TW-37 is an nonpeptide inhibitor to recombinant Bcl-2, Bcl-xL and Mcl-1 (Ki: 0.29/1.11/0.26 μM).

MCL1:0.26 μM(Ki), BCL2:0.29 μM(Ki), BCL-XL:1.11 μM(Ki)

TW-37针对Bcl-2的BH3结合凹槽，这是促凋亡Bcl-2蛋白结合的位置，并且对Bcl-2和Mcl-1显示出比Bcl-xL更高的亲和力和选择性，Ki值分别为0.29 μM、0.26 μM和1.11 μM。[1] 在体外研究中，TW-37在WSU-DLCL2淋巴瘤细胞系和来自淋巴瘤患者的原代细胞中显示出显著的抗增殖和促凋亡效果，而对正常外周血淋巴细胞没有影响。[1] TW-37在内皮细胞中展现了对细胞生长和细胞死亡的抑制作用，IC50约为1.8 μM，对同一浓度范围内的成纤维细胞没有影响。此外，TW37还在MCF-7、LNCaP和SLK肿瘤细胞系中展示了抗增殖效果，并且所需浓度范围等同或低于抑制内皮细胞生长所需的浓度。[2]

TW-37在单独使用时，对重组免疫缺陷（SCID）小鼠进行三次静脉注射，最大耐受剂量（MTD）为40 mg/kg，并能增强环磷酰胺-多柔比星-长春新碱-强的松（CHOP）方案的抑瘤效果。[1] 通过静脉（i.v.）给药，TW-37能通过降低功能性人类微血管密度，在重组免疫缺陷小鼠的人类血管生成模型中产生抗血管生成效应。[2] TW-37与MEK抑制剂的联合使用，通过显著减少肿瘤体积和肿瘤质量，协同阻断小鼠中的黑色素瘤细胞生长。[3]

Fluorescence polarization-based binding assay for recombinant Bcl-2, Bcl-XL, and Mcl-1 protein : For this assay, the 21-residue BH3 peptide QEDIIRNIARHLAQVGDSMDR derived from Bid labeled with 6-carboxyfluorescein succinimidyl ester (FAM-Bid) and recombinant proteins derived from human Bcl-2,Bcl-X L,and Mcl-1 are employed. It is determined that FAM-Bid has a Ki of 11 nM to Bcl-2 protein,25 nM to Bcl-XL protein,and 5.7 nM to Mcl-1 protein. The competitive binding assay for Bcl-XL is same as that for Bcl-2 with the following exceptions: 30 nM Bcl-XL protein and 2.5 nM FAM-Bid peptide in the following assay buffer [50 mM Tris-Bis (pH 7.4) and 0.01% bovine gamma-globulin].

The sulforhodamine B (SRB) cytotoxicity assay is used as described. Briefly, optimal cell density for cytotoxicity assay is determined by growth curve analysis. HDMECs are seeded in a 96-well plate and allowed to adhere overnight. Drug or control is diluted in EGM2-MV and layered onto cells, which are allowed to incubate for times as indicated in the figures. Alternatively, HDMECs are coincubated with TW37 and 0 to 100 ng/mL recombinant human VEGF (rhVEGF)165 or 0 to 100 ng/mL recombinant human CXCL8. Cells are fixed on the plates by addition of cold trichloroacetic acid (10% final concentration) and incubation for 1 hour at 4 °C. Cellular protein is stained by addition of 0.4% SRB in 1% acetic acid and incubation at room temperature for 30 minutes. Unbound SRB is removed by washing with 1% acetic acid and the plates are air dried. Bound SRB is resolubilized in 10 mM unbuffered Tris-base and absorbance is determined on a microplate reader at 560 nm. Test results are normalized against initial plating density and drug-free controls. Data are obtained from triplicate wells per condition and are representative of at least three independent experiments(Only for Reference)