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Copyright by Gayle Diane Burstein 2014 Copyright by Gayle Diane Burstein 2014 The Dissertation Committee for Gayle Diane Burstein Certifies that this is the approved version of the following dissertation: An Investigation of the Irreversible Inhibition of Human Nω, Nω- Dimethylarginine Dimethylaminohydrolase (DDAH1) Committee: Walter Fast, Supervisor Christian Whitman Yan Jessie Zhang Brent Iverson David Hoffman An Investigation of the Irreversible Inhibition of Human Nω, Nω- Dimethylarginine Dimethylaminohydrolase (DDAH1) by Gayle Diane Burstein, B.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 2014 Dedication For my loving family Acknowledgements Thank you to my advisor, Dr. Walter Fast. Dr. Fast was always available to talk through my scientific and personal difficulties. He believed in me even when I didn’t believe in myself and I could not be more thankful for that. Thank you to my friends and colleagues in the Fast lab, past and present, for all your insights, thoughts, and our scientific collaborations. Thank you to Dr. Art Monzigo and the Robertus lab for our collaborative efforts and their X-ray crystallographic work. Thank you to my friends that have helped me and pushed me to pursue my degree, your encouragement is always appreciated. Finally, thank you to Jordan Teitelbaum for pushing me to work hard and being there for me during the difficult times (and Lazer Wolf for his purrs). v An Investigation of the Irreversible Inhibition of Human Nω, Nω- Dimethylarginine Dimethylaminohydrolase (DDAH1) Gayle Diane Burstein, Ph.D. The University of Texas at Austin, 2014 Supervisor: Walter Fast Nitric oxide synthases (NOS) are responsible for the production of nitric oxide (NO), an essential cell-signaling molecule, in mammals. There are three isoforms of NOS with widely different tissue distribution. The overproduction of NO is marked in many human disease states and cancers, however due to the similarities of the enzyme isoforms, targeting NOS for inhibition has proven challenging. Endogenously, the methylated arginines, Nω-monomethyl-L-arginine (NMMA) and asymmetric Nω, Nω- dimethyl-L-arginine (ADMA), inhibit NOS. Nω, Nω-Dimethylarginine dimethylaminohydrolase (DDAH1) metabolizes these methylated arginines and thus relieves NOS inhibition. The role of DDAH1 in the regulation of diseases such as cancer and septic shock is still being elucidated. It is thought that targeting DDAH1 for inhibition rather than NOS may circumvent many of the current problems with the treatment of NO overproduction such as isoform selectivity. My PhD studies focus on the synthesis of a series of irreversible inhibitors of DDAH1, an extensive study of their in vitro mode of inhibition, a comparison of analytical fitting methods, and the viability and efficacy of the inactivators in a human cell line. I also studied a potential endogenous inactivator of DDAH1, nitroxyl (HNO), a one-electron reduction product of NO. vi Table of Contents List of Tables ...........................................................................................................x List of Figures ........................................................................................................ xi List of Schemes .................................................................................................... xiii Chapter 1: Background and Significance ................................................................1 1.1: The Dual Nature of Nitric Oxide (NO) ....................................................1 1.1.1: Nitric Oxide (NO) is an important cell signaling molecule .........1 1.1.2: NO overproduction is implicated in many diseases states. ..........5 1.2: Dimethylarginine Dimethylaminohydrolase as a Means to Control NO Production ..............................................................................................7 1.2.1: Methylated arginines inhibit NOS ...............................................7 1.2.2: Dimethylarginine Dimethylaminohydrolase (DDAH) contributes to the regulation of NO production ...................................................9 1.2.3: Manipulation of DDAH can alter NO levels .............................13 1.3 Towards the irreversible inhibition of Dimethylarginine Dimethylamniohydrolase .....................................................................14 1.4 References ...............................................................................................18 Chapter 2: Optimizing Chloroacetamidine Inactivators of DDAH1 and a Comparison of Fitting Methods ........................................................................................26 2.1 Introduction .............................................................................................26 2.2 Materials and Methods ............................................................................28 General Synthesis .................................................................................28 DDAH1 Expression and Purification ...................................................30 Time-dependent Inactivation of DDAH1 by Irreversible Inhibitors ...31 Partition ratio .......................................................................................33 Crystal Structure ..................................................................................33 Arginase Assay ....................................................................................34 In cell IC50 value determination ...........................................................34 In Cell Single Point Assay ...................................................................36 vii 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)- 2-(4- sulfophenyl)-2H-tetrazolium (MTS) Assay ................................36 2.3 Results and Discussion ...........................................................................37 Kinetic Analysis of DDAH1 Time-dependent Inactivation by Chloroacetamidines. ....................................................................39 Cl-NIL acts as a mechanism based inhibitor .......................................49 Cl-NIL forms a covalent bond with the active-site cysteine ................50 Cl-NIL is selective for DDAH1 over Mn2+-containing human arginase I (Mn-hArgI) .................................................................................56 Compounds inhibit DDAH1 within cultured cells ...............................57 2.4 Conclusions .............................................................................................63 2.5 References ...............................................................................................63 Chapter 3: Inhibition of Dimethylarginine Dimethylaminohydrolase by Angeli’s Salt .......................................................................................................................67 3.1 Introduction .............................................................................................67 3.2 Materials and Methods ............................................................................70 Materials ..............................................................................................70 Expression and Purification of DDAH1 ..............................................70 Time-dependent Inactivation of DDAH1 by Angeli’s Salt .................71 Time-dependent Inactivation by Decomposed Angeli’s Salt ..............72 Time-dependent Inactivation by Nitrite ...............................................72 Time-dependent Inactivation by GSH Incubated Angeli’s Salt ..........72 Acid Quench of Inactive DDAH1 ........................................................72 LC-ESI-MS Analysis ...........................................................................73 In Cell Inhibition of DDAH1 by Angeli’s Salt ....................................74 3.3 Results and Discussion ...........................................................................75 Angeli’s Salt Covalently Inactivates DDAH1 .....................................75 HNO is the Predominant Inactivating Species of DDAH1 by Angeli’s Salt .....................................................................................................79 GSH Prevents DDAH1 Inactivation by Angeli’s Salt in vitro. ...........82 Angeli’s Salt does not Inactivate DDAH1 in HEK293T Cells. ...........84 viii 3.4 Conclusions .............................................................................................86 3.5 References ...............................................................................................87 Appendix: Kinetics of Chloroacetamidines ...........................................................90 Bibliography ........................................................................................................108 Vita ....................................................................................................................120 ix List of Tables Table 1.1 Representative DDAH inhibitors ...........................................................16 Table 2.1 Inactivation constants for DDAH1 inactivators .....................................40 Table 2.2 Rate constants obtained during global fitting to the model in scheme 2.1 ...........................................................................................................46 Table 2.3 Crystallographic Data ............................................................................51 Table A.1
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