Methods for making and using amber-specific phage display libraries

Abstract

Embodiments of the present disclosure relate to methods for constructing phage display libraries, wherein at least 90% of the combined regions in the phage display library include at least one in-frame amber codon. Further embodiments of the present disclosure relate to the formed phage display libraries. Other embodiments of the present disclosure relate to methods for selecting peptides or proteins that bind to a desired target (such as a ligand binding site of a desired target) by utilizing the phage display libraries of the present disclosure. Further embodiments of the present disclosure relate to peptides screened from the phage display libraries and methods of the present disclosure, such as inhibitors of sirtuin 2 or inhibitors of ENL.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 813,340, filed on March 4, 2019. The entire contents of the above application are incorporated herein by reference.

[0003] A note about federally funded research

[0004] This invention was made with government support under Grant No. R01CA161158 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0005] Development of this invention was supported in part by the Welch Foundation under Grant No. A-1715. Background Art

[0006] Phage display technology has many limitations, including the limited ability to combine phage display and chemical modification when generating peptide libraries, and the inclusion of non-canonical amino acids in cell surface display. Various embodiments of the present disclosure address the above limitations. Summary of the invention

[0007] In some embodiments, the present disclosure relates to a method for constructing a phage display library by the following steps: (a) providing an original A phage display library, wherein the original phage display library comprises a plurality of nucleic acids, wherein the plurality of nucleic acids comprises nucleic acids having a phage capsid protein gene comprising a combined region, wherein the combined region of at least some of the nucleic acids comprises at least one in-frame amber codon; (b) transforming the original phage display library into a bacterial host cell; (c) inducing expression of the phage capsid protein gene in the transformed bacterial host cell, wherein expression of the phage capsid protein from the nucleic acid comprising only in-frame sense codons in the phage capsid protein gene renders the bacterial host cell immune to infection by a helper phage; (d) infecting the transformed bacterial host cell with a helper phage, wherein the helper phage encodes and expresses a gene that renders the infected bacteria resistant to an antibiotic; (e) growing the infected and uninfected bacterial host cells in a medium containing an antibiotic, wherein (f) extracting the nucleic acid from the bacterial cell host cell; (g) transforming the extracted nucleic acid into an amber-suppressive bacterial host strain, wherein the amber-suppressive bacterial host strain is co-infected with a knockout helper phage that does not express the phage capsid protein gene, and wherein the transformation allows for the selective production of phage particles from cells containing nucleic acid for the phage capsid protein gene with at least one in-frame amber codon in the combined region; and (h) purifying the nucleic acid from the produced phage particles to provide a phage display library, wherein at least 90% of the combined regions in the phage display library include at least one in-frame amber codon.

[0008] Other embodiments of the present disclosure relate to phage display libraries comprising a plurality of nucleic acids. The plurality of nucleic acids include nucleic acids having a phage capsid protein gene containing a combination region, wherein at least 90% of the combination regions in the phage display library include at least one in-frame amber codon. Further embodiments of the present disclosure relate to methods for selecting peptides or proteins that bind to a desired target by the following steps: (a) providing a phage display library of the present disclosure; (b) transforming the phage display library into a bacterial host cell, wherein the bacterial host cell is capable of translating the combination region of the phage capsid protein gene so that at least one in-frame amber codon encodes a desired non-classical amino acid, wherein the bacterial host cell is co-infected with a knockout helper phage that does not express the phage capsid protein gene, wherein the transformation allows the production of phage particles containing a phage capsid protein having a desired non-classical amino acid in the combination region; (c) screening phage particles for the desired target, wherein the screening results in the selection of phage particles having a phage capsid protein combination region that binds to the desired target; and (d) identifying the amino acid sequence of the selected phage particle combination region.

[0009] Other embodiments of the present disclosure relate to methods for selecting a peptide that binds to a ligand binding site of a desired target by the following steps: (a) providing a phage display library having a plurality of nucleic acids, wherein the plurality of nucleic acids includes a nucleic acid having a phage capsid protein gene, the gene comprising a combined region, wherein at least 90% of the combined regions in the phage display library include at least one in-frame amber codon, and the at least one in-frame amber codon is located within a coding region for a peptide that binds to a ligand binding site of a desired target; (b) transforming the phage display library into a bacterial host cell, wherein the bacterial host cell is capable of translating the combined regions of the phage capsid protein gene such that at least one in-frame amber codon is located within a coding region for a peptide that binds to a ligand binding site of a desired target; The invention relates to a method for producing a phage particle comprising a phage capsid protein having a desired non-classical amino acid in a combined region; (c) screening the phage particles against a desired target, wherein the screening results in the selection of phage particles having a combined region of a phage capsid protein that binds to a ligand binding site of the desired target, wherein the non-classical amino acid acts as a ligand to guide the combined region of the phage capsid protein to the ligand binding site of the desired target; and (d) identifying the amino acid sequence of the combined region of the selected phage particle.

[0010] Other embodiments of the present disclosure relate to peptides screened from the phage display libraries and methods of the present disclosure. For example, in some embodiments, the present disclosure relates to compositions comprising sirtuin 2 inhibitors. In some embodiments, the present disclosure relates to compositions comprising ENL inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 provides a method for making a phage display library ( Figure 1A ) and using phage display libraries to select peptides or proteins containing non-classical amino acids that bind to targets ( Figure 1B ) method scheme.

[0012] Figure 1C An embodiment of active site directed ligand evolution using phage display is illustrated. Figure A shows the target and ligand binding to form a protein-ligand complex. The target is a biomacromolecule, which is RNA or protein. Figure B illustrates phage-assisted active site directed ligand evolution to identify high affinity ligands for protein targets. Non-classical amino acids containing ligands are incorporated into the randomized peptide library displayed by phage to promote binding to the protein target. Amino acid residues flanking the non-classical amino acids containing ligands provide additional interactions with the protein target to improve binding.

[0013] Figure 1D The structures of various non-classical amino acids are shown.

[0014] Figure 2 Schematic diagram of the method for constructing an amber-codon-obligate phage display library by superinfection selection. After cloning, the original phagemid library is used to transform Escherichia coli with F pili ( Figure 2 ). The expression of pIII was induced with IPTG, and shortly thereafter, the cells were superinfected with CM13 helper phage ( Figure 2 Expression of pIII in cells containing only a copy of the library containing sense codons renders these cells immune to superinfection ( Figure 2 ). The medium was changed to one containing kanamycin, allowing the selective growth of cells with copies of the library containing the in-frame amber codon ( Figure 2 ). Cells carrying copies of the library with deleterious mutations will also pass this selection. Purification of phagemid libraries ( Figure 2 ) and used to transform the amber suppressor strain DH5α of Escherichia coli ( Figure 2 ). Complementation of the phagemid with a pill-knockout helper phage in E. coli DH5α allowed production of phagemid particles only from cells that had copies of the peptide-pIII library containing an in-frame amber codon.

[0015] Figure 3 illustrates the results of superinfection selection. Figure 3A Shown is the percentage of a population susceptible to superinfection (i.e., clones containing amber codons) monitored by growth on kanamycin during two rounds of superinfection selection. An increase in the number of cells susceptible to superinfection between the first and second rounds indicates successful selection of library clones containing amber codons. Figure 3B Sequencing analysis after the second round of superinfection selection is shown, confirming the presence of an in-frame amber codon in each clone.

[0016] FIG. 4 illustrates the genetic incorporation of non-canonical amino acids (ncAAs) into phage-displayed peptide libraries. Figure 4A A schematic diagram of the three-plasmid system used to incorporate ncAAs into the peptide library is provided. Figure 4B The structures of phenylalanine derivatives 1-4 are shown. Figure 4C Phage yields in the presence and absence (NA) of 4 mM ncAA 1-4 are shown. Yields are shown in millions of colony forming units per milliliter of culture medium. Error bars represent one standard deviation of the mean of at least three independent experiments. Figure 4D The structures of lysine derivatives 5-7 are shown. Figure 4E Phage production is shown in the presence and absence (NA) of lysine derivatives 5 and 6. Production is shown in millions or hundreds of thousands of colony forming units for compounds 5 and 6, respectively. Error bars represent one standard deviation of the mean of three independent experiments.

[0017] FIG. 5 illustrates the amino acid sequences of selected ncAA-containing peptides. Figure 5A Amino acid sequences of peptides isolated after the third and fourth rounds of selection against streptavidin are shown. Figure 5B The amino acid sequences of peptides isolated after the third round of selection against SIRT2 are shown. X represents the position of the ncAA mBrF or BuK, which is determined by the position of the amber codon in the DNA sequence.

[0018] FIG. 6 illustrates molecular dynamics simulations and inhibition of human sirtuins (SIRT) in vitro and in vivo. Fig. 6A Molecular dynamics simulation of SIRT2 in complex with peptide S2P03 is shown (frame 95 / 1000). Hydrophobic residues in SIRT2 that interact with residues flanking thBuK in the peptide are shown in orange. Figure 6B Inhibition of human sirtuins 1-3 by S2P04 (left) and TB (right) is shown. Error bars represent one standard deviation of the mean of at least two independent experiments. Figure 6C Shown are representative immunoblots of α-tubulin acetylation and total α-tubulin in MCF-7 and HEK 293T cells treated with 50 μM S2P04, TB, or vehicle (0.25% DMF) for 24 hours.

[0019] FIG. 7 illustrates a method for selecting an ENL inhibitor. Fig. 7A Schematic diagrams illustrating the interactions between protein targets and ligands are provided. Figure 7B Genetic incorporation of ligand-fused ncAAs into phage-displayed peptides for active site-directed binding to protein targets, followed by stringent washing and elution to select phages with high affinity and selectivity, is shown. Figure 7C The crystal structure of the H3K9cr peptide in complex with ENL from PDB entry 5HJB is shown. CrK, cyan, in complex between F59 and Y78, green.

[0020] Figure 8 provides the structures and properties of various ENL inhibitors. Fig. 8A The structure of 7CrKEnl1 selected from phage display is shown. Figure 8B The structure of 7CrKEnl2 selected from phage display is shown. Figure 8C Fluorescence polarization analysis of 5-FAM-conjugated 7CrkENL1 binding to ENL is shown. Fig.8D Fluorescence polarization analysis of 5-FAM-conjugated 7CrkENL2 binding to ENL is shown. The table shows the percentage of first and last round ligands, the enrichment ratio and the dissociation constant (Kd) of the two ligands. DETAILED DESCRIPTION

[0021] It should be understood that the above general description and the following detailed description are illustrative and explanatory and do not limit the subject matter for which patent protection is claimed. In this application, the use of the singular includes the plural, the word "a" or "a" means "at least one / a", and the use of the word "or" means "and / or", unless otherwise specifically stated. In addition, the use of the term "including" and other forms, such as "including" and "containing" are not restrictive. At the same time, unless otherwise specifically stated, terms such as "element" or "component" include elements or components of one unit and elements or components including more than one unit.

[0022] The section headings used herein are for organizational purposes and should not be construed as limiting the contents. All documents or portions of documents cited in this application, including but not limited to patents, patent applications, articles, books, and treaties, are hereby incorporated by reference in their entirety for any purpose. When one or more of the combined documents and similar materials conflict with the definition of a term in this application, the application shall prevail.

[0023] Phage display has several advantages over traditional random screening methods used in drug discovery. These advantages include simplicity, cost-effectiveness, and speed.

[0024] For example, a library can be rapidly screened for binding to a target and the 'selectants' identified by DNA sequencing. Similarly, using standard and rapid molecular biology methods, a wide variety of foreign peptide or protein libraries can be displayed on the surface of phages, rather than using genetically engineered protein or peptide variants alone.

[0025] Peptide phage display using large libraries accompanied by high-throughput screening has played an important role in the development of clinically useful peptides and peptide mimetics. For example, in clinical applications such as endocrinology, oncology, urology, and obstetrics and gynecology, peptides are now widely used as therapeutic drugs and diagnostic methods. In addition, peptides often have many advantages over proteins (including antibodies) in terms of manufacturing cost, activity, stability, immunogenicity, and organ penetration efficiency.

[0026] In fact, the current annual market size of peptide drugs is $300-500 million, with an annual growth rate of 25%. In addition, several peptide drugs developed using phage display have been approved for clinical use or are undergoing clinical trials.

[0027] The first marketed peptibody, Nplate1 (Romiplostim, AMG 531), is a thrombopoietin receptor agonist used to treat immune thrombocytopenic purpura. The peptide component of this peptibody has undergone extensive development, but it originated from a phage-displayed peptide library.

[0028] The search for high affinity peptides and proteins useful for diagnostic and therapeutic purposes can also be achieved by alternative display methods that have their origins in the original phage display concept. These methods can be broadly classified as cell surface systems or cell-free display systems. However, cell-based methods are limited by the size of the library due to DNA conversion efficiency and any toxicity of the displayed molecules to the host cells.

[0029] Chemical modification strategies have also been used to improve the pharmacokinetics of peptides. For example, functional groups (such as phosphates) are added to the N-terminus of peptides to improve binding to serum albumin or other serum proteins and extend half-life. However, there is no effective technology that can combine phage display and chemical modification to generate peptide libraries. In addition, there is currently no effective strategy to incorporate non-classical amino acids into cell surface display.

[0030] The above limitations become more evident in areas such as oncology, where potential ligand targets must also carry epigenetic modifications in addition to the 20 natural amino acids. Various embodiments of the present disclosure address the above limitations.

[0031] In some embodiments, the present disclosure relates to amber-obligated phage display libraries that can be used to efficiently produce peptides or proteins containing non-classical amino acids. In some embodiments, the present disclosure relates to methods for constructing the above-mentioned amber-obligated phage display libraries. Other embodiments of the present disclosure relate to methods for selecting peptides or proteins that bind to a desired target by utilizing the phage display libraries of the present disclosure. Further embodiments of the present disclosure relate to peptides selected by utilizing the methods of the present disclosure, such as compositions comprising one or more sirtuin 2 or ENL inhibitors.

[0032] In some embodiments, the present disclosure relates to a general strategy for incorporating non-classical amino acids that are ligands of a desired target (or that are chemically modified to form ligands) into a phage display library. In some embodiments, the present disclosure relates to the construction of a phage display library, wherein the incorporated non-classical amino acids direct the binding of phage-displayed peptides to the ligand binding site of the desired target and facilitate the selection of potent ligands that bind directly to the ligand binding site of the desired target.

[0033] Further embodiments of the present disclosure relate to the inclusion of non-classical amino acids. In some embodiments, the included non-classical amino acids are the same as post-translationally modified amino acids that directly interact with epigenetic enzymes and epigenetic reader proteins. In some embodiments, the included non-classical amino acids are the same as unnatural amino acids that bind to active sites of cellular proteins.

[0034] Amber obligate phage display library

[0035] In some embodiments, the present disclosure relates to a phage display library having a plurality of nucleic acids. The plurality of nucleic acids includes nucleic acids having a phage capsid protein gene containing a combinatorial region. In some embodiments, at least 90% of the combinatorial regions in the phage display library include at least one in-frame amber codon.

[0036] Other embodiments of the present disclosure relate to methods for constructing a phage display library of the present disclosure. Figure 1A In some embodiments described in the claims, the methods of the present disclosure include: (a) providing an original phage display library, wherein the original phage display library includes a plurality of nucleic acids having phage capsid protein genes, the nucleic acids including combined regions, wherein the combined regions of at least some of the nucleic acids include at least one in-frame amber codon (step 10); (b) transforming the original phage display library into a bacterial host cell (step 12); (c) inducing expression of the phage capsid protein gene in the transformed bacterial host cell, wherein expression of the phage capsid protein from the nucleic acid containing only in-frame sense codons in the phage capsid protein gene renders the bacterial host cell immune to infection by a helper phage (step 14); (d) infecting the transformed bacterial host cell with a helper phage, wherein the helper phage encodes and expresses a gene that renders the infected bacteria resistant to an antibiotic (step 16); (e) infecting the infected and uninfected bacterial host cells with a helper phage. (f) extracting nucleic acid from the bacterial cell host cell (step 20); (g) transforming the extracted nucleic acid into an amber-suppressive bacterial host strain (step 22), wherein the amber-suppressive bacterial host strain is co-infected with a knockout helper phage that does not express the phage capsid protein gene, and wherein the transformation allows for the selective production of phage particles from cells containing nucleic acid whose combined region comprises a phage capsid protein gene having at least one amber codon in frame (step 24); and (h) purifying nucleic acid from the produced phage particles to provide a phage display library of the present disclosure (step 26).

[0037] The methods of the present disclosure can have many embodiments. For example, in some embodiments, the original phage display library is prepared by site-directed mutagenesis.

[0038] In some embodiments, the phage display library comprises a plurality of phage. In some embodiments, the plurality of nucleic acids in the phage display library comprises nucleic acids of phage.

[0039] In some embodiments, the phage display library comprises a plurality of phagemids. In some embodiments, the nucleic acids in the phage display library comprise nucleic acids of phagemids.

[0040] In some embodiments, the bacteriophage capsid protein gene is a PIII gene. In some embodiments, the bacterial host cell comprises Escherichia coli with F pili.

[0041] In some embodiments, the bacteriophage capsid protein gene is located near an IPTG-inducible promoter. In some embodiments, the bacteriophage capsid protein is expressed by exposing the bacterial host cell to IPTG.

[0042] In some embodiments, the helper phage is a CM13 helper phage. In some embodiments, the helper phage encodes and expresses a gene that renders the infected bacteria resistant to kanamycin. Thus, in some embodiments, selective growth occurs in a culture medium containing kanamycin.

[0043] The phage display libraries of the present disclosure can also have many embodiments. For example, in some embodiments, more than 90% of the combined regions in the phage display library include at least one in-frame amber codon. In some embodiments, more than 95% of the combined regions in the phage display library include at least one in-frame amber codon. In some embodiments, more than 99% of the combined regions in the phage display library include at least one in-frame amber codon.

[0044] In some embodiments, the combined region in at least some of the nucleic acids in the phage display library comprises a plurality of in-frame amber codons. In some embodiments, the plurality of in-frame amber codons are randomly distributed throughout the combined region. In some embodiments, at least one in-frame amber codon is within a region of a peptide that binds to a ligand binding site of a protein.

[0045] In some embodiments, the combined regions encode a peptide. In some embodiments, the combined regions encode a protein.

[0046] Methods for selecting peptides or proteins that bind to a desired target

[0047] Other embodiments of the present disclosure relate to methods for selecting peptides or proteins that bind to a desired target by utilizing the phage display library of the present disclosure. Figure 1BIn some embodiments described in the claims, the method of the present disclosure comprises: (a) providing a phage display library of the present disclosure (step 30); (b) transforming the phage display library into a bacterial host cell (step 32), wherein the bacterial host cell is capable of translating the combined region of the phage capsid protein gene so that the in-frame amber codon in the combined region encodes the desired non-classical amino acid, wherein the bacterial host cell is co-infected with a knockout helper phage that does not express the phage capsid protein gene, and, wherein the transformation allows the production of phage particles containing phage capsid proteins with the desired non-classical amino acid in the combined region (step 34); (c) screening the phage particles against the desired target (step 36), wherein the result of the screening is the selection of phage particles having the combined region of the phage capsid protein that binds to the desired target (step 38); and (d) identifying the amino acid sequence of the selected phage particle combined region to identify the selected peptide or protein (step 40).

[0048] In a more specific embodiment, the present disclosure relates to a method for selecting a peptide that binds to a ligand binding site of a desired target by the following steps: (a) providing a phage display library comprising a plurality of nucleic acids, wherein the plurality of nucleic acids comprises a nucleic acid having a phage capsid protein gene, the gene comprising a combined region, wherein at least 90% of the combined regions in the phage display library comprise at least one in-frame amber codon, and the at least one in-frame amber codon is located within the coding region of a peptide that binds to a ligand binding site of a desired target; (b) transforming the phage display library into a bacterial host cell, wherein the bacterial host cell is capable of translating the combined regions of the phage capsid protein gene such that at least one in-frame amber codon is located within the coding region of the peptide that binds to the ligand binding site of the desired target; The internal amber codon encodes a desired non-classical amino acid, wherein the bacterial host cell is co-infected with a knockout helper phage that does not express the phage capsid protein gene, and, wherein the transformation allows the production of phage particles containing the phage capsid protein with the desired non-classical amino acid in the combination region; (c) screening the phage particles against the desired target, wherein the screening results in the selection of phage particles having the phage capsid protein combination region that binds to the ligand binding site of the desired target, wherein the non-classical amino acid acts as a ligand to guide the phage capsid protein combination region to the ligand binding site of the desired target; and (d) identifying the amino acid sequence of the combination region of the selected phage particles.

[0049] The selection methods disclosed herein can also be implemented in many ways. For example, in some embodiments, the screening step is performed by affinity selection for a desired target. In some embodiments, the screening is performed by the following steps: (1) incubating phage particles with the desired target immobilized on a surface; (2) separating unbound phage particles from phage particles that bind to the desired target; and (3) isolating the bound phage particles. In some embodiments, the separation step may be performed by washing unbound phage particles away from phage particles that bind to the desired target.

[0050] In some embodiments, the desired target can be biotinylated and immobilized on a streptavidin surface. In some such embodiments, screening can be performed by: (1) incubating phage particles with the desired target immobilized on a streptavidin surface; (2) separating unbound phage particles from phage particles bound to the desired target by a washing step; and (3) separating bound phage particles by competitively eluting bound phage particles with biotin or by adding an acidic buffer (e.g., pH 2) to release bound phage particles.

[0051] In some embodiments, the screening can be performed multiple times. For example, in some embodiments, the screening step further comprises: (1) transforming the selected phage particles into bacterial host cells to allow production of additional phage particles; and (2) rescreening the phage particles according to step (c) (as outlined above and described in detail below). Figure 1B In some embodiments, this further screening can be repeated multiple times.

[0052] In some embodiments, the screening results in the selection of phage particles having a phage capsid protein combinatorial region that binds to a ligand binding site of a desired target. In some embodiments, non-classical amino acids serve as ligands to direct the phage capsid combinatorial region to the ligand binding site of a desired target. In some embodiments, the ligand inhibits the activity of one or more epigenetic enzymes or epigenetic reader proteins.

[0053] A variety of methods can also be used to identify the amino acid sequence of the combined regions of the selected phage particles. For example, in some embodiments, identification is performed by sequencing the combined regions of the selected phage particles. In some embodiments, identification is performed by: (1) purifying the selected phage particles; (2) isolating the nucleic acid from the selected phage particles; and (3) sequencing the combined regions of the nucleic acid.

[0054] The methods of the present disclosure can also utilize various bacterial host cells. For example, in some embodiments, the bacterial host cell is an amber suppressor bacterial host strain. In some embodiments, the bacterial host cell comprises an amber suppressor tRNA that has been aminoacylated by a desired non-classical amino acid by a homologous aminoacyl-tRNA synthetase. In some embodiments, the desired non-classical amino acid is incorporated during translation by the amber suppressor tRNA in response to an in-frame amber codon.

[0055] In a more specific embodiment, the bacterial host cell is a bacterium that has been transformed with three plasmids: (1) a plasmid encoding a bacteriophage capsid protein gene having at least one in-frame amber codon in the combining region; 2) a plasmid encoding an amber suppressor tRNA and a cognate aminoacyl-tRNA synthetase capable of attaching a desired non-classical amino acid to the suppressor tRNA; and (3) a helper phage encoding all essential phage proteins except the phage capsid protein containing the combining region.

[0056] Various non-classical amino acids can be incorporated into the combination regions of the phage capsid protein using the methods disclosed herein. For example, in some embodiments, the desired non-classical amino acids include, but are not limited to, phenylalanine-derived non-classical amino acids, lysine-derived non-classical amino acids, and combinations thereof. In some embodiments, the desired non-classical amino acids include, but are not limited to, S-2-amino-8-oxononanoic acid, S-amino-8-oxonadecanoic acid, N 6 -Acetyl-L-lysine, N 6 -Propionyl-L-lysine, N 6 -Butyryl-L-lysine, N 6 -crotonyl-L-lysine, N 6 -(prop-2-yn-1-yl)-lysine, N 6 -Methyl-N 6 -N 6 -(prop-2-yn-1-yl)-lysine, and combinations thereof. The structures of the above non-classical amino acids are as follows Figure 1D shown.

[0057] The method disclosed herein can be used to screen peptides or proteins in the combined regions of phage capsid proteins for a variety of desired targets. For example, in some embodiments, the desired targets include, but are not limited to, peptides, proteins, enzymes, small molecules, cell receptors, antigens, ligand binding sites of proteins, active sites of desired targets, active sites of proteins, and combinations thereof. In some embodiments, the desired target is a ligand binding site of a protein. In some embodiments, the ligand binding site is an active site of a protein. In some embodiments, the ligand binding site is an allosteric site of a desired protein. In some embodiments, the desired target is an enzyme, such as sirtuin 2.

[0058] Sirtuin 2 inhibitors

[0059] Other embodiments of the present disclosure relate to peptide inhibitors of sirtuin 2 selected by utilizing the methods of the present disclosure. More specific embodiments of the present disclosure relate to compositions comprising sirtuin 2 inhibitors. In some embodiments, inhibitors of sirtuin 2 include, but are not limited to: (a) CTVXTSL; (b) TCTVXIG; (c) CTFXVPT; (d) CTVXI; (e) ZTVXI; or (f) combinations thereof. In some embodiments, X is a non-classical amino acid. In some embodiments, Z is a non-classical amino acid or aminobutyric acid.

[0060] In some embodiments, the inhibitor of sirtuin 2 is NH2-CTVXTSL-NH2 (S2P03). In some embodiments, the inhibitor of sirtuin 2 is NH2-TCTVXIG-NH2 (S2P04). In some embodiments, the inhibitor of sirtuin 2 is NH2-CTFXVPT-NH2 (S2P07). In some embodiments, the inhibitor of sirtuin 2 is Ac-CTVXI-NH2 (Ac-S2P04-5). In some embodiments, the inhibitor of sirtuin 2 is NH2-ZTVXI-NH2, wherein Z is aminobutyric acid (S2P04(Abu)-5).

[0061] Inhibitors of SNL

[0062] Further embodiments of the present disclosure relate to peptide inhibitors of SNL selected by utilizing the methods of the present disclosure. More specific embodiments of the present disclosure relate to compositions comprising SNL inhibitors. In some embodiments, inhibitors of SNL include, but are not limited to: (a) DIWCFXG; (b) SXDIWCF; or (c) combinations thereof. In some embodiments, X is a non-classical amino acid.

[0063] In some embodiments, the inhibitor of SNL is DIWCFXG. In some embodiments, the inhibitor of SNL is SXDIWCF.

[0064] Applications and Advantages

[0065] The methods and phage display libraries disclosed herein can have various advantages and applications. For example, in some embodiments, the methods disclosed herein allow for efficient incorporation of non-classical amino acids into displayed peptides to expand the structural and functional diversity of phage display libraries. In addition, the methods disclosed herein can be used to generate libraries suitable for iterative rounds of selection and amplification required for directed evolution, thereby allowing for rapid identification of novel peptide ligands and monoclonal antibodies from large polypeptide libraries carrying functional groups not limited to those found in the 20 classical amino acids.

[0066] In more specific embodiments, the phage display libraries and selection methods disclosed herein can be used for ligand binding site directed ligand evolution to identify inhibitors containing non-classical amino acids that are active both in vitro and in vivo. In some embodiments, the inhibitors may include zinc-dependent and NAD-dependent HDAC inhibitors that bind to the active site region of the enzyme with high specificity.

[0067] In some embodiments, the phage display libraries and selection methods of the present disclosure can be used to generate peptide libraries to screen and discover lead molecules with increased drug efficacy in epigenetic regulation.

[0068] Reference will now be made to more specific embodiments of the present disclosure and to experimental data supporting these embodiments. However, applicants note that the following disclosure is for illustrative purposes only and is not intended to limit the scope of the claimed subject matter in any way.

[0069] Example 1. Amber-codon-specific phage display system with active site-directed ligand evolution Assisting in drug discovery

[0070] Phage display technology enables the rapid identification of novel peptide ligands and single-molecule antibodies from huge peptide libraries. However, these libraries are limited to twenty classical amino acids. Using orthogonal aminoacyl-tRNA synthetases and tRNAs, the repertoire can be expanded to include non-classical amino acids (ncAAs). However, inefficient ncAA inclusion biases phage libraries, limiting the widespread adoption of this technology.

[0071] In this embodiment, the applicant reports a method known as superinfection-selection, which is used to construct a phage display library in which each clone contains an in-frame amber codon-encoded ncAA. This amber-codon-specific library eliminates the bias caused by the inefficient inclusion of ncAA in the display peptide. The applicant proves that these libraries are suitable for the selection and amplification of the iterative rounds required for directed evolution. Using this technology, the applicant has generated a peptide library containing a genetically encoded lysine post-translational modification, and the library is used for the active site directed ligand evolution of peptides that inhibit sirtuin 2 in vitro and in human cancer cells.

[0072] Phage display is a widely used tool for selecting peptide ligands and monoclonal antibodies from large combinatorial peptide libraries. In this technique, a DNA library is cloned as a genetic fusion to a gene encoding a bacteriophage capsid protein. When the fusion protein is expressed and integrated into the viral capsid, the peptide library is 'displayed' on the surface of the virus. This configuration establishes a physical link between the displayed peptide and its encoding DNA, allowing for amplification of the library in a bacterial host and facile peptide sequence determination after several rounds of target-directed selection. Phage display libraries have been used for many applications, from epitope mapping to identifying inhibitors of enzymes and protein-protein interactions, and a variety of phage-derived peptides and antibodies have been approved for pharmaceutical use or are in late-stage clinical trials. However, because phage display libraries are expressed in Escherichia coli, the peptides in these libraries are restricted to the 20 canonical amino acids, which greatly limits the sequence space they can be used to explore.

[0073] Using orthogonal aminoacyl-tRNA synthetase (aaRS) and tRNA pairs, researchers have been able to artificially expand the genetic code to incorporate many non-canonical amino acids (ncAAs), assuming various functions, across a variety of host organisms. In this technology, the ncAA is incorporated during translation in response to a redefined nonsense codon by an orthogonal suppressor tRNA that has been selectively aminoacylated with the ncAA by its cognate aaRS. Applying nonsense suppression to phage display, previous studies have demonstrated the genetic incorporation of several ncAAs into proteins and peptides displayed on the surface of phages. However, the selection of ncAA-containing peptides has proven to be extremely challenging, primarily due to the propagation bias of ncAA-containing phage clones. This bias is caused by inefficient stop codon suppression, which limits the production of peptide-capsid fusion proteins, thereby reducing the propagation rate of ncAA-containing clones relative to those containing only canonical amino acids. Although ncAA-containing peptides and proteins have been selected from these types of libraries, the requirement for extensive optimization of efficient aaRS-tRNA pairs and expression conditions has prevented widespread adoption of this technology.

[0074] In order to overcome the challenge of expression bias, the applicant has developed a general method for constructing a phage display library in which each clone contains an in-frame amber codon-encoded ncAA. The applicant has demonstrated in this embodiment that this 'amber codon-specific' library is suitable for displaying peptides containing various ncAAs, thereby greatly expanding the application of phage display. The applicant has verified the method of library construction by generating an amber-codon-specific heptapeptide library displayed at the N-terminus of the minor coat protein (pIII) of the M13 phage. The applicant further demonstrated that this type of library is suitable for amplification and selection of iterative rounds of ncAAs derived from phenylalanine and derived from lysine. Finally, the applicant uses a library containing genetically encoded post-translational modifications of lysine to select peptides that bind to the active site of the lysine deacylase deacetylase 2. The applicant has demonstrated that the derivatives of these peptides are effective inhibitors of lysine deacetylation catalyzed by deacetylase 2 in vitro and in vivo.

[0075] Example 1.1. Construction of an amber-codon-specific phage display library

[0076] A phage display library capable of displaying peptides containing ncAAs was constructed by site-directed saturation mutagenesis using degenerate primers covering 32 codons (including one amber codon) and encoding all 20 classical amino acids. Previous methods for constructing amber-codon-specific libraries utilized primer pairs in which the position of the amber codon (and thus the ncAA) was fixed and all other positions were randomized. Applicants sought to construct a more diverse library in which the position of the amber codon could be randomly distributed throughout the peptide sequence. In order to construct such a library, a primer pair combination could theoretically be used in which the position of the amber codon was altered to cover every position in the sequence. In addition to the possibility that the library may be biased by unequal mixing of cloning products, this primer combination approach does not address common problems associated with primer-based DNA library construction. Synthesis errors, such as nucleotide deletions, are inevitable in oligonucleotide primers, despite their low frequency of occurrence.

[0077] Therefore, when using this method, it is inevitable to introduce clones without amber codons into the library. These clones that do not rely on stop codon suppression will have a significant growth advantage over amber-encoded clones. During phage production, this propagation bias will cause these clones to be enriched and gradually mask the amber-encoded clones. Applicants observed this problem in parallel studies involving fixation of an amber-encoding site.

[0078] In view of the lack of alternative methods to solve this problem, the applicant has developed a universal method for constructing amber-encoded-specific phage libraries that allow random distribution of ncAA. The method utilizes a phenomenon of filamentous (Ff) phage biology, known as superinfection immunity. In short, superinfection immunity describes the resistance of infected bacteria to further infection (superinfection) of the same type of phage. For E. coli infected with Ff phage, superinfection immunity is conferred by phage coat protein pill, which is expressed by resident phage in the host. Endogenously expressed pill combines with TolA, which is an E. coli cell surface protein and is used as a receptor for host adsorption by Ff phage. The combination of endogenous pill and TolA blocks the adsorption of competitive phages, and therefore also blocks their superinfection.

[0079] Applicants' strategy for constructing an amber-codon-specific phage display library includes two steps. In the first step, Applicants constructed the original library as a genetic fusion with the phage coat protein pill. The library was constructed with degenerate primers and, therefore, included sequences with amber codons and sequences with only sense codons. In the second step, referred to as "superinfection selection," Applicants used the principle of superinfection immunity to select library members containing amber codons and remove those library members containing only sense codons from the library pool.

[0080] The applicant first constructed an original combinatorial heptapeptide library fused to pill. To clone the library, the applicant introduced seven randomized (NNK) codons into the N-terminal genetic fusion of pill on the pADL-10b phagemid by site-directed saturation mutagenesis. After cloning, the applicant transformed electrocompetent E. coli Top10 F' with the purified library, producing 1.4 x 10 9 unique transformants (theoretical peptide diversity = 1.8 x 10 9 ).

[0081] Next, the applicant performed superinfection selection to select library clones containing in-frame amber codons. The applicant grew E. coli Top10 F' with the phagemid library to early logarithmic phase in medium supplemented with ampicillin and tetracycline, at which time the applicant induced the expression of pill by adding IPTG ( Figure 2 Under these conditions, full-length pill was expressed in cells with only the sense codon copy of the library. However, translation termination prevented pill expression in cells with the amber codon-containing copy of the library ( Figure 2 ).

[0082] Since endogenous pill confers superinfection immunity, the former are immune to superinfection and the latter are susceptible to superinfection. After inducing pill expression, the applicant infected the culture with CM13 helper phage carrying a gene that confers kanamycin resistance. After superinfection with CM13, the applicant changed the culture medium to a medium containing 25 μg mL -1 Kanamycin-containing medium allows the selective growth of cells that are susceptible to superinfection (i.e., those cells that have a copy of the library containing an in-frame amber codon) ( Figure 2 ).

[0083] After a round of super infection selection, the applicant sequenced clones in several libraries. The sequencing results showed that multiple clones containing harmful mutations in the peptide or pIII coding region and clones containing amber codons were also enriched. These mutations prevented the expression of functional pIII, thereby allowing the clones to pass the selection.

[0084] To eliminate these undesirable clonal populations, the applicant purified the phagemid library and used the purified library to transform E. coli DH5α. DH5α is an E. coli supE strain that incorporates glutamine in response to the amber codon. Therefore, expressing the library in this strain allows the production of full-length pill in clones containing the amber codon, but not in clones containing deleterious mutations.

[0085] The applicant used M13KO7 (pIII -) supplemented phagemid library, M13KO7 (pIII - ) is a helper phage that contains a nonsense (TAA) mutation in gene III and is therefore unable to produce its own pill. - ) complementation resulted in selective expression of phagemid particles from clones containing amber codons in the peptide sequence ( Figure 2 ). To confirm successful removal of harmful clones, the applicant sequenced several phagemids after expression. None of the clones expressed in DH5α that the applicant sequenced contained harmful mutations that would prevent pill expression. After passage through DH5α, the applicant purified the phagemid particles and used them to infect E. coli Top10 F' for another round of superinfection selection.

[0086] In two rounds of superinfection selection, the applicant monitored the enrichment of amber codons by measuring the percentage of the E. coli population that was susceptible to superinfection. In the first round, 18% of the population was susceptible to superinfection with the CM13 helper phage. This is consistent with theoretical calculations predicting that 19.9% ​​of the clones in a library of 7 NNK codons contain at least one amber codon (based on a binomial distribution, using an amber codon probability of 1 / 32). After the second round of selection, greater than 98% of the population was susceptible to superinfection, indicating that there was a large increase in the number of clones containing amber codons between rounds 1 and 2 ( Figure 3A ).

[0087] Sequencing of the library after the second round of superinfection selection confirmed the success of the selection, as all clones sequenced by the applicant (n=10) contained an amber codon in the random sequence ( Figure 3B ). These data demonstrate that superinfection selection is an effective strategy for generating amber-codon-obligatory phage display libraries.

[0088] Example 1.2. Genetic incorporation of ncAAs into phage-displayed peptides

[0089] The applicant used derivatives of pyrrolysyl-tRNA synthetase (PylRS) and their cognate amber suppressor tRNA In combination, the genetic inclusion of phenylalanine and lysine derivatives into the amber-codon-obligatory library was tested. Applicants first tested the inclusion of phenylalanine derivatives containing ortho- and meta-substituted phenyl rings. To include these ncAAs into the peptide library, Applicants utilized a previously reported PylRS mutant (PhdRS) containing alanine mutations at positions N346 and C348. To express phagemid particles, Applicants transformed E. coli Top10 with three plasmids: 1) pADL(NNK)7gIII, encoding the amber-codon-obligatory peptide-pIII library, 2) pEDF-PhdRS, containing the encoding PhdRS and genes, and 3) M13KO7 (pIII – ), encoding all essential phage proteins except pill ( Figure 4A ). The applicant supplemented with 4 mM of one of the four phenylalanine derivatives 1-4 ( Figure 4B When transformed cells were cultured in 2xYT medium supplemented with ncAA, phagemid particle production increased 36- to 83-fold compared to growth medium without ncAA supplementation ( Figure 4C ).

[0090] ncAA-dependent increase in particle production and The suppression of amber codons was consistent, indicating that the ncAA was successfully incorporated into the phage-displayed peptide library. The low level of background phagemid production in the absence of the ncAA is likely due to the incorporation of phenylalanine, as Applicants have shown that, albeit to a small extent, Mis-aminoacylation of phenylalanine is a common phenomenon in evolved PylRS.

[0091] After the applicant successfully incorporated phenylalanine derivatives, the applicant next tested the inclusion of lysine derivatives into the peptide library. The applicant selected two lysine derivatives to test their inclusion: N ε -Butyryl-lysine (5,BuK) and N ε -crotonyl-lysine (6,CrK) Figure 4D ). These two ncAAs represent natural post-translational modifications of lysine. Therefore, peptide libraries containing these ncAAs could prove useful for studying enzymes that naturally interact with these modifications.

[0092] To incorporate BuK and CrK, the applicant used a previously reported PylRS mutant (BuKRS) containing a tryptophan mutation at position Y384. The applicant has demonstrated that BuKRS Together, they can promote the efficient incorporation of BuK and CrK in response to amber codons in E. coli. - ) in E. coli Top10, which resulted in minimal particle production in the absence of ncAAs. However, addition of 5 mM BuK or CrK to the growth medium resulted in a nearly 100-fold increase in particle production, indicating that these ncAAs were successfully incorporated into the phage-displayed peptide library ( Figure 4E ).

[0093] Example 1.3. Maintaining library integrity during multiple rounds of amplification

[0094] Directed evolution experiments based on phage display require robust libraries that can withstand iterative rounds of amplification and targeted selection. If the superinfection-selection strategy used for library construction does not successfully remove all clones containing only sense codons, or if acquired mutations lead to the loss of amber codons during amplification, a reproductive bias favoring these sequences will hamper selection efforts.

[0095] To investigate the integrity of our amber-codon-specific library in the face of iterative rounds of amplification and selection, we performed a simulated selection against streptavidin. We expressed a phage-displayed heptapeptide library (approximately 5 x 10 10 cfu) were incubated with immobilized streptavidin target, unbound phagemid particles were removed by washing, and specifically bound particles were competitively eluted with 0.1 mM biotin. After each round of selection, the applicant amplified the eluted E. coli Top10 containing pEDF-PhdRS and M13KO7 (pIII - ) phagemid particles for further rounds of selection.

[0096] Applicants performed a total of four rounds of amplification and selection. Phagemid clones were sequenced after the third (n=6) and fourth (n=12) rounds. Notably, even after four rounds of amplification, all clones sequenced contained mBrF in the peptide sequence, as indicated by the presence of an in-frame amber codon ( Figure 5A ). These data demonstrate that the superinfection selection approach generates robust libraries that can withstand the iterative rounds of amplification required for directed evolution.

[0097] Example 1.4. Selecting N ε -Butyryl-lysine peptides as inhibitors of sirtuin 2

[0098] Sirtuins are a group of NAD + -dependent lysine deacylase, which catalyzes the + and N ε -acyl-lysine is converted to lysine and O-acyl-ADP ribose. As regulators of lysine acylation levels of proteins, sirtuins are considered key regulators of various cellular processes ranging from gene transcription and DNA repair to metabolism and aging. Therefore, sirtuins have become promising targets for therapeutic intervention in human diseases. Recently, inhibitors of sirtuin 2 (SIRT2), a sirtuin isoform that is primarily localized in the cytoplasm, have been shown to have significant anticancer activity in cellular and animal models of human cancer. SIRT2 inhibitors also show promise in the treatment of age-related neurodegenerative diseases. Therefore, there is currently great interest in the development of potent and selective SIRT2 inhibitors.

[0099] The applicants envision that phage-displayed peptide libraries containing genetically encoded acyl-lysine ncAAs can be used for active site-directed ligand evolution to reveal peptide sequences that bind to the SIRT2 active site. To this end, the applicants used an amber-codon-specific library to express a library of more than 300 million peptides containing BuK residues and selected sequences that bind to SIRT2 in this library.

[0100] Using pEDF-BuKRS and M13KO7 (pIII - ) E. coli strain, the applicant expressed the amber-codon-specific library and subjected the purified phagemid library to affinity selection against recombinant human SIRT2. In order to avoid catalytic removal of the butyryl modification during the selection process, the applicant expressed the amber-codon-specific library in the absence of NAD + The applicants isolated phagemid clones eluted after the third round of affinity selection and sequenced their DNA (n=20). Figure 5B Shown are peptide sequences of clones isolated after the third round of selection against SIRT2.

[0101] Phagemid sequencing after affinity selection for SIRT2 showed that the population had converged on a consensus sequence involving residues primarily adjacent to the BuK residue. Consistent with two previous studies screening modified peptide libraries for SIRT2, the applicant observed an enrichment of valine and leucine residues adjacent to the N-terminal position of BuK (-1 position). The applicant also observed a strong enrichment of phenylalanine at this position. The applicant observed a strong enrichment of amino acids with aliphatic side chains adjacent to the C-terminus of BuK (+1 position), with more than 50% of the isolated clones having isoleucine, valine, leucine or alanine at this position.

[0102] Together, these findings suggest a substrate preference for SIRT2 for sequences with hydrophobic residues adjacent to the modified lysine. To further understand how the selected peptides interact with the enzyme, the applicant performed molecular dynamics simulations of the peptides that form a complex with SIRT2. The simulation results showed that the flanking residues of the acyl lysine residues have hydrophobic interactions with the Phe235 and Leu239 residues of SIRT2 ( Fig. 6A ). This observation provides a possible explanation for why the applicants and others have observed a preference for hydrophobic residues at these positions in peptide substrates. Also consistent with previous findings, the applicants observed an enrichment of serine and threonine residues at the +3 position. It has been demonstrated that the threonine at the +3 position interacts with Gln265 of SIRT2 in a hydrogen bonding interaction, providing a reasonable explanation for this observation. Of all the sequenced clones selected by the applicants, 25% contained the consensus motif Cys-Thr-Val / Phe-BuK-Val / Ile.

[0103] It has been demonstrated that replacing the acyl lysine amide moiety of sirtuin substrates with slowly hydrolyzed thioamide analogs is an effective strategy for inhibiting sirtuins. ε -thiobutyryl-lysine (7,thBuK) instead of BuK, chemically synthesized each selected peptide containing the Cys-Thr-Val / Phe-Buk-Val / Ile motif, and tested the inhibition of SIRT2 by the synthetic peptides. Applicants used TB (Table 1) as a reference compound, which is a recently reported potent SIRT2 selective inhibitor.

[0104]

[0105] The inhibitory activities of selected peptides and TB are summarized in Table 1. Values ​​are expressed as mean ± SD of two independent experiments.

[0106] All three peptides tested by the applicants showed significantly better inhibition of SIRT2 than TB (up to 45-fold), with IC 50 The values ​​ranged from 68 to 101 nM (Table 1). Since the consensus motif found in the applicant's selection only included five of the seven residues in the library, the applicant also synthesized a truncated form of one of the peptides (S2P04-5) that contained only five residues of the consensus motif. The IC50 of S2P04-5 for inhibiting SIRT2 was 136 ± 29 nM, slightly higher than that of its parent peptide S2P04. This observation suggests that although the identity of the residues at the distal position of BuK may not be critical, interactions such as backbone hydrogen bonding may still contribute to the binding of the enzyme to the ligand.

[0107] Applicants tested the selectivity of S2P04 for SIRT2 by measuring its inhibitory properties against two other deacetylases that also recognize lysine butyrylation (SIRT1 and SIRT3). S2P04's inhibitory effect on SIRT2 was 2.7 times and 152 times stronger than SIRT1 and SIRT3, respectively ( Figure 6B ). This selectivity was comparable to that measured for TB, however, for each enzyme, S2P04 was at least 33-fold more potent than TB.

[0108] Finally, the applicant tested the ability of S2P04 (a selected peptide with moderate potency) to inhibit SIRT2 in human embryonic kidney cells (HEK293T) and breast cancer cells (MCF-7). To test the inhibitory effect on SIRT2, the applicant incubated the cell culture in medium containing 50 μM S2P04, TB or vehicle control for 24 hours. After incubation with the inhibitor, the applicant measured the acetylation level of lysine 40 of α-tubulin, which is a known deacetylation substrate of SIRT2.

[0109] Applicants observed that acetylation of α-tubulin increased in both MCF-7 and HEK 293T cells treated with S2P04, but the effect was greatest in the MCF-7 cell line ( Figure 6C ). MCF-7 cells treated with TB also showed increased α-tubulin acetylation compared to the control group. However, the effect was lower than that observed in S2P04. This observation is consistent with the low efficacy of TB in vitro. No increase in α-tubulin acetylation was observed in HEK 293T cells treated with TB.

[0110] After observing that S2P04 inhibited SIRT2 in vivo, the applicant synthesized two shortened derivatives of the peptide: Ac-S2P04-5 and S2P04(Abu)-5. The former contains an N-terminal acetylation, which may contribute to cell permeability and proteolytic stability, and the latter contains an aminobutyric acid (Abu) to replace cysteine, which is more redox stable. Although both peptides inhibited SIRT2 in vitro (Table 1), neither peptide resulted in a greater increase in acetylation levels in vivo compared to S2P04. These data demonstrate that phage display libraries constructed by superinfection selection methods can be used for active site-directed ligand evolution to identify inhibitors containing ncAAs with in vitro and in vivo activity.

[0111] Example 1.5. Discussion

[0112] Applicants have developed a method for constructing a phage-displayed peptide library, wherein each member comprises a ncAA encoded by amber codons in response to random distribution. Although the applicant's focus in this embodiment is to construct a short peptide library, larger proteins (such as antibodies) are also usually displayed on the surface of filamentous phage. Due to the inherent characteristics of superinfection selection that depends on Ff phage biology, this method is not subject to library design or size restrictions. Therefore, in theory, superinfection selection can be used to generate much larger peptide and protein libraries under the universal constraints displayed by pill.

[0113] The applicant validated the method of library construction by generating an amber-codon-specific heptapeptide library fused to the N-terminus of the pill protein displayed on M13 phage. The applicant further demonstrated that the expression of phagemid particles from the library depended on the addition of ncAA in the growth medium. Previous studies involving the expansion of the genetic code of phage libraries were limited to efficient aaRS / ncAA pairs to minimize the expression bias of library clones containing ncAA. However, the amber-codon-specific library eliminates this bias. Therefore, despite their different incorporation efficiencies, every ncAA tested by the applicant is easily incorporated into the phage-displayed peptides without the need for optimization.

[0114] To date, more than 150 ncAAs containing natural post-translational modifications, bioorthogonal functional groups, spectroscopic probes, and other unique chemical and structural motifs have been genetically encoded by nonsense suppression. The methods reported in this example provide a means to easily generate peptide libraries containing these diverse ncAAs, for which numerous applications can be envisioned.

[0115] As a demonstration of the utility of this technology, the applicant generated a peptide library containing naturally occurring post-translational modifications and used this library for active site-directed ligand evolution of peptides that bind to SIRT2. Phage particles were sequenced after three rounds of selection and the sequences of the peptides were found to be consistent with previous library screening against SIRT2, validating the applicant's results.

[0116] The selected peptides synthesized by replacing BuK with thBuK were shown to be effective inhibitors of lysine deacetylation catalyzed by SIRT2. Although the selected peptides retained isotype selectivity comparable to TB, their higher potency resulted in stronger inhibition of SIRT2 in vivo. Since SIRT1 and SIRT2 have high sequence homology around their active sites and peptide binding clefts, it is not surprising that the deacetylase selectivity of S2P04 and TB is similar. BuK contains a chemical part that can naturally guide the peptide to bind to the SIRT2 active site. This active site directed ligand evolution can be extended to other genetically encoded ncAAs (such as methyl lysine) for the identification of histone demethylase ligands. Although not reported in this embodiment, small molecule ligands identified by high throughput screening can be covalently attached to ncAAs and displayed on phagemids, allowing the identification of organic peptide ligands. In summary, the applicant believes that amber-codon-specific phage display technology combined with active site directed ligand evolution will have great application in the field of drug discovery.

[0117] Example 1.6. Molecular cloning

[0118] Phagemid library construction

[0119] The phagemid pADL-10b containing the gene encoding pill was purchased from Antibody Design Labs. To introduce a library of seven sites at the N-terminus of pill, the phagemid was PCR amplified with primers pADL-g3-P1: 5'-CATGCCATGGCC(NNK)7GCGGCGAAAGCGG-3' and pADL-g3-P2: 5'-CATGCCATGGCCGGCTGGGCCGC-3'. The PCR product was digested with NcoI, ligated, and used to transform electrocompetent E. coli Top10 F' (Thermo Fisher). A small aliquot of the transformed cells was inoculated onto an agar selection plate to quantify the transformation efficiency, and the remaining culture was diluted in fresh 2xYT medium and grown overnight at 37°C. The next day, cells were harvested by centrifugation, resuspended in 1 / 10 volume of 2xYT containing 20% ​​glycerol, and aliquots were stored at -80°C for later superinfection selection.

[0120] M13KO7(pIII - )

[0121] Helper phage M13KO7 was purchased from New England Biolabs. To produce a pill-deficient derivative of M13KO7 for phagemid complementation, the applicant introduced a TAA mutation at position K10 of gene 3 by site-directed mutagenesis. The phage vector was amplified using primers M13gIII-1: 5'-CCTTCCCTCCCTTAATCGGTTGAATG-3' and M13gIII-2: 5'-CATTCAACCGATTAAGGGAGGGAAGG-3' to obtain M13KO7 (piIII – ) carrier.

[0122] pEDF-PylRS

[0123] In order to encode The origin of replication was changed from p15A to CloDF in the pEVOL construct with the PylRS mutation. The CloDF origin was amplified from plasmid pCDF-1 (EMD Millipore) using primer CloDFori-1: 5'-TTGGCGCGCCCAATTAGCTAGCTCACTCGGTC-3' and primer CloDFori-2: 5'-TGTTCCTAGGGATAAATTGCACTGAAATCTAG-3'. The PCR product was cloned into the AscI and AvrII sites that had been introduced into the previously reported pEVOL-PhdRS and pEVOL-BuKRS constructs using primer pEVOLori-1: 5′-TGTTCCTAGGTCTTCAAATGTAGCACCTGAAG-3′ and primer pEVOLori-2: 5′-TTGGCGCGCCCCTTTTTTCTCCTGCCACATG-3′ to obtain pEDF-PhdRS and pEDF-BuKRS, respectively.

[0124] Example 1.7. Superinfection selection for amber codon enrichment

[0125] For the first round of superinfection selection, E. coli Top10 F′ containing the original phage library was incubated at 37°C in 100 μg mL -1 Ampicillin and 10 μg mL -1 Cultured in tetracycline-free 2xYT medium. 600 After ≈0.3, the expression of pIII was induced by adding 0.2 mM IPTG. After 30 min of induction, CM13 helper phage (Antibody Design Laboratory, 1x 10 11 pfu, MOI ≈ 15) and incubated at 37°C for another 40 min. After superinfection, cells were pelleted by centrifugation and resuspended in 100 μg mL -1 Ampicillin, 2 5μg·mL -1 The cells were cultured in fresh 2xYT medium containing kanamycin and 1 mM IPTG and grown overnight at 37°C. The next day, the cells were pelleted and plasmids were purified from the cells using a commercial plasmid extraction kit (Epoch Life Sciences, Inc.). The phagemid library was separated from the helper phage and purified by agarose gel electrophoresis.

[0126] After the first round of superinfection selection, the phagemids were passed through E. coli DH5α to remove clones containing nonfunctional pill. E. coli DH5α (Lucigen) containing the M13KO7 (pIII-) helper phage was transformed with the purified phagemid library, recovered in 2xYT medium for 1 h, and then in 2xYT medium containing 100 μg mL -1 Ampicillin, 25 μg mL -1 Grow overnight in 2xYT with kanamycin and 1 mM IPTG. The next day, pellet the cells and add 1 / 5 volume of 5x phage precipitation solution (2.5M NaCl, 20% PEG 8,000) on ice to precipitate phage particles from the supernatant. Pellet the precipitated particles by centrifugation and then resuspend in 1 / 10 volume of PBS. Clarify the resuspension by centrifugation and incubate the supernatant at 65°C for 15 minutes to kill residual E. coli.

[0127] For the second round of superinfection selection, purified phagemid particles (3.7 x 10 9 cfu, MOI≈0.1) was added to 100 mL and supplemented with 10 μg mL -1 A mid-logarithmic phase culture of E. coli Top10 F' was grown in 2xYT medium containing tetracycline. The culture was incubated at 37 °C for 1 h and then incubated with 100 μg mL -1 Dilute 1:1 fresh 2xYT with ampicillin and 0.5 mM IPTG. Incubate the culture at 37 °C until OD 600 ≈0.4, then take out 60mL and add CM13 to help phage (1x10 11 pfu, MOI≈10). The culture was incubated for another 45 min, and then the cells were pelleted and resuspended in 500 mL of 5% paraformaldehyde supplemented with 100 μg mL -1 Ampicillin, 25 μg mL -1 The culture was then incubated at 37°C overnight, and the phagemid library was extracted and purified the next day as described above.

[0128] Example 1.8. Phagemid particles and phage quantification

[0129] In all experiments, phagemid particles and phages were quantified by colony forming unit assay. In this assay, serial dilutions of phage solution were prepared in 2xYT medium, and 10 μL of each dilution was added to 90 μL of logarithmic phase E. coli Top10 F'. After adding the phage dilutions, the culture was incubated at 37 °C for 45 min, and 10 μL was then spotted onto a plate containing 100 μg mL-1 Ampicillin (phagemid) or 25 μg mL -1 The cells were plated on agar selective plates containing kanamycin (phage) and cultured overnight at 37° C. The next day, colonies in each inoculation spot were counted and the numbers were used to calculate the number of colony forming units in the solution.

[0130] Example 1.9. Phagemid particle expression and purification

[0131] To express an amber-codon-obligatory phagemid library with phenylalanine derivatives, Applicants used a previously reported pyrrolysyl-tRNA synthetase mutant containing alanine mutations at positions N346 and C348 (PhdRS). PhdRS and its cognate tRNA ( The amber-codon-specific library from the second round of superinfection selection was used to transform E. coli Top10 containing M13KO7 (pIII-) and pEDF-PhdRS, producing 6.92 x 10 8 transformants. -1 Ampicillin, 34 μg mL -1 Chloramphenicol and 25 μg mL -1 The transformed cells were cultured in 200 mL 2xYT medium containing kanamycin at 37 °C until OD 600 ≈0.5, at which point 1 mM IPTG and 0.2% arabinose were added to induce protein expression, and the culture was split into two flasks. One of the phenylalanine derivatives 1-4 was added to one flask at a final concentration of 4 mM. After induction, the temperature was changed to 30°C and the phagemid particles were expressed for 18-24 hours.

[0132] To express phagemid particles carrying lysine derivatives, Applicants used a previously reported PylRS mutant (BuKRS) containing the Y384W mutation. 19 BuKRS and The amber-codon-specific library was used to transform electrocompetent E. coli Top10 containing M13KO7(pIII-) and pEDF-BuKRS to produce 3.1×10 9 Expression of the particles was exactly as described for the phenylalanine derivatives, except that 5 mM nicotinamide was added to the growth medium upon induction.

[0133] To purify ncAA-incorporated phagemid particles, pellet the cells by centrifugation and transfer the top 80 mL of medium to a new tube. Add 20 mL of 5x phage precipitation solution to the clarified medium and incubate the mixture overnight at 4°C. The next day, pellet the particles by centrifugation, decant the supernatant, and resuspend the pellet in 10 mL of PBS, pH 7.4. Pellet the remaining cells by centrifugation and pellet the phagemid particles again by incubating in 1x phage precipitation solution overnight. After the second precipitation, pellet the particles by centrifugation and resuspend the pellet in 1 mL of PBS. Pellet the remaining cells by centrifugation and incubate the solution at 65°C for 15 minutes to kill any remaining E. coli. Purified phagemid particles were quantified as described above and used immediately in selection experiments.

[0134] Example 1.10. Recombinant protein preparation

[0135] Sirtuin 1

[0136] E. coli BL21 (DE3) containing plasmid pQE80-His6-SIRT1 was cultured at 37°C in 3 L supplemented with 100 μg mL -1 Grow in 2xYT medium containing ampicillin to an OD of 600 The pH was 0.7, at which time 0.5 mM IPTG was added to induce protein expression. After 4 hours of induction, cells were collected by centrifugation and the cell pellets were stored at -80°C until purification.

[0137] The cell pellet was resuspended in 50 mL of lysis buffer (50 mM Tris pH 8.0, 250 mM NaCl, 10 mM imidazole, 0.1 mM phenylmethanesulfonyl chloride (PMSF), 1% Triton X-100) and mixed with 1 mg mL -1Lysozyme (hen egg white) was incubated on ice for 30 minutes. After incubation, the resuspension was sonicated twice (1 second on, 1 second off, 1 minute total, 60% output) and clarified by centrifugation. 4 mL of a 50% slurry of high affinity Ni-filled resin (Genscript) was added to the clarified supernatant, and the mixture was incubated at 4°C with end-over-end rotation for 30 minutes. After 30 minutes, the mixture was filtered through a disposable column, and the resin was washed with 40 mL of lysis buffer containing 0.1% Triton X-100, and then washed with 40 mL of lysis buffer without Triton X-100. The bound protein was eluted with 7 mL of elution buffer (lysis buffer containing 250 mM imidazole) and loaded onto a HiPrep 26 / 10 desalting column (GE Healthcare) that had been pre-equilibrated with Source 15Q buffer A (20 mM Tris pH 8.0, 50 mM NaCl, 0.2 mM DTT, 10% glycerol). The desalted protein was concentrated to 1 mL (10 kDa MWCO) by ultrafiltration and applied to a 10 mL Source 15Q column (GE Healthcare) pre-equilibrated with Source 15Q buffer A. The column was purified by 3 CV (2 mL min -1 The column was washed with Source 15Q buffer A (20 mM Tris pH 8.0, 1 M NaCl, 0.2 mM DTT, 10% glycerol; 1 mL min -1 ) was eluted for 5 CV. Fractions containing SIRT1 were concentrated and stored at -80°C. The extinction coefficient was calculated from the absorbance at 280 nm using 40,340 M -1 cm -1 Determine the enzyme concentration.

[0138] Sirtuin 2

[0139] E. coli Top10 containing plasmid pHEX-His6-SIRT2 was incubated at 37°C in 2 L containing 100 μg mL -1 Grow in 2xYT medium containing ampicillin to an OD of 600 The expression of SIRT2 was induced by adding 0.1 mM IPTG at this time. After 2 hours of induction, the cells were collected by centrifugation and the cell pellets were stored at -80°C until purification.

[0140] Thaw the cell pellet and resuspend in 40 mL of lysis buffer containing 50 mM NaH2PO4 pH 8.0, 300 mM NaCl, 10 mM imidazole, 0.1 mM PMSF. The resuspended pellet was concentrated to 1 mg mL -1 Incubate on ice for 30 minutes in the presence of lysozyme. After incubation, the resuspension was sonicated twice (1 second on, 1 second off, 1 minute total, 60% output) and clarified by centrifugation. 4 mL of 50% slurry of high affinity Ni-filled resin (Genscript) was added to the clarified supernatant, and the mixture was incubated for 30 minutes at 4°C with end-over-end rotation. After 30 minutes, the mixture was filtered through a disposable column, the resin was washed with 40 mL of lysis buffer, and the bound protein was eluted with 7 mL of elution buffer (lysis buffer containing 250 mM imidazole). Subsequent purification steps depend on whether the protein is used for biotinylation and affinity selection or inhibition experiments.

[0141] For affinity selection, the eluted protein was concentrated to 1 mL by ultrafiltration (10 kDa MWCO), diluted 1:9 with Q Sepharose buffer A (50 mM NaH2PO4 pH 8.0, 50 mM NaCl, 0.1 mM DTT), and concentrated again to 1 mL. The concentrated sample was applied to a 25 mL Q Sepharose column (GE Healthcare) pre-equilibrated with Q Sepharose buffer A. The column was then eluted with 2 CV (2 mL min -1 ) Buffer A was used to wash the column, and the protein was eluted with a 0-100% linear gradient of Buffer B (50 mM NaH2PO4 pH 8.0, 1 M NaCl, 0.1 mM DTT) for 3 CV (2 mL min -1 ). The main peak fractions were combined and concentrated to 1 mL for biotinylation.

[0142] For inhibition assays, the eluted protein was loaded onto a HiPrep 26 / 10 desalting column (GE Healthcare) that had been pre-equilibrated with Source 15Q Buffer A (20 mM Tris pH 8.0, 50 mM NaCl, 0.2 mM DTT, 10% glycerol). The desalted protein was concentrated to 1 mL (10 kDa MWCO) by ultrafiltration and applied to a 10 mL Source 15Q column (GE Healthcare) pre-equilibrated with Source 15Q Buffer A. The column was purified by 3 CV (2 mL min -1The column was washed with Source 15Q buffer A (20 mM Tris pH 8.0, 1 M NaCl, 0.2 mM DTT, 10% glycerol; 1 mL min -1 ) was eluted for 5 CV. Fractions containing pure SIRT2 were concentrated and stored at -80°C. The extinction coefficient was calculated from the absorbance at 280 nm using 32,470 M -1 cm -1 Determine the enzyme concentration.

[0143] Sirtuin 3

[0144] E. coli Top10 containing plasmid pGEX4T-GST-SIRT3 was cultured at 37 °C in 2 L supplemented with 100 μg mL -1 Grow in 2xYT medium containing ampicillin to an OD of 600 The pH was 0.6, at which time 2 mM IPTG was added to induce protein expression. After 2 hours of induction, cells were collected by centrifugation and the cell pellets were stored at -80°C until purification.

[0145] The cell pellet was resuspended in 50 mL of lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.1 mM PMSF, 1% Triton X-100) and diluted in 1 mg mL -1 Incubate on ice for 30 minutes in the presence of lysozyme. After incubation, the resuspension was sonicated twice (1 second on, 1 second off, 1 minute in total, 60% output) and clarified by centrifugation. 3 mL of 50% immobilized glutathione resin (Genscript) slurry was added to the clarified supernatant, and the mixture was incubated upside down at 4 ° C for 30 minutes. After 30 minutes, the mixture was filtered through a disposable column and the resin was washed with 50 mL of lysis buffer containing 0.1% Triton X-100. After washing, the bound protein was eluted with 10 mL of lysis buffer containing 10 mM reduced glutathione but without Triton X-100. The eluate was loaded directly onto a HiPrep 26 / 10 desalting column (GE Healthcare), which had been pre-equilibrated with 50 mM Tris pH 7.5, 150 mM NaCl, 0.2 mM DTT and 10% glycerol. The desalted protein was concentrated and stored at -80°C. The extinction coefficient was calculated from the absorbance at 280 nm using 80,790 M -1 cm -1 Determine the enzyme concentration.

[0146] Example 1.11. Biotinylation of Sirtuin 2

[0147] SIRT2 (53 μM) was biotinylated in PBS pH 7.4 with a 10-fold molar excess of EZ-LinkTM NHS-biotin (Thermo Fisher Scientific) with gentle shaking on ice for 2 hours. After the reaction was completed, the solution was directly loaded onto a Superdex 75 10 / 300GL size exclusion column (GE Healthcare) pre-equilibrated with 50 mM Tris pH 8.0, 250 mM NaCl, 0.2 mM DTT, and 10% glycerol. The eluted protein was concentrated by ultrafiltration (10 kDa MWCO), flash frozen, and stored at -80 ° C. As previously described, 2 μg of biotinylated SIRT2 was used to confirm the biotinylation of the protein by capture on magnetic streptavidin beads (Genscript), followed by SDS-PAGE and Coomassie staining.

[0148] Example 1.12. Affinity selection for streptavidin

[0149] Lyophilized streptavidin (Chem-Impex International, Inc.) was dissolved in 0.1 M NaHCO3, (pH 8.6) at a concentration of 25 μg mL -1 . 1.5 mL of protein solution was coated on a polystyrene dish (60 x 15 mm) and incubated overnight at 4° in a sealed humidified chamber. After removing the protein solution, 5 mg mL -1 BSA and 0.1 μg mL -1 The plate was blocked with streptavidin in 0.1 M NaHCO3 at 4°C for 2 hours. The plate was washed 6 times with 2 mL PBST (PBS containing 0.1% Tween-20, pH 7.5), and then the purified phage library (2 x 10 10 -4x 10 11 cfu) were incubated with the target at room temperature for 1 hour (rounds 1-3) or 30 minutes (round 4). After incubation with ncAA-incorporated phagemid particles, the plate was washed 10 times with PBST and then incubated with 1 mL of 0.1 mM D-biotin solution for 30 minutes to competitively elute the bound phagemid particles.

[0150] Example 1.13. Affinity selection for sirtuin 2

[0151] For affinity selection, biotinylated SIRT2 was captured by streptavidin-coated magnetic beads (rounds 1 and 3) or immobilized on neutravidin-coated polystyrene plates (round 2). For rounds 1 and 3 of selection, 100 μL of streptavidin-coated magnetic beads (50% slurry, GenScript) were washed twice with PBS and then divided into two tubes. 400 μL of biotinylated SIRT2 (10 μg) in PBS was added to one tube and an equal volume of buffer was added to the other tube. Both tubes were incubated at room temperature for 20 min. After incubation, the supernatant was removed and washed with 1 mL of 5 mg mL -1 The beads were blocked with BSA in 0.1 M NaHCO3 pH 8.6 for 30 minutes at room temperature with rotation. After blocking, the supernatant was removed from the tube without SIRT2, washed three times with PBST, washed once with PBS, and incubated with purified phage library (2.9 x 10 10 -6.2x 10 10 cfu) in 1 mL PBS with slow rotation for 30 minutes for negative selection. After negative selection, the beads were precipitated and the supernatant was transferred to beads containing SIRT2, which had been washed as described above. The phage library was incubated with the immobilized target for 30 minutes at room temperature with rotation. After selection, the supernatant was removed and the beads were washed 10 times with PBST. After the third, sixth and ninth washes, the beads were transferred to a new test tube and the phagemid particles displaying peptides bound to the polypropylene tube were removed. After washing, the bound particles were eluted by incubation in 100 μL of 50mM glycine pH2.2 for exactly 10 minutes. After 10 minutes, the supernatant was removed and neutralized with 50 μL 1M Tris pH 8.0.

[0152] For the second round of selection, 8 wells of a pre-blocked neutravidin-coated polystyrene plate (Pierce) were washed three times with 250 μL PBS, and then 100 μL of biotinylated SIRT2 (1.25 μg) in PBS was added to each well. The plate was incubated at room temperature in a sealed humidified chamber for 30 minutes to allow the biotinylated SIRT2 target to be captured. The plate was then washed three times with PBST and once with PBS, and the phage library (5.4 x 10 9 cfu) were evenly distributed into each well. The phagemid particles were incubated with the immobilized target for 30 minutes, then the supernatant was removed and each well was washed 10 times with 250 μL PBST. After washing, the bound phagemid particles were eluted by incubating in 100 μL of 50 mM glycine pH 2.2 for exactly 10 minutes. After 10 minutes, the eluted particles were pooled from each well and neutralized with 400 μL 1M Tris pH 8.0.

[0153] Example 1.14. Phagemid amplification

[0154] After each round of selection, the eluted phagemid particles were passed through a cell culture medium containing M13KO7 (pIII - ) helper phage and plasmid pEDF-PhdRS (for incorporation of mBrF) or pEDF-BuKRS (for incorporation of BuK) were passaged for amplification. 10 μL of eluted phagemid particles were retained for titration, and the remaining solution was used to infect 20 mL of E. coli Top10 F' culture at 37°C for 45 minutes. After infection, the cells were pelleted and resuspended in 100-300 mL of 100 μg mL -1 The phagemid library was purified using a commercial plasmid extraction kit and used to transform electrocompetent E. coli Top10 containing M13KO7 (pIII-) and the appropriate pEDF-aaRS construct. A small portion of the transformation was titrated to ensure that the number of transformants exceeded the number of phagemid particles eluted in the previous round by at least 100 times. The transformed cells were directly used to express phagemid particles for the next round of selection.

[0155] Example 1.15. Molecular dynamics simulation

[0156] General information

[0157] Based on the crystal structure PDB 4X3O, the interaction of peptides with NAD was studied by simulating different peptides at positions similar to the inhibitor. + The protein environment after the reaction. (2017-4 version) 39 The Desmond software was used for all simulations. Each simulation to study protein-peptide interactions followed the steps described below.

[0158] Protein Preparation Module

[0159] There are several structures of SIRT2 available in the PDB. By overlaying these entries, it is apparent that SIRT2 is a conformationally flexible protein with many of its loops. However, the domain to which the peptide inhibitors bind is a somewhat inflexible region. The 4X3O crystal structure was chosen as the template structure because it contains similar thiomyristoyl-lysine peptides that were modified into the target peptides (S2P03, S2P04, S2P07, S2P04-5, and Ac-S2P04-5) and because it has the fewest missing residues and the highest resolution among the available crystal structures. Using The Protein Preparation Wizard in the software was used to prepare the 4X30.pdb structure and simulated at a pH of 7.5±0. Crystal structure water was maintained. The PrimeLoop algorithm was used to fill in missing residues (Met299-Gly304). After the initial preparation, the H-bonding network was optimized using the PROPKA tool at a pH of 7.5±0, the orientation of water molecules was sampled, and all atoms were constrained and minimized using the OPLS3 force field.

[0160] System Builder Module: Preparation for Molecular Dynamics Simulations

[0161] Use the System Builder tool in the Desmond module (Note: This is distributed with Desmond The model for Desmond MD-simulation was prepared in the software Maestro interface. The original thiomyristoyl-lysine peptide was transformed into the target peptide before the solvent box was applied. A distance of 1.5 m was constructed around the peptide-protein assembly. The orthorhombic solvent box was used. The size of the solvent box was minimized before applying explicit water molecules to the system. The TIP4P water model was used for explicit water molecules and the OPLS3 force field was used for all other atoms. Physiological NaCl at a concentration of 0.15 M was used for charge neutralization of the protein-peptide assembly.

[0162] Desmond module: molecular dynamics simulation

[0163] All runs were performed using the Desmond simulation (multisim) molecular dynamics program, using the NPT suite (1.0135 bar, 310.15 K), 10 ns production runs, snapshots taken every 10 ps, ​​and 1000 frames of trajectory. Prior to the production run, the system was placed in a relaxation scheme and the Desmond It is implemented in the software Maestro interface and is outlined as follows:

[0164] 1. Constrained minimization step

[0165] 2. Brownian dynamics NVT, T = 10K, small time step, constraint on solute heavy atoms, 100ps

[0166] 3. NVT, T = 10K, small time step, constraints on solute heavy atoms, 12ps

[0167] 4. NVT, T = 10K, restraint of solute heavy atoms, 12ps

[0168] 5. NVT, restraint of solute heavy atoms, 12ps

[0169] 6. NPT and no constraints, 24ps

[0170] 7. Production operation

[0171] Protein-peptide interaction maps

[0172] Import MD-simulation results and then distribute them using Desmond The ligand interaction graph module in the software generates a ligand interaction table. The results show that in the peptide inhibitor, only the residues adjacent to the thBuK residues make hydrophobic interactions. The attached figure shows the frames where interactions occur (orange bars) out of 1000 frames.

[0173] Example 1.16. Peptide synthesis

[0174] The peptides were synthesized by manual Fmoc-based solid phase peptide synthesis on Rink-amide resin using N,N,N',N'-tetramethyl-O-(1H-benzotriazol-1-yl)uranium hexafluorophosphate (HBTU) as coupling agent. Rink-amide MBHA resin (Novabiochem, 100 mg, substitution: 0.78 mmol g -1 ) was swollen in 10 mL DMF for 1 hour. Deprotection, coupling and cleavage reactions were carried out in a glass reaction vessel at room temperature and stirred upside down under the following conditions:

[0175] Deprotection

[0176] A solution of 20% piperidine in DMF (v:v, 5 mL) was added to the swollen resin for 5 minutes. The resin was washed (3 x 5 mL DMF) and the deprotection reaction was repeated for 15 minutes. After the second deprotection reaction, the resin was washed again (5 x 5 mL DMF, 2 x 5 mL DCM).

[0177] Coupling

[0178] Each coupling reaction contained Fmoc-protected amino acid (312 μmol, 4 equivalents), HBTU (304.2 μmol, 3.9 equivalents) and N, N-diisopropylethylamine (DIPEA, 780 μmol, 10 equivalents) dissolved in 5 mL DMF. The Fmoc-protected amino acid was preactivated for 5 minutes with HBTU and DIPEA and then added to the resin for 40 minutes. After 40 minutes, the resin was washed again (5x 5 mL DMF, 2x 5 mL DCM).

[0179] N-terminal acetylation

[0180] To synthesize Ac-S2PO4-5 with N-terminal acetylation modification, after the last Fmoc deprotection, the resin was washed (5x 5 mL DMF, 3x 5 mL DCM) and then incubated with a solution of acetic anhydride, pyridine and DMF (1:2:3, v:v:v) at room temperature with rotation for 1 hour. The peptide was then cleaved and the side chain deprotection was performed simultaneously as described below.

[0181] Cleavage and deprotection

[0182] After the final Fmoc deprotection, the resin was washed (5x 5mL DMF, 5x 5mL DCM), and the peptide was cleaved from the resin while using 7mL cleavage mixture (containing 88% trifluoroacetic acid (TFA), 5% phenol, 5% H2O and 2% triisopropylsilane (v:w:v:v)) for two hours of side chain deprotection under nitrogen. After two hours, the resin was filtered and washed (2x1mL cleavage mixture), and the combined filtrate was reduced to about 4mL with a nitrogen stream. The concentrated solution was added dropwise to cold ether (40mL) and incubated on ice for 10 minutes. The precipitated peptide was collected by centrifugation, washed twice with cold ether (20mL), dried under a nitrogen stream, and stored at -20°C.

[0183] purification

[0184] The crude peptide was dissolved in DMF and purified on a C18 column (Alltech, 10 x 250 mm, 10 μm, Hole or Supelco, 10x 250mm, 5μm, Semi-preparative RP-HPLC purification was performed on 400 μl (10 μl) wells. Solvent A: water with 0.1% TFA, solvent B: acetonitrile with 0.1% TFA. The peptide was eluted with a linear gradient from 20-50% solvent B in 30 minutes. The fractions containing the purified peptide were combined and lyophilized to obtain a fluffy white powder. The quality of the peptide was confirmed by MALDI-TOF mass spectrometry. The purity of the peptide was determined based on the HPLC trace at 215 nm, and the purity of all peptides used was greater than 90%.

[0185] Example 1.17. Deacetylase inhibitor test

[0186] Inhibition assays for SIRT1-3 were performed using a recently reported fluorescent universal sirtuin substrate (JPT Peptide Technologies, GmbH). Inhibition reactions were performed in a volume of 50 μL at 37°C in black half-area 96-well plates. Sirtuins (0.25 μM) were incubated with different concentrations of synthetic peptides (0.0064-50 μM for SIRT1 and SIRT2, 0.5-100 μM for SIRT3) or TB (0.16-150 μM for SIRT1-3) and 1 mM NAD + Preincubate for 15 min in assay buffer (20 mM Tris pH 7.8, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT). After incubation, the reaction was initiated by adding substrate (5 μM) and product formation was monitored by measuring fluorescence intensity every 20 s at λEx = 320 nm, λEm = 408 nm (gain = 130, read height = 7 mm) for 5 min (SIRT2) or 30 min (SIRT1 and SIRT3) in a BioTek Synergy H1 microplate reader. The initial rate of product formation was determined by linear regression of fluorescence intensity versus time and normalized to 0% activity (maximum inhibition) and 100% activity (no inhibition) controls. IC was determined by nonlinear regression of the normalized initial rate versus inhibitor concentration curve using GraphPad Prism. 50 value.

[0187] Example 1.18. Cell culture and Western blotting

[0188] MCF-7 (HTB-22) and HEK 293T (CRL-11268) cells purchased from the American Type Culture Collection were grown in Dulbecco's modified E. coli medium (Invitrogen) containing 10% fetal bovine serum (Invitrogen) at 37°C in a 5% CO2 atmosphere. 5Cells were seeded at 100 viable cells / well and grown for 24-48 hours to 60-70% confluence. The medium was aspirated and replaced with medium containing 50 μM synthetic peptides, TB or vehicle control (0.25% DMF). After incubation with inhibitors for 24 hours, the medium was removed and the cells were washed twice with ice-cold PBS (Invitrogen). Cells were lysed on ice for 30 minutes in a buffer containing 25mM Tris pH 7.4, 150mM NaCl, 10% glycerol, 4mM MgCl2, 0.2mM DTT, 1% NP-40, 25mM nicotinamide, 5μM trichostatin A and a 1:100 dilution of a protease inhibitor cocktail (Sigma-Aldrich). The lysate was clarified by centrifugation, quantified by BCA assay (Pierce), and aliquots were stored at -80°C. Cell lysates (15 or 20 μg) were resolved by SDS-PAGE on 12% acrylamide gels under denaturing conditions and then transferred to 0.2 μm nitrocellulose membranes (Bio-Rad). Acetylated (K40) α-tubulin and total α-tubulin were probed with primary antibody clones 6-11B-1 and B-5-1-2, respectively, purchased from Sigma-Aldrich (1:2,000 dilution). The primary antibody was probed with HRP-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc., 1:10,000 dilution). Chemiluminescent signals were detected using SuperSignal TM ELISA Pico chemiluminescent substrate (Thermo Fisher Scientific) was generated and visualized using the Bio-Rad ChemiDoc XRS+ imaging system.

[0189] Example 2. Identification of ENL inhibitors

[0190] Acute myeloid leukemia (AML), a disease characterized by the uncontrolled proliferation of abnormal myeloid cells, is the most commonly detected and deadly subtype of leukemia. In the United States, an estimated 21,450 people are diagnosed with AML and 10,920 will die from the disease each year, accounting for nearly half of all deaths from all leukemia subtypes.

[0191] It is now clear that the survival of several types of AML cells is linked to ENL, an epigenetic reader of histones. ENL protein binds to acylated histone H3 through its N-terminal YEATS domain and then recruits RNA polymerase II to proto-oncogenes that are highly expressed in leukemic cells. Knockout or mutation of ENL protein in leukemic cells significantly inhibits their growth and increases their differentiation.

[0192] Therefore, if the binding of ENL to acetylated histones is inhibited, the transcription of proto-oncogenes in leukemic cells should be downregulated, leading to potential targeted therapy for AML. In this example, the applicant describes a phage-assisted, active site-directed ligand evolution technology that can easily screen potential inhibitors with nanomolar affinity.

[0193] Peptide-based inhibitors have advantages over small molecules, providing satisfactory affinity and selectivity for binding to proteins involving large, relatively flat surfaces. Peptides are also easy to screen. For example, using phage display technology, a researcher may be able to screen more than 10 10 A library of unique peptides.

[0194] However, because phage-displayed peptides are expressed in E. coli, this limits the coding capacity of bacterial translation to only 20 canonical amino acid (cAA) building blocks, restricting the sequences and chemistries that can be developed. In addition, traditional phage display often relies on the use of randomized libraries and is often not associated with any known protein-ligand interactions, which may limit the ability to mimic strong interactions.

[0195] Therefore, to expand the chemical diversity of the library and exploit unique chemical interactions not possessed by the 20 cAAs, Applicants incorporated non-canonical amino acids (ncAAs) into the phage display system. Fig. 7A -B shows the proposed phage-assisted, active site-directed ligand evolution method, in which warheads with known ability to bind to target proteins are installed in ncAAs, which can be genetically incorporated into a phage-displayed sequence-randomized peptide library. In the phage-displayed peptide, the warhead is expected to interact with the binding site and bind to the ligand-binding cavity of the target enzyme as an anchor, while the amino acid residues of the ncAA flanking the ligand-fused ncAA provide additional interactions in the peptide-binding groove to improve affinity and selectivity.

[0196] Recent studies have found that two aromatic residues F59 and Y78 in the ligand binding site of the YEATS domain can bind to N ε -crotonyl lysine (CrK, Figure 7C ) forms a π-π-π stacking effect. Inspired by this discovery, the applicant decided to incorporate CrK into the phage display system described in Example 1 and use the π-π-π interaction generated by ncAA to guide the peptide library to the active site of the protein containing the YEATS domain.

[0197] In this example, the applicant first constructed a 21 base pair DNA library encoding 7 random amino acids from the NNK triplet nucleotide. In order to genetically incorporate CrK, the applicant applied the superinfection immunity-based technology described in Example 1 to construct an amber-specific phagemid library. The resulting amber-specific library can be expressed using the PylRS amber suppression system previously evolved by the applicant (ACS chemical biology 2013, 8 (8), 1664-70).

[0198] To prove the concept of active site ligand evolution, a displayed peptide library was panned against ENL that had been biotinylated and immobilized on streptavidin-coated magnetic beads. Once bound to the target protein, the expressed peptide library was washed to remove nonspecific binding, eluted with acidic denaturing conditions, and amplified in E. coli. The selection process was performed four times to screen for inhibitors with high affinity and selectivity. Over the four rounds of selection, phage yield increased significantly, with a 540-fold difference between the first and fourth rounds.

[0199] Additionally, after four rounds of selection, the library regions were analyzed using Illumina next generation sequencing. This yielded two strong consensus sequences, 7CrKEnl1 and 7CrKEnl2 ( Fig. 8A -B), after the fourth round of selection, these two sequences accounted for 98% of the total phage, with enrichments of 540-fold and 300-fold between the first and fourth rounds, respectively. In order to determine the affinity of 7CrKEnl1 and 7CrKEnl2 for ENL, the applicant synthesized 5-FAM-coupled peptides and then characterized their binding to ENL using fluorescence polarization analysis. The results showed that the dissociation constant of 7CrKEnl1 was 94nM and the dissociation constant of 7CrKEnl2 was 90nM ( Figure 8C -D). In summary, the results in Figure 8 demonstrate the successful application of our genetically encoded phage-assisted active site-directed ligand evolution in identifying potent peptide inhibitors for therapeutic protein targets.

[0200] Without further elaboration, it is believed that those skilled in the art can make the most of the contents of the present invention through the description herein. The embodiments described herein should be understood to be illustrative and not to limit the remainder of the disclosure in any way. Although embodiments are shown and described, many changes and modifications may be made thereto by those skilled in the art without departing from the spirit and teachings of the present invention. Therefore, the scope of protection is not limited by the above description, but only by the claims, including all equivalents of the subject matter of the claims. The disclosures of all patents, patent applications, and publications listed herein are incorporated herein by reference to the extent that they provide procedural or other details consistent with and supplement those described herein. 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Xaa Ile 1 5 <210> 5 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Sirtuin 2 inhibitors <220> <221> MISC_Features <222> (1) <223> X is a non-classical amino acid or GABA <220> <221> MISC_Features <222> (4) <223> X is a non-classical amino acid <400> 5 Xaa Thr Val Xaa Ile 1 5 <210> 6 <211> 7 <212> PRT <213> Artificial sequence <220> <223> SNL inhibitors <220> <221> MISC_Features <222> (6) <223> X is a non-classical amino acid <400> 6 Asp Ile Trp Cys Phe Xaa Gly 1 5 <210> 7 <211> 7 <212> PRT <213> Artificial sequence <220> <223> SNL inhibitors <220> <221> MISC_Features <222> (2) <223> X is a non-classical amino acid <400> 7 Ser Xaa Asp Ile Trp Cys Phe 1 5 <210> 8 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Partial plasmid encoding amber-codon-specific peptide-pIII library <220> <221> misc_features <222> (1)..(2) <223> n is a, c, g or t <220> <221> misc_features <222> (4)..(5) <223> n is a, c, g or t <220> <221> misc_features <222> (7)..(8) <223> n is a, c, g or t <220> <221> misc_features <222> (10)..(11) <223> n is a, c, g or t <220> <221> misc_features <222> (13)..(14) <223> n is a, c, g or t <220> <221> misc_features <222> (16)..(17) <223> n is a, c, g or t <220> <221> misc_features <222> (19)..(20) <223> n is a, c, g or t <400> 8 nnknnknnkn nknnknnkn k 21 <210> 9 <211> 5 <212> PRT <213> Artificial sequence <220> <223> S2P04-5 <220> <221> MISC_Features <222> (4) <223> X is a non-classical amino acid <400> 9 Cys Thr Val Xaa Ile 1 5 <210> 10 <211> 5 <212> PRT <213> Artificial sequence <220> <223> S2P04(Abu)-5 <220> <221> MISC_Features <222> (1) <223> X is for Abu <220> <221> MISC_Features <222> (4) <223> X is a non-classical amino acid <400> 10 Xaa Thr Val Xaa Ile 1 5 <210> 11 <211> 46 <212> DNA <213> Artificial sequence <220> <223> pADL-g3-P1 Primer <220> <221> misc_features <222> (13)..(14) <223> n is a, c, g or t <220> <221> misc_features <222> (16)..(17) <223> n is a, c, g or t <220> <221> misc_features <222> (19)..(20) <223> n is a, c, g or t <220> <221> misc_features <222> (22)..(23) <223> n is a, c, g or t <220> <221> misc_features <222> (25)..(26) <223> n is a, c, g or t <220> <221> misc_features <222> (28)..(29) <223> n is a, c, g or t <220> <221> misc_features <222> (31)..(32) <223> n is a, c, g or t <400> 11 catgccatgg ccnnknnknn knnknnknnk nnkgcggcga aagcgg 46 <210> 12 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> pADL-g3-P2 Primer <400> 12 catgccatgg ccggctgggc cgc 23 <210> 13 <211> 26 <212> DNA <213> Artificial sequence <220> <223> M13gIII-1 primer <400> 13 ccttccctcc cttaatcggt tgaatg 26 <210> 14 <211> 26 <212> DNA <213> Artificial sequence <220> <223> M13gIII-2 primer <400> 14 cattcaaccg attaagggag ggaagg 26 <210> 15 <211> 32 <212> DNA <213> Artificial sequence <220> <223> CloDFori-1 primers <400> 15 ttggcgcgcc caattagcta gctcactcgg tc 32 <210> 16 <211> 32 <212> DNA <213> Artificial sequence <220> <223> CloDFori-2 primers <400> 16 tgttcctagg gataaattgc actgaaatct ag 32 <210> 17 <211> 32 <212> DNA <213> Artificial sequence <220> <223> pEVOLori-1 Primers <400> 17 tgttcctagg tcttcaaatg tagcacctga ag 32 <210> 18 <211> 31 <212> DNA <213> Artificial sequence <220> <223> pEVOLori-2 Primers <400> 18 ttggcgcgcc ccttttttct cctgccacat g 31 <210> 19 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> In-frame amber codon fragments from phage display <400> 19 tcgttgtagt tgcagcattc t 21 <210> 20 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> In-frame amber codon fragments from phage display <400> 20 tagagtgcta ttacgtattg t 21 <210> twenty one <211> twenty one <212> DNA <213> Artificial sequence <220> <223> In-frame amber codon fragments from phage display <400> twenty one atttagattc atccgtattt t 21 <210> twenty two <211> twenty one <212> DNA <213> Artificial sequence <220> <223> In-frame amber codon fragments from phage display <400> twenty two tatagtatta agtggtaggt t 21 <210> twenty three <211> twenty one <212> DNA <213> Artificial sequence <220> <223> In-frame amber codon fragments from phage display <400> twenty three ggtgcgtaga ggccggggat g 21 <210> twenty four <211> twenty one <212> DNA <213> Artificial sequence <220> <223> In-frame amber codon fragments from phage display <400> twenty four cgtatgcatt aggcgttttt t 21 <210> 25 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> In-frame amber codon fragments from phage display <400> 25 actacttata ctggtgatta g 21 <210> 26 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> In-frame amber codon fragments from phage display <400> 26 ggttcgttgc atgttcatta g 21 <210> 27 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> In-frame amber codon fragments from phage display <400> 27 tagttgcatc cggggatgcg t 21 <210> 28 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> In-frame amber codon fragments from phage display <400> 28 tattagactg attctgatgc t 21 <210> 29 <211> 7 <212> PRT <213> Artificial sequence <220> <223> SAP01 <220> <221> MISC_Features <222> (2) <223> X is a non-classical amino acid <400> 29 Lys Xaa Asn Phe Gly Tyr Tyr 1 5 <210> 30 <211> 7 <212> PRT <213> Artificial sequence <220> <223> SAP02 <220> <221> MISC_Features <222> (5) <223> X is a non-classical amino acid <400> 30 Lys Trp Lys Val Xaa Thr Ser 1 5 <210> 31 <211> 7 <212> PRT <213> Artificial sequence <220> <223> SAP03 <220> <221> MISC_Features <222> (3) <223> X is a non-classical amino acid <400> 31 Thr Trp Xaa Lys Ser Asn Trp 1 5 <210> 32 <211> 7 <212> PRT <213> Artificial sequence <220> <223> SAP04 <220> <221> MISC_Features <222> (7) <223> X is a non-classical amino acid <400> 32 Met Ala Lys Pro Gln Arg Xaa 1 5 <210> 33 <211> 7 <212> PRT <213> Artificial sequence <220> <223> SAP05 <220> <221> MISC_Features <222> (6) <223> X is a non-classical amino acid <400> 33 Arg Glu His Lys Pro Xaa Asn 1 5 <210> 34 <211> 7 <212> PRT <213> Artificial sequence <220> <223> SAP06 <220> <221> MISC_Features <222> (3) <223> X is a non-classical amino acid <400> 34 Lys Leu Xaa Lys His Tyr Pro 1 5 <210> 35 <211> 7 <212> PRT <213> Artificial sequence <220> <223> SAP09 <220> <221> MISC_Features <222> (3) <223> X is a non-classical amino acid <400> 35 Lys Leu Xaa Lys His Tyr Pro 1 5 <210> 36 <211> 7 <212> PRT <213> Artificial sequence <220> <223> SAP10 <220> <221> MISC_Features <222> (3) <223> X is a non-classical amino acid <400> 36 Lys Leu Xaa Lys His Tyr Pro 1 5 <210> 37 <211> 7 <212> PRT <213> Artificial sequence <220> <223> SAP11 <220> <221> MISC_Features <222> (3) <223> X is a non-classical amino acid <400> 37 Lys Leu Xaa Lys His Tyr Pro 1 5 <210> 38 <211> 7 <212> PRT <213> Artificial sequence <220> <223> SAP12 <220> <221> MISC_Features <222> (3) <223> X is a non-classical amino acid <400> 38 Lys Leu Xaa Lys His Tyr Pro 1 5 <210> 39 <211> 7 <212> PRT <213> Artificial sequence <220> <223> SAP13 <220> <221> MISC_Features <222> (3) <223> X is a non-classical amino acid <400> 39 Lys His Xaa Lys Ala Ile Pro 1 5 <210> 40 <211> 7 <212> PRT <213> Artificial sequence <220> <223> SAP14 <220> <221> MISC_Features <222> (3) <223> X is a non-classical amino acid <400> 40 Lys Met Xaa Pro Gln Arg Asn 1 5 <210> 41 <211> 7 <212> PRT <213> Artificial sequence <220> <223> SAP15 <220> <221> MISC_Features <222> (3) <223> X is a non-classical amino acid <400> 41 Lys Met Xaa Pro Gln Arg Asn 1 5 <210> 42 <211> 7 <212> PRT <213> Artificial sequence <220> <223> SAP16 <220> <221> MISC_Features <222> (3) <223> X is a non-classical amino acid <400> 42 Lys Met Xaa Pro Gln Arg Asn 1 5 <210> 43 <211> 7 <212> PRT <213> Artificial sequence <220> <223> SAP17 <220> <221> MISC_Features <222> (3) <223> X is a non-classical amino acid <400> 43 Lys Met Xaa Pro Gln Arg Asn 1 5 <210> 44 <211> 7 <212> PRT <213> Artificial sequence <220> <223> SAP18 <220> <221> MISC_Features <222> (3) <223> X is a non-classical amino acid <400> 44 Lys Met Xaa Pro Gln Arg Asn 1 5 <210> 45 <211> 7 <212> PRT <213> Artificial sequence <220> <223> SAP19 <220> <221> MISC_Features <222> (4) <223> X is a non-classical amino acid <400> 45 Lys Trp Cys Xaa Asn Cys Arg 1 5 <210> 46 <211> 7 <212> PRT <213> Artificial sequence <220> <223> SAP20 <220> <221> MISC_Features <222> (6) <223> X is a non-classical amino acid <400> 46 Ser Ala Ala Lys Thr Xaa Ile 1 5 <210> 47 <211> 7 <212> PRT <213> Artificial sequence <220> <223> S2P01 <220> <221> MISC_Features <222> (2) <223> X is a non-classical amino acid <400> 47 Leu Xaa Met Thr Ser Ile Leu 1 5 <210> 48 <211> 7 <212> PRT <213> Artificial sequence <220> <223> S2P02 <220> <221> MISC_Features <222> (3) <223> X is a non-classical amino acid <400> 48 His Leu Xaa Thr Phe Phe Tyr 1 5 <210> 49 <211> 7 <212> PRT <213> Artificial sequence <220> <223> S2P03 <220> <221> MISC_Features <222> (4) <223> X is a non-classical amino acid <400> 49 Cys Thr Val Xaa Thr Ser Leu 1 5 <210> 50 <211> 7 <212> PRT <213> Artificial sequence <220> <223> S2P04 <220> <221> MISC_Features <222> (5) <223> X is a non-classical amino acid <400> 50 Thr Cys Thr Val Xaa Ile Gly 1 5 <210> 51 <211> 7 <212> PRT <213> Artificial sequence <220> <223> S2P05 <220> <221> MISC_Features <222> (5) <223> X is a non-classical amino acid <400> 51 Thr Cys Thr Val Xaa Ile Gly 1 5 <210> 52 <211> 7 <212> PRT <213> Artificial sequence <220> <223> S2P06 <220> <221> MISC_Features <222> (5) <223> X is a non-classical amino acid <400> 52 Thr Cys Thr Val Xaa Ile Gly 1 5 <210> 53 <211> 7 <212> PRT <213> Artificial sequence <220> <223> S2P07 <220> <221> MISC_Features <222> (4) <223> X is a non-classical amino acid <400> 53 Cys Thr Phe Xaa Val Pro Thr 1 5 <210> 54 <211> 7 <212> PRT <213> Artificial sequence <220> <223> S2P08 <220> <221> MISC_Features <222> (5) <223> X is a non-classical amino acid <400> 54 Trp Ser Gly Phe Xaa Ala Pro 1 5 <210> 55 <211> 7 <212> PRT <213> Artificial sequence <220> <223> S2P09 <220> <221> MISC_Features <222> (5) <223> X is a non-classical amino acid <400> 55 Trp Ser Gly Phe Xaa Ala Pro 1 5 <210> 56 <211> 7 <212> PRT <213> Artificial sequence <220> <223> S2P10 <220> <221> MISC_Features <222> (2) <223> X is a non-classical amino acid <400> 56 Phe Xaa Leu Glu Ser Phe Leu 1 5 <210> 57 <211> 7 <212> PRT <213> Artificial sequence <220> <223> S2P11 <220> <221> MISC_Features <222> (5) <223> X is a non-classical amino acid <400> 57 Ser Asn Val Phe Xaa Val Ile 1 5 <210> 58 <211> 7 <212> PRT <213> Artificial sequence <220> <223> S2P12 <220> <221> MISC_Features <222> (3) <223> X is a non-classical amino acid <400> 58 Ala Phe Xaa His Met Thr Val 1 5 <210> 59 <211> 7 <212> PRT <213> Artificial sequence <220> <223> S2P13 <220> <221> MISC_Features <222> (5) <223> X is a non-classical amino acid <400> 59 Gln Met Arg Phe Xaa Pro Ile 1 5 <210> 60 <211> 7 <212> PRT <213> Artificial sequence <220> <223> S2P14 <220> <221> MISC_Features <222> (5) <223> X is a non-classical amino acid <400> 60 Gln Met Arg Phe Xaa Pro Ile 1 5 <210> 61 <211> 7 <212> PRT <213> Artificial sequence <220> <223> S2P15 <220> <221> MISC_Features <222> (1) <223> X is a non-classical amino acid <400> 61 Xaa Val Cys Ser Cys Tyr Ala 1 5 <210> 62 <211> 7 <212> PRT <213> Artificial sequence <220> <223> S2P16 <220> <221> MISC_Features <222> (1) <223> X is a non-classical amino acid <400> 62 Xaa Val Cys Ser Cys Tyr Ala 1 5 <210> 63 <211> 7 <212> PRT <213> Artificial sequence <220> <223> S2P17 <220> <221> MISC_Features <222> (5) <223> X is a non-classical amino acid <400> 63 Cys Trp Trp Cys Xaa Val Ser 1 5 <210> 64 <211> 7 <212> PRT <213> Artificial sequence <220> <223> S2P18 <220> <221> MISC_Features <222> (6) <223> X is a non-classical amino acid <400> 64 Thr Glu Ser Asn His Xaa Gly 1 5 <210> 65 <211> 7 <212> PRT <213> Artificial sequence <220> <223> S2P19 <220> <221> MISC_Features <222> (7) <223> X is a non-classical amino acid <400> 65 Leu Phe Leu Trp Met Pro Xaa 1 5 <210> 66 <211> 7 <212> PRT <213> Artificial sequence <220> <223> S2P20 <220> <221> MISC_Features <222> (4) <223> X is a non-classical amino acid <400> 66 Ser Pro Met Xaa Asn Lys Val 1 5 <210> 67 <211> 7 <212> PRT <213> Artificial sequence <220> <223> ENL inhibitors <220> <221> MISC_Features <222> (6) <223> X is CrK <400> 67 Asp Ile Trp Cys Phe Xaa Gly 1 5 <210> 68 <211> 7 <212> PRT <213> Artificial sequence <220> <223> ENL inhibitors <220> <221> MISC_Features <222> (2) <223> X is CrK <400> 68 Ser Xaa Asp Ile Trp Cys Phe 1 5

Claims

1. A method for selecting a peptide or protein that binds to a desired target, the method comprising: (a) providing a filamentous phage display library comprising a plurality of nucleic acids, wherein the plurality of nucleic acids includes a nucleic acid having a bacteriophage capsid protein gene containing a combined region, wherein the bacteriophage capsid protein gene is a pill gene, wherein at least 90% of the combined regions in the phage display library include at least one in-frame amber codon; (b) transforming the phage display library into bacterial host cells; wherein the bacterial host cell is capable of translating the combined regions of the bacteriophage capsid protein gene such that at least one in-frame amber codon encodes the desired non-classical amino acid, wherein the bacterial host cell is co-infected with a knockout helper phage that does not express the phage capsid protein gene, and wherein said transformation allows for the production of a phage particle comprising said phage capsid protein having said desired non-canonical amino acids in the combined regions; (c) screening the phage particles against the desired target, wherein the screening results in the selection of phage particles having a combination of phage capsid protein regions that bind to the desired target; and (d) Identifying the amino acid sequence of the combined regions of the selected phage particles.

2. The method of claim 1, wherein the screening is performed by affinity selection against the desired target.

3. The method of claim 1, wherein the screening is performed by: (a) incubating the phage particles with the desired target, wherein the desired target is immobilized on a surface; (b) separating unbound phage particles from phage particles bound to the desired target; and (c) isolating the bound phage particles.

4. The method of claim 1, wherein: The screening further includes: (a) transforming the selected phage particle into the bacterial host cell to allow for production of further phage particles; and (b) According to step (c) of claim 1, further screening the phage particles.

5. The method of claim 4, wherein: This further screening was repeated multiple times.

6. The method of claim 1, wherein the screening result is the selection of phage particles having a phage capsid protein combination region that binds to a ligand binding site of a desired target, and wherein the non-classical amino acids act as ligands to guide the phage capsid combination region to the ligand binding site of the desired target.

7. The method of claim 1, wherein the identification is performed by sequencing combinatorial regions of the selected phage particles.

8. The method of claim 1, wherein the identifying comprises: (a) purifying the selected phage particles; (b) separating the nucleic acid from the selected phage particles; and (c) sequencing said combined regions of said nucleic acid.

9. The method of claim 1, wherein the desired target is selected from the group consisting of a peptide, a protein, a cell receptor, an antigen, and combinations thereof.

10. The method of claim 1, wherein the desired target is an enzyme or an active site of a desired target.

11. The method of claim 1, wherein the desired target is selected from the group consisting of a ligand binding site of a desired target, an active site of a protein, an allosteric site of a protein, and combinations thereof.

12. The method of claim 1, wherein the bacterial host cell is an amber-suppressive bacterial host strain.

13. The method of claim 1, wherein the bacterial host cell comprises an amber suppressor tRNA that has been aminoacylated with a desired non-classical amino acid by a cognate aminoacyl-tRNA synthetase.

14. The method of claim 1, wherein the desired non-classical amino acid is selected from the group consisting of S-2-amino-8-oxononanoic acid, S-amino-8-oxodecanoic acid, N 6 -Acetyl-L-lysine, N 6 -Propionyl-L-lysine, N 6 -Butyryl-L-lysine, N 6 -crotonyl-L-lysine, N 6 -(prop-2-yn-1-yl)-lysine, N 6 -Methyl-N 6 -N 6 -(Prop-2-yn-1-yl)-lysine, and combinations thereof.

15. The method of claim 1, wherein the at least one in-frame amber codon is within a region of the peptide that binds to a ligand binding site of a protein.

16. The method of claim 1, wherein the phage display library comprises a plurality of phage, and wherein the plurality of nucleic acids in the phage display library comprises nucleic acids of the phage.

17. The method of claim 1, wherein the phage display library comprises a plurality of phagemids, and wherein the plurality of nucleic acids in the phage display library comprises nucleic acids of the phagemids.

18. A method for selecting a peptide that binds to a ligand binding site of a desired target, the method comprising: (a) providing a filamentous phage display library comprising a plurality of nucleic acids, wherein the plurality of nucleic acids includes a nucleic acid having a bacteriophage capsid protein gene containing a combined region, wherein the bacteriophage capsid protein gene is a pill gene, wherein at least 90% of the combined regions in the phage display library include at least one in-frame amber codon, wherein the at least one in-frame amber codon is within the coding region for a peptide that binds to a ligand binding site of the desired target; (b) transforming the phage display library into bacterial host cells; wherein the bacterial host cell is capable of translating the combined regions of the bacteriophage capsid protein gene such that at least one in-frame amber codon encodes the desired non-classical amino acid, wherein the bacterial host cell is co-infected with a knockout helper phage that does not express the phage capsid protein gene, and wherein said transformation allows for the production of a phage particle comprising said phage capsid protein having said desired non-canonical amino acids in the combined regions; (c) screening the phage particles against the desired target, wherein the screening results in the selection of phage particles having a combination of regions of phage capsid proteins that bind to the ligand binding site of the desired target, and wherein the non-classical amino acids act as ligands to direct the phage capsid protein combination region to the ligand binding site of the desired target; and (d) Identifying the amino acid sequence of the combined regions of the selected phage particles.

19. The method of claim 18, wherein the screening is performed by affinity selection against the desired target.

20. The method of claim 18, wherein the screening is performed by: (a) incubating the phage particles with the desired target, wherein the target is immobilized on a surface; (b) separating unbound phage particles from phage particles bound to the desired target; and (c) isolating the bound phage particles.

21. The method of claim 18, wherein: The screening further includes: (a) transforming the selected phage particle into the bacterial host cell to allow for production of further phage particles; and (b) According to step (c) of claim 13, further screening the phage particles.

22. The method of claim 21, wherein: This further screening was repeated multiple times.

23. The method of claim 18, wherein the identification is performed by sequencing combinatorial regions of the selected phage particles.

24. The method of claim 18, wherein the identifying comprises: (a) purifying the selected phage particles; (b) separating the nucleic acid from the selected phage particles; and (c) sequencing said combined regions of said nucleic acid.

25. The method of claim 18, wherein the bacterial host cell is an amber suppressive bacterial host strain.

26. The method of claim 18, wherein the bacterial host cell comprises an amber suppressor tRNA that has been aminoacylated with a desired non-classical amino acid by a cognate aminoacyl-tRNA synthetase.

27. The method of claim 18, wherein the desired non-classical amino acid is selected from the group consisting of S-2-amino-8-oxononanoic acid, S-amino-8-oxodecanoic acid, N 6 -Acetyl-L-lysine, N 6 -Propionyl-L-lysine, N 6 -Butyryl-L-lysine, N 6 -crotonyl-L-lysine, N 6 -(prop-2-yn-1-yl)-lysine, N 6 -Methyl-N 6 -N 6 -(Prop-2-yn-1-yl)-lysine, and combinations thereof.

28. The method of claim 18, wherein the ligand binding site of the desired target is an active site of a desired protein, an allosteric site of a desired protein, or a combination thereof.

29. The method of claim 18, wherein the ligand inhibits the activity of one or more epigenetic enzymes or epigenetic reader proteins.

30. The method of claim 18, wherein the phage display library comprises a plurality of phage, and wherein the plurality of nucleic acids in the phage display library comprises nucleic acids of the phage.

31. The method of claim 18, wherein the phage display library comprises a plurality of phagemids, and wherein the plurality of nucleic acids in the phage display library comprises nucleic acids of the phagemids.

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