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Publication Number
US-11085926-B2
Publication Date
2021-08-10
Expiration Date
Abstract
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.
Core Innovation
The invention relates to a SEP-based high-throughput live-cell phenotypic screening approach for identifying previously unknown target proteins and protein interaction sites that modulate mammalian-cell phenotypes. It exposes a population of in vitro cultured mammalian cells capable of displaying a phenotype to a library of short peptides, each less than 150 amino acids, expressed by a nucleic acid encoding a diverse population of short peptides.
The short peptide library comprises bespoke short peptides designed in silico, including library and sourcing constraints grounded in in silico designs and proteome sources, such as mammalian sORFome/SEPome and proteome-consensus libraries, as well as bespoke cDNA-derived subdomains. After detecting an alteration in said phenotype, the approach selects cells undergoing a phenotypic change and identifies a short peptide of the library that alters the phenotype of the selected cells.
The disclosure further provides said short peptide and identifies a cellular protein that binds to said short peptide, where the cellular protein is a target protein that modulates the phenotype of the selected cells. A key aspect is that the target protein is not identified prior to exposing the population of mammalian cells to the library of short peptides.
The disclosure further links SEP-driven phenotypic modulation to compound discovery by identifying compounds that bind the target protein and displace or block SEP binding. In disclosed examples, the phenotypic readouts include reporter systems and pathway signaling outputs that enable target identification and later identification of compounds that modulate SEP-target interactions.
Claims Coverage
The partial content provides two independent claims. Both claims share a core workflow: express a nucleic-acid encoded library of short peptides less than 150 amino acids in mammalian cells, detect phenotypic alteration, identify an effective peptide and its binding target protein that was not known prior to screening, and, in the second independent claim, identify a compound that displaces or blocks short peptide binding.
In silico designed short peptide library for unknown target identification
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, and wherein exposing said population comprises expressing a library of nucleic acids encoding a diverse population of short peptides, wherein the library comprises bespoke short peptides designed in silico; identifying an alteration in said phenotype following exposure; selecting cells undergoing a phenotypic change and identifying a short peptide that alters the phenotype; providing said short peptide and identifying a cellular protein that binds to said short peptide, the cellular protein being a target protein that modulates the phenotype; wherein the target protein is not identified prior to exposing the population.
Compound identification that displaces or blocks short peptide binding to an unknown target
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; comprising exposing mammalian cells to a nucleic-acid-expressed library of short peptides less than 150 amino acids comprising bespoke short peptides designed in silico; identifying a cell displaying an alteration in said phenotype; identifying a short peptide that alters the phenotype; identifying a cellular protein that binds to said short peptide as an as-yet-unidentified target protein; and identifying the compound that binds to said target protein and displaces or blocks binding of said short peptide.
Across both independent claims, the inventive coverage centers on expressing an in silico designed, nucleic-acid encoded library of short peptides in mammalian cells to induce and detect phenotypic changes, then using the effective peptide’s binding to identify an initially unknown target protein. The second independent claim extends this by identifying compounds that bind the identified target protein and displace or block the short peptide’s binding while modulating the phenotype.
Stated Advantages
Identifies a target protein that modulates a phenotype of a mammalian cell where the target protein is not identified prior to exposing the population to the library.
Enables identification of a compound that binds to a target protein and displaces or blocks binding of a short peptide, with the compound modulating the phenotype.
Documented Applications
SEP-based phenotypic screening to identify previously unknown target proteins and protein interaction sites that modulate mammalian-cell phenotypes, including disease-pathway phenotype readouts.
Disclosed example of phenotypic screening for NF-kappaB and Notch protein activity using reporter readouts and short peptides expressed from human sORFs.
Disclosed pooled screens for 6-thioguanine resistance and Hippo/YAP signaling, with selection/enrichment and subsequent Next Generation Sequencing analysis of peptide libraries.
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