Methods of producing conformationally constrained peptides

Abstract

The present invention relates to methods of producing a conformationally constrained peptide in cellulo using a pnictogen-containing crosslinker. The present invention also relates to conformationally constrained peptides obtainable by said methods and a method for screening peptide libraries for transcription factor agonists that tests whether said conformationally constrained peptide inhibits association between two candidate binding partners. The present invention also relates to the use of a pnictogen-containing cross-linker in a method of producing a conformationally constrained peptide in cellulo. The present invention also relates to recombinant peptides comprising three thiol- / selenol-containing residues linked to a pnictogen-containing cross-linker. The present invention also relates to a kit for use in said methods.

Description

[0001] Methods of producing conformationally constrained peptides This application claims priority from GB2418996.1 filed 23 December 2024, the contents and elements of which are herein incorporated by reference for all purposes.

[0002] Field of the Invention

[0003] The present invention relates to methods of producing conformationally constrained peptides and particularly, although not exclusively, to a method of producing a conformationally constrained peptide in cellulo.

[0004] Background

[0005] Proteins are essential for the function of a variety of biological processes. Protein function, such as engagement in particular protein-protein interactions, is usually linked to its three-dimensional structure. The tertiary structure of a protein is formed by an arrangement of secondary structures, such as alpha helices and beta sheets, which are the result of specific intramolecular and intermolecular interactions between amino acid residues in the primary amino acid sequence. Many protein applications require stabilisation of these tertiary structures to prevent unfolding under harsh conditions involving chemical and thermal stressors (Haim et al., 2021).

[0006] Protein structures are stabilised by disulfide bridges and many chemical strategies take advantage of existing cysteines to generate conformationally constrained peptides. For example, conventional scaffolds such as 1 ,3-bis(bromomethyl)benzene (DBMB) can be used in vitro to crosslink cysteine residues in a peptide (Timmerman et al., 2005). Cysteine residues can also be introduced into different secondary structures and these can subsequently be coordinated onto synthetic crosslinks to stabilise the tertiary structure in vitro (Pelay-Gimeno et al., 2018).

[0007] Three cysteine thiol groups in different secondary structures have also been reacted with a C3 symmetric, tris-electrophilic agent composed of a central core structure projecting three electrophilic groups, and this macrocyclisation strategy has been shown to stabilise the resulting structure (Neubacher et al., 2020). Based on ability of Bismuth(lll) to bind cysteine residues in peptides, a trivalent bismuth salt can also be used for in vitro cyclisation of peptides with three cysteine residues to produce stable constrained peptides (Matzapetakis et al., 2006; Cun et al., 2008; Voss et al. 2022). In addition to the improved stability of the constrained peptides, phage selection of peptide-bismuth crosslinked peptides has been shown to result in cyclic peptides with 100-fold improved affinity compared with linear peptides, further underlying the potential of bismuth-based crosslinking in improving peptide affinity to target proteins (He et al., 2024; Ullrich et al., 2024).

[0008] Notably, to ensure a biocompatible approach when using bismuth(lll), an aqueous solution is typically used at physiological pH, and, as most BiIHforms insoluble oxido / hydroxido species in water, a highlyconcentrated stock of BiBr3 in dimethylsulfoxide (DMSO), which is toxic to cells (Verheijen et al., 2019), must be used that is directly added to this aqueous solution (Voss et al., 2022).

[0009] The present invention has been devised in light of the above considerations.

[0010] Summary of the Invention

[0011] The present inventors surprisingly found that a pnictogen-containing crosslinker is able to cross the cell membrane and coordinate and form covalent bonds with thiol-containing residues present within different secondary structures in a peptide within a living cell. To the best of the inventors’ knowledge, this is the first time that a pnictogen-containing thiol-reactive crosslinker was shown to form intermolecular covalent bonds in cellulo. This ability to enter cells to introduce post-translational peptide modifications intracellularly offers a significant advantage over in vitro approaches as it negates the need to purify individual peptides which can be used immediately in downstream intracellular screening assays, e.g. to test whether the crosslinked peptide is able to disrupt protein-protein interactions within the presence of the host proteome. The presently disclosed method is thus more efficient than the above-described prior art method of in vitro cyclisation and simplifies the process for identifying conformationally constrained peptides that can be applied in downstream processes.

[0012] Accordingly, in a first aspect there is provided a method of producing a conformationally constrained peptide in cellulo, the method comprising the steps of:

[0013] i. providing a cell containing a recombinant peptide, the recombinant peptide comprising at least two thiol-containing residues or selenol-containing residues,

[0014] ii. contacting the cell with a pnictogen-containing crosslinker, wherein the pnictogen-containing crosslinker is thiol-reactive and / or a selenol-reactive,

[0015] Hi. culturing the cells in the presence of the crosslinker, such that a covalent bond is formed between each of the at least two thiol-containing or selenol-containing residues and a pnictogen of the pnictogen-containing crosslinker, thereby producing a conformationally constrained peptide.

[0016] In some embodiments, the thiol-containing residues are cysteines, or the selenol-containing residues are selenocysteines.

[0017] In some embodiments, the recombinant peptide comprises three thiol-containing residues or selenol-containing residues. In some embodiments, the recombinant peptide comprises six thiol-containing residues or selenol-containing residues.

[0018] In some embodiments, the pnictogen-containing crosslinker is non-planar.

[0019] The present inventors have surprisingly found that non-planar crosslinkers are able to cross the cell membrane and facilitate the generation of conformationally constrained peptides in cellulo. This was unexpected as it was previously believed that small molecules most likely to accumulate within cells are planar (i.e. have low globularity), as described in Richter et al., 2017.In some embodiments, the pnictogen is bismuth or a pharmaceutically acceptable salt thereof. The pnictogen-containing crosslinker may be trivalent. The pnictogen-containing crosslinker may comprise a trihalide such as chlorine, bromine, or iodine.

[0020] In some embodiments, the pnictogen-containing crosslinker comprises BiBr3.

[0021] BiBr3 provides a stable, sustainable, and non-toxic linker for in cellulo production of conformationally constrained peptides. In addition, the three bromide groups arrange around the central bismuth atom to form a three-dimensional, pyramidal shape. The inventors surprisingly found that a non-planar molecule, such as BiBr3, was able to cross the cell membrane and effectively facilitate the formation of intermolecular covalent bonds in a peptide in cellulo.

[0022] In some embodiments, the pnictogen-containing crosslinker comprises one or more of bismuth tripotassium citrate, bismuth citrate, ammonium bismuth citrate, bismuth acetate, and bismuth subsalicylate. In some embodiments, the pnictogen-containing thiol reactive crosslinker is a threefold-symmetric. In some embodiments, the pnictogen-containing crosslinker comprises bismuth tripotassium citrate.

[0023] In some embodiments, the pnictogen-containing crosslinker comprises a functional moiety, optionally wherein the functional moiety is an affinity tag (e.g. a His tag) , and / or a detectable label (e.g. a GFP tag), and / or a degradation tag (e.g. a SSRA tag), and / or a proteolysis targeting chimera (PROTAC), and / or another peptide (e.g. a cell penetrating peptide (CPP); and / or a fatty acid or lipid; and / or a therapeutic agent.

[0024] In some embodiments, the culturing step Hi. (e.g. where the crosslinker is BiBr3) is performed in a culture medium comprising butan-2-one.

[0025] The present inventors found, surprisingly, that adding butan-2-one significantly increased the yield of the recombinant peptide. This advantageously provides for a more efficient and sustainable method for producing a conformationally constrained peptide.

[0026] In some embodiments, the culture medium comprises between 0.010% and 0.50% butan-2-one, optionally wherein the culture medium comprises between 0.025% and 0.25% butan-2-one.

[0027] In some embodiments, the concentration of pnictogen-containing crosslinker in the culture medium is between 10 and 300 pM, optionally wherein the concentration of the crosslinker is about 25 pM, 50 pM, 75 pM, 100 pM, 150 pM, 200 pM, or 250 pM.

[0028] In some embodiments, the concentration of pnictogen-containing crosslinker in the culture medium is between 10 pM and 1 mM, optionally wherein the concentration of the crosslinker is about 25 pM, 50 pM, 75 pM, 100 pM, 150 pM, 200 pM, 250 pM, 500 pM or 1 mM.

[0029] In some embodiments, the pnictogen-containing crosslinker comprises BiBr3 and the concentration of BiBr3 in the culture medium is between 25 and 150 pM, such as about 75 pM.In some embodiments, the pnictogen-containing crosslinker comprises bismuth tripotassium citrate and the concentration of bismuth tripotassium citrate in the culture medium is between 150 and 500 pM, such as about 250 pM.

[0030] Advantageously, the method provided herein enables the use of lower concentrations of crosslinker, and thus provides a cheaper and more sustainable process compared with prior art crosslinkers that must be used at higher concentrations.

[0031] In some embodiments, the culture medium further comprises a reducing agent, optionally wherein the reducing agent is tris(2-carboxyethyl)phosphine (TCEP).

[0032] In some embodiments, the cell is cultured in the presence of the crosslinker for at least 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 18 hours, 20 hours, 24 hours, 36 hours, or 48 hours.

[0033] In some embodiments, the recombinant peptide is over-expressed in the cell. Advantageously, overexpressed protein reacts with the compound and prevents it reacting with other thiols in the proteasome.

[0034] In some embodiments, the recombinant peptide has previously been modified to introduce one or more of the at least two thiol-containing residues or selenol-containing residues, or wherein the thiol-containing residues or selenol-containing residues are native thiol-containing residues.

[0035] In some embodiments, the recombinant peptide has a length of at least 10, at least 100, or at least 500 amino acids.

[0036] In some embodiments, the cell is a prokaryotic cell, optionally a bacterial cell, or a eukaryotic cell, optionally a yeast cell, plant cell, insect cell, or mammalian cell.

[0037] In some embodiments, the step of providing the cell containing the recombinant peptide comprises delivering a nucleic acid encoding the recombinant peptide to the cell, such that the cell expresses the recombinant peptide.

[0038] In some embodiments, the method is performed at a temperature of about 37°C and / or a pH of about 7.4. In some embodiments, the method further comprises isolating the crosslinked peptide from the cell. In some embodiments, the cross-linked peptide has increased resistance to thermal denaturation compared with a peptide not contacted with the crosslinker, optionally wherein the increased resistance to denaturation is determined by circular dichroism.

[0039] In a second aspect, there is provided a conformationally constrained peptide obtainable by a method according to the first aspect of the invention.

[0040] In a third aspect, there is provided a recombinant peptide comprising three thiol-containing residues, wherein each of the three thiol-containing residues is covalently linked to a pnictogen-containing crosslinker.In some embodiments, the recombinant peptide comprises two sets of three thiol-containing or selenol-containing residues, wherein each of the three thiol-containing or selenol-containing residues in a set is covalently linked to a pnictogen-containing crosslinker.

[0041] In a fourth aspect, there is a provided the use of a pnictogen-containing thiol reactive crosslinker in a method of producing a conformationally constrained peptide in cellulo.

[0042] In a fifth aspect, there is provided the recombinant peptide of the third aspect of the invention, or the use of the fourth aspect of the invention, wherein the thiol-containing residues are cysteines.

[0043] In some embodiments, the pnictogen is bismuth.

[0044] In a sixth aspect, there is provided a method for screening peptide libraries fortranscription factor antagonists, the method comprising:

[0045] a) providing a cell, wherein the cell comprises a conformationally constrained peptide obtained according to the method of the first aspect of the invention, and a first and second candidate binding partner, and

[0046] b) assaying whether the conformationally constrained peptide is able to inhibit association between the first and second candidate binding partner.

[0047] In some embodiments, assaying for whether the conformationally constrained peptide is able to inhibit association between the first and second candidate binding partner comprises determining whether the conformationally constrained peptide is able to modulate expression and / or activity of a reporter protein. In some embodiments, association of the first and second candidate binding partners forms a DNA binding complex that binds to one or more binding sites in a nucleic acid encoding the reporter protein, wherein binding of the DNA-binding complex to the binding site inhibits expression of the reporter protein, optionally wherein some or all of the binding sites are located in the transcribed sequence of the nucleic acid encoding the reporter protein.

[0048] In some embodiments, the first and second candidate binding partners are transcription factors (e.g. human transcription factors).

[0049] In some embodiments, the transcription factor is a bHLH transcription factor, optionally wherein the bHLH transcription factor is c-Myc; a bZIP transcription factor, optionally wherein the bZIP transcription factor is c-Jun, CREB, and / or BZLF1 ; a DLX transcription factor, optionally wherein the DLX transcription factor is DLX-1 , DLX-2, DLX-3, DLX-4, DLX-5, and / or DLX-6.

[0050] In a seventh aspect, there is provided a kit for use in the method according to the first aspect of the invention, wherein the kit comprises:

[0051] i. a nucleic acid encoding a recombinant peptide, wherein the recombinant peptide comprises at least two thiol-containing residues or selenol-containing residues, andii. a pnictogen-containing crosslinker, wherein the pnictogen-containing crosslinker is thiol-reactive or a selenol-reactive.

[0052] The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

[0053] Summary of the Figures

[0054] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

[0055] Figure 1 shows microbial growth of E. Coli expressing a test peptide construct. (A) shows the ODeoonm of E. Coli containing plasmids for expression of the test construct grown in 20 mL LB medium, containing ampicillin and IPTG and indicated concentrations of BiBre (50 pM, 75 pM, 100 pM, 250 pM, 500 pM, and butan-2-one (0.05%, 0.075%, 0.1%, 0.25%, and 0.5%). (B) shows E. Coli containing plasmid for expression of the test construct grown in 20 mL LB + Amp + 75 pM BiBr3 with or without the addition of IPTG.

[0056] Figure 2 shows the LC-MS results of partially purified test peptides grown (A) in the absence of BiBr3; or in media supplemented with (B) 25 pM BiBr3, 0.025% butan-2-one; (C) 50 pM Bi Br3, 0.05% butan-2-one; (D) 75 pM BiBr3, 0.075% butan-2-one; (E) 100 pM BiBr3, 0.1% butan-2-one; (F) 250 pM BiBr3, 0.1% butan-2-one.

[0057] Figure 3 shows the percentage of cyclised peptide versus bacterial growth measured by ODeoonm.

[0058] Figure 4 shows the results of a mini protein cyclisation experiment. (A) shows Alphafold3 predicted structure of the mini protein shown from two angles. The three cysteines are predicted to be held in proximity to each other with two of them forming a disulphide. (B) shows the mass spectrometry results of the linear (top) and bismuth-cyclised (bottom) mini proteins. (C) shows that CD spectra of linear and cyclised 3xhelixV1 with or without the addition of reducing agent TCEP (tris(2-carboxyethyl)phosphine) at 20°C. (D) shows CD thermal denaturation experiments of linear and cyclised 3xhelixV1 with or without the addition of TCEP.

[0059] Figure 5 shows yeast growth in the presence of the indicated compounds and solvents. (A) yeast extract peptone dextrose (YPD) + Ampicillin (AMP), (B) YPD + Amp + 0.25% butanone, (C) YPD + Amp + 0.5% butanone, (D) YPD + Amp + 1% butanone; (E) YPD + Amp +0.25% butanone +75 pM BiBrs, (F) YPD + Amp + 0.25% butanone + 250 pM BiBrs, (G) YPD + Amp +75 pM Gastrodenol, (H) YPD + Amp + 250 pM Gastrodenol, (I) YPD + Amp + 0.25% butanone + 75 pM Bi acetate, (J) YPD + Amp + 0.25% butanone + 250 pM Bi acetate.

[0060] Figure 6 shows microbial growth of E. coli expressing a test peptide construct. (A) shows the ODeoonm of E. coli containing plasmids for expression of the test construct grown in 20 mL LB medium, containing ampicillin and indicated concentrations of BiBre (0 pM, 25 pM, 50 pM, 75 pM, 100 pM and 250 pM) and butan-2-one (0.0%, 0.025%, 0.05%, 0.075%, 0.1% and 0.25%). (B) shows the ODeoonm of E. colicontaining plasmids for expression of the test construct grown in 20 mL LB medium, containing ampicillin and indicated concentrations of BiK3[citrate]2 (0 pM, 50 pM, 100 pM, 250 pM, 500 pM and 1 mM).

[0061] Figure 7 shows microbial growth of E. coli expressing a test peptide construct. (A) shows the ODeoonm of E. Coli containing plasmids for expression of the test construct grown in the same conditions as Fig 6A but in the presence of IPTG. (B) shows the ODeoonm of E. coli containing plasmids for expression of the test construct grown in the same conditions as Fig 6B but in the presence of IPTG.

[0062] Figure 8 shows the LC-MS results of purified test peptide expressed in bacterial media supplemented with (A) 75 pM BiBrs, 0.075% butan-2-one or (B) 250 pM BiK3[citrate]2.

[0063] Figure 9 shows microbial growth of E. coli expressing a test peptide construct. (A) shows the ODeoonm of E. coli containing plasmids for expression of the test construct grown in 20 mL LB medium, containing either ampicillin, 75 pM of BiBrs and 0.075% butan-2-one, or ampicillin and 250 pM BiKs [citrate] 2, with or without IPTG. (B) shows the ODeoonm of E. coli grown with 250 pM BiK3[citrate]2, with or without IPTG, from (A). (C) shows the ODeoonm of E. coli grown with 75 pM of BiBrs and 0.075% butan-2-one, with or without IPTG, from (A).

[0064] Figure 10 shows microbial growth of E. coli expressing a test peptide construct and the ratio of cyclised-to-linear peptides determined by LC-MS with time for E. coli grown with (A) 75 pM of BiBrsand 0.075% butan-2-one (B) 250 pM BiK3[citrate]2.

[0065] Figure 11 shows Alphafold3 predictions of miniprotein structures compared to the original gHHH_06 NMR structure (PDB ID: 2ND2).

[0066] Figure 12 shows the mass spectrometry results of linear and BiK3[citrate]2-cyclised miniproteins (A) 1 , (B) 2 and (C) 3 synthesised in vitro.

[0067] Figure 13 shows the CD spectroscopy results of linear and BiK3[citrate]2-cyclised miniproteins (A) 1 , (B) 2 and (C) 3, either with or without TCEP.

[0068] Figure 14 shows the CD thermal denaturation results of linear and BiK3[citrate]2-cyclised miniproteins 1 , 2 and 3 in the presence of TCEP.

[0069] Figure 15 shows the fraction of linear or BiK3[citrate]2-cyclised miniproteins 1 , 2 and 3 remaining in human serum with incubation time. Data were collected in triplicate and are plotted as an average with error bars shown as one standard deviation

[0070] Figure 16 shows the CD spectroscopy results of synthetic BiK3[citrate]2-cyclised miniproteins.

[0071] Figure 17 shows the mass spectrometry results of linear and BiK3[citrate]2-cyclised SUMO-miniproteins (A) 1 and (B) 3 expressed and purified from E. coli.

[0072] Figure 18 shows the CD spectroscopy results of synthesised and recombinant (A) BiK3[citrate]2-cyclised miniproteinl and (B) BiK3[citrate]2-cyclised miniprotein3.Detailed Description of the Invention

[0073] 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.

[0074] Described herein are methods of producing conformationally constrained peptides in cellulo, comprising the steps of:

[0075] i. providing a cell containing a recombinant peptide, the recombinant peptide comprising at least two thiol-containing residues or selenol-containing residues,

[0076] ii. contacting the cell with a pnictogen-containing crosslinker, wherein the pnictogen-containing crosslinker is thiol-reactive and / or selenol-reactive,

[0077] Hi. culturing the cells in the presence of the crosslinker, such that a covalent bond is formed between each of the at least two thiol-containing or selenol-containing residues and a pnictogen of the pnictogen-containing crosslinker, thereby producing a conformationally constrained peptide.

[0078] The method as described herein is performed in cellulo, i.e. within a living cell. The term “in cellulo”, or intracellularly, is intended to encompass experiments that take place involving cells and may be in cultured cells or cells or tissues taken from an organism. The methods described herein are not practiced on the human or animal body.

[0079] The cell used in the methods described herein may be a prokaryotic or eukaryotic cell. Typically, cells are isolated cells.

[0080] The cell used in methods described herein may be a bacterial cell, such as an Escherichia coli cell, for example BL21 (DE3), XL-1 , RV308, Shuffle T7, or DH5alpha cells. Methods where the cell is a bacterial cell may involve culturing the bacterial cell in suitable media. Such techniques are well known to those of skill in the art.

[0081] Alternatively, the cell is a eukaryotic cell such as a yeast cell (such as Saccharomyces cerevisiae or Pichia pastoris), a plant cell, insect cell or a mammalian cell. In some embodiments, the cell is a mammalian cell, for example a human cell. Mammalian cells, especially human cells, may be somatic cells. Screening methods where the cell is a eukaryotic cell may involve culture or fermentation of the eukaryotic cell. The culture or fermentation may be performed in a bioreactor provided with an appropriate supply of nutrients, air / oxygen and / or growth factors. Culture, fermentation and separation techniques are well known to those of skill in the art.

[0082] As used herein, a conformationally constrained peptide refers to a peptide with a restricted flexibility, such as cyclised peptides. Assays for determining whether the methods described herein result in a conformationally constrained peptides are well known to the skilled person. For example, the presence of a cross-link in a conformationally-constrained peptide may be determined by mass spectrometry, such asliquid chromatography mass spectrometry (LC-MS) spectrometry as cyclised protein confirmations have an increased mass compared to their linear form.

[0083] The pnictogen-containing crosslinker may stabilise the tertiary structure of a peptide, i.e. it may form covalent bonds with thiol-containing or selenol-containing residues in different secondary structures of the recombinant peptide, i.e., the pnictogen of the pnictogen-containing thiol reactive crosslinker may coordinate the formation of intermolecular bonds. The recombinant peptide may comprise helical conformations, i.e. two or more alpha helices, and / or beta-sheets that can be stabilised by the pnictogencrosslinker. In some embodiments, the recombinant peptide comprises two or more secondary structures, for example two or more alpha helices and / or beta sheets, or other secondary structures such as beta turns and / or omega loops. In some embodiments, the recombinant peptide comprises two or more alpha helices. In some embodiments, the recombinant peptide comprises three alpha helices. In such embodiments, each of the three alpha helices may comprise one thiol-containing residue or selenol-containing residue that forms a covalent bond with the pnictogen of the pnictogen-containing crosslinker. In these instances, the crosslinker is an interhelical crosslinker and coordinates at least two thiol-containing residues, such as cysteines, i.e. one residue in each helix, so that the at least two helices in the recombinant peptide form a conformationally constrained peptide. Helicity may be determined by measuring the mean residue ellipticity (MRE) using circular dichroism spectroscopy, with a relative decrease in MRE being indicative of increased helicity compared with a linear (non-constrained) peptide. In some embodiments, the recombinant peptide comprises two or more beta sheets. For example, the crosslinker may conformationally restrain two or more beta sheets, such as three beta sheets, i.e. the crosslinker may coordinate three thiol-containing residues, such as cysteines, so that the three beta sheets in the recombinant peptide form a conformationally constrained peptide.

[0084] The pnictogen-containing crosslinker may stabilise the quaternary polypeptide structures, i.e. it may form covalent bonds with thiol-containing residues in different polypeptide chains or subunits.

[0085] The cell contains a recombinant peptide, that is recombinant peptide is expressed and remains entirely localised within the cell’s cytoplasm during the production of the conformationally constrained peptide. When the cell is contacted with the pnictogen-containing crosslinker, the peptide is localised within the cell, and not, for example, expressed on the surface of the cell. Methods of determining whether the recombinant peptide is localised within the cell are known in the art. For example, cells expressing the peptide can be lysed and separated into different fractions using differential centrifugation allowing for the identification of the peptide in the cytosol fraction using standard techniques.

[0086] The recombinant peptide comprises at least two thiol-containing (-SH) residues, or selenol-containing (HSe) (e.g. selenocysteine), which may be natural or non-natural amino acids. In some embodiments, the recombinant peptide comprises a mixture of thiol-containing and selenol-containing residues. In preferred embodiments, the thiol-containing residue is a cysteine. The thiol-containing residues may be native cysteines or non-native cysteines, i.e. the recombinant peptide has previously been modified to introduce one or more of the at least two cysteine residues. In some embodiments, the recombinant peptide consists of three thiol-containing (-SH) residues. In some embodiments, the recombinant peptide comprises three thiol-containing residues. In some embodiments, the recombinant peptide comprisesthree selenol-containing residues. In some embodiments, the recombinant peptide consists of three selenol-containing residues. In some embodiments, the recombinant peptide consists of six thiol-containing (-SH) residues. In some embodiments, the recombinant peptide comprises six thiol-containing residues. In some embodiments, the recombinant peptide comprises six selenol-containing residues. In some embodiments, the recombinant peptide consists of six selenol-containing residues.

[0087] The pnictogen-containing cross-linker used in the methods of the invention is capable of accessing the cytosol of the cell in order to react with the thiol-containing residues present in the recombinant peptide localised within the cell, i.e. the pnictogen-containing crosslinker is thiol reactive. This results in the formation of covalent bonds between the thiol groups and the pnictogen of the pnictogen-containing crosslinker, thus conformationally restraining the peptide. A pnictogen as used herein refers to an element in group 15 of the periodic table, also known as the nitrogen group, and includes nitrogen (N), phosphorus (P), arsenic (Ar), antimony (Sb), and bismuth (Bi), or a pharmaceutically acceptable salt thereof. In some embodiments, the pnictogen is selected from the group bismuth, antimony, or arsenic, or a pharmaceutically acceptable salt thereof. In preferred embodiments, the pnictogen is bismuth.

[0088] In some embodiments, the pnictogen-containing crosslinker is non-planar. “Non-planar” as used herein refers to a molecule that is not organised in a single plane, but instead forms in a three- dimensional shape, such as a pyramidal shape.

[0089] The shape of a compound may also be described by referring to “g lobu larity”, a term which provides information on the three-dimensionality of compounds, where a completely flat, or planar, compound has a globu larity of 0 and a spherical compound has a globularity of 1. Therefore, in some embodiments, the pnictogen-containing thiol reactive crosslinkers have a globularity of > 0.

[0090] In some embodiments, the pnictogen-containing thiol-reactive crosslinker comprises a carboxylate ligand. In some embodiments, the pnictogen-containing crosslinker is trivalent, that is it contains three thiolreactive (or selenol reactive) ends.

[0091] In some embodiments the crosslinker is a trihalide, that is a compound containing 3 atoms of halogen combined with an element, such as bismuth. The halogen may be selected from chlorine (Cl), bromine (Br) or iodine (I). In preferred embodiments, the crosslinker comprises the trihalide, BiBr3. In some embodiments, one of the thiol (or selenol) reactive ends of the crosslinker comprises a functional moiety as described herein. For example, in the instance where the crosslinker is a trihalide, one of the three reactive ends that would normally react with a halogen is now connected to (i.e. fused or covalently attached) to a functional moiety. This means that the crosslinker would react with two thiol (or selenol)-containing groups instead of three because one of the reactive ends is masked by the functional moiety. For example, in instances where the pnictogen-containing crosslinker is BiBr3, one of the bromines is replaced with a functional moiety as described herein.

[0092] In alternative embodiments, the crosslinker comprises bismuth tripotassium dicitrate (also known as Gastrodenol or bismuth tripotassium citrate or Bi Ksfcitratek); bismuth citrate ([O2CCH2C(OH)(CO2)CH2CO2]Bi); ammonium bismuth citrate; bismuth acetate (Bi(CH3COO)3); bismuthsubsalicylate (C HsBiO^, also known under the generic name “pink bismuth”. In some embodiments, the crosslinker comprises bismuth tripotassium dicitrate.

[0093] The crosslinker may be three-fold rotationally symmetric, or “three-fold symmetric”. Three-fold rotational symmetry as used herein allows for the selective coordination of the three thiol-containing residues so that one type of cross-linked recombinant peptide is generated.

[0094] The method involves contacting and culturing the cell in the presence of the crosslinker. For example, the crosslinker may be present in or added to the culture media (e.g. a solid, liquid or semi-solid media containing components such as nutrients and antibiotics to support cell growth) that the cell is being cultured in, wherein the cross-linker is capable of permeating the cell such that it moves from the media into the cytosol of the cell (i.e. through the cell membrane, and cell wall if present) where it can react with the thiol-containing residues present in the intracellularly-localised recombinant protein. If the cells are being grown on a solid growth medium such as an agar plate, the crosslinker may be included in the solid growth medium. If the cells are being grown in a liquid medium, the cross-linker may be included or added to the liquid medium.

[0095] The skilled person will be familiar with suitable culture media to use depending on the cells that are being cultured. For example, bacterial cells (e.g. E. Coli) may be cultured in LB media. Yeast cells may be cultured in YPD medium. In some cases, the culture medium comprises an antibiotic, such as Ampicillin. In some embodiments, e.g. where the crosslinker is BiBr3, the culture medium comprises butan-2-one. The solvent butan-2-one is also known as methyl ethyl ketone (MEK), and has the molecular formula CH3COCH2CH3. The concentration of butan-2-one in the culture medium may be between 0.010% and 0.50%,

[0096] The concentration of butan-2-one may be about 0.010% 0.015%, 0.020%, 0.025%, 0.030% ,0.035%, 0.040%, 0.045%, 0.050%, 0.055%, 0.060%; 0.065%, 0.070%, 0.075%, 0.080%, 0.085%, 0.090%, 0.095%, 0.10%, 0.15%, 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, 0.45%, 0.50%.

[0097] In some embodiments, the concentration of the cross linker in the culture medium is at most 1 mM. In some embodiments, the concentration of the cross linker in the culture medium is at most 500 pM. In some embodiments, the concentration of the cross-linker is at most 450 pM, 400 pM, 350 pM, 300 pM, 250 pM, 200 pM, 150 pM, 100 pM, or 50 pM. In some embodiments, the concentration of the crosslinker is between 10 pM and 500 pM. In some embodiments, e.g., where the crosslinker comprises BiBrs, the concentration of crosslinker in the culture medium is between 10 pM and 300 pM, or between 25 pM and 300 pM, or between 25 pM and 150 pM, or between 50 pM and 100 pM, such as about 75 pM. In some embodiments the concentration of the crosslinker in the culture medium is between 10 pM and 275 pM; 10 pM and 250 pM, 10 pM and 225 pM, 10 pM and 200 pM; 10 pM and 175 pM, 10 pM and 150 pM, 10 pM and 125 pM, 10 pM and 100 pM, 10 pM and 75 pM, 10 pM and 50 pM, 10 pM and 25 pM, 25 pM and 300 pM, 25 pM and 275 pM, 25 pM and 250 pM, 25 pM and 225 pM, 25 pM and 200 pM, 25 pM and 175 pM, 25 pM and 150 pM, 25 pM and 125 pM, 25 pM and 100 pM, 25 pM and 75 pM, 25 pM and 50 pM, 50 pM and 300 pM, 50 pM and 275 pM, 50 pM and 250 pM, 50 pM and 225 pM, 50 pM and 200 pM, 50 pM and 175 pM, 50 pM and 150 pM, 50 pM and 125 pM, 50 pM and 100 pM, 50 pM and 75 pM, 50 pM and70 pM, 75 pM and 300 pM,75 pM and 275 pM, 75 pM and 250 pM, 75 pM and 225 pM, 75 pM and 200 pM, 75 pM and 175 pM, 75 pM and 150 pM, 75 pM and 125 pM, 75 pM and 100 pM, 100 pM and 300 pM, 100 pM and 275 pM, 100 pM and 250 pM, 100 pM and 250 pM, 100 pM and 225 pM, 100 pM and 200 pM, 100 pM and 175 pM, 100 pM and 150 pM, 100 pM and 125 pM, 125 pM and 300 pM, 125 pM and 275 pM, 125 pM and 250 pM, 125 pM and 225 pM, 125 pM and 200 pM, 125 pM and 175 pM, 125 pM and 150 pM, 150 pM and 300 pM, 150 pM and 275 pM, 150 pM and 250 pM, 150 pM and 225 pM, 150 pM and 200 pM, 150 pM and 175 pM, 175 pM and 300 pM, 175 pM and 275 pM, 175 pM and 250 pM, 175 pM and 225 pM, 175 pM and 200 pM, 200 pM and 300 pM, 200 pM and 275 pM, 200 pM and 250 pM, 200 pM and 225 pM, 225 pM and 300 pM, 225 pM and 275 pM, 225 pM and 250 pM, 250 pM and 300 pM, 250 pM and 275 pM, 275 pM and 300 pM. In preferred embodiments, the concentration of the crosslinker in the culture medium is between 50 pM and 70 pM.

[0098] In some embodiments, the concentration of the crosslinker in the culture medium is about 10 pM, about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM, about 45 pM, about 50 pM, about 55 pM, about 60 pM, about 65 pM, about 70 pM, about 75 pM, about 80 pM, about 85 pM, about 90 pM, about 95 pM, about 100 pM, about 105 pM, about 110 pM, about 115 pM, about 120 pM, about 125 pM, about 130 pM, about 135 pM, about 140 pM, about 145 pM, about 150 pM, about 155 pM, about 160 pM, about 165 pM, about 170 pM, about 175 pM, about 180 pM, about 185 pM, about 190 pM, about 195 pM, about 200 pM, about 205 pM, about 210 pM, about 215 pM, about 220 pM, about 225 pM, about 230 pM, about 235 pM, about 240 pM, about 245 pM, about 250 pM, about 255 pM, about 260 pM, about 265 pM, about 270 pM, about 275 pM, about 280 pM, about 285 pM, about 290 pM.

[0099] In some embodiments, e.g., where the crosslinker comprises bismuth tripotassium citrate, the concentration of crosslinker in the culture medium is between 50 pM and 500 mM, or between 100 pM and 500 pM, or between 150 pM and 400 pM, or between 200 pM and 300 pM, such as about 250 pM. In some embodiments, the cell may be cultured in the presence of a reducing agent in addition to the cross-linker. Examples of suitable reducing agents include tris(2-carboxyethyl) phosphine (TCEP), dithiothreitol (DTT), 2-mercaptoethanol, and 2-mercaptothylamine. In some embodiments, the cell is cultured in the presence of TCEP.

[0100] In some embodiments, the cell may be cultured in the presence of the crosslinker for at least 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 18 hours, 20 hours, 24 hours, 36 hours, or 48 hours. In some embodiments, the cell may be cultured in the presence of the crosslinker for up to 24 hours, 18 hours, 12 hours, 8 hours or 7 hours. In some embodiments, the cell is kept in the dark while it is being cultured in presence of the cross-linker.

[0101] In some embodiments, the ratio of cyclised-to-linear peptides in the conformationally constrained peptide produced by the method is at least 10, optionally wherein the ratio of cyclised-to-linear peptides is determined according to the methods described in Example 4.4,.

[0102] “Recombinant peptide” as used herein refers to peptides made by recombinant DNA techniques familiar to the person skilled in the art. In some embodiments, the recombinant peptide is over-expressed in the cell, that is the recombinant peptide is expressed at increased quantity compared with a cell thatexpresses a native, i.e. non-recombinant, form of the peptide, or that does not express the peptide. In instances where the gene encoding the recombinant peptide is under the control of a lac operon, the overexpression may be due to the addition of IPTG to the culture medium.

[0103] In some embodiments, the distance between the thiol-containing residues, such as cysteines, in the recombinant peptide, is between 1 and 10 amino acids, optionally between 1 and 5 amino acids.

[0104] In some embodiments, the recombinant peptide comprises the sequence CGITKDCVNEAGC, (SEQ ID NO: 1) or a variant thereof comprising one, two or three amino acid modifications.

[0105] In some embodiments, the recombinant peptide has a length of at least 10 amino acids, at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at least 60 amino acids, at least 70 amino acids, at least 80 amino acids, at least 90 amino acids, at least 100 amino acids, at least 120 amino acids, at least 140 amino acids, at least 160 amino acids, at least 180 amino acids, at least 200 amino acids, at least 220 amino acids, at least 240 amino acids, at least 260 amino acids, at least 280 amino acids, at least 300 amino acids, at least 320 amino acids, at least 340 amino acids, at least 360 amino acids, at least 380 amino acids, at least 400 amino acids, at least 420 amino acids, at least 440 amino acids, at least 460 amino acids, at least 480 amino acids, at least 500 amino acids, at least 520 amino acids, at least 540 amino acids, at least 560 amino acids, at least 580 amino acids, at least 600 amino at least 620 amino acids, at least 640 amino acids, at least 660 amino acids, at least 680 amino acids, at least 700 amino acids, at least 720 amino acids, at least 740 amino acids, at least 760 amino acids, at least 780 amino acids, at least 800 amino acids, at least 820 amino acids, at least 840 amino acids, at least 860 amino acids, at least 880 amino acids, at least 900 amino acids, at least 920 amino acids, at least 940 amino acids, at least 960 amino acids, at least 980 amino acids, at least 1000 amino acids, at least 1500 amino acids, at least 2000 amino acids, at least 2500 amino acids, at least 3000 amino acids, at least 3500 amino acids, at least 4000 amino acids, at least 4500 amino acids, at least 5000 amino acids, at least 5500 amino acids, at least 6000 amino acids, at least 6500 amino acids, at least 7000 amino acids, at least 7500 amino acids, at least 8000 amino acids, at least 8500 amino acids, at least 9000 amino acids, at least 9500 amino acids, at least 10000 amino acids.

[0106] In some embodiments, the recombinant peptide has a length of between 10 and 100 amino acids, such as between 10 and 90 amino acids, between 10 and 80 amino acids, between 10 and 70 amino acids, between 10 and 60 amino acids, between 10 and 50 amino acids, between 10 and 40 amino acids, between 10 and 30 amino acids, between 10 and 20 amino acids, between 20 and 100 amino acids, between 20 and 90 amino acids, between 20 and 80 amino acids, between 20 and 70 amino acids, between 20 and 60 amino acids, between 20 and 50 amino acids, between 20 and 40 amino acids, between 20 and 30 amino acids, between 30 and 100 amino acids, between 30 and 90 amino acids, between 30 and 80 amino acids, between 30 and 70 amino acids, between 30 and 60 amino acids, between 30 and 50 amino acids, between 30 and 40 amino acids, between 40 and 100 amino acids, between 40 and 90 amino acids, between 40 and 80 amino acids, between 40 and 70 amino acids, between 40 and 60 amino acids, between 40 and 50 amino acids, between 50 and 100 amino acids, between 50 and 90 amino acids, between 50 and 80 amino acids, between 50 and 70 amino acids, between 50 and 60 amino acids, between 60 and 100 amino acids, between 60 and 90 amino acids,between 60 and 80 amino acids, between 60 and 70 amino acids, between 70 and 100 amino acids, between 70 and 90 amino acids, between 70 and 80 amino acids, between 80 and 100 amino acids, between 80 and 90 amino acids, between 90 and 100 amino acids.

[0107] In some embodiments, the recombinant peptide comprises more than 10 amino acids, more than 50 amino acids, more than 100 amino acids, more than 500 amino acids, or more than 1000 amino acids. In some embodiments the peptide has a length of about 15 amino acids, about 20 amino acids, about 30 amino acids, about 35 amino acids, about 40 amino acids, about 45 amino acids, about 50 amino acids, about 55 amino acids, about 60 amino acids, about 65 amino acids, about 70 amino acids, about 75 amino acids, about 80 amino acids, about 85 amino acids, about 90 amino acids, about 95 amino acids. “Amino acid” as used herein may be any natural or non-natural amino acid.

[0108] In some embodiments the recombinant peptide is a mini-protein, that is a protein with a molecular weight below 10 kDa.

[0109] In some embodiments, the recombinant peptide is expressed intracellularly from a nucleic acid. For example, the nucleic acid encoding the recombinant peptide may be an expression cassette (also termed a “recombinant peptide expression cassette”), which may be delivered to the cell, optionally as part of an expression vector, or may be incorporated into the genome of the cell.

[0110] Typically, an expression cassette comprises a promoter operably linked to a protein coding sequence. The term “operably linked” includes the situation where a selected coding sequence and promoter are covalently linked in such a way as to place the expression of the protein coding sequence under the influence or control of the promoter. Thus, a promoter is operably linked to the protein coding sequence if the promoter is capable of effecting transcription of the protein coding sequence. In some embodiments, the expression cassette may further comprise further components of a eukaryotic or prokaryotic gene, such as one or more selected from a list consisting of: an intron, an enhancer, a silencer, a 5’ UTR, a 3’ UTR, and a regulator.

[0111] Any suitable promoter known in the art may be used in the expression cassette providing it functions in the cell type being used. For example, where the cell is a bacterial cell, expression may be under control of the Tryptophan (trp), L-arabinose (ara), or lac operon. In cases where the promotor is under control of the lac operon, the cell may also contain a lac repressor protein, whereby expression can be controlled by the introduction of isopropyl p-D-1 -thiogalactopyranoside (IPTG). The promoter may be endogenous to the cell in which the method is being carried out. Where multiple expression cassettes are used, each coding sequence may be independently operably linked to its own promoter. Alternatively, the coding sequence for one or more of the expression cassettes may be operably linked to the same promoter. The expression cassettes described herein may be part of one or more expression vector(s). An “expression vector” as used herein is a DNA molecule used for expression of foreign genetic material in a cell. Any suitable vectors known in the art may be used. Suitable vectors include plasmids, binary vectors, viral vectors and artificial chromosomes (e.g. yeast artificial chromosomes). Alternatively, the expression cassettes described herein may be incorporated into the genome of the cell.The methods described herein may comprise delivering (or “administering”) one or more nucleic acids described herein to the cell. Molecular biology techniques suitable for administering nucleic acids and producing peptides such as the recombinant peptide described herein in cells are well known in the art, such as those set out in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989.

[0112] Techniques for expressing peptides such that they are localised intracellularly are well known in the art. In some embodiments, the method is performed at a temperature of about 25 °C about 26 °C about 27 °C about 28 °C about 29°C about 30 °C , about 31 °C, about 32 °C, about 33 °C, about 34 °C, about 35 °C, about 36 °C, about 37 °C, about 38 °C, about 39 °C, about 40 °C. In some preferred embodiments, the method is performed at a temperature of about 37°C

[0113] In some embodiments, the method is performed at a pH of about 6.5. about 6.6, about 6.7, about 6.8, about 6.9, about 7, about 7.1 , about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9. In preferred embodiments, the method is performed at a pH of about 7.4.

[0114] In some embodiments, the method is performed at physiological pH, i.e. a pH of between 7.35 and 7. 45.

[0115] In some embodiments, the method described herein further comprises a step of isolating the crosslinked peptide from the cell. Isolated crosslinked peptides and cells identified by the methods described herein are therefore also disclosed herein.

[0116] In some embodiments, the cross-linked recombinant peptide has one or more improved functional properties relative to the recombinant peptide not contacted with the crosslinker, such as improved stability, increased affinity for cognate binding partners, improved enzyme activity, increased resistance to protease degradation, and enhanced cell penetrance. Increased resistance to thermal denaturation of the conformationally constrained peptide relative to a peptide not contacted with a crosslinker is indicative of increased stability. Increased resistance to thermal denaturation may be determined using thermal melt circular dichroism (CD), or a thermal shift assay, or isothermal titration calorimetry. Increased affinity for binding partners may be determined by bio-layer interferometry (BLI)

[0117] In some embodiments, the crosslinker may comprise a functional moiety, for example by conjugation. Examples of functional moieties include, but are not limited to, affinity tags, detectable labels, degradation tags, cell penetrating molecules, and therapeutic agents.

[0118] In some embodiments, the recombinant peptide may comprise a functional moiety, for example by conjugation. Examples of functional moieties include, but are not limited to, affinity tags, detectable labels, degradation tags, cell penetrating molecules, and therapeutic agents.

[0119] “Affinity tag” as used herein refers to tags that help with protein purification by separating the molecules to which the affinity tag binds from those that do not. Examples of affinity tags include, biotin, hexahistidine (His) tag, chitin binding protein (CBP), maltose binding protein (MBP), Strep-tag and glutathione-S-transferase (GST).“Detectable label” as used herein refers to a tag that is able to generate a detectable signal either directly or indirectly. Examples include but are not limited to fluorophores (e.g. a fluorescent dye such as green fluorescent protein (GFP); red fluorescent protein (RFP); 5-Carboxyfluorescein (5-FAM); 6-Carboxyfluorescein (6-FAM); Anthranilyl, 2-Aminobenzoyl (Abz); 7-Methoxycoumarinyl-4-acetyl (Mca); Cy5; Cy3; 5-[(2-Aminoethyl)amino] naphthalene-1 -sulfonic acid (EDANS); 5-(Dimethylamino) naphthalene-1 -sulfonyl (Dansyl)) or isotopic labels, such as radiolabels (e.g. Tritium; lodine-125; Carbon-11 , fluorine). Methods for labelling peptides using fluorophores or radioactive labels are well-known in the art.

[0120] “Degradation tag” as used herein refers to a tag that acts as a degradation signal and targets peptides for proteolysis by the ubiquitin-proteasome system. The degradation signal may be present in the peptide sequence or added by covalent modification. Examples include but are not limited to SSRA tags, PROTACs, ligand-directed degraders (LDDs), and molecular glues.

[0121] “Cell penetrating molecule” as used herein refers to molecular entities attached to cell-penetrating peptides (CPPs). CPPs facilitate cellular uptake of the molecular entity or “cargo”. These molecular entities may be associated with the CPP through covalent or non-covalent interactions. Examples of CPPs include, but are not limited to, a TAT peptide (GRKKRRQRRRPQ SEQ ID NO: 2); oligo-arginine containing CPPs such as R9, R4, R6, R8, R10; MPG; PEP-1 ; EB1 , transportan, penetratin, and lactoferrin. Further examples of CPPs are described in Reissmann, 2014. Examples of molecular entities associated with the CPP include but are not limited to nanoparticles, antisense oligonucleotides, small interfering RNA, double stranded RNA, liposomes. In some embodiments, the first and / or second peptide sequence is fused to a TAT peptide. In some embodiments the TAT peptide is conjugated to detectable label, such as a FAM label. In some embodiments, the TAT peptide comprises one or more unnatural amino acid residues as described above.

[0122] “Therapeutic agent” as used herein refers to a drug, protein, peptide, gene, compound or other pharmaceutically active ingredient.

[0123] Pnictogen-containing thiol reactive crosslinkers modified by the functional moiety may be prepared using standard techniques known to those skilled in the art.

[0124] Also described is a conformationally constrained peptide obtainable by the method described herein.

[0125] A recombinant peptide comprising at least two thiol-containing residues, wherein each of the at least two thiol-containing residues is covalently linked to a pnictogen of the pnictogen-containing cross-linker as described elsewhere herein is also described. In some embodiments, the recombinant peptide comprises two sets of three thiol-containing or selenol-containing residues, wherein each of the three thiol-containing or selenol-containing residues in a set is covalently linked to a pnictogen of the pnictogen-containing crosslinker.

[0126] In some embodiments, the peptide may be a retro-inverso form of a peptide described elsewhere herein. Generally, retro-inverso forms of peptides are more resistant to proteolysis and are therefore notdegraded as easily as the original peptide. When applied to a given amino acid sequence, the term "retro-inverso" is used to indicate an alternative form containing the same residues, in the opposite configuration (L or D), and in which the order of the residues from N- to C-terminus is reversed. The term is often used to refer to a reversed-sequence all-D version of a conventional peptide consisting of L-form residues. In the context of the present invention, a “retro-inverso” form of a peptide containing both L-and D-amino acids would have the reverse N- to C- orientation, with L-amino acids exchanged for D-amino acids and D-amino acids exchanged for L-amino-acids.

[0127] In some embodiments, at least one amino acid residue of the peptide is a D-form amino acid residue, optionally wherein all the amino acid residues are D-form amino acid residues. In some embodiments, at least one amino acid residue of the peptide is an L-form amino acid residue, optionally wherein all the amino acid residues are L-form amino acid residues.

[0128] Further described is the use of a pnictogen-containing thiol reactive crosslinker in a method of producing a conformationally constrained peptide in cellulo. The methods may be as described above.

[0129] The crosslinked peptides generated using the methods described herein may be used in a method of screening peptide libraries fortranscription factor antagonists. The method comprises providing a cell, wherein the cell comprises a conformationally constrained peptide according to the methods described herein, and first and second candidate binding partners, followed by assaying whether the crosslinked peptide is able to inhibit association between the first and second candidate binding partners. One approach to functional antagonists of transcription factors is the Transcription Block Survival (TBS) assay described in W02020128015, incorporated herein by reference in its entirety.

[0130] The candidate binding partners can be any peptides that associate with one another (or are expected to do so). The first and second binding partners may have an identical amino acid sequence (e.g. they may homodimerise with each other). Alternatively, the first and second binding partners may have different amino acid sequences (e.g. they may heterodimerise with each other).

[0131] In some embodiments, the candidate binding partners form a DNA-binding complex upon association. Suitable candidate binding partners that form a DNA-binding complex upon association include transcription factors (e.g. human transcription factors), such as those of the basic leucine zipper (bZIP), basic helix-loop helix (bHLH) or bHLH leucine zipper (bHLH-Zip), and DLXtranscription factor families. bHLH and bHLH-Zip transcription factors are exclusively eukaryotic proteins that bind to sequencespecific double-stranded DNA as homodimers or heterodimers to either activate or repress gene transcription. bZIP transcription factors also contain leucine repeats through which the proteins dimerize to form a coiled coil, which is required for functional activity of the protein. As well as human proteins, certain viral proteins such as BZLF1 form part of the bZIP family. Details of bZIP, bHLH and bHLH-ZIP transcription factors and their consensus sequences are provided in Vinson et al. (2002), Newman & Keating (2003) and Rodriguez-Martinez et al. (2017). Typically, the DNA-binding fragment of these transcription factors will be (or will comprise) the basic portions of the transcription factors which physically interact with DNA.Exemplary bHLH transcription factors include AT0H1 , AhR, AHRR, ARNT, ASCL1 , BHLH2, BHLH3, BHLH9, ARNTL, ARNTL2, CLOCK, EPAS1, FIGLA, HAND1, HAND2, HES5, HES6, HEY1, HEY2, HEYL, HES1, HIF1A, HIF3A, ID1, ID2, ID3, ID4, LYL1, MESP2, MXD4, MYCL1, MYCN, MyoD, Myogenin, MYF5, MYF6, Neurogeninl, Neurogenin2, Neurogenin3, NeuroDI, NeuoD2, NPAS1, NPAS2, NPAS3, OLIG1 , OLIG2, Pho4, Scleraxis, SIM1 , SIM2, TAL1 , TAL2, Twist and USF1. Exemplary bHLH-ZIP transcription factors include AP-4, Max, MXD1, MXD3, MITF, MNT, MLX, MLXIPL, MXI1, Myc, SREBP1 and SREBP2. In particular embodiments, the bHLH-ZIP transcription factor used may be c-Myc or Max, or a heterodimer between c-Myc and Max (c-Myc-Max). bHLH and bHLH-ZIP transcription factors typically bind to a consensus sequence called an E-box, which can have the sequence CANNTG (‘N’ being any nucleotide) and in particular cases has the sequence CACGTG. The candidate DNA binding partner may be or may comprise any of these bHLH or bHLH-Zip transcription factors.

[0132] Exemplary DLX transcription factors include DLX-1, DLX-2, DLX-4, DLX-5, and DLX-6.

[0133] Exemplary human bZIP transcription factor subfamilies, the nucleotide sequences of their binding sites and examples of proteins of these subfamilies are set forth in Table 1 below. The candidate binding partners may be or may comprise any of these human bZIP proteins. For example, the first and second candidate binding partners may be proteins of the Fos / Jun bZIP family that form a DNA-binding complex upon association.

[0134]

[0135] Table 1: Exemplary human bZIP transcription factor subfamilies, the nucleotide sequences of their binding sites and examples of proteins of these subfamilies.In other examples, the candidate binding partners may form protein aggregates, or may be expected to do so. Protein aggregates are typically formed where multiple misfolded proteins accumulate and clump together and their presence is associated with a number of diseases, in particular neurodegenerative diseases such as Alzheimer’s Disease (AD), Parkinson’s disease (PD) and prion disease (also known as transmissible spongiform encephalopathy). In some embodiments, the presence of an aggregate of the candidate binding partners in a human patient is associated with a disease or other pathological condition, such as a neurodegenerative disease.

[0136] Examples of peptides and polypeptides that are capable of forming protein aggregates include those that are capable of aggregating to form amyloids, as well as those capable of aggregating to form amorphous or native-like deposits. In some embodiments, the candidate binding partners are amyloid-p (Ap) peptides, a-synuclein (aS) polypeptides, tau proteins, or prion proteins.

[0137] In other examples, PPI between the candidate binding partners may be associated with an intracellular signalling pathway. Numerous intracellular signalling pathways are associated with the interaction (which may be covalent or non-covalent) between one or more proteins, e.g. an enzyme such as a kinase.

[0138] Accordingly, one of the candidate binding partners could be an enzyme such as a kinase and the other candidate binding partner be a protein that interacts (e.g. binds to) the enzyme. For example, guanine nucleotide exchange factors (GEFs) are proteins or protein domains that associate with small GTPases to induce catalytic activity of the GEF. Exemplary methodology for designing and producing peptides that can target and modulate helical PPIs associated with intracellular signalling pathways is provided in Yoo etal. 2020.

[0139] In some embodiments, the candidate binding partners are expressed intracellularly from one or more nucleic acids. For example, one or more nucleic acids encoding the candidate binding partners may be an expression cassette (also termed a “candidate binding partner expression cassette”), which may be delivered to the cell, optionally as part of an expression vector, or may be incorporated into the genome of the cell. Where the first and second candidate binding partners have an identical amino acid sequence, both binding partners may be expressed from the name nucleic acid.

[0140] Assaying for whether the conformationally constrained peptide is able to inhibit association between the first and second candidate binding partners may involve determining whether the conformationally constrained peptide is able to modulate activity and / or expression of a reporter protein.

[0141] A reporter protein as used herein is any protein that provides a phenotypic readout. Examples of reporter proteins include cell survival proteins, cell reproduction proteins, fluorescence proteins, bioluminescence proteins, enzymes that act on a substrate to produce a colorimetric signal, protein kinases, proteases, transcription factors, and regulatory proteins such as ubiquitin. The use of suitable reporter proteins in assays for determining PPIs is described, for example, in Wehr and Rossner (2016).

[0142] In these assays, activity of the reporter protein is controlled by the association of the first and second candidate protein. This can be achieved in several ways. For example, the reporter protein may be split into a first and second fragments of the reporter protein, such that the first and second fragments need to be brought into sufficient proximity (e.g. non-covalently interact) in order to reconstitute activity of thereporter protein. Reporter proteins that can be split into fragments in this way can be termed “split reporters”. Several split reporters are known in the art and include beta-lactamase, dihydrofolate reductase (DHFR), focal adhesion kinase (FAK), Gal4, GFP (split-GFP), horseradish peroxidase, infrared fluorescent protein IFP1.4, an engineered chromophore-binding domain (CBD), LacZ (betagalactosidase), luciferase, TEV (Tobacco etch virus protease) and ubiquitin

[0143] In some embodiments, the first candidate binding partner is linked (e.g. fused) to a first fragment of the reporter protein and the second candidate binding partner is linked (e.g. fused) to the second fragment of the reporter protein, where association (e.g. dimerisation) of the first and second candidate binding partners reconstitutes reporter protein activity. This assay may be termed the protein-fragment complementation assay, or PCA and is well known in the art. In cases where association of the first and second candidate binding partners reconstitutes reporter protein activity, the additional presence in the cell of a peptide that inhibits association between the first and second candidate binding partners will decrease activity of the reporter protein.

[0144] Other suitable assays may make use of a DNA-binding complex to inhibit or promote expression of the reporter protein as a way of controlling activity of the reporter protein. In these assays, the cell may further comprise a nucleic acid encoding the reporter protein. The nucleic acid encoding the reporter protein may be an expression cassette (also termed a “reporter protein expression cassette”), which may be delivered to the cell, optionally as part of an expression vector, or may be incorporated into the genome of the cell. The nucleic acid encoding the reporter protein comprises a binding site that the DNA-binding complex binds to and inhibits or promotes expression of the reporter protein. This DNA-binding based assay can be used in embodiments where the first and second candidate binding partners form the DNA-binding complex. Additionally, this DNA-binding based assay can be used in embodiments where the first and second candidate binding partners are linked to components that form a DNA-binding complex when brought into sufficient proximity (i.e. though association of the first and second candidate binding partners).

[0145] The DNA-binding complex may comprise any of the proteins of a particular bZIP family set forth in Table 1 above and the binding site in the nucleic acid encoding the reporter protein may the binding site associated with that bZIP family set forth in Table 1 above.

[0146] In some embodiments, one or more binding sites are located in the promoter or enhancer region of the nucleic acid encoding the reporter protein. In these embodiments, the DNA-binding complex typically has transcriptional activation or transcriptional repression activity such that upon binding to the binding site(s) it is capable of promoting or inhibiting expression of the reporter protein.

[0147] In other embodiments, some or all binding sites are located in the transcribed sequence (e.g. the coding sequence) of the nucleic acid encoding the reporter protein. In these embodiments, binding of the DNA-binding complex to the binding site(s) inhibits transcription of the reporter protein.

[0148] Accordingly in preferred embodiments, the cell comprises the first and second candidate binding partners and a nucleic acid encoding a reporter protein, where association of the first and second candidate binding partners form a DNA-binding complex that binds to one or more binding sites in the nucleic acidencoding the reporter protein such that binding of the DNA-binding complex to the binding site(s) inhibits expression of the reporter protein. In these embodiments an increase in expression of the reporter protein in the presence of the ligated peptide indicates that the ligated peptide is capable of inhibiting association of the first and second candidate binding partners.

[0149] Monitoring the activity and / or expression of the reporter protein will depend on the reporter protein used. For example, where the reporter protein is a cell survival protein, then inhibition of expression and / or activity of the cell survival protein will result in cell death. Cell death can be determined by one of a number of techniques known to the person skilled in the art, e.g. the observing of morphological changes such as cytoplasmic blebbing, cell shrinkage, internucleosomal fragmentation and chromatin condensation. Use of a cell survival protein as a reporter protein can be advantageous as it gives a simple binary readout, i.e. the cell is either dead or alive. Methods using cell survival proteins as reporter proteins in screening for inhibitors that disrupt PPIs are known. See, for example, Park et al. (2007), which describes methods involving beta-lactamase in a fragmentation complementation strategy.

[0150] If the reporter protein is a cell reproduction protein, then inhibition of expression and / or activity of the cell reproduction protein will result in the cell being unable to proliferate and therefore unable to form progeny. Cell proliferation can be determined by one of a number of techniques known to the person skilled in the art, e.g. by counting of individual cells, foci or colonies, measuring metabolic activity using dyes such as MTT and WST-1, using nucleoside analogues such as bromodeoxyuridine (BrdU) and measuring incorporation of this analogue in the cells, staining dividing cells using reagents such as succinimidyl ester of carboxyfluorescein diacetate, and detecting proliferation markers such as PCNA, poisomerase IIB or phosphohistone H3. Inhibition of cell proliferation may also result in cell death, which can be measured as described above.

[0151] As a further example of a reporter protein that provides an observable phenotype, the reporter protein can be a fluorescent protein, a bioluminescent protein, or an enzyme that acts on a substrate to produce a colorimetric signal. In these cases, activity of the reporter proteins results in an observable signal when active that can therefore be monitored.

[0152] A conformationally constrained peptide that is able to modulate expression and / or activity of the reporter protein may be able to modulation expression and / or activity by at least 50%, by at least 2-fold, by at least 5-fold, or by at least 10-fold when compared to reporter protein expression and / or activity in an equivalent cell that lacks the conformationally constrained peptide. For example, where association of the first and second candidate binding partners results in a decrease in expression and / or activity of the reporter protein (e.g. a cell survival protein such as DHFR), an increase by at least 50% in expression and / or activity of the reporter protein (e.g. at least 50% more living cells) in the presence of the conformationally constrained peptide may indicate the constrained peptide is capable of modulating expression and / or activity of the reporter protein.

[0153] The conformationally constrained peptide may elicit a greater modulation (e.g. at least 50% greater modulation, at least 2-fold greater modulation, at least 5-fold greater modulation, or at least 10-fold greater modulation) of expression and / or activity of the reporter protein when compared to the ability anun-ligated peptide to modulate expression and / or activity of the reporter protein. This may indicate that conformationally constrained peptide increases its ability to disrupt PPIs between the first and second candidate binding partner (e.g. the conformationally constrained peptide binds its target with a higher affinity). Thus, in some embodiments, the method further comprises determining whether the conformationally constrained peptide elicits greater modulation of the expression and / or activity of the reporter protein compared to a peptide that is not conformationally constrained. This may involve measuring the reporter protein expression and / or activity before and after the cell is contacted and cultured with the conformationally constrained peptide.

[0154] Also described is a kit for use in any of the methods described above, the kit comprising:

[0155] i. a nucleic acid encoding a recombinant peptide, wherein the recombinant peptide comprises at least two thiol-containing residues or selenol-containing residues, and

[0156] ii. a pnictogen-containing crosslinker, wherein the pnictogen-containing crosslinker is thiol-reactive and / or selenol-reactive.

[0157] The pnictogen-containing thiol-reactive crosslinker may be as defined above.

[0158] The kit may further comprise a cell for expressing the recombinant protein. The cell may be as defined above.

[0159] ***

[0160] 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.

[0161] 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.

[0162] 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.

[0163] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0164] 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%.

[0165] Numbered Statements of Invention

[0166] 1. A method of producing a conformationally constrained peptide in cellulo, the method comprising the steps of:

[0167] i. providing a cell containing a recombinant peptide, the recombinant peptide comprising at least two thiol-containing residues or selenol-containing residues,

[0168] ii. contacting the cell with a pnictogen-containing crosslinker, wherein the pnictogen-containing crosslinker is thiol-reactive and / or selenol-reactive,

[0169] Hi. culturing the cells in the presence of the crosslinker, such that a covalent bond is formed between each of the at least two thiol-containing or selenol-containing residues and a pnictogen of the pnictogen-containing crosslinker, thereby producing a conformationally constrained peptide.

[0170] 2. The method of statement 1 , wherein the thiol-containing residues are cysteines, or wherein the selenol-containing residues are selenocysteines.

[0171] 3. The method of statements 1-2, wherein the pnictogen-containing crosslinker is non-planar.

[0172] 4. The method of statements 1-3, wherein the pnictogen is bismuth or a pharmaceutically acceptable salt thereof.

[0173] 5. The method according to any preceding statement, wherein the pnictogen-containing crosslinker is trivalent.

[0174] 6. The method according to any preceding statement, wherein the pnictogen-containing crosslinker comprises a trihalide.

[0175] 7. The method according to statement 6, wherein the trihalide comprises chlorine, bromine, or iodine.

[0176] 8. The method according to any preceding statement, wherein the pnictogen-containing crosslinker comprises BiBr3.

[0177] 9. The method of statements 1 to 5, wherein the pnictogen-containing crosslinker comprises one or more of bismuth tripotassium citrate, bismuth citrate, ammonium bismuth citrate, bismuth acetate, and bismuth subsalicylate.10. The method according to any preceding statement, wherein the pnictogen-containing crosslinker is threefold-symmetric.

[0178] 11. The method of any of statements 1-5 or statement 9, wherein the pnictogen-containing crosslinker comprises a functional moiety, optionally wherein the functional moiety is an affinity tag (e.g. a His tag) , and / or a detectable label (e.g. a GFP tag), and / or a degradation tag (e.g. a SSRA tag), and / or a proteolysis targeting chimera (PROTAC), and / or another peptide (e.g. a cell penetrating peptide (CPP); and / or a fatty acid or lipid; and / or a therapeutic agent.

[0179] 12. The method according to any preceding statement, wherein the culturing step Hi. is performed in a culture medium comprising butan-2-one.

[0180] 13. The method according to statement 12, wherein the culture medium comprises between 0.010% and 0.50% butan-2-one, optionally wherein the culture medium comprises between 0.025% and 0.25% butan-2-one.

[0181] 14. The method according to statements 12 or 13, wherein the concentration of pnictogen-containing crosslinker in the culture medium is between 10 and 300 pM, optionally wherein the concentration of the crosslinker is about 25 pM, 50 pM, 75 pM, 100 pM, 150 pM, 200 pM, or 250 pM.

[0182] 15. The method according to statements 11 to 13, wherein the culture medium further comprises a reducing agent, optionally wherein the reducing agent is tris(2-carboxyethyl)phosphine (TCEP).

[0183] 16. The method according to any preceding statement, wherein the cell is cultured in the presence of the crosslinker for at least 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 18 hours, 20 hours, 24 hours, 36 hours, or 48 hours.

[0184] 17. The method according to any preceding statement, wherein the recombinant peptide is overexpressed in the cell.

[0185] 18. The method according to any preceding statement, wherein the recombinant peptide has previously been modified to introduce one or more of the at least two thiol-containing residues and / or selenol-containing residues, or wherein the thiol-containing residues and / or selenol-containing residues are native residues.

[0186] 19. The method according to any preceding statement wherein the recombinant peptide has a length of at least 10, at least 100, or at least 500 amino acids.

[0187] 20. The method according to any preceding statement, wherein the cell is a prokaryotic cell, optionally a bacterial cell, or a eukaryotic cell, optionally a yeast cell, plant cell, insect cell, or mammalian cell.21. The method according to any preceding statement, wherein providing the cell containing the recombinant peptide comprises delivering a nucleic acid encoding the recombinant peptide to the cell, such that the cell expresses the recombinant peptide.

[0188] 22. The method according to any preceding statement, wherein the method is performed at a temperature of about 37°C and / or a pH of about 7.4.

[0189] 23. The method according to any preceding statement, wherein the method further comprises isolating the crosslinked peptide from the cell.

[0190] 24. The method according to any preceding statement, wherein the cross-linked peptide has increased resistance to thermal denaturation compared with a peptide not contacted with the crosslinker, optionally wherein the increased resistance to denaturation is determined by circular dichroism.

[0191] 25. A conformationally constrained peptide obtainable by a method according to any one of statements 1 to 24.

[0192] 26. A recombinant peptide comprising three thiol-containing or selenol-containing residues, wherein each of the three thiol-containing or selenol-containing residues is covalently linked to a pnictogencontaining cross-linker.

[0193] 27. Use of a pnictogen-containing crosslinker in a method of producing a conformationally constrained peptide in cellulo.

[0194] 28. The recombinant peptide of statement 26, or the use of statement 27, wherein the thiol-containing residues are cysteines, or wherein the selenol-containing residues are selenocysteines.

[0195] 29. The recombinant peptide of statement 26 or 28, or the use of statement 27 or 28, wherein the pnictogen is bismuth.

[0196] 30. A method for screening peptide libraries for transcription factor antagonists, the method comprising:

[0197] a) providing a cell, wherein the cell comprises a conformationally constrained peptide obtained according to the method of statements 1 to 24, and a first and second candidate binding partner, and b) assaying whether the conformationally constrained peptide is able to inhibit association between the first and second candidate binding partner.

[0198] 31. The method according to statement 30, wherein assaying for whether the conformationally constrained peptide is able to inhibit association between the first and second candidate binding partner comprises determining whether the conformationally constrained peptide is able to modulate expression and / or activity of a reporter protein.32. The method according to statements 30 or 31 , wherein association of the first and second candidate binding partners forms a DNA binding complex that binds to one or more binding sites in a nucleic acid encoding the reporter protein, wherein binding of the DNA-binding complex to the binding site inhibits expression of the reporter protein, optionally wherein some or all of the binding sites are located in the transcribed sequence of the nucleic acid encoding the reporter protein.

[0199] 33. The method according to statements 30 to 32, wherein the first and second candidate binding partners are transcription factors (e.g. human transcription factors).

[0200] 34. The method according to statement 33, wherein the transcription factor is a bHLH transcription factor, optionally wherein the bHLH transcription factor is c-Myc; a bZIP transcription factor, optionally wherein the bZIP transcription factor is c-Jun, CREB, and / or BZLF1 ; a DLX transcription factor, optionally wherein the DLX transcription factor is DLX-1, DLX-2, DLX-3, DLX-4, DLX-5, and / or DLX-6.

[0201] 35. A kit for use in any of the methods of statements 1 to 24, wherein the kit comprises:

[0202] i. a nucleic acid encoding a recombinant peptide, wherein the recombinant peptide comprises at least two thiol-containing residues or selenol-containing residues, and

[0203] ii. a pnictogen-containing crosslinker, wherein the pnictogen-containing crosslinker is thiol-reactive and / or selenol-reactive.Sequences

[0204]

[0205] Examples

[0206] EXAMPLE 1- cellular tolerability of BiBr3

[0207] An experiment was carried out to determine the tolerability and toxicity of BiBrs on E. coli cell growth.

[0208] Design of a test construct- A His-SUMO-tagged peptide with three cysteine residues for coordination was designed with the following sequence (SEQ ID NO: 4):

[0209] MGHHHHHGSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQG KEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGASCGITKDCVNEAGCGAP

[0210] A plasmid encoding this peptide was generated for expression in E. coli.

[0211] Tolerability of BIBrs - To determine the tolerability of BiBrs on E. coli, cells were grown in media comprising varying concentrations of BiBrs and butan-2-one. The effect of BiBrs on cell growth was determined by measuring optical density (OD) at regular intervals using standard spectrophotometry techniques.

[0212] E. coli cells were transformed to express the peptide encoded plasmid using standard techniques. These cells were grown overnight at 37°C from a glycerol stock under standard conditions. A 100mM stock of BiBrs was prepared in butan-2-one. Autoclaved liquid broth (LB) media was prepared comprising Ampicillin (Amp), IPTG and indicated concentrations of BiBrs (0 pM, 50 pM, 75 pM, 100 pM, 250 pM and 500 pM) and butan-2-one (0.0%, 0.05%, 0.075%, 0.1%, 0.25% and 0.5%).

[0213] 20mL of this media was inoculated with the overnight E. coli culture and grown to ODeoo = 0.1. These samples were incubated at 37°C, 180 rpm shaking. ODeoo readings were taken at 60 minute intervals as shown in Fig 1A. Overall, a significant reduction in cellular tolerability was observed at higher concentrations of BiBrs. In the presence of low concentration BiBrs (50 - 75 pM) cell growth was reduced but remained relatively well tolerated. At higher concentrations (100 - 250 pM), an initial lag in cell growth was observed followed by an increase at -300 minutes. At the highest test concentration of 500 pM BiBrs, no discernible cell growth was observed in this experiment. These data suggest concentrations below 500 pM BiBrs can be tolerated by E. coli cells in a concentration dependent manner.

[0214] Toxicity of BiBr3 - Based on the same conditions as described above, E. coli cultures were grown in 75 pM BiBrs in the presence and absence of IPTG which induces expression of the test construct. E. coli growth was improved in the presence of IPTG as shown in Fig 1 B. These data show IPTG decreases the toxicity of BiBrs in E. coli through overexpression of the test construct. This provides cysteines to reactwith BiBrs reducing off-target BiBrs reactions with other thiols present in the proteosome. Thus, intracellular overexpression of cysteine comprising proteins reduces the toxicity of BiBrs.

[0215] EXAMPLE 2 - in cell peptide cyclisation using BiBrj- Peptide structure characterisation - The presence of a cross-link in a conformationally-constrained peptide can be determined using mass spectrometry as cyclised protein confirmations have an increased mass compared to their linear form.

[0216] Under the same conditions as described above, E. coli cultures were inoculated in media (LB or M9) comprising indicated concentrations of BiBrs (0 pM, 25 pM, 50 pM, 75 pM, 100 pM and 250 pM) and butan-2-one (0%, 0.025%, 0.05%, 0.075%, 0.1%, 2.5%). These cultures were incubated at 37°C, 180 rpm shaking for 24 hours. Cells were collected by centrifugation and the peptide encoded by the test construct was purified by immobilised metal affinity chromatography (IMAC) using standard techniques. Following this, LC-MS of the partially purified sample was performed using standard techniques. The Counts of linear and cyclised peptide structure at varying BiBrs concentrations were identified, as shown in Fig 2.

[0217] These data show that the proportion of peptide observed in linear and cyclised structures vary depending on the concentration of BiBrs. As summarised in Table 2, in the absence of BiBrs, no protein was observed in a cyclised structure whereas at increasing BiBrs concentrations, higher percentages of cyclised peptides were observed.

[0218]

[0219] Table 2. Percentage of observed peptide cyclised (%) for indicated concentrations of BiBr3

[0220] These data show the presence of BiBrs in E. coli culture media enables in cell peptide cyclisation in a concentration dependent manner.

[0221] Utility of intracellular cyclisation - To demonstrate the advantage of in cell cyclisation, E. coli cells were cultured in conditions as described for Fig 1 A with 75 pM BiBrs, 0.075 % butan-2-one. Growth was started at ODeoo = 0.1 and samples were taken over time at indicated ODeoo values for purification. Thepercentage of cyclised peptide observed was determined by LC-MS, as shown in Fig 3. These data illustrate the utility of the intracellular cyclisation method for use in transcriptional block survival (TBS) assays as peptide cyclisation efficiency remains high over the growth window required for the assay.

[0222] To further confirm BiBrs was inducing protein helicity, mass spectrometry and circular dichroism assays were conducted on miniprotein 3xhelixV1 comprising the following sequence with cysteines for coordination (SEQ ID NO: 5):

[0223] APAEDLKERLKKLGCSEECRQRLEKMAKEGTSEDAERMARNCES

[0224] Alphafold3 was used to predict the structure of the miniprotein from two different angles, as shown in Fig 4A. The three coordinated cysteines were predicted to be held in proximity to each other with two of the three forming a disulphide bond.

[0225] Mass spectrometry was conducted on 3xhelixV1 expressed and purified from E. coli, to determine the effect of BiBrs on the m / z ratio. As shown in Fig 4B, the absence of BiBrs resulted in a lower m / z ratio indicative of a linear 3xhelixV1 structure whilst the presence of BiBrs resulted in a higher m / z ratio indicative of cyclisation. These data further confirm the ability of BiBrs to induce intracellular cyclisation of cysteine comprising peptides through cross-linking.

[0226] Stability of intracellular cyclised peptides - To determine the stability of BiBrs cyclised peptides, CD spectroscopy was conducted on 3xhelixV1 expressed and purified from E. coli. The effect of BiBrs and reducing agent TCEP (20°C) on peptide helicity was measured using MRE, as shown in Fig 4C. These data show the presence of BiBrs resulted in a reduction in MRE indicating increased 3xhelixV1 helicity as compared to the absence of BiBrs. Furthermore, the addition of TCEP in the presence of BiBrs did not alter the increased helicity of 3xhelixV1 but did reduce the helicity of linear 3xhelixV1 as TCEP prevents the stabilisation of disulphide bond formation. These data show that BiBrs generates cross-linked peptides with improved stability compared to disulfide-bonded peptides, and that the cross-linking reaction was not affected by disulfide reduction.

[0227] This stabilisation was further confirmed by CD thermal denaturation experiments of linear and BiBrs cyclised 3xhelixV1 in the presence and absence of TCEP. By measuring MRE, altered melting behaviour was observed, as shown in Fig 4D. These data show the presence of TCEP with linear 3xhelixV1 causes a reduction in thermal stability whilst BiBrs cyclised 3xhelixV1 remains stable in the presence of this reducing agent. BiBrs therefore alters the melting behaviour of thiol containing proteins, resulting in increased stability.EXAMPLE 3 - Bismuth-* are tolerated in

[0228] Controlled numbers of yeast were plated out on agar plates containing yeast extract peptone dextrose (YPD) media, antibiotic (ampicillin), the solvent butanone, and Bismuth-containing compounds BiBr3, I Gastrodenol, or Bi acetate. The results are shown in Fig 5.

[0229] Fig 5 shows that yeast cells tolerate Bismuth-containing compounds, including BiBr3 (see Figs 5E and F), Gastrodenol (Figs 5G and H), and Bismuth acetate (Figs 5I and 5J) at concentrations of 75 pm and 250 pm.

[0230] Based on these data, it is expected that intracellular peptide cyclisation would work well in yeast cells.

[0231] EXAMPLE 4 - in cell peptide cyclisation with BiBr^and BiKsfcitrate

[0232] 4.1 Design of a test construct - A His-SUMO-tagged peptide with three cysteine residues for coordination was designed with the following sequence (SEQ ID NO: 4):

[0233] MGHHHHHGSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQG KEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGASCGITKDCVNEAGCGAP

[0234] A plasmid encoding this peptide was generated for expression in E. coli.

[0235] 4.2 Tolerability of BiBrs and BIK3[citrate]2- o determine the tolerability of BiBrs and BiK3[citrate]2 on E. coli, cells were grown in media comprising varying concentrations of BiBrs (with butan-2-one) or BiK3[citrate]2. The effect of BiBrs and BiK3[citrate]2 on cell growth was determined by measuring optical density (OD) at regular intervals using standard spectrophotometry techniques.

[0236] E. coli cells were transformed to express the peptide encoded plasmid using standard techniques. These cells were grown overnight at 37°C from a glycerol stock under standard conditions. A 100 mM stock of BiBrs was prepared in butan-2-one. Autoclaved liquid broth (LB) media was prepared comprising Ampicillin (Amp) and indicated concentrations of BiBrs (0 pM, 25 pM, 50 pM, 75 pM, 100 pM and 250 pM) and butan-2-one (0.0%, 0.025%, 0.05%, 0.075%, 0.1% and 0.25%). A 10 mM stock of BiKs[citrate]2 was prepared in water. Autoclaved LB media was prepared comprising Ampicillin (Amp) and indicated concentrations of BiKs[citrate]2 (0 pM, 50 pM, 100 pM, 250 pM, 500 pM and 1 mM).

[0237] The E. coli culture was grown as described in Example 1 , except the culture was grown to ODeoo = 0.05. ODeoo readings were taken at time intervals as shown in Fig 6.

[0238] Overall, a significant reduction in cellular tolerability was observed at higher concentrations of BiBrs in the absence of IPTG (Fig 6). In the presence of low concentration BiBrs (25 - 75 pM) cell growth wasreduced but remained relatively well tolerated. At higher concentrations of BiBrs (100 - 250 pM), cell growth was significantly reduced. These data suggest concentrations below 250 pM BiBrs can be tolerated by E. coli cells in a concentration dependent manner.

[0239] Also, a significant reduction in cellular tolerability was observed at higher concentrations of BiK3[citrate]2 in the absence of IPTG (Fig 6). In the presence of low concentration BiK3[citrate]2 (50 - 100 pM) cell growth was reduced but remained relatively well tolerated. At higher concentrations of BiK3[citrate]2 (250 pM - 1 mM), cell growth was significantly reduced. These data suggest concentrations below 1 mM BiK3[citrate]2 can be tolerated by E. coli cells in a concentration dependent manner.

[0240] 4.3 Toxicity of Bi Brs and BiKs[citrate]2 - Based on the same conditions as described above, E. coli cultures were grown with BiBrs or BiK3[citrate]2 in the presence of IPTG which induces expression of the test construct. For E. coli grown with BiBrs or BiK3[citrate]2, E. coli growth was improved in the presence of IPTG as shown in Fig 7. These data show IPTG decreases the toxicity of BiBrs and BiK3[citrate]2 in E. coli through overexpression of the test construct. This provides cysteines to react with BiBrs or BiK3[citrate]2, reducing off-target BiBr3 / BiK3[citrate]2 reactions with other thiols present in the proteosome Thus, intracellular overexpression of cysteine comprising proteins reduces the toxicity of BiBrsand BiK3[citrate]2.

[0241] 4.4 Peptide structure characterisation - The presence of a cross-link in a conformationally-constrained peptide can be determined using mass spectrometry as cyclised protein confirmations have an increased mass compared to their linear form.

[0242] E. coli cultures were inoculated in media with indicated concentrations of BiBrs (25 pM, 50 pM, 75 pM, 100 pM, and 250 pM) and butan-2-one (0.0%, 0.025%, 0.05%, 0.075%, 0.1% and 0.25%), as described in Example 2, or with indicated concentrations of Bi K3 [citrate] 2 (50 pM, 100 pM, 250 pM, 500 pM and 1000 pM). These cultures were incubated at 37°C, 180 rpm shaking for 24 hours. Cells were collected by centrifugation and the peptide encoded by the test construct was purified by immobilised metal affinity chromatography (IMAC) using standard techniques. Following this, LC-MS of the partially purified sample was performed using standard techniques. The ratio of cyclised-to-linear peptides at varying BiBrs and BiK3[citrate]2 concentrations were identified, as shown in Table 3.

[0243]

[0244]

[0245] Table 3. Ratio of cyclised / linear peptides for indicated concentrations of BiBrs and BiK3[citrate]2.

[0246] These data show that the proportion of peptide observed in linear and cyclised structures vary depending on the concentration of BiBrs and BiK3[citrate]2. At increasing BiBrs and BiK3[citrate]2 concentrations, a higher proportion of cyclised peptides were observed.

[0247] From the effects of varying BiBrs and BiK3[citrate]2 concentrations on E. coli cell growth (Figs 6 and 7) and intracellular cyclisation efficiency (Table 3), the optimal concentrations to maximise cyclised peptide yield were found to be 75 pM BiBrs and 250 pM BiK3[citrate]2.

[0248] The LC-MS of purified test peptide expressed in bacterial media supplemented with 75 pM BiBrs (Fig 8A) or 250 pM BiK3[citrate]2 (Fig 8B) shows a mass change corresponding to reaction with a Bi atom.

[0249] 4.5 Tolerability and toxicity of optimal concentrations of BiBrs and BiKs[citrate]2 - The effects of 75 pM BiBrs and 250 pM BiK3[citrate]2 on E. coli growth, as determined in Example sections 4.2 and 4.3, are shown in Fig 9.

[0250] E. coli growth at 75 pM BiBrs and 250 pM BiKs[citrate]2 in the absence of IPTG was similar, indicating that E. coli has a similar tolerability to these concentrations of BiBrs and BiKs[citrate]2 (Fig 9A). For E. coli grown with BiBrs or BiKs[citrate]2, E. coli growth was improved in the presence of IPTG, with a more significant improvement in growth observed for E. coli grown with BiKs[citrate]2 than for E. coli grown with BiBrs (Fig 9A-C). These data show IPTG decreases the toxicity of BiBrs and BiKs[citrate]2 in E. coli through overexpression of the test construct, as discussed in example section 4.3, and that when the cysteine-containing test construct is expressed, 250 pM BiKs [citrate] 2 is less toxic to E. coli than 75 pM BiBrs.4.6 Peptide cyclisation efficiency at optimal concentrations of BiBrs and BiK3[citrate]2 - Under the same E. coli growth conditions as described in example section 4.3, samples were taken and the degree of intracellular cyclisation of the test peptide was determined. For E. coli grown with 75 pM BiBrs or 250 pM BiK3[citrate]2, a decrease in cyclisation ratio was observed with increasing time in cell culture (Fig 10). However, the cyclisation ratio remained above 10 for all samples, which is sufficiently high for use in intracellular assays such as the TBS assay. The cyclisation ratio and cell growth rate were higher for 250 pM BiK3[citrate]2 than for 75 pM Bi Brs, indicating that 250 pM BiK3[citrate]2 causes a higher degree of peptide cyclisation and has a lower toxicity than 75 pM BiBrs.

[0251] EXAMPLE 5 - in vitro and in cell cyclisation of three-helix miniproteins with BiK- citrateh- 5.1 Design of miniproteins - Using a computationally-designed three helix miniprotein (gHHH_06; PDB ID: 2ND2) which contains two cysteine pairs, three protein constructs were designed that contained either three or six cysteine residues for bismuth cyclisation.

[0252] The miniprotein gHHH_06 (PDB ID: 2ND2) has the following sequence (SEQ ID NO: 6):

[0253] APCEDLKERLKKLGMSEECRQRLEKMCKEGTSEDAERMARNCES

[0254] The three protein constructs were designed with the following sequences:

[0255] Miniprotein 1: APAEDLKERLKKLGCSEECRQRLEKMAKEGTSEDAERMARNCES (SEQ ID NO: 5) Miniprotein 2: APAEDLKERLKKLGSSEEARQRLEKCAKEGTSECAERCARNAES (SEQ ID NO: 7) Miniprotein 3: ACAEDLKERLKKLGCSEECRQRLEKMCKEGTCEDAERMARNCES (SEQ ID NO: 8)

[0256] For Miniprotein 1 , one cysteine pair was removed and an additional cysteine was added in proximity to the remaining cysteine pair to introduce a new cysteine triad for cyclisation by Bi. For Miniprotein 2, both cysteine pairs were removed and a new triad was introduced, with two cysteines introduced at i ->i+4 positions on one helix and a third cysteine placed on a neighbouring helix. For Miniprotein 3, two triads were introduced by adding two additional cysteines in proximity to the existing cysteine pairs. Alphafold3 was used to predict the structure of the miniproteins compared to gHHH_06 (Fig 11).

[0257] To confirm that BiK3[citrate]2 could induce cyclisation of the three designed miniproteins, LC-MS was conducted on the three designed miniproteins. The miniproteins were synthesised in vitro in the presence of Bi K3 [citrate] 2 at a 1.2:1 bismuth:miniprotein molar ratio. As shown in Fig 12, for all miniproteins, the absence of BiK3[citrate]2 resulted in a lower mass whilst the presence of BiK3[citrate]2 resulted in a higher mass indicative of cyclisation by Bi. These data further confirm the ability of BiK3[citrate]2 to induce in vitro cyclisation of cysteine-comprising proteins through cross-linking.5.2 Stability of in vitro cyclised miniproteins - To determine the stability of BiK3[citrate]2-cyclised miniproteins, CD spectroscopy (37°C) was conducted on synthetic miniproteins 1 , 2 and 3. The effect of BiK3[citrate]2 and reducing agent TCEP (5 mM) on peptide helicity was measured using MRE (Fig 13). These data show that, for all three synthetic miniproteins, the presence of BiK3[citrate]2 resulted in a reduction in MRE, indicating increased miniprotein helicity as compared to the absence of BiK3[citrate]2. Furthermore, the addition of TCEP in the presence of BiK3[citrate]2 did not alter the helicity of cyclised miniproteins but did reduce the helicity of linear miniproteins as TCEP prevents the stabilisation of disulphide bond formation. These data show that BiK3[citrate]2 generates cross-linked peptides with improved stability compared to disulfide-bonded peptides, and that the cross-linking reaction was not affected by disulfide reduction.

[0258] This stabilisation was further confirmed by CD thermal denaturation experiments of linear and BiK3[citrate]2-cyclised miniproteins in the presence of TCEP. By measuring MRE, altered melting behaviour was observed (Fig 14). These data show that the linear miniproteins and BiK3[citrate]2-cyclised miniprotein 2 have reduced thermal stability compared to BiK3[citrate]2-cyclised miniproteins 1 and 3. For BiK3[citrate]2-cyclised miniproteins 1 and 3, BiK3[citrate]2 alters the melting behaviour of thiol containing proteins, resulting in increased stability.

[0259] The serum stability of the synthetic miniproteins was also tested. Miniprotein stocks (250 pM) were prepared in water and 120 pL was added to 480 pL human serum (Merck) before incubation at 37°C. 50 pL aliquots were removed at designated timepoints and added to 200 pL acetonitrile+1%TFA. Samples were vortexed for 30 seconds, incubated at 4°C for 10 minutes and then centrifuged (15000 xg, 10 minutes). The supernatant was analysed by LC-MS to quantify intact miniprotein. Data were collected in triplicate and are plotted as an average with error bars shown as one standard deviation (Fig 15). The serum half-life of the synthetic miniproteins were calculated (Table 4). Cyclised miniproteins had significantly higher serum half-lives than their linear counterparts, indicating that BiK3[citrate]2-induced cyclisation increases the stability of the miniproteins. Of the cyclised miniproteins, cyclised Miniprotein 3 had the highest half-life whilst cyclised Miniprotein 2 had the lowest half-life.

[0260]

[0261] Table 4. Serum half-life of synthetic miniproteins (linear and cyclised).The stability of synthetic cyclised miniproteins 1 , 2 and 3 were compared by CD spectroscopy (37°C) and it was found that miniprotein 3 was the most helical, whilst miniprotein 2 was the least helical (Fig 16).

[0262] 5.3 Intracellular miniprotein cyclisation with BiK3[citrate]2 - For in cellulo expression of the Miniproteins 1 and 3, SUMO-tagged miniprotein constructs were designed with the following sequences:

[0263] SUMO-Miniprotein 1: MGHHHHH-SUMO- APAEDLKERLKKLGCSEECRQRLEKMAKEGTSEDAERMARNCES (SEQ ID NO: 9) SUMO-Miniprotein 3: MGHHHHH-SUMO- ACAEDLKERLKKLGCSEECRQRLEKMCKEGTCEDAERMARNCES (SEQ ID NO: 10) where the SUMO sequence is the following:

[0264] GSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRF LYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG.

[0265] E. coli cells were transformed to express the SUMO-Miniprotein 1 encoded plasmid or SUMO-Miniprotein 3 encoded plasmid using standard techniques. These cells were grown overnight at 37°C from a glycerol stock under standard conditions. BiK3[citrate]2 stock was prepared in water. Autoclaved LB media was prepared comprising Ampicillin (Amp) and 250 pM BiK3[citrate]2. 20mL of this media was inoculated with the overnight E. coli culture and was incubated at 37°C, 180 rpm shaking for 24 hours. Cells were collected by centrifugation and the SUMO-miniproteins were purified by immobilised metal affinity chromatography (IMAC) using standard techniques.

[0266] LC-MS was conducted on the SUMO-miniproteins expressed and purified from E. coli. For both SUMO-miniproteins, the absence of BiK3[citrate]2 resulted in a lower mass whilst the presence of BiK3[citrate]2 resulted in a higher mass indicative of cyclisation by one Bi atom for SUMO-Miniprotein 1 and two Bi atoms for SUMO-Miniprotein 3, corresponding to one Bi atom for each of the two cysteine triads in SUMO-Miniprotein 3 (Fig 17). These data further confirm the ability of BiK3[citrate]2 to induce in cellulo cyclisation of cysteine-comprising proteins through cross-linking.

[0267] The secondary structures of the synthesised BiK3[citrate]2-cyclised miniproteins 1 and 3 and recombinantly expressed BiK3[citrate]2-cyclised miniproteins 1 and 3 were compared by CD spectroscopy (Fig 18). It was found that for both cyclised Miniproteins 1 and 3, the synthesised and recombinantly-expressed miniproteins had highly similar secondary structures indicating that in vivo cyclisation produces a miniprotein with the same folded structure as production through in vitro synthesis.References

[0268] A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. The entirety of each of these references is incorporated herein.

[0269] &

[0270]

[0271]

[0272] For standard molecular biology techniques, see Sambrook, J., Russel, D.W. Molecular Cloning, A Laboratory Manual. 3 ed. 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.

Claims

ClaimsI . A method of producing a conformationally constrained peptide in cellulo, the method comprising the steps of:

1. providing a cell containing a recombinant peptide, the recombinant peptide comprising at least two thiol-containing residues or selenol-containing residues,ii. contacting the cell with a pnictogen-containing crosslinker, wherein the pnictogen-containing crosslinker is thiol-reactive and / or selenol-reactive,Hi. culturing the cells in the presence of the crosslinker, such that a covalent bond is formed between each of the at least two thiol-containing or selenol-containing residues and a pnictogen of the pnictogen-containing crosslinker, thereby producing a conformationally constrained peptide.

2. The method according to claim 1 , wherein the thiol-containing residues are cysteines, or wherein the selenol-containing residues are selenocysteines.

3. The method according to claim 1 or 2, wherein the recombinant peptide comprises three thiol- containing residues or three selenol-containing residues.

4. The method according to any one of the preceding claims, wherein the recombinant peptide comprises six thiol-containing residues or six selenol-containing residues.

5. The method according to any one of the preceding claims, wherein the pnictogen-containing crosslinker is non-planar.

6. The method according to any one of the preceding claims, wherein the pnictogen is bismuth or a pharmaceutically acceptable salt thereof.

7. The method according to any one of the preceding claims, wherein the pnictogen-containing crosslinker is trivalent.

8. The method according to any one of the preceding claims, wherein the pnictogen-containing crosslinker comprises a trihalide.

9. The method according to claim 8, wherein the trihalide comprises chlorine, bromine, or iodine.

10. The method according to any one of the preceding claims, wherein the pnictogen-containing crosslinker comprises BiBr3.I I. The method according to any one of claims 1 to 7, wherein the pnictogen-containing crosslinker comprises one or more of bismuth tripotassium citrate, bismuth citrate, ammonium bismuth citrate, bismuth acetate, and bismuth subsalicylate.

12. The method according to any one of the preceding claims, wherein the pnictogen-containing crosslinker is threefold-symmetric.

13. The method according to any one of claims 1 -7 or 11 , wherein the pnictogen-containing crosslinker comprises bismuth tripotassium citrate.

14. The method according to any one of claims 1-7 and 11-12, wherein the pnictogen-containing crosslinker comprises a functional moiety, optionally wherein the functional moiety is an affinity tag (e.g. a His tag) , and / or a detectable label (e.g. a GFP tag), and / or a degradation tag (e.g. a SSRA tag), and / or a proteolysis targeting chimera (PROTAC), and / or another peptide (e.g. a cell penetrating peptide (CPP); and / or a fatty acid or lipid; and / or a therapeutic agent.

15. The method according to any preceding claim, wherein the culturing step Hi. is performed in a culture medium comprising butan-2-one.

16. The method according to claim 15, wherein the culture medium comprises between 0.010% and 0.50% butan-2-one, optionally wherein the culture medium comprises between 0.025% and 0.25% butan-2-one.

17. The method according to any one of the preceding claims, wherein the concentration of pnictogen-containing crosslinker in the culture medium is between 10 pM and 1 mM, optionally wherein the concentration of the crosslinker is about 25 pM, 50 pM, 75 pM, 100 pM, 150 pM, 200 pM, 250 pM, 500 pM or 1 mM.

18. The method according to any one of the preceding claims, wherein the pnictogen-containing crosslinker comprises BiBr3 and the concentration of BiBr3 in the culture medium is between 25 and 150 pM.

19. The method according to any one of the preceding claims, wherein the pnictogen-containing crosslinker comprises bismuth tripotassium citrate and the concentration of bismuth tripotassium citrate in the culture medium is between 150 and 300 pM.

20. The method according to any one of claims 14-19, wherein the culture medium further comprises a reducing agent, optionally wherein the reducing agent is tris(2-carboxyethyl)phosphine (TCEP).

21. The method according to any one of the preceding claims, wherein the cell is cultured in the presence of the crosslinker for at least 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 18 hours, 20 hours, 24 hours, 36 hours, or 48 hours.

22. The method according to any one of the preceding claims, wherein the recombinant peptide is over-expressed in the cell.

23. The method according to any one of the preceding claims, wherein the recombinant peptide has previously been modified to introduce one or more of the at least two thiol-containing residues and / or selenol-containing residues, or wherein the thiol-containing residues and / or selenol- containing residues are native residues.

24. The method according to any one of the preceding claims, wherein the recombinant peptide has a length of at least 10, at least 100, or at least 500 amino acids.

25. The method according to any one of the preceding claims, wherein the cell is a prokaryotic cell, optionally a bacterial cell, or a eukaryotic cell, optionally a yeast cell, plant cell, insect cell, or mammalian cell.

26. The method according to any one of the preceding claims, wherein providing the cell containing the recombinant peptide comprises delivering a nucleic acid encoding the recombinant peptide to the cell, such that the cell expresses the recombinant peptide.

27. The method according to any one of the preceding claims, wherein the method is performed at a temperature of about 37°C and / or a pH of about 7.4.

28. The method according to any one of the preceding claims, wherein the method further comprises isolating the crosslinked peptide from the cell.

29. The method according to any one of the preceding claims, wherein the cross-linked peptide has increased resistance to thermal denaturation compared with a peptide not contacted with the crosslinker, optionally wherein the increased resistance to denaturation is determined by circular dichroism.

30. A conformationally constrained peptide obtainable by a method according to any one of the preceding claims.

31. A recombinant peptide comprising three thiol-containing or selenol-containing residues, wherein each of the three thiol-containing or selenol-containing residues is covalently linked to a pnictogen-containing crosslinker.

32. The recombinant peptide of claim 31 , wherein the recombinant peptide comprises two sets of three thiol-containing or selenol-containing residues, wherein each of the three thiol-containing or selenol-containing residues in a set is covalently linked to a pnictogen-containing crosslinker.

33. Use of a pnictogen-containing crosslinker in a method of producing a conformationally constrained peptide in cellulo.

34. The recombinant peptide of claim 31 or 32, or the use of claim 33, wherein the thiol-containing residues are cysteines, or wherein the selenol-containing residues are selenocysteines.

35. The recombinant peptide of any one of claims 31 , 32 and 34, or the use of claim 33 or 34, wherein the pnictogen is bismuth.

36. A method for screening peptide libraries for transcription factor antagonists, the method comprising:a) providing a cell, wherein the cell comprises a conformationally constrained peptide obtained according to the method of any one of claims 1 to 29, and a first and second candidate binding partner, andb) assaying whether the conformationally constrained peptide is able to inhibit association between the first and second candidate binding partner.

37. The method according to claim 36, wherein assaying for whether the conformationally constrained peptide is able to inhibit association between the first and second candidate binding partner comprises determining whether the conformationally constrained peptide is able to modulate expression and / or activity of a reporter protein.

38. The method according to claim 36 or 37, wherein association of the first and second candidate binding partners forms a DNA binding complex that binds to one or more binding sites in a nucleic acid encoding the reporter protein, wherein binding of the DNA-binding complex to the binding site inhibits expression of the reporter protein, optionally wherein some or all of the binding sites are located in the transcribed sequence of the nucleic acid encoding the reporter protein.

39. The method according to any one of claims 36-38, wherein the first and second candidate binding partners are transcription factors (e.g. human transcription factors).

40. The method according to claim 39, wherein the transcription factor is a bHLH transcription factor, optionally wherein the bHLH transcription factor is c-Myc; a bZIP transcription factor, optionally wherein the bZIP transcription factor is c-Jun, CREB, and / or BZLF1 ; a DLX transcription factor, optionally wherein the DLX transcription factor is DLX-1 , DLX-2, DLX-3, DLX-4, DLX-5, and / or DLX-6.

41. A kit for use in any of the methods of claims 1 to 29, wherein the kit comprises:i. a nucleic acid encoding a recombinant peptide, wherein the recombinant peptide comprises at least two thiol-containing residues or selenol-containing residues, and ii. a pnictogen-containing crosslinker, wherein the pnictogen-containing crosslinker is thiolreactive and / or selenol-reactive.

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