title: Potent Allosteric Dengue Virus NS5 Polymerase Inhibitors: Mechanism of Action and Resistance Profiling.
abstract: Flaviviruses comprise major emerging pathogens such as dengue virus (DENV) or Zika virus (ZIKV). The flavivirus RNA genome is replicated by the RNA-dependent-RNA polymerase (RdRp) domain of non-structural protein 5 (NS5). This essential enzymatic activity renders the RdRp attractive for antiviral therapy. NS5 synthesizes viral RNA via a "de novo" initiation mechanism. Crystal structures of the flavivirus RdRp revealed a "closed" conformation reminiscent of a pre-initiation state, with a well ordered priming loop that extrudes from the thumb subdomain into the dsRNA exit tunnel, close to the "GDD" active site. To-date, no allosteric pockets have been identified for the RdRp, and compound screening campaigns did not yield suitable drug candidates. Using fragment-based screening via X-ray crystallography, we found a fragment that bound to a pocket of the apo-DENV RdRp close to its active site (termed "N pocket"). Structure-guided improvements yielded DENV pan-serotype inhibitors of the RdRp de novo initiation activity with nano-molar potency that also impeded elongation activity at micro-molar concentrations. Inhibitors exhibited mixed inhibition kinetics with respect to competition with the RNA or GTP substrate. The best compounds have EC50 values of 1-2 μM against all four DENV serotypes in cell culture assays. Genome-sequencing of compound-resistant DENV replicons, identified amino acid changes that mapped to the N pocket. Since inhibitors bind at the thumb/palm interface of the RdRp, this class of compounds is proposed to hinder RdRp conformational changes during its transition from initiation to elongation. This is the first report of a class of pan-serotype and cell-active DENV RdRp inhibitors. Given the evolutionary conservation of residues lining the N pocket, these molecules offer insights to treat other serious conditions caused by flaviviruses.
authors: Novartis Institute for Tropical Diseases, Singapore.
authors: Siew Pheng
authors: SP
authors: Lim
authors: Novartis Institute for Tropical Diseases, Singapore.
authors: Christian Guy
authors: CG
authors: Noble
authors: Novartis Institute for Tropical Diseases, Singapore.
authors: Cheah Chen
authors: CC
authors: Seh
authors: Novartis Institute for Tropical Diseases, Singapore.|School of Biological Sciences, Nanyang Technological University, Singapore.
authors: Tingjin Sherryl
authors: TS
authors: Soh
authors: School of Biological Sciences, Nanyang Technological University, Singapore.
authors: Abbas
authors: A
authors: El Sahili
authors: School of Biological Sciences, Nanyang Technological University, Singapore.
authors: Grace Kar Yarn
authors: GK
authors: Chan
authors: School of Biological Sciences, Nanyang Technological University, Singapore.|UPMC UMRS CR7-CNRS ERL 8255-INSERM U1135 Centre d'Immunologie et des Maladies Infectieuses, Centre Hospitalier Universitaire Pitié-Salpêtrière, Faculté de Médecine Pierre et Marie Curie, Paris, France.
authors: Julien
authors: J
authors: Lescar
authors: Novartis Institute for Biomedical Research, Cambridge, Massachusetts, United States of America.
authors: Rishi
authors: R
authors: Arora
authors: Novartis Institute for Biomedical Research, Cambridge, Massachusetts, United States of America.
authors: Timothy
authors: T
authors: Benson
authors: Novartis Institute for Tropical Diseases, Singapore.
authors: Shahul
authors: S
authors: Nilar
authors: Novartis Institute for Tropical Diseases, Singapore.
authors: Ujjini
authors: U
authors: Manjunatha
authors: Novartis Institute for Tropical Diseases, Singapore.
authors: Kah Fei
authors: KF
authors: Wan
authors: Novartis Institute for Tropical Diseases, Singapore.
authors: Hongping
authors: H
authors: Dong
authors: Novartis Institute for Tropical Diseases, Singapore.
authors: Xuping
authors: X
authors: Xie
authors: Novartis Institute for Tropical Diseases, Singapore.
authors: Pei-Yong
authors: 0000-0001-5553-1616
authors: PY
authors: Shi
authors: Novartis Institute for Tropical Diseases, Singapore.
authors: Fumiaki
authors: F
authors: Yokokawa
mesh_terms: D000072283:A549 Cells; D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology*; D018360:Crystallography, X-Ray; D003715:Dengue; D003716:Dengue Virus / Q000187:drug effects*; D015195:Drug Design; D006801:Humans; D019384:Nucleic Acid Synthesis Inhibitors / Q000737:chemistry / Q000494:pharmacology; D000072417:Protein Domains; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors* / Q000737:chemistry; D020349:Surface Plasmon Resonance; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry
chemical_list: D000998:Antiviral Agents; C582223:NS5 protein, dengue virus; D019384:Nucleic Acid Synthesis Inhibitors; D017361:Viral Nonstructural Proteins; D012324:RNA-Dependent RNA Polymerase