Evaluating Blood–Brain Barrier Permeability, Cytotoxicity, and Activity of Potential Acetylcholinesterase Inhibitors: In Vitro and In Silico Study

Abstract

ABSTRACT Acetylcholinesterase inhibitors (AChEIs) remain the first‐line treatment for Alzheimer's disease. However, these drugs are largely symptomatic and often associated with adverse effects. This study aimed to evaluate novel pharmacophores for their in vitro AChEI activity, blood–brain barrier (BBB) permeability, and cytotoxic potential, hypothesizing that a combination of AChEIs could enhance symptom management while minimizing toxicity. A library of 1453 synthetic pharmacophores was assessed using in vitro and in silico methods to determine their feasibility as an inhibitor of the AChE enzyme. An in‐house miniaturized Ellman's assay determined acellular AChEI activities, while pharmacokinetic properties were evaluated using the SwissADME web tool. The combinational effects of in silico BBB‐permeable pharmacophores and donepezil were examined using a checkerboard AChEI assay. Cytotoxicity of active compounds and their synergistic combinations was assessed in SH‐SY5Y neuroblastoma and bEnd.5 cells using the sulforhodamine B assay. Cellular AChEI activity of active in silico BBB‐permeable predicted compounds was determined using an SH‐SY5Y AChE‐based assay. An in vitro BBB model was used to assess the effect of compounds on the integrity of the bEnd.5 monolayer. Out of the screened compounds, 12 demonstrated 60% AChEI activity at 5 μM, with compound A51 showing the lowest IC50 (0.20 μM). Five compounds were identified as BBB‐permeable, with the donepezil‐C53 combination at ¼IC50 exhibiting the strongest synergy (CI = 0.82). Compounds A136 and C129, either alone or with donepezil, showed cytotoxicity. Notably, compound C53, both alone and in combination with donepezil, demonstrated high AChEI activity and promising BBB permeability, warranting further investigation.

Keywords

• acetylcholinesterase inhibition
• blood–brain barrier
• cytotoxicity
• synthetic compounds