Controlling nitric oxide (NO) overproduction : N[omega], N[omega]-dimethylarginine dimethylaminohydrolase (DDAH) as a novel drug target

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dc.contributor.advisorFast, Walter L.en
dc.creatorWang, Yun, 1981-en
dc.date.accessioned2011-11-01T19:44:57Zen
dc.date.accessioned2017-05-11T22:23:38Z
dc.date.available2011-11-01T19:44:57Zen
dc.date.available2017-05-11T22:23:38Z
dc.date.issued2010-08en
dc.descriptiontexten
dc.description.abstractNitric oxide (NO) overproduction is correlated with numerous human diseases, such as arthritis, asthma, diabetes, inflammation and septic shock. The enzyme activities of both NO synthase (NOS) and dimethylarginine dimethylaminohydrolase-1 (DDAH-1) promote NO production. DDAH-1 mainly colocalizes in the same tissues as the neuronal isoform of NOS and catabolizes the endogenously-produced competitive inhibitors of NOS, N[omega]-monomethyl-L-arginine (NMMA) and asymmetric N[omega], N[omega]-dimethyl-L-arginine (ADMA). Inhibition of DDAH-1 leads to elevated concentrations of NMMA and ADMA, which subsequently inhibit NOS. To better understand DDAH-1, I first characterized the catalytic mechanism of human DDAH-1, where Cys274, His173, Asp79 and Asp127 form a catalytic center. Particularly, Cys274 is an active site nucleophile and His173 plays a dual role in acid/base catalysis. I also studied an unusual mechanism for covalent inhibition of DDAH-1 by S-nitroso-L-homocysteine (HcyNO), where an N-thiosulfoximide adduct is formed at Cys274. Using a combination of site directed mutagenesis and mass spectrometry, we found that many residues that participate in catalysis also participate in HcyNO mediated inactivation. Following these studies, I then screened a small set of known NOS inhibitors as potential inhibitors of DDAH-1. The most potent of these, an alkylamidine, was selected as a scaffold for homologation. Stepwise lengthening of the alkyl substituent changes an NOS-selective inhibitor into a dual-targeted NOS/DDAH-1 inhibitor then into a DDAH-1 selective inhibitor, as seen in the inhibition constants of N5-(1-iminoethyl)-, N5-(1-iminopropyl)-, N5-(1-iminopentyl)- and N5-(1-iminohexyl)-L-ornithine for neuronal NOS (1.7, 3, 20, >1,900 [mu]M, respectively) and DDAH-1 (990, 52, 7.5, 110 [mu]M, respectively). X-ray crystal structures suggest that this selectivity is likely due to active site size differences. To rank the inhibitors' in vivo potency, we constructed a click-chemistry based activity probe to detect inhibition of DDAH-1 in live mammalian cell culture. In vivo IC50 values for representative alkylamidine based inhibitors were measured in living HEK293T cells. Future application of this probe will address the regulation of DDAH-1 activity in pathophysiological states. In summary, this work identifies a versatile scaffold for developing DDAH targeted inhibitors to control NO overproduction and provides useful biochemical tools to better understand the etiology of endothelial dysfunction.en
dc.description.departmentPharmacyen
dc.format.mediumelectronicen
dc.identifier.urihttp://hdl.handle.net/2152/14048en
dc.language.isoengen
dc.rightsCopyright is held by the author. Presentation of this material on the Libraries' web site by University Libraries, The University of Texas at Austin was made possible under a limited license grant from the author who has retained all copyrights in the works.en
dc.subjectNitric oxide (NO)en
dc.subjectOverproductionen
dc.subjectNO synthase (NOS)en
dc.subjectDimethylarginine dimethylaminohydrolase-1 (DDAH-1)en
dc.subjectCatalytic mechanismen
dc.subjectInhibitionen
dc.subjectClick-chemistry based activity probeen
dc.subjectLiving HEK293T cellsen
dc.titleControlling nitric oxide (NO) overproduction : N[omega], N[omega]-dimethylarginine dimethylaminohydrolase (DDAH) as a novel drug targeten

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