Identification of a novel small molecule chaperon of rhodopsin and its therapeutic potential in retinal degeneration

Abstract

Purpose : P23H rhodopsin mutation disrupts rhodopsin proteostasis which leads to autosomal dominant retinitis pigmentosa (adRP) that is currently untreatable. We hypothesis that a small-molecule chaperon of rhodopsin will stabilize the P23H rhodopsin mutant and delay vision loss by restoring rhodopsin homeostasis in the retinal.

Methods : We performed a small-molecule high-throughput screening using a cell-based beta-galactosidase assay to quantify the rescued P23H opsin on the plasma membrane. A total of 78,520 molecules from a collection of three small molecule libraries were tested for their activities on rescuing the transport of P23H opsin and YC-001 was identified as an active lead compound. To characterize the mechanism of action of YC-001, we tested: 1) if YC-001 binds to rod opsin using a Trp fluorescence assay; 2) if YC-001 improves the glycosylation profile of P23H opsin by immunoblots; 3) if YC-001 protects retina from bright light-induced degeneration in Abca4^-/- Rdh8^-/- mice by administering 50 or 200 mg/kg by weight of YC-001 before exposing mice to bright light (10,000 lux, 30 min) followed by spectral domain optical coherence tomography (SD-OCT) and retinal histology imaging seven days later.

Results : YC-001 showed an EC₅₀ at 7.8 μM with its efficacy at 310% compared to the effect of 5 μM 9-cis-retina in the beta-galactosidase fragment complementation assay. YC-001's effect on rescuing P23H opsin transport was confirmed by the immunostaining and high-content imaging assay showing an EC₅₀ at 3.2 μM. YC-001 reversibly binds with the rod opsin in the bovine disc membrane with a Kd at 1 μM. Treatment with YC-001 improved the glycosylation profile of P23H opsin mutant in a dose-dependent manner, demonstrated as shifted bands in immunoblots. Mice preconditioned with YC-001 at 200 mg/kg by weight were resistant to bright light-induced retinal damage, revealed by SD-OCT and H&E retinal staining images with an outer nuclear layer comparable to that from mice without bright-light damage, whereas vehicle control group showed significant reduction of ONL thickness.

Conclusions : YC-001 is a novel small molecule chaperone of rhodopsin that prevents bright light-induced retinal degeneration in Abca4^-/- Rdh8^-/- mice, revealing its therapeutic potential of preventing retinal degenerations. Long-term treatment to P23H/+ mice will show if YC-001 will delay retinal degeneration for adRP.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.