Ligand-enzyme thermodynamic bindings provide an efficient and microscale evaluating approach for α-amylase inhibition...

Inhibiting the activity of essential carbohydrate-hydrolyzing α-amylase is considered as a promising approach in regulating postprandial blood-glucose level. The enzyme inhibition effect is usually determined through substrate hydrolysis, which brings a challenge regarding inhibitory activity fluctuation as substrate enzymolysis modes cause difference in α-amylase inhibition. Herein, we innovatively established a thermodynamics-inhibition model for evaluating α-amylase inhibitors that was afterwards independent of substrate digestion. During model establishment, the direct inhibitor-enzyme binding affinity (1/K_d) was determined by microscale thermophoresis (MST) using a specific histidine-labelling tag based on ligand-receptor binding behaviors, along with critical inhibiting parameters through inhibition analysis including inhibitory activity (IC₅₀) and competitive inhibition coefficient (1/K_ic). Then, the regularly positive correlation between thermodynamic binding affinity and enzyme inhibition was specifically found across a weak-to-strong inhibiting range. This confidently provided the model with an ability in evaluating α-amylase inhibition of a potential inhibitor, based on which the landmark 1/K_d threshold for gradient inhibitory activity compared to positive control was obtained. Through this model, galloyl moiety, especially with configurational freedom, was indicated as a promising active moiety in natural product discovering and pharmaceutical synthesis for α-amylase inhibitors. Conclusively, the thermodynamics-inhibition model provided an efficient approach in assessing α-amylase inhibitors in a substrate-free mode.