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Inhibitor Studies and Photoaffinity Labeling

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Inhibitor Studies and Photoaffinity Labeling Human Steroid Sulfatase: Inhibitor Studies and Photoaffinity Labeling by Chau-Minh Phan A thesis presented to the University of Waterloo in fulfillment of the thesis requirement for the degree of Master of Science in Chemistry Waterloo, Ontario, Canada, 2010 © Chau-Minh Phan 2010 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 Steroid sulfatase (STS) is considered to be one of the key enzymes contributing to the development of breast cancer. It catalyzes the hydrolysis of inactive sulfated steroids such as estrone sulfate (ES) to inorganic sulfate active steroids such as estrone (E1), a precursor to estradiol (E2), a key stimulator for breast cancer development. Inhibitors of STS are currently being pursued in both academia and industry as potential drugs for treating breast cancer. A series of 4-substituted estrone and estradiol derivatives were examined as inhibitors of STS. Inhibition of STS with 4-FE1, an irreversible inhibitor of STS previously studied in the Taylor group, can be enhanced by introducing a hydrophobic benzyl group at the 17-positon of 4-FE1. As with 4-FE1, the inhibition was concentration and time-dependent. Only 14% of the activity could be recovered after extensive dialysis. Introducing substituents at the 2-position of 4-formyl estrogen derivatives resulted in loss of concentration and time-dependent inhibition and a considerable decrease in inhibitor affinity. Studies with estrogen derivatives substituted at the 4-position with groups other than a formyl revealed that a relatively good reversible inhibitor can be obtained simply by introducing an electron withdrawing group at this position. These types of inhibitors are non-competitive inhibitors suggesting an alternative steroid binding site. A series of estrone derivatives were examined as photoaffinity labels of STS. 4- azidoestrone suflate and 4-azidoestrone phosphate exhibited properties that are suitable for photoaffinity labeling studies with STS. These labels may be useful for ascertaining pathways of substrate entry into the STS active site. 16-diazoestrone phosphate was not a photoaffinity label of STS. 2- and 4-azido estrone and 16-diazoestrone all acted as photoaffinity labels of STS. These compounds may be useful for ascertaining pathways of product release from the STS active site. iii Acknowledgements I am greatly indebted to my supervisor, Dr. Scott Taylor, whose encouragement, guidance and support from the initial to the final level enabled me to develop an understanding of the subject. Lastly, I offer my regards and thanks to my committee members, Dr. Joseph Guillemette and Dr. Thorsten Dieckmann, and all of those who supported me in any respect during the completion of the project. iv Table of Contents List of Tables viii List of Figures ix List of Schemes xv List of Abbreviations xvi Chapter 1 – Steroid Sulfatase: Function, Structure, Inhibitors, and Substrate Inhibition 1.1 Introduction 1.1.1 Estrogen Dependent Breast cancer 1 1.1.2 Estrogen Biosynthesis 2 1.1.3 Aromatase 2 1.1.4 17β-Hydroxysteroid Dehydrogenase 3 1.1.5 Estrone sulfotransferase 4 1.1.6 Steroid Sulfatase 4 1.2 Steroid Sulfatase 1.2.1 STS Expression and Location 5 1.2.2 Crystal Structure of STS 6 1.2.3 Substrate Movement 7 1.2.4 Post-Translational Modification of Aryl Sulfatases 9 1.2.5 Proposed mechanism for Aryl Sulfatases 10 1.2.6 STS Specific for Sulfate-Esters 12 1.3 STS Inhibitors 1.3.1 Reversible Inhibitors 13 1.3.2 Irreversible Inhibitors 16 1.4 Substrate Inhibition 1.4.1 4-DFMES unique irreversible inhibitor 19 1.4.2 Substrate Inhibition 21 1.4.3 Evidence for Substrate Inhibition in STS 23 1.5 Research Objectives 23 Chapter 2 – Purification and Assaying of Steroid Sulfatase 2.1 Introduction 24 2.2 Results and Discussion 2.2.1. Placental Purification of STS 25 2.2.2 2-Nitroestrone sulfate as a potential chromogenic substrate for STS 27 2.3 Conclusions and Future Work 35 2.4 Experimental v 2.4.1 Materials 36 2.4.2 STS purification 36 2.4.3 STS activity in purification 38 2.4.4 Protein determination 38 2.4.5 Spectral data 38 2.4.6 HPLC studies 39 Chapter 3 – 4-Substituted Estrogens as Reversible and Irreversible Inhibitors of STS 3.1 Introduction 40 3.2 Results and Discussion 3.2.1 Inhibition of STS with 17-α-benzyl-4-formyl estradiol 41 3.2.2 Reversible Inhibition of STS with 4-substituted estrogen derivatives 46 3.3 Conclusions and Future Work 52 3.4 Experimental 3.4.1 Materials 53 3.4.2 IC 50 determinations 53 3.4.3 Ki determination 54 3.4.4 Determination of time and concentration dependent inhibition of STS with 3.4 54 3.4.5 Time and concentration-dependent inhibition of STS in the presence of estrone phosphate (EP) (protection experiments) 55 3.4.6 Dialysis Experiment 55 Chapter 4 – Photoaffinity Labeling to Reveal Mode of Substrate and Product Transport in STS 4.1 Introduction 4.1.1 Photoaffinity labeling 56 4.1.2 Photaffinity Probes 57 4.2 Results and Discussion 4.2.1 Studies with compounds 4.1 and 4.2 59 4.2.2 Studies with compounds 4.3-4.5 71 4.2.3 Deglycosylation of STS 84 4.3 Conclusions and Future Work 84 4.4 Experimental 4.4.1 Materials 85 4.4.2 Effect of 350nm light on STS activity 85 4.4.3 Spectrophotometric studies with compounds 4.1 -4.8 86 4.4.4 Photoaffinity labeling procedure 86 4.4.5 HPLC Studies with 4.1 and 4.2 87 4.4.6 IC 50 and K i determinations 88 vi 4.4.7 Deglycosylation of STS 88 REFERENCES 89 APPENDICES Appendix A. IC 50 plots for compounds 3.3. 3.7-3.9, 3.11, 3.16-3.24, E1 and E2 and replots of the data in figures 3.10-3.12. 96 Appendix B.IC 50 plots for compounds 4.1-4.5. 4.8 and EP and replots of the data in figures 4.12, 4.13, 4.26, 4.27, 4.29 106 vii List of Tables Chapter 1 – Steroid Sulfatase: Function, Structure, Inhibitors, and Substrate Inhibition Table 1.1 . Estrone-3-sulfate mimics as reversible inhibitors of STS ..........................................14 Table 1.2 . 17-substituted analogs as reversible inhibitors of STS ...............................................14 Chapter 2 – Purification and Assaying of Steroid Sulfatase Table 2.1. Spectra of 2-NE1 at various pH. Concentrations of 2-NE1 ........................................30 Table 2.2 Spectra of 2-NES at pH 8 .............................................................................................33 Chapter 3 – 4-Substituted Estrogens as Reversible and Irreversible Inhibitors of STS Table 3.1. Inhibition of STS with 2-substituted 4-FE1 Derivatives..............................................46 Table 3.2 . Inhibition of STS with 4-substituted estrogen derivatives. .........................................47 Table 3.3. Ki’s for compounds 3.19 , 3.20 and 3.24 .....................................................................51 Chapter 4 – Photoaffinity Labeling to Reveal Mode of Substrate and Product Transport in STS Table 4.1. Summary of results with compounds 4.1 -4.8 ..............................................................83 viii List of Figures Chapter 1 – Steroid Sulfatase: Function, Structure, Inhibitors, and Substrate Inhibition Figure 1.1 Biosynthesis of estradiol from dehydroepiandrosterone ..............................................2 Figure 1.2. STS crystal structure revealing the tertiary mushroom-shape structure .......................6 Figure 1.3. Structure depicting the electrostatic potential of STS and its association with the RER. ...........................................................................................................................................7 Figure 1.4. Structure of STS showing the three flexible loops in red which may allow substrates to enter and leave the active site .....................................................................................8 Figure 1.5. The post-translational modification found in all aryl sulfatases ................................10 Figure 1.6. The active site of steroid sulfatase showing a sulfated formylglycine hydrate residue .........................................................................................................................................11 Figure 1.7. Vanadate, sulfite, and sulfate as initial inhibitors of STS .........................................13 Figure 1.8. General structures for madurahydroxylacetone thiosemicarbazones, aryl piperizines and arylsulfonylureas. .................................................................................................15 Figure 1.9 . Structures of two potent STS inhibitors: EMATE and 667 COUMATE ..................17 Figure 1.10. Time- and concentration-dependent inactivation of STS with 4-DFMES at concentrations 10 µM or less. ........................................................................................................20 Figure 1.11. Time- and concentration-dependent inactivation of STS with 4-DFMES at concentrations of 10 µM and greater .............................................................................................20 Figure 1.12. The kinetics associated with substrate inhibition are slightly different than that of normal Michaelis-Menten kinetics ................................................................................................21 Chapter 2 – Purification and Assaying of Steroid Sulfatase Figure 2.1. Elution profile of STS activity by DEAE chromotography .......................................26 Figure 2.2. Elution profile
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