Rowlands Martin, McAndrew Craig, Prodromou Chris, Pearl Laurence, Kalusa Andrew, Jones Keith, Workman Paul, Aherne Wynne
Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, Haddow Laboratories, Sutton, Surrey, United Kingdom.
J Biomol Screen. 2010 Mar;15(3):279-86. doi: 10.1177/1087057109360253. Epub 2010 Feb 10.
The molecular chaperone heat shock protein 90 (Hsp90) is required for the correct folding and stability of a number of client proteins that are important for the growth and maintenance of cancer cells. Heat shock protein 72 (Hsp72), a co-chaperone of Hsp90, is also emerging as an attractive cancer drug target. Both proteins bind and hydrolyze adenosine triphosphate (ATP), and ATPase activity is essential for their function. Inhibition of Hsp90 ATPase activity leads to the degradation of client proteins, resulting in cell growth inhibition and apoptosis. Several small-molecule inhibitors of the ATPase activity of Hsp90 have been described and are currently being evaluated clinically for the treatment of cancer. A number of methods for the measurement of ATPase activity have been previously used, but not all of these are ideally suited to screening cascades in drug discovery projects. The authors have evaluated the use of commercial reagents (Transcreener ADP) for the measurement of ATPase activity of both yeast and human Hsp90 (ATP K(m) approximately 500 microM) and human Hsp72 (ATP K(m) ~1 microM). The low ATPase activity of human Hsp90 and its stimulation by the co-chaperone Aha1 was measured with ease using reduced incubation times, generating robust data (Z' = 0.75). The potency of several small-molecule inhibitors of both Hsp90 and Hsp72 was determined using the Transcreener reagents and compared well to that determined using other assay formats.
分子伴侣热休克蛋白90（Hsp90）对于许多对癌细胞生长和维持至关重要的客户蛋白的正确折叠和稳定性是必需的。热休克蛋白72（Hsp72）作为Hsp90的共伴侣，也正在成为一个有吸引力的癌症药物靶点。这两种蛋白都能结合并水解三磷酸腺苷（ATP），且ATP酶活性对其功能至关重要。抑制Hsp90的ATP酶活性会导致客户蛋白降解，从而抑制细胞生长并诱导凋亡。已经描述了几种Hsp90 ATP酶活性的小分子抑制剂，目前正在进行临床评估以用于癌症治疗。以前已经使用了多种测量ATP酶活性的方法，但并非所有这些方法都理想地适用于药物发现项目中的筛选级联。作者评估了使用商业试剂（Transcreener ADP）来测量酵母和人Hsp90（ATP Km约为500 microM）以及人Hsp72（ATP Km约为1 microM）的ATP酶活性。使用缩短的孵育时间轻松测量了人Hsp90的低ATP酶活性及其被共伴侣Aha1的刺激，产生了可靠的数据（Z' = 0.75）。使用Transcreener试剂测定了几种Hsp90和Hsp72小分子抑制剂的效力，与使用其他测定形式测定的结果相比良好。
