Design and Evaluation of Protein Inhibitors Against the KRAS G12D Mutation in Colorectal Cancer via Computer-Aided Drug Design Techniques

Objective: KRAS mutations are among the most common oncogenic drivers in colorectal cancer (CRC), with the G12D variant posing a particularly formidable challenge due to the protein's smooth surface and lack of druggable pockets. Despite the success of covalent inhibitors for KRAS G12C, equivalent strategies for G12D remain underdeveloped. This study aimed to design and evaluate peptide-based inhibitors capable of selectively targeting the GDP-binding pocket of KRAS G12D using computer-aided drug design (CADD) techniques. Methods: A peptide library consisting of 108 decameric sequences was rationally designed based on structure-activity relationship (SAR) insights and residue preferences for protein-protein interactions. Flexible docking was performed using the CABS-dock platform to simulate peptide binding to the KRAS G12D structure (PDB ID: 5US4). The top-ranked peptides were subjected to molecular mechanics/generalized Born surface area (MM/GBSA) calculations to estimate binding free energies. Key molecular interactions were evaluated using the Protein Interactions Calculator (PIC), and structural validity was confirmed via Ramachandran plot analysis. Results: Nine peptides showed strong and reproducible binding within the GDP-binding pocket. Ligand 3 (DCWRHRLCID) demonstrated the most favorable interaction, with a binding free energy of –57.59 kcal/mol, followed by Ligand 34 (NCWRRHLCIN). Both ligands engaged in ionic, hydrogen bonding, and π-π stacking interactions with critical KRAS residues, notably Asp12. Ramachandran analysis showed 98% of residues within favored regions, indicating conformational stability. Conclusion: This study identifies Ligands 3 and 34 as promising peptide-based inhibitors of KRAS G12D. These findings offer a foundation for future experimental validation and therapeutic development targeting mutant KRAS in CRC. Keywords: Colorectal cancer; G12D mutation; KRAS; MM/GBSA; Molecular docking; Peptide inhibitors