Drug Discovery Department, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida 33612, USA.
ACS Chem Biol. 2011 May 20;6(5):492-501. doi: 10.1021/cb100410m. Epub 2011 Feb 22.
Cyclin-dependent kinases (CDKs) are key regulatory enzymes in cell cycle progression and transcription. Aberrant activity of CDKs has been implicated in a number of medical conditions, and numerous small molecule CDK inhibitors have been reported as potential drug leads. However, these inhibitors exclusively bind to the ATP site, which is largely conserved among protein kinases, and clinical trials have not resulted in viable drug candidates, attributed in part to the lack of target selectivity. CDKs are unique among protein kinases, as their functionality strictly depends on association with their partner proteins, the cyclins. In an effort to identify potential target sites for disruption of the CDK-cyclin interaction, we probed the extrinsic fluorophore 8-anilino-1-naphthalene sulfonate (ANS) with human CDK2 and cyclin A using fluorescence spectroscopy and protein crystallography. ANS interacts with free CDK2 in a saturation-dependent manner with an apparent K(d) of 37 μM, and cyclin A displaced ANS from CDK2 with an EC(50) value of 0.6 μM. Co-crystal structures with ANS alone and in ternary complex with ATP site-directed inhibitors revealed two ANS molecules bound adjacent to one another, away from the ATP site, in a large pocket that extends from the DFG region above the C-helix. Binding of ANS is accompanied by substantial structural changes in CDK2, resulting in a C-helix conformation that is incompatible for cyclin A association. These findings indicate the potential of the ANS binding pocket as a new target site for allosteric inhibitors disrupting the interaction of CDKs and cyclins.
细胞周期蛋白依赖性激酶（CDKs）是细胞周期进程和转录的关键调节酶。CDKs 的异常活性与许多医学病症有关，并且已经报道了许多小分子 CDK 抑制剂作为潜在的药物先导。然而，这些抑制剂专门结合到 ATP 结合位点，该位点在蛋白激酶中基本保守，临床试验并未产生可行的候选药物，部分原因是缺乏靶标选择性。CDKs 在蛋白激酶中是独特的，因为它们的功能严格依赖于与其伴侣蛋白 cyclin 的结合。为了鉴定破坏 CDK-cyclin 相互作用的潜在靶标位点，我们使用荧光光谱法和蛋白质晶体学方法用人类 CDK2 和 cyclin A 探测外生荧光团 8-苯胺-1-萘磺酸盐（ANS）。ANS 以饱和依赖性方式与游离 CDK2 相互作用，表观 K(d)为 37 μM，并且 cyclin A 以 0.6 μM 的 EC(50)值从 CDK2 上置换 ANS。与 ANS 单独和与 ATP 位点定向抑制剂的三元复合物的共晶结构显示，两个 ANS 分子彼此相邻结合，远离 ATP 结合位点，位于从 DFG 区域延伸到 C-螺旋上方的大口袋中。ANS 的结合伴随着 CDK2 的大量结构变化，导致 C-螺旋构象与 cyclin A 结合不兼容。这些发现表明 ANS 结合口袋作为一种新的靶标位点的潜力，用于变构抑制剂破坏 CDKs 和 cyclins 的相互作用。
