Syntheses and applications of small molecule inhibitors of miRNAs miR-21 and miR-122 by Méryl Thomas Bachelor of sciences, ESCPE Lyon, 2009 Master degree, ESCPE Lyon, 2012 Submitted to the Graduate Faculty of the Dietrich School of Arts and Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2015 UNIVERSITY OF PITTSBURGH Dietrich School of Arts and Sciences This thesis was presented by Méryl Thomas It was defended on July 24th, 2015 and approved by Dr. Andrew W. Duncan, Assistant Professor, Department of Pathology Dr. Seth Horne, Associate Professor, Department of Chemistry Dr. Kabirul Islam, Assistant Professor, Department of Chemistry Committee Chair: Dr. Alexander Deiters, Professor, Department of Chemistry ii Copyright © by Méryl Thomas 2015 iii Syntheses and applications of small molecule inhibitors of miRNA miR-21 and miR-122 Méryl Thomas, PhD University of Pittsburgh, 2015 MicroRNAs (miRNAs) are regulatory RNA molecules of 22 nucleotides that (in part) control up to 60% of all genes in humans. They act by binding to the 3' untranslated regions of target messenger RNAs, leading either to translational repression or mRNA degradation. In addition to being involved in the regulation of several fundamental cellular processes, the misregulation of miRNAs has been linked to a wide range of diseases including cancer. Particularly, miR-21 is significantly upregulated in nearly all types of human cancers, and its overexpression is often associated with poor prognosis. The downregulation of miR-122 is found in more than 70% of hepatocellular carcinoma cases and miR-122 is a required factor for the replication of the HCV virus. The modulation of miRNA function is commonly achieved using oligonucleotide agents. However, compared to oligonucleotides, small molecules have several advantages, such as fast activity, systemic delivery, and excellent cell permeability. Taking advantage of luciferase-based reporters, two separate high-throughput screens of >300,000 compounds each, were conducted to discover new small molecule inhibitors of miR-21 or miR-122. Several hit compounds were re- synthesized, their ability to inhibit miR-21 was validated, and the most promising compounds were investigated by SAR studies, which revealed two additional, structurally diverse classes of miR-21 inhibitors. Similarly, extensive SAR studies of previously discovered miR-122 inhibitors were performed in order to better understand the molecular requirements for the miR-122 inhibitory activity. The hit compounds identified in the HTS were analyzed through secondary assays that led to the identification of two new promising miR-122 inhibitors. Furthermore, the iv knowledge gained during the SAR studies was further used to synthesize several small molecule miR-21/miR-122 inhibitors as probes to explore their mechanisms of action. MicroRNAs represent promising, novel drug targets, and small molecule miRNA inhibitors provide tools to study the molecular mechanisms of miRNA biogenesis and have the potential to be new therapeutic agents for the treatment of cancers and viral infections. v TABLE OF CONTENTS PREFACE ............................................................................................................................... XXII 1.0 INTRODUCTION TO MICRORNAS ....................................................................... 1 1.1 MIRNA BIOGENESIS AND FUNCTION ........................................................ 2 1.2 MIRNAS AS POTENTIAL DRUG TARGETS................................................ 5 1.3 ENDOGENOUS REGULATION OF MIRNAS ............................................... 8 1.4 MODULATING MIRNA FUNCTION WITH OLIGONUCLEOTIDES.... 13 1.4.1 Clinical Development of miRNA Therapeutics ........................................... 20 1.5 MODULATING MIRNA FUNCTION WITH SMALL MOLECULES ..... 22 1.5.1 Small Molecule General Modulators of siRNA and miRNA Pathways ... 23 1.5.2 Regulation of miRNA Function with Approved Drugs .............................. 28 1.5.3 Specific Small Molecule Modifiers of miRNA Function ............................ 31 2.0 SYNTHESIS OF SMALL MOLECULE INHIBITORS OF MIR-21 ................... 36 2.1 INTRODUCTION TO MIR-21 ........................................................................ 36 2.2 PREVIOUS WORK: DEVELOPMENT OF A REPORTER ASSAY FOR MIR-21 FUNCTION AND ITS APPLICATION TO THE DISCOVERY OF THE FIRST SMALL MOLECULE INHIBITORS OF MIR-21 ............................................ 38 2.2.1 Structure Activity Relationship Studies of the Previously Discovered miR- 21 Inhibitor 24 ............................................................................................................ 40 vi 2.3 DISCOVERY OF SMALL MOLECULE MIR-21 INHIBITORS THROUGH HIGH-THROUGHPUT SCREENING ...................................................... 43 2.3.1 Validation of Potential Ether-amide miR-21 Inhibitors ............................ 45 2.3.2 Validation of Potential N-Acylhydrazone miR-21 Inhibitors .................... 49 2.3.3 Validation of Potential Ether-amide miR-21 Inhibitors ............................ 55 2.3.4 Validation of Potential Aryl-amide miR-21 Inhibitors (53 & 54) ............. 65 2.3.5 Validation of the Potential Aryl-amide miR-21 Inhibitor 55 .................... 67 2.4 INVESTIGATION INTO THE MODE OF ACTION OF MIRNA INHIBITOR 51 ................................................................................................................... 72 2.4.1 Recovery of Inhibition by Transfection of Precursor miR-21 ................... 72 2.4.2 Target Identification Experiments for the Oxadiazole Inhibitor 51 ......... 74 2.5 THERAPEUTIC EVALUATION OF THE SMALL MOLECULE MIR-21 INHIBITORS IN LIVER CANCER CELLS................................................................... 84 2.6 SUMMARY AND OUTLOOK ......................................................................... 87 2.7 EXPERIMENTALS .......................................................................................... 89 3.0 DEVELOPMENT OF SMALL MOLECULE INHIBITORS OF MIR-122 ...... 136 3.1 INTRODUCTION TO MIRNA-122 .............................................................. 136 3.1.1 Implication of miR-122 in HCC ................................................................. 139 3.1.2 Role of miR-122 in HCV ............................................................................. 141 3.2 PREVIOUS WORK......................................................................................... 146 3.3 SYNTHESIS OF SMALL MOLECULE MIR-122 INHIBITORS ............. 153 3.3.1 SAR Studies of Inhibitor 30 through Modifications of the trans- decahydroquinoline .................................................................................................. 153 vii 3.3.2 SAR Studies of Inhibitor 30 through Modifications of the Sulfonamide Linkage ...................................................................................................................... 160 3.3.3 SAR Studies of Inhibitor 30 through Modifications of the Tetrahydroquinoline ................................................................................................ 161 3.4 HIGH-THROUGHPUT SCREENING FOR SMALL MOLECULE MIR- 122 INHIBITORS ............................................................................................................. 168 3.4.1 Investigation of Hit Compounds Identified in the Primary Screen ........ 171 3.4.2 Preliminary SAR studies of the Hit Compounds 278-279 Identified in the HTS ................................................................................................................ 176 3.4.3 Validation of the Hit Compound 278 Identified in the HTS ................... 180 3.4.4 Validation of the Hit Compound 279 Identified in the HTS ................... 183 3.4.5 Re-examination of the 34 Hit Compounds Identified in the HTS ........... 188 3.5 SUMMARY AND OUTLOOK ....................................................................... 193 3.6 EXPERIMENTALS ........................................................................................ 196 4.0 MODE OF ACTION STUDIES OF THE MIR-122 INHIBITORS .................... 237 4.1 PREVIOUS WORK: RECOVERY OF INHIBITION BY TRANSFECTION OF PRECURSOR PRE-MIR-122 ................................................................................... 237 4.2 INVESTIGATION INTO THE PROTEIN TARGETED BY THE SMALL MOLECULE MIR-122 INHIBITOR ............................................................................. 240 4.2.1 Synthesis of Small Molecule Probes ........................................................... 240 4.2.2 Target Identification Experiments with the Inhibitor 30 ........................ 249 4.3 INVESTIGATION INTO MIR-122 EPIGENETIC REGULATION ........ 271 viii 4.4 ANALYSIS OF THE SMALL MOLECULE INTERACTION WITH THE MIR- 122 PROMOTER ............................................................................................................... 274 4.4.1 Development of a Reporter Construct to Study miR-122 Promoter Activity ...................................................................................................................... 274 4.4.2 Analysis of Potential Interaction between the Inhibitor 30 and known miR-122 Transcription Factors ........................................................................... 277 4.5 SUMMARY AND OUTLOOK ............................................................................ 282 4.6 EXPERIMENTALS .......................................................................................