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Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. ProQuest Information and Learning 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 800-521-0600 UMI* DEVELOPMENT OF PHOTOREVERSIBLE COVALENT INHIBITORS FOR PROTEIN TYROSINE PHOSPHATASES DISSERTATION Presented in Partial Fulfillment of the Requirements for The Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Gulnur Arabaci, M.S. ***** The Ohio State University 2001 Dissertation Committee: Professor Dehua Pei, Adviser Approved by Professor Ross E. Dalbey Professor Ming-Daw Tsai Adviser Department of Chemistry Professor Don W. Miller UMI Number: 3022438 UMI UMI Microform 3022438 Copyright 2001 by Bell & Howell Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. Bell & Howell Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT Reversible protein tyrosine phosphorylation is a key determinant in eukaryotic signaling pathways. The level of phosphorylation is balanced by the opposing activities of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Although a large number of PTPs have been identified, elucidating the in-vivo function of any one specific PTP is still a difficult task due to the lack of specific PTP agents. This dissertation describes the development of photo-reversible and covalent inhibitors for specific PTPs. These inhibitors are a-haloacetophenone derivatives and inactive PTPs by alkylating the conserved active site catalytic cysteine residue. The inactivated PTPs can be conveniently reactivated by irradiation with UV light. Further, this class of PTP inhibitors can permeate human cells and affect tyrosine phosphorylation levels in certain proteins. Initially, the a-haloacetophenone inhibitors tested possessed similar inhibition potency towards different PTPs. Previous results indicated that PTP IB inhibition by cinnamates could be improved by attaching peptide based specificity elements. First, a variety of a-haloacetophenones were tested for SHP-1(ASH2) and PTP IB inhibition. u A combinatorial approach was then developed to screen for specificity elements that improved PTP inhibition. This involved attaching a combinatorial peptide library to the inhibitor core and screening to identify elements that improved SHP-1(ASH2) and PTP IB inhibition. Ill Dedicated to my parents IV ACKNOWLEDGMENTS I would like to acknowledge my advisor. Dr. Dehua Pei for guidance and encouragement throughout my graduate study. Dr. Mark K. Coggeshall is thanked for carrying out the in-vivo PTP inhibition experiments. I would also like to thank the members of my dissertation committee, Dr. Ross E. Dalbey and Dr. Ming-Daw Tsai. I extend thanks to all members of the Pei group (past and present) for their help and also for providing a great work environment. Their support and assistance were crucial in my success. Special thanks are due to Dr. Kirk D. Beebe for providing enzymes, intellectual discussions about research and life, and in general, being a very good fiiend. I acknowledge the help of Kari Green-Church and Nanette Kleinholz with mass spectrometric experiments. On a more personal note, I would like to thank my close friends, Latife Sahin, Zebra Ayhan, Eser Tufekci, Elizabeth Guo and P.T. Ravi Rajagopalan. Their support and encouragement has always been there when I needed it the most. Finally, I thank my parents, my brother and his family for their love and encouragement of my quest for higher education. VITA January 26, 1970 .................................Bom - Istanbul, Turkey. June, 1991............................................B.S. Chemistry, Istanbul Technical University, Istanbul, Turkey. 1992-1993............................................Graduate student, Istanbul Technical University, Istanbul, Turkey. 1995-1997............................................ M.S. Chemistry, The Ohio State University. 1997-present ........................................ Graduate student. The Ohio State University. PUBLICATIONS 1. Arabaci, G., Guo, X-C., Beebe, K. D., Coggeshall, K. M., and Pei, D. (1999) J. Am. Chem. Soc. 121, 5085-5086. 2. Beebe, K. D., Wang, P., Arabaci, G. and Pei, D. (2000) Biochemistry 39, 13251- 13260. 3. Wang, P., Arabaci, G. and Pei, D. (2001) J. Comb. Chem. in press. FIELDS OF STUDY Major Field: Chemistry VI TABLE OF CONTENTS Page Abstract ........................................................................................................................ ii Dedication .................................................................................................................... iv Acknowledgments ........................................................................................................... v Vita ............................................................................................................................... vi List of Tables ............................................................................................................... ix List of Figures .............................................................................................................. x List of Abbreviations, Symbols and Nomenclature ................................................... xii Chapters: 1. Introduction ......................................................................................................... 1 1.1 Phosphorylation in Signal Transduction ................................................. 1 1.2 Classification of Kinases and Phosphatases ......................................... 2 1.3 Structure and Mechanism of Protein Tyrosine Phosphatase ................ 5 1.4 Autoregulation and Biological Functions of SHP-1 and SHP-2 ............ 9 1.5 Inhibition of Protein Tyrosine Phosphatases ......................................... 12 2. Characterization of Photoreversible and Covalent Inhibition of Protein Tyrosine Phosphatases ..................................................................................... 23 2.1 Introduction ................................................................................................ 23 2.2 Experimental procedures ........................................................................... 25 2.2.1 Materials ....................................................................................... 25 2.2.2 Purification of SHP-1 and SHP-1 (ASH2) ................................. 25 2.2.3 Purification ofVHR phosphatase ............................................... 27 2.2.4 Activity assay for phosphatases ................................................. 28 2.2.5 Time dependent inhibition of phosphatases .............................. 28 2.2.6 Determination of the reaction rate of Ic with free thiols ........ 31 vu 2.2.7 Mass spectrometric analysis of SHP-1 (ASH2) .......................... 31 2.2.8 PTP reactivation ......................................................................... 33 2.2.9 Photolysis product determination .............................................. 34 2.2.10 Inhibition of B Cell PTPs ..................................................... 35 2.3 Results ....................................................................................................... 36 2.3.1 Kinetics of time dependent inactivation of PTPs by a-haloaceto phenones ............................................................................. 36 2.3.2 Mode of PTP inhibition by a-bromoacetophenones .................. 38 2.3.3 Photo-reactivation of a-bromoacetophenone inactivated PTPs . 40 2.3.4 Analysis of photolytic reactivation ............................................. 41 2.3.5 Membrane permeability of a-bromoacetophenone .................... 43 2.4 Discussion ................................................................................................. 44 3. Developing Specific PTP Inhibitors .................................................................. 62 3.1 Introduction ............................................................................................... 62 3.2 Experimental procedures .......................................................................... 63 3.2.1 Materials and general methods ..................................................... 63 3.2.2 Biotinylation of proteins