Tanespimycin (KOS 953) (17-AAG) is an inhibitor of Hsp90 that selectively inhibits BT474 tumor cell Hsp90 (IC50: 5 nM).

HSP90:5 nM (cell free)

源自肿瘤细胞的Hsp90与Tanespimycin的结合亲和力是正常细胞中Hsp90的100倍。体外重组Hsp90伴侣复合物后，其与Tanespimycin的结合亲和力增强，且ATP酶活性增加[1]。Tanespimycin能促进HER2、Akt以及突变型和野生型AR的降解，并导致依赖视网膜母细胞瘤的G1期生长阻滞在前列腺癌细胞中[2]。Tanespimycin与Trastuzumab联合处理ErbB2过表达的乳腺癌细胞系，增强了ErbB2的泛素化、从细胞表面的下调及溶酶体降解[3]。

在非毒性剂量下，Tanespimycin导致前列腺癌异种移植瘤中AR、HER2和Akt表达水平呈剂量依赖性下降。这种下降速度很快，HER2和AR表达在4小时内分别下降了97%和80% [2]。相比之下，接受了Tanespimycin（5至40 mg/kg）治疗的小鼠的脾脏明显变小，脾脏中渗透的淋巴瘤细胞减少，转移至其他器官的癌细胞也显著降低，与接受车辆对照组治疗的小鼠相比。此外，接受Tanespimycin治疗的小鼠比仅接受车辆处理的小鼠存活时间显著延长 [4]。

Purified native Hsp90 protein or cell lysates in lysis buffer (20 mM HEPES, pH 7.3, 1 mM EDTA, 5 mM MgCl2, 100 mM KCl) were incubated with or without 17-AAG for 30 min at 4 °C, and then incubated with biotin-GM linked to streptavidin magnetic beads for 1 h at 4 °C. Tubes were placed on a magnetic rack, and the unbound supernatant removed. The magnetic beads were washed three times in lysis buffer and heated for 5 min at 95 °C in SDS–PAGE sample buffer. Samples were analyzed on SDS protein gels, and western blots done using indicated antibodies. Bands in the western blots were quantified, and the percentage inhibition of binding of Hsp90 to the biotin-GM was calculated. The IC50 reported is the concentration of 17-AAG needed to cause half-maximal inhibition of binding. For in vitro reconstitution, 5 μM of purified Hsp90 was combined with 1 μM each of Hsp70, Hsp40, p23, and Hop purified proteins [1].

Cells were seeded in 96-well plates at 2,000 cells per well in a final culture volume of 100 μl for 24 h before the addition of increasing concentrations of 17-AAG that was incubated for 5 days. Viable cell number was determined using the Celltiter 96 AQueous Nonradioactive Cell Proliferation Assay. The value of the background absorbance at 490 nm (A490) of wells not containing cells was subtracted. Percentage of viable cells ? (A490 of 17-AAG treated sample/A490 untreated cells) × 100. The IC50 was defined as the concentration that gave rise to 50% viable cell number [1].

B10.BR mice were inoculated with 5×10^5 lymphoma cells through intraperitoneal injection. Seven days following tumor implantation, the mice were I.P. injected with 17-AAG or vehicle (10% DMSO + 40% Cremophor EL: Ethanol (3:1) (v/v) + 50 % PBS) every other day for three weeks. At the cessation of treatment, mice were monitored up to 80 days post tumor cell injection. To determine the effects of 17-AAG on lymphoma initiation in vivo, secondary B10.BR recipient mice were implanted by intraperitoneal injection of 1×10^5 lymphoma cells from the spleens of first-round mice that had been treated with 17-AAG or vehicle. These mice were followed up to 160 days post tumor cell injection to monitor differences in tumor initiation between the mice [4].