5jjs | pdb_00005jjs
Dengue 3 NS5 protein with compound 27
Structural highlights
Function[Q6DLV0_9FLAV] Envelope protein E binding to host cell surface receptor is followed by virus internalization through clathrin-mediated endocytosis. Envelope protein E is subsequently involved in membrane fusion between virion and host late endosomes. Synthesized as a homodimer with prM which acts as a chaperone for envelope protein E. After cleavage of prM, envelope protein E dissociate from small envelope protein M and homodimerizes (By similarity).[SAAS:SAAS000336_004_099774] Publication Abstract from PubMedFlaviviruses 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 muM 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. Potent Allosteric Dengue Virus NS5 Polymerase Inhibitors: Mechanism of Action and Resistance Profiling.,Lim SP, Noble CG, Seh CC, Soh TS, El Sahili A, Chan GK, Lescar J, Arora R, Benson T, Nilar S, Manjunatha U, Wan KF, Dong H, Xie X, Shi PY, Yokokawa F PLoS Pathog. 2016 Aug 8;12(8):e1005737. doi: 10.1371/journal.ppat.1005737., eCollection 2016 Aug. PMID:27500641[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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