation of malignant neoplastic cells, such as tumors, neoplasms, carcinomas, sarcomas, leukemias, lymphomas and the like. For example, cancers include, but are not limited to, mesothelioma, leukemias and lymphomas such as cutaneous T-cell lymphomas (CTCL), noncutaneous peripheral T-cell lymphomas, lymphomas associated with human T-cell lymphotrophic virus (HTLV) such as adult T-cell leukemia/lymphoma (ATLL), B-cell lymphoma, acute nonlymphocytic leukemias, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, lymphomas, and multiple myeloma, non-Hodgkin lymphoma, acute lymphatic leukemia (ALL), chronic lymphatic leukemia (CLL), Hodgkin's lymphoma, Burkitt lymphoma, adult T-cell leukemia lymphoma, acute-myeloid leukemia (AML), chronic myeloid leukemia (CML), or hepatocellular carcinoma. Further examples include myelodysplastic syndrome, childhood solid tumors such as brain tumors, neuroblastoma, retinoblastoma, Wilms' tumor, bone tumors, and soft-tissue sarcomas, common solid tumors of adults such as head and neck cancers (e.g., oral, laryngeal, nasopharyngeal and esophageal), genitourinary cancers (e.g., prostate, bladder, renal, uterine, ovarian, testicular), lung cancer (e.g., small-cell and non-small cell), breast cancer, pancreatic cancer, melanoma and other skin cancers, stomach cancer, brain tumors, tumors related to Gorlin syndrome (e.g., medulloblastoma, meningioma, etc.), and liver cancer. Additional exemplary forms of cancer which may be treated by the subject compounds include, but are not limited to, cancer of skeletal or smooth muscle, stomach cancer, cancer of the small intestine, rectum carcinoma, cancer of the salivary gland, endometrial cancer, adrenal cancer, anal cancer, rectal cancer, parathyroid cancer, and pituitary cancer.

In some embodiments, the cancer is lung cancer, colon cancer, breast cancer, prostate cancer, liver cancer, pancreas cancer, brain cancer, kidney cancer, ovarian cancer, stomach cancer, skin cancer, bone cancer, gastric cancer, breast cancer, pancreatic cancer, glioma, glioblastoma, hepatocellular carcinoma, papillary renal carcinoma, head and neck squamous cell carcinoma, leukemias, lymphomas, myelomas, or solid tumors. In further embodiments, the disease is lung cancer, breast cancer, ovarian cancer, glioma, squamous cell carcinoma, or prostate cancer. In some embodiments, the cancer is breast cancer, colorectal cancer, pancreatic cancer, gastric cancer, or uterine cancer. In some embodiments, the cancer is a hematological malignancy. In some embodiments, the cancer is acute myeloid leukemia, chronic myelogenous leukemia, Hodgkin's lymphoma, or diffuse large B-cell lymphoma. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is a non-small cell lung cancer.

In some embodiments, the covalent protein dimes of this disclosure are useful for treating cancer, such as colorectal, thyroid, breast, and lung cancer; and myeloproliferative disorders, such as polycythemia vera, thrombocythemia, myeloid metaplasia with myelofibrosis, chronic myelogenous leukemia, chronic myelomonocytic leukemia, hypereosinophilic syndrome, juvenile myelomonocytic leukemia, and systemic mast cell disease. In some embodiments, the covalent protein dimers of this disclosure are useful for treating hematopoietic disorders acute-myelogenous leukemia (AML), chronic-myelogenous leukemia (CML), acute-promyelocytic leukemia, and acute lymphocytic leukemia (ALL).

In one aspect, the present disclosure provides for the use of one or more covalent protein dimers of the disclosure in the manufacture of a medicament for the treatment of cancer, including without limitation the various types of cancer disclosed herein.

Formulations and Dosages

The covalent protein dimers described herein will generally be administered to a subject as a pharmaceutical composition. The terms “patient” and “subject”, as used herein, include humans and non-human animals. The covalent protein dimers described herein may be employed therapeutically, under the guidance of a physician.

The compositions comprising the covalent protein dimers of the instant disclosure may be conveniently formulated for administration with any pharmaceutically acceptable carrier(s). For example, the covalent protein dimers may be formulated with an acceptable medium such as water, buffered saline, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), dimethyl sulfoxide (DMSO), oils, detergents, suspending agents or suitable mixtures thereof. The concentration of the covalent protein dimers in the chosen medium may be varied and the medium may be chosen based on the desired route of administration of the pharmaceutical composition. Except insofar as any conventional media or agent is incompatible with the covalent protein dimers to be administered, its use in the pharmaceutical composition is contemplated.

The dose and dosage regimen of the covalent protein dimers disclosed herein that are suitable for administration to a particular subject may be determined by a physician considering the subject's age, sex, weight, general medical condition, and the specific condition for which the covalent protein dimer(s) is being administered and the severity thereof. The physician may also take into account the route of administration, the pharmaceutical carrier, and the covalent protein dimers' biological activity.

Selection of a suitable pharmaceutical composition will also depend upon the mode of administration chosen. For example, the covalent protein dimers of the invention may be administered by direct injection to a desired site (e.g., tumor). In this instance, a pharmaceutical composition comprising the covalent protein dimers is dispersed in a medium that is compatible with the site of injection. Covalent protein dimers of the instant disclosure may be administered by any method. For example, the covalent protein dimers of the instant disclosure can be administered, without limitation parenterally, subcutaneously, orally, topically, pulmonarily, rectally, vaginally, intravenously, intraperitoneally, intrathecally, intracerbrally, epidurally, intramuscularly, intradermally, or intracarotidly.

Pharmaceutical compositions containing a covalent protein dimer of the present disclosure as the active ingredient in intimate admixture with a pharmaceutically acceptable carrier can be prepared according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., intravenous, oral, direct injection, intracranial, and intravitreal.

A pharmaceutical composition of the disclosure may be formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to a physically discrete unit of the pharmaceutical preparation appropriate for the patient undergoing treatment. Each dosage should contain a quantity of active ingredient calculated to produce the desired effect in association with the selected pharmaceutical carrier. Procedures for determining the appropriate dosage unit are well known to those skilled in the art.

Dosage units may be proportionately increased or decreased based on the weight of the subject. Appropriate concentrations for alleviation of a particular pathological condition may be determined by dosage concentration curve calculations, as known in the art.

In accordance with the present disclosure, the appropriate dosage unit for the administration of covalent protein dimers may be determined by evaluating the toxicity of the molecules or cells in animal models. Various concentrations of covalent protein dimers in pharmaceutical preparations may be administered to mice, and the minimal and maximal dosages may be determined based on the beneficial results and side effects observed as a result of the treatment. Appropriate dosage units may also be determined by assessing the efficacy of the covalent protein dimers in combination with other standard drugs. The dosage units of covalent protein dimers may be determined individually or in combination with each treatment according to the effect detected.

The pharmaceutical compositions comprising the covalent protein dimers may be administered at appropriate intervals, for example, at least twice a day or more until the pathological symptoms are reduced or alleviated, after which the dosage may be reduced to a maintenance level. The appropriate interval in a particular case would normally depend on the condition of the subject.

EXAMPLES

Synthesis of Lysine-Linked Covalent Protein Dimers

Manual Preparation of Peptidyl Resins 1 and 2

Preparation of ChemMatrix® Rink amide resin (loading 0.18 mmol/g, typical scale: 100 mg, 0.02 mmol) was loaded into a fritted syringe (6 mL), swollen in DMF (4 mL) for 5 minutes and then drained. Each Na-Fmoc protected amino acid (0.2 mmol, 10 equiv.) was dissolved in DMF containing 0.39 M HATU (0.5 mL). Immediately before the coupling, DIEA (100 μL, 30 equiv.) was added to the mixture to activate the amino acid. After 15 seconds preactivation, the mixture was added to the resin and reacted for 10 min, with occasional stirring. After completion of the coupling step, the syringe was drained, and the resin was washed with DMF (3×5 mL). Fmoc deprotection was performed by addition of piperidine (20% in DMF, 3 mL) to the resin (1×1 min+1×5 min), followed by draining and washing the resin with DMF (5×5 mL). For peptidyl resin 1 the coupling cycles were performed sequentially with Fmoc-Lys (Alloc)—OH, Fmoc-βAla-OH, and Fmoc-Lys (Fmoc)—OH; for peptidyl resin 2 the coupling cycles were performed sequentially with Fmoc-Lys (Boc)—OH, Fmoc-βAla-OH and Fmoc-Lys (Alloc)—OH.

Automated Flow Peptide Synthesis (AFPS)

Covalent MAX-MAX and Omomyc-Omomyc homodimers were prepared via parallel single-shot fast-flow solid-phase synthesis from peptidyl resin 1. Each step involved the parallel coupling and subsequent deprotection of two amino acids simultaneously. The synthesis time for each homodimer was about 3.5 hours (MAX-MAX (3), 164 residues; Omomyc-Omomyc (4), 184 residues). After cleavage and side-chain deprotection, LC-MS analysis indicated the desired products as the major component of both crude reaction mixtures. Upon preparative HPLC purification, pure MAX-MAX (3) and Omomyc-Omomyc (4) were obtained in 6% and 8% yield, respectively.

Covalent MYC-MAX and Omomyc-MAX heterodimers were prepared by consecutive single-shot fast flow solid phase synthesis. With the fast flow synthesizer, MAX was assembled from the α-amine of the lysine linker of peptidyl resin 2. For the last amino acid, Boc-glycine was added, and the Alloc protection was removed from the NE of the lysine linker. On this amine, MYC or Omomyc were assembled to provide 5 and 6, respectively. The synthesis time for each heterodimer amounted to about 8 hours (MYC-MAX (5), 167 residues; Omomyc-MAX (6), 175 residues). Both heterodimers were observed as the main component of the crude product mixture obtained from cleavage and side-chain deprotection. Upon preparative HPLC purification, pure MYC-MAX (5) and Omomyc-MAX (6) dimers were obtained in 4% and 5% yield, respectively.

All peptides were synthesized on two automated-flow systems depicted inFIG.1and as described in Mijalis, A. J. et al.Nat. Chem. Biol.13, 464-466 (2017). The synthesis conditions used are according to Hartrampf, N. et al.Science368, 980-987 (2020). Flow-rate=40 mL/min, temperature =90° C. (loop 1), 70° C. (loop 2; used for histidine) and 85-90° C. (reactor). The 50 ml/min pump head pumps 400 μL of liquid per pump stroke; the 5 mL/min pump head pumps 40 μL of liquid per pump stroke. The standard synthetic cycle involved a first step of prewashing the resin at elevated temperatures for 60 s at 40 mL/min. During the coupling step, three HPLC pumps were used: a 50 mL/min pump head pumped the activating agent, a second 50 ml/min pump head pumped the amino acid, and a 5 mL/min pump head pumped DIEA. The first two pumps were activated for 8 pumping strokes in order to prime the coupling agent and amino acid before the DIEA pump was activated. The three pumps were then actuated together for a period of 7 pumping strokes, after which the activating agent pump and amino acid pump were switched using a rotary valve to select DMF. The three pumps were actuated together for a final 8 pumping strokes, after which the DIEA pump was shut off and the other two pumps continued to wash the resin for another 40 pump strokes. During the deprotection step, two HPLC pumps were used. Using a rotary valve, one HPLC pump selected deprotection stock solution and DMF. The pumps were activated for 13 pump strokes. Both solutions were mixed in a 1:1 ratio. Next, the rotary valves selected DMF for both HPLC pumps, and the resin was washed for an additional 40 pump strokes. The coupling-deprotection cycle was repeated for all additional monomers.

For heterodimers 5 and 6, prior to site-selective modification via the Alloc protected lysine, the protein N-termini was blocked with Boc-Gly-OH: Peptidyl resin (˜10 μmol theoretical loading) was loaded into a fritted syringe (6 mL), swollen in DMF (4 mL) for 5 minutes and then drained. Boc-Gly-OH (18 mg, 100 μmol) and HATU (54 mg, 90 μmol) were dissolved in DMF (250 μL), activated with DIEA (38 mg, 52 μL, 300 μmol), added to the peptidyl resin and incubated for 15 minutes. After this time, the resin was drained, washed with DMF (3×5 mL) and used for the next step.

The peptidyl resin (˜10 μmol theoretical loading) was washed with dichloromethane (3×5 mL) and then treated with Pd(PPh3)4(11.0 mg, 10 μmol, 1 equiv) in dichloromethane/piperidine (8:2, 1 mL) for 30 minutes at room temperature under exclusion of light. The resin was then drained and washed with dichloromethane (3×5 mL).

Mach3 and TAMRA Conjugation

Starting from homodimer 3 or 4, N-termini Boc-protection and Alloc deprotection of the C-terminal lysine was performed according to the protocols above. Mach3 (SEQ ID NO: 4) was installed via AFPS from the resulting free amine. For TAMRA installation, the peptidyl resin (˜10 μmol theoretical loading) was loaded into a fritted syringe (6 mL), swollen in DMF (4 mL) for 5 minutes and then drained. 5-Carboxytetramethylrhodamine (5-TAMRA, 22 mg, 50 μmol, 5 equivalents) and HATU (17 mg, 45 μmol, 4.5 equivalents) were dissolved in DMF (500 μL), activated with DIEA (19 mg, 26 μL, 150 μmol), added to the peptidyl resin and incubated for 30 minutes under exclusion of light. After this time, the resin was drained, washed with DMF (3×5 mL), and stored until cleavage.

Cleavage Protocol

After synthesis, the peptidyl resin was washed with dichloromethane (3×5 mL) and dried. Approximately 8 mL of cleavage solution (82.5% TFA, 5% water, 5% phenol, 5% thioanisole, 2.5% EDT) was added to the peptidyl resin inside the fritted syringe. The cleavage was kept at room temperature for 4 h, with occasional shaking. After this time, the cleavage mixture was transferred to a falcon tube (through the syringe frit, keeping the resin in the syringe), and the resin washed with an additional 2 mL of cleavage solution. Ice cold diethyl ether (45 mL) was added to the cleavage mixture and the precipitate was collected by centrifugation and triturated twice more with cold diethyl ether (45 mL). The supernatant was discarded. Residual ether was allowed to evaporate, and the peptide was dissolved in 50% acetonitrile in water with 0.1% TFA (long peptides were dissolved 70% acetonitrile in water with 0.1% TFA). The peptide solution was filtrated with a Nylon 0.22 um syringe filter and frozen and then lyophilized until dry.

Characterization of Lysine-Linked Covalent Protein Dimers

Biophysical characterization confirmed the folding and DNA-binding activity of the four covalent protein dimers 3, 4, 5, and 6. The dimers were first analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). All dimer constructs had bands at the expected height of ˜20 kDa, and the monomers MYC, MAX, and Omomyc (synthesized by AFPS) were observed at ˜10 kDa. The refolding of the protein dimers did not require special procedures. The lyophilized dimers were dissolved in folding buffer (MES 10 mM, KCl 150 mM, MgCl21 mM, TCEP 1 mM, glycerol 10%, pH=6.5) and all four dimers displayed defined α-helical signatures, as determined by circular dichroism (CD). Next an electrophoretic mobility shift assay (EMSA) was performed to determine the dimers' DNA binding activity. At a DNA concentration of 1 μM and protein concentration of 2 μM, all dimers formed complexes with the E-Box DNA, as observed by the shift retardation on the gel. Monomeric MYC, tested as a negative control (4 μM), did not bind to E-box DNA.

Covalently linked dimers have stabilized structures in aqueous buffer compared to their non-covalent analogs. CD signals at 221 nm between 4 and 89° C. were recorded to determine melting temperatures (Tm). Using this method, the non-covalent MAX/MAX and Omomyc/Omomyc dimers were compared to the four synthetic covalent protein dimers (3, 4, 5, and 6). Protein melting temperature measurements were also performed in the presence of equimolar E-Box DNA. Overall the DNA stabilized the protein complexes' structures. The covalent linkage showed a significant stabilizing effect on the MAX dimers: The Tm of the non-covalent MAX structure was determined to be 29° C. while the Tm of the covalent dimer was 38° C. Omomyc complexes, overall, displayed higher structural stability than the other dimers tested. A significant Tm difference was not observed for non-covalent Omomyc compared to covalent Omomyc-Omomyc (4). This observation might be explained by the greater stability of the Omomyc leucine zipper. The most stable complex of all structures tested was the covalent Omomyc-Omomyc dimer (4) in the presence of DNA, with a Tm of 67° C. Finally, the proteolytic stability of dimer 4 was tested. After 1 h incubation in human serum (5% in PBS) at 37° C. 91% of intact protein dimer was found.

SDS-PAGE analysis was performed using Bolt™ 4-12% Bis-Tris Plus Gels (10-wells) at 165 V for 36 min utilizing pre-stained Invitrogen SeeBlue™ Plus2 molecular weight standard. Bolt™ LDS Sample Buffer (4×) was added to each protein sample (1 μg) for loading on the gel. The bands were visualized by Coomassie blue staining.

Electrophoretic Mobility Shift Assay (EMSA)

The E-Box DNA probe (2 μM in binding buffer) was heated to 95° C. for 5 minutes and then let cool down to room temperature over 15 minutes for double-strand annealing. Protein dimer (4 μM in binding buffer: MES 10 mM, KCl 150 mM, MgCl21 mM, TCEP 1 mM, glycerol 10%, pH=6.5) was added to the DNA (final concentrations: 2 μM protein and 1 μM DNA) and the mixture was incubated for 1 h at room temperature. During the incubation, a 10% polyacrylamide gel was prerun (1 h, 4° C., 100 V) in 1× TBE buffer. After that time, DNA protein mixture (20 μL) was mixed with 6× DNA Loading Dye (4 μL)) and loaded on the gel, which was run at 75 V, for 90 min at 4° C. The gel was washed with water for 20 seconds and then stained with 0.02% ethidium bromide in 1xTBE buffer for 15 min at room temperature. Bands were visualized on a Biorad Gel imager.

Lyophilized samples were dissolved in folding buffer (MES 10 mM, KCl 150 mM, MgCl21 mM, TCEP 1 mM, glycerol 10%, pH=6.5) at a final protein concentration of 0.1 mg/mL. The circular dichroism (CD) spectra were obtained using an AVIV 420 circular dichroism spectrometer with a 1 mm path length quartz cuvette. 300 μL sample were used for each measurement. For full wavelength scans the CD spectra were recorded from 250 to 200 nm at 4° C. with three seconds averaging times at each wavelength. Y-axis values are reported in molar ellipticity. For melting temperature determination CD spectra were recorded at 221 nm from 4 to 89° C., with +5 degree steps and equilibration times of 60 seconds at each temperature. For measurements in of DNA/protein complexes equimolar E-Box DNA was added to the proteins in folding buffer; the mixtures were heated to 95° C. for 5 minutes, let cool down to room temperature over 15 minutes and then analyzed.

Cell Penetration of Covalent Protein Dimers

Covalent dimers 7, 8, and 9 were used to assess cell penetration via microscopy and flow cytometry. To evaluate uptake, Hela cells were treated with fluorophore-labeled dimers (7, 8, and 9) and fluorescence was measured via flow cytometry. All three analogs are taken up into cells in a dose-dependent manner after a brief (15 min) incubation (FIG.4). Addition of (4′,6-Diamidin-2-phenylindol) DAPI as a membrane-impermeable viability dye showed no staining of the gated population of TAMRA-fluorescent cells, suggesting that the constructs entered cells without compromising the membrane. These findings were confirmed by fluorescent microscopy. Treatment of Hela cells for 15 min with the covalent dimers followed by 1 h incubation in fresh media and imaging via confocal microscopy revealed intense, punctate fluorescence, in agreement with previous observations for monomeric Omomyc (FIG.5). However, treatment with 9 resulted in punctate fluorescence as well as diffuse fluorescence in the nucleus, indicating endosomal escape and nuclear localization (FIG.5). These experiments show that the dimeric transcription factors are rapidly taken up into cells, and their nuclear localization can be improved with the addition of a non-natural targeting sequence.

Cell Culture

Flow Cytometry

Hela cells were plated at 10,000 cells per well in a 96-well plate the night before the experiment. On the day of, cells were treated with the indicated concentrations of TAMRA-Omomyc, 7, 8, or 9 for 15 minutes in serum-containing culture medium, washed once with PBS, and treated with 0.25% trypsin-EDTA for 30 minutes to digest membrane-bound protein, at 37° C. and 5% CO2. Cells were then washed with PBS, incubated in PBS containing 1× DAPI for three minutes, and then resuspended in PBS containing 2% FBS. Cells were then immediately analyzed on a BD FACS LSR II using DAPI and PE channels.

Microscopy

Hela cells were plated at 10,000 cells/well in a 96-well 30 mm glass-bottom plate the night before the experiment. On the day of, cells were treated with TAMRA-Omomyc, 7, 8, or 9 (5 μM) in complete medium for 15 minutes, washed twice with fresh medium, and incubated at 37° C. and 5% CO2for 1 h before imaging. Micrographs were obtained in the W.M. Keck microscopy facility on an RPI Spinning Disk Confocal microscope on RFP setting (561 nm 100 mW OPSL excitation laser, 605/70 nm emission) and DAPI setting (405 nm 100 mW OPSL excitation laser, 450/50 nm emission).

Inhibition of Cancer Cell Proliferation and MYC-Driven Transcription Upon Omomyc-Omomyc Treatment

The covalent protein dimers inhibit the proliferation of cancer cells. MYC is known to drive cell proliferation in the majority of human cancers. The bioactivity of all compounds was tested in three cell lines with a range of MYC expression levels (see Example 3 for cell culture protocols); HeLa contains high MYC levels, A549 contains mid-level, and H441 has low MYC expression. The cells were treated for 72 hours with covalent protein dimers and the proliferation was measured with a CellTiter-Glo® (CTG) assay. Cell proliferation inhibition followed the expected trend according to MYC expression levels; the most substantial inhibition was observed in Hela cells and the weakest in H441 cells. All synthetic dimers demonstrated inhibitory activity, with 4 having the highest activity with an EC50of 4 μM. This observation is in line with the structural stability data. Moreover, 9 further decreased the EC50in each cell line (2 μM in Hela cells), indicating that the nuclear-targeting moiety assists the transcription factor in reaching its target and imparts enhanced activity.

The covalent protein dimers interfere with MYC-driven gene expression, as determined by RNA-sequencing (RNA-seq) and gene set enrichment analysis (GSEA). To evaluate whether the compounds' bioactivity is related to the suppression of MYC-driven expression, RNA-seq was performed on A549 cells treated with 4. Compared to the control cells, downregulation of 431 and upregulation of 297 genes was observed, indicating that the covalent protein dimers have an effect on gene expression (FIG.13). Among the downregulated genes, several genes involved in KRas signaling pathways were found, which are known to drive cancer development in A549 non-small lung cancer cells. This finding is in accordance with previous reports showing that MYC is a dominant effector of KRas mutation-positive lung cancer pathogenesis. GSEA of the RNA-seq data shows a negative enrichment of MYC-target gene set in the 4-treated condition, further corroborating that the covalent protein dimers interfere with MYC-driven gene expression programs (FIG.14).

Cell Proliferation Inhibition Assay

Cells were plated at 5,000 cells/well in a 96-well plate the day before the experiment. Covalent protein dimers were prepared at varying concentrations in complete media and transferred to the plate. Cells were incubated at 37° C. and 5% CO2for 72 h and cell proliferation was measured using the CellTiter-Glo assay quantified by luminescence.

In a 6 well plate, 125,000 A549 cells were plated into each well. The following day, the cells were treated with 4 (12.5 μM) in F12K media supplemented with 10% FBS and 1% pen/strep and incubated for 72 h. RNA was isolated using the Qiagen RNeasy Plus Mini Kit (74136) followed by DNAse treatment (AM1906). KAPAHyperRiboErase libraries were prepared and sequenced on a Hi-seq 2500 instrument. Reads from sequencing were aligned using HISAT2 htseq-count function. Differential gene expression analysis between treated and control cells was performed using DESEQ2 package in R on raw aligned read counts. The differentially expressed genes were ranked by their log2FC and adjusted p-value. Pre-ranked Gene Set Enrichment Analysis (GSEA) was performed using gene sets in Molecular Signatures Database (MSigBD) to identify MYC-target gene sets.

Synthesis of MAX, MYC, and Omomyc Analogs

Stepwise automated fast-flow solid-phase synthesis (as described in Example 1) enabled rapid high-fidelity synthesis of Max (10), Myc (11), and Omomyc (12) analogs (83 to 91 residues in length;FIG.15). The synthesis time for each protein amounted to ˜3.5 hours, and the three proteins were generated in one working day. A C-terminal cysteine residue was incorporated in the three analogs to allow subsequent cross-coupling reactions through palladium mediated S-arylation chemistry. In-line UV-vis detection of the Fmoc deprotection step after each coupling cycle indicated efficient incorporation of all monomers (FIG.16). High quality syntheses of the three analogs was confirmed by LC-MS analysis of the crude products, after trifluoroacetic acid (TFA) cleavage and ether precipitation (FIG.17). Analogs 10, 11, and 12 were purified via reversed-phase flash-chromatography, obtaining tens of milligrams of each of the three purified analogs in 38%, 44%, and 40% isolated yield, respectively. These results indicate that flow-based synthesis enables the generation of DNA-binding domains of Myc, Max, and Omomyc proteins.

The three synthetic analogs 10, 11, and 12 can form non-covalent dimers and bind to the target E-box DNA (5′-CCGGCTGACACGTGGTATTAAT-3′). The DNA-binding activity of 10, 11, and 12 toward the canonical E-box sequence was determined by combining the analogs in all possible binary combinations (Max+Max, Myc+Myc, Omomyc+Omomyc, Myc+Max, Omomyc+Max, and Omomyc+Myc (FIG.18). Each of the resulting six solutions was incubated individually with a 22 bp double-stranded DNA E-box sequence and the DNA-binding activity was examined by electrophoretic mobility shift assay (EMSA,FIG.19). The synthetic proteins, with the exception of Myc 10, complexed with the E-Box DNA probe as indicated by a significant upward shift. As expected, Myc 10 alone does not bind to the E-box DNA probe because it cannot homodimerize. This DNA-binding assay suggests that each monomer is able to dimerize as expected and form functional protein complexes with E-box DNA. However, it is not possible to determine which dimeric species form in a solution containing two different monomers. For instance, upon mixing Max (11) and Omomyc (12), three different dimers (Max/Max, Omomyc/Omomyc and Max/Omomyc) can form and potentially bind to DNA (FIG.18). To assess the activity of these protein complexes in a reliable manner, access to well-defined covalent protein dimers is critical.

Synthesis and Characterization of Dithiobenzene-Linked Covalent Protein Dimers

Cross-Coupling of TF Monomers Using Bifunctional Palladium OACs

Bifunctional palladium oxidative addition complexes (OACs) enabled on-demand synthesis of homo- and heterodimeric analogs of the proteins 10, 11, and 12 to generate all possible covalent dimeric combinations. The dimerization strategy is shown inFIG.20: the reaction of bifunctional Pd OAC with a protein monomer and subsequent palladium reinsertion into the aryl-iodide bond results in a protein-OAC that can then react with the cysteine of a second protein monomer, forming the final dimer. A single-flask protocol was used to form the homodimeric analogs. Each of the proteins 10, 11, and 12 was independently reacted with Pd OAC in 10% DMF, 20 mM Tris, 150 mM NaCl buffer (pH 7.5) at room temperature for 60 min (FIG.21) to obtain the protein homodimers, as confirmed by SDS-PAGE. The homodimers were then purified via RP-HPLC and characterized by LC-MS analysis, affording Myc-Myc (13), Max-Max (14), and Omomyc-Omomyc (15) in 37%, 40%, and 38% isolated yield, respectively (FIG.22).

To prepare the heterodimeric analogs, a two-step procedure was used by isolating the intermediate protein-OACs. The proteins 10, 11, and 12 were reacted with five equivalents of Pd OAC at room temperature for 60 min (FIG.20) and the resulting protein-OAC intermediates were isolated via RP-HPLC. 10-OAC, 11-OAC, and 12-OAC were obtained in 45%, 54%, and 43% isolated yield, respectively. Next, each intermediate was reacted with the desired analog (10-OAC with 12, 11-OAC with 10, and 12-OAC with 11,FIG.21). Finally, the heterodimer products were purified by RP-HPLC to provide the Myc-Max (16), Omomyc-Max (17), and Omomyc-Myc (18) analogs in 7%, 16%, and 6% isolated yield, respectively. The identity and purity of all six dimers were confirmed by LC-MS (FIG.22) and SDS-PAGE analysis (FIG.23). Next, the chemical stability of the S-aryl linkage was investigated. Protein dimer 14 (25 μM) was incubated in phosphate-buffered saline (PBS, pH 7.5) at 37° C. LC-MS analysis showed that no degradation had occurred after 24 h.

The covalent protein dimers exhibited α-helical character and displayed higher thermal stability compared to the monomeric analogs. The folding and stability of the dimeric analogs (13, 14, 15, 16, 17, and 18) was characterized via circular dichroism (CD) spectroscopy (see Example 2 for protocol). Strong double minima at 207 and 222 nm indicate an α-helical character of the dimeric analogs (FIG.24). Analysis of the melting temperature (Tm) showed the dimers formed more thermodynamically stable complexes, indicated by the increase in Tm compared to the monomeric analogs (FIG.25andFIG.26). Interestingly, the Omomyc-Max dimer 17 showed the highest Tm of 63° C., followed by Myc-Max 16 (53° C.) and Max-Max 14 (40° C.), compared to Max monomer 11, which was found to be 30° C. Omomyc-Omomyc 15, however, showed a similar Tm as the Omomyc monomer 12 at 59° C., likely due to the high propensity for homodimerization of the Omomyc protein. Overall, these results show that the S-aryl linkage can result in a structural stabilization of the dimeric protein complexes, compared to the monomeric analogs.

General Strategy for Pd-Mediated Homodimer Synthesis

To a 1.5 mL Eppendorf tube was added Protein-Cys monomer (300 μL, 10.0 mg/mL, 1.0 equiv) as a solution in 20 mM Tris, 150 mM NaCl (pH 7.5), 234 μL 20 mM Tris, 150 mM NaCl (pH 7.5), 30.5 μL DMF and Pd OAC 4 (28.5 μL, 10.0 mg/mL, 1.0 equiv) as a solution in DMF (titrated over one minute). The final reaction concentrations of the major reaction components were the following: 2 (500 μM); 4 (500 μM). The Eppendorf tube was closed, vortexed, and incubated at room temperature for 60 min. A small aliquot was taken from the reaction mixture for analysis by SDS-PAGE. Finally, the reaction was quenched by DTT (10 μl, 1 M in H2O) and kept at room temperature for 5 min, then purified by RP-HPLC.

General Strategy for Protein-OAC Synthesis

To a Falcon 15 ml Conical Centrifuge Tube was added Protein monomer (9.0 mL, 1.1 mg/ml, 1.0 equiv) as a solution in 20 mM Tris, 150 mM NaCl (pH 7.5), and Pd OAC (1.0 mL, 4.8 mg/mL, 5.0 equiv) as a solution in DMF. The final reaction concentrations of the major reaction components were the following: 10 (100 μM); Pd OAc (500 μM). The Falcon Tube was closed vortexed and incubated at room temperature for 30 min. A small aliquot was taken from the reaction mixture for analysis by LC-MS. Finally, the reaction was purified by RP-HPLC.

General Strategy for Pd-Mediated Heterodimer Synthesis

To a 5.0 mL Eppendorf tube was added protein-OAC (500 μL, 6.0 mg/mL, 1.0 equiv) as a solution in 20 mM Tris, 150 mM NaCl (pH 7.5), 260 μl 20 mM Tris, 150 mM NaCl buffer (pH 7.5), 185 μl DMF, and protein-Cys monomer (905 μl, 6.0 mg/ml, 2.0 equiv) as a solution in 20 mM Tris, 150 mM NaCl buffer (pH 7.5). The final reaction concentrations of the major reaction components were the following: protein-OAC (150 μM); protein-Cys (300 μM). The Eppendorf tube was closed, vortexed and incubated at room temperature for 60 min. A small aliquot was taken from the reaction mixture for analysis by SDS-PAGE. Finally, the reaction was quenched by DTT (10 μl, 1 M in H2O) and kept at room temperature for 5 min, then purified by RP-HPLC.

DNA-Binding Activity and Biophysical Characterization of the Protein Dimers

The S-aryl crosslinked protein dimers displayed DNA-binding activity to the E-box sequence. By EMSA, DNA association of Max-Max 14, Myc-Max 16, and Omomyc-Max 17 was observed (FIG.27). No DNA binding was detected with negative control Myc-Myc 13. Also, Omomyc-Myc 18 showed no association to DNA, suggesting that this dimers' inhibitory activity might be related to its sequestering endogenous Myc into an inactive form. Finally, the dissociation constant of Max-Max 14 to the E-box DNA probe was measured by bio-layer interferometry (BLI) (FIG.28). A KDof 50±11 nM was determined, which is in good agreement with previous reports showing low nanomolar KDvalues for E-box Max/Max complexes. Together these experiments demonstrate that the dimeric proteins form complexes with the E-box DNA with similar efficiency as the non-covalent analogs. Specifically, Max-Max 14 was identified as the closest analog to the natural Myc inhibitor Max/Max, as a potent binder for the E-box DNA.

Cell-Permeability and Anti-Proliferative Activity of Covalent Protein Dimer 14

Cell-based studies revealed that the Max-Max 14 covalent protein dimer is intrinsically cell-permeable. To study Max-Max 14 bioactivity, the cross-coupling reaction was scaled up to generate ˜10 mg pure material for cellular studies. To assess the cell permeability, a Max-Max analog labeled with a single carboxytetramethylrhodamine (TAMRA) fluorophore (TAMRA-Max-Max (19) was prepared. Next, the cellular uptake of TAMRA-Max-Max 19 at varying concentrations was determined via flow-cytometry (see Example 3 for protocols). It was found that treatment with dimer 19 produced a dose-dependent increase in fluorescence, indicating that the dimer is taken up into cells (FIG.29). Inclusion of DAPI as a viability stain did not result in a population of stained cells, indicating that the cells treated with TAMRA-Max-Max 19 did not suffer from membrane permeabilization. Further investigation of the uptake of the construct in Hela cells by confocal fluorescence microscopy demonstrated strong internalized fluorescence after a short incubation time (see Example 3 for protocols) (FIG.30). These results establish that the synthetic dimer does not require further engineering to be directly delivered into the cell.

In addition to entering cells, Max-Max 14 also inhibits the proliferation of Myc-dependent cancer cell lines. In some cancer cell lines, such as HeLa, high levels of Myc drive robust cell proliferation. Covalent dimer 14 was tested in in Hela cells, which contain high levels of Myc, and cell proliferation was measured after 72 h. Covalent dimer 14 was found to inhibit HeLa cell proliferation in a dose-dependent manner with an EC50of 6 μM. The EC50of Max-Max 14 is in line with recent studies reporting small molecules for stabilizing endogenous Max dimer in cancer cell lines. Remarkably, in addition to its cell permeability, Max-Max 14 has comparable activity to small molecule-based inhibitors for Myc. Max-Max 14 was also found to inhibit the proliferation of lung adenocarcinoma cells A549 and H441 with EC50of 19 μM for both. Both of these lung cancer cell lines are known to have lower Myc levels compared to HeLa, which might explain the lower antiproliferative effect of Max-Max 14 in these cells assuming equivalent cell penetration. Taken together, these experiments suggest that Max-Max 14 enters the cells and inhibits cancer cell proliferation potentially by occupying the E-box site and blocking Myc-dependent gene transcription.

RNA-Sequencing and Gene Set Enrichment Analysis for Cancer Cells Treated with Max-Max

RNA-sequencing analysis revealed Max-Max 14 selectively downregulates Myc-target genes in cancer cells. Myc is known to drive cell proliferation by triggering the expression of pro-proliferative genes through binding to the E-box DNA sequences. To assess the antiproliferative activity of Max-Max 14 through regulating Myc-driven genes, lung adenocarcinoma cells A549 were treated with Max-Max 14 for 72 h, and the RNA was extracted for RNA-sequencing analysis (see Example 4 for protocol). It was found that 14 directly interferes with gene transcription by downregulating 160 genes and upregulating 70 genes (FIG.32). The identified down- and up-regulated genes are in agreement with previous reports of Myc inhibition. Remarkably, Max-Max 14 was found to downregulate the expression of several genes involved in KRas signaling pathways that often progresses cancer. The selectivity of 14 toward Myc-related genes was further confirmed by gene set enrichment analysis (GSEA) of the RNA-sequencing data with several Myc target gene sets. Taken together, these results confirm that synthetic complex 14 is capable of downregulating Myc-driven gene signatures.

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51. Amati, B. et al. Transcriptional activation by the human c-Myc oncoprotein in yeast requires interaction with Max.Nature359, 423-426 (1992).