KW-2478 is a non-ansamycin HSP90 inhibitor with IC50 of 3.8 nM.

HSP90:3.8 nM

KW-2478 通过IC50为3.8 nM来抑制bRD与Hsp90α的结合。该化合物能降解Hsp90的客体蛋白，包括FGFR3、IGF-1Rβ和c-Raf-1。KW-2478减少磷酸化Erk1/2的水平，并通过PARP的裂解触发U266细胞中的凋亡，PARP是caspase-3的底物。KW-2478抑制细胞增殖活动存在时间依赖性，连续暴露至少12小时是必要的以发挥强大的抗肿瘤活性。KW-2478降低IgH位点的易位产物，并主要通过抑制Cdk9的功能来抑制c-Maf和cyclin D1基因的转录。文献[1]显示，KW-2478对各种细胞系具有强大且广泛的生长抑制活性，对B细胞性淋巴瘤、套细胞淋巴瘤和多发性骨髓瘤细胞系的癌细胞生长抑制，EC50分别为101-252 nM、81.4-91.4 nM和120-622 nM。KW-2478还对原发性CLL和NHL细胞显示出强大的生长抑制活性，EC50分别为40-170 nM和200-400 nM。文献[2]。

KW-2478在剂量达到或超过100 mg/kg时，通过诱导肿瘤中客体蛋白的降解，抑制NCI-H929 s.c.接种模型中的肿瘤生长。同样剂量下，KW-2478亦能降低OPM-2/GFP i.v.接种小鼠模型中的血清M蛋白水平和骨髓中的多发性骨髓瘤（MM）肿瘤负荷。[1]

Hsp90 Binding Assay: Human Hsp90α solution (0.5 μg/mL) is fixed on 96-well plates, followed by blocking with TBS containing 1% bovine serum albumin. KW-2478 solutions is added to the wells, and bRD is added to a concentration of 0.1 μmol/L. After removal of solution, poly-HRP streptavidin solution dilutes with poly-HRP dilution buffer is added to the wells. After removal of solution, equal volumes of TMB peroxidase substrate and peroxidase solution B are added to the wells. To stop the HRP reaction, 2 mol/L H2SO4 are added, followed by measurement of absorbance at 450 nm using a microplate spectrophotometer.

To measure the IC50, OPM-2/green fluorescent protein (GFP) cells, KMS-11 cells, OPM-2/GFP and other cells are plated into 96-well plates and treat with KW-2478. After 72 hours of cultivation, cell viability is determined using Cell Proliferation Reagent WST-1. WST reagent is added to the wells, followed by incubation for 4 hours at 37 °C. After that, the absorbance at 450 nm with reference at 650 nm is measured with a microplate spectrophotometer. To examine time dependency of antiproliferative activity of KW-2478, the cells are plated into 96-well V-bottomed plates and treated with KW-2478. After 0 hour and at intervals from 3 to 72 hours at 37 °C, the supernatant is aspirated. After drug-free medium is added to the wells, the supernatant is aspirated again. Finally, drug-free medium is added to the wells, and the plates are further incubated for the remainder of the 72-hour period, followed by measurement of cell viabil(Only for Reference)