Copyright by Yun Wang 2010

Copyright by Yun Wang 2010 The Dissertation Committee for Yun Wang Certifies that this is the approved version of the following dissertation: Controlling Nitric Oxide (NO) Overproduction: Nω, Nω- Dimethylarginine Dimethylaminohydrolase (DDAH) as a Novel Drug Target Committee: Walter L. Fast, Supervisor Christian P. Whitman Jon D. Robertus George Georgiou Sean M. Kerwin Controlling Nitric Oxide (NO) Overproduction: Nω, Nω- Dimethylarginine Dimethylaminohydrolase (DDAH) as a Novel Drug Target by Yun Wang, B.S.; M.S. Dissertation Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy The University of Texas at Austin August, 2010 Dedication To all people who have given me generous help when I am in Austin, including professors, colleagues and friends. To my beloved parents in P.R.China, who always motivate me to pursue my dream. Acknowledgements First and foremost, I would like to thank my supervisor, Dr. Walt Fast, for his generous support and for giving me an opportunity to work in his lab four years ago at the University of Texas at Austin. Walt gives me lots of freedom to explore scientific problems and provides a wonderful working environment along with my lovely colleagues. He always gives me useful directions when I meet difficulties. I appreciate every scientific conversation with him. They were especially important to me during the beginning of my Ph.D. study. I wouldn’t have accomplished as much as today without his help. In addition, I’d like to thank colleagues in Fast lab for their generous help during these years. I also want to thank professors in the Medicinal Chemistry Division who have helped me, especially my dissertation committee members. Finally, I give my special thanks to my dear friends in Austin and my parents in China, for their support when I was down. I am proud of myself that I finally stand at the point of finishing my Ph.D. degree. Despite troubles along the way, all’s well that ends well. v Controlling Nitric Oxide (NO) Overproduction: Nω, Nω- Dimethylarginine Dimethylaminohydrolase (DDAH) as a Novel Drug Target Publication No._____________ Yun Wang, Ph.D. The University of Texas at Austin, 2010 Supervisor: Walter L. Fast 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, Nω-monomethyl-L-arginine (NMMA) and asymmetric Nω, Nω-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 vi 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 µM, respectively) and DDAH-1 (990, 52, 7.5, 110 µM, 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. vii Table of Contents List of Tables ....................................................................................................... xii List of Figures ..................................................................................................... xiii List of Schemes ................................................................................................... xvi Chapter 1 Background and Significance.................................................................1 1.1 Nitric oxide (NO) - a double-edged sword ..............................................1 1.1.1 NO is an important cellular second-messenger ...........................1 1.1.2 NO overproduction is a serious problem in a number of severe diseases ........................................................................................4 1.1.3 The dual effects of NO .................................................................8 1.2 Dimethylarginine dimethylaminohydrolase (DDAH) as a novel drug target to control NO overproduction ...............................................................9 1.2.1 Nω-methyl-L-arginine and Nω,Nω-dimethyl-L-arginine are endogenous NOS inhibitors .........................................................9 1.2.2 Nω,Nω-dimethyl-L-arginine dimethylaminohyrolase, a novel target for NO regulation........................................................................12 1.3 Towards finding inhibitors to control diseases marked by NO overproduction .....................................................................................18 Chapter 2 Catalytic participation of human DDAH-1 in its own inactivation by S- nitroso-L-homocysteine ................................................................................26 2.1 Introduction.............................................................................................26 2.2 Material and Methods .............................................................................30 Construction of human DDAH-1 (hDDAH-1) variants ......................30 Expression and purification of hDDAH-1 variants ............................31 Steady-state kinetic studies .................................................................32 Preparation of L-homocysteine ...........................................................33 Preparation of S-nitroso-L-homocysteine ...........................................33 Reaction of hDDAH-1 variants with HcyNO .....................................34 Acid quench of covalently modified hDDAH-1 variants ...................34 LC-ESI-Mass spectrometry analysis ...................................................35 viii MALDI-TOF analysis of modified peptides in human DDAH-1 and Pseudomonas aeruginosa DDAH ..............................................35 UV-Vis spectroscopy of HcyNO modified hDDAH-1 .......................36 2.3 Results and Discussion ...........................................................................36 hDDAH-1 uses D79, D127, H173 and C274 to form a catalytic center .....................................................................................................36 Other active site amino acids play assisting roles in hDDAH-1 catalysis .....................................................................................................40 Trapped reaction intermediates during catalysis turnover reveals active site residues' participation in different catalysis steps ......................42 Proposed hDDAH-1 catalytic mechanism ..........................................46 Covalent inhibition of hDDAH-1 variants by S-nitroso-L-homocysteine yields two different adducts .......................................................47 Chapter 3 Developing dual and specific inhibitors of dimethylarginine dimethylaminohydrolase-1 and nitric oxide synthase: Toward a targeted polypharmacology to control nitric oxide.....................................................57 3.1 Introduction.............................................................................................57 3.2 Material and Methods .............................................................................60 Materials .............................................................................................60 General procedure for synthesis of N5-(1-iminoalkyl)-L-ornithines ...60 Cloning of recombinant human DDAH-1 ...........................................61 Site-directed mutagenesis to generate L30A, E78A and L271G muations .....................................................................................................63 Expression and purification of DDAH-1 ............................................64 Steady-state kinetic studies .................................................................66 Survey of selected NOS

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