Unraveling the binding interaction and kinetics of a prospective anti-HIV drug with a model transport protein: results and challenges

Abstract

The present contribution reports a detailed characterization of the binding interaction of a potential anticancer, anti-HIV drug 1-phenylisatin (1-PI) with a model transport protein Bovine Serum Albumin (BSA) using fluorescence spectroscopic techniques. The thermodynamic parameters e.g., ΔH, ΔS and ΔG for the binding phenomenon have been evaluated on the basis of the van't Hoff equation to reveal that the binding process is principally driven by ionic interactions mediated by charge transfer interaction. This line of argument has been substantiated by frontier molecular orbital analysis of 1-PI. However, the drug-induced quenching of the intrinsic tryptophanyl fluorescence of the protein is found not to abide by a linear Stern-Volmer regression (displaying an upward curvature) when an extensive time-resolved fluorescence spectroscopic characterization of the quenching process has been undertaken to unveil the actuating quenching mechanism. Based on the constancy of the fluorescence lifetime of the protein as a function of drug concentration the observed quenching is inferred to proceed through a static mechanism between the quenching partners. Constant wavelength synchronous fluorescence, excitation-emission matrix fluorescence and circular dichroic (CD) spectroscopic techniques have been exploited to unravel the tertiary and secondary conformational changes in the protein (BSA) induced by drug (1-PI)-binding. The probable binding location of the drug molecule within the protein cavity (hydrophilic subdomain I) has been explored by AutoDock-based blind docking simulation and the inference is further substantiated by site-competitive replacement experiments with specific site-markers. Light is also cast on the drug-protein binding kinetics using the stopped-flow fluorescence technique which reveals an association rate constant of k(a) (± 5%) = 1.471 × 10(-3) s(-1) for the interaction of 1-PI with BSA.