Identification of a Pyridoxine-Derived Small-Molecule Inhibitor Targeting Dengue Virus RNA-Dependent RNA Polymerase

Abstract

The viral RNA-dependent RNA polymerase (RdRp) activity of the dengue virus (DENV) NS5 protein is an attractive target for drug design. Here, we report the identification of a novel class of inhibitor (i.e., an active-site metal ion chelator) that acts against DENV RdRp activity. DENV RdRp utilizes a two-metal-ion mechanism of catalysis; therefore, we constructed a small library of compounds, through mechanism-based drug design, aimed at chelating divalent metal ions in the catalytic site of DENV RdRp. We now describe a pyridoxine-derived small-molecule inhibitor that targets DENV RdRp and show that 5-benzenesulfonylmethyl-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylic acid hydroxyamide (termed DMB220) inhibited the RdRp activity of DENV serotypes 1 to 4 at low micromolar 50% inhibitory concentrations (IC 50s of 5 to 6.7 M) in an enzymatic assay. The antiviral activity of DMB220 against DENV infection was also verified in a cell-based assay and showed a 50% effective concentration (EC 50 ) of <3 M. Enzyme assays proved that DMB220 was competitive with nucleotide incorporation. DMB220 did not inhibit the enzymatic activity of recombinant HIV-1 reverse transcriptase and showed only weak inhibition of HIV-1 integrase strand transfer activity, indicating high specificity for DENV RdRp. S600T substitution in the DENV RdRp, which was previously shown to confer resistance to nucleoside analogue inhibitors (NI), conferred 3-fold hypersusceptibility to DMB220, and enzymatic analyses showed that this hypersusceptibility may arise from the decreased binding/incorporation efficiency of the natural NTP substrate without significantly impacting inhibitor binding. Thus, metal ion chelation at the active site of DENV RdRp represents a viable anti-DENV strategy, and DMB220 is the first of a new class of DENV inhibitor. D engue virus (DENV) belongs to the family Flaviviridae, a group of enveloped positive-sense single-stranded RNA viruses that includes the genera Hepacivirus (prototype, hepatitis C virus [HCV]), Flavivirus (prototype, yellow fever virus), and Pestivirus (prototype, bovine viral diarrhea virus) (1). Distinct from the hepaciviruses and pestiviruses that are not arthropodborne, the flaviviruses are transmitted by mosquitos and ticks. Dengue, the most prevalent arthropod-borne viral disease of humans, is caused by four serotypes (DENV1 to -4) and has had a major impact on global public health (2-4).Infection with any of the DENV serotypes may result in a wide spectrum of clinical symptoms ranging from a mild flu-like syndrome (known as dengue fever [DF]) to the most severe forms of the disease, which are characterized by coagulopathy, increased vascular fragility, and permeability (dengue hemorrhagic fever [DHF]). The latter may progress to hypovolemic shock (dengue shock syndrome [DSS]) (3, 5). Among the four serotypes, DENV2 is the most prevalent on a global scale, followed by DENV3, DENV1, and DENV4 (6). Dengue is endemic in over 100 tropical and subtropical countries, and the global incidence of dengue has grown ...

Sophoraflavenone G Restricts Dengue and Zika Virus Infection via RNA Polymerase Interference

“…The inhibitory effect of SFG on DENV and ZIKV polymerases was evaluated by measuring the amount of radiolabeled GMP incorporated in newly synthesized RNA (please see the supplementary methods for details regarding protein expression and purification and references [ 15 , 16 ] for polymerase validation). This was accomplished by employing homopolymeric poly(rC) as an RNA template/rG 13 primer (T/P).…”

Dengue Virus and Its Inhibitors: A Brief Review

“…ST-148, reported as a potent DENV capsid inhibitor (EC 50 =0.052 µM), enhanced capsid-protein interaction and caused steric hindrance and/or structural rigidity that inhibited both the entry and assembly or release of infectious virions. 71,72) 5.6. RdRp Inhibitors To inhibit DENV viral RdRp, several approaches were introduced: nucleosides or their analogs that terminate the replication of RNA; non-nucleosides that block allosteric sites and affect the activities of RdRp (e.g., N-sulfonylanthranilic acid derivatives [NITD-1 and NITD-2], sofosbuvir [SOF], and HeE1-2Tyr, 66E2); and metal chelating agents that target essential ions in the catalytic process (DMB220).…”

Flaviviridae virus nonstructural proteins 5 and 5A mediate viral immune evasion and are promising targets in drug development

“…divalent metal ions in the catalytic site of DENV RdRp and inhibits replication of DENV serotypes 1-4 in vitro (Xu et al, 2015) (see Table 3). Notably, a novel drug-target, named the "N pocket", was identified in the DENV RdRp domain through fragment-based screening via X-ray crystallography (Lim et al, 2016;Noble et al, 2016).…”