Inhibitors of cyclin-dependent kinases as cancer therapeutics

Abstract

Over the past two decades there has been a great deal of interest in the development of inhibitors of the cyclin-dependent kinases (CDKs). This attention initially stemmed from observations that different CDK isoforms have key roles in cancer cell proliferation through loss of regulation of the cell cycle, a hallmark feature of cancer. CDKs have now been shown to regulate other processes, particularly various aspects of transcription. The early non-selective CDK inhibitors exhibited considerable toxicity and proved to be insufficiently active in most cancers. The lack of patient selection biomarkers and an absence of understanding of the inhibitory profile required for efficacy hampered the development of these inhibitors. However, the advent of potent isoform-selective inhibitors with accompanying biomarkers has re-ignited interest. Palbociclib, a selective CDK4/6 inhibitor, is now approved for the treatment of ER+/HER2- advanced breast cancer. Current developments in the field include the identification of potent and selective inhibitors of the transcriptional CDKs; these include tool compounds that have allowed exploration of individual CDKs as cancer targets and the determination of their potential therapeutic windows. Biomarkers that allow the selection of patients likely to respond are now being discovered. Drug resistance has emerged as a major hurdle in the clinic for most protein kinase inhibitors and resistance mechanism are beginning to be identified for CDK inhibitors. This suggests that the selective inhibitors may be best used combined with standard of care or other molecularly targeted agents now in development rather than in isolation as monotherapies.

Keywords: Cell cycle; Cyclin-dependent kinase; Inhibitor; Transcription.

Figure 1. The evolutionary relationships between human CDK subfamilies determined by phylogenetic analysis based on gene sequence similarity.

Conserved domains are color-coded: green, kinase domain; pink, arginine/serine-rich domain; blue, glutamic acid-rich domain; yellow, glutamine-rich domain; red, proline-rich domain. CDK11 is encoded by two separate genes, CDK11A and CDK11B, which each encode two isoforms (adapted from (Malumbres, 2014). Cyclins required for CDK activation are also indicated.

Figure 2. A simplified model of the mammalian cell cycle.

Mitogenic stimulation leads to the synthesis of D-type cyclins, activating CDK4/6 and ultimately CDK2. CDKs4/6 phosphorylate the RB protein (the dotted lines indicate phosphorylation or dephosphorylation), releasing histone deacetylase1 (HDAC), which relieves repression of the transcription factor E2F1. Cyclin E is transcribed, activating CDK2, enabling further phosphorylation of RB, allowing DNA synthesis to occur. S phase is terminated when CDK2/cyclin A phosphorylates E2F1, blocking its DNA-binding ability. CDK1/cyclin B activation triggers mitosis and RB is dephosphorylated by protein phosphatase 1 (PP1). The INK4 and CIP/KIP proteins that modulate CDK activity are also indicated. The CDKs are also regulated by two families of small inhibitory proteins, INK4 and CIP/KIP, which generally act by interfering with cyclin binding (Sherr & Roberts, 1999), for example, binding of p16^INK4A, p15^INK4B, p18^INK4C and p19^INK4D to CDK4 blocks the interaction with cyclin D.

Figure 3. A simplified model of the transcriptional cycle of initiation, elongation and termination.

RNA polymerase II undergoes multiple rounds of phosphorylation and dephosphorylation in order to coordinate its activity and bring about the synthesis of mRNAs. CDK8 has positive and negative roles in regulating transcription through effects on specific transcription factors, super-enhancers and other transcriptional CDKs. CDK7 and CDK9 are involved in the elongation of mRNAs, while DSIF acts to block elongation and SCP1 promotes termination through dephosphorylation of Ser5 of RNA polymerase II.

Figure 4. Simplified schematic of the role of the Mediator complex and CDK8 in the initiation of transcription.

The preinitiation complex forms following binding of the Mediator complex, TFIID and other general transcription factors in a step-wise manner that eventually recruits RNA polymerase II, and finally TFIIH, to the complex. The helicase activity of TFIIH opens the DNA to initiate transcription, and CDK7 activity contributes to promoter escape by breaking interactions with some factors through phosphorylation of RNA polymerase II CTD Ser5, and also Ser7. The RNA polymerase transcribes around 20-100 bases downstream of the promoter before pausing and in another regulatory process, following recruitment of the CDK8 kinase and CDK9 activation, phosphorylation of CTD Ser2 and other substrates, that loses the remaining components of the initiation complex, yielding a fully functional elongation complex (adapted from Allen & Taatjes, 2015).

Figure 5. Structure and activity of pan- or multitarget-CDK inhibitors.

Table indicates IC₅₀ (nM) values for each compound, with the exception of compound 4 for which K_i (nM) values are given.

Figure 6. Structure and activity of selective CDK4/6 inhibitors.

Table indicates IC₅₀ (nM) values.

Figure 7. Structure and activity of selective CDK7 and CDK9 inhibitors.

Table indicates IC₅₀ (nM) values.

Figure 8. Structures of CDK8/19 inhibitors.

Figure 9. Type I and Type II inhibitor binding to CDK8/Cyclin C.

Diagram shows sorafenib (green structure, cyan compound) and CCT251545 (blue structure, magenta compound) bound to the CDK8/cyclin C complex. The DMG motif is shown in orange and is flipped “out” when bound to sorafenib and “in” when bound to CCT251545.