Mechanism of action of a novel series of inhibitors of the Hsp90 molecular chaperone

Swee Sharp, Alison Maloney, Paul A. Clarke, Wynne Aherne, Suzanne Eccles, Kathy Boxall, Martin Rowlands, Florence Raynaud, Bernard Nutley, Nicola Smith, Angela Hayes, Anthea Hardcastle, Laurence Pearl, Christomos Prodromou, Karen E. Ball, Jack Cheung, Thomas P. Matthews, Edward MacDonald, Martin Drysdale, Paul Workman

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Inhibition of the molecular chaperone Hsp90 is a highly attractive anticancer strategy because of its potential to provide combinatorial blockade of multiple oncogenic pathways and simultaneous antagonism of all of the malignant hallmarks of cancer. Although the geldanamycin analogue 17AAG shows promise in early clinical trials, the natural product inhibitors have liabilities that would best be overcome by identifying synthetic small molecule agents. We previously described the discovery by high throughput screening of an early lead compound designated CCT018159 (Aherne et al Proc. AACR 44 Abstract #4002). We now report the biological properties of a series of closely related analogues of this diarylpyrazole lead structure. Improved activity and potency has been observed against the yeast and human recombinant Hsp90 ATPase activity using a malachite green colorimetric assay for inorganic phosphate. Anticancer activity in cell culture has been demonstrated in a range of tumour types, including HCT116 colon carcinoma as well as various melanoma cell lines. The molecular signature of Hsp90 inhibition, defined in details by proteomic and gene expression microarray analysis, has been confirmed using western blotting and ELISA for elevation of Hsp70 and Aha1 and depletion of C-Raf-1, ErbB2 and phospho-ERK1/2, together with other client proteins and co-chaperones. Cell-line dependent G1 and G2M arrest has been demonstrated by dual-stain Hoechst-propidium iodide flow cytometry and apoptosis has been confirmed by sub-G1 peak, morphology, PARP and caspage cleavage. Activity is retained in a P-glycoprotein positive cell line made resistant to doxorubicin and also in cells made resistant to platinum agents. Pharmacokinetic properties are promising, with glucuromide conjugation of the resorcinol ring being a major clearance mechanism. In summary, this series of compounds shows significant promise for development as Hsp90 inhibitors with potential for broad spectrum anticancer activity. Supported by Cancer Research UK, the Wellcome Trust and Vernalis.
Original languageEnglish
JournalProceedings of the American Association for Cancer Research
Publication statusPublished - 2004


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