Selective small-molecule inhibitor reveals critical mitotic functions of human CDK1

Abstract

CDK1 is a nonredundant cyclin-dependent kinase (CDK) with an essential role in mitosis, but its multiple functions still are poorly understood at a molecular level. Here we identify a selective small-molecule inhibitor of CDK1 that reversibly arrests human cells at the G 2͞M border of the cell cycle and allows for effective cell synchronization in early mitosis. Inhibition of CDK1 during cell division revealed that its activity is necessary and sufficient for maintaining the mitotic state of the cells, preventing replication origin licensing and premature cytokinesis. Although CDK1 inhibition for up to 24 h is well tolerated, longer exposure to the inhibitor induces apoptosis in tumor cells, suggesting that selective CDK1 inhibitors may have utility in cancer therapy.apoptosis ͉ cancer therapy ͉ cell cycle ͉ cytokinesis ͉ replication origin T he cell cycle is a precisely controlled set of biochemical and morphological events driven by the sequential activation of cyclin-dependent kinases (CDKs) (1). Three CDKs and their activating cyclins (A, B, D, and E) play key roles in mammalian cell cycle regulation (2). It has been established that CDK4͞ cyclin D and CDK2͞cyclin E͞A promote the passage through G 1 and S phases, whereas CDK1͞cyclin B regulates the transition through late G 2 and mitosis (3). However, recent genetic and RNA interference studies in mammalian cells have revealed that CDK2 and CDK4 are not essential for cell cycle progression, thus leaving CDK1 as the only nonredundant cell cycle driver (4-6). Genetic studies in yeast and mammalian cells have established the critical role of CDK1 in mitosis, but the long list of its putative substrate proteins still is growing, and its precise functions in the context of the dividing cell are poorly understood (7,8). This deficiency is due, in part, to the lack of specific molecular tools for reversible modulation of CDK1 activity in vivo. Several potent small-molecule inhibitors have been reported, but their activity and cell cycle profiles are not consistent with specific CDK1 inhibition (9, 10). Here, we identify a selective and reversible inhibitor of the catalytic activity of human CDK1͞cyclin B1 and CDK1͞cyclin A complexes that allows synchronization of proliferating cells in the late G 2 phase and probing of CDK1 function in the cellular context. Results and DiscussionWe screened a diverse library of organic compounds for their ability to inhibit the catalytic activity of human CDK1͞cyclin B1. The hits then were tested for selectivity against CDK2͞cyclin E and CDK4͞cyclin D. A class of thiazolinone analogs emerged as a source of ATP-competitive CDK1 inhibitors that were then further optimized for potency, selectivity, and ability to modulate CDK1 in proliferating cells. One quinolinyl thiazolinone derivative, RO-3306, showed good potency, in vitro selectivity, and a cell cycle profile (G 2 ͞M arrest) consistent with CDK1 inhibition (Fig. 1A). RO-3306 inhibited CDK1͞cyclin B1 activity with K i of 35 nM, nearly 10-fold selectivity relative to CDK2͞ cyclin ...

Cyclin B2 suppresses mitotic failure and DNA re-replication in human somatic cells knocked down for both cyclins B1 and B2

“…However, DNA, even if it begins to segregate between these daughter cells, seems to be trapped in the midzone. This phenomenon is readily comparable to the phenotype described after CDK1 chemical inhibition by BMI-1026 or RO-3306 (Seong et al, 2003;Niiya et al, 2005;Vassilev et al, 2006). However, in our case, the chromosomes eventually collapse, giving rise to a single tetraploid nucleus.…”

“…Several years ago, it was proposed that CDK1 took part in the negative control of premature S-phase entry in mammals, because CDK1 lacking human HT2-19 cells became polyploid (Itzhaki et al, 1997). However, more recently, despite the number of CDK1 inhibitors tested in the literature, only RO-3306-mediated CDK1 inhibition was shown to lead to re-replication in HeLa cells (Vassilev et al, 2006). Geminin is another possible key component that negatively regulates re-replication in mammalian cells by inhibiting the replication factor Cdt1.…”

“…It has recently been shown that chemical inhibition of CDK1 after metaphase arrest promotes a rapid mitosis exit and premature abnormal cytokinesis (Seong et al, 2003;Niiya et al, 2005;Vassilev et al, 2006).…”

Determinants of Mitotic Catastrophe on Abrogation of the G2 DNA Damage Checkpoint by UCN-01

“…1B). As expected, the increase in histone H3 Ser10 phosphorylation and G 1 population could be suppressed by inhibition of CDK1 with RO3306 (31). As a positive control, downregulation of CHK1 with siRNA also promoted G 1 entry (Fig.…”

The dNTP triphosphohydrolase activity of SAMHD1 persists during S-phase when the enzyme is phosphorylated at T592

“…Then we examined the timing of SAMHD1 dephosphorylation during synchronized mitotic exit obtained by a two-step synchronization protocol [27, 28]. Briefly, cultures were first enriched of mitotic cells by overnight treatment with nocodazole.…”