以药找靶、药物与蛋白结合验证：TPP+CETSA+MST+分子对接+HDX-MS揭示tutin作用靶点

具体方法如下：将原代海马神经元与5μM tutin或PBS原位孵育3h，然后在不同温度下热处理3min（37-67℃），随即通过液氮反复冻融的方式提取总蛋白，再经高速离心去除沉淀后（15000×g，30min），取上清进行质谱鉴定。在鉴定到的蛋白质中，CNA（钙调磷酸酶）表现出了明显的热稳定改变，并且有报道称CNA可共同参与癫痫，并调节GABA和NMDA受体的活性，提示CNA可能是tutin的一个可能靶点。

为了验证tutin和CN的直接相互作用，作者进行了CETSA（细胞热迁移）实验，证明tutin能提高CN的热稳定性（图3a）；通过MST（微量热泳动）实验，验证了二者的直接相互作用，KD值为0.28±0.27 um（图3c）；同时为了探索tutin结合CN的氨基酸位点，作者采用了HDX-MS（氢氘交换质谱）技术，证明tutin和CN的结合，同时在质谱鉴定结果中鉴定到了7个同位素存在差异的肽段，作者对这7个多肽的氘摄取谱进行了分析，发现230-259，232-242，243-258的H/D交换速率明显变化，而这三部分肽段都位于CN催化亚基的活性位点（图3d）；根据HDX-MS的结果，作者使用薛定谔软件进行了分子对接，结果显示CN的Arg254、Ala283能和tutin形成氢键作用（图3e、3f）。

根据此结果作者构建了R254K和A283V两个突变体，MST实验发现，两个突变体和tutin的亲和力显著降低。综上所述，CN是tutin发挥作用的直接靶点，且CN的Arg254和Ala283对于二者的结合十分重要。

Neurons were heated at different temperatures (50-70℃) for 3 min and lysed, which were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and incubated with CNA antibody

MST实验方法

The proteins were labeled using the Monolith His-tag Labeling Kit RED-NHS 2nd Generation Kit. Tutin was diluted in a series of concentration. The labeled CN was diluted to working assay buffer (40nM, 0.05% Tween-20). The mixture was incubated and then loaded into Monolith standard-treated capillaries. The thermophoresis was detected by A Monolith NT.115 instrument (Nano Temper Technologies) and KD values was calculated by NT Analysis software (Nano Temper Technologies).

HDX-MS方法

Before 2 h of HDX analysis, the compound tutin (200 μM) was added into the sample, with the control sample adding an equal volume of tutin buffer. For deuterium labeling, CN (4 μM) in the buffer (20 mM Tris-HCl, 1 mM CaCl2, 0.5 mM TCEP, and 150 mM NaCl, in H2O, pH 7.5) in the presence or absence of 200 μM tutin was diluted 10-fold by the labeling buffer containing 20 mM TrisHCl, 1 mM CaCl2, 0.5 mM TCEP, and 150 mM NaCl, in 100% D2O at pD 7.4. After incubation for 30, 90 or 300 seconds at 25 °C, the same volume of ice-cold quench buffer containing 4 M guanidine hydrochloride, 500 mM TECP and 200 mM citric acid in water solution at pH 1.8, 100% H2O, was added to quench deuterium uptake. The sample was digested with pepsin (Promega) on ice for 5 min, and removed by centrifugation. An ACQUITY UPLC BEH C18 column (2.1 μm, 1.0 mm × 50 mm, Waters) equipped with an Ultimate 3000 UPLC system (Thermo Scientific) were used for the obtained peptides separation. A Q Exactive mass spectrometer was used for mass spectrometry analysis of the peptides. Mass spectrometry data were compared with Proteome Discoverer (Thermo Scientific) to match the corresponding peptide in CN. XCALIBUR (Thermo Scientific) was used to inspected peptide peaks. In order to estimate the max deuterium uptake of peptides, a repeated experiment was performed extending incubation in D2O for 24 h.

分子对接方法

The docking study was performed by using Schrodinger’s soft. The protein coordinates were retrieved from the Protein Data Bank (PDB code: 6NUU). The structures of tutin were generated and energy-minimized. Both the protein and the ligands were prepared by adding polar hydrogen atoms

首先作者通过体外体内实验，证明tutin能激活CN蛋白酶（图4a）；随后通过CN抑制剂FK506与tutin联用，小鼠实验中发现FK506能拮抗tutin诱导的癫痫（图4b）；最后敲低小鼠大脑中的CNA基因表达，发现敲低CNA基因表达可降低tutin诱导的癫痫和神经元损伤（图4c）。

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