Abstract 1486 Development of Activity-Based Protein Profiling Assays for the Human Rhomboid Proteases

Rhomboid intramembrane proteases (RIPs) are a family of serine hydrolases distinguished by their membrane-embedded active sites and proposed involvement in metabolic and neurodegenerative diseases as well as cancer. Despite the ubiquity of these enzymes in all of kingdoms of life, our knowledge of the enzymatic functions of RIPs is still quite limited. Consequently, the development of suitable substrate-based activity assays for these proteases has proved challenging due to the lack of known substrates. Activity-based protein profiling (ABPP) represents an alternative approach for studying the activity of these enzymes; in these assays, a small-molecule probe is used to engage the active enzyme by binding covalently to its active site. Here, we present our progress on the development of ABPP assays for the human RIPs. We expressed all five human RIPs (hRHBDL1, hRHBDL2, hRHBDL3, hRHBDL4, and hPARL), along with their inactive mutants, in HEK293T cells. We then screened a library of small molecule probes, including fluorophosphonates, β-lactams, and benzoxazinones, for their ability to engage the active enzymes, but not their inactive mutants, in a complex proteome. Probe labeling was visualized by performing the azide-alkyne Huisgen cycloaddition reaction with a functionalized rhodamine on probe-treated proteome followed by gel electrophoresis (SDS-PAGE). Through these efforts, we have identified activity-based probes for several of the human RIPs and observed differences in probe engagement in lysate versus intact cells. Our findings provide encouraging precedent for the development of suitable ABPP assays for each of the human RIPs as well as insight into the types of chemical scaffolds that could be used to generate inhibitors for these enzymes. This work has been supported through a grant from the National Institute of General Medical Sciences (R15GM146210).