Identification of small molecule compounds that alleviate retinal ciliopathy phenotype

Abstract

Purpose : Ciliopathy mutations interfering with opsin trafficking through the connecting cilium of photoreceptors account for a significant proportion of hereditary retinal dystrophies including Leber congenital amaurosis (LCA). Currently there are no approved medications available for the treatment of most ciliopathies. We aimed to identify lead compounds for the treatment of CEP290-related retinal ciliopathies through a cell-based compound library screening.

Methods : We established a CEP290^null RPE1 cell line mimicking mutations associated with LCA using CRISPR/Cas9 system. Next, libraries of synthetic and natural compounds were screened for the identification of lead compounds that can rescue the ciliogenesis defect caused by CEP290 loss. Studies including immunofluorescence, western blotting, co-immunoprecipitation, and electroretinography (ERG) were conducted to identify molecular targets of lead compounds and to test phenotype rescue effects using a LCA mouse model (rd16) which harbors a Cep290 gene mutation.

Results : Ciliogenesis was defective in CEP290^null RPE1 cells. Among 2800 synthetic and natural compounds, we identified 6 compounds that significantly increase the number of cells developing primary cilia in the context of CEP290-null. The compounds showing the strongest rescue effect were four structural analogs: compound-A, B, C, and D. The compound-A relieved ciliogenesis and ciliary receptor delivery defects resulting from deletion of CEP290, a gene mutated in several ciliopathies. We found that the compound-A can improve the function of the ciliary transition zone damaged by CEP290 loss. Remarkably, in compound-A treated rd16 homozygotes, photopic ERGs showed a reproducible partial recovery, and opsin trafficking to the outer segment of cone photoreceptors was substantially recovered.

Conclusions : We identify compound-A as a lead compound for developing medication for CEP290-related ciliopathies involving retinal degeneration. Our screening strategy provides a useful platform for developing small molecule drugs for ciliopathies.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

 

(A) The loss of CEP290 in RPE1 cells. (B) Defective ciliogenesis in CEP290^null RPE1 cells. (C) Results of compound library screening. (D) Dose-response curve. (E) Photopic electroretinogram of rd16^het and rd16^homo mice after compound-A treatment. (F) Fluorescence micrographs visualizing M-opsin in the retina of rd16^het and rd16^homo mice after compound-A treatment.