Peptide display systems and method for the identification of modulators of mammalian receptors

Abstract

Provided herein are systems and methods for the identification of modulators of mammalian receptors and modulators identified therefrom. For example, the technology provides a mammalian peptide display system combined with a cell-based assay with functional readout that simultaneously permits the screening and characterization of large numbers of candidate modulators.

Description

[0001] STDU2-44084.601

[0002] PEPTIDE DISPLAY SYSTEMS AND METHOD FOR THE IDENTIFICATION OF MODULATORS OF MAMMALIAN RECEPTORS

[0003] The present application claims priority to United States Provisional Patent Application serial number 63 / 728,951, filed December 6, 2024, the disclosure of which is herein incorporated by reference in its entirety.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under AI148623 and AI143757 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0006] SEQUENCE LISTING

[0007] The text of the computer readable sequence listing filed herewith, titled “STDU2_44084_601_SequenceListing.xml”, created December 5, 2025, having a file size of 95,512 bytes, is hereby incorporated by reference in its entirety.

[0008] FIELD

[0009] Provided herein are systems and methods for the identification of modulators of mammalian receptors and modulators identified therefrom. For example, the technology provides a mammalian peptide display system combined with a cell-based assay with functional readout that simultaneously permits the screening and characterization of large numbers of candidate modulators.

[0010] BACKGROUND

[0011] Identifying variants of important peptide activated mammalian receptors, such as GPCRs, can be difficult. Many peptide-agonized GPCRs are important in diseases and clinical syndromes, such as obesity. For example, GLP-1R, the glucagon-like receptor, can be targeted by the peptide hormone GLP-1 and this modulates hunger and satiety. Identifying more potent STDU2-44084.601 agonists or antagonists of these receptors could have significant clinical utility. Existing methods to screen potential agonists / antagonists are plagued by low throughput (e.g. arrayed screens) and high costs of peptide synthesis; or are screens for binding (e.g. phage display) instead of functional activity of a receptor. New and more effective and efficient systems are needed.

[0012] SUMMARY

[0013] G protein-coupled receptors (GPCRs) are the largest class of membrane proteins in the human genome, composed of over 800 receptors. 398 of these receptors are non-olfactory GPCRs integral in regulating numerous biological functions, including sensory perception, immune response, and neuro transmission (6). Non-olfactory G protein-coupled receptors (GPCRs) regulate essential physiological functions and are the targets of approximately 34% of FDA-approved drugs (1). Most of the GPCRs targeted by FDA-approved drugs are aminergic with small molecule ligands (1,6). However, in an interesting shift in focus, more than half of the emerging GPCR targets that have reached clinical trials but have not yet been approved are peptide receptors (1,6). The expansion in the number of peptide receptors in clinical trials has likely been empowered by exciting technological advances. These include more comprehensive annotation of natural peptide ligands and emerging novel strategies such as half-life extension platforms and enhancing resistance to proteolysis, which have improved pharmacokinetics and oral bioavailability (1,7). Current methods for studying GPCR-peptide interactions at the amino acid level are resource-intensive and challenging to scale, making it difficult to simultaneously investigate the effects of thousands of peptides on receptor activation. To overcome these limitations, the present technology provides a high-throughput peptide display platform that uses a cell surface peptide display system coupled with a functional read-out.

[0014] Melanocortin-4 receptor (MC4R), a key player in energy homeostasis and appetite control, was used to demonstrate the effectiveness of the system by utilizing a P-arrestin-based MC4R reporter. Activation of MC4R by its natural ligand, P-melanocyte-stimulating hormone (P-MSH), reduces food intake (2), and dysfunction in this pathway leads to obesity in various species (3-5). These findings underscore the critical role of the MC4R and P-MSH pathway in energy regulation and illustrate the clinical significance of characterizing GPCR-peptide interactions in molecular detail. The screening system was leveraged to screen a peptide library comprised of over two thousand P-MSH peptide point mutants and identify variants that STDU2-44084.601 significantly impact MC4R activation. Notably, this system identified a novel D5H mutation of 0-MSH with an increased ability to activate MC4R. Together, these results demonstrate an innovative approach that allows one to directly link the functional activation of a GPCR to a specific peptide, all within a single cell.

[0015] In some embodiments, provided herein are method for screening peptides, comprising: expressing a plurality of different candidate peptides (e.g., regulatory peptides, i.e., peptides that regulate the activity of one or more biological pathways, for example via binding to a cell surface receptor) in a plurality of mammalian cells, wherein said cells express a heterologous receptor reporter (i.e., a receptor combined with a reporter system that is not natively found in the cell) that generates a detectable signal (e.g., creation of signal, loss of signal, increase in signal, decrease in signal) when a peptide regulates (e.g., activates or inhibits) the receptor. In some embodiments, the receptor is a G protein-coupled receptor.

[0016] The plurality of different candidate peptides can include related or unrelated peptides sequences or structures, hi some embodiments, the different candidate peptides are variants of one or more specific reference polypeptides. The reference polypeptide may comprise, for example, a wild type GPCR-interacting sequence such as a ligand, or any other optimizable sequence wherein the receptor-regulating properties thereof are sought to be modulated or improved. The variants may comprise a collection of mutations, e.g. an amino acid substitution, deletion, or addition relative to that of the reference sequence. The polypeptides may be any desirable length (e.g., 3 or more, 5 or more, 10 or more, 15 or more. 20 or more, 30 or more, 50 or more, 100 or more amino acids; 3-100, 5-50, 10-30, etc.). In some embodiments, the collection of variants of a polypeptide includes at least one mutation at every amino acid position of the reference polypeptide sequence. Each variant may have a single mutation relative to the reference polypeptide or may have two or more mutations. In some embodiments, the collection of variants includes every possible amino acid change at a given position (or two or more positions) of the polypeptide. In some embodiments, the nucleic acid molecules used to express the candidate peptides include nucleic acid variations to account for any or each of the possible codon variations for a given amino acid. The term “peptide library” refers to the group of different candidate peptides that are analyzed together either simultaneously or sequentially to determine their function (e.g.. their ability to regulate a cell surface receptor). STDU2-44084.601

[0017] In some embodiments, the cells are human cells, although other mammalian cells may be employed. In some embodiments, the cells are immortalized cultured cells. In some embodiments, the cells are primary cells. In some embodiments, the cells are tumor cells. In some embodiments, the cells are stem cells. In various embodiments, the cells may comprise HEK293 cells, HEK293T, HEK293F, HeLa, A549, MCF-7, U20S, and CHO cells.

[0018] In some embodiments, the cells are engineered to express one or more heterologous proteins. For example, in some embodiments, the cells are engineered to express one or more heterologous cell surface receptors (e.g., G-protein coupled receptors). In some embodiments, the cells are engineered to express a heterologous fluorescent or luminescent molecule (e.g., luciferase) or other detectable marker. A “heterologous” protein refers to a protein expressed in the cell synthetically. Heterologous proteins include proteins that are not native to the cell or those that may be native to the cell, but that are expressed synthetically, e.g., using a non-native promoter, added in multiple copies, located in a non-native position within the genome, etc.

[0019] Cell Reporter Systems

[0020] In one implementation, the cells comprise a receptor reporter cell line. The receptor reporter cell line comprises a cell line engineered to express a heterologous reporter system wherein a selected cell receptor is expressed and wherein activation of the receptor by a ligand or other interactor generates a detectable signal. In a primary embodiment, the receptor is a GPCR.

[0021] In one implementation, the GPCR reporter system comprises a Presto-TANGO reporter system (20). For example, in one embodiment, each GPCR is modified to include a “Tango” protein fragment at the 3’ end, comprising a Tobacco Etch Virus (TEV) protease cleavage site and a tetracycline controlled transactivator (tTA). This synthetic GPCR is expressed in a selected cell line stably expressing a luciferase reporter and a synthetic beta-arrestin fused to the TEV protease. Upon activation by a ligand, the GPCR triggers the recruitment of a synthetic betaarrestin fused to the TEV protease and releases the tTA (25). Once the tTA is freed, it translocates into the nucleus and turns on the reporter leading to luciferase expression (FIG. la). In some embodiments, the Presto-TANGO system comprises activator domains and mutations in the TetR protein to reduce leakage (27,28). In some embodiments, the “Tango” component comprises tTA Advance (27), a tetracycline-controlled transactivator with improved stability and minimal background expression. In some embodiments, the VP 16 activator comprises the STDU2-44084.601 compact human activator combination, NFZ, as described in (29), which has strong activation and when overexpressed and is less toxic to human cells (FIG. la). In some embodiments, different combinations of stronger and weaker activators may be employed to fine-tune the signal-to-noise ratio and sensitivity, as desired.

[0022] In various embodiments, the GPCR reporter system may comprise any GPCR reporter system which generates a signal in response to interaction of a ligand with a selected GPCR. In various embodiments, the GPCR reporter system may comprise an assay responsive to GPCR activation by conformational changes, G-protein recruitment, 0-arrestin recruitment, GTPyS binding, cAMP generation, IP3 / IP1 generation , Ca2+ flux, and reporter gene expression. Conformational assays include, for example, Nb80-GFP, dLight and GRAB -DA assays. 0- arrestin based assays include, for example BRET / FRET / NanoBRET™ (Promega), Transfluor™ (Molecular Devices), and PathHunter™ (DiscoverX) assays. Reporter genes include, for example, genes expressing luciferase, alkaline phosphatase, 0-galactosidase, 0-lactamase or fluorescent proteins.

[0023] In some embodiments, internalization of a GPCR is detected using an antibody that is conjugated to a marker. Upon activation, GPCRs may relocate from the cell membrane to the endosomal compartment within the cell. This process can be monitored by using an antibody that specifically targets the GPCR of interest. The antibody can be conjugated to a fluorescent marker or targeted by a secondary antibody that is conjugated to a marker, allowing for visualization of this movement.

[0024] Peptide Display Elements

[0025] The system of the invention provides a modular peptide display element that allows the expression and localization of any peptide of interest to the cell membrane. A library of candidate peptides may be expressed in the peptide display elements. Localization of candidate peptides to the cell surface facilitates interactions (e.g. GPCR activation) by effective peptide variants, wherein the peptide activation of the receptor results in generation of detectable signal, allowing selection of cells comprising effective peptide variants and their identification by subsequent sequencing of selected cells. STDU2-44084.601

[0026] In some embodiments, the peptide display expression element is an expression vector that codes for the peptide display element. In some embodiments, the peptide display expression element comprises: a coding sequence for one or more cleavable membrane localization signals; a peptide cloning site wherein a candidate peptide coding sequence can be or has been inserted; a linker sequence; and a transmembrane domain that anchors the linked candidate peptide to the plasma membrane and displays it on the cell surface for accessibility to the receptor of the receptor reporter system. The expression element may further comprise the coding sequence of one or more detectable elements. In some embodiments, the peptide display expression element is transfected into the reporter cell, wherein the peptide display element is expressed and localized to the membrane. Cleavage of the one or more localization signals results in a peptide display element comprising a membrane bound candidate peptide, comprised of the transmembrane membrane anchoring element, the linker sequence, and the candidate peptide, and optionally, one or more detectable elements. In one embodiment, the peptide display expression element is in a configuration as depicted in FIG. la (top expression vector) and the peptide display element is in a configuration as depicted in FIG. la in the membrane.

[0027] In one embodiment, the membrane localization element comprises a cleavable signal peptide derived from pre-pro-trypsin (e.g.. comprising sequence MSALLILALVGAAVA (SEQ ID NO:1)).

[0028] Mammalian signal peptides that direct the peptide display construct to the cell membrane include, but are not limited to, human OSM (MGVLLTQRTLLSLVLALLFPSMASM, SEQ ID NO: 14), VSV-G (MKCLLYLAFLFIGVNC, SEQ ID NO: 15), mouse Ig Kappa (METDTLLLWVLLLWVPGSTGD, SEQ ID NO: 16), mouse Ig Heavy (MGWSCIILFLVATATGVHS, SEQ ID NO: 17), BM40 (MRAWIFFLLCLAGRALA, SEQ ID NO: 18), Secrecon (MWWRLWWLLLLLLLLWPMVWA, SEQ ID NO: 19), Human IgKVIII (MDMRVPAQLLGLLLLWLRGARC, SEQ ID NO:20), CD33 (MPLLLLLPLLWAGALA, SEQ ID NO:21), tPA (MDAMKRGLCCVLLLCGAVFVSPS, SEQ ID NO:22), human chymotrypsinogen (MAFLWLLSCWALLGTTFG, SEQ ID NO:23), human trypsinogen-2 (MNLLLILTFVAAAVA, SEQ ID NO:24), human IL-2 (MYRMQLLSCIALSLALVTNS, SEQ ID NO:25), Gaussia luc (MGVKVLF ALICIA VAEA, SEQ ID NO:26), albumin (MKWVTFISLLFSSAYS, SEQ ID NO:27), Influenza Haemagglutinin STDU2-44084.601

[0029] (MKTIIALSYIFCLVLG, SEQ ID NO:28), human insulin

[0030] (MALWMRLLPLLALLALWGPDPAAA, SEQ ID NO:29), and silkworm fibroin LC

[0031] (MKPIFLVLLVVTSAYA, SEQ ID NO:30).

[0032] In one embodiment, the linker sequence comprises any flexible linker, for example, comprising 2-100 amino acids, for example 5-30 amino acids for example, 10-20 amino acids. Linkers may comprise flexible linkers such as glycine and serine rich linkers such as polyglycine-serine linkers as known in the art. In one embodiment, the linker comprises one or more repeats of the XTEN linker (e.g., comprising amino acid sequence SGSETPGTSESATPES (SEQ ID NO:31)). As described herein, XTEN provided high quality results.

[0033] In one embodiment, the membrane- anchoring element comprises a PDGFR-0 transmembrane domain (e.g., comprising amino acid sequence NAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISL1ILIMLWQKKPR (SEQ ID NO:32)).

[0034] In some embodiments, a detectable element is present in the peptide display element. In one embodiment, the detectable element is disposed between the C-terminus of the linker and the N -terminus of the membrane-anchoring element, for example, as depicted in FIG la. In one embodiment, the detectable element comprises a Myc Tag (e.g., comprising the amino acid sequence EQKLISEEDL (SEQ ID NO:33)). In alternative embodiments, the one or more detectable elements may comprise any of a HisTag, FLAG Tag, HA Tag, or fluorescent protein.

[0035] In some embodiments, the library of candidate peptides is generated using the “alLby-all” deep mutational scanning methodology described herein. Alternative methods of generating candidate peptide libraries are known in the art, including, for example, random mutagenesis (for example, by error-PCR), recombination and gene shuffling (for example, by SCHEMA), and targeted mutagenesis approaches (for example, site-directed, site-saturation and combinatorial saturation mutagenesis).

[0036] In some embodiments, the coding sequences of the candidate peptide further comprises a unique oligonucleotide identification element to facilitate identification of effective peptide variants by selection and sequencing of reporter cells that generate sufficient reporter signal. As described herein, unique oligo sequences of 120 base pairs were included in the peptide STDU2-44084.601 expression elements. In alternative embodiments, any number of barcode or other unique sequence identifiers may be used.

[0037] In some embodiments, the detectable signal is generated if a candidate regulatory peptide activates the receptor. In some embodiments, the detectable signal is generated if a candidate regulatory peptide inhibits the receptor.

[0038] In some embodiments, the heterologous receptor reporter comprises one or more reporter molecules fused to the receptor, wherein the one or more reporter molecules directly or indirectly generate the detectable signal when the receptor is activated. In some embodiments, the one or more reporter molecules comprise a transcription factor linked to the receptor by a cleavage site. In some embodiments, the one or more reporter molecules further comprise a cleavage enzyme linked to a P-arrestin molecule, wherein upon activation of the receptor, said cleavage enzyme is mobilized to cleave the cleavage site and release the transcription factor from the receptor. In some embodiments, the cleavage enzyme is a TEV protease. In some embodiments, the transcription factor is an artificial transcription factor. In some embodiments, the cell comprises a luciferase reporter gene and the artificial transcription factor activates expression of the luciferase reporter gene to generate the detectable signal.

[0039] In some embodiments, positive and / or negative control peptides are utilized. For example, in some embodiments, a positive control peptide is a peptide sequence known to activate a target receptor. By expressing the positive control peptide (e.g., as part of a transmembrane fusion construct) in at least one cell, to generate a detectable signal, the function of the system can be assessed. Likewise, by including at least one negative control peptide known to not activate a target receptor, a background level associated with no activity can be established.

[0040] The methods find use with both small and large peptide libraries and can be utilized as part of an efficient, high-throughput process. In some embodiments, 10 or more (e.g., 100 or more. 200 or more, 500 or more, 1000 or more, 1500 or more. 2000 or more; between 10-5000, 100-2000; etc.) different candidate regulatory peptides may be tested.

[0041] In some embodiments, the candidate regulatory peptides are expressed from a vector. In some embodiments, the vector is integrated into a genome of the cell. In some embodiments, the STDU2-44084.601 vector is expressed episomally. Tn some embodiments, the vector comprises a selection marker (e.g., to allow for the selection of cells that successfully integrate or express a transmembrane fusion construct containing the candidate peptide).

[0042] Also provided herein are systems and kits for conducting any of the methods described herein. For example, in some embodiments, a kit comprises one or more or each of: a) a first expression vector comprising a cloning site, for receiving a sequence encoding a polypeptide of interest, fused to a linker, fused to a transmembrane domain; b) a second expression vector encoding a transmembrane receptor fused to one or more reporter proteins (e.g., transcription factors) that generate a detectable signal (e.g., directly or indirectly) when a peptide regulates said receptor; c) mammalian cells comprising a heterologous receptor reporter that generates a detectable signal when a peptide regulates said receptor; d) cell culture media; e) cell culture hardware and / or instrumentation; f) data collection and / or analysis software; g) cloning reagents (e.g., restriction enzymes); and h) instructions for use. In some embodiments, the first expression vector further comprises a sequence encoding a detectable tag and / or a selection marker. In some embodiments, the mammalian cells comprise a luciferase gene that generates the detectable signal. Uses of such kits are also provided (e.g., to identify receptor modulators).

[0043] Further provided herein are screening systems, comprising: a) a plurality of expression vectors, each comprising a sequence encoding a different candidate regulatory peptide, fused to a linker, fused to a transmembrane domain; and b) mammalian cells comprising a heterologous receptor reporter that generates a detectable signal if a candidate regulatory peptide regulates said receptor. In some embodiments, the plurality of expression vectors encode variants of a polypeptide. In some embodiments, the variants comprise codon-altered or -optimized variants. Uses of such screening systems are also provided (e.g., to identify receptor modulators).

[0044] Also provided herein are active peptides identified by the methods or system described herein. For example, in some embodiments, provided herein are peptide regulators of MC4R, compositions comprising such peptides (e.g., pharmaceutical compositions), and uses of such peptides and compositions (e.g., methods of managing energy homeostasis and / or appetite). In some embodiments, the peptide comprises AEKKXEGPYRMEHRFWGSPPKD (SEQ ID NO:3), wherein X is not D. In some embodiments, X is H.

[0045] Definitions STDU2-44084.601

[0046] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not. As used herein, comprising a certain sequence or a certain SEQ ID NO usually implies that at least one copy of said sequence is present in recited peptide or polynucleotide. However, two or more copies are also contemplated.

[0047] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0048] Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0049] As used herein, a “nucleic acid” or a “nucleic acid sequence” refers to a polymer or oligomer of pyrimidine and / or purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L. Lehninger, Principles of Biochemistry, at 793- 800 (Worth Pub. 1982)). The present technology contemplates any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases, and the like. The polymers or oligomers may be heterogenous or homogenous in composition and may be isolated from naturally occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single- stranded or double- stranded form, including homoduplex, heteroduplex, and hybrid states. In some embodiments, a nucleic acid or nucleic acid sequence comprises other STDU2-44084.601 kinds of nucleic acid structures such as, for instance, a DNA / RNA helix, peptide nucleic acid (PNA), morpholino nucleic acid (see, e.g., Braasch and Corey, Biochemistry, 41(14): 4503-4510 (2002)) and U.S. Pat. No. 5,034,506), locked nucleic acid (LNA; see Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 97: 5633-5638 (2000)), cyclohexenyl nucleic acids (see Wang, J. Am. Chem. Soc., 122: 8595-8602 (2000)), and / or a ribozyme. Hence, the term “nucleic acid” or “nucleic acid sequence” may also encompass a chain comprising non-natural nucleotides, modified nucleotides, and / or non- nucleotide building blocks that can exhibit the same function as natural nucleotides (e.g., “nucleotide analogs”); further, the term “nucleic acid sequence” as used herein refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single or doublestranded, and represent the sense or antisense strand. The terms “nucleic acid,” “polynucleotide,” “nucleotide sequence,” and “oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.

[0050] A “peptide” or “polypeptide” is a linked sequence of two or more amino acids linked by peptide bonds. The peptide or polypeptide can be natural, synthetic, or a modification or combination of natural and synthetic. Polypeptides include proteins such as binding proteins, receptors, and antibodies. The proteins may be modified by the addition of sugars, lipids or other moieties not included in the amino acid chain. The terms “polypeptide” and “protein,” are used interchangeably herein.

[0051] As used herein, the term “percent sequence identity” refers to the percentage of nucleotides or nucleotide analogs in a nucleic acid sequence, or amino acids in an amino acid sequence, that is identical with the corresponding nucleotides or amino acids in a reference sequence after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Hence, in case a nucleic acid according to the technology is longer than a reference sequence, additional nucleotides in the nucleic acid, that do not align with the reference sequence, are not taken into account for determining sequence identity. A number of mathematical algorithms for obtaining the optimal alignment and calculating identity between two or more sequences are known and incorporated into a number of available software programs. Examples of such programs include CLUSTAL-W, T-Coffee, and ALIGN (for STDU2-44084.601 alignment of nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST 2.1 , BL2SEQ, and later versions thereof) and FASTA programs (e.g., FASTA3x, FAS™, and SSEARCH) (for sequence alignment and sequence similarity searches). Sequence alignment algorithms also are disclosed in, for example, Altschul et al., J. Molecular Biol., 215(3): 403-410 (1990), Beigert et al., Proc. Natl. Acad. Sci. USA, 106(10): 3770-3775 (2009), Durbin et al., eds., Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009), Soding, Bioinformatics, 21(7): 951-960 (2005), Altschul et al.. Nucleic Acids Res., 25(17): 3389-3402 (1997), and Gusfield. Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK (1997)).

[0052] The term “amino acid” or “any amino acid” as used here refers to any and all amino acids, including naturally occurring amino acids (e.g., a-amino acids), unnatural amino acids, modified amino acids, and non-natural amino acids. It includes both D- and L-amino acids. Natural amino acids include those found in nature, such as, e.g., the 23 amino acids that combine into peptide chains to form the building-blocks of a vast array of proteins. These are primarily L stereoisomers, although a few D-amino acids occur in bacterial envelopes and some antibiotics. The “non-standard,” natural amino acids include, for example, pyrolysine (found in methanogenic organisms and other eukaryotes), selenocysteine (present in many non-eukaryotes as well as most eukaryotes), and N-formylmethionine (encoded by the start codon AUG in bacteria, mitochondria, and chloroplasts). “Unnatural” or “non-natural” amino acids are non- proteinogenic amino acids (e.g., those not naturally encoded or found in the genetic code) that either occur naturally or are chemically synthesized. Over 140 unnatural amino acids are known and thousands of more combinations are possible. Examples of “unnatural” amino acids include 0-amino acids (03 and 02), homo-amino acids, proline and pyruvic acid derivatives, 3-substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids, diamino acids, D-amino acids, alpha-methyl amino acids and N-methyl amino acids. Unnatural or non-natural amino acids also include modified amino acids. “Modified” amino acids include amino acids (e.g., natural amino acids) that have been chemically modified to include a group, groups, or chemical moiety not naturally present on the amino acid. STDU2-44084.601

[0053] The terms “non-naturally occurring,” “engineered,” and “synthetic” are used interchangeably and indicate the involvement of the hand of man. The terms, when referring to nucleic acid molecules or polypeptides mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and as found in nature.

[0054] A “vector” or “expression vector” is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, e.g., an “insert,” may be attached or incorporated so as to bring about the replication of the attached segment in a cell.

[0055] A cell has been “genetically modified.” “transformed,” or “transfected” by exogenous DNA, e.g., a recombinant expression vector, when such DNA has been introduced inside the cell. The presence of the exogenous DNA results in permanent or transient genetic change. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones that comprise a population of daughter cells containing the transforming DNA. A “clone” is a population of cells derived from a single cell or common ancestor by mitosis. A “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.

[0056] The term “contacting” as used herein refers to bring or put in contact, to be in or come into contact. The term “contact” as used herein refers to a state or condition of touching or of immediate or local proximity. Contacting a system to a target destination, such as. but not limited to, an organ, tissue, cell, or tumor, may occur by any means of administration known to the skilled artisan.

[0057] As used herein, the terms “providing,” “administering,” “introducing.” are used interchangeably herein and refer to the placement of the systems, polypeptides (e.g., pharmaceutical compositions comprising a polypeptide), or nucleic acids of the disclosure into a cell, organism, or subject by a method or route which results in at least partial localization of the system to a desired site. STDU2-44084.601

[0058] A “subject” or “patient” may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such a mouse model. Likewise, patient may include either adults or juveniles (e.g., children). Moreover, patient may mean any living organism, preferably a mammal (e.g., human or non-human) that may benefit from the administration of compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. In one embodiment of the methods and compositions provided herein, the mammal is a human.

[0059] Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0060] Description of Figures

[0061] FIG 1 . Development of a peptide display system to identify agonists of G-protein coupled receptors (GPCRs): a. Schematic illustrating a cell-based platform to identify peptide agonists against human GPCRs. The exemplary system comprises: 1) HTLA reporter cells that expressed P-Arrestin fused to Tobacco Etch Virus (TEV) protease and a tetracycline responsive element (TRE) driving luciferase expression; 2) A synthetic GPCR with a TEV protease cleavage site linked to a Tet Repressor (TetR)-NFZ tripartite activator; and 3) A peptide expression vector with a type IIS restriction site fused to a flexible linker, MYC tag, and platelet derived growth factor receptor beta (PDGFRP) transmembrane (TM) domain. Proper co-expression of a GPCR with its cognate peptide agonist triggers the cleavage of the TetR-NFZ activator and turns on the luciferase reporter, b. Cell surface measurement of P-MSH peptide expression in MC4R HTLA cells using an anti-myc tag conjugated antibody. A glycine and asparagine (GN9) repeat linker was used. Untransfected MC4R HTLA cells are stained in parallel as a negative control, c. Luciferase reporter assay measuring the relative activation of the MC4R in HTLA cells co- STDU2-44084.601 expressing synthetic MC4R and its cognate peptide, P-MSH. MC4R HTLA cells transduced with NPY are the negative control, while MC4R HTLA cells treated with synthetic P-MSH are the positive control. All measurements were normalized to the mean of MC4R HTLA cells treated with DMSO. d. Relative luciferase reporter activity of HTLA cells co-expressing synthetic MC4R and P-MSH fused to various flexible linkers. MC4R HTLA cells transduced with neuropeptide Y fused to the corresponding linkers are used as the negative control. MC4R HTLA cells treated with synthetic P-MSH are used as the positive control. All measurements were normalized to the mean luciferase activity of MC4R HTLA cells treated with DMSO. e. Measurement of P-MSH protein abundance in MC4R HLTA cells transduced with P-MSH tethered to various flexible linkers. Untransduced MC4R HTLA cells are used as a negative gating control.

[0062] FIG. 2. Deep mutational scan of the P-MSH peptide in MC4R HTLA cells using the peptide display system, a. Schematic illustrating the composition of the P-MSH peptide library and the deep mutagenesis screening workflow. b. Reproducibility from two biological replicates. In green are wild type P-MSH peptides, in dark gray are random peptides, in yellow are WT P- MSH peptides, in light gray are single nucleotide mutants of P-MSH, in red are high activity P- MSH mutants, and in blue are low activity P-MSH mutants. Dash lines represent two standard deviations from the mean of the Log2(TOP15:BTM15) values of the wild type P-MSH peptides, c. Heat map of the normalized z-scores of the average Log2(TOP15:BTM15) values of mutants at each position. The z-scores are normalized to the mean and standard deviation of the wild type P-MSH Log2(TOP15:BTM15) values, d. Whisker plot of the Log2(TOP15:BTM15) values of all oligos encoding top ten high, neutral, and low activity mutants. The dotted line indicates the threshold value that corresponds to the mean of the wild type P-MSH peptides Log2(TOP15:BTM15) values. The whiskers represent the range of Log2(TOP15) values for each point mutation.

[0063] FIG. 3. Identification and validation of a P-MSH mutant with enhanced ability to activate MC4R. a. Diagram of the amino acid sequence of the signal peptide (red), P-MSH peptide (blue), and Xten linker sequence (green). The arrows indicate predicted protease cleavage sites, b. Single-well validation of low-activity P-MSH mutants in MC4R HTLA reporter cells. Two nucleotide variants are tested for each P-MSH mutant, and every experiment is done in triplicate. STDU2-44084.601

[0064] All luciferase measurements are normalized to the average luciferase level of the empty vector control, c. Dose-response curve of luciferase activity from MC4R HTLA cells treated with wild type P-MSH and shortened wild type P-MSH. MC4R HTLA cells treated with random 22-mer peptide are used as a negative control. All values are normalized to DMSO-treated cells, d. Single-well validation of high-activity P-MSH mutants in MC4R HTLA reporter cells. Two nucleotide variants are tested for each P-MSH mutant, and every experiment is done in triplicate. All luciferase measurements are normalized to the average luciferase level of the empty vector control, e. Dose-response curve of luciferase activity from MC4R HTLA cells treated with wild type P-MSH and D5H P-MSH mutant. MC4R HTLA cells treated with random 22-mer peptide are used as a negative control. All values are normalized to DMSO-treated cells.

[0065] FIG. 4. FACS gating strategy of the P-MSH peptide screen in MC4R HTLA reporter cells. Scatter plots show MC4R HTLA reporter cells differentiated by size (FSS / SCC), singlets (FSCA / FSC-H), and peptide library expression (mCherry +). Distribution plot shows luciferase expression (FITC) levels in peptide library expressing and non-expressing cells.

[0066] FIG. 5A. Luciferase reporter assay measuring the relative activation of GPCR reporter cells co-expressing different synthetic GPCR receptors and their cognate peptides.

[0067] FIG. 6. pAL63_MC4R_TTA_ADV_NFZ vector; a. structure; and b. sequence.

[0068] FIG. 7. pAL57_mcherry_hygro_peptide_library vector; a. structure; and b. sequence.

[0069] DETAILED DESCRIPTION

[0070] A better understanding of peptide-GPCR interactions is important in advancing GPCR biology and facilitating drug discovery. In some embodiments, the systems and methods described herein provide a high-throughput peptide display platform that directly links the functional activation of receptors (e.g., G protein-coupled receptors (GPCRs)) to specific peptide variants. This system, when combined with a mutational screen, identifies peptide regulators of the receptor.

[0071] For example, using this system, as describe below, a deep mutational screen of the p- MSH peptide and from a pool of 2500 peptide variants, identified peptides that significantly impact the activation of the MC4R, a key regulator of energy homeostasis and appetite control STDU2-44084.601

[0072] (2). Notably, mutations of P-MSH were discovered that exhibit an increased ability to activate MC4R. This work aligns with previous findings that certain point mutations in the P-MSH peptide can influence MC4R activation (36) and highlights the value of the platform to identify peptide alterations that can modulate receptor activity more effectively.

[0073] Several approaches exist to study GPCR-peptide interaction; however, existing methods have some key limitations that limit their utility in the systematic study of GPCR-peptide interactions. Phage display, a widely used assay for identifying proteins that bind to specific receptors, has been critical in the development of biological drugs (14-17). In this process, peptide libraries are displayed on the surface of phages and screened against a target protein. After screening, the binding clones are isolated and sequenced to identify putative peptide hits (18). However, because phage display is a binding-based assay, further steps such as testing the effect of synthesized candidate peptides in functional assays are necessary. The workload to identify functional binders from this pool of physical binders is significant, and many clones selected as strong binders from natural peptide libraries are nonfunctional (19). To address this, cell-based functional assays have been developed in which cells express a reporter system to indicate the activity of GPCRs of interest (20). Panels of peptides can be tested in an arrayed manner on the reporter cell lines leading to the identification of novel agonists of GPCRs (20,21). This approach enables direct identification of functional agonists or antagonists of GPCRs. However, these assays are limited by the scale and cost of plate-based robotics and assays, as well as the high cost of peptide synthesis. Lastly, while genetic association studies have advanced our understanding of the clinical relevance of peptide and receptor variants (3,22,23), they are limited in their ability to identify rare variants that might not be represented in the sampled populations.

[0074] Compared with previous methods for studying GPCR-peptides, many of which have been critical in furthering our understanding of GPCR biology, the functional readout of the reporter system provided herein, combined with the use of peptide libraries, allows for higher throughput screening while minimizing the workload for hit identification (15,17,20,21). The use of reporter systems with direct and specific read-outs (e.g., P-arrestin-based reporter) with the peptide display system provides a direct and specific readout of GPCR activation, allowing one to accurately link peptide binding to functional receptor responses within a cell. Integration with STDU2-44084.601 oligonucleotide library synthesis allows the rapid generation of a vast library of peptide variants, facilitating comprehensive screening that was previously difficult in cell-based assays. The platform allows parallel testing of numerous peptides, significantly increasing throughput while reducing time and resource requirements. Variations in peptide expression levels, influenced by codon usage, can affect the activation of the reporter system (See FIG. 2D). The platform provided herein can address this challenge by including multiple codon variants of the same peptide in the library.

[0075] Provided herein are peptide display systems and methods that provide high-throughput identification of peptide ligands that agonize a specific GPCR. In some embodiments, this technology integrates the pooled synthesis of oligonucleotide libraries encoding variants of the peptides of interest, a peptide display system that expresses and localizes these peptides on the cell surface, and a synthetic cell recruitment reporter for readout (e.g., P-arrestin-based). By utilizing this cell-based assay with a direct functional GPCR reporter readout, the approach minimizes false positives often arising from non-functional interactions in other screening methods. The system is illustrated in the Example section below using MC4R and its known ligand, -MSH. A library was created to perform deep mutational scanning of the P-MSH peptide, simultaneously screening over two thousand peptide elements in a single pooled experiment. This identified novel single amino acid mutations in P-MSH that significantly enhance or diminish the ability of P-MSH to activate MC4R. Linking the mutant P- MSH peptides to publicly available clinical data, it was discovered that one of the identified loss of function mutants is prevalent in children with obesity. Additionally, a novel D5H mutation in P- MSH was identified that enhances its ability to activate MC4R. Together, these results demonstrate a single-cell approach that allows the simultaneous measurement of activities for thousands of peptide variants to identify potent agonists of GPCRs.

[0076] In some embodiments, peptides are tested for their ability to modulate the activity of two or more receptors. In some embodiments, at least one of the receptors is a GPCR. In some embodiments, each of the receptors is a GPCR.

[0077] In some embodiments, the GPCR is a GPCR that is naturally activated by a peptide (i.e., a peptide is an endogenous ligand of the receptor). In other embodiments, the GPCR is a GPCR that is not naturally activated by a peptide. In some embodiments, the GPCR is a class A STDU2-44084.601

[0078] (rhodopsin-like) GPCR. Tn some embodiments, the GPCR is a class B (secretin (e.g., calcitonin, corticotropin-releasing factor, glucagon, parathyroid hormone, vasoactive intestinal peptide (VIP) or pituitary adenylate cyclase-activating peptide (PACAP) family)) GPCR. In some embodiments, the GPCR is one or more of: angiotensin II type 2 (AT2) receptor, beta-2 adrenergic receptor, cholecystokinin 2 (CCK2) receptor, endothelin receptor (ETA or ETB), ghrelin receptor, gonadotropin-releasing hormone receptor (e.g.. GnRHi), a melanocortin receptor (e.g., MCi, MC2, MC4), a neuropeptide Y receptor (NPY2, NPY4), delta opioid receptor (5), kappa opioid receptor (K), nociceptin / orphanin FQ (N / OFQ) peptide receptor (NOP), protease- activated receptor 1 (PARI), relaxin family peptide receptor 1 (RXFP1), a somatostatin receptor (e.g., SSTi, SST2, SST3, SST5), thyrotropin-releasing hormone receptor 1 (TRHi), oxytocin receptor (OT), 5-HT2A receptor, a chemokine receptor (e.g., CXCR4), a corticotropinreleasing factor receptor (e.g., CRFi, CRF2), calcitonin receptor (CT), calcitonin gene-related peptide receptor (CGRP), gastric inhibitory polypeptide receptor (GIP), growth-hormone- releasing hormone receptor (GHRH), glucagon receptor, a glucagon-like peptide receptor (e.g.. GLP-1, GLP-2), vasoactive intestinal peptide receptor 1 (VPACi), parathyroid hormone type 1 receptor (PTH1), secretin receptor, or calcium-sensing receptor (CaS). Other GPCRs and their ligands, targets, and associated diseases and conditions are found at: https: / / www.guidetopharmacology.org / GRAC / ReceptorFamiliesForward7type-GPCR.

[0079] Nucleic acid sequences encoding candidate polypeptides (e.g., a transmembrane fusion constructs comprising candidate polypeptides) or heterologous receptor reporters may be introduced into cells using a vector. When introduced into a cell, the vectors may be maintained as an autonomously replicating sequence or extrachromosomal element or may be integrated into host DNA.

[0080] Conventional viral and non-viral based gene transfer methods can be used to introduce the nucleic acids into cells (e.g., into cultured cells). Non-viral vector delivery systems include DNA plasmids, cosmids, RNA, including nucleic acid complexed with a delivery vehicle. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. A variety of viral constructs may be used to deliver the present nucleic acids to the cells, tissues and / or a subject. Viral vectors include, for example, retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex viral vectors. Nonlimiting examples STDU2-44084.601 of such recombinant viruses include recombinant adeno-associated virus (AAV), recombinant adenoviruses, recombinant lentiviruses, recombinant retroviruses, recombinant herpes simplex viruses, recombinant poxviruses, phages, etc. The present disclosure provides vectors capable of integration in the host genome, such as retrovirus or lentivirus. See, e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989; Kay, M. A., et al., 2001 Nat. Medic. 7(l):33-40; and Walther W. and Stein U„ 2000 Drugs, 60(2): 249-71. incorporated herein by reference.

[0081] Methods of delivering vectors to cells are well known in the art and may include DNA or RNA electroporation, transfection reagents such as liposomes or nanoparticles to delivery DNA or RNA; delivery of DNA, RNA, or protein by mechanical deformation (see, e.g., Sharei et al. Proc. Natl. Acad. Sci. USA (2013) 110(6): 2082-2087, incorporated herein by reference); or viral transduction. In some embodiments, the vectors are delivered to host cells by viral transduction. Nucleic acids can be delivered as part of a larger construct, such as a plasmid or viral vector, or directly, e.g.. by electroporation, lipid vesicles, viral transporters, microinjection, and biolistics (high-speed particle bombardment). Similarly, the construct containing the one or more transgenes can be delivered by any method appropriate for introducing nucleic acids into a cell. In some embodiments, the construct or the nucleic acid encoding the components of the present system is a DNA molecule. In some embodiments, the nucleic acid encoding the components of the present system is a DNA vector and may be electroporated to cells. In some embodiments, the nucleic acid encoding the components of the present system is an RNA molecule, which may be electroporated to cells.

[0082] Additionally, delivery vehicles such as nanoparticle- and lipid-based delivery systems can be used. Further examples of delivery vehicles include lentiviral vectors, ribonucleoprotein (RNP) complexes, lipid-based delivery system, gene gun, hydrodynamic, electroporation or nucleofection microinjection, and biolistics. Various gene delivery methods are discussed in detail by Nayerossadat et al. (Adv Biomed Res. 2012; 1: 27) and Ibraheem et al. (Int J Pharm. 2014 Jan 1 ;459(l-2):70-83), incorporated herein by reference.

[0083] A number of suitable mammalian and human host cells are known in the art, and many are available from the American Type Culture Collection (ATCC, Manassas, Va.). Examples of suitable mammalian cells include, but are not limited to, Chinese hamster ovary cells (CHO) (ATCC No. CCL61), CHO DHFR-cells (Urlaub et al., Proc. Natl. Acad. Sci. USA, 97: 4216- STDU2-44084.601

[0084] 4220 (1980)), human embryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573), and 3T3 cells (ATCC No. CCL92). Other suitable mammalian cell lines are the monkey COS-1 (ATCC No. CRL1650) and COS-7 cell lines (ATCC No. CRL1651), as well as the CV-1 cell line (ATCC No. CCL70). Further exemplary mammalian host cells include primate, rodent, and human cell lines, including transformed cell lines. Normal diploid cells, cell strains derived from in vitro culture of primary tissue, as well as primary explants, are also suitable. Other suitable mammalian cell lines include, but are not limited to, mouse neuroblastoma N2A cells, HeLa, HEK (e.g., HEK293), A549, HepG2, mouse L-929 cells, and BHK or HaK hamster cell lines, or cells derived therefrom (e.g., HTLA).

[0085] Peptides identified by the screening technologies provided herein find use in research, diagnostic, and therapeutic fields.

[0086] As research reagents, the peptides, or modified versions thereof, may be used to study receptor function. In some embodiments, the peptides find use as competitive activators or inhibitors.

[0087] In some embodiments, the peptides, or modified versions thereof, are used therapeutically, for example, to modulate diseases or conditions associated with receptor activity, or lack thereof. In some embodiments, the peptides are included in therapeutic compositions that include the peptide and one or more other components (e.g., carriers, excipients, delivery systems, additional active agents, etc.). Peptides may be modified to enhance their therapeutic properties, such as stability, longevity, and resistance to proteolysis, or otherwise to improve pharmacokinetics or bioavailability via a desired delivery approach (e.g., oral, intranasal, injection, etc.). Modifications to peptides include, but are not limited to, formation of cyclic or conformationally constrained structures, attachment to a brain-penetrant peptide, and use of unnatural amino acid.

[0088] Unnatural amino acids include, without limitation, amino acid analogs, amino acid mimetics, synthetic amino acids, N-substituted glycines, and N-methyl amino acids in either the L- or D-configuration that function in a manner similar to the naturally-occurring amino acids. For example, “amino acid analogs” are unnatural amino acids that have the same basic chemical structure as naturally-occurring amino acids, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, but have modified R (i.e., side-chain) groups. STDU2-44084.601

[0089] Non-limiting examples of unnatural amino acids include 1 -aminocyclopentane- 1- carboxylic acid (Acp), 1-aminocyclobutane-l-carboxylic acid (Acb), 1 -aminocyclopropane- 1- carboxylic acid (Acpc), citrulline (Cit), homocitrulline (HoCit), a-aminohexanedioic acid (Aad), 3-(4-pyridyl)alanine (4-Pal), 3-(3-pyridyl)alanine (3-Pal), propargylglycine (Pra), a- aminoisobutyric acid (Aib), a- aminobutyric acid (Abu), norvaline (Nva), a, p -diaminopropionic acid (Dpr), a.y-diaminobutyric acid (Dbu), a-tert-butylglycine (Bug), 3,5-dinitrotyrosine (Tyr(3,5-di NO2)), norleucine (Nle), 3-(2-naphthyl)alanine (Nal-2), 3-(l-naphthyl)alanine (Nal- 1), cyclohexylalanine (Cha), di-n-propylglycine (Dpg), cyclopropylalanine (Cpa), homoleucine (Hie), homoserine (HoSer), homoarginine (Har), homocysteine (Hey), methionine sulfoxide (Met(O)), methionine methylsulfonium (Met (S-Me)), a-cyclohexylglycine (Chg), 3-benzo- thienylalanine (Bta). taurine (Tau), hydroxyproline (Hyp), O-benzyl-hydroxyproline (Hyp(Bzl)), homoproline (HoPro), P-homoproline (PHoPro), thiazolidine-4-carboxylic acid (Thz), nipecotic acid (Nip), isonipecotic acid (IsoNip), 3-carboxymethyl-l-phenyl-l,3,8-triazaspiro[4,5]decan-4- one (Cptd). tetrahydro-isoquinoline-3-carboxylic acid (3-Tic), 5H-thiazolo [3,2-a]pyridine-3- carboxylic acid (Btd), 3 -aminobenzoic acid (3-Abz), 3-(2-thienyl)alanine (2-Thi), 3-(3- thienyl)alanine (3-Thi), a-aminooctanedioc acid (Asu), diethylglycine (Deg), 4-amino-4- carboxy-l,l-dioxo-tetrahydrothiopyran (Acdt), 1 -amino- l-(4-hydroxycyclohexyl) carboxylic acid (Ahch), 1 -amino- l-(4-ketocyclohexyl)carboxylic acid (Akch), 4-amino-4- carboxytetrahydropyran (Actp), 3-nitrotyrosine (Tyr(3-NO2)), 1-amino-l-cyclohexane carboxylic acid (Ach), 1 -amino- l-(3-piperidinyl)carboxylic acid (3-Apc), 1 -amino- 1 -(4- piperidinyl)carboxylic acid (4-Apc), 2-amino-3-(4-piperidinyl) propionic acid (4-App), 2- aminoindane-2-carboxylic acid (Aic), 2-amino-2-naphthylacetic acid (Ana), (2S.5R)-5- phenylpyrrolidine-2-carboxylic acid (Ppca), 4-thiazoylalanine (Tha), 2-aminooctanoic acid (Aoa), 2-aminoheptanoic acid (Aha), ornithine (Om), azetidine-2-carboxylic acid (Aca), a- amino-3-chloro-4,5-dihydro-5-isoazoleacetic acid (Acdi), thiazolidine-2-carboxylic acid (Thz(2- COOH)), allylglycine (Agl), 4-cyano-2-aminobutyric acid (Cab), 2-pyridylalanine (2-Pal), 2- quinoylalanine (2-Qal), cyclobutylalanine (Cba), a phenylalanine analog, derivatives of lysine, ornithine (Orn) and a,y-diaminobutyric acid (Dbu), stereoisomers thereof, and combinations thereof (see, e.g., Liu et al., Anal. Biochem., 295:9-16 (2001)). As such, the unnatural a-amino acids are present either as unnatural L-a-amino acids, unnatural D-ot-amino acids, or combinations thereof. STDU2-44084.601

[0090] “Amino acid mimetics” are chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally-occurring amino acid. Suitable amino acid mimetics include, without limitation, P- amino acids and y-amino acids. In -amino acids, the amino group is bonded to the P-carbon atom of the carboxyl group such that there are two carbon atoms between the amino and carboxyl groups. In y-amino acids, the amino group is bonded to the y-carbon atom of the carboxyl group such that there are three carbon atoms between the amino and carboxyl groups. Suitable R groups for p- or y-amino acids include, but are not limited to, side-chains present in naturally-occurring amino acids and unnatural amino acids.

[0091] As used herein, the term “administering” includes oral administration, topical contact, administration as a suppository, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal, or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc.

[0092] In some embodiments, a therapeutic peptide is attached to or contained within a delivery system. Delivery systems include, but are not limited to. nanoparticles (e.g., mesoporous silica nanoparticles, gold nanoparticles, chitosan nanoparticles, virus-like nanoparticles, etc.), hydrogels, liposomes, micelles, polyethylene glycol (PEG), dendrimers, and the like.

[0093] The term “therapeutically effective amount” refers to the amount of a pharmaceutical composition that is capable of achieving a therapeutic effect in a subject in need thereof. For example, a therapeutically effective amount of a composition comprising a peptide can be the amount that is capable of preventing or relieving one or more symptoms associated with a disease or disorder.

[0094] Depending on the indication, severity, and administration route, a suitable dose may be selected accordingly. For example, for acute indications, fewer treatments with a higher dose in each treatment are administered; while for chronic indications requiring frequent and long-term STDU2-44084.601 treatment, a lower dose treatment is administered. Dosage may be determined by the effective amount of peptide and / or pharmaceutical composition to improve symptoms or molecular outcomes. It is to be understood that, for the subject, specific dosage regimes should be adjusted over time according to the severity of disease or symptoms associated with disease of the subject in need thereof. For example, the dosage of the peptide and / or pharmaceutical composition may be increased if the lower dose does not provide sufficient therapeutic activity. Typical dosage ranges are from 0.01 to 1000 mg / dose (e.g., per day) (e.g., 0.1 to 100 mg; 1 to 50 mg).

[0095] In some embodiments, the peptide is a modulator of melanocortin-4 receptor (MC4R). The melanocortin-4 receptor (MC4R) is one of five human melanocortin receptors (MC1-5R) that belong to a subgroup of class A GPCRs (2). The melanocortin receptors were originally named for their earliest known function in melanogenesis and have since been shown to play roles in energy homeostasis, cardiovascular function, immune regulation, and sexual functions (8-10). MC4R plays a central role in regulating energy balance and appetite (2). Given its role in regulating satiety, MC4R is one of the major peptide GPCRs being targeted by novel therapeutics designed to counteract obesity (11,12). Activation of MC4R in neurons of the paraventricular nucleus of the hypothalamus by its natural agonist, P-melanocyte- stimulating hormone (P-MSH), leads to reduced appetite and decreased food intake (2). Loss of function of MC4R in other species such as rats, mice, and canines increases appetite and weight gain, demonstrating the conservation of the MC4R-P-MSH axis in regulating energy homeostasis and food motivation (3-5). Furthermore, naturally occurring mutations in the MC4R and P-MSH pathway are the most frequent cause of monogenic obesity in humans, with a prevalence of around 6% in children with severe, early-onset obesity (13). These findings underscore the critical role of the MC4R and P-MSH pathway in energy regulation and illustrate the clinical significance of characterizing the interaction of P-MSH with MC4R.

[0096] In some embodiments, one or more of the following peptides is used to alter MC4R activity.

[0097] Table 1 STDU2-44084.601

[0098] It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of present technology, and it is understood that such equivalent embodiments, are to be included herein. The following examples are intended to illustrate various embodiments of the present technology. As such, the specific embodiments discussed are not to be constructed as limitations on the scope of the present technology.

[0099] EXAMPLES

[0100] Material and Methods Cell line and cell culture

[0101] HTLA cells, a HEK293-derived cell line with stable integrations of a tTA-dependent luciferase reporter and a P-arrestin2-TEV fusion gene, obtained from Dr. Jonathan Long’s Laboratory at Stanford University (25). All cells were cultured in a humidity-controlled incubator at 37°C and 5% CO2 in DMEM supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, catalog no. F6765-500ML), 2 mM L-glutamine (Gibco, catalog no. 25030081), and 100 U / ml Penicillin-Streptomycin (Gibco, catalog no. 15-140-163).

[0102] Plasmids

[0103] All primers and gene fragments used in this work were purchased from Integrated DNA Technologies (IDT, Table 4). Plasmids were constructed using Golden Gate Assembly (New STDU2-44084.601

[0104] England Biolabs, catalog no. E1602L) and transformed into One Shot Stbl3 chemically competent E. coli cells (ThermoFisher Scientific, catalog no. C737303). The reporter plasmid for the MC4R-tTA Advance-NFZ gene was derived from the MC4R vector in the Presto-Tango system (Addgene, Kit #1000000068). Briefly, a tetracycline-controlled transactivator with improved stability and reduced noise (tTA Advance) was fused to the MC4R gene (27). To reduce cellular toxicity and increase the measurable signal of MC4R activation, a compact human activator combination NFZ was used in the construct instead of VP 16 (29). The resulting fusion gene was cloned into a lentiviral vector under an EF- la promoter with a blasticidin resistance gene. For the construction of the plasmid carrying the peptide display vector, DNA fragments encoding a preprotrypsin-derived secretory signal peptide sequence

[0105] (MS ALLIL ALVGAAVA (SEQ ID NO:1)), p-melanocyte-stimulating hormone (MSH), a myc- tag, a linker sequence, and the transmembrane domain of the platelet-derived growth factor receptor p were assembled and cloned into a lentiviral vector with mCherry and hygromycin resistance genes. The peptide display gene is under an EF-la promoter. To simplify the protocol for cloning peptide libraries, the peptide display vector was later modified to include two typellS BsmBI enzyme recognition sites between the secretory signal and the Myc tag.

[0106] Table 4 STDU2-44084.601

[0107] Plasmid transfection and transduction

[0108] HEK293T cells were transfected using the polyethyleneimine (PEI) (ThermoFisher

[0109] Scientific, catalog no. NC1014320) (40). Viral supernatants were collected 48 and 72 hours post- transfection, filtered through a 0.45 pm syringe, and applied directly to cells with 4 pg / mL polybrene (ThermoFisher Scientific, catalog no. TR1003G) or stored at -80°C for later use.

[0110] Stable MC4R reporter cell line generation

[0111] 100,000 HTLA cells were plated in a well of a 6-well plate, allowed to attach overnight, and then transduced with lentiviral particles carrying expression vectors for the MC4R-tTA Advance-NFZ (Addgene #228457) fusion gene. After a media change and three days of recovery, the cells were moved into a 10-cm plate and selected with lOpg / ml of blasticidin S HC1

[0112] T1 STDU2-44084.601

[0113] (ThermoFisher Scientific, A1 13903). Tn parallel, untransduced HTLA cells were treated with equal concentrations of blasticidin to determine when selection was complete. Post selection, the expression of MC4R TetR-NFZ was validated by treating the HTLA MC4R reporter cells with 500nm of synthetic 0-MSH peptide (Genscript), the putative agonist of the G-protein coupled receptor. MC4R activation was measured using the ONE-Glo™ Luciferase Assay (Promega, catalog no. E6110).

[0114] Peptide Display Assay

[0115] To measure MC4R activation by the cis-displayed peptides, 100,000 MC4R HTLA reporter cells were plated in each well of a 6-well plate and allowed to attach overnight. In parallel, lentivirus encoding peptides of interest on the display vector were titered on HEK293T cells and volumes equating to equal multiplicity of infection (MOI) were calculated. After attaching, MC4R HTLA cells were transduced with the peptide display constructs at equal MOI. Media was changed the next day and cells were given 48 hours to recover. MC4R activation was measured using the ONEGlo™ Luciferase Assay (Promega, catalog no. E6110).

[0116] Cell surface staining to measure peptide expression

[0117] Surface staining against the Myc tag was performed to confirm the membrane localization of the peptide display construct. HTLA cells were transfected or transduced with the peptide display vector at least 48 hours before staining and proper mCherry expression was confirmed. To measure peptide expression, one million cells were incubated in lOOul of staining buffer with 5pl of Human TruStain FcX (BioLegend, cat no. 422301) for 5-10 minutes at room temperature to block the FC-receptor. Cells were probed using Alexa 488 conjugated anti-Myc tag antibody (lul per sample, AbCam, cat no. ab202008). Surface protein expression was measured using flow cytometry (Attune NxT) and gated on mCherry-positive cells. The data was analyzed using the Cell Engine software.

[0118] Intracellular staining of luciferase protein

[0119] To prepare the cells for intracellular staining, the following steps were taken: Cells were trypsinized, pelleted at 350g at 4°C, and washed in staining buffer (BioLegend, catalog no. 420201). Cells were then fixed and permeabilized with 200pL of FixPerm solution (BD Bioscience, catalog no. 554714). After a 30-minute incubation at room temperature, the cells STDU2-44084.601 were centrifuged and washed twice with 500pL of Permwash buffer. Concurrently, the antifirefly luciferase antibody was diluted 1:200 in Permwash buffer (BD Bioscience, catalog no. 554714) and added to each sample following the second wash. After a one-hour incubation, excess antibodies were removed with further washes in Permwash buffer. Cells were then incubated with a secondary anti-rabbit conjugated FITC antibody at a 1:500 dilution for another hour (ThermoFisher Scientific, catalog no. 31583). Luciferase expression was quantified using flow cytometry (Attune NxT), focusing on mCherry-positive cells, and data was analyzed using CellEngine software. This protocol was designed for up to 5 million cells and was scaled proportionally to accommodate larger cell numbers in the library screen.

[0120] Design of P-MSH mutagenesis library

[0121] An all-by-all deep mutational scan of the 22 amino acid P-MSH peptide sequence (AEKKDEGPYRMEHFRWGSPPKD (SEQ ID NO:2)) was performed to generate 418 mutant peptides. Each peptide was then encoded 5 times with varied codon usage, for a total of 2090 mutant elements. Primer binding and typellS BsmBI enzyme recognition sites were concatenated to the 5’ and 3’ ends of each mutant element. Random DNA padding sequences were generated and added between the 5’ primer binding and 5’ enzyme recognition sites to increase oligo size to 260 bp. 30 wild type encodings and 30 random encodings were created as positive and negative controls, respectively. The final library contained 2150 elements, each 260 bp in length. The first 120 bp of each oligo was designed to be unique to ensure that it would be possible to distinguish every library element with short-read sequencing. Additionally. 7xC homopolymers and internal BsmBI sites were removed, 20-75% GC content was utilized in every 50 bp window, and human codon usage was optimized in every oligo using DNAchisel (35).

[0122] Peptide library cloning

[0123] Oligonucleotides encoding the peptides were synthesized in the form of a pooled library through Twist Biosciences. PCR amplification was conducted in a controlled PCR hood environment to prevent unintentional amplification of contaminating DNA. For each of the six reactions, 5 ng of template was used, 0.1 ml of each 100 mM primer, 1 ml of Herculase II polymerase (from Agilent), 1 ml of DMSO, 1 ml of 10 nM dNTPs, and 10 ml of 5x Herculase buffer. The thermocycling protocol involved an initial step at 98°C for 3 minutes, followed by a series of cycles at 98°C for 20 seconds, 61°C for 20 seconds, 72°C for 30 seconds, and a final STDU2-44084.601 extension step at 72°C for 3 minutes. The default cycle number was set at 29, with adjustments made to determine the minimum number of cycles that obtained a clean and visible product for subsequent gel extraction (typically, 25 cycles proved to be the minimum). Following PCR, the resulting double- stranded DNA libraries underwent gel extraction, where the desired bands at approximately 250 base pairs were excised from a 2% TBE gel (four lanes), and a QIAgen gel extraction kit was employed for purification. Due to the nature of the varying length of the peptide encoding oligos, the PCR product was not a clear single band but a smear ranging from 400 to 200 base pairs and therefore, the whole smear was extracted. These libraries were subsequently cloned into the lentiviral peptide display vector (Addgene #228456) using a total of 4x10 ml GoldenGate reactions, which included 75 ng of pre-digested and gel-extracted backbone plasmid, 5 ng of library (in a 2: 1 molar ratio of insert to backbone), 0.13 ml of T4 DNA ligase (NEB, 20000 U / ml), 0.75 ml of Esp3I-HF (NEB), and 1 ml of lOx T4 DNA ligase buffer. These reactions underwent 30 cycles of digestion at 37 °C and ligation at 16°C for 5 minutes each, followed by a final 5-minute digestion at 37°C and heat inactivation at 70°C for 20 minutes. The resulting reactions were pooled and purified using MinElute columns (QIAgen), with an elution volume of 6 ul of ddH2O. Subsequently, 2 ml per tube was transformed into two tubes containing 50 ml of Endura electrocompetent cells (Lucigen, catalog no. #60242-2) following the manufacturer’s instructions. After the recovery process, the cells were plated on 3 - 7 large 10” x 10” LB plates with carbenicillin. Following overnight growth at 37°C, the bacterial colonies were scraped into a collection bottle, and plasmid pools were extracted using a HiSpeed Plasmid Maxiprep kit (QIAgen). In parallel, 2 - 3 small plates were prepared with diluted transformed cells to count colonies and confirm the transformation efficiency, ensuring at least 30x library coverage was maintained. To assess the quality of the libraries, 20 - 30 colonies from the transformations underwent Sanger sequencing (Quintara) and the peptides were amplified from the plasmid pool for premium PCR (Primordium) to estimate cloning efficiency and the proportion of empty backbone plasmids within the pools. The PCR and sequencing protocols were consistent with those described below for sequencing from genomic DNA. The resulting sequencing datasets were analyzed, as detailed below, to assess coverage uniformity and synthesis quality of the libraries.

[0124] High throughput P-MSH peptide mutagenesis screen STDU2-44084.601

[0125] Large-scale lentivirus production and infection of MC4R HTLA cells were performed as follows: To generate sufficient lentivirus to infect the libraries into MC4R HTLA cells, HEK293T cells were plated on two 15 -cm tissue culture plates. On each plate, 9 million HEK293T cells were plated in 30 mL of DMEM, grown overnight, and then transfected with 8 mg of an equimolar mixture of the three third-generation packaging plasmids (pMD2.G, psPAX2, pMDLg / pRRE) and 8 mg of P-MSH peptide library vectors using 50 mL of polyethylenimine (PEI, Polysciences #23966). pMD2.G (Addgene, plasmid #12259), psPAX2 (Addgene, plasmid #12260), and pMDLg / pRRE (Addgene plasmid #12251). After 48 hours and 72 hours of incubation, lentivirus was harvested and filtered through a 0.45-mm PVDF filter (Millipore) to remove any cellular debris. For the P-MSH peptide screen, approximately 5 million MC4R HTLA cells were seeded into each 15-cm tissue culture plate. Four plates were prepared, constituting two biological replicates. The cells were allowed to attach overnight before being infected with lentiviruses encoding the peptide library at an MOI of approximately 0.3. This infection rate was chosen to ensure a higher-than-standard coverage of over 1000 for each library element, to offset potential losses during subsequent FACs sorting, library preparation, and synthesis errors. After three days of recovery, the infected cells were trypsinized, and mCherry measurements were taken to confirm the efficiency of the infection. The cells were then replated at a density of 5 million cells per 15-cm plate and allowed to grow for an additional two days. Subsequently, the cells were split and stained for luciferase protein expression in preparation for fluorescence-activated cell sorting (FACS). FACS was conducted at the Stanford FACS facility, where cells were initially gated on mCherry to isolate those expressing the peptide library. Further gating was done based on luciferase expression, as indicated by the intensity of the FITC signal. Cells falling within the top or bottom 15 percent based on FITC signal intensity were sorted into separate bins. Cells were pelleted and frozen at negative 20 degrees Celsius until genomic DNA extractions were performed.

[0126] Library preparation and sequencing

[0127] Genomic DNA was extracted with the QIAgen Blood Maxi Kit following the manufacturer’s instructions. DNA was eluted in EB and not AE to avoid subsequence PCR inhibition. A test PCR was performed using 2.5 ug of genomic DNA in a 25 uL (half size) reaction to verify if the PCR conditions would result in a visible band at the expected size for STDU2-44084.601 each sample. After determining the optimal PCR condition, 50 uL reactions were prepared in 96 well plates with the number of reactions depending on the amount of genomic DNA available in each experiment. 5 ug of genomic DNA, 0.25 uL of each 100 mM primer, and 25 uL of NEBnext 2x Master Mix (NEB) were used in each reaction. The reactions were set up on ice in a clean PCR environment to prevent contamination. The PCR protocol involved preheating the thermocycler to 98°C. followed by a 3-minute initial denaturation at 98°C, and then 27 cycles of denaturation at 98°C for 10 seconds, annealing at 63°C for 30 seconds, extension at 72°C for 30 seconds, and a final extension at 72°C for 2 minutes. All subsequent procedures were carried out outside the PCR hood. The PCR reactions were combined, and the pooled material was loaded onto a 2% TBE gel, alongside a 100-bp ladder. Gel electrophoresis was conducted until clear ladder separation was observed, and the peptide library band around 200bp was excised. DNA purification was performed using the QIAquick Gel Extraction Kit (QIAgen). DNA was elution into non-stick tubes (Ambion). A follow-up gel analysis confirmed the removal of small fragments. These libraries were then quantified using a Qubit HS kit (ThermoFisher Scientific) and sequencing using Illumina NextSeq using a single-end forward read (150 cycles) and 8-cycle index reads (Novogene).

[0128] Peptide screen analysis

[0129] The reads were trimmed using AdapterRemoval (41). The subsequent steps follow the standard workflow of the HT-recruit analysis (www.github.com / bintulab / HT-recruit-Analyze). First, a Bowtie reference was created using the designed library sequences via the 'makeindices. py' script, and the reads were aligned allowing for zero mismatches using the 'makeCounts. py' script. The enrichment differences for each peptide in the top and bottom 15% bins were calculated using the 'makeRhos.py' script. Peptides with fewer than five reads in both samples of a particular replicate were excluded from that replicate and set to zero counts. If peptides had fewer than five reads in one sample, those reads were adjusted to five to prevent the inflation of enrichment values due to low sequencing depth. For all peptide screens, peptides with a count of five or fewer in both replicates for a given condition were removed from downstream analysis. For each peptide, the enrichment difference between the top and bottom 15% bins was log2- transformed. High (and low) activity mutants were defined as having an enrichment score higher (or lower) than two standard deviations above (or lower) the mean for STDU2-44084.601 wild type 0-MSH peptides. Agreement between replicates were tested using Fisher’s exact test for the identification of high and low activity mutants. Additionally, the Z prime factor test was used for signal-to-noise based on the random 22-mers included in the screen (42). Given the high concordance between the two biological replicates (see FIG. 2B), values were averaged across the two biological replicates. For residue-based analyses (see FIG. 2CD), the enrichment scores for all five codon variants were averaged and z-scores were computed based on the mean and standard deviation for wild type P-MSH peptides.

[0130] Peptide activation assay

[0131] All the peptides used in this study were synthesized by Genscript. To quantify relative levels of activation of MC4R by a particular peptide, 50,000 MC4R HTLA were seeded in a 24- well plate with 500uL of culturing media. Cells were allowed to attach overnight and fresh media was added to each well. The highest concentration of the peptide was added onto the first column of the plate and then four ten-fold serial dilutions were performed across the plate. After 24 hours of treatment, luciferase reporter activation was quantified using the ONE-Glo™ Luciferase Assay (Promega, catalog no. E6110).

[0132] Example 1 - Design of a peptide display platform to identify GPCR Agonist

[0133] To develop a high throughput platform to identify peptide agonists of GPCRs, we first sought to: 1) devise a strategy that would allow the expression of a library of peptides on the cell membrane and 2) develop a cell-based reporter that would respond when a peptide ligand agonizes the GPCR of interest. To express a library of peptides that can interact with GPCRs on the cell surface, we created a modular peptide display system that allows the expression and localization of any peptide of interest to the cell membrane. At the 5' end of the peptide display construct, we incorporated a cleavable signal peptide derived from pre-pro-trypsin (24) to promote membrane localization, and a peptide cloning site for efficient cloning. At the 3' end, we added a PDGFR transmembrane domain to embed the construct into the cell membrane, and a MYC epitope tag to facilitate monitoring of cell surface expression by flow cytometry. The components are connected by a linker to ensure proper domain folding (FIG. la).

[0134] To ensure the scalability and generalizability of the platform across a broad range of GPCRs, we paired our peptide display system with a modified version of the PRESTO-Tango STDU2-44084.601

[0135] GPCR reporter assay (20). Briefly, each GPCR is modified to include a “Tango” protein fragment at the 3’ end, comprising a Tobacco Etch Virus (TEV) protease cleavage site and a tetracycline controlled transactivator (tTA). This synthetic GPCR is transfected into HTLA cells, a HEK293-derived reporter cell line stably expressing a luciferase reporter and a synthetic betaarrestin fused to the TEV protease. Upon activation by a ligand, the GPCR triggers the recruitment of a synthetic beta-arrestin fused to the TEV protease and releases the tTA (25). Once the tTA is freed, it translocates into the nucleus and turns on the reporter leading to luciferase expression (FIG. la). It has been noted in the literature that Tet-based reporter systems experience leakiness and high expression of VP16, which can lead to cellular toxicity (26,27). Significant advances have been made to address these challenges, including the development of new activator domains and mutations in the TetR protein to reduce leakage (27,28). To improve the signal-to-noise ratio of the reporter, we modified the “Tango” component by replacing the original tTA with tTA Advance (27), a tetracycline-controlled transactivator with improved stability and minimal background expression. Furthermore, we replaced the VP 16 activator with a more compact human activator combination, NFZ, that we developed (29), which has stronger activation and when overexpressed, is less toxic to human cells (FIG. la). This modified GPCR “Tango” construct was encoded within a lentiviral vector and transduced into HTLA cells to create stable reporter cell lines.

[0136] Example 2- Validation and optimization of the GPCR peptide display platform

[0137] To assess the ability of the peptide display construct to localize peptides of interest to the cell membrane and to determine if co-expression of a GPCR and peptide ligand is sufficient to activate the reporter, preliminary tests of our peptide display platform were performed using a known peptide-GPCR pair: the melanocortin 4 receptor (MC4R) and its natural peptide ligand, P-melanocyte-stimulating hormone (MSH) (30). We lentivirally transduced and selected for a pure population of MC4R stably expressing HTLA cells with peptide display constructs encoding P-MSH. We measured peptide expression at the cell surface through anti-Myc staining and found that more than 90.54% of the transduced cells stained positive for cell surface expression of P-MSH post-transduction (FIG. lb). After confirming proper localization of the peptide to the membrane, we then assayed whether the peptide display of P-MSH is sufficient to activate MC4R and induce reporter expression of luciferase. We found that cells expressing STDU2-44084.601 peptide-di splayed 0-MSH were able to induce similar levels of luciferase activity as MC4R cells treated with 1 pM of the synthetic form of the peptide (FIG. 1c). Importantly, MC4R HTLA cells expressing a negative control peptide expected to have no activity on these cells, neuropeptide Y (NPY), did not induce significant luciferase expression. Next, we sought to understand how the linker sequence in the peptide display vector influences peptide activation of the GPCR as protein linkers have previously been reported to influence protein folding and stability (31). Our experiments were conducted using a (GN)9 linker, which was extended by testing three additional linkers (Table 2) (31,32). We found that the XTEN linker increased the abundance of P-MSH present at the cell surface and led to a significant increase in MC4R activation compared to the other linkers (FIG. Id, le). These results are consistent with previous observations that the XTEN linker can improve the stability of fusion proteins (32,33). However, the incorporation of amino acids that should increase linker flexibility, such as glycines and serines, upstream and downstream of the XTEN linker decreased peptide abundance at the cell surface and reduced reporter activation. The inclusion of hyperflexible linkers composed of only glycine and serines decreased cell surface P-MSH abundance by more than twofold and luciferase reporter activation by more than threefold (FIG. Id-e). Based on these results. XTEN was selected as the linker for the exemplary peptide display system.

[0138] Table 2 STDU2-44084.601

[0139] Example 3 - Designing a deep mutational scan of the P-MSH peptide

[0140] We next aimed to explore whether the peptide display platform could be expanded to perform high-throughput screening and identify peptide agonists of GPCRs in a pooled fashion. To test this, we designed an ‘all-by-aU’ deep mutational scanning library of the P-MSH peptide, testing every single possible point mutation across all 22 positions of the peptide. Each mutant was encoded using five different oligonucleotide sequences, varying codon usage to enable unique determination of each sequence by deep sequencing (34). We also generated 50 oligos each encoding the wild type P-MSH peptide and 250 22-mer random peptides (35). In total, the library was composed of 2500 elements encoding 740 unique peptides (Table 3). We transduced the library into MC4R expressing HTLA reporter cells (FIG. 2a). Then, the cells were permeabilized, stained with an anti-luciferase antibody, and sorted into high (top 15% expression, TOP15) and low (bottom 15% expression, BTM15) luciferase expression level bins. We sorted a minimum of 1 million cells into each bin to maintain a coverage of 400x per element. We sequenced the two bins and calculated the enrichment of the peptides in each sorted population. We observed an increase in luciferase levels in the MC4R-expressing HTLA cells that expressed the peptide library compared to the cells that did not. For example, in both biological replicates, we saw a two-fold increase in luciferase-positive cells in the population expressing the peptide library compared to non-transduced cells (FIG. 2c, FIG. 4).

[0141] After filtering out peptide oligos with low reads, we conservatively called peptides that fall two standard deviations above and below the mean of the log2(TOP15:BTM15) score of all the wild type P-MSH peptides as high and low activity mutants, respectively (FIG. 2b). We found that over 74% of the random negative control peptides did not significantly activate MC4R, with their activity falling at least two standard deviations below the mean of the wildtype peptide in at least one biological replicate. Based on the enrichment scores of all the wildtype positive control peptides and random sequence negative control peptides, we determined STDU2-44084.601 that our screen had an excellent signal-to-noise ratio (Z prime value = 0.89 for both biological replicates). We compared the overlap of significant hits between the two biological replicates to assess the reproducibility of the screens. The results showed significant overlap in hits (Fisher’s exact test p = 9.52e-22), indicating good reproducibility. Overall, the consistent reproducibility, excellent signal-to-noise ratio, and accurate identification of expected controls across both biological replicates showed that the GPCR peptide display platform yielded reliable results.

[0142] Example 4 - Validating novel P-MSH mutants

[0143] While we found that most point mutations do not significantly impact the ability of 0- MSH to activate MC4R, we did observe a striking enrichment in loss of function mutations in the first four amino acids of the 0-MSH peptide (FIG. 2c, 2d). In fact, all of the top ten ranking low activity mutants identified in the screen bore mutations in the first four positions of the peptide (FIG. 2d). We further validated our findings by transducing four of the candidate loss of function mutants into MC4R HTLA cells in a low throughput experiment and measured luciferase activity. We found that reporter cells expressing all of these mutants failed to activate MC4R and induce significant luciferase expression (FIG. 3b). Interestingly, the glutamic acid [E] to glycine [G] mutation in the second position of the peptide is more prevalent in children with obesity, though the mechanism by which this missense mutation causes obesity had not been identified (36). 0-MSH is derived from a precursor polypeptide, pro-opiomelanocortin, through multiple cleavage events at dibasic amino acids, and mutations in those cleavage positions have been previously reported to contribute to obesity in children (37,38). Using signalP 6.0. we found two predicted cleavage sites within the first four amino acids for the wild type 0-MSH peptide (39). We hypothesized that these four positions are important for peptide cleavage and conversion of the 0-MSH peptide from the pro-form into the active form (FIG. 3a). To test this hypothesis, we treated MC4R-expressing HTLA cells with wild type 0-MSH and a shortened version of 0-MSH without the first four amino acids. There was no significant difference in MC4R reporter activation between the two peptide treatments (FIG. 3c). This suggests that the first four amino acids are not important for binding to MC4R and that the glutamic acid to glycine SNP mutation in the second position likely disrupts the proteolytic processing of wild type 0-MSH. STDU2-44084.601

[0144] Additionally, mutations that significantly enhance P-MSH’ s ability to activate MC4R are rare (FIG. 2c). Nine out of the ten high-activity mutants bear mutations in the 5th and 6th position of the P-MSH peptide (FIG. 2d). We transduced the D5H, D5V, and E6K mutants into MC4R-expressing HTLA cells and found that they indeed were able to activate MC4R more potently compared to the wild type. Notably, an aspartic acid [D] to histidine [H] substitution in the fifth position increased MC4R activation by more than 50% when expressed on a peptide display construct (FIG. 3c). To eliminate the possibility that the enhanced reporter activation is due to a unique property of the peptide being expressed on the display construct, we also treated reporter cells with a chemically synthesized version of the D5H mutant peptide. We observed an increase in MC4R activation in the D5H mutant peptide treated cells compared to the wild type P-MSH (FIG. 3d). These findings illuminate the important roles of specific amino acid residues in P-MSH in regulating MC4R function, and identify enhanced activity peptides and core residues for generating enhanced activity peptides.

[0145] Example 5 - Multiple receptor types FIG. 5A shows luciferase reporter assay measuring the relative activation of GPCR reporter cells co-expressing different synthetic receptors (MC4R, NPY5R, MLNR, and NPFF1) and their cognate peptides, demonstrating the generalizability of the system.

[0146] STDU2-44084.601

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Claims

STDU2-44084.601CLAIMSWe claim:

1. A method for screening peptides, comprising: expressing a plurality of different candidate regulatory peptides in a plurality of mammalian cells, wherein said cells express a heterologous receptor reporter that generates a detectable signal when a peptide regulates said receptor.

2. The method of claim 1, wherein said receptor is a G protein-coupled receptor.

3. The method of claim 1, wherein said plurality of different candidate regulatory peptides comprise a plurality of variants of a polypeptide.

4. The method of claim 3, wherein said plurality of different candidate regulatory peptides comprise a library of variants of said polypeptide comprising at least one sequence variant at each amino acid position of said polypeptide.

5. The method of claim 1, wherein said cells comprise human cells.

6. The method of claim 1, wherein said expressing comprises expressing said plurality of different candidate regulatory peptides in a transmembrane fusion construct.

7. The method of claim 6. wherein said transmembrane fusion construct comprises a transmembrane domain, a linker, and a candidate regulatory peptide.

8. The method of claim 7, wherein said candidate regulatory peptide is positioned extracellularly when said transmembrane domain is integrated into a membrane of said cell.

9. The method of claim 8, wherein said transmembrane fusion construct further comprises a detectable tag.

10. The method of claim 9, wherein said detectable tag is a Myc tag.

11. The method of claim 1, wherein said detectable signal is generated if a candidate regulatory peptide activates said receptor.

12. The method of claim 1, wherein said detectable signal is generated if a candidate regulatory peptide inhibits said receptor.STDU2-44084.60113. The method of claim 1 , wherein said heterologous receptor reporter comprises one or more reporter molecules fused to said receptor, wherein said one or more reporter molecules directly or indirectly generate said detectable signal when said receptor is activated.

14. The method of claim 13, wherein said one or more reporter molecules comprise a transcription factor linked to said receptor by a cleavage site.

15. The method of claim 14, wherein said one or more reporter molecules further comprise a cleavage enzyme linked to a P-arrestin molecule, wherein upon activation of said receptor, said cleavage enzyme is mobilized to cleave said cleavage site and release said transcription factor from said receptor.

16. The method of claim 15, wherein said cleavage enzyme is a TEV protease.

17. The method of claim 15, wherein said transcription factor is an artificial transcription factor.

18. The method of claim 17, wherein said cell comprises a luciferase reporter gene and said artificial transcription factor activates expression of said luciferase reporter gene.

19. The method of claim 1, further comprising expression a positive control peptide in at least one cell.

20. The method of claim 1, further comprising expression a negative control peptide in at least one cell.

21. The method of claim 1, wherein at least 100 different candidate regulatory peptides are expressed in said plurality of mammalian cells.

22. The method of claim 1, wherein at least 1000 different candidate regulatory peptides are expressed in said plurality of mammalian cells.

23. The method of claim 1, wherein said candidate regulatory peptides are expressed from a vector.

24. The method of claim 23, wherein said vector comprises a selection marker.

25. A kit comprising:STDU2-44084.601 a) a first expression vector comprising a cloning site, for receiving a sequence encoding a polypeptide of interest, fused to a linker fused to a transmembrane domain; and b) a second expression vector encoding a transmembrane receptor fused to one or more reporter proteins that generate a detectable signal when a peptide regulates said receptor; or c) mammalian cells comprising a heterologous receptor reporter that generates a detectable signal when a peptide regulates said receptor.

26. The kit of claim 25, wherein said first expression vector further comprises a sequence encoding a detectable tag and / or a selection marker.

27. The kit of claim 25, wherein said mammalian cells comprise a luciferase gene that generates said detectable signal.

28. A screening system comprising: a) a plurality of expression vectors, each comprising a sequence encoding a different candidate regulatory peptide, fused to a linker fused to a transmembrane domain; and b) mammalian cells comprising a heterologous receptor reporter that generates a detectable signal if a candidate regulatory peptide regulates said receptor.

29. The system of claim 28, wherein plurality of expression vectors encode variants of a polypeptide.

30. The system of claim 29, wherein said variants comprise codon-optimized variants.

31. An isolated polypeptide comprising AEKKXEGPYRMEHRFWGSPPKD, werein X is not D.

32. The isolated polypeptide of claim 31, wherein X is H.

33. A pharmaceutical composition comrising the isolated polypeptide of claim 31 orSTDU2-44084.60134. A method for altering expression of melanocortin-4 receptor (MC4R), comprising administering the pharmaceutical composition of claim 33 to a subject.

35. Use of a kit of any of claims 25-27.

36. Use of a kit of any of claims 25-27 to identify a receptor modulator.

37. Use of a screening system of any of claims 28-30.

38. Use of a screening system of any of claims 28-30 to identify a receptor modulator.

39. Use of an isolated polypeptide of claim 31 or 32.40 Use of a pharmaceutical composition of claim 33.

41. Use of a pharmaceutical composition of claim 33 for managing energy homeostasis and / or appetite.

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