A highly selective Hsp90 affinity chromatography resin with a cleavable linker

Hughes, Philip F.; Barrott, Jared J.; Carlson, David A.; Loiselle, David R.; Speer, Brittany L.; Bodoor, Khaldon; Rund, Laurie A.; Haystead, Timothy A.J.

doi:10.1016/j.bmc.2012.03.043

Cited by 29 publications

(51 citation statements)

References 47 publications

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“…Despite some reduction in affinity, the addition of the tethered components was found to increase specificity by eliminating binding to Grp94 (Figure S2B). Previous work had also shown that the addition of the tether at the ortho -position of the parent ligand reduced the specificity towards recombinant and native Trap-1 (Hughes et al, 2012). These findings suggest that the added steric bulk due to the presence of the tether and added fluor, for example, interferes with the ATP-binding site of Grp94 and Trap-1, but not Hsp90.…”

Section: Resultsmentioning

confidence: 98%

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Optical and Radioiodinated Tethered Hsp90 Inhibitors Reveal Selective Internalization of Ectopic Hsp90 in Malignant Breast Tumor Cells

Barrott

Hughes

Osada

et al. 2013

Chemistry & Biology

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Summary Hsp90 inhibitors have demonstrated unusual selectivity for tumor cells despite its ubiquitous expression. This phenomenon has remained unexplained but could be influenced by ectopically expressed Hsp90 in tumors. We have synthesized novel Hsp90 inhibitors that can carry optical or radioiodinated probes via a PEG tether. We show that these tethered inhibitors selectively recognize cells expressing ectopic Hsp90 and become internalized. The internalization process is blocked by Hsp90 antibodies, suggesting that active cycling of the protein is occurring at the plasma membrane. In mice, we show exquisite accumulation of the fluor-tethered versions within breast tumors at very sensitive levels. Cell-based assays with the radiolabeled version showed picomolar detection in cells that express ectopic Hsp90. Our findings show that fluor-tethered or radiolabeled inhibitors targeting ectopic Hsp90 can be used to detect breast cancer malignancies through non-invasive imaging.

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Section: Resultsmentioning

confidence: 98%

“…We recently reported the development of a cleavable tethered Hsp90 inhibitor and demonstrated its use as an affinity resin(Hughes et al, 2012). When bound to the tethered ligand, Hsp90 could be recovered along with one of its established oncogenic clients, Her2, in a competitive manner.…”

Section: Resultsmentioning

confidence: 99%

Optical and Radioiodinated Tethered Hsp90 Inhibitors Reveal Selective Internalization of Ectopic Hsp90 in Malignant Breast Tumor Cells

Barrott

Hughes

Osada

et al. 2013

Chemistry & Biology

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“…Specifically, the histidine-tagged yeast Hsp90 was loaded onto an affinity column [12] and was subsequently tested with these natural product. Mass spectrum analysis of the eluted solutions of proteins resulted in the identification of the compounds bound to Hsp90.…”

Section: Introductionmentioning

confidence: 99%

FW-04-806 inhibits proliferation and induces apoptosis in human breast cancer cells by binding to N-terminus of Hsp90 and disrupting Hsp90-Cdc37 complex formation

Huang

Zhang

et al. 2014

Mol Cancer

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BackgroundHeat shock protein 90 (Hsp90) is a promising therapeutic target and inhibition of Hsp90 will presumably result in suppression of multiple signaling pathways. FW-04-806, a bis-oxazolyl macrolide compound extracted from China-native Streptomyces FIM-04-806, was reported to be identical in structure to the polyketide Conglobatin.MethodsWe adopted the methods of chemproteomics, computational docking, immunoprecipitation, siRNA gene knock down, Quantitative Real-time PCR and xenograft models on the research of FW-04-806 antitumor mechanism, through the HER2-overexpressing breast cancer SKBR3 and HER2-underexpressing breast cancer MCF-7 cell line.ResultsWe have verified the direct binding of FW-04-806 to the N-terminal domain of Hsp90 and found that FW-04-806 inhibits Hsp90/cell division cycle protein 37 (Cdc37) chaperone/co-chaperone interactions, but does not affect ATP-binding capability of Hsp90, thereby leading to the degradation of multiple Hsp90 client proteins via the proteasome pathway. In breast cancer cell lines, FW-04-806 inhibits cell proliferation, caused G2/M cell cycle arrest, induced apoptosis, and downregulated Hsp90 client proteins HER2, Akt, Raf-1 and their phosphorylated forms (p-HER2, p-Akt) in a dose and time-dependent manner. Importantly, FW-04-806 displays a better anti-tumor effect in HER2-overexpressed SKBR3 tumor xenograft model than in HER2-underexpressed MCF-7 model. The result is consistent with cell proliferation assay and in vitro apoptosis assay applied for SKBR-3 and MCF-7. Furthermore, FW-04-806 has a favorable toxicity profile.ConclusionsAs a novel Hsp90 inhibitor, FW-04-806 binds to the N-terminal of Hsp90 and inhibits Hsp90/Cdc37 interaction, resulting in the disassociation of Hsp90/Cdc37/client complexes and the degradation of Hsp90 client proteins. FW-04-806 displays promising antitumor activity against breast cancer cells both in vitro and in vivo, especially for HER2-overexpressed breast cancer cells.

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“…1B and S1A). To determine if the induction of Hsp70i was the result of a general cellular stress in response to DENV infection or likely to be facilitated by the virus itself, we induced expression of the protein in uninfected cells using the Hsp90 inhibitor, HS10 (30). Induction of Hsp70i expression is a signature response to all Hsp90 inhibitors targeting the ATP binding domain of the protein (34–36).…”

Section: Resultsmentioning

confidence: 99%

“…HS-72 (27) and HS-10 (30) were synthesized as previously described and Ribavirin was purchased from Sigma-Aldrich.…”

Section: Methodsmentioning

confidence: 99%

An inducible heat shock protein 70 small molecule inhibitor demonstrates anti-dengue virus activity, validating Hsp70 as a host antiviral target

et al. 2016

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An estimated three billion people are at risk of Dengue virus (DENV) infection worldwide and there are currently no approved therapeutic interventions for DENV infection. Due to the relatively small size of the DENV genome, DENV is reliant on host factors throughout the viral life cycle. The inducible form of Heat Shock Protein 70 (Hsp70i) has been implicated as a host factor in DENV pathogenesis, however the complete role remains to be elucidated. Here we further illustrate the importance of Hsp70i in dengue virus pathogenesis and describe the antiviral activity of the allosteric small molecule inhibitor that is selective for Hsp70i, called HS-72. In monocytes, Hsp70i is expressed at low levels preceding DENV infection, but Hsp70i expression is induced upon DENV infection. Targeting Hsp70i with HS-72, results in a dose dependent reduction in DENV infected monocytes, while cell viability was maintained. HS-72 works to reduce DENV infection by inhibiting the entry stage of the viral life cycle, through disrupting the association of Hsp70i with the DENV receptor complex. This work highlights Hsp70i as an antiviral target and HS-72 as a potential anti-DENV therapeutic agent.

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A highly selective Hsp90 affinity chromatography resin with a cleavable linker

Cited by 29 publications

References 47 publications

Optical and Radioiodinated Tethered Hsp90 Inhibitors Reveal Selective Internalization of Ectopic Hsp90 in Malignant Breast Tumor Cells

Optical and Radioiodinated Tethered Hsp90 Inhibitors Reveal Selective Internalization of Ectopic Hsp90 in Malignant Breast Tumor Cells

FW-04-806 inhibits proliferation and induces apoptosis in human breast cancer cells by binding to N-terminus of Hsp90 and disrupting Hsp90-Cdc37 complex formation

An inducible heat shock protein 70 small molecule inhibitor demonstrates anti-dengue virus activity, validating Hsp70 as a host antiviral target

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