Devkota Ashwini K, Warthaka Mangalika, Edupuganti Ramakrishna, Tavares Clint D J, Johnson William H, Ozpolat Bulent, Cho Eun Jeong, Dalby Kevin N
1Texas Screening Alliance for Cancer Therapeutics, The University of Texas at Austin, TX, USA.
J Biomol Screen. 2014 Mar;19(3):445-52. doi: 10.1177/1087057113505204. Epub 2013 Sep 27.
eEF-2 kinase is a potential therapeutic target for breast cancer, gliomas, and depression. No potent inhibitors of eEF-2K have been reported, and thus development of high-throughput assay systems may expedite the process. Two high-throughput assays are described for eEF-2K using recombinant, tag-free enzyme purified from bacteria. The first is a fluorescence-based assay that uses the phosphorylation of a Sox-based peptide substrate by eEF-2K, which results in a 5-fold increase in fluorescence emission, allowing for continuous monitoring of the kinase activity. The second is a luminescence-based assay that produces a luminescence signal, which correlates with the amount of adenosine triphosphate remaining in the kinase reaction. Both assays have been optimized and miniaturized for a 384-well plate format and validated in screens. In conclusion, we demonstrated that a traditional radiolabeled assay can be readily transferred to universal spectroscopic assays that are robust and will facilitate high-throughput screening of larger size libraries for the identification of small-molecule inhibitors and significantly contribute to the development of therapies for targeting eEF2K.
真核延伸因子2激酶（eEF-2 kinase）是乳腺癌、神经胶质瘤和抑郁症的潜在治疗靶点。目前尚未报道有强效的eEF-2K抑制剂，因此开发高通量检测系统可能会加快这一进程。本文描述了两种使用从细菌中纯化的重组无标签酶对eEF-2K进行的高通量检测方法。第一种是基于荧光的检测方法，该方法利用eEF-2K对基于Sox的肽底物进行磷酸化，导致荧光发射增加5倍，从而可以连续监测激酶活性。第二种是基于发光的检测方法，该方法产生与激酶反应中剩余三磷酸腺苷量相关的发光信号。这两种检测方法均已针对384孔板形式进行了优化和小型化，并在筛选中得到验证。总之，我们证明了传统的放射性标记检测方法可以很容易地转化为通用的光谱检测方法，这些方法稳健且将有助于对更大规模文库进行高通量筛选以鉴定小分子抑制剂，并显著促进针对eEF2K的治疗方法的开发。
