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Publication Number
US-11821904-B2
Publication Date
2023-11-21
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 provides a method of preparing a library of nucleic acid molecules for intracellular expression of short peptides having lengths of fewer than 150 amino acids in mammalian cells. The method includes designing, in silico, a diverse population of bespoke short peptides by rationally selecting fragments from larger proteins based on bioinformatics predictions of domain structures, and the short peptides are designed to only use expressed proteins from a proteome consensus database.
The invention further includes constructing the library from nucleic acid molecules that encode the short peptides by cloning the nucleic acid molecules into an expression vector adapted for intracellular expression of the short peptides in mammalian cells. This library preparation framework is tied to phenotypic screening using mammalian encoded short expressed peptides (SEPs), including sORF-derived peptides and bespoke cDNA-derived protein fragments.
In the disclosed phenotypic screening context, the prepared SEP libraries are used to identify SEP-mediated phenotype-altering cells. The screening is also described as enabling identification of cellular target proteins binding SEPs and druggable interaction sites by subsequent compound-displacement/competition approaches.
Claims Coverage
The independent claim is directed to an in silico design and nucleic-acid library construction method for intracellular expression of bespoke short peptides (<150 amino acids) in mammalian cells. Two inventive features are identified: rational fragment selection based on predicted domain structures and construction of the nucleic-acid library by cloning into a mammalian-expression-adapted vector.
Bespoke short peptide library design under 150 amino acids in mammalian-expression context
Designing, in silico, a diverse population of bespoke short peptides having lengths of fewer than 150 amino acids by rationally selecting fragments from larger proteins based on bioinformatics predictions of domain structures, wherein the short peptides are designed to only use expressed proteins from a proteome consensus database.
Cloning-constructed nucleic acid library for intracellular expression in mammalian cells
Constructing the library from nucleic acid molecules that encode the short peptides by cloning the nucleic acid molecules into an expression vector adapted for intracellular expression of the short peptides in mammalian cells.
The coverage centers on rational in silico peptide design from expressed-protein sources and on library construction by cloning encoding nucleic acids into an expression vector adapted for intracellular expression in mammalian cells.
Stated Advantages
Enables identification of SEP-mediated phenotype-altering cells.
Enables identification of cellular target proteins binding SEPs.
Enables identification of druggable interaction sites by subsequent compound-displacement/competition approaches.
Documented Applications
Protein-i phenotypic screening examples for NF-κB signalling and Notch signalling.
Pooled resistance described with 6-thioguanine and mismatch repair.
Hippo/YAP phenotypic screening using YAP/TEAD-GFP.
Diseased-cell differential expression screening as described in diseased cell models.
Screening across diseased cell models including cancer, autoreactive T-cells, inflamed cartilage, and insulin-resistant cells.
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