The NS2B-NS3 protease (NS2B-NS3pro) is regarded as an interesting molecular target for drug design, discovery, and development because of its essential role in the Zika virus (ZIKV) cycle. Although no NS2B-NS3pro inhibitors have reached clinical trials, the employment of drug-like scaffolds can facilitate the screening process for new compounds. In this study, we performed a combination of ligand-based and structure-based in silico methods targeting two known non-peptide small-molecule scaffolds with micromolar inhibitory activity against ZIKV NS2B-NS3pro by a virtual screening (VS) of promising compounds. Based on these two scaffolds, we selected 13 compounds from an initial library of 509 compounds from ZINC15’s similarity search. These compounds exhibited structural modifications that are distinct from previously known compounds yet keep pertinent features for binding. Despite promising outcomes from molecular docking and initial enzymatic assays against NS2B-NS3pro, confirmatory assays with a counter-screening enzyme revealed an artifactual inhibition of the assessed compounds. However, we report two compounds, 9 and 11, that exhibited antiviral properties at a concentration of 50 μM in cellular-based assays. Overall, this study provides valuable insights into the ongoing research on anti-ZIKV compounds to facilitate and improve the development of new inhibitors.

Background/Objectives: Zika fever is a disease caused by the Zika virus (ZIKV). Symptomatic cases may be associated with neurological disorders in adults, as well as congenital Zika syndrome and other birth defects during pregnancy. In 2016, Zika fever was considered a public health problem by the World Health Organization (WHO), highlighting the need to develop new therapies against the disease. Currently, there is no antiviral or vaccine available to treat or prevent severe cases. Due to the lack of available therapeutics and few promising hit molecules, we computationally screened the well-described ZIKV protease (NS3^pro) as a drug target to revisit the small-molecule database Brazilian Compound Library (BraCoLi) and select potential inhibitors. Methods: We employed a consensus docking screening of a library of 1176 compounds using GOLD and DockThor. We selected 28 hits based on predicted binding affinity, and only the remnants of three compounds were available in the library at the time of this study for experimental validation. The hits were evaluated for their cytotoxic (CC₅₀) and effective concentrations (EC₅₀) for their potential antiviral activity in Vero cells. Results: The three hit compounds presented modest CC₅₀ values of 89.15 ± 3.72, >100, and 29.67 ± 1.01 μM, with the latter, a carbohydrate derivative, having an EC₅₀ value of >12.5 μM (~40% inhibition) against ZIKV PE243. Additionally, the essentially non-toxic compound, an arylfuran derivative, also inhibited the ZIKV NS3^pro with an IC₅₀ value of 17 μM but presented evidence of acting through a promiscuous mechanism for enzyme inhibition. Conclusion: This study highlights the relevance of revisiting existing small-molecule assets to identify novel therapeutic starting points against ZIKV, aiming for potential lead candidates in the future.