Molecular insights into vasicine and butyrylcholinesterase interactions: A complimentary biophysical, multi-spectroscopic, and computational study.

Butyrylcholinesterase (BChE) plays a pivotal role in regulating acetylcholine (ACh) levels during the progression of Alzheimer's disease (AD), so emerged as an attractive target in AD treatment. Vasicine, a naturally occurring pyrroloquinazoline alkaloid, was identified as a natural BChE inhibitor (IC50 = 1.47 ± 0.37 μM) from Traditional Chinese Medicine database. No any detailed research concerning the binding behavior of BChE with small molecule. As the first case, the inhibitory mechanism of vasicine on BChE was investigated using multi-spectroscopic methods (including fluorescence quenching, ANS fluorescence probe, three-dimensional fluorescence, time-resolved fluorescence, circular dichroism), isothermal titration calorimetry, surface plasmon resonance, and computational approaches. As a reversible and mixed inhibitor, vasicine displayed moderate affinity for BChE with an affinity constant KD of 2.111 μM, its binding process was characterized as a spontaneous exothermic reaction with reduced entropy, primarily driven by hydrogen bonding interactions. Vasicine quenched the fluorescence of BChE through both static and dynamic quenching mechanisms, leading to an increase in the α-helix content and surface hydrophobicity of BChE. Furthermore, the fluctuation of the skeleton atoms in the vasicine-BChE complex system remained stable, indicating good stability within the simulated physiological environment. In addition, vasicine exerted good safety for PC12 cells. Above findings provide molecular insights into the inhibitory mechanism of vasicine against BChE for the first time, and offer valuable information for future structure modification and therapeutic applications of vasicine as a BChE inhibitor.