Structural Basis for Chk1 Inhibition by UCN-01

Abstract

Chk1 is a serine-threonine kinase that plays an important role in the DNA damage response, including G2/M cell cycle control. UCN-01 (7-hydroxystaurosporine), currently in clinical trials, has recently been shown to be a potent Chk1 inhibitor that abrogates the G2/M checkpoint induced by DNA-damaging agents. To understand the structural basis of Chk1 inhibition by UCN-01, we determined the crystal structure of the Chk1 kinase domain in complex with UCN-01. Chk1 structures with staurosporine and its analog SB-218078 were also determined. All three compounds bind in the ATP-binding pocket of Chk1, producing only slight changes in the protein conformation. Selectivity of UCN-01 toward Chk1 over cyclin-dependent kinases can be explained by the presence of a hydroxyl group in the lactam moiety interacting with the ATP-binding pocket. Hydrophobic interactions and hydrogen-bonding interactions were observed in the structures between UCN-01 and the Chk1 kinase domain. The high structural complementarity of these interactions is consistent with the potency and selectivity of UCN-01. Chk1 is a serine-threonine kinase that plays an important role in the DNA damage response, including G2/M cell cycle control. UCN-01 (7-hydroxystaurosporine), currently in clinical trials, has recently been shown to be a potent Chk1 inhibitor that abrogates the G2/M checkpoint induced by DNA-damaging agents. To understand the structural basis of Chk1 inhibition by UCN-01, we determined the crystal structure of the Chk1 kinase domain in complex with UCN-01. Chk1 structures with staurosporine and its analog SB-218078 were also determined. All three compounds bind in the ATP-binding pocket of Chk1, producing only slight changes in the protein conformation. Selectivity of UCN-01 toward Chk1 over cyclin-dependent kinases can be explained by the presence of a hydroxyl group in the lactam moiety interacting with the ATP-binding pocket. Hydrophobic interactions and hydrogen-bonding interactions were observed in the structures between UCN-01 and the Chk1 kinase domain. The high structural complementarity of these interactions is consistent with the potency and selectivity of UCN-01. The cellular response to DNA damage involves cell cycle delays, increased repair, and apoptosis (1Zhou B.-B. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2645) Google Scholar). Although many effective cancer therapies work by causing cell death due to DNA damage, resistance to these therapeutic agents remains a significant limitation in the treatment of cancer. One important mechanism of drug resistance has been attributed to cell cycle delays, also called checkpoints, which provide opportunities for cells to repair DNA damage (2Hartwell L.H. Weinert T.A. Science. 1989; 246: 629-634Crossref PubMed Scopus (2431) Google Scholar). Among different kinases mediating the DNA damage response, Chk1 kinase was shown to be involved in G2/M cell cycle control responding to DNA damage and to play an essential although less understood function during normal cell cycle function (3Liu 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. Genes Dev. 2000; 14: 1448-1459Crossref PubMed Scopus (199) Google Scholar, 4Takai H. Tominaga K. Motoyama N. Minamishima Y.A. Nagahama H. Tsukiyama T. Ikeda K. Nakayama K. Nakanishi M. Nakayama K. Genes Dev. 2000; 14: 1439-1447PubMed Google Scholar). It is likely that Chk1 inhibitors, which abrogate these pathways, would have clinical significance. Recently, UCN-01 1The abbreviations used for: UCN-01, 7-hydroxystaurosporine; CDKs, cyclin-dependent kinases; AEBSF, 4-(2-Aminoethyl)benzenesulfonyl fluoride; DTT, dithiothreitol; GST, glutathione S-transferase; AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate; r.m.s., root mean square. and another staurosporine-like inhibitor, SB218078, were shown to be potent Chk1 inhibitors (5Jackson J.R. Gilmartin A. Imburgia C. Winkler J.D. Marshall L.A. Roshak A. Cancer Res. 2000; 60: 566-572PubMed Google Scholar, 6Graves P.R. Yu L. Schwarz J.K. Gales J. Sausville E.A. O'Connor P.M. Piwnica-Worms H. J. Biol. Chem. 2000; 275: 5600-5605Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar, 7Busby E.C. Leistritz D.F. Abraham R.T. Karnitz L.M. Sarkaria J.N. Cancer Res. 2000; 60: 2108-2112PubMed Google Scholar). UCN-01 is undergoing clinical trials for cancer treatment as a kinase inhibitor (8Sausville E.A. Arbuck S.G. Messmann R. Headlee D. Bauer K.S. Lush R.M. Murgo A. Figg W.D. Lahusen T. Jaken S. Jing X. Roberge M. Fuse E. Kuwabara T. Senderowicz A.M. J. Clin. Oncol. 2001; 19: 2319-2333Crossref PubMed Scopus (300) Google Scholar). UCN-01 potentiates the cytotoxicity of a variety of anticancer agents, including cisplatin, camptothecin, and ionizing radiation (9Wang Q. Fan S. Eastman A. Worland P.J. Sausville E.A. O'Connor P.M. J. Natl. Cancer Inst. 1996; 88: 956-965Crossref PubMed Scopus (450) Google Scholar, 10Bunch R.T. Eastman A. Clin. Cancer Res. 1996; 2: 791-797PubMed Google Scholar, 11Shao R.G. Cao C.X. Shimizu T. O'Connor P.M. Kohn K.W. Pommier Y. Cancer Res. 1997; 57: 4029-4035PubMed Google Scholar), as expected for a Chk1 inhibitor. The latter findings have encouraged the investigation of UCN-01 in combination therapy for cancer treatment. Compared with staurosporine, UCN-01 is a more selective Chk1 inhibitor and is less potent against CDK1 (5Jackson J.R. Gilmartin A. Imburgia C. Winkler J.D. Marshall L.A. Roshak A. Cancer Res. 2000; 60: 566-572PubMed Google Scholar). However, a structural explanation for this selectivity has not been previously available. The x-ray structure for Chk1 kinase domain has recently been solved to 1.7 Å (12Chen P. Luo C. Deng Y. Ryan K. Register J. Margosiak S. Tempczyk-Russell A. Nguyen B. Myers P. Lundgren K. Kan C.C. O'Connor P.M. Cell. 2000; 100: 681-692Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). A crystal structure is also available for staurosporine-liganded CDK2, a close homolog of CDK1 (13Lawrie A.M. Noble M.E. Tunnah P. Brown N.R. Johnson L.N. Endicott J.A. Nat. Struct. Biol. 1997; 4: 796-801Crossref PubMed Scopus (246) Google Scholar). To understand how UCN-01 binds to Chk1 and the structural basis for the selectivity of UCN-01 against CDKs, we determined the x-ray crystal structures of Chk1 in complex with UCN-01 and its analogs staurosporine and SB218078 and compared them with that of the CDK2·staurosporine complex. The structural information from these Chk1 binary complexes should increase our understanding of the structure-activity relationship for kinase inhibitors and its applications to structure-based drug design. The Chk1 kinase domain region, which contains the conserved kinase domain (residues 1–265), part of the linker region (residues 266–289) between the kinase domain and the FHA domain, and 6 His residues at the C terminus (12Chen P. Luo C. Deng Y. Ryan K. Register J. Margosiak S. Tempczyk-Russell A. Nguyen B. Myers P. Lundgren K. Kan C.C. O'Connor P.M. Cell. 2000; 100: 681-692Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar), was subcloned into a pFastBac1 vector at theBamHI and XhoI sites for baculovirus expression. The resulting plasmid was transformed into competent DH10BacEscherichia coli cells according to the Bac-to-Bac method of Invitrogen to generate the high molecular weight bacmid DNA, which was then transfected into Spodoptera frugiperda (Sf9) cells. High titer stocks of the virus were generated and used to infect the cells for expression at 25 °C for 36 h. Harvested cells were suspended in the lysis buffer (50 mmHEPES, pH 7.5, containing 0.34 m sucrose, 10 mm2-mercaptoethanol, 1 mm benzamidine, 1 mmphenylmethylsulfonyl fluoride, and 1 mm AEBSF), homogenized with a Tekmar Tissumizer, and disrupted with a model 110Y fluidizer (Microfluidics Corp., Newton, MA). The Chk1 kinase domain was purified from insect cells by sequential chromatography steps, including Q-Sepharose FastFlow, nickel-nitrilotriacetic acid-agarose, Source 15 Phenyl, and Sephacryl S200 columns at 4 °C. The solution buffer for the final column step was 25 mm Na-HEPES, pH 7.5, containing 0.5 m NaCl, 10% glycerol, 0.05% octylglucoside, 5 mm dithiothreitol (DTT), and 1 mm EDTA. Fractions containing a monomeric form of Chk1 were pooled and concentrated to about 7 mg/ml for crystallization. N-terminal sequencing and liquid chromatography/mass spectrometry analysis revealed that the N-terminal Met of purified Chk1 was cleaved. The Chk1 assay was carried out in 50-μl volumes in the following buffer: 20 mm Na-HEPES, pH 7.4, 50 mm KCl, 1 mm EGTA, 10 mm MgCl2, and 1 mm DTT. A synthetic peptide with a sequence flanking Ser-216 of human Cdc25C, KVSRSGLYRSPSMPENLNRK, was used as its substrate. 2R. Johanson and B. Zhou, unpublished results. Chk1 was used at 5 nm, the peptide was used at 20 μm, and [γ-32P]ATP was used at 10 μm (final concentration). Reactions were incubated at 37 °C for 20 min and stopped by adding 5 μl of acetic acid containing 10 mmATP. Thirty-microliter samples were spotted on 2-cm phosphocellulose filter disks, which were washed four times, 5 min each, with 0.5% phosphoric acid and counted by liquid scintillation. The apparentK m for ATP in the Chk1 assay was about 120 μm. CDK1 and CDK2 assays utilized the peptide Biotin-aminohexyl-ARRPMSPKKKA-NH2 as a phosphoryl group acceptor. CDK1 and CDK2 were expressed in a baculovirus expression system and were partially purified. Cyclin A was purified separately and mixed with CDK1 or CDK2 before assays. Typically, assays were performed by incubating enzyme (2 nm), in the presence or absence of inhibitor, 1.5 mm peptide substrate, 1.4 μm [γ-32P]ATP, and 10 mmMg2+ for periods of time generally within the range of 15 min. Reactions were terminated with equal volumes of either 20% acetic acid or 50 mm EDTA buffered to pH 7 (substrate consumption < 20%). The buffer employed in the enzyme assays was 30 mm HEPES, pH 7.4, containing 0.15 m NaCl and 5% Me2SO. Detection of peptide phosphorylation was accomplished by scintillation counting following collection of the peptide onto phosphocellulose The apparentK m for ATP in CDK1 and CDK2 assays about μm. and were expressed in a baculovirus expression system and purified. The of the protein was by the phosphorylation of a protein (residues to The assay were mm HEPES, pH 7.5, μm μm 5 mm 1 mm DTT, mg/ml enzyme in a final of 50 Reactions were incubated for 30 min at 30 °C and terminated by the of 50 μl of mm mm pH Detection of protein phosphorylation was accomplished by the protein onto phosphocellulose The apparentK m for ATP in the assay was 25 μm. were determined by a to the UCN-01, staurosporine, and SB-218078 bind to the ATP of the kinase and inhibitors, the was on the Although the Chk1 kinase domain structure has been solved (12Chen P. Luo C. Deng Y. Ryan K. Register J. Margosiak S. Tempczyk-Russell A. Nguyen B. Myers P. Lundgren K. Kan C.C. O'Connor P.M. Cell. 2000; 100: 681-692Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar), the were not available to at the time of the solved the structures of the and the complexes The Chk1 kinase domain and its complexes with UCN-01, staurosporine, and SB218078 were m and glycerol, at from previously (12Chen P. Luo C. Deng Y. Ryan K. Register J. Margosiak S. Tempczyk-Russell A. Nguyen B. Myers P. Lundgren K. Kan C.C. O'Connor P.M. Cell. 2000; 100: 681-692Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). To with inhibitors, Chk1 was mixed with inhibitors 1 before The at 20 °C to mm in All were the at the at the Source in the x-ray the were into a solution containing and 20% and were in a at °C. The crystal to the group with cell a Å and and contains The crystal to All three and also to the group and to or the and structural analysis of collection and structure is the of an observed and is the of by of of of is from a of of the that not used in structural is the of an observed and is the of by is from a of of the that not used in structural in a The for Chk1 were by molecular of kinase Noble M.E. Johnson L.N. J. 1997; PubMed Scopus Google as a model in the Scholar). The kinase model was by residues with model a and function solution for Chk1 and with an of for between 10 and Å at the of the model The the for in the and for in the N-terminal domain and for residues between and in the domain was The of model and and model were with the T.A. M. PubMed Scopus Google and to of model were carried out in Scholar). were in the and the was used to and the were used as to residues and The model of the was the with the against between 20 and The final model of the kinase domain residues in and a the from the sequence was not in the residues in the between and and the residues at the C terminus were also of the peptide within the or generally of the as in J. Google Scholar). The three complex structures were solved and with the the model for The from was the in which residues were in with the enzyme model was the inhibitors were into the model on for and were the and by with The in not by in the of resulting in for the structure to protein the Chk1 kinase domain is of an N-terminal domain and a domain. The N-terminal domain is of the domain is and by an linker region (residues The is at the between these Science. PubMed Scopus Google Scholar). The for and the of residues as the in the of The protein in the three with a of Å for from which not changes in Chk1 (12Chen P. Luo C. Deng Y. Ryan K. Register J. Margosiak S. Tempczyk-Russell A. Nguyen B. Myers P. Lundgren K. Kan C.C. O'Connor P.M. Cell. 2000; 100: 681-692Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar), three inhibitors a slight of the ATP-binding pocket. The in the (residues in the between and and between and in the N-terminal domain, the was with the binary complexes Compared with the the for in is the enzyme more as by the of the and The is in an with the of toward the inhibitor. the binary the of three in the N-terminal domain (residues and compared with the of the as from The of Chk1 (residues is and into an and The of the of the in the and in the complex only in a the previously Chk1 structure (12Chen P. Luo C. Deng Y. Ryan K. Register J. Margosiak S. Tempczyk-Russell A. Nguyen B. Myers P. Lundgren K. Kan C.C. O'Connor P.M. Cell. 2000; 100: 681-692Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar), an peptide was to form an with the for the and the binary previously for was observed in the by the at the of N-terminal domain However, the of the is not to in the The between the of this peptide and the of in the model is 37 peptide be a region at the of the C residues from our the between the of this peptide and the of from a is only is also that this peptide from the C terminus of the and to the protein in the Compared with staurosporine, UCN-01 structures in was to be a more selective Chk1 inhibitor and less potent against CDK1 J.R. Gilmartin A. Imburgia C. Winkler J.D. Marshall L.A. Roshak A. Cancer Res. 2000; 60: 566-572PubMed Google Scholar, 6Graves P.R. Yu L. Schwarz J.K. Gales J. Sausville E.A. O'Connor P.M. Piwnica-Worms H. J. Biol. Chem. 2000; 275: 5600-5605Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar, 7Busby E.C. Leistritz D.F. Abraham R.T. Karnitz L.M. Sarkaria J.N. Cancer Res. 2000; 60: 2108-2112PubMed Google Scholar). these and as in B. we these compounds against CDK2 and and from results. UCN-01, staurosporine, and SB-218078 bind to the ATP-binding pocket of the enzyme in the the the inhibitor binds to the pocket between the and with the in a One of the of the many with the and of residues from the N-terminal domain, which and The of the interactions with the of and from the and The of the in the of the with the and of and from The group in the pocket by and The group of UCN-01 on the lactam moiety a to the of the of the inhibitor a from the of in the linker hydrogen-bonding is in many protein complexes L.N. Noble M.E. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). The and with in the A has also been in CDK2 structures (13Lawrie A.M. Noble M.E. Tunnah P. Brown N.R. Johnson L.N. Endicott J.A. Nat. Struct. Biol. 1997; 4: 796-801Crossref PubMed Scopus (246) Google Scholar). The hydroxyl group in the of the pocket toward the domain from the structure of the The hydroxyl group of UCN-01 a with the of and a with more and them with the of and the of and The between UCN-01 and Chk1 the group of the of the inhibitor. group is and to form with the group of and the of 4 To UCN-01, residues in the ATP-binding and either to or to with the inhibitor. The of is in the with has a inhibitors The of in the binary complex changes from that in the that its can a with UCN-01. The interactions between UCN-01 and the N-terminal domain residues in the by a in the of this for the of the is in the is in the UCN-01 binary complex. UCN-01 the more and changes in the of residues is for the of in the its is in the binary complex. The of this toward the inhibitor interactions and with the enzyme as 4 It has the structure for the of the hydroxyl group on the lactam moiety The staurosporine group a to the of the of the inhibitor a from the of in the linker The of from the inhibitor to to UCN-01 and staurosporine, SB218078 a group and has on either of the SB218078 binds in the pocket as UCN-01, although its binary complex from the UCN-01 complex in the of residues and SB218078 with and The hydrogen-bonding interactions of the moiety with the of and in the linker region the as of UCN-01. Compared with the binary SB218078 from the pocket to close between the and the of a the between the group of SB218078 and the of is in the the between and the in the of from Å to more The complementarity between a and a protein has been as the of the of the protein by the of and the of the by the of the protein J. C. J. Biol. Chem. Full Text PDF PubMed Google Scholar). this the between and protein a with the close not and inhibitors, the complementarity is expected to be high and has been at for ATP in complex with CDK2 Worland P.J. Sausville E. Natl. S. A. 1996; PubMed Scopus Google Scholar). inhibitor complexes with CDK2 have shown complementarity between and N. L. C. L. Chem. Google Scholar). not only the of the to a which is to the protein kinase also the changes the protein in response to the The complementarity and for the staurosporine, and SB-218078 complexes in these high complementarity with Chk1 of Chk1 and in Chk1 in in in in in a the structure of the Chk1 kinase domain is to of CDK2 and kinase with which and sequence the acid residues in the have the structure and interactions as in The between Chk1 and CDK2 is in the at the Chk1 a (residues and CDK2 has a of residues to The between from residues of Chk1 and CDK2 kinase has an of only to the in the and the is in the CDK2·staurosporine model due to in the region (13Lawrie A.M. Noble M.E. Tunnah P. Brown N.R. Johnson L.N. Endicott J.A. Nat. Struct. Biol. 1997; 4: 796-801Crossref PubMed Scopus (246) Google Scholar). The of the ATP-binding pocket for Chk1 is to that of conserved residues have the and in the Chk1 structure and in the CDK2 in Chk1 is also conserved in CDK2 as with a different conformation. Chk1, the of is the that the group at the of the toward the ATP-binding from as in the CDK2 The on the group of with and of the with the of a acid in the Chk1 ATP-binding pocket and which by and in also in the which is more in Chk1 in The of in Chk1 has a different from that of the in CDK2, causing the of the to be in a different a this is more that in The crystal structures of the Chk1 kinase domain and its complexes not only the structural basis for UCN-01 inhibition also structural of Chk1 is the that the Chk1 kinase domain an with a in the absence of phosphorylation or (12Chen P. Luo C. Deng Y. Ryan K. Register J. Margosiak S. Tempczyk-Russell A. Nguyen B. Myers P. Lundgren K. Kan C.C. O'Connor P.M. Cell. 2000; 100: 681-692Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). in the kinase domain not increase its kinase in (12Chen P. Luo C. Deng Y. Ryan K. Register J. Margosiak S. Tempczyk-Russell A. Nguyen B. Myers P. Lundgren K. Kan C.C. O'Connor P.M. Cell. 2000; 100: 681-692Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). either UCN-01, staurosporine, or SB218078 to Chk1 only a slight of the and of the (12Chen P. Luo C. Deng Y. Ryan K. Register J. Margosiak S. Tempczyk-Russell A. Nguyen B. Myers P. Lundgren K. Kan C.C. O'Connor P.M. Cell. 2000; 100: 681-692Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). that Chk1 kinase is not by phosphorylation of its kinase domain or by It is likely that Chk1 is different from the mechanism of of CDK1 and CDK2, which involves the phosphorylation and of changes in or Chk1 phosphorylation its cellular or The high x-ray structures of human Chk1 in complex with UCN-01 and its analogs staurosporine and SB218078 provide the structural basis for understanding the potency of this of three inhibitors bind to the ATP-binding pocket of the The lactam group from UCN-01 and staurosporine, and the group from SB218078, the hydrogen-bonding interactions of the ATP It is to that the and in these inhibitors with in the the of this linker region has a to with the of in these is the hydroxyl group of UCN-01 a with in is in the staurosporine and the SB-218078 complex between UCN-01 or staurosporine and Chk1 the group in the of these group is to form with the of in Chk1, in CDK2, and to the of in Chk1, in to UCN-01 and staurosporine, SB218078 has a and not have the However, SB218078 is as potent as staurosporine in Chk1 and CDK2 that the of these to inhibitor is is consistent with the that structural changes in the group generally have less on the potency of staurosporine analogs H. 1996; Scopus Google Scholar). the potency of staurosporine analogs from the between the lactam moiety and the linker region and from the of between the and the of the The revealed the of the interactions between Chk1 and UCN-01 or its of the that UCN-01 is The is for staurosporine and for SB218078 The for the has been to be PubMed Scopus Google Scholar). the of in the an can be for complex. The at and with the for Chk1 inhibition The that the of these can be explained in with only is consistent with the of this of inhibitors, and also that the of Chk1 not by these UCN-01 has a of the lactam in The of a to by a hydroxyl group changes the kinase selectivity against cyclin-dependent kinases the CDKs, that with the of UCN-01 and staurosporine from at in It is that the to in Chk1 is by in CDK2 and in Although this not the of the the would with the inhibitor a group hydroxyl is the hydroxyl group of UCN-01 with in Chk1, which is by in CDK2, and The that UCN-01 is a inhibitor staurosporine be partially due to these the selectivity of SB-218078 is more complex due to changes in to Chk1, UCN-01 and its analogs a slight of the ATP-binding The of residues that with the inhibitors with to in the changes to UCN-01 and its residues in the and inhibitor Although these changes compared with should be in structure-activity relationship analysis and drug design. the structures of Chk1 in complex with UCN-01 and its analogs provide structural information for Chk1 and Hydrophobic interactions and hydrogen-bonding interactions observed in the structures between the inhibitors and the enzyme and provide a structural explanation for the potency of UCN-01 against selectivity of Chk1 inhibitors over can be by changes to the lactam group interacting with the ATP-binding pocket. our the of of drug at more inhibitors of Chk1 and for on the and for and Johnson for and during the of this