A high-throughput screening method for small-molecule pharmacologic chaperones of misfolded rhodopsin.

PURPOSE Many mutations in rhodopsin, including P23H, result in misfolding and mislocalization of the protein. It has been demonstrated that pharmacologic chaperones are effective in assisting the proper folding and targeting of P23H opsin. This study was designed to investigate a high-throughput screening strategy for identification of pharmacologic chaperones by using a combination of in silico, cell-based, and in vitro METHODS methods. A library of 24,000 drug-like small molecules was screened by in silico molecular docking with DOCK5.1. The top hits were assayed in an in vitro competition assay. The selected compound was then assayed for pharmacologic chaperoning activity in stable cell lines expressing wild-type and P23H opsin. RESULTS Beta-ionone was easily identified by the high-throughput screen. It strongly inhibits rhodopsin formation and, when incubated in cells expressing P23H opsin, resulted in a 2.5-fold rescue of P23H opsin. The screen also identified compound NSC45012 [1-(3,5-dimethyl-1H-pyrazol-4-yl)ethanone], a weak inhibitor of opsin regeneration and resulted in a 40% rescue of the mutant opsin. The level of rescue correlated well with the extent of inhibition. CONCLUSIONS A combination of in silico and cell-based screening provides a useful tool for identifying pharmacologic chaperones for P23H opsin. This approach identified both potent and weak pharmacologic chaperones. Both types of molecules may be potential candidates for treatment of opsin-related RP.