A Chemical Genetic Strategy to Study Vip1 in Inositol Pyrophosphate Signaling

Abstract

The water-soluble and highly phosphorylated inositol pyrophosphates (PP-IPs) are a conserved class of signaling molecules. Genetic studies in yeast have implicated the PPIPs in a variety of roles, including nutrient sensing and response to environmental stress, but have largely proven ineffective in decoding their mechanisms of action. This thesis focuses on developing a chemical genetic approach for an in-depth analysis of the signaling roles of Vip1, an ATP-grasp kinase, and its corresponding PP-IP products in Saccharomyces cerevisiae. The technique relies on strategically introducing mutations into the kinase active site and developing orthogonal ATP analogs. I report the successful identification and engineering of analog sensitive alleles of Vip1 (Vip1-AS) that maintain functionality both biochemically and in yeast. Furthermore, I use a structure-guided approach to develop a small class of biorthogonal ATP-competitive pyridopyrimidines, and demonstrate their potency against and selectivity for Vip1-AS in vitro. Though inhibition studies in yeast are thus far incomplete, preliminary results provide promising evidence for the successful creation of an appropriate chemical genetic system for Vip1. Once verified and optimized, the system can be not only applied to investigate Vip1’s role in transcriptional and metabolic regulation, but also extended to examine homologues in higher-order eukaryotes and structurally similar enzymes within the ATPgrasp superfamily.