Modified proteins

Abstract

A protein is disclosed herein. The protein comprises: (i) a polypeptide having an alpha helical DNA binding domain, wherein the polypeptide has 50 or more amino acid residues, and wherein the alpha helical DNA binding domain comprises a first amino acid and a second amino acid, wherein the second amino acid is non-adjacent to the first amino acid; and (ii) a peptide staple covalently linking a side chain of the first amino acid to a side chain of the second amino acid. Also described is a pharmaceutical composition comprising the protein, as well as methods and uses of the protein dimer. Also described is a method of preparing the protein.

Description

MODIFIED PROTEINSFIELD OF THE INVENTION

[0001] The invention relates to a protein. The invention also relates to a pharmaceutical composition comprising the protein, as well as methods and uses of the protein, particularly in the treatment of cancer. The invention also provides a method of preparing the protein.BACKGROUND

[0002] Transcription factors (TFs) orchestrate the delicate equilibrium between gene activation and repression and are crucial for cellular function.1TFs mainly act via protein-protein and protein-nucleic acid interaction and display extensive intrinsically disordered regions used to recruit components of the transcriptional machinery.2These traits make TFs, despite their significance in health and disease, challenging drug targets,3highlighting the need for innovative approaches in drug discovery and design.

[0003] MYC is a prime example of an undruggable TF. MYC is involved in the pathomechanisms of over 50% of all human cancers.4-6To exert its activity MYC heterodimerizes with its partner MAX and as a complex they bind to enhancer box (E-Box) DNA.7MYC’s intrinsically disordered transactivation domain (TAD) then recruits the transcriptional machinery and activates gene programs related to cell growth, proliferation and survival.8MAX can also homodimerize and occupy the same E-Box sequence as a non-productive inhibitory complex.9-12Under healthy conditions MAX / MAX homodimers and MYC / MAX heterodimers are well balanced, while MYC overexpression is commonly involved in cancer. The MYC / MAX, MAX / MAX belong to the bHLH- Lz protein family, where the Leucine zipper facilitates dimerization, and the basic helix mediates DNA binding. Dimeric configurations are requisite for DNA interaction, whereas monomers lack such affinity. Situated between the basic helix and Leucine zipper, the loop helix domain stabilizes tertiary and quaternary structures, aligning the basic helices appropriately with the major grooves of their target E-Box DNA. The basic helix loop helix leucine zipper motif (bHLH-Lz) of MYC and its intrinsically disordered TAD do not offer any obvious binding sites for therapeutic compounds.

[0004] Only recently, a few small molecules with innovative modes of action have shown potential in initial investigations. KI-MS2-008, developed in the Koehler lab, acts as a stabilizer for the MAX / MAX complex, reinforcing this endogenous inhibitor.13EN4, discovered by the Nomura lab, is a covalent inhibitor binding to a cysteine in the intrinsically disordered domain of MYC and leading to MYC destabilization and degradation.14In addition to these approaches, a MYC-targeting bicyclic peptide was recently discovered from a combinatorial library selection.15

[0005] An intriguing strategy to target MYC is based on the use of proteins that mimic the inhibitory activity of MAX and occupy E-Box DNA.16-22However, while in general, protein based modalities hold immense potential in targeting ‘undruggable’ interactions, their usage is usually limited to extracellular targets, because they cannot cross cell membranes.23,24Indeed, also the protein-based MYC inhibitors, of which Omomyc is the most studied representant, have been mainly explored as research tools and utilized via ectopic expression in cells or xenografts.16 18However, recently, the Soucek group discovered that purified Omomyc has some intrinsic cell penetrating activity, propelling it from tool compound to a viable therapeutic modality.25An Omomyc variant (Omo-103) is currently being investigated in human clinical trials for the treatment of various forms of tumors.26

[0006] The intrinsic cell penetration of Omomyc makes it a promising therapeutic modality, but at the same time, cell penetration remains its biggest limitation. Based on the microscopy analysis of both fluorescently labeled monomeric Omomyc25and dimeric Omomyc variants21,22only very limited compound reaches the cell nucleus, while most of the protein is sequestered in endosomes. Moreover, Wang et al. showed that Omomyc exhibits negligible cellular uptake unless conjugated to a cell-penetrating modality.27These observations correlate with the fact that while Omomyc has a low nanomolar binding affinity for E-Box DNA (~20 nM), the reported cellular activities are usually 100-1000-fold weaker.

[0007] The Moellering group recently showed that miniaturized proteomimetics derived from the MAX bHLH domain can tightly bind to E-Box (3.5 nM) and show improved cell penetration compared to full length MAX.28However, these compounds required > 1000-fold higher concentrations (20 μM) in order to enable significant effects in cellular assays (e.g. reporter gene). Missing a stable dimerization domain, these compounds might not be able to properly dimerize under physiological conditions inside cells.

[0008] There is thus a need for the development of an improved synthetic miniprotein that has high affinity for E-Box DNA and can inhibit MYC driven transcription with great potency.SUMMARY OF THE INVENTION

[0009] The invention provides a protein. The protein comprises:(i) a polypeptide having an alpha helical DNA binding domain, wherein the polypeptide has 50 or more amino acid residues, and wherein the alpha helical DNA binding domain comprises a first amino acid and a second amino acid, wherein the second amino acid is non-adjacent to the first amino acid; and(ii) a peptide staple covalently linking a side chain of the first amino acid to a side chain of the second amino acid.

[0010] The protein is for inhibiting DNA transcription, preferably MYC driven transcription.

[0011] The protein shows improved cell activity, as evidenced by a significant improvement in bioactivity in cellular assays, particularly when compared to an analogous protein that does not include the chemical modifications. The protein of the invention may have improved binding affinity to target DNA. It includes one or more chemical modifications that can have a dramatic effect on the intracellular activity of protein therapeutics. Certain chemical modifications of the protein may improve cell penetration.

[0012] The invention further provides a pharmaceutical composition. The pharmaceutical composition comprises a protein in accordance with the invention and a pharmaceutically acceptable excipient.

[0013] A further aspect of the invention relates to the protein or the pharmaceutical composition for use in a method of therapy and / or as a medicament.

[0014] Another aspect of the invention relates to the protein or the pharmaceutical composition for use in the treatment or prevention of a proliferative disorder.

[0015] The invention further a provides a method for the treatment or prevention of a proliferative disorder. The method comprises administering to a subject a protein or a pharmaceutical composition in accordance with the invention.

[0016] Also provided is a method of preparing the protein of the invention. The method comprises:(a) recombinantly expressing the polypeptide; and(b) modifying the polypeptide to include the peptide staple.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The invention is further described hereinafter with reference to the accompanying drawings.

[0018] Figure 1 shows that the present invention, ReCH EM binant proteins, can inhibit MYC driven oncogenic gene programs, a) Schematic overview of MYC function and inhibitor activity, b) ReCH EM binant workflow including rational design - site directed mutagenesis - bacterial expression and purification - synthetic functionalization and validation.

[0019] Figure 2 shows the sequences of Omomyc variants and stapling sites.

[0020] Figure 3 shows LCMS traces and DNA binding Gels of Omomyc and reCHEMbinant variants.

[0021] Figure 4 shows a histogram with the results from a MYC reporter gene assay (HEK293 cells). EN4 is a small molecule MYC inhibitor used as positive control.

[0022] Figure 5 shows the sequences of Omomyc variants and stapling sites.

[0023] Figure 6 shows LCMS traces of purified, expressed proteins and final stapled proteins.

[0024] Figure 7 shows the results of electrophoretic mobility shift assays (EMSAs), which the show the binding of the proteins to E-Box DNA.

[0025] Figure 8 shows a histogram with results demonstrating that the proteins impair cell proliferation of HeLa cells.DETAILED DESCRIPTIONDefinitions

[0026] The term “MYC” as used, herein refers to a family of transcription factors which includes c-Myc, N-Myc and L-Myc. Myc protein activates expression of many genes through binding on consensus sequence CACGTG (Enhancer Box sequences or E-boxes and recruiting histone acetyl-transferases, or HATs). However, MYC can also act as a transcriptional repressor. By binding the Miz-1 transcription factor and displacing p300 co-activator, it inhibits expression of Mix-1 target genes. MYC also has a direct role in the control of DNA replication.

[0027] The term “subject” as used herein refers to a mammal, particularly a human.

[0028] The term “therapeutically effective amount” as used herein refers to an amount of the protein or the pharmaceutical composition comprising the protein that (a) inhibits, or provides a clinical improvement in, a particular disease, condition or disorder; (b) attenuates, ameliorates or eliminates one or more symptoms of a particular disease, condition or disorder; (c) or delays the onset of one or more symptoms of a particular disease, condition or disorder described herein. It should be understood that the terms “therapeutic” and “therapeutically effective” encompass any one of the aforementioned effects (a)-(c), either alone or in combination with any of the others (a)-(c). It should be understood that in, for example, a human or other mammal, a therapeutically effective amount can be determined experimentally in a laboratory or clinical setting, or a therapeutically effective amount may be the amount required by the guidelines of the United States Food and Drug Administration (FDA) or equivalent foreign regulatory body, for the particular disease and subject being treated. It should be appreciated that determination of proper dosage forms, dosage amounts, and routes of administration is within the level of ordinary skill in the pharmaceutical and medical arts.

[0029] The terms “treatment” or “treating” as used herein refer to curative uses and results as well as uses and results that diminish or reduce the severity of a particular condition,characteristic, symptom, disorder, or disease described herein. For example, treatment can include diminishment of several symptoms of a condition or disorder or complete eradication of said condition or disorder.

[0030] The terms “prevention” or “preventing” as used herein refers to prophylactic uses and results where the administration of the protein diminishes the likelihood or seriousness of a condition, symptom, or disease state, and / or delays the onset of a condition, symptom, or disease state for a period of time.

[0031] The various hydrocarbon-containing moieties described herein may be described using a prefix designating the minimum and maximum number of carbon atoms in the moiety, e.g “Ca- Cb”. For example, (Ca-Cb)alkyl indicates an alkyl moiety having the integer “a” to the integer “b” number of carbon atoms, inclusive.

[0032] The terms “alkylene” and “alkylene group” as used herein, by themselves or in conjunction with another term or terms, refers to a branched or unbranched saturated hydrocarbon chain. Representative examples include, but are not limited to, methylene (-CH2-), ethylene (e.g. -CH(CH3)- or -CH2CH2-), propylene (e.g. -CH(CH3)CH2-, -CH(CH2CH3)-, - C(CH3)3-, or -CH2CH2CH2-).

[0033] The term “alkenylene” and “alkylene group” as used herein, by themselves or in conjunction with another term or terms, refers to a branched or unbranched hydrocarbon chain containing a double bond. Representative examples include, but are not limited to, -CH=CH- or -CH2CH=CH2-.

[0034] The terms “arylene” and “arylene group” as used herein, by themselves or in conjunction with another term or terms, refer to a phenylene (-C6H4-), bis-phenylene or a 7 to 15 membered bicyclic or tricyclic hydrocarbon ring systems, including bridged, spiro, and / or fused ring systems, in which at least one of the rings is aromatic. Arylene groups can be substituted or unsubstituted. In some embodiments, an arylene group may contain 6 (i.e. , phenylene) ring atoms or be a ring system containing 9 to 15 atoms; such as 9 to 11 ring atoms; or 9 or 10 ring atoms. Arylene groups can be substituted or unsubstituted.

[0035] The terms “halo” and “halogen” as used herein include fluorine, chlorine, bromine and iodine atoms and substituents. It is preferred that the “halo” group or substituent is fluorine or chlorine, more preferably fluorine.Protein

[0036] The protein of the invention is for binding to DNA, preferably for binding to E-box DNA. Additionally or alternatively, the protein may inhibit MYC driven transcription.

[0037] The protein comprises a polypeptide having an alpha helical DNA binding domain. The polypeptide has 50 or more amino acid residues, preferably 55 or more amino acid residues, more preferably 60 or more amino acid residues.

[0038] Longer polypeptide chains, such as polypeptides including at least a loop domain and optionally also a helix domain, may show a stronger binding affinity to DNA. However, longer polypeptides generally show poor cell penetration and are typically more difficult and costly to prepare.

[0039] In one example, the polypeptide has no more than 110 amino acid residues, preferably no more than 100 amino acid residues, such as no more than 90 amino acid residues, more preferably no more than 80 amino acid residues.

[0040] In general, the polypeptide is a recombinant polypeptide.

[0041] The alpha helical DNA binding domain has at least two non-adjacent amino acids, each with a side chain that is covalently linked by a peptide staple. Thus, the alpha helical DNA binding domain comprises a first amino acid and a second amino acid, where the second amino acid is non-adjacent to the first amino acid. A peptide staple covalently links a side chain of the first amino acid to a side chain of the second amino acid.

[0042] The presence of the peptide staple in the alpha helical DNA binding domain of the polypeptide can improve binding of the peptide to DNA.

[0043] Typically, the protein is a basic helix-loop-helix leucine zipper (bHLH-Zip) transcription factor. It is preferred that the protein is a MYC bHLH-Zip transcription factor, a MAX bHLH-Zip transcription factor, an Omomyc or an Omomyc derived bHLH-Zip transcription factor or an E- Box binding bHLH-Zip transcription factor. More preferably, the protein is a MYC basic helix-loop- helix leucine zipper (bHLH-Zip) transcription factor,

[0044] The first amino acid is located at position i of the alpha helical DNA binding domain. The second amino acid is preferably located at either (a) position i + 4 of the alpha helical DNA binding domain or (b) position i + 7 of the alpha helical DNA binding domain. More preferably, the second amino acid is located at position i + 7 of the alpha helical DNA binding domain. Peptide staples are known to link the side chain of a first amino acid at position i to the side chain of a second amino acid at either position i + 4 or position i + 7 within the same peptide chain.

[0045] In principle, the peptide staple may be formed in a variety of ways.

[0046] For example, the peptide staple may be obtained or is obtainable by a reaction selected from a ring closing metathesis reaction, a copper catalyzed azide alkyne cycloaddition reaction, a lactamization reaction, a cysteine-xylene reaction, cysteine-perfluorobenzene reaction, a thiolyne or thiol-ene Click chemistry reaction, a selenocysteine reaction, a tryptophan condensationreaction, a C-H activation reaction and a 1 ,3-dipolar cycloaddition reaction. These reactions have previously been used to form peptide staples.

[0047] It is preferred that the peptide staple may be obtained or is obtainable by a reaction selected from a ring closing metathesis reaction, a copper catalyzed azide alkyne cycloaddition reaction, a lactamization reaction, a cysteine-xylene reaction, cysteine-perfluorobenzene reaction, a thiol-yne or thiol-ene Click chemistry reaction and, a selenocysteine reaction. More preferably, the peptide staple may be obtained or is obtainable by a cysteine-xylene reaction or a cysteine-perfluorobenzene reaction, even more preferably a cysteine-xylene reaction.

[0048] In a first example, the peptide staple may comprise an alkene group, such as when the peptide staple is obtained or is obtainable from a ring closing metathesis reaction. Typically, the ring closing metathesis is performed between (i) a terminal alkene in the side chain of the first amino acid and (ii) a terminal alkene in the side chain of the second amino acid. A First Generation Grubbs Catalyst, a Second Generation Grubbs Catalyst or a Schrock Catalyst may be used to perform the ring closing metathesis reaction. In this embodiment, the first amino acid and the second amino acid

[0049] In a second example, the peptide staple comprises a triazole group, preferably a 1 ,4- disubstituted 1 ,2,3-triazole ring, such as when the peptide staple is obtained or is obtainable from a copper catalyzed azide alkyne cycloaddition reaction (also known as Huisgen Cycloaddition or Click-Chemistry). The copper catalyzed azide alkyne cycloaddition reaction is performed between (i) an azide group on the side chain of one of the first amino acid and the second amino acid, and (ii) a terminal alkyne group in the side chain of the other one of the first amino acid and the second amino acid (e.g. if the azide group on the side chain of the first amino acid, then the terminal alkyne group is in the side chain of the second amino acid group or vice-versa).

[0050] In a third example, the peptide staple comprises an amide group. As the amide group will be part of the peptide staple that forms a ring with the peptide chain, the resulting moiety is referred to as a lactam. The peptide staple comprising an amide group may be obtained or is obtainable from a lactamization reaction.

[0051] The lactamization reaction is a condensation reaction performed between (i) a primary or secondary amine group on a side chain of one of the first amino acid and the second amino acid, and (ii) a carboxylic acid or a carboxylic ester on a side chain of the other one of the first amino acid and the second amino acid, optionally in the presence of a condensing agent. The condensing agent may, for example, be a carbodiimide (e.g. DCC and DIG), a phosphonium salt (e.g. BOP or PyBOP), a uronium salt (e.g. HBTU) or a thiouronium salt (e.g. TOTT).

[0052] When the peptide staple is formed by a lactamization reaction, it is preferred that the first amino acid and the second amino acid are natural amino acids. One of the first amino acid and the second amino acid may be aspartic acid or glutamic acid and the other one of the first amino acid and the second amino acid may be lysine.

[0053] In a fourth example, the peptide staple comprises a methylene-(C6-C18-arylene)- methylene group, preferably a methylene-(C6-C12-arylene)-methylene group.

[0054] The peptide staple may comprise a methylene-(C12-arylene)-methylene group, such as a staple of formula (1):wherein each asterisk (*) in formula (1) above indicates a carbon atom that is covalently linked to a sulfur atom in a cysteine residue of one of the first amino acid or the second amino acid.

[0055] When the peptide staple comprises a staple of formula (1), then the first amino acid is located at position i of the alpha helical DNA binding domain and the second amino acid is located at position i + 7 of the alpha helical DNA binding domain.

[0056] The peptide staple may comprise a methylene-(C6-arylene)-methylene group, such as a staple of formula (2):wherein each asterisk (*) in formula (2) above indicates a carbon atom that is covalently linked to a sulfur atom in a cysteine residue of one of the first amino acid or the second amino acid.

[0057] When the peptide staple comprises a staple of formula (2), then the first amino acid is located at position i of the DNA binding domain and the second amino acid is located at position i + 4 of the DNA binding domain.

[0058] It is preferred that the peptide staple comprises a staple of formula (1).

[0059] The peptide staple comprising a staple of formula (1) or formula (2) may be obtained or is obtainable from a cysteine-xylene reaction.

[0060] The cysteine-xylene reaction is a double nucleophilic substitution reaction between (i) a thiol group, preferably a terminal thiol group, on the side chain of each of the first amino acid and the second amino acid, and (ii) a bis(halomethyl)-C6-C18-arylene compound, such as 1 ,4-bis(halomethyl)benzene or 4, 4’-bis(halomethyl)-1 ,1 ’-biphenyl. It is preferred that the bis(halomethyl)-C6-C18-arylene compound is a bis(bromomethyl)-C6-C18-arylene compound, such as 1 ,4-bis(bromomethyl)-benzene or 4,4’-bis(bromomethyl)-1 , 1 ’-biphenyl. Even though the name of the reaction refers to xylene, it is to be understood that the reaction is not limited to peptide staples derived from only xylene, as can be seen by the reference to the bis(halomethyl)-C6-C18- arylene compound.

[0061] When the peptide staple comprises a staple of formula (1) or formula (2), it is preferred that the first amino acid and the second amino acid are cysteine residues.

[0062] In a fifth example, the peptide staple comprises a tetrafluorobenzene group, such as a staple of formula (3):wherein each asterisk (*) in formula (3) indicates a carbon atom that is covalently linked to a sulfur atom in a cysteine residue of one of the first amino acid or the second amino acid.

[0063] When the peptide staple comprises a staple of formula (3), then the first amino acid is located at position i of the DNA binding domain and the second amino acid is located at position i + 4 of the DNA binding domain.

[0064] The peptide staple comprising a staple of formula (3) may be obtained or is obtainable from a cysteine-perfluorobenzene reaction.

[0065] The cysteine-perfluorobenzene reaction is a double aromatic nucleophilic substitution reaction between (i) a thiol group, preferably a terminal thiol group, on the side chain of each of the first amino acid and the second amino acid, and (ii) hexafluorobenzene.

[0066] When the peptide staple comprises a staple of formula (3), it is preferred that the first amino acid and the second amino acid are cysteine residues.

[0067] In a sixth example, the peptide staple comprises a C2-alkenylene group (e.g. formed from a thiol-yne Click chemistry reaction) or a C2-alkylene group (e.g. formed from a thiol-ene Click chemistry reaction).

[0068] The thiol-yne Click chemistry reaction is performed between (i) a thiol group on the side chain of one of the first amino acid and the second amino acid, and (ii) a terminal alkyne group inthe side chain of the other of the first amino acid and the second amino acid, in the presence of a radical initiator or with UV irradiation in the presence of a photoinitiator.

[0069] The thiol-ene Click chemistry reaction is performed between (i) a thiol group on the side chain of one of the first amino acid and the second amino acid, and (ii) a terminal alkene group in the side chain of the other of the first amino acid and the second amino acid, in the presence of a radical initiator or with UV irradiation in the presence of a photoinitiator.

[0070] It is preferred that one of the first amino acid and the second amino acid is cysteine.

[0071] In a seventh example, the peptide staple comprises an C2-C4-alkylene group. The peptide staple comprising a staple of formula (3) may be obtained or is obtainable from a selenocysteine reaction.

[0072] The selenocysteine reaction is performed between (i) a selenol group, preferably a terminal selenol group, on the side chain of each of the first amino acid and the second amino acid, and (ii) a dihalo-C2-C4-alkylene compound, such as 1 ,3-dihalopropylene. It is preferred that the dihalo-C2-C4-alkylene compound is a dibromo-C2-C4-alkylene compound.

[0073] The first amino acid and the second amino acid are preferably selenocysteine.

[0074] In general, it is preferred that the polypeptide having an alpha helical DNA binding domain comprises only natural amino acid residues. More preferably, the protein of the invention is a natural recombinant protein. The natural recombinant protein comprises only natural amino acid residues.

[0075] When the polypeptide comprises only natural amino acid residues or the protein is a natural recombinant protein, then the peptide staple may be obtained or is obtainable by a cysteine-xylene reaction or cysteine-perfluorobenzene reaction, preferably a cysteine-xylene reaction.

[0076] Typically, the polypeptide having an alpha helical DNA binding domain has a degree of identity of at least 70% with respect to SEQ ID NO: 1 . The degree of identity may be at least 80% with respect to SEQ ID NO: 1 , preferably at least 86% with respect to SEQ ID NO: 1 , such as at least 90% with respect to SEQ ID NO: 1 , more preferably at least 93% with respect to SEQ ID NO: 1.

[0077] Typically, the first amino acid is derived from an amino acid having a side chain for forming a peptide staple and is a replacement of one amino acid in SEQ ID NO: 1. Additionally, or alternatively, the second amino acid is derived from an amino acid having a side chain for forming a peptide staple and is a replacement of one amino acid in SEQ ID NO: 1 .

[0078] When the peptide staple comprises a staple of any one of formulas (1) to (3), then the amino acid having a side chain for forming a peptide staple for both the first amino acid and the second amino acid is cysteine.

[0079] Typically, the first amino acid is at an amino acid position of from 1 to 15 of SEQ ID NO: 1 , preferably at amino acid position 1 , 5 or 12 of SEQ ID NO: 1. The position of the second amino acid will depend on the peptide staple that is used.

[0080] In one example, the polypeptide having an alpha helical DNA binding domain has a degree of identity of at least 70% with respect to either SEQ I D NO: 6 or SEQ I D NO: 7, preferably a degree of identity may be at least 80% with respect to either SEQ ID NO: 6 or SEQ ID NO: 7, preferably at least 86% with respect to SEQ ID NO: 6 or SEQ ID NO: 7, such as at least 90% with respect to SEQ ID NO: 6 or SEQ ID NO: 7, more preferably at least 93% with respect to SEQ ID NO: 6 or SEQ ID NO: 7. Even more preferably, the polypeptide having an alpha helical DNA binding domain comprises, or consists of, a sequence represented by SEQ ID NO: 6 or SEQ ID NO: 7. In this example, the peptide staple preferably comprises a staple of formula (1).

[0081] In another example, the polypeptide having an alpha helical DNA binding domain has a degree of identity of at least 70% with respect to either SEQ I D NO: 8 or SEQ I D NO: 9, preferably a degree of identity may be at least 80% with respect to either SEQ ID NO: 8 or SEQ ID NO: 9, preferably at least 86% with respect to SEQ ID NO: 8 or SEQ ID NO: 9, such as at least 90% with respect to SEQ ID NO: 8 or SEQ ID NO: 9, more preferably at least 93% with respect to SEQ ID NO: 8 or SEQ ID NO: 9. Even more preferably, the polypeptide having an alpha helical DNA binding domain comprises, or consists of, a sequence represented by SEQ ID NO: 8 or SEQ ID NO: 9. In this example, the peptide staple preferably comprises a staple of formula (2) or formula (3).

[0082] In general, the polypeptide may include loop domain. Typically, the loop domain is covalently linked to the C-terminus of the alpha helical DNA binding domain.

[0083] When the polypeptide includes a loop domain, then generally the polypeptide may further comprise a helix domain. The helix domain is covalently linked to the C-terminus of the loop domain.

[0084] The presence of the loop domain by itself or in combination with the helix domain may aid or improve the binding of the protein to DNA.

[0085] The loop domain and the helix domain may have a degree of identity of at least 70% with respect to SEQ ID NO: 2, preferably a degree of identity of at least 80% with respect to SEQ ID NO: 2, such as a degree of identity of at least 83% with respect to SEQ ID NO: 2, more preferablyat least 92% with respect to SEQ ID NO: 2. Even more preferably, the loop domain and the helix domain comprise, or consist of, a sequence represented by SEQ ID NO: 8 or SEQ ID NO: 9.

[0086] Typically, the loop domain (e.g. of the combined loop and helix domain) is covalently linked to the C-terminus of the alpha helical DNA binding domain.

[0087] The polypeptide may further include a coiled domain. The coiled domain is preferably covalently linked to the C-terminus of the helix domain.

[0088] In one example, the coiled domain has a degree of identity of at least 50% with respect to SEQ ID NO: 10, preferably a degree of identity may be at least 61 % with respect to either SEQ ID NO: 10, preferably at least 75% with respect to SEQ ID NO: 10, such as at least 86% with respect to SEQ ID NO: 10, more preferably at least 92% with respect to SEQ ID NO: 10. Even more preferably, the coiled domain comprises, or consists of, a sequence represented by SEQ ID NO: 10.

[0089] In another example, the coiled domain has a degree of identity of at least 50% with respect to SEQ ID NO: 11 , preferably a degree of identity may be at least 61% with respect to either SEQ ID NO: 11 , preferably at least 75% with respect to SEQ ID NO: 11 , such as at least 86% with respect to SEQ ID NO: 11 , more preferably at least 92% with respect to SEQ ID NO:11. Even more preferably, the coiled domain comprises, or consists of, a sequence represented by SEQ ID NO: 11.

[0090] An amino acid in the sequence represented by SEQ ID NO: 10 or SEQ ID NO: 11 may be replaced by an arginine residue. It is preferred that a plurality of amino acids in in the sequence represented by SEQ ID NO: 10 or SEQ ID NO: 11 are replaced by a plurality of arginine residues. The replacement is a one-for-one replacement (e.g. one amino acid in SEQ ID NO: 10 or SEQ ID NO: 11 is replaced with one arginine residue). For the avoidance of doubt, each amino acid that is replaced in the sequence represented by SEQ ID NO: 10 or SEQ ID NO: 11 is not an arginine residue.

[0091] The inclusion of one or more arginine residues in the coiled domain may, surprisingly, improve cell penetration of the protein.

[0092] In another example, the coiled domain has a degree of identity of at least 50% with respect to SEQ ID NO: 12, preferably a degree of identity may be at least 61% with respect to either SEQ ID NO: 12, preferably at least 75% with respect to SEQ ID NO: 12, such as at least 86% with respect to SEQ ID NO: 12, more preferably at least 92% with respect to SEQ ID NO:12. Even more preferably, the coiled domain comprises, or consists of, a sequence represented by SEQ ID NO: 12.

[0093] In yet another example, the coiled domain has a degree of identity of at least 50% with respect to SEQ ID NO: 13, preferably a degree of identity may be at least 61% with respect to either SEQ ID NO: 13, preferably at least 75% with respect to SEQ ID NO: 13, such as at least 86% with respect to SEQ ID NO: 13, more preferably at least 92% with respect to SEQ ID NO:13. Even more preferably, the coiled domain comprises, or consists of, a sequence represented by SEQ ID NO: 13.

[0094] In a further example, the coiled domain has a degree of identity of at least 50% with respect to SEQ ID NO: 14, preferably a degree of identity may be at least 61% with respect to either SEQ ID NO: 14, preferably at least 75% with respect to SEQ ID NO: 14, such as at least 86% with respect to SEQ ID NO: 14, more preferably at least 92% with respect to SEQ ID NO:14. Even more preferably, the coiled domain comprises, or consists of, a sequence represented by SEQ ID NO: 14.

[0095] The coiled domain may be artificial, such as the polypeptide represented by SEQ ID NO: 14. An artificial coiled domain may comprise, or consist essentially of, natural amino acids.

[0096] The artificial coiled domain may comprise a polypeptide having no more than 30 amino acid residues, preferably no more than 28 amino acid residues, more preferably no more than 26 amino acid residues, particularly no more than 24 amino acid residues. A shorter artificial coiled domain is advantageous because it may have higher selectivity for homodimerization, may have lower immunogenicity and / or it may have better proteolytic stability.

[0097] The polypeptide may have a degree of identity of at least 70% with respect to either SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, preferably an identity of at least 80% with respect to either SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, more preferably an identity of at least 85% with respect to either SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, such as an identity of at least 90% with respect to either SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, more preferably at least 95.5% with respect to either SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. Even more preferably, the polypeptide comprises, or consists of, a sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0098] Each of the sequences represented by SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO:5 include an alpha helical DNA binding domain, a loop domain, a helix domain and a coiled domain.

[0099] When the polypeptide has a degree of identity of at least 70%, at least 80%, at least 85%, at least 90% or at least 95.5% with respect to SEQ ID NO: 3 or SEQ ID NO:4, then at least one arginine residue replaces an amino acid at an amino acid position of from 55 to 85 in SEQ ID NO: 3 or from 64 to 94 in SEQ ID NO: 4.

[0100] The polypeptide may have a degree of identity of at least 70% with respect to one of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23, SEQ ID NO:25, SEQ I D NO: 27, SEQ I D NO: 29 or SEQ I D NO: 31 , preferably an identity of at least 80% with respect to one of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29 or SEQ ID NO: 31 , more preferably an identity of at least 85% with respect to one of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29 or SEQ ID NO: 31 , such as an identity of at least 90% with respect to one of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29 or SEQ ID NO: 31. Even more preferably, the polypeptide is one of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29 or SEQ ID NO: 31 .

[0101] Typically, the polypeptide has a degree of identity of at least 70% with respect to one of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO:26, SEQ ID NO: 28 or SEQ ID NO: 30, preferably an identity of at least 80% with respect to one of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ I D NO: 28 or SEQ I D NO: 30, more preferably an identity of at least 85% with respect to one of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28 or SEQ ID NO: 30, such as an identity of at least 90% with respect to one of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28 or SEQ ID NO: 30. Even more preferably, the polypeptide is one of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28 or SEQ ID NO: 30.

[0102] Each of the sequences represented by SEQ ID NO: 15 to SEQ ID NO: 31 include an alpha helical DNA binding domain, a loop domain, a helix domain and a coiled domain.Pharmaceutical composition

[0103] A pharmaceutical composition of the invention may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), for topical use (for example as creams, ointments, gels, or aqueous or oily solutions or suspensions), for administration by inhalation (for example as a finely divided powder or a liquid aerosol), for administration by insufflation (for example as a finely divided powder) or for parenteral administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular, intraperitoneal or intramuscular dosing or as a suppository for rectal dosing).

[0104] The compositions of the invention may be obtained by conventional procedures using conventional pharmaceutical acceptable excipients, well known in the art, such as one or more colouring, sweetening, flavouring and / or preservative agents.

[0105] The pharmaceutically acceptable excipient may be selected from a stabilizer, a carrier and a preservative agent.

[0106] A therapeutically effective amount of the protein of the present invention for use in therapy is an amount sufficient to treat or prevent a proliferative condition referred to herein, slow its progression and / or reduce the symptoms associated with the condition.Methods and uses

[0107] The protein or a pharmaceutical composition comprising the protein is for the treatment or prevention of a proliferative disorder. The proliferative disorder is typically associated with MYC dysregulation.

[0108] It is preferred that the proliferative disorder is cancer, e.g. a cancer associated with MYC dysregulation.

[0109] The use or method of the invention typically comprises administering a therapeutically effective amount of the protein to the subject (e.g. a subject in need thereof).

[0110] The uses and methods of the invention are useful for the treatment of local and malignant tumours.

[0111] The cancer may be breast cancer; biliary tract cancer; bladder cancer; brain cancer including glioblastomas and medulloblastomas; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematological neoplasms including acute lymphocytic and myelogenous leukemia: T-cell acute lymphoblastic leukemia / lymphoma; hairy cell leukemia; chronic myelogenous leukemia, multiple myeloma; AIDS-associated leukemias and adult T-cell leukemia / lymphoma; intraepithelial neoplasms including Bowen's disease and Paget's disease; liver cancer; lung cancer; lymphomas including Hodglun's disease and lymphocytic lymphomas; neuroblastomas; oral cancer including squamous cell carcinoma; ovarian cancer including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells; pancreatic cancer: prostate cancer; rectal cancer; sarcomas including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma; slun cancer including melanoma, Merkel cell carcinoma, Kaposi's sarcoma, basal cell carcinoma, and squamous cell cancer; testicular cancer including germinal tumors such as seminoma, non-seminoma (teratomas, choriocarcinomas), stromal tumors, and germ cell tumors; thyroid cancer including thyroid adenocarcinoma and medullar carcinoma; and renal cancer including adenocarcinoma and Wilms tumor.

[0112] The protein or the pharmaceutical composition may be administered by any type of suitable route, such as by oral route, topical route, by inhalation or parenteral route.Method of preparation

[0113] The invention provides a method of preparing a protein. The protein is a protein in accordance with the invention.

[0114] The method comprises:(a) recombinantly expressing the polypeptide; and(b) modifying the polypeptide to include the peptide staple.

[0115] The protein of the invention may be recombinantly expressed using any methods known in the art. For recombinant expression, host cells such as Escherichia coli (E.coli), such as ArcticXpress DE3 RIL cells from Agilent Technologies may be employed. E. coli cells used to produce the polypeptide described in this invention may be cultured in suitable growth or cell culture media in which the promoters can be induced as described generally, e.g., in Sambrook, J., et al., in Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press (1989). Host cells may be transformed with one or more vectors encoding any polypeptide of the invention using methods known in the art. For example, host cells can be transformed by heat shock in a water bath for 20 seconds at 42 °C. Transformed host cells are then cultured under conditions that enable expression of polypeptides. For example, transformed cells may be incubated initially in approximately 0.9 ml SOC cell culture media at 37 °C, 250 rpm for 1 hour. Subsequently, the cells may be pelleted and resuspended in media for plating on an agarose plate containing relevant antibiotics to select for host cells that have successfully been transformed with vectors encoding the polypeptide of interest, the vectors of which also conferring resistance to said antibiotics. Single colonies of transformed cells can then be cultured overnight at for example, 37 °C and 200 rpm in 100 mL of growth or cell culture media such as LB media containing the relevant antibiotics. Transformed host cells can be further grown or expanded as desired, for example to achieve small scale or large scale production of the polypeptide described in the invention. For example, 25 ml of the aforementioned overnight culture can be further grown in 2L of LB broth in a 5L vessel prior to induction at 37 °C, 200 rpm until an OD of 0.8 is reached.

[0116] The induction of expression of the nucleic acid encoding the polypeptide is preferably carried out by adding an inducer to the culture. The inducer is preferably IPTG. Typically, the inducer is added after the cells are cultured until a certain optical density has been reached, e.g. an OD600 of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 or more. Preferably the OD600 is 0.8. For example, the temperature of the cell culture containing transformed host cells can be set to14 °C and protein expression induced by addition of 100 μM of IPTG. Protein expression can then be conducted overnight for 18 hours.

[0117] A variety of techniques may be employed to facilitate the preparation of intracellular proteins from E.coli. Typically, the initial steps in these techniques involve lysis or rupture of the bacterial cells, to disrupt the bacterial cell wall and allow release of the intracellular proteins into the extracellular milieu. Following this release, the desired proteins are purified from the extracts, typically by a series of chromatographic steps.

[0118] The polypeptides can then be harvested from the host cells using any means known in the art. For example, the host cells may be pelleted by centrifugation and lysed using lysis buffer e.g., 20 mM Tris-HCI pH 8, 0.5 M NaCI, 10 mM imidazole, 3 mM MgCh, 1x complete EDTA-free protease inhibitor cocktail tablet (Roche) and 0.05-0.1% DNase. Cell Debris can be removed by ultracentrifugation e.g., (35,000 rpm, 4°C, 45 min).

[0119] The polypeptides of the invention can be purified using any means known in the art. For example, the polypeptides may be tagged with a 6xHis-tag and the 6xHis-tagged protein may be purified using immobilized metal affinity chromatography (IMAC) such as a HisTrap HP His tag protein purification column (from Cytiva). The purification column may be washed with wash buffer (e.g., (20 mM Tris-HCI pH 8, 0.5 M NaCI, 10 mM imidazole) to remove unbound proteins and the polypeptide as described in the invention may be eluted e.g., using a gradient of 10 mM to 500 mM imidazole.

[0120] As used herein, culturing or cultured refer to the bulk growing of cells in a growth or cell culture medium. This includes an exponential phase, characterized by cell doubling, therefore bulk growth and a stationary phase, where the growth rate and death rate of cells is equal often due to a growth-limiting factor such as the depletion of an essential nutrient, and / or the formation of an inhibitory product such as an organic acid.

[0121] The culturing step may take place under conditions of high cell density, that is, generally at a cell density of about 15 to 150 g dry weight / litre, such as at least about 40, more preferably about 40-150, most preferably about 40 to 100. In addition, the culturing can be accomplished using any scale, even very large scales of 100,000 litres. Preferably, the scale is about 100 litres or greater, more preferably at least about 500 litres, and most preferably from about 500 litres to 100,000 litres.

[0122] The vector encoding the polypeptide described by the invention may be any vector suitable for protein expression. For example, the vector may be a pET-30a vector including any other vectors disclosed herein. E. coli cells may be transformed with the above-describedexpression vector(s) and cultured in conventional growth / cell culture media modified as appropriate for inducing the various promoters if induction is carried out.

[0123] Recombinant cells or host cells for use in accordance with the invention may be prepared by recombinant DNA techniques that are familiar to one or ordinary skill in the art (see e.g., Kotewicz, M. L., et al., Nucl. Acids Res. 16:265 (1988); Soltis, D. A., and Skalka, A. M., Proc. Natl. Acad. Sci. USA 85:3372-3376 (1988)). Such recombinant cells may be grown according to standard microbiological techniques, using culture media and incubation conditions suitable for growing active cultures of the particular species that are well-known to one of ordinary skill in the art (see, e.g., Brock, T. D., and Freeze, H., J. Bacteriol. 98(1):289-297 (1969); Oshima, T., and Imahori, K., Int. J. Syst. Bacteriol. 24(1):102-112 (1974)).

[0124] Transformation means introducing DNA into an organism so that the DNA is replicable, either as an extrachromosomal element or as chromosomal integration. Depending on the host cell used, transformation is done using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), is generally used for bacterial cells that contain substantial cell-wall barriers. Another method for transformation employs polyethylene glycol / DMSO, as described in Chung and Miller, Nucleic Acids Res., 16: 3580 (1988). Yet another method is the use of the technique termed electroporation.

[0125] As used herein “Escherichia coli" or “E.coli" refers to a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus Escherichia. E.coli host strains for recombinant protein expression are well known in the art. The polypeptide described in the invention can be used in conjunction with any E.coli host strains known in the art e.g., E. coli B (such as BL21 (DE3) or its derivatives.

[0126] The polypeptide may be modified to include a peptide staple by a reaction selected from a ring closing metathesis reaction, a copper catalyzed azide alkyne cycloaddition reaction, a lactamization reaction, a cysteine-xylene reaction, cysteine-perfluorobenzene reaction, a thiolyne or thiol-ene Click chemistry reaction and, a selenocysteine reaction. More preferably, the peptide staple may be obtained or is obtainable by a cysteine-xylene reaction or a cysteine- perfluorobenzene reaction, even more preferably a cysteine-xylene reaction.

[0127] The peptide staple, the first amino acid and the second amino acid of the polypeptide are as described herein, particularly as described above in connection with the protein.ExamplesIntroduction

[0128] Protein therapeutics are potent tools for targeting difficult drug targets, but their efficacy is limited to extracellular targets due to their inability to cross cell membranes. Here, the inventors introduce chemically modified protein therapeutics, termed reCH EM binant proteins, designed to penetrate cells and inhibit MYC-driven oncogenic gene programs in the nucleus.

[0129] MYC is a key transcription factor implicated in various cancers, but targeting it with traditional drugs has been challenging. Omomyc, a miniprotein, shows promise in inhibiting MYC function but struggles to efficiently enter cells. Here, Omomyc variants were engineered with chemical staples to enhance cell penetration. The inventor’s approach combined structure-based design, mutagenesis, expression, purification, and synthetic modification. These reCHEMbinant variants of the invention exhibit improved affinity for DNA and surpass Omomyc in cell assays, highlighting their potential for targeting intracellular drug targets effectively.Example 1Materials and MethodsGeneral

[0130] All reactions were carried out using commercially available reagents, unless noted otherwise. Reagents and solvents were purchased from Sigma-Aldrich (Merck), Fisher Scientific, or VWR chemicals.

[0131] Plasmid pET-30a with 6-his-tagged Omomyc was donated by the group of Prof. Dr. Eilers (University of Wurzburg, Germany) and sequenced before being used. Primers for site-directed DNA mutagenesis were purchased from IDT (Leuven, Belgium).LC-MS

[0132] LC-MS chromatograms and associated mass spectra were acquired using a Shimazu LCMS-2020 system (Method A) or, for high resolution mass spectrometry data, a Sciex X500b QTOF ESI-QToF mass spectrometer coupled to a Shimadzu Nexera UHPLC LC40DX3 (Method B). Mobile phases used for LC-MS analysis are solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile).

[0133] The following LCMS methods were used:Method A

[0134] Column: Kinetex® 2.6μm XB-C18 100 Å LC Column (50 x 3 mm) UV detector: 214 nM.

[0135] LC Method: 0% solvent B over 1 min, followed by a linear gradient 0% to 70% solvent B over 10 min, followed by 70% solvent B over 1 .5 min, followed by 70% to 0% solvent B over 4.5, flowrate 0.55 mL / minMethod B (high resolution)

[0136] Column: Phenomenex Synergi™ 4 μm Fusion-RP 80 Å LC Column (50 x 2 mm)

[0137] LC Method: 0% solvent B over 1 min, followed by a linear gradient 0% to 60% solvent B over 3.5 min, followed by a linear gradient 60% to 95% solvent B over 0.1 min, followed by 95% solvent B over 0.4 min, followed by a linear gradient 95% to 0% solvent B over 0.5 min, followed by 0% solvent B over 1.5 min, flowrate 0.5 mL / min.

[0138] MS parameters: General parameters: Method duration: 5 min; Total scan time: 0.276 sec; Estimated cycles: 1086; Intact protein mode: False; Decrease detector voltage: False; Large protein (>70 kDa): False; Ion Source: Source name: TurbolonSpray; Curtain gas: 35 psi; Ion source gas 1 : 60 psi; Ion source gas 2: 60 psi; Temperature: 500 °C; Experiment: Scan type: TOF MS; Polarity: Positive; Spray voltage: 5500 V; CAD gas: 7; Time bins to sum: 4; Channel 1-4: True; TOF start mass: 350 Da; TOF stop mass 1500 Da; Accumulation time: 0.25; Declustering potential: 80V; Declustering potential spread: 0 V; Collision energy: 10V; Collision energy spread: 0 V; Override Qjet RF value: FalseChemistryStapling reactionsStapling with i, i+7 staple (4,4'-Bis(bromomethvl)biphenvl)

[0139] The respective protein was dissolved at 20x the final concentration in water (2 mM) and then diluted into stapling buffer (NH4HCO3, 100 mM, pH= 8). 4,4'-Bis(bromomethyl)biphenyl was dissolved at 4x the final concentration in ACN (500 μM) and then added to the protein in stapling buffer. Final concentrations used were 100 μM (1 eq.) for the protein and 125 μM (1.25 eq.) for the staple. Final ACN content was 25%.

[0140] When the stapling reaction was completed as seen by LC-MS, precipitate formed during reaction was dissolved by adding water and ACN with 0.1% TFA content to the reaction mixture and the product was purified using a Biotage® Selekt Flash Purification System equipped with a Biotage® Sfar C18 D - Duo 10g column on a gradient of water in ACN, both containing 0.1% TFA. The product containing fractions were lyophilized yielding the proteins as TFA salts.

[0141] Yield Omomyc C91A V7C V14C stapled: 1.94 mg (67%)

[0142] Yield Omomyc C91A V14C N21C stapled: 0.85 mg (53%)Stapling with i, i+4 staple (a.a'-Dibromo-m-xylene)

[0143] The respective protein was dissolved at 20x the final concentration in water (2 mM) and then diluted into stapling buffer (NH4HCO3, 100 mM, pH= 8). TCEP was dissolved at 100x the final concentration in water (20 mM) and added to the stapling reaction. α,α'-Dibromo-m-xylene was dissolved at 4x the final concentration in ACN (500 μM) and then added to the protein instapling buffer. Final concentrations used were 100 μM (1 eq.) for the protein, 125 μM (1.25 eq.) for the staple and 200 μM (2 eq.) for TCEP. Final ACN content was 25%.

[0144] When the stapling reaction was completed as seen by LC-MS, precipitate formed during reaction was dissolved by adding water and ACN with 0.1% TFA content to the reaction mixture and the product was purified using a Biotage® Selekt Flash Purification System equipped with a Biotage® Sfär C18 D - Duo 10g column on a gradient of water in ACN, both containing 0.1 % TFA. The product containing fractions were lyophilized yielding the proteins as TFA salts.

[0145] Yield Omomyc C91A T3C V7C stapled: 0.27 mg (13%)

[0146] Yield Omomyc C91A V14C N21C stapled: 0.32 mg (16%)Site directed mutagenesis

[0147] For site directed mutagenesis the QuikChange II site-directed mutagenesis kit (Agilent Technologies) was used and primers were designed using the QuikChange® Primer Design Program provided by Agilent Technologies. Mutagenesis was performed according to the manufacturer’s protocol In brief, PCRs reactions were prepared using pET-30a with Omnomyc as template (0.5 μL, ~25 ng), forward and reverse primers (0.6 μL each, 10 μM), MQ water (18.8 μL) as well as the contents provided by the kit: NTP mix (1 μL), 10x reaction buffer (2.5 μL) and PfuUltra high fidelity DNA polymerase. In some cases, when the formation of primer dimers was seen, primer concentration was reduced and 1 μL of dmso added. PCR was performed for 22 cycles (95 °C, 30s; 55 °C, 1 minute; 68 °C, 10 minutes) followed by Dpn I restriction for 2 hours at 37 °C(addition of 0.5 μL at 10 U / μL)

[0148] Mutated plasmids were then incorporated into XL-1 blue supercompetent. Cells were thawed on ice and 8 μL of the Dpn / -treated DNA were added to 50 μL of cells. The mixture was incubated on ice for 30 minutes, followed by a 45 second heat pulse at 42 °C and incubation on ice for another 2 minutes. Subsequently, 0.5 mL SOC-medium were added and the cells shaken at 250 rpm, 37 °C for 1 hour. For selection 100 μL of the mixture were plated on one half of an LB agar plate containing gentamicin and kanamycin. The rest of the cells was spun down, the supernatant removed except for 100 μL, the cells resuspended in these 100 μL and plated on the other half of the LB agar plate. The plate was incubated at 37 °C overnight.

[0149] About 5-10 colonies were picked from selection and grown overnight in 5 mL LB media supplemented with gentamicin and kanamycin and plasmid DNA was isolated using the QIAprep Spin Miniprep kit (Qiagen, Venlo, NL) according to the manufacturer’s protocol. The isolated plasmid DNA was sequenced using a sanger sequencing service and analyzed using Benchling.Primers used for site-directed mutagenesisExpression of different Omomyc variants

[0150] Plasmids used were either obtained from site-directed mutagenesis of pET-30a with the omomyc gene sequence (for stapling variants) or genes as well as insertion of them by express cloning were ordered from GenScript and the plasmids used as delivered (for artificial coiled-coil and mutations in the coiled-coil, see annex for plasmid sequence).

[0151] Plasmids were transformed into competent ArcticXpress DE3 RIL cells by heat shock. 2 μL of 10% β mercaptoethanol were mixed with 100 μL of competent cell suspension thawed on ice and incubated for 10 minutes on ice. Next, 25 ng of plasmid DNA were added and the cells incubated for another 30 minutes on ice. The cells were then heat-shocked in a water bath for 20 seconds at 42 °C and subsequently incubated on ice for 2 minutes followed by the addition of 0.9 mL SOC media and incubation at 37 °C, 250 rpm for 1 h. Cells were then pelleted by centrifugation, 0.9 mL of the supernatant decanted and the pellet resuspended in the remaining 100 μL of media. Cells were plated for selection on LB agar with kanamycin and gentamicin and incubated at 37 °C overnight.

[0152] Single colonies were picked from the plate and cultured overnight at 37 °C and 200 rpm in 100 mL of LB media containing kanamycin and gentamycin. Next, 3x2 L of LB media containing kanamycin and gentamycin in 5 L Erlenmeyer flasks were inoculated with 25 mL of preculture and grown at 37 °C, 200 rpm until an OD of 0.8 was reached. The temperature was then set to14 °C and protein expression induced by addition of 100 μM of IPTG. Protein expression was conducted overnight for 18 h after which the cells were harvested by centrifugation (5,000g, 4 °C, 12 min.), the pellet resuspended in lysis buffer (20 mM Tris-HCI pH 8, 0.5 M NaCI, 10 mM imidazole, 3 mM MgCl2, freshly added 1 complete EDTA-free protease inhibitor cocktail tablet and 0.05-0.1 % DNase) and the cells lysed by pressure lysis. Cell debris was removed by ultracentrifugation (35,000 rpm, 4 °C, 45 min.) Purification of his-tagged proteins

[0153] The supernatant after ultracentrifugation was purified using an ÄKTA start protein purification system equipped with a 5 mL HisTrap HP His tag protein purification column (Cytiva). After washing out unbound protein with wash buffer (20 mM Tris-HCI pH 8, 0.5 M NaCI, 10 mM imidazole) the protein was eluted using a gradient of 10 mM to 500 mM imidazole in the same buffer over 40-50 column. Fractions with protein were analyzed for protein content by SDS-PAGE. General protocol for SDS-PAGE

[0154] Samples were mixed with the appropriate amount of 4x LB buffer containing β- mercaptoethanol and heated to 95 °C for 5 minutes to denature proteins. Samples were then loaded onto acrylamide gels of the desired percentage (made using acrylamide 37.5:1 acrylamide / Bis) and ran at room temperature at 150V for one hour. If desired, gels were stained for at least 20 minutes with coomassie blue staining solution followed by at least three rounds of destaining with destaining solution (40% MeOH, 50 % MQ water, 10% AcOH). Stained gels were scanned on a Bio-Rad ChemiDoc MP machine.General protocol for buffer exchange

[0155] The combined fractions obtained from Ni-column purification were incubated with TCEP (500 μM-1mM) to break any possible formed disulfide bonds. The buffer was then exchanged by subjecting the protein to column chromatography on a Biotage® Selekt Flash Purification System equipped with a Biotage® Sfär C18 D - Duo 25g or 50g column and a stepwise gradient of 0 % ACN in water, 50 to 100% ACN water). The combined fractions containing the protein were lyophilized, yielding the his-tagged or final protein as TFA salt.Tag cleavage with enterokinase and subseguent purification

[0156] Lyophilized protein was dissolved at 2 mg / mL in EK cleavage buffer (200 mM Tris-HCI, pH 7.4, 0.5 M NaCI, 20 mM CaCI2) and after addition of 10 u / mL enterokinase (previously diluted in dilution buffer, 20 mM Tris-HCI, pH 7.4, 200 mM NaCI, 2 mM CaCI2, 50% glycerol), the protein was incubated overnight. The protein was then purified using an ÄKTA start protein purification system equipped with a 5 mL HisTrap HP His tag protein purification column (Cytiva) applying a gradient of 10 mM to 500 mM imidazole in elution buffer (20 mM Tris-HCI pH 8, 0.5 M NaCI, 10-500 mM imidazole). The fractions were analyzed by SDS-PAGE (see above) and the buffer of the combined protein containing fractions was exchanged as described above, yielding the final proteins as TFA salts after lyophilization.Electromobility shift assay (EMSA)

[0157] For EMSAs, 14 μL (15 μL for no protein control) water were mixed with 1 μL of 20x FAM- labelled DNA construct (IRD700-ACC CCA CCA CGT GGT GCC T), 1 μL of 20x protein in water and 4 μL of 5x EMSA buffer(final buffer concentration: 20 mM HEPES, pH 8.0, 150 mM NaCI, 5% glycerol, 1 mM EDTA, 2 mM MgCI2, 0.5 mg / mL of BSA, 1 mM DTT and 0.05% NP-40). The samples were incubated for 30 minutes at room temperature, placed on ice and incubated for another 15 minutes after which 15 μL of the samples were loaded onto a 10% native acrylamide TBE gel which was pre run before for 1h at 100 V at 4 °C in 0.5x TBE. Samples were run for 20 minutes at 120 V followed by 40 minutes at 100 V at 4 °C in 0.5x TBE and subsequently scanned on aBio-Rad ChemiDoc MP machine.Cell culture

[0158] Cells were cultured at 37°C in 5% CO2atmosphere. Cell lines were cultured in ATCC recommended media and split twice a week before confluency was reached. Reporter gene assay

[0159] The reporter gene assay was performed using Cignal reporter assay (CCS-012L, Qiagen) according to the manufacturer’s protocol. In brief, HEK293T cells were harvested and resuspended in OptiMEM media containing 5% FBS and 1% non-essential amino acids. 40,000 cells were seeded per well in a 96-well plate and 50 μL transfection cocktail of either cignal reporter or positive or negative control reporter along with attractene transfection reagent in OptiMEM without additives was added and the cells incubated overnight. Next, media was changed to assay media (OptiMEM, 0.5% FBS, 1% NEAA, pen / strep) and the cells were incubated for 8 hours after which the media was replaced by 75 μL assay media containing the different proteins at the required concentration and the cells were incubated for 24 hours .

[0160] Luciferase assay was then performed using a luciferase assay kit (E2940, Promega). Cells were lysed by addition of 75 μL of DualGlo Luciferase assay reagent and incubated for 15 minutes after which Luciferase luminescence was measured on an Agilent EnVision system. Subsequently, 75 μL of DualGlo Stop & Gio reagent were added and the Renilla luciferase luminescence measured after 15 minutes of incubation time. Signal was normalized against cell number by calculating the ratio of firefly and renilla luminescence and eventually these signals were normalized against the untreated control. All experiments were done in technical triplicates. Data was analyzed using GraphPad Prism 9.0.0.Cell proliferation assay with Hela and HCT 116 cells

[0161] Cells were seeded in 100 μL of their respective media (5000 cells / well in MEME for Hela cells and 2000 cells / well in McCoy’s 5A for HCT 116 cells) in a white opaque 96-well plate and let attach overnight. Media was then changed to 100 μL of media with the required protein at the appropriate concentration, the plate covered with a membrane to prevent them from drying out and the cells incubated with the proteins for 72 hours. Cell proliferation was then assessed using CellTiter-Glo (Promega) reading luminescence on an Agilent EnVision system. All experiments were done in technical triplicates. Data was analyzed using GraphPad Prism 9.0.0.Example 2Results

[0162] The present invention, (also termed “ReCHEMbinant proteins”), can inhibit MYC driven oncogenic gene programs. Figure 1 shows a schematic overview of MYC function and inhibitor activity. Site directed mutagenesis was performed to produce the ReCHEMbinant proteins described herein. Various stapling sites based on Omomyc variants were designed by the inventors as shown in Figure 2.

[0163] LCMS traces and DNA binding Gels of Omomyc and reCHEMbinant variants (Omomyc, V14C / N21C, V7C / V14C, T3C / V7C, V7C / T11C, Q64R / D71 R / Q86R, and artificial coiled-coil) were obtained determining that the proteins of the invention have EC50 values ranging from 154 nM to 7.6 nM, as shown in Figure 3.

[0164] Next, a MYC reporter gene assay (HEK293 cells) was performed using varying amounts of the proteins of the invention. EN4, a small molecule MYC inhibitor, was used as positive control. As a negative control, no treatment was used. Various proteins of the invention demonstrated MYC inhibitory properties surpassing the positive control as shown in Figure 4.Conclusion

[0165] The inventors have shown that carefully designed chemical modification can have a dramatic effect on the intracellular activity of protein therapeutics. The i, i+7 biphenyl staples mainly improved the cell activity of the reCHEMbinant variants (as shown by reporter gene assay of Figure 4).Example 3

[0166] Proteins were expressed and purified as all other HeloMYC proteins were.Stapling of Helomyc protein fused to NLS domain

[0167] The expressed and purified protein (5 mg, 329 nM, 1 eq.) was dissolved in water (164 μL, 2 mM) and then diluted into stapling buffer (2.3 mL, NH4HCO3, 100 mM, pH= 8). 4,4'- Bis(bromomethyl)biphenyl was dissolved at 4x the final concentration in acetonitrile (500 μM) andthen added (823 μL, 0.14 mg, 411 nmol, 1.25 eq.) to the protein in stapling buffer. Final concentrations used were 100 μM (1 eq.) protein and 125 μM (1.25 eq.) staple. Final acetonitrile content was 25%.

[0168] When the stapling reaction was completed as seen by LC-MS the product was purified using a Biotage(RTM)Selekt Flash Purification System equipped with a Biotage(RTM)Sfär C18 D - Duo 10g column on a gradient of water in ACN (0-100%), both containing 0.1 % TFA. The product containing fractions were lyophilized yielding HeloMYC-NLS (3.02 mg, 196 nmol, 60%) as TFA salt.Stapling of NLS-Helomyc

[0169] The expressed and purified protein (8 mg, 526 nM, 1 eq.) was dissolved in water (263 μL, 2 mM) and then diluted into stapling buffer (3.68 mL, NH4HCO3, 100 mM, pH= 8). 4,4'- Bis(bromomethyl)biphenyl was dissolved at 4x the final concentration in acetonitrile (500 μM) and then added (1.3 mL, 0.22 mg, 650 nmol, 1.25 eq.) to the protein in stapling buffer. Final concentrations used were 100 μM (1 eq.) protein and 125 μM (1.25 eq.) staple. Final acetonitrile content was 25%.

[0170] When the stapling reaction was completed as seen by LC-MS the product was purified using a Biotage(RTM)Selekt Flash Purification System equipped with a Biotage(RTM)Sfär C18 D - Duo 10g column on a gradient of water in ACN (0-100%), both containing 0.1 % TFA. The product containing fractions were lyophilized yielding NLS-HeloMYC (3.71 mg, 241 nmol, 46%) as TFA salt.Results

[0171] Figure 5 shows the position of the staples in the NLS-modified HeloMYC proteins.

[0172] The LCMS traces of purified, expressed proteins and final stapled proteins are shown in Figure 6.

[0173] Figure 7 shows the results of electrophoretic mobility shift assays (EMSAs), which the show the binding of the proteins to E-Box DNA.

[0174] Figure 8 shows a histogram with results demonstrating that the proteins impair cell proliferation of HeLa cells. A one-Way ANOVA was performed to compare the effect of the indicated treatment with dmso vehicle (F(14,28)=49.17, p<0.0001). Dunnett’s test was used for multiple comparisons to compare each treatment with dmso. *p<0.05, **p<0.01 , ***p<0.001 , ****p<0.0001. Sequences

[0175] SEQ ID NO: 1: TEENVKRRTHNVLERQRRNELKRSFFALRD

[0176] SEQ ID NO: 2: QIPELENNEKAPKVVILKKATAYI

[0177] SEQ ID NO: 3:TEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAETQKLI SEIDLLRKQNEQLKHKLEQLRNSCA

[0178] SEQ ID NO: 4:AMADIGSMATEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAETQKLISEIDLLRKQNEQLKHKLEQLRNSCA

[0179] SEQ ID NO: 5:AMADIGSMATEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVKREIAALKREIAALKREIAALKRE

[0180] SEQ ID NO: 6: TEENCKRRTHNCLERQRRNELKRSFFALRD

[0181] SEQ ID NO: 7: TEENVKRRTHNCLERQRRCELKRSFFALRD

[0182] SEQ ID NO: 8: CEENCKRRTHNVLERQRRNELKRSFFALRD

[0183] SEQ ID NO: 9: TEENCKRRCHNVLERQRRNELKRSFFALRD

[0184] SEQ ID NO: 10: LSVQAETQKLISEIDLLRKQNEQLKHKLEQLRNSCA

[0185] SEQ ID NO: 11: LSVQAETQKLISEIDLLRKQNEQLKHKLEQLRNSAA

[0186] SEQ ID NO: 12: LSVQAETRKLISEIRLLRKQNEQLKHKLERLRNSCA

[0187] SEQ ID NO: 13: LSVQAETRKLISEIRLLRKQNEQLKHKLERLRNSAA

[0188] SEQ ID NO: 14: KREIAALKREIAALKREIAALKRE

[0189] SEQ ID NO: 15 (Omomyc):AMADIGSMATEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAET QKLISEIDLLRKQNEQLKHKLEQLRNSCA

[0190] SEQ ID NO: 16:Enterokinase cleavage site shown withMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDK | AMADIGSMATEENVKRRTHNVLERQR RNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAETQKLISEIDLLRKQNEQLKHKLEQLRN SCA

[0191] SEQ ID NO: 17 (Omomyc T3C V7C C91A):AMADIGSMACEENCKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAET QKLISEIDLLRKQNEQLKHKLEQLRNSAA

[0192] SEQ ID NO: 18:Enterokinase cleavage site shown withMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDK | AMADIGSMACEENCKRRTHNVLERQR RNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAETQKLISEIDLLRKQNEQLKHKLEQLRN SAA

[0193] SEQ ID NO: 19 (Omomyc V7C T11C C91A):AMADIGSMATEENCKRRCHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAET QKLISEIDLLRKQNEQLKHKLEQLRNSAA

[0194] SEQ ID NO: 20:Enterokinase cleavage site shown withMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDK | AMADIGSMATEENCKRRCHNVLERQR RNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAETQKLISEIDLLRKQNEQLKHKLEQLRN SAA

[0195] SEQ ID NO: 21 (Omomyc V7C V14C C91A):AMADIGSMATEENCKRRTHNCLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAET QKLISEIDLLRKQNEQLKHKLEQLRNSAA

[0196] SEQ ID NO: 22:Enterokinase cleavage site shown withMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDK | AMADIGSMATEENCKRRTHNCLERQR RNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAETQKLISEIDLLRKQNEQLKHKLEQLRN SAA

[0197] SEQ ID NO: 23 (Omomyc V14C N21C C91A):AMADIGSMATEENVKRRTHNCLERQRRCELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAET QKLISEIDLLRKQNEQLKHKLEQLRNSAA

[0198] SEQ ID NO: 24:Enterokinase cleavage site shown withMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDK | AMADIGSMATEENVKRRTHNCLERQRRCELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAETQKLISEIDLLRKQNEQLKHKLEQLRN SAA

[0199] SEQ ID NO: 25 (Omomyc Q64R D71R Q86R C91A):AMADIGSMATEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAETRKLISEIRLLRKQNEQLKHKLERLRNSAA

[0200] SEQ ID NO: 26:Enterokinase cleavage site shown withMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDK | AMADIGSMATEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAETRKLISEIRLLRKQNEQLKHKLERLRN SAA

[0201] SEQ ID NO: 27 (NLS-Helomyc):AMADIGSPAAKRVKLDMATEENVKRRTHNCLERQRRCELKRSFFALRDQIPELENNEKAPKVVILKKATA YILSVQAETQKLISEIDLLRKQNEQLKHKLEQLRNSAA

[0202] SEQ ID NO: 28:Enterokinase cleavage site shown withMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDK | AMADIGSPAAKRVKLDMATEENVKRRTHNCLERQRRCELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAETQKLISEIDLLRKQNEQL KHKLEQLRNSAA

[0203] SEQ ID NO: 29 (Helomyc-NLS):AMADIGSMATEENVKRRTHNCLERQRRCELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAETQKLISEIDLLRKQNEQLKHKLEQLRNSAAPAAKRVKLD

[0204] SEQ ID NO: 30:Enterokinase cleavage site shown withMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDK | AMADIGSMATEENVKRRTHNCLERQRRCELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAETQKLISEIDLLRKQNEQLKHKLEQLRN SAAPAAKRVKLD

[0205] SEQ ID NO: 31 (Omomyc with artificial coiled-coil):MHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDK | AMADIGSMATEENVKRRTHNVLERQR RNE L KRS F F AL RDQIPELENNEKAP KV V I L KKAT AY ILS VKRE I AALKRE I AALKRE I AALKREPlasmid sequences

[0206] SEQ ID NO: 32 (pET-30a with Omomyc):TCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCT

[0207] SEQ ID NO: 33 (pET-30a with artificial coiled-coil):

[0208] SEQ ID NO: 34 (pET-30a with natural zipper domain Q64R D71 R Q86R):References

[0209] The following references are cited in this disclosure.1. Lee, T. I. & Young, R. A. Transcriptional regulation and its misregulation in disease. Cell 152, 1237-1251 (2013).2. Boija, A. et al. Transcription Factors Activate Genes through the Phase-Separation Capacity of Their Activation Domains. Cell 175, 1842-1855. e16 (2018).3. Bushweller, J. H. Targeting transcription factors in cancer — from undruggable to reality. Nat. Rev. Cancer 19, 611-624 (2019).4. Meyer, N. & Penn, L. Z. Reflecting on 25 years with MYC. Nat. Rev. Cancer 8, 976-990 (2008).5. Madden, S. K., de Araujo, A. D., Gerhardt, M., Fairlie, D. P. & Mason, J. M. Taking the Myc out of cancer: toward therapeutic strategies to directly inhibit c-Myc. Mol. Cancer 20, 1-18 (2021).6. Das, S. K., Lewis, B. A. & Levens, D. MYC: a complex problem. Trends Cell Biol. 33, 235-246 (2023).7. Blackwood, E. M. & Eisenman, R. N. Max: A helix-loop-helix zipper protein that forms a sequence-specific DNA-binding complex with Myc. Science (80-. ). 251, 1211-1217 (1991).8. Kalkat, M. et al. MYC Protein Interactome Profiling Reveals Functionally Distinct Regions that Cooperate to Drive Tumorigenesis. Mol. Cell 72, 836-848. e7 (2018).9. Blackwood, E. M. & Eisenman, R. N. Max : A Helix-Loop-Helix Zipper Protein That Complex with Myc. Science (80-. ). 251, 1211-1217 (1991).10. Nithun, R. V. et al. Deciphering the Role of the Ser-Phosphorylation Pattern on the DNA- Binding Activity of Max Transcription Factor Using Chemical Protein Synthesis. Angew. Chemie - Int. Ed. 62, (2023).11. Ferre-D’Amare, A. R., Prendergast, G. C., Ziff, E. B. & Burley, S. K. Recognition by Max of its cognate DNA through a dimeric b / HLH / Z domain. Nature 363, 38-45 (1993).12. Nair, S. K. & Burley, S. K. X-ray structures of Myc-Max and Mad-Max recognizing DNA: Molecular bases of regulation by proto-oncogenic transcription factors. Cell 112, 193-205 (2003).13. Struntz, N. B. et al. Stabilization of the Max Homodimer with a Small Molecule Attenuates Myc-Driven Transcription. Cell Chem. Biol. 26, 711-723. e14 (2019).14. Boike, L. et al. Discovery of a Functional Covalent Ligand Targeting an Intrinsically Disordered Cysteine within MYC. Cell Chem. Biol. 1-10 (2020) doi:10.1016 / j.chembiol.2020.09.001.15. Li, Z. et al. MYC-Targeting Inhibitors Generated from a Stereodiversified Bicyclic Peptide Library. (2023) doi:10.1021 / jacs.3c09615.16. Soucek, L. et al. Design and properties of a Myc derivative that efficiently homodimerizes. Oncogene 17, 2463-2472 (1998).17. Jung, L. A. et al. OmoMYC blunts promoter invasion by oncogenic MYC to inhibit gene expression characteristic of MYC-dependent tumors. Oncogene 36, 1911-1924 (2017).18. Lustig, L. C. et al. Inhibiting MYC binding to the E-box DNA motif by ME47 decreases tumour xenograft growth. Oncogene 36, 6830-6837 (2017).19. Montagne, M. et al. The max b-HLH-LZ can transduce into cells and inhibit c-Myc transcriptional activities. PLoS One 7, 2-10 (2012).20. Lin, X., Harel, O. & Jbara, M. Chemical Engineering of Artificial Transcription Factors by Orthogonal Palladium(ll)-Mediated S-Arylation Reactions. Angew. Chemie - Int. Ed. 202317511, (2023).21. Pomplun, S. et al. Parallel Automated Flow Synthesis of Covalent Protein Complexes That Can Inhibit MYC-Driven Transcription. ACS Cent. Sci. 7, 1408-1418 (2021).22. Jbara, M. et al. Engineering Bioactive Dimeric Transcription Factor Analogs via Palladium Rebound Reagents. J. Am. Chem. Soc. 143, 11788-11798 (2021).23. Harel, O. & Jbara, M. Chemical Synthesis of Bioactive Proteins. Angew. Chemie - Int.Ed. 62, (2023).24. Leader B, Baca Q J & Golan D E. Protein therapeutics: a summary and pharmacological classification. Nat. Rev. Drug Discov. 7, 21-39 (2008).25. Beaulieu, M. E. et al. Intrinsic cell-penetrating activity propels omomyc from proof of concept to viable anti-myc therapy. Sci. Transl. Med. 11, 1-14 (2019).26. Garralda, E. et al. MYC targeting by OMO-103 in solid tumors: a phase 1 trial. Nat. Med. (2024) doi:10.1038 / s41591-024-02805-1.27. Wang, E. et al. Tumor penetrating peptides inhibiting MYC as a potent targeted therapeutic strategy for triple-negative breast cancers. Oncogene 38, 140-150 (2019).28. Speltz, T. E. et al. Targeting MYC with modular synthetic transcriptional repressors derived from bHLH DNA-binding domains. Nat. Biotechnol. 41, (2023).

[0210] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety and to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law).

[0211] All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way.

[0212] The use of any and all examples, or exemplary language (e.g. “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise paragraphed. No language in the specification should be construed as indicating any non-paragraphed element as essential to the practice of the invention.

[0213] The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability, and / or enforceability of such patent documents.

[0214] This invention includes all modifications and equivalents of the subject matter recited in the paragraphs appended hereto as permitted by applicable law.

Claims

CLAIMS1. A protein comprising:(i) a polypeptide having an alpha helical DNA binding domain, wherein the polypeptide has 50 or more amino acid residues, and wherein the alpha helical DNA binding domain comprises a first amino acid and a second amino acid, wherein the second amino acid is non-adjacent to the first amino acid; and(ii) a peptide staple covalently linking a side chain of the first amino acid to a side chain of the second amino acid, wherein the peptide staple is obtained or is obtainable by a reaction selected from a ring closing metathesis reaction, a copper catalyzed azide alkyne cycloaddition reaction, a lactamization reaction, a cysteine-xylene reaction, cysteine-perfluorobenzene reaction, a thiolyne or thiol-ene Click chemistry reaction, a selenocysteine reaction, a tryptophan condensation reaction, a C-H activation reaction and a 1 ,3-dipolar cycloaddition reaction.

2. The protein according to claim 1 , wherein the peptide staple comprises a staple of formula (1):wherein each asterisk (*) in formula (1) indicates a carbon atom that is covalently linked to a sulfur atom in a cysteine residue of one of the first amino acid or the second amino acid, and wherein the first amino acid is located at position i of the alpha helical DNA binding domain and the second amino acid is located at position i + 7 of the alpha helical DNA binding domain.

3. The protein according to claim 1 or claim 2, wherein the alpha helical DNA binding domain has a degree of identity of at least 70% with respect to SEQ ID NO: 1.

4. The protein according to any one of the preceding claims, wherein the first amino acid is at an amino acid position of from 1 to 15 of SEQ ID NO: 1 , preferably at amino acid position 1 , 5 or 12 of SEQ ID NO: 1.

5. The protein according to any one of the preceding claims, wherein the polypeptide further comprises a loop domain and a helix domain, wherein the loop domain and the helix domain havea degree of identity of at least 70% with respect to SEQ ID NO: 2, and is covalently linked to the C-terminus of the alpha helical DNA binding domain.

6. The protein according to any one of the preceding claims, wherein the polypeptide has a degree of identity of at least 70% with respect to SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO:5.

7. The protein according to claim 6, wherein the polypeptide has a degree of identity of at least 70% with respect to SEQ ID NO: 3 or SEQ ID NO:4, wherein at least one arginine residue replaces an amino acid at an amino acid position of from 55 to 85 in SEQ ID NO: 3 or from 64 to 94 in SEQ ID NO: 4.

8. The protein according to any one of the preceding claims, wherein the protein is a basic helix-loop-helix leucine zipper transcription factor.

9. The protein according to any one of the preceding claims, wherein the first amino acid is located at position i of the alpha helical DNA binding domain and the second amino acid is located at either (a) position i + 4 of the alpha helical DNA binding domain or (b) position i + 7 of the alpha helical DNA binding domain, preferably the second amino acid is located at position i + 7 of the alpha helical DNA binding domain.

10. The protein according to any one of the preceding claims, wherein the peptide staple is obtained or is obtainable by a cysteine-xylene reaction or a cysteine-perfluorobenzene reaction.

11. The protein according to any one of the preceding claims, wherein the first amino acid is a cysteine residue and the second amino acid is a cysteine residue.

12. The protein according to any one of the preceding claims, wherein the polypeptide is a recombinant polypeptide.

13. A pharmaceutical composition comprising the protein of any one of the preceding claims and a pharmaceutically acceptable excipient.

14. The protein according to any one of claims 1 to 12 for use in a method of therapy and / or as a medicament.

15. The protein according to any one of claims 1 to 12 for use in the treatment or prevention of a proliferative disorder, preferably wherein the proliferative disorder is cancer.

16. The protein for use according to claim 15, wherein the cancer is associated with MYC dysregulation.

17. A method of preparing the protein of any one of claims 1 to 12 comprising(a) recombinantly expressing the polypeptide; and(b) modifying the polypeptide to include the peptide staple.

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