Discovery of hepatitis C virus NS3 helicase inhibitors by a multiplexed, high-throughput helicase activity assay based on graphene oxide.

Worldwide, over 170 million people have hepatitis C virus (HCV) infections. Chronic infection with HCV leads to liver diseases such as cirrhosis and hepatocarcinoma and is the major reason of liver transplantation. Currently, the standard treatment for hepatitis C relies on a combination of interferon-a and ribavirin (an immune booster and a general inhibitor of virus replication, respectively) which is associated with serious side effects including hemolytic anaemia, depression, fatigue, flu-like symptoms, and birth defects. The standard of care is frequently ineffective in clearing HCV infections and the virus often survives and thrives even under the treatment. To develop direct-acting antiviral agents for hepatitis C treatment, studies have been focused on the discovery of inhibitors of viral enzymes, specifically nonstructural protein 3 (NS3) serine protease and NS5B RNA-dependent RNA polymerase (RdRp). Last year, new drugs for treating hepatitis C, Telaprevir (Vertex) and Boceprevir (Merck) , which are NS3 serine protease inhibitors, were approved by the U.S. Food and Drug Administration (FDA), and over 30 drug candidates targeting the same protease or NS5B RdRp are currently in clinical trials. Although phase III clinical trials of Telaprevir and Boceprevir showed notable increases in the cure rate, nearly every patient still suffered from at least one side effect of the new therapy. In addition, HCV has strong drug resistance due to its high mutability. Thus, further therapeutic options are urgently needed to treat HCV infections more effectively. The C-terminal two thirds of HCV NS3 forms a helicase, which has the ability to unwind double-stranded DNA (dsDNA) into single-stranded DNA (ssDNA) and is fueled by nucleoside triphosphates (NTPs) hydrolyzed by its NTPase domain. NS3 helicase is one of the essential enzymes of HCV along with NS3 serine protease and NS5B RdRp for processing HCV proteins and replication of HCV. Thus, the inhibition of helicase activities is an important strategy for treating HCV infections. However, discovery of helicase inhibitors has been much slower compared to that of other HCV drug targets. To date, only a few classes of NS3 helicase inhibitors have been reported, partly because high-throughput screens have yielded only a few successful hits. For example, the compounds discovered in the NIH screen based on the molecular-beacon-based helicase assay (MBHA) showed poor activity in cells and turned out to interfere with the assay, even though another MBHA screening identified compounds that inhibited RNA replication in cells. Therefore, there is an urgent need for new assays to measure helicase activity that are suitable for high-throughput screens. Herein, we developed a multiplexed helicase assay based on graphene oxide (GO) for high-throughput screening of inhibitors of HCV NS3 helicase and severe acute respiratory syndrome coronavirus (SARS CoV) helicase. Previously, we reported a GO-based helicase assay (GOHA), which relied on the preferential binding of ssDNA over dsDNA to the GO surface and subsequent quenching of the fluorescently labeled ssDNA by energy transfer from the dyes to the GO, and we validated the assay using SARS CoV helicase. Herein, we show that the GOHA can be used for measuring the activities of HCVNS3 helicase and SARS CoV helicase in a single mixed solution using two distinct DNA substrates tethered to different fluorophores, and furthermore, for multiplexed high-throughput screening to discover highly selective small-molecule inhibitors of these helicases (Figure 1). One round of screening the chemical library using the multiplexed GOHA (mGOHA) revealed three classes of inhibitors—specific inhibitors of HCVNS3 helicase, specific inhibitors of SARS CoV helicase, and general inhibitors of both helicases. To date, GO has been used to develop various biosensors and enzyme assays, but concerns about the heterogeneity of the chemical structure and the physical dimensions of GO and nonspecific binding of biomolecules to GO hamper the application of GO-based [*] S.-R. Ryoo, Y.-K. Kim, Prof. Dr. D.-H. Min Department of Chemistry, Seoul National University Seoul, 151-747 (Korea) E-mail: [email protected]