Department of Chemistry and Physics, Louisiana State University, Shreveport, LA 71115, USA.
Department of Inorganic Chemistry, Faculty of Chemistry, Razi University, Kermanshah 6714414971, Iran.
Molecules. 2020 Nov 19;25(22):5410. doi: 10.3390/molecules25225410.
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.
小分子药物与血浆血清白蛋白的相互作用很重要，因为这种相互作用会影响这些治疗剂的药代动力学。5-氨基咪唑-4-甲酰胺核苷（AICAR）是一种候选药物，最近因其作为抗癌剂的有前途的临床应用而受到关注。本研究揭示了 AICAR 药代动力学的一些关键方面，这些方面尚未得到很好的理解。我们在模拟生化条件下，使用 HSA 的配体依赖性荧光灵敏度，对 AICAR 与人类血清白蛋白（HSA）的相互作用进行了深入的实验和计算结合分析。这使我们能够表征结合的强度和模式、荧光猝灭的机制、FRET 的验证以及 AICAR-HSA 复合物的分子间相互作用。我们确定 AICAR 和 HSA 形成两个稳定的低能量复合物，导致蛋白质荧光的构象变化和猝灭。荧光数据的 Stern-Volmer 分析还揭示了 AICAR-HSA 复合物形成时荧光猝灭的非碰撞独立静态机制。使用 HSA 结合位点（I、II 和 III）的已知位点特异性配体进行的配体竞争性置换实验表明，AICAR 能够结合到 HSA 的位点 I（华法林结合位点，亚域 IIA）和位点 II（氟芬酸结合位点，亚域 IIIA）。计算分子对接实验证实了这些位点竞争实验，揭示了稳定两个 AICAR-HSA 复合物的关键氢键相互作用，再次证实 AICAR 结合到位点 I 和位点 II。
