c-Jun antagonist peptides

Abstract

c-Jun antagonists are described. Compositions comprising these c-Jun antagonists, as well as methods, including methods of treatment, and therapeutic uses of the c-Jun antagonists are also described.

Description

[Technical field]

[0001] This application claims priority to UK Patent No. 2203399.7, filed March 11, 2022, the contents and elements of which are incorporated herein by reference for all purposes.

[0002] The present invention relates to peptides that antagonize c-Jun, nucleic acids encoding peptides that antagonize c-Jun, pharmaceutical preparations containing peptides that antagonize c-Jun, and the use of the antagonist peptides in the treatment of c-Jun mediated diseases. [Background technology]

[0003] Transcription factors (TFs) play a key role in determining cell function and fate. A wide range of upstream signals converge on TFs, which, through specific DNA site recognition, transduce live cell signaling processes into transcriptional outputs. As a result, of the roughly 1600 TFs in the human genome, more than 300 are associated with disease phenotypes. TF dysfunction leads to a wide range of adverse outcomes, including cancer, diabetes, and cardiovascular disease (Lee et al., 2013; Lambert et al., 2018). Selective TF antagonism is therefore a potential therapeutic pathway for the treatment of these diseases.

[0004] c-Jun is a transcription factor associated with a wide range of human diseases (Eferl et al., 2003;Yung et al., 2010;Shiozawa et al., 2009). c-Jun is a member of the activator protein-1 (AP-1) family of dimeric transcription factors. AP-1 proteins bind to DNA recognition elements via their basic leucine zipper (bZIP) domain, which consists of a leucine zipper (LZ) that promotes dimerization and a DNA binding domain (DBD) that promotes DNA sequence recognition (Glover et al., 1995;Risse et al., 1989). c-Jun binds to 12-O-tetradecanoylphorbol-13-acetate response elements (TREs) and directly affects cellular processes such as differentiation, proliferation, and survival (Shaulian et al., 2001;Eferl et al., 2003;Eckert et al., 2013;Alani et al., 1991). Thus, dysregulation of these functions promotes hallmarks of cancer cell behavior, making c-Jun a focus for cancer therapy.

[0005] TF function relies on protein-protein interactions (PPIs) and protein-DNA interactions that form many contact points on their large surfaces. Small molecules (SMs) are typically unable to abolish these types of interactions due to the lack of tractable pockets.

[0006] Planar protein-protein interactions are inaccessible to many drugs, including small molecules, but peptides have great potential as high affinity and selective inhibitors when designed to complement the broad target surface. Various methods have produced peptide c-Jun antagonists that target the broad LZ binding interface (Boysen et al., 2002;Mason et al., 2006;Kaplan et al., 2014;Baxter et al., 2017;Lathbridge et al., 2018). However, it is difficult to predict whether LZ binding will lead to functional antagonism, as the cJun DBD remains unbound and can bind to TRE DNA (Seldeen et al., 2008;Szaloki et al., 2015). Rationally designed peptides have been shown to target the c-Jun DBD, but exhibit lower potency than LZ antagonists and there are concerns regarding specificity due to the high sequence similarity across AP-1 family DBDs ( Tsuchida et al., 2004 ).

[0007] One approach to circumvent the potential downsides of existing methods is to utilize longer peptides that target the full-length c-Jun bZIP domain with selective but high-affinity interactions, while simultaneously blocking both DNA binding and LZ dimerization. Olive et al. used this approach to generate A-Fos, which combines the wild-type (WT) cFos LZ (known to heterodimerize with c-Jun) and a rationally designed Glu-rich acidic chain (Olive et al., 1997). The A-Fos design principle postulates that the LZ interaction is extended N-terminally, resulting in a DBD-acidic chain interaction facilitated by the incorporation of a Leu residue at the putative d-position of the acidic chain.

[0008] For peptides to act as functionally active c-Jun antagonists, they must not only bind to c-Jun, but target binding must also result in loss of function. One recently described method for identifying functional antagonists of transcription factors is the Transcription Block Survival (TBS) assay described in WO2020128015. Summary of the Invention [Problem to be solved by the invention]

[0009] There remains a need for c-Jun antagonists that act as functional antagonists, particularly those that exhibit desirable pharmacokinetic properties for therapeutic use. [Means for solving the problem]

[0010] The present invention has been devised in light of the above considerations. The present inventors have identified novel peptide inhibitors that are demonstrated to bind to c-Jun and antagonize its DNA binding function.

[0011] In the work leading to the present invention, the inventors used a library-based approach in which the hinge region spanning the acidic chain and leucine zipper regions of the c-Jun antagonist was semi-randomized. A peptide library was produced on this scaffold, randomized across the core system of residues, and tested using a TBS screening platform. This led to the identification of a recombinantly produced functional c-Jun antagonist, called "HingeW", from a library of approximately 130,000 peptides. The HingeW peptide is demonstrated in the Examples to bind c-Jun preferentially and with high affinity compared to the c-Jun antagonist from which HingeW is derived, and to effectively antagonize the c-Jun / TRE DNA interaction. The nature of the broad, shallow helix-binding surface of c-Jun supports the use of longer peptides, such as those identified by TBS.

[0012] The binding epitopes of bZIP antagonists are presented on one side of a single α-helix, so that target binding requires the peptide to adopt this secondary structure. Recognizing that a fundamental step in the development of therapeutic peptides is the reduction of the peptide to the smallest functional unit required for efficient binding, the inventors introduced stepwise truncations to the identified antagonist peptides. The reduction tends to reduce the α-helix of the peptide, since both the interaction interface and the extended internal hydrogen bond network are reduced, and water competes with these interactions, shifting the folding equilibrium to random coil. Reducing the peptide to increase drug-like characteristics such as stability and membrane permeability must therefore be balanced against the reduced affinity resulting from the reduction of the α-helix of the peptide. As demonstrated herein, various optimized and truncated forms of the HingeW peptide have been developed that retain functional activity while improving the drug-like characteristics of the peptide.

[0013] In a first aspect, the present invention provides an antibody comprising an extended hinge region having an amino acid sequence of LV[X1]EE[X2][X3]LE[X4]E (SEQ ID NO: 1); and a leucine zipper (LZ) region C-terminal to the extended hinge region, [where: X1 is selected from V, D, K, C and R; X2 is selected from D, K, C and R; X3 is selected from V, D, C, K and R; and X4 is selected from E, D, C, K and R. The present invention provides a c-Jun antagonist comprising:

[0014] In some embodiments: X1 is selected from V, D, K, and C; X2 is selected from D, K, and C; X3 is selected from V, D, and C; and X4 is selected from E, D, and C.

[0015] In some embodiments, the extended hinge region has the amino acid sequence of EA[X5][X6] (SEQ ID NO:2): [where: X5 is selected from E, K or C; and X6 is selected from E or D] It further comprises an N-terminal acidic chain having the following structure:

[0016] In some embodiments, the acidic chain has the amino acid sequence of EAEE (SEQ ID NO:3). In some embodiments, X1 is V. In some embodiments, X3 is V. In some embodiments, X1 and X3 are V.

[0017] In some embodiments: (i) X1 is V, X2 is D, X3 is V, and X4 is E; (ii) X1 is V, X2 is K, X3 is V, and X4 is D; or (iii) X1 is V, X2 is C, X3 is V, and X4 is C.

[0018] In some embodiments, the LZ region comprises the amino acid sequence IEQLEERNYALR[X7]E[X8]K[X9]L[X10]D[X11] (SEQ ID NO: 29) or IEQLEERNYALR[X7]E[X8]C[X9]L[X10]C[X11] (SEQ ID NO: 30), {where [X7] is K, L, S, W, P, Q, R, M, T, V, A, E, or G; [X8] is I, V, or L; [X9] and [X10] are any amino acid residues, [X11] is Q, E, or K} Includes.

[0019] In some embodiments, the LZ region comprises: IEQLEERNYALRSEICSLQCQ (SEQ ID NO:66); or IEQLEERNYALRKEICELSCQ (SEQ ID NO:67); or IEQLEERNYALRAEICNLSCQ (SEQ ID NO:68); or IEQLEERNYALRTEICSLMCK (SEQ ID NO:69); or IEQLEERNYALRAEICSLQCQ (SEQ ID NO: 70), or a variant thereof containing 1, 2 or 3 amino acid alterations.

[0020] In some embodiments, the LZ region comprises: IEQLEERNYALRKEIEDLQKQ (SEQ ID NO:4); IEQLEEKNKALKDEIEDLQKQ (SEQ ID NO:5); IKQLEDRNYALRKEIEDLQKQ (SEQ ID NO:6); IEQLEERNYALRKEIKDLQDQ (SEQ ID NO: 7); IEQLEEKNKALKDEIEDLY (SEQ ID NO:8); IEQLEERNYALRKEIEDLQ (SEQ ID NO: 9); and IEQLEERNYALRKEICDLQCQ (SEQ ID NO:27), or a variant thereof containing 1, 2 or 3 amino acid alterations.

[0021] In some embodiments, the LZ region comprises the amino acid sequence of IEQLEERNYALRKEIKDLQDQ (SEQ ID NO: 7), or a variant thereof comprising one, two or three amino acid alterations; Optionally, the amino acid residues at positions corresponding to positions 16 (b position of a heptad) and 20 (f position of a heptad) of SEQ ID NO:7 of the variant are K and D amino acid residues, respectively.

[0022] In some embodiments, the LZ region comprises the amino acid sequence of IEQLEERNYALRKEICDLQCQ (SEQ ID NO: 27), or a variant thereof comprising one, two or three amino acid alterations; Optionally, the amino acid residues at positions corresponding to positions 16 (position b of a heptad) and 20 (position f of a heptad) of SEQ ID NO: 27 of the variant are both C amino acid residues.

[0023] In some embodiments, the c-Jun antagonist has a length of between 30 and 70 amino acids, between 30 and 60 amino acids, between 30 and 50 amino acids, or between 30 and 40 amino acids, In certain embodiments, the c-Jun antagonist has a length of 36 amino acids.

[0024] In some embodiments, the c-Jun antagonist is EAEELVVEEDVLEEEIEQLEERNYALRKEIKDLQDQ (SEQ ID NO: 10); EAEELVVEEKVLEDEIEQLEERNYALRKEIKDLQDQ (SEQ ID NO: 11); or EAEELVVEEDVLEEEIEQLEERNYALRKEICDLQCQ (SEQ ID NO:28); EAEELVVEEDVLEEEIEQLEERNYALRSEICSLQCQ (SEQ ID NO:37); EAEELVVEEDVLEEEIEQLEERNYALRKEICELSCQ (SEQ ID NO: 38); or EAEELVVEEDVLEEEIEQLEERNYALRAEICNLSCQ (SEQ ID NO: 39); or EAEELVVEEDVLEEEIEQLEERNYALRTEICSLMCK (SEQ ID NO: 40); or EAEELVVEEDVLEEEIEQLEERNYALRAEICSLQCQ (SEQ ID NO: 41) or variants thereof that contain 1, 2 or 3 amino acid alterations.

[0025] In some embodiments, the c-Jun antagonist comprises at least one covalent amino acid residue crosslinker. Such peptides may be referred to herein as "helix-constrained c-Jun antagonists". In some embodiments, the c-Jun antagonist peptide comprises at least one covalent i to i+4 or i to i+7 amino acid residue crosslinker. As demonstrated herein, the introduction of a covalent amino acid residue crosslinker can increase the helicity and increase the antagonist activity of the peptide. This is beneficial because it can be used to derive functionally active peptide antagonists with similar binding affinity and function as the parent protein, with the same amino acid sequence conferring specificity while retaining similar stability and solubility as small molecule therapeutics. Although this has been demonstrated for the KD lactam crosslinker as the crosslinker, similar results are expected when other crosslinkers are used, such as an alkyl crosslink formed between two C residues via cysteine ​​alkylation, such as DBMB.

[0026] In some embodiments, the c-Jun antagonist peptide comprises at least one covalent i to i+4 amino acid residue crosslinker. For example, the c-Jun antagonist peptide may comprise two covalent i to i+4 amino acid crosslinkers. Preferably, the covalent i to i+4 amino acid crosslinker(s) is / are at the b to f or f to c positions of a heptad. Preferably, the covalent i to i+4 amino acid crosslinker(s) is / are a KD lactam bridge(s) or an alkyl bridge bond (cysteine ​​alkylation) formed between two C residues.

[0027] In a second aspect, the present invention provides a nucleic acid encoding a c-Jun antagonist peptide according to the first aspect of the invention. In a third aspect, the present invention provides a conjugate comprising a c-Jun antagonist peptide according to the first aspect of the invention conjugated to a lipid, a polymer or a second peptide.

[0028] In a fourth aspect, the present invention provides a pharmaceutical composition comprising a c-Jun antagonist according to the first aspect of the invention, a nucleic acid according to the second aspect of the invention, or a conjugate according to the third aspect of the invention, in combination with a physiologically acceptable vehicle or carrier.

[0029] In a fifth aspect, the present invention provides a c-Jun antagonist peptide according to the first aspect of the invention, a nucleic acid according to the second aspect of the invention, a conjugate according to the third aspect of the invention, or a pharmaceutical composition according to the fourth aspect of the invention for use as a medicament.

[0030] In a sixth aspect, the present invention provides a method of inhibiting c-Jun in vitro against a cell containing or expressing a c-Jun peptide comprising a peptide according to the first aspect of the invention, a nucleic acid according to the second aspect of the invention, or a conjugate according to the third aspect of the invention.

[0031] Also provided herein is a method for producing the c-Jun antagonist peptide according to the first aspect of the present invention. The method for producing the c-Jun antagonist peptide may comprise synthesizing the c-Jun antagonist peptide using solid-phase or liquid-phase peptide synthesis, or may comprise producing the c-Jun antagonist peptide by recombinant expression. The method may further comprise contacting the c-Jun antagonist peptide with a cross-linker to produce a helix-constrained c-Jun antagonist peptide. Also provided herein is a method for producing a helix-constrained c-Jun antagonist peptide, comprising contacting the c-Jun antagonist peptide according to the first aspect of the present invention with a cross-linker.

[0032] The present invention includes combinations of the embodiments and preferred features described, except where such combinations are clearly unacceptable or clearly avoided. These and other aspects of the invention are described in more detail below.

[0033] BRIEF DESCRIPTION OF THE DRAWINGS Embodiments and experiments illustrating the principles of the present invention will now be discussed with reference to the accompanying drawings. [Brief description of the drawings]

[0034] [Figure 1A] TRE DNA-bound cJun structure and cJun antagonist design. (A) The DNA-bound cJun homodimer crystal structure (PDB:2H7H) is shown highlighting the LZ and DBD components required for dimerization and DNA binding. (B) Schematic of the acidic tether design principle (A-FosW, HingeW). This utilizes a region known to bind to the cJun LZ, to which a Glu-rich tether is added to interact with the cJun DBD. [Figure 1B] TRE DNA-bound cJun structure and cJun antagonist design. (A) The DNA-bound cJun homodimer crystal structure (PDB:2H7H) is shown highlighting the LZ and DBD components required for dimerization and DNA binding. (B) Schematic of the acidic tether design principle (A-FosW, HingeW). This utilizes a region known to bind to the cJun LZ, to which a Glu-rich tether is added to interact with the cJun DBD. [Diagram 2] Protein and DNA sequences of TRE-mDHFR showing the introduction of 15 TRE sites into the gene. The amino acids and DNA bases mutated from the WT mouse protein are shown in red, and the TRE DNA sites where these mutations were added are bolded and underlined. Shown in green are the NheI and HindIII sites used to subclone the gene into the pES300d vector. [Figure 3A]mDHFR retains activity upon introduction of TRE sites. (A) Fifteen TRE sites were introduced into the mDHFR gene (two silent and thirteen substitutions) allowing for a block of cJun-induced transcription. The substitutions were mapped (green) onto the mDHFR structure (PDB code: 1U72) showing surface exposure distal to the active site with the substrate DHF (shown is the competitive inhibitor methotrexate (MTX) bound to the DHF binding site) and the bound cofactor NADPH. The change in absorbance at 340 nm was measured to determine the rate of turnover of NADPH by (B) WT-mDHFR and (C) TRE-mDHFR with or without the substrate DHF. Reactions repeated in the presence of TMP are also shown, showing that, as expected, TRE-mDHFR is partially inhibited and activity of both enzymes is retained. Specific activities were calculated from linear initial velocities (WT-mDHFR, first 2.5 min; TRE-mDHFR, first 10 min; +NADPH only reaction blank was subtracted). Data are shown with error as one standard deviation and are averages from three experiments. MTX showed broader inhibition than TMP, inhibiting both eukaryotic and prokaryotic DHFR enzymes and thus both WT- and TRE-mDHFR. [Figure 3B]mDHFR retains activity upon introduction of TRE sites. (A) Fifteen TRE sites were introduced into the mDHFR gene (two silent and thirteen substitutions) allowing for a block of cJun-induced transcription. The substitutions were mapped (green) onto the mDHFR structure (PDB code: 1U72) showing surface exposure distal to the active site with the substrate DHF (shown is the competitive inhibitor methotrexate (MTX) bound to the DHF binding site) and the bound cofactor NADPH. The change in absorbance at 340 nm was measured to determine the rate of turnover of NADPH by (B) WT-mDHFR and (C) TRE-mDHFR with or without the substrate DHF. Reactions repeated in the presence of TMP are also shown, showing that, as expected, TRE-mDHFR is partially inhibited and activity of both enzymes is retained. Specific activities were calculated from linear initial velocities (WT-mDHFR, first 2.5 min; TRE-mDHFR, first 10 min; +NADPH only reaction blank was subtracted). Data are shown with error as one standard deviation and are averages from three experiments. MTX showed broader inhibition than TMP, inhibiting both eukaryotic and prokaryotic DHFR enzymes and thus both WT- and TRE-mDHFR. [Figure 3C]mDHFR retains activity upon introduction of TRE sites. (A) Fifteen TRE sites were introduced into the mDHFR gene (two silent and thirteen substitutions) allowing for a block of cJun-induced transcription. The substitutions were mapped (green) onto the mDHFR structure (PDB code: 1U72) showing surface exposure distal to the active site with the substrate DHF (shown is the competitive inhibitor methotrexate (MTX) bound to the DHF binding site) and the bound cofactor NADPH. The change in absorbance at 340 nm was measured to determine the rate of turnover of NADPH by (B) WT-mDHFR and (C) TRE-mDHFR with or without the substrate DHF. Reactions repeated in the presence of TMP are also shown, showing that, as expected, TRE-mDHFR is partially inhibited and activity of both enzymes is retained. Specific activities were calculated from linear initial velocities (WT-mDHFR, first 2.5 min; TRE-mDHFR, first 10 min; +NADPH only reaction blank was subtracted). Data are shown with error as one standard deviation and are averages from three experiments. MTX showed broader inhibition than TMP, inhibiting both eukaryotic and prokaryotic DHFR enzymes and thus both WT- and TRE-mDHFR. [Figure 4A]TRE-mDHFR is expressed in the soluble fraction and can be purified for further studies. (A) SDS-PAGE analysis of E. Coli cell lysates from cells before and after induction of TRE-mDHFR plasmid expression with IPTG (1 mM, 18 h, 30° C.). Total (T) samples are taken immediately after lysis and soluble (S) samples are taken after centrifugation of the lysate. This shows the appearance of a protein band in the induced sample with equal band intensity in the T and S fractions, indicating that the protein is folded and soluble. TRE-mDHFR can be bound to an immobilized metal affinity chromatography column via its 6×His tag and subsequently eluted with an imidazole gradient (B) to yield pure protein as determined by SDS-PAGE (C) of the combined concentrated fractions shown to contain the induced protein band. Although TRE-mDHFR did not migrate through the polyacrylamide gel as predicted by the protein marker lane (M), running at an apparently higher molecular weight, its identity was confirmed by electrospray ionization mass spectrometry (D). [Figure 4B]TRE-mDHFR is expressed in the soluble fraction and can be purified for further studies. (A) SDS-PAGE analysis of E. Coli cell lysates from cells before and after induction of TRE-mDHFR plasmid expression with IPTG (1 mM, 18 h, 30° C.). Total (T) samples are taken immediately after lysis and soluble (S) samples are taken after centrifugation of the lysate. This shows the appearance of a protein band in the induced sample with equal band intensity in the T and S fractions, indicating that the protein is folded and soluble. TRE-mDHFR can be bound to an immobilized metal affinity chromatography column via its 6×His tag and subsequently eluted with an imidazole gradient (B) to yield pure protein as determined by SDS-PAGE (C) of the combined concentrated fractions shown to contain the induced protein band. Although TRE-mDHFR did not migrate through the polyacrylamide gel as predicted by the protein marker lane (M), running at an apparently higher molecular weight, its identity was confirmed by electrospray ionization mass spectrometry (D). [Figure 4C]TRE-mDHFR is expressed in the soluble fraction and can be purified for further studies. (A) SDS-PAGE analysis of E. Coli cell lysates from cells before and after induction of TRE-mDHFR plasmid expression with IPTG (1 mM, 18 h, 30° C.). Total (T) samples are taken immediately after lysis and soluble (S) samples are taken after centrifugation of the lysate. This shows the appearance of a protein band in the induced sample with equal band intensity in the T and S fractions, indicating that the protein is folded and soluble. TRE-mDHFR can be bound to an immobilized metal affinity chromatography column via its 6×His tag and subsequently eluted with an imidazole gradient (B) to yield pure protein as determined by SDS-PAGE (C) of the combined concentrated fractions shown to contain the induced protein band. Although TRE-mDHFR did not migrate through the polyacrylamide gel as predicted by the protein marker lane (M), running at an apparently higher molecular weight, its identity was confirmed by electrospray ionization mass spectrometry (D). [Figure 4D]TRE-mDHFR is expressed in the soluble fraction and can be purified for further studies. (A) SDS-PAGE analysis of E. Coli cell lysates from cells before and after induction of TRE-mDHFR plasmid expression with IPTG (1 mM, 18 h, 30° C.). Total (T) samples are taken immediately after lysis and soluble (S) samples are taken after centrifugation of the lysate. This shows the appearance of a protein band in the induced sample with equal band intensity in the T and S fractions, indicating that the protein is folded and soluble. TRE-mDHFR can be bound to an immobilized metal affinity chromatography column via its 6×His tag and subsequently eluted with an imidazole gradient (B) to yield pure protein as determined by SDS-PAGE (C) of the combined concentrated fractions shown to contain the induced protein band. Although TRE-mDHFR did not migrate through the polyacrylamide gel as predicted by the protein marker lane (M), running at an apparently higher molecular weight, its identity was confirmed by electrospray ionization mass spectrometry (D). [Diagram 5] Optimization of the TMP concentration required to provide selectivity between E. coli expressing TRE-mDHFR and E. coli showing transcriptionally blocked TRE-mDHFR expression by cJun bZIP. Controlled numbers of E. coli cells expressing the indicated proteins were plated on selective media at various TMP concentrations. 4 μM TMP provided the optimal difference in colony numbers between TRE-mDHFR only and TRE-mDHFR+cJun bZIP plates. [Figure 6] Bacterial DHFR is inhibited by TMP and its activity is replaced by induction of TRE-mDHFR expression. E. coli cells containing a plasmid for TRE-mDHFR only grow differentially on different agar media, according to the design principles of the TBS assay. On M9 agar (1), a lawn of colonies is produced as free bacterial growth; upon addition of TMP to the medium (2), bacterial DHFR is inhibited and cells cannot grow; further addition of IPTG (3) leads to expression of TRE-mDHFR and restores cell survival to some extent. [Figure 7] Both WT-mDHFR and TRE-mDHFR are inhibited by the broad spectrum DHFR inhibitor MTX. The change in absorbance at 340 nm was measured to determine the rate of NADPH turnover by WT-mDHFR and TRE-mDHFR with and without the substrate DHF. The reaction was also shown repeated in the presence of MTX, showing a clear inhibition of the reaction as expected, indicating DHFR activity. [Figure 8A] Transcription Block Survival (TBS) assay driving functionally active cJun inhibitors. (A) Schematic showing the design and operation of the TBS. (B) Controlled numbers of E. coli expressing the indicated proteins were plated on selective media and growth rates were calculated by counting colony forming units. (1) WT-mDHFR expression can replace ecDHFR and is not inhibited by TMP, resulting in significant growth. (2) A small effect on colony numbers is observed when cJun bZIP is additionally expressed. (3) TRE-mDHFR can replace the inhibited ecDHFR with fewer colonies than WT as expected. (4) The cJun LZ domain (lacking the DBD) does not affect TRE-mDHFR transcription and colony formation, whereas (5) the cJun bZIP domain (with the DBD) binds to the TRE site and blocks the transcription of TRE-mDHFR, resulting in reduced bacterial survival. (6) cFos LZ and (7) FosW are known cJun binders, but they are unable to effectively dissociate cJun bZIP from TRE DNA. However, (8) A-FosW and the TBS-derived hit (9) HingeW remove the TRE-mDHFR transcription block and restore cell viability. Bar graphs represent the mean of three replicate experiments. Errors are shown as one standard deviation. Selected P values ​​from t-tests are shown with values ​​for all possible comparisons within the bar graphs reported in Figure S7 (*P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001). Serial dilutions were used to quantitate colony numbers when necessary. Representative plate images and a schematic showing the effect on TRE-mDHFR transcription are also shown. [Figure 8B] Transcription Block Survival (TBS) assay driving functionally active cJun inhibitors. (A) Schematic showing the design and operation of the TBS. (B) Controlled numbers of E. coli expressing the indicated proteins were plated on selective media and growth rates were calculated by counting colony forming units. (1) WT-mDHFR expression can replace ecDHFR and is not inhibited by TMP, resulting in significant growth. (2) A small effect on colony numbers is observed when cJun bZIP is additionally expressed. (3) TRE-mDHFR can replace the inhibited ecDHFR with fewer colonies than WT as expected. (4) The cJun LZ domain (lacking the DBD) does not affect TRE-mDHFR transcription and colony formation, whereas (5) the cJun bZIP domain (with the DBD) binds to the TRE site and blocks the transcription of TRE-mDHFR, resulting in reduced bacterial survival. (6) cFos LZ and (7) FosW are known cJun binders, but they are unable to effectively dissociate cJun bZIP from TRE DNA. However, (8) A-FosW and the TBS-derived hit (9) HingeW remove the TRE-mDHFR transcription block and restore cell viability. Bar graphs represent the mean of three replicate experiments. Errors are shown as one standard deviation. Selected P values ​​from t-tests are shown with values ​​for all possible comparisons within the bar graphs reported in Figure S7 (*P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001). Serial dilutions were used to quantitate colony numbers when necessary. Representative plate images and a schematic showing the effect on TRE-mDHFR transcription are also shown. [Figure 9]Target and antagonist peptide sequences, and TBS library design. The cJun target sequence is shown and compared to the related off-target cFos. To facilitate optimization of cJun binding, nine residues within a 10-residue stretch (e4-g5) of the A-FosW sequence were selected for variation in the library, providing acidic, polar and hydrophobic options, resulting in a library of 131,072 members. Screening using TBS produced the "HingeW" sequence. The DBD and acidic chain regions are shown in blue or red, respectively, with the selected library option for the winner peptide highlighted in green. Residues are named by heptad number and position within a given heptad repeat. [Figure 10] TBS selection pressure shifts the presentation of library members in DNA pool sequencing towards selection of HingeW as the assay winning sequence. Sequence logos showing the relative abundance of amino acids in the first selection plate (9 colonies sequenced), first passage (6 colonies sequenced) and first winning sequence (HingeW; the only sequence present in the DNA pool and 5 colonies). [Figure 11A]The TBS winner peptide HingeW binds cJun preferentially over A-FosW. CD spectra (20 °C) show binding of cJun to either (A) HingeW or (B) A-FosW. In both cases, the heterodimer spectra show an increase in α-helical character compared to the average of the component peptides. However, the effect is greater with HingeW, indicating a greater increase in the helicity of the peptide. Similarly, thermal denaturation of cJun bound to either (C) HingeW or (D) A-FosW is shifted to the right from the average of the component peptide denaturation profiles. HingeW / cJun shows a larger ΔTm of binding than A-FosW / cJun due to the lower Tm of the HingeW homodimer (indicated by arrows). CD dimer exchange spectra show that (E) when HingeW is mixed with A-FosW / cJun heterodimers, helicity increases because HingeW exchanges with A-FosW due to preferential binding of cJun to HingeW, and (F) when A-FosW is mixed with HingeW / cJun heterodimers, no shift from the average is observed, indicating no change in dimer population. Arrows are shown to highlight the shift from the average at 190 and 222 nm. In all experiments, total sample peptide concentration was fixed at 10 μM using equimolar concentrations of each component peptide to rule out concentration-dependent effects. [Figure 11B]The TBS winner peptide HingeW binds cJun preferentially over A-FosW. CD spectra (20 °C) show binding of cJun to either (A) HingeW or (B) A-FosW. In both cases, the heterodimer spectra show an increase in α-helical character compared to the average of the component peptides. However, the effect is greater with HingeW, indicating a greater increase in the helicity of the peptide. Similarly, thermal denaturation of cJun bound to either (C) HingeW or (D) A-FosW is shifted to the right from the average of the component peptide denaturation profiles. HingeW / cJun shows a larger ΔTm of binding than A-FosW / cJun due to the lower Tm of the HingeW homodimer (indicated by arrows). CD dimer exchange spectra show that (E) when HingeW is mixed with A-FosW / cJun heterodimers, helicity increases because HingeW exchanges with A-FosW due to preferential binding of cJun to HingeW, and (F) when A-FosW is mixed with HingeW / cJun heterodimers, no shift from the average is observed, indicating no change in dimer population. Arrows are shown to highlight the shift from the average at 190 and 222 nm. In all experiments, total sample peptide concentration was fixed at 10 μM using equimolar concentrations of each component peptide to rule out concentration-dependent effects. [Figure 11C]The TBS winner peptide HingeW binds cJun preferentially over A-FosW. CD spectra (20 °C) show binding of cJun to either (A) HingeW or (B) A-FosW. In both cases, the heterodimer spectra show an increase in α-helical character compared to the average of the component peptides. However, the effect is greater with HingeW, indicating a greater increase in the helicity of the peptide. Similarly, thermal denaturation of cJun bound to either (C) HingeW or (D) A-FosW is shifted to the right from the average of the component peptide denaturation profiles. HingeW / cJun shows a larger ΔTm of binding than A-FosW / cJun due to the lower Tm of the HingeW homodimer (indicated by arrows). CD dimer exchange spectra show that (E) when HingeW is mixed with A-FosW / cJun heterodimers, helicity increases because HingeW exchanges with A-FosW due to preferential binding of cJun to HingeW, and (F) when A-FosW is mixed with HingeW / cJun heterodimers, no shift from the average is observed, indicating no change in dimer population. Arrows are shown to highlight the shift from the average at 190 and 222 nm. In all experiments, total sample peptide concentration was fixed at 10 μM using equimolar concentrations of each component peptide to rule out concentration-dependent effects. [Figure 11D]The TBS winner peptide HingeW binds cJun preferentially over A-FosW. CD spectra (20 °C) show binding of cJun to either (A) HingeW or (B) A-FosW. In both cases, the heterodimer spectra show an increase in α-helical character compared to the average of the component peptides. However, the effect is greater with HingeW, indicating a greater increase in the helicity of the peptide. Similarly, thermal denaturation of cJun bound to either (C) HingeW or (D) A-FosW is shifted to the right from the average of the component peptide denaturation profiles. HingeW / cJun shows a larger ΔTm of binding than A-FosW / cJun due to the lower Tm of the HingeW homodimer (indicated by arrows). CD dimer exchange spectra show that (E) when HingeW is mixed with A-FosW / cJun heterodimers, helicity increases because HingeW exchanges with A-FosW due to preferential binding of cJun to HingeW, and (F) when A-FosW is mixed with HingeW / cJun heterodimers, no shift from the average is observed, indicating no change in dimer population. Arrows are shown to highlight the shift from the average at 190 and 222 nm. In all experiments, total sample peptide concentration was fixed at 10 μM using equimolar concentrations of each component peptide to rule out concentration-dependent effects. [Figure 11E]The TBS winner peptide HingeW binds cJun preferentially over A-FosW. CD spectra (20 °C) show binding of cJun to either (A) HingeW or (B) A-FosW. In both cases, the heterodimer spectra show an increase in α-helical character compared to the average of the component peptides. However, the effect is greater with HingeW, indicating a greater increase in the helicity of the peptide. Similarly, thermal denaturation of cJun bound to either (C) HingeW or (D) A-FosW is shifted to the right from the average of the component peptide denaturation profiles. HingeW / cJun shows a larger ΔTm of binding than A-FosW / cJun due to the lower Tm of the HingeW homodimer (indicated by arrows). CD dimer exchange spectra show that (E) when HingeW is mixed with A-FosW / cJun heterodimers, helicity increases because HingeW exchanges with A-FosW due to preferential binding of cJun to HingeW, and (F) when A-FosW is mixed with HingeW / cJun heterodimers, no shift from the average is observed, indicating no change in dimer population. Arrows are shown to highlight the shift from the average at 190 and 222 nm. In all experiments, total sample peptide concentration was fixed at 10 μM using equimolar concentrations of each component peptide to rule out concentration-dependent effects. [Figure 11F]The TBS winner peptide HingeW binds cJun preferentially over A-FosW. CD spectra (20 °C) show binding of cJun to either (A) HingeW or (B) A-FosW. In both cases, the heterodimer spectra show an increase in α-helical character compared to the average of the component peptides. However, the effect is greater with HingeW, indicating a greater increase in the helicity of the peptide. Similarly, thermal denaturation of cJun bound to either (C) HingeW or (D) A-FosW is shifted to the right from the average of the component peptide denaturation profiles. HingeW / cJun shows a larger ΔTm of binding than A-FosW / cJun due to the lower Tm of the HingeW homodimer (indicated by arrows). CD dimer exchange spectra show that (E) when HingeW is mixed with A-FosW / cJun heterodimers, helicity increases because HingeW exchanges with A-FosW due to preferential binding of cJun to HingeW, and (F) when A-FosW is mixed with HingeW / cJun heterodimers, no shift from the average is observed, indicating no change in dimer population. Arrows are shown to highlight the shift from the average at 190 and 222 nm. In all experiments, total sample peptide concentration was fixed at 10 μM using equimolar concentrations of each component peptide to rule out concentration-dependent effects. [Figure 12] CD thermal denaturation profile showing the interaction of FosW with cJun bZIP. The thermal denaturation profile of the FosW / cJun bZIP heterodimer is shifted from the average of the two component peptide curves. It shows an increase in helicity and Tm (54 °C for the heterodimer), indicative of a binding interaction. [Figure 13] The TBS winner peptide HingeW does not interact with cFos. CD spectra and thermal denaturation curves show no interaction between HingeW and cFos when the measured heterodimer spectrum / thermal denaturation curve is overlaid with the average of the individual component spectra. [Figure 14A]Isothermal titration calorimetry data show a six-fold higher affinity for HingeW / cJun compared to A-FosW / cJun. ITC profiles of cJun binding to (A) HingeW and (B) A-FosW show the titration-corrected potency plot in the top graph and the integrated data points and single-site model fit (MicroCal ORIGIN software) in the bottom graph. [Figure 14B] Isothermal titration calorimetry data show a six-fold higher affinity for HingeW / cJun compared to A-FosW / cJun. ITC profiles of cJun binding to (A) HingeW and (B) A-FosW show the titration-corrected potency plot in the top graph and the integrated data points and single-site model fit (MicroCal ORIGIN software) in the bottom graph. [Figure 15A] HingeW antagonizes cJun / TRE DNA interactions more effectively than A-FosW. (A) CD spectra showing a shift in the TRE DNA peak at ~281 nm upon addition of cJun that is reversed by titration of HingeW to the sample, as HingeW sequesters cJun into a nonfunctional heterodimer. (B) The relative peak shift from bound to free TRE is plotted for varying concentrations of HingeW and A-FosW showing greater cJun / TRE DNA inhibition of HingeW at all concentrations. EMSA showing (C) unbound TRE DNA band shift upon addition of cJun and subsequent recovery of unbound DNA band intensity upon titration of either (D) HingeW or (E) A-FosW. (F) For both CD and EMSA, data are averaged from three independent experiments and plotted error bars indicate one standard deviation. [Figure 15B]HingeW antagonizes cJun / TRE DNA interactions more effectively than A-FosW. (A) CD spectra showing a shift in the TRE DNA peak at ~281 nm upon addition of cJun that is reversed by titration of HingeW to the sample, as HingeW sequesters cJun into a nonfunctional heterodimer. (B) The relative peak shift from bound to free TRE is plotted for varying concentrations of HingeW and A-FosW showing greater cJun / TRE DNA inhibition of HingeW at all concentrations. EMSA showing (C) unbound TRE DNA band shift upon addition of cJun and subsequent recovery of unbound DNA band intensity upon titration of either (D) HingeW or (E) A-FosW. (F) For both CD and EMSA, data are averaged from three independent experiments and plotted error bars indicate one standard deviation. [Fig. 15CDE] HingeW antagonizes cJun / TRE DNA interactions more effectively than A-FosW. (A) CD spectra showing a shift in the TRE DNA peak at ~281 nm upon addition of cJun that is reversed by titration of HingeW to the sample, as HingeW sequesters cJun into a nonfunctional heterodimer. (B) The relative peak shift from bound to free TRE is plotted for varying concentrations of HingeW and A-FosW showing greater cJun / TRE DNA inhibition of HingeW at all concentrations. EMSA showing (C) unbound TRE DNA band shift upon addition of cJun and subsequent recovery of unbound DNA band intensity upon titration of either (D) HingeW or (E) A-FosW. (F) For both CD and EMSA, data are averaged from three independent experiments and plotted error bars indicate one standard deviation. [Figure 15F]HingeW antagonizes cJun / TRE DNA interactions more effectively than A-FosW. (A) CD spectra showing a shift in the TRE DNA peak at ~281 nm upon addition of cJun that is reversed by titration of HingeW to the sample, as HingeW sequesters cJun into a nonfunctional heterodimer. (B) The relative peak shift from bound to free TRE is plotted for varying concentrations of HingeW and A-FosW showing greater cJun / TRE DNA inhibition of HingeW at all concentrations. EMSA showing (C) unbound TRE DNA band shift upon addition of cJun and subsequent recovery of unbound DNA band intensity upon titration of either (D) HingeW or (E) A-FosW. (F) For both CD and EMSA, data are averaged from three independent experiments and plotted error bars indicate one standard deviation. [Figure 16] CD antagonism data showing the shift in DNA spectra upon addition of FosW to cJun-bound DNA. The relative shift from the cJun-bound to free TRE DNA peak is monitored at 281 nm when FosW is added in turn. Data were averaged from three independent experiments. [Figure 17] HingW and A-FosW do not interact with TRE DNA. CD spectra showing no interaction between TRE DNA and either HingeW or A-FosW. Proteins do not absorb in this wavelength range and the observed CD signal arises from DNA structure and is not perturbed upon addition of either protein. [Figure 18A] Stepwise N-terminal truncation of HingeW reduces c-Jun binding and antagonism. (A) Thermal denaturation profiles of stepwise truncated peptide (5 μM) / c-Jun (5 μM) heterodimer samples. (B) CD antagonism data generated by monitoring the shift in DNA-specific peaks, providing a direct readout of cJun-induced DNA binding. [Figure 18B]Stepwise N-terminal truncation of HingeW reduces c-Jun binding and antagonism. (A) Thermal denaturation profiles of stepwise truncated peptide (5 μM) / c-Jun (5 μM) heterodimer samples. (B) CD antagonism data generated by monitoring the shift in DNA-specific peaks, providing a direct readout of cJun-induced DNA binding. [Figure 19] Tm values ​​correlate broadly with IC50 values. As peptide-cJun heterodimer (i.e., binary complex formation) Tm increases, a direct correlation is observed with improved cJun / TRE DNA antagonism (antagonism of DNA binding where ternary complex formation is blocked) as shown by lower IC50 values. [Figure 20] Thermodynamic parameters ITC data showing cJun peptide interactions. [Figure 21] Lactamization results in enhanced serum stability. The amount of peptide detected by LC-MS is plotted relative to the starting point, showing that the linear peptide degrades faster than lactamization, with doubly lactamized 24 exhibiting the highest stability. [Figure 22A] Peptide optimization quantified by CD to determine peptide helicity, c-Jun target binding and c-Jun / TRE DNA antagonism. Biophysical characteristics of selected peptides are shown to illustrate exemplary data and the effects observed through the optimization process. (A) Spectra of selected peptides (10 μM) showing increased helicity from N-terminal truncation, decreased helicity from C-terminal truncation and increased helicity through lactamization. (B) Thermal denaturation profile of selected antagonist (5 μM) / cJun (5 μM) heterodimer samples. (C) CD spectrum showing a shift in the TRE DNA peak at ~281 nm upon addition of cJun that is restored by titration of HingeW into the sample, as HingeW sequesters cJun into a non-functional heterodimer. (D) The relative peak shift from bound to free TRE is plotted at various peptide concentrations. [Figure 22B]Peptide optimization quantified by CD to determine peptide helicity, c-Jun target binding and c-Jun / TRE DNA antagonism. Biophysical characteristics of selected peptides are shown to illustrate exemplary data and the effects observed through the optimization process. (A) Spectra of selected peptides (10 μM) showing increased helicity from N-terminal truncation, decreased helicity from C-terminal truncation and increased helicity through lactamization. (B) Thermal denaturation profile of selected antagonist (5 μM) / cJun (5 μM) heterodimer samples. (C) CD spectrum showing a shift in the TRE DNA peak at ~281 nm upon addition of cJun that is restored by titration of HingeW into the sample, as HingeW sequesters cJun into a non-functional heterodimer. (D) The relative peak shift from bound to free TRE is plotted at various peptide concentrations. [Figure 22C] Peptide optimization quantified by CD to determine peptide helicity, c-Jun target binding and c-Jun / TRE DNA antagonism. Biophysical characteristics of selected peptides are shown to illustrate exemplary data and the effects observed through the optimization process. (A) Spectra of selected peptides (10 μM) showing increased helicity from N-terminal truncation, decreased helicity from C-terminal truncation and increased helicity through lactamization. (B) Thermal denaturation profile of selected antagonist (5 μM) / cJun (5 μM) heterodimer samples. (C) CD spectrum showing a shift in the TRE DNA peak at ~281 nm upon addition of cJun that is restored by titration of HingeW into the sample, as HingeW sequesters cJun into a non-functional heterodimer. (D) The relative peak shift from bound to free TRE is plotted at various peptide concentrations. [Figure 22D]Peptide optimization quantified by CD to determine peptide helicity, c-Jun target binding and c-Jun / TRE DNA antagonism. Biophysical characteristics of selected peptides are shown to illustrate exemplary data and the effects observed through the optimization process. (A) Spectra of selected peptides (10 μM) showing increased helicity from N-terminal truncation, decreased helicity from C-terminal truncation and increased helicity through lactamization. (B) Thermal denaturation profile of selected antagonist (5 μM) / cJun (5 μM) heterodimer samples. (C) CD spectrum showing a shift in the TRE DNA peak at ~281 nm upon addition of cJun that is restored by titration of HingeW into the sample, as HingeW sequesters cJun into a non-functional heterodimer. (D) The relative peak shift from bound to free TRE is plotted at various peptide concentrations. [Diagram 23] Optimization of bis-alkylated HingeW peptide variants. CD antagonism data generated by monitoring the shift from bound to free TRE provides a direct readout of cJun-induced DNA binding for circularized (mDBMBW circularized and 0W circularized) versus linear HingeW (mDBMBW linear and 0W linear) variants tested. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] Aspects and embodiments of the present invention will now be discussed with reference to the accompanying drawings. Further aspects and embodiments will be apparent to those skilled in the art. All texts mentioned in this text are incorporated herein by reference.

[0036] The c-Jun antagonists described herein typically comprise an extended hinge region having the amino acid sequence of V[X1]EE[X2][X3]LE[X4]E, more preferably LV[X1]EE[X2][X3]LE[X4]E (SEQ ID NO: 1); and a leucine zipper (LZ) region C-terminal to the extended hinge region, where X1 is V, D, K, C, or R, X2 is K, D, C, or R, X3 is V, D, C, K, or R, and X4 is selected from E, D, C, K, or R. The c-Jun antagonists are also referred to herein as 'c-Jun antagonist peptides.

[0037] Extended hinge region As used herein, the term "hinge region" is intended to mean a 10-residue amino acid stretch. The amino acid residues within the stretch may be acidic (D / E) and hydrophobic (V) residues, and K residues may be present in some cases. The "hinge region" of a c-Jun antagonist is so named because it corresponds to the "hinge"-forming portion of both the DBD and LZ domains of c-Jun, and thus can interact with both of these domains of c-Jun. The presence of the hinge region provides the peptide with the ability to bind c-Jun as well as antagonize its DNA binding activity. It is also believed that the predominant negative charge of the hinge region creates a favorable interaction with the positive charge within the c-Jun DNA binding domain (DBD).

[0038] The extended hinge region comprises a hinge region and an L residue at its N-terminal portion. The extended hinge region has an amino acid sequence of LV[X1]EE[X2][X3]LE[X3]E (SEQ ID NO: 1), where X1 is selected from V, D, K, C, and R, X2 is selected from D, K, C, and R, X3 is selected from V, D, C, K, and R, and X4 can be selected from E, D, C, K, and R. In some embodiments, X1 is selected from V, D, K, and C, X2 is selected from K, D, and C, X3 is selected from V, D, or C, and X4 is selected from E, D, C, or R. It is believed that the additional negatively charged amino acid residues favor the interaction with the positively charged DBD of c-Jun. Variants of the extended hinge region sequences described herein are also contemplated, and the variants contain one, two, or three amino acid modifications.

[0039] In some embodiments, X1 is V and / or X3 is V. In some embodiments, X2 is D and X4 is E. For example, the extended hinge region can comprise the amino acid sequence of LVVEEDVLEEE (SEQ ID NO: 31).

[0040] In other embodiments, X2 is K and X4 is D. For example, the extended hinge region can comprise the amino acid sequence of LVVEEKVLEDE (SEQ ID NO: 32). Such an amino acid sequence can be used, for example, to introduce a KD lactam bridge from i to i+4 at the bf heptad position of the hinge region of the antagonist.

[0041] In other embodiments, X1 is K and X3 is D. For example, the extended hinge region can comprise the amino acid sequence of LVKEEDDLEEE (SEQ ID NO: 33). Such an amino acid sequence can be used, for example, to introduce a KD lactam bridge from i to i+4 at the fc heptad position of the hinge region of the antagonist.

[0042] In other embodiments, X2 is C and X4 is C. For example, the extended hinge region can comprise the amino acid sequence of LVVEECVLECE (SEQ ID NO: 34). Such an amino acid sequence can be used, for example, to introduce an i to i+4 alkyl bridge bond at the bf heptad position of the hinge region of the antagonist.

[0043] In other embodiments, X1 is C and X3 is C. For example, the extended hinge region can comprise the amino acid sequence of LVCEEDCLEEE (SEQ ID NO: 35). Such an amino acid sequence can be used, for example, to introduce an i to i+4 alkyl bridge bond at the fc heptad position of the hinge region of the antagonist.

[0044] In other embodiments, X1 is C and X4 is C. For example, the extended hinge region can comprise the amino acid sequence of LVCEEDVLECE (SEQ ID NO: 36). Such an amino acid sequence can be used, for example, to introduce an i to i+7 alkyl bridge bond at the ff heptad position of the hinge region of the antagonist.

[0045] In some embodiments, one or more of X1, X2, X3, and X4 are R or K. In one embodiment, X 4 is R or K. For example, the extended hinge region may comprise the amino acid sequence of LVVEEKVLERE (SEQ ID NO: 71). As explained below, introducing arginine or lysine into solvent exposed positions may increase the cell permeability of the peptide.

[0046] acidic chain According to the invention, the extended hinge region may further comprise an N-terminal acidic chain having an amino acid sequence of EA[X5][X6] (SEQ ID NO:2), where X5 is selected from E, K and C, and X6 is selected from E, D and C. In some embodiments, X6 is selected from E and D (e.g., X6 is E).

[0047] The N-terminal acidic chain is thought to generate electrostatic repulsion, advantageously reducing the tendency of the peptide to homodimerize, thereby making the peptide antagonist more available for heterodimerization with c-Jun. The negative charge across the N-terminal domain of the acidic chain works well with the positive charge of the c-Jun DBD.

[0048] In some embodiments, X1 (in the extended hinge region) is D and X5 (in the acidic chain) is K. Such amino acid sequences can be used, for example, to introduce a KD lactam bridge from i to i+4 spanning the acidic chain (heptad position b) and the hinge region (heptad position f) of the antagonist.

[0049] In some embodiments, X1 (in the extended hinge region) is C and X5 (in the acidic chain) is C. Such amino acid sequences can be used, for example, to introduce an i to i+4 alkyl bridge bond across the acidic chain (heptad position b) and hinge region (heptad position f) of the antagonist.

[0050] According to the invention, the acidic chain may have the amino acid sequence EAEE (SEQ ID NO: 3), which is believed to induce helicity and stabilize the dipole of the molecule. An additional advantage of the EAEE sequence is that its two central residues AE occur in positions on cJun that correspond to the interaction with DNA, thereby forming a direct block between c-Jun and DNA.

[0051] LZ area The LZ region of the antagonist of the present invention is located C-terminal to the hinge region and can interact with the leucine zipper of c-Jun.

[0052] According to the present invention, the LZ region is IEQLEERNYALRKEIEDLQKQ (SEQ ID NO:4); IEQLEEKNKALKDEIEDLQKQ (SEQ ID NO:5); IKQLEDRNYALRKEIEDLQKQ (SEQ ID NO:6); IEQLEERNYALRKEIKDLQDQ (SEQ ID NO: 7); IEQLEEKNKALKDEIEDLY (SEQ ID NO: 8); and IEQLEERNYALRKEIEDLQ (SEQ ID NO: 9), or may comprise or consist of an amino acid sequence selected from the group consisting of these variants. The variant may comprise one or more amino acid modifications. For example, the variant may comprise 1, 2, 3, 4, or 5 amino acid modifications. For example, the variant may comprise 1, 2, or 3 amino acid modifications.

[0053] In some embodiments, the LZ region comprises or consists of the amino acid sequence of IEQLEERNYALRKEIKDLQDQ (SEQ ID NO:7), or a variant containing 1, 2, or 3 modifications. In some embodiments, the amino acid residues at positions corresponding to positions 16 (position b of a heptad) and 20 (position f of the same heptad) of SEQ ID NO:7 in the variant are K and D amino acid residues, respectively (i.e., the amino acid modification(s) are at positions other than those corresponding to positions 16 and 20 of SEQ ID NO:7).

[0054] In some embodiments, the LZ region comprises or consists of the amino acid sequence of IEQLEERNYALRKEICDLQCQ (SEQ ID NO:27), or a variant containing 1, 2, or 3 modifications. In some embodiments, the amino acid residues at positions corresponding to positions 16 (position b of a heptad) and 20 (position f of the same heptad) of SEQ ID NO:27 in the variant are both C amino acid residues (i.e., the amino acid modification(s) are at positions other than those corresponding to 16 and 20 of SEQ ID NO:27).

[0055] In some embodiments, the LZ region is: IEQLEERNYALR[X7]E[X8]K[X9]L[X 10 ]D[X 11 ] (SEQ ID NO: 29) or IEQLEERNYALR[X7]E[X8]C[X9]L[X 10 ]C[X 11] (SEQ ID NO: 30) {where: [X7] is K, L, S, W, P, Q, R, M, T, V, A, E, or G; [X8] is I, V, or L; [X9] and [X 10 ] is any amino acid residue; [X 11 ] is Q, E, or K} Includes.

[0056] In some embodiments, the LZ region is: IEQLEERNYALRSEICSLQCQ (SEQ ID NO:66); or IEQLEERNYALRKEICELSCQ (SEQ ID NO:67); or IEQLEERNYALRAEICNLSCQ (SEQ ID NO:68); or IEQLEERNYALRTEICSLMCK (SEQ ID NO:69); or IEQLEERNYALRAEICSLQCQ (SEQ ID NO: 70) Includes.

[0057] Such LZ regions are suitable for bis-alkylation, as described in more detail below. In some embodiments, the LZ region comprises one or more lysine(s) and / or arginine(s) at the b, c, and / or f positions of the heptads of the LZ region. In some embodiments, the lysine(s) or arginine(s) are located at positions other than those used to introduce cross-links (e.g., lactam bridges or bis-alkylations). As described in more detail below, introduction of positively charged amino acids to the solvent-exposed surface of an α-helical peptide can improve cell permeability.

[0058] Thus, in some embodiments, the LZ region comprises or consists of the amino acid sequence of IRRLERRNRALRKEIKDLQDQ (SEQ ID NO: 74), or a variant comprising one, two, or three modifications. In some embodiments, the amino acid residues at positions corresponding to positions 16 (position b of a heptad) and 20 (position f of the same heptad) of SEQ ID NO: 74 of the variant are K and D amino acid residues, respectively (i.e., the amino acid modification(s) are at positions other than those corresponding to positions 16 and 20 of SEQ ID NO: 74). In some embodiments, the amino acid residues at positions corresponding to positions 2 (position b of a heptad) and 3 (position c of the same heptad) and 9 (position b of a heptad) of SEQ ID NO: 74 of the variant are K or R (optionally R) (i.e., the amino acid modification(s) are at positions other than those corresponding to positions 2, 3, and 9 of SEQ ID NO: 74). In some embodiments, the amino acid modification(s) are at positions other than those corresponding to positions 2, 3, 9, 16, and 20 of SEQ ID NO: 74.

[0059] In other embodiments, the LZ region comprises or consists of the amino acid sequence of IERLERRNYRLRREIKDLQDQ (SEQ ID NO: 75), or a variant containing 1, 2, or 3 modifications. In some embodiments, the amino acid residues at positions corresponding to positions 16 (position b of a heptad) and 20 (position f of the same heptad) of SEQ ID NO: 75 of the variant are K and D amino acid residues, respectively (i.e., the amino acid modification(s) are at positions other than those corresponding to positions 16 and 20 of SEQ ID NO: 75). In some embodiments, the amino acid residues at positions corresponding to positions 3 (position c of a heptad), 10 (position c of a heptad), and 13 (position f of a heptad) of SEQ ID NO: 75 of the variant are K or R (optionally R) (i.e., the amino acid modification(s) are at positions other than those corresponding to positions 3, 10, and 13 of SEQ ID NO: 75). In some embodiments, the amino acid modification(s) is at a position other than positions corresponding to 3, 10, 13, 16 and 20 of SEQ ID NO:75.

[0060] Antagonists and Properties The c-Jun antagonists described herein are peptidic and can be in D or L form. As used herein, "peptidic" includes compounds that are composed of or contain a linear chain of amino acids linked by peptide bonds, and may be any peptide, polypeptide or protein. The amino acid residues that form the peptidic antagonist may be composed of D or L form amino acid residues, or a mixture of both. In this specification, peptidic compounds are typically referred to as peptides.

[0061] The c-Jun antagonists described herein may be isolated, meaning free from contaminants such as other polypeptides and / or cellular components. The c-Jun antagonists described herein can be in the free form or in any pharmacologically acceptable salt form, such as an acid salt, a metal salt, an alkaline earth metal salt, or an amine salt.

[0062] The c-Jun antagonist may be between 10 and 100 amino acid residues in length. The c-Jun antagonist may be less than 70 amino acids in length, preferably less than 60 amino acids in length, more preferably less than 55 amino acids in length, even more preferably less than 50 amino acids in length, even more preferably less than 45 amino acids in length, even more preferably less than 40 amino acids in length. The c-Jun antagonist may be between 30 and 70 amino acid residues in length, between 30 and 60 amino acid residues in length, between 30 and 50 amino acid residues in length, or between 30 and 40 amino acid residues in length. For example, the c-Jun antagonist can have a length of 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 amino acids. In certain embodiments, the c-Jun antagonist has a length of 36 amino acids.

[0063] The c-Jun antagonist can be a HingeW peptide, or a variant thereof. The HingeW peptide comprises the amino acid sequence LEQRAEELARENEELEKEAEELVVEEDVLEEEIEQLEERNYALRKEIEDLQKQLEKL (SEQ ID NO: 12).

[0064] The HingeW peptide may further comprise one or more of the following: an N-terminal MAS, a C-terminal GAP, and a C-terminal 6xHis tag (HHHHHH) (SEQ ID NO: 53). As described herein, the HingeW peptide has been demonstrated to bind to the target c-Jun protein with high affinity and antagonize the DNA binding function of c-Jun, and thus is a functional antagonist of c-Jun.

[0065] c-Jun antagonists can be truncated forms of HingeW peptide or variants thereof.As described herein, various truncated HingeW peptides have been developed and demonstrated to be functional antagonists of HingeW.Although the functional antagonism of these truncated forms is reduced compared to HingeW peptide, the truncated peptides appear to exhibit more drug-like characteristics compared to full-length HingeW peptide, indicating that these truncated forms also present effective therapeutic candidates for antagonizing c-Jun function.

[0066] As used herein, a "functional antagonist" of c-Jun is a peptide compound that can bind to c-Jun and inhibit its DNA binding activity. Methods for identifying functional antagonist peptides include the Transcription Block Survival (TBS) assay described in Example 1. Briefly, in TBS, the coding region of the essential gene dihydrofolate reductase (DHFR) is mutated to introduce a TRE site, and introduction of c-Jun into this gene in E. coli results in a transcription block that abrogates cell proliferation. The c-Jun molecule bound to the TRE site sterically prevents RNA polymerase from transcribing the essential gene, which can only be restored upon introduction of an effective c-Jun / TRE antagonist. As a result, the survival of a particular cell is controlled by the ability of peptide library members to remove the transcription block of c-Jun and thus restore DHFR activity. Thus, TBS facilitates the identification of therapeutically valuable sequences. Further details of the TBS assay are provided in WO2020128015, which is incorporated by reference in its entirety.

[0067] Other methods for determining c-Jun antagonism include circular dichroism (CD) assays, as described in Example 2. Briefly, this assay involves preparing samples containing the peptide and the TRE-DNA construct (GTCAGTCAGTGACTCAATCGGTCA) (SEQ ID NO: 51), and measuring the signal between 265-320 nm. The TRE-DNA construct produces a positive CD peak at ~281 nm, the intensity of which decreases upon c-Jun binding. If the peptide is able to antagonize c-Jun DNA binding activity, increasing the concentration of the peptide will shift the peak back to the free TRE-DNA peak. Thus, the peak shift can be used to quantitate the ability of the peptide to antagonize c-Jun DNA binding. This method determines the IC by fitting the titration data to the Hill equation. 50 Allows values ​​to be calculated.

[0068] In some embodiments, the c-Jun antagonist is capable of inhibiting the DNA binding activity of c-Jun within 10-fold of the ability of HingeW to inhibit the DNA binding activity of c-Jun (e.g., the c-Jun antagonist has a reduced ability to inhibit the DNA binding activity of c-Jun that is within 10-fold of that determined for HingeW). In some embodiments, the c-Jun antagonist is capable of inhibiting the DNA binding activity of c-Jun within 9-fold, preferably within 8-fold, more preferably within 7-fold, even more preferably within 6-fold, and even more preferably within 5-fold of the ability of HingeW to inhibit the DNA binding activity of c-Jun. The ability of the c-Jun antagonist and HingeW to inhibit DNA binding activity can be determined by measuring the IC using a TBS assay (e.g., by quantitating the number of colonies) or using the circular dichroism assay described herein. 50 The activity can be measured by determining the activity of c-Jun antagonists. Optionally, the c-Jun antagonists can have this activity when cross-linked. Methods for cross-linking peptides are described in more detail below.

[0069] In some embodiments, the c-Jun antagonist has the following amino acid sequence: LEQRAEELARENEELEKEAEELVVEEDVLEEEIEQLEERNYALRKEIEDLQKQLEKL (SEQ ID NO: 12); LARENEELEKEAEELVVEEDVLEEEIEQLEERNYALRKEIEDLQKQLEKL (SEQ ID NO: 13); LEKEAEELVVEEDVLEEEIEQLEERNYALRKEIEDLQKQLEKL (SEQ ID NO: 14); EAEELVVEEDVLEEEIEQLEERNYALRKEIEDLQKQLEKL (SEQ ID NO: 15); LVVEEDVLEEEIEQLEEKNKALKDEIEDLQKQLEKLY (SEQ ID NO: 16); LVVEEDVLEEEIEQLEERNYALRKEIEDLQKQLEDL (SEQ ID NO: 17); EAEELVVEEDVLEEEIEQLEERNYALRKEIEDLQKQ (SEQ ID NO: 18); KEAEDLVVEEDVLEEEIEQLEERNYALRKEIKDLQDQ (SEQ ID NO: 19); EAKELVDEEDVLEEEIEQLEERNYALRKEIEDLQKQ (SEQ ID NO:20); EAEELVVEEKVLEDEIEQLEERNYALRKEIEDLQKQ (SEQ ID NO:21); EAEELVVEEDVLEEEIKQLEDRNYALRKEIEDLQKQ (SEQ ID NO:22); EAEELVVEEDVLEEEIEQLEEKNKALKDEIEDLQKQY (SEQ ID NO:23); EAEELVVEEDVLEEEIEQLEERNYALRKEIKDLQDQ (SEQ ID NO: 10); EAEELVVEEKVLEDEIEQLEERNYALRKEIKDLQDQ (SEQ ID NO: 11); EAEELVVEEDVLEEEIEQLEERNYALRKEIEDL (SEQ ID NO:24); EAEELVVEEDVLEEEIEQLEEKNKALKDEIEDLY (SEQ ID NO:25); EAEELVVEEDVLEEEIEQLEERNYALRKEIEDLQ (SEQ ID NO:26); EAEELVVEEDVLEEEIEQLEERNYALRKEICDLQCQ (SEQ ID NO:28); EAEELVVEEDVLEEEIEQLEERNYALRSEICSLQCQ (SEQ ID NO:37); EAEELVVEEDVLEEEIEQLEERNYALRKEICELSCQ (SEQ ID NO:38); EAEELVVEEDVLEEEIEQLEERNYALRAEICNLSCQ (SEQ ID NO:39); EAEELVVEEDVLEEEIEQLEERNYALRTEICSLMCK (SEQ ID NO: 40); or EAEELVVEEDVLEEEIEQLEERNYALRAEICSLQCQ (SEQ ID NO: 41); EAEELVVEEKVLEREIRRLERRNRALRKEIKDLQDQ (SEQ ID NO: 72); or EAEELVVEEKVLEDEIERLERRNYRLRREIKDLQDQ (SEQ ID NO: 73), or comprises or consists of any one of these variants containing 1, 2 or 3 amino acid alterations, optionally wherein the 1, 2 or 3 amino acid alterations are in the LZ region.

[0070] As described herein, the c-Jun antagonist amino acid sequence can be modified to introduce covalent i to i+4 crosslinker(s) or i to i+7 crosslinker(s) into the c-Jun antagonist. The covalent i to i+4 amino acid crosslinker(s) can be KD lactam bridge(s) or alkyl bridge bond (cysteine ​​alkylation) formed between two C residues. Preferably, the i to i+4 amino acid residue crosslink is introduced at the solvent exposed bf (within one heptad) or fc (spanning two heptads) heptad position to prevent the destruction of the binding surface of the helix. Herein, the heptad number refers to the position of a particular amino acid residue within a heptad repeat, a structural motif consisting of a repeating pattern of seven amino acids. The position of the heptad repeat is generally indicated by lowercase letters a to g, typically abcdefg. Table 5 below shows how the heptad numbers correspond to the HingeW amino acid sequence.

[0071] Thus, provided herein is a c-Jun antagonist comprising a modified version of the amino acid sequence EAEELVVEEDVLEEEIEQLEERNYALRKEIEDLQKQ (SEQ ID NO: 18), the modification being as follows: (i) the amino acid residues at positions corresponding to positions 3 (position b of a heptad) and 7 (position f of the same heptad) of SEQ ID NO:18 are K and D amino acid residues, respectively, or both are C amino acid residues; (ii) the amino acid residues at positions corresponding to positions 10 (position b of a heptad) and 14 (position f of the same heptad) of SEQ ID NO:18 are K and D amino acid residues, respectively, or both are C amino acid residues; (iii) the amino acid residues at positions corresponding to positions 17 (position b of a heptad) and 21 (position f of the same heptad) of SEQ ID NO:18 are K and D amino acid residues, respectively, or both are C amino acid residues; (iv) the amino acid residues at positions corresponding to 24 (position b of a heptad) and 28 (position f of the same heptad) of SEQ ID NO: 18 are K and D amino acid residues, respectively, or both are C amino acid residues; and (v) the amino acid residues at positions corresponding to positions 31 (position b of a heptad) and 35 (position f of the same heptad) of SEQ ID NO:18 are K and D amino acid residues, respectively, or both are C amino acid residues; or variants thereof that contain one, two, three or four amino acid alterations outside the recited positions (i.e., outside the b and f positions of each heptad).

[0072] Also provided herein is a c-Jun antagonist comprising a modified version of the amino acid sequence EAEELVVEEDVLEEEIEQLEERNYALRKEIEDLQKQ (SEQ ID NO: 18), the modification being as follows: (i) the amino acid residues at positions corresponding to positions 7 (position f of a heptad) and 11 (position c of the following heptad) of SEQ ID NO: 18 are K and D amino acid residues, respectively, or both are C amino acid residues; (ii) the amino acid residues at positions corresponding to positions 14 (position f of a heptad) and 18 (position c of the following heptad) of SEQ ID NO:18 are K and D amino acid residues, respectively, or both are C amino acid residues; (iii) the amino acid residues at positions corresponding to 21 (position f of a heptad) and 25 (position c of the next heptad) of SEQ ID NO: 18 are K and D amino acid residues, respectively, or both are C amino acid residues; and (iv) the amino acid residues at positions corresponding to 28 (position f of a heptad) and 32 (position c of the following heptad) of SEQ ID NO:18 are K and D amino acid residues, respectively, or both are C amino acid residues; or variants thereof that contain one, two, three or four amino acid modifications outside the recited positions (i.e., outside the f position of one heptad and the c position of the following heptad).

[0073] Also provided are c-Jun antagonists whose amino acid sequence has been modified to introduce a i to i+7 crosslinker(s) into the c-Jun antagonist. The covalent i to i+7 amino acid crosslinker(s) can be an alkyl crosslink (cysteine ​​alkylation) formed between two C residues. Preferably, the i to i+7 amino acid residue crosslink is introduced into the solvent exposed bb, cc or ff (spanning two heptads) heptad position.

[0074] Thus, provided herein is a c-Jun antagonist comprising a modified version of the amino acid sequence EAEELVVEEDVLEEEIEQLEERNYALRKEIEDLQKQ (SEQ ID NO: 18), the modification being as follows: (i) the amino acid residues at positions corresponding to positions 3 (position b of the heptad) and 10 (position b of the following heptad) of SEQ ID NO:18 are both C amino acid residues; (ii) the amino acid residues at positions corresponding to positions 10 (position b of the heptad) and 17 (position b of the following heptad) of SEQ ID NO:18 are both C amino acid residues; (iii) the amino acid residues at positions corresponding to positions 17 (position b of a heptad) and 24 (position b of the following heptad) of SEQ ID NO:7 are both C amino acid residues; (iv) the amino acid residues at positions corresponding to 24 (position b of a heptad) and 31 (position b of the following heptad) of SEQ ID NO:7 are both C amino acid residues; (v) the amino acid residues at positions corresponding to positions 4 (the c position of a heptad) and 11 (the c position of the following heptad) of SEQ ID NO:18 are both C amino acid residues; (vi) the amino acid residues at positions corresponding to positions 11 (the c position of a heptad) and 18 (the c position of the following heptad) of SEQ ID NO:18 are both C amino acid residues; (vii) the amino acid residues at positions corresponding to positions 18 (the c position of a heptad) and 25 (the c position of the subsequent heptad) of SEQ ID NO:18 are both C amino acid residues; (viii) the amino acid residues at positions corresponding to 25 (position c of a heptad) and 32 (position c of the subsequent heptad) of SEQ ID NO:18 are both C amino acid residues; (ix) the amino acid residues at positions corresponding to positions 7 (position f of a heptad) and 14 (position f of the following heptad) of SEQ ID NO:18 are both C amino acid residues; (x) the amino acid residues at positions corresponding to positions 14 (position f of a heptad) and 21 (position f of the following heptad) of SEQ ID NO:18 are both C amino acid residues; (xi) the amino acid residues at positions corresponding to 21 (position f of a heptad) and 28 (position f of the following heptad) of SEQ ID NO: 18 are both C amino acid residues; and (xii) the amino acid residues at positions corresponding to 28 (position f of a heptad) and 35 (position f of the following heptad) of SEQ ID NO:18 are both C amino acid residues; or variants thereof that contain 1, 2, 3 or 4 amino acid alterations outside the recited positions (i.e., therefore outside positions b, c or f).

[0075] In a preferred embodiment, the c-Jun antagonist is EAEELVVEEDVLEEEIEQLEERNYALRKEIEDLQKQLEKL (SEQ ID NO: 15); EAEELVVEEDVLEEEIEQLEERNYALRKEIEDLQKQ (SEQ ID NO: 18); EAEELVVEEDVLEEEIEQLEERNYALRKEIKDLQDQ (SEQ ID NO: 10); EAEELVVEEKVLEDEIEQLEERNYALRKEIKDLQDQ (SEQ ID NO: 11); or EAEELVVEEDVLEEEIEQLEERNYALRKEICDLQCQ (SEQ ID NO: 28), or comprises or consists of the amino acid sequences of these variants containing 1, 2 or 3 amino acid alterations, optionally wherein the 1, 2 or 3 amino acid alterations are in the LZ region.

[0076] In a more preferred embodiment, the c-Jun antagonist is EAEELVVEEDVLEEEIEQLEERNYALRKEIKDLQDQ (SEQ ID NO: 10); EAEELVVEEKVLEDEIEQLEERNYALRKEIKDLQDQ (SEQ ID NO: 11); or EAEELVVEEDVLEEEIEQLEERNYALRKEICDLQCQ (SEQ ID NO: 28), or comprises or consists of the amino acid sequences of these variants containing 1, 2 or 3 amino acid alterations, optionally wherein the 1, 2 or 3 amino acid alterations are in the LZ region.

[0077] For example, in embodiments in which the c-Jun antagonist comprises a KD lactam bridge, the c-Jun antagonist is: EAEELVVEEDVLEEEIEQLEERNYALRKEIKDLQDQ (SEQ ID NO: 10) or comprising or consisting of an amino acid sequence of a variant thereof comprising one, two or three amino acid alterations, optionally wherein the one, two or three amino acid alterations are present in the LZ region, and optionally wherein the amino acid residues at positions corresponding to positions 31 (position b of a heptad) and 35 (position f of a heptad) of SEQ ID NO: 10 of the variant are K and D amino acid residues, respectively.

[0078] As another example, in embodiments in which the c-Jun antagonist comprises two KD lactam bridges, the c-Jun antagonist may be EAEELVVEEKVLEDEIEQLEERNYALRKEIKDLQDQ (SEQ ID NO: 11) or comprising or consisting of the amino acid sequence of a variant thereof comprising one, two or three amino acid alterations, optionally in the LZ region; Further optionally, the amino acid residues at positions corresponding to 10 (position b of the first heptad) and 14 (position f of the first heptad) of SEQ ID NO:11 of the variant are K and D amino acid residues, respectively, and the amino acid residues at positions corresponding to 31 (position b of the second heptad) and 35 (position f of the second heptad) of SEQ ID NO:11 of the variant are K and D amino acid residues, respectively.

[0079] As another example, in embodiments in which the c-Jun antagonist comprises an alkyl cross-link, the c-Jun antagonist may be EAEELVVEEDVLEEEIEQLEERNYALRKEICDLQCQ (SEQ ID NO: 28) or comprises or consists of an amino acid sequence of a variant thereof comprising one, two or three amino acid alterations, optionally wherein the one, two or three amino acid alterations are present in the LZ region, and optionally wherein the amino acid residues at positions corresponding to positions 31 (position b of a heptad) and 35 (position f of the same heptad) of the variant of SEQ ID NO:28 are both C amino acid residues.

[0080] As another example, in embodiments in which the c-Jun antagonist comprises an alkyl cross-link, the c-Jun antagonist can be: EAEELVVEEDVLEEEIEQLEERNYALRSEICSLQCQ (SEQ ID NO: 37); or EAEELVVEEDVLEEEIEQLEERNYALRKEICELSCQ (SEQ ID NO: 38); or EAEELVVEEDVLEEEIEQLEERNYALRAEICNLSCQ (SEQ ID NO: 39); or EAEELVVEEDVLEEEIEQLEERNYALRTEICSLMCK (SEQ ID NO: 40); or EAEELVVEEDVLEEEIEQLEERNYALRAEICSLQCQ (SEQ ID NO: 41) or comprises or consists of the amino acid sequences of these variants containing 1, 2 or 3 amino acid alterations, optionally wherein the amino acid alterations are in the LZ region.

[0081] Amino acid modification may be insertion, substitution or deletion.In some embodiments, amino acid modification is the substitution of amino acid residue with any other amino acid residue.Substituted amino acid residue may be D or L form, and may be naturally occurring or non-naturally occurring amino acid residue.

[0082] Naturally occurring residues can be divided into classes based on common side chain properties: 1) Non-polar, aliphatic (hydrophobic): glycine (G), methionine (M), alanine (A), valine (V), leucine (L), isoleucine (I); 2) polar, uncharged: cysteine ​​(C), serine (S), threonine (T), asparagine (N), glutamine (Q), proline (P); 3) Acidic (negative charge); aspartic acid (D), glutamic acid (E); 4) Basic (positive charge); histidine (H), lysine (K), arginine I; 5) Aromatic; tryptophan (W), tyrosine (Y), phenylalanine (F).

[0083] The amino acid substitution may be a conservative amino acid substitution. A conservative amino acid substitution may involve the exchange of a member of one of these classes with another member of the same class. For example, a conservative amino acid substitution may be the substitution of the acidic amino acid aspartic acid (D) with the acidic amino acid glutamic acid (E).

[0084] Amino acid substitutions (e.g., conservative amino acid substitutions) may include non-naturally occurring amino acid residues that are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. Suitable non-natural amino acids include 3-cyclohexylalanine (Cha), norleucine (NLe) and ornithine (Orn). Other examples of non-natural amino acids include citrulline (Cit), hydroxyproline (Hyp), 3-nitrotyrosine, nitroarginine naphthylalanine (Nal), Abu, DAB, methionine sulfoxide and methionine sulfone.

[0085] In some embodiments, the amino acid modification results in the introduction of hydrophobic and charged surface patches of the peptide. Hydrophobic and charged surface patches can be introduced by inserting a cluster of hydrophobic and / or positively charged amino acid residues (e.g., at least three consecutive residues), as described for example in Perry et al., 2018. For example, the amino acid modification described herein can produce a c-Jun antagonist that contains at least three consecutive amino acid residues that are either lysine or leucine (e.g., an extended hinge region and / or leucine region).

[0086] In cross-linked c-Jun antagonists, any amino acid modifications (e.g., substitutions) are typically located outside the relevant positions used for cross-linking. That is, antagonists containing a bf (in one heptad) amino acid residue cross-link may have the amino acid modification(s) at a, c, d, e, or g positions within that heptad. Similarly, antagonists containing a fc (across two heptads) amino acid residue cross-link may have the amino acid modification(s) at any of the a, b, c, d, or e positions within the first heptad and the a, b, d, e, f, or g positions within the second heptad.

[0087] It is known in the art that the introduction of positively charged amino acids into the solvent-exposed surface of α-helical peptides improves cell permeability (see, e.g., Smith et al., 2008 and Perry et al., 2018). This can be achieved by the introduction of arginine residues at specific positions to generate arginine substitution patterns known to promote cell permeability, as described in Smith et al., 2008.

[0088] Thus, in some embodiments, the peptides described herein comprise one or more arginine or lysine substitutions. In some embodiments, the extended hinge region and / or LZ region comprise one or more arginine or lysine modifications, i.e., arginine or lysine substitution patterns can be introduced into the peptides described herein. In some embodiments, these arginine modifications are located at heptad b, c, and / or f positions, i.e., on the solvent-exposed surface of the α-helical peptide.

[0089] In some embodiments, the c-Jun antagonist peptides described herein comprise a modified version of the amino acid sequence set forth in SEQ ID NO:11, wherein the modification is as follows: (i) the amino acid residues at positions corresponding to 14 (position f of a heptad), 17 (position b of a heptad), 18 (position c of a heptad), and 24 (position b of a heptad) are K or R residues (optionally R). For example, the c-Jun antagonist peptide may have the amino acid sequence EAEELVVEEKVLEREIRRLERRNRALRKEIKDLQDQ (SEQ ID NO: 72); or (ii) the amino acid residues at positions corresponding to 18 (c position of a heptad), 25 (c position of a heptad), and 28 (f position of a heptad) are K or R residues (optionally R). For example, a c-Jun antagonist can have the amino acid sequence EAEELVVEEKVLEDEIERLERRNYRLRREIKDLQDQ (SEQ ID NO: 73); or variants thereof that contain one or more (e.g., one or two) of the above, or one, two, three or four amino acid modifications outside the recited positions. Optionally, the amino acid residues at positions corresponding to positions 31 (position b of a heptad) and 35 (position f of a heptad) of SEQ ID NO:72 or 73 of the variants are K and D amino acid residues, respectively (i.e., the amino acid modification(s) are at positions other than those corresponding to positions 14, 17, 18, 31 and 35 of SEQ ID NO:72, or at positions other than those corresponding to positions 18, 25, 28, 31 and 35 of SEQ ID NO:73).

[0090] Alternatively or additionally, the c-Jun antagonist may have an amino acid sequence having a particular degree of sequence identity to one of SEQ ID NOs: 12-26. The particular degree of sequence identity may be at least 60% to 100% sequence identity. More preferably, the particular degree of sequence identity may be at least one of 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0091] Antagonist production The c-Jun antagonist peptides described herein may be provided using synthetic or recombinant techniques that are standard in the art. Conveniently, the c-Jun peptides described herein may be produced by solid-phase synthesis. Peptides are typically synthesized by stepwise solid-phase synthesis from the C-terminus to the N-terminus. In the first step, an N-protected amino acid is covalently attached to an insoluble solid support via its carbonyl group. Suitable N-protecting groups for amino acids include 9-fluoromethyloxycarbonyl (Fmoc) and t-butyloxycarbonyl (Boc). After covalent attachment of the N-protected amino acid, the N-protecting group is removed and deprotected, and the NH2 group of the attached amino acid is reacted with the carboxylic acid group of the next N-protected amino acid to generate a nascent peptide that includes two amino acids covalently attached to the solid phase. This process is repeated until the complete peptide sequence is assembled on the solid phase. In some embodiments, protecting groups may be used to prevent the reaction of functional groups of the side chains of amino acids with new N-protected amino acids. These side chain protecting groups may be present throughout the synthesis of the peptide and may be removed in a final deprotection step.

[0092] A method for producing a c-Jun antagonist peptide can include synthesizing a peptide comprising SEQ ID NO:1 by solid-phase or solution-phase peptide synthesis. Methods for solid phase peptide synthesis are well established in the art (see, e.g., Coin et al Nature Protocols 2, 3247-3256 (2007); Stawikowski (2002) Curr Protoc Protein Sci. 2002 Unit-18.1. oi:10.1002 / 0471140864. ps1801s26; Chan and White; Fmoc Solid Phase Peptide Synthesis - A Practical Approach. Oxford University Press, 2000; Stewart, JM; Young, JD Solid-Phase Peptide Synthesis (2nd ed.), Pierce Chemical Co., Rockford, IL, 1984; Atherton, E.; Sheppard, RC, Solid-Phase Peptide Synthesis: A Practical Approach. Oxford University Press: New York City, 1989; M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis. Synthesis, Springer Verlag, New York (1984); JH Jones, The Chemical Synthesis of Peptides. Oxford University Press, Oxford 1991; in Applied Biosystems 430A User's Manual, ABI Inc., Foster City, California; GA Grant, (Ed.) Synthetic Peptides, A User's Guide. WH Freeman & Co., New York 1992, and GB Fields, (Ed.) Solid-Phase Peptide Synthesis (Methods in Enzymology Vol. 289).Academic Press, New York and London 1997); see Merrifield, J. Amer. Chem. Soc. 85:2149-54(1963). Methods for solution phase peptide synthesis are also well established in the art (U.S. Pat. No. 5,516,891).

[0093] The c-Jun antagonist peptides described herein can be produced using recombinant expression. Recombinant techniques for producing peptides are standard in the art, for example, as described in Sambrook, J., Russel, DW Molecular Cloning, A Laboratory Manual. 3 ed. 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press. The c-Jun antagonist peptides can be capped, for example, at the N-terminus with a MAS residue and at the C-terminus with a GAP residue. The c-Jun antagonist peptides described herein can be further His-tagged (for example, 6xHis-tagged).

[0094] c-Jun The antagonist peptide described herein antagonizes c-Jun. c-Jun is involved in many cellular processes, including differentiation, proliferation, and survival (Shaulian et al., 2001; Eferl et al., 2003; Eckert et al., 2013; Alani et al., 1991). Human c-Jun has been well characterized in the art and can have, for example, the amino acid sequence of (UniProt accession P05412, version 2.).

[0095] Peptide Cross-Linking Agents Residues present on the surface of proteins responsible for PPIs associate with protein secondary structure motifs, such as alpha-helices, beta-sheets, and beta-turns. Of note, alpha-helices are thought to comprise approximately 60% of all secondary structures in protein complexes (Jochim and Arora, 2010). Thus, alpha-helices have been shown to mediate many important therapeutically relevant PPI interfaces, 60% of which bind to one surface of the helix (Raj et al., 2013). Alpha-helices contain hydrogen bonds between the carbonyl group (C=O) of a given amino acid and the amino group (NH) of an amino acid 3 or 4 residues away.

[0096] It has been reported that constraining peptides into a helical conformation using cross-linking agents confers advantages including enhanced protease resistance, intracellular stability, increased cellular uptake, enhanced biophysical properties, and is expected to bind to their targets with higher potency compared to wild-type peptide sequences (Azzarito et al. 2013). As a result, peptides containing constrained alpha-helices (also called "helix-constrained peptides") have received great attention for the identification of PPI inhibitors (Robertson and Spring, 2018).

[0097] Thus, in some embodiments, the c-Jun antagonist peptide compound is a helix-constrained peptide. The term "helix-constrained peptide" is intended to mean a peptide having at least one chemical modification that results in an intermolecular cross-link between two amino acids to produce a stable alpha-helix. Typically, the cross-link extends over the length of one or two helical turns (i.e., about 3-3.6 or about 7 amino acids). Thus, amino acids located at i and one of i+3, i+4, and i+7 are ideal candidates for cross-linking. Thus, for example, a peptide may have the sequence ...N1, N2, N3, N4, N5, N6, N7, N8, N9..., where amino acid N is selected independently for each position, and cross-links between N1 and N4, or between N1 and N5, or between N1 and N8 are useful, as are cross-links between N2 and N5, or between N2 and N6, or between N2 and N9, etc. The use of multiple cross-links (e.g., 2, 3, 4 or more) is also contemplated. Thus, as used herein, a helix-constrained peptide comprises at least one cross-linker between two amino acid residues.

[0098] Chemical modifications include those that incorporate molecular tethers, such as hydrocarbon staples, and those that promote the formation of disulfide bonds. The cross-links may be ionic, covalent, or hydrogen bonds that link two residues together, preferably the cross-links are covalent bonds.

[0099] The presence of a stabilized alpha-helix can be determined using methods such as circular dichroism spectroscopy of alpha-helices, for example, as described in Jo et al. (2012) as an example herein. Circular dichroism is used to measure the increase in helicity, i.e., from linear to cyclic. In situations where cross-linking occurs by the formation of a disulfide bond between two thiol groups, such as between two cysteine ​​residues, the presence of a stable alpha-helix can also be determined using an assay that determines whether the thiol in the sample is free or conjugated. For example, free thiols can be assayed via reaction with Ellman's reagent (5,5'-dithiobis(2-nitrobenzoic acid; DNTB) (Sigma)) and by monitoring absorbance at 412 nm.

[0100] Methods for inducing cross-links between amino acids are well known and include methods for inducing cross-links between the peptide backbone, e.g., between the carbonyl and amino groups as in natural alpha-helices, as well as between the side chains of the peptide.

[0101] Crosslinkers include disulfide bonds (e.g., as described in Leduc et al. (2003)), hydrogen bond surrogates (e.g., as described in Wang et al. (2005)), ring-closing metathesis (e.g., as described in Walensky et al. (2004)), cysteine ​​alkylation using α-haloacetamide derivatives (e.g., as described in Woolley (2005)) or biaryl halides (e.g., as described in Muppidi et al. (2011)), lactam rings (e.g., as described in Fujimoto et al. (2008)), hydrazine bonds (e.g., as described in Cabezas & Satterthwait (1999)), oxime bonds (e.g., as described in Haney et al. (2011)), metal chelators (e.g., as described in Ruan et al. (1990)), and "click" chemistry (e.g., as described in Holland-Nell & Meldal (2011)).

[0102] Cross-linking agents can be used to cross-link cysteine ​​residues. Thus, a peptide can contain cysteines (C) at positions i and i+4, or i and i+7 of its amino acid sequence. As described in Jo et al. (2012), the introduction of cysteine ​​residues at positions i and i+4 is useful because this spacing brings two thioether residues close to the alpha-helix. Suitable cross-linking agents for stabilizing alpha-helices in peptides containing cysteines (C) at positions i and i+4 are described in Jo et al. (2012). For example, the cross-linking agent can be a cross-linking agent selected from the group consisting of alkyl bromides, alkyl iodides, benzyl bromides, aryl bromides, maleimides, and electrophilic difluorobenzenes. Suitable cross-linking agents are known in the art for cross-linking cysteines (see, for example, Fairlie & Dantas de Araujo, 2016 and Jo et al., 2012).

[0103] In some embodiments, the cross-linking agent is m-xylene-based, o-xylene-based, or p-xylene-based benzyl bromide, more preferably m-xylene-based benzyl bromide.

[0104] In some embodiments, the crosslinker has Formula 1:

[0105] [ka]

[0106] [In the formula, n is an integer selected from 1 to 3; m is an integer selected from 0 to 2; A is C 2~6 -Alkenylene, C 5~12 -Arylene and C 5~12 -heteroarylene; Y is a covalent bond, C 1~6 -alkylene or -N(H)C(=O)CH2-; R 1is selected from Cl, Br, I, or F; and Each L is -C(=O)-, -C≡C-, -N≡N-, C 1~6 and independently selected from an alkylene and a covalent bond. It is a compound of the formula:

[0107] R 1 The A group provides a reactive group (e.g., a leaving group) for reaction with cysteine. The A group provides a linker with a suitable structure to conformationally constrain the peptide as required when crosslinked via two derivatizable amino acid residues. For example, the A group can be conformationally constrained into a shape suitable for linking two derivatizable amino acid residues. In some embodiments, R 1 In some embodiments, A is C 5~12 -Arylene and C 5~12 -heteroarylene. In some embodiments, m is 0. In some embodiments, Y is methylene. In some embodiments, L is a covalent bond.

[0108] In some preferred embodiments, the cross-linking agent has the following chemical formula:

[0109] [ka]

[0110] The compound having the formula: 1,3-dibromomethylbenzene (DBMB). DBMB can be used to react with derivatizable amino acid residues at i and i+3 or i and i+4 of the amino acid sequence of the peptide.

[0111] In some preferred embodiments, the cross-linking agent has the following chemical formula:

[0112] [ka]

[0113] and 4,4'-bisbromomethyl-biphenyl (Bpy), having the formula: Bpy can be used to react with derivatizable amino acid residues at i and i+7 of the amino acid sequence of a peptide.

[0114] Cross-linking of cysteine ​​residues of peptides may be performed using known methods, such as those described in Timmerman et al., 2005 or WO2021 / 260074. Briefly, the method may include reacting the peptide with a cross-linker (e.g., DBMB) in the presence of tris(2-carboxyethyl)phosphine (TCEP) and ammonium bicarbonate, at pH 8.0 and room temperature in the dark for 4-5 hours. The method may be performed in vitro or in cellulo. In cellulo methods may include providing cells (e.g., bacterial cells, such as E. coli cells, or eukaryotic cells, such as human cells) containing the recombinant peptide, contacting the cells with a cross-linker (e.g., as part of a cell culture medium), and culturing the cells in the presence of the cross-linker. The cross-linker may be present at a concentration between 1 μM and 1 mM (e.g., between 10 μM and 100 μM) for a period of at least 20 minutes (e.g., between 20 minutes and 10 hours). Further details of suitable in cellulo cross-linking methods are provided, for example, in WO2021 / 260074.

[0115] The crosslinker is a c-Jun antagonist having the structure:

[0116] [ka]

[0117] The thioether bridges at least one pair of cysteines so that the thioether bridges may include Y, L, R 1 , n, m and A are as defined in Formula 1. 1a represents a bond or a CH2-CH2- linker derived from the appropriate R1 group of formula 1.

[0118] In embodiments where the alkyl bridge bond formed between the two C residues is formed by DBMB, the c-Jun antagonist has the structure:

[0119] [ka]

[0120] may include. A cross-linking agent may be used to cross-link lysine (K) and aspartic acid (D) of a peptide. Thus, a peptide may contain lysine (K) and aspartic acid (D) at positions i and i+4 of its amino acid sequence. That is, position i is lysine (K) and position i+4 is aspartic acid (D), or position i is aspartic acid (D) and position i+4 is lysine (K). For example, position b of one heptad may be K and position f is D, or position f of one heptad may be K and position c of the following heptad may be D.

[0121] Lactamization is useful in terms of in vivo stability due to the constraint of providing an additional steric block and avoiding backbone access (Tyndall JD et al., 2005), as well as potential bioavailability and membrane permeability due to the lipophilicity of the constraint, since proteases generally recognize β-strands. The lactam bridge of a peptide refers to the side chain of a lysine (K) that forms an amide bond with the side chain of a glutamic acid (E) or aspartic acid (D), typically aspartic acid (D). Methods for performing KD lactamization are described in the Examples herein and, for example, in Araujo et al. (2014).

[0122] As mentioned above, the cross-links may be formed between the amino acids at positions i and i+3, i and i+4, or i and i+7 of the amino acid sequence of the peptide. In some embodiments, the cross-links are between the cysteine ​​(C) residues located at these positions. In other embodiments, the cross-links are between the lysine (K) and aspartic acid (D) residues at these positions. Preferably, the cross-links are formed between the amino acids at positions i and i+4.

[0123] nucleic acid As used herein, a nucleic acid encoding a c-Jun antagonist peptide can be any nucleic acid (DNA or RNA).

[0124] Conjugates In some embodiments, the c-Jun antagonist may be conjugated, optionally by a linker, to another moiety, such as a fatty acid or other lipid, a polymer, or another peptide sequence (e.g., a cell-penetrating peptide (CPP)). Such a conjugate may retain the functional antagonist properties of the c-Jun antagonist and may have one or more improved properties, such as stability, in vivo half-life, or potency, or cell permeability, compared to the unconjugated c-Jun antagonist. The moiety may be conjugated to the c-Jun antagonist by the N- or C-terminus, or any other site of the peptide.

[0125] In some embodiments, the peptide can be conjugated to a cell penetrating peptide (CPP). CPPs are a class of peptides that can penetrate the cell membrane of mammalian cells and transport compounds of many types and molecular weights through the membrane. When CPPs are chemically linked or fused to other proteins, the resulting polypeptides can enter cells. The linkage to the CPP can be direct (e.g., as part of a fusion protein) or through a linker (e.g., a short peptide linker). CPPs are generally peptides of less than 30 amino acids and are derived from natural or non-natural proteins or chimeric sequences. Examples of CPPs include tat (PGRKKRRQRRPPQ) (SEQ ID NO: 54), penetratin (RQIKIWFQNRRMKWKK) (SEQ ID NO: 55), transportan (GWTLNSAGYLLGKINLKALAALAKKIL) (SEQ ID NO: 56), VP-22 (DAATATRGRSAASRPTERPRAPARSASRPRRPVD) (SEQ ID NO: 57), Pep-1 (KETWWETWWTEWSQPKKKRKV) (SEQ ID NO: 58), MAP (KALA Suitable CPPs include KALAKALA (SEQ ID NO: 59), SAP (VRLPPPVRLPPPVRLPPP) (SEQ ID NO: 60), oligoarginine (RRRRRRRR (SEQ ID NO: 61) or RRRRRRRRR (SEQ ID NO: 62)), calcitonin (LGTYTQDFNKTFPQTAIGVGAP) (SEQ ID NO: 63), SynB (RGGRLSYSRRRFSTSTGR (SEQ ID NO: 64)), and Pvec (LLIILRRRIRKQAHAHSK (SEQ ID NO: 65)). These and other suitable CPPs are described in Heitz et al. 2009.

[0126] In some embodiments, peptides can be conjugated to lipids. Peptide ligation is an effective strategy to modify the pharmacokinetics, pharmacodynamics and cell permeability of peptide therapeutics and has been shown to be successful with some therapeutic peptides. Cholesterol and fatty acids of various chain lengths, such as C8-caprylic acid, C12-lauric acid and C16-palmitic acid, are often utilized as lipid motifs that are covalently linked to peptide inhibitors via ester, ether, amide or carbamate bonds. Examples of peptide ligation are described in Kowalczyk et al. 2017.

[0127] Pharmaceutical Compositions Functionally active antagonists of c-Jun of the present invention may be useful in inhibiting c-Jun in a therapeutic setting.Accordingly, the c-Jun antagonist peptides of the present invention may be formulated into pharmaceutical compositions.

[0128] A pharmaceutical composition is a formulation that includes one or more active agents (e.g., a c-Jun antagonist peptide or conjugate described herein) and one or more pharma- ceutically acceptable excipients. A pharmaceutical composition may be capable of eliciting a therapeutic effect.

[0129] A pharmaceutical composition can include a c-Jun antagonist peptide or conjugate of the invention and a pharma- ceutically acceptable excipient or carrier. A method of making a pharmaceutical composition may comprise the step of mixing the c-Jun antagonist peptide or conjugate described above with a pharma- ceutically acceptable excipient.

[0130] The term "pharmacologically acceptable" refers to compounds, substances, compositions, and / or dosage forms that are, within the scope of veterinary or medical judgment, suitable for use in contact with the tissues of a subject (e.g., a human or other mammal) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable risk-benefit ratio. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

[0131] Suitable excipients and carriers include, but are not limited to, water, saline, buffered saline, phosphate buffer, alcoholic / aqueous solutions, emulsions or suspensions. Other conventionally used diluents, adjuvants, and excipients can be added according to conventional techniques. Such carriers can include ethanol, polyols and suitable mixtures thereof, vegetable oils, and injectable organic esters. Buffers and pH adjusters may be used, including, but are not limited to, salts prepared from organic acids or bases. Representative buffers include, but are not limited to, organic acid salts, such as citric acid (e.g., citrate salts), ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, phthalic acid salts, Tris, trimethylamine hydrochloride, or phosphate buffers. Parenteral carriers can include sodium chloride solution, Ringer's dextrose, dextrose, trehalose, sucrose, lactated Ringer's, or fixed oils. Intravenous carriers may include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents (e.g., EGTA; EDTA), inert gases, and the like, may also be provided in the pharmaceutical carrier. The pharmaceutical compositions described herein are not limited by the selection of the carrier. The preparation of these pharma- ceutical acceptable compositions, from the above ingredients, with appropriate pH, isotonicity, stability and other conventional characteristics is within the skill of the art.

[0132] Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts, such as Remington's Pharmaceutical Sciences and The Handbook of Pharmaceutical Excipients, 4th edit., eds. RC Rowe et al, APhA Publications, 2003.

[0133] The term "carrier" refers to diluents, binders, lubricants and disintegrants. Those skilled in the art are familiar with such pharmaceutical carriers and how to formulate pharmaceutical compositions using such carriers. Pharmaceutical composition can be conveniently presented in unit dosage form and can be prepared by any method well known in the field of pharmacy.Such method includes the step of combining peptide with the above-mentioned carrier or excipient that can constitute one or more accessory components.Generally, formulation is prepared by uniformly and intrinsically combining active compound with liquid carrier or finely divided solid carrier or both.

[0134] The pharmaceutical compositions described herein may be produced in various forms depending on the route of administration. The pharmaceutical compositions may be prepared for administration to a subject in the form of, for example, liquid, powder, aerosol, tablet, capsule, enteric-coated tablet or capsule, or suppository. The pharmaceutical compositions may be in the form of suspensions, solutions, emulsions in oily or aqueous media, pastes, and implantable sustained release or biodegradable preparations. Compositions for sustained release or implantation may contain pharma- ceutical acceptable polymeric or hydrophobic substances, such as emulsions, ion exchange resins, sparingly soluble polymers, or sparingly soluble salts. The pharmaceutical compositions may be made in the form of sterile aqueous solutions or dispersions suitable for injectable use, or may be made in lyophilized form using lyophilization techniques. Lyophilized pharmaceutical compositions are typically kept at about 4°C and may be reconstituted in a stable liquid, such as saline or HEPES, with or without adjuvants. The pharmaceutical compositions may be made in the form of suspensions or emulsions.

[0135] The pharmaceutical compositions may be presented in unit-dose or multi-dose hermetically sealed containers, for example, ampoules and vials and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of sterile liquid carriers, for example water for injections immediately prior to use.

[0136] The pharmaceutical composition may be administered to a subject by any conventional route of administration.In some embodiments, administration is by systemic route, including oral, or more preferably by parenteral route.For example, the pharmaceutical composition may be administered by intravenous, intraperitoneal or subcutaneous injection.

[0137] Treatment of Disease c-Jun plays a role in many cellular processes, such as differentiation, proliferation, and survival, and dysregulation of this transcription factor can therefore lead to a wide range of human diseases.Therefore, the c-Jun antagonist peptide, nucleic acid, conjugate, or pharmaceutical composition described herein may be for use in a method of treating c-Jun-mediated diseases in an animal or human body, such as an individual in need thereof.

[0138] An individual with a c-Jun-mediated disease may show at least one identifiable sign, symptom, or laboratory finding that is sufficient for the diagnosis of a c-Jun-mediated disorder according to clinical criteria known in the art. Examples of such clinical criteria may be found in medical textbooks, such as Harrison's Principles of Internal Medicine, 15th Ed., Fauci AS et al., eds., McGraw-Hill, New York, 2001. In some embodiments, an individual may be previously identified or diagnosed as having a c-Jun-mediated disorder, or the method of the present invention may include identifying or diagnosing the presence of a c-Jun-mediated disorder in an individual, prognosing a c-Jun-mediated disorder, or assessing the risk of developing a c-Jun-mediated disorder in an individual.

[0139] Treatment may be any treatment or therapy of a human or animal (e.g., veterinary application) in which some desired therapeutic effect is achieved, e.g., inhibition or slowing of the progression of a c-Jun mediated disease, including slowing the rate of progression, halting the rate of progression, alleviating a c-Jun mediated disease, curing or ameliorating (partially or in full) a c-Jun mediated disease, preventing, slowing, reducing or arresting one or more symptoms and / or signs of a c-Jun mediated disease, or prolonging the survival of a subject or patient beyond that expected in the absence of treatment.

[0140] Treatment as a preventative measure (i.e., prevention) is also included (e.g., treatment before the onset of a condition in an individual to reduce the risk of developing the condition in the individual; delay its onset; or reduce its severity after onset). For example, an individual who is susceptible to or at risk of developing or recurring a c-Jun-mediated disease, such as cancer, can be treated as described herein. Such treatment can prevent or delay the onset or recurrence of a c-Jun-mediated disease, or one or more symptoms thereof, in an individual.

[0141] The c-Jun antagonist peptides may be used in methods of treatment of any one of the following diseases: cancer, diabetes, cardiovascular disease, autoimmune disease, joint disorders (eg, arthritis), and neurodegenerative diseases.

[0142] "Cancer" includes the following: acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), adrenal cortical carcinoma, anal cancer, bladder cancer, blood cancer, bone cancer, brain tumor, breast cancer, cancer of the female reproductive system, cancer of the male reproductive system, central nervous system lymphoma, cervical cancer, childhood rhabdomyosarcoma, childhood sarcoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), colorectal cancer, colon cancer, endometrial cancer, endometrial sarcoma, esophageal cancer, eye cancer, gallbladder cancer, stomach cancer, gastrointestinal cancer, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, Hodgkin's disease, hypopharyngeal cancer, Kaposi's sarcoma, kidney cancer, pharyngeal cancer, leukemia, liver cancer, lung cancer, malignant fibromyalgia, The cancers may include any one or more of the following: fibrous histiocytoma, malignant thymoma, melanoma, mesothelioma, multiple myeloma, myeloma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, nervous system cancer, neuroblastoma, non-Hodgkin's lymphoma, oral cavity cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pituitary tumors, plasmacytoma, primary CNS lymphoma, prostate cancer, rectal cancer, respiratory system, retinoblastoma, salivary gland cancer, skin cancer, small intestine cancer, soft tissue sarcoma, gastric cancer, stomach cancer, testicular cancer, thyroid cancer, urinary system cancer, uterine sarcoma, vaginal cancer, vasculature, Waldenstrom's macroglobulinemia, and Wilms' tumor.

[0143] The cancer may be of a particular type. Examples of cancer types include astrocytoma, carcinoma (e.g., adenocarcinoma, hepatocellular carcinoma, medullary carcinoma, papillary carcinoma, squamous cell carcinoma), glioma, lymphoma, medulloblastoma, melanoma, myeloma, meningioma, neuroblastoma, sarcoma (e.g., angiosarcoma, chrondrosarcoma, osteosarcoma).

[0144] In some embodiments, the individual may have minimal residual disease (MRD) after an initial cancer treatment. Cancer treatment may include inhibiting cancer growth, including complete cancer remission, and / or inhibiting cancer metastasis. Cancer growth generally refers to any one of many indicators that indicate changes in cancer to a more developed form. Thus, indicators that measure inhibition of cancer growth include reduced cancer cell survival, reduced tumor volume or morphology (e.g., as determined using computed tomography (CT), ultrasound, or other imaging methods), slowed tumor growth, disruption of tumor vasculature, improved performance in delayed-type skin hypersensitivity test, increased activity of T cells, and reduced levels of tumor-specific antigens.

[0145] In some embodiments, the c-Jun antagonist peptides may be useful in inhibiting or reducing metastasis of cancer. For example, a method of reducing or inhibiting metastasis in an individual having cancer comprises administering to the individual a therapeutically effective amount of a c-Jun peptide.

[0146] An individual suitable for the above treatment may be a mammal, such as a rodent (e.g., guinea pig, hamster, rat, mouse), murine (e.g., mouse), canine (e.g., dog), feline (e.g., cat), equine (e.g., horse), primate, simian (e.g., monkey or ape), monkey (e.g., marmoset, baboon), ape (e.g., gorilla, chimpanzee, orangutan, gibbon), or human.

[0147] In some preferred embodiments, the individual is a human. In other preferred embodiments, a non-human mammal may be used, particularly a mammal that is conventionally used as a model to demonstrate therapeutic effects in humans (e.g., a mouse, a primate, a pig, a dog, or a rabbit animal).

[0148] The method according to the present invention may be carried out or the product may be present in vitro, ex vivo, or in vivo. The term "in vitro" is intended to include experiments with materials, biological substrates, cells and / or tissues in laboratory conditions or in culture, while the term "in vivo" is intended to include experiments and procedures with intact multicellular organisms. "Ex vivo" refers to something that exists or occurs outside of an organism, for example, outside the human or animal body, and may be tissues (e.g., whole organs) or cells taken from an organism.

[0149] When the method is performed in vitro, it may involve high throughput screening assays.The test compounds used in the method may be obtained from a synthetic combinatorial peptide library or may be synthetic peptides or peptibomimetic molecules.

[0150] The features in the foregoing description, or disclosed in the following claims, or in the accompanying drawings, set forth in terms of their specific forms or means for performing the disclosed functions, or methods or processes for obtaining the disclosed results, may, as appropriate, be utilized separately or in any combination of such features to realize the invention in its diverse forms.

[0151] Although the present invention has been described in conjunction with the above exemplary embodiments, many equivalent modifications and variations will be apparent to those skilled in the art in view of this disclosure. Accordingly, the above described exemplary embodiments of the present invention are considered to be illustrative and not limiting. Various modifications to the described embodiments may be made without departing from the spirit and scope of the present invention.

[0152] For the avoidance of doubt, any theoretical explanations provided herein are provided for the purpose of improving the understanding of the reader, and the inventors do not wish to be bound by any of these theoretical explanations.

[0153] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Throughout this specification, including the claims which follow, unless the context requires otherwise, the terms "comprise" and "include", as well as variations such as "comprises", "comprising", and "including", will be understood to include the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps.

[0154] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when a value is expressed as approximately, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. With respect to numerical values, the term "about" is optional and means, for example, + / - 10%.

[0155] array

[0156] [Table 1-1]

[0157] [Table 1-2]

[0158] [Table 1-3] EXAMPLES

[0159] Particular aspects and embodiments are now illustrated, by way of example, and with reference to the above-mentioned drawings. Example 1 –Identification of functional peptide antagonists Many rational design approaches, library screening, and selection systems exist and have led to successful identification of molecules capable of binding to a given TF target, but a significant challenge remains ensuring that target binding leads to loss of function (Brennan et al., 2020;Baxter et al., 2014). Various methods have produced peptide-based c-Jun antagonists that target the broad LZ binding interface (Boysen et al., 2002;Mason et al., 2006;Kaplan et al., 2014;Baxter et al., 2017;Lathbridge et al., 2018). However, it is difficult to predict whether LZ binding will lead to functional antagonism, as the c-Jun DBD remains unbound and can bind to TRE DNA (Seldeen et al., 2008;Szaloki N et al., 2015). Rationally designed peptides have been shown to target the c-Jun DBD, but they exhibit lower potency than LZ antagonists, and there are concerns regarding specificity due to the high sequence similarity across AP-1 family DBDs (Tsuchida et al., 2004). Similarly, a broad range of SMs have been developed to target TRE DNA (Dai et al., 2004; Fanjul et al., 1994), but these too are less potent and may produce off-target effects. This is because multiple TFs typically bind to any given DNA element, although some bZIP / DNA combinations are known to promote anticancer outcomes (Eferl et al., 2003; Rodriguez-Martinez JA et al., 2017). One approach to circumvent the potential downsides of these methods is to utilize longer peptides that target the full-length c-Jun bZIP domain with selective but high-affinity interactions, while simultaneously blocking both DNA binding and LZ dimerization.Olive et al. used this approach to generate A-Fos, which combines the wild-type (WT) cFos LZ (known to heterodimerize with c-Jun) and a rationally designed Glu-rich acidic chain (Figure 1) (Olive et al., 1997). The A-Fos design principle is premised on the LZ interaction being extended N-terminally, resulting in a DBD acidic chain interaction facilitated by the incorporation of a Leu residue at the putative d-position of the acidic chain. An intracellular Transcription Block Survival (TBS) library screening assay was developed and validated to search for functional TF antagonists, with cell survival occurring only if TF activity was abolished. Furthermore, bacterial growth rates correlate with antagonist efficiency, allowing comparison and competition between TF antagonists. Here we present an approach using a large peptide library (131,027 members) that demonstrates that they can be screened within the TBS platform for functional c-Jun antagonism. Selected peptides are evaluated using a wide range of biophysical techniques showing clear improvements over the parent peptide in target binding and c-Jun / TRE DNA antagonism, particularly driven by reduced homodimer stability. The following methods were used: 1.1 Method Plasmid constructs and protein production: TRE-mDHFR (Figure 2) and WT-mDHFR DNA constructs were subcloned into the pQE16 derivative plasmid pES300d; c-Jun LZ and c-Jun bZIP DNA constructs were subcloned into the pQE16 derivative plasmid pES230d; and cFos LZ and A-FosW DNA constructs were subcloned into pET24a. The human c-Jun bZIP domain is encoded by the Arg 252 From Leu 308 The LZ domain is located in the Ile 277 From Leu 308 The human cFos bZIP domain spans Glu 137 From Leu 193 The LZ domain is 162 From Leu 193A-FosW has the following sequence: LEQRAEELARENEELEKEAEELEQELDELQAEIEQLEERNYALRKEIEDLQKQLEKL (bold is the FosW sequence). All constructs are capped at the N-terminus with an AS residue and at the C-terminus with a GAP residue and are also 6xHis tagged, except for the WT or TRE-mDHFR constructs which are only 6xHis tagged. A complete list of sequences is provided in Table 1.

[0160] [Table 2]

[0161] Proteins were purified by subcloning their DNA sequences into either the pET21-His-SUMO plasmid (c-Jun bZIP, cFos bZIP) or the pET24a plasmid (HingeW, A-FosW, FosW) using the NheI and AscI sites. An overnight culture of E. coli containing the relevant plasmid was used to inoculate LB medium at a dilution ratio of 1:1000. The cultures reached an OD 600nmThe cultures were incubated with shaking (37°C, 200 rpm) until the NA reached 0.7. Protein overexpression was induced by addition of IPTG (1 mM) before incubation with shaking (25°C, 200 rpm) overnight. Cells were then harvested from the cultures by centrifugation. The cell pellet was resuspended in HisTrap binding buffer (20 mM potassium phosphate, 500 mM NaCl, 40 mM imidazole, 5 mM DTT, pH 7.4), sonicated, and loaded onto a HisTrap HP 5 mL preloaded column. The column was washed with binding buffer before eluting the protein sample with a binding buffer:elution buffer (20 mM potassium phosphate buffer, 500 mM NaCl, 400 mM imidazole, 5 mM DTT, pH 7.4) gradient. Using this method, we also produced a ∼80% pure sample of His-tagged ULP1 protease for use in the SUMO cleavage step. SUMO-tagged proteins were buffer exchanged into standard buffer (20 mM Tris.HCl, 2 mM DTT, pH 8.0). A 10:1 mixture of SUMO-tagged protein:ULP1 was incubated at 30°C for 16 hours. SUMO-tagged constructs were His-tagged at the N-terminus of SUMO, and the cleavage reaction was diluted 1:5 in binding buffer and then passed through a HisTrap column to remove cleaved SUMO tag and His-tagged ULP1. The HisTrap flow-through was finally purified to >98% purity by using a RP-HPLC equipped with a Jupiter Proteo column (particle size 4 μm, pore size 90 Å, 250 × 10 mm; Phenomenex) using a water:acetonitrile gradient (0.1% TFA). Peptides without SUMO tags were enriched after HisTrap elution and HPLC purification. Peptide purity and identity were assessed by SDS-PAGE and electrospray ionization mass spectrometry.

[0162] DHFR activity assay: The activity of purified DFHR enzyme was measured using a colorimetric kit (Sigma CD0340). WT or TRE-mDHFR (100 nM in the reaction) and NADPH (60 μM in the reaction) were mixed with assay buffer alone or with the DHFR inhibitors TMP or Mtx (1 μM in the reaction). The reaction was started by the addition of DHF (50 μM in the reaction + blank reaction without DHF) and the absorbance of the samples at 340 nm was measured using a Varian Cary 50 UV-Vis spectrophotometer. Specific activity was calculated using the following formula:

[0163]

number

[0164] Library construction and TBS assay: Library inserts were generated using a PCR fill in reaction from synthetic primers (Sigma) with degenerate codons at the desired positions to generate the correct residue options. The library was subcloned into a pET24a plasmid containing A-FosW using SacI and AscI sites. The primers used were cJun-Hinge-Lib-F: 5'-GAAGAGCTCSWGSWGSWGSWTSWGCTGSWGGMASWGATTGAACAGCTGGAAGAACGCAACTATGCC-3' (SEQ ID NO: 49) and cJun-Hinge-Lib-R: 5'-TGAGGCGCGCCCAGTTTCTCCAGCTGTTTCTGGAGGTCTTCGATCTCTTTGCGCAAGGCATAGTTGCGTTC-3' (SEQ ID NO: 50). Library DNA was transformed into NEB-10 beta electrocompetent E. coli cells. The following formula was used:

[0165]

number

[0166] Library coverage was determined by the number of single colonies using the formula: where E is the percentage of library loss, m is the number of colonies recovered, and n is the library size. This indicated that 99.9% of the Hinge library was covered from 2,155,000 library colonies recovered. The quality of the library DNA was assessed by sequencing both the DNA pool and many single colonies, which showed degenerate codons at the correct positions in the pool, indicating diverse library members from the single colonies. The library DNA pool was transformed into BL21 Gold cells already containing pES300d-TRE-mDHFR and pES230d-cJun bZIP.

[0167] Selection pressure is applied by growing bacteria in M9 mineral medium containing TMP (2-4 μM) along with ampicillin, kanamycin and chloramphenicol to maintain the required plasmids, and IPTG (1 mM) to induce protein expression. Library transformants are first plated onto selective agar plates (2 μM TMP) and grown at 37 °C for 72-96 h. Optimization experiments (Figure 5) show that 4 μM TMP is optimal for selection, although low stringency should be used initially prior to selection and increased in subsequent steps. Colonies from this first stage selection are pooled and assayed at a starting OD 600 Grown in liquid culture at a stepwise rate of OD 0.05 600The cultures were grown at 37°C with shaking at 200 rpm until an OD of 0.6 was reached. The TMP concentration was 2 μM in the first liquid culture passage before being increased to an optimal 4 μM in the next passage. Bacteria containing the most effective functional antagonists would produce high levels of TRE-mDHFR providing a growth advantage; these would dominate in the culture. At each passage step, samples of the cultures were plated on LB agar (which supplements antibiotics and maintains the plasmid), individual colonies were selected and sequenced, and DNA pools were also sequenced. This allows the emergence of library members to be monitored while winning sequences are selected. The assay evaluation experiments utilized a modified assay method whereby overnight cultures from glycerol stocks of the cultures (Table 2) reached an OD 600 = 0.5 and 50 μL was plated onto selective M9 minimal medium agar plates.

[0168] [Table 3]

[0169] Circular dichroism (CD): Applied Photophysics Chirascan was used for CD measurements with 200 μL of sample in a CD cell with 1 mm path length. Protein / DNA samples were suspended in 150 mM potassium phosphate, 150 mM potassium fluoride, and 5 mM TCEP, pH 7.4, and equilibrated for 30 min before measurement. For all spectra, three scans between 190 and 260 nm (265–320 nm for DNA binding experiments) were collected with a bandwidth of 1 nm and were then measured for 0.5 s. -1 The data were sampled at a rate of 0.01 μm / s. These scans were averaged and converted to molar residue ellipticity (MRE). Thermal denaturation experiments were performed by measuring the ellipticity at 222 nm over a gradient from 1 to 90 °C in 1 °C increments. Post-melting scans at 20 °C confirmed that the transition was reversible if they overlapped within 10% of the pre-melting scan. The resulting thermal denaturation curves were converted to MREs and fitted to a two-state model derived from a modification of the Gibbs-Helmholtz equation to estimate the melting temperature (T m) was determined (Mason et al., 2007).

[0170] Isothermal titration calorimetry (ITC): Peptides were tested in ITC buffer consisting of 10 mM potassium phosphate, 150 mM potassium fluoride, and 5 mM TCEP at pH 7.4. Using a MicroCal VP-ITC instrument (Malvern), 10 μL injections of antagonist peptides (HingeW or A-FosW) at 10 μM were injected into cells containing 1 μM cJun. Heat changes upon addition were recorded and analyzed using MicroCal Origin software. Control experiments included injection of antagonist peptide samples into cells containing ITC buffer only to determine the heat of dilution that was subtracted. The resulting binding data were fitted to a one site binding model to determine the enthalpy change of binding (ΔH) and the equilibrium binding constant (K D ) were extracted from which the free energy change of binding (ΔG) and the entropy change of binding (ΔS) were calculated (Wiseman et al., 1989). Thermodynamic parameters are presented as the average of two independent experiments, with errors given as one standard deviation.

[0171] Electrophoretic mobility shift analysis (EMSA): The following double-stranded oligonucleotide sequences were used: TRE: 5'-GTCAGTCAGTGACTCAATCGGTCA (SEQ ID NO: 51), control non-TRE: 5'-CCTGCGTAGTTCCATAAGGATAGC (SEQ ID NO: 52) (Sigma). Complementary single strands of DNA were purchased (Sigma), mixed in a 1:1 ratio, and then heated to 95°C for 20 minutes before slowly cooling to room temperature to form DNA duplexes. Protein / DNA samples for electrophoresis were incubated in binding buffer (150 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, 10 mM Tris, 10 mM MgCl2, pH 8) for 30 minutes at 4°C before being run on a 1.3% agarose gel in 0.5xTBE buffer (supplemented with 10 mM MgCl2). SYBR® Green stain was included in the gel and running buffer to stain DNA and imaged on a transilluminator before adding SYPRO® Ruby and incubating for 3 hours to stain proteins. Gels were destained in a 10% methanol, 7% acetic acid solution for 1 hour before imaging on a transilluminator.

[0172] 1.2 Creation of active mDHFR from TRE-containing genes that promote cJun forced transcription block Transcription Block Survival (TBS) is an intracellular assay that utilizes cell survival as a readout. It allows protein-DNA interaction antagonists to be screened and the most active ones to be identified by their ability to remove the transcription block to exogenous mouse dihydrofolate reductase (DHFR). This enzyme is absolutely essential for survival, as it is required for the production of purines required for DNA and amino acid synthesis. Endogenous E. coli DHFR (ecSHFR) is selectively inhibited by trimethoprim (TMP), meaning that cells grown in M9 minimal medium become dependent on exogenous mouse DHFR (mDHFR) activity for their survival (Matthews et al., 1985). We generated the mDHFR gene by rational design introducing 15 TRE sites into the coding DNA sequence (Figures 2 and 3), allowing a strong cJun transcription block with minimal changes to the expressed protein (TRE-mDHFR). In particular, the resulting TRE-mDHFR construct was produced with two silent mutations and 13 conservative mutations. Using the WT-mDHFR crystal structure (PDB code: 1U72) (Cody et al., 2005) as a design guide, residues thought to be important for 7,8-dihydrofolate (DHF) substrate or nicotinamide adenine dinucleotide phosphate (NADPH) cofactor binding (e.g., A10, L23, W25, and R71) (Cody et al. 2005; Thillet et al. 1988) were not altered. The accessible surface areas of all other amino acid residues were calculated using the 'Accessible Surface Area and Accessibility Calculation for Protein' tool (http: / / cib.cf.ocha.ac.jp / bitool / ASA / ). A cutoff score of 20 Å2 was implemented, below which residues were considered buried from solvent exposure and therefore more likely to cause disruption if altered. Of the remaining non-essential, surface-exposed residues, mutations were only tolerated if the R group changes were relatively conservative.The exact substitutions allowed for inclusion in order to incorporate a TRE site were as follows: F32S, T40Q, S42D, G46S, K64S, R78Q, Q103S, M112S, N127T, R138Q, L154S, Y163S and E169Q.

[0173] 1.3 Establishment of Transcription Block Survival Assay We first wanted to confirm whether the novel TRE-mDHFR construct could replace the TMP-inhibited ecDHFR by confirming that it was expressed, folded, and catalytically active. This was accomplished by i) SDS-PAGE analysis of cell lysates (Figure 4), confirming that the protein was expressed in the soluble fraction upon isopropyl β-D-1-thiogalactopyranoside (IPTG) induction, ii) plating E. coli containing the TRE-mDHFR plasmid onto M9 agar supplemented with TMP [no growth was observed (4 μM TMP, optimized in Figure 5), and growth was restored upon induction of TRE-mDHFR expression with IPTG (Figures 5 and 8B-3)], and iii) monitoring purified recombinant WT- and TRE-mDHFR activity by following the decrease in NADPH at 340 nm in the presence of DHF substrate (Figure 3). Specific activities calculated from these reactions showed a 24-fold decrease in activity of TRE-mDHFR compared to WT. Furthermore, TRE-mDHFR showed a ∼1.8-fold decrease in specific activity in the presence of TMP, whereas WT-mDHFR was unaffected. Despite the predicted decrease in activity resulting from the 13 amino acid substitutions, TRE-mDHFR retained its ability to restore DHF and confer survival (ecDHFR was impaired), confirming its suitability for TBS.

[0174] Having established that TRE-mDHFR is active and absolutely required for cell survival under selection conditions (M9 minimal medium + 4 μM Tmp + 1 mM IPTG), we then expressed the cJun bZIP domain in cells containing the TRE-mDHFR plasmid, resulting in a 21-fold reduction in colony numbers (P≦0.0001; FIG. 8B-5). Expression of cJun bZIP in the presence of WT-mDHFR (i.e., lacking the necessary TRE-binding site) reduced bacterial growth (P≦0.05, FIG. 8B-1 vs. 8B-2). This is likely due to nonspecific binding of overexpressed cJun to plasmid DNA. However, the transcription block is strongly TRE site specific, as shown by the 1.3-fold reduction in the absence of the TRE site. As an additional control, we also introduced a cJun LZ-only construct in which the 25-residue DBD was deleted. This peptide was unable to initiate DNA binding and, as expected, did not affect bacterial colony formation (P=0.1, Figure 8B-4 vs. 8B-3). Together, this specifically correlates the interaction between the cJun bZIP and TRE sites with the loss of bacterial growth in the TBS system, and confirms that any subsequent increase in bacterial growth is due to inhibition of this interaction.

[0175] Next, peptides known to bind cJun were introduced into the system to establish whether they could affect cJun function, i.e., sequester cJun bZIP as a non-functional heterodimer, thus preventing DNA binding and rescuing TRE-mDHFR transcription. Here, we used two peptides targeting the cJun LZ domain: cFos LZ and FosW [optimized sequences identified from protein fragment complementation assays (PCA) that readily bind cJun in the absence of DNA with nM affinity] (Mason et al., 2006; Worral et al., 2011). Despite their known interactions with cJun, both peptides were shown to be ineffective in restoring TRE-mDHFR expression and activity, and did not result in a significant increase in colony numbers from transcriptionally blocked cells (P>0.05 in both cases, Figure 8B-6 or 8B-7 vs. 8B-5). This important finding indicates that FosW outcompetes cJun dimers to form nonfunctional heterodimers but is unable to release DNA-bound cJun from the TRE site in TRE-mDHFR.

[0176] To address this, we turned to the work of Olive et al., where antagonism of cJun was achieved using Acidic-cFos (A-Fos), whereby a rationally designed acidic chain was added to the cFos LZ (Olive et al., 1997). Since FosW was shown to have improved binding to the cJun LZ compared to the WT cFos LZ sequence in the absence of DNA (Mason et al., 2006), an improved hybrid construct was rationally designed. This blended the two previously published components by adding a rationally designed acidic chain to the N-terminus of the FosW LZ sequence, generating A-FosW (Figure 9). This protein was designed to act as a template for peptide library design and optimization using TBS screening. To reassure, the template peptide was able to antagonize the cJun / TRE DNA interaction, restoring colony numbers by 60% compared to TRE-mDHFR alone (Figure 8B-8 vs. 8B-5). Importantly, all above experimental variations were plasmid matched with appropriate dummy constructs to adjust for possible differences in antibiotic stress (Table 3). The TBS design is summarized in Figure 8A.

[0177] [Table 4]

[0178] 1.4 Hinge library design The acidic strand design principle is the most successful method in the literature to target full-length bZIP domains of various proteins (Olive et al., 1997; Ahn et al., 1998; Chen et al., 2011). However, incomplete recovery of colonies using A-FosW indicated that transcription remained partially hindered by cJun binding across the 15 TRE sites. This allowed us to use TBS to screen peptide libraries for further improvement of cJun / TRE DNA antagonism using A-FosW as a design template. The library design utilized semi-randomized positions within the hinge region spanning the acidic strand and the LZ domain (Figure 9). Optimization of the hinge region was expected to induce a significant increase in functional antagonism by high affinity binding that simultaneously disrupts both cJun LZ dimerization, and by scrambling in the region between the two domains that provides a more effective block of DBD-DNA interactions. Previous studies have generally attempted to inhibit binding at either the DBD or the LZ, but here we aimed to target both regions and generate improved antagonists compared to targeting either domain separately. Of the nine semi-randomized hinge positions, four options were included across eight positions (e4, f4, g4, a4, b4, c4, e5, f5 and g5): two hydrophobic (V / L), one acidic (E or D) and one polar (Q or H). The A / E option was included in f5 as the f position of the LZ region, these positions are not likely to be involved in direct target interactions and therefore these residues have shown assistance by solubility or high helicity to enhance PPI by entropy pre-assembly. Experimental limitations do not allow for greater amino acid diversity to be incorporated at many positions, but these selections will allow for testing of common amino acid preferences. At every position, the parent residue of A-Fos was incorporated into the library, and this functionally active benchmark protein was included in the screen. Limitations from the genetic method used to generate the library result in different mixtures of residues at different positions.The Leu at d5 in the middle semi-randomized position of this chain was left unchanged since the change was not expected to be favored or other coiled-coil structures might be favored over the desired parallel dimer. Together, this produced a peptide library of 131072 members (Figure 9). 1.5 TBS library screening During the TBS library screening process, E. coli was transformed with pooled DNA plasmid libraries such that each cell expressed a given member. The cells were plated on M9 selective medium and incubated until colonies expressing TBS-active library members formed. These colonies were pooled and repeatedly passaged in liquid culture under selective conditions to compete the library members against each other and enrich for the most TBS-active sequences. At each stage of the assay, DNA sequencing was used to monitor the presence of TBS-active sequences in the culture and the information that a residue was selected at each position until one alternative DNA sequence, termed HingeW, was detected in the culture (Figure 9). The residues selected in the winning HingeW peptides were generally found to be either acidic (D / E) or hydrophobic (V). Six of the nine selected residues that deviated from the parent A-FosW sequence with both V (E to V at e4 and c4, Q to V at f4) and E (L to E at a4, Q to E at e5, A to E at f5) were newly selected at three positions. During the competition, the shift in the ratio of library options at each position provides information on how the selection may proceed for a particular preferred residue or residue type (Figure 10). Two residues were selected after the first passage and therefore considered to be particularly important for the optimization of this interaction: V at f4 (Q in the parent sequence) and E at g4 (unchanged). At position f5, there is a slight preference for either option at the end of the second passage, with E being selected over A (A in the parent sequence), indicating less impact on target binding. Although the sequence of A-FosW is included in the library, further confirmation of the TBS selection preference of HingeW over the parent sequence was performed by direct competition in liquid culture. In this experiment, equal numbers of TBS cells containing either A-FosW or HingeW were mixed in selective M9 medium and subjected to competitive selection. After three passages, only HingeW was observed by DNA sequencing, with A-FosW-containing cells losing the competition.This was further supported by TBS colony counting experiments, which showed a 10% increase in colony number for HingeW compared with A-FosW ( P = 0.009, Fig. 8B-9 vs. 8B-B ), up to 66% of the theoretical maximum colony number observed with TRE-mDHFR alone ( Fig. 8B-3 ).

[0179] 1.6 HingeW binds c-Jun preferentially to A-FosW Experiments were next performed to compare the binding of A-FosW and TBS-optimized HingeW to cJun bZIP. CD spectroscopy was utilized to measure the global secondary structure of homo- and heterodimeric peptide samples, providing information on the overall α-helicity and thermal melting temperature (Tm). HingeW / cJun spectra have 82% higher α-helical content compared to the average of the spectra of the two components expected to have no interaction (Figure 11A). Although less abundant, the same trend was observed from the A-FosW / cJun spectrum, where the α-helicity was 39% higher than the average (Figure 11B). The greater α-helicity obtained upon HingeW / cJun binding implies a higher affinity interaction. Of note, HingeW in the isolate is 12.9% less helical compared to A-FosW. The AGADIR helix propensity calculator was used to calculate predicted helix scores of 14.5 and 13.2 for A-FosW and HingeW, respectively (Munoz et al. 1994). The observed difference in helicity of the heterodimeric peptides may be explained in part by A-FosW being more helical in nature, but it is likely that the larger than predicted observed effect is explained by the homodimer preference of A-FosW compared to HingeW.

[0180] Thermal denaturation analysis of HingeW / cJun showed a Tm of 71.2 °C for the HingeW / cJun heterodimer after loss of signal at 222 nm, a clear increase from the Tm of the denaturation profile of the two components (Figure 11C). For A-FosW / cJun, the Tm was observed as 69.9 °C (Figure 11D). This represented a slight 1.3 °C increase in the heterodimer Tm, but there was a crucially larger ΔTm of HingeW / cJun compared to the component peptide denaturation profile of A-FosW / cJun. The lower thermal stability of the HingeW homodimer did not result in an observable lower baseline before the transition, and the Tm of this component, and therefore the average, could not be determined. However, this ΔTm can be estimated to be ~40 °C, compared to 27.5 °C for A-FosW / cJun. The TBS screen therefore resulted in an optimized decrease in homodimerization rather than an increase in heterodimerization with the target. This confirms that more antagonist is available as free monomer in solution and therefore in the target-dimerization receptive state. Another difference between the two denaturation profiles is the presence of a double transition in the A-FosW / cJun heterodimer, with the smaller first transition occurring at ~30°C. Jain et al. previously reported a double transition in a similar acidic chain antagonist / bZIP denaturation profile, showing that the lower temperature transition occurs due to the fraying of the N-terminal acidic chain / DBD interaction, while the higher temperature transition corresponds to the dissociation of the LZ region (Jain P et al., 2017). Importantly, these two novel antagonists have significantly higher target heterodimer Tm values ​​than FosW (Tm=54°C, Figure 12). This demonstrates a clear advantage from including the acidic chain, which is absent in FosW. Importantly, due to sequence differences between cJun and cFos (Fig. 9 ), optimization of cJun binding means that HingeW shows no interaction with the cFos bZIP domain (Fig. 13 ).

[0181] 1.7 HingeW outcompetes A-FosW for cJun binding Direct competition between HingeW and A-FosW for cJun binding was observed using CD dimer exchange experiments, where solutions containing one antagonist / cJun mixture were combined with the other antagonist to observe possible changes in α-helicity as an indication of changes in the cJun dimerization partner. In this case, when HingeW was mixed with preformed A-FosW / cJun heterodimers, a 17% increase in helicity was observed when measured at 222 nm, compared to the average, indicating a change in binding (Figure 11E). There was also a clear increase in the peak at 190 nm, compared to the average of the two component peptide spectra. Reversing the experiment, and mixing A-FosW with preformed HingeW / cJun heterodimers, produced measured spectra that overlaid with the average of the components, indicating that no dimer exchange occurs (Figure 11F). Combined with the TBS growth competition data, the dimer exchange experiments strongly indicate preferential binding of cJun to HingeW compared to A-FosW when competed.

[0182] 1.8 Isothermal titration calorimetry shows improved binding affinity to HingeW The binding interactions of cJun with HingeW and A-FosW were further studied by ITC to provide information on thermodynamic parameters. Data generated from injection of HingeW into cJun (Figure S14A) were fitted to a single-site binding model (N=1.05±0.05) with a KD of 14.4±3.7nM and a ΔH of -85.4±4.5KJ / mol (TΔS=-39.2±4.5KJ / mol). A-FosW binding to cJun (Figure S14B) was also fitted to a single-site model (N=1.04±0.08) with a KD of 88.3±17.6nM and a ΔH of -152.6±4.1KJ / mol (TΔS=-112.6±4.1KJ / mol). This confirms the predicted 1:1 binding stoichiometry for both interactions, demonstrating a six-fold increase in binding affinity upon TBS optimization of A-FosW for HingeW. Both interactions are entropically driven with negative entropy contributions, the latter being less favorable for the HingeW interaction than the former.

[0183] 1.9 HingeW effectively antagonizes the c-Jun / TRE DNA interaction. Binding of cJun to TRE DNA can be observed by monitoring the DNA absorbance peak in the CD spectrum centered at ~281 nm (John M et al., 1996). None of the peptides in the isolate (cJun, HingeW or A-FosW) absorb at this wavelength, meaning that any change in the spectrum in this region corresponds to a shift in DNA structure. Addition of cJun (20 μM) to TRE DNA (5 μM) reduced this DNA peak by 55% (Figure 15A) as cJun binds to its target TRE site and alters DNA structure. Subsequent titration of HingeW into this bound cJun / TRE DNA mixture reverses the peak shift, with the peak increasing as DNA is released. This occurs in a dose-dependent manner until the signal overlays the free DNA spectrum at HingeW concentrations of 50 and 100 μM, indicating complete antagonism of the cJun / TRE interaction. Plotting and fitting of the relative peak shifts to the Hill equation (OriginPro) yielded an IC50 of 13.4 ± 0.6 μM, which can be compared to the equivalent data for A-FosW antagonism, which produced an IC50 of 16.0 ± 0.4 μM (Figure 15B). This shows a marked improvement in both cases to FosW, which lacks the acidic strand, showing an IC50 of 119.8 ± 1.1 μM (Figure 16). Control experiments showed that both HingeW and A-FosW had no interaction with DNA (Figure 17). To provide further evidence of functional antagonism, electrophoretic mobility shift assays (EMSA) were used. First, cJun bZIP (20 μM) was mixed with the TRE DNA construct (2 μM), resulting in a marked decrease in the free DNA band intensity compared to DNA alone (Figure 15C). No bound cJun / TRE DNA band was observed because the bulk of this complex prevented its entry into the gel. Antagonism was therefore best observed by monitoring the intensity of the free DNA band. A concentration-dependent increase in the free DNA band intensity was observed upon addition of HingeW to cJun / TRE DNA (Figure 15D). The same trend was observed upon increasing concentrations of A-FosW with cJun / TRE DNA (Figure 15E). In close agreement with the CD DNA peak analysis, the data were fitted to the Hill equation (OriginPro) and IC50 values ​​of 9.6 ± 0.8 μM for HingeW and 12.1 ± 1.9 μM for A-FosW could be determined. In close agreement with the CD DNA peak analysis, the data were fitted to the Hill equation (OriginPro) and IC50 values ​​of 9.6 ± 0.8 μM for HingeW and 12.1 ± 1.9 μM for A-FosW could be determined (Figure 15F).

[0184] A summary of the thermodynamic parameters of the interaction between c-Jun and either the rationally designed A-Fos template or the TBE library derived HingeW is provided in Table 4 below:

[0185] [Table 5]

[0186] 1.10 Conclusion Although there are many screening platforms for driving high affinity PPIs, none guarantee target binding resulting in the desired loss of function of the target protein. Using cJun / TRE as an example, we developed a Transcription Block Survival assay that can be used as a generalized approach for the derivation of peptides capable of abolishing TF activity. We engineered a "molecular dial" into a bacterial system whereby cJun / TRE DNA interaction inversely correlated with cell proliferation. By introducing a cJun / TRE antagonist into this system, cell proliferation becomes a direct readout of the ability of the antagonist to functionally block many cJun / TRE interactions, dialing up the molecule. The most potent rationally designed acidic antagonists were then utilized as parent sequences to design a semi-randomized library that was successfully screened in the TBS platform to produce the in vitro validated assay hit HingeW.

[0187] Establishing the TBS system required the production of a mutated DHFR gene (TRE-mDHFR) that maintains its enzymatic activity upon introduction of 15 TRE sites into its DNA sequence, resulting in 13 amino acid substitutions. This allowed for cJun-inducible transcription block when TFs bind to the TRE sites on the TRE-mDHFR plasmid DNA. For the resulting lack of TRE-mDHFR activity, there is an absolute requirement for both the TF DBD and the TRE site within the mDHFR gene, confirming specificity in the TBS system. The bacterial growth rate phenotype is directly linked to the genotype of the antagonist sequence expressed upon containment of the system in a single cell. The bacterial growth rate phenotype is directly linked to the genotype of the antagonist sequence expressed upon containment of the system in a single cell. Bacterial cells are ideal for this process due to their fast growth rate, durability, ease of use, and low cost. Importantly, it also allows for the direct measurement of cJun interacting with the TRE sites in the absence of any relevant eukaryotic TFs that would interfere with the assay.

[0188] TBS facilitates high-throughput genotype-phenotype screening and competition of peptide libraries to isolate those that result in loss of function of cJun DNA-binding activity from those that bind but have little or no effect on target activity (or no binding at all). This distinction is important because it means that antagonists must not only be able to bind to the target free in solution, but also meet the much more difficult challenge of releasing the TF from DNA, which is known to be more stable (Seldeen et al., 2011). Finally, all of the above takes place within the complex environment of the cytoplasm, eliminating molecules that are toxic, nonspecific, insoluble, or protease-sensitive from consideration in the initial screening stages, rather than determining this at a later hit evaluation or clinical trial stage. These factors are particularly important for long peptides that need to bind to the large, shallow cJun bZIP surface, as such peptides tend to lack these important properties. TBS improves relevant protein-fragment complementation assays as well as in vitro screening platforms such as phage display or ribosome display by completely removing any requirement for large protein fusions or hydrophobic / aromatic tags that can interfere with relevant assay interactions and result in failed readouts.

[0189] A major advantage of TBS is that assay hits are required to prevent binding of TFs to their consensus DNA sequence, as exemplified by the composite design of A-FosW, a hybrid containing domains from both A-Fos (Olive et al., 1997) and FosW PCA hits (Mason et al., 2006). In A-FosW, the LZ targets antagonists to cJun bZIP with high affinity and selectivity, and an acidic tether is added to aid in the step of functionally antagonizing the cJun / TRE DNA interaction by blocking the cJun DBD. The LZ domains of bZIP proteins tend to exhibit more sequence diversity than the DBD, making them useful for therapeutic targeting of specific AP-1 family members, providing better regulation and less potential for adverse events (Eferl et al., 2003). Although it is not clear whether A-FosW binds cJun by forming a single continuous LZ interaction as designed, increased binding around the hinge region of cJun was predicted to increase helicity and therefore affinity in either orientation. Furthermore, a focus on the hinge region was supported in the original work of Olive et al., where a point mutation in this region of A-Fos (N26L at position a4 of A-FosW) resulted in a significant increase in cJun binding affinity and subsequent cJun / TRE antagonism (Olive M et al., 1997). Optimization of the acidic chain by rational design was hampered by a lack of design rules for guidance, as is the case for LZ domains with known structures and prescriptive tools that result in high affinity interactions (Boysen RI et al., 2002;Kaplan JB et al., 2014). Furthermore, no library-based approach has been used previously to optimize binding within this region of cJun. Using A-FosW as a design template and including library options in the hinge region was the obvious next step resulting in TBS selection of HingeW with an affinity of 14 nM to the target cJun protein (a 6-fold improvement over A-FosW).HingeW supports the approach of Olive et al. by containing one more acidic residue than A-FosW and a dominant-negative charge in the N-terminal domain that interacts favorably with the positive charge in the cJun DBD. However, the exact selection pattern was more subtle than simply producing a block of negatively charged residues. The properties of HingeW suggested another advantage of the TBS library screening approach, where directed evolution of antagonists provided improvements by reducing homodimerization. TBS was extremely useful for exploring novel sequence space by producing protein sequences that could not have been predicted without using this library screening approach.

[0190] TBS opens new possibilities for semi-rational PPI design, where both affinity and activity are selected simultaneously. This offers great potential for expanding the TBS approach to both novel libraries and targets, where previous studies could generate potential antagonists that were later found to lack functional activity. In principle, the approach could be fully expanded to any DNA binding protein that recognizes a discontinuous consensus sequence, or even any dimeric system to which a DBD is added. The method could be envisioned as generalizable, since any DNA consensus sequence could be incorporated into the DHFR DNA sequence and transcriptionally blocked by co-expression of the relevant TF. Although this would require an iterative DHFR design process and subsequent testing and optimization of each system, the main principles were shown herein to be valid. Screening of exogenous molecules could also allow for the simultaneous profiling of both cell-penetrating agents and functionally active inhibitors. Furthermore, libraries with different design principles and expansion options would provide a good guarantee for the production of peptide hits across a broad range of targets where pathogenic TFs are implicated. 10 6 ~10 7Library sizes of are possible using standard techniques and readily available reagents that may allow the development of a broad peptide diversity and further optimization. Further TBS screening of a broad range of TF targets will produce both non-genetic tools and probes of disease pathways, but there is also considerable potential for de novo generation of optimized functional antagonists and clinical leads.

[0191] Example 2 – Optimization of a functional c-Jun antagonist This example shows the optimization of hits from the peptide library screening of Example 1, designed to target the full-length cJun bZIP domain in an attempt to simultaneously block both cJun dimerization and DNA binding. TBS screening of a 130,000-member peptide library yielded the HingeW sequence (HW1). HingeW was developed to be able to bind across the full-length cJun bZIP domain for more effective functional antagonism of TRE binding compared to DBD-only or LZ-only cJun inhibitors. The nature of the broad, shallow helix-binding surface supports the use of longer peptides, such as Hinge. However, it was unclear whether the full length of the sequence was necessary to achieve functional antagonism. HW1 was recombinantly produced and biophysically characterized as a 69 amino acid long, capped (MAS at the N-terminus, GAP at the C-terminus), C-terminally 6xHis-tagged protein construct with significant overall negative charge. Optimization of the peptides was performed with the goal of improving their drug-like characteristics.

[0192] 2.1 Method Peptide synthesis and purification. All peptides were synthesized using a Liberty Blue microwave peptide synthesizer (CEM) on a 0.1 mmol scale on ChemMatrix Rink amide resin using standard Fmoc solid phase methods. Coupling was performed using 5x amino acids, 4.5x PyBOP and 10x diisopropylethylamine in dimethylformamide (DMF, 5 mL). Deprotection was performed using 20% ​​piperidine in DMF. Peptides were N-terminally capped by a final reaction with 3x acetic anhydride, 4.5x diisopropylethylamine in DMF at 90°C for 5 min. In lactamized peptides, the relevant K and D positions were orthogonally protected by the use of Lys (Mtt) and Asp (O-2-PhiPr). The side chains of these residues were selectively deprotected by washing the resin with dichloromethane (DCM) × 3, 2% trifluoroacetic acid (TFA) in DCM × 10, DCM × 3, then DMF × 3. The newly deprotected side chains were coupled in PyBOP (1 mL), diisopropylethylamine (1 mL) and DMF (3 mL) for 5 h at 60 °C. The resin was dried and the same reagents were added for a second reaction at 60 °C for 16 h. Incubation in cleavage mixture (95% TFA, 2.5% triisopropylsilane, 2.5% H2O, 10 mL) for 4 h at room temperature cleaves the peptide from the resin and removes the side chain protecting groups. The resin was removed by filtration and the cleaved peptide was precipitated in diethyl ether at -80 °C and centrifuged. The pellet was washed a further 4 times with diethyl ether and then dried overnight at room temperature. Peptides were resuspended in 3:1 water:acetonitrile and then purified using RP-HPLC on a Jupiter Proteo column (4 μm particle size, 90 Å pore size, 250 × 10 mm; Phenomenex) using a water:acetonitrile gradient (0.1% TFA). Peptide mass and purity (>95%) were confirmed by electrospray ionization mass spectrometry.

[0193] Circular dichroism (CD): An Applied Photophysics Chirascan was used for CD measurements with 200 μL of sample in a 1 mm path length CD cell. Protein / DNA samples were suspended in 150 mM potassium phosphate, 150 mM potassium fluoride, and 5 mM TCEP, pH 7.4, and equilibrated for 30 min before measurement. For every spectrum, three scans between 190 and 260 nm (265–320 nm for DNA binding experiments) were collected with a 1 nm bandwidth, and data were sampled at a rate of 0.5 s-1. These scans were averaged and converted to molar residue ellipticity (MRE). Thermal denaturation experiments were performed by measuring the ellipticity at 222 nm over a gradient from 1 to 90 °C in 1 °C increments. Post-melt scans at 20 °C confirmed that the transition was reversible when they overlapped within 10% of the pre-melt scan. The resulting thermal metamorphic curves were converted to MREs and fitted to a two-state model derived from a modification of the Gibbs-Helmholtz equation to obtain melting temperatures (T m ) was determined (Mason et al., 2007).

[0194] Serum stability: Peptide stocks (600 μM) were prepared in water and 50 μL was added to 950 μL of human serum (Merck) followed by incubation at 37°C. Aliquots of 100 μL were removed at the indicated time points and added to 300 μL of 3:1 acetonitrile:water and centrifuged (18000×g, 15 min). Supernatants were analyzed by LC-MS and quantified using the sum of the two highest charge state intensities (1:9+,10+; 23,24:3+,4+).

[0195] Isothermal titration calorimetry (ITC): Peptides were tested by ITC using a PEAQ-ITC (Malvern Instruments) with an ITC buffer consisting of 10 mM potassium phosphate, 150 mM potassium fluoride, and 5 mM TCEP at pH 7.4. 2 μL injections of antagonist peptides at 25–200 μM were injected into cells containing cJun at 2.5–20 μM. The heat change upon addition was recorded and analyzed using software. A control experiment included injection of antagonist peptide samples into cells containing ITC buffer only to determine the heat of dilution that was subtracted. The resulting binding data were fitted to a one-site binding model to extract the enthalpy change of binding (ΔH) and equilibrium binding constant (KD), from which the free energy change of binding (ΔG) and entropy change of binding (ΔS) were calculated (Wiseman et al., 1989). Thermodynamic parameters are presented as the mean of two independent experiments and errors are given as one standard deviation.

[0196] 2.2 Acidic / N-terminal truncation A summary of the various peptides used and described in this section is provided in Table 5 below.

[0197] [Table 6]

[0198] The exact nature of the interaction between the cJun DBD and the rationally designed acidic domain of 1 is unknown. Therefore, the acidic domain was initially focused on for optimization. 1 was repeatedly truncated (2–6) to examine the effect on c-Jun binding and c-Jun / TRE DNA antagonism. The helicity of the peptides was determined by quantitating the CD signal at 222 nm of peptide-only samples. Thermal denaturation experiments were then used to determine the T of the peptide-c-Jun heterodimers. mwas determined and used as an approximate measure of target binding. CD was also used to examine the ability of the peptides to functionally activity antagonize c-Jun / TRE DNA interaction. The TRE-DNA construct used produced a positive CD peak at ~281 nm (there is no c-Jun absorbance at this wavelength allowing a direct measurement of DNA upon binding) that decreased in intensity upon c-Jun binding. This provided an unambiguous and direct measure of the percentage of DNA binding. As increasing concentrations of antagonist peptide were added to the sample, the peak shifted back into overlay with free TRE-DNA. This was determined by fitting the titration data to the Hill equation to determine the IC 50 The higher concentrations required in these experiments (due to the signal detected by CD) allowed the calculation of IC 50 The values ​​are similarly high, with 20 μM cJun in the experimental conditions, with the lowest IC50 falling to 10 μM. Therefore, these values ​​are incorporated into the text and should not be compared with values ​​produced by different methods, but are instead used herein for comparison between the antagonists tested. The same 60 amino acid c-Jun bZIP construct was used for all experiments, regardless of antagonist peptide length. These experiments allowed both target binding and subsequent target antagonism to be rapidly characterized in vitro.

[0199] The thermal denaturation (T m ), functional activity (IC 50 A summary of the CD (μM), helicity (fH (%)) results is provided in Table 6 below.

[0200] [Table 7]

[0201] All N-terminal truncations in the series (2-6) reduced peptide / target binding and antagonistic effects, indicating that the full-length acidic chain contributes to antagonism of the c-Jun / TRE interaction (Figure 18). However, each truncation in the series resulted in an increasingly greater effect on antagonism per residue removed. Inspection of truncations 1-2, 2-3, and 3-5, representing three full heptad N-terminal deletions, reveals a 1.2-, 2-, and 2.3-fold reduction in antagonism, respectively. As expected, the percentage reduction increases as each subsequent heptad is deleted, and thus becomes more significant in antagonism. Furthermore, the final N-terminal truncation examined, 5-6, reduced antagonism 1.7-fold, despite removing only four residues. Peptide 6 (NΔ28) represents only the LZ portion of the HingeW molecule. To test the possibility that the acidic domain alone might antagonize c-Jun, this moiety was also tested alone and showed no binding (data not shown), indicating that the acidic chain and the LZ domain of HingeW operate synergistically to bind and antagonize c-Jun.

[0202] N-terminal truncations also resulted in a progressive increase in helical fraction (fH), rising from ~27% in 1 to ~47% in 5, indicating that the deleted region is less helical compared to the LZ region. The LZ domain of 1 was the most unchanged from its parent sequence, FosW, which is known to homodimerize (Mason et al., 2006). The negative charges of the acidic chains produce electrostatic repulsions and thus reduce the tendency to homodimerize, and their removal increases homodimerization-induced helical character. However, further truncation from 5 to 6 reverses this trend, decreasing fH to ~38.0%, but removing one acidic and three hydrophobic residues, leaving an LZ-only domain. There is a slight additional electrostatic repulsion that is reduced, but this region appears to contribute to homodimerization by direct binding or helix induction.

[0203] Overall, removal of the acidic strand domain (1–6) reduced cJun / TRE DNA antagonism by 9.7-fold. Previously, we observed a similar IC as observed with peptide 6 (130 μM). 50 We observed a very significant decrease in peptide activity during TBS screening of peptides with IC50 values, implying that 6 does not fully compete for cJun / TRE DNA binding. This further supports the rationale for the acidic tether design, and 5 (IC50=78 μM, 5.8× lower antagonism than 1 and 1.7× better than 6) can be considered the largest feasible truncation proceeding from the N-terminus.

[0204] 2.3 Lactamization of N-terminal truncations Peptide 5 was then optimized by incorporating an i→i+4 (K to D) lactam bridge. The lactam bridge was incorporated through the use of orthogonal protecting groups (Lys(Mtt) and Asp(O-2-PhiPr)), selectively deprotected (2% trifluoroacetic acid in DCM) and could be reacted using typical solid-phase chemistry while the peptide remained bound to the resin. The success of the reaction was confirmed using mass spectrometry (MS), observing a reduction in mass from the absence of water molecules compared to the linear unreacted peptide. In this example, lactams were only introduced at the solvent-exposed bf or fc heptad positions to prevent disruption of the binding surface of the helix. Point mutations into the sequences were required to incorporate the bridge of K and D residues in both the linear (7,9) and cyclic (8,10) versions of each sequence produced by split batch synthesis.

[0205] Cyclic peptides 8 and 10 increase antagonism by 1.8- and 1.9-fold, respectively, compared to their linear counterpart 5. Side-chain lactamization results in increased helicity leading to high affinity binding. T m The binding, as indicated by the corresponding low IC 50It is interesting to note that these peptides do not produce any T m As expected, the IC 50 Although it can be assumed that the T is inversely correlated with the T, there is a level of trend change (Figure 19). Peptides 2 and 10, for example, have similar T m Although 10 has a higher binding affinity than 2, 2 antagonizes the cJun / TRE interaction 4-fold more effectively. 10 is truncated by 14 more N-terminal residues than 2. This supports the importance of the rational design principles used for HingeW, whereby inhibition of both domains of the cJun bZIP produces the most effective antagonism, such that most of the acidic chain is removed in peptides 5 and 7-10, and all of it in peptide 6. This results in peptides that can be optimized to bind strongly to the cJun LZ, but are limited in their ability to functionally antagonize cJun by also blocking the DBD.

[0206] Truncation of 4 to 5 removed the block of negative charges (EAEE) and resulted in a 1.7-fold reduction in antagonism. This represents a region important for interacting with the positively charged cJun DBD surface as well as inducing helicity and potentially stabilizing the dipolarity of the molecule (Pace et al., 1998; Sali et al., 1988). This has implications for studies of cJun DBD interactions with TRE DNA, which indicate specific residues that directly interact with DNA. The central two residues of this added block (E AE E - 10 to 11) occur at positions that are responsible for the interaction of cJun with DNA, i.e., they help form a direct block between cJun and DNA. The NΔ20 truncation can therefore be considered an optimal balance between downsizing and retention of functional activity.

[0207] 2.4 C-Terminal Truncation Peptide 11 was therefore the next step in optimization utilizing an NΔ20 truncation and also truncating at the C-terminus. Removal of the four C-terminal residues from 4 to 11 reduces antagonism to 1.8-fold, but further truncation at the C-terminus to produce 25 greatly reduced antagonism to 14.8-fold compared to 11 and 26.7-fold compared to 4. Attempts to optimize 25 by lactamization to produce 27 and 29 were effective as they significantly improved antagonism, but they still produced 8.8-fold and 10.4-fold reductions in antagonism compared to 1. Although these lactamizations had a much larger effect on the peptides, they were still considered ineffective for further study.

[0208] 2.5 Optimization of 11 Peptide 11 was considered as a scaffold for further optimization, with almost half the number of residues compared to 1, but retaining a high level of functional activity. The KD lactam bridge from i to i+4 positions was systematically incorporated at different sites to explore which region was most amenable to helical constraints, resulting in improved affinity and inhibition. Also, point mutations tuning the bridging K and D residues produced both linear and cyclic peptides. Heterodimer ΔT from lactamization m The ΔT ranges from ~2°C for 14 / 15 to ~9°C for 22 / 23. Comparison with the original parental sequences is also useful, and the heterodimer ΔT m ranged from ~1°C on 11 / 13 to ~8°C on 11 / 23. c-Jun / TRE antagonism was the most important measurement in this study, but only 23 showed IC 50 It was shown that 23 provided a significant improvement in antagonism compared with 11, 1.6-fold lower (P = 0.003). By use of a lactam, 23 restores the loss of antagonism caused by truncating the four C-terminal residues of 4.

[0209] Information regarding the suitability of each site for lactamization can be gleaned from this data. The change in peptide helicity ranges from ∼1% in 12 / 13 to ∼8% in 22 / 23. There is a relationship between the increase in peptide helicity induced by lactamization at specific sites and the increased cJun / TRE antagonism observed.

[0210] Lactamization of 17 did not result in a significant increase in antagonism (P=0.24), but did result in an ∼5% increase in peptide helicity and a fold increase in the cJun heterodimer T from its linear form 16. m Due to the distance between this site and the lactamization site in 23, the double lactamized peptide produced 24. The double lactamization was ∼4% more helical compared to the best single lactamized peptide, resulting in a ∼9°C increase in the T m There is no significant change in IC 50 There is a significant decrease in values ​​(P=0.02) for the peptide.

[0211] From this study, a selection of the most effective peptides were examined by ITC to quantify the thermodynamic parameters of their interaction with the target cJun (Figure 20, Table 7). The two single lactam peptides that were most effective in CD experiments and a bilactam utilizing both of these were tested and compared to the parent sequence. 1 / cJun was previously examined by ITC and showed low K D It is reported herein that all interactions examined were dominated by an enthalpic component, with a smaller unfavorable entropic contribution. This unfavorable entropic component was largest for the 24 / cJun interaction and smallest for 17 / cJun.

[0212] [Table 8]

[0213] 1, 23 and 24 were also tested for their serum stability and showed the effect of truncation and lactamization (Figure 21). At 24 hours, 1 was 67% degraded, 23 was 21% degraded and 24 was 7% degraded. Only 1 showed the exponential decay required to fit the model of exponential decay, indicating a half-life of 18.9 hours. After 96 hours, no peak corresponding to 1 was observed by MS. At this time point, the peak intensities of 23 were still at 17% of their starting values ​​and 24 was at 35%. This clearly shows the increased stability provided by the non-native lactam bridge and the effect is cumulative with 24 being more stable than 23 (P=0.038).

[0214] 2.5 Conclusion Through a systematic study of truncation and helix-guided lactamization, a series of rationally designed peptides were synthesized to explore and optimize HingeW (1). 1 proved to be inaccessible by SPPS using standard techniques (i.e., without native chemical ligation), and only 2 and 3 were produced in significantly lower yields. All remaining small peptides were produced in similar and higher yields by SPPS. The increase in production efficiency derived from the transition from recombinant expression to SPPS should not be underestimated. The process of repeated truncation and characterization clarifies the effectiveness of different parts of the parent sequence and allows careful consideration of how much the peptide should be truncated to derive the minimal effective sequence. In this example, the key consideration is whether the peptide binding to the target c-Jun can outcompete dimeric c-Jun bound to the TRE. This interaction is driven by the K DVariously established to have ~100-200 nM, maintaining an interaction stronger than this was considered an important criterion (Seldeen et al., 2011). Comparison of the CD antagonism data of 1 and 6 (Figure 18B) illustrates this important consideration, as 1 is able to restore the TRE DNA peak to its unbound intensity, indicating that no DNA binds to cJun at high peptide concentrations. However, 6 inhibits the interaction, but the line does not tend toward 1, and some c-Jun / TRE DNA complex may remain regardless of the amount of 6 present in the sample. Although the balance between efficacy and loss of gain of drug-like characteristics versus synthetic efficiency is difficult to quantify, the data suggested truncation of 1 to 4, removing 20 residues from the N-terminus. Truncation at the C-terminus of 4 to 11 was also supported, as truncation beyond that weakens peptide efficacy to the extent that even lactamization cannot restore binding sufficient for the peptide to be able to compete for the c-Jun / TRE DNA interaction.

[0215] The tolerable degree of truncation at the N-terminus appears to go beyond binding affinity considerations, since this acidic domain is important for blocking the c-Jun DBD. Peptides 5-10 can no longer effectively prevent DNA binding to the c-Jun DBD, i.e., cJun can still bind to the TRE site as a monomer when these antagonist peptides are bound to its LZ domain. However, effective antagonism does not require binding to the full length of the cJun DBD. Specific contacts between certain c-Jun DBD amino acid side chains and TRE DNA bases are known, and it can be hypothesized that antagonists binding to any of these cJun residues would significantly reduce c-Jun / TRE binding, and that binding to the full length DBD would not be required to maintain a high level of inhibition (Glover et al., 1995;Seldeen et al., 2011). Thus, peptide 4 appears to be the largest truncation tolerated, extending the N-terminus far enough to directly block the interaction of the c-Jun DBD with DNA and, crucially, presenting a block of negatively charged residues in this region that attract the DBD and repel the DNA. With the addition of EAEE residues, 4 has slightly reduced homodimerization and better antagonism compared to 5.

[0216] Nearly half the length of 1 was removed to produce 24, which retained a high level of efficacy. Due to the nature of the α-helical coiled-coil structure and hydrogen bond network, residues that are not involved in the binding of the peptide to its dimeric partner may still affect binding by introducing helicity. Furthermore, the helicity of the peptides studied here must be considered in the equilibrium of various dimerizations, e.g., between homodimers, monomers and heterodimers, with any dimerization event tending to result in increased helicity. Reducing the length of the α-helix tends to reduce its helicity, as long as the removed residue does not disrupt the structure (e.g., if it contains proline or glycine) or repel any dimeric binding partner (e.g., electrostatic repulsion).

[0217] A 20% increase in peptide helicity from 1 to 24 resulted in an increase in peptide serum stability. The N-terminal region of full-length 1 appears to be particularly non-helical and does not improve target antagonism, and their removal increased stability without significantly affecting peptide potency. Cyclization increases peptide helicity, reduces protease recognition, and increases peptide serum stability. Addition of a second lactam in 24 increases this stability further, but the increase in stability is smaller than the addition of the first lactam. Peptide degradation of lactamized peptides is too slow to be fit to an exponential decay function.

[0218] Certain lactam flanking residues may be more suitable for lactam accommodation and adoption of a helical structure than others at any given position. How the peptide folds to adopt a helical structure also affects the effectiveness of lactams, for example, helical folds appear to increase from one end of the peptide to the other, and the effectiveness of lactams then appears to vary depending on how close to this locus of folding they are located.

[0219] The thermodynamic parameters of binding observed by ITC show a slightly unexpected result with respect to the entropy component. It is usually assumed that the introduction of lactam bridges increases the binding affinity by pre-assembling the peptide molecule into its helical structure that can bind to the target with an entropic penalty. All peptides examined by ITC have an unfavorable entropy component, but 17 has a significantly lower contribution from this component. In this case, the entropy penalty of binding is reduced by pre-assembling the peptide into a helical fold that is complementary to the cJun binding surface. However, for 23, and especially 24, there is a large unfavorable contribution from the entropy component. This indicates that the increase in target binding affinity from side chain cyclization can also result from improved enthalpic interactions.

[0220] Example 3 – HingeW Bis-alkylation To identify additional peptide HingeW variants that could be cyclized using bis-alkylation as opposed to lactamization, variants of the cJun antagonist sequence EAEELVVEEDVLEEEIEQLEERNYALRKEICDLQCQ (SEQ ID NO:28) were screened using the TBS library screen described in Example 1. The screen identified the sequences set forth in Table 8 below.

[0221] [Table 9]

[0222] In addition to the five sequences listed in Table 8, 0W and meta-DBMBW were cyclized using bis-alkylation with mDBMB, and the functional activity of the linear and cyclic forms was tested in a CD assay, with the results and CD curves shown in Figure 23. Figure 23 shows that cyclic meta-DBMBW and 0W have improved c-Jun target binding compared to their linear counterparts, which is reflected in the lower IC50 of the cyclic variants, as shown in Table 9.

[0223] [Table 10]

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Claims

1. LV[X １ ]EE[X ２ ] [X ３ ]LE[X ４ An extended hinge region having the amino acid sequence of ]E (SEQ ID NO: 1); and The leucine zipper (LZ) region at the C-terminus of the extended hinge region, [Here, X １ It is selected from V, D, K, C and R, X ２ is selected from D, K, C, and R, X ３ is selected from V, D, C, K and R, and X ４ [Selected from E, D, C, K, and R] c-Jun antagonists, including

2. The extended hinge region has the amino acid sequence of EA[X ５ [X ６ (SEQ ID NO: 2), [Here, X ５ is selected from E, K, or C, and X ６ [Selected from E or D] The c-Jun antagonist according to claim 1, further comprising an N-terminal acidic chain having the above.

3. The c-Jun antagonist according to claim 2, wherein the acidic chain has the amino acid sequence EAEE (SEQ ID NO: 3).

4. X １ However, V is and / or X ３ However, the c-Jun antagonist according to claim 1 is V.

5. (i) X １ V is X ２ D is X ３ is V, and X ４ is E; (ii) X １ V is X ２ is K, and X ３ is V, and X ４ is D; or (iii)X １ V is X ２ C is X ３ is V, and X ４ C is The c-Jun antagonist according to claim 1.

6. The c-Jun antagonist according to claim 1, wherein the LZ region comprises IEQLEERNYALRKEIKDLQDQ (SEQ ID NO: 7), or the amino acid sequence of this variant comprising 1, 2, 3, 4, or 5 amino acid modifications.

7. (i) The LZ region contains IEQLEERNYALRKEIKDLQDQ (SEQ ID NO: 7), or the amino acid sequence of this variant including 1, 2, or 3 amino acid modifications. Optionally, the amino acid residues at positions 16 and 20 of variant SEQ ID NO: 7 are K and D amino acid residues, respectively; or (ii) The LZ region contains the amino acid sequence of this variant including IEQLEERNYALRKEICDLQCQ (SEQ ID NO: 27), or 1, 2, or 3 amino acid modifications. Optionally, the amino acid residues at positions 16 (heptad b-position) and 20 (heptad f-position) of variant SEQ ID NO: 27 are both C amino acid residues. The c-Jun antagonist according to claim 6.

8. The LZ region is IEQLEERNYALR[X ７ ]EX[X ８ ]K[X ９ ]L[X １０ ]D[X １１ ] (Sequence No. 29) or IEQLEERNYALR[X ７ ]EX[X ８ ]C[X ９ ]L[X １０ ]C[X １１ The amino acid sequence contains the sequence of ] (SEQ ID NO: 30), {Here, [X ７ ] is K, L, S, W, P, Q, R, M, T, V, A, E, or G; [X ８ ] is I, V, or L; [X ９ ] and [X １０ ] is any amino acid residue, [X １１ ] is Q, E, or K. The c-Jun antagonist according to claim 1.

9. The LZ region is IEQLEERNYALRSEICSLQCQ (Sequence ID 66); or IEQLEERNYALRKEICELSQ (Sequence ID 67); or IEQLEERNYALRAEICNLSCQ (Sequence ID 68); or IEQLEERNYALRTEICSLMCK (SEQ ID NO: 69); or IEQLEERNYALRAEICSLQCQ (Sequence ID 70), The c-Jun antagonist according to claim 8, comprising an amino acid sequence selected from the group consisting of these variants including one, two, or three amino acid modifications.

10. The antagonist, (i) EAEELVVEEDVLEEEEIEQLEERNYALRKEIKDLQDQ (Sequence ID 10), or any variant thereof comprising one, two, or three amino acid modifications, wherein optionally the amino acid residues at positions 31 and 35 of Sequence ID 10 of the variant are K and D amino acid residues, respectively; (ii) EAEELVVEEKVLEDEIEQLEERNYALRKEIKDLQDQ (Sequence ID 11), or a variant thereof comprising one, two, or three amino acid modifications, wherein optionally the amino acid residues at positions 10 and 14 of Sequence ID 11 of the variant are K and D amino acid residues, respectively, and the amino acid residues at positions 31 and 35 of Sequence ID 11 of the variant are K and D amino acid residues, respectively; (iii) EAEELVVEEDVLEEEIEQLEERNYALRKEICDLQCQ (Sequence ID 28), or a variant of this variant comprising one, two, or three amino acid modifications, wherein optionally, the amino acid residues at positions 31 and 35 of Sequence ID 28 of the variant are both C amino acid residues; (iv) EAEELVVEEDVLEEEIEQLEERNYALRSEICSLQCQ (Sequence ID 37), or a variant of this variant comprising one, two, or three amino acid modifications, wherein optionally, the amino acid residues at positions 31 and 35 of Sequence ID 28 of the variant are both C amino acid residues; or (v) EAEELVVEEDVLEEEEIEQLEERNYALRAEICNLSCQ (SEQ ID NO: 39), or a variant thereof comprising one, two, or three amino acid modifications, wherein optionally, the amino acid residues at positions 31 and 35 of SEQ ID NO: 28 of the variant are both C amino acid residues. A c-Jun antagonist according to claim 1, having the amino acid sequence.

11. The c-Jun antagonist according to claim 1, wherein the antagonist has a length between 30 and 70 amino acids, between 30 and 60 amino acids, between 30 and 50 amino acids, or between 30 and 40 amino acids.

12. The c-Jun antagonist according to claim 1, which can inhibit the DNA binding activity of c-Jun to within 10 times the ability of HingeW (SEQ ID NO: 48) to inhibit the DNA binding activity of c-Jun.

13. The c-Jun antagonist according to claim 1, comprising at least one amino acid residue crosslinking agent.

14. The c-Jun antagonist according to claim 1, comprising at least one covalently bonded i to i+4 or i to i+7 amino acid residue crosslinking agent.

15. The c-Jun antagonist according to claim 14, comprising at least one covalent i to i+4 amino acid residue crosslinking agent, and optionally comprising two covalent i to i+4 amino acid residue crosslinking agents in the peptide.

16. The c-Jun antagonist according to claim 14 or claim 15, wherein a covalently bonded amino acid crosslinking agent (or more) from i to i+4 is present from the b position to the f position or from the f position to the c position of the heptade.

17. The c-Jun antagonist according to claim 14 or claim 15, wherein the covalently bonded amino acid residue crosslinking agent (multiple) from i to i+4 is a K-D lactam crosslink (multiple) or an alkyl crosslink formed between a pair (multiple) of C residues (multiple).

18. The c-Jun antagonist according to claim 13, wherein the alkyl crosslink bond formed between the two C residues is formed by 1,3-dibromomethylbenzene (DBMB).

19. A nucleic acid encoding the c-Jun antagonist according to Claim 1.

20. A conjugate comprising the c-Jun antagonist according to claim 1, conjugated to a lipid, polymer, or second peptide, wherein optionally the second peptide is a cell-permeable peptide.

21. A pharmaceutical composition comprising a c-Jun antagonist according to claim 1, a nucleic acid according to claim 19, or a conjugate according to claim 20, in combination with a pharmaceutically acceptable excipient or carrier.

22. A c-Jun antagonist according to claim 1, a nucleic acid according to claim 19, or a conjugate according to claim 20, for use as a pharmaceutical.

23. The pharmaceutical composition according to claim 21 for use as a pharmaceutical.

24. A c-Jun antagonist according to claim 1, a nucleic acid according to claim 19, or a conjugate according to claim 20 for use in a method of treating a disease selected from the group consisting of cancer, diabetes, cardiovascular disease, autoimmune disease, joint disorder, and neurodegenerative disease.

25. The pharmaceutical composition according to claim 21 for use in a method of treating a disease selected from the group consisting of cancer, diabetes, cardiovascular disease, autoimmune disease, joint disorder, and neurodegenerative disease.

26. A method for inhibiting c-Jun, comprising the step of administering the c-Jun antagonist according to claim 1, the nucleic acid according to claim 19, or the conjugate according to claim 20 in vitro to cells containing or expressing c-Jun.

27. Use of the c-Jun antagonist according to claim 1, the nucleic acid according to claim 19, or the conjugate according to claim 20 for the manufacture of a pharmaceutical product for treating a disease selected from the group consisting of cancer, diabetes, cardiovascular disease, autoimmune disease, joint disorder, and neurodegenerative disease.

28. Use of the pharmaceutical composition according to claim 21 for the manufacture of a pharmaceutical for treating a disease selected from the group consisting of cancer, diabetes, cardiovascular disease, autoimmune disease, joint disorder, and neurodegenerative disease.

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