Dbf4-Cdc7 (DDK) Inhibitor PHA-767491 Displays Potent Anti-Proliferative Effects via Crosstalk with the CDK2-RB-E2F Pathway

by Selleckchem | Oct 28 2023

Precise regulation of DNA replication complex assembly requires cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK) activities to activate the replicative helicase complex and initiate DNA replication. Chemical probes have been essential in the molecular analysis of DDK-mediated regulation of MCM2-7 activation and the initiation phase of DNA replication. Here, the inhibitory activity of two distinct DDK inhibitor chemotypes, PHA-767491 and XL-413, were assessed in cell-free and cell-based proliferation assays. PHA-767491 and XL-413 show distinct effects at the level of cellular proliferation, initiation of DNA replication and replisome activity. XL-413 and PHA-767491 both reduce DDK-specific phosphorylation of MCM2 but show differential potency in prevention of S-phase entry. DNA combing and DNA replication assays show that PHA-767491 is a potent inhibitor of the initiation phase of DNA replication but XL413 has weak activity. Importantly, PHA-767491 decreased E2F-mediated transcription of the G1/S regulators cyclin A2, cyclin E1 and cyclin E2, and this effect was independent of CDK9 inhibition. Significantly, the enhanced inhibitory profile of PHA-767491 is mediated by potent inhibition of both DDK and the CDK2-Rb-E2F transcriptional network, that provides the molecular basis for its increased anti-proliferative effects in RB+ cancer cell lines.

Comments:

The paragraph you provided describes a study that investigated the effects of two different chemical inhibitors, PHA-767491 and XL-413, on the regulation of DNA replication mediated by Dbf4-dependent kinase (DDK). The study employed cell-free and cell-based assays to assess the inhibitory activity of these compounds.

The researchers found that PHA-767491 and XL-413 had distinct effects on cellular proliferation, initiation of DNA replication, and replisome activity. Both inhibitors were able to reduce DDK-specific phosphorylation of MCM2, a component of the replicative helicase complex required for DNA replication. However, they exhibited different potencies in preventing S-phase entry, which is the phase of the cell cycle where DNA replication occurs.

Further experiments using DNA combing and DNA replication assays revealed that PHA-767491 was a potent inhibitor of the initiation phase of DNA replication, while XL-413 had weaker activity in this regard. This suggests that PHA-767491 specifically targets the early stages of DNA replication.

Interestingly, PHA-767491 was also found to decrease the transcriptional activity of E2F, a transcription factor involved in the regulation of genes required for progression from the G1 to the S phase of the cell cycle. Specifically, it reduced the transcription of cyclin A2, cyclin E1, and cyclin E2, which are regulators of the G1/S transition. Importantly, this effect was independent of CDK9 inhibition, indicating that PHA-767491 acts through a different mechanism.

The study concluded that the enhanced inhibitory profile of PHA-767491, compared to XL-413, was attributed to its potent inhibition of both DDK and the CDK2-Rb-E2F transcriptional network. This dual inhibition likely underlies the increased anti-proliferative effects of PHA-767491 observed in cancer cell lines that possess a functional retinoblastoma protein (RB+).

In summary, the study demonstrates that PHA-767491 and XL-413 are distinct inhibitors of DDK, with PHA-767491 exhibiting stronger inhibitory effects on DNA replication initiation and E2F-mediated transcription. These findings contribute to our understanding of the molecular mechanisms involved in the regulation of DNA replication and provide insights into potential therapeutic strategies targeting RB+ cancer cells.