Intriguing the significance of hydrophobic groove by the inhibitory mechanism of mineralocorticoid receptor bound with spironolactone through mutations using long-range molecular dynamics simulations in conjunction with statistical analysis.

CONTEXT: Elucidating the role of the hydrophobic groove in mineralocorticoid receptor (MR)-spironolactone interactions is important for structure-based drug design, receptor modulation, and the development of more selective MR antagonists. Despite the clinical importance of spironolactone, the contribution of the hydrophobic groove, particularly residues M807, F829, M845, C849, and M852, remains underexplored. Here, we demonstrate through molecular dynamic simulations that these hydrophobic residues, together with polar residue N770, stabilize the thioacetyl moiety of spironolactone. Binding free energy calculations of the hydrophobic groove, both with the complete binding site and with the groove alone, demonstrate the impact of the groove's hydrophobicity along with the polar residues N770, Q776, and R817. Simulation results, supported by statistical analysis, highlight the groove's structural and energetic significance. Site-directed mutagenesis targeting residues F829, M845, and C849 further clarifies their role in the binding mechanism, offering insights for rational drug design and biomarker development.

METHODS

The crystal structure of the MR-spironolactone complex (PDB ID: 3VHU) was retrieved and mutated using COOT. Mutant complexes were constructed and subjected to 1 μs molecular dynamics simulations using GROMACS. Binding free energies were calculated via MM/PBSA. Residue-ligand interactions were analyzed from MD trajectories using LigPlot + and GROMACS tools. Statistical significance of residue contributions was assessed using ANOVA, comparing polar and hydrophobic residue mutations across simulated complexes.