PDF(Pubmed)
Abstract：
Ketoprofen (KTF) and ketorolac (KTL) are among the most primarily used non-steroidal anti-inflammatory drugs (NSAIDs) in humans to alleviate moderate pain and to treat inflammation. Their binding affinity with albumin (the main globular protein responsible for the biodistribution of drugs in the bloodstream) was previously determined by spectroscopy without considering some conventional pitfalls. Thus, the present work updates the biophysical characterization of the interactions of HSA:KTF and HSA:KTL by 1H saturation-transfer difference nuclear magnetic resonance (1H STD-NMR), ultraviolet (UV) absorption, circular dichroism (CD), steady-state, and time-resolved fluorescence spectroscopies combined with in silico calculations. The binding of HSA:NSAIDs is spontaneous, endothermic, and entropically driven, leading to a conformational rearrangement of HSA with a slight decrease in the α-helix content (7.1% to 7.6%). The predominance of the static quenching mechanism (ground-state association) was identified. Thus, both Stern-Volmer quenching constant (KSV) and binding constant (Kb) values enabled the determination of the binding affinity. In this sense, the KSV and Kb values were found in the order of 104 M-1 at human body temperature, indicating moderate binding affinity with differences in the range of 0.7- and 3.4-fold between KTF and KTL, which agree with the previously reported experimental pharmacokinetic profile. According to 1H STD-NMR data combined with in silico calculations, the aromatic groups in relation to the aliphatic moiety of the drugs interact preferentially with HSA into subdomain IIIA (site II) and are stabilized by interactions via hydrogen bonding and hydrophobic forces. In general, the data obtained in this study have been revised and updated in comparison to those previously reported by other authors who did not account for inner filter corrections, spectral backgrounds, or the identification of the primary mathematical approach for determining the binding affinity of HSA:KTF and HSA:KTL.
摘要：
酮洛芬（KTF）和酮咯酸（KTL）是人类最主要使用的非甾体抗炎药（NSAID），用于缓解中度疼痛和治疗炎症。它们与白蛋白（负责药物在血液中的生物分布的主要球状蛋白）的结合亲和力先前通过光谱学确定，而不考虑一些常规陷阱。因此，本工作通过1H饱和转移差分核磁共振（1HSTD-NMR）更新了HSA：KTF和HSA：KTL相互作用的生物物理表征，紫外线（UV）吸收，圆二色性（CD），稳态,以及时间分辨荧光光谱法与计算机计算相结合。HSA：NSAIDs的结合是自发的，吸热,熵驱动，导致HSA的构象重排，α-螺旋含量略有降低（7.1％至7.6％）。确定了静态猝灭机制（基态缔合）的优势。因此，Stern-Volmer猝灭常数(KSV)和结合常数(Kb)值均能够确定结合亲和力。在这个意义上,在人体温度下，发现KSV和Kb值大约为104M-1，表明适度的结合亲和力，KTF和KTL之间的差异范围为0.7倍和3.4倍，这与先前报道的实验药代动力学特征一致。根据1HSTD-NMR数据结合计算机计算，与药物的脂肪族部分相关的芳族基团优先与HSA相互作用进入亚结构域IIIA（位点II），并通过氢键和疏水性力的相互作用而稳定。总的来说,在这项研究中获得的数据进行了修订和更新，与以前报告的其他作者没有考虑内部滤波器校正的数据相比，光谱背景,或用于确定HSA:KTF和HSA:KTL的结合亲和力的主要数学方法的鉴定。
