IDENTIFICATION AND CHARACTERIZATION OF NOVEL ANTIRETROVIRAL COMPOUNDS: FROM SMALL MOLECULE LIBRARY SCREENING TO RATIONALLY DESIGNED COMPOUNDS A dissertation submitted to Kent State University in cooperation with the Lerner Research Institute, Cleveland Clinic Foundation in partial fulfillment of the requirements For the degree of Doctor of Philosophy By Oyebisi Jegede August, 2007 Dissertation written by Oyebisi Jegede M.B.B.S., University of Ilorin, 1998 Ph.D., Kent State University, 2007 Approved by _______________, Dr. Miguel Quiñones-Mateu, Chair, Doctoral Dissertation Committee _______________, Dr. Chun-Che Tsai, Co-Chair, Doctoral Dissertation Committee _______________, Dr. Philip Pellett, Member, Doctoral Dissertation Committee _______________, Dr. Gail Fraizer, Member, Doctoral Dissertation Committee _______________, Dr. Arvind Bansal, Outside Discipline Member _______________, Dr. Mary Cismowski, Graduate Faculty Representative Accepted by _______________, Dr. Robert V. Dorman, Director, School of Biomedical Sciences _______________, Dr. John Stalvey, Dean, College of Arts and Sciences ii TABLE OF CONTENTS LIST OF FIGURES…………………………………..………….…….……..............…vi LIST OF TABLES………………………………………………………...….....……....ix ABBREVIATIONS……………………………………………………….……………...x ACKNOWLEDGEMENTS …………………………………..……………….……....xv DEDICATION………………………………………………..……………….……....xvii CHAPTER I: GENERAL INTRODUCTION………………….……………...…...…1 CHAPTER II: IDENTIFICATION AND CHARACTERIZATION OF NOVEL ANTIRETROVIRAL COMPOUNDS FROM HIGH-THROUGHPUT SCREENING OF SMALL MOLECULE LIBRARIES…………….……………......48 INTRODUCTION………………….…………………………………………...…..48 MATERIALS AND METHODS…………………………………..……….….…..50 RESULTS….………………………….……………….……………...……..….…...72 HIV-1 susceptibility of hits……………………….…….…..….…….......………73 Hits were neither cytotoxic nor cytostatic……….…………..……………..…….73 Pre vs. post viral transcription inhibition of HIV-1 replication…….....…..…..…78 Direct effect of hits on HIV-1B-HXB2 virions…………………….….……...….….79 Time of addition ………………………………………………….……...………81 Inhibition of HIV-1 coreceptors, CCR5 and CXCR4……………..........…..……82 iii In vitro inhibition of HIV-1 reverse transcriptase…...…………..………………84 In vitro inhibition of HIV-1 integrase…………………..….……….….……...…85 In vitro inhibition of HIV-1 protease……………………...…................………...89 Drug susceptibility of primary and resistant HIV subtypes…......…………....…90 In vitro selection for resistant strains……...………………...........…….…….….94 Effect of CBL 26 on other HIV-1 gene targets…………………..……..…...….100 DISCUSSION…………………………………..………...……………..…...….…103 CHAPTER III: CELL-BASED SCREENING OF A SMALL MOLECULE LIBRARY FOR NEW ANTIRETROVIRAL COMPOUNDS PARTICULARLY HIV-1 CORECEPTOR ANTAGONISTS……………………………..…..……..….109 INTRODUCTION……………………………………….…………….......………109 MATERIALS AND METHODS……………………..…...………………….......111 RESULTS……………………………..……………………………..…….……….121 A novel assay to identify HIV-1 inhibitors……………………………………..121 Effect of known antiretroviral drugs on fluorescent viruses……...…….……... 124 Small molecule library screening…………………………...……….….............125 Time-of-addition…………………………………..…...……...………....……...129 DISCUSSION………………………....……………..…………………...…..……131 CHAPTER IV: KST 201 AND KST 301: ANALOGUES OF RESVERATROL AS NOVEL ANTIRETROVIRAL COMPOUNDS…………………………...………...137 INTRODUCTION………………..……………………………………….……….137 iv MATERIALS AND METHODS………………………………....…….……..….139 RESULTS……………………………………..........................................................144 DISCUSSION……………………………………………….……..…..…....……..151 CHAPTER V: DIVALENT METAL ION CHELATING SMALL MOLECULES AS NOVEL HIV INTEGRASE INHIBITORS……………………………..……….154 INTRODUCTION……………………………....………………….………...……154 MATERIALS AND METHODS…………………….………………...................155 RESULTS……………………………………………..............................................160 DISCUSSION…………………………………..……………....……………….....174 SUMMARY AND PERSPECTIVES…….……………….…………….........………176 REFERENCES……………….…………………………………….………….………180 v LIST OF FIGURES CHAPTER I Fig 1.1 Global view of HIV-1 distribution…………………………..…..………..……....3 Fig 1.2 Geographical distribution of HIV-1 genetic forms…………………..……………5 Fig 1.3 Geographical distribution of HIV-1 subtypes…………………..………….….….7 Fig 1.4 Mature HIV-1 virion……………………………………..………………..………8 Fig 1.5 HIV-1 linear genome………………. ………………………………..……….…10 Fig 1.6 HIV-1 lifecycle…………………………………………….…………….………12 Fig 1.7 Native trimeric state of HIV-1 envelope glycoproteins……………...……….….13 Fig 1.8 Multi-step process of HIV-1 entry………………………………….....…………14 Fig 1.9 HIV-1 reverse transcription……………………………….…………....……..…20 Fig 1.10 HIV-1 integration………………………………….……...…………...……….21 Fig 1.11 Assembly and maturation of HIV-1 virion……………………………..………25 Fig 1.12 HAART………………………………...…...………….……………….……...27 Fig 1.13 Targets of antiretroviral drugs…………………………………….……………29 Fig 1.14 Structures of nucleoside reverse transcriptase inhibitors……………..…….….31 Fig 1.15 Structure of tenofovir disoproxil fumarate………………..…..……….….…....34 Fig 1.16 Structures of non-nucleoside reverse transcriptase inhibitors…………….……36 Fig 1.17 Structure of protease inhibitors………………………………..….……….……38 Fig 1.18 Structure of fusion inhibitor…………………………….………….……….….40 CHAPTER II Fig 2.1 Small molecule library screening protocol…………………..………..…………51 Fig 2.2 Antiviral assays……………………………………………..………….....…..….54 Fig 2.3 Cellular toxicity assays………………………………………………...…..…….56 vi Fig 2.4 Inhibition of HIV-1 coreceptors assay ………………………………….……….59 Fig 2.5 High-throughput integrase inhibition assay…………………………..........…….61 Fig 2.6 In vitro selection for resistant strains ……………………………….……….…..65 Fig 2.7 Cellular toxicity of hits by cell viability…………………………........….……...74 Fig 2.8 Viral susceptibility of hits…………..………………………..…..………….…...75 Fig 2.9 Selectivity indices of hits………………………….……….….………....….…...76 Fig 2.10 Pre vs. post viral transcription inhibition……………………………..…..…….78 Fig 2.11 Direct effect of hits on HIV-1 virions…….….…………………....….……..…79 Fig 2.12 Time of addition assay………….……………...…………………………...…..81 Fig 2.13 Effect on HIV-1 coreceptors….…………………...………………...…..….…..83 Fig 2.14 HIV-1 integrase high-throughput assay………….…………………...……...…86 Fig 2.15 In vitro HIV-1 integrase gel-based electrophoresis……….………….……...…86 Fig 2.16 Effect of CBL 4.3 on HIV-1 integrase in Mg2+………….……………………..87 Fig 2.17 Effect of CBL 4.3 on HIV-1 integrase in Mn2+….………………………..……87 Fig 2.18 In vitro HIV-1 protease inhibition assay………….……………….…...…...….89 Fig 2.19 Selection for resistant HIV-1 strains….……………………….....……….……96 Fig 2.20 Viral growth kinetics………….……………….…………….…………....……99 Fig 2.21 Site directed mutagenesis…….…………...……………..….….………….….101 Fig 2.22 Susceptibility of SDM virus and wild type viruses to CBL 26…….….......….102 CHAPTER III Fig 3.1 Fluorescent viruses……….……………………….………………...…...…..…112 Fig 3.2 Verification of coreceptor usage assay.……….………………...………...……113 Fig 3.3 Plate design ………….………………………………………...…………...…..114 Fig 3.4 Protocol for screening small molecule library….………………………...……115 Fig 3.5 Confirmation of susceptibility of B-HXB2 and C5 to hits….…………….……117 Fig 3.6 Cellular toxicity in U87.CD4.CCR5 and U87.CD4.CXCR4….……….…....…118 Fig 3.7 Result of coreceptor usage verification ….……………………….…………....122 Fig 3.8 Admixtures of multiplicities of HIV-1 fluorescent viruses………….…………123 vii Fig 3.9 Antiviral activity of known drugs…………….……………………..…….….124 Fig 3.10 Results of the cell-based small library screening….…………………….…..125 Fig 3.11 Time of addition assay …….………………………….………………...…..129 CHAPTER IV Fig 4.1 Structure of Resveratrol……………….…………………….....……………….144 Fig 4.2 Cellular toxicity in Ghost X4/R5……….…………………...…..…...…...….....145 Fig 4.3 Antiviral activity determined by EGFP expression…………….….…………...148 Fig 4.4 Inhibition of HIV-1 ……………….………………………...…..…………...…149 Fig 4.5 Inhibition of HIV-l coreceptors, CCR5 and CXCR4…….………...…….....….150 CHAPTER V Fig 5.1 Pre vs. post viral transcription inhibition…….…………………....................…164 Fig 5.2 Effect of BFX 1001-BFX 1004 on HIV-1 coreceptors ….…………...……..…165 Fig 5.3 PCR-based single cycle assays…………….…………...…….…………….......166 Fig 5.4 Passages of B-HXB2 in increasing concentration of BFX compounds….….…167 Fig 5.5 Restriction analysis by Southern blot on BFX treated acutely infected cells…..169 Fig 5.6 Inhibition of luciferase expression by BFX compounds….….…………...……171 Fig 5.7 Inhibition of strand transfer reaction by drug controls…………………………173 viii LIST OF TABLES CHAPTER II Table 1 In vitro inhibition of HIV-1 reverse transcriptase……………..……..….…..….84 Table 2 Antiviral activity of hits against primary HIV-1 &-2 isolates……………….….92 Table 3 Antiviral profile of hits against recombinant MDR viruses………….…..….….93 Table 4 Drug susceptibility of CBL resistant viruses…………………….…….………..98 CHAPTER III Table 1 Summary of the antiviral and cellular toxicity profiles………………...…..….128 Table 2 Cellular viability, cellular proliferation and antiviral activity…………....……128 CHAPTER IV Table 4.1 Antiviral activity and cellular toxicity……..……………………..…..……...146 Table 4.2 IC of Resveratrol, KST 201 and KST 301 against HIV-1 and HIV-2...…...149 50 CHAPTER V Table 5.1 CC50 and IC50 of BFX 1001 – BFX 1008……………..………………....…..161 Table 5.2 Summary of the IC50 of BFX 1001 – BFX 1004……………………….....…162 Table 5.3 The IC50 of BFX 1012, BFX 1025 and BFX 1028………………...….…..…173 ix ABBREVIATIONS 1 or 2-LTR circles circular DNA with either 1 or 2- long terminal repeats 5′-LTR 5′- long terminal repeat 3′-OH 3′- Hydroxyl 5′-PO4 5′- Phosphate A Alanine Abacavir