Discovery of Novel Allosteric Inhibitor Hits for Insulin-Regulated Aminopeptidase Provides Insights on Enzymatic Mechanism

Insulin-regulated aminopeptidase (IRAP) is a transmembrane zinc metalloprotease with various important biological roles, including fibrosis, septic thrombosis, cognitive functions, and immune system regulation. As a result, IRAP is an emerging pharmacological target for several diseases. However, the development of selective inhibitors that specifically regulate its activity remains challenging due to its high sequence and functional homology with many other enzymes that have highly conserved active sites. To circumvent this limitation, we targeted the malate allosteric site, a site that has yielded highly selective inhibitors of the homologous enzyme ERAP1. We performed virtual screening to discover drug-like compounds that bind with high affinity to this allosteric site in IRAP. A database of 38 million diverse, drug-like compounds from ENAMINE was employed for screening at three conformations of the targeted site. A subset of the top-ranked compounds was subsequently evaluated using molecular dynamics simulations and comparative MM/GBSA free energy calculation, from which 17 were selected for further <i>in vitro</i> evaluation of their inhibitory activity for IRAP by two orthogonal assays. Three hits, one for each enzyme conformation and substrate class, were selected for further mechanistic evaluation revealing substrate-dependent uncompetitive or noncompetitive mechanisms of action, consistent with the conformationally sensitive nature of the allosteric site. Our results support the tractability of the malate site for the discovery of novel selective IRAP inhibitors, establish novel hits for further development, and suggest that it may be possible to target specific biological functions of IRAP by targeting distinct conformations of the enzyme by allosteric inhibitors.