Improving the Yeast Three-Hybrid System for High-Throughput Target Discovery

Abstract

High-throughput screening and rational design can be used to create bioactive compounds with high affinity for selected therapeutic targets. However, a significant challenge in preclinical drug development is the identification of off-target proteins that contribute to phenotypic effects. This limits the understanding of the molecular basis of such effects, thus subverting rational drug design and hindering the identification of new therapeutic targets. Most previous strategies for proteome-wide target identification (target ID) have involved incubating cell lysates with compound-conjugated affinity resins. Despite their simplicity, such approaches can subject the proteome to conditions that prevent the detection of small molecule-protein interactions. The yeast three-hybrid system is an attractive alternative that uses genetic tools to screen for protein-small molecule interactions in cellulo. This thesis describes efforts to improve the utility of the yeast three-hybrid system to screen for drug targets. The proposed improvements utilize 1) the native fluorescence of green fluorescent protein (GFP) to identify interactions by flow-cytometry and fluorescence-activated cell sorting (FACS) and 2) the extreme affinity of streptavidin to search the mammalian proteome with biotinylated probes. The first objective required improvements to the sensitivity and dynamic range of a reporter vector encoding a popular GFP spectral variant. A new reporter vector was constructed and shown to exhibit better fluorescent properties compared to an existing reporter in a yeast one-hybrid assay. This reporter was also used to detect ligand dependent dimerization of the estrogen receptor β and progesterone receptor proteins. The second goal involved efforts to create a reduced valency streptavidin to enhance sensitivity for detection of biotinylated molecules in yeast three-hybrid systems. Circular permutations of wild-type and a low affinity mutant of streptavidin were constructed and fused to create dimeric streptavidins with variable valency. These constructs were tested with yeast three-hybrid assays using a GFP variant reporter, and shown to have altered profiles in fluorescence-based assays.