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Use of Structure-And Ligand-Based Drug Design Tools for The University of Mississippi eGrove Electronic Theses and Dissertations Graduate School 2011 Use of Structure-And Ligand-Based Drug Design Tools for the Discovery of Small Molecule Inhibitors of Cysteine Proteases for the Treatment of Malaria and Sars Infection Falgun Shah Follow this and additional works at: https://egrove.olemiss.edu/etd Part of the Pharmacy and Pharmaceutical Sciences Commons Recommended Citation Shah, Falgun, "Use of Structure-And Ligand-Based Drug Design Tools for the Discovery of Small Molecule Inhibitors of Cysteine Proteases for the Treatment of Malaria and Sars Infection" (2011). Electronic Theses and Dissertations. 260. https://egrove.olemiss.edu/etd/260 This Dissertation is brought to you for free and open access by the Graduate School at eGrove. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of eGrove. For more information, please contact [email protected]. USE OF STRUCTURE-AND LIGAND-BASED DRUG DESIGN TOOLS FOR THE DISCOVERY OF SMALL MOLECULE INHIBITORS OF CYSTEINE PROTEASES FOR THE TREATMENT OF MALARIA AND SARS INFECTION A Dissertation Presented for the Doctor of Philosophy In Pharmaceutical Sciences The University of Mississippi Falgun Shah November 2011 Copyright © 2011 by Falgun Shah All rights reserved ABSTRACT A wide array of molecular modeling tools were utilized to design and develop inhibitors against cysteine protease of P. Falciparum Malaria and Severe Acute Respiratory Syndrome (SARS). A number of potent inhibitors were developed against cysteine protease and hemoglobinase of P. falciparum, referred as Falcipains (FPs), by the structure-based virtual screening of the focused libraries enriched in soft-electrophiles containing compounds. Twenty one diverse, non-peptidic, low micromolar hits were identified. A combined data mining and combinatorial library synthesis approach was performed to discover analogs of virtual screening hits and establish the structure-activity relationships (SAR). However, the resulting SAR of the identified hits was unusually steep in some cases and could not be explained by a traditional analysis of the interactions (electrostatics, van der Waals or H-bond). To gain insights, a statistical thermodynamic analysis of explicit solvent in the ligand binding domain of FP-2 and FP-3 was performed that explained some of the complex trends in the SAR. Furthermore, the moderate potency of a subset of FP-2 hits was elucidated using quantum mechanics calculations that showed reduced reactivity of the electrophilic center of these hits. In addition, solvent thermodynamics and reactivity analysis also helped to elucidate the complex trends in SAR of peptidomimetic inhibitors of FP-2 and FP-3 synthesized in our laboratory. Multi nanosecond explicit solvent molecular dynamics simulations were carried out using the docking poses of the known inhibitors in the binding site of SARS-3CLpro, a cysteine protease important for replication of SARS virus, to study the overall stability of the binding site interactions as well as ii identify important changes in the interaction profile that were not apparent from the docking study. Analysis of the simulation studies led to the identification of certain protein-ligand interaction patterns which would be useful in further structure based design efforts against cysteine protease (3CLpro) of SARS. iii DEDICATION To my loving mom, dad and sister iv LIST OF ABBREVIATIONS 3Clpro = 3C-like protease, C=Chymotrypsin ADME = Absorption, Distribution, Metabolism, Elimination Boc = t-butyloxycarbonyl Cat = Cathepsins Cbz = Carboxybenzyl DMF = Dimethylformamide EDCI = 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide FCPI = Focused cysteine protease inhibitor library Gscore = Glide score HOBT =Hydroxybenzotriazole HOMO = Highest occupied molecular orbital HRMS = High resolution mass spectroscopy HTS = High throughput screening FMK = Fluoro methyl ketone FP-2 = Falcipain-2 FP-3 = Falcipain-3 LBD = Ligand binding domain LUMO = Lowest unoccupied molecular orbital MD = Molecular dynamics v M pro = Main protease MLR = Multiple linear regression MM-GBSA = Molecular mechanics Generalized Born surface area MM-PBSA = Molecular mechanics Poission-Boltzmann surface area NMR = Nuclear magnetic resonance ORF = Open reading frames OPLS = Optimized potential for liquid simulation PDB = Protein data bank P. falciparum = Plasmodium falciparum PP = polyprotein QSAR = Quantitative structure activity relationships RMSD = Root mean square deviation SAR = Structure-activity relationships SARS = Severe acute respiratory syndrome SBVS = Structure-based virtual screening TEA = triethylamine W2 = Chloroquine-resistant strain of P. falciparum vi ACKNOWLEDGMENTS I thank Dr. Mitchell A. Avery, my research mentor, for presenting me with multiple opportunities to work in a diverse medicinal chemistry projects in his group, for providing me a comprehensive training in the field of synthetic organic chemistry, medicinal chemistry and computational chemistry and for funding my research for five years. He let me pursue my own ideas in malaria project and helped me grow as a capable researcher. In addition to giving leadership for ongoing cross-disciplinary projects in his group, Dr. Avery also allowed me to pursue several research projects outside the university campus in the form of summer internships, giving me an opportunity to prove myself in the challenging research environments of both, pharmaceutical industry and renowned academic institutions. I am thankful to Dr. Stephen J. Cutler for his constant motivation and appreciations most often via brief notes whenever I received internal or external awards, for allowing me to attend the scientific meetings and external training at times when our group had limited funding and for keeping me on tract to conclude my dissertation. I would like to thank Dr. Babu Tekwani for his help with biological assays against cultured malaria parasites and his valuable insights in my research projects. I am thankful to Dr. Robert J. Doerksen for teaching fundamentals of molecular modeling as a part of my training as a computational chemist and his willingness to help in solving problems pertaining to computational chemistry. I also appreciate efforts from all committee members for scheduling my defense on a very short notice. I thank Dr. John S. Williamson for providing up to date information with graduate vii curriculum requirement, for his kind recommendation for the ACS Olemiss section Graduate Student Research Award and for his advices on the career selection. I thank Dr. Christopher R. McCurdy for revising my concepts of medicinal chemistry in a very elegant way during the first year of my Ph.D. and imprinting the importance of networking in individual‟s scientific career. I appreciate efforts from Dr. John M. Rimoldi for teaching me the fundamentals of bioorganic medicinal chemistry thoroughly, for his constructive comments during my original research proposal and most importantly, for notifying me five years back that I am accepted as a graduate student at the Department of Medicinal Chemistry at Olemiss. I deeply appreciate efforts of my internship mentors, Dr. Alexander Tropsha at the University of North Carolina, Chapel Hill, Dr. Jack A. Bikker at Pfizer Groton site and Dr. Ruben Abagyan at the University of California, San Diego for providing me extensive training in diverse disciplines of computational chemistry. Also, I highly appreciate efforts of Dr. Philip Rosenthal, for clearing my concepts of parasites biology, for his help in biological assays of falcipains and his critical evaluation and extensive edits for my first authored manuscripts. I also appreciate efforts of our collaborators Dr. Woody Sherman from Schrodinger Inc., and Dr. Sanjeev Krishna from St. George University of London, UK for sharing their expertise in the advancement of my research project on Malaria. I am deeply indebted to Dr. Prasenjit Mukherjee, my immediate senior, a lab mate and a very close friend, for sharing his scientific acumen, for helping me understand principles of computational chemistry more closely and precisely, for his advise on a professional and viii personal front. I thank Dr. Ronak Patel, an excellent post-docotral fellow in Dr. Doerksen‟s research group, a very close friend and a great teacher, for his help in solving technical problems related to computational chemistry in my projects. I thank Dr. Yakambram Pedurri, Dr. Amar Chittiboyina, Dr. Yunshan Wu and Dr. Swapnil Kulkarni for helping me to polish my synthetic chemistry skills and giving me a sufficient confidence that I as a computational chemist am capable of making real molecules when required. I would also like to thank other members of Avery‟s research group for their continued support. Dr. Rohit Bhat, a close friend, my roommate and a constructive critic and appreciator of lot of my activities deserves a special vote of thanks. Last but not least, I am grateful to my mother (Mrs. Jyotiben H. Shah), my father (Mr. Hemantbhai D. Shah) and my sister (Mrs. Foram N. Parmar) for their selfless love, continuous support and encouragement to overcome toughest hurdles in my life as I face it. ix TABLE OF CONTENTS CHAPTER PAGE 1. TARGETING CYSTEINE PROTEASE OF P. FALCIPARUM MALARIA AND SARS FOR DRUG DISCOVERY…………………………………………………..…………...1 1.1. INTRODUCTION……………………………………………………………………2 1.2. MALARIAL CYSTEINE PROTEASE “FALCIPAIN”: GENESIS, FUNCTION, AND STRUCTURAL REQUIREMENTS…………………………………………..5 1.3. DRUG DESIGN APPROACHES AGAINST FPS
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