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7156208 
Journal Article 
Discovery of a small molecule inhibitor targeting dengue virus NS5 RNA-dependent RNA polymerase 
Shimizu, H; Saito, A; Mikuni, J; Nakayama, EmiE; Koyama, H; Honma, T; Shirouzu, M; Sekine, SI; Shioda, T; , 
2019 
1 
PLoS Neglected Tropical Diseases
ISSN: 1935-2727
EISSN: 1935-2735 
PUBLIC LIBRARY SCIENCE 
SAN FRANCISCO 
31738758 
WOS:000503278100021 
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 mu M 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.Author summary 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.