Jelinska_2026_Int.J.Mol.Sci_27_

Acetylcholinesterase (AChE) inhibition remains a key therapeutic strategy in the management of neurodegenerative disorders such as Alzheimer's disease. In this study, the inhibitory potential of the gastric pentadecapeptide BPC-157 and two newly designed hybrid analogs, CIARA-1 and CIARA-2, was investigated for the first time. The hybrid peptides were rationally designed by combining a BPC-157-derived fragment with an arginine-containing C-terminal sequence to enhance interactions with the enzyme's active and peripheral binding sites. Enzyme kinetics were evaluated using a modified Ellman assay, and inhibition parameters were determined through Lineweaver-Burk analysis. All tested compounds exhibited a competitive mechanism of inhibition, as evidenced by increased Michaelis-Menten constant (K(m)) values with unchanged maximum velocity (V(max)), indicating competition with the substrate at the catalytic site of AChE. Among the tested compounds, CIARA-1 demonstrated the highest inhibitory potency, reflected by the lowest inhibition constant (K(i) = 0.24 mM) and IC(50) value (2.52 mM), followed by CIARA-2 (K(i) = 0.29 mM; IC(50) = 2.73 mM) and BPC-157 (K(i) = 0.48 mM; IC(50) = 2.80 mM). These findings were consistent with molecular modeling predictions, supporting stronger binding interactions for CIARA-1. Despite significantly lower potency compared to clinically used AChE inhibitors, the studied peptides represent a promising scaffold for further optimization. Overall, this work demonstrates that BPC-157 and its hybrid analogs act as reversible competitive AChE inhibitors, with enhanced activity observed for structurally modified derivatives. The results highlight the potential of peptide-based hybrid molecules as multifunctional candidates in the development of novel therapeutics targeting cholinergic dysfunction.