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Abstract:
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Nitric 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 . |