Design, synthesis and biological activity evaluation of novel allosteric inhibitors of the DENV NS2B–NS3 protease

Abstract

The dengue virus (DENV) is a mosquito-borne flavivirus transmitted through the infected mosquito, primarily of the Aedes genus. Over recent decades, the significant emergence of DENV infections has posed growing challenges for control efforts. Extensive research indicates that the dengue NS2B–NS3 protease, a non-structural viral protein, is the most promising target in dengue drug research. Therefore, our study synthesized two series of novel allosteric inhibitors of the NS2B–NS3 protease to explore previous allosteric inhibitors of the protease. Meanwhile, enzyme inhibitory activities of all compounds are measured and IC₅₀ values of the optimized compounds are obtained by utilizing a fluorescence-based assay. Notably, compound A24 demonstrates the highest enzyme inhibitory activity with an IC₅₀ value of 16 μM. Further enzyme kinetic experiments confirm that the optimized compound A24 functions as a non-competitive inhibitor of the NS2B–NS3 protease. Through molecular docking studies, compound A24 showed a binding energy of −5.6 kcal mol⁻¹, comparable to the lead compounds, which preliminarily explains its similar inhibitory activity. In addition, compound A24 forms a stable complex with the protein primarily through hydrogen bonds and π–π interaction, with subsequent dynamics simulations confirming the stable binding mode. In conclusion, we designed and synthesized 59 novel scaffold compounds and identified compound A24 with the highest activity through pharmacological experiments and computer-aided drug design, which targets the allosteric sites and contributes significantly to the prevention of the dengue virus.