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Inhibition of the G2 DNA Damage Checkpoint and of Protein Kinases Chk1 and Chk2 by the Marine Sponge Alkaloid Debromohymenialdisine*

Open AccessPublished:May 25, 2001DOI:https://doi.org/10.1074/jbc.M100728200
      Cells can respond to DNA damage by activating checkpoints that delay cell cycle progression and allow time for DNA repair. Chemical inhibitors of the G2 phase DNA damage checkpoint may be used as tools to understand better how the checkpoint is regulated and may be used to sensitize cancer cells to DNA-damaging therapies. However, few inhibitors are known. We used a cell-based assay to screen natural extracts for G2checkpoint inhibitors and identified debromohymenialdisine (DBH) from a marine sponge. DBH is distinct structurally from previously known G2 checkpoint inhibitors. It inhibited the G2checkpoint with an IC50 of 8 μm and showed moderate cytotoxicity (IC50 = 25 μm) toward MCF-7 cells. DBH inhibited the checkpoint kinases Chk1 (IC50 = 3 μm) and Chk2 (IC50 = 3.5 μm) but not ataxia-telangiectasia mutated (ATM), ATM-Rad3-related protein, or DNA-dependent protein kinase in vitro, indicating that it blocks two major branches of the checkpoint pathway downstream of ATM. It did not cause the activation or inhibition of different signal transduction proteins, as determined by mobility shift analysis in Western blots, suggesting that it inhibits a narrow range of protein kinases in vivo.
      ATM
      ataxia-telangiectasia mutated protein
      DBH
      debromohymenialdisine
      HPLC
      high pressure liquid chromatography
      mp53
      mutant p53
      MTT
      3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
      PK
      protein kinase
      DNA-PK
      DNA-dependent protein kinase
      SAPK
      stress-activated protein kinase
      ATR
      ATM-Rad3-related protein
      DNA damage activates signal transduction pathways called checkpoints, which delay cell cycle progression and allow more time to repair DNA (
      • Lowndes N.F.
      • Murguia J.R.
      ,
      • Clarke D.J.
      • Gimenez-Abian J.F.
      ,
      • Zhou B.B.
      • Elledge S.J.
      ). Checkpoints arrest cells in the G1 phase to prevent replication of damaged DNA and in the G2 phase to prevent the segregation of damaged chromosomes during mitosis.
      The G2/M transition is controlled by the Cdc2 protein kinase. During G2 arrest, Cdc2 is inactivated through phosphorylation of Thr-14 and Tyr-15 in its ATP-binding site by protein kinases including Wee1 and Myt1 (
      • Parker L.L.
      • Piwnica-Worms H.
      ,
      • Mueller P.R.
      • Coleman T.R.
      • Kumagai A.
      • Dunphy W.G.
      ). Entry into mitosis requires dephosphorylation of these sites by Cdc25 phosphatases. According to our current understanding of the G2 checkpoint, DNA damage activates the ATM,1 and ATR members of the phosphoinositide kinase family (
      • Cliby W.A.
      • Roberts C.J.
      • Cimprich K.A.
      • Stringer C.M.
      • Lamb J.R.
      • Schreiber S.L.
      • Friend S.H.
      ,
      • Wright J.A.
      • Keegan K.S.
      • Herendeen D.R.
      • Bentley N.J.
      • Carr A.M.
      • Hoekstra M.F.
      • Concannon P.
      ). A signal then is transmitted through the downstream protein kinases Chk1 and Chk2 (
      • Cliby W.A.
      • Roberts C.J.
      • Cimprich K.A.
      • Stringer C.M.
      • Lamb J.R.
      • Schreiber S.L.
      • Friend S.H.
      ,
      • Wright J.A.
      • Keegan K.S.
      • Herendeen D.R.
      • Bentley N.J.
      • Carr A.M.
      • Hoekstra M.F.
      • Concannon P.
      ,
      • Savitsky K.
      • Bar S.A.
      • Gilad S.
      • Rotman G.
      • Ziv Y.
      • Vanagaite L.
      • Tagle D.A.
      • Smith S.
      • Uziel T.
      • Sfez S.
      • Ashkenazi M.
      • Pecker I.
      • Frydman M.
      • Harnik R.
      • Patanjali S.R.
      • Simmons A.
      • Clines G.A.
      • Sartiel A.
      • Gatti R.A.
      • Chessa L.
      • Sanal O.
      • Lavin M.F.
      • Jaspers N.G.J.
      • Taylor A.M.R.
      • Arlett C.F.
      • Miki T.
      • Weissman S.M.
      • Lovett M.
      • Collins F.S.
      • Shiloh Y.
      ,
      • Matsuoka S.
      • Huang M.
      • Elledge S.J.
      ,
      • Sanchez Y.
      • Wong C.
      • Thoma R.S.
      • Richman R.
      • Wu Z.
      • Piwnica-Worms H.
      • Elledge S.J.
      ,
      • Chaturvedi P.
      • Eng W.K.
      • Zhu Y.
      • Mattern M.R.
      • Mishra R.
      • Hurle M.R.
      • Zhang X.
      • Annan R.S.
      • Lu Q.
      • Faucette L.F.
      • Scott G.F.
      • Li X.
      • Carr S.A.
      • Johnson R.K.
      • Winkler J.D.
      • Zhou B.B.
      ), which are able to phosphorylate Cdc25 on Ser-216. This phosphorylation is thought to directly prevent Cdc25 from activating Cdc2 kinase (
      • Blasina A.
      • Van de Weyer I.
      • Laus M.C.
      • Luyten W.H.M.L.
      • Parker A.E.
      • McGowan C.
      ) or to separate Cdc25 from Cdc2 kinase by promoting the association of Cdc25 with 14-3-3 proteins (
      • Sanchez Y.
      • Wong C.
      • Thoma R.S.
      • Richman R.
      • Wu Z.
      • Piwnica-Worms H.
      • Elledge S.J.
      ,
      • Peng C.-Y.
      • Graves P.R.
      • Thoma R.S.
      • Wu Z.
      • Shaw A.S.
      • Piwnica-Worms H.
      ,
      • Yang J.
      • Winkler K.
      • Yoshida M.
      • Kornbluth S.
      ,
      • Dalal S.N.
      • Schweitzer C.M.
      • Gan J.
      • DeCaprio J.
      ,
      • Zeng Y.
      • Piwnica-Worms H.
      ). Chk1 and Chk2 also can phosphorylate and activate Wee1, a kinase that catalyzes Cdc2 inhibitory phosphorylation (
      • O'Connell M.J.
      • Raleigh J.M.
      • Verkade H.D.
      • Nurse P.
      ,
      • Raleigh J.M.
      • O'Connell M.J.
      ). Chk1 is required for initiating G2 arrest (
      • Takai H.
      • Tominaga K.
      • Motoyama N.
      • Minamishima Y.A.
      • Nagahama H.
      • Tsukiyama T.
      • Ikeda K.
      • Nakayama K.
      • Nakanishi M.
      • Nakayama K.
      ,
      • Liu Q.
      • Guntuku S.
      • Cui X.-S.
      • Matsuoka S.
      • Cortez D.
      • Tamai K.
      • Luo G.
      • Carattini-Rivera S.
      • DeMayo F.
      • Bradley A.
      • Donehower L.A.
      • Elledge S.J.
      ). Chk2 can phosphorylate p53 on Ser-20in vitro (
      • Hirao A.
      • Kong Y.-Y.
      • Matsuoka S.
      • Wakeman A.
      • Ruland J.
      • Yoshida H.
      • Liu D.
      • Elledge S.J.
      • Mak T.W.
      ,
      • Chehab N.H.
      • Malikzay A.
      • Appel M.
      • Halazonetis T.D.
      ,
      • Shieh S.Y.
      • Ahn J.
      • Tamai K.
      • Taya Y.
      • Prives C.
      ), and p53 targets such as p21 and 14-3-3ς have roles in maintaining G2 arrest (
      • Bunz F.
      • Dutriaux A.
      • Lengauer C.
      • Waldman T.
      • Zhou S.
      • Brown J.P.
      • Sedivy J.M.
      • Kinzler K.W.
      • Vogelstein B.
      ,
      • Chan T.
      • Hermeking H.
      • Lengauer C.
      • Kinzler K.W.
      • Vogelstein B.
      ). These results as well as experiments with knockout mice (
      • Hirao A.
      • Kong Y.-Y.
      • Matsuoka S.
      • Wakeman A.
      • Ruland J.
      • Yoshida H.
      • Liu D.
      • Elledge S.J.
      • Mak T.W.
      ) are consistent with a role of Chk2 in maintaining G2 arrest.
      Our understanding of checkpoints stems mainly from genetic studies in yeast and mice, in vitro studies with amphibian egg extracts, and studies of human syndromes associated with predisposition to cancer. Compounds that inhibit the G2 checkpoint may be useful additional tools to study the checkpoint mechanism in mammalian systems. G2 checkpoint inhibitors also may be valuable in cancer therapy to enhance the effectiveness of DNA-damaging agents in tumors with a defective G1 DNA damage checkpoint, such as those with mutated p53 (
      • Weinert T.
      • Lydall D.
      , ,
      • Kao G.D.
      • McKenna W.G.
      • Maity A.
      • Blank K.
      • Muschel R.J.
      ,
      • Roberge M.
      • Berlinck R.G.S.
      • Xu L.
      • Anderson H.J.
      • Lim L.Y.
      • Curman D.
      • Stringer C.M.
      • Friend S.H.
      • Davies P.
      • Haggarty S.J.
      • Kelly M.T.
      • Britton R.
      • Piers E.
      • Andersen R.J.
      ). However, few G2 checkpoint inhibitors are known. Those found so far include caffeine and 1-substituted caffeine analogs (
      • Walters R.A.
      • Gurley L.R.
      • Tobey R.A.
      ,
      • Busse P.M.
      • Bose S.K.
      • Jones R.W.
      • Tolmach L.J.
      ,
      • Rowley R.
      • Zorch M.
      • Leeper D.B.
      ,
      • Zampetti-Bosseler F.
      • Scott D.
      ,
      • Schlegel R.
      • Pardee A.B.
      ,
      • Jiang X.
      • Lim L.Y.
      • Daly J.W.
      • Li A.H.
      • Jacobson K.A.
      • Roberge M.
      ), 2-aminopurine, and 6-dimethylaminopurine (
      • Andreassen P.R.
      • Margolis R.L.
      ), staurosporine, 7-hydroxystaurosporine, SB-218078 (
      • Tam S.W.
      • Schlegel R.
      ,
      • Wang Q.
      • Fan S.
      • Eastman A.
      • Worland P.J.
      • Sausville E.A.
      • O'Connor P.M.
      ,
      • Jackson J.R.
      • Gilmartin A.
      • Imburgia C.
      • Winkler J.D.
      • Marshall L.A.
      • Roshak A.
      ), and isogranulatimide (
      • Roberge M.
      • Berlinck R.G.S.
      • Xu L.
      • Anderson H.J.
      • Lim L.Y.
      • Curman D.
      • Stringer C.M.
      • Friend S.H.
      • Davies P.
      • Haggarty S.J.
      • Kelly M.T.
      • Britton R.
      • Piers E.
      • Andersen R.J.
      ). All have been shown to enhance the cytotoxicity of DNA-damaging agents (
      • Roberge M.
      • Berlinck R.G.S.
      • Xu L.
      • Anderson H.J.
      • Lim L.Y.
      • Curman D.
      • Stringer C.M.
      • Friend S.H.
      • Davies P.
      • Haggarty S.J.
      • Kelly M.T.
      • Britton R.
      • Piers E.
      • Andersen R.J.
      ,
      • Jackson J.R.
      • Gilmartin A.
      • Imburgia C.
      • Winkler J.D.
      • Marshall L.A.
      • Roshak A.
      ). Staurosporine is a broad specificity protein kinase inhibitor, and 7-hydroxystaurosporine is an in vitro inhibitor of several protein kinases (
      • Akinaga S.
      • Gomi K.
      • Morimoto M.
      • Tamaoki T.
      • Okabe M.
      ,
      • Mizuno K.
      • Noda K.
      • Ueda Y.
      • Hanaki H.
      • Saido T.C.
      • Ikuta T.
      • Kuroki T.
      • Tamaoki T.
      • Hirai S.
      • Osada S.
      • Ohno S.
      ,
      • Kawakami K.
      • Futami H.
      • Takahara J.
      • Yamaguchi K.
      ) including Chk1 (
      • Busby E.C.
      • Leistritz D.F.
      • Abraham R.T.
      • Karnitz L.M.
      • Sarkaria J.N.
      ,
      • Graves P.R.
      • Yu L
      • Schwarz J.K.
      • Gales J.
      • O'Connor P.M.
      • Piwnica-Worms H.
      ). 7-Hydroxystaurosporine is being evaluated in phase I clinical trials for the treatment of cancer (
      • Sausville E.A.
      • Arbuck S.G.
      • Messmann R.
      • Headlee D.
      • Lush R.D.
      • Bauer K.
      • Murgo A.
      • Figg W.D.
      • Lahusen T.
      • Jaken S.
      • Roberge M.
      • Fuse E.
      • Kuwabara T.
      • Senderowicz A.M.
      ). Caffeine and caffeine analogs have many pharmacological activities (
      • Jiang X.
      • Lim L.Y.
      • Daly J.W.
      • Li A.H.
      • Jacobson K.A.
      • Roberge M.
      ) including in vitroinhibition of ATM and ATR protein kinase activity (
      • Zhou B.B.
      • Elledge S.J.
      ,
      • Blasina A.
      • Van de Weyer I.
      • Laus M.C.
      • Luyten W.H.M.L.
      • Parker A.E.
      • McGowan C.
      ,
      • Sarkaria J.N.
      • Busby E.C.
      • Tibbetts R.S.
      • Roos P.
      • Taya Y.
      • Karnitz L.M.
      • Abraham R.T.
      ,
      • Hall-Jackson C.A.
      • Cross D.A.E.
      • Morrice N.
      • Smythe C.
      ,
      • Zhou B.B.
      • Chaturvedi P.
      • Spring K.
      • Scott S.P.
      • Johanson R.A.
      • Mishra R.
      • Mattern M.R.
      • Winkler J.D.
      • Khanna K.K.
      ), but they are not considered drug candidates.
      To find new G2 checkpoint inhibitors, we used a cell-based assay (
      • Roberge M.
      • Berlinck R.G.S.
      • Xu L.
      • Anderson H.J.
      • Lim L.Y.
      • Curman D.
      • Stringer C.M.
      • Friend S.H.
      • Davies P.
      • Haggarty S.J.
      • Kelly M.T.
      • Britton R.
      • Piers E.
      • Andersen R.J.
      ) to screen marine invertebrate extracts. From the spongeStylissa flabeliformis, we have isolated debromohymenialdisine (DBH), a compound structurally distinct from previously known G2 checkpoint inhibitors. We have characterized the G2 checkpoint inhibitory activity of DBH and analogs, and we describe its effects on checkpoint and signal transduction protein kinases.

      DISCUSSION

      This study identifies DBH and hymenialdisine as new G2checkpoint inhibitors. These compounds show no obvious structural resemblance to previously described checkpoint inhibitors. The bulky bromine substituent at position 2 in hymenialdisine that is absent in DBH does not influence G2 checkpoint inhibition significantly, the two compounds having IC50 values of 6 and 8 μm, respectively (Table I). Both the pyrrololactam and the aminoimidazole moieties are required because 2-aminoimidazole and 2-amino-4,5-imidazole-dicarbonitrile (which lack the pyrrolactam moiety) and debromopyrrololactam (which lacks the aminoimidazole moiety) are without G2 checkpoint inhibition activity (Table I). Debromoaxinohydantoin is very similar structurally to DBH (Fig. 1) but has negligible activity (Table I), indicating that a precise orientation of the carbonyl and amino groups in aminoimidazolidinone is important.
      Hymenialdisine is a protein kinase inhibitor in vitro (
      • Meijer L.
      • Thunissen A.-M.W.H.
      • White A.W.
      • Garnier M.
      • Nikolic M.
      • Tsai L.-H.
      • Walter J.
      • Cleverley K.E.
      • Salinas P.C.
      • Wu Y.-Z.
      • Biernat J.
      • Mandelkow E.-M.
      • Pettit G.R.
      ), and our in vivo structure-activity data for G2checkpoint inhibition are consistent with inhibition of a protein kinase as the mechanism of action. A crystal structure of a complex of hymenialdisine and the protein kinase Cdk2 shows multiple hydrogen bonds and Van der Waals contacts between the aminoimidazolidinone and residues in the ATP binding pocket of the kinase (
      • Meijer L.
      • Thunissen A.-M.W.H.
      • White A.W.
      • Garnier M.
      • Nikolic M.
      • Tsai L.-H.
      • Walter J.
      • Cleverley K.E.
      • Salinas P.C.
      • Wu Y.-Z.
      • Biernat J.
      • Mandelkow E.-M.
      • Pettit G.R.
      ). These would not occur with the analog axinohydantoin, which is two orders of magnitude less potent as an in vitro protein kinase inhibitor than hymenialdisine (
      • Meijer L.
      • Thunissen A.-M.W.H.
      • White A.W.
      • Garnier M.
      • Nikolic M.
      • Tsai L.-H.
      • Walter J.
      • Cleverley K.E.
      • Salinas P.C.
      • Wu Y.-Z.
      • Biernat J.
      • Mandelkow E.-M.
      • Pettit G.R.
      ). This agrees with our finding that debromoaxinohydantoin shows no G2 checkpoint inhibition activity. In the Cdk2·hymenialdisine complex, the bromine at position 2 faces the entrance of the ATP binding pocket, where it contributes somewhat to binding affinity but would not be required for binding (
      • Meijer L.
      • Thunissen A.-M.W.H.
      • White A.W.
      • Garnier M.
      • Nikolic M.
      • Tsai L.-H.
      • Walter J.
      • Cleverley K.E.
      • Salinas P.C.
      • Wu Y.-Z.
      • Biernat J.
      • Mandelkow E.-M.
      • Pettit G.R.
      ). This agrees with our finding that hymenialdisine and DBH has similar G2 checkpoint inhibition activities.
      Hymenialdisine inhibits a number of kinases in vitro: cyclin-dependent kinases, GSK-3, and CK1 at nanomolar concentrations and several more kinases at micromolar concentrations (
      • Meijer L.
      • Thunissen A.-M.W.H.
      • White A.W.
      • Garnier M.
      • Nikolic M.
      • Tsai L.-H.
      • Walter J.
      • Cleverley K.E.
      • Salinas P.C.
      • Wu Y.-Z.
      • Biernat J.
      • Mandelkow E.-M.
      • Pettit G.R.
      ). However, we show that in vivo neither hymenialdisine nor DBH have such broad activity. First, although hymenialdisine can inhibit Cdc2 kinase at nanomolar concentrations in vitro,in vivo hymenialdisine and DBH are checkpoint inhibitors and actually activate Cdc2 kinase (Fig. 3 and data not shown). Second, although hymenialdisine can inhibit Cdk2, -3, -4, -5, and -6 at submicromolar concentrations in vitro, in vivoDBH is only a poor inhibitor of cell proliferation (IC50 = 25 μm, Fig. 2), indicating that it does not inhibit these kinases in vivo. Third, DBH has no effect on the phosphorylation state of 24 signal transduction kinases in vivo. Different factors could explain higher selectivity in vivo than in vitro. For example, DBH could reach different concentrations in different subcellular compartments, resulting in preferential inhibition of kinases within certain compartments. Certain protein kinases also may be part of macromolecular complexes that facilitate or hinder DBH binding.
      We propose that the checkpoint kinases Chk1 and Chk2 are the targets of DBH involved in the G2 checkpoint. Preliminary data indicate that DBH has little or no effect on the activity of Wee1in vitro at concentrations up to 10 μm (data not shown). However, Myt1 cannot be excluded as a target because it was not tested. The concentration of DBH required to inhibit Chk1 and Chk2in vitro (IC50 = 3 and 3.5 μm,respectively) is close to that required for inhibition of the checkpoint in vivo (IC50 = 8 μm). Both kinases probably cooperate in maintaining Cdc25C phosphorylated at Ser-216 after DNA damage, and it seems likely that DBH owes its high efficacy as a checkpoint inhibitor to inhibition of both kinases. Structural and functional redundancy of kinases is common in mammalian signal transduction pathways. It may be generally the case that agents with broader specificity will have more impact on complex signaling pathways than highly selective agents.
      Irradiation and DBH treatment caused no noticeable alteration of the cellular distribution of the major cell cycle regulatory proteins Cdc2, Cdc25C, cyclin A, cyclin B, Cdk2, Cdc25B, Wee1, and 14-3-3 in MCF-7 mp53 cells. In addition, we saw no evidence of irradiation causing a physical separation of Cdc2-cyclin B from Cdc25C or of DBH reversing such a separation. This may indicate that the major function of the phosphorylation of Ser-216 in Cdc25C by Chk1 and Chk2 is not to separate Cdc25C from Cdc2 kinase physically.
      The evidence that DBH inhibits two checkpoint kinases in vitro and is a more specific kinase inhibitor in vivothan in vitro may be relevant to its consideration as a drug candidate. DBH showed moderate cytotoxicity (IC50 = 25 μm) toward cells not exposed to DNA damage, roughly 3-fold higher than the concentration required for G2checkpoint inhibition (IC50 = 8 μm). This difference may be sufficient to provide the therapeutic window required for achieving G2 checkpoint inhibition in animal models without excessive toxicity. Indeed, hymenialdisine has been reported to slow down joint deterioration and cartilage degradation-associated osteoarthritis in animal models (
      • Barrios Sosa A.C.
      • Yakushijin K.
      • Horne D.A.
      ), implying that concentrations sufficiently high to modulate cellular responses are achievable in animals. If hymenialdisine and DBH can be shown to modulate checkpoint function in animal models, they may merit consideration as drug candidates for cancer therapy.

      Acknowledgments

      We thank Mike LeBlanc for collecting sponges in Papua New Guinea, Steven Pelech and Kinexus Bioinformatics for immunoblot assays, Ruiqiong Ye for excellent technical assistance, and Hilary Anderson for critical evaluation of the manuscript.

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