VER-82576 (NVP-BEP800), a synthetic HSP90β inhibitor (IC50: 58 nM), exhibits >70-fold selectivity against Hsp90 family members Trap-1 and Grp94.

HSP90 β:58 nM

在A375恶性黑色素移植瘤模型中,NVP-BEP800（15或 30 mg/kg/day,p.o.）处理15天,可剂量依赖性降低B-Raf和Akt磷酸化水平,且有抗癌活性,NVP-BEP800（15和30 mg/kg/day）处理,T/C值分别为53%和6%,每天30 mg/kg则几乎抑制全部肿瘤.在BT-474乳腺癌移植瘤中,NVP-BEP800处理可提高Hsp90-p23复合体解离,并降低稳定态ErbB2,p-Akt和p-S6水平,,该作用呈剂量依赖性,NVP-BEP800（30 mg/kg/day）处理可使肿瘤衰退38%.每天15 mg/kg处理则T/C为36%.

NVP-BEP800有效抑制包括A375细胞（GI50：38 nM）和PC3细胞（GI50：1.05 μM）在内的多种肿瘤细胞系增殖。在A2058和A549细胞中,NVP-BEP800（GI50的5倍）可提高G2-M期百分数。在BT-474细胞中,NVP-BEP800可浓度依赖性地引起Akt和ErbB2去磷酸化、ErbB2降解和Hsp70诱发（IC50：218/39.5/137/207 nM）。Hsp70浓度为10 μM时, NVP-BEP800对密切相关的GHKL ATP酶、拓扑异构酶 II和结构不相关的ATP酶均无明显抑制效果。

Competitive binding fluorescent polarization assay: Recombinant Hsp90β, TAMRA-radicicol, or various concentrations of NVP-BEP800 is added in assay buffer (50 mM TRIS pH 7.4, 5 mM MgCl2, 150 mM KCl, and 0.1% CHAPS), mixed, and incubated at room temperature for 30 to 45 minutes prior to reading. The 2D-FIDA-based HTS assay based on confocal technologies monitors the decreased fluorescence polarization on displacement of the high affinity ligand TAMRA-radicicol from Hsp90β by NVP-BEP800. The concentration of NVP-BEP800 which inhibits Hsp90β by 50% is determined from the competition curve.

Cells are exposed to NVP-BEP800 for 24 hours. Cell proliferation is determined using either sulforhodamine B for adherent cells or MTS assay for suspension cells or those showing low adherence. Cell death is determined using a ToxiLight nondestructive cytotoxicity bioassay kit. Cell cycle progression is determined by RNase A/propidium iodide staining following fixation in 70% ethanol. Caspase-3/7 activity is determined using a homogeneous caspase activity kit.(Only for Reference)