Targeting atherosclerosis by inhibiting CD40-CD40L Protein-Protein interaction via novel protein design strategies

Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of plaque within arterial walls, often leading to cardiovascular-related concerns. The CD40(Cluster of differentiation 40)-CD40L (Cluster of differentiation 40 ligand) interaction plays a pivotal role in the pathogenesis of atherosclerosis, contributing to endothelial cell activation and macrophage infiltration. In this study, a novel therapeutic protein was designed to inhibit the CD40-CD40L interaction by targeting CD40 protein with the novel protein. In-silico protein design methods were used, and the structure of CD40L was initially employed as a template for modeling the interaction between CD40 and its ligand (CD40L). In this study, 23 critical binding residues were identified at the protein-protein interface and subsequently mutated in the docked conformation to improve binding affinity and specificity for CD40. Afterwards, molecular dynamics (MD) simulations were performed to evaluate the stability of the mutated complex, ensuring that the introduced mutations did not destabilize the protein structure. However, the native form showed higher stability in the complex than the mutated form, in an order of 9 kcal/mol. Consequently, further modifications were employed in the protein structure at the non-interfacial residues. This resulted in a stable protein scaffold designed by integrating the optimized mutations into a new protein framework that exhibited enhanced interaction with CD40. The novel protein showed a binding free energy of ΔG of -26.90 kcal/mol compared to native CD40L with ΔG of -14.47 kcal/mol in the last stable 20 ns time frame during the simulation. The study shows that protein engineering can lead to develop smart protein that can target protein-protein interaction.