Genetically encoded bicyclic peptide library

Abstract

To provide a bicyclic peptide library, a method for producing a library of the bicyclic peptide, and use of the library in various assays.SOLUTION: The present invention relates to a bicyclic peptide complex including a peptide structure, where the structure contains: (i) a polypeptide having a free terminal (N or C); (ii) optionally, a nucleic acid encoding polypeptide; and (iii) a two-fold symmetric linker (TSL) compound bonded to the polypeptide in which the linker is bonded to a terminal of the polypeptide and at least two separate side chains of the peptide via covalent bond. The present invention also relates to a method for manufacturing a library and a composite, and a method for screening by using the compound.SELECTED DRAWING: None

Description

Technical Field

[0001] This application relates to the fields of receptor-ligand interactions and molecular recognition. More particularly, this application relates to bicyclic peptide libraries, methods for producing libraries of bicyclic peptides, and the use of such libraries in various assays.

Background Art

[0002] The generation of libraries of small molecules and the selection of those molecules that uniquely bind to a target of interest are important for drug discovery. The production of genetically encoded libraries in which each library member is linked to an information template such as DNA or RNA allows large chemical libraries to be processed without separating individual library members into individual solutions or reaction vessels. A target molecule can be selected from a mixture of genetically encoded molecules and the information template used to identify or amplify the selected molecule of interest.

[0003] Peptides that contain intramolecular covalent bonds and exhibit a bicyclic topology are known to be more stable to proteolysis and other forms of degradation than monocyclic or linear peptides. They are also known to exhibit stronger binding interactions with protein targets; the increased interaction is hypothesized to be due to a reduced conformational penalty for the constrained bicyclic system compared to linear or cyclic systems.

[0004] Phage display is an example of a well-known technique used for the analysis, display, and production of genetically encoded libraries of peptides and proteins (Scott et al, 1990). Phage display is a process in which phage are engineered so that they expose or "display" different peptides or proteins, such as human antibodies, on their surface. Through genetic engineering, the peptide or protein of interest is individually linked to the phage cell surface protein molecule (usually gene III protein or g3p). In such a phage population (phage library), each phage carries the gene for a different fusion peptide or protein of g3p and exposes it on its surface. Through various selection procedures, phage that "display" binders to a specific target molecule of interest can be identified and isolated. These binders can include protein interaction partners for determining new functions or functional mechanisms of proteins, peptides that recognize and bind antigens (e.g., for use in diagnostic and therapeutic targets), and proteins involved in protein-DNA interactions (e.g., novel transcription factors).

[0005] Phage display can be very useful for the discovery and development of pharmaceutical and / or diagnostic polypeptides. In phage display, the entire phage can bind and elute from an immobilized target molecule. Since the phage remains infectious, its DNA can be injected into bacterial cells for amplification. The phage display method is usually limited to the production of libraries that can be encoded directly by DNA-RNA-protein information transfer. These methods are typically limited to linear sequences of peptides made only from 20 natural amino acids or cyclic peptides cross-linked through disulfide bonds.

[0006] RNA and ribosome display are other techniques known in the art that enable the display of peptides made naturally on an information template. Amplification of a library of peptides bound to RNA requires an in vitro translation system to generate or re-amplify the library. Suga et al (US20100168380 A1) teach the production of cyclic peptides containing N-methyl amino acids and other special (non-standard) amino acids by encoding unnatural amino acids in an RNA sequence. There are no examples of the production of bicyclic libraries by direct encoding and translation only of bicyclic peptides in RNA.

[0007] It is known to produce a library of bicyclic peptides or a display of bicyclic peptides on phage, DNA, or RNA by modifying the encoded display of molecules derived from peptides via chemical post-translational modification (cPTM). Typically, these methods use organic synthesis of peptides to create peptide derivatives. It is known that an entire peptide library can be modified by uniform chemical modification. Selection from the modified library and DNA sequencing yields peptide sequences that can produce modified peptide derivatives. There are several methods that involve the conversion of peptide libraries, libraries of phage-displayed polypeptides, and libraries of RNA-displayed polypeptides to libraries of peptide derivatives.

[0008] Suga et al. described a methodology for synthesizing bicyclic peptides displayed on RNA by using cysteine (Cys) and three different non-proteinogenic amino acids, Cab, Aha, and Pgl, simultaneously incorporated into the peptide chain. The first cyclization occurred in situ during translation between the chloroacetyl group of Cab and the sulfhydryl group in Cys, and the second cyclization at the side chain of Aha-Pgl was carried out via Cu(I)-catalyzed azide-alkyne cycloaddition. (J. Am. Chem. Soc., 2008, 130(23), pp 7232-7234). Hartman and co-workers used a different approach that combined two Cys with non-proteinogenic amino acids, azidohomoalanine (AzHA), and p-ethynylphenylalanine (F-yne) (ACS Chem. Biol. 2017, 12, 795-804). The first cyclization occurred by cross-linking cysteine with dibromo-m-xylene, and the second cyclization at the side chain of AzHA-F-yne was carried out via Cu(I)-catalyzed azide-alkyne cycloaddition. Special non-natural translation systems are required to express non-natural amino acids (UAAs) such as Aha, Pgl, F-yne; the expression of multiple non-natural side chains is difficult in display systems such as phage display, and the incorporation efficiency of UAAs is low. Therefore, it is interesting to develop methods using peptides composed of natural amino acid residues.

[0009] In US Patent Publication WO2004 / 077062, a method for modifying a plurality of unprotected peptides consisting of natural amino acid residues in water with a symmetric linker to produce a library of cyclic and bicyclic peptides is described. This method can be extended to other three-fold symmetric linkers having three thiol-reactive groups, but the number of such highly symmetric linkers is limited. Examples of the prior art (WO2009098450A2, WO2004077062, WO2011018227A2) and peer-reviewed literature ((Nat.Chem.Biol.2009, 5, 502-507; Angew.Chem.Int.Ed.2014, 53, 1602-1606) describe the synthesis of bicyclics limited to three-fold symmetric connector compounds having three identical reactive groups such as sulfur-containing side chains and tris-(bromomethyl)benzene (TBMB). The production of bicyclic peptides with lower symmetry (two-fold) linkers is not apparent from such prior art. Examples of the prior art using two-fold symmetric linkers to modify a plurality of unprotected peptides consisting of natural amino acids are limited to the production of monocyclic peptides (ACS Chem.Biol.2016, 11, 1422-1427; J.Am.Chem.Soc.2014, 136, 5880-5883; Bioconj.Chem.2016, 27, 509-514; Chem.Sci.2016, 7, 3785-3790; Org.Biomol.Chem.2016, 14, 5539-5545).

[0010] To access a broad chemical diversity space and maximize the potential of finding bicyclicity with desired chemical or biological properties, it is interesting to generate libraries that use linkers with low symmetry, such as two-fold symmetric linkers. Although it has been reported that low-symmetry modifications are applied to unprotected peptides, all examples in the art demonstrate that low-symmetry linkers, when applied to the modification of unprotected peptides, produce complex mixtures of bicyclic peptides. Heinis and co-workers (Nature Chemistry 2018, 10, 715) have specifically demonstrated that using a two-fold symmetric linker that reacts with amino acid side chains produces a complex mixture of multiple bicyclic structures. Such mixtures may not be separable by chromatography techniques or other techniques. Liu, Heinis et al. have demonstrated that a two-fold symmetric linker applied to side-chain modification produces a complex mixture of products (Angew. Chem. Int. Ed. 2017, 56, 4458), and that it is necessary to use special amino acids with non-natural side chains to avoid the production of such heterogeneous mixtures. What is generally emphasized in these and other examples is that modifying three or more similar reactive groups in a peptide using a modification factor with two-fold or less symmetry often results in the production of a complex mixture of products whose reactions are not controlled.

[0011] Special methods for synthesizing bicyclic peptides are known by methods that obligate the use of one or more of the following factors: (i) amino acids containing special reactive groups not found in natural amino acids, (ii) protecting groups for amino acids, (iii) solid supports for organic synthesis, (iv) organic solvents, and (v) reaction conditions incompatible with biomolecules such as DNA, RNA and biomolecular complexes such as phages. There does not appear to be an example of the use of a low-symmetry linker for the synthesis of bicyclic peptides that involves the modification of peptides consisting of natural amino acids without protecting groups. Such methods may be interesting because they may provide an unobstructed route to the synthesis of a wide variety of genetically encoded bicyclic structures.

[0012] US Patent Publication No. WO2009098450 A3 describes a method for modifying a genetically encoded peptide library displayed on a phage using a three-fold symmetric linker. This method produces a library of bicyclic peptides having free amino termini and obligates the use of a three-fold symmetric linker having a thiol-reactive group. The difficulty of generating a phage library having an odd number of cysteines (here, three) is known in the art.

[0013] The above-described methods described in the prior art are thought to each produce a library of bicyclic peptides having free amino termini. This terminus is known to be susceptible to proteolytic cleavage. Methodologies for producing polycyclic peptides without a free N-terminus are known, but such methods require the incorporation of non-proteinogenic amino acids containing an N-chloroacetyl (ClAc) group. Methods for producing a genetically encoded bicyclic library with an N-terminally blocked natural amino acid are not known.

[0014] This background information is provided for the purpose of making known information that may be relevant to the present invention. It is not necessarily intended that any of the foregoing information constitutes prior art against the present invention, nor should it be so construed. SUMMARY OF THE INVENTION

[0015] In general terms, the present invention includes bicyclic peptides having no N-terminal residue, produced by modifying a peptide or peptides (library) with a linker. Preferably, the peptide or peptide library includes a polypeptide comprising or consisting of unprotected natural proteinogenic amino acids.

[0016] In some embodiments, the invention includes a genetically encoded bicyclic peptide library made by modifying a linker genetically encoded peptide library, having no N-terminal residue.

[0017] Accordingly, in some embodiments, the invention is (a) a polypeptide; (b) a nucleic acid encoding the polypeptide, and optionally, an identifying tag that uniquely identifies the polypeptide; and (c) a bicyclic structure resulting from attaching a linker having a first terminal reactive group (A) and a second terminal having two reactive groups (B2, B2) to the polypeptide, wherein the first terminal is ligated to the terminal of the polypeptide by a covalent bond, and both reactive groups of the second terminal of the linker are covalently bound to side chain residues of the polypeptide and may include a genetically encoded peptide construct.

[0018] In some embodiments, the linker has two-fold symmetry in that the two second terminal reactive groups are identical. In some embodiments, the polypeptide terminus is the N-terminus and the side chain residues are each cysteine, lysine, or tyrosine. Alternatively, the polypeptide terminus is the C-terminus and the side chain residues are each cysteine, lysine, or tyrosine. The first terminal reactive group of the linker is then a group having unique reactivity towards the C-terminus such as C-terminal selective photoredox decarboxylation conjugate addition at acidic pH (Org. Lett., 2015, 17, 4830-4833 and Nature Chemistry 2018, 10, 205-211). In some embodiments, the first terminal reactive group of the linker is an aldehyde reactive group such as an oxime, hydrazine, 2-aminobenzamidoxime, phosphonium ylide, sulfur ylide, nitrogen ylide, or any other carbon nucleophile and carbenoid reagent known to be stable in an aqueous environment and reactive with an aldehyde.

[0019] In some embodiments, the second terminal reactive group of the linker is an electrophilic group reactive with thiol, amine, or phenol. Examples of thiol-reactive groups include halo ketones, haloacetamides, halobenzyls, maleimides, acryloylates, carbonyl acrylate reagents, 3-arylpropionitrile, arenamide fluoroarenes, chlorotetrazines, Julia-Kocienski-like reagents, 2-azidoacrylates, organometallic palladium reagents, Michael acceptors containing conjugated C-C double bonds including organogold(I) reagents, conversion of thiol to dehydroalanine (Dha), followed by conjugate addition to Dha, any other method that specifically reacts with thiol residues in water, etc. Examples of groups that specifically react with lysine amine residues in water include N-hydroxysuccinimide esters, aryl esters, perfluoroaryl esters, perfluoroarenes, ketenes, ortho-phthalaldehyde strain-release amine modifiers, or other groups that specifically react with lysine amine residues in water. Examples of groups that react with tyrosine or other aromatic side chains include allyl palladium (J. Am. Chem. Soc. 2006, 128, 1080-1081), diazodicarboxylate (J. Am. Chem. Soc. 2010, 132, 1523-1525), diazonium salts, aniline-formaldehyde hemiaminals, rhodium carbenoids, dirhodium metallopeptide catalysts, manganese-catalyzed C-H alkynylation, Wacker reagents, 1-[(triisopropylsilyl)ethynyl]-1,2-benziodoxol-3(1H)-one (TIPS-EBX), selective ruthenium-(II)-catalyzed C-H activation under gold(I) catalysis, palladium(II)-acetate-catalyzed C-H activation by aryl iodides in water, and other reagents known in the art for specifically modifying the phenol of tyrosine residues (Biochemistry 2017, 56, 3863-3873).

[0020] In some embodiments, the polypeptide comprises two cysteine residues and an N-terminal serine or threonine residue, and the N-terminal serine or threonine is first converted to an aldehyde by selective oxidation for reaction with a first terminal reactive group of a linker, and the second terminal reactive group of the linker is covalently bound to the cysteine residue.

[0021] In some embodiments, the complex can be bound to a carrier such as a phage particle that externally holds the polypeptide and contains a nucleic acid encoding the polypeptide. Alternatively, the complex can be an RNA display compound that holds the polypeptide, encodes the polypeptide, and contains an RNA sequence linked to the polypeptide. Alternatively, the complex can be a DNA display compound, wherein the DNA display contains DNA encoding the polypeptide that holds the polypeptide and is linked to the polypeptide. Alternatively, the complex can be a polypeptide linked to a polymer or protein carrier together with an identification tag such as another peptide.

[0022] In another aspect, the present invention can include a method for producing a phage display complex, comprising: (i) providing a phage particle containing a polypeptide having a terminus; (ii) providing a linker having a first terminal reactive group and a second terminus having two reactive groups, and ligating the first terminal reactive group of the linker to the polypeptide terminus to form an intermediate complex by forming a covalent bond (the "ligation step"); and (iii) forming a bicyclic structure from the intermediate complex by reacting both reactive groups at the second terminus of the linker with side chain residues of the polypeptide (the "bicyclization step"). The ligation step and the bicyclization step are preferably separate and consecutive steps and / or are preferably performed at different pHs.

[0023] In some embodiments, the terminus of the polypeptide is the N-terminus, which is oxidized to an aldehyde prior to the ligation step (the "oxidation step"), and the ligation step comprises mixing a polypeptide comprising an N-terminal aldehyde with a linker in an aqueous buffer at acidic pH. Preferably, the intermediate complex produced after the ligation step is purified by size exclusion purification, e.g., gel filtration or dialysis in an aqueous buffer at acidic pH, preferably below about pH 5.

[0024] In some embodiments, the intermediate complex that can be purified is reduced with a reducing agent such as TCEP prior to the dicyclization step (the "reduction step"). The reduced intermediate complex may then be exposed to an alkaline pH (>7) to induce dicyclization, and the second terminal reactive groups each comprise thiol-reactive groups that react with two cysteine residues in the polypeptide.

[0025] In some embodiments, the peptide is modified to introduce a first cycle and an orthogonally reactive diketone group. The diketone group can then be used to perform a reaction (dicyclization reaction) to create a second cycle. The intermediate complex containing the first cycle and the diketone group can be purified and stored without degrading the reactivity of the diketone, which can be a 1,3-diketone. The intermediate complex can then be subjected to many reactions that utilize the unique reactivity of 1,3-diketone with hydrazine under biocompatible aqueous conditions.

[0026] In some embodiments, the 1,3-diketone functional group is introduced by reaction of a peptide containing two cysteine residues with 1,5-dichloropentadione-2,5 under neutral aqueous conditions (from about pH 7 to about pH 8) to form a monocyclic peptide having a 1,3-diketone functional group.

[0027] In some embodiments, the second cyclic structure is formed by combining a monocyclic peptide containing a 1,3-diketone functional group with a molecule containing a hydrazine functional group and a functional group having unique reactivity with respect to the N-terminus or C-terminus of the peptide. By the reaction between these molecules, a connection is formed between the 1,3-ketone functional group and the end of the peptide, and a second cycle is formed in the peptide. Alternatively, hydrazine can be reacted with 1,3-diketone to present a new functional group or reactive group, which can then be linked to the N-terminus or C-terminus of the peptide by a linear linker.

[0028] In another aspect, the present invention provides a method for measuring the yield of a reaction after at least one chemical reaction step described herein, the method comprising exposing a complex or polypeptide to a capture reagent that is reactive with unreacted polypeptide but not with reacted polypeptide, and measuring the uptake of the capture agent by an affinity reagent. The capture agent includes a reactive group and an affinity handle paired with an affinity reagent. The affinity handle-affinity reagent pair can include any affinity ligand pair known to have a specific binding high enough to enable quantification of the binding pair. Exemplary pairs include biotin-streptavidin, FLAG peptide-anti-FLAG antibody, sulfonamide-carbonic anhydrase, methotrexate-dehydrofolate reductase (DHFR). Preferably, the affinity handle or affinity reagent is immobilized on a solid support such as agarose beads.

[0029] In some embodiments, the capture agent reactive group is an aldehyde reactive group such as an aminooxy group, or a thiol reactive group such as iodoacetamide.

[0030] In some embodiments, the measured reaction is an oxidation step, and the phage or complex is exposed to aminooxybiotin (AOB) after the oxidation step, which reacts with the N-terminal group oxidized to an aldehyde, dilutes the reactant by at least one order of magnitude, adds streptavidin beads to retain the reacted complex, measures the difference in the number of phages retained on the streptavidin beads compared to the number of phages not retained, and determines the yield of the oxidation step (the proportion of phage particles that have acquired aldehyde groups).

[0031] In some embodiments, the measured reaction is a ligation step, and the phage or complex is exposed to aminooxybiotin (AOB) before and / or after the ligation step, dilutes the reactant by at least one order of magnitude, adds streptavidin beads to retain the phage or complex with unligated aldehyde groups, measures the difference in the number of phage particles retained on the beads compared to the number of phages not retained, and determines the yield of the ligation step (the proportion of phage particles that have lost aldehyde groups).

[0032] In some embodiments, the measured reaction is a reduction step, and the phage or complex is exposed to biotin-iodoacetamide (BIA) before and / or after the reduction step, dilutes the reactant by at least one order of magnitude, adds streptavidin beads to retain the phage or complex reduced to expose thiol groups, measures the difference in the number of phage particles retained on the beads compared to the number of phages not retained, and determines the yield of the reduction step (the proportion of phage particles that have acquired thiol groups during the reduction step).

[0033] In some embodiments, the measured reaction is a bicyclization step, and the phage or complex is exposed to biotin-iodoacetamide (BIA) before and / or after the bicyclization step to dilute the reactants by at least one order of magnitude, add streptavidin beads to retain the phage or complex with unreacted thiol groups, and measure the difference in the number of phage particles retained on the beads compared to the number of phage not retained, to determine the yield of the bicyclization step (the percentage of phage particles that lost thiol groups during the bicyclization step). Alternatively, the phage or complex may be exposed to biotin-thiol (BSH) that reacts with benzyl chloride (or other thiol-reactive groups) before and after the bicyclization step, (i) dilute the reactants by at least one order of magnitude, (ii) add streptavidin beads, and (iii) measure the number of phage particles remaining after capture, whereby the number of phage retained on the streptavidin beads constitutes the yield of the bicyclization step (the percentage of phage particles that lost thiol-reactive groups during the bicyclization step).

[0034] In another aspect, the invention may include a library of genetically encoded ligands comprising a plurality of different polypeptide sequences, each forming a linker and a bicyclic structure as described herein. In some embodiments, at least two polypeptides P1 and P2 are separately encoded by DNA sequences. The plurality of different polypeptides may be modified with the same or different linkers. Each linker may be associated with a unique identification tag such as a silent gene barcode.

[0035] A mixed library of genetically encoded complexes can be formed by pooling libraries together to produce a library of complexes described herein. The mixed library can include at least two different peptide sequences (P1 and P2), each separately modified with at least two different linkers (L1, L2), forming at least four distinct complexes (P1L1, P1L2, P1L2, and P2L2). In some embodiments, linker L1 can react with two peptides associated with a predefined nucleic acid code B1, and linker L2 reacts with two peptides containing a predefined nucleic acid code B2. The nucleic acid code can include silent gene barcodes as described in WO2016061695-A1.

[0036] In another aspect, the invention is a method for identifying a complex described herein, wherein a polypeptide bicyclic structure can bind to a ligand, comprising: (i) contacting a library of complexes with a ligand; (ii) selecting those complexes that bind to the ligand; and (iii) identifying the structure of the binding complex by sequencing the nucleic acid encoding the structure of the polypeptide and / or the linker. In some embodiments, the library of complexes can include at least four distinct bicyclic structures, P1L1, P1L2, P1L2, P2L2, as described above.

[0037] In another aspect, the invention includes a Nodal antagonist peptide comprising a bicyclic complex formed from a polypeptide comprising SPCQRGHMFC or SYCKRAHKNC and a linker comprising TSL6.

[0038] To better understand the present invention, as well as other aspects and further features thereof, reference is made, together with the description, to the drawings that form a part of this specification. BRIEF DESCRIPTION OF THE DRAWINGS

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DETAILED DESCRIPTION OF THE INVENTION

[0059] In one aspect, the present invention includes a method for synthesizing a bicyclic peptide library comprising two separate modification steps. The first step includes creating an intermediate complex by ligating a first end of a linker to a terminus of a peptide displayed on a carrier by ligation chemistry in an aqueous environment. The peptide terminus can be an N-terminus or a C-terminus. After an optional purification step, the second step includes exposing the intermediate complex to reaction conditions to induce an intramolecular bicyclization reaction, wherein two reactive groups at the second end of the linker each independently react with side chains of amino acids in the peptide sequence.

[0060] In some embodiments, the bicyclic carrier can include a phage, mRNA, DNA, ribosome, bacterium, yeast, beads made of a synthetic polymer such as PEG or polystyrene, or any other genetically encoded biological display technology or synthetically encoded peptide library technology known in the art. In some embodiments, the carrier includes a display set of gene sequences encoding a random peptide library of different chemical compositions. In another embodiment, the display set of gene sequences includes a focused gene library encoding a subset of peptide sequences of different chemical compositions that are focused, such as those generated by random mutagenesis.

[0061] The display set of gene sequences can be paired with a specific linker such that a second set is linked to the chemical structure of a linker molecule such as a linker of different size or composition, or a linker that is a stereoisomer, diastereomer, or enantiomer.

[0062] Either or both of the two steps of ligation and bicyclization can be chemical or enzymatic modification of the peptide. In some embodiments, both modifications are chemical conjugation techniques specific to the N-terminus or a specific N-terminal amino acid and a separate set of amino acids in the peptide. For example, chemical modification used for ligation can form an oxime with an oxidized N-terminal serine. Ligation can use other N-terminal or C-terminal specific chemical actions known in the art.

[0063] A schematic diagram of a carrier having a peptide and a linker is shown in FIG. 1A. The peptide terminus Z, which can be either the N-terminus or the C-terminus, reacts with the first terminus A of the linker L. The peptide has reactive side chains X1 and X2 that react with two functional groups B1 and B2 on the second terminus of the linker. The linker L can include an aliphatic chain and can also include esters, phenyl, amines, and the like.

[0064] In some embodiments, the first terminus A is first ligated to the peptide terminus Z, resulting in the linear intermediate complex identified in Figure 1A, followed by bicyclization. The ligation reaction can include reactions that modify oxidized serine (oxaloyl) through a carbon-carbon bond formation process such as the Wittig reaction. One preferred embodiment of the bicyclization step includes alkylation of cysteine or any other suitable method for modifying a peptide or protein at specific locations.

[0065] In alternative embodiments, schematically shown in Figures 1C and 1D, the first step includes a reaction with a diketone that forms a first ring and simultaneously introduces its own reactive group. The diketone is preferably a 1,3-diketone. The intermediate monocyclic peptide formed in this reaction has its own stability and can be purified and stored without degrading the reactive group. The intermediate monocyclic peptide bearing the diketone group can then undergo chemical or enzyme-catalyzed ligation at one end with a 1,3-diketone reactive group such as alkyl or aryl hydrazine and at the other end with the terminus of the peptide to react with a linker molecule containing another group capable of inducing the formation of a second ring ("bicyclization"). Ligation at the peptide terminus can occur first, resulting in a linear intermediate. Alternatively, the reaction with the diketone can occur first, resulting in a branched intermediate.

[0066] For example, a display peptide having two internal cysteine residues in an array can be reacted with 1,5-dichloropentadione-2,4 at an alkaline pH, such as about pH 8 or 9, to introduce a 1,3-diketone group into the peptide. The 1,3-diketone group can then be reacted with a linker containing an alkyl or aryl hydrazine, thereby introducing a functional group that completes a subsequent bicyclization reaction with the N-terminus of the peptide. For example, the peptide containing the 1,3-diketone group can be modified at the N-terminus to introduce a hydrazine linker and then bicyclized via an intramolecular reaction between the N-terminal hydrazine and the 1,3-diketone ligated to the side chain. Alternatively, the order of the reactions is reversed and a linker containing an N-terminal reactive group reacts with the 1,3-diketone at an acidic pH. Then, terminal oxidation at neutral pH and a change in the environment to acidic pH 4.5 cause bicyclization via reaction with the N-terminus.

[0067] In all cases, it is preferred that the two steps of ligation and bicyclization occur under conditions independent of each other. Such independence allows for purification of the intermediate product and minimizes side reactions. Some embodiments involve the use of reactions that require different pH values. For example, formation of an oxime at the oxidized N-terminal serine occurs at an acidic pH of about pH 3 and can be catalyzed by 0.1% trifluoroacetic acid (TFA). These conditions are tolerated by other genetically encoded peptide libraries such as phage-displayed libraries and RNA-displayed libraries. The subsequent bicyclization reaction can be any intermolecular reaction with the side chains of amino acids that occurs at a higher pH. Suitable reactions can include nucleophilic substitution between a thiol and a thiol-reactive group, such as halobenzyl, haloacetamide, nucleophilic aromatic substitution, or Michael addition of a thiol to a conjugated alkene and / or allene amide. It is also possible to use other known reactions that occur at an alkaline pH with side chain residues of Lys, Tyr or other amino acid side chains.

[0068] In an alternative embodiment, the ligation and bicyclization steps are separated by use of a protecting - deprotecting reaction. For example, formation of a C - C bond at the N - terminal serine oxidized via a Wittig reaction occurs at about pH 7 to about pH 8. The Wittig reaction is known not to disrupt S - S disulfides that protect thiol residues. The Wittig reaction is tolerated by other genetically - encoded peptide libraries such as phage - displayed libraries and RNA - displayed libraries. After ligation and purification, the bicyclization reaction is triggered by reduction of the disulfide and ligation between the thiol and a thiol - reactive group, which can occur at about pH 7 to about pH 8. Many linkers can be designed that combine the stabilized ylide used in the Wittig ligation reaction with a thiol - reactive group for nucleophilic substitution, nucleophilic aromatic substitution, or Michael addition, or other well - known reactions of the bicyclization step.

[0069] In some embodiments, the carrier comprises an identifier, preferably a variable nucleic acid code identifier. The identifier can be silent such that it does not encode any peptide carried on the outside of the carrier. Alternatively, the identifier can be such that all variants of the identifier encode the same or substantially similar peptides. In this latter case, it is called "silent barcode" technology, and this method involves producing a bacteriophage display system on particles that contain DNA of different compositions inside bacteriophage particles and display peptides of the same composition. In some embodiments, the carrier is a virus or bacteriophage virion of the same external chemical composition containing a variable nucleic acid code that includes degenerate DNA tags within the genome, packaged inside these particles. The genome of the virus or phage is engineered in such a way that it does not cause changes in the chemical composition of the virion coat, such as the use of degenerate codons in the virion coat coding region, changes in the DNA sequence encoding excised sequences, changes in the DNA sequence that does not encode the expressed protein sequence, or changes in the DNA sequence encoding components not incorporated into the virion coat. Thus, a library of carriers (such as phages or viruses) can be provided, all of which are chemically identical externally (e.g., before modification of any ligand binding), but contain distinct nucleic acid identifiers therein.

[0070] In another aspect, the invention may include a method of selecting genetically encoded modifications of a peptide library by using unique identifiers associated with specific bicyclic structures, preferably silent gene barcodes. In some embodiments, a library of carriers is produced, each having a unique silent gene barcode and each displaying a polypeptide. Each library is then modified with a different linker as described herein to produce a unique bicyclic structure for the linker used. The libraries are then combined to produce a mixed library, and the bicyclic structure specific to each linker can be identified by the barcode. The mixed library can then be screened to select peptides having the desired sequence and bicyclic topology, which can then be identified by sequencing the gene barcode (or otherwise identifying the identifier).

[0071] For example, as schematically shown in FIG. 2, the first carrier carries a first silent nucleic acid code (barcode) and a DNA sequence encoding a peptide. The second carrier carries a second silent nucleic acid code different from the first code, and a DNA sequence encoding a peptide that may be the same as or different from the first peptide. The first carrier peptide is modified with a first linker (TSL1), and the second carrier is modified with a different linker (TSL2). After ligation and cyclization, the resulting two different bicyclic structures are distinguishable by the first and second nucleic acid codes.

[0072] In another aspect, the invention may include a method of identifying drug candidates that includes preparing a library of bicyclic peptide mixtures encoded by genes as described herein, and screening the library with a putative receptor molecule to identify those bicyclic peptides that bind to the receptor molecule. The bicyclic peptides that bind to the receptor are then identified by enriching and sequencing the sequences encoding the silent nucleic acid code and the peptide.

[0073] In another aspect, the invention includes inserting into a plurality of independent vectors in a substrate a redundant set of gene sequences encoding a peptide linker such that the gene sequences produce peptide sequences (a "linker") that are identical or closely related upon translation; inserting into each vector a second set of gene sequences encoding genetically diverse inserts such that a diverse set of peptides (a "library") is expressed upon translation; expressing and amplifying the first and second gene sequences such that the translation products include a non-variable linker and a variable peptide library is synthesized; and modifying each peptide library with a distinct TSL and combining the plurality of modified libraries to produce a library in which chemical modifications are genetically encoded. The method may include a method of synthesizing a genetically encoded chemically bicyclic peptide library.

[0074] U.S. Patent Publication 2013 / 050083 to Derda et al., the entire contents of which are incorporated herein by reference if permitted, describes new strategies for quantifying chemical modifications of genetically encoded peptide libraries and for selecting effective modifications. These methods can be used to quantify the yield of any one or all of the reactive steps described herein based on the presence or absence of reactive groups consumed in the reaction.

[0075] The technique of genetic encoding of chemical post-translational modifications of phage-displayed libraries is described in PCT Patent Application No. WO2016061695A1, the entire contents of which are incorporated herein by reference if permitted.

[0076] Example To better understand the invention described herein, the following examples are provided. It should be understood that these examples are for illustrative purposes only. Therefore, they should not limit the scope of the invention in any way.

[0077] Example 1.Synthesis of linkers for bicyclization. As shown in Figure 4A, crosslinkers 1 (TSL1), 2 (TSL3), and 3 (TSL6) were designed based on oxime formation and cysteine S-alkylation chemistry. These reactions are fast, biocompatible, clean, and high-yielding. The synthetic steps are outlined in Figures 4B and 4C. The three differences are mainly the number of carbon atoms in the linking chain.

[0078] Example 2. Modification of the reduced peptide SHCVWWDC with the TSL6 linker. The details of the modification are outlined in Figures 5A and 5B. The ligation step occurs at low pH. Size-exclusion chromatography enables purification after the aldehyde step or ligation. The low pH in the reduction step maximizes the yield of the bicyclization reaction. The increase in pH after reduction results in a clean bicyclic product with no visible unreacted starting material or by-products by LCMS. The identity of all intermediates at each step was confirmed by LCMS characterization.

[0079] Example 3. Modification of the reduced peptide SVCFDNGC with the TSL6 linker. The details of the modification are outlined in Figure 5. Key details of the chemistry: low pH in the ligation step. Size-exclusion chromatography enables purification after the aldehyde step or ligation. The low pH in the reduction step maximizes the yield of the bicyclization reaction. The increase in pH after reduction results in a clean bicyclic product with no visible unreacted starting material or by-products by LCMS. The identity of all intermediates at each step was confirmed by LCMS characterization.

[0080] Example 4.Modification of diverse sets of peptides with several TSL linkers. A series of peptide sequences having a serine residue at the N-terminus and two cysteines at downstream positions were selected. Using oxime ligation-based chemistry, the oxoaldehyde generated from periodate-mediated oxidation of the N-terminal serine was recognized, and then the two cysteines were captured by a cross-linking strategy, and finally conformationally rigid peptide macrocyclization was formed using the conditions described in Example 2 or 3. As described in FIGS. 8A-8G, a summary of the reactions with multiple different peptides confirms that ligation and dicyclization chemistry occur effectively with various peptide sequences. Modification with a TSL linker with a reduced number of atoms proceeded effectively even when the resulting second ring was formed with only one amino acid (FIG. 7B). These results indicate that there are no constraints on the size of the peptide loop and almost no constraints on the geometry of the TSL.

[0081] Example 5. Stability of dicyclicity in buffers and biological media. The dicyclic peptide formed from the sequence SHCVWWDC by modification with the TSL3 linker was incubated at room temperature for 1 month in buffer media at pH 4, 7, and 8.5 (FIG. 9). The inventors observed no LCMS changes indicating no degradation of the dicyclic product under any of these conditions. SHCVWWDC-TSL3 dicyclicity was incubated with fetal bovine serum (FBS) at 37° C., and the integrity of the dicyclicity was tested by LCMS. Using ion-selective LCMS, the dicyclicity did not change in FBS even after incubation at 37° C. for 3 days. In contrast, ion-selective LCMS demonstrated that 90% of the cyclic peptide disulfide SHCVWWDC was degraded by FBS after 300 minutes (FIG. 10A).

[0082] In another stringent stability test, the linear sequence SWDYRECYLEC, its disulfide derivatives, and two bicyclic derivatives of this sequence modified with TSL1 and TSL6 linkers were digested using an aggressive cocktail of endoprotease and exoprotease (Pronase) (Figure 10B). After incubation at 37 °C for 5 h, only 0.4 ± 0.1% of the linear peptide and 0.9 ± 0.4% of the linear and disulfide peptides remained undigested. Under the same conditions, after 5 h of proteolytic digestion, 68 ± 14% of the TSL6-SWDYRECYLEC bicyclicity and 82 ± 13% of the TSL1-SWDYRECYLEC bicyclicity remained intact (Figure 10B). The latter observation estimates the half-life stability of the TSL1-SWDYRECYLEC bicyclicity to be approximately 24 h.

[0083] Figure 11 demonstrates the generality of this observation in 12 other bicyclic peptide compositions under two different degradation conditions. Figure 11a explains the sequences and abbreviations of the reagents used for modification (TSL-1, TSL-3, and TSL-6 correspond to Structures 1, 3, and 6 in Figure 4A). Alphanumeric notations are used to describe the products. Figure 11b shows examples of time-resolved measurements of stability at 36 °C for 5 h and endpoint measurements of stability after 5 h. Figure 11c summarizes the endpoint measurements (5 h at 36 °C) under two different proteolytic conditions of Pronase and fresh mouse serum. Figures 11c and 11d compare the stability of the bicyclic peptides to some of the cyclic and bicyclic structures known in the art. This comparison shows the surprising non-obvious advantages of Compound 14f, a compound produced by the TSL-6 modifying factor reported herein, and of the known state-of-the-art compound 16j produced from a similar peptide sequence by the TBMB modifier (J. Med. Chem. 2018, 61(7), 2823-2836). The bicyclic compound 14f is 10-fold more stable to Pronase treatment than 16j. Another comparison between 13i and 13e demonstrates again the advantage of TSL-6 when compared to cyclization known in the art through perfluoroarenes as described in WO2014052650A2.

[0084] Example 6. Generation of a genetically encoded bicyclic library by modification of a phage-displayed peptide library and verification of the modification by a scavenger.

[0085] A library of phage-displayed peptides having the structure SxCxxxC where S is serine, C is cysteine, and x is a random amino acid was used as the starting point for the generation of genetically encoded bicyclic libraries. Figure 12 illustrates an example of the modification of clones from such a phage library using the TSL6 linker. Figures 13 and 14 illustrate examples of the modification of large-scale phage libraries SxCxxxxxxC using the TSL1, TSL3, or TSL6 linker, demonstrating the generality of this approach for any library and any linker geometry. The inventors used conditions optimized for the ligation of synthetic peptide sequences: specifically, the library was exposed to an ice-cold solution of 60 micromolar sodium periodate in PBS for 8 minutes, and a 0.5 mM solution of methionine was added to stop the reaction. The oxidized library was exposed to a solution of 1 mM TSL6 linker in 0.1% aqueous trifluoroacetic acid at room temperature for 1 hour. The ligated library can be purified by size-exclusion chromatography using pH 4 buffer as the eluent, such as Zeba™ Spin Desalting Columns, 7K MWCO. The purified library was exposed to TCEP at pH 4 to reduce disulfide bonds. Finally, Tris buffer medium was added to the solution to raise the pH to 8 and promote bicyclization (Figure 13A). Each step of the reaction can be effectively monitored using various capture reagents (Figure 13B). For example, to quantify the oxidation step, the inventors mixed the oxidized library with aminooxybiotin (AOB) in aniline acetate buffer and measured the phage titers before and after exposure to streptavidin-coated beads (Figures 13C, F). AOB capture demonstrated that 73% of the library was oxidized. Only 10% of the phage population contained AOB-reactive aldehydes after exposure to TSL6, indicating that 87% of the oxidized library was ligated to TSL6. The same procedure can be used to test the integrity of the reactive groups.For example, by modification with AOB followed by capture, it was demonstrated that aldehydes maintain reactivity when the library was incubated with 0.1% TFA without a TSL linker (Figure 13F). Similarly, the number of thiols can be quantified using exposure to BIA and capture with streptavidin (“BIA capture”), and the proportion of the library containing benzyl chloride groups with thiol reactivity can be quantified using exposure to BSH and subsequent capture with streptavidin (“BSH capture”). Exposure to BIA (Figure 13G) after ligation and after dicyclization (Figure 13H) indicates that thiols are present in the library after ligation but disappear after dicyclization. Similarly, exposure to BSH can be used to check for the presence or absence of benzyl chloride groups. Exposure to BSH can also be used to check the integrity of the benzyl chloride groups. For example, exposing the ligated library to pH 7 for a long time or purifying it with a pH 7 buffer leads to hydrolysis of the benzyl chloride groups as determined by BSH capture. This observation was used to select specific conditions for purifying the library ligated to TSL6. It is not clear how the purification and integrity of the library modified with TSL7 can be checked without performing such quantification. Optimization of other peptide sizes and linker sizes follows the same steps (Figures 11, 12) and shows the same observations.

[0086] Example 7:A synthetic peptide or a phage-displayed peptide having two Cys residues in the array can be reacted with 1,5-dichloropentadione-2,4 at pH 8.5 to quantitatively introduce a 1,3-diketone group into the peptide. In the synthetic peptide, LCMS confirms the completion of the reaction in multiple diverse peptide sequences. In a phage library displayed on phage, the presence of 1,3-diketone in the displayed peptide is confirmed by the reaction of the 1,3-diketone-modified product with hydrazine-biotin and subsequent capture by streptavidin beads. The 1,3-diketone group in the peptide or phage-displayed peptide can then be modified with an alkyl or aryl hydrazine under controlled conditions such as ammonium acetate buffer pH 4.5 (Figure 15A). This reactivity can be used to introduce functional groups such as aldehyde-reactive ylides that complete the reaction with the N-terminus and bicyclization in subsequent steps (Figure 15B). For example, the peptide containing the 1,3-diketone group can be modified at the N-terminus to introduce an aldehyde, and the change in the environment to acidic pH induces bicyclization via an intramolecular reaction between the N-terminal hydrazine and the 1,3-diketone ligated to the side chain (right pathway in Figure 15B). In another example, the order of the reaction can be reversed, and a linker containing an N-terminal reactive group is reacted with 1,3-diketone at acidic pH. Oxidation of the terminus at neutral pH causes bicyclization via reaction with the N-terminus. (Left pathway in Figure 15B).

[0087] Example 8: Generation of a genetically encoded bicyclic library by modification of a phage-displayed peptide library and selection of bicyclic peptides that bind to a target from this library. The screening step was performed using the complete sequence described in the representative human protein Nodal: GenBank: BC104976.1; Proc. Natl. Acad. Sci. U.S.A., 2002, 99(26), 16899-16903. Specifically, the 40-338 amino acid sequence PLAYMLSLY[..]VLLDHHKD encoded by BC104976 of hexahistidine-tagged Nodal (His-Nodal) catalog number ag21882 from Proteintech was used. The selection can be readily performed against any other target using techniques known for the selection of phage-displayed libraries. The results of screening, panning, and validation of SXCX6C modified with TSL-6 are shown in Figure 13.

[0088] Targets immobilized on agarose beads functionalized with nitrilotriacetic acid (NTA) were panned using a mixed bicyclic library modified with a TSL-6 (6-carbon) linker (Figure 16B-A). After washing the beads with KingFisher Duo, the beads were boiled in water to release Nodal, and the bound ligand / phage was subjected to PCR and amplification. After 3 rounds of selection, the desired convergence was observed: the bicyclic library at round 3 (R3) was specifically enriched with Nodal immobilized on agarose beads when compared to the R1 and R2 libraries. No enrichment was observed with blank beads or beads with a control His-tagged target. Importantly, the unmodified R3 library panned on Nodal-modified beads did not show enrichment, confirming that the selected peptide sequences bind to Nodal only when constrained to the bicyclic scaffold (Figure 16A). The dsDNA amplicons from each round of selection were sequenced using Illumina NextSeq, and informatics analysis suggested that a number of sequences (Figure 16B) and at least 3 sequence motif families (Figure 16C) were potential ligands for Nodal. A list of peptide sequences that bind to Nodal is shown in Table 1. [Table 1]

[0089] To verify the binding ability of the predicted ligand, the inventors tested the bicyclic ability to inhibit known signaling events induced by Nodal. Specifically, the inventors detected Nodal-induced phosphorylation of the effector protein Smad2 in embryonic carcinoma P19 cells using Western blot with an anti-phosphorylated Smad antibody. Figure 17a describes that when P19 cells are treated with 100 ng / mL of Nodal for 1 hour in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 2.5% fetal bovine serum (FBS) + 7.5% bovine serum albumin (BSA), the intensity of pSmad2 increases. Incubation of P19 cells with 100 ng / mL of Nodal and the known kinase inhibitor SB-431542 suppresses the increase in Nodal induction in pSmad. Similarly, co-treatment of 100 ng / mL of Nodal with 100 μM of bicyclic peptides 2 (SPCKAGTGQC), 3 (SPCKGPSATC), 4 (SPCKGRHHNC), 5 (SPCKKAHGAC), 7 (SPCQRGHMFC), and 11 (SYCKRAHKNC) does not increase pSmad2 phosphorylation beyond the background level. The bicyclic peptides function as antagonists of Nodal at a concentration of 100 micromolar. Figure 17b explains that only 2 out of 6 bicyclic peptides retain potency at a concentration of 10 μM: bicyclic 7 (SPCQRGHMFC) and 11 (SYCKRAHKNC) inhibit pSmad2 phosphorylation, while bicyclic 2, 3, 4, and 5 do not inhibit pSmad2 phosphorylation at a concentration of 10 μM. Figures 17c-d explain the dose response of bicyclic 7 and 11, suggesting that the half-maximal inhibitory concentration for antagonism of Nodal signaling is 1 - 3 μM.

[0090] SPCQRGHMFC-TSL6, SYCKRAHKNC-TSL6, and derivative compounds have the ability to antagonize the signaling function of the protein Nodal in cancer cells. Derivative compounds of SPCQRGHMFC-TSL6 and SYCKRAHKNC-TSL6 retain the similar structural features of these compounds and exhibit similar or enhanced ability to antagonize the function of the Nodal protein. The only known Nodal antagonist peptide is the anti-human Nodal monoclonal antibody 3D1 (WO2016057683A2). Small molecule compounds that can antagonize Nodal include SB431542 (described as SB in Figure 17) and its derivatives. However, these compounds do not interact with Nodal and are inhibitors of ALK5, ALK4, and ALK7 kinases that act downstream of Nodal. References

[0091] All publications, patents, and patent applications mentioned herein are indicative of the level of skill of those of ordinary skill in the art to which the present invention pertains, and, if permitted, are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. JPEG2025090596000002.jpg214154 JPEG2025090596000003.jpg140154

Claims

1. A peptide construct comprising a bicyclic structure resulting from attaching a linker having a first end and a second end comprising two reactive groups to a polypeptide, wherein the first end is ligated to a terminus of the polypeptide by a covalent bond and both reactive groups at the second end of the linker each react with a side chain residue of the polypeptide.

2. The construct of claim 1 , wherein the two second terminal reactive groups are identical.

3. 3. The construct of claim 1 or 2, wherein the polypeptide terminus is an N-terminus and the side chain residue is a cysteine, lysine, or tyrosine.

4. The construct of any one of claims 1 to 3, wherein the linker reactive group is an electrophilic group reactive to thiols, amines, or phenols.

5. 5. The construct of any one of claims 1 to 4, wherein the first end of the linker comprises an aldehyde-reactive group such as an oxime, hydrazine, 2-aminobenzamidoxime, phosphonium ylide, sulfur ylide, nitrogen ylide, or any other carbon nucleophile and carbenoid reagent known to be reactive with aldehydes and stable in an aqueous environment.

6. 6. The construct of any one of claims 1 to 5, wherein the polypeptide comprises two cysteine ​​residues and an N-terminal serine or threonine residue, the N-terminal serine or threonine being first converted to an aldehyde by selective oxidation, and the second terminal reactive group being covalently attached to the cysteine ​​residue.

7. A construct according to any one of claims 1 to 6, which carries the polypeptide externally and is bound to a carrier such as a phage particle comprising a nucleic acid encoding the polypeptide.

8. The construct according to any one of claims 1 to 6, which is an RNA display compound, said RNA display comprising an RNA sequence carrying said polypeptide and encoding said polypeptide and linked to said polypeptide.

9. The construct according to any one of claims 1 to 6, which is a DNA display compound, said DNA display comprising a polypeptide and encoding said polypeptide and linked to said polypeptide.

10. 2. The construct of claim 1, wherein the polypeptide terminus is the C-terminus, the side chain residue is a cysteine ​​or lysine, a first terminus of the linker contains a reactive group that is reactive towards the C-terminus at acidic pH, and a second terminus of the linker contains two electrophilic groups that react specifically with thiol or amine residues.

11. The construct of claim 10, wherein the reactive group reactive to the C-terminus at acidic pH is a C-terminus selective photoredox decarboxylative conjugate addition.

12. 11. The construct of claim 4 or 10, wherein the linker electrophilic group is reactive with thiols in water, such as haloketones, haloacetamides, halobenzyls, maleimides, actylates, carbonylacryl reagents, 3-arylpropiolonitriles, alenamidofluoroarenes, chlorotetrazines, Julia-Kocienski-like reagents, 2-azidoacrylates, Michael acceptors containing conjugated C-C double bonds, including organometallic palladium reagents, organogold(I) reagents, conversion of the thiol to dehydroalanine (Dha) followed by conjugate addition to Dha, and the like.

13. 11. The construct of claim 4 or 10, wherein the linker electrophilic group is reactive with amines in water, such as N-hydroxysuccinimide esters, aryl esters, perfluoroaryl esters, perfluoroarenes, ketenes, orthophthalaldehyde, or strain-releasing amine modifiers.

14. 11. The construct of claim 4 or 10, wherein the linker electrophilic group is reactive with phenols in water, such as allylpalladium, diazodicarboxylates, diazonium salts, aniline-formaldehyde hemiaminal, rhodium carbenoids, dirhodium metallopeptide catalysts, manganese catalyzed C-H alkynylation, Wother's reagent, 1-[(triisopropylsilyl)ethynyl]-1,2-benziodoxol-3(1H)-one (TIPS-EBX), selective ruthenium-(II) catalyzed C-H activation under gold(I) catalysis, palladium(II) acetate catalyzed C-H activation with aryl iodides in water, and the like.

15. The construct of claim 1 , wherein the polypeptide comprises a terminal serine residue and two cysteine ​​residues separated by at least 2, 3, 4, or 5 amino acid residues.

16. The construct of any one of claims 1 to 15, wherein the linker comprises an alkyl chain having 1 to 6 carbon atoms.

17. The construct according to any one of claims 1 to 16, wherein said polypeptide comprises only naturally occurring proteogenic amino acids and / or does not comprise a free N-terminus.

18. A method for producing a phage display complex, comprising: (i) providing a phage particle comprising a polypeptide having an end; (ii) providing a linker having a first end and a second end comprising two reactive groups, forming an intermediate complex by ligating the first end of the linker to the polypeptide end to form a covalent bond; and (iii) forming a bicyclic structure from the intermediate complex by reacting both reactive groups at the second end of the linker to side chain residues of the polypeptide.

19. 20. The method of claim 18, wherein steps (ii) and (iii) are independent and sequential.

20. 20. The method of claim 18 or 19, wherein steps (ii) and (iii) are carried out at different pH.

21. 21. The method of any one of claims 18 to 20, wherein the terminus of the polypeptide is N-terminal and is oxidized to an aldehyde prior to the ligation step, and the ligation step comprises mixing a polypeptide comprising an N-terminal aldehyde with a linker in an aqueous buffer at an acidic pH.

22. 22. The method of any one of claims 18 to 21, wherein the intermediate complex is purified, such as by size exclusion purification, such as by gel filtration or dialysis in an aqueous buffer at an acidic pH, such as below about pH 5.

23. 23. The method of claim 22, wherein the purified intermediate conjugate is reduced with a reducing agent such as TCEP prior to the bicyclization step ("reduction step").

24. 24. The method of claim 23, wherein the reduced intermediate complex is exposed to a pH of 7 or greater to induce dicyclization, the second terminal reactive groups each comprise a thiol reactive group, and the polypeptide comprises two cysteine ​​residues.

25. A method for measuring the yield of a reaction after at least one step according to any one of claims 18 to 24, comprising exposing said polypeptide to a capture reagent reactive to unreacted polypeptide but not to reacted polypeptide, and measuring the uptake of the capture agent by the affinity reagent.

26. 26. The method of claim 25, wherein the capture agent comprises an affinity handle paired with a reactive group and an affinity reagent such as biotin-streptavidin, FLAG peptide-anti-FLAG antibody, sulfonamide-carbonic anhydrase, methotrexate-dehydrofolate reductase (DHFR), and preferably the affinity handle or the affinity reagent is immobilized on a solid support such as agarose beads.

27. 27. The method of claim 26, wherein the capture agent reactive group is an aldehyde-reactive group, such as an aminooxy group, or a thiol-reactive group, such as an iodoacetamide group.

28. 28. The method of claim 21, wherein at least one step comprises (i) exposing the phage to aminooxybiotin (AOB) after the oxidation step, (ii) diluting the reaction by at least one order of magnitude, (iii) adding streptavidin beads, and (iv) measuring the number of phage particles remaining after capture, whereby the difference in the number of phage retained on the streptavidin beads constitutes the yield of the oxidation step (proportion of phage particles that have acquired an aldehyde group).

29. 28. The method of any one of claims 25 to 27, wherein the ligation step of claim 21 comprises at least one step: (i) exposing the phage to aminooxybiotin (AOB) before and after the ligation step, (ii) diluting the reaction by at least one order of magnitude, (iii) adding streptavidin beads, and (iv) measuring the number of phage particles remaining after capture, whereby the difference in the number of phage retained on the streptavidin beads constitutes the yield of the ligation step (proportion of phage particles that have lost an aldehyde group).

30. 28. The method of any one of claims 25 to 27, wherein the reduction step according to claim 23, at least one step comprises (i) exposing phage to biotin-iodoacetamide (BIA) before and after the reduction step, (ii) diluting the reaction by at least one order of magnitude, (iii) adding streptavidin beads, and (iv) measuring the number of phage particles remaining after capture, the difference in the number of phage retained on the streptavidin beads constituting the yield of the reduction step (proportion of phage particles that have acquired a thiol group during the reduction step).

31. 28. The method of any one of claims 25 to 27, wherein at least one step comprises (i) exposing phage to biotin-iodoacetamide (BIA) or biotin-thiol (BSH) before and after the bicyclization step, (ii) diluting the reaction by at least one order of magnitude, (iii) adding streptavidin beads, and (iv) measuring the number of phage particles remaining after capture, whereby the number of phage retained on the streptavidin beads constitutes the yield of the bicyclization step (proportion of phage particles that have lost a thiol group during the bicyclization step).

32. A library of genetically encoded ligands comprising a plurality of different peptide sequences that form a bicyclic structure as described in claim 1.

33. 33. The library of claim 32, wherein the at least two peptides P1 and P2 are separately encoded by DNA sequences.

34. 34. The library of claim 33, comprising at least two different peptide sequences P1 and P2, each separately modified with at least two different linkers L1 and L2, forming at least four separate constructs P1L1, P1L2, P1L2, and P2L2.

35. 35. The mixed library of claim 34, wherein linker L1 reacts with two peptides associated with predefined nucleic acid code B1 and linker L2 reacts with two peptides associated with predefined nucleic acid code B2.

36. 36. The mixed library of claim 35, wherein the nucleic acid code is a silent genetic barcode.

37. 37. A method for identifying a complex according to any one of claims 1 to 36 capable of binding to a ligand, comprising: (i) contacting a library of complexes with said ligand; (ii) selecting those complexes which bind to said ligand; and (iii) identifying the structure of the binding complex by sequencing nucleic acids encoding the peptide and / or linker structure.

38. The method of claim 37, wherein the library of complexes comprises at least four separate constructs P1L1, P1L2, P1L2, P2L2 as described in claim 34, and the complexes are as described in any one of claims 1 to 17.

39. 2. A Nodal antagonist peptide comprising the complex of claim 1 formed from a polypeptide comprising SPCQRGHMFC or SYCKRAHKNC, and a linker comprising TSL6.

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