Discovery of a small molecule inhibitor targeting dengue virus NS5 RNA-dependent RNA polymerase

Abstract

Dengue is a mosquito-borne viral infection that has spread globally in recent years. Around half of the world’s population, especially in the tropics and subtropics, is at risk of infection. Every year, 50–100 million clinical cases are reported, and more than 500,000 patients develop the symptoms of severe dengue infection: dengue haemorrhagic fever and dengue shock syndrome, which threaten life in Asia and Latin America. No antiviral drug for dengue is available. The dengue virus (DENV) non-structural protein 5 (NS5), which possesses the RNA-dependent RNA polymerase (RdRp) activity and is responsible for viral replication and transcription, is an attractive target for anti-dengue drug development. In the present study, 16,240 small-molecule compounds in a fragment library were screened for their capabilities to inhibit the DENV type 2 (DENV2) RdRp activities in vitro. Based on in cellulo antiviral and cytotoxity assays, we selected the compound RK-0404678 with the EC₅₀ value of 6.0 μM for DENV2. Crystallographic analyses revealed two unique binding sites for RK-0404678 within the RdRp, which are conserved in flavivirus NS5 proteins. No resistant viruses emerged after nine rounds of serial passage of DENV2 in the presence of RK-0404678, suggesting the high genetic barrier of this compound to the emergence of a resistant virus. Collectively, RK-0404678 and its binding sites provide a new framework for antiviral drug development. Dengue is a mosquito-borne infection caused by dengue viruses (DENV), and is currently a major public health concern worldwide. No antiviral drug for dengue is available. To develop a potent inhibitor of the DENV NS5 RNA-dependent RNA polymerase (RdRp), we performed a high-throughput screening of a fragment library. We identified RK-0404678 as a potent inhibitor of the DENV RdRp. Interestingly, we found that RK-0404678 binds to two distinct sites in the DENV RdRp domains. Site 1 lies in the thumb domain, which is distant from the active site, and Site 2 is located in the active site of the RdRp domain. RK-0404678 binding to Site 2 induces a conformational change around the Tyr607 residue. These are unique features among the known RdRp inhibitors. In addition, our adaptation experiment demonstrated that this compound imposed a high genetic barrier to the emergence of an RK-0404678-resistant virus. These characteristics of RK-0404678 suggest that this inhibitor is a promising lead compound for the development of anti-dengue therapeutics.