微量热泳动技术(MST)分享：验证中药单体与靶点蛋白的结合

分子对接技术 (Molecular docking)：

通过计算机计算出药物与蛋白结合的最佳的空间结合构象；能量匹配则是分子间保持稳定结合的基础，通过对受体与配体复合物间的弱相互作用(包括氢键、范德华力、π-π相互作用等)进行计算，找到受体与配体形成的复合物结合能最低，也就是最稳定的构象。

CETSA-WB的核心原理基于小分子-靶蛋白相互作用引发的热稳定性改变。当药物分子与靶蛋白特异性结合时，可通过构象调节作用增强或削弱其热力学稳定性。

实验通过温度梯度诱导蛋白质变性(变性沉淀)：

将样本在预设温度加热后，未与药物结合的游离蛋白会在临界变性温度下聚集沉淀，而小分子-靶蛋白复合物因稳定性变化表现出不同的抗变性能力。通过离心分离沉淀与上清组分，利用Western blot等检测手段对可溶性靶蛋白进行定量分析，通过比较处理组与对照组的蛋白稳定性差异，定性检测小分子与靶蛋白的结合。

MST采用微量热泳动原理，基于蛋白结合配体后构象、水化层和大小改变导致分子在温度梯度场中泳动速度的变化进行亲和力检测。将靶蛋白(Target)进行荧光标记，再将配体分子梯度稀释，靶蛋白与稀释后的配体共孵育后进行MST检测，从而检测靶蛋白与配体之间的亲和力。

高分文章分享

图1 6-姜酚和8-姜酚与GPX4分子对接结果

为了进一步验证6-姜酚和8-姜酚与GPX4的相互作用，作者通过细胞热迁移分析(CETSA)检测GPX4蛋白的热稳定性变化。

实验时，作者采用液氮反复冻融法裂解细胞，离心获取蛋白上清液，获取的蛋白样品在37℃条件下分别与6-姜酚(10 μM)和8-姜酚(10 μM)或溶剂对照(DMSO)共孵育1小时，然后在40°C-70°C的温度梯度下将样品孵育四分钟，室温冷却四分钟，离心取上清进行WB检测。

结果表明6-姜酚、8-姜酚均可增强GPX4蛋白的热稳定性，6-姜酚、8-姜酚可特异性结合GPX4蛋白(图2)。

图3 微量热泳动(MST)实验验证6-姜酚和8-姜酚与GPX4的相互作用

此外，作者还通过酶活实验，发现6-姜酚、8-姜酚与GPX4存在相互作用。并且6-姜酚和8-姜酚在体内和体外模型中均观察到GPX4表达增加。这种同时增强GPX4活性和表达的双重机制，突出了这些化合物在恢复GPX4功能方面的治疗潜力。

Molecular docking

Molecular docking was conducted using Accelrys Discovery Studio (version 3.5, Accelrys, San Diego, CA, USA) to elucidate the binding modes of compounds with the GPX4 protein. The X-ray crystal structure of GPX4 (PDB ID: 2OBI) was retrieved from the Protein Data Bank and used as the template. Prior to docking, the protein was prepared by removing water molecules and adding hydrogen atoms, followed by the application of the CHARMM force field. The reported allosteric site of GPX430 was identified using Accelrys Discovery Studio. The compounds from the ChemDiv compound library, provided in SDF format, underwent 3D conformer generation and energy minimization using the CHARMM force field. Subsequently, the compounds were docked to GPX4 using the LibDock module with default parameters.

Cellular thermal shift assay

To assess the effect of 6-, 8-Gingerol on GPX4 binding, cells were lysed by three cycles of freeze-thawing in liquid nitrogen and then divided into two equal aliquots. One aliquot was incubated with 6-, 8-Gingerol (10 μM) and the other aliquot with DMSO as a control for 1 hour at 37 °C. The lysates were then subjected to different heat shock temperatures (40-70°C) for 4 minutes each, followed by 4 minutes of cooling at room temperature. The samples were centrifuged at 12, 000 × g for 10 minutes at 4 °C to separate the soluble and insoluble fraction. The soluble fractions were collected and analyzed by western blot.

Microscale thermophoresis assay

To measure the binding affinity of 6- or 8-Gingerol to GPX4, the compounds were dissolved in microscale thermophoresis assay buffer (50 mM HEPES, 150 mM NaCl, 10 mM MgCl₂, 0.05 %Tween-20, pH 7.0). The compound and GPX4 enzyme mixtures were incubated at room temperature for 30 minutes and then subjected to microscale thermophoresis (NanoTemper Monolith Instrument NT.115). The data were analyzed by MO Affinity Analysis v2.3.

参考文献

[1] Zhang Q Y, Gong H B, Jiang M Y, et al. Regulation of enzymatic lipid peroxidation in osteoblasts protects against postmenopausal osteoporosis[J]. Nature Communications, 2025, 16(1): 758.