The molecular chaperone Heat Shock Protein 90 (HSP90) has emerged as an exciting biological target in cancer therapy. HSP90 regulates the conformation, stability and activity of several client proteins such as ERBB2, BRAF, CRAF, AKT and mutant p53, many of which are associated with the six hallmarks of cancer. Inhibition of HSP90 results in the degradation of these clients by the 26S proteasome, leading to cell cycle arrest and apoptosis. Resorcinylic isoxazole amide HSP90 inhibitors show considerable promise and NVP-AUY922 is currently in Phase I clinical trials. Our previous studies have reported that one or both of the phenolic groups on the resorcinol ring are important for HSP90 inhibitory activity (Brough et al J Med Chem 51 196-218 2008). To explore the mechanisms underlying the importance of the phenol groups on protein binding and biological activity, a series of monomethoxy- and dimethoxy-substituted resorcinylic 5′-ethyl isoxazole amides were synthesized and compared with the 2′,4′-dihydroxy compound by protein X-ray crystal structure and biological techniques. The most potent HSP90 inhibitors in this series in terms of binding to the HSP90 target and inhibiting cancer cell proliferation was the 2′,4′-dihydroxy-5′ethyl isoxazole CCT239215, followed by the 2′-hydroxy-4′methoxy-5′ethyl isoxazole CCT078722. In contrast, 2′,4′-dimethoxy and 2′-methoxy, 4′-hydroxy derivatives showed no binding to HSP90 or antiproliferative activity. This structure-activity relationship was also maintained with respect to biomarkers of HSP90 target inhibition, as measured by depletion of HSP90 client proteins and induction of HSP72. In HCT116 human colon cells treatment with 350nM of CCT239215 and CCT078722 for 4hr caused a loss of HSP90 from P23 immunoprecipitates, demonstrating dissociation between P23 and HSP90, thereby confirming on-target effects. No dissociation was observed with the inactive 2′-methoxy, 4′-hydroxy or 2′,4′-dimethoxy analogs. These results confirm that the hydroxy group at the 2′ position on the resorcinol ring is crucial for HSP90 inhibitory activity. X-ray crystal structures showed that for CCT078722, the 4′-methoxy group of the resorcinol ring disrupts the hydrogen bonding that is seen with 2′,4′-dihydroxy CCT239215 and with the clinical candidate NVP-AUY922, and instead is involved in van der Waals contacts. Thus the results provide a structural explanation for the reduced but still significant activity of the 2′-methoxy-subtituted analog and the lack of activity of the 2′,4′-dimethoxy-substituted compound.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2677.

©2010 American Association for Cancer Research
2010