A fluorescence polarization assay for inhibitors of Hsp90

Howes, Ruth; Barril, Xavier; Dymock, Brian; Grant, Kate; Northfield, Christopher J.; Robertson, Alan; Surgenor, A.E.; Wayne, Joanne; Wright, Lisa; James, Karen; Matthews, Thomas P.; Cheung, Kwai-Ming J.; McDonald, Edward; Workman, Paul; Drysdale, Martin J.

doi:10.1016/j.ab.2005.12.023

Cited by 78 publications

(69 citation statements)

References 46 publications

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“…This correlated with the K d of 0.79 ± 0.38 μM, which was determined by filter binding assay for saturation binding and also correlated with that reported value for the K d of 17AAG for both the N-terminal domain of HSP90 and full-length HSP90. In contrast, we found Ub0 and decyl-Ub have no affinity for the N-terminal domain of HSP90 in filter binding assays at concentrations of 10 −4 to 10 −11 M. In addition, radicicol, a known inhibitor of HSP90 function, showed strong affinity toward the N-terminal domain of HSP90 by competitive binding experiments with a K i of 21.5 ± 6.8 nM, in line with that previously reported (20)(21)(22). These analyses suggest that GA compounds, due to the benzoquinone moiety, can bind to mitochondrial VDAC and that benzoquinones influence mitochondria directly.…”

supporting

confidence: 69%

Benzoquinone ansamycin 17AAG binds to mitochondrial voltage-dependent anion channel and inhibits cell invasion

Xie

Wondergem

Shen

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Geldanamycin and its derivative 17AAG [17-(Allylamino)-17-demethoxygeldanamycin, telatinib] bind selectively to the Hsp90 chaperone protein and inhibit its function. We discovered that these drugs associate with mitochondria, specifically to the mitochondrial membrane voltage-dependent anion channel (VDAC) via a hydrophobic interaction that is independent of HSP90. In vitro, 17AAG functions as a Ca 2+ mitochondrial regulator similar to benzoquinone-ubiquinones like Ub0. All of these compounds increase intracellular Ca 2+ and diminish the plasma membrane cationic current, inhibiting urokinase activity and cell invasion. In contrast, the HSP90 inhibitor radicicol, lacking a bezoquinone moiety, has no measurable effect on cationic current and is less effective in influencing intercellular Ca 2+ concentration. We conclude that some of the effects of 17-AAG and other ansamycins are due to their effects on VDAC and that this may play a role in their clinical activity.

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supporting

confidence: 69%

Benzoquinone ansamycin 17AAG binds to mitochondrial voltage-dependent anion channel and inhibits cell invasion

Xie

Wondergem

Shen

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…Competitive binding of inhibitors to full-length human HSP90h was measured using a fluorescent pyrazole resorcinol probe (41). Relative binding affinities for HSP90 in lysates from cancer and nontumorigenic cell lines were also measured by fluorescence polarization assay.…”

Section: Methodsmentioning

confidence: 99%

“…As before (15), 17-AAG was much less active in the BE human colon tumor cells, which carry an inactivating NQO1 polymorphism, compared with HT29 counterparts with naturally high NQO1 levels (P < NOTE: (A) Enzyme assays were carried out using full-length yeast Hsp90 or human HSP90h, the latter in the presence of the activating protein AHA1, using the malachite green method (34). The fluorescence polarization competitive binding assay was carried out with human HSP90h using a fluorescent pyrazole resorcinol probe (41). Cell growth inhibitory activities are also shown; these were carried out in HCT116 human colon cancer cells using the SRB endpoint.…”

Section: Cancer Researchmentioning

confidence: 99%

In vitro Biological Characterization of a Novel, Synthetic Diaryl Pyrazole Resorcinol Class of Heat Shock Protein 90 Inhibitors

et al. 2007

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The molecular chaperone heat shock protein 90 (HSP90) has emerged as an exciting molecular target. Derivatives of the natural product geldanamycin, such as 17-allylamino-17-demethoxy-geldanamycin (17-AAG), were the first HSP90 ATPase inhibitors to enter clinical trial. Synthetic smallmolecule HSP90 inhibitors have potential advantages. Here, we describe the biological properties of the lead compound of a new class of 3,4-diaryl pyrazole resorcinol HSP90 inhibitor (CCT018159), which we identified by high-throughput screening. CCT018159 inhibited human HSP90B with comparable potency to 17-AAG and with similar ATP-competitive kinetics. X-ray crystallographic structures of the NH 2 -terminal domain of yeast Hsp90 complexed with CCT018159 or its analogues showed binding properties similar to radicicol. The mean cellular GI 50 value of CCT018159 across a panel of human cancer cell lines, including melanoma, was 5.3 Mmol/L. Unlike 17-AAG, the in vitro antitumor activity of the pyrazole resorcinol analogues is independent of NQO1/ DT-diaphorase and P-glycoprotein expression. The molecular signature of HSP90 inhibition, comprising increased expression of HSP72 protein and depletion of ERBB2, CDK4, C-RAF, and mutant B-RAF, was shown by Western blotting and quantified by time-resolved fluorescent-Cellisa in human cancer cell lines treated with CCT018159. CCT018159 caused cell cytostasis associated with a G 1 arrest and induced apoptosis. CCT018159 also inhibited key endothelial and tumor cell functions implicated in invasion and angiogenesis. Overall, we have shown that diaryl pyrazole resorcinols exhibited similar cellular properties to 17-AAG with potential advantages (e.g., aqueous solubility, independence from NQO1 and P-glycoprotein). These compounds form the basis for further structure-based optimization to identify more potent inhibitors suitable for clinical development. [Cancer Res 2007;67(5):2206-16]

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“…Fluorescence Polarization Assay Binding of HSP90 inhibitors to human full-length recombinant HSP90h was determined by a competitive binding fluorescence polarization assay, using a fluorescent pyrazole resorcinol probe (34).…”

Section: Methodsmentioning

confidence: 99%

Inhibition of the heat shock protein 90 molecular chaperone in vitro and in vivo by novel, synthetic, potent resorcinylic pyrazole/isoxazole amide analogues

Sharp

Prodromou

Boxall

et al. 2007

Molecular Cancer Therapeutics

139

103

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Although the heat shock protein 90 (HSP90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) shows clinical promise, potential limitations encourage development of alternative chemotypes. We discovered the 3,4-diarylpyrazole resorcinol CCT018159 by high-throughput screening and used structure-based design to generate more potent pyrazole amide analogues, exemplified by VER-49009. Here, we describe the detailed biological properties of VER-49009 and the corresponding isoxazole VER-50589. X-ray crystallography showed a virtually identical HSP90 binding mode. However, the dissociation constant (K d ) of VER-50589 was 4.5 F 2.2 nmol/L compared with 78.0 F 10.4 nmol/L for VER-49009, attributable to higher enthalpy for VER-50589 binding. A competitive binding assay gave a lower IC 50 of 21 F 4 nmol/L for VER-50589 compared with 47 F 9 nmol/L for VER-49009. Cellular uptake of VER-50589 was 4-fold greater than for VER-49009. Mean cellular antiproliferative GI 50 values for VER-50589 and VER-49009 for a human cancer cell line panel were 78 F 15 and 685 F 119 nmol/L, respectively, showing a 9-fold potency gain for the isoxazole. Unlike 17-AAG, but as with CCT018159, cellular potency of these analogues was independent of NAD(P)H:quinone oxidoreductase 1/DT-diaphorase and Pglycoprotein expression. Consistent with HSP90 inhibition, VER-50589 and VER-49009 caused induction of HSP72 and HSP27 alongside depletion of client proteins, including C-RAF, B-RAF, and survivin, and the protein arginine methyltransferase PRMT5. Both caused cell cycle arrest and apoptosis. Extent and duration of pharmacodynamic changes in an orthotopic human ovarian carcinoma model confirmed the superiority of VER-50589 over VER-49009. VER-50589 accumulated in HCT116 human colon cancer xenografts at levels above the cellular GI 50 for 24 h, resulting in 30% growth inhibition. The results indicate the therapeutic potential of the resorcinylic pyrazole/isoxazole amide analogues as

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A fluorescence polarization assay for inhibitors of Hsp90

Cited by 78 publications

References 46 publications

Benzoquinone ansamycin 17AAG binds to mitochondrial voltage-dependent anion channel and inhibits cell invasion

Benzoquinone ansamycin 17AAG binds to mitochondrial voltage-dependent anion channel and inhibits cell invasion

In vitro Biological Characterization of a Novel, Synthetic Diaryl Pyrazole Resorcinol Class of Heat Shock Protein 90 Inhibitors

Inhibition of the heat shock protein 90 molecular chaperone in vitro and in vivo by novel, synthetic, potent resorcinylic pyrazole/isoxazole amide analogues

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