Screening of Chemical Libraries Using a Yeast Model of Retinal Disease
Benjamin M. Scott
Retinitis pigmentosa (RP) is a degenerative retinal disease, often caused by mutations in the G protein-coupled receptor rhodopsin. The majority of pathogenic rhodopsin mutations cause rhodopsin to misfold, including P23H, disrupting its crucial ability to respond to light. Previous screens to discover pharmacological chaperones of rhodopsin have primarily been based on rescuing rhodopsin trafficking and localization to the plasma membrane. Here, we present methods utilizing a newly developed yeast-based assay to screen for compounds that rescue the ability of rhodopsin to activate an associated downstream G protein signaling cascade. We engineered a yeast strain in which human rhodopsin variants were genomically integrated, and were able to demonstrate functional coupling to the yeast mating pathway, leading to fluorescent protein expression. We confirmed that a known pharmacological chaperone, 9-cis retinal, could partially rescue light- dependent activation of a disease-associated rhodopsin mutation (P23H) expressed in yeast. These novel yeast strains were used to perform a phenotypic screen of 4280 compounds from the LOPAC1280 library, and a peptidomimetic library, to discover novel pharmacological chaperones of rhodopsin. The fluorescence-based assay was robust in a 96-well format, with a Z'-factor of 0.65, and a signal- to-background ratio of above 14. One compound was selected for additional analysis, but it did not appear to rescue rhodopsin function in yeast. The methods presented here are amenable to future screens of small molecule libraries, as they are robust and cost effective. We also discuss how these methods could be further modified or adapted to perform screens of more compounds in the future.