University of Tennessee Health Science Center UTHSC Digital Commons Theses and Dissertations (ETD) College of Graduate Health Sciences 12-2008 Structure- and Ligand-Based Design of Novel Antimicrobial Agents Kirk Edward Hevener University of Tennessee Health Science Center Follow this and additional works at: https://dc.uthsc.edu/dissertations Part of the Medicinal and Pharmaceutical Chemistry Commons, and the Pharmaceutics and Drug Design Commons Recommended Citation Hevener, Kirk Edward , "Structure- and Ligand-Based Design of Novel Antimicrobial Agents" (2008). Theses and Dissertations (ETD). Paper 351. http://dx.doi.org/10.21007/etd.cghs.2008.0136. This Dissertation is brought to you for free and open access by the College of Graduate Health Sciences at UTHSC Digital Commons. It has been accepted for inclusion in Theses and Dissertations (ETD) by an authorized administrator of UTHSC Digital Commons. For more information, please contact [email protected]. Structure- and Ligand-Based Design of Novel Antimicrobial Agents Document Type Dissertation Degree Name Doctor of Philosophy (PhD) Program Pharmaceutical Sciences Research Advisor Richard E. Lee, Ph.D. Committee Duane D. Miller, Ph.D. Bob M. Moore II, Ph.D. Brien L. Neudeck, Pharm.D. Stephen W. White, Ph.D. DOI 10.21007/etd.cghs.2008.0136 This dissertation is available at UTHSC Digital Commons: https://dc.uthsc.edu/dissertations/351 STRUCTURE- AND LIGAND-BASED DESIGN OF NOVEL ANTIMICROBIAL AGENTS A Dissertation Presented for The Graduate Studies Council The University of Tennessee Health Science Center In Partial Fulfillment Of the Requirements for the Degree Doctor of Philosophy From The University of Tennessee By Kirk Edward Hevener December 2008 Portions of Chapter 5 © 2008 by Elsevier Ltd. All other material © 2008 by Kirk Edward Hevener. ii DEDICATION This work is dedicated with love to my family: to my parents, Eugene and Ellen, my sisters Michelle and Kara, and my brother, Kevin; for their patience, support, and encouragement; to the brothers of the Omega Chapter of Phi Delta Chi, for always being there; and to Michael Cox, a truer friend one could not hope for. iii ACKNOWLEDGMENTS I would like to express my sincerest gratitude to my mentor and research advisor, Dr. Richard E. Lee for his support, guidance, and encouragement during the course ofmy graduate studies. I also sincerely appreciate the guidance and advice of my committee members: Dr. Duane Miller, Dr. Bob Moore, Dr. Brien Neudeck, and Dr. Stephen White. I thank the members of the Lee lab for their friendship and companionship over the years and acknowledge the work of Robin Lee, Dr. Elizabeth Carson, and Dr. Julian Hurdle for performing MIC studies to measure the whole cell activity of the compounds discussed in this work. I also acknowledge the organic synthesis work of Dr. Kris Virga, Dr. Rajendra Tangallapally, Dr. Raghunandan Yendapally, and Dr. Jianjun Qi (former Lee lab members) for their efforts on design and synthesis of many of the compounds discussed in this work. I acknowledge and thank the members of the White lab for their collaborative efforts on the DHPS research project. I would like to particularly acknowledge the work of Dr. Kerim Babaoglu, who solved the majority of the DHPS crystal structures I used in my research project. I also thank Dr. Iain Kerr and Kate Ayers for the subsequent crystallography work they performed in the DHPS project. Dr. Mi-Kyun Yun’s work in implementing and running the DHPS enzyme assay was instrumental to this research and her efforts are also gratefully acknowledged. I thank Dr. John Buolamwini in the Department of Pharmaceutical Science at the University of Tennessee for his assistance and guidance with the GOLD docking program and his valuable assistance with the 3D-QSAR studies. I would also like to express my sincere appreciation to David Ball, my friend and colleague, for his tireless assistance with this research and his help in the preparation of the manuscripts generated from the work presented herein. Funding for this research was provided by the National Institutes of Health, ALSAC, and the University of Tennessee, College of Pharmacy. I gratefully acknowledge the American Foundation for Pharmaceutical Education for the pre- doctoral fellowship I was provided during my final two years of graduate course work as well as the UT College of Pharmacy’s Feurt Scholarship and the University of Tennessee’s National Alumni Association’s Andy Holt Scholarship for the funding and support I received during and after my pharmacy training. iv ABSTRACT The use of computer based techniques in the design of novel therapeutic agents is a rapidly emerging field. Although the drug-design techniques utilized by Computational Medicinal Chemists vary greatly, they can roughly be classified into structure-based and ligand-based approaches. Structure-based methods utilize a solved structure of the design target, protein or DNA, usually obtained by X-ray or NMR methods to design or improve compounds with activity against the target. Ligand-based methods use active compounds with known affinity for a target that may yet be unresolved. These methods include Pharmacophore-based searching for novel active compounds or Quantitative Structure-Activity Relationship (QSAR) studies. The research presented here utilized both structure and ligand-based methods against two bacterial targets: Bacillus anthracis and Mycobacterium tuberculosis. The first part of this thesis details our efforts to design novel inhibitors of the enzyme dihydropteroate synthase from B. anthracis using crystal structures with known inhibitors bound. The second part describes a QSAR study that was performed using a series of novel nitrofuranyl compounds with known, whole-cell, inhibitory activity against M. tuberculosis. Dihydropteroate synthase (DHPS) catalyzes the addition of p-amino benzoic acid (pABA) to dihydropterin pyrophosphate (DHPP) to form pteroic acid as a key step in bacterial folate biosynthesis. It is the traditional target of the sulfonamide class of antibiotics. Unfortunately, bacterial resistance and adverse effects have limited the clinical utility of the sulfonamide antibiotics. Although six bacterial crystal structures are available, the flexible loop regions that enclose pABA during binding and contain key sulfonamide resistance sites have yet to be visualized in their functional conformation. To gain a new understanding of the structural basis of sulfonamide resistance, the molecular mechanism of DHPS action, and to generate a screening structure for high- throughput virtual screening, molecular dynamics simulations were applied to model the conformations of the unresolved loops in the active site. Several series of molecular dynamics simulations were designed and performed utilizing enzyme substrates and inhibitors, a transition state analog, and a pterin-sulfamethoxazole adduct. The positions of key mutation sites conserved across several bacterial species were closely monitored during these analyses. These residues were shown to interact closely with the sulfonamide binding site. The simulations helped us gain new understanding of the positions of the flexible loops during inhibitor binding that has allowed the development of a DHPS structural model that could be used for high-through put virtual screening (HTVS). Additionally, insights gained on the location and possible function of key mutation sites on the flexible loops will facilitate the design of new, potent inhibitors of DHPS that can bypass resistance mutations that render sulfonamides inactive. Prior to performing high-throughput virtual screening, the docking and scoring functions to be used were validated using established techniques against the B. v anthracis DHPS target. In this validation study, five commonly used docking programs, FlexX, Surflex, Glide, GOLD, and DOCK, as well as nine scoring functions, were evaluated for their utility in virtual screening against the novel pterin binding site. Their performance in ligand docking and virtual screening against this target was examined by their ability to reproduce a known inhibitor conformation and to correctly detect known active compounds seeded into three separate decoy sets. Enrichment was demonstrated by calculated enrichment factors at 1% and Receiver Operating Characteristic (ROC) curves. The effectiveness of post-docking relaxation prior to rescoring and consensus scoring were also evaluated. Of the docking and scoring functions evaluated, Surflex with SurflexScore and Glide with GlideScore performed best overall for virtual screening against the DHPS target. The next phase of the DHPS structure-based drug design project involved high- throughput virtual screening against the DHPS structural model previously developed and docking methodology validated against this target. Two general virtual screening methods were employed. First, large, virtual libraries were pre-filtered by 3D pharmacophore and modified Rule-of-Three fragment constraints. Nearly 5 million compounds from the ZINC databases were screened generating 3,104 unique, fragment-like hits that were subsequently docked and ranked by score. Second, fragment docking without pharmacophore filtering was performed on almost 285,000 fragment-like compounds obtained from databases of commercial vendors. Hits from both virtual screens with high predicted affinity for the pterin binding pocket, as determined by docking score, were selected for in vitro testing. Activity and structure- activity relationship