Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Structural Insights and Biological Effects of Glycogen Synthase Kinase 3-specific Inhibitor AR-A014418

2003; Elsevier BV; Volume: 278; Issue: 46 Linguagem: Inglês

10.1074/jbc.m306268200

ISSN

1083-351X

Autores

Ratan V. Bhat, Yafeng Xue, Stefan von Berg, Sven Hellberg, Mats Ormö, Yvonne Nilsson, Ann‐Cathrin Radesäter, Eva Jerning, Per-Olof Markgren, Thomas Borgegård, Martin Nylöf, Alfredo Giménez-Cassina, Félix Hernández, José J. Lucas, Javier Dı́az-Nido, Jesús Ávila,

Tópico(s)

Alzheimer's disease research and treatments

Resumo

Glycogen synthase kinase 3 (GSK3) is a serine/threonine kinase that has been implicated in pathological conditions such as diabetes and Alzheimer's disease. We report the characterization of a GSK3 inhibitor, AR-A014418, which inhibits GSK3 (IC50 = 104 ± 27 nM), in an ATP-competitive manner (K i = 38 nM). AR-A014418 does not significantly inhibit cdk2 or cdk5 (IC50 > 100 μM) or 26 other kinases demonstrating high specificity for GSK3. We report the co-crystallization of AR-A014418 with the GSK3β protein and provide a description of the interactions within the ATP pocket, as well as an understanding of the structural basis for the selectivity of AR-A014418. AR-A014418 inhibits tau phosphorylation at a GSK3-specific site (Ser-396) in cells stably expressing human four-repeat tau protein. AR-A014418 protects N2A neuroblastoma cells against cell death mediated by inhibition of the phosphatidylinositol 3-kinase/protein kinase B survival pathway. Furthermore, AR-A014418 inhibits neurodegeneration mediated by β-amyloid peptide in hippocampal slices. AR-A014418 may thus have important applications as a tool to elucidate the role of GSK3 in cellular signaling and possibly in Alzheimer's disease. AR-A014418 is the first compound of a family of specific inhibitors of GSK3 that does not significantly inhibit closely related kinases such as cdk2 or cdk5. Glycogen synthase kinase 3 (GSK3) is a serine/threonine kinase that has been implicated in pathological conditions such as diabetes and Alzheimer's disease. We report the characterization of a GSK3 inhibitor, AR-A014418, which inhibits GSK3 (IC50 = 104 ± 27 nM), in an ATP-competitive manner (K i = 38 nM). AR-A014418 does not significantly inhibit cdk2 or cdk5 (IC50 > 100 μM) or 26 other kinases demonstrating high specificity for GSK3. We report the co-crystallization of AR-A014418 with the GSK3β protein and provide a description of the interactions within the ATP pocket, as well as an understanding of the structural basis for the selectivity of AR-A014418. AR-A014418 inhibits tau phosphorylation at a GSK3-specific site (Ser-396) in cells stably expressing human four-repeat tau protein. AR-A014418 protects N2A neuroblastoma cells against cell death mediated by inhibition of the phosphatidylinositol 3-kinase/protein kinase B survival pathway. Furthermore, AR-A014418 inhibits neurodegeneration mediated by β-amyloid peptide in hippocampal slices. AR-A014418 may thus have important applications as a tool to elucidate the role of GSK3 in cellular signaling and possibly in Alzheimer's disease. AR-A014418 is the first compound of a family of specific inhibitors of GSK3 that does not significantly inhibit closely related kinases such as cdk2 or cdk5. Alzheimer's disease (AD) 1The abbreviations used are: AD, Alzheimer's disease; NFT, intraneuronal neurofibrillary tangle; PHF, paired helical filament; GSK, glycogen synthase kinase; APP, amyloid precursor protein; DTT, dithiothreitol; PS, presenilin; GST, glutathione S-transferase; PI, propidium iodide; SPA, scintillation proximity assay; MOPS, 4-morpholinepropanesulfonic acid; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; Aβ, amyloid β; PBS, phosphate-buffered saline; eIF2B, eukaryotic initiation factor 2B; PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B. affects more than 18 million people worldwide. The onset is insidious, and the loss of memory is an early indicator of the disease. As the disease progresses, there are psychiatric abnormalities and motor deficits and death occurs within 7-8 years after diagnosis. The pathological hallmarks of AD include extracellular amyloid plaques and intraneuronal neurofibrillary tangles (NFTs) (1Alzheimer A. Z. Gesamte Neurol. Psychiat. 1911; 4: 356-385Crossref Scopus (551) Google Scholar, 2Yankner B.A. Neuron. 1996; 16: 921-932Abstract Full Text Full Text PDF PubMed Scopus (920) Google Scholar). Neurode-generation is also evident in the limbic regions of the brain, and brain atrophy occurs at the later stages. The density of NFTs correlates extremely well with the clinical severity of the disease, and the distribution follows a characteristic pattern of expansion as the disease progresses (3Braak H. Braak E. Acta Neuropathol. (Berl.). 1991; 82: 239-259Crossref PubMed Scopus (11641) Google Scholar). This provides a compelling argument that strategies to inhibit factors leading to NFT development and neuronal death would be useful in the treatment of mid-moderate stage AD. NFTs are found in the neuronal cell bodies and apical dendrites and comprise aggregates of paired helical filaments (PHFs), which are assembled from hyperphosphorylated forms of the microtubule-associated protein tau (4Avila J. FEBS Lett. 2000; 476: 89-92Crossref PubMed Scopus (76) Google Scholar, 5Goedert M. Spillantini M.G. Biochem. Soc. Symp. 2001; 67: 59-71Crossref Scopus (49) Google Scholar, 6Lee V.M. Goedert M. Trojanowski J.Q. Annu. Rev. Neurosci. 2001; 24: 1121-1159Crossref PubMed Scopus (2145) Google Scholar). Pathologic alterations in the microtubule-associated protein tau leading to PHFs and subsequently NFT have been implicated in a number of neurodegenerative disorders, particularly dementias, including AD, dementia pugilistic, progressive supranuclear palsy, frontotemporal dementia, and amyotrophic lateral sclerosis dementia (6Lee V.M. Goedert M. Trojanowski J.Q. Annu. Rev. Neurosci. 2001; 24: 1121-1159Crossref PubMed Scopus (2145) Google Scholar). GSK3β, also called tau phosphorylating kinase I (7Ishiguro K. Shiratsuchi A. Sato S. Omori A. Arioka M. Kobayashi S. Uchida T. Imahori K. FEBS Lett. 1993; 325: 167-172Crossref PubMed Scopus (366) Google Scholar), is a serine/threonine kinase that has highest abundance in brain and is localized primarily in neurons (8Woodgett J.R. EMBO J. 1990; 9: 2431-2438Crossref PubMed Scopus (1154) Google Scholar, 9Hanger D.P. Hughes K. Woodgett J.R. Brion J.P. Anderton B.H. Neurosci. Lett. 1992; 147: 58-62Crossref PubMed Scopus (656) Google Scholar, 10Takahashi M. Tomizawa K. Kato R. Sato K. Uchida T. Fujita S.C. Imahori K. J. Neurochem. 1994; 63: 245-255Crossref PubMed Scopus (128) Google Scholar). It is highly conserved throughout evolution, and its expression is decreased in the adult (10Takahashi M. Tomizawa K. Kato R. Sato K. Uchida T. Fujita S.C. Imahori K. J. Neurochem. 1994; 63: 245-255Crossref PubMed Scopus (128) Google Scholar). Aberrant increase in GSK3β expression and activity in adult brain has been directly linked to several of the key neuropathological mechanisms of AD (11Shiurba R.A. Ishiguro K. Takahashi M. Sato K. Spooner E.T. Mercken M. Yoshida R. Wheelock T.R. Yanagawa H. Imahori K. Nixon R.A. Brain Res. 1996; 737: 119-132Crossref PubMed Scopus (65) Google Scholar, 12Pei J.J. Braak E. Braak H. Grundke-Iqbal I. Iqbal K. Winblad B. Cowburn R.F. J. Neuropathol. Exp. Neurol. 1999; 58: 1010-1019Crossref PubMed Scopus (430) Google Scholar, 51Phiel C.J. Wilson C.A. Lee V.M. Klein P.S. Nature. 2003; 423: 435-439Crossref PubMed Scopus (1091) Google Scholar). GSK3 has two major isoforms (α and β), which are 98% identical within the catalytic domain. GSK3 phosphorylates tau at most sites, which becomes abnormally hyperphosphorylated in PHFs, both in cells (13Lovestone S. Reynolds C.H. Latimer D. Davis D.R. Anderton B.H. Gallo J.M. Hanger D. Mulot S. Marquardt B. Stabel S. Curr. Biol. 1994; 4: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar) and in rodents (14Hong M. Chen D.C. Klein P.S. Lee V.M. J. Biol. Chem. 1997; 272: 25326-25332Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar, 15Munoz-Montano J.R. Moreno F.J. Avila J. Diaz-Nido J. FEBS Lett. 1997; 411: 183-188Crossref PubMed Scopus (311) Google Scholar), and has been linked to the production of β-amyloid, the chief constituent of amyloid plaques (51Phiel C.J. Wilson C.A. Lee V.M. Klein P.S. Nature. 2003; 423: 435-439Crossref PubMed Scopus (1091) Google Scholar). The most crucial pathological link with AD has been its association with PHFs (16Grundke-Iqbal I. Iqbal K. Tung Y.C. Quinlan M. Wisniewski H.M. Binder L.I. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4913-4917Crossref PubMed Scopus (2877) Google Scholar, 17Lee V.M. Balin B.J. Otvos Jr., L. Trojanowski J.Q. Science. 1991; 251: 675-678Crossref PubMed Scopus (1252) Google Scholar). GSK3 has also been linked to the downstream effects of β-amyloid. Exposure of cortical and hippocampal primary neuronal cultures to β-amyloid induces the activation of GSK3β (18Takashima A. Noguchi K. Michel G. Mercken M. Hoshi M. Ishiguro K. Imahori K. Neurosci. Lett. 1996; 203: 33-36Crossref PubMed Scopus (244) Google Scholar) and cell death (19Busciglio J. Lorenzo A. Yeh J. Yankner B.A. Neuron. 1995; 14: 879-888Abstract Full Text PDF PubMed Scopus (565) Google Scholar, 20Takashima A. Noguchi K. Sato K. Hoshino T. Imahori K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7789-7793Crossref PubMed Scopus (380) Google Scholar). Blockade of GSK3β expression by antisense oligonucleotides (20Takashima A. Noguchi K. Sato K. Hoshino T. Imahori K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7789-7793Crossref PubMed Scopus (380) Google Scholar) or its activity by lithium (21Alvarez G. Munoz-Montano J.R. Satrustegui J. Avila J. Bogonez E. Diaz-Nido J. FEBS Lett. 1999; 453: 260-264Crossref PubMed Scopus (262) Google Scholar) inhibits Aβ-induced neurodegeneration of cortical and hippocampal primary cultures. Presenilin-1 (PS-1), a protein influencing APP metabolism and linked to familial AD, has been shown to directly bind GSK3β and tau in co-immunoprecipitation experiments from human brain samples (22Takashima A. Murayama M. Murayama O. Kohno T. Honda T. Yasutake K. Nihonmatsu N. Mercken M. Yamaguchi H. Sugihara S. Wolozin B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9637-9641Crossref PubMed Scopus (400) Google Scholar). Thus, the ability of PS-1 to bring GSK3β and tau into close proximity suggests that PS-1 may regulate phosphorylation of tau by GSK3β. Mutant PS-1 results in increased PS-1/GSK3β association and increased phosphorylation of tau (22Takashima A. Murayama M. Murayama O. Kohno T. Honda T. Yasutake K. Nihonmatsu N. Mercken M. Yamaguchi H. Sugihara S. Wolozin B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9637-9641Crossref PubMed Scopus (400) Google Scholar). Furthermore, PS-1 forms a complex with the GSK3β substrate β-catenin in transfected cells (23Yu G. Chen F. Levesque G. Nishimura M. Zhang D.M. Levesque L. Rogaeva E. Xu D. Liang Y. Duthie M. St. George-Hyslop P.H. Fraser P.E. J. Biol. Chem. 1998; 273: 16470-16475Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar, 24Murayama M. Tanaka S. Palacino J. Murayama O. Honda T. Sun X. Yasutake K. Nihonmatsu N. Wolozin B. Takashima A. FEBS Lett. 1998; 433: 73-77Crossref PubMed Scopus (150) Google Scholar) and in vivo (23Yu G. Chen F. Levesque G. Nishimura M. Zhang D.M. Levesque L. Rogaeva E. Xu D. Liang Y. Duthie M. St. George-Hyslop P.H. Fraser P.E. J. Biol. Chem. 1998; 273: 16470-16475Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar, 25Zhang Z. Hartmann H. Do V.M. Abramowski D. Sturchler-Pierrat C. Staufenbiel M. Sommer B. van de Wetering M. Clevers H. Saftig P. De Strooper B. He X. Yankner B.A. Nature. 1998; 395: 698-702Crossref PubMed Scopus (473) Google Scholar), and this interaction increases β-catenin stability (25Zhang Z. Hartmann H. Do V.M. Abramowski D. Sturchler-Pierrat C. Staufenbiel M. Sommer B. van de Wetering M. Clevers H. Saftig P. De Strooper B. He X. Yankner B.A. Nature. 1998; 395: 698-702Crossref PubMed Scopus (473) Google Scholar). Pathogenic PS-1 mutations reduce the ability of PS-1 to stabilize β-catenin, which in turn results in decreased β-catenin levels in brains from AD patients with PS-1 mutations (25Zhang Z. Hartmann H. Do V.M. Abramowski D. Sturchler-Pierrat C. Staufenbiel M. Sommer B. van de Wetering M. Clevers H. Saftig P. De Strooper B. He X. Yankner B.A. Nature. 1998; 395: 698-702Crossref PubMed Scopus (473) Google Scholar). GSK3β phosphorylates pyruvate dehydrogenase and inhibits it in response to β-amyloid; as a consequence, a reduction in ATP and ACh synthesis has been described in hippocampal neurons (26Hoshi M. Takashima A. Noguchi K. Murayama M. Sato M. Kondo S. Saitoh Y. Ishiguro K. Hoshino T. Imahori K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2719-2723Crossref PubMed Scopus (226) Google Scholar). Taken together, these data indicate that GSK3β is located at the convergence of pathways involved in AD-like tau hyperphosphorylation, β-amyloid-induced toxicity, PS-1 mutations, and ACh metabolism, all of which are relevant to AD. Recent evidence using lithium and short interfering RNAs directed toward GSK3α block the production of Aβ by interfering with APP at the γ-secretase step but not Notch processing (51Phiel C.J. Wilson C.A. Lee V.M. Klein P.S. Nature. 2003; 423: 435-439Crossref PubMed Scopus (1091) Google Scholar). The effects of the GSK3 inhibitor lithium was observed in cells, as well as in transgenic mice that overproduce APP. An increase in GSK3 levels or activity in AD post mortem tissue, compared with non-diseased human brain, has been described by several researchers. Using active site antibodies, GSK3 has been shown to localize to pretangle neurons, dystrophic neurites, and NFTs in AD brain (12Pei J.J. Braak E. Braak H. Grundke-Iqbal I. Iqbal K. Winblad B. Cowburn R.F. J. Neuropathol. Exp. Neurol. 1999; 58: 1010-1019Crossref PubMed Scopus (430) Google Scholar). Neurons actively undergoing granulovacular degeneration are also immunopositive for active GSK3 (27Leroy K. Boutajangout A. Authelet M. Woodgett J.R. Anderton B.H. Brion J.P. Acta Neuropathol. (Berl.). 2002; 103: 91-99Crossref PubMed Scopus (171) Google Scholar). A spatial and temporal pattern of increased active GSK3β expression coinciding with the progression of NFT and neurodegeneration has been reported (11Shiurba R.A. Ishiguro K. Takahashi M. Sato K. Spooner E.T. Mercken M. Yoshida R. Wheelock T.R. Yanagawa H. Imahori K. Nixon R.A. Brain Res. 1996; 737: 119-132Crossref PubMed Scopus (65) Google Scholar). Taken together, these studies provide evidence that the active form of GSK3β is increased in AD brain. Because active GSK3β triggers signal transduction events that participate in cell death (28Bhat R.V. Shanley J. Corell M.P. Fieles W.E. Keith R.A. Scott C.W. Lee C.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11074-11079Crossref PubMed Scopus (354) Google Scholar, 29Hetman M. Cavanaugh J.E. Kimelman D. Xia Z. J. Neurosci. 2000; 20: 2567-2574Crossref PubMed Google Scholar), and loss of neuronal and synaptic plasticity (30Salinas P.C. Hall A.C. Bipolar Disord. 1999; 1: 87-90Crossref PubMed Scopus (21) Google Scholar), a significant part of the AD pathology could result from an abnormal increase in GSK3β expression and activity. When GSK3β transgene expression is induced in brain, adult mice developed hyperphosphorylated tau at the PHF-1 site (phospho-epitope at Ser-396/404) (31Lucas J.J. Hernandez F. Gomez-Ramos P. Moran M.A. Hen R. Avila J. EMBO J. 2001; 20: 27-39Crossref PubMed Scopus (813) Google Scholar), a site critical for PHF formation and represents increased phosphorylation in AD brain (32Otvos Jr., L. Feiner L. Lang E. Szendrei G.I. Goedert M. Lee V.M. J. Neurosci. Res. 1994; 39: 669-673Crossref PubMed Scopus (408) Google Scholar). These mice also developed pretangle structures in the hippocampus, as well as increased neuronal death, gliosis, and spatial learning deficits in the Morris water maze (33Hernandez F. Borrell J. Guaza C. Avila J. Lucas J.J. J. Neurochem. 2002; 83: 1529-1533Crossref PubMed Scopus (297) Google Scholar). Overexpression of human tau, in combination with its phosphorylation by the Drosophila GSK3β homologue Shaggy, exacerbates neurodegeneration induced by tau overexpression alone and gives rise to NFT pathology in the fly (34Jackson G.R. Wiedau-Pazos M. Sang T.K. Wagle N. Brown C.A. Massachi S. Geschwind D.H. Neuron. 2002; 34: 509-519Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar). Taken together, these studies provide strong evidence that increased GSK3β expression and activity are necessary for the early events leading to pretangle and neurodegenerative pathology that is associated with AD. Accordingly, several groups have identified small molecule GSK3 inhibitors (35Coghlan M.P. Culbert A.A. Cross D.A. Corcoran S.L. Yates J.W. Pearce N.J. Rausch O.L. Murphy G.J. Carter P.S. Roxbee Cox L. Mills D. Brown M.J. Haigh D. Ward R.W. Smith D.G. Murray K.J. Reith A.D. Holder J.C. Chem. Biol. 2000; 7: 793-803Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar, 36Hers I. Tavare J.M. Denton R.M. FEBS Lett. 1999; 460: 433-436Crossref PubMed Scopus (136) Google Scholar, 37Smith D.G. Buffet M. Fenwick A.E. Haigh D. Ife R.J. Saunders M. Slingsby B.P. Stacey R. Ward R.W. Bioorg. Med. Chem. Lett. 2001; 11: 635-639Crossref PubMed Scopus (163) Google Scholar, 38Meijer L. Thunnissen A.M. 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. Kim S.H. Pettit G.R. Chem. Biol. 2000; 7: 51-63Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar, 39Leclerc S. Garnier M. Hoessel R. Marko D. Bibb J.A. Snyder G.L. Greengard P. Biernat J. Wu Y.Z. Mandelkow E.M. Eisenbrand G. Meijer L. J. Biol. Chem. 2001; 276: 251-260Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar, 40Martinez A. Alonso M. Castro A. Perez C. Moreno F.J. J. Med. Chem. 2002; 45: 1292-1299Crossref PubMed Scopus (421) Google Scholar). However, the majority of the reported inhibitors are not selective against GSK3 and also affect cdk2, a kinase that shares 33% amino acid identity with GSK3, or cdk5, another protein kinase involved in tau phosphorylation. In addition, there is a lack of basic information about the structural basis of the interaction between GSK3β and these inhibitors, as co-crystallization studies have not been reported. Here, we report the identification and characterization of a thiazole, N-(4-methoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-yl)urea (AR-A014418), which is a selective and potent inhibitor of GSK3 able to decrease tau phosphorylation and cell death. Furthermore, the co-crystallization of AR-A014418 with the GSK3β protein provides the structural information required to understand the high selectivities of this inhibitor. Propidium iodide (PI) was purchased from Sigma and calcein-AM from Molecular Probes. Antibody Tau5 was purchased from Research Diagnostics Inc., and tau Ser(P)-396 was from BIOSOURCE International. Anti-GSK3β was from Transduction Laboratories. The peptide β-amyloid 25-35 was from Bachem and was prepared as described previously (48Studer L. Spenger C. Luthman J. Seiler R.W. J. Comp. Neurol. 1994; 340: 281-296Crossref PubMed Scopus (35) Google Scholar, 49Luthman J. Radesäter A.-C. Öberg C. Amino Acids. 1998; 14: 263-269Crossref PubMed Scopus (20) Google Scholar). The biotinylated eukaryotic initiation factor 2B (eIF2B) peptide, biotin-AAEELDSRAGS(PO3H2)PQL, was synthesized at AstraZeneca R&D Lund (Lund, Sweden). A peptide based on glycogen synthase, RRRPASVPPSPSLSRHS-S(PO4)-HQRR, was produced by Auspep Pty. Ltd. (Parkville, Australia). The biotinylated peptide biotin-AKKPKTPKKAKKL-OH used in cdk5 assay was produced by Bachem AG (Bubendorf, Switzerland). Glutathione S-transferase (GST)-retinoblastoma expressed in a GST expression system and cdk5/p25 (co-transfected 2:5) were supplied by AstraZeneca Biotech Laboratory (Södertälje, Sweden). Recombinant human GSK3 was obtained from Dundee University (Dundee, Scotland, United Kingdom) and contained a mixture of GSK3β and GSK3α to a minor extent. Baculoviral cdk2/cyclin E enzyme was supplied by Enabling Science & Technology Biology Department, AstraZeneca R&D, Alderley Park, United Kingdom. [γ-33P]ATP, protein A, and streptavidin-coated scintillation proximity assay (SPA) beads were purchased from Amersham Biosciences UK. The synthesis of AR-A014418 is outlined in Scheme I. In brief, commercially available 2-amino-5-nitrothiazole and 4-methoxybenzylisocyanate was mixed in N,N-dimethylformamide to give AR-A014418 in 22% yield. The aqueous solubility of AR-A014418 was determined to be 136 μm. A mixture of 2-amino-5-nitrothiazole (0.89 g, 6.13 mmol) and 4-methoxybenzylisocyanate (1 g, 6.13 mmol) in N,N-dimethylformamide (6 ml) was heated at 100 °C under nitrogen atmosphere for 15 h. The mixture was allowed to cool and was partitioned between water and ethyl acetate. The aqueous layer was extracted with another portion of ethyl acetate. The combined organic layers were washed with brine, dried (MgSO4), and evaporated to give 2.5 g of a semisolid crude product. Most of the material was dissolved in chloroform/ethanol (98:2, approximately 15 ml) and triethylamine (3 ml), followed by filtration. The dissolved crude product was purified on a silica gel column using chloroform/ethanol (95:5) as the eluent to give 408 mg (22% yield) of the title compound as a yellowish solid: mp >190 °C (decomposition); 1H NMR (Me2SO-d 6, 400 MHz) δ 11.64 (br s, 1 H), 8.50 (s, 1 H), 7.25-7.23 (m, 3 H), 6.92-6.89 (m, 2 H), 4.30 (d, J = 5.9 Hz, 2 H), 3.73 (s, 3 H); 13C NMR (Me2SO-d 6, 100 MHz) δ 164.42, 158.43, 153.48, 143.47, 140.80, 130.82, 128.72, 113.82, 55.08, 42.60; MS(ES) m/z 309 (M+ + 1). Full-length human GSK3β was cloned into the NdeI and EcoRI restriction sites of pET28a(+) (Novagen) via the PCR Script vector (Stratagene) using the PCR primers 5′-CATCATATGTCAGGGCGGCCCAGAACC and 5′-TCATCAGGTGGAGTTGGAAGCTGATGC. An NcoI-EcoRI gene fragment was subsequently subcloned into pFASTBAC HTb (Invitrogen) resulting in a construct with an N-terminal double histidine tag with the following amino acid sequence: MSYYHHHHHHDYDIPTTENLYFQGAMGSSHHHHHHSSGLVPRGSH-. Briefly, Sf21 cells were transfected with recombinant bacmid DNA using Superfect (Qiagen) and incubated for 3 days at 27 °C. The virus particles were amplified in Sf21 cells for 7 days and titrated using the Clontech titration kit. High Five cells were grown at 27 °C in a 20-liter cell culture bioreactor (BBI) in Expression High Five SFM medium (Invitrogen), supplemented with 12 mm l-glutamine, 5 mm asparagine, and 0.1% NaCl. The culture was infected with the recombinant baculovirus with a multiplicity of infection of 2 at a cell density of 2.12 × 106 cells/ml. The cells were harvested 46 h after infection using a Contifug Stratos centrifuge. The cells were washed with PBS, frozen, and stored in -80 °C prior to purification. The cells were thawed, resuspended in lysis buffer (20 mm HEPES, pH 7.0, 500 mm NaCl, 10 mm imidazole, 10% (v/v) glycerol, 1 mm Tris (2-carboxyethyl) phosphine, and EDTA-free Complete Protease Inhibitor, Roche Diagnostics) to a density of 20 × 106 cells/ml, and then lysed by using an Ultra-Turrax T50 (Janke & Kunkel). The cell debris was removed by centrifugation for 30 min at 3 °C and 35,000 × g using an Avanti J-20 with a JLA 16.250 rotor (Beckman). Chelating Sepharose Fast Flow (Amersham Biosciences), previously loaded with 0.5 bed volume of 0.1 m Ni2SO4, was added in a ratio of 2 ml of Sepharose/109 cells, and GSK3β was allowed to adsorb to the matrix in a batch fashion for 60 min at 4 °C. All subsequent steps were performed at room temperature. Unbound protein was removed with Buffer A (lysis buffer without protease inhibitor) using a sintered glass filter funnel, and the resin was packed into a column, further washed with Buffer A, and then washed with Buffer A containing 250 mm imidazole. GSK3β protein was finally eluted with Buffer A containing 500 mm imidazole. GSK3β-containing fractions were pooled and loaded onto a Sephadex G25 column equilibrated with Buffer B (20 mm HEPES, pH 7.0, 350 mm NaCl, 5% (v/v) glycerol, and 1 mm Tris (2-carboxyethyl) phosphine. The final protein was aliquoted, frozen, and stored in -80 °C. Approximately 8 mg of >95% pure GSK3β was produced per liter of cultivation. The histidine tag was removed by thrombin cleavage using 500 units of thrombin (Amersham Biosciences)/70 mg of protein for 4 h at room temperature and cleavage at a second site (after arginine 6) was observed. A residual thrombin activity remained despite later purification steps, and the protein preparation used for crystallization was a mix of full length and the 7-420 species. Unphosphorylated and phosphorylated GSK3β was separated using a 6-ml Resource S column (Amersham Biosciences), equilibrated in 20 mm HEPES, pH 7.0, 80 mm NaCl, 2 mm DTT, and 5% glycerol, using a 20-column volume gradient from 80 to 350 mm NaCl. Full-length unphosphorylated GSK3β eluted at approximately 200 mm NaCl, and the peak fractions were pooled and concentrated to 6.3 mg/ml (λ280 = 34,960 m-1 cm-1). Crystals were grown using the hanging drop method. A 1.5 + 1.5-μl drop consisting of protein solution (130 μm GSK3β, 250 μm AR-A014418, 20 mm HEPES, pH 7.0, 200 mm NaCl, 2 mm DTT, 5% glycerol, and 1.3% Me2SO) and well solution (17% polyethylene glycol 3350, 50 mm BisTris propane, pH 6.5, and 5 mm urea) was placed over 500 μl of well solution at 20 °C. Crystals grew within a few days to a size of 100 μm and were soaked in cryo solution (15% glycerol, 10% polyethylene glycol 3350, 100 mm NaCl, and 50 mm BisTris propane, pH 6.5) before being frozen in a 100 K nitrogen stream. All the structure descriptions in this paper are based on the refined structure using the synchrotron data. Details of data collection and refinement statistics are recorded in Table I. The current R-factor/R free is 0.22/0.24. Data were first collected using in-house x-ray source (CuKα radiation; wavelength = 1.54 Å) to a resolution of 3.1 Å on a Mar-Research 345-mm image-plate detector system. X-ray radiation was generated by a Rigaku RU300HB rotating anode operated at 50 kV and 100 mA. The crystal belongs to orthorhombic space group P212121, with cell parameters of a = 82.028 Å, b = 84.215 Å, and c = 178.091 Å. Molecular replacement was carried out with program MOLREP using overlaid structures of cdk2, ERK, and p38g as the search model. To prepare the search model, these three structures were first superimposed using program MAPS, and then each was trimmed manually in the program O to remove some loop regions to ∼260 residues, respectively. MOLREP found two molecules after rotation and translation search with R-factor/correlation coefficient of 0.514/0.280. Phase improvement with solvent flattening (solvent content 0.622) and 2-fold density averaging was performed using the program DM. The solution was verified by inspection of the calculated electron density map in program O after phase improvement. Subsequently the cdk2 model from molecular replacement was used for initial refinement and model building. A high resolution data set (1.94 Å) was later collected at beamline ID29 (wavelength of 1.0064 Å) in ESRF (Grenoble, France).Table ICrystallography: data collection and refinement statisticsData collectionIn-house data (λ = 1.54 Å)Synchrotron data, ID29 European Synchotron Radiation Facility (λ = 1.0064 Å)Space groupP212121P212121Cell parameter (Å)82.03, 84.21, 178.0982.58, 84.91, 178.42Solvent content (%)62.963.5Resolution (last shell) (Å)3.11 (3.11-3.30)1.94 (1.94-2.04)Unique reflections21,48278,815R merge (all/last shell)0.290/0.6690.071/0.431I/I s (all/last shell)2.4/1.17.1/1.7Completeness (%) (all/last shell)94.3/93.484.4/58.9Redundancy3.77.2Structure refinementResolution (last shell) (Å)1.94 (1.94-2.06)Number of reflections used (last shell)78,753 (9134)Fraction of test set for calculating R free (%)3.4No. of reflections in the test set (last shell)2670 (310)R work (last shell)0.222 (0.274)R free (last shell)0.243 (0.291)Root mean square deviation bond lengths (Å)/bond angles (°)0.006/1.2Estimated coordinate error (Luzzati plot/SigmaA) (Å)0.26/0.21B values (mean B value/Wilson plot) (Å2)40.4/41.3Number of atoms: final model (protein/waters/ligand)5521/373/42 Open table in a new tab GSK3 Scintillation Proximity Assay—The competition experiments were carried out in duplicate with 10 concentrations of the inhibitor in clear-bottomed microtiter plates. The biotinylated peptide substrate, biotin-AAEELDSRAGS(PO3H2)PQL, was added at a final concentration of 2 μm in an assay buffer containing 6 milliunits of recombinant human GSK3 (equal mix of both α and β), 12 mm MOPS, pH 7.0, 0.3 mm EDTA, 0.01% β-mercaptoethanol, 0.004% Brij 35, 0.5% glycerol, and 0.5 μg of bovine serum albumin/25 μl and preincubated for 10-15 min. The reaction was initiated by the addition of 0.04 μCi of [γ-33P]ATP and unlabeled ATP in 50 mm Mg(Ac)2 to a final concentration of 1 μm ATP and assay volume of 25 μl. Blank controls without peptide substrate were used. After incubation for 20 min at room temperature, each reaction was terminated by the addition of 25 μl of stop solution containing 5 mm EDTA, 50 μm ATP, 0.1% Triton X-100, and 0.25 mg of streptavidin-coated SPA beads corresponding to ∼35 pmol of binding capacity. After 6 h the radioactivity was determined in a liquid scintillation counter (1450 MicroBeta Trilux, Wallac). cdk2 Scintillation Proximity Assay—The competition experiments were carried out in duplicate with 10 concentrations of the inhibitor in clear-bottomed microtiter plates. cdk2/cyclin E enzyme was added at a concentration corresponding to a 80× dilution of the partially purified baculovirus-infected insect cell lysate in a buffer containing 50 mm HEPES, 10 mm MnCl2, 1 mm DTT, 100 μm NaF, 100 μm sodium O-vanadate, 10 mm sodium glycerophosphate, 5 μg/ml aprotinin, 2.5 μg/ml leupeptin, and 100 μm phenylmethylsulfonyl fluoride. Blank controls without enzyme were used. The reaction was initiated by the addition of 1.25 μg of GST-retinoblastoma (part of the retinoblastoma gene (792-928) expressed in a

Referência(s)