Using structural and chemical biology to understand the roles and mechanisms of CDKs: generating hypotheses for drug discovery

细胞的行为取决于它所表达的基因，以及它处于休眠状态还是增殖状态。细胞周期蛋白依赖性激酶（CDK）与细胞周期蛋白家族成员结合形成复合物，调节基因表达和细胞增殖。转录描述了基因序列转化为mRNA的过程。转录CDK主要通过控制合成mRNA的酶的活性来调节这一过程。转录CDK还调节RNA加工事件。控制细胞周期的CDK响应于生长促进信号而被激活，并控制基因组复制的时机及其随后的分离，以在细胞分裂时产生两个相同的拷贝。正如CDK-细胞周期蛋白在正常细胞中很重要一样，当它们功能不正常时，它们也会促进疾病的发展。我们研究计划的第一部分是促进对含CDK复合物的结构和功能的理解。我们选择了CDK-细胞周期蛋白进行研究，基于它们在特定癌症发展中的作用。我们的目标是找到这些CDK-细胞周期蛋白结合的蛋白质，发现它们的3D结构，并阐明CDK活性如何在这些结构中调节。然后，我们可以研究这些CDK复合物在功能不正常时如何促进疾病的发展。X射线晶体学技术和近年来的低温电子显微镜技术使我们能够对蛋白质复合物进行原子细节的成像，我们将同时使用这两种方法。我们使用细菌、昆虫或培养的哺乳动物细胞来产生蛋白质，以通过晶体学或冷冻电镜进行研究。这些蛋白质也可以用于功能测定，例如，它们相互结合的紧密程度，以及突变对其特性的影响。我们的第二个目标是利用对CDK-细胞周期蛋白复合物的深入了解，以产生如何更好地被抑制剂靶向的想法。从历史上看，CDK抑制剂的开发已经靶向CDK ATP结合位点。这些抑制剂竞争胜过ATP，一种CDK通常使用的辅因子，从而阻断CDK的催化活性。这种方法无法区分其CDK靶点的不同活性，这可能取决于它们所处的复合物。因此，ATP竞争性抑制剂可能具有限制其作为药物使用的不利影响。第一代混合CDK4/6抑制剂开创了CDK抑制剂在癌症治疗中的应用，但肿瘤已经对这些抑制剂产生了耐药性。为了提高安全性并增加对CDK抑制剂临床反应的稳健性，我们的计划将寻找抑制不靶向ATP结合位点的CDK的机会;（i）首先确定CDK和细胞周期蛋白上的热点，通过这些热点它们与其他蛋白质相互作用伙伴，然后开发阻断这些热点的抑制剂（所谓的蛋白质-蛋白质相互作用抑制剂或PPI）;和（ii）通过利用我们对伴随CDK活化的结构变化的理解来设计防止CDK活化的"变构抑制剂"。我们将使用一组名为“FragLites”的小分子，旨在通过X射线晶体学筛选找到蛋白质上潜在的相互作用热点。我们还将鉴定选择性结合的环肽，并与我们的CDK-细胞周期蛋白靶点具有高亲和力。我们将开发和验证我们的FragLite和环肽，以确定更有效的PPI和变构抑制剂。总的来说，我们的计划将使我们能够解决基础科学和药物发现小组可以采用的验证项目之间的障碍。这些方法将为药物设计提供新的生物学见解和抑制CDK的新方法的可行方法。

The behaviour of a cell depends on the genes it expresses and on its commitment to either a dormant or a proliferating state. The cyclin-dependent kinases (CDKs) bind to members of the cyclin protein family to form complexes that regulate both the expression of genes and cell proliferation. Transcription describes the process by which a gene sequence is converted into mRNA. Transcriptional CDKs regulate this process, mostly by controlling the activity of an enzyme that synthesizes the mRNA. Transcriptional CDKs also regulate RNA processing events. CDKs that control the cell cycle are activated in response to growth promoting signals and control the timing of the duplication of the genome and its subsequent segregation to generate two identical copies when the cell divides. Just as CDK-cyclins are important in normal cells, so they can also contribute to the development of disease when they do not function properly. The first part of our research programme is to advance understanding of the structures and functions of CDK-containing complexes. We have selected CDK-cyclins to study based on their roles in the development of specific cancers. We aim to find proteins that these CDK-cyclins bind to, discover their 3D structures, and characterise how CDK activity is regulated within these structures. We can then study how these CDK complexes contribute to the development of disease when they do not function correctly. The techniques of X-ray crystallography and, in recent years, cryo-electron microscopy allow us to image protein complexes in atomic detail and we will use both methods. We use bacteria, insect or cultured mammalian cells to generate the proteins for study by crystallography or cryoEM. The proteins can also be used in functional assays to determine, for example, how tightly they bind to one another, and what effect mutations have on their properties.Our second aim is to exploit insight into CDK-cyclin complexes to generate ideas for how they may better be targeted by inhibitors. Historically the development of CDK inhibitors has targeted the CDK ATP-binding site. These inhibitors outcompete ATP, a cofactor that CDKs normally use, and thereby block the CDK's catalytic activity. This approach cannot distinguish the different activities of their CDK target, which may depend on the complexes in which they are found. Consequently, ATP-competitive inhibitors can have unwanted effects that limit their use as drugs. The use of CDK inhibitors in cancer therapy has been pioneered by a first generation of mixed CDK4/6 inhibitors, but tumours are already developing resistance to these inhibitors. To improve the safety and increase the robustness of clinical response to CDK inhibitors, our programme will identify opportunities to inhibit CDKs that do not target the ATP-binding site; (i) by first identifying hotspots on the CDKs and cyclins through which they interact with other protein partners and then developing inhibitors that block those hotspots (so-called protein-protein interaction inhibitors or PPIs); and (ii) by exploiting our understanding of the structural changes that accompany CDK activation to design "allosteric inhibitors" that prevent CDK activation. We will use a set of small molecules called "FragLites" that are designed to find potential interaction hotspots on a protein through an X-ray crystallographic screen. We will also identify cyclic peptides that bind selectively and with high affinity to our CDK-cyclin targets. We will develop and characterise both our FragLite and cyclic peptides to identify more potent PPIs and allosteric inhibitors. Overall, our programme will allow us to address a barrier between basic science and validated projects that drug discovery groups can adopt. The approaches will deliver both novel biological insight, and actionable approaches to novel ways of inhibiting CDKs for drug design.