Chen_2026_Biophys.Chem_334_107617

Current in vitro enzyme inhibition assays often involve subjective data analysis based on the researcher's experience. In this study, we developed a multi-dimensional quantitative integration platform (MDQIP) that uses a model to objectively calculate and rank compound activities, addressing the limitations of traditional "experience-driven" evaluations, accelerates the screening and evaluation of potential AChE inhibitors from Red Gastrodia elata, offering a more efficient approach to drug discovery. Ultrafiltration-LC screening identified parishin A as having the most stable binding, with binding degree and recovery rates of 98.85% and 99.39%, respectively. Molecular docking revealed that parishins A and C were the strongest AChE inhibitors, exhibiting stable binding through hydrogen bonds, Pi-alkyl, and Pi-Pi interactions. Molecular dynamics simulations confirmed the stability of these compounds, with binding energies of -82.65 +/- 4.24 and - 80.69 +/- 4.19 kcal/mol. Enzyme kinetics showed that parishins A and C are mixed-type inhibitors, with IC(50) values of 0.0145 and 0.0148 mM, respectively, outperforming other compounds. Lineweaver-Burk analysis demonstrated that these compounds inhibit AChE by binding to both the free enzyme and enzyme-substrate complex, effectively increasing acetylcholine levels, which is mechanistically relevant to cholinergic signaling associated with cognitive processes. Using the MDQIP for screening and evaluation, parishins A and C were selected as promising candidates. Network pharmacology analysis revealed that these compounds target multiple AD-related genes (e.g., AChE, BACE1, PTGS2, IL2) and pathways (e.g., AD, AGE-RAGE, HIF-1, JAK-STAT), suggesting broad neuroprotective effects. In conclusion, parishins A and C exhibit strong AChE inhibition and warrant further investigation in the context of AD-related cholinergic mechanisms. Further studies should focus on their pharmacokinetics and in vivo efficacy.