Dataset Information

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 ?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.

SUBMITTER: Shimizu H

PROVIDER: S-EPMC6886872 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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

Shimizu Hideaki H Saito Akatsuki A Mikuni Junko J Nakayama Emi E EE Koyama Hiroo H Honma Teruki T Shirouzu Mikako M Sekine Shun-Ichi SI Shioda Tatsuo T

PLoS neglected tropical diseases 20191118 11

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-st ...[more]

PMID: 31738758

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Project description:Background:Dengue fever has rapidly manifested into a serious global health concern. The emergence of various viral serotypes has prompted the urgent need for innovative drug design techniques. Of the viral non-structural enzymes, the NS5 RNA-dependent RNA polymerase has been established as a promising target due to its lack of an enzymatic counterpart in mammalian cells and its conserved structure amongst all serotypes. The onus is now on scientists to probe further into understanding this enzyme and its mechanism of action. The field of bioinformatics has evolved greatly over recent decades, with updated drug design tools now being publically available. Methods:In this study, bioinformatics tools were used to provide a comprehensive sequence and structural analysis of the two most prominent serotypes of Dengue RNA-dependent RNA polymerase. A list of popular flavivirus inhibitors were also chosen to dock to the active site of the enzyme. The best docked compound was then used as a template to generate a pharmacophore model that may assist in the design of target-specific Dengue virus inhibitors. Results:Comparative sequence alignment exhibited similarity between all three domains of serotype 2 and 3.Sequence analysis revealed highly conserved regions at residues Meth530, Thr543 Asp597, Glu616, Arg659 and Pro671. Mapping of the active site demonstrated two highly conserved residues: Ser710 and Arg729. Of the active site interacting residues, Ser796 was common amongst all ten docked compounds, indicating its importance in the drug design process. Of the ten docked flavivirus inhibitors, NITD-203 showed the best binding affinity to the active site. Further pharmacophore modeling of NITD-203 depicted significant pharmacophoric elements that are necessary for stable binding to the active site. Discussion:This study utilized publically available bioinformatics tools to provide a comprehensive framework on Dengue RNA-dependent RNA polymerase. Based on docking studies, a pharmacophore model was also designed to unveil the crucial pharmacophoric elements that are required when constructing an efficacious DENV inhibitor. We believe that this study will be a cornerstone in paving the road toward the design of target-specific inhibitors against DENV RdRp.

Project description:UnlabelledThe nucleolar subcompartment of the nucleus is increasingly recognized as an important target of RNA viruses. Here we document for the first time the ability of dengue virus (DENV) polymerase, nonstructural protein 5 (NS5), to accumulate within the nucleolus of infected cells and to target green fluorescent protein (GFP) to the nucleolus of live transfected cells. Intriguingly, NS5 exchange between the nucleus and nucleolus is dynamically modulated by extracellular pH, responding rapidly and reversibly to pH change, in contrast to GFP alone or other nucleolar and non-nucleolar targeted protein controls. The minimal pH-sensitive nucleolar targeting region (pHNTR), sufficient to target GFP to the nucleolus in a pH-sensitive fashion, was mapped to NS5 residues 1 to 244, with mutation of key hydrophobic residues, Leu-165, Leu-167, and Val-168, abolishing pHNTR function in NS5-transfected cells, and severely attenuating DENV growth in infected cells. This is the first report of a viral protein whose nucleolar targeting ability is rapidly modulated by extracellular stimuli, suggesting that DENV has the ability to detect and respond dynamically to the extracellular environment.ImportanceInfections by dengue virus (DENV) threaten 40% of the world's population yet there is no approved vaccine or antiviral therapeutic to treat infections. Understanding the molecular details that govern effective viral replication is key for the development of novel antiviral strategies. Here, we describe for the first time dynamic trafficking of DENV nonstructural protein 5 (NS5) to the subnuclear compartment, the nucleolus. We demonstrate that NS5's targeting to the nucleolus occurs in response to acidic pH, identify the key amino acid residues within NS5 that are responsible, and demonstrate that their mutation severely impairs production of infectious DENV. Overall, this study identifies a unique subcellular trafficking event and suggests that DENV is able to detect and respond dynamically to environmental changes.

Dataset Information

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

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

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