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bioRxiv · 10.1101/2022.12.20.521302

Inhibition of the DENV2 and ZIKV RNA polymerases by Galidesivir triphosphate measured using a continuous fluorescence assay

Deshpande, S.·Huo, W.·Shrestha, R.·Sparrow, K.·Evans, G. B.·Harris, L. D.·Kingston, R. L.·Bulloch, E. M. M.
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Abstract

Millions of people are infected by the Dengue and Zika viruses each year, which can result in serious illness, permanent disability or death. There are currently no FDA-approved antivirals for treating infection by these viruses. Galidesivir is an adenosine nucleoside analog which can attenuate flavivirus replication in cell-based and animal models of infection. Galidesivir is converted to the triphosphorylated form by host kinases, and subsequently incorporated into viral RNA by viral RNA-dependent RNA polymerases, leading to the termination of RNA synthesis via an unknown mechanism. Here we report the direct in vitro testing of the effects of Galidesivir triphosphate on RNA synthesis by the polymerases of Dengue-2 and Zika virus. Galidesivir triphosphate was chemically synthesized and inhibition of RNA synthesis followed using a continuous fluorescence-based assay. Galidesivir triphosphate was equipotent against the polymerase activity of Dengue-2 and Zika, with IC50 values of 42 {+/-} 12 M and 47 {+/-} 5 M, respectively. This modest potency in vitro is consistent with results previously obtained in cell-based antiviral assays and suggests that the binding affinity for Galidesivir triphosphate is similar to the natural ATP substrate that it closely mimics. The inhibition assay we have developed will allow the rapid screening of Galidesivir and related compounds against other flavivirus polymerases, and the availability of Galidesivir triphosphate will allow detailed analysis of its mechanism of action. HighlightsO_LIGalidesivir triphosphate was chemically synthesized. C_LIO_LIA continuous assay detecting double-stranded RNA formation was optimized for polymerase inhibition studies. C_LIO_LIGalidesivir triphosphate has moderate potency against DENV2 and ZIKA polymerase activity. C_LIO_LIThe availability of Galidesivir triphosphate will facilitate study of its mechanism of action. C_LI

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Deshpande, S., Huo, W., Shrestha, R., Sparrow, K., Evans, G. B., Harris, L. D., Kingston, R. L., Bulloch, E. M. M.. 2022-12-22. Inhibition of the DENV2 and ZIKV RNA polymerases by Galidesivir triphosphate measured using a continuous fluorescence assay. https://doi.org/10.1101/2022.12.20.521302

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