mRNA display libraries and methods of use

Abstract

Disclosed herein are mRNA display libraries comprising peptides with non-canonical amino acids and methods of making the mRNA display libraries which may include a plurality of mRNA-linker-macrocyclic peptide molecules comprising AcTyr-[amino acid]m-[PhSec]-[amino acid]n-[Cys]-[linker]-[mRNA]; wherein m and n are each an integer from 0 to 30; and wherein each macrocyclic peptide comprises a thioether linkage and is according to formula I: wherein R comprises [linker]-[mRNA].

Description

mRNA DISPLAY LIBRARIES AND METHODS OF USESTATEMENT OF PRIORITY

[0001] This application claims the benefit, under 35 U.S.C. §119(e), of U.S. Provisional Application No. 63 / 511,283, filed June 30, 2023, the entire contents of which is incorporated by reference herein in its entirety.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant Number GM125005 awarded by the National Institutes of Health. The government has certain rights in the invention.STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

[0003] A Sequence Listing in XML format, entitled 5470-955WO-l_ST26.xml, 135,100 bytes in size, generated on June 28, 2024 and filed herewith, is hereby incorporated by reference into the specification for its disclosures.FIELD OF THE INVENTION

[0004] The invention relates to mRNA display libraries and methods of making same. The invention further relates to methods of using mRNA display libraries for identification of cyclic peptide inhibitors.BACKGROUND OF THE INVENTION

[0005] mRNA display is revolutionizing peptide drug discovery through its ability to quickly identify potent peptide binders of therapeutic protein targets. Peptides have historically been underutilized for covalent inhibitor discovery despite their unique abilities to interact with protein surfaces and interfaces. This is in part due to a lack of methods for the screening and identification of covalent peptide ligands.

[0006] Covalent inhibition has regained favor as a drug discovery strategy in recent years, as exemplified by a substantial increase in FDA approved covalent drugs in the last three decades. Many covalent inhibitors to date have relied on structural information to retrofit electrophiles onto reversible binders, optimize naturally occurring covalent ligands, or design covalentsubstrate analogs of a given protein target. However, when no opportunistically positioned substrates can be used to inform inhibitor design, de novo covalent inhibitors must be discovered. In these endeavors, high throughput screening of electrophilic libraries can enable initial hit discovery. For instance, a small molecule acrylamide library screened against the G12C mutant of KRAS, which otherwise lacks distinct pockets for reversible ligand engagement, led to the discovery of a previously unknown binding site — exploitation of which enabled the development of now FDA-approved sotorasib (AMG-510). This success further validates the use of electrophilic libraries for covalent drug development, and applications of this approach have continued to expand in recent years.

[0007] Incorporating electrophilic handles into genetically encoded libraries (GELs), such as those in phage and mRNA display, could provide several added benefits for covalent inhibitor discovery. GELs can be exceedingly large (>1013molecules), are synthesized quickly and accurately by the ribosome, and are increasingly approaching the chemical space of natural products. Resulting peptide inhibitors have greater propensity to occupy shallower binding pockets, disrupt protein-protein interactions, and have high selectivity for their protein target; with high selectivity having potential to be particularly desirable in covalent drug discovery, where non-specific reactivity is a unique concern. Despite these advantages, there are relatively few examples of electrophilic GELs, with a handful of reports employing phage display, but still none in in vitro display methods like mRNA display.

[0008] Methods to expand the chemical diversity of display libraries are continually needed to increase the likelihood of identifying clinically relevant peptide ligands.SUMMARY OF THE INVENTION

[0009] The present invention is based, in part, on the development of methods for generating cyclic mRNA display libraries. These libraries can be used to identify targeting ligands (e.g., covalent binders) of relevant targets.

[0010] One embodiment of the invention is an mRNA display library comprising a plurality of mRNA-linker-macrocyclic peptide molecules comprising:AcTyr-[amino acid]m-[PhSec]-[amino acid]n-[Cys]-[linker]-[mRNA]; wherein m and n are each an integer from 0 to 30; and wherein each macrocyclic peptide comprises a thioether linkage and is according to formula I:wherein R comprises [linker]-[mRNA.]

[0011] A further embodiment of the invention is an mRNA display library comprising a plurality of mRNA-linker-macrocyclic peptide molecules comprising:AcTyr-[amino acid]m-[dAla]-[amino acid]n-[linker]-[mRNA]; wherein m and n are each an integer from 0 to 30; and wherein each macrocyclic peptide comprises a thioether linkage and is according to formula II:wherein R comprises [linker]-[mRNA],

[0012] Another embodiment of the invention is a method of preparing an mRNA display library comprising a plurality of mRNA-linker-macrocyclic peptide molecules, the methodcomprising the steps of providing a randomized library of mRNA molecules comprising a plurality of molecules according to a formula according to one of (Illa) -(Illf) :AUG-(NiWW)m-TAG-(NiWW)n -Cys-[linker] (Illa) (SEQ ID NO:1) AUG-(NiN2K)m-TAG-(NiN2K)n-Cys-[linker] (nib) (SEQ ID NO:2) AUG-Cys-(NiWW)m-TAG-(NiWW)n-Cys-[linker] (IIIc) (SEQ ID NO:3) AUG-Cys-(NiN2K)m-TAG-(NiN2K)n-Cys-[linker] (Illd) (SEQ ID NO:4) AUG-Cys-(NiWW)m-TAG-(NiWW)n-AUG-[linker] (Ille) (SEQ ID NO:5) AUG-Cys-(NiN2K)m-TAG-(NiN2K)n-AUG-[linker] (inf) (SEQ ID NO:6) wherein Ni andN2are independently any nucleotide, each W is independently A or U, and each K is independently G or U, and m and n are each an integer from 0 to 30; performing in vitro translation of each of the plurality of molecules in the mRNA library with a promiscuous orthogonal aminoacyl-tRNA synthetase (ORS) in the presence of phenylselenocysteine (PhSec) to thereby produce a plurality of linear molecules, the ORS incorporating a PhSec at the TAG or other non-competitive codon; cyclizing each of the plurality of linear molecules; forming a dehydroalanine via oxidative elimination at the PhSec in each of the plurality of cyclized molecules; thereby forming an mRNA library of a plurality of mRNA-linker-macrocyclic peptide molecules comprising a dehydroalanine.

[0013] Methods for screening for a targeting cyclic peptide are provided, the methods comprising producing an mRNA display library or providing an mRNA display library of the present invention, bringing the mRNA display library molecules into contact with the target substance, incubating the library molecules and target substance; and selecting cyclic peptides that bind the target substance.

[0014] These and other aspects of the invention are set forth in more detail in the description of the invention below.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIGS. 1A-1B. FIG. 1A) Strategy for dehydroalanine (Dha) incorporation in mRNA display. FIG. IB) Adjustments to standard selection process to facilitate identification of covalent binders.

[0016] FIGS. 2A-2D. FIG. 2A) NGS % composition of CCP1 tracked over selection rounds. FIG. 2B) Matrix-assisted laser desorption / ionization-mass spectrometry (MALDI-MS) of CIB1when incubated with CCP1 , its L / D-Al a mutants, or CCP1 + CTB1 C134A mutant. FIG. 2C) Time-dependent Time-resolved Forster's resonance energy transfer (TR-FRET) between CIB1 and AF647-phage peptide in the presence of CCP1, its L / D-Ala mutants, and non-covalent CIB 1 inhibitors UNCI 0245131 and UNCI 0245231. FIG. 2D) Western blot of biotin-CCPl titration in MDA-MB-453 cell lysates spiked with CIB1 (6 pM). Detection enabled by neutravidin-HRP.

[0017] FIGS. 3A-3C. FIG. 3A) MALDI-MS spectra of MAGE- A4 when incubated with each peptide (MCP1, MCP2, MCP3), its L / D-Ala mutants, or when peptides are incubated with MAGE-A4 C80A. FIG. 3B) Time-dependent TR-FRET between AF647-MTP-1 and MAGE-A4 or MAGE-A4 C80A in the presence of covalent peptides. FIG. 3C) Western blots of MAGE- A4 co-immunoprecipitation with RAD 18 at 10 pM peptides, including an MTP-1 Y5A negative control.

[0018] FIG. 4. Overview of generation of mRNA library and selection of targeting ligands.

[0019] FIG. 5. SDS-PAGE gel of exemplary recombinant proteins used in Example 1.

[0020] FIGS. 6A-6C. MALDI-MS of PhSec IVT, conversion to DHA, and conjugate addition with model thiol. FIG. 6A) Schematic of model IVT gene containing TAG codon for PhSec translation, DHA formation, and conjugate addition by model thiol -mercaptoethanol (BME). FIG. 6B) Sequence of model IVT peptide (Gl; SEQ ID NO: 108), where X = TAG codon. FIG. 6C) MALDI mass spectra of model IVT peptide when translated with PhSec, converted to DHA, and reacted with BME.

[0021] FIGS. 7A-7B. mRNA display compatibility with guanidine denaturation. FIG. 7A) cDNA percent recovery of model mRNA display gene (G3) translated with biotin-Phe, incubated with streptavidin beads, and exposed to guanidine (+G), compared to non-treated control. Error bars report standard deviation (N=3). FIG. 7B) cDNA percent recovery of non-covalent CIB1 inhibitor, UNC10245129 (G2),12in mRNA display with (+G) and without (-G) exposure to guanidine after selection. Error bars report standard deviation (N=3).

[0022] FIG. 8. MALDI-MS of trypsin digested CIB1-CCP1 adduct. Table of cysteine- containing CIB1 fragments when digested with trypsin and corresponding masses upon CCP1 modification and MALDI mass spectra of trypsin-digested CIB1 alone (top), CIB1 + CCP1 (middle), and CIB1 + non-specific electrophile iodoacetamide (IAA).

[0023] FIG. 9. CIB1 C134A TR-FRET. Time-dependent TR-FRET between CIB1 C134A and AF647-phage peptide1in the presence of CCP1, its L / D-Ala mutants, and known CIB1 inhibitorsUNC10245131 and UNCI 0245231.12Inhibition curves were measured at 1 h (filled shape and solid line), 16 h (half-filled shape and long dash) and 24 h (shape outline and short dash).FIGS. 10A-10B. FIG. 10A) Co-crystal structure of CIB 1 and phage peptide with Cysl34 highlighted.1FIG. 10B) Distance of Cl 34 thiol from surface residues of the CIB1 C-lobe hydrophobic pocket that phage peptide occupies.

[0024] FIGS. 11A-11B. Titration of biotin-CCPl in MDA-MB-453 lysates spiked with 6 pM CIB1 detected through FIG. 11 A) Neutravidin-HRP western blot or FIG. 11B) Ponceau S staining.

[0025] FIG. 12. mRNA display recovery of CCP1 under varied selection conditions. Percent recovery of CCP1 from CIB1 selection in mRNA display under various conditions. Selection time varied from Ih, 2h, to overnight (O / N). Selection temperatures varied from 4 °C, RT, to 37 °C. Error bars report standard deviation (n=2).

[0026] FIGS. 13A-13B. MCP modifications of A4-MHD and CIB 1. MALDI-MS spectra of MHD (FIG. 13A) or CIB1 (FIG. 13B) when incubated with the top peptides from the DHA MAGE-A4 +guanidine selections (MCP 1-3).

[0027] FIG. 14. MALDI-MS of GluC-digested MAGE-A4 covalent peptide adducts. Table of cysteine-containing MAGE-A4 fragments when digested with GluC, and corresponding masses upon covalent peptide modification. MALDI mass spectra of GluC-digested MAGE-A4 alone (top), MAGE-A4 + MCP1 (middle), and MAGE-A4 + MCP2 (bottom).

[0028] FIGS. 15A-15B. Mass-dilution TR-FRET with MCP1 and MAGE-A4 for determination of covalent kinetic parameters. FIG. 15A) Calculated percent inhibition of fluorescent MTP-1 and MAGE-A4 interaction in the presence of increasing concentrations of MCP1. FIG. 15B) Determined kinact and Ki values from rate of inhibition measured in part FIG.15A.

[0029] FIGS. 16A-16C. FIGS. 16A-16B. Method includes Dha-containing libraries prepared by desulfurization of internal cysteines by means of dibromovalerate (DBV) or similar reagents. FIG. 16C. MALDI for desulfurization of representative peptide library members.DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention will now be described in more detail with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. Thisinvention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. In addition, any references cited herein are incorporated by reference in their entireties.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents, patent publications and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and / or paragraph in which the reference is presented.

[0032] Amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either the one-letter code, or the three-letter code, both in accordance with 37 C.F.R. §1.831 and established usage.

[0033] Except as otherwise indicated, standard methods known to those skilled in the art may be used for cloning genes, amplifying and detecting nucleic acids, and the like. Such techniques are known to those skilled in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 4th Ed. (Cold Spring Harbor, NY, 2012); Ausubel et al. Current Protocols in Molecular Biology (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

[0034] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination.

[0035] Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted.

[0036] To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0037] As used in the description of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0038] Also as used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0039] The term “about,” as used herein when referring to a measurable value such as an amount of polypeptide, dose, time, temperature, enzymatic activity or other biological activity and the like, is meant to encompass variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount.

[0040] As used herein, the transitional phrase “consisting essentially of’ (and grammatical variants) is to be interpreted as encompassing the recited materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term “consisting essentially of’ as used herein should not be interpreted as equivalent to “comprising.”

[0041] The term “consists essentially of’ (and grammatical variants), as applied to a polypeptide or polynucleotide sequence of this invention, means a polypeptide or polynucleotide that consists of both the recited sequence (e.g., SEQ ID NO) and a total of ten or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional amino acids on the N-terminal and / or C-terminal ends of the recited sequence or additional nucleotides on the 5’ and / or 3’ ends of the recited sequence such that the function of the polypeptide or polynucleotide is not materially altered. The total of ten or less additional amino acids or nucleotides includes the total number of additional amino acids or nucleotides on both ends added together. The term “materially altered,” as applied to polypeptides of the invention, refers to an increase or decrease in biological activities / properties (e.g., binding activity or inhibition activity) of at least about 50% or more as compared to the activity of a polypeptide consisting of the recited sequence.

[0042] As used herein, the term “polypeptide” encompasses both peptides and proteins, unless indicated otherwise.

[0043] The terms “polynucleotide”, “nucleic acid,” “nucleic acid molecule,” and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), guide RNA(gRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, genomic DNA, chimeras of RNA and DNA, isolated DNA of any sequence, isolated RNA of any sequence, synthetic DNA of any sequence (e.g., chemically synthesized), synthetic RNA of any sequence (e.g., chemically synthesized), nucleic acid probes and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs or derivatives (e.g, inosine or phosphorothioate nucleotides). Such nucleotides can be used, for example, to prepare nucleic acid molecules that have altered base-pairing abilities or increased resistance to nucleases.

[0044] The term “modulate,” “modulates,” or “modulation” refers to enhancement (e.g., an increase) or inhibition (e.g., a decrease) in the specified level or activity.

[0045] The term “enhance” or “increase” refers to an increase in the specified parameter of at least about 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, twelve-fold, or even fifteen-fold and / or can be expressed in the enhancement and / or increase of a specified level and / or activity of at least about 1%, 5%, 10%, 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more.

[0046] The term “inhibit” or “reduce” or grammatical variations thereof as used herein refers to a decrease or diminishment in the specified level or activity of at least about 1, 5, 10, 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more. In particular embodiments, the inhibition or reduction results in little or essentially no detectible activity (at most, an insignificant amount, e.g., less than about 10% or even 5%).

[0047] The term “contact” or grammatical variations thereof refers to bringing two or more substances in sufficiently close proximity to each other for one to exert a biological effect on the other.

[0048] Grammatical variations of “administer,” “administration,” and “administering” to a subject include any route of introducing or delivering to a subject an agent. Administration can be carried out by any suitable route, including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injections or infusion techniques), and the like. “Concurrent administration,”“administration in combination,” “simultaneous administration,” or “administered simultaneously” as used herein, means that the compounds are administered at the same point in time, overlapping in time, or one following the other. In the latter case, the two compounds are administered at times sufficiently close that the results observed are indistinguishable from those achieved when the compounds are administered at the same point in time. “Systemic administration” refers to the introducing or delivering to a subject an agent via a route which introduces or delivers the agent to extensive areas of the subject’s body (e.g., greater than 50% of the body), for example through entrance into the circulatory or lymph systems. By contrast, “local administration” refers to the introducing or delivery to a subject an agent via a route which introduces or delivers the agent to the area or area immediately adjacent to the point of administration and does not introduce the agent systemically in a therapeutically significant amount. For example, locally administered agents are easily detectable in the local vicinity of the point of administration but are undetectable or detectable at negligible amounts in distal parts of the subject's body. Administration includes self-administration and the administration by another.

[0049] A “subject” may be any vertebrate organism in various embodiments. A subject may be individual to whom an agent is administered, e.g., for experimental, diagnostic, and / or therapeutic purposes or from whom a sample is obtained or on whom a procedure is performed. In some embodiments a subject is a mammal, e.g., a human, non-human primate, lagomorph (e.g., rabbit), or rodent (e.g., mouse, rat). In some embodiments a human subject is a neonate, child, adult, or geriatric subject. In some embodiments a human subject is at least 50, 60, 70, 80, or 90 years old.

[0050] “ Treat,” “treating” and similar terms as used herein in the context of treating a subject refer to providing medical and / or surgical management of a subject. Treatment may include, but is not limited to, administering an agent or composition (e.g., a pharmaceutical composition) to a subject. Treatment is typically undertaken in an effort to alter the course of a disease (which term is used to indicate any disease, disorder, syndrome, or undesirable condition warranting or potentially warranting therapy) in a manner beneficial to the subject. The effect of treatment may include reversing, alleviating, reducing severity of, delaying the onset of, curing, inhibiting the progression of, and / or reducing the likelihood of occurrence or recurrence of the disease or one or more symptoms or manifestations of the disease. A therapeutic agent may be administered to asubject who has a disease or is at increased risk of developing a disease relative to a member of the general population. In some embodiments a therapeutic agent may be administered to a subject who has had a disease but no longer shows evidence of the disease. The agent may be administered e.g., to reduce the likelihood of recurrence of evident disease. A therapeutic agent may be administered prophylactically, i.e., before development of any symptom or manifestation of a disease. “Prophylactic treatment” refers to providing medical and / or surgical management to a subject who has not developed a disease or does not show evidence of a disease in order, e.g., to reduce the likelihood that the disease will occur, delay the onset of the disease, or to reduce the severity of the disease should it occur. The subject may have been identified as being at risk of developing the disease (e.g., at increased risk relative to the general population or as having a risk factor that increases the likelihood of developing the disease.

[0051] When any variable (e.g., Ri) occurs more than one time in any constituent or formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with one or more Ri moieties, then Ri at each occurrence is selected independently from the Markush group recited for Ri. Also, combinations of substituents and / or variables are permissible, but only if such combinations result in stable compounds within a designated atom’s normal valency.

[0052] The present invention relates to mRNA display libraries and methods of preparing mRNA display libraries that comprise cyclized peptides wherein each of the cyclized peptides comprises dehydroalanine (dAla or Dha). The present invention utilizes the mRNA display libraries to screen for cyclic peptides that bind target substances. In some embodiments, the mRNA display libraries are useful to screen for cyclic peptides that covalently bind the target substance.

[0053] An amino acid, as described herein, includes both naturally occurring and / or non- naturally occurring amino acids. Naturally occurring amino acids include those encoded by the genetic code and those amino acids that are later modified, for example, carboxyglutamate and hydroxyproline. Amino acid analogs comprise a structure similar to a naturally occurring amino acid but have modified R groups, such as norleucine or norvaline, or modified peptide backbones. Such non-canonical amino acids may include amino acid analogs and amino acid mimetics that function similar to naturally occurring amino acids. Exemplary amino acids, analogs and mimetics include, without limitation, dehydroalanine (Dha), P-alanine (P-Ala), N-a-methyl -alanine (Me-Ala), aminobutyric acid (Abu), a-aminobutyric acid (y-Abu), aminohexanoic acid (s-Ahx), aminoisobutyric acid (Aib), aminomethylpyrrole carboxylic acid, aminopiperidinecarboxylic acid, aminoserine (Ams), aminotetrahydropyran-4-carboxylic acid, arginine N-methoxy-N-methyl amide, P-aspartic acid (P-Asp), azetidine carboxylic acid, 3-(2- benzothiazolyl)alanine, a-tert-butylglycine, 2-amino-5-ureido-n-valeric acid (citrulline, Cit), P- Cyclohexylalanine (Cha), acetamidomethyl-cysteine, diaminobutanoic acid (Dab), diaminopropionic acid (Dpr), dihydroxyphenylalanine (DOPA), dimethylthiazolidine (DMTA), y-Glutamic acid (y-Glu), homoserine (Hse), hydroxyproline (Hyp), isoleucine N-methoxy-N- methyl amide, methyl-isoleucine (Melle), isonipecotic acid (Isn), methyl-leucine (MeLeu), methyl-lysine, dimethyl-lysine, trimethyl-lysine, methanoproline, methionine-sulfoxide (Met(O)), methionine-sulfone (Met(O2)), norleucine (Nle), methyl-norleucine (Me-Nle), norvaline (Nva), ornithine (Om), para-aminobenzoic acid (PABA), penicillamine (Pen), methylphenylalanine (MePhe), 4-Chlorophenylalanine (Phe(4-Cl)), 4-fluorophenylalanine (Phe(4-F)), 4-nitrophenylalanine (Phe(4-NO2)), 4-cyanophenylalanine ((Phe(4-CN)), phenylglycine (Phg), piperidinylalanine, piperidinylglycine, 3,4-dehydroproline, pyrrolidinylalanine, sarcosine (Sar), selenocysteine (Sec), U-Benzyl-phosphoserine, 4-amino-3- hydroxy-6-methylheptanoic acid (Sta), 4-amino-5-cyclohexyl-3-hydroxypentanoic acid (ACHPA), 4-amino-3-hydroxy-5-phenylpentanoic acid (AHPPA), 1, 2,3,4, -tetrahydro- isoquinoline-3-carboxylic acid (Tic), tetrahydropyranglycine, thienylalanine (Thi), U-Benzyl- phosphotyrosine, O-Phosphotyrosine, methoxytyrosine, ethoxytyrosine, O-(bis-dimethylamino- phosphono)-tyrosine, tyrosine sulfate tetrabutylamine, methyl-valine (MeVal), 1-amino-l- cyclohexane carboxylic acid (Acx), aminovaleric acid, beta-cyclopropyl-alanine (Cpa), propargylglycine (Prg), allylglycine (Alg), 2-amino-2-cyclohexyl-propanoic acid (2-Cha), tertbutylglycine (Tbg), vinylglycine (Vg), 1 -amino- 1 -cyclopropane carboxylic acid (Acp), 1- amino-1 -cyclopentane carboxylic acid (Acpe), alkylated 3-mercaptopropionic acid, and 1-amino- 1-cyclobutane carboxylic acid (Acb). In embodiments, the amino acid is a phenylalanine derivative or other electrophilic non-canonical amino acid. In embodiments, the phenylalanine derivative is / wa-functionalized, which may be, for example, / ?-CN-pyridylalanine; / ?-CN-Phe;?-N.3-PHe, / ?-CCH-Phe, / 2-biphenyl-Ala, / ?-NH2-Phe; / ?-CH-Phe; / 2-iPr-Phe; / ?-F-Phe; or p- SChbsTHPhe. D and L isomers of the naturally occurring or non-naturally occurring amino acids are contemplated within the term amino acid. In certain instances, a charged amino acid can besubstituted with another charged amino acid, a neutral amino acid with another neutral amino acid. In some instances, an amino acid can be substituted with an amino acid of a different charge to modify the interactions of the peptide targeting ligand. Molecules of the invention, including peptides and nucleic acid / peptide conjugates, may comprise a cap on the N-terminus of an amino acid, including formyl, pyroglutamyl, acetyl, fatty acids, urea, alkylamine and sulfonamide.

[0054] In some embodiments, an mRNA display library is provided comprising a plurality of mRNA-linker-macrocyclic peptide molecules comprisingAcTyr-[amino acid]m-[PhSec]-[amino acid]n-[Cys]-[linker]-[mRNA]; wherein m and n are each an integer from 0 to 30; and wherein each macrocyclic peptide comprises a thioether linkage and is according to formula I:wherein R comprises [linker]-[mRNA], In some embodiments, m+n is between 7 and 15. In some embodiments, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, n is i, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0055] In some embodiments, an mRNA display library is provided comprising a plurality of mRNA-linker-macrocyclic peptide molecules comprising:AcTyr-[amino acid]m-[dAla]-[amino acid]n-[linker]-[mRNA]; wherein m and n are each an integer from 0 to 30 and wherein each macrocyclic peptide comprises a thioether linkage and is according to formula II:wherein R comprises [linker]-[mRNA], In some embodiments, m+n is between 7 and 15. In some embodiments, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0056] In some embodiments, the cyclized peptide can be further oxidized to comprise a pyridine-thiazole bridge. Substitutions of the amino acids can be made, and may be according to desired function, including, for example, charge, aromaticity, and R group functionality, as detailed elsewhere herein.

[0057] The mRNA display libraries can be used to identify one or more targeting ligands, e.g., cyclic peptides, useful for binding a target of interest. The targeting ligand can, in some embodiments, be provided as a cyclized molecule, which can be according to formula I or formula II.

[0058] Another embodiment of the invention provides a method of preparing an mRNA display library comprising a plurality of mRNA-linker-macrocyclic peptide molecules, the method comprising the steps of providing a randomized library of mRNA molecules comprising a plurality of molecules according to a formula according to one of (Illa) -(Illf) :AUG-(NiWW)m-TAG-(NiWW)n -Cys-[linker] (Illa) (SEQ ID NO:1) AUG-(NiN2K)m-TAG-(NiN2K)n -Cys-[linker] (nib) (SEQ ID NO:2) AUG-Cys-(NiWW)m-TAG-(NiWW)n-Cys-[linker] (IIIc) (SEQ ID NO:3) AUG-Cys-(NiN2K)m-TAG-(NiN2K)n-Cys-[linker] (Illd) (SEQ ID NO:4) AUG-Cys-(NiWW)m-TAG-(NiWW)n-AUG-[linker] (Ille) (SEQ ID NO:5) AUG-Cys-(NiN2K)m-TAG-(NiN2K)n-AUG-[linker] (Illf) (SEQ ID NO:6)wherein Ni andN2 are independently any nucleotide, each W is independently A or U, and each K is independently G or U, and m and n are each an integer from 0 to 30; performing in vitro translation of each of the plurality of molecules in the mRNA library with a promiscuous orthogonal aminoacyl-tRNA synthetase (ORS) in the presence of phenylselenocysteine (PhSec) to thereby produce a plurality of linear molecules, with the ORS incorporating a PhSec at the TAG or other non-competitive codon; cyclizing each of the plurality of linear molecules; forming a dehydroalanine via oxidative elimination at the PhSec in each of the plurality of cyclized molecules; thereby forming an mRNA library of a plurality of mRNA-linker- macrocyclic peptide molecules comprising a dehydroalanine.

[0059] ORSs can incorporate Dha at other codons as long as they are "vacated" in the translation system. Accordingly, in some embodiments, one or more Dhas may be incorporated using one or more mutually orthogonal synthetases. Mutually orthogonal synthetases refers to two non-native synthetases that do not crosstalk with one another (i.e., the ORSs do not accept each other’s amino acids or tRNAs).

[0060] In some embodiments, one or more mRNA libraries of linear molecules according to: Cys-[amino acid]m-[PhSec]-[amino acid]n-[Cys]-[linker]-[mRNA], Cys-[amino acid]m-[PhSec]-[amino acid]n-[Cys]-[linker]-[mRNA], or ClAcTyr-[amino acid]m-[PhSec]-[amino acid]n-[Cys]-[linker]-[mRNA] are provided which can be cyclized according to methods described herein, and subsequently treated with H2O2 to generate a dAla (Dha) at the PhSec position.

[0061] In some embodiments, m+n is between 7 and 15. In some embodiments, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0062] In some embodiments, the method comprises performing in vitro translation with chloroacetyl-Tyr (ClAcTyr) initiator tRNA to thereby produce a plurality of linear ClAcTyr- [amino acid]m-[PhSec]-[arnino acid]n-[Cys]-[linker]-[mRNA] and wherein cyclization occurs via formation of a thioether linkage between AcTyr and cysteine.

[0063] In some embodiments, the method comprises performing in vitro translation of a plurality of molecules according to formula IIIc and / or Illd to thereby produce a plurality of linear molecules AUG-Cys-[amino acid]m-[PhSec]-[amino acid]n-[Cys]-[linker]-[mRNA], and the step of cyclizing comprises treating the plurality of linear molecules with a dihaloalkane or atrihaloalkane thereby cyclizing via a thioether linkage at two or more cysteines in each of the plurality of linear molecules. Example dihaloalkanes include, but are not limited to, a, a’ dibromoxylenes (DBX), e.g., a, a’ dibromo-o-oxylene, a, a’ dibromo- / ?-xylene, a, a’ dibromo-zw- xylene. Example trihaloalkanes include l,3,5-tris(bromomethyl)-2,4,6-trimethylbenzene and l,3,5-tris(chloromethyl)-2,4,6-trimethylbenzene. Methods of cyclizing cysteine residues with dihaloalkanes and trihaloalkanes are described, for example, in Timmerman etal., ChemBioChem, 2005, 6, 821-824; doi: 10.1002 / cbic.200400374, incorporated herein by reference in its entirety.

[0064] In some embodiments, the method comprises performing in vitro translation of a plurality of molecules according to Formula Ille and / or Formula Illf with a p- or zzz- cyanopyridylalanine-loaded elongation tRNA (e.g., Met elongation tRNA), to thereby produce a plurality of linear molecules according to AUG-Cys-[amino acid]m-[PhSec]-[amino acid]n- CNpyrA-[linker]-[mRNA], and optionally cyclizing comprises treating the plurality of linear molecules with peptide deformylase (PDF) and Met aminopeptidase (MAP) to remove N- terminal fMet, thereby cyclizing via thiazoline formation (pyridine-thiazoline macrocyclization). Exemplary methods are described in Iskander etal., J. Am. Chem. Soc. 2023, 145, 1512-1517, incorporated herein by reference in its entirety.

[0065] In some embodiments of the method, removing the N-terminal fMet is performed by one or more proteases. One or more proteases can comprise peptide deformylase and / or methionine aminopeptidase. Other proteases can be utilized, with proteases and their recognition sequences known in the art, see, e.g., Barrett A., et al. Handbook of proteolytic enzymes Academic Press (1998); see also the PeptideCutter program available at Expasy, identifying enzymes and their cleavage sites within peptides. In some embodiments, a recognition sequence may be included between the N-terminal formyl methionine and the cysteine to provide a cleavage site for a particular protease. A linker also may be provided between the formylmethionine and the recognition sequence when a recognition sequence is present.

[0066] In some embodiments, the method further comprises the step of performing in vitro translation with chloroacetyl-Tyr (ClAcTyr) initiator tRNA to thereby produce a plurality of linear ClAcTyr-[amino acid]m-[PhSec]-[arnino acid]n-[Cys] [linker]-[mRNA] and wherein cyclization occurs via formation of a thioether linkage between AcTyr and cysteine.

[0067] In some embodiments, the linker according to formula Illa, formula Illb, formula IIIc, formula Illd, formula Ille, or formula Illf is a Gly-Ser linker which may comprise from two to six repeats. In one aspect, the Gly-Ser linker is GSGSGS (SEQ ID NO: 121).

[0068] In some embodiments, (NiWW) or (N1N2K) of any one of Formula Illa-Formula Illf does not encode a cysteine. In some embodiments, at least one of (NiWW) or (N1N2K) of any one of Formula Illa-Formula Illf encodes for a cysteine. In some embodiments, cyclizing comprises a cysteine-based cyclization. Crosslinking reagents (such as dihaloalkanes or trihaloalkanes) can react with a cysteine residue encoded at (NiWW) or (N1N2K) and the cysteine residue at the C-terminal of any one of Formula Illa-IIIf form thioether linked macrocyclic peptides. In an embodiment, the N-terminal and C-terminal cysteine residues according to formula IIIc or formula Illd are crosslinked to form thioether linked macrocyclic peptides. In some embodiments, the cyclizing can comprise dibromoxylene mediated thioether cyclization.

[0069] In some embodiments, a method comprising the step of forming a dehydroalanine comprises treating each of the plurality of molecules with H2O2 to thereby generate the dehydroalanine at the PhSec position. Other methods of revealing Dhas through oxidative elimination can be utilized. Incorporation of the PhSec can be performed by a promiscuous orthogonal ami noacyl -tRN A synthetase (ORS). In some embodiments, the ORS is evolved to incorporate phenylselenocysteine. In some embodiments, the ORS is phenylselenocysteine tRNA synthetase (PhSec-RS).

[0070] Methods of making the mRNA display library can further comprise the step of reacting one or more encoded cysteines with an alkylating agent to incorporate a non-canonical amino acid side chain. In some embodiments, the alkylating agent is an alkyl halide. Alkylating agents for the addition of side chains to cysteine are known in the art, see, e.g., Deming, Chem. Rev. 2016, 116, 3, 786-808, doi: 10.1021 / acs.chemrev.5b00292.

[0071] In some embodiments, a method for conjugation comprises reacting exogenous cysteine-containing chemical reagents with linear peptides comprising non-canonical amino acid, e.g., >-cyanopyridylalanine for conjugation to mRNA display libraries.

[0072] Methods for screening for a targeting cyclic peptide, also referred to herein as a targeting ligand, are provided. Methods comprise producing an mRNA display library according to the methods described herein or providing an mRNA display library as described herein,bringing the mRNA display library molecules into contact with the target substance, incubating the library molecules and target substance; and selecting cyclic peptides that bind the target substance.

[0073] Exemplary methods for screening for a targeting ligand as provided herein are described in the working examples for an example target, melanoma-associated antigen 4 (MAGE-A4). An additional exemplary method for screening includes the example target calcium and integrin- binding protein 1 (CIB1). In some embodiments, the targeting ligand is a MAGE-A4 targeting ligand peptide. In some embodiments, the targeting ligand is provided as a cyclized molecule. The cyclized targeting ligand may comprise a bond between an N-terminal AcTyr and a C- terminal cysteine. In an aspect, the cyclized targeting ligand comprises a thioether linkage bridge between the N-terminal AcTyr and the C-terminal cysteine. In some embodiments, the method can further comprise denaturing the target substance subsequent to incubating the library molecules and target substance. In an aspect, the denaturing comprises contacting the target with guanidine or another denaturing agent. Without being bound by theory, the step of denaturation (e.g., guanidine denaturation) prevents enrichment of non-covalent polycycles and / or to bias an mRNA display selection toward covalent ligands. In some embodiments, the method further comprises multiple rounds of selecting cyclic peptides that bind the target substance and incubating the selected cyclic peptides and the target substance, optionally with a step of denaturation. In some embodiments, the cyclic peptide covalently binds the target substance.

[0074] In some embodiments, a targeting ligand identified by the methods disclosed herein can be used for treatment of a disease or disorder. Accordingly, the targeting ligands identified by the methods provided herein can further comprise a cargo associated with the ligand. Accordingly, in some embodiments, the cargo can be associated at the N-terminus or C-terminus of the targeting ligand, or on a side chain of an amino acid, for example a cysteine, for example conjugated via covalent bonding. In an aspect, the cargo is associated with the targeting ligand via a cleavable linker, the linker covalently bonded to the targeting ligand at the N-terminus or C-terminus or side chain of an amino acid. The cargo can comprise a detectable label, biologically active agent, imaging agent, and / or therapeutic agent. In embodiments, the cargo comprises a nucleic acid, a protein, a complex of a nucleic acid and a protein, a carbohydrate, a lipid, or a small molecule. The cargo can be selected for the treatment of a disease or disorder, for the detection of a disease or disorder, or for monitoring of a disease or disorder.

[0075] A further aspect of the invention relates to kits for use in the methods of the invention. Kits may comprise an mRNA display library comprising a plurality of mRNA display molecules, each mRNA display molecule comprising one or more amber codons, a promiscuous orthogonal aminoacyl-tRNA synthetase (ORS), one or more non-canonical amino acids, or a combination thereof. Kits may comprise H2O2 for generating DhAla at the PhSec position, and / or guanidine or other denaturing agent for denaturation and recovery of covalent ligands identified in screening methods. Kits may comprise one or more targeting ligands; and / or instructions for use, in any combination. Kits can further comprise cargo, carriers, buffers, containers, devices for administration of the components, and the like. The kit can further comprise labels and / or instructions for assay selection and execution. Such labeling and / or instructions can include, for example, information concerning the amount, and methods of administration, detection and quantification for assays detailed herein.

[0076] Having described the present invention, the same will be explained in greater detail in the following examples, which are included herein for illustration purposes only, and which are not intended to be limiting to the invention.EXAMPLESExample 1.

[0077] Dehydroalanines (Dhas) are biologically occurring electrophiles2930that have previously been incorporated in mRNA display as handles for peptide cyclization, side chain functionalization, or heterocycle formation.17’31,32Dhas might also serve as competent cysteine electrophiles for covalent inhibitor discovery in mRNA display. Dhas can be accessed through protected precursors like phenylselenocysteine (PhSec), which can be revealed after peptide cyclization to allow compatibility with virtually all cysteine-based cyclization methods that are common in mRNA display. Thus, it was envisioned there may be a route to cyclic electrophilic mRNA display libraries involving 1) translation of PhSec with an orthogonal aminoacyl-tRNA synthetase (ORS),332) cyclization of libraries through the robust Flexizyme-mediated thioether linkage,34,35and 3) post-translational reveal of Dhas through oxidative elimination (FIG. 1A; FIG. 5, FIGS. 6A-6B). Importantly, this strategy allows positional variation and overrepresentation of the electrophile through strategic design of the mRNA library (FIG. IB).which contrasts prior phage display approaches where the chemistry restricts electrophile incorporation to a single position, immediately flanked by cysteines. Lastly, denaturing the selection targets with guanidine after incubation with the electrophilic libraries was utilized to encourage recovery of covalent ligands, as previously reported in phage display. Herein, the realization of this selection strategy against two model targets, calcium and integrin-binding protein 1 (CIB1) and melanoma-associated antigen 4 (MAGE-A4) is described.

[0078] First, the compatibility of mRNA display with target denaturation conditions using CIB1 was established. CIB1 is a model protein with two surface-exposed cysteines that is highly selectable in mRNA display.365 M guanidine had minimal effects on cDNA amplification (FIGS. 7A-7B). Additionally, guanidine exposure was effective in ablating the recovery of a previously selected non-covalent inhibitor of CIB1 in mRNA display (FIGS. 7A-7B).37In the first selection, a 9-mer library was designed with a cysteine-deficient NWW randomization to prevent potential consumption of Dhas in smaller macrocycles or poly cycles. Notably, this randomization also removes several amino acid residues and the TAG codon, resulting in a reduced theoretical library diversity of ~2E8 molecules. The TAG codon was scanned across all positions of the randomized library (FIG. IB), resulting in library members each with one TAG codon and thus Dha.

[0079] Five selection rounds were conducted with guanidine denaturation against CIB1 and results analyzed by next generation sequencing (NGS). Significant family convergence was observed in later rounds, with one sequence comprising >20% of NGS reads at round four and >80% of NGS reads by round five (Table 1, FIG. 2A).Table 1. Top families from round 4 of Dha selections with guanidine denaturation against CIB1.B=TAG

[0080] This peptide was synthesized as CCP1 ([M+H]=1408.6354 m / z) and its ability to covalently modify CIB1 tested via MALDI-MS.CCP1 (SEQ ID NO: 14)

[0081] Pleasingly, a dose-dependent shift in CIB1 mass was observed that roughly corresponds to CCP1 addition (FIG. 2B). CIB1 modification was not observed with either the non-specific electrophile, iodoacetamide (FIG. 8), or when the Dha of CCP1 was mutated to non-reactive L- or D-Ala. These results suggest specificity toward CIB1 and implicate the Dha in covalent modification. Trypsin-MALDI-MS of the CIB1-CCP1 complex suggested modification of a protein fragment containing Cysl34 (FIG. 8), which was confirmed via MALDI-MS assay of CCP1 against the CIB1 C134A mutant (FIG. 2B).

[0082] Next, it was assessed whether CCP1 can inhibit interactions in the key C-terminal binding pocket of CIB138To test this activity, a previously developed TR-FRET assay between CIB1 and a known phage display peptide was used.39In this assay, CCP1 showed modest inhibitory activity after a 1 hour pre-incubation with CIB1, with an ICso of ~0.5 pM (Table 2, FIG. 2C). In contrast, non-covalent cyclic peptide inhibitors37UNC 10245131 and UNC 10245231 displayed low nanomolar ICsos at this time point.Table 2. Calculated ICsos from TR-FRET in FIG. 2C.

[0083] The potency of CCP1 increased significantly over time — a hallmark of covalent inhibitors — with the IC50 improving almost ten-fold after 24 hours. In contrast, the non-covalent inhibitors largely sustained potency on longer incubation. L- and D-Ala mutants of CCP1 did not display any inhibition in this assay, and inhibitory activity of CCP1 was ablated against the CIB1 C134A mutant (FIG. 9). Notably, Cl 34 is located ~7A from the surface of the phage peptide binding pocket (FIGS. 10A-10B).39Thus, the lack of inhibition against C134A suggests that CCP1 may induce a conformational change in CIB1 after covalent modification that prevents access to the phage peptide binding site, or else that CCP1 is a much weaker binder without its electrophile. The ability of CCP1 to label CIB1 when spiked into cellular lysates (MDA-MB- 453) was also evaluated; CCP1 only labeled CIB1 with no additional off-target modifications, demonstrating its selectivity toward CIB1 in a complex environment (FIG. 2D; FIGS. 11A- 11B)

[0084] To demonstrate the robustness of this covalent selection strategy, a second selection against another protein target, MAGE-A4, was performed. In light of the CIB1 selection, changes to the protocol were made for the MAGE-A4 campaign. Namely, incubation times in the first two rounds of selection were extended because these conditions appeared to improve reaction between CCP1 and CIB1 (FIG. 12), and an NNK library randomization which includes all 20 amino acids and the TAG codon was also employed, because guanidine denaturation appeared sufficient to prevent enrichment of non-covalent polycycles. Additionally, it was hypothesized that the lower NWW library diversity might be partly responsible for the moderate activity of CCP1. With these adjustments, parallel selections with (+G) or without (-G) guanidine denaturation were performed against MAGE-A4, and after four rounds NGS data revealed convergence upon several families (Table 3). The top families in each selection differed significantly, and enrichment of any multi-Cys families in the +G NGS data was not observed. Top families in the -G selection appeared similar to a previous non-covalent selection againstMAGE-A4,24demonstrating the ability of guanidine to vastly change the emergent enrichment landscape.Table 3. Top families from +G (round 4) or -G (round 3) Dha selections.B=TAG

[0085] Three representative peptides, MCP1 ([M+H] = 1512.7317 m / z), MCP2 ([M+H] = 1444.7518 m / z), and MCP3 ([M+H] = 1381.6358 m / z), from the +G selections were synthesized, all of which are distinct from previously selected MAGE-A4 inhibitors.MCP3 (SEQ ID NO: 55)

[0086] All peptides modified MAGE-A4 by MALDI-MS, with MCP1 and MCP2 performing more efficiently than MCP3 (FIG. 3A). MCP3 also seemed to modify a second site over an extended time, though the emergent peak is several hundred Daltons heavier than MCP3. To crudely gauge peptide selectivity, testing if any of the MCPs modify CIB1 was performed, and only significant labeling by MCP3 was observed (FIGS. 13A-13B). The more selective and complete labeling efficiencies of MCP1 and MCP2 correlate well with their increased enrichment over MCP3 in the NGS data. L- and D-Ala mutants of MCP1 and MCP2 did not modify MAGE-A4 by MALDI-MS, confirming their modifications as Dha-dependent.

[0087] MAGE-A4 has four cysteines: two within the MAGE homology domain (MHD) and two in the structurally undefined N-terminal region.40Initial testing if any of the covalent peptides modify the MHD alone was performed, but substantial modification by MALDI-MS was not observed (FIGS. 13A-13B). GluC-MALDI-MS digests of MAGE- A4 adducts with MCP1 or MCP2 suggested that the peptides modify a protein fragment containing Cys80 (FIG. 14), which was confirmed when MCP1 and MCP2 failed to label a MAGE-A4 C80A mutant (FIG. 3A). Some modification of C80A by MCP3 was still observed, further suggesting that MCP3 may be capable of modifying two sites over time.

[0088] Despite covalently modifying the N-terminal region, it remains possible that the MCPs are still able to block access to the MHD. Thus, it was tested if the MCPs could inhibit theassociation of a previously selected non-covalent MHD binder, MTP-1, in TR-FRET.24Indeed, MCP1 and MCP2 exhibited single digit nanomolar IC50S of 9.0 and 6.4 nM, respectively, after 1 hour incubation times (FIG. 3C, Table 4).Table 4. Calculated ICsos from TR-FRET curves in FIG. 3B.

[0089] As expected, MCP3 exhibited much weaker inhibitory activity. The L- and D- Ala mutants of MCP1 and MCP2 were much less potent than their Dha-containing counterparts. However, the D-Ala mutants of both peptides were more potent than L-Ala, suggesting that D- Ala stereochemistry may be adopted upon nucleophilic attack of the Dha by C80. A small yet significant increase in potency over time was observed only for MCP1 and MCP2, which could perhaps be further differentiated in an assay with sub-nanomolar sensitivity. In the presence of MAGE-A4 C80A, ICsos of MCP1 and MCP2 fell in between those of their L- and D-Ala mutants, while MCP3 activity was ablated (FIGS. 3B). With validated TR-FRET inhibition,lastly it was tested whether the MCPs could disrupt MAGE-A4 engagement with one of its E3 ligase-binding partners, RADI 8, in a cellular context. In a co-immunoprecipitation of the RAD 18-MAGE-A4 complex, all MCPs were able to disrupt MAGE-A4 pulldown, with MCP3 being slightly less effective than MCP1 and MCP2 (FIG. 3C). In contrast, none of the peptides disrupted the interaction between E2 ligase RAD6 and RAD 18, showcasing some selectivity for MAGE-A4.

[0090] In summary, multiple library diversification techniques were combined — including ORSs, post-translational chemistry, and Flexizymes — to incorporate electrophiles into macrocyclic mRNA display libraries for the selection of covalent binders. This strategy is compatible with both co- and post-translational cysteine-based cyclizations, including chemical cyclizations such as dibromoxylene. Dhas were established as electrophiles for covalent inhibitor discovery and the ability of guanidine to bias an mRNA display selection toward covalent ligands was demonstrated. Additionally, this method can identify covalent inhibitors that exhibit selectivity in complex environments and can disrupt known protein-protein interactions.

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Structures of Two Melanoma-Associated Antigens Suggest Allosteric Regulation of Effector Binding. PLoS One 2016, 77 (2). doi.org / 10.1371 / journal.pone.0148762.Materials and MethodsMaterials

[0092] Reagents for molecular biology were purchased from New England BioLabs (NEB),Thermo Fisher Scientific, and Fisher Scientific. Specifically, PURExpress kits (E6850Z - custom for mRNA display), T7 RNA Polymerase (M0251L), Q5 DNA Polymerase (M0491L) were purchased from New England Biolabs. T4 RNA ligase I (PR-M1051), RQ1 DNase (PR-M6101), and M-MLV ReverseTranscriptase, RNase H Minus, Point Mutant (PRM3683) were purchased from Promega through Fisher Scientific. Anti-HA magnetic beads (88836), HA synthetic peptide (26184), and M-280 Streptavidin Dynabeads (11205D) were purchased from Thermo Fisher Scientific. Bulk solvents were purchased from Fisher Scientific. Fmoc-amino acids and materials for peptide synthesis were purchased from Chemlmpex unless otherwise mentioned. DNA gene fragments and all purchased plasmids were purchased from Twist Bioscience. Primers were purchased from Integrated DNA Technologies. Next Generation Sequencing (NGS) was performed by Azenta using their Amplicon-EZ (150-500 bp) service. NMR spectra were recorded at RT on a Varian Inova 400 NMR (400 MHz). Chemical shifts are reported in 8 (ppm) relative units to residual solvent peaks CD3OD (3.31 ppm (XH), 49.00 ppm (13C)). Splitting patterns are assigned as s (singlet), d (doublet), t (triplet), q (quartet), quint (quintet), multiplet (m), and dd (doublet of doublets).Instrumentation

[0093] Preparatory high pressure liquid chromatography (HPLC) was performed with an Agilent G7161 A 1260 Prep Bin Pump and G1364E 1260 FC-PS with a G7115A diode array detector set to 220 and 254nm and over a 250 x 21.2 mm Phenomenex LUNA 10 pm C18 100 A AXIA semi -preparatory column at a flow rate of 25 mL / min. For purification a two solvent system (Solvent A: 0.1% Trifluoracetic acid and water; Solvent B: 0.1% Trifluoracetic acid and acetonitrile) was used on a gradient from A to B using the following gradient:Table 5A. Time: Solvent GradientWhere X = 45 - 60

[0094] Liquid chromatography mass spectrometry (LCMS) measurements were recorded using an Agilent 6520 Accurate-Mass Q-TOF, fitted with an ESI ion source. Samples were run in positive ion polarity and high-resolution mode, with 350 °C gas temperature and 250 Vfragmentor voltage. Separation was achieved on either a 50 x 2.1 mm Kinetex 2.6 pm Cl 8 100 A column or a 100 x 2 mm Gemini 5 pm NX-C18 100 A column. Predicted masses were identified within ± 20 ppm. The LC portion was performed using a two solvent system consisting of 0.1% formic acid in water (Solvent A) and 0.1% formic acid in acetonitrile (Solvent B) at 0.5 mL / min from A to B using the following gradient:Table 5B. Time:Solvent Gradient

[0095] MALDI-TOF analysis was performed on an Applied Biosystems (AB) Sciex TOF / TOF 5800 system in reflector positive mode. An AB ViiA 7 Real-Time PCR System (qPCR) was used for amplification and quantitative analysis after mRNA display experiments. TR-FRET signals were measured using an EnVision 2105 multimode plate reader.Software

[0096] Software used for data analysis comprised of Agilent Masshunter Qualitative Analysis Navigator B.08.00 (LCMS analysis), Qiagen CLC Main Workbench (NGS analysis), AB Data Explorer v4.5 (MALDI-TOF analysis), Graphpad Prism v9.4.1, and Microsoft office suite. Adobe Illustrator was used to assemble figures. In-house python scripts were used to analyze NGS data.Protein Expression and PurificationPhenylselenocysteine tRNA synthetase (PhSec-RS)

[0097] pMCSG7 PhSec-RS was transformed into BL21 cells and a 5mL saturated culture was used to inoculate IL of Luria-Bertani (LB) medium supplemented with 100 pg / mL ampicillin. The culture was incubated at 37 °C and shaken at 220 rpm until reaching an O.D. of 0.55, at which point the medium was supplemented with 0.2 mM IPTG. The culture was grown at 37 °C for 3 hours, then pelleted and stored at -80 °C until purification. Pellets were resuspended in 30 mL of lysis buffer (50 mM HEPES pH 7.9, 500 mM NaCl, 10 mM imidazole, 10% glycerol,0.1% Triton) supplemented with 1 .5 mM PMSF, 0.25 mg / mL lysozyme, and 80 units of DNAse. The cells were lysed by sonication and the soluble protein was recovered by pelleting the cell debris by centrifugation at 4 °C, 15,000 rpm for 20 mins. The supernatant was filtered through a 0.45 pm sterile syringe filter, loaded onto a 5 mb HisTrap (Ni2+) IMAC column, and washed with ~5 column volumes (CV) of wash buffer (20 mM HEPES pH 7.6, 300 mM NaCl, 30 mM imidazole). Protein was eluted with elution buffer (20 mM HEPES pH 7.6, 300 mM NaCl, 250 mM imidazole) over a stepwise gradient 0-100%. Fractions containing purified protein were collected and concentrated to 2.5 mL using a Centricon (10,000 Da MWCO) concentrator (EMD Millipore®). The concentrated protein was then buffer exchanged using a PD-10 column (GE Healthcare Life Sciences®) into storage buffer (20mM Tris pH 8.0, lOOmM NaCl, 7mM MgCh, 5 mM P-mercaptoethanol, 10% glycerol) and stored at -80 °C. 1 L culture yielded ~30 mg of protein.General Protocol for Biotinylated Proteins (CIB1 WT / C 134A, MAGE-A4 (WT / C80A)

[0098] Expressions of biotinylated wild-type CIB1, CIB1 point mutant C134A, wild-type MAGE-A4, and MAGE-A4 point mutant C80A were performed as previously described.1,2Modified pET28a vectors with each protein sequence and an N-terminal hexahistidine tag and Avi tag sequence (GLNDIFEAQKIEWHE; SEQ ID NO:56) were purchased from Twist Biosciences. Each construct was co-transformed into Rosetta2 BL21(DE3)pLysS competent cells (MilliporeSigma) along with an expression plasmid for E. coli biotin ligase, BirA (pET21a-BirA was a gift from Alice Ting (Addgene plasmid # 20857)).3A I L culture was grown to mid log phase at 37 °C at which time the temperature was lowered to 18 °C and protein expression was induced by addition of 0.5 mM IPTG. D-biotin was added to the culture at this time to a final concentration of 50 pM. Expression was allowed to continue overnight. Cells were harvested by centrifugation and pellets were stored at -80°C.

[0099] Biotinylated proteins were purified by resuspending thawed cell pellets in 30 mL of lysis buffer (50 mM sodium phosphate pH 7.2, 500 mM NaCl, 30 mM imidazole, IX EDTA free protease inhibitor cocktail (Roche) per liter of culture. Cells were lysed on ice by sonication with a Branson Digital 450 Sonifier (Branson Ultrasonics) at 40% amplitude for 12 cycles with each cycle consisting of a 20 second pulse followed by a 40 second rest. The cell lysate was clarified by centrifugation and loaded onto a HisTrap FF column (Cytiva) that had been preequilibrated with 10 column volumes of binding buffer (50 mM sodium phosphate pH 7.2, 500 mM NaCl, 30mM imidazole) using an AKTA FPLC (Cytiva). The column was washed with 15 column volumes of binding buffer and protein was eluted in a linear gradient to 100% elution buffer (50 mM sodium phosphate pH 7.2, 500 mM NaCl, 500 mM imidazole) over 20 column volumes. Peak fractions containing the desired protein were pooled and concentrated to 2 mL in Ami con Ultra-15 concentrators 10,000 MWCO (MilliporeSigma). Biotinylated protein was isolated by passing concentrated protein over a 2 mL monomeric avidin column by gravity flow following manufacturer’s recommendations (Thermo Scientific). Bound protein was eluted in 12 mL PBS containing 2 mM D-biotin. Each biotinylated protein was concentrated in Amicon Ultra- 15 concentrators 10,000 MWCO and further purified by loading concentrated protein onto a HiLoad 26 / 60 Superdex 200 prep grade column (Cytiva) that had been preequilibrated with 1.2 column volumes of sizing buffer (25 mM Tris pH 7.5, 250 mM NaCl, 2mM DTT, 5% glycerol) using an ATKA FPLC (Cytiva). Protein was eluted isocratically in sizing buffer over 1.3 column volumes at a flow rate of 2 ml / min collecting 3 mL fractions. Peak fractions were analyzed for purity by SDS-PAGE and those containing pure protein were pooled and concentrated to less than 2 mL using Amicon Ultra- 15 concentrators 10,000 MWCO. Concentrated proteins were aliquoted and stored at -80 °C.MAGE-A4MHD

[0100] Expression and purification of the MHD of MAGE- A4 was performed as previously described.2Briefly, pMCSG47 A4-MHD was transformed into BL21 (DE3) cells. Cells supplemented with 100 pg / mL ampicillin were grown until the Aeoo reached 0.6 - 0.8, after which protein expression was induced with 0.2 mM IPTG and allowed to proceed at 18 °C overnight. Cells were pelleted, lysed, and soluble material was collected via centrifugation. Clarified cellular lysates were purified over a nickel column and subsequent size-exclusion chromatography, then recovered in 50 mM HEPES, 500 mM NaCl, 0.5 mM TCEP, 5% glycerol, pH 7.5 for storage at -80 °C.Protein SequencesPhSec-RS:MHHHHHHSSGVDLGTENLYFQSNADEFEMIKRNTSEIISEEELREVLKKDEKSAWIGFEP SGKIHLGHYLQIKKMIDLQNAGFDIIIHLADLGAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSENSLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQV NSIHYEGVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIA VDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMRLKNAVAEELIKILEPIRKRL (SEQ ID NO: 57)CIB1MGSSHHHHHHSSGLVPRGSHMGLNDIFEAQKIEWHEGSGGSGSRLSKELLAEYQDLTFL TKQEILLAHRRFCELLPQEQRSVESSLRAQVPFEQILSLPELKANPFKERICRVFSTSPAKD SLSFEDFLDLLSVFSDTATPDIKSHYAFRIFDFDDDGTLNREDLSRLVNCLTGEGEDTRLSASEMKQLIDNILEESDIDRDGTINLSEFQHVISRSPDFASSFKIVL (SEQ ID NO:58)CIB1 (C134A)MGSSHHHHHHSSGLVPRGSHMGLNDIFEAQKIEWHEGSGGSGSRLSKELLAEYQDLTFL TKQEILLAHRRFCELLPQEQRSVESSLRAQVPFEQILSLPELKANPFKERICRVFSTSPAKDSLSFEDFLDLLSVFSDTATPDIKSHYAFRIFDFDDDGTLNREDLSRLVNALTGEGEDTRLSASEMKQLIDNILEESDIDRDGTINLSEFQHVISRSPDFASSFKIVL (SEQ ID NO:59)MAGE-A4MGS SHHHHHHS SGLVPRGSHMGLNDIFEAQKIEWHEGSMS SEQKSQHCKPEEGVEAQE EALGLVGAQAPTTEEQEAAVS S S SPLVPGTLEEVPAAESAGPPQSPQGAS ALPTTISFTC WRQPNEGSSSQEEEGPSTSPDAESLFREALSNKVDELAHFLLRKYRAKELVTKAEMLER VIKNYKRCFPVIFGKASESLKMIFGIDVKEVDPTSNTYTLVTCLGLSYDGLLGNNQIFPKT GLLIIVLGTIAMEGDSASEEEIWEELGVMGVYDGREHTVYGEPRKLLTQDWVQENYLEY RQVPGSNPARYEFLWGPRALAETSYVKVLEHVVRVNARVRIAYPSLREAALLEEEEGVV (SEQ ID NO:60)MAGE-A4 (C80A)MGS SHHHHHHS SGLVPRGSHMGLNDIFEAQKIEWHEGSMS SEQKSQHCKPEEGVEAQE EALGLVGAQAPTTEEQEAAVS S S SPLVPGTLEEVPAAESAGPPQSPQGAS ALPTTISFTA WRQPNEGSSSQEEEGPSTSPDAESLFREALSNKVDELAHFLLRKYRAKELVTKAEMLER VIKNYKRCFPVIFGKASESLKMIFGIDVKEVDPTSNTYTLVTCLGLSYDGLLGNNQIFPKT GLLIIVLGTIAMEGDSASEEEIWEELGVMGVYDGREHTVYGEPRKLLTQDWVQENYLEYRQVPGSNPARYEFLWGPRALAETSYVKVLEHVVRVNARVRIAYPSLREAALLEEEEGVV (SEQ ID NO:61)MAGE-A4MHDMHHHHHHSSGVDLGTENLYFQSNADAESLFREALSNI<VDELAHFLLRKYRAI<ELVTI<A EMLERVIKNYKRCFPVIFGKASESLKMIFGIDVKEVDPTSNTYTLVTCLGLSYDGLLGNN QIFPKTGLLIIVLGTIAMEGDSASEEEIWEELGVMGVYDGREHTVYGEPRKLLTQDWVQ ENYLE YRQ VPGSNP ARYEFLWGPRAL AET S YVK VLEHVVRVNARVRIA YP SLRE A ALLEEEEGV (SEQ ID NO:62)Table 6. List of Primers, Genes, and LibrariesTable 7. Peptide Characterizations:Gene Fragments and LibrariesMj-tRNAcuA sequence (assembled from primers):GGCGTAATACGACTCACTATAGCCGGCGGTAGTTCAGCAGGGCAGAACGGCGGACTCTAAATCCGCATGGCAGGGGTTCAAATCCCCTCCGCCGGACCAm (SEQ ID NO: 101)G1 - Model IVT peptide for PhSec incorporation:TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGAAGCCCTCACAACGTGACACTGTAGAGTAGTATGCGAAAAAATACCCATATGATGTACCCGACTATGCTTAATGACGCATTGGAAGTGGATAACGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACC (SEQ ID NO: 102)G2 - CIB1 clone (UNC10245129) for mRNA display clonal assays (assembled from P7-9):TAATACGACTCACTATAGGGTTAACTTTAAGAAGGAGATATACATATGACAGGACCTTGGACCATCCGTTGGTGCGGCAGCGGCAGCGGCAGCTAGGACGGGGGGCGGAAA(SEQ ID NO: 103)G3 - Model mRNA display sequence for Biotin-Phe pulldowns: TAATACGACTCACTATAGGGTTAACTTTAAGAAGGAGATATAAATATGAAGCCCAG TCAGCGCGACACTGTTGAATGGTATGCTAAGAAGGGGTCAGGCTCATATCCTTATGA TGTTCCCGATTATGCCGGCAGCGGCAGCGGCAGCTAGGACGGGGGGCGGAAA (SEQID NO: 104)G4 - CCP1 clone for mRNA display clonal assays: TAATACGACTCACTATAGGGTTAACTTTAAGAAGGAGATATACATATGGACATTTAG ATCTATCATTTGAATTTTTGCGGATCAGGCTCCTATCCATACGATGTGCCCGATTATG CCGGCAGCGGCAGCGGCAGCTGAGACGGGGGGCGGAAA (SEQ ID NO: 105)LI - NWW8 TAG mRNA display library (assembled from primers)*:*below is indicative of TAG at position 1. In full library, TAG is scanned across all 9 randomized positions.TAATACGACTCACTATAGGGTTAACTTTAAGAAGGAGATATACATATGTAGNWWN WWNWWNWWNWWNWWNWWNWWTGCGGGTCAGGCTCATATCCTTATGATGTTCC CGATTATGCCGGCAGCGGCAGCGGCAGCTGAGACGGGGGGCGGAAA (SEQ IDNO: 106)L2 - NNK8 TAG mRNA display library (assembled from primers)*:*below is indicative of TAG at position 1. In full library, TAG is scanned across all 9 randomized positions.TAATACGACTCACTATAGGGTTAACTTTAAGAAGGAGATATACATATGTAGNNKNN KNNKNNKNNKNNKNNKNNKTGCGGGTCAGGCTCATATCCTTATGATGTTCCCGATT ATGCCGGCAGCGGCAGCGGCAGCTGAGACGGGGGGCGGAAA (SEQ ID NO: 107)DNA and RNA PreparationGene fragment and randomized library PCR

[0101] Gene fragments for IVT and mRNA display were first resuspended to a final concentration of 10 ng / pL. PCR amplification was carried out under standard Q5 DNA polymerase (NEB M0491) protocols. Pl and P2 were used to amplify gene fragments for IVT; P7 and P28 were used to amplify sequences for mRNA display (G2-4, NWW = Pl 0-18; NNK = Pl 9-27). Amplification was performed under conditions listed in the below table and confirmed by DNA gel (3% agarose gel supplemented with 1.5% ethidium bromide). DNA was then purified by a PCR purification kit, isolated in MQ-H2O, and concentration was determined bynanodrop. TAG sub-libraries for mRNA display were adjusted to equal concentrations and combined to a total DNA concentration of 300 ng / uL. Gene fragments and libraries were stored at -20 °C until further use.Table 8. Gene fragment PCR conditions:**Primer annealing temperatures were determined by NEB Tm calculatorMethanococcus jannaschii-tRNA (Mj-tRNA) PCR

[0102] Mj-tRNA PCR was performed as previously described.4An initial extension reaction (100 pL) was prepared containing lx Q5 Reaction buffer (NEB M0491), 0.25 mM dNTPs, 0.5 pM forward primer (P3), 0.5 pM reverse primer (P4), 2 units Q5 DNA Polymerase (NEB M0491). Extension occurred for 5 cycles on a thermocycler at conditions listed in below extension reaction table. Once complete, a 1 mL PCR amplification was set up containing all of the initial extension reaction, lx Q5 Reaction buffer, 2.5 mM MgCh, 0.25 mM dNTPs, 0.5 pM forward primer (P5), 0.5 pM reverse primer (P6), 15 units Q5 DNA Polymerase (NEB M0491), and diluted in MQ-H2O. Amplification was performed under conditions listed in the below PCR- 2 amplification table. Amplification was confirmed by DNA gel. Then, lx volume of phenol / chloroform / isoamyl alcohol (PCI) was added to the solution, mixed vigorously, and centrifuged. The top layer was recovered, transferred to a new tube, and PCI extraction was performed once more. To the extracted aqueous layer, 1 / 10 volume of 3M NaCl and 2x volume of 100% ethanol were added and mixed vigorously. This mixture was centrifuged for 15 mins at 13,000 rpm to pellet the DNA. Once pelleted, the DNA was washed with excess 70% ethanol, then allowed to dry at RT. The DNA was then solubilized in a 1 / 10 volume (100 pL) of the PCR reaction.Table 9. Extension reaction conditions:Table 10. PCR-2 amplification conditions:General RNA transcription

[0103] A 1 mL transcription reaction (adapted from NEB T7 RNA polymerase protocols) containing amplified DNA (100 pL), lx T7 RNA Polymerase Buffer, 1 mM Dithiothreitol (DTT), 16.5 mM MgCb, 5 mM rNTPs, and 5 units / pL T7 RNA Polymerase (NEB M0251) was incubated overnight at 37 °C. Upon successful transcription, magnesium pyrophosphate precipitates and the solution appears cloudy. Then, 115 pL of lOx DNase I buffer and 30 pL of DNase I (Promega PR-M6101) was added to the solution and incubated at 37 °C for Ih. After DNase incubation, EDTA, NaCl, and 100% isopropyl alcohol were added to final concentrations of 37.5 mM, 150 mM, 45%, respectively, upon which the solution became clear. The solution was then centrifuged at 4,000 rpm for 15 mins to pellet the RNA. The supernatant was removed, and the pellet was washed with excess 70% ethanol. This solution was briefly centrifuged at 4,000 rpm, the supernatant was removed, and the RNA pellet was allowed to dry at RT. The RNA pellet was then solubilized in 100 pL of MQ-H2O and an equal volume (100 pL) of 2x RNA loading dye. The sample was heated at 95 °C for 2 mins, then run on a large scale 12% Urea-PAGE gel at 230 V for 1.5 h in a Tris-Borate-EDTA (TBE) buffer. The desired RNA band was then visualized under UV illumination at 254 nm on a silica-coated thin-layer chromatography plate and excised from the gel. The excised band was crushed into fine pieces and the RNA was extracted with 0.3 M NaCl (2 x 1 h incubation at RT). To collect the RNA, the gel was pelleted by centrifugation at 4,000 rpm and the supernatant isolated. The supernatant wasthen passed through a 0.45 pm filter before addition of 2x volume of 100% ethanol. The solution was mixed vigorously, then was centrifuged at 4,000 rpm for 25 mins to pellet the RNA. The pellet was washed with excess 70% ethanol, briefly centrifuged at 4,000 rpm, and then allowed to dry at RT. The RNA was solubilized in MQ-H2O and the concentration was determined by nanodrop and adjusted as necessary. Purity was confirmed via an analytical scale 12% Urea- PAGE gel. RNA was stored at -80 °C until use.Phenyselenocysteine (PhSec) Synthesis (SI)

[0104] Boc-PhSec was synthesized according to previously reported procedures?6To an oven- dried round bottom flask under inert atmosphere was added a solution of tert-butyl (S)-(2- oxooxetan-3-yl)carbamate (0.485 g, 1.1 Eq, 2.59 mmol) in anhydrous DMF (5mL) and a solution of benzeneselenol (1 Eq, 2.35 mmol) in anhydrous DMF (5mL). The reaction was stirred at 80 °C for 2 h with progress monitored by TLC (9: 1 DCM / MeOH). Once complete, the reaction was quenched with 0.3 M NaOH and washed with diethyl ether (lx), after which the aqueous layer was acidified to pH 3.0 with 1 M HC1. The acidified solution was partitioned between ethyl acetate (3x), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to afford a pale yellow oil. The crude material was dried under vacuum, then purified via silica gel flash chromatography (DCM / MeOH, 0-10% MeOH) to afford the Boc-protected intermediate. This material was dissolved in DCM (3 mL) and TFA (2 mL) was added dropwise at RT. Reaction was then stirred at RT for 2 h with progress monitored by TLC (9: 1 DCM / MeOH). Once complete, the reaction was evaporated under reduced pressure to afford the desired product (R)-2-amino-3-(phenylselanyl)propanoic acid (0.503 g, 2.06 mmol, 87.6%) as an off-white / pale yellow solid.

[0105] ’H NMR (400 MHz, Methanol-^) 6 7.65 - 7.61 (m, 2H), 7.36 - 7.31 (m, 3H), 4.05 (dd, J = 7.8, 4.5 Hz, 1H), 3.51 (dd, J= 13.7, 4.5 Hz, 1H), 3.30 - 3.25 (m, 1H).

[0106] 13C NMR (101 MHZ, Methanol-^) 8 170.39, 134.87, 130.58, 129.28, 53.86, 28.11.PhSec IVT with PhSec-RS, DHA formation, and thiol conjugate addition

[0107] Translations were performed using a custom NEB PURExpress® kit (-aa, -tRNA, - RF123) -E6850Z, supplemented with 10 pM Mj-tRNA, 10 pM PhSec-RS, 5 mM PhSec, and 0.2x of RF2 and RF3 (supplied as lOx stock). Translations were incubated at 37 °C for 0.5 h. Then, the crude IVT solution was diluted 1 : 1 with 2 M H2O2 in 50 mM HEPES pH 8 (final concentration 1 M H2O2), then incubated on ice for 0.5 h. The reaction was quenched with 500 mM TCEP and shaken at 37 °C for 1 h. The entire solution was briefly centrifuged to pellet any precipitant. The supernatant was collected, diluted by lOx with 50 mM HEPES pH 8, supplemented with 100 mM P-mercaptoethanol, and allowed to mix at RT for 1 h. Translated peptides were purified from the IVT solution using Pierce™ C18 Spin tips (Pierce PI84850) and eluted with a half-saturated solution of a-cyano-4-hydroxycinnamic acid in 80% acetonitrile and 0.5% acetic acid in water onto a MALDI-TOF plate for analysis. MALDI-TOF analysis was performed on an AB Sciex TOF / TOF 5800 system in reflector positive mode. mRNA DisplayPuromycin Linking

[0108] RNA of single sequences or randomized libraries were covalently linked to puromycin via an adapted Y-ligation method.7A solution with 1 pM RNA, 20% DMSO, 1.5 pM puromycin linker (P29), lx T4 RNA ligase buffer, and 1 unit / pL T4 RNA ligase I (PR-M1051) in MQ-H2O was incubated at 37 °C for 0.5 h. Then, lx volume of precipitation solution (0.6 M NaCl, and 50 mM EDTA), 0.02x volume of 5 mg / mL glycogen, and 2x volume 100% ethanol were added. This mixture was briefly vortexed, then centrifuged at 13,000 rpm for 15 mins to pellet the puromycin-linked RNA. The supernatant was removed, washed excess 70% ethanol, and briefly centrifuged at 16,000 x g. The supernatant was removed and the pellet was allowed to dry at RT. Once dry, the puromycin-linked RNA pellet was reconstituted in 1 / 10ththe volume of the p- linking reaction (10 pM RNA). The efficiency of the reaction was determined by 12% Urea- PAGE Gel (National Diagnostics, EC-833).Translation and Reverse Transcription

[0109] Flexizyme was used to aminoacylate chloroacetyl-Tyr (ClAcY) or Biotin-Phe onto optimized initiator tRNA (ini-tRNA) according to standard protocols.8,9Translation of puromycin-linked RNA was performed using a custom NEB PURExpress® kit (-aa, -tRNA, -RF123) -E6850Z. Single sequences were translated with 25 pM ClAcY-ini-tRNA (G2) or 100 qM biotin-Phe-ini-tRNA (G3). Libraries containing TAG codons (LI -2) were translated with 10 qM Mj-tRNA, 10 qM PhSec-RS, 5 mM PhSec, and 25 qM ClAcY-ini-tRNA at 10-40 qL total translation volume depending on the selection round. Translations were incubated at 37 °C for 30 mins, followed by a 12 min incubation at RT to facilitate fusion of peptide to its mRNA strand. EDTA was then added to a final concentration of 17 mM to dissociate the ribosome, and the mixture was incubated at 37 °C for 30 mins. Then, complementary DNA was appended by a reverse transcription reaction containing all the translation product, 0.6 mM dNTPs, 5 M reverse primer, 62.5 mM Tris-HCl pH 8.3, 37.5 mM Mg(OAc)2, 25 mM KOH, 2.5 x M-MLV reverse transcriptase H (-) point mutant (Promega, M3681, supplied at 40x), and MQ-H2O (to reach final volume). This solution was incubated at 42 °C for Ih.DHA Formation

[0110] After reverse transcription, libraries containing PhSec were treated with hydrogen peroxide to a final concentration of 1 M H2O2 in TBS-T (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween-20) and incubated on ice for 0.5 h. Once complete, libraries were HA purified.HA Purification[0U1] Fusions containing an HA peptide sequence were diluted by lOx with TBS-T and added to anti-HA magnetic beads (ThermoFisher catalog number: 88836) at a 4: 1 ratio of bead slurry to initial IVT volume. The bead slurry rotated at RT for 30 min, after which beads were washed 3x with TBS-T. 2 mg / mL HA peptide in covalent selection buffer (25 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM TCEP (fresh), 0.05% Tween-20) was then added to the beads, and fusions were eluted by rotation at RT for 1 h. The HA elution volume should be calculated such that immobilized protein is at 200 nM during the selection.Biotin-Phe SA Bead Pulldown

[0112] Fusions containing biotin-Phe for probing guanidine compatibility with biotinstreptavidin were added to an excess of Dynabeads™ M-280 Streptavidin beads (ThermoFisher catalog number: 11205D) and incubated with rotation for 30 mins at 4 °C. The beads were then washed 3x with selection buffer before guanidine exposure (see below).CIB1 / MAGE-A4 Streptavidin Immobilization

[0113] Biotinylated CIB1 or MAGE-A4 was immobilized onto Dynabeads™ M-280 Streptavidin beads (ThermoFisher catalog number: 11205D). Excess CIB 1 / MAGE-A4 in covalent selection buffer was added to streptavidin beads such that, when combined with the volume of HA eluted fusions, enough pmol protein was immobilized to arrive at a final concentration of 200 nM. A separate streptavidin immobilization assay was performed prior to this to determine the quantity of beads needed to immobilize this concentration of each protein. Immobilization occurred at 4 °C for 30 mins. Then, free biotin in H2O was added to a final concentration of 25 pM to occupy any remaining biotin binding sites. The solution rotated for an additional 15 mins at 4 °C. Once complete, protein-immobilized SA beads were washed 3x with selection buffer.Selection, Guanidine Denaturation, and cDNA Elution

[0114] HA eluted fusions were added to the prepared CIB1-SA or MAGE-A4-SA beads. For CIB1, the NWW library was incubated for 1 h at 4 °C over all rounds of selection. For MAGE- A4, the NNK library was incubated at 4 °C overnight for the first two rounds, then 1 h for all subsequent rounds. After incubation, the supernatant was removed and protein immobilized-SA beads were washed 3x with covalent selection buffer. Then, the beads were suspended in a solution of 5 M guanidine-HCl in TBS-T and incubated at RT for 0.5 h with rotation, after which the guanidine was removed and beads were further washed 3x with TBS-T. Protein- SA beads were then resuspended in 50 pL lx Q5 Reaction buffer (NEB B9027S) and heated at 95 °C for 5 mins to elute cDNA. The supernatant containing the eluted cDNA was recovered, analyzed via qPCR, and amplified for the next round of selection (see below). qPCR

[0115] qPCR was performed with an Applied Biosystems Viia7 qPCR Real-Time qPCR Instrument. Each qPCR sample contained 0.25 pM forward and reverse primers (P7, P28) and 1 pL of experimental sample in lx SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, 172- 5271). qPCR standards were prepared by reverse transcription of a known quantity of RNA into cDNA (assuming 100% yield) and prepared in 10-fold serial dilutions to 2e9, 2e8, 2e7, 2e6, 2e5molecules. For amplification, initial heating began at 50 °C for 2 min followed by 10 min 95 °C incubation. Cycling then occurred between 95 °C for 15 sec and 60 °C for 1 min, with cycler heating acceleration held at 1.6 °C between each step. A total of 40 cycles were performed in each qPCR run. During each elongation step, SYBR green fluorescence was measured with ROXas a passive reference. Following the run, standard curves were generated and used to calculate cDNA quantities.PCR

[0116] Simultaneously, a small-scale PCR reaction amplifying 5 pL of cDNA elution was performed according to standard Q5 polymerase amplification protocols. All PCR reactions were performed alongside a no template control. Amplification was performed until bands could be observed via DNA gel, usually 15-30 cycles. A large-scale PCR with the remaining eluted cDNA was performed for the determined number of amplification cycles and confirmed via DNA gel.Table 11. PCR conditions for library amplification:Rounds 2+

[0117] Beginning in the second round of selection, libraries were incubated three subsequent times with biotinylated Dynabeads™ M-280 streptavidin beads before incubation with immobilized protein to eliminate bead binders.NGS of Eluted cDNA

[0118] Recovered DNA from each round of selection was prepared for NGS sequencing by PCR amplification using forward and reverse primers containing NGS adaptor sequences provided by Azenta (P30-31). After PCR cleanup, 25 pL of 500 ng PCR product in water was sent to Azenta for Amplicon EZ sequencing. Sequences were translated by their open reading frame and analyzed for enriched families using CLC Sequence Viewer™.Table 12. NGS PCR ConditionsSolid Phase Peptide SynthesisGeneral Peptide Synthesis

[0119] Peptides were prepared using 50-100 mg of Rink Amide ChemMatrix resin (0.48 mmol / g) following standard fluorenylmethoxycarbonyl (Fmoc) protocols. Resin was swollen at RT on a Bohdan Miniblock shaking at 550 rpm for 1 h in dichloromethane (DCM). Linear peptide synthesis was carried out through cycles of deprotection and coupling. For Fmoc deprotection, 2 ml of 20% piperidine in dimethyl formamide (DMF) was added and shaken at RT for 10 mins. The vial was drained, washed with DCM (4 x 5 mL) and DMF (4 x 5 mL). For amino acid coupling, Fmoc-AA-OH (5.0 equiv., 0.5 M in DMF), O-(lH-6-Chlorobenzotriazole- l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HCTU) (5.0 equiv., 0.5 M in DMF), and N,N-diisoproplyethylamine (DIPEA) (10 eq, 1.0 M in DMF) were pre-mixed during a deprotection step. The activated amino acid was then added to the vial and shaken at RT for 15 mins, after which the vial was drained and thoroughly washed with DCM (4 x 5 mL) and DMF (4 x 5 mL). This process was repeated until the full peptide sequence is prepared. All canonical amino acids were protected with standard acid labile protecting groups. DHA precursors were incorporated as Fmoc-Cys(StBu)-OH.

[0120] After deprotection of the final residue, triethylamine (50 equiv.) was added to chloroacetic anhydride (25 equiv.) dissolved in 1 :1 DMF / DCM (~2mL). This entire solution was swiftly added to the resin and allowed to shake for 3 minutes, after which the vial was drained and washed. Then, the resin was dried and cleaved with a cleavage cocktail (TFA / TIPS / H2O 95:2.5:2.5) at 37 °C for 1 h. The resin was then filtered from the cleavage cocktail solution and the cleavage cocktail was evaporated under a stream of nitrogen. Cooled diethyl ether (>10x volume of residual TFA) was added to crash out the crude peptide. The crude peptide was pelleted by centrifugation (10 min at 4,000 rpm at 4 °C) and the diethyl ether was decanted.

[0121] Once cleaved and pelleted, crude peptides were dissolved in 1 : 1 DMSO:H2O to a final concentration of ~1 mM, and the pH of the solution was increased to >8 via addition of tri ethylamine. The reaction stirred at RT for 1-2 h to enable cyclization. Once complete, the solution was acidified with dropwise addition of TFA until pH <6 was reached. Reactions werefurther diluted with 1 : 1 acetonitrile:H2O) and purified by reverse phase preparatory HPLC following the general gradient.Cysteine Conversion to DHA (CCP1, Biotin-CCPl, MCP1, MCP2, MCP3)

[0122] DHA formations from cysteine were performed as previously described.10,11Once cyclized and purified, peptides containing Cys-StBu were dissolved in 1 : 1 DMF:H2O supplemented with TCEP (20 equiv.). The reactions where shaken in a 37 °C incubator at -250 rpm for 5 h, after which K2CO3 (50 equiv.) and methyl 2,5-dibromopentanoate (ChemScene CS- 0150917, 50 equiv.) were added. The reactions were allowed to shake at 37 °C for an additional 3 h, after which the crude peptides were diluted with MeCN:H2O (1 : 1) and purified by reverse phase preparatory HPLC following the general gradient.Synthesis of Biotin-CCPl

[0123] The peptide sequence ClAc-YDIC(StBu)IYHLNFCGAGK(N3)A-NH2(SEQ ID NO: 122) was synthesized, cleaved, and cyclized following the general peptide synthesis protocols. DBCO-PEG4-biotin (Click Chemistry Tools, A105-5, 1.2 equiv.) dissolved in DMSO was added to cyclized peptide in 1 :1 ACN / H2O with up to 10% DMSO. The reaction stirred at RT for 1 h, after which the mixture was injected directly for purification via HPLC. Desired fractions were collected and lyophilized, then converted into the DHA according to the procedure described above.MALDI-MS of Covalently Modified Proteins & Protease DigestsCovalent Peptide Modification

[0124] DMSO stocks of peptides or iodoacetamide (1-20 equiv.) were added to CIB1 or MAGE-A4 (10 pM) in 25 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM TCEP (fresh) and incubated at RT with rotation for 30 mins (up to 4 h for MCP3). MAGE-A4 samples were diluted 2:1 with H2O after the incubation period for adequate visualization via MALDI. Then, 0.6 pL of modified protein was mixed with 0.6 pL of a saturated sinapinic acid solution in 80% acetonitrile and 0.5% acetic acid in water and spotted directly onto a MALDI-TOF plate for analysis. MALDI-TOF analysis was performed on an AB Sciex TOF / TOF 5800 system in high mass linear positive mode.Protease Digests

[0125] After peptide modification, proteins were either digested with trypsin (CIB 1) or GluC (MAGE-A4).

[0126] CIB1 : Modified CIB1 (~2.5 pg) was diluted 5x in 50 mM NH4HCO3, 1 mM CaCh, and 20 mM TCEP (fresh). The protein was heat denatured at 95 °C for 10 minutes and allowed to cool to RT before addition of trypsin (0.32 pg, Pierce 90057). The digest was incubated overnight at 37 °C, then purified using Pierce™ C18 Spin tips (Pierce PI84850) and eluted with a half-saturated solution of a-cyano-4-hydroxycinnamic acid in 80% acetonitrile and 0.5% acetic acid in water onto a MALDI-TOF plate for analysis. MALDI-TOF analysis was performed on an AB Sciex TOF / TOF 5800 system in reflector positive mode.

[0127] MAGE-A4: Modified MAGE-A4 (~1.5 pg) was diluted in 2x GluC buffer (NEB P8100S), heat denatured at 95 °C for 10 mins, then allowed to cool to RT before addition of GluC (0.6 pg, NEB P8100S). The digest was incubated at 37 °C overnight, then purified using Pierce™ Cl 8 Spin tips (Pierce PI84850) and eluted with a half-saturated solution of a-cyano-4- hydroxy cinnamic acid in 80% acetonitrile and 0.5% acetic acid in water onto a MALDI-TOF plate for analysis. MALDI-TOF analysis was performed on an AB Sciex TOF / TOF 5800 system in reflector positive mode.CCP1 CIB1 Cell Lysate Labeling

[0128] MDA-MB-453 cells (UNC Lineberger Cancer Center tissue culture facility) were grown in high glucose DMEM supplemented with 10% fetal bovine serum (FBS) and 1% non- essential amino acids at 5% CO2 and 37°C. Cells were lysed in 25 mM Tris, pH 8.0, 150 mM NaCl, 1 mM TECP, 10 mM CHAPS, 20 mM sodium fluoride, 0.1 mM sodium pervanadate, and 1 :200 protease inhibitor cocktail (Sigma) and incubated 20 min on ice, followed by centrifugation at 13,000 x g for 5 min. Protein concentration was adjusted to - 500 pg / ml and 6 pM recombinant His-CIBl was added prior to dividing into 100 pl aliquots. DMSO or CCP1 peptide (5, 10, 25, and 50 pM) was added to appropriate aliquots and incubated Ih at RT. Samples were prepared by adding an equal volume of non-reduced SDS-PAGE sample buffer and heated 1 min at 90 °C. Proteins were separated by SDS-PAGE electrophoresis and transferred to PVDF membranes. To visualize total protein, membranes were washed 3x with water and incubated 5 min with Ponceau S, washed and true color images were captured on an Azure 600 imaging system (Azure Biosystems). The membranes were then destained with 0.1% v / v NaOH and blocked with 3% BSA in TBS-Tween 20 (TBS-T; 25 mM Tris, pH 7.5, 150 mM NaCl, and 0.1% Tween-20) for 30 min. Membranes were probed with Neutravidin-HRP(1 : 10,000; Pierce) for 1 h, washed 3X with TBS-T, and enhanced chemiluminescence (ECL-2; Pierce) images captured with Azure 600.CIB1 and MAGE-A4 TR-FRETCIB1

[0129] CIB1 TR-FRET was performed as previously described.12Peptide inhibitors were dispensed into 384-well plates using a Mosquito HTS nanoliter instrument (TTP LABTech) as 3- fold serial dilutions (lOOx in DMSO, 0.1 pL) for final concentrations ranging from 10 pM - 0.5 nM. Biotinylated CIB1 was diluted to a final concentration of 3 nM in assay buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM CaCl, 1 mM CHAPS, and 1 mM DTT (fresh). 5 pL of diluted protein was added to the wells of the assay plate using a Multidrop Combi Reagent Dispenser (Thermo Scientific) and incubated at RT for 20 min protected from light. 5 pL detection solution containing 2 nM Lance Eu- Streptavidin (Perkin Elmer) and 30 nM AF647-CIB1 -phage peptide1diluted in assay buffer was added to the wells and incubated for 1 h at RT protected from light.MAGE-A4

[0130] MAGE-A4 TR-FRET was performed as previously described.2Peptide inhibitors were dispensed into 384-well plates using a Mosquito HTS nanoliter instrument (TTP LABTech) as 3- fold serial dilutions (lOOx in DMSO, 0.1 pL) for final concentrations ranging from 10 pM - 0.2 nM. Biotinylated MAGE-A4 and Lance Eu- Streptavidin (PerkinElmer) were diluted to 3 nM and 2 nM, respectively, in assay buffer (20 mM Tris pH 8, 150 mM NaCl, 2 mM DTT (fresh), 0.05% Tween-20). 5 pL of the diluted MAGE-A4-SA-Eu solution was added to the wells of the assay plate using a Multidrop Combi Reagent Dispenser (Thermo Scientific) and incubated at RT for 20 min protected from light. 5 pL detection solution containing 3 nM AF647-MTP-12diluted in assay buffer was added to the wells and incubated 1 h at RT protected from light.TR-FRET Readout

[0131] TR-FRET signals were measured after 1 h (filled shape, solid line), 16 h (half-filled shape, long dashed line), and 24 h (shape outline, short dashed line) using an Envision Multilabel Plate Reader (PerkinElmer; Eu excitation 320 nm, Eu emission 615 nm, Alexa dye emission 665 nm). TR-FRET signal is measured as the ratio of 665 nm / 615 nm emission and percent inhibition can be calculated using two sets of control wells: biotinylated protein and detection solution in the presence (100% inhibition, positive control), or absence (0% inhibition, negative control) ofinhibitor. Inhibition curves were analyzed using a non-linear regression curve fit in GraphPad Prism 9.5.1.Mass dilution MAGE-A4 TR-FRET for covalent labelins kinetics

[0132] Mass dilution TR-FRET assays were run using the white, high volume, flat-bottom, nonbinding, 384-well OptiPlate™ from PerkinElmer (Part No. 6007290) containing a total assay volume of 100 pL per well. All experiments were run at room temperature with the same buffer composition and final concentrations of protein and FRET reagents as the TR-FRET methods described above.

[0133] The test peptides and MAGE-A4 protein were diluted in Eppendorf tubes for incubation at the desired testing concentrations (3.33X). In the OptiPlate, the remaining components necessary for the FRET assay were dispensed at 70 pL (1 ,4X) per well in preparation for mass dilution. Starting at time point zero, 30 pL aliquots of the protein-peptide stock were transferred from the Eppendorf tube to the Optiplate for mass dilution every three minutes for 30 minutes total (11 total wells per concentration, 100 pL total). The dilution factor (MCP1 : 3.33) was predetermined so the mass dilution step reduces the peptide concentration below the reported ICso. All TR-FRET components are diluted to the final concentrations listed in the TR-FRET methods above (IX). A DMSO control sample was also included for data normalization at each time point tested. 100% (saturated with known inhibitor) and 0% (no inhibitor present) inhibition controls were incorporated to calculate the percentage of inhibition for all data points. Following mass dilution, each time point was incubated for 15 minutes to allow for any reversible compound binding to be diluted off. Then, each data point was read on an EnVision 2103 Multilabel Plate Reader (PerkinElmer) using a 320 nm excitation filter and 615 and 665 nm emission filters. Emission signals were measured simultaneously using a dual mirror D400 / D630 and 100 psec delay. The TR-FRET output signal was expressed as a ratio of acceptor / donor (665 nm / 615 nm) emission counts. The normalized percent inhibition for each concentration tested was plotted at each time point. The data was fitted using a linear fit, and the slope (kobs) of each concentration was plotted against the different peptide concentrations tested. From this, the max kobs from these experiments is taken as the kinact, while the concentration at which ’Akinactis observed is taken as the Ki. From here, the inactivation efficiency can be calculated using kinact / Ki. GraphPad Prism 9.0 was used for data visualization, fitting, and figure preparation.MAGE-A4 Co-Immunoprecipitation with RAD18Cell culture and expression plasmids

[0134] H1299 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS), 100 U / ml penicillin, and 100 pg / ml streptomycin as described previously.2N-terminal HA-tagged MAGE-A4 was expressed using the pINDUCER20 vector2and C-terminal Myc-tagged RAD 18 was expressed using the pCAGGS vector as described previously.13Plasmid transfections were performed using Lipofectamine 2000 (Invitrogen) according to manufacturer’s protocol, except concentrations of DNA and Lipofectamine 2000 used in each reaction were decreased by approximately 50%.Immunoprecipitation and immunoblotting

[0135] After 48 hours transfection, cultured cells were lysed using cold cytoskeletal buffer (CSK) as described.2Lysates were incubated with 250U / ml nuclease (Pierce 88702) on ice for 30 minutes, then centrifuged at 16,000 g for 10 minutes.

[0136] For immunoprecipitation of Myc-RAD18, 425 pg cell extract was incubated with indicated peptide concentration or equivalent volume DMSO for 2 hours at 4°C using rotating racks, in a total volume of 200 pl. 25 pl Anti-c-Myc magnetic beads (Pierce 88842) were washed 2x with 1 mL CSK, resuspended in 50 pl CSK buffer and incubated with samples overnight, rotating at 4 °C. Beads were washed 3x with 1 mL CSK (5 minutes rotating at 4 °C per wash), before being boiled in protein loading buffer for 5 minutes.

[0137] For immunoblotting, samples were separated by SDS-PAGE and transferred to nitrocellulose membranes before being incubated overnight with the following primary antibodies: HA (A190-138A), RAD6 (A300-281A) from Bethyl Laboratories; Myc (sc-40), GAPDH (sc-32233) from Santa Cruz Biotech. Antibody dilutions used for immunoblotting were 1 : 1000 for RAD6 and Myc, 1 : 3000 for HA and GAPDH.References for Materials and Methods:(1) Puhi, A. C.; Bogart, J. W .; Haberman, V. A.; Larson, J. E.; Godoy, A. S.; Norris-Drouin, J. L.; Cholensky, S. H.; Leisner, T. M.; Frye, S. V.; Frye, S. V.; Parise, L. V.; Parise, L.V.; Bowers, A. A.; Bowers, A. A.; Bowers, A. A.; Pearce, K. H.; Pearce, K. H. Discovery and Characterization of Peptide Inhibitors for Calcium and Integrin Binding Protein 1.ACS Chem. Biol. 2020, 75 (6), 1505-1516. doi.org / 10.1021 / acschembio.0c00144.(2) Fleming, M. C.; Chiou, L. F.; Tumbale, P. P.; Droby, G. N.; Lim, J.; Norris-Drouin, J. L.; Williams, J. G.; Pearce, K. H.; Williams, R. S.; Vaziri, C.; Bowers, A. A. Discovery and Structural Basis of the Selectivity of Potent Cyclic Peptide Inhibitors of MAGE-A4. J.Med. Chem. 2022, 65 (10), 7231-7245. doi.org / 10.1021 / acs.jmedchem.2c00185.(3) Howarth, M.; Takao, K.; Hayashi, Y.; Ting, A. Y. Targeting Quantum Dots to Surface Proteins in Living Cells with Biotin Ligase. Proc. Natl. Acad. Sci. 2005, 102 (21), 7583- 7588. doi.org / 10.1073 / pnas.0503125102.(4) Iskandar, S. E.; Pelton, J. M.; Wick, E. T.; Bolhuis, D. L.; Baldwin, A. S.; Emanuele, M.I.; Brown, N. G.; Bowers, A. A. Enabling Genetic Code Expansion and Peptide Macrocyclization in MRNA Display via a Promiscuous Orthogonal Aminoacyl -TRNA Synthetase. J. Am. Chem. Soc. 2023, 145 (3 1512-1517. doi.org / 10.1021 / jacs.2cl 1294.(5) Mori, T.; Higashibayashi, S.; Goto, T.; Kohno, M.; Satouchi, Y.; Shinko, K.; Suzuki, K.; Suzuki, S.; Tohmiya, H. Total Synthesis of Siomycin A : Construction of Synthetic Segments. Chem. - An Asian J. 2008, 3, 984-1012. doi.org / 10.1002 / asia.200800032.(6) Fleming, S. R.; Bartges, T. E.; Vinogradov, A. A.; Kirkpatrick, C. L.; Goto, Y.; Suga, H; Hicks, L. M.; Bowers, A. A. Flexizyme-Enabled Benchtop Biosynthesis of Thiopeptides.J. Am. Chem. Soc. 2019, 141, 758-762. doi.org / 10.1021 / jacs.8bl l521.(7) Sakai, K.; Passioura, T.; Sato, H.; Ito, K.; Furuhashi, H.; Umitsu, M.; Takagi, J.; Kato, Y.; Mukai, H.; Warashina, S.; Zouda, M.; Watanabe, Y.; Yano, S.; Shibata, M.; Suga, H.; Matsumoto, K. Macrocyclic Peptide-Based Inhibition and Imaging of Hepatocyte Growth Factor. Nat. Chem. Biol. 2019, 15 (6), 598-606. doi.org / 10.1038 / s41589-019-0285-7.(8) Goto, Y.; Katoh, T.; Suga, H. Flexizymes for Genetic Code Reprogramming. Nat. Protoc. 2011, 6 (6), 779-790. doi.org / 10.1038 / nprot.2011.331.(9) Ohuchi, M.; Murakami, H.; Suga, H. The Flexizyme System: A Highly Flexible TRNA Aminoacylation Tool for the Translation Apparatus. Cnrr. Opin. Chem. Biol. 2007, 11 (5), 537-542. doi.org / 10.1016 / j.cbpa.2007.08.011.(10) Bogart, J. W.; Kramer, N. J.; Turlik, A.; Bleich, R. M.; Catlin, D. S.; Schroeder, F. C.; Nair, S. K.; Williamson, R. T.; Houk, K. N.; Bowers, A. A.; Bowers, A. A. Interception of the Bycroft-Gowland Intermediate in the Enzymatic Macrocyclization of Thiopeptides. J. Am. Chem. Soc. 2020, 142 (30), 13170-13179. doi.org / 10.1021 / jacs.0c05639.(1 1) Rice, A. I.; Pelton, J. M.; Kramer, N. J.; Catlin, D. S.; Nair, S. K.; Pogorelov, T. V.; Mitchell, D. A.; Bowers, A. A. Enzymatic Pyridine Aromatization during Thiopeptide Biosynthesis. J. Am. Chem. Soc. 2022, 144 (46), 21116-21124. doi.org / 10.1021 / jacs.2c07377.(12) Haberman, V. A.; Fleming, S. R.; Leisner, T. M.; Puhi, A. C.; Feng, E.; Xie, L.; Chen, X.; Goto, Y.; Suga, H.; Parise, L. V.; Kireev, D.; Pearce, K. H.; Bowers, A. A. Discovery and Development of Cyclic Peptide Inhibitors of CIB1. ACS Med. Chem. Lett. 2021, 12 (11), 1832-1839. doi.org / 10.1021 / acsmedchemlett.lc00438.(13) Watanabe, K.; Tateishi, S.; Kawasuji, M.; Tsurimoto, T.; Inoue, H.; Yamaizumi, M.Radi 8 Guides Poleta to Replication Stalling Sites through Physical Interaction and PCNA Monoubiquitination. EMBO J. 2004, 23 (19), 3886-3896. doi.org / 10.1038 / sj.emboj.7600383.Alternate Cyclization

[0138] Dha may be also incorporated by means of desulfurization of an internal Cys residue. In this embodiment, cyclization would be achieved by site selective- (e.g., N-terminal Cys, FIGS. 16A-16C) or Cys-independent chemistry.

[0139] The foregoing examples are illustrative of the present invention and are not to be construed as limiting thereof. Although the invention has been described in detail with reference to preferred embodiments, variations and modifications exist within the scope and spirit of the invention as described and defined in the following claims.

Claims

CLAIMSWhat is claimed is:

1. An mRNA display library comprising a plurality of mRNA-linker-macrocyclic peptide molecules comprisingAcTyr-[amino acid]m-[PhSec]-[amino acid]n-[Cys]-[linker]-[mRNA]; wherein m and n are each an integer from 0 to 30; and wherein each macrocyclic peptide comprises a thioether linkage and is according to the formula I:wherein R comprises [linker]-[mRNA],2. An mRNA display library comprising a plurality of mRNA-linker-macrocyclic peptide molecules comprising:AcTyr-[amino acid]m-[dAla]-[amino acid]n-[linker]-[mRNA]; wherein m and n are each an integer from 0 to 30 and wherein each macrocyclic peptide comprises a thioether linkage and is according to the formula II:wherein R comprises [linker]-[mRNA].

3. The mRNA display library of claim 1 or 2, wherein m+n is between 7 and 15.

4. The mRNA display library of any one of claims 1-3, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 15.

5. The mRNA display library of any one of claims 1-3, wherein wherein n is 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, or 156. A method of preparing an mRNA display library comprising a plurality of mRNA-linker- macrocyclic peptide molecules comprising: providing a randomized library of mRNA molecules comprising a plurality of molecules according to a formula according to one of (Illa) -(IIIF):AUG-(NiWW)m-TAG-(NiWW)n -Cys-[linker] (Illa) (SEQ ID NO:1)AUG-(NiN2K)m-TAG-(NiN2K)n-Cys-[linker] (Illb) (SEQ ID NO:2)AUG-Cys-(NiWW)m-TAG-(NiWW)n-Cys-[linker] (IIIc) (SEQ ID NO:3) AUG-Cys-(NiN2K)m-TAG-(NiN2K)n-Cys-[linker] (Illd) (SEQ ID NO:4)AUG-Cys-(NiWW)m-TAG-(NiWW)n-AUG-[linker] (Ille) (SEQ ID NO:5)AUG-Cys-(NiN2K)m-TAG-(NiN2K)n-AUG-[linker] (III!) (SEQ ID NO:6)wherein Ni andN are independently any nucleotide, each W is independently A or U, and each K is independently G or U, and m and n are each an integer from 0 to 30; performing in vitro translation of each of the plurality of molecules in the mRNA library with a promiscuous orthogonal aminoacyl-tRNA synthetase (ORS) in the presence of phenylselenocysteine (PhSec) to thereby produce a plurality of linear molecules, the ORS incorporating a PhSec at the TAG or other non-competitive codon of each molecule; cyclizing each of the plurality of linear molecules; and forming a dehydroalanine via oxidative elimination at the PhSec in each of the plurality of cyclized molecules; thereby forming an mRNA library of a plurality of mRNA-linker-macrocyclic peptide molecules comprising a dehydroalanine.

7. The method of claim 6, wherein m+n is between 7 and 15.

8. The method of claim 6 or 7, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

9. The method of claim 6 or 7, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

10. The method of any one of claims 6-9, wherein the ORS is evolved to incorporate phenylsel enocysteine.

11. The method of any one of claims 6-10, comprising performing in vitro translation with chloroacetyl-Tyr (ClAcY) initiator tRNA to thereby produce a plurality of linear molecules according to ClAcTyr-[amino acid]m-[PhSec]-[arnino acid]n-[Cys]-[linker]- [mRNA] and wherein cyclization occurs via formation of a thioether linkage between AcTyr and cysteine.

12. The method of any one of claims 6-10, comprising performing in vitro translation of a plurality of molecules according to formula IIIc and / or Illd to thereby produce a plurality of linear molecules according to AUG-Cys-[amino acid]m-[PhSec]-[amino acid]n-[Cys]- [linker]-[mRNA], and the step of cyclizing comprises treating the plurality of linearmolecules with a dihaloalkane or a trihaloalkane thereby cyclizing via a thioether linkage at two or more cysteines in each of the plurality of linear molecules.

13. The method of claim 12, wherein the dihaloalkane is a, a’ dibromoxylene (DBX).

14. The method of any one of claims 6-10, comprising performing in vitro translation of a plurality of molecules according to Formula Ille and / or Formula Illf with a p- or m- cyanopyridylalanine-loaded elongation tRNA, to thereby produce a plurality of linear molecules according to AUG-Cys-[amino acid]m-[PhSec]-[amino acid]n-CNpyrA-[linker]- [mRNA], thereby cyclizing via thiazoline formation.

15. The method of any one of claims 6-11, wherein (NiWW) of Formula Illa or (N1N2K) of Formula Illb does not encode a cysteine.

16. The method of claim 6, wherein at least one of (NiWW) of Formula Illa or (N1N2K) of Formula Illb encodes for a cysteine.

17. The method of claim 16, wherein cyclizing comprises a cysteine-based cyclization.

18. The method of claim 17, wherein cyclizing comprises dibromoxylene mediated thioether cyclization.

19. The method of any one of claims 6-18, wherein forming a dehydroalanine comprises treating each of the plurality of molecules with H2O2 to thereby generate the dehydroalanine at the PhSec position.

20. A method for screening for a targeting cyclic peptide, the method comprising: a. providing an mRNA display library according to any one of claims 1-5 or producing an mRNA display library according to the method of any one of claims 6-19,b. bringing the mRNA display library into contact with a target substance, and incubating the library and target substance; and c. selecting cyclic peptides that bind the target substance.

21. The method of claim 20, further comprising after step b, a step of denaturing the target substance.

22. The method of claim 21, comprising denaturing with guanidine.

23. The method of any one of claims 20-22, wherein the cyclic peptide covalently binds the target substance.

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