A Novel Compound, ML336, Inhibits VEEV Replication by Interfering with Viral RNA Synthesis

Abstract

AbstractVenezuelan equine encephalitis virus (VEEV) is an alphavirus that is endemic to Central and South America. VEEV is known to cause periodic outbreaks of encephalitis in both humans and equids. There are currently no treatments or preventatives for VEEV disease. Our group has previously reported on the development of a novel VEEV inhibitor, ML336, which showed a potent antiviral effect in cell culture models. However, the mechanism of action had yet to be elucidated. Based on the discovery of mutations conferring resistance within nonstructural proteins, we hypothesized that ML336 inhibits viral RNA synthesis. We found that ML336 was able to inhibit VEEV RNA synthesis with an IC50value of 1.1 nM in a metabolic labelling assay. ML336 marginally affected cellular transcription at levels 20,000-fold above the IC50, and did not show any cytotoxicity up to 50 µM. Using a combination of fluorography, strand-specific qRT-PCR, and a metabolic labelling assay, we found that ML336 inhibits the synthesis of all forms of VEEV RNA. Structural analogues of ML336 showed a correlation between their RNA synthesis inhibitory activity and their antiviral activity in cells, leading us to propose that the primary mechanism of action of this class of compounds is viral RNA synthesis inhibition. The activities of ML336 were highly specific to VEEV, without measurable activity against Chikungunya virus. ML336 was efficacious even in a cell-free viral RNA synthesis assay, suggesting a direct interaction with viral proteins.ImportanceVenezuelan equine encephalitis virus (VEEV) is a pathogenic alphavirus that circulates in the Americas which can cause a lethal encephalitis in humans and equids. There are currently no licensed treatments or vaccines for VEEV. Due to the high potential for aerosol infection and severe outcomes, it is classified as an NIAID Category B agent. To address the unmet need for VEEV antivirals, we continue to advance a novel amidine compound, ML336, through medicinal chemistry and mechanism of action (MOA) studies. Here, we present the molecular MOA by which ML336 inhibits VEEV replication using cellular and biochemical approaches. Our data suggest that ML336 is a direct-acting antiviral that inhibits viral RNA synthesis by interfering with the viral replicase complex. Our studies provide new insights into approaches for the development of novel RNA virus replication inhibitors and the molecular mechanism of alphavirus RNA synthesis.