Discovery of Indazole-Based Dual-Pharmacophore Inhibitors of Dengue and Zika Proteases as Promising Antivirals

Abstract Flavivirus outbreaks caused by dengue virus (DENV) and Zika virus (ZIKV) remain a major global health challenge, with no FDA-approved antivirals available. Herein, we designed, synthesized, and evaluated indazole-based dual pharmacophore motifs targeting NS2B-NS3 serine proteases, essential for viral replication. Enzymatic assays revealed nanomolar to sub-micromolar potency, with strong selectivity over host serine proteases, minimizing off-target risks. Biophysical studies showed ligand-induced thermal stabilization, while kinetic and dialysis analyses confirmed a reversible, competitive, non-covalent inhibition mechanism. In DENV-infected Vero CCL-81 cells, several leads exhibited potent antiviral activity with low cytotoxicity. Notably, compound C8 demonstrated low-nanomolar enzyme inhibition against both viruses, translating to complete viral suppression in cells (EC50 = 0.5 μM) with a good selectivity index, underscoring its therapeutic potential. Molecular docking and molecular dynamics simulations supported these findings, revealing key active-site interactions consistent with potency and specificity. Collectively, these results highlight indazole-based dual-pharmacophore inhibitors as promising anti-flaviviral leads.