Nitric oxide (NO) is reactive, radical gas that is involved in a myriad of cellular signaling pathways including the regulation of blood flow and immunodefense. NO is produced from the oxidation of L-arginine to L-citrulline by nitric oxide synthase (NOS). The activity of NOS and by default, the production of NO, is regulated by the arginine derivatives N[omega],N[omega]-dimethyl-L-arginine (ADMA) and N[omega]-monomethyl-L-arginine (NMMA) which arise from the proteolytic degradation of post translationally methylated proteins. The cellular concentrations of ADMA and NMMA are regulated by the enzyme dimethylarginine dimethylaminohydrolase (DDAH), which catabolizes these compounds to L-citrulline and dimethylamine or methyl amine, respectively. Because over and under production of NO has been implicated in several pathophysiological states, compounds that control NO production by inhibiting NOS or DDAH may prove useful as treatments. In this study, the crystal structures of human DDAH-1 with the inhibitors N⁵-(1-iminopentyl)-L-ornithine (L-IPnO) and ebselen were solved to 2.9 and 2.0 Å resolution, respectively. L-IPnO was observed to inhibit DDAH-1 in essentially the same manner as another amidino-containing inhibitor: docking to the enzyme via hydrogen bond and ion pair interactions and forming a covalent adduct with the active site cysteine. Ebselen was also observed to covalently attach to the active site cysteine, however, the docking mechanism was absent of hydrogen bond and ion pair interactions. The work presented here contributes to the design of compounds that may effectively regulate the production of NO for therapeutic purposes. Translin is a highly conserved mammalian RNA and DNA binding protein known to be involved in DNA recombination and repair, RNA trafficking in neurons, and post-transcriptional regulation of gene expression in male germ cells. Although crystal structures of the mouse and human orthologs of translin have been solved, they do not provide details on the structure-function relationship of the protein. Studies have identified a partner protein for translin, translin associated factor x (trax), which is believed to have a crucial role in assisting translin with its cellular functions. It is believed that trax regulates translin’s affinity for certain RNA and DNA sequences. In this work the binding affinities of translin and the translin●trax complex were investigated. It was observed that translin preferentially binds to G-rich RNA sequences, most likely recognizing a secondary structure intrinsic to these sequences, whereas translin●trax preferentially binds G-rich DNA sequences. The results from these experiments provide insight into the cellular functions of translin and trax and their respective roles in mRNA trafficking. / text

Dimethylarginine dimethylaminohydrolase (DDAH) indirectly regulates nitric oxide production by hydrolyzing methylated arginines, which are endogenous nitric oxide synthase inhibitors. This enzyme is a member of the mechanistically diverse pentein superfamily, which contains hydrolase, dihydrolase, and amidinotransferase enzymes. These enzymes are proposed to use the same first catalytic step, followed by partitioning into their respective activities. Here, variants of DDAH that can catalyze the dihydrolase and amidinotransfer reactions are presented, as well as a variant of succinylarginine dihydrolase which catalyzes a single hydrolysis reaction. The results experimentally demonstrate that the proposed common catalytic intermediate can be used for several different reactions. The results suggest that enzymes in the pentein superfamily may have evolved divergently from a catalytically promiscuous ancestor. The control DDAH asserts over nitric oxide production makes it an attractive drug target for disease states marked by pathological overproduction of nitric oxide. Only a limited number of inhibitors different from substrate are reported, due in part to lack of robust assays for high-throughput screening of compound libraries. Therefore, high-throughput assays were developed, optimized, and validated to screen for inhibitors of Pseudomonas aeruginosa DDAH and human DDAH-1. These assays were used to screen three commercial libraries totaling 6,466 compounds. One drug in phase III clinical trials, ebselen, was identified and characterized as a bioavailable, rapid covalent inactivator of DDAH both in vitro and in cultured cells. Four "fragment-sized" inhibitors were also identified and characterized in the screening, including 4-halopyridines and benzimidazole-like compounds. The 4-halopyridines, not previously known to modify proteins, act as quiescent affinity labels to selectively inactivate DDAH, and the benzimidazole-like compounds are competitive, rapidly reversible inhibitors of DDAH. These diverse molecules serve as starting points for the development of molecular probes and therapeutic drugs to reduce pathological overproduction of nitric oxide. / text

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. / text