Ivermectin is a potent inhibitor of flavivirus replication specifically targeting NS3 helicase activity: new prospects for an old drug

Abstract

Objectives: Infection with yellow fever virus (YFV), the prototypic mosquito-borne flavivirus, causes severe febrile disease with haemorrhage, multi-organ failure and a high mortality. Moreover, in recent years the Flavivirus genus has gained further attention due to re-emergence and increasing incidence of West Nile, dengue and Japanese encephalitis viruses. Potent and safe antivirals are urgently needed.

Methods: Starting from the crystal structure of the NS3 helicase from Kunjin virus (an Australian variant of West Nile virus), we identified a novel, unexploited protein site that might be involved in the helicase catalytic cycle and could thus in principle be targeted for enzyme inhibition. In silico docking of a library of small molecules allowed us to identify a few selected compounds with high predicted affinity for the new site. Their activity against helicases from several flaviviruses was confirmed in in vitro helicase/enzymatic assays. The effect on the in vitro replication of flaviviruses was then evaluated.

Results: Ivermectin, a broadly used anti-helminthic drug, proved to be a highly potent inhibitor of YFV replication (EC₅₀ values in the sub-nanomolar range). Moreover, ivermectin inhibited, although less efficiently, the replication of several other flaviviruses, i.e. dengue fever, Japanese encephalitis and tick-borne encephalitis viruses. Ivermectin exerts its effect at a timepoint that coincides with the onset of intracellular viral RNA synthesis, as expected for a molecule that specifically targets the viral helicase.

Conclusions: The well-tolerated drug ivermectin may hold great potential for treatment of YFV infections. Furthermore, structure-based optimization may result in analogues exerting potent activity against flaviviruses other than YFV.

Figure 1.

In silico docking of flavivirus helicase. (a) Model of the helicase/RNA interaction for WNV helicase domain. The crystal structure of free WNV helicase (PDB 2QEQ) was used to identify a potential ssRNA access site to the enzyme active site by superposition with the DNA-bound bacterial helicase PcrA (PDB 3PJR). The respective region is located between helices α2 in subdomain II (red) and α9 in subdomain III (green) of the WNV helicase, forming an α-helical gate for the entering RNA substrate (orange worm/sticks). This putative helicase ssRNA access site (enclosed in black) was then chosen as the target site for the in silico ligand search. The amino acids selected for mutation (Asp409 and Thr410) are shown in yellow. The close-up box shows a potential docking conformation of ivermectin in the ssRNA access site. The location of the molecule's macro ring is well established, having the two sugar rings of the compound located inside the protein. The region occupied by the inhibitor is conserved in all binding modes of the different docked conformations. (b) Chemical structure of ivermectin (B1a form; Sigma-Aldrich). Figures created using PyMol (http://www.pymol.org). This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Figure 2.

Kinetic studies of the mechanism of helicase inhibition by ivermectin. Double-reciprocal plot of the rate of dsRNA unwinding versus substrate concentration (dsRNA) during the inhibition of (a) YFV helicase, (b) DENV helicase and (c) WNV helicase by an increasing amount of ivermectin (I). HEL, helicase.

Figure 3.

YFV-17D-induced CPE. (a) YFV-17D-induced CPE: concentration-dependent inhibition by ivermectin. At concentrations of >3 nM complete protection against virus-induced CPE formation (at 6 days post-infection) is observed. VC, virus-infected control without drug; CC, cell control (uninfected/untreated). Cells are shown at ×100 magnification. (b) Effect of ivermectin on the viability of uninfected host cells and on virus yield determined by RT-qPCR. Both results are reported as percentages of untreated controls. Arrows indicate the concentrations at which the reduction (‘log reduction’) in viral RNA levels was determined. Mean values of at least three independent experiments ± SD.

Figure 4.

Time-dependent inhibition of YFV replication. (a) Kinetics of intracellular YFV-17D RNA synthesis following infection of Vero cells. Onset of viral RNA synthesis is detected at 14 h post-infection. (b) Effect of time of addition of ivermectin (left-hand panel) or ribavirin (right-hand panel) to YFV-17D-infected Vero cells. Both molecules gradually lost their protective activity when added at a timepoint (or later) that coincided with onset of viral replication in the control cultures.