Identification of membrane proteins targeted by small-molecule compounds using nanomagnetic beads.

Abstract

In drug discovery research, it is important to identify target proteins of bioactive small-molecule compounds and analyse their functions. In this study, we examined whether target membrane proteins could be captured by compounds that bind to membrane proteins on the cell surface. For this purpose, we performed affinity purification using the compound-immobilized nanomagnetic beads. Affinity purification with nanomagnetic beads is known to be effective for determining the protein binding partners of small molecules. However, most previous studies have targeted proteins in the cytoplasm. As a model compound, we chose BMS-1166 (a representative small-molecule compound from Bristol Myers Squibb), a PD-1/PD-L1 immune checkpoint inhibitor that binds to PD- L1 and promotes PD-L1 dimerization. BMS-1166-immobilized beads were manufactured and incubated with extracts of cells with high PD-L1 protein expression. The bound protein was confirmed by western blotting and proteomic analysis to be PD-L1. BMS-1166-immobilized nano-magnetic beads were able to specifically bind and capture the membrane protein PD-L1. In addition, high-purity protein could be obtained from cell extracts in a single step. This is the first report of the purification of a membrane protein to high purity with nanobeads. Nanomagnetic beads with immobilized compounds are an effective tool for identifying the protein binding partners of small molecules, especially when the targets are membrane proteins.