Molecular Biology and Nanotechnology Laboratory (MolBNL@UniTS), DEA, University of Trieste, Piazzale Europa 1, 34127 Trieste, Italy.
Molecular Biology and Nanotechnology Laboratory (MolBNL@UniTS), DEA, University of Trieste, Piazzale Europa 1, 34127 Trieste, Italy.
Eur J Pharm Sci. 2023 Oct 1;189:106550. doi: 10.1016/j.ejps.2023.106550. Epub 2023 Jul 30.
The utilization of BRAF and MEK inhibitors in combination therapy has demonstrated superior outcomes in the treatment of melanoma as compared to monotherapy. In the present scenario, the combination therapy of Encorafenib (ENC), a BRAF inhibitor, and Binimetinib (BINI), a MEK inhibitor, has been identified as one of the most efficacious treatment modalities for this malignancy. Investigations of protein binding, particularly with human serum albumin (HSA), are essential to understand drug performance and enhance therapeutic outcomes. The investigation of the interplay between small molecule drugs and HSA is of paramount importance, given that such interactions can exert a substantial influence on the pharmacokinetics of these therapeutic agents. The present study aims to bridge these lacunae by implementing a comprehensive approach that integrates fluorescence spectroscopy (FS), isothermal titration calorimetry (ITC), far-ultraviolet circular dichroism (far-UV CD), and molecular simulations. Through analysis of the fluorescence quenching of HSA at three distinct temperatures, it was ascertained that the association constants for the complexes formed between drugs and HSA were of the magnitude of 10 M. This suggests that the interactions between the compounds and albumin were moderate and comparable. Simultaneously, the investigation of fluorescence indicated a contrasting binding mechanism for the two inhibitors: ENC predominantly binds to HSA through enthalpic interaction, while BINI/HSA is stabilized by entropic contributions. The data obtained was confirmed through experimental procedures conducted using the ITC method. The results of ligand-competitive displacement experiments indicate that ENC and BINI can bind to HSA within subdomain IIA, specifically Sudlow site I. However, far-UV CD studies show that there are no notable alterations in the structure of HSA upon binding with either of the two inhibitors. Ultimately, the results were supported by computational molecular analysis, which identified the key interactions that contribute to the stabilization of the two ligand/HSA complexes.
BRAF 和 MEK 抑制剂联合治疗在黑色素瘤治疗中的疗效优于单药治疗。在当前情况下，BRAF 抑制剂恩考芬尼（ENC）和 MEK 抑制剂比美替尼（BINI）的联合治疗已被确定为治疗这种恶性肿瘤最有效的治疗方法之一。研究蛋白质结合，特别是与人血清白蛋白（HSA）的结合，对于了解药物性能和提高治疗效果至关重要。鉴于小分子药物与 HSA 之间的相互作用会对这些治疗剂的药代动力学产生重大影响，因此研究小分子药物与 HSA 之间的相互作用至关重要。本研究旨在通过实施综合方法来填补这些空白，该方法结合了荧光光谱（FS）、等温滴定量热法（ITC）、远紫外圆二色性（far-UV CD）和分子模拟。通过在三个不同温度下分析 HSA 的荧光猝灭，确定了药物与 HSA 形成的复合物的结合常数在 10 M 的数量级。这表明化合物与白蛋白之间的相互作用是中等强度且相当的。同时，荧光研究表明两种抑制剂的结合机制不同：ENC 主要通过焓相互作用与 HSA 结合，而 BINI/HSA 则通过熵贡献稳定。通过使用 ITC 方法进行的实验程序验证了获得的数据。配体竞争性置换实验的结果表明，ENC 和 BINI 可以在亚域 IIA 内，即 Sudlow 位点 I 与 HSA 结合。然而，远紫外 CD 研究表明，两种抑制剂中的任何一种与 HSA 结合时，HSA 的结构都没有明显变化。最终，计算分子分析支持了这些结果，该分析确定了导致两种配体/HSA 复合物稳定的关键相互作用。
