| 包装 | 价格(元) |
| 5mg | 电议 |
| 10mg | 电议 |
| 50mg | 电议 |
Kinase experiment: | The Hsp90 FP competition assays are carried out in black 96-well micro-plates in a total volume of 100 μL in each well. A stock of 10 μM cy3B-GM and PU-FITC3 is prepared in DMSO and diluted with Felts buffer (20 mM Hepes (K), pH 7.3, 50 mM KCl, 2 mM DTT, 5 mM MgCl2, 20 mM Na2MoO4 and 0.01% NP40 with 0.1 mg/mL BGG). To each well is added the fluorescent dye-labeled Hsp90 ligand (6 nM cy3B-GM for Hsp90α, Hsp90β and Grp94 and 3 nM PU-FITC3 for Trap-1), protein (10 nM Hsp90α, 10 nM Hsp90β, 10 nM Grp94, 30 nM Trap-1) and tested inhibitor (including PU-WS13, initial stock in DMSO) in a final volume of 100 μL Felts buffer. Compounds are added in duplicate or triplicate wells. For each assay, background wells (buffer only), tracer controls (free, fluorescent dye-labeled Hsp90 ligand only) and bound controls (fluorescent dye-labeled Hsp90 ligand in the presence of protein) are included on each assay plate. The assay plate is incubated on a shaker at 4℃ for 24 h, and the FP values (in mP) are measured. The fraction of fluorescent dye-labeled Hsp90 ligand bound to Hsp90 is correlated to the mP value and plotted against values of competitor concentrations. The inhibitor concentration at which 50% of bound fluorescent dye-labeled Hsp90 ligand is displaced is obtained by fitting the data. For cy3B-GM, an excitation filter at 530 nm and an emission filter at 580 nm are used with a dichroic mirror of 561 nm. For PU-FITC3, an excitation filter at 485 nm and an emission filter at 530 nm are used with a dichroic mirror of 505 nm. All of the experimental data are analyzed, and binding affinity values are given as relative binding affinity values (EC50, concentration at which 50% of fluorescent ligand is competed off by compound)[1]. |
Cell experiment: | Cells are treated for 72 h with inhibitors (including PU-WS13) or transfected with Grp94 siRNA or control siRNA, and their viability is assessed using CellTiter-Glo luminescent Cell Viability Assay. The method determines the number of viable cells in culture based on quantification of the ATP present, which signals the presence of metabolically active cells[1]. |
| 产品描述 | The Hsp90 family of molecular chaperones regulates and maintains cell homeostasis under proteotoxic stress and pathogenic pressure. In humans, Hsp90α and Hsp90β in the cytoplasm, Grp94 in the endoplasmic reticulum and Trap-1 in the mitochondria are the four Hsp90 paralogs. PU-WS13 is a potent, Grp94-specific Hsp90 inhibitor of the purine scaffold class. In vitro: Brief treatment of SKBr3 cells with PU-WS13 disrupted the circular architecture of HER2 at the plasma membrane, resulting in a ‘shredded’ HER2 pattern. The effect of PU-WS13 on the HER2 surface architecture is mediated through Grp94. Upon treatment of cells with PU-WS13, a rapid accumulation of cells in the sub-G1 phase, PARP cleavage and a substantial increase in markers of early- and late-stage apoptosis were noted [1]. In vivo: Potent and durable anti-tumor effects in TNBC xenografts, including complete response and tumor regression, without toxicity to the host are achieved with PUH71, a chemical analogue of PU-WS13. Notably, TNBC tumors respond to retreatment with PUH71 for several cycles extending for over 5 months without evidence of resistance or toxicity [2]. Clinical trial: Up to now, PU-WS13 is still in the preclinical development stage. Reference: |
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