Simons Peter C, Young Susan M, Gibaja Veronica, Lee Wen-Cherng, Josiah Serene, Edwards Bruce S, Sklar Larry A
New Mexico Molecular Libraries Screening Center, University of New Mexico, Health Sciences Center, Albuquerque, New Mexico 87131, USA.
Cytometry A. 2007 Jul;71(7):451-9. doi: 10.1002/cyto.a.20398.
Many cellular signal transduction cascades have protein kinases as critical components. Small molecule protein kinase inhibitors can be effective as laboratory probes and drugs. Methods that allow two or more kinases to be evaluated simultaneously for inhibition by a small molecule would allow unequivocal tests of specificity and selectivity of action of the small molecule.
Two hexahistidine-tagged activin receptor-like kinases were expressed in E. coli, purified, and bound to nickel beads. A fluorescent kinase ligand (F-KL) that binds to the ATP-binding site of these kinases with nanomolar affinity was developed. Binding of F-KL with kinase on the bead made the beads bright, and inhibitors decreased the brightness.
A test panel of 17 nonfluorescent kinase inhibitors, spanning two orders of magnitude affinity for the kinases, gave K(d) values for the kinases that correlated well with a fluorescence polarization assay. Results were obtained for the kinases in duplex, using an autosampler to send beads from a 96-well plate to a flow cytometer in a format suitable for high throughput screening.
Inhibitors of kinases can be measured in duplex in a high throughput format by flow cytometry, if a suitable fluorescent ligand is available.
许多细胞信号转导级联反应都以蛋白激酶作为关键组成部分。小分子蛋白激酶抑制剂可作为有效的实验室探针和药物。能够同时评估两种或更多种激酶被小分子抑制情况的方法，将能对小分子作用的特异性和选择性进行明确测试。
两种带有六组氨酸标签的激活素受体样激酶在大肠杆菌中表达、纯化并与镍珠结合。开发了一种荧光激酶配体（F-KL），它以纳摩尔亲和力与这些激酶的ATP结合位点结合。F-KL与珠上激酶的结合使珠子发光，而抑制剂会降低发光强度。
一组包含17种非荧光激酶抑制剂的测试面板，其对激酶的亲和力跨度达两个数量级，所得激酶的解离常数（K(d)）值与荧光偏振分析结果高度相关。使用自动进样器将96孔板中的珠子以适合高通量筛选的形式送入流式细胞仪，对两种激酶同时进行了检测。
如果有合适的荧光配体，激酶抑制剂可以通过流式细胞术以高通量形式进行双份检测。
Zotero 插件