STUDIES IN BIOCHEMISTRY: STEROID SYNTHESIS, P-450 ENZYMOLOGY AND RENAL LITHOGENESIS By JEREMY D. SELENGUT Submitted to the Department of Chemistry in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY in Biological Chemistry at the Massachusetts Institute of Technology February 1994 © 1994 Massachusetts Institute of T o6hn'ogy All rigLireserved Signature of Author vf' _Dpartment of Chemistry -- pecember 28, 1993 Certified by , , I I · I \/William H. Orme-Johnson Professor of Chemistry Thesis Supervisor -vr v /- Accepted--I by J IJ I MAssCt '-'/ Glenn A. Berchtold o-r TsrljT, ,NSTTyUhairman, Departmental Committee on Graduate Students MAR 2 1 1994 LIBRARlES :.-.ence This doctoral thesis has been examined by a Committee of the Department of Chemistry as follows: Professor_- -~ JoAnne Stubbe . .. .. I _- , - Chairman Professor William H. Orme-Johnson -, . , I, va ,, -. - ResearckSupervisor Professor Peter Lansbury. -47 11-11%I-, ,- - Z Professor Scott Virgil L_ ,/ //Lo///L.v, .L.,7 ,//1I - fJ 2 STUDIES IN BIOCHEMISTRY: STEROID SYNTHESIS, P-450 ENZYMOLOGY AND RENAL LITHOGENESIS By JEREMY D. SELENGUT Submitted to the Department of Chemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biological Chemistry, February 1994 ABSTRACT I. A method for the synthesis of lanosterol (Lanosta-8,24-dien-3p-ol) is developed such that isotopes of hydrogen can be specifically introduced into the C-32 position of the molecule. Lanosterol labeled with tritium in this way is suitable for use in recently developed assay methods for lanosterol-14ox-demethylase, a P-450 enzyme responsible for the common step in the synthesis of cholesterol and ergosterol from lanosterol. This enzyme is known to be the target of an important class of anti-fungal drugs and therefore has been the focus of considerable industrial, clinical and scientific interest. This synthesis is the first to preserve both of the double bonds in the lanosterol molecule and thus provide specifically labeled samples of the natural substrate for the mammalian and yeast enzymes, and additionally provides access to new molecules modified at the C-32 position and posessing the proper side-chain configuration. The synthesis is based on previous syntheses of C-32-modified dihydrolanosterol (Lanost-8-en-30-ol) and significant improvements to several literature methods are provided. II. The inhibitor (20R)-20-(1,5-hexadiynyl)-pregn-5-en-31,20-diol has been shown to be a mechanism-based inactivator of the cholesterol side-chain cleavage enzyme, P-450scc, which is responsible for the conversion of cholesterol into pregnenolone in steroidogenic tissues. Furthermore, this inactivator has been shown to covalently bind to the protein during the course of this inactivation. These properties have been confirmed in the present work. Preliminary studies towards the identification of the site or sites of covalent linkage between the steroid inactivator and the putative active-site peptide(s) are presented. Evidence suggests that the steroid is attached to the 42 tryptic fragment of the protein which is believed to contain the heme- and substrate-binding domain. Technical difficulties with further progress are analyzed. Studies with related mechanism-based inactivators having only one alkynyl group (such as (20R)-20-(1-hexynyl)-pregn-5-en- 3P,20-diol), indicate that these molecules do not covalently bind to the protein. Speculations on the chemical differences between the inactivation modes of these two classes of molecule are discussed. 3 III. The granulocyte protein, calprotectin, is extracted from an unusual matrix stone (a large-volume, amorphous, soft kidney stone) and identified by N-teminal peptide sequencing and immunochemical (ELISA) methods. This protein has recently been identified with numerous inflammatory diseases including cystic fibrosis, rheumatoid arthritis, psoriasis, and skin cancers. Additionally, calprotectin has been found to posess considerable antimicrobial properties versus several important human infectious strains of fungi as well as bacteria. Struvite kidney stones, which are strongly associated with urinary tract infection, are analyzed for calprotectin content and found to contain the protein in considerable amounts. Speculations on the origin of this material and its significance for the process of renal lithogenesis are discussed and proposals for future research are outlined. Thesis Supervisor: Dr. William H. Orme-Johnson Title: Professor of Chemistry 4 DEDICATION This thesis is dedicated to my family to whom I am indebted for the opportunity to have reached this goal To my mother, the artist, who taught me reading from the New York Times, algebra on her knee at the kitchen table and philosophy ever since. To my father, the engineer, who gave me my first chemistry set, would not accept a poorly-reasoned answer, and kept me pondering for my entire seventh year when he asked me what was different about the way sight and sound and smell reached my eyes and ears and nose. To my grandparents, you are my inspiration. To you I owe my love of words, books, music, history, biology and building something beautiful where before there was nothing at all. 5 A CKNOWLEDGEMENTS If my time at MIT has been successful and rewarding it is due in large portion to those who were around to help and support me, to you all a hearty thanks. Professor Orme-Johnson (Bill, to all) has provided much more than the means and motivations for my graduate work. He has always been true to his avowed philosophy of placing the responsibility for fundamental research directions in his students hands - if only we dare to take it. His curiosity is infectious and he inspired me strike out in the disparate directions that are recounted in this thesis. Where I fell upon dead ends his first concern was always that I not lose self-confidence, and where I succeeded his faith has borne fruit. Bill's sense of humor and humanity has kept me going in hard times; I would be truly dissapointed if I left a group meeting without one of his anecdotes and, so far, I have not been dissapointed. I want to thank Bill in particular for respecting my committment to remaining in Providence, R.I. to be with my wife and being that most unlikely of creatures, a commuter graduate student I would also like to thank Professor Fred Greene, for whom I was a teaching assistant my first year. His door has always been open to me with wise insights and helpful, practical suggestions (not to mention a warm smile and an encouraging pat on the back) and I have appreciated his assistance a great deal. My fellow basement denizens, David Wright, Patti Christie, Rob Pollock, Normand Cloutier and (recently graduated) "Uncle" Subhendu Joardar, have been partners in all my efforts and good friends even if I haven't been around to socialize with them after hours very much. They have always been willing to listen when I came by with my (very likely all to frequent) "Got a minute?" Their insights and suggestions have headed off many an error, developed many an interesting new idea, and pleasantly dispensed with many an afternoon. I'd like to particularly thank Normand Cloutier, my lab mate of four years, an indispensible resource on so many practical matters, my favorite debating opponant and, as I now appreciate, a fine editor. Also deserving of thanks in the basement are all the members of the Kemp research group, who have participated in the Kemp-O.J. chemical and glassware superstore, and helped answer so many questions about organic chemistry over the years. I'd like to specially thank Sherri Oslick for her excellent birthday cakes and other pastry over the years, as well as her steadfast leadership of that great team of underdogs, the Bargain Basement Bozos volleyball team (without which I would have had neither exercise nor sunlight). Professor John Essigman and his entire lab get big thank you's for putting up with my incessant borrowing of their scintillation counter over the last year; they welcomed me into their lounge like a member of the family. Dr. Steve Dretler was instrumental in focusing our attention on the problem of the protein componant of kidney stones (the subject of chapter 4 of this thesis), and has provided 6 critical support for this new research project. I only hope that, should I fall ill, I'm attended to by a doctor with such dedication to his chosen speciality. Credit is also due to Dr. Jeff Bennett, a resident at the Massachusetts General Hospital, who provided much appreciated research support on the kidney stone project. I wish to thank Dr. Hirotaka Asakura for his very generous patience and support over the last six months as I tied up the various loose ends of my graduate career prior to joining him in further work on kidney stone proteins. I owe a debt of gratitude to the several educators who inspired me to come this far. Robert "Merf" Merrifield was the high school biology teacher, not terribly long out of college, who routinely handed out "Future Nobel Prizes" to students who asked questions of particular depth and which were then scientific terra incognita. One by one I have watched as those questions have found their answers. They have been like mileposts along the road, constantly reminding me of the possibilities. Professor Peter Heywood let me into his junior Cell Biology class as a freshman at Brown and took me under his wing (as I later learned he has done for dozens of grateful young scientists). An inspiring teacher, he encouraged my growing understanding that evolution could be found in biology at scales as small as you were clever enough to see it. Prof. Heywood is the one who got me my first summer research job, and one thing has just lead to another.