Bridging In Silico Design and Experimental Validation: Virtual Screening and In Vitro Assessment of Biomimetic Anticancer Peptides Inspired by Horseshoe Crab Hemolymph Proteins

• A multi-step virtual screening pipeline, integrating six machine learning predictors and multi-criteria ranking, identified two hemocyanin-derived anticancer peptides. • Peptide H22 exhibited moderate but favorable selectivity against HT-29 colon cancer cells (IC₅₀ = 39.83 µM, Selectivity Index = 1.83). • Structural analysis revealed H22 possesses balanced amphipathicity (hydrophobicity = 0.271, hydrophobic moment = 0.278) and an extended α-helical conformation. • Mechanistically, H22 induced expression of p53, Bax, Caspase-9, and Caspase-7, suggesting activation of the intrinsic apoptotic pathway. • The integrated computational-experimental workflow provides a reproducible proof-of-concept framework for peptide discovery with favorable safety and mechanistic profiles. Colorectal cancer (CRC) remains a leading cause of cancer-related mortality, necessitating the development of more selective and safer therapeutic options. Biomimetic peptides derived from natural sources offer promising alternatives to conventional chemotherapeutics, yet their discovery through experimental screening alone is time-consuming and resource-intensive. To integrate computational modeling and AI-driven virtual screening with experimental validation for the discovery of novel anticancer peptides from the hemolymph of Tachypleus tridentatus . A multi-step in silico pipeline comprising tryptic digestion, six machine learning predictors, safety profiling (toxicity, hemolysis, allergenicity), and multi-criteria ranking was employed. Top candidates were synthesized and validated using MTT assays, AO/PI staining, and qRT-PCR. Structural analysis was performed using HeliQuest and PEP-FOLD 4.0. Computational screening prioritized two hemocyanin-derived peptides, H10 and H22. H22 exhibited balanced amphipathicity (hydrophobicity = 0.271, hydrophobic moment = 0.278) and an extended α-helical structure. In vitro , H22 demonstrated moderate cytotoxicity against HT-29 cells (IC₅₀ = 39.83 ± 0.59 µM) with favorable selectivity (Selectivity Index = 1.83) compared to doxorubicin (SI = 0.12). Mechanistically, qRT-PCR revealed that H22 upregulated p53 (19.3-fold), Bax (4.7-fold), Caspase-9 (4.6-fold), and Caspase-7 (49.4-fold), suggesting apoptotic inductive mechanism via the intrinsic pathway. This integrated approach successfully identified H22 as a hemocyanin-derived peptide with moderately potent anticancer activity and promising selectivity. The workflow provides a reproducible proof‑of‑concept framework for computational-guided peptide, warranting further optimization and experimental validation.