Elucidating the degradation mechanism of neuropeptides by aminopeptidase-B (APB): An in-vitro and in-silico perspective

Aminopeptidase-B (APB) plays a critical role in modulating the bioavailability of neuroactive peptides and is known to exhibit inhibitory interactions with anti-inflammatory and antihypertensive agents. The present study aimed to identify and characterize bioactive peptides susceptible to degradation by APB. The APB was purified to homogeneity from goat brain tissue and incubated with various neuropeptides and immunomodulatory peptides. Hydrolytic activity was assessed by utilized a thin-layer chromatography (TLC) and visualized using ninhydrin staining. Bioinformatics techniques including molecular modelling, molecular docking simulations, and protein–protein interaction (PPI) network analysis were employed to validate peptide-APB interactions and to map potential binding affinities. Wet lab experiments and in-silico findings revealed that APB efficiently hydrolyzed substance-P, bradykinin, neurotensin, and thymopentin. Among these, substance-P exhibited the most favorable binding energy (-334.80 kcal/mol), followed by bradykinin (-249.07 kcal/mol), thymopentin (-227.02 kcal/mol), and neurotensin (-214.64 kcal/mol). Notably, substance-P and bradykinin are capable of crossing the blood-brain barrier (BBB) showed significant degradation, underscoring APB potential involvement in neuropeptide regulation within the central nervous system. This integrative biochemical and computational approach offers novel insights into specificity of substrate of APB and highlights its possible therapeutic implications in neurodegenerative and immunological disorders.