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