Abstract

The interactions of small molecule drugs with plasma serum albumin are important because of the influence of such interactions on the pharmacokinetics of these therapeutic agents. 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) is one such drug candidate that has recently gained attention for its promising clinical applications as an anti-cancer agent. This study sheds light upon key aspects of AICAR’s pharmacokinetics, which are not well understood. We performed in-depth experimental and computational binding analyses of AICAR with human serum albumin (HSA) under simulated biochemical conditions, using ligand-dependent fluorescence sensitivity of HSA. This allowed us to characterize the strength and modes of binding, mechanism of fluorescence quenching, validation of FRET, and intermolecular interactions for the AICAR–HSA complexes. We determined that AICAR and HSA form two stable low-energy complexes, leading to conformational changes and quenching of protein fluorescence. Stern–Volmer analysis of the fluorescence data also revealed a collision-independent static mechanism for fluorescence quenching upon formation of the AICAR–HSA complex. Ligand-competitive displacement experiments, using known site-specific ligands for HSA’s binding sites (I, II, and III) suggest that AICAR is capable of binding to both HSA site I (warfarin binding site, subdomain IIA) and site II (flufenamic acid binding site, subdomain IIIA). Computational molecular docking experiments corroborated these site-competitive experiments, revealing key hydrogen bonding interactions involved in stabilization of both AICAR–HSA complexes, reaffirming that AICAR binds to both site I and site II.

Highlights

  • Human serum albumin (HSA) is the most abundant blood serum protein [1]

  • Fluorescence spectroscopy has been widely used to investigate the intermolecular interactions between small ligands and proteins, leading to various ligand-induced changes in protein biophysical properties, including fluorescence quenching, binding affinity, energy transfer, and conformational dynamics [12]

  • Fluorescence quenching refers to any process that decreases the protein fluorescence intensity because of the changes in the fluorophore’s microenvironment

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Summary

Introduction

Human serum albumin (HSA) is the most abundant blood serum protein [1]. It transports and interacts with many endogenous substances (water, small cations, fatty acids, hormones, bilirubin) and exogenous ligands, including small bioactive drugs [1,2]. HSA is an ideal drug-delivery and transport protein, mainly because of its extraordinary ligand binding properties, excellent water solubility, and stability profiles (pH range of 4–9 and temperature up to 60 ◦ C for 10 h) [2,3]. HSA is often used as an appropriate in vitro model transport protein for studying the structural and biochemical basis of drug–protein interactions and the evaluation of drug-like properties related to distribution, bioavailability, and efficacy [1]

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