Chemical Modulation and Enzymatic Characterization of MESH1

Abstract

MESH1 (Metazoan SpoT Homologue 1) is the human homologue of the bacterial stringent response enzyme SpoT, which degrades the alarmone (p)ppGpp. Humans do not have significant levels of (p)ppGpp, and the function of MESH1 in humans remained a mystery for decades. Previously, work from our group demonstrated that MESH1 can act as an NADPH phosphatase that can regulate ferroptosis, a regulated form of cell death driven by iron accumulation and lipid peroxidation.Building upon these findings, this dissertation identifies 4,5,6,7-tetrabromo-1H-benzotriazole (TBB) as the first small molecule inhibitor of MESH1. The compound was identified in chemical screen and found to inhibit the NADPH phosphatase activity of MESH1. X-ray crystallography and structure activity relationships of TBB analogs revealed the molecular determinants of binding. In cellular models, pharmacological inhibition of MESH1 with TBB suppresses ferroptosis, establishing MESH1 as a druggable enzyme and validating chemical inhibition as a strategy to regulate ferroptosis. In parallel, this work expanded upon the known enzymatic scope of MESH1, revealing broad substrate promiscuity towards multiple nucleotide-based metabolites including Coenzyme A (CoA), phosphoadenosine-5’phosphosulfate (PAPS), and phosphoadenosine-5’phosphate (PAP). Biochemical and structural studies demonstrate these metabolites are bona fide MESH1 substrates, implicating MESH1 in regulating antioxidant capacity, sulfation metabolism and nucleotide homeostasis. Together these findings, establish MESH1 as a multifunctional metabolic enzyme and provides a framework for understanding how its diverse enzymatic activities contribute to cellular physiology.