# Development of Novel Proline- and Pipecolic Acid-Based Allosteric Inhibitors of Dengue and Zika Virus NS2B/NS3 Protease

## TL;DR

This study develops new small molecule inhibitors targeting the NS2B/NS3 protease of dengue and Zika viruses, showing promising antiviral activity and selectivity.

## Contribution

The paper introduces novel proline- and pipecolic acid-based allosteric inhibitors with improved cellular efficacy and selectivity for flaviviral proteases.

## Key findings

- S-proline derivatives with trifluoromethyl groups showed high potency against dengue virus NS2B/NS3 protease.
- R-configured pipecolic acid derivatives demonstrated strong cellular efficacy in a dengue virus reporter gene assay.
- All compounds were non-toxic and selective for the viral protease over other serine proteases.

## Abstract

Background: In this study, we report a novel series of proline- and pipecolic acid-based small molecules designed as allosteric inhibitors of the NS2B/NS3 serine proteases from dengue and Zika viruses, key targets in antiviral drug discovery. Results: Enzymatic studies revealed that S-proline derivatives bearing electron-withdrawing substituents on the aromatic ring, particularly that with a trifluoromethyl group in meta position (i.e., compound 3, IC50 = 5.0 µM), were the most potent against DENV NS2B/NS3, while nitro-substituted inhibitors were mostly effective only against the ZIKV protease. R-configured pipecolic acid-based derivatives were the only ones active against DENV NS2B/NS3, even if the mid-micromolar range; however, they demonstrated improved cellular efficacy since inhibitors 24 and 27 exhibiting strong activity in a DENV2 protease reporter gene assay (EC50 = 5.2 and 5.1 µM, respectively). All compounds showed no cytotoxicity (CC50 > 100 µM) and were selective for the viral protease over off-target serine proteases. Structure-based approaches were exploited to map the druggable allosteric site close to Asn152. Conclusions: Our findings led us to identify proline and pipecolic acid-based inhibitors as promising leads for the development of selective flaviviral NS2B/NS3 allosteric inhibitors.