title: Identifying initiation and elongation inhibitors of dengue virus RNA polymerase in a high-throughput lead-finding campaign.
abstract: Dengue virus (DENV) is the most significant mosquito-borne viral pathogen in the world and is the cause of dengue fever. The DENV RNA-dependent RNA polymerase (RdRp) is conserved among the four viral serotypes and is an attractive target for antiviral drug development. During initiation of viral RNA synthesis, the polymerase switches from a "closed" to "open" conformation to accommodate the viral RNA template. Inhibitors that lock the "closed" or block the "open" conformation would prevent viral RNA synthesis. Herein, we describe a screening campaign that employed two biochemical assays to identify inhibitors of RdRp initiation and elongation. Using a DENV subgenomic RNA template that promotes RdRp de novo initiation, the first assay measures cytosine nucleotide analogue (Atto-CTP) incorporation. Liberated Atto fluorophore allows for quantification of RdRp activity via fluorescence. The second assay uses the same RNA template but is label free and directly detects RdRp-mediated liberation of pyrophosphates of native ribonucleotides via liquid chromatography-mass spectrometry. The ability of inhibitors to bind and stabilize a "closed" conformation of the DENV RdRp was further assessed in a differential scanning fluorimetry assay. Last, active compounds were evaluated in a renilla luciferase-based DENV replicon cell-based assay to monitor cellular efficacy. All assays described herein are medium to high throughput, are robust and reproducible, and allow identification of inhibitors of the open and closed forms of DENV RNA polymerase.
authors.affiliation: Center for Proteomic Chemistry, Novartis Institutes for BioMedical Research, Inc., Cambridge, MA, USA thomasm.smith@novartis.com.
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Smith
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore.
authors.forename: Siew Pheng
authors.initials: SP
authors.lastname: Lim
authors.affiliation: Center for Proteomic Chemistry, Novartis Institutes for BioMedical Research, Inc., Cambridge, MA, USA.
authors.forename: Kimberley
authors.initials: K
authors.lastname: Yue
authors.affiliation: Center for Proteomic Chemistry, Novartis Institutes for BioMedical Research, Inc., Cambridge, MA, USA.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Busby
authors.affiliation: Center for Proteomic Chemistry, Novartis Institutes for BioMedical Research, Inc., Cambridge, MA, USA.
authors.forename: Rishi
authors.initials: R
authors.lastname: Arora
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore.
authors.forename: Cheah Chen
authors.initials: CC
authors.lastname: Seh
authors.affiliation: Center for Proteomic Chemistry, Novartis Institutes for BioMedical Research, Inc., Cambridge, MA, USA.
authors.forename: S Kirk
authors.initials: SK
authors.lastname: Wright
authors.affiliation: Center for Proteomic Chemistry, Novartis Institutes for BioMedical Research, Inc., Cambridge, MA, USA.
authors.forename: Razvan
authors.initials: R
authors.lastname: Nutiu
authors.affiliation: Maveta Co., Ltd., Bangkok, Thailand.
authors.forename: Pornwaratt
authors.initials: P
authors.lastname: Niyomrattanakit
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore.
authors.forename: Kah Fei
authors.initials: KF
authors.lastname: Wan
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore.
authors.forename: David
authors.initials: D
authors.lastname: Beer
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore.
authors.forename: Pei-Yong
authors.initials: PY
authors.lastname: Shi
authors.affiliation: Center for Proteomic Chemistry, Novartis Institutes for BioMedical Research, Inc., Cambridge, MA, USA.
authors.forename: Timothy E
authors.initials: TE
authors.lastname: Benson
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D002853:Chromatography, Liquid; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D003716:Dengue Virus / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D055808:Drug Discovery / Q000379:methods / Q000592:standards; D004353:Drug Evaluation, Preclinical / Q000379:methods* / Q000592:standards; D057166:High-Throughput Screening Assays / Q000379:methods* / Q000592:standards; D006801:Humans; D020128:Inhibitory Concentration 50; D013058:Mass Spectrometry; D008826:Microbial Sensitivity Tests / Q000379:methods* / Q000592:standards; D015203:Reproducibility of Results; D054852:Small Molecule Libraries
chemical_list: D000998:Antiviral Agents; D054852:Small Molecule Libraries; D012321:DNA-Directed RNA Polymerases
keywords: Flaviviridae; LCMS; RNA-dependent RNA polymerase; RdRp; assay; dengue virus; inhibitor