Potent Allosteric Dengue Virus NS5 Polymerase Inhibitors: Mechanism of Action and Resistance Profiling

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.
Author Summary Dengue virus (DENV) is the world’s most prevalent mosquito-borne viral disease and nearly 40% of the world’s population is at risk of infection. Currently, no specific drugs are available to treat dengue or other flaviviral diseases. DENV NS5 is a large protein of 900 amino acids composed of two domains with key enzymatic activities for viral RNA replication in the host cell and constitutes a prime target for the design of anti-viral inhibitors. We performed a fragment-based screening by X-ray crystallography targeting the DENV NS5 polymerase and identified an allosteric binding pocket at the base of the thumb subdomain close to the enzyme active site. Potent inhibitors active in both DENV polymerase biochemical and cell-based assays were developed through structure-guided design. Resistant virus replicons grown in the presence of the inhibitor, harbored amino acid changes that mapped to the compound binding site. The proposed mode of action for this class of inhibitors is by impeding RdRp protein conformational changes during the transition from initiation to elongation phase of enzyme activity.