4-Phenylpyrrolo[3,4-c]carbazole-1,3(2H,6H)-dione Inhibitors of the Checkpoint Kinase Wee1. Structure−Activity Relationships for Chromophore Modification and Phenyl Ring Substitution

Palmer, Brian D.; Thompson, A. M.; Booth, Roger J.; Dobrusin, Ellen M.; Kraker, Alan J.; Lee, Ho H.; Lunney, Elizabeth A.; Mitchell, Lorna H.; Ortwine, Daniel F.; Smaill, Jeff B.; Swan, Leesa M; Denny, William A.

doi:10.1021/jm0512591

Cited by 101 publications

(84 citation statements)

References 23 publications

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“…In transient expression studies, each mutant was expressed at levels comparable to the wild-type protein except M2, which displayed lower levels (data not shown). Examination of the crystal structure of the Wee1 kinase domain (46,51) suggests that the RXL2 leucine is involved in packing interactions. Loss of these interactions might render the protein unstable.…”

Section: Resultsmentioning

confidence: 99%

A Bifunctional Regulatory Element in Human Somatic Wee1 Mediates Cyclin A/Cdk2 Binding and Crm1-Dependent Nuclear Export

Andrake

Dunbrack

et al. 2010

Molecular and Cellular Biology

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Sophisticated models for the regulation of mitotic entry are lacking for human cells. Inactivating human cyclin A/Cdk2 complexes through diverse approaches delays mitotic entry and promotes inhibitory phosphorylation of Cdk1 on tyrosine 15, a modification performed by Wee1. We show here that cyclin A/Cdk2 complexes physically associate with Wee1 in U2OS cells. Mutation of four conserved RXL cyclin A/Cdk binding motifs (RXL1 to RXL4) in Wee1 diminished stable binding. RXL1 resides within a large regulatory region of Wee1 that is predicted to be intrinsically disordered (residues 1 to 292). Near RXL1 is T239, a site of inhibitory Cdk phosphorylation in Xenopus Wee1 proteins. We found that T239 is phosphorylated in human Wee1 and that this phosphorylation was reduced in an RXL1 mutant. RXL1 and T239 mutants each mediated greater Cdk phosphorylation and G 2 /M inhibition than the wild type, suggesting that cyclin A/Cdk complexes inhibit human Wee1 through these sites. The RXL1 mutant uniquely also displayed increased nuclear localization. RXL1 is embedded within sequences homologous to Crm1-dependent nuclear export signals (NESs). Coimmunoprecipitation showed that Crm1 associated with Wee1. Moreover, treatment with the Crm1 inhibitor leptomycin B or independent mutation of the potential NES (NESm) abolished Wee1 nuclear export. Export was also reduced by Cdk inhibition or cyclin A RNA interference, suggesting that cyclin A/Cdk complexes contribute to Wee1 export. Somewhat surprisingly, NESm did not display increased G 2 /M inhibition. Thus, nuclear export of Wee1 is not essential for mitotic entry though an important functional role remains likely. These studies identify a novel bifunctional regulatory element in Wee1 that mediates cyclin A/Cdk2 association and nuclear export.

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Section: Resultsmentioning

confidence: 99%

A Bifunctional Regulatory Element in Human Somatic Wee1 Mediates Cyclin A/Cdk2 Binding and Crm1-Dependent Nuclear Export

Andrake

Dunbrack

et al. 2010

Molecular and Cellular Biology

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“…A commercial WEE1 inhibitor sensitized a representative panel of cell lines to Ara-C in the range of 2-to 25-fold (supplemental Figure 5A) as calculated by a shift in the EC 50 of Ara-C. Because the commercial WEE1 inhibitor targets other kinases, including CHEK1, at higher concentrations at which maximal sensitization occurred, 23 we aimed to confirm results with the selective WEE1 kinase inhibitor MK1775. 21,22 MK1775 is the first WEE1 kinase inhibitor in clinical trials.…”

Section: Wee1 Inhibitors Potentiate the Antileukemic Activity Of Ara-mentioning

confidence: 95%

RNAi screening of the kinome with cytarabine in leukemias

Tibes

Bogenberger

Chaudhuri

et al. 2012

Blood

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To identify rational therapeutic combinations with cytarabine (Ara-C), we developed a high-throughput, small-interference RNA (siRNA) platform for myeloid leukemia cells. Of 572 kinases individually silenced in combination with Ara-C, silencing of 10 (1.7%) and 8 (1.4%) kinases strongly increased Ara-C activity in TF-1 and THP-1 cells, respectively. The strongest molecular concepts emerged around kinases involved in cell-cycle checkpoints and DNA-damage repair. In confirmatory siRNA assays, inhibition of WEE1 resulted in more potent and universal sensitization across myeloid cell lines than siRNA inhibition of PKMYT1, CHEK1, or ATR. Treatment of 8 acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic myeloid leukemia (CML) cell lines with commercial and the first-in-class clinical WEE1 kinase inhibitor MK1775 confirmed sensitization to Ara-C up to 97-fold. Ex vivo, adding MK1775 substantially reduced viability in 13 of 14 AML, CML, and myelodysplastic syndrome patient samples compared with Ara-C alone. Maximum sensitization occurred at lower to moderate concentrations of both drugs. Induction of apoptosis was increased using a combination of Ara-C and MK1775 compared with using either drug alone. WEE1 is expressed in primary AML, ALL, and CML specimens. Data from this first siRNA-kinome sensitizer screen suggests that inhibiting WEE1 in combination with Ara-C is a rational combination for the treatment of myeloid and lymphoid leukemias.

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“…These compounds are based on pyrimidine and pyrrolo-carbazole derivatives, and their working mechanism aims to abolish CDK1 phosphorylation at Y15 (5,(36)(37)(38)(39). PD0166285 is a pyrido-pyrimidine derivative that is a potent but nonselective inhibitor of WEE1 …”

Section: Selectivity Of Wee1 Inhibitionmentioning

confidence: 99%

WEE1 Kinase Targeting Combined with DNA-Damaging Cancer Therapy Catalyzes Mitotic Catastrophe

Hamer

Mir

Noske

et al. 2011

Clinical Cancer Research

218

193

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WEE1 kinase is a key molecule in maintaining G 2 -cell-cycle checkpoint arrest for premitotic DNA repair. Whereas normal cells repair damaged DNA during G 1 -arrest, cancer cells often have a deficient G 1 -arrest and largely depend on G 2 -arrest. The molecular switch for the G 2 -M transition is held by WEE1 and is pushed forward by CDC25. WEE1 is overexpressed in various cancer types, including glioblastoma and breast cancer. Preclinical studies with cancer cell lines and animal models showed decreased cancer cell viability, reduced tumor burden, and improved survival after WEE1 inhibition by siRNA or small molecule inhibitors, which is enhanced by combination with conventional DNAdamaging therapy, such as radiotherapy and/or cytostatics. Mitotic catastrophe results from premature entry into mitosis with unrepaired lethal DNA damage. As such, cancer cells become sensitized to conventional therapy by WEE1 inhibition, in particular those with insufficient G 1 -arrest due to deficient p53 signaling, like glioblastoma cells. One WEE1 inhibitor has now reached clinical phase I studies. Dose-limiting toxicity consisted of hematologic events, nausea and/or vomiting, and fatigue. The combination of DNA-damaging cancer therapy with WEE1 inhibition seems to be a rational approach to push cancer cells in mitotic catastrophe. Its safety and efficacy are being evaluated in clinical studies.

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4-Phenylpyrrolo[3,4-c]carbazole-1,3(2H,6H)-dione Inhibitors of the Checkpoint Kinase Wee1. Structure−Activity Relationships for Chromophore Modification and Phenyl Ring Substitution

Cited by 101 publications

References 23 publications

A Bifunctional Regulatory Element in Human Somatic Wee1 Mediates Cyclin A/Cdk2 Binding and Crm1-Dependent Nuclear Export

A Bifunctional Regulatory Element in Human Somatic Wee1 Mediates Cyclin A/Cdk2 Binding and Crm1-Dependent Nuclear Export

RNAi screening of the kinome with cytarabine in leukemias

WEE1 Kinase Targeting Combined with DNA-Damaging Cancer Therapy Catalyzes Mitotic Catastrophe

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