The Potent Cdc7-Dbf4 (DDK) Kinase Inhibitor XL413 Has Limited Activity in Many Cancer Cell Lines and Discovery of Potential New DDK Inhibitor Scaffolds

Abstract

Cdc7-Dbf4 kinase or DDK (Dbf4-dependent kinase) is required to initiate DNA replication by phosphorylating and activating the replicative Mcm2-7 DNA helicase. DDK is overexpressed in many tumor cells and is an emerging chemotherapeutic target since DDK inhibition causes apoptosis of diverse cancer cell types but not of normal cells. PHA-767491 and XL413 are among a number of potent DDK inhibitors with low nanomolar IC50 values against the purified kinase. Although XL413 is highly selective for DDK, its activity has not been extensively characterized on cell lines. We measured anti-proliferative and apoptotic effects of XL413 on a panel of tumor cell lines compared to PHA-767491, whose activity is well characterized. Both compounds were effective biochemical DDK inhibitors but surprisingly, their activities in cell lines were highly divergent. Unlike PHA-767491, XL413 had significant anti-proliferative activity against only one of the ten cell lines tested. Since XL413 did not effectively inhibit DDK in multiple cell lines, this compound likely has limited bioavailability. To identify potential leads for additional DDK inhibitors, we also tested the cross-reactivity of ∼400 known kinase inhibitors against DDK using a DDK thermal stability shift assay (TSA). We identified 11 compounds that significantly stabilized DDK. Several inhibited DDK with comparable potency to PHA-767491, including Chk1 and PKR kinase inhibitors, but had divergent chemical scaffolds from known DDK inhibitors. Taken together, these data show that several well-known kinase inhibitors cross-react with DDK and also highlight the opportunity to design additional specific, biologically active DDK inhibitors for use as chemotherapeutic agents.

DDK has a primary role in processing stalled replication forks to initiate downstream checkpoint signaling

“…We found that 5 µM of DDKi and a pre-treatment time of 4 hours were required to substantially block CHK1 activation by HU in HCC1954 cells (Supplementary Figure 1A). HCC1954 is a breast cancer cell line that expresses high levels of both DDK subunits and exhibits robust decrease in MCM2 phosphorylation in response to the prototype DDK inhibitor, PHA-767491 (DDKi) [18]. As shown previously, CHK1 activation was almost completely eliminated by pre-treatment with the DDK inhibitor PHA-767491 ( Figure 1A).…”

“…The DDK inhibitors, PHA-767491 and XL413, were synthesized as described previously [18]. ATR inhibitor (VE-821, #A2521) and Camptothecin (#A2877) was from APExBIO.…”

“…To confirm that this effect is DDK-specific we also tested the more selective biochemical DDK inhibitor, XL413 [45]. In HCC1954 cells and many other cell lines, XL413 is a poor in vivo inhibitor of DDK activity and has little effect on cell growth even at high inhibitor concentrations [18]. We therefore used relatively high 10µM and 20µM concentrations of XL413 to moderately inhibit DDK in HCC1954 cells but not induce apoptosis.…”

Reversal of DDK-Mediated MCM Phosphorylation by Rif1-PP1 Regulates Replication Initiation and Replisome Stability Independently of ATR/Chk1

“…However, extended XL413 treatment in HeLa cells seems to merely delay progression through S phase rather than completely shut down replication. This perhaps provides a mechanistic explanation for why XL413 fails to induce apoptosis efficiently in cancer cells (Sasi et al., 2014). It is also important to note that several drug screens have been carried out using Mcm2 S40 phosphorylation as a marker of DDK activity.…”

O Cdc7 kinase where art thou?

“…Because it has been difficult to map mammalian origins of replication and there is no consensus binding site for known origins (Leonard and Mechali 2013), application of the Calling Card method to human DDK and to other proteins should identify functional origins that are used over many cell cycles. Furthermore, our studies are highly significant because inhibitors of DDK are in clinical trials against cancer and the Calling Card system could be used to help determine their effects on cells (Sasi, et al 2014, Swords, et al 2010). …”