Development of the high-throughput screening fluorescence polarization assay for HSP90 inhibitor / 中国生化药物杂志

Objective To establish the high-throughput screening fluorescence polarization assay for HSP90 inhibitor.Methods E.coli strain BL21 ( DE3) competent cells were transformed with pET24α( +)-HSP90αplasmid.The cell lysate supernatant was induced to product the soluble protein and purified with Ni-NTA agarose.Western blot analysis was used to identify whether the purified protein is HSP90α.The fluorescence polarization assay for screening HSP90 inhibitors was established and optimized using varying concentrations of recombinant HSP90 protein and molecular probe VER00051001.Meanwhile, the binding activity of GA and NVP-AUY922 for HSP90αwas measured by fluorescence polarization assay.Results HSP90αwas induced expression and purified successfully.The fluorescence polarization assay was performed using 80 nM probe VER00051001 and 2.01μg/mL HSP90α, with the Z factor of 0.83.GA and NVP-AUY922 competed with the probes VER00051001 for binding sites of HSP90, with IC50 of 55 nM and 13 nM, respectively.Conclusion A reliable model was established using fluorescence polarization assay for screening HSP90 inhibitors.

The advances of the HSP90 inhibitor NVP-AUY922:applied alone or in combination with anti-tumor treatment / 实用肿瘤学杂志

Heat shock protein 90(Hsp90)is a highly conserved protein which have been proved to play an important role in the development and progression of malignant transformation .As one of small molecule inhibi-tors that has been detected to have potent antitumor activity in a wide range of malignancies ,NVP-AUY922 is a pyrazole scaffold drug with many advantages such as low toxicity and stable structure .As a result of this,NVP-AUY922 is extensively considered as a new promising kind of anti -tumor drug .This review intends to update the reader on advances made over the past four years in the clinical development of NVP -AUY922 in advanced cancers.

In Vitro Antifungal Activity and Mode of Action of 2',4'-Dihydroxychalcone against Aspergillus fumigatus

2',4'-Dihydroxychalcone (2',4'-DHC) was identified from a heat shock protein 90 (Hsp90)-targeting library as a compound with Hsp90 inhibitory and antifungal effects. In the presence of 2',4'-DHC (8 microg/mL), radial growth of Aspergillus fumigatus was inhibited 20% compared to the control, and green pigmentation was completely blocked. The expression of the conidiation-associated genes abaA, brlA, and wetA was significantly decreased (approximately 3- to 5-fold) by treatment with 2',4'-DHC. The expression of calcineurin signaling components, cnaA and crzA, was also significantly reduced. The inhibitory effects of 2',4'-DHC on metabolic activity and mycelial growth were significantly enhanced by combination treatment with itraconazole and caspofungin. Docking studies indicated that 2',4'-DHC bind to the ATPase domain of Hsp90. These results suggest that 2',4'-DHC act as an Hsp90-calcinurin pathway inhibitor.

Heat Shock Protein 90 (HSP90) and Immune Regulation

Heat shock protein 90 (HSP90) is involved in conformational and structural maturation of signalling molecules and transcription factors in immune reaction. HSP90 inhibitors induce immune modulation via anti-inflammatory effect, regulating humoral and cellular immune responses. Therefore, HSP90 inhibitors potentially useful target for the autoimmune disease and chronic inflammatory diseases.

The effects of heat shock protein 90 inhibitor 17-AAG on the proliferation and cell cycles of LoVo cells / 中国癌症杂志

Background and purpose:Hsp90 is cell chaperone protein that interacts with many proteins,but itself can not degrade its client proteins.Hsp90 inhibitor can inhibit tumor cell proliferation,induce cell apoptosis,cell growth arrest and increase the degradation of Hsp90 client proteins like Survivin.In order to explore the co-effects of Hsp90 inhibitor 17-AAG and Survivin on LoVo cells and the possible mechanisms,we observed the effects of 17-AAG on proliferation and cycles of LoVo cells and the protein level of Survivin.Methods:LoVo cells were treated with 17-AAG.The cell proliferation inhibition rate was evaluated by MTT assay.The cell cycle was detected by ? ow cytometry.The expression of Hsp90 client protein Survivin was detected by Western blot.Results:17-AAG time-dose-dependently inhibit the proliferation of LoVo cells,after 100 ng/ml,500 ng/ml and 800 ng/ml 17-AAG exposure for 24 hrs,the cell proliferation inhibition rate was 21.00%,40.81%,60.34% respectively,after exposure for 48 hrs,the cell proliferation inhibition rate was increased to 27.29%,48.17%,80.97% respectively,after exposure for 72 hrs,the cell proliferation inhibition rate was to 34.45%,67.81%,88.42%;17-AAG arrested cell cycle,when LoVo cells were exposed to 100 ng/ml 17-AAG for 72 hrs,the cell ratio of G0/G1 phase was(61?3)%,when to 500 ng/ml for 72 hrs,cell ratio of G0/G1 phase was increased to(74?3)%,compared to(48.2?0.8)% LoVo cells without 17-AAG(P