Drug Discovery Department, Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, USA.
Chembiochem. 2012 Sep 24;13(14):2128-36. doi: 10.1002/cbic.201200316. Epub 2012 Aug 14.
In an attempt to identify novel small-molecule ligands of cyclin-dependent kinase 2 (CDK2) with potential as allosteric inhibitors, we have devised a robust and cost-effective fluorescence-based high-throughput screening assay. The assay is based on the specific interaction of CDK2 with the extrinsic fluorophore 8-anilino-1-naphthalene sulfonate (ANS), which binds to a large allosteric pocket adjacent to the ATP site. Hit compounds that displace ANS directly or indirectly from CDK2 are readily classified as ATP site binders or allosteric ligands through the use of staurosporine, which blocks the ATP site without displacing ANS. Pilot screening of 1453 compounds led to the discovery of 12 compounds with displacement activities (EC(50) values) ranging from 6 to 44 μM, all of which were classified as ATP-site-directed ligands. Four new type I inhibitor scaffolds were confirmed by X-ray crystallography. Although this small compound library contained only ATP-site-directed ligands, the application of this assay to large compound libraries has the potential to reveal previously unrecognized chemical scaffolds suitable for structure-based design of CDK2 inhibitors with new mechanisms of action.
为了鉴定细胞周期蛋白依赖性激酶 2（CDK2）的新型小分子配体，这些配体可能作为别构抑制剂，我们设计了一种强大且经济高效的基于荧光的高通量筛选测定法。该测定法基于 CDK2 与外生荧光团 8-苯胺-1-萘磺酸（ANS）的特异性相互作用，ANS 结合到邻近 ATP 结合位点的大别构口袋中。通过使用可阻断 ATP 结合位点而不置换 ANS 的 staurosporine，直接或间接从 CDK2 中置换 ANS 的命中化合物可轻松分类为 ATP 结合位点结合物或别构配体。对 1453 种化合物的初步筛选发现了 12 种具有置换活性（EC50 值）在 6 到 44 μM 之间的化合物，它们均被归类为 ATP 位点定向配体。通过 X 射线晶体学证实了四个新的 I 型抑制剂支架。尽管该小化合物文库仅包含 ATP 结合位点定向配体，但将该测定法应用于大型化合物文库有可能揭示以前未被识别的化学支架，这些支架适合基于结构的 CDK2 抑制剂设计，具有新的作用机制。
