Journal
MOLECULES
Volume 25, Issue 22, Pages -Publisher
MDPI
DOI: 10.3390/molecules25225410
Keywords
AICAR; human serum albumin (HSA); static fluorescence quenching; synchronous fluorescence; 3D fluorescence; FRET; Trp214; fluorophore microenvironment; molecular docking; MOE
Funding
- Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health [5 P20 GM103424-18, 3 P20 GM103424-15S1]
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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.
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