Improved albumin binding properties of Isoguvacine upon esterification as characterized by biophysical and computational tools

The prevalence of epilepsy, affecting millions across all age groups, has intensified the need for novel therapeutic interventions. Despite advances in antiseizure medications (ASMs), challenges persist, particularly drug resistance and significant psychiatric and behavioral adverse effects. Isoguvacine (IGV), a potent γ-aminobutyric acid type A receptor agonist, holds promise for epilepsy treatment but suffers from poor blood-brain barrier (BBB) permeability due to its zwitterionic nature. To address this limitation, two ester prodrugs: E7 (heptyl 1,2,3,6-tetrahydropyridine-4-carboxylate) and E14 (tetradecyl 1,2,3,6-tetrahydropyridine-4-carboxylate) were synthesized to enhance BBB penetration and central nervous system (CNS) delivery. This study investigates the interactions of IGV and its derivatives with human serum albumin (HSA), a key plasma transport protein, using a suite of biophysical and computational techniques. UV spectroscopic analysis confirmed protein-ligand complexation, with E7 and E14 exhibiting enhanced binding compared to IGV. Isothermal titration calorimetry revealed that E14 demonstrated superior binding affinity (K_a = 2.42 × 10⁶ M⁻¹) compared to E7 (K_a = 3.41 × 10⁴ M⁻¹), while IGV showed negligible interaction. Atomic force microscopy and circular dichroism spectroscopy indicated that the enhanced hydrophobicity of E7 and E14 promoted stable protein-ligand interactions without perturbing the secondary and tertiary structures of HSA. Molecular docking studies corroborated the superior binding affinity of E14 towards HSA, while molecular dynamics simulations confirmed the enhanced stability of the E14-HSA complex. These results highlight the potential of E14 as a promising prodrug candidate for CNS-targeted epilepsy therapy and underscore the importance of albumin binding in modulating drug bioavailability and distribution.