Rosenberg Laura H, Lafitte Marie, Grant Wayne, Chen Weimin, Cleveland John L, Duckett Derek R
Department of Molecular Therapeutics, Scripps Florida, Jupiter, FL, USA.
Department of Tumor Biology, The Moffitt Cancer Center and Research Institute, Tampa, FL, USA.
J Biomol Screen. 2015 Aug;20(7):913-20. doi: 10.1177/1087057115579391. Epub 2015 Apr 7.
A rapidly accumulating body of work suggests the autophagy pathway is an attractive therapeutic target for neurodegenerative diseases and cancer. To validate autophagy as an anticancer strategy and to assess if systemic inhibition of the pathway will have deleterious effects on normal tissues and physiology, highly selective autophagy inhibitors are needed. While several inducers and inhibitors of autophagy are known, all are nonspecific and none target the enzymes that execute the pathway. A central upstream regulator of the autophagy pathway is the serine/threonine kinase Ulk1 (UNC-51-like kinase-1). Selective molecular probes that function as Ulk1-specific inhibitors are needed to improve our understanding of the autophagy pathway. To identify inhibitors of Ulk1 kinase activity, we developed an HTS-compatible, homogeneous biochemical assay using AlphaScreen technology. This novel assay design uses purified stress-activated Ulk1 and monitors phosphorylation of its full-length native substrate, Atg13. This assay was optimized and validated in a 384-well format by screening the Sigma LOPAC library. Here we report that the Ulk1 AlphaScreen assay is robust and reproducible, with a Z' factor value of 0.83 ± 0.02 and a signal to background ratio of 20 ± 1.2. Thus, this assay can be used to screen large chemical libraries to discover novel inhibitors of Ulk1.
大量迅速积累的研究表明，自噬途径是神经退行性疾病和癌症颇具吸引力的治疗靶点。为了验证自噬作为一种抗癌策略，并评估对该途径的全身性抑制是否会对正常组织和生理机能产生有害影响，需要高选择性的自噬抑制剂。虽然已知几种自噬诱导剂和抑制剂，但它们均不具有特异性，且没有一种靶向执行该途径的酶。自噬途径的一个核心上游调节因子是丝氨酸/苏氨酸激酶Ulk1（UNC-51样激酶-1）。需要能够作为Ulk1特异性抑制剂发挥作用的选择性分子探针，以增进我们对自噬途径的理解。为了鉴定Ulk1激酶活性的抑制剂，我们利用AlphaScreen技术开发了一种与高通量筛选兼容的均相生化检测方法。这种新颖的检测设计使用纯化的应激激活型Ulk1，并监测其全长天然底物Atg13的磷酸化。通过筛选西格玛LOPAC文库，该检测方法在384孔板形式下得到了优化和验证。在此我们报告，Ulk1 AlphaScreen检测方法稳健且可重复，Z'因子值为0.83±0.02，信号与背景比为20±1.2。因此，该检测方法可用于筛选大型化学文库，以发现Ulk1的新型抑制剂。
