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.

The structure of Zika virus NS5 reveals a conserved domain conformation

“…A previous study, through fragment-based crystallography method, identified a pocket near to the active site of the DENV3 RdRp domain, termed ‘N pocket', which binds to a small-molecule that inhibits DENV3 NS5-mediated RNA initiation and elongation (Fig. 4a)25. Detailed analysis of this inhibitor-binding site revealed that the critical residues for the inhibitor binding are also conserved in ZIKV NS5, arranged in a similar structural environment (Fig.…”

Structure and function of the Zika virus full-length NS5 protein

“…These results suggest that inhibitors of viral MT activity and/or RNA synthesis can be developed to inhibit ZIKV replication. In fact, nucleoside analogues that can inhibit the polymerases of Dengue virus, West Nile virus and yellow fever virus have recently been shown to affect RNA synthesis by ZIKV NS5 (refs 5, 21, 22, 23, 24). Sofosbuvir, which is highly effective in treating hepatitis C, also has modest inhibitory activity for the replication of the ZIKV25.…”

“…Nonstructural protein 5 (NS5) is essential for the replication of the flaviviral RNA genome456. The N-terminal portion of NS5 contains a methyltransferase (MT), followed by a short linker that connects to the RNA-dependent RNA polymerase (RdRp).…”

Crystal structure of Zika virus NS5 RNA-dependent RNA polymerase

“…The ZIKV thumb, palm and fingers domains are also well superimposed on the equivalent domains of DENV2 and DENV3 (r.m.s.d. of 1.17 Å and 1.19 Å, respectively)612. Nevertheless, a close inspection at the priming loop revealed structural elements that differ from DENV homologues and might impact on inhibitor discovery.…”

“…Although nucleoside polymerase inhibitors (NPIs) have achieved clinical success in the case of Hepatitis C virus infections (for example, sofosbovir), they depend on activation by host kinases and are potentially subjected to toxicity problems5. Therefore, non-NPIs have been actively sought as inhibitors of flaviviral NS5 RdRp, particularly targeting the so-called priming loop, that regulates RNA-template binding and polymerization6. Recently, several groups reported the discovery of novel RdRp inhibitors with pan-serotype activity against dengue viruses (DENV).…”