Discovery of XL413, a potent and selective CDC7 inhibitor

Koltun, Elena S.; Tsuhako, Amy Lew; Brown, David S.; Aay, Naing; Arcalas, Arlyn; Chan, Vicky; Du, Hongwang; Engst, Stefan; Ferguson, Kim; Franzini, Maurizio; Galan, Adam; Holst, Charles R.; Huang, Ping; Kane, Brian; Kim, Moon H.; Li, Jia; Markby, David W.; Mohan, Manisha; Noson, Kevin; Płonowski, Artur; Richards, Steven; Robertson, Scott; Shaw, Kenneth J.; Stott, Gordon M.; Stout, Thomas J.; Young, Jenny; Yu, Peiwen; Zaharia, Cristiana A.; Zhang, Wentao; Zhou, Peiwen; Nuss, John M.; Xu, Wei; Kearney, Patrick C.

doi:10.1016/j.bmcl.2012.04.024

Cited by 80 publications

(86 citation statements)

References 19 publications

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“…The phosphorylation of Mcm2 on S40 and S53 has been exploited as a readout for Cdc7 kinase activity and has been used in screens for the development of small-molecule inhibitors of Cdc7 (Montagnoli et al., 2008, Koltun et al., 2012). We used Xenopus egg extracts to evaluate the hyper-phosphorylation of Mcm4 and the phosphorylation of Mcm2 at the Xenopus orthologous S40 and S53 residues (S25 and S38, respectively) as readouts for the DDK activity required for DNA replication (Figure 1A).…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2017

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SummaryDbf4-dependent kinases (DDKs) are required for the initiation of DNA replication, their essential targets being the MCM2-7 proteins. We show that, in Xenopus laevis egg extracts and human cells, hyper-phosphorylation of DNA-bound Mcm4, but not phosphorylation of Mcm2, correlates with DNA replication. These phosphorylations are differentially affected by the DDK inhibitors PHA-767491 and XL413. We show that DDK-dependent MCM phosphorylation is reversed by protein phosphatase 1 (PP1) targeted to chromatin by Rif1. Loss of Rif1 increased MCM phosphorylation and the rate of replication initiation and also compromised the ability of cells to block initiation when challenged with replication inhibitors. We also provide evidence that Rif1 can mediate MCM dephosphorylation at replication forks and that the stability of dephosphorylated replisomes strongly depends on Chk1 activity. We propose that both replication initiation and replisome stability depend on MCM phosphorylation, which is maintained by a balance of DDK-dependent phosphorylation and Rif1-mediated dephosphorylation.

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Section: Resultsmentioning

confidence: 99%

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

et al. 2017

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“…The defects in mitosis observed after 24-hour treatment with 1µM DDKi (PHA) were not a by-product of apoptosis as the same effect was seen both with and without co-treatment with the pan-caspase inhibitor zVAD. 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].…”

Section: Low Dose Ddki Causes Aberrant Mitotic Structuresmentioning

confidence: 99%

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

Sasi

Coquel

Lin

et al. 2017

Preprint

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Abstract:CDC7-DBF4 kinase (DDK) is required to initiate DNA replication in eukaryotes by activating the replicative MCM helicase. DDK has also been reported to have diverse and sometimes conflicting roles in the replication checkpoint response in various organisms but the underlying mechanisms are far from settled. Here we show that human DDK promotes limited resection of newly synthesized DNA at stalled replication forks or sites of DNA damage to initiate replication checkpoint signaling. DDK is also required for efficient fork restart and G2/M cell cycle arrest. DDK exhibits genetic interactions with the ssDNA exonuclease EXO1, and we show that EXO1 is also required for nascent strand degradation following exposure to HU, raising the possibility that DDK regulates EXO1 directly. Thus, DDK has a primary and previously undescribed role in the replication checkpoint to promote ssDNA accumulation at stalled forks, which is required to initiate a robust checkpoint response and cell cycle arrest to maintain genome integrity.Keywords: DNA replication checkpoint/ replication fork/ fork resection/ fork restart/ DDK peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/207266 doi: bioRxiv preprint first posted online 3 DDK is a two-subunit kinase that is essential to initiate DNA replication at individual replication origins by phosphorylating and activating the MCM2-7 replicative helicase. The regulatory subunit DBF4 binds to CDC7 and is required for its kinase activity [1]. DDK also has roles in replication checkpoint signaling that are less well understood [1]. In budding yeast, DDK is a target of the checkpoint effector kinase Rad53 that is activated following replication stress [2]. Rad53-mediated phosphorylation of Dbf4 modestly reduces DDK activity [2] and also inhibits late origin firing [3,4], but there is also evidence that DDK is required for the complete activation of Rad53 kinase [5]. In fission yeast, DDK subunits Hsk1 and Dfp1 are phosphorylated upon HU treatment by the checkpoint kinase Cds1, orthologous to budding yeast Rad53 and mammalian CHK2 [6][7][8]. Deletion of either Cds1 or its activating protein Mrc1 partially rescues the temperature sensitivity of hsk1-1312 mutants suggesting that Cds1 (like Rad53) inhibits DDK activity [8,9]. Cds1 activation in response to HU, however, was reduced significantly in hsk1(ts) strains at restrictive temperature and the cell cycle arrest was also aberrant as seen by an increased population of 'cut' cells (indicative of mitosis without complete DNA replication) [8,10]. These paradoxical results in yeast could be explained by a negative feedback loop where DDK first helps to initiate replication checkpoint activation and then the checkpoint pathway subsequently alters DDK activity to inhibit late origin firing.Initial studies in human cells showed that the replication checkpoint inhibits DDK activity, presumably to inhibit origin firing [11,...

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“…5D), was synthesized, tested for inhibition of human Cdc7 in vitro and in vivo, and advanced into clinical trials as a potential chemotherapeutic agent (42). To determine whether Drosophila Cdc7 is relevant as a model for human Cdc7 function, we tested whether XL413 could inhibit the kinase activity of Drosophila Cdc7 in vitro.…”

Section: Drosophila Contains Two Cdc7mentioning

confidence: 99%

“…BLU502A250UC), substrate (0.5 g of Mcm4-GST or Mcm2-GST or 2 g of purified HeLa core histones), and Cdc7 (6, 12, or 24 ng) or DDK (6, 12, 24 ng Cdc7 with co-purified ChiffonN) were incubated at 30°C for 30 min (Mcm2/4 as substrate) or 60 min (core histones as substrate). The XL413 inhibitor was synthesized by us as described previously (42) and solubilized in DMSO. Reaction mixtures were separated by SDS-PAGE (10% gel, Mcm2/4; 15% gel, HeLa core histones), stained with Coomassie, imaged, dried, and exposed to a phosphor screen (GE Healthcare).…”

Section: Cloning and Purification Of Mcm2-gst And Mcm4-gst-mentioning

confidence: 99%