INHIBITION AND MECHANISM STUDIES OF SECRETED PHOSPHOLIPASE A2, KYNURENINE AMI

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. NEW INHIBITORS OF SECRETED PHOSPHOLIPASE A2 (sPLA2) sPLA2 enzymes regulate cytokine-mediated inflammatory pathways. Exogenous Group IIA sPLA2(sPLA2-IIA)can enhance prostaglandin E2(PGE2)production in fibroblast-like synovial cells from RA patients. Furthermore sPLA2-IIA can induce the proliferation of prostate cancer cells. sPLA2-IIA exhibits both enzymatic and activity independent actions and we have developed inhibitors that block both. We now routinely achieve datasets to 1.6 Ǻ resolution the inhibitor enzyme complexes. We are developing more potent inhibitors and seek to confirm their predicted mechanism. HUMAN KYNURENINE AMINOTRANSFERASE-I (hKAT-I) hKAT-I catalyses the formation of kynurenic acid from kynurenine. Kynurenic acid is found in elevated levels in the brains and CSF of patients with schizophrenia. This is a consequence of significantly higher brain hKAT-I activity and is compelling evidence that such an inhibitor would be an efficacious anti-psychotic agent, however there are currently no known specific inhibitors of hKAT-I. The preliminary results suggest that these compounds exhibit strong binding and potent inhibition of hKAT-I. The structure of the hKAT-I inhibitor complexes will elucidate the mechanism of inhibition and aid in the design more potent analogues. CATALYSIS AND ALLOSTERIC REGULATION OF TRANSGLUTAMINASE 2 (TG2) TG2 is a ubiquitous multi-functional protein. Its cross-linking (TG) activity is important in fibrotic disorders, coeliac disease and neuropathies; its GTPase activity mediates cell signalling through its function as a receptor-coupled G-protein. Catalytic details of TG activity in terms of substrate access to the buried active site well as the mechanism of allosteric regulation of TG2 by calcium (to activate TG activity) and GTP (to inhibit TG activity) remain poorly understood. We propose to determine the structures of TG-inactive (GTP-bound) and TG-activated (calcium-bound with or without substrate) TG2.