A simple and widely applicable hit validation strategy for protein-protein interaction inhibitors based on a quantitative ligand displacement assay

作者：Sameshima Tomoya; Miyahisa Ikuo; Homma Misaki; Aikawa Katsuji; Hixon Mark S^*; Matsui Junji

来源：Bioorganic & Medicinal Chemistry Letters, 2014, 24(24): 5836-5839.

DOI：10.1016/j.bmcl.2014.09.073

• High-throughput screening
• Protein-protein interaction inhibitor
• Mcl-1
• Bcl-xL
• Hit compound validation

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.

• 出版日期2014-12-15