Abstract
Purpose::
To develop non-retinoid pharmacological chaperones for misfolded P23H opsin.
Methods::
Docking calculations were performed with the development version of DOCK, v5.1.0. The coordinates for the crystal structure of rhodopsin, PDB code 1GZM, was used in the molecular docking calculations. ß-ionone, a known inhibitor of opsin regeneration, showed high degree of fit in the docking model. Wild-type (WT) opsin was regenerated in vitro with 11-cis retinal either in the presence or absence of the identified compounds to determine their ability to compete for the retinal binding pocket. To test the effect of the selected compound on the yields of folded P23H rhodopsin, the protein was expressed in a tetracycline-inducible HEK293 cell line. After 48h the cells were harvested and incubated with 11-cis retinal, the folded P23H rhodopsin was immunoaffinity purified and quantitated by UV-visible spectroscopy.
Results::
Five non-retinoid compounds were identified from a total of 200,000 small molecules screened based on their ability to fit into the retinal binding pocket of opsin. One compound, SN001, competed with 11-cis retinal for the retinal binding pocket of WT opsin. This inhibition was significantly less than that observed with ß-ionone, a known inhibitor, and a compound that also fits well in the retinal binding pocket based on our in silico modeling. When mutant P23H opsin was expressed in the presence of ß-ionone or SN001, a 2.5 fold or 30-35% increase in folded P23H rhodopsin was observed respectively. The increased yield of folded protein correlated well with the extent of in vitro inhibition of the compounds.
Conclusions::
We demonstrate that in silico modeling of the retinal binding pocket can be successfully used in high-throughput screening of large chemical libraries. This method has yielded one novel compound that can assist the folding and stabilization of P23H opsin and confirmed that the known retinal inhibitor, ß-ionone, can also serve as a pharmacological chaperone.
Keywords: opsins • mutations • chaperones