Methods of screening
The invention provides a method of identifying a peptide interaction site on a target protein wherein the target protein modulates the phenotype of a mammalian cell, using mammalian encoded peptides (SEPs) such as short open reading frame (sORF) encoded peptides. The invention further provides a method for the identification of new therapeutic targets and protein interaction sites for use in drug discovery.
1. A method of identifying a target protein that modulates a phenotype of a mammalian cell, said method comprising:
exposing a population of in vitro cultured mammalian cells capable of displaying said phenotype to a library of short peptides, wherein each short peptide is less than 150 amino acids and is expressed by a nucleic acid, wherein exposing said population of mammalian cells to the library of short peptides comprises expressing a library of nucleic acids encoding a diverse population of short peptides in said population of mammalian cells, and wherein the library of short peptides comprises bespoke short peptides designed in silico;
identifying in said population of mammalian cells an alteration in said phenotype following said exposure;
selecting cells undergoing a phenotypic change and identifying a short peptide of the library of short peptides that alters the phenotype of the selected cells; and
providing said short peptide and identifying a cellular protein that binds to said short peptide, said cellular protein being a target protein that modulates the phenotype of the selected cells;
wherein the target protein is not identified prior to exposing the population of mammalian cells to the library of short peptides.
2. The method according to claim 1 , wherein the library of short peptides comprises human short peptides.
3. The method according to claim 1 , wherein the short peptide is a peptide of 6-130 amino acids.
4. The method according to claim 1 , wherein the cellular protein and the short peptide are used in an assay to identify ligands that bind to said cellular protein and disrupt short peptide binding.
5. A method of identifying a compound which binds to a target protein and displaces or blocks binding of a short peptide, wherein the compound modulates a phenotype of a mammalian cell, said method comprising the steps:
i. exposing a population of in vitro cultured mammalian cells capable of displaying said phenotype to a library of short peptides, wherein each short peptide is less than 150 amino acids and is expressed by a nucleic acid, wherein exposing said population of mammalian cells to the library of short peptides comprises expressing a library of nucleic acids encoding a diverse population of short peptides in said population of mammalian cells, and wherein the library of short peptides comprises bespoke short peptides designed in silico;
ii. identifying a cell in the population of mammalian cells that displays an alteration in said phenotype following said exposure;
iii. identifying a short peptide of the library of short peptides that alters said phenotype of the cell;
iv. identifying a cellular protein that binds to said short peptide, said cellular protein being a target protein that modulates said phenotype of the mammalian cell, wherein the target protein is not identified prior to exposing the population of mammalian cells to the library of short peptides; and
v. identifying the compound that binds to said target protein and displaces or blocks binding of said short peptide.
6. The method according to claim 1 , wherein the phenotype of the selected mammalian cells is associated with a disease-associated pathway.
7. The method according to claim 6 , wherein the disease-associated pathway is an activated cell signalling pathway; and/or a disease-associated pathway selected from the group consisting of differentiation, transcriptional activity, protein expression, resistance to infection, permeability, and proliferation.
8. The method according to claim 1 , wherein the library of short peptides includes rationally selected fragments from larger proteins.
9. The method according to claim 8 , wherein the library of short peptides includes predefined subdomains of mammalian peptides or micropeptides.
10. The method according to claim 8 , wherein the library of short peptides includes rationally selected fragments from larger proteins based on bioinformatic predictions of domain structures.
11. The method according to claim 1 , wherein the library of short peptides is in silico designed to only use expressed proteins from a proteome consensus database.
12. The method according to claim 1 , wherein the short peptides in the library are of constant size.
13. The method according to claim 1 , wherein the library of nucleic acids comprises bacterial-derived sequences in combination with non-bacterial sequences.
14. The method according to claim 1 , wherein the library of nucleic acids comprises mammalian-derived sequences.
15. The method according to claim 1 , wherein the library of nucleic acids comprises short fragments of nucleotide sequences from one or more mammalian nucleic acids cloned into expression vectors.
16. The method according to claim 1 , wherein the library of nucleic acids comprises short fragments of nucleotide sequences generated through bioinformatics based design of fragments followed by DNA synthesis.
17. The method according to claim 1 , wherein the library of short peptides comprises 1×10 5 or more different amino acid sequences.
18. The method according to claim 1 , wherein the cellular protein is an intracellular binding partner.
19. The method according to claim 1 , wherein the phenotype is cell death.