Abstract 4749: Insights into the molecular mechanism of HSP90 binding of methoxy-substituted resorcinylic isoxazole amide inhibitors reveal different isoform selectivity profiles
Authors: Sharp Swee Y. , Prodromou Chrisostomos , Cheung Kwai-Ming , Roe Mark , Lynch Christopher , Pearl Laurence , Collins Ian , McDonald Edward , Jones Keith , Workman Paul 
Published: August 2012

Abstract
The molecular chaperone Heat Shock Protein 90 (HSP90) regulates the conformation, stability and activity of several oncogenic client proteins (eg ERBB2, ALK, BRAF, CRAF and AKT). Inhibition of HSP90 results in the degradation of these clients, leading to cell cycle arrest and apoptosis. The resorcinylic isoxazole amide HSP90 inhibitor NVP-AUY922 is currently in Phase I/II clinical trials and other related agents are in development. We previously reported that one or both of the phenolic groups on the resorcinol ring are important for HSP90 inhibitory activity. To further characterise the importance of the phenol groups on protein binding and biological activity, a series of mono- and dimethoxy-substituted resorcinylic 5′-ethyl isoxazole amides were synthesized and compared with the 2′,4′-dihydroxy compound by X-ray crystal structure and biological techniques. The most potent HSP90 inhibitors in terms of binding to the HSP90α target was the 2′,4′-dihydroxy-5′-ethyl isoxazole CCT239215 (Kd=11nM), followed by the 2′-hydroxy-4′-methoxy analogue CCT078722 (Kd=36nM). In contrast, the 2′-methoxy-4′-hydroxy and 2′,4′-dimethoxy derivatives showed no binding to HSP90. 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 NVP-AUY922, and instead is involved in van der Waals contacts. CCT239215 also showed the greatest cellular growth inhibitory potency (GI50=5-16nM in a panel of human cancer cell lines), followed by CCT078722 (GI50=59-588nM). As predicted, CCT078723 and CCT078721 were inactive in the cells (GI50&gt;10µM). 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, CCT239215 and CCT078722 caused a loss of HSP90 from immunoprecipitates of the HSP90 co-chaperone, demonstrating dissociation between P23 and HSP90, further confirming their on-target effects. No such dissociation was observed with the inactive analogs. These results explain how the hydroxy group at the 2′-position on the resorcinol ring is crucial for HSP90 inhibitory activity. Interestingly, the 2′-hydroxy-4′-methoxy-5′-ethyl isoxazole CCT078722 exhibited 55-fold less affinity for GRP94 when compared to HSP90α/α, indicating that these compounds show potential for different isoform selectivity profiles. Modelling studies suggest that IIe247 in GRP94 negatively impacts on the binding of CCT078722. Our findings offer insights into the molecular mechanism of HSP90 binding by resorcinylic isoxazole inhibitors, provide active/inactive probe compounds for mechanistic studies and suggest potential approaches to isoform selectivity.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4749. doi:1538-7445.AM2012-4749