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Mentions: We analyzed the inhibitor binding affinity and efficacy of reducing the cell survival endpoints through monitoring reduction in activated Akt-1 levels. It was observed that with 1 μM concentration of the inhibitor with Ki of 4.9E−2, active Akt-1 levels were reduced by almost 24-folds when the ATPase activity was 7.5 s−1 (50-fold) as compared to only 12-folds with an ATPase activity of 1.5 s−1 (30-fold; Fig. 6a). This data clearly indicates that the inhibitor would be more efficacious when ATPase activity for Hsp90 is higher, as is seen in cancer cells (Kamal et al. 2003) as compared to cells with a lower ATPase activity (normal cells). Virtual prototyping studies reveal that the inhibitor will be more effective or sensitive in a cancer condition marked by a sequential increase in the ATPase activity for Hsp90 (Fig. 6a).Fig. 6
Virtual prototyping study shows increased ATPase activity of Hsp90 to be the key determinant of cancer phenotype
Bottom Line: A tenfold increase in ATPase activity of Hsp90 often seen in cancer cells increases the levels of active client proteins such as Akt-1, Raf-1 and Cyclin D1 amongst others to about 12-, 8- and 186-folds respectively.Additionally we studied the effect of a competitive inhibitor of Hsp90 activity on the reduction in the client protein levels.The online version of this article (doi:10.1007/s11693-009-9046-3) contains supplementary material, which is available to authorized users.
Unlabelled: Hsp90 is an ATP-dependent molecular chaperone that regulates key signaling proteins and thereby impacts cell growth and development. Chaperone cycle of Hsp90 is regulated by ATP binding and hydrolysis through its intrinsic ATPase activities, which is in turn modulated by interaction with its co-chaperones. Hsp90 ATPase activity varies in different organisms and is known to be increased in tumor cells. In this study we have quantitatively analyzed the impact of increasing Hsp90 ATPase activity on the activities of its clients through a virtual prototyping technology, which comprises a dynamic model of Hsp90 interaction with clients involved in proliferation pathways. Our studies highlight the importance of increased ATPase activity of Hsp90 in cancer cells as the key modulator for increased proliferation and survival. A tenfold increase in ATPase activity of Hsp90 often seen in cancer cells increases the levels of active client proteins such as Akt-1, Raf-1 and Cyclin D1 amongst others to about 12-, 8- and 186-folds respectively. Additionally we studied the effect of a competitive inhibitor of Hsp90 activity on the reduction in the client protein levels. Virtual prototyping experiments corroborate with findings that the drug has almost 10- to 100-fold higher affinity as indicated by a lower IC(50) value (30-100 nM) in tumor cells with higher ATPase activity. The results also indicate a 15- to 25-fold higher efficacy of the inhibitor in reducing client levels in tumor cells. This analysis provides mechanistic insights into the links between increased Hsp90 ATPase activity, tumor phenotype and the hypersensitivity of tumor Hsp90 to inhibition by ATP analogs.
Electronic supplementary material: The online version of this article (doi:10.1007/s11693-009-9046-3) contains supplementary material, which is available to authorized users.