Screening of Chemical Libraries Using a Yeast Model of Retinal Disease

Abstract

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 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.

A GPCR-based yeast biosensor for biomedical, biotechnological, and point-of-use cannabinoid determination

“…In previous work, it has been possible to hijack the yeast pheromone pathway by replacing the pheromone receptor with a GPCR of interest and monitor the pathway's downstream response using a reporter gene 4,5,[24][25][26] . This has been broadly exploited for example, in the study of specific receptors 27,28 , deorphanization of uncharacterized receptors 25 , study of cell-cell communication 29 guiding metabolic engineering efforts 30 and detection of fungal pathogens 24 . However, specialized yeast biosensors capable of performing low-cost highthroughput bioactivity characterization, bioprospecting, and, especially, out-of-lab applications, have yet to be introduced.…”

Pre-mRNA Processing Factors and Retinitis Pigmentosa: RNA Splicing and Beyond

“…While Spp81 was identified as suppressor of PRP8 mutation ( Jamieson et al, 1991 ). And the pharmacological chaperone, 9- cis retinal, could partially rescue light-dependent activation of a disease-associated rhodopsin mutation (Pro23His) expressed in yeast ( Scott et al, 2019 ).…”

A GPCR-based yeast biosensor for biomedical, biotechnological, and point-of-use cannabinoid determination

“…In previous work, it has been possible to hijack the yeast pheromone pathway by replacing the pheromone receptor with a GPCR of interest and monitor the pathway's downstream response using a reporter gene 4,5,[28][29][30] . This has been broadly exploited for example, in the study of specific receptors 31,32 , deorphanization of uncharacterized receptors 29 , study of cell-cell communication 33 , guiding metabolic engineering efforts 34 , and detection of fungal pathogens 28 . However, specialized yeast biosensors capable of performing lowcost high-throughput bioactivity characterization, bioprospecting, and, especially, out-of-lab applications, have yet to be introduced.…”