肿瘤细胞的重要特征之一就是正常细胞凋亡程序“关闭”，相关信号通路上的成员因此成为潜在的药物靶点。而“信号通路的开关”与“信号通路的传导”一样，是通过通路上蛋白与蛋白之间的相互作用来完成，将靶点机制设定为信号通路上两个蛋白结合的抑制剂可以更精准的控制调节下游基因的表达。通过Bcl-2/Bcl-XL、IAP或MDM2-p53的PPI抑制剂重启肿瘤细胞的凋亡程序，这样的设计相对精妙。此类靶点的药物研发长期受到罗氏、诺华、安进、艾伯维等国际巨头关注。下文列举了此类药物在细胞水平和动物实验中使用MSD技术检测信号通路的案例。

Bcl-2抑制剂 – FDA首个蛋白相互作用抑制剂Venetoclax

2016年，FDA批准了艾伯维和罗氏旗下基因泰克的Bcl-2抑制剂Venetoclax（商品名Venclexta），用于一种叫做p17缺失型CLL的二线疗法。这类病人通常预后较差，没有什么治疗选择。在一个107人参与的二期临床中，80%复发p17缺失型CLL病人对Venetoclax应答，其中85%病人应答维持至少一年。Venetoclax是第一个真正意义上的蛋白-蛋白相互作用（PPI）抑制剂，也是第一个诱导细胞死亡（apoptosis）药物，所以在科学上是个重要进展。Venetoclax目前正在多种血液癌症的临床试验中。

MDM2-P53 抑制剂

癌症的发病率越来越高，研发抗肿瘤药物迫在眉捷。随着科技的进步，医学研究者对作用机制的不断深入研究，抗癌药物有了很大的进展。针对抑癌基因p53和MDM2之间的调控机制，人们发现通过干扰两者的相互作用，肿瘤细胞的活性会明显减弱。因此，设计p53-MDM2相互作用的抑制剂成为了治疗癌症的重要方向。根据近几年的研究报道，主要有两类：一类为化学合成的多肽及其类似物；另一类是利用基因工程对野生型p53进行结构改造所得的药物包含肽类或非肽类小分子抑制物等等。

案例一：BCL-2抑制剂Venetoclax使用MSD检测MCL-1-BIM complexes 

Expression Profile of BCL-2, BCL-XL, and MCL-1 Predicts Pharmacological Response to the BCL-2 Selective Antagonist Venetoclax in Multiple Myeloma Models 

图一. Evaluation of BCL-2:BIM complexes in HMCLs pre- and post-treatment with venetoclax. 

BCL-2:BIM Complex Evaluation: BCL-2:BIM complexes were measured using an immunoassay platform. Briefly, 15 million MM cells were exposed to DMSO vehicle or 1 μM venetoclax for 24 hours and then processed for evaluation of the disruption of BCL-2:BIM complexes. Streptavidin coated plates was blocked for 16-24 hours with 40-150 μL blocking buffer (Mesoscale Discovery) while protein concentrations of cell lysates were adjusted to 4 mg/ml using cell lysis buffer. Anti-BCL-2 monoclonal antibody (Invitrogen) was labeled with 6:1 molar challenge ratio with ruthenium. Anti-BIM monoclonal antibody (Epitomics) was labeled with biotin at a 20:1 molar challenge ratio. Ruthenium-tagged anti-BCL-2 antibody was diluted to 1 μg/ml in incubation buffer and added to streptavidin-coated plates. An equal volume of cell lysates or BCL-2:BIM standard protein complexes were added to the plates and incubated at room temperature. Plates were washed with 0.5% polysorbate 20 in PBS followed by incubation with 1 μg/ml biotinylated anti-BIM antibody for 90 minutes at room temperature. Electrochemiluminescent signal intensity was measured on the Meso Scale Discovery SECTOR Imager 6000. Concentration of BCL-2:BIM complexes in samples treated with DMSO and venetoclax was determined by four-parameter fit logistic regression analysis based on the respective standard curves.

案例二：MCL-1抑制剂Navitoclax使用MSD检测MCL-1-BIM complexes检测

Potent and selective small-molecule MCL-1 inhibitors demonstrate on-target cancer cell killing activity as single agents and in combination with ABT-263 (navitoclax). 

图二. MCL-1 inhibitor A-1210477 disrupts of MCL-1–BIM complexes.

Electrochemiluminescent ELISA.

After compound treatments, cells were lysed in buffer containing 1% CHAPS, 10 mM HEPES, and 150 mM NaCl with protease inhibitor cocktail (Roche, Indianapolis, IN, USA). In all, 100 μg of cell lysate was added to each well of a Meso Scale Discovery (Rockville, MD, USA) 96-well plate precoated with biotinylated anti-MCL-1 antibody (clone RC13) from Thermo Fisher Scientific. MCL-1-BIM complexes were detected with rabbit anti-BIM antibody (clone Y36) from Abcam, followed by Sulfo-tagged anti-rabbit antibody (Meso Scale Discovery; cat no. R32AB-5) and Meso Scale Discovery reading buffer with surfactant (cat. no. R92TC-2) according to the manufacturer’s recommendations. Each step was followed by washing three times with PBS-T (PBS+0.1% Tween-20).

案例三： MCL-1抑制剂S63845使用MSD检测cleaved PARP

The MCL1 inhibitor S63845 is tolerable and effective in diverse cancer models (发表在Nature）

图三. S63845 induces apoptosis of sensitive tumour derived cell lines.

Immunodetection of cleaved PARP by MesoScale discovery assay. Twentyfour hours after seeding, cells were treated with the indicated compounds for 6 h and harvested in lysis buffer (10 mM HEPES pH 7.4, 142.5 mM KCl, 5 mM MgCl2, 1 mM EDTA, 1% NP40, protease and phosphatase inhibitors cocktails (Calbiochem)). Cleared lysates (5 μ g protein) were prepared for immunodetection of cleaved PARP (a marker of apoptosis) by using the MSD apoptosis panel whole cell lysate kit (MSD) in 96-well plates according to manufacturer’s instructions, and were analysed on the Sector Image 2400.

案例四：MDM2抑制剂SAR405838 使用MSD检测MDM2 , p21 ,  p53, Cleaved Caspase 3, cleaved PARP

SAR405838: A novel and potent inhibitor of the MDM2:p53 axis for the treatment of dedifferentiated liposarcoma.  

图四. Antitumor effects of SAR405838 in DDLPS xenograft models

For Mesoscale Discovery assays, protein analysis was performed after appropriate dilutions with following kits: MDM2 (K152FID), p21 (N45ZA-1), p53, Cleaved Caspase 3 and cleaved PARP (K15102D-1) with a detection on Sector Imager 2400. Results were normalized with total protein concentration.