title: A simple and widely applicable hit validation strategy for protein-protein interaction inhibitors based on a quantitative ligand displacement assay.
abstract: Identification of inhibitors for protein-protein interactions (PPIs) from high-throughput screening (HTS) is challenging due to the weak affinity of primary hits. We present a hit validation strategy of PPI inhibitors using quantitative ligand displacement assay. From an HTS for Bcl-xL/Mcl-1 inhibitors, we obtained a hit candidate, I1, which potentially forms a reactive Michael acceptor, I2, inhibiting Bcl-xL/Mcl-1 through covalent modification. We confirmed rapid reversible and competitive binding of I1 with a probe peptide, suggesting non-covalent binding. The advantages of our approach over biophysical assays include; simplicity, higher throughput, low protein consumption and universal application to PPIs including insoluble membrane proteins.
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan.
authors.forename: Tomoya
authors.initials: T
authors.lastname: Sameshima
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan; Enzymology and Biophysical Chemistry, Takeda California, Inc., 10410 Science Center Drive, San Diego, CA 92121, United States.
authors.forename: Ikuo
authors.initials: I
authors.lastname: Miyahisa
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan.
authors.forename: Misaki
authors.initials: M
authors.lastname: Homma
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan.
authors.forename: Katsuji
authors.initials: K
authors.lastname: Aikawa
authors.affiliation: Enzymology and Biophysical Chemistry, Takeda California, Inc., 10410 Science Center Drive, San Diego, CA 92121, United States.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Hixon
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan.
authors.forename: Junji
authors.initials: J
authors.lastname: Matsui
mesh_terms: D001667:Binding, Competitive; D002087:Butyrates / Q000737:chemistry / Q000378:metabolism; D057166:High-Throughput Screening Assays; D007651:Keto Acids / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D064549:Myeloid Cell Leukemia Sequence 1 Protein / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs; D051020:bcl-X Protein / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D002087:Butyrates; D007651:Keto Acids; D008024:Ligands; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D051020:bcl-X Protein
keywords: Bcl-xL; High-throughput screening; Hit compound validation; Mcl-1; Protein–protein interaction inhibitor