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

点击翻译按钮获取中文摘要

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.