Identification of Novel AChE-Targeting Neuroprotective Peptides from Pacific Oyster (Crassostrea gigas): An Integrated Pipeline of Peptidomics, Molecular Dynamics, and Cellular Validation

Although the Pacific oyster (Crassostrea gigas) is a premium marine protein source, its neuroprotective peptidome remains largely uncharacterized. This study established an integrated in silico and in vitro pipeline to discover acetylcholinesterase (AChE)-targeting peptides with cellular AChE-regulating and neuroprotective peptides from simulated gastrointestinal digests of oyster. Peptidomic profiling identified 18,292 sequences, which were filtered down to seven candidates predicted to have favorable blood–brain barrier (BBB) permeability and to be non-toxic and non-allergenic (VPYPR, VPVHF, HHTF, PVHF, GPKPW, HWF, and KYW) via multi-step virtual screening. In cellular assays, simulated H2O2 injury (500 μM) reduced PC12 cell viability to 47.53 ± 4.53%. Compared with the model group, pretreatment with the three most potent candidates—HHTF, VPYPR, and VPVHF (200 μM)—significantly rescued injured cells, restoring cell viability to 88.31 ± 7.83%, 85.12 ± 3.35%, and 82.00 ± 3.47%, respectively (p < 0.05). These peptides effectively fortified cellular antioxidant defenses by increasing glutathione (GSH) levels to 24.24, 30.11, and 26.83 nmol/mg protein (from 20.22 nmol/mg protein in the model group) and superoxide dismutase (SOD) activity to 151.41, 153.97, and 151.96 U/mg protein (from 119.33 U/mg protein), while suppressing malondialdehyde (MDA) accumulation to 0.088, 0.064, and 0.086 nmol/mg protein (from 0.193 nmol/mg protein). Crucially, the peptides alleviated cholinergic dysfunction by normalizing the H2O2-induced elevation of intracellular AChE activity (11.39 nmol/min/mg protein) down to 7.02, 6.22, and 7.14 nmol/min/mg protein, respectively. Specifically, VPYPR (200 μM) restored AChE activity to a level (6.22 nmol/min/mg protein) that was not significantly different from that in the normal control group (p > 0.05). Molecular dynamics (MD) simulations (100 ns) and molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) calculations identified VPYPR as the leading candidate with a remarkably low binding free energy of −49.74 ± 3.58 kcal/mol. This study demonstrates that oyster gastrointestinal digests are valuable reservoirs of multi-target neuroprotective ingredients and provides an efficient strategy for marine bioactive peptide discovery.