Novel small-molecule chaperones to overcome opsin misfolding, mistrafficking and aggregation in retinal blinding diseases

Abstract

Purpose : Opsin misfolding and its incorrect trafficking represent the main biomolecular causes of photoreceptor death leading to retinal degeneration in Leber’s congenital amaurosis (LCA) and Retinitis pigmentosa (RP). This study aims to develop novel small-molecule chaperones binding opsin in order to promote its proper folding and trafficking to the membrane with consequent reduction of the endoplasmic reticulum (ER) stress caused by protein accumulation that leads to cell death.

Methods : Small-molecule chaperon candidates were identified through in silico studies of the retinal-binding pocket based on the available opsin crystal structures and subsequently purchased or synthetized in-house. The compounds ability to bind wild-type opsin isolated from bovine eyes was evaluated through competitive binding assay by measuring via spectrophotometer the rate of formation of isorhodopsin in presence of 9-cis-retinal and the compounds in comparison to 9-cis-retinal alone. The compound ability to rescue opsin trafficking and proper subcellular localization was evaluated by immunofluorescence microscopy in a cell-based model expressing P23H mutant human opsin – an intrinsically instable mutant previously demonstrated to be accumulated in the ER and partially rescued by 9-cis-retinal.

Results : 20 opsin-binding candidates were selected based on the structure-based virtual screening of a commercially available compound library, and further 55 novel chemical entities have been rationally designed to improve binding site occupancy and subsequently synthetized. The competitive binding test on a total of 67 compounds has revealed that multiple small molecules were able to decrease the rate of isorhodopsin formation by over 10%. Furthermore, multiple compounds visibly improved P23H mutant opsin proper localization on the cell surface reducing the opsin accumulation in the ER observed in the vehicle control.

Conclusions : This work has led to the design, synthesis and identification of novel opsin-binding small molecules that improve opsin correct localization in cell-based model and could serve as a strategy to prevent photoreceptor death in opsin-related blinding diseases. Additional confirmation of these results is needed and the synthesis and development of further chaperone candidates is ongoing.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.