Crystal Structures of Catalytic Subunit of cAMP-dependent Protein Kinase in Complex with Isoquinolinesulfonyl Protein Kinase Inhibitors H7, H8, and H89

Abstract

The discovery of several hundred different protein kinases involved in highly diverse cellular signaling pathways is in stark contrast to the much smaller number of known modulators of cell signaling. Of these, the H series protein kinase inhibitors (1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H8) N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89)) are frequently used to block signaling pathways in studies of cellular regulation. To elucidate inhibition mechanisms at atomic resolution and to enable structure-based drug design of potential therapeutic modulators of signaling pathways, we determined the crystal structures of corresponding complexes with the cAPK catalytic subunit. Complexes with H7 and H8 (2.2 Å) and with H89 (2.3 Å) define the binding mode of the isoquinoline-sulfonamide derivatives in the ATP-binding site while demonstrating effects of ligand-induced structural change. Specific interactions between the enzyme and the inhibitors include the isoquinoline ring nitrogen ligating to backbone amide of Val-123 and an inhibitor side chain amide bonding to the backbone carbonyl of Glu-170. The conservation of the ATP-binding site of protein kinases allows evaluation of factors governing general selectivity of these inhibitors among kinases. These results should assist efforts in the design of protein kinase inhibitors with specific properties. The discovery of several hundred different protein kinases involved in highly diverse cellular signaling pathways is in stark contrast to the much smaller number of known modulators of cell signaling. Of these, the H series protein kinase inhibitors (1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H8) N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89)) are frequently used to block signaling pathways in studies of cellular regulation. To elucidate inhibition mechanisms at atomic resolution and to enable structure-based drug design of potential therapeutic modulators of signaling pathways, we determined the crystal structures of corresponding complexes with the cAPK catalytic subunit. Complexes with H7 and H8 (2.2 Å) and with H89 (2.3 Å) define the binding mode of the isoquinoline-sulfonamide derivatives in the ATP-binding site while demonstrating effects of ligand-induced structural change. Specific interactions between the enzyme and the inhibitors include the isoquinoline ring nitrogen ligating to backbone amide of Val-123 and an inhibitor side chain amide bonding to the backbone carbonyl of Glu-170. The conservation of the ATP-binding site of protein kinases allows evaluation of factors governing general selectivity of these inhibitors among kinases. These results should assist efforts in the design of protein kinase inhibitors with specific properties. INTRODUCTIONIsoquinolinesulfonamide protein kinase inhibitors of the H series are among the most widely used inhibitors of Ser/Thr kinases and are indispensable in cellular and signal transduction research. Protein phosphorylation (central to cellular regulation) is mediated by the individual action of several hundred different protein kinases. Recent progress in the understanding of the highly complex cellular signaling networks depends largely on the availability and quality of specific agents that interfere with the pathways under investigation. The usefulness of a protein kinase inhibitor is defined by its ability to permeate cell membranes, its solubility, and its relative degree of specificity. Each year hundreds of publications describe work using isoquinolinesulfonamide inhibitors, despite the fact that the mode of specific inhibition by these compounds is not completely understood.We have used the cAMP-dependent protein kinase (cAPK), 1The abbreviations used are: cAPKcAMP-dependent protein kinaseH71-(5-isoquinolinesulfonyl)-2-methylpiperazineH8N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamideH89N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamideCKi-1truncated form of protein kinase CK-1CKI-7N-[2-aminoethyl]-5-chloro-isoquinoline-8-sulfonamideMES4-morpholineethanesulfonic acidChaps3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diolPKIprotein kinase inhibitor. which binds many H series inhibitors, as a model system to investigate the factors governing inhibitor binding and specificity. Three of the most frequently used members of this class are H7, H8 (1Hidaka H. Inagaki M. Kawamoto S. Sasaki Y. Biochemistry. 1984; 23: 5036-5041Crossref PubMed Scopus (2321) Google Scholar), and H89 (2Chijiwa T. Mishima A. Hagiwara M. Sano M. Hayashi K. Inoue T. Naito K. Toshioka T. Hidaka H. J. Biol. Chem. 1990; 265: 5267-5272Abstract Full Text PDF PubMed Google Scholar) (Fig. 1). They all act in competition to ATP but not to substrate. The highest selectivity and affinity is found with H89, which has a Ki of 48 nM for cAPK, whereas H8 has a moderate affinity for both cGPK and cAPK, and H7 inhibits protein kinase C in addition to cGPK and cAPK (Table I). The inhibitors have enabled assignment of specific roles of these protein kinases in numerous regulatory interrelations (3Denisenko N. Burighel P. Citi S. J. Cell. Sci. 1994; 107: 969-981PubMed Google Scholar, 4Malinow R. Schulman H. Tsien R.W. Science. 1989; 245: 862-866Crossref PubMed Scopus (1034) Google Scholar, 5Malenka R.C. Kauer J.A. Perkel D.J. Mauk M.D. Kelly P.T. Nicoll R.A. Waxham M.N. Nature. 1989; 340: 554-557Crossref PubMed Scopus (770) Google Scholar, 6Dubois M.-F. Nguyen V.T. Bellier S. Bensaude O. J. Biol. Chem. 1994; 269: 13331-13336Abstract Full Text PDF PubMed Google Scholar, 7Konda Y. Gantz I. DelValle J. Shimoto Y. Miwa H. Yamada T. J. Biol. Chem. 1994; 269: 13162-13166Abstract Full Text PDF PubMed Google Scholar, 8Guizouarn H. Borgese F. Pellissier B. Garcia-Romeu F. Motais R. J. Biol. Chem. 1993; 268: 8632-8639Abstract Full Text PDF PubMed Google Scholar). They are, however, not only valuable for the cell biologist. Pharmacologists are increasingly interested in the possibility of interfering with cellular signaling, especially in the area of anticancer drug discovery. There is evidence that isoquinolinesulfonyl inhibitors may be promising in this field (9Findik D. Song Q. Hidaka H. Lavin M. J. Cell. Biochem. 1995; 57: 12-21Crossref PubMed Scopus (51) Google Scholar, 10Traganos F. Knuttihotz J. Hotz M. Gorczyca W. Ardelt B. Darzynkiewicz Z. Int. J. Oncol. 1993; 2: 47-59PubMed Google Scholar, 11Felipo V. Minana M.D. Cabedo H. Perezminguez F. Llombartbosch A. Grisolia S. Eur. J. Can. 1994; 30: 525-527Abstract Full Text PDF Scopus (5) Google Scholar, 12May W.S. Tyler P.G. Ito T. Armstrong D.K. Qatsha K.A. Davidson N.E. J. Biol. Chem. 1994; 269: 26865-26870Abstract Full Text PDF PubMed Google Scholar, 13Honma Y. Okabe Kado J. Kasukabe T. Hozumi M. Kajigaya S. Suda T. Miura Y. Cancer Res. 1991; 51: 4649-4655PubMed Google Scholar, 14Kim S.H. Park J.I. Chung B.S. Kang C.D. Hidaka H. Cancer Lett. 1993; 74: 37-41Crossref PubMed Scopus (28) Google Scholar).Table IInhibitory constants of H series protein kinase inhibitorsCompoundKi (μM)Ref.cGPKcAPKMLCKPKCCK-1CK-21-(3-Isoquinollnesulfonyl)-2-methylpiperazine (H7)5.83.0976.01007801N-[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide (H8)0.481.268151339501N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89)0.48 ± 0.130.048 ±0.00828.3 ± 17.531.7 ± 15.938.3 ± 6.0136.7 ± 17.02 Open table in a new tab Recently, crystal structures from several different protein kinases have been solved (for review see 15Morgan D.O. Debondt H.L. Curr. Opin. Cell Biol. 1994; 6: 239-246Crossref PubMed Scopus (91) Google Scholar, 16Goldsmith E.J. Cobb M.H. Curr. Opin. Struct. Biol. 1994; 4: 833-840Crossref PubMed Scopus (63) Google Scholar, 17Bossemeyer D. FEBS Lett. 1995; 369: 57-61Crossref PubMed Scopus (90) Google Scholar, 18Taylor S.S. Radzioandzelm E. Structure. 1994; 2: 345-355Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 19Cox S. Radzioandzelm E. Taylor S.S. Curr. Opin. Struct. Biol. 1994; 4: 893-901Crossref PubMed Scopus (128) Google Scholar). The structures confirmed not only the high degree of structural conservation of the highly homologous catalytic kinase core (20Hanks S.K. Quinn A.M. Methods Enzymol. 1991; 200: 38-81Crossref PubMed Scopus (1079) Google Scholar) but simultaneously showed how subtle differences are responsible for individual properties of protein kinases. Most of the residues that are highly conserved or invariant in the protein kinase family line the active site, and many of them interact with ATP (21Zheng J.H. Knighton D.R. ten-Eyck L.F. Karlsson R. Xuong N. Taylor S.S. Sowadski J.M. Biochemistry. 1993; 32: 2154-2161Crossref PubMed Scopus (512) Google Scholar, 22Bossemeyer D. Engh R.A. Kinzel V. Ponstingl H. Huber R. EMBO J. 1993; 12: 849-859Crossref PubMed Scopus (371) Google Scholar). Despite high similarity of Km values for ATP binding among protein kinases, the H inhibitors have remarkably different Ki values. The crystal structures of kinase bound inhibitor molecules described here now show the mode of inhibitory action and the factors governing selectivity and will provide a firm basis for the design of new protein kinase inhibitors. INTRODUCTIONIsoquinolinesulfonamide protein kinase inhibitors of the H series are among the most widely used inhibitors of Ser/Thr kinases and are indispensable in cellular and signal transduction research. Protein phosphorylation (central to cellular regulation) is mediated by the individual action of several hundred different protein kinases. Recent progress in the understanding of the highly complex cellular signaling networks depends largely on the availability and quality of specific agents that interfere with the pathways under investigation. The usefulness of a protein kinase inhibitor is defined by its ability to permeate cell membranes, its solubility, and its relative degree of specificity. Each year hundreds of publications describe work using isoquinolinesulfonamide inhibitors, despite the fact that the mode of specific inhibition by these compounds is not completely understood.We have used the cAMP-dependent protein kinase (cAPK), 1The abbreviations used are: cAPKcAMP-dependent protein kinaseH71-(5-isoquinolinesulfonyl)-2-methylpiperazineH8N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamideH89N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamideCKi-1truncated form of protein kinase CK-1CKI-7N-[2-aminoethyl]-5-chloro-isoquinoline-8-sulfonamideMES4-morpholineethanesulfonic acidChaps3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diolPKIprotein kinase inhibitor. which binds many H series inhibitors, as a model system to investigate the factors governing inhibitor binding and specificity. Three of the most frequently used members of this class are H7, H8 (1Hidaka H. Inagaki M. Kawamoto S. Sasaki Y. Biochemistry. 1984; 23: 5036-5041Crossref PubMed Scopus (2321) Google Scholar), and H89 (2Chijiwa T. Mishima A. Hagiwara M. Sano M. Hayashi K. Inoue T. Naito K. Toshioka T. Hidaka H. J. Biol. Chem. 1990; 265: 5267-5272Abstract Full Text PDF PubMed Google Scholar) (Fig. 1). They all act in competition to ATP but not to substrate. The highest selectivity and affinity is found with H89, which has a Ki of 48 nM for cAPK, whereas H8 has a moderate affinity for both cGPK and cAPK, and H7 inhibits protein kinase C in addition to cGPK and cAPK (Table I). The inhibitors have enabled assignment of specific roles of these protein kinases in numerous regulatory interrelations (3Denisenko N. Burighel P. Citi S. J. Cell. Sci. 1994; 107: 969-981PubMed Google Scholar, 4Malinow R. Schulman H. Tsien R.W. Science. 1989; 245: 862-866Crossref PubMed Scopus (1034) Google Scholar, 5Malenka R.C. Kauer J.A. Perkel D.J. Mauk M.D. Kelly P.T. Nicoll R.A. Waxham M.N. Nature. 1989; 340: 554-557Crossref PubMed Scopus (770) Google Scholar, 6Dubois M.-F. Nguyen V.T. Bellier S. Bensaude O. J. Biol. Chem. 1994; 269: 13331-13336Abstract Full Text PDF PubMed Google Scholar, 7Konda Y. Gantz I. DelValle J. Shimoto Y. Miwa H. Yamada T. J. Biol. Chem. 1994; 269: 13162-13166Abstract Full Text PDF PubMed Google Scholar, 8Guizouarn H. Borgese F. Pellissier B. Garcia-Romeu F. Motais R. J. Biol. Chem. 1993; 268: 8632-8639Abstract Full Text PDF PubMed Google Scholar). They are, however, not only valuable for the cell biologist. Pharmacologists are increasingly interested in the possibility of interfering with cellular signaling, especially in the area of anticancer drug discovery. There is evidence that isoquinolinesulfonyl inhibitors may be promising in this field (9Findik D. Song Q. Hidaka H. Lavin M. J. Cell. Biochem. 1995; 57: 12-21Crossref PubMed Scopus (51) Google Scholar, 10Traganos F. Knuttihotz J. Hotz M. Gorczyca W. Ardelt B. Darzynkiewicz Z. Int. J. Oncol. 1993; 2: 47-59PubMed Google Scholar, 11Felipo V. Minana M.D. Cabedo H. Perezminguez F. Llombartbosch A. Grisolia S. Eur. J. Can. 1994; 30: 525-527Abstract Full Text PDF Scopus (5) Google Scholar, 12May W.S. Tyler P.G. Ito T. Armstrong D.K. Qatsha K.A. Davidson N.E. J. Biol. Chem. 1994; 269: 26865-26870Abstract Full Text PDF PubMed Google Scholar, 13Honma Y. Okabe Kado J. Kasukabe T. Hozumi M. Kajigaya S. Suda T. Miura Y. Cancer Res. 1991; 51: 4649-4655PubMed Google Scholar, 14Kim S.H. Park J.I. Chung B.S. Kang C.D. Hidaka H. Cancer Lett. 1993; 74: 37-41Crossref PubMed Scopus (28) Google Scholar).Table IInhibitory constants of H series protein kinase inhibitorsCompoundKi (μM)Ref.cGPKcAPKMLCKPKCCK-1CK-21-(3-Isoquinollnesulfonyl)-2-methylpiperazine (H7)5.83.0976.01007801N-[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide (H8)0.481.268151339501N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89)0.48 ± 0.130.048 ±0.00828.3 ± 17.531.7 ± 15.938.3 ± 6.0136.7 ± 17.02 Open table in a new tab Recently, crystal structures from several different protein kinases have been solved (for review see 15Morgan D.O. Debondt H.L. Curr. Opin. Cell Biol. 1994; 6: 239-246Crossref PubMed Scopus (91) Google Scholar, 16Goldsmith E.J. Cobb M.H. Curr. Opin. Struct. Biol. 1994; 4: 833-840Crossref PubMed Scopus (63) Google Scholar, 17Bossemeyer D. FEBS Lett. 1995; 369: 57-61Crossref PubMed Scopus (90) Google Scholar, 18Taylor S.S. Radzioandzelm E. Structure. 1994; 2: 345-355Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 19Cox S. Radzioandzelm E. Taylor S.S. Curr. Opin. Struct. Biol. 1994; 4: 893-901Crossref PubMed Scopus (128) Google Scholar). The structures confirmed not only the high degree of structural conservation of the highly homologous catalytic kinase core (20Hanks S.K. Quinn A.M. Methods Enzymol. 1991; 200: 38-81Crossref PubMed Scopus (1079) Google Scholar) but simultaneously showed how subtle differences are responsible for individual properties of protein kinases. Most of the residues that are highly conserved or invariant in the protein kinase family line the active site, and many of them interact with ATP (21Zheng J.H. Knighton D.R. ten-Eyck L.F. Karlsson R. Xuong N. Taylor S.S. Sowadski J.M. Biochemistry. 1993; 32: 2154-2161Crossref PubMed Scopus (512) Google Scholar, 22Bossemeyer D. Engh R.A. Kinzel V. Ponstingl H. Huber R. EMBO J. 1993; 12: 849-859Crossref PubMed Scopus (371) Google Scholar). Despite high similarity of Km values for ATP binding among protein kinases, the H inhibitors have remarkably different Ki values. The crystal structures of kinase bound inhibitor molecules described here now show the mode of inhibitory action and the factors governing selectivity and will provide a firm basis for the design of new protein kinase inhibitors.