[Frontiers in Bioscience 13, 3857-3868, May 1, 2008] 3857 4-Thiazolidinones: a novel class of hepatitis C virus NS5B polymerase inhibitors Neerja Kaushik-Basu 1 , Alain Bopda-Waffo 1 , Tanaji T. Talele 2 , Amartya Basu 1 , Ye Chen 1 , S. Guniz Kucukguzel 3 1 Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103, 2 Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, St. John's University, Jamaica, NY 11439, 3 Marmara University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Haydarpasa, 34668 Istanbul, Turkey TABLE OF CONTENTS 1. Abstract 2. Introduction 3. Materials and Methods 3.1. Synthesis of inhibitors 3.2. Purification of recombinant replicase proteins 3.3. RNA-dependent RNA polymerase (RdRp) assay 3.4. Cross-linking of NS5B to TP 3.5. Determination of the Ki and mode of inhibition 3.6. Molecular modeling 4. Results 4.1. Expression and purification of recombinant replicase proteins 4.2. Identification of 4-Thiazolidinones as inhibitors of HCV NS5B RdRp activity 4.3. Mechanism of inhibition 4.4. Molecular docking and analysis of the binding mode of compound 6 5. Discussion 6. Acknowledgement 7. References 1. ABSTRACT In a quest to identify novel compounds targeting HCV viral replicase, we evaluated a new series of 4- thiazolidinone derivatives (18 compounds). Our in vitro NS5B RdRp inhibition analysis with a series of 2′,4′- difluoro-4-hydroxybiphenyl-3-carboxylic acid [2-(5-nitro- 2-furyl / substituted phenyl)-4-thiazolidinone-3-yl] amides (1-7) yielded IC 50 values ranging between 45-75 microM. Of these, lead compound 6: 2′,4′-difluoro-4- hydroxybiphenyl-3-carboxylic acid[2-(2-fluorophenyl)-4- thiazolidinone-3-yl]amide exhibited an IC 50 value of 48 microM and inhibited NS5B non-competitively with respect to UTP and exhibited a mixed mode of inhibition with respect to RNA. Molecular docking of thiazolidinone derivatives within the allosteric site of NS5B yielded significant correlation between their calculated binding affinity and IC 50 values. Taken together, these data suggest that the 4-thiazolidinone scaffold may be optimized for generating new analogues with improved anti-NS5B potency. 2. INTRODUCTION Hepatitis C virus (HCV), identified in 1989 as the etiological agent of parenteral non-A non-B hepatitis, often causes the development of malignant chronic disease including liver cirrhosis and hepatocellular carcinoma frequently resulting in death (1-3). With an estimated 3% of the global population infected with HCV, including 4.1 million in the United States alone, and no protective vaccine available at present, this disease has emerged as a serious global health problem (4,5). Although significant advances have been made in the development of treatments for chronic hepatitis C, their efficacy is not universal and only 50% success has been reported in achieving a sustained viral response for the current combination therapy with new pegylated (PEG) forms of interferon plus ribavirin (6-9). Moreover, this therapy has considerable liabilities including significant adverse side effects and high cost, thus highlighting the need to develop improved therapeutic options to combat HCV infections (10).