Dynamic claw of dengue protease unveils druggability potential with high affinity allosteric inhibitors

Abstract Several enzymes receive functional signals from allosteric site to the active site through conformational dynamics while domain dynamics can also play a significant role. Current investigation identifies a claw-like regulatory region near the active site of dengue protease, which remains in dynamic equilibrium as active (open) and inactive (closed) forms. Enhanced sampling with metadynamics showed that the flexibility of the T[RK][SN]G loop of the claw region is crucial for the protease activity as it helps in the opening of the claw-like structure near the active site, and allows the C-terminal hydrophilic part of NS2B cofactor to enter the claw and transform the protease into its active conformation. Binding kinetics and thermodynamics studies further reveal that allosteric modulator epigallocatechin-3-gallate (EGCG) binds to the enzyme in a biphasic manner and disrupts the active/inactive conformational dynamics and locks the enzyme in an inactive conformation. The inhibitor binding in this region introduces an energy barrier in the free energy landscape of NS2B cofactor binding. In the stronger mode, EGCG could directly bind into the claw, induces conformational changes, locking the dynamic loops in a closed position, preventing NS2B binding and rendering the enzyme inactive. Whereas in the weaker mode, EGCG binds to the T[RK][SN]G loop and perturbs its flexibility driving the claw structure into a more closed conformation and maintaining the protease in an inactive state. The understanding of the mechanism, therefore, would help researchers design more potent inhibitors targeting dengue protease and homologous enzymes.