Reversible and Mechanism-Based Irreversible Inhibitor Studies on Human Steroid Sulfatase and Protein Tyrosine Phosphatase 1B

Reversible and Mechanism-Based Irreversible Inhibitor Studies on Human Steroid Sulfatase and Protein Tyrosine Phosphatase 1B by F. Vanessa Ahmed A thesis presented to the University of Waterloo in fulfilment of the thesis requirement for the degree of Doctor of Philosophy in Chemistry Waterloo, Ontario, Canada, 2009 © F. Vanessa Ahmed 2009 Author’s Declaration I hereby declare that I am the sole author of this thesis. This is a true copy of the thesis, including any required final revisions, as accepted by my examiners. I understand that my thesis may be made electronically available to the public. ii Abstract The development of reversible and irreversible inhibitors of steroid sulfatase (STS) and protein tyrosine phosphatase 1B (PTP1B) is reported herein. STS belongs to to the aryl sulfatase family of enzymes that have roles in diverse processes such as hormone regulation, cellular degradation, bone and cartilage development, intracellular communication, and signalling pathways. STS catalyzes the desulfation of sulfated steroids which are the storage forms of many steroids such as the female hormone estrone. Its crucial role in the regulation of estrogen levels has made it a therapeutic target for the treatment of estrogen-dependent cancers. Estrone sulfate derivatives bearing 2- and 4- mono- and difluoromethyl substitutions were examined as quinone methide-generating suicide inhibitors of STS with the goal of developing these small molecules as activity- based probes for proteomic profiling of sulfatases. Kinetic studies suggest that inhibition by the monofluoro derivatives is a result of a quinone methide intermediate that reacts with active-site nucleophiles. However, the main inhibition pathway of the 4- difluoromethyl derivative involved an unexpected process in which initially formed quinone methide diffuses from the active site and decomposes to an aldehyde in solution which then acts as a potent, almost irreversible STS inhibitor. This is the first example where this class of inactivator functions by in situ generation of an aldehyde. 6- and 8- mono- and difluoromethyl coumarin derivatives were also examined as quinone methide- generating suicide inhibitors of STS. The 6-monofluoromethyl derivative acted as a classic suicide inhibitor. The partition ratio of this compound was found to be very large indicating that this class of compounds is not likely suitable as an activity-based probe for proteomic profiling of sulfatases. Boronic acids derived from steroid and coumarin iii platforms were also examined as STS inhibitors with the goal of improving our understanding of substrate binding specificity of STS. Inhibition constants in the high nanomolar to low micromolar range were observed for the steroidal derivatives. The coumarin derivatives were poor inhibitors. These results suggest that the boronic acid moiety must be attached to a platform very closely resembling a natural substrate in order for it to impart a beneficial effect on binding affinity compared to its phenolic analog. The mode of inhibition observed was reversible and kinetic properties corresponding to the mechanism for slow-binding inhibitors were not observed. PTP1B catalyzes the dephosphorylation of phosphotyrosine residues in the insulin receptor kinase and is a key enzyme in the down regulation of insulin signaling. Inhibitors of PTP1B are considered to have potential as therapeutics for treating type II diabetes mellitus. The difluoromethylenesulfonic (DFMS) acid group, one of the best monoanionic phosphotyrosine mimics reported in the literature, was examined as a phosphotyrosine (pTyr) mimic in a non-peptidyl platform for PTP1B inhibition. The DFMS-bearing inhibitor was found to be an approximately 1000-fold poorer inhibitor than its phosphorus analogue. It was also found that the fluorines in the DFMS inhibitor contributed little to inhibitory potency. In addition, [sulfonamido(difluoromethyl)]- phenylalanine (F 2Smp) was examined as a neutral pTyr mimic in commonly used hexapeptide and tripeptide platforms. F 2Smp was found to be a poor pTyr mimic. These inhibition studies also revealed that the tripeptide platform is not suitable for assessing pTyr mimics for PTP1B inhibition. iv Taken together, the kinetic data on the inhibition of STS and PTP1B provide valuable information relevant for future design of inhibitors of these two therapeutic targets. v Acknowledgements I would foremost like to thank my dedicated supervisor, Dr. Scott D. Taylor for his guidance in overseeing my development from an undergraduate to a graduate researcher. I am grateful for his support, mentorship and encouragement during my doctoral research, which has been a fulfilling, rewarding journey and a true privilege. I extend sincere appreciation to the members of my advisory committee for their advice, constructive criticism and helpful discussions throughout the stages of my graduate research. I thank Drs. Guy Guillemette, Elizabeth Meiering, and Richard Manderville for their support and insight. I am indebted to my coworkers in the lab who made instrumental contributions to my project, in particular Yong Liu, Bryan Hill and Munawar Hussain, who synthesized many of the compounds integral toward my study of steroid sulfatase and protein tyrosine phosphatase 1B. I also thank them and other members in the lab for their support and contributions to my professional development, particularly, Jason Trinh, Cassandra Silvestro, Dustin Little, Andrea Dupont, Laura Ingram, Latifeh Navidpour, Farzad Mirzai, Mehdi Ispahany and Jennifer Lapierre. vi Table of Contents List of Tables xi List of Figures xii List of Abbreviations xviii CHAPTER 1 – STEROID SULFATASE : STRUCTURE , FUCTIO AD IHIBITOR DEVELOPMET Page 1.1 Introduction 1 1.1.1 The sulfatase family of enzymes 2 1.1.2 Formylglycine 4 1.2 Steroid sulfatase 10 1.2.1 STS expression and location 10 1.2.2 STS and hormone-dependent breast cancer 12 1.2.3 STS action in prostate cancer 16 1.2.4 STS and skin 17 1.2.5 STS and neurofunction 17 1.2.7 Sulfatase deficiencies 18 1.2.8 Crystal structure of STS 21 1.2.9 Comparison of tertiary structures of sulfatases 26 1.2.10 Comparison of active site structures of sulfatases 28 1.2.11 Catalytic mechanism of sulfatases 30 1.3 STS inhibitor development 34 1.3.1 Reversible inhibitors 34 1.3.2 Irreversible inhibitors 40 1.4 Research Objectives 48 CHAPTER 2 – PURIFICATIO OF STEROID SULFATASE 2.1 Introduction 50 2.2 Results and Discussion 57 2.2.1 Study of the effect of neutralization of pH of STS after 60 immunoaffinity purification 2.2.2 Evaluation of kinetic properties and comparison to literature 63 2.3 Conclusions 70 2.4 Experimental 71 2.4.1 Materials 71 2.4.2 Methods 71 2.4.2.1 Activity Assay 71 2.4.2.2 Homogenization and Chromatography 72 2.4.2.4 Protein concentration determination 74 2.4.2.5 Determining kinetic properties 75 2.4.2.6 Determining pH-rate profile 75 vii CHAPTER 3 – QUIOE METHIDE -GEERATIG ACTIVE SITE -DIRECTED MECHAISM - BASED IRREVEVERSIBLE IHIBITORS OF STEROID SULFATASE 3.1 Introduction 76 3.1.1 Mechanism-based enzyme inhibitors 77 3.1.2 Quinone methide-generating suicide inhibitors 80 3.1.3 Objectives 83 3.2 Results and Discussion 84 3.2.1 General criteria for SIs 84 3.2.2 Evaluation of time- and concentration-dependence of compounds 85 3.10 and 3.11 3.2.3 Active site protection and trapping experiments for compounds 88 3.10 and 3.11 3.2.4 Irreversibility of inhibition of STS by compounds 3.10 and 3.11 . 92 3.2.5 Evaluation of time- and concentration-dependence for compound 93 3.9 . 3.2.6 Evaluation of time- and concentration-dependence, effect of 96 exogenous nucleophiles and active site protection for compound 3.12 3.2.7 Monitoring reaction products and intermediates produced by 100 incubation of compound 3.12 and STS by HPLC 3.2.8 Examination of 4-FE1 as a time- and concentration dependent 101 inhibitor of STS 3.2.9 Specificity of inhibition of STS by formylated steroids 102 3.2.10 2- and 4-Hydroxymethylestrone as STS inhibitors 108 3.2.11 Screening of coumarin derivatives 3.13 -3.16 as time-dependent 112 STS inhibitors 3.2.12 Kinetic studies with compound 3.14 113 3.2.13 Determination of the partition coefficient of 3.14 115 3.2.14 Potential for quinone methide-generating SIs as activity-based 118 proteomic profiling probes 3.3 Conclusions and Future Work 121 3.4 Experimental 124 3.4.1 General 124 3.4.2 Preliminary screening of compounds 3.13 -3.16 125 3.4.3 General procedure for the determination of time and 125 concentration-dependent inhibition of STS 3.4.4 Lineweaver-Burk analysis of compound 3.9 126 3.4.5 Time and concentration-dependent inhibition of STS in the 126 presence of estrone-3-O-phosphate (E1P) (protection experiments) 3.4.6 Time and concentration-dependent inhibition of STS in the 127 presence of β-mercaptoethanol ( β-ME) (trapping experiments) 3.4.7 Dialysis experiments 128 3.4.8 Effect of NaBH 4 on STS activity 129 3.4.9 Generation of standard curve for coumarin 3.28 129 viii 3.4.10 Determination of partition coefficient for compound 3.14 130 3.4.11 Monitoring the reactions of compounds 3.9 -3.12 with STS by 131 HPLC CHAPTER 4 – BOROIC ACIDS AS IHIBITORS OF STEROID SULFATASE 4.1 Introduction 133 4.1.1 Boronic acids as enzyme inhibitors 133 4.1.2 Objectives 136 4.2 Results and Discussion 138 4.3 Conclusions and Future Work 146 4.4 Experimental 148 4.4.1 General 148 4.4.2 Determination of Ki and αKi values for compounds 4.1 , 4.6 , 4.7 148 and 4.10 4.4.3 Determination of IC 50 values for compounds 4.8 , 4.9 , and 4.10 149 CHAPTER 5 – A ASSESSMET

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