Chemical Institute, Kremlevskaya 18, Kazan Federal University, 420008 Kazan, Russia.
Chemical Institute, Kremlevskaya 18, Kazan Federal University, 420008 Kazan, Russia.
Int J Pharm. 2020 Jun 15;583:119362. doi: 10.1016/j.ijpharm.2020.119362. Epub 2020 Apr 22.
There is a demand in rapid and robust methods to determine the affinity of drugs to receptors, enzymes, and transport proteins. Differential scanning calorimetry (DSC) is a common method to prove the existence of ligand-protein binding from the shift of denaturation peak, but it is rarely used to obtain the binding constant values. The work is aimed to prove that the DSC experiments can be a source of reliable values of the binding constants and information on the stoichiometry of drug-albumin binding. DSC thermograms of bovine serum albumin denaturation in the presence of several drugs with different affinity and stoichiometry of binding to albumin: naproxen, warfarin, ibuprofen, and isoniazid were recorded. The dependences of the denaturation peak maximum temperature and area on the molar drug/protein ratio, which varied from 0 to 100, were considered. With the help of numerical modeling of the DSC curves, these dependences were predicted using the binding parameters determined in independent experiments and a simple two-state model of denaturation. The DSC data at relatively small concentrations of ligands are in good agreement with the calculation results. The deviations from the model predictions at high ligand concentrations in the cases of naproxen and ibuprofen indicate that albumin is able to bind several additional molecules of these drugs with its low-affinity sites. The fit was improved by using a sequential binding model with two binding constants K = 1.0 × 10 and K = 1.0 × 10 for naproxen and a cooperative binding model for ibuprofen. The stoichiometry of drug-albumin complexes fully saturated with drug ligand was calculated from the dependence of the denaturation temperature on the drug concentration. In the case of isoniazid, DSC thermograms indicated very weak binding to albumin.
人们对快速、稳健的方法有着强烈需求，以便测定药物与受体、酶和转运蛋白的亲和力。差示扫描量热法（DSC）是一种从变性峰位移证明配体-蛋白结合存在的常用方法，但很少用于获得结合常数值。本工作旨在证明 DSC 实验可以成为获得可靠的结合常数值和药物-白蛋白结合化学计量学信息的来源。记录了在几种具有不同亲和力和与白蛋白结合化学计量的药物存在下牛血清白蛋白变性的 DSC 热谱：萘普生、华法林、布洛芬和异烟肼。考虑了变性峰最大温度和面积随药物/蛋白质摩尔比（从 0 到 100）的变化。借助 DSC 曲线的数值建模，使用在独立实验中确定的结合参数和简单的变性两态模型对这些依赖性进行了预测。在配体浓度相对较低的情况下，DSC 数据与计算结果吻合良好。在萘普生和布洛芬的情况下，在高配体浓度下偏离模型预测表明白蛋白能够与其低亲和力部位结合这些药物的额外几个分子。通过使用具有两个结合常数 K=1.0×10 和 K=1.0×10 的顺序结合模型和用于布洛芬的协同结合模型，对萘普生的拟合进行了改进。从变性温度随药物浓度的依赖性计算出药物-白蛋白复合物的药物配体完全饱和的化学计量。在异烟肼的情况下，DSC 热谱表明与白蛋白的结合非常弱。
