Patent ID: 9182408
Filing Date: 2015-11-10
Classification: G01N

Abstract:
1. A method of selecting test compounds having the ability to modulate the biological activity of a target protein by modifying the conformation of said target protein, said method consisting essentially of: A) providing a combinatorial library of peptides, wherein said library is expressed in a plurality of yeast cells and said cells collectively express all members of said library, and wherein each cell co-expresses said target protein or a ligand-binding protein moiety thereof and one member of said library; and wherein each cell further comprises a signal producing system comprising: a protein-bound component which is fused to said target protein or to said ligand-binding protein moiety thereof, so as to provide a chimeric protein; and a peptide-bound component which is fused to said peptide so as to provide a chimeric peptide, where one of said components is a DNA-binding domain (DBD) and the other component is a complementary transactivation domain (AD), and the signal producing system further comprises at least one reporter gene operably linked to an operator bound by said DBD, the binding of the peptide to the target protein or protein-binding moiety thereof resulting in the constitution of a functional transactivation activator protein which activates expression of said reporter gene and a signal is produced by activation of the expression of a reporter gene when the peptide-bound and protein-bound components are brought into physical proximity as a result of the binding of the peptide to said target protein or moiety thereof; B) screening said library for the ability of its members to bind said target protein in a native conformation and selecting the peptides binding to said target protein in native-conformation; C) providing a known ligand of said target protein, wherein the binding of said known ligand to the target protein induces a conformational change in said target protein; screening the peptides selection selected in B) and selecting among the peptides selected in B) those having a decreased or no ability to bind to said target protein in presence of said known ligand compared to the ability of said peptides to bind to said target protein in native conformation; wherein step C) is further carried out with at least another known ligand; and wherein a conformation-sensitive peptide is selected for having a decreased or no ability to bind to said target protein in presence of the known ligands compared to the ability of said conformation-sensitive peptide to bind to said target protein in native conformation, said conformation-sensitive peptide thereby presenting the ability to identify conformational changes induced by more than one known ligand; and D) screening a library of test compounds in a human cell line for their ability to alter the binding of a single conformation-sensitive peptide of those selected in step C) to said target protein in native conformation; and selecting among said library of test compounds those test compound(s) that alter the binding of said single conformation- sensitive peptide, wherein the presence of said test compound(s) results in a decrease or an absence of binding of said single conformation-sensitive peptide to the target protein; and wherein said cell line in step D) co-expresses: a) said target protein, or a ligand-binding protein moiety thereof, and b) said single conformation-sensitive peptide selected in step C); said cell further comprising a signal producing system comprising: a protein-bound component which is fused to said target protein or to said ligand-binding protein moiety thereof, so as to provide a chimeric protein; and a peptide-bound component which is fused to said peptide so as to provide a chimeric peptide, wherein one of said components is a DNA-binding domain (DBD) and the other component is a complementary transactivation domain (AD), and the signal producing system further comprises at least one reporter gene operably linked to an operator bound by said DBD, the binding of the peptide to the target protein or protein-binding moiety thereof resulting in the constitution of a functional transactivation activator protein which activates expression of said reporter gene and a signal is produced by activation of the expression of a reporter gene when the peptide-bound and protein-bound components are brought into physical proximity as a result of the binding of the peptide to said target protein or moiety thereof.