Abstract
Purpose :
Sustained vision relies on a metabolic pathway that continuously regenerates 11-cis-retinal (11-cisRAL) from its all-trans-retinal (atRAL) isomer. During intense light exposure, the elevated generation of atRAL leads to inadequate clearance and then toxic atRAL condensation products, which accumulate over age and can contribute to retinal degeneration. The underlying cellular mechanisms leading to retinal degeneration remain uncertain, but previous studies have shown that atRAL promotes intracellular calcium influx associated with cell apoptosis. Here, we aim to identify compounds that mitigate the effects of atRAL toxicity that otherwise instigate photoreceptor degeneration using a high-throughput screen (HTS) of 50,000 compounds.
Methods :
We developed an image-based HTS assay that can detect changes in intracellular calcium levels in U2OS cells after exposure to atRAL. This assay takes advantage of a cell-permeable dye, fluo-3 AM, which exhibits an increase in fluorescence upon binding Ca2+. A total of 50,000 compounds were screened, and the protective effect of hit compounds was further confirmed in Abca4-/-Rdh8-/- double knockout mice exposed to light that normally induces retinal damage.
Results :
The screen identified an initial 323 hit compounds, which reduced the rise in intracellular calcium concentration following treatment with atRAL with an activity score ≥ 55 %. Following the triplicate validation of the 323 compounds, 100 compounds were identified as potential candidates and evaluated for drug-induced cytotoxicity, narrowing the number to 32 compounds. Among them, 16 hit compounds demonstrated dose-dependent effects on maintaining intracellular calcium homeostasis, and two of them showed protection against light-induced damage in the Abca4-/-Rdh8-/- double knockout mouse model.
Conclusions :
We demonstrate the development and utilization of an image-based assay for high throughput screening and identification of compounds that mitigate the effects of atRAL-induced calcium influx and toxicity in photoreceptor degeneration. Results were validated by both in vitro and in vivo studies. We believe the identified hit compounds will have broad application against retinal degenerative diseases in humans associated with the abnormal accumulation of atRAL condensation products.
This is a 2020 ARVO Annual Meeting abstract.