| Autor: |
Lim SP; Novartis Institute for Tropical Diseases, Singapore., Noble CG; Novartis Institute for Tropical Diseases, Singapore., Seh CC; Novartis Institute for Tropical Diseases, Singapore., Soh TS; Novartis Institute for Tropical Diseases, Singapore.; School of Biological Sciences, Nanyang Technological University, Singapore., El Sahili A; School of Biological Sciences, Nanyang Technological University, Singapore., Chan GK; School of Biological Sciences, Nanyang Technological University, Singapore., Lescar J; 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., Arora R; Novartis Institute for Biomedical Research, Cambridge, Massachusetts, United States of America., Benson T; Novartis Institute for Biomedical Research, Cambridge, Massachusetts, United States of America., Nilar S; Novartis Institute for Tropical Diseases, Singapore., Manjunatha U; Novartis Institute for Tropical Diseases, Singapore., Wan KF; Novartis Institute for Tropical Diseases, Singapore., Dong H; Novartis Institute for Tropical Diseases, Singapore., Xie X; Novartis Institute for Tropical Diseases, Singapore., Shi PY; Novartis Institute for Tropical Diseases, Singapore., Yokokawa F; Novartis Institute for Tropical Diseases, Singapore. |
| Abstrakt: |
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. |
