Computational and Experimental Studies of Inhibitor Design for Aldolase A

Glycolytic enzyme fructose-bisphosphate aldolase A is an emerging therapeutic target in cancer. Recently, we have solved the crystal structure of murine aldolase in complex with naphthalene-2,6-diyl bisphosphate (ND1) that served as a template of the design of bisphosphate-based inhibitors. In this work, a series of ND1 analogues containing difluoromethylene (-CF₂), methylene (-CH₂), or aldehyde substitutions were designed. All designed compounds were studied using molecular dynamics (MD) simulations with the AMOEBA force field. Both energetics and structural analyses have been done to understand the calculated binding free energies. The average distances between ligand and protein atoms for ND1 were very similar to those for the ND1 crystal structure, which indicates that our MD simulation is sampling the correct conformation well. CF₂ insertion lowers the binding free energy by 10-15 kcal/mol, while CF₂ substitution slightly increases the binding free energy, which matches the experimental measurement. In addition, we found that NDB with two CF₂ insertions, the strongest binder, is entropically driven, while others including NDA with one CF₂ insertion are all enthalpically driven. This work provides insights into the mechanisms underlying protein-phosphate binding and enhances the capability of applying computational and theoretical frameworks to model, predict, and design diagnostic strategies targeting cancer.