Patent Application
20250188151
The application herein incorporates by reference in its entirety the sequence listing material submitted concurrently with the specification as an XML file, with a filename of “to EPO-ST 26 Sequence listing”, a production date of Apr. 12, 2023, and a size of 86,016 bytes. The ST26 Sequence Listing is part of the specification and is incorporated in its entirety by reference herein.
The present invention relates to peptides that antagonise c-Jun, nucleic acids encoding peptides that antagonise c-Jun, pharmaceutical preparations comprising peptides that antagonise c-Jun, and the use of the antagonist peptides in the treatment of c-Jun-mediated diseases.
Transcription factors (TFs) play crucial roles in the determination of cell function and fate. A range of upstream signals converge upon TFs, converting vital cell signalling processes into transcriptional outputs via specific DNA site recognition. Consequently, of the ˜1600 TFs in the human genome, >300 are associated with a disease phenotype. TF dysfunction leads to a range of detrimental outcomes including cancer, diabetes, and cardiovascular disease (Lee et al., 2013; Lambert et al., 2018). Selective TF antagonism is therefore a compelling therapeutic route for the treatment of these diseases.
c-Jun is a transcription factor that is implicated in a 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) to facilitate dimerisation and a DNA-binding domain (DBD) to facilitate DNA sequence recognition (Glover et al., 1995; Risse et al., 1989). c-Jun binds to 12-O-tetradecanoylphorbol-13-acetate response elements (TREs), directly influencing cellular processes such as differentiation, proliferation, and survival (Shaulian et al., 2001; Eferl et al., 2003; Eckert et al., 2013; Alani et al., 1991). Dysregulation of these functions therefore promotes hallmark cancer cell behaviour, rendering cJun a focal point for cancer therapy.
TF function relies on protein-protein interactions (PPIs) and protein-DNA interactions which form many points of contact over their large surfaces. Small molecules (SMs) typically fail to abrogate these types of interactions due to the lack of tractable pockets.
While the flat protein-protein interactions are inaccessible to many pharmaceuticals, including small molecules, peptides have the potential to excel as high-affinity and selective inhibitors when designed to complement the broad target surface. Various methodologies 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 if LZ binding will translate into functional antagonism as the cJun DBD remains unbound and capable of binding TRE DNA (Seldeen et al., 2008; Szalóki et al., 2015). A rationally designed peptide has been shown to target the c-Jun DBD but exhibits lower potency than LZ antagonists, with concerns over specificity due to high sequence similarity across the AP-1 family DBDs (Tsuchida et al., 2004).
One approach to circumvent the potential downsides of existing methods is to utilise longer peptides that target the full c-Jun bZIP domain with a selective yet high affinity interaction, simultaneously blocking both DNA binding and LZ dimerisation. Olive et al. took this approach to produce A-Fos, which combined the wild-type (WT) cFos LZ (known to heterodimerise with c-Jun) and a rationally designed Glu-rich acidic extension (Olive et al., 1997). The A-Fos design principle postulated that the LZ interaction is extended N-terminally generating a DBD-acidic extension interaction facilitated by the incorporation of Leu residues into putative d positions in the acidic extension.
For peptides to be able to act as functionally active c-Jun antagonists they must not only bind to c-Jun but target binding must also result in ablation of function. One recently described approach to identify functional antagonists of transcription factors is the Transcription Block Survival (TBS) assay described in WO2020128015.
There remains a need for c-Jun antagonists that act as functional antagonists, especially those that exhibit desirable pharmacokinetic properties for therapeutic use.
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 c-Jun and antagonise its DNA-binding function. In work leading up to the present invention, the inventors used a library-based approach in which the hinge region that straddles the acidic extension and leucine zipper region of a c-Jun antagonist was semi-randomised. A peptide library was produced upon this scaffold and was randomised across a central tract of residues and tested using the TBS screening platform. This led to the identification of a recombinantly produced functional c-Jun antagonist termed ‘HingeW’, from a library of ˜130,000 peptides. The HingeW peptide is demonstrated in the examples to bind to c-Jun preferentially and with a higher affinity compared to the c-Jun antagonist from which HingeW was derived, and effectively antagonises the c-June/TRE DNA interaction. The nature of the broad, shallow helical binding surface of c-Jun supports the use of longer peptides such as the one identified by TBS.
The binding epitope of bZIP antagonists is presented on one side of a single α-helix and as such target binding requires the peptide to adopt this secondary structure. Recognising that a fundamental step in the development of therapeutic peptides is downsizing of the peptide towards the smallest functional unit required for effective binding, the inventors introduced iterative truncations in the identified antagonist peptide. Downsizing tends to reduce the α-helicity of the peptide as both the interaction interface and extended internal hydrogen bonding network becomes reduced and water competes for these interactions, shifting the folding equilibrium towards a random coil. Downsizing peptides to increase drug-like characteristics such as stability and membrane permeability therefore needs to be balanced against reduced affinity resulting from a reduction in the α-helicity of the peptide. As demonstrated herein, various optimised and truncated forms of the HingeW peptide were developed that retain functional activity, while improving on the peptide's drug-like characteristics.
In a first aspect, the present invention provides a c-Jun antagonist 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,
wherein
In some embodiments, the extended hinge region further comprises an N-terminal acidic extension having an amino acid sequence of EA[X5][X6] (SEQ ID NO: 2), wherein
In some embodiments, the acidic extension has an 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.
In some embodiments:
In some embodiments the LZ region comprises amino acid sequence
IEQLEERNYALR[X7] E [X8]K[X9]L[X10]D[X11] (SEQ ID NO: 29) or
IEQLEERNYALR[X7]E[X8][X9]L[X10]C[X11] (SEQ ID NO: 30) wherein
In some embodiments, the LZ region comprises an amino acid sequence selected from the group consisting of:
or a variant thereof comprising 1, 2, or 3 amino acid modifications.
In some embodiments, the LZ region comprises an amino acid sequence selected from the group consisting of:
IEQLEERNYALRKEICDLQCQ (SEQ ID NO: 27), or a variant thereof comprising 1, 2 or 3 amino acid modifications.
In some embodiments, the LZ region comprises an amino acid sequence of
IEQLEERNYALRKEIKDLQDQ (SEQ ID NO: 7), or a variant thereof comprising 1, 2 or 3 amino acid modifications,
In some embodiments, the LZ region comprises an amino acid sequence of IEQLEERNYALRKEICDLQCQ (SEQ ID NO: 27), or a variant thereof comprising 1, 2 or 3 amino acid modifications,
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 particular embodiments, the c-Jun antagonists have a length of 36 amino acids.
In some embodiments, the c-Jun antagonist has the amino acid sequence of
In some embodiments, the c-Jun antagonist comprises at least one covalent amino acid residue cross-linker. 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 cross-linker. As demonstrated herein, introducing a covalent amino acid residue cross-linker increases helicity and can increase antagonist activity of the peptide. This is beneficial, as it can be used to derive functionally active peptide antagonists that have similar binding affinity and function as parental proteins, with the same amino acid sequences that confer specificity, while retaining stability and solubility akin to small molecule therapeutics. While this is demonstrated for K to D lactam bridge as cross-linkers, similar results are expected if other cross-linkers are used, such as alkyl cross-links formed between two C residues via cystine alkylation, such as DBMB.
In some embodiments, the c-Jun antagonist peptide comprises at least one covalent i to i+4 amino acid residue cross-linker. For example, the c-Jun antagonist peptide may comprise two covalent i to i+4 amino acid cross-linkers. Preferably, the covalent i to i+4 cross-linker(s) are present at heptad locations b-to-f or f-to-c. Preferably, the covalent i to i+4 amino acid cross-linker(s) are K to D lactam bridge(s), or an alkyl cross-link formed between two C residues (cysteine alkylation).
In a second aspect, the invention provides a nucleic acid encoding the c-Jun antagonist peptide according to the first aspect of the invention.
In a third aspect, the invention provides a conjugate comprising the c-Jun antagonist peptide according to the first aspect of the invention conjugated to a lipid, a polymer, or a second peptide.
In a fourth aspect, the invention provides a pharmaceutical composition comprising the c-Jun antagonist according to the first aspect of the invention, nucleic acid according to the second aspect of the invention, or conjugate according to the third aspect of the invention in combination with a physiologically acceptable vehicle or carrier.
In a fifth aspect, the invention provides the c-Jun antagonist peptide according to the first aspect of the invention, a nucleic acid according to the second aspect of the invention, 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.
In a sixth aspect, the invention provides a method of inhibiting c-Jun comprising a peptide according to the first aspect of the invention, a nucleic acid according to the second aspect of the invention, or conjugate according to the third aspect of the invention, in vitro to a cell comprising or expressing c-Jun peptide.
Also provided herein are methods of producing the c-Jun antagonist peptide according to the first aspect of the invention. A method of producing a c-Jun antagonist peptide may comprise synthesising the c-Jun antagonist peptide using solid 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. Additionally provided herein are methods of producing a helix constrained c-Jun antagonist peptide, comprising contacting the c-Jun antagonist peptide according to the first aspect of the invention with a cross-linker.
The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.
These and other aspects of the invention are described in more detail below.
Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:
Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.
The c-Jun antagonist described herein typically comprises a hinge region of V[X1]EE[X2][X3]LE[X4]E, more preferably 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, wherein 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, Kor R. The c-Jun antagonists are also referred to herein as ′c-Jun antagonist peptides.
In this specification the term “hinge region” is intended to mean a ten-residue amino acid tract. The amino acid residues in the tract may be acidic (D/E) and hydrophobic residues (V), and, optionally, a K residue may be present. The “hinge region” of the c-Jun antagonist is so named because it corresponds to the “hinge” forming parts of both the DBD domain and LZ domain of c-Jun, and therefore is capable of interacting with both these domains in c-Jun. The presence of a hinge region provides a peptide with the ability of binding c-Jun as well as antagonising its DNA-binding activity. The dominant negative charge of the hinge region is also believed to result in a favourable interaction with the positive charge within the c-Jun DNA-Binding Domain (DBD).
The extended hinge region comprises the hinge region as well as an L residue at its N-terminal portion. The extended hinge region may have an amino acid sequence of LV[X1]EE[X2][X3]LE[X3]E (SEQ ID NO: 1) wherein 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. 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. The extra negatively charged amino acid residue is believed to favour the interaction with the positively charged DBD of c-Jun. Also contemplated are variants of the extended hinge region sequences described herein, wherein the variant contains 1, 2 or 3 amino acid modifications.
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 may comprise an amino acid sequence of LVVEEDVLEEE (SEQ ID NO: 31)
In other embodiments, X2 is K and X4 is D. For example, the extended hinge region may comprise an amino acid sequence of LVVEEKVLEDE (SEQ ID NO: 32). Such amino acid sequences can be used, for example, to introduce an i to i+4 K to D lactam bridge in the b-to-f heptad positions of the hinge region of the antagonist.
In other embodiments, X1 is K and X3 is D. For example, the extended hinge region may comprise an amino acid sequence of LVKEEDDLEEE (SEQ ID NO: 33). Such amino acid sequences can be used, for example, to introduce an i to i+4 K to D lactam bridge in the in the f-to-c heptad positions of the hinge region of the antagonist.
In other embodiments, X2 is C and X4 is C. For example, the extended hinge region may comprise an amino acid sequence of LVVEECVLECE (SEQ ID NO: 34). Such amino acid sequences can be used, for example, to introduce an i to i+4 alkyl cross-link in the in the b-to-f heptad positions of the hinge region of the antagonist.
In other embodiments, X1 is C and X3 is C. For example, the extended hinge region may comprise an amino acid sequence of LVCEEDCLEEE (SEQ ID NO: 35). Such amino acid sequences can be used, for example, to introduce an i to i+4 alkyl cross-link in the in the f-to-c heptad positions of the hinge region of the antagonist.
In other embodiments, X1 is C and X4 is C. For example, the extended hinge region may comprise an amino acid sequence of LVCEEDVLECE (SEQ ID NO: 36). Such amino acid sequences can be used, for example, to introduce an i to i+7 alkyl cross-link in the in the f-to-f heptad positions of the hinge region of the antagonist.
In some embodiments, one or more of X1, X2, X3, and X4 is an R or a K. In one embodiment, X4 is R or K. For example, the extended hinge region may comprise an amino acid sequence of LVVEEKVLERE (SEQ ID NO: 71). As explained below, introducing an arginine or lysine at solvent exposed positions can increase cell permeability of the peptide.
According to the present invention the extended hinge region may further comprise an N-terminal acidic extension having an amino acid sequence of EA[X5][X6] (SEQ ID NO: 2), wherein 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).
The N-terminal acidic extension is believed to produce electrostatic repulsion, advantageously reducing the tendency of the peptide to homodimerize thereby making more peptide antagonist available for heterodimerisation with c-Jun. The negative charge throughout the N-terminal domain of the acidic extension acts favourably with the positive charge of the c-Jun DBD.
In some embodiments, X1 (in the extended hinge region) is D and X5 (in the acidic extension) is K. Such amino acid sequences can be used, for example, to introduce an i to i+4 K to D lactam bridge that spans the acidic extension (heptad position b) and hinge region (heptad position f) of the antagonist.
In some embodiments, X1 (in the extended hinge region) is C and X5 (in the acidic extension) is C. Such amino acid sequences can be used, for example, to introduce an i to i+4 alkyl cross-link that spans the acidic extension (heptad position b) and hinge region (heptad position f) of the antagonist.
According to the present invention, the acidic extension may have an amino acid sequence of EAEE (SEQ ID NO:3). This amino acid sequence is believed to induces helicity and stabilises the dipole of the molecule. A further advantage of the EAEE sequence is that its two central residues, AE, occur at positions corresponding to interaction with DNA on c-Jun, thereby forming a direct block between c-Jun and DNA.
The LZ region of the antagonist of the invention is located C-terminal to the hinge region and is capable of interacting with the leucine zipper of c-Jun.
According to the present invention the LZ region may comprise or consist of an amino acid sequence selected from the group consisting of:
or a variant thereof. 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 comprises 1, 2 or 3 amino acid modifications.
In some embodiments, the LZ region comprises or consists of an amino acid sequence of IEQLEERNYALRKEIKDLQDQ (SEQ ID NO: 7), or a variant comprising 1, 2, or 3 modifications. In some embodiments, amino acid residues at positions corresponding to position 16 (position b in a heptad) and position 20 (position f in 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 the positions corresponding to 16 and 20 of SEQ ID NO: 7).
In some embodiments, the LZ region comprises or consists of an amino acid sequence of IEQLEERNYALRKEICDLQCQ (SEQ ID NO: 27), or a variant comprising 1, 2, or 3 modifications. In some embodiments, amino acid residues at positions corresponding to position 16 (position b in a heptad) and position 20 (position fin 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 the positions corresponding to 16 and 20 of SEQ ID NO: 27).
In some embodiments, the LZ region comprises:
In some embodiments, the LZ region comprises:
Such LZ regions are suitable for bisalkylation, as explained in more detail below.
In some embodiments, the LZ region comprises one or more lysine(s) and/or arginine(s) at heptad positions b, c, and/or f in the LZ region. In some embodiments, the lysine(s) or arginine(s) are located at positions other than the positions being used to introduce the cross-link (e.g. lactam bridge or bisalkylation). As explained in more detail below, the introduction of positively charged amino acids at a solvent exposed face of an α-helical peptide may improve cell penetrance.
Accordingly, in some embodiments, the LZ region comprises or consists of an amino acid sequence of IRRLERRNRALRKEIKDLQDQ (SEQ ID NO: 74), or a variant comprising 1, 2, or 3 modifications. In some embodiments, amino acid residues at positions corresponding to position 16 (position b in a heptad) and position 20 (position f in the same heptad) of SEQ ID NO: 74 in the variant are K and D amino acid residues, respectively (i.e. the amino acid modification(s) are at positions other than the positions corresponding to 16 and 20 of SEQ ID NO: 74). In some embodiments, amino acid residues at positions corresponding to position 2 (position b in a heptad) and position 3 (position c in a heptad) and position 9 (position b a heptad) of SEQ ID NO: 74 in the variant are K or R (optionally R) (i.e. the amino acid modification(s) are at positions other than the positions corresponding to 2, 3, and 9 of SEQ ID NO: 74). In some embodiments, the amino acid modification(s) are at positions other than the positions corresponding to positions 2, 3, 9, 16 and 20 of SEQ ID NO: 74.
In other embodiments, the LZ region comprises or consists of an amino acid sequence of IERLERRNYRLRREIKDLQDQ (SEQ ID NO: 75), or a variant comprising 1, 2, or 3 modifications. In some embodiments, amino acid residues at positions corresponding to position 16 (position b in a heptad) and position 20 (position f in the same heptad) of SEQ ID NO: 75 in the variant are K and D amino acid residues, respectively (i.e. the amino acid modification(s) are at positions other than the positions corresponding to 16 and 20 of SEQ ID NO: 75). In some embodiments, amino acid residues at positions corresponding to position 3 (position c in a heptad), position 10 (position c in a heptad) and position 13 (position f in a heptad) of SEQ ID NO: 75 in the variant are K or R (optionally R) (i.e. the amino acid modification(s) are at positions other than the positions corresponding to 3, 10 and 13 of SEQ ID NO: 75). In some embodiments, the amino acid modification(s) are at positions other than the positions corresponding to positions 3, 10, 13, 16 and 20 of SEQ ID NO: 75.
The c-Jun antagonist as described herein is peptidic and may be in the D-or L-form. “Peptidic” as used herein includes compounds that are composed of or comprise 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 antagonists may be comprised of D-or L-form amino acid residues, or a mixture of both. In this specification, the peptidic compounds are typically referred to as peptides.
A c-Jun antagonist as described herein may be isolated, in the sense of being free from contaminants, such as other polypeptides and/or cellular components.
The c-Jun antagonist as described herein may be in the free form, or any pharmacologically acceptable salt form, for example, a form of acid salt, metal salt, alkaline earth metal salt, or amine salt.
The c-Jun antagonist may be between 10 and 100 amino acid residues long. The c-Jun antagonist may be less than 70, preferably less than 60, more preferably less than 55, even more preferably less than 50, yet more preferably less than 45, still more preferably less than 40 amino acids long. The c-Jun antagonist may be between 30 and 70, 30 and 60, 30 and 50, or 30 and 40 amino acid residues long. For example, the c-Jun antagonist may have a length of 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.
The c-Jun antagonist may be the HingeW peptide, or a variant thereof. The HingeW peptide comprises an amino acid sequence of
LEQRAEELARENEELEKEAEELVVEEDVLEEEIEQLEERNYALRKEIEDLQKQLEKL (SEQ ID NO: 12). The HingeW peptide may further comprise one or more of the following: MAS at the N-terminus, GAP at the C-terminus, and a 6xHis tag (HHHHHH) (SEQ ID NO: 53) at the C-terminus. As described herein, the HingeW peptide was demonstrated to bind to the target c-Jun protein with a high affinity and antagonise the DNA-binding function of c-Jun, and hence is demonstrated to be a functional antagonist of c-Jun.
The c-Jun antagonist may be a truncated form of the HingeW peptide, or a variant thereof. As described herein, various truncated HingeW peptides were developed and demonstrated to be functional antagonists of HingeW. Although the functional antagonism of these truncated forms was reduced compared to the HingeW peptide, the truncated peptides are believed to exhibit more drug-like characteristics compared to the full-length HingeW peptide, suggesting that these truncated forms also represent effective therapeutic candidates for antagonising c-Jun function.
As used herein, a ‘functional antagonist’ of c-Jun is a peptidic compound that is capable of binding 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 for the essential gene dihydrofolate reductase (DHFR) is mutated to incorporate TRE sites so that introduction of c-Jun to this gene inside E. coli produces a transcriptional block that abrogates cell proliferation. The TRE site-bound c-Jun molecules sterically prevent RNA polymerase transcribing the essential gene and this can only be restored upon introduction of an effective c-Jun/TRE antagonist. Consequently, the survival of a particular cell is controlled by the ability of a peptide library member to remove the c-Jun transcriptional block and therefore to restore DHFR activity. TBS thus facilitates the identification of therapeutically valuable sequences. Further details for the TBS assay are provided in WO2020128015, which is incorporated herein by reference in its entirety. Other methods of determining antagonism of c-Jun includes a circular dichroism (CD) assay, as described in Example 2. Briefly, this assay involves preparing a sample containing the peptide and a 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, which decreases in intensity upon c-Jun binding. If peptide is capable of antagonising c-Jun DNA binding activity, increasing concentrations of the peptide will shift the peak back to the free TRE-DNA peak. Hence, peak shift can be used to quantify the ability of the peptide to antagonise c-Jun DNA binding. This method allows for the calculation of an IC50 value by fitting the titration data to a Hill equation. In some embodiments, the c-Jun antagonist is able to inhibit 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 able to inhibit 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, yet 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 the DNA-binding activity may be measured using the TBS assay (e.g. by quantifying the number of colonies) or by determining the IC50 using a circular dichroism assay described herein. Optionally, the c-Jun antagonist may have this activity when cross-linked. Methods for cross-linking peptides are described in more detail below.
In some embodiments, the c-Jun antagonist comprises or consists of any one of the following amino acid sequences:
or a variant thereof comprising 1, 2 or 3 amino acid modifications, optionally wherein the 1, 2 or 3 amino acid modifications are present in the LZ region.
As described herein, the c-Jun antagonist amino acid sequence may be modified in order to introduce covalent i to i+4 cross-linker(s) or i to i+7 cross-linker(s) into the c-Jun antagonist. The covalent i to i+4 amino acid cross-linker(s) may be K to D lactam bridge(s), or an alkyl cross-link formed between two C residues (cysteine alkylation). Preferably, i to i+4 amino acid residue cross-links are introduced at solvent exposed b-to-f (in one heptad) or f-to-c (spanning two heptads) heptad positions in order to prevent disruption of the binding surface of the helix. In this specification, heptad numbering refers to the positioning of a specific amino acid residue within a heptad repeat, which is a structural motif that consists of a repeating pattern of seven amino acids. The positions of the heptad repeat are commonly denoted by the lowercase letters a to g, typically abcdefg. Table 5 below illustrates how heptad number corresponds to the HingeW amino acid sequence.
Hence, provided herein is a c-Jun antagonist comprising a modified version of the amino acid sequence EAEELVVEEDVLEEEIEQLEERNYALRKEIEDLQKQ (SEQ ID NO: 18), wherein the modifications include one or more (e.g. one or two) of the following:
Also provided herein is a c-Jun antagonist comprising a modified version of the amino acid sequence EAEELVVEEDVLEEEIEQLEERNYALRKEIEDLQKQ (SEQ ID NO: 18), wherein the modifications include one or more (e.g. one or two) of the following:
Also provided are c-Jun antagonists where the amino acid sequence has been modified in order to introduce i to i+7 cross-linker(s) into the c-Jun antagonist. The covalent i to i+7 amino acid cross-linker(s) may be alkyl cross-link formed between two C residues (cysteine alkylation). Preferably, i to i+7 amino acid residue cross-links are introduced at solvent exposed b-to-b, c-to-c or f-to-f (spanning two heptads) heptad positions.
Hence, provided herein is a c-Jun antagonist comprising a modified version of the amino acid sequence EAEELVVEEDVLEEEIEQLEERNYALRKEIEDLQKQ (SEQ ID NO: 18), wherein the modifications include one or more (e.g. one or two) of the following:
In preferred embodiments, the c-Jun antagonist comprises or consists of the amino acid sequence of:
or a variant thereof comprising 1, 2 or 3 amino acid modifications, optionally wherein the 1, 2 or 3 amino acid modifications are present in the LZ region.
In more preferred embodiments, the c-Jun antagonist comprises or consists of the amino acid sequence of:
or a variant thereof comprising 1, 2 or 3 amino acid modifications, optionally wherein the 1, 2 or 3 amino acid modifications are present in the LZ region.
For example, in embodiments where the c-Jun antagonist comprises a K to D lactam bridge, the c-Jun antagonist may comprise or consist of the amino acid sequence of:
or a variant thereof comprising 1, 2 or 3 amino acid modifications, optionally wherein the 1, 2 or 3 amino acid modifications are present in the LZ region, further optionally wherein amino acid residues at positions corresponding to position 31 (position b in the heptad) and position 35 (position f in the heptad) of SEQ ID NO: 10 in the variant are K and D amino acid residues, respectively.
As another example, in embodiments where the c-Jun antagonist comprises two K to D lactam bridges, the c-Jun antagonist may comprise or consist of the amino acid sequence of:
or a variant thereof comprising 1, 2 or 3 amino acid modifications, optionally wherein the 1, 2 or 3 amino acid modifications are present in the LZ region,
further optionally wherein amino acid residues at positions corresponding to position 10 (position b in a first heptad) and position 14 (position f in the first heptad) of SEQ ID NO: 11 in the variant are K and D amino acid residues, respectively, and wherein amino acid residues at positions corresponding to position 31 (position b in a second heptad) and position 35 (position fin the second heptad) of SEQ ID NO: 11 in the variant are K and D amino acid residues, respectively.
As another example, in embodiments where the c-Jun antagonist comprises an alkyl cross-link, the c-Jun antagonist may comprise or consist of the amino acid sequence of
or a variant thereof comprising 1, 2 or 3 amino acid modifications, optionally wherein the 1, 2 or 3 amino acid modifications are present in the LZ region, further optionally wherein amino acid residues at positions corresponding to position 31 (position b in a heptad) and position 35 (position f in the same heptad) of SEQ ID NO: 28 in the variant are both C amino acid residues.
As another example, in embodiments where the c-Jun antagonist comprises an alkyl cross-link the c-Jun antagonist may comprise or consist of the amino acid sequence of
EAEELVVEEDVLEEEIEQLEERNYALRAEICSLOCQ (SEQ ID NO: 41) or a variant thereof comprising 1, 2, or 3 amino acid modifications, optionally wherein the amino acid modifications are present in the LZ region.
An amino acid modification may be an insertion, a substitution, or a deletion. In some embodiments, the amino acid modification is a substitution of an amino acid residue to any other amino acid residue. The substituted amino acid residue may be in the D-or L-form and may be a naturally occurring amino acid residue or a non-naturally occurring amino acid residue.
Naturally occurring residues may be divided into classes based on common side chain properties:
The amino acid substitution may be a conservative amino acid substitution. Conservative amino acid substitutions may involve 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 a substitution of the acidic amino acid glutamic acid (E) for the acidic amino acid aspartic acid (D).
Amino acid substitutions (e.g. conservative amino acid substitutions) may encompass non-naturally occurring amino acid residues, which 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 naphtylalanine (Nal), Abu, DAB, methionine sulfoxide and methionine sulfone.
In some embodiments, the amino acid modifications result in the introduction of hydrophobic and charged surface patches in the peptide. Hydrophobic and charged surface patches can be introduced by inserting clusters of amino acid residues (e.g. at least 3 contiguous residues) that are hydrophobic and/or positively charged, as described for example in Perry et al., 2018. For example, the amino acid modifications described herein may produce a c-Jun antagonist that contains at least 3 contiguous amino acid residues that are either lysine or leucine (e.g. in the extended hinge region and/or leucine region).
For c-Jun antagonists that are cross-linked, any amino acid modifications (e.g. substitutions) are typically located outside of the relevant positions that are being used for cross-linking. That is, for antagonists comprising b-to-f (in one heptad) amino acid residue cross-links, the amino acid modification(s) may be at positions a, c, d, e, or g in that heptad. Similarly, for antagonists comprising f-to-c (spanning two heptads) amino acid residue cross-links, the amino acid modification(s) may be at any of positions a, b, c, d or e in the first heptad and a, b, d, e, for g, in the second heptad.
It is known in the art that the introduction of positively charged amino acids at a solvent exposed face of an α-helical peptide improves cell penetrance (see for example, Smith et al., 2008 and Perry et al., 2018). This may be achieved by the introduction of arginine residues at specific positions in order to generate an arginine substitution pattern known to promote cell permeability as described in Smith et al., 2008.Accordingly, 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 comprises one or more arginine or lysine modifications, i.e. an arginine or lysine substitutions pattern may be introduced in the peptides described herein. In some embodiments, these arginine modifications are located at heptad positions b, c, and/or f, i.e. on the solvent exposed face of an α-helical peptide.
In some embodiments, the c-Jun antagonist peptide described herein comprises a modified version of the amino acid sequence according to SEQ ID NO: 11, wherein modifications include one or more (e.g one or two) of the following:
or a variant thereof comprising 1, 2, 3 or 4 amino acid modifications outside of the stated positions. Optionally, the amino acid residues at positions corresponding to position 31 (position b in a heptad) and position 35 (position fin a heptad) of SEQ ID NO: 72 or 73 in the variant are K and D amino acid residues, respectively (i.e. the amino acid modification(s) are at positions other than positions corresponding to positions 14, 17, 18, 31 and 35 of SEQ ID NO: 72, or at positions other than positions corresponding to positions 18, 25, 28, 31 and 35 of SEQ ID NO: 73.
Alternatively or additionally, a c-Jun antagonist may have an amino acid sequence having a specified degree of sequence identity to one of SEQ ID Nos 12 to 26. The specified degree of sequence identity may be from at least 60% to 100% sequence identity. More preferably, the specified degree of sequence identity may be one of at least 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
A c-Jun antagonist peptide as described herein may be provided using synthetic or recombinant techniques which are standard in the art. Conveniently, a c-Jun peptide as described herein may be produced by solid phase synthesis. Peptides are typically synthesized by solid phase synthesis in a stepwise fashion from the C terminus to the N terminus. In an initial step, an N protected amino acid is covalently attached to an insoluble solid support via its carbonyl group. Suitable groups for N protecting the amino acid include 9-fluorenylmethyloxycarbonyl group (Fmoc) and t-butyloxycarbonyl (Boc). Following covalent attachment of the N protected amino acid, the N protecting group is removed and the deprotected 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 comprising 2 amino acids that is covalently attached to the solid phase. This process is repeated until the complete peptide sequence is built up on the solid phase. In some embodiments, protecting groups may be employed to prevent functional groups in the side chains of amino acids from reacting with an incoming 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.
A method of producing a c-Jun antagonist peptide may comprise synthesising a peptide comprising SEQ ID NO: 1 by solid or liquid phase peptide synthesis.
Methods of solid phase peptide synthesis are well-established in the art (see for example 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, J. M.; Young, J. D. Solid-Phase Peptide Synthesis (2nd ed.), Pierce Chemical Co., Rockford, IL, 1984; Atherton, E.; Sheppard, R. C., Solid-Phase Peptide Synthesis: A Practical Approach. Oxford University Press: New York City, 1989; M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984); J. H. Jones, The Chemical Synthesis of Peptides. Oxford University Press, Oxford 1991; in Applied Biosystems 430A User's Manual, ABI Inc., Foster City, California; G. A. Grant, (Ed.) Synthetic Peptides, A User's Guide. W. H. Freeman & Co., New York 1992, and G. B. Fields, (Ed.) Solid-Phase Peptide Synthesis (Methods in Enzymology Vol. 289). Academic Press, New York and London 1997); Merrifield, J. Amer. Chem. Soc. 85:2149-54 (1963)). Methods of liquid phase peptide synthesis are also well-established in the art (U.S. Pat. No. 5,516,891).
A c-Jun antagonist peptide as described herein may be produced using recombinant expression. Recombinant techniques for producing peptides are standard in the art, for example as described in Sambrook, J., Russel, D. W. Molecular Cloning, A Laboratory Manual. 3 ed. 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press. The c-Jun antagonist peptide may be capped, for example it may be capped at the N-terminus with MAS residues and at the C-terminus with GAP residues. The c-Jun antagonist peptide as described herein may further also be His-tagged (e.g. 6xHis-tagged).
c-Jun
An antagonist peptide described herein antagonises c-Jun. C-Jun is involved in a number of 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-characterised in the art and may, for example, have the amino acid sequence of (UniProt accession P05412, version 2.).
The residues present on the surface of a protein that are responsible for PPIs are associated with protein secondary structure motifs, such as alpha-helix, 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). Additionally, alpha-helices have been shown to mediate a large number of key therapeutically relevant PPI interfaces, of which 60% bind to one face of the helix (Raj et al., 2013). Alpha-helices contain a hydrogen bond between the carbonyl group (C═O) of a given amino acid and the amino group (NH) of an amino acid three or four residues away.
Constraining peptides in a helical conformation using a cross-linker has been reported to confer benefits that include enhancing protease resistance, stability in cells, increases cellular uptake, enhanced biophysical properties and are anticipated to bind their targets with higher potency in comparison to wild-type peptide sequences (Azzarito et al. 2013). As a result, peptides that contain constrained alpha-helices (also termed “helix-constrained peptides”) have been of great interest for identifying PPI inhibitors (Robertson and Spring, 2018).
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 intramolecular cross-link between two amino acids in order to produce a stabilised alpha-helix. Generally, the cross-link extends across the length of one or two helical turns (i.e. about 3-3.6 or about 7 amino acids). Accordingly, amino acids positioned at i and one of: i+3, i+4, and i+7 are ideal candidates for cross-linking. Thus, for example, where a peptide has the sequence . . . N1, N2, N3, N4, N5, N6, N7, N8, N9 . . . , and the amino acid N is independently selected for each position, 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. Hence, as used herein, a helix-constrained peptide comprises at least one cross-linker between two amino acid residues.
Chemical modification includes a chemical modification to incorporate a molecular tether, such as a hydrocarbon staple, and a chemical modification to promote the formation of a disulphide bridge. The cross-link can be an ionic, covalent or hydrogen bond that links the two residues together, preferably the cross-link is a covalent bond.
The presence of a stabilised alpha-helix can be determined using methods such as circular dichroism spectroscopy for an alpha-helix, for example as described in Jo et al. (2012) as in the examples herein. Circular dichroism be used to measure a helicity increase, i.e. linear to cyclic. In situations where the cross-linking occurs through the formation of a disulphide bridge between two thiol groups, such as between two cysteine residues, the presence of a stabilised alpha-helix can also be determined using an assay that determining if thiols in the sample are 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 monitoring absorbance at 412 nm.
Methods of inducing cross-links between amino acids are well known and include methods that induce cross-links between the peptide backbone, e.g. between the carbonyl group and amino group as in natural alpha-helices, as well as between side-chains of the peptides.
Cross linkers 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 linkage (e.g. as described in Cabezas & Satterthwait (1999)), oxime linkage (e.g. as described in Haney et al. (2011)), metal chelation (e.g. as described in Ruan et al. (1990)), and “click” chemistry (e.g. as described in Holland-Nell & Meldal (2011)).
The cross-linker may be used to cross-link cysteine residues. Hence, the peptide may comprise a cysteine (C) at positions i and i+4, or i and i+7, in its amino acid sequence. As described in Jo et al. (2012), the introduction of cysteine residues at i and i+4 positions is useful because this spacing brings two thioether residues into proximity when in the alpha-helix. Suitable cross-linking agents for stabilising the alpha-helix within the peptide containing a cysteine (C) at position i and i+4 are described in Jo et al. (2012). For example, the cross-linking agent could be a cross-linker selected from the group consisting of an alkyl bromide, an alkyl iodide, a benzyl bromide, an allyl bromide, a maleimide, and an electrophilic difluorobenzene. Suitable cross-linkers are known in the art for crosslinking cysteine (see for example: Fairlie & Dantas de Araujo, 2016 and Jo et al., 2012).
In some embodiments, the cross-linking agent is an m-xylene based, o-xylene based, or p-xylene based benzyl bromide, more preferably a m-xylene based benzyl bromide.
In some embodiments, the cross-linker is a compound of formula 1:
wherein
The R1 groups provide reactive groups (e.g. leaving groups) for reaction with the cysteine. The A groups provide the linkers with structures suitable for conformationally constraining a peptide in a call when cross inked via the two derivatisable amino acid residues. For example, the A group may be conformational constrained into a geometry suitable for linking the two derivatisable amino acid residues. In some embodiments, R1 is Br. In some embodiments A is selected from C5-12-arylene and C5-12-heteroarylene. In some embodiments m is 0. In some embodiments Y is methylene. In some embodiments L is a covalent bond.
In some preferred embodiments, the cross-linking agent is 1,3-dibromomethylbenzene (DBMB) having the following chemical formula:
DBMB can be used to react with derivatisable amino acid residues at the i and i+3 or i and i+4 in the amino acid sequence of the peptide.
In some preferred embodiments, the cross-linking agent is 4,4′-bisbromomethyl-biphenyl (Bpy) having the following chemical formula:
Bpy can be used to react with derivatisable amino acid residues at the i and i+7 in the amino acid sequence of the peptide.
Cross-linking cysteine residues in peptides can be carried out using known methods, such as those described in Timmerman et al., 2005 or WO 2021/260074. Briefly, this method may comprise reacting the cross-linker (e.g. DBMB) with the peptide in the presence of tris (2-carboxyethyl) phosphine (TCEP) and ammonium bicarbonate, and reacted at pH 8.0 and room temperature for 4 to 5 hours in the dark. The method may be carried out in vitro or in cellulo. in cellulo methods may comprise providing a cell (e.g. a bacterial cell, such as an E. coli cell, or a eukaryotic cell, such as a human cell) containing a recombinant peptide, contacting the cell with the cross-linker (e.g. as part of the cell culture media) and culturing the cell in the presence of the cross-linker. The cross-linker may be present at a concentration of between 1 μM and 1 mM (e.g. between 10 μM and 100 μM), and for a period of at least 20 minutes (e.g. between 20 minutes and 10 hours). Further details of a suitable in cellulo cross-linking method are provided for example in WO 2021/260074.
The crosslinker forms thioether cross-links with the at least a pair of cysteines such that the c-Jun antagonist may comprise the structure:
Y, L, R1, n, m and A are as defined for formula 1. R1a represents a bond or CH2-CH2- linker derived from the appropriate R1 group in formula 1.
In embodiments where the alkyl cross-link formed between two C residues is formed by DBMB, the c-Jun antagonist may comprise the structure:
The cross-linker may be used to cross-link lysine (K) and aspartic acid (D) in the peptide. Hence, the peptide may comprise a lysine (K) and aspartic acid (D) at i and i+4 positions in its amino acid sequence.
That is, position i is a lysine (K) and position i+4 is an aspartic acid (D), or position i is an aspartic acid (D) and position i+4 is a lysine (K). For example, the K may be at b and the D at f in one heptad, or the K may be at f in one heptad and the D at c in the subsequent heptad.
Lactamisation is useful in terms of biostability since proteases universally recognise β-strands, with the constraint providing a further steric block, denying access to the backbone (Tyndall J D et al., 2005), and potentially bioavailability and membrane permeability owing to the lipophilic nature of the constraint. A lactam bridge in a peptide refers to the side chain of lysine (K) forming an amide bond with the side chain of glutamic acid (E) or aspartic acid (D), typically aspartic acid (D). Methods of carrying out K-D lactamisation are described in the examples herein and, for example, in de Araujo et al. (2014). As noted above, the cross-link may be formed between amino acids at positions i and i+3, i and i+4, or i and i+7 in the amino acid sequence of the peptide. In some embodiments, the cross-link is between cysteine (C) residues located at these positions. In other embodiments, the cross-link is between lysine (K) and aspartic acid (D) residues at these positions. Preferably, the cross-link is formed between amino acids at positions i and i+4.
In this specification, a nucleic acid encoding a c-Jun antagonist peptide may be any nucleic acid (DNA or RNA).
In some embodiments, the c-Jun antagonist may be conjugated, optionally through a linker, to another moiety, such as a fatty acid or other lipid, a polymer, or another peptide sequence (e.g. a cell penetrating peptides (CPPs). Such conjugates retain the functional antagonist property of the c-Jun antagonist, and may have one or more improved properties, such as stability, in vivo half-life, or potency, or cell penetrance relative to unconjugated c-Jun antagonist. The moiety may be conjugated to the c-Jun antagonist through the N- or C-terminus, or any other site of the peptide.
In some embodiments, the peptide may be conjugated to a cell penetrating peptides (CPP). CPPs are a class of peptides capable of penetrating the plasma membrane of mammalian cells and of transporting compounds of many types and molecular weights across the membrane. When CPPs are chemically linked or fused to other proteins, the resulting polypeptides are able to enter cells. The linkage to the CPP may be direct (e.g. as part of a fusion protein), or may be via a linker (e.g. a short peptide linker). CPPs are generally peptides of less than 30 amino acids, derived from natural or unnatural protein 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 (KALAKALAKALA) (SEQ ID NO: 59), SAP (VRLPPPVRLPPPVRLPPP) (SEQ ID NO: 60), oligoarginine (RRRRRRRR (SEQ ID NO: 61) or RRRRRRRRR (SEQ ID NO: 62)), calcitonin (LGTYTQDENKTFPQTAIGVGAP) (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.
In some embodiments, the peptide may be conjugated to a lipid. Peptide lipidation is an effective strategy to modify the pharmacokinetic, pharmacodynamic and cell penetrance properties of peptide therapeutics and has proven to be successful with several therapeutic peptides. Cholesterol and fatty acids of various chain lengths such as C8-caprylic, C12-lauric, and C16-palmitic are often utilized as lipid motifs that are covalently attached to a peptide inhibitor via ester, ether, amide or carbamate bonds. Examples of peptide lipidation are described in Kowalczyk et al. 2017.
Functionally active antagonists of c-Jun of the present invention may be useful in inhibiting c-Jun in a therapeutic setting. Thus, the c-Jun antagonist peptides of the invention may be formulated in a pharmaceutical composition.
A pharmaceutical composition is a formulation comprising one or more active agents (e.g. the c-Jun antagonist peptides or conjugates described herein) and one or more pharmaceutically acceptable excipients. The pharmaceutical composition may be capable of eliciting a therapeutic effect.
A pharmaceutical composition may comprise the c-Jun antagonist peptide or conjugate of the invention and a pharmaceutically acceptable excipient or carrier.
A method of making a pharmaceutical composition may comprise; admixing a c-Jun antagonist peptide or conjugate as described above with a pharmaceutically acceptable excipient.
The term “pharmaceutically acceptable” relates to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound veterinary or medical judgement, suitable for use in contact with the tissues of a subject (e.g. human or other mammal) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.
Suitable excipients and carriers include, without limitation, water, saline, buffered saline, phosphate buffer, alcoholic/aqueous solutions, emulsions or suspensions. Other conventionally employed diluents, adjuvants, and excipients may be added in accordance with conventional techniques. Such carriers can include ethanol, polyols, and suitable mixtures thereof, vegetable oils, and injectable organic esters. Buffers and pH-adjusting agents may also be employed, and include, without limitation, salts prepared from an organic acid or base. Representative buffers include, without limitation, organic acid salts, such as salts of citric acid (e.g., citrates), ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, phthalic acid, 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 can include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives such as, for example, antimicrobials, antioxidants, chelating agents (e.g., EGTA; EDTA), inert gases, and the like may also be provided in the pharmaceutical carriers. The pharmaceutical compositions described herein are not limited by the selection of the carrier. The preparation of these pharmaceutically-acceptable compositions, from the above-described components, having appropriate pH, isotonicity, stability and other conventional characteristics, is within the skill of the art.
Suitable carriers, excipients, etc. may be found in standard pharmaceutical texts, for example, Remington's Pharmaceutical Sciences and The Handbook of Pharmaceutical Excipients 4th edit., eds. R. C. Rowe et al, APhA Publications, 2003.
The term “carrier” refers to diluents, binders, lubricants and disintegrants. Those with skill in the art are familiar with such pharmaceutical carriers and methods of compounding pharmaceutical compositions using such carriers.
A pharmaceutical composition may conveniently be presented in unit dosage form and may be prepared by any methods well-known in the art of pharmacy. Such methods include the step of bringing the peptide into association with a carrier or excipient as described above which may constitute one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both.
Pharmaceutical compositions described herein may be produced in various forms, depending upon the route of administration. The pharmaceutical compositions may be prepared for administration to subjects in the form of, for example, liquids, powders, aerosols, tablets, capsules, enteric-coated tablets or capsules, or suppositories. Pharmaceutical compositions may also be in the form of suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials, such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt. Pharmaceutical compositions may be made in the form of sterile aqueous solutions or dispersions, suitable for injectable use, or made in lyophilized forms using freeze-drying techniques. Lyophilized pharmaceutical compositions are typically maintained at about 4° C., and can be reconstituted in a stabilizing solution, e.g., saline or HEPES, with or without adjuvant. Pharmaceutical compositions can also be made in the form of suspensions or emulsions.
Pharmaceutical compositions may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections immediately prior to use.
The pharmaceutical composition may be administered to a subject by any convenient route of administration. In some embodiments, administration is by systemic routes, including oral, or more preferably parenteral routes. For example, the pharmaceutical composition may be administered by intravenous, intraperitoneal or subcutaneous injection.
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 range of human diseases. Accordingly, a c-Jun antagonist peptide, nucleic acid, conjugate, or pharmaceutical composition as described herein may be for use in a method of treatment of the animal or human body, for example a c-Jun-mediated disease in an individual in need thereof.
An individual with a c-Jun-mediated disease may display at least one identifiable sign, symptom, or laboratory finding that is sufficient to make a diagnosis of a c-Jun-mediated disorder in accordance with clinical standards known in the art. Examples of such clinical standards can be found in textbooks of medicine such as Harrison's Principles of Internal Medicine, 15th Ed., Fauci A S et al., eds., McGraw-Hill, New York, 2001. In some embodiments, the individual may have been previously identified or diagnosed with a c-Jun-mediated disorder or a method of the invention may comprise identifying or diagnosing the presence of a c-Jun-mediated disorder in the individual, prognosing a c-Jun-mediated disorder or assessing the risk of onset of a c-Jun-mediated disorder in the individual.
Treatment may be any treatment and therapy, whether of a human or an animal (e.g. in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition or delay of the progress of the c-Jun-mediated disease, and includes a reduction in the rate of progress, a halt in the rate of progress, amelioration of the c-Jun-mediated disease, cure or remission (whether partial or total) of the c-Jun-mediated disease, preventing, delaying, abating or arresting one or more symptoms and/or signs of the c-Jun-mediated disease or prolonging survival of a subject or patient beyond that expected in the absence of treatment.
Treatment as a prophylactic measure (i.e. prophylaxis) is also included (e.g. treatment before the onset of a condition in an individual to reduce the risk of the condition occurring in the individual; delay its onset; or reduce its severity after onset). For example, an individual susceptible to or at risk of the occurrence or re-occurrence of a c-Jun-mediated disease, such as cancer may be treated as described herein. Such treatment may prevent or delay the occurrence or re-occurrence of a c-Jun-mediated disease or one or more symptoms thereof in the individual.
The c-Jun antagonist peptide may be used in a method of treatment of any one of the following diseases: cancer, diabetes, cardiovascular disease, autoimmune disease, joint disorders (such as arthritis), and neurodegenerative disease.
A “cancer” can comprise any one or more of the following: acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical cancer, anal cancer, bladder cancer, blood cancer, bone cancer, brain tumor, breast cancer, cancer of the female genital system, cancer of the male genital system, central nervous system lymphoma, cervical cancer, childhood rhabdomyosarcoma, childhood sarcoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), colon and rectal cancer, colon cancer, endometrial cancer, endometrial sarcoma, esophageal cancer, eye cancer, gallbladder cancer, gastric cancer, gastrointestinal tract cancer, hairy cell leukemia, head and neck cancer, hepatocellular cancer, Hodgkin's disease, hypopharyngeal cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leukemia, leukemia, liver cancer, lung cancer, malignant 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 tumor, plasma cell neoplasm, primary CNS lymphoma, prostate cancer, rectal cancer, respiratory system, retinoblastoma, salivary gland cancer, skin cancer, small intestine cancer, soft tissue sarcoma, stomach cancer, stomach cancer, testicular cancer, thyroid cancer, urinary system cancer, uterine sarcoma, vaginal cancer, vascular system, Waldenstrom's macroglobulinemia and Wilms' tumor.
Cancers may be of a particular type. Examples of types of cancer 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).
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 a number of indices that indicate change within the cancer to a more developed form. Thus, indices for measuring an inhibition of cancer growth include a decrease in cancer cell survival, a decrease in tumour volume or morphology (for example, as determined using computed tomographic (CT), sonography, or other imaging method), a delayed tumour growth, a destruction of tumour vasculature, improved performance in delayed hypersensitivity skin test, an increase in the activity of T cells, and a decrease in levels of tumour-specific antigens.
In some embodiments, a c-Jun antagonist peptide may be useful in inhibiting or reducing the metastasis of a cancer. For example, a method of reducing or inhibiting metastasis in an individual with cancer may comprise administering therapeutically effective amounts of a c-Jun peptide to the individual
An individual suitable for treatment as described above may be a mammal, such as a rodent (e.g. a guinea pig, a hamster, a rat, a mouse), murine (e.g. a mouse), canine (e.g. a dog), feline (e.g. a cat), equine (e.g. a horse), a primate, simian (e.g. a monkey or ape), a monkey (e.g. marmoset, baboon), an ape (e.g. gorilla, chimpanzee, orang-utan, gibbon), or a human.
In some preferred embodiments, the individual is a human. In other preferred embodiments, non-human mammals, especially mammals that are conventionally used as models for demonstrating therapeutic efficacy in humans (e.g. murine, primate, porcine, canine, or rabbit animals) may be employed.
Methods according to the present invention may be performed, or products may be present, in vitro, ex vivo, or in vivo. The term “in vitro” is intended to encompass experiments with materials, biological substances, cells and/or tissues in laboratory conditions or in culture whereas the term “in vivo” is intended to encompass experiments and procedures with intact multi-cellular organisms. “Ex vivo” refers to something present or taking place outside an organism, e.g. outside the human or animal body, which may be on tissue (e.g. whole organs) or cells taken from the organism.
Where the method is performed in vitro it may comprise a high throughput screening assay. Test compounds used in the method may be obtained from a synthetic combinatorial peptide library, or may be synthetic peptides or peptide mimetic molecules.
The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.
While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.
For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations.
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 word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply 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.
It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents 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 values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example +/−10%.
Certain aspects and embodiments will now be illustrated by way of example and with reference to the figures described above.
Many rational design approaches, library screens, and selection systems exist and have resulted in the successful identification of molecules capable of binding to given TF targets, but a key challenge remains in ensuring that target binding will translate into ablation of function (Brennan et al., 2020; Baxter et al., 2014). Various methodologies 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 if LZ binding will translate into functional antagonism as the c-Jun DBD remains unbound and capable of binding TRE DNA (Seldeen et al., 2008; Szalóki N et al., 2015). A rationally designed peptide has been shown to target the cJun DBD but exhibits lower potency than LZ antagonists, with concerns over specificity due to high sequence similarity across the AP-1 family DBDs (Tsuchida et al 2004). Similarly, a range of SMs targeting TRE DNA have been developed (Dai et al., 2004; Fanjul et al., 1994) but these are also lower potency and have the potential to produce off-target effects since multiple TFs typically bind to any given DNA element, with some bZIP/DNA combinations known to promote anti-oncogenic outcomes (Eferl et al., 2003; Rodriguez-Martinez J A et al., 2017). One approach to circumvent the potential downsides of these methods is to utilise longer peptides that target the full c-Jun bZIP domain with a selective yet high affinity interaction, simultaneously blocking both DNA binding and LZ dimerisation. Olive et al. took this approach to produce A-Fos, which combined the wild-type (WT) cFos LZ (known to heterodimerise with c-Jun) and a rationally designed Glu-rich acidic extension (
Plasmid Constructs and Protein Production: The TRE-mDHFR (
LEQRAEELARENEELEKEAEELEQELDELQAEIEQLEERNYALRKEIEDLQKQLEKL (FosW sequence in bold). All constructs are capped at the N-terminus with AS residues and at the C-terminus with GAP residues and are also 6×His-tagged, other than the WT-or TRE-mDHFR constructs which are only 6×His-tagged. A full list of sequences is provided in Table 1.
Proteins were purified by subcloning their DNA sequences into either a pET21-His-SUMO plasmid (cJun bZIP, cFos bZIP) or a pET24a plasmid (HingeW, A-FosW, FosW) using NheI and AscI sites. An overnight culture of E. coli containing the relevant plasmid was used to inoculate LB media at a dilution factor of 1:1000. This culture was incubated with shaking (37° C., 200 rpm) until the OD600nm reached 0.7. Protein over-expression was induced by the addition of IPTG (1 mM) before incubation with shaking (25° C., 200 rpm) overnight. Cells were then harvested from the culture by centrifugation. Cell pellets were resuspended in Histrap Binding Buffer (20 mM potassium phosphate, 500 mM NaCl, 40 mM imidazole, 5 mM DTT, pH 7.4), sonicated and loaded on a HisTrap HP 5 mL pre-loaded column. The column was washed with Binding Buffer before eluting protein samples on a Binding Buffer: Elution Buffer (20 mM potassium phosphate buffer, 500 mM NaCl, 400 mM imidazole, 5 mM DTT, PH 7.4) gradient. This methodology was also used to produce 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 h. As the SUMO-tagged construct was N-terminally His-tagged on the SUMO, the cleavage reaction was diluted 1 in 5 in Binding Buffer and then passed through the HisTrap column to remove the cleaved SUMO tag and the His-tagged ULP1. The HisTrap flowthrough was finally purified to >98% purity by using RP-HPLC with a Jupiter Proteo column (4-um particle size, 90 Å pore size, 250×10 mm; Phenomenex) using a water: acetonitrile gradient (0.1% TFA). Peptides without a SUMO tag, were concentrated after Histrap elution and HPLC purified. Peptide purity and identity were verified by SDS-PAGE and electrospray ionisation mass spectrometry.
DHFR Activity Assay: A colorimetric assay kit (Sigma CD0340) was used to measure the activity of purified DHFR enzymes. WT- or TRE-mDHFR (100 nM in reaction) and NADPH (60 μM in reaction) were mixed in assay buffer only, or with DHFR inhibitors TMP or Mtx (1 μM in reaction). Reactions were initiated by the addition of DHF (50 μM in reaction plus a blank reaction with no DHF) and the absorbance at 340 nm of samples was measured using a Varian Cary 50 UV-Vis spectrophotometer. The specific activity was calculated using the following equation. Specific activity=(ΔOD/minsample−ΔOD/minblank)/12.3×mg protein
Library Construction and TBS Assay: Library inserts were produced using PCR fill-in reactions from synthesised primers (Sigma) with degenerate codons at the desired positions to produce the correct residue options. The library was subcloned using SacI and AscI sites into the pET24a plasmid containing A-FosW. The primers used were cJun-Hinge-Lib-F: 5′-GAAGAGCTCSWGSWGSWGSWGSWTSWGCTGSWGGMASWGATTGAACAGCTGGAAGAACGCAAC TATGCC-3′ (SEQ ID NO: 49) and cJun-Hinge-Lib_R: 5′-TGAGGCGCGCCCAGTTTCTCCAGCTGTTTCTGGAGGTCTTCGATCTCTTTGCGCAAGGCATAGTTGC GTTC-3′ (SEQ ID NO: 50). The library DNA was transformed into NEB 10-beta electrocompetent E. coli cells. The following equation was utilised to determine library coverage by the number of single colonies: E=100×(1−1/n)m where E is the percentage of the library missing, m is the number of colonies collected and n is the library size. This showed that from 2155000 library colonies collected, 99.9% of the Hinge library was covered. Library DNA quality was assessed by sequencing both the DNA pool and a number of single colonies to show degenerate codons in the correct positions in the pool and to show a diversity of library members from single colonies. The pool of library DNA was transformed into BL21 Gold cells already containing pES300d-TRE-mDHFR and pES230d-cJun bZIP.
Selective pressure is applied by growing the bacteria in M9 minimal media with TMP (2-4 μM) alongside ampicillin, kanamycin and chloramphenicol to maintain the required plasmids, and IPTG (1 mM) to induce protein expression. The library transformants were first plated out onto selective agar plates (2 μM TMP) and grown at 37° C. for 72-96 h. Optimisation experiments (
Circular Dichroism (CD): An Applied Photophysics Chirascan was used for CD measurements, with a 200 μL 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 at pH 7.4 and were equilibrated for 30 minutes before measurement. For full spectra, three scans between 190 and 260 nm (265-320 nm for DNA binding experiments) were collected with a bandwidth of 1 nm and data sampled at a rate of 0.5 s−1. These scans were averaged and converted to molar residue ellipticities (MRE). Thermal denaturation experiments were performed by measuring the ellipticity at 222 nm over a 1 to 90° C. gradient at 1° C. increments. Post-melt scans at 20° C. confirmed the transitions were reversible as they overlaid within 10% of the pre-melt scan. The resulting thermal denaturation curves were converted to MRE and fitted to a two-state model, derived via modification of the Gibbs-Helmholtz equation to determine the melting temperature (Tm) (Mason et al., 2007).
Isothermal Titration Calorimetry (ITC): Peptides were studied in an 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 peptide (HingeW or A-FosW) at 10 μM were injected into the cell containing cJun at 1 μM. MicroCal Origin software was used to record and analyse the heat change upon addition. Control experiments involved the injection of the antagonist peptide sample into the cell containing ITC buffer alone to determine the heat of dilution which was subtracted. The resulting binding data were fit to a one site binding model to extract the enthalpy change of binding (ΔH) and the equilibrium binding constant (KD), from which the free energy change of binding (ΔG) and the entropy change of binding (AS) was calculated (Wiseman et al., 1989). Thermodynamic parameters are presented as an average of two independent experiments with errors given as one standard deviation.
Electrophoretic Mobility Shift Assay (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) and mixed at a 1:1 ratio, then heated to 95° C. for 20 minutes before cooling slowly to room temperature to form DNA duplexes. Protein/DNA samples for electrophoresis were incubated at 4° C. for 30 minutes in binding buffer (150 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, 10 mM Tris, 10 mM MgCl2, pH 8) before running on a 1.3% agarose gel in 0.5×TBE buffer (supplemented with 10 mM MgCl2). SYBR® Green stain was included in the gel and running buffer to stain for DNA which was imaged on a transilluminator before SYPRO® Ruby was added and incubated for 3 h to stain for protein. The gel was destained in a 10% methanol, 7% acetic acid solution for 1 h before imaging on a transilluminator.
1.2 Creation of an Active mDHFR from a TRE-Containing Gene to Facilitate a c-Jun Imposed Transcriptional Block
Transcription block survival (TBS) is an intracellular assay that utilises cell survival as a readout. This allows protein-DNA interaction antagonists to be screened, and the most active identified by their ability to remove a transcriptional block on exogenous murine dihydrofolate reductase (DHFR). This enzyme is absolutely essential for survival since it is required for the production of purines needed for DNA and amino acid synthesis. Endogenous E.coli DHFR (ecDHFR) can be selectively inhibited by trimethoprim (TMP), meaning that cells grown in M9 minimal media are rendered dependent on exogenous murine DHFR (mDHFR) activity for their survival (Matthews et al., 1985). We produced a mDHFR gene (
We first sought to confirm whether the new TRE-mDHFR construct could replace the TMP-inhibited ecDHFR by confirming it expresses, folds, and is catalytically active. This was achieved via i) SDS-PAGE analysis of cell lysate, confirming that the protein is expressed within the soluble fraction upon isopropyl β-D-1-thiogalactopyranoside (IPTG) induction (
Having established that TRE-mDHFR is active and absolutely required for cell survival under selective conditions (M9 minimal media+4 μM Tmp+1 mM IPTG), we next expressed the cJun bZIP domain in cells containing the TRE-mDHFR plasmid, which resulted in a 21-fold reduction in colony counts (P≤0.0001;
TRE binding sites) reduced bacterial proliferation (P≤0.05,
Next, peptides known to bind to cJun were introduced into the system, to establish whether they can impact upon cJun function—i.e. sequester the cJun bZIP as a non-functional heterodimer therefore preventing DNA-binding and rescuing TRE-mDHFR transcription. Here, we used two peptides targeting the cJun LZ domain: cFos LZ and FosW, an optimised sequence identified from a protein-fragment complementation assay (PCA) that readily binds to cJun in the absence of DNA at 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, producing no significant increase in colony numbers from the transcriptionally blocked cells (P>0.05 in both cases,
The acidic extension design principle is the most successful methodology in the literature to target the full bZIP domain of various proteins (Olive et al., 1997; Ahn et al., 1998; Chen et al., 2011). However, incomplete restoration of colonies using A-FosW indicated that transcription remained partially hampered by cJun binding across the 15 TRE sites. This allowed us to employ TBS to screen a peptide library, using A-FosW as a design template, towards further improvement in cJun/TRE DNA antagonism. The library design utilised semi-randomised positions within the hinge region that straddles the acidic extension and LZ domains (
During the TBS library screening process, E. coli were transformed with the pooled DNA plasmid library such that each cell expressed a given member. Cells were plated onto M9 selective media and incubated until colonies expressing TBS active library members had formed. These colonies were pooled and repeated liquid culture passages were undertaken under selective conditions to compete library members against each other, enriching 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 inform on which residues were being selected at each position until one discrete DNA sequence was detected in the culture, referred to as HingeW (
1.6 HingeW Binds c-Jun Preferentially Over A-FosW
Experiments were next undertaken to compare the binding of A-FosW and TBS-optimised HingeW to the cJun bZIP. CD spectroscopy was utilised to measure the global secondary structure of homo-and heterodimeric peptide samples, providing information on overall α-helicity and thermal melting temperatures (Tm). The HingeW/cJun spectrum was 82% higher in α-helical content relative to the average of the two component spectra expected for no interaction (
Thermal denaturation analysis of HingeW/cJun, following the loss of signal at 222 nm, displayed a Tm of 71.2° C. for the HingeW/cJun heterodimer, representing a clear increase from the Tm of the two component denaturation profiles (
1.7 HingeW Outcompetes A-FosW for c-Jun Binding
Direct competition between HingeW and A-FosW for cJun binding was observed using CD dimer exchange experiments in which a solution containing one antagonist/cJun mixture was combined with the other antagonist to observe potential changes in α-helicity, as an indicator of a change in cJun dimerisation partner. In this case, when HingeW was mixed with the preformed A-FosW/cJun heterodimer a 17% increase in helicity was observed, as measured at 222 nm, relative to the average, indicating a change in binding (
The binding interactions of cJun with HingeW and A-FosW were further studied by ITC, to provide information on the thermodynamic parameters. The data produced from the injection of HingeW into cJun (
1.9 HingeW Effectively Antagonises the c-Jun/TRE DNA Interaction
The binding of cJun to TRE DNA can be observed by monitoring a DNA absorbance peak in the CD spectrum centred at ˜281 nm (John M et al., 1996). Peptides (cJun, HingeW or A-FosW) in isolation do not absorb at this wavelength meaning that all changes in the spectrum in this region correspond to shifts in DNA conformation. Addition of cJun (20 μM) to TRE DNA (5 μM) decreases this DNA peak by 55% as the cJun engages its target TRE site and alters the DNA structure (
To provide further evidence of functional antagonism, an electrophoretic mobility shift assay (EMSA) was employed. Firstly, cJun bZIP (20 μM) was mixed with the TRE DNA construct (2 μM), resulting in a significant reduction in the free DNA band intensity relative to DNA alone (
A summary of the thermodynamic parameters for the interactions between c-Jun and either the rationally designed A-FosW template or the TBS library-derived HingeW is provided in Table 4 as follows:
There are many screening platforms in place to derive high affinity PPIs but none that guarantee target binding will lead to the desired loss-of-function of the target protein. Using cJun/TRE as an exemplar, we have developed a Transcription Block Survival assay that can be used as a generalised approach for the derivation of peptides capable of ablating TF activity. We have engineered a ‘molecular dial’ into a bacterial system whereby the cJun/TRE DNA interaction is inversely correlated with cell proliferation. By introducing cJun/TRE antagonists into this system, cellular growth becomes a direct readout for the ability of the antagonist to functionally block the numerous cJun/TRE interactions, turning the molecular dial up. The most effective rationally designed acidic antagonist was next utilised as a parental sequence to design a semi-randomised library that was successfully screened in the TBS platform, to produce the in vitro validated assay hit HingeW.
Establishing the TBS system required the production of a mutant DHFR gene (TRE-mDHFR) which retained its enzymatic activity upon introduction of 15 TRE sites into its DNA sequence, leading to 13 amino acid substitutions. This allowed for a cJun-induced transcriptional block when the TF binds to the TRE sites on the TRE-mDHFR plasmid DNA. For loss of TRE-mDHFR activity to take place there is an absolute requirement for both the TF DBD and the TRE sites within the mDHFR gene, confirming specificity in the TBS system. The phenotype of bacterial growth rate is directly linked to the genotype of the antagonist sequence expressed by virtue of the systems containment in a single cell. The phenotype of bacterial growth rate is directly linked to the genotype of the antagonist sequence expressed by virtue of the system's containment within a single cell. Bacterial cells are ideal for this process owing to their fast growth rate, durability, ease of use and low cost. Crucially, they also allow for the direct measurement of cJun interacting with TRE sites in the absence of any related eukaryotic TFs that might interfere with the assay.
TBS facilitates high-throughput genotype to phenotype screening and competition of peptide libraries to isolate those that result in functional loss of cJun DNA binding activity from those that bind but have little or no effect upon target activity (or those that do not bind at all). The distinction is important since it means that an antagonist must not only bind to the target free in solution but must also be capable of meeting the much more demanding task of liberating the TF from DNA, which is known to be more stable (Seldeen et al., 2011). Lastly, all the above is undertaken within the complex environment of the cytoplasm, removing molecules that are toxic, non-specific, insoluble, or protease susceptible from consideration at the initial screening stage, rather than determining this at later hit validation or clinical trial stages. These factors are particularly important for longer peptides, as required to bind to the large and shallow cJun bZIP surface, which tend to lack these important qualities. TBS improves upon the related protein-fragment complementation assay, as well as in vitro screening platforms such as phage display or ribosome display, by the complete removal of any requirement for bulky protein fusions or hydrophobic/aromatic tags, which can interfere with the relevant assay interactions and lead to false readouts.
The central advantage of TBS is the requirement for assay hits to prevent TFs from binding to their consensus DNA sequence as exemplified by the combined design of A-FosW, a hybrid containing domains from both A-Fos (Olive et al., 1997) and the FosW PCA hit (Mason et al., 2006). In A-FosW the LZ targets the antagonist to the cJun bZIP with high affinity and selectivity, with the acidic extension added to assist in functionally antagonising the cJun/TRE DNA interaction by blocking the cJun DBD. The LZ domains of bZIP proteins tend to display more sequence diversity than the DBD, which is useful for therapeutic targeting of specific AP-1 family members, which provides better control and potentially fewer side effects (Eferl et al., 2003). Although it is unclear if A-FosW binds cJun by forming a single continuous LZ interaction as designed, increased binding around the hinge region of cJun was anticipated to propagate increased helicity and therefore affinity in either direction. Further, focusing 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) produced a significant increase in cJun binding affinity and subsequent cJun/TRE antagonism (Olive M et al., 1997). Optimisation of the acidic extension through rational design is hampered by the lack of design rules for guidance, as is the case for the LZ domain which has known structure and predicative tools to produce high affinity interactions (Boysen R I et al., 2002; Kaplan J B et al., 2014). Additionally, no library-based approach had previously been used to optimise binding within this region of cJun. Using A-FosW as a design template and including library options in the hinge region was a clear next step which resulted in the TBS selection of HingeW, with 14 nM affinity for the target cJun protein (a 6-fold improvement over A-FosW). HingeW included one more acidic residue than A-FosW, supporting the Olive et al. methodology of including dominant negative charge throughout the N-terminal domain to interact favourably with positive charge within the cJun DBD. However, the precise selection pattern was more nuanced than simply producing a block of negative charged residues. The nature of HingeW suggests another benefit of the TBS library screening approach, in which directed evolution of the antagonist led to an improvement by reducing homodimerisation. TBS has provided considerable utility in the exploration of novel sequence space by producing a protein sequence which could not have been predicted without the use of this library screening approach.
TBS opens a new capability in semi-rational PPI design where both affinity and activity are co-selected for. This offers significant potential to expand the TBS approach to both new libraries and targets where previous work may have produced potential antagonists which were later found to lack functional activity. In principle, the approach can be fully expanded to any DNA-binding protein that recognises a discrete consensus sequence, or even any dimeric system to which a DBD is appended. The method can be assumed to be generalizable, since any DNA consensus sequence can be incorporated into the DHFR DNA sequence and can be transcriptionally blocked by co-expression of the relevant TFs. This will require the DHFR design process to be iterated and subsequent testing and optimization for each system, however, the central principle has been shown here to be valid. It also potentially permits for the screening of exogenous molecules to allow concomitant profiling of both cell penetrant and functionally active inhibitors. Moreover, libraries with different design principles and expanded options harbour considerable additional promise in producing peptide hits across a broad range of targets in which pathogenic TFs are implicated. Library sizes of 106-107 are possible using standard techniques and readily available reagents which may allow the exploitation of a broader range of peptide diversity and further optimisation. Further TBS screening of a range of TF targets will produce both non-genetic tools and probes of disease pathways, but there is also considerable potential for a new generation of optimised functional antagonists and clinical leads.
This example shows the optimisation of the peptide library screen-derived hit of example 1, designed to target the full cJun bZIP domain in an attempt to simultaneously block both cJun dimerisation and DNA-binding. TBS screening of a 130,000-member peptide library resulted in the HingeW sequence (HW1). HingeW was developed to be capable of binding across the full cJun bZIP domain for more effective functional antagonism of TRE binding, relative to DBD-only or LZ-only cJun inhibitors. The nature of the broad, shallow helical binding surface supports the use of longer peptides such as Hinge. However it was unclear whether the full length of the sequence was required to achieve functional antagonism. HW1 was recombinantly produced and biophysically characterised as a capped (MAS at the N-terminus, GAP at the C-terminus) and C-terminally 6×His-tagged protein construct of 69 amino acids in length, with significant negative charge throughout. Optimisation of the peptides was carried out with the aim to improve their drug-like characteristics.
Peptide synthesis and purification: All peptides were synthesised using a Liberty Blue microwave peptide synthesiser (CEM) at a 0.1 mmol scale on ChemMatrix Rink amid resin using standard Fmoc solid-phase methodology. Coupling was performed using 5× amino acid, 4.5× PyBOP and 10× diisopropylethylamine in dimethylformamide (DMF, 5 mL). Deprotection was performed using 20% piperidine in DMF. Peptides were capped at the N-terminus by a final reaction with 3× acetic anhydride, 4.5× diisopropylethylamine in DMF for 5 min at 90° C. For lactamised peptides, the relevant K and D positions were orthogonally protected by the use of Lys (Mtt) and Asp (O-2-PhiPr). The sidechains 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 sidechains were coupled in PyBOP (1 mL), diisopropylethylamine (1 mL) and DMF (3 mL) for 5 hours at 60° C. The resin was dried, and the same reagents added for a second reaction for 16 hours at 60° C. Incubation in a 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 side chain protecting groups. The resin was removed by filtration and cleaved peptides were precipitated in diethyl ether at −80° C. and centrifuged. This pellet was washed a further four times with diethyl ether before it was dried overnight at room temperature. Peptides were resuspended in 3:1 water: acetonitrile before purification using RP-HPLC with a Jupiter Proteo column (4-μm particle size, 90 A pore size, 250×10 mm; Phenomenex) using a water: acetonitrile gradient (0.1% TFA). Peptide masses and purity (>95%) were verified by electrospray ionisation mass spectrometry.
Circular Dichroism (CD): An Applied Photophysics Chirascan was used for CD measurements, with a 200 μL 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 at pH 7.4 and were equilibrated for 30minutes before measurement. For full spectra, three scans between 190 and 260 nm (265-320 nm for DNA binding experiments) were collected with a bandwidth of 1 nm and data sampled at a rate of 0.5 s−1.These scans were averaged and converted to molar residue ellipticities (MRE). Thermal denaturation experiments were performed by measuring the ellipticity at 222 nm over a 1 to 90° C. gradient at 1° C. increments. Post-melt scans at 20° C. confirmed the transitions were reversible as they overlaid within 10% of the pre-melt scan. The resulting thermal denaturation curves were converted to MRE and fitted to a two-state model, derived via modification of the Gibbs-Helmholtz equation to determine the melting temperature (Tm) (Mason et al., 2007).
Serum Stability: Peptide stocks (600 μM) were prepared in water and 50 μL added to 950 μL human serum (Merck) before incubation at 37° C. 100 μL aliquots were removed at designated timepoints and added to 300 μL 3:1 acetonitrile: water and centrifuged (18000×g, 15 minutes). The supernatant was analysed by LC-MS and quantified using the sum of the two largest charge state intensities (1: 9+, 10+; 23,24: 3+, 4+).
Isothermal Titration calorimetry (ITC): Peptides were studied by ITC using a PEAQ-ITC (Malvern Instruments) using 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 peptide at 25-200 μM were injected into the cell containing cJun at 2.5-20 μM. Software was used to record and analyse the heat change upon addition. Control experiments involved the injection of the antagonist peptide sample into the cell containing ITC buffer alone to determine the heat of dilution which was subtracted. The resulting binding data were fit to a one site binding model to extract the enthalpy change of binding (ΔH) and the equilibrium binding constant (KD), from which the free energy change of binding (ΔG) and the entropy change of binding (ΔS) was calculated (Wiseman et al., 1989). Thermodynamic parameters are presented as an average of two independent experiments with errors given as one standard deviation.
A summary of the various peptides used and described in this section is provided in the Table 5 as follows:
The precise nature of the interaction between the c-Jun DBD and the rationally designed acidic domain of 1 is unknown. Therefore, the acidic domain was the initial focus for optimisation. 1 was iteratively truncated (2-6) to investigate the effect on c-Jun binding and c-Jun/TRE DNA antagonism. Peptide helicity was determined by quantifying the CD signal at 222 nm of peptide only samples. Thermal denaturation experiments were then used to determine the Tm of peptide-c-Jun heterodimers, which was used as an approximate measure of target engagement. CD was also used to investigate the ability of the functional activity of peptides in antagonising the c-Jun/TRE DNA interaction. The TRE-DNA construct used produces a positive CD peak at ˜281 nm (no c-Jun absorbance at this wavelength allows for direct measurement of DNA upon binding) which decreases in intensity upon c-Jun binding. This provided a clear and direct measurement of the proportion of DNA bound. As increasing concentrations of antagonist peptides were added to the sample, the peak shifts back to overlay with the free TRE-DNA. This allowed for the calculation of an IC50 value by fitting the titration data to a Hill equation. Due to the higher concentrations required in these experiments (for signal to be detected in the CD) the IC50 values are corresponding higher, as there is 20 μM cJun in the experimental conditions the lowest the IC50 could drop is 10 μM. Therefore, these values should be taken in context and not compared with values produced by different methodologies but instead used here for comparison amongst antagonists tested. The same 60 amino acid c-Jun bZIP construct was used for all experiments, regardless of antagonist peptide length. Through these experiments, the target binding and subsequent target antagonism were both rapidly characterised in vitro.
A summary of the thermal denaturation (Tm), functional activity (IC50 CD (μM)), helicity (fH (%)) results obtained for each of the peptides tested is provided in Table 6 as follows:
All N-terminal truncations in the series (2-6) reduced peptide/target binding and antagonism efficacy, indicating that the full length of the acidic extension contributes to antagonism of the c-Jun/TRE interaction (
Truncating the N-terminus also leads to sequential increases in fraction helicity (fH), rising from ˜27% for 1 to ˜47% for 5, indicating that deleted regions are of lower helicity relative to the LZ region. The LZ domain of 1 was mostly unchanged from its parent sequence, FosW, which is known to homodimerize (Mason et al., 2006). The negative charge of the acidic extension produces electrostatic repulsion and therefore decreases the propensity to homodimerse, with their removal increasing homodimerisation-induced helicity. However, further truncation from 5 to 6 reverses this trend, reducing fH to ˜38.0% while removing one acidic and three hydrophobic residues, to leave the LZ-only domain. There is little further electrostatic repulsion to be reduced but this region likely contributes to homodimerisation through direct binding or helix induction.
Overall, removal of the acidic extension domain (1 to 6) reduced cJun/TRE DNA antagonism 9.7-fold. Previously, we observed a highly significant reduction in peptide activity during TBS screening for peptides with IC50 values similar to that observed for peptide 6 (130 μM), meaning that 6 may be unable to fully outcompete cJun/TRE DNA binding. This further supported the design rationale of the acidic extension and suggested that 5 (IC50=78 uM, 5.8× lower antagonism than 1, and 1.7× better than 6) may be considered the maximum viable truncation from the N-terminus to be taken forward.
Peptide 5 was next optimised by incorporating i→i+4 (K-to-D) lactam bridges. Lactam bridges can be incorporated through the use of orthogonal-protecting groups (Lys(Mtt) and Asp(O-2-PhiPr)), which can be selectively deprotected (2% trifluoroacetic acid in DCM) and reacted using typical solid phase chemistry while the peptide is still attached to the resin. The success of the reaction can be confirmed using mass spectrometry (MS) to observe the decreased mass from the loss of a water molecule, compared with the linear unreacted peptide. In this example lactams were only introduced at solvent exposed b-to-f or f-to-c heptad positions to prevent disruption of the binding surface of the helix. Point mutations to the sequence were required to incorporate the bridging K and D residues, with both linear (7,9) and cyclised (8,10) versions of each sequence produced by split batch synthesis.
Cyclised peptides 8 and 10 increase antagonism relative to the linear counterpart 5, 1.8-fold and 1.9-fold respectively. The side chain lactamisation reactions lead to increased helicity which translate to higher affinity binding. It is interesting to note in these peptides that the binding indicated by Tm values of ˜67 and ˜68° C. do not produce correspondingly low IC50 values. Tm can be seen to inversely correlate with IC50 for each peptide as predicted, however there is a level of variability in the trend (
Truncation from 4 to 5 removed a block of negative charge (EAEE) and resulted in a 1.7× reduction in antagonism. This suggests the regions importance for interacting with the positively charged cJun DBD surface, as well as inducing helicity and potentially stabilising the dipole of the molecule (Pace et al., 1998; Sali et al., 1988). This correlates with work on the interaction of the cJun DBD with TRE DNA that has shown the particular residues which interact directly with the DNA. The central two residues of this added block (EAEE—moving from 10 to 11) occur at positions corresponding to interaction with DNA on cJun i.e.
these assist in form a direct block between cJun and DNA. The NΔ20 truncation may therefore be considered as an optimal balance between downsizing and retaining functional activity.
Peptide 11 was therefore the next step in optimisation which utilises the NΔ20 truncation whilst also truncating at the C-terminus. The removal of the four C-terminal residues from 4 to 11 reduced antagonism 1.8× but further truncation at the C-terminus to produce 25 vastly reduced antagonism 14.8× compared to 11, and 26.7× compared to 4. Attempts to optimise 25 by lactamisation to produce 27 and 29 were effective in that they significantly improved antagonism however they still produce 8.8× and 10.4× reductions in antagonism relative to 1. Although these lactamisations produced a much larger impact on the peptides, they were still considered to be too ineffective for further study.
Peptide 11 was considered as a scaffold for further optimisation which has almost half the number of residues compared with 1 whilst retaining a high level of functional activity. K-to-D lactam bridges at i to i+4 positions were systematically incorporated at different sites to investigate which regions were most amenable to the helix constraint, and which produced improvements in affinity and inhibition. Again, due to point mutations to accommodate the bridging K and D residues, both linear and cyclised peptides were produced. The heterodimer ΔTm from lactamisation ranges from ˜2° C. for 14/15 to ˜9° C. for 22/23. It is also useful to compare to the original parent sequence where the heterodimer ΔTm ranges from ˜1° C. for 11/13 to ˜8° C. for 11/23. C-Jun/TRE antagonism is the most important measure in this work however and only 23 produced a significant improvement in IC50, shown to be 1.6× lower than for 11 (P=0.003). By the use of a lactam, 23 restores the reduction in antagonism caused by truncating the four C-terminal residues of 4.
Information regarding the suitability of each site for lactamisation can be gathered from this data. As the change in fraction helicity of the peptides ranges from ˜1% for 12/13 to ˜8% for 22/23. There is a correlation between the increase in peptide helicity induced by lactamisation at a particular site and the increase in cJun/TRE antagonism observed.
Although the lactamisation within 17 did not lead to a significant increase in antagonism (P=0.24), it did produce ˜5% increase in peptide helicity and ˜9° C. increase in cJun heterodimer Tm from its linear form 16. Due to the distance between this site and the lactamisation site of 23, a double lactamised peptide was produced, 24. The double lactamisation produces a peptide that is ˜4% more helical, no significant change in heterodimer Tm but a significant decrease in IC50 value (P=0.02), compared to the best single lactamised peptide.
A selection of the most effective peptides from this work were investigated by ITC to quantify the thermodynamic parameters of their interaction with target cJun (
1, 23 and 24 were also tested for their serum stability to illustrate the effect of truncation and lactamisation (
A rationally designed series of peptides was synthesised to investigate and optimise HingeW (1), through the systematic study of truncation and helix-inducing lactamisation. 1 proved to be inaccessible via SPPS using standard techniques (i.e. without native chemical ligation), with 2 and 3 only produced at strikingly low yields. The remaining smaller peptides were all produced to a similar and high yield via SPPS. The increased production efficiency derived through moving from recombinant expression to SPPS should not be understated. The process of iterative truncation and characterisation elucidates the efficacy of different portions of the parent sequence and allowed careful consideration of how much peptide to truncate to derive a minimal effective sequence. In this example, the important consideration is whether a peptide binding to the target c-Jun will be able to outcompete dimeric c-Jun bound to TRE. This interaction has been variously established as having a KD˜100-200 nM so maintaining a stronger interaction than this was considered an important benchmark (Seldeen et al., 2011). Comparing the CD antagonism data of 1 and 6 (FIG. 18B) illustrates this important consideration as 1 is able to restore the TRE DNA peak to its unbound intensity, indicating that no DNA is bound to cJun at high peptide concentration. However, whilst 6 is inhibiting the interaction, it may be that the line does not tend towards 1 and that regardless of the amount of 6 present in the sample some c-Jun/TRE DNA complex will remain. The balance between this loss of efficacy and the gains in drug-like characteristics and synthesis efficiency are difficult to quantify but the data suggested the truncation from 1 to 4, which removed 20 residues from the N-terminus. Truncation at the C-terminus from 4 to 11 was also supported as in more truncation weakened the peptides efficacy to a degree whereby even lactamisation could not restore binding to the point where the peptide can outcompete the c-Jun/TRE DNA interaction.
The acceptable degree of truncation at the N-terminus appears to go beyond binding affinity considerations as this acidic domain is crucial for blocking the c-Jun DBD. Peptides 5-10 are no longer able to effectively prevent DNA binding to the c-Jun DBD, i.e. cJun may still bind to TRE sites as a monomer whilst these antagonist peptides are bound to its LZ domain. However, effective antagonism might not require binding to the full length of the c-Jun DBD. Specific contacts between particular c-Jun DBD amino acid side chains and TRE DNA bases are known so it can be assumed that an antagonist that binds any of these c-Jun residues will significantly reduce c-Jun/TRE binding, so binding to the full DBD may not be required to maintain high levels of inhibition (Glover et al., 1995; Seldeen et al., 2011). So peptide 4 appears to be the maximum acceptable truncation as it extends N-terminally to a point where it can directly block the c-Jun DBD from interacting with DNA, and crucially presents a block of negatively charged residues in this region to attract the DBD and repel DNA. 4, by virtue of the addition of EAEE residues, has slightly reduced homodimerisation compared to 5 and better antagonism.
Almost half of the length of 1 has been removed to produce 24, with a high level of efficacy retained. Due to the nature of the α-helical coiled coil structure and hydrogen bond networks, residues which are not involved in binding to the peptide's dimer partner may still affect binding by the induction of helicity. Further, the helicity of peptides studied here must be considered in their various dimerisation equilibria e.g. between homodimer, monomer and heterodimer, with increases in either dimerisation event tending to lead to increases in helicity. Reducing the length of an α-helix will tend to reduce its helicity unless the removed residues disrupt the structure (e.g. if it contains proline or glycine) or repel either dimer binding partner (e.g. electrostatic repulsion).
The 20% increase in peptide helicity from 1 to 24 resulted in increased peptide serum stability. The N-terminal regions of the full length 1 in particular appear to be non-helical and don't improve target antagonism so their removal increases stability without a significant impact on peptide efficacy. Cyclisation increases peptide helicity, which reduces protease recognition and increases peptide serum stability. Adding the second lactam in 24 does increase this stability further though the increase in stability is smaller than for the addition of the first lactam. Peptide degradation for the lactamised peptides cannot be fit to an exponential decay function as it occurs too slowly.
Particular lactam flanking residues, at any given position, may be better suited to accommodating the lactam and adopting a helix structure than others. How the peptide folds to adopt the helical structure may also influence the effect of a lactam, as for example if the helix fold is propagated from one end of the peptide to the other then the effect of a lactam will likely vary depending on how close to this locus of folding they are located.
The thermodynamic parameters of binding observed by ITC show a slightly unexpected result in terms of the entropic component. It is usually asserted that introduction of lactam bridges increases binding affinity by preorganising the peptide molecule into its helical structure which can bind to the target with an entropic penalty. All of the peptides investigated by ITC have an unfavourable entropic component however 17 has a significantly lower contribution from this component. In this case it does appear that the entropic penalty of binding is being reduced by preorganising the peptide into a helical fold that is complimentary to the cJun binding surface. However, for 23 and particularly 24 there is large unfavourable contribution from the entropic component. This illustrates that increased target binding affinity from sidechain cyclisation can also occur due to improved enthalpic interactions.
To identify further peptide HingeW variants that can be cyclised using bisalkylation, as opposed to lactamisation, variants of the c-Jun antagonist sequence
EAEELVVEEDVLEEEIEQLEERNYALRKEICDLQCQ (SEQ ID NO: 28) were screened using the TBS library screen described in Example 1. The screen identified the sequences set out in Table 8 below.
Out of the five sequences listed in Table 8, 0W and metaDBMBW were cyclised using bisalkylation with mDBMB and the functional activity of the linear and cyclised forms were tested in a CD assay and results and the CD curves are shown in
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| Number | Date | Country | Kind |
|---|---|---|---|
| 2203399.7 | Mar 2022 | GB | national |
This application is a 371 National Stage filing and claims the benefit under 35 U.S.C. § 120 to International Application No. PCT/EP2023/056243, filed 10 Mar. 2023, which claims priority to Great Britain Application No. 2203399.7 filed on 11 Mar. 2022, the contents and elements of each of which are herein incorporated by reference in their entirety for all purposes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2023/056243 | 3/10/2023 | WO |
