# NS5-targeting nucleoside analogs inhibit dengue virus and other flaviviruses

## TL;DR

Scientists found two nucleoside analogs that effectively inhibit dengue and related viruses by targeting their RNA polymerase.

## Contribution

Identification of two broad-spectrum nucleoside analogs with potent antiviral activity against all four dengue serotypes and other flaviviruses.

## Key findings

- UPGNUC255 and UPGNUC558 reduced viral load by over 10-fold across DENV serotypes and related flaviviruses.
- Both compounds target the NS5 RNA-dependent RNA polymerase domain of flaviviruses.
- Resistance mutations S604T and R355Q were identified for UPGNUC558 and UPGNUC255, respectively.

## Abstract

Dengue virus (DENV) is a mosquito-transmitted flavivirus that circulates globally as four distinct serotypes and poses a substantial threat to public health. There are an estimated ~96 million symptomatic infections yearly, including severe cases of dengue fever, underscoring the urgency of identifying effective therapeutics targeting all four serotypes. Nucleoside analogs, which mimic endogenous nucleosides to inhibit viral RNA replication, offer a promising strategy for broad-spectrum antiviral development. Here, we conducted a high-throughput screen of 1,101 nucleoside analogs against DENV serotype 2 (DENV2) in a panel of human cell models, including human epithelial cells, hepatocytes, and fibroblasts. Candidates that were active against DENV2 were screened against all four serotypes. Since flaviviruses including West Nile virus and Zika virus are also important human pathogens, we screened these compounds for activity and identified compounds that were broadly active in these cellular and viral models. We further evaluated antivirals in primary human keratinocytes and fibroblasts, which are early targets of mosquito-transmitted DENV infection. From this screen, we identified 23 nucleoside analogs with broad antiviral activity against DENV and focused on two purine analogs UPGNUC255 and UPGNUC558, that demonstrated potent pan-flaviviral activity achieving >10-fold viral load reduction across all four DENV serotypes and other flaviviruses across cell models. Mechanistic studies revealed that both compounds target the viral RNA-dependent RNA polymerase (RdRp) domain of NS5. Resistance to UPGNUC558 was associated with a conserved S604T substitution, conferring cross-resistance to other 2′C-substituted nucleoside analogs. Resistance to UPGNUC255 was linked to a previously unknown R355Q mutation, located near the catalytic GDD motif of RdRp. These findings highlight UPGNUC255 and UPGNUC558 as promising leads for the development of broad-spectrum antiviral agents against flaviviruses.

Dengue virus (DENV) infects millions of people each year, yet no specific antiviral treatment is available. Nucleoside analogs are a broad class of potentially broad-spectrum antivirals that mimic natural RNA building blocks. Therefore, we screened over a thousand nucleoside analogs, to identify inhibitors that are effective across cell types and against all four serotypes of DENV as well as related flaviviruses. We discovered two purine analogs, UPGNUC255 and UPGNUC558, that potently blocked replication of all four DENV serotypes and other flaviviruses including West Nile virus and Zika virus. These compounds target the viral RNA polymerase, a key enzyme required for viral RNA synthesis, as mutations near the catalytic site of this enzyme alter sensitivity. Our findings uncover promising broad-spectrum antiviral candidates and valuable insights into mechanisms of resistance among flaviviruses paving the way for next-generation broad-spectrum antivirals.