Alvespimycin hydrochloride (BMS 826476) is a potent HSP90 inhibitor with IC50 of 62 nM. Phase 2.

HSP90:62 nM

Alvespimycin相比17-AAG对人类Hsp90的活性约高出2倍，IC50分别为62 nM和119 nM。在过表达Hsp90客体蛋白Her2的SKBR3和SKOV3细胞中，Alvespimycin分别以EC50 8 nM和46 nM下调Her2表达，并分别以EC50 4 nM和14 nM诱导Hsp70表达，从而导致显著细胞毒性。[1] 与vorinostat共用，Alvespimycin在培养的MCL细胞和原发性MCL细胞中协同诱导凋亡，通过显著减少cyclin D1和CDK4的水平，以及c-Myc、c-RAF和Akt的水平，其效果比单独使用任一化合物更为强大。[3] 与仅在慢性淋巴细胞性白血病(CLL)细胞中对IKKβ活跃的17-AAG相比，Alvespimycin有效地导致Hsp90客体蛋白的耗尽，从而减少NF-κB p50/p65 DNA结合、降低NF-κB目标基因的转录，并通过caspase依赖的方式诱导凋亡。通过靶向NF-κB家族，Alvespimycin选择性地介导对CLL细胞的剂量和时间依赖的细胞毒性，但不影响对免疫监视重要的正常T细胞或NK细胞。[5]

Alvespimycin以5 mg/kg或25 mg/kg剂量，每周三次处理，显著抑制TMK-1异种移植瘤的生长，主要通过显著减少血管面积和增殖中的肿瘤细胞数量实现。[2] 一致地，通过抑制体内FAK信号传导，每周三次以25 mg/kg剂量的Alvespimycin处理显著抑制了ME180和SiHa异种移植瘤在小鼠中的生长和转移。[4] 在TCL1-SCID移植小鼠模型中，连续16天以10 mg/kg剂量给予Alvespimycin显著减少白细胞计数，并延长存活时间。[5]

Fluorescence polarization (FP)-based competition binding assay: This assay utilizes a boron difluoride dipyrromethene (BODIPY) labeled geldanamycin analogue (BODIPY-AG) as a probe and measured fluorescence polarization upon binding of the probe to a protein. Native human Hsp90 protein (α + β isoforms) is isolated from HeLa cells. BODIPY-AG solution is freshly prepared in FP assay buffer (20 mM HEPES-KOH, pH 7.3, 1.0 mM EDTA, 100 mM KCl, 5.0 mM MgCl2, 0.01% NP-40, 0.1 mg/mL fresh bovine γ-globulin (BGG), 1.0 mM fresh DTT, and protease inhibitor from stock solution in DMSO. Competition curves are obtained by mixing 10 μL each of a solution containing BODIPY-AG and Hsp90, and a serial dilution of 17-DMAG freshly prepared in FP assay buffer from stock solution in DMSO. Final concentrations are 10 nM BODIPY-AG, 40 or 60 nM Hsp90, varying concentration of 17-DMAG (0.10 nM-10 μM), and ≤0.25% DMSO in a 384-well microplate. After 3 hours incubation at 30 °C, fluorescence anisotropy (γEx = 485 nm, γEm = 535 nm) is measured on an EnVision 2100 multilabel plate reader. IC50 value of 17-DMAG is obtained from the competition curves.

Cells are exposed to various concentrations of 17-DMAG for 24, or 48 hours. For the assessment of cytotoxicity, MTT reagent is then added, and plates are incubated for an additional 24 hours before spectrophotometric measurement. Apoptosis is determined by staining with annexin V-fluorescein isothiocyanate and propidium iodide (PI).(Only for Reference)