Project Summary Endoplasmic reticulum aminopeptidase 1 (ERAP1) and insulin-regulated aminopeptidase (IRAP) belong to the oxytocinase subfamily of M1 aminopeptidases (M1APs), which are a diverse family of metalloenzymes involved in a wide range of functions including cell maintenance, development, and immune defense, and have been implicated in various chronic and infectious diseases of humans. Structure and mechanism of the conserved catalytic domain, termed peptidase_M1 domain, have been well studied. As a result, many research groups have begun assessing their potential as therapeutic targets for various diseases, such as cancer, autoimmunity, Alzheimer's and Parkinson's diseases, hypertension, and viral infections. However, targeting any M1AP to treat human diseases is complicated because there are nine characterized and closely related M1APs in humans. This poses a dilemma situation to deal with off-target side effects. Nonetheless, current inhibitor- developing strategies rely heavily on Bestatin derivatives or phosphinic pseudopeptides to occupy the S1 and/or S1’ binding pockets of the highly conserved peptidase_M1 site. Although potent, these inhibitors also inhibit many other essential M1AP members, lead to undesired side effects. Our lab has previously determined a novel ERAP1 C-terminal regulatory domain structure, now classified as the ERAP1_C like domain. This ERAP1_C fold, separated from the conserved peptidase_M1 domain, is found in all available structures of the M1AP family, but their sequences vary substantially among family members, likely to harbor distinct specificity subsites. Indeed, our recent results on four structures of ERAP1_C domain in complex with various substrate C-termini have revealed specificity subsites (e.g., SC’ subsite) embedded in this ERAP1_C domain to recognize ERAP1- specific anchor residue at the substrate carboxyl-end, a critical feature of ERAP1 to act as a molecular ruler in generating antigens with a correct size. We thus hypothesize that each M1AP carries distinct specificity subsites in its special version of ERAP1_C domain to accommodate distal parts of their cognate substrates. To test this hypothesis, we propose in this R15 project to investigate structural details of IRAP specificity subsites for recognizing the distal ends of its cognate substrates, and to exploit newly identified specificity subsites for targeted screenings to identify modulators that are both potent and selective against ERAP1 or IRAP over other M1APs. The proposed research will provide insights into both common and distinct interactions between specificity subsites of different M1APs and their cognate substrates, and is thus critical to improve our capability to develop specific and selective modulators or inhibitors to fight against pathogens or chronic diseases. Furthermore, this AREA research will provide continuous research opportunities for undergraduate and graduate students at UMass Lowell, and help the University to enhance its research environment.

Project Narrative This application is to perform structural and comparative analyses of two human aminopeptidases, ERAP1 and IRAP, in order to enhance our understanding of their distinct substrate specificities. It will also carry out large-scale computational and biochemical screenings for identifying lead compounds to selectively modulate or inhibit ERAP1 or IRAP over close-related aminopeptidases. This knowledge is critical to improve our capability to develop specific and selective modulators or inhibitors as human therapeutics to fight against pathogens or chronic diseases, and is thus critical to public health.