MST微量热泳动——分子互作实验服务

药物作用靶点是指存在于机体细胞、可与药物直接结合并发生相互作用，从而赋予药物生物学效应的特定分子。药物靶点的发现对于理解药物作用机制和新药开发至关重要。微量热泳动技术(Microscale Thermophoresis，MST)由于其无须固定蛋白，在一定程度上保证了蛋白的稳定性，并且灵敏度高、样品用量少，广泛应用于研究化合物与生物分子的相互作用中，成为药物靶点研究中的重要工具。

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MST技术原理

MST实验结果

案例一：小分子药物与靶蛋白结合验证

表面等离子共振（SPR）结合常数（KD）为278nM，表明藤黄酸（GBA）与靶蛋白CNPY3之间存在强相互作用（图C）。

微量热泳动（MST）：检测GBA和CNPY3的稳定结合曲线，KD为200nM（图D ）。

细胞热迁移实验（CETSA）：GBA处理在52-58℃的温度范围内提高了DU145细胞中CNPY3的热稳定性（图E-F ）。

这些结果联合证实了GBA和靶蛋白CNPY3之间的特定相互作用，表明 CNPY3 是 GBA 的细胞靶标[1]。

The equilibrium dissociation constant (KD) values were measured using the Monolith NT.115 instrument. The CNPY3 proteins were fluorescently labeled according to the manufacturer’s protocol. GBA was diluted to the indicated concentration and incubated with the labeled CNPY3 protein for 30 min in the dark. The samples were loaded into the NanoTemper glass capillaries, and microthermophoresis was carried out using 80% light emitting diode power and 80% MST. The KD values were calculated using the mass action equation via the NanoTemper software from duplicate reads of measurements.

案例二：关键结合氨基酸位点验证

利用pull down-WB、细胞热迁移实验（CETSA）证实野马追内酯B（EB）可以直接靶向BCAT1。分子对接结果表明EB可以与BCAT1的Cys335残基相互作用，对Cys335残基进行点突变，突变体和野生型rhBCAT1进行微量热泳动（MST）实验，结果表明BCAT1-C335A-突变体与EB的Kd值明显高于BCAT1-WT，表明Cys335有助于EB与rhBCAT1之间的亲和力(图3H)。文章整体研究结果证明EB可以直接与BCAT1蛋白结合并共价修饰BCAT1的Cys335残基[2]。

Human BCAT1 was cloned into the pET-28a vector for purification. BCAT1 were induced using 0.4 mM IPTG for 16 hours. Cells were cultured and resuspended in PBS containing 5 mM imidazole and lysed by sonication. The protein was purified using Ni-NTA beads and washed with buffer containing 25 mM imidazole added to lysis buffer. Finally, the protein was eluted in elution buffer containing 250 mM imidazole added to lysis buffer. In the BCAT1 mutations, Cys335 (TGT) is mutated to Ala (AAA). Monolith NT.115 (Nanotemper Technologies) was used to determine the binding affinity of the wild type and mutant rhBCAT1 to EB as previously described . Data analysis was performed using MO. Affinity Analysis v.2.3 software, and the Kd value is within the specified range.

案例三：蛋白与蛋白互作验证

通过 Co-IP 、GST pull-down实验发现咽峡炎链球菌病原体表面蛋白 TMPC 与胃黏膜上皮细胞受体 Annexin A2 (ANXA2) 存在直接相互作用；利用微量热泳动 (MST) 验证 TMPC 与 ANXA2 的结合亲和力，Kd值为 57.4 μM。文章整体研究表明 TMPC 与 Annexin A2 发生结合，通过 MAPK 信号通路促进胃癌发生[2]。

MST was performed using the Monolith NT.115 (NanoTemper Technologies). Recombinant Annexin A2 was served as target and labeled with fluorescent by His-Tag Labeling Kit Red-tris-NTA (MO-L018, NanoTemper Technologies). Recombinant GST-TMPC was employed as the ligand. Proteins were prepared in MST buffer (1 X PBS, pH 7.2, 0.05% Tween-20, 2.5 mM DTT). Samples were loaded into NT.115 standard capillaries. Analysis was performed at 25 °C, 40% excitation power, 40% MST power and a constant Annexin A2 concentration of 50 nM. Apparent Kd value was calculated using MO Affinity Analysis v.3.0.5.

MST技术在进行互作亲和力检测时，不依赖于分子量的变化，即使是几十个道尔顿的离子和蛋白的亲和力也可以轻松胜任。此外，MST亲和力检测范围宽（pM-mM），可研究不同强度亲和力的互作。MST可与其他分子互作技术如表面等离子体共振技术（SPR）、细胞热位移（CETSA）等联用，相互印证结果后继续进行细胞及动物实验，为药物与靶点相互作用的研究提供有力工具。

集思慧远致力于为医药类客户提供ABPP/TPP小分子药物靶标鉴定，虚拟筛选、分子对接、动力学模拟分析，SPR/MST/CETSA等相关分子互作验证实验和蛋白表达纯化服务，可为您提供一站式的药物发现解决方案！欢迎来询。

参考文献：

[1]Zhang, Xiao-Wen., Li, Ling., Liao, Min., Liu, Dan.,  & Rehman, Asma.. (2024). Thermal Proteome Profiling Strategy Identifies CNPY3 as a Cellular Target of Gambogic Acid for Inducing Prostate Cancer Pyroptosis. Journal of medicinal chemistry.

[2]Huang, Ling., Li, Guanjun., Zhang, Ying., Zhuge, Ruishen.,  & Qin, Shijie.. (2024). Small-molecule targeting BCAT1-mediated BCAA metabolism inhibits the activation of SHOC2-RAS-ERK to induce apoptosis of Triple-negative breast cancer cells. Journal of advanced research.

[3]Fu, Kaili., Cheung, Alvin Ho Kwan., Wong, Chi Chun., Liu, Weixin.,  & Zhou, Yunfei.. (2024). Streptococcus anginosus promotes gastric inflammation, atrophy, and tumorigenesis in mice. Cell.

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