Abstract

Direct FXa inhibitors are an important class of bioactive molecules (rivaroxaban, apixaban, edoxaban, and betrixaban) applied for thromboprophylaxis in diverse cardiovascular pathologies. The interaction of active compounds with human serum albumin (HSA), the most abundant protein in blood plasma, is a key research area and provides crucial information about drugs' pharmacokinetics and pharmacodynamic properties. This research focuses on the study of the interactions between HSA and four commercially available direct oral FXa inhibitors, applying methodologies including steady-state and time-resolved fluorescence, isothermal titration calorimetry (ITC), and molecular dynamics. The HSA complexation of FXa inhibitors was found to occur via static quenching, and the complex formation in the ground states affects the fluorescence of HSA, with a moderate binding constant of 10(4) M-1. However, the ITC studies reported significantly different binding constants (10(3) M-1) compared with the results obtained through spectrophotometric methods. The suspected binding mode is supported by molecular dynamics simulations, where the predominant interactions were hydrogen bonds and hydrophobic interactions (mainly pi-pi stacking interactions between the phenyl ring of FXa inhibitors and the indole moiety of Trp214). Finally, the possible implications of the obtained results regarding pathologies such as hypoalbuminemia are briefly discussed.