Aloisines, a new family of CDK/GSK-3 inhibitors. SAR study, crystal structure in complex with CDK2, enzyme selectivity, and cellular effects.

Cyclin-dependent kinases (CDKs) regulate the cell cycle, apoptosis, neuronal functions, transcription, and exocytosis. The observation of CDK deregulations in various pathological situations suggests that CDK inhibitors may have a therapeutic value. In this article, we report on the identification of 6-phenyl[5H]pyrrolo[2,3-b]pyrazines (aloisines) as a novel potent CDK inhibitory scaffold. A selectivity study performed on 26 kinases shows that aloisine A is highly selective for CDK1/cyclin B, CDK2/cyclin A-E, CDK5/p25, and GSK-3 alpha/beta; the two latter enzymes have been implicated in Alzheimer's disease. Kinetic studies, as well as the resolution of a CDK2-aloisine cocrystal structure, demonstrate that aloisines act by competitive inhibition of ATP binding to the catalytic subunit of the kinase. As observed with all inhibitors reported so far, aloisine interacts with the ATP-binding pocket through two hydrogen bonds with backbone nitrogen and oxygen atoms of Leu 83. Aloisine inhibits cell proliferation by arresting cells in both G1 and G2.

[1]  M. Barbacid,et al.  To cycle or not to cycle: a critical decision in cancer , 2001, Nature reviews. Cancer.

[2]  D. Lane,et al.  Inhibitors of cyclin-dependent kinases as anti-cancer therapeutics. , 2000, Current medicinal chemistry.

[3]  M. Saunders,et al.  3-Anilino-4-arylmaleimides: potent and selective inhibitors of glycogen synthase kinase-3 (GSK-3). , 2001, Bioorganic & medicinal chemistry letters.

[4]  L Meijer,et al.  Multiple modes of ligand recognition: Crystal structures of cyclin‐dependent protein kinase 2 in complex with ATP and two inhibitors, olomoucine and isopentenyladenine , 1995, Proteins.

[5]  L Meijer,et al.  Discovery and initial characterization of the paullones, a novel class of small-molecule inhibitors of cyclin-dependent kinases. , 1999, Cancer research.

[6]  Michael L. Davis,et al.  Reactions of β-(lithiomethyl)azines with nitriles as a route to pyrrolo-pyridines, -quinolines, -pyrazines, -quinoxalines and -pyrimidines , 1992 .

[7]  L. Johnson,et al.  Protein kinase inhibition by staurosporine revealed in details of the molecular interaction with CDK2 , 1997, Nature Structural Biology.

[8]  P. Traxler,et al.  ATP site‐directed competitive and irreversible inhibitors of protein kinases , 2000, Medicinal research reviews.

[9]  L. Meijer,et al.  Paullones, a series of cyclin-dependent kinase inhibitors: synthesis, evaluation of CDK1/cyclin B inhibition, and in vitro antitumor activity. , 1999, Journal of medicinal chemistry.

[10]  D. W. Fry,et al.  Cell Cycle and Biochemical Effects of PD 0183812 , 2001, The Journal of Biological Chemistry.

[11]  J W Yates,et al.  Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription. , 2000, Chemistry & biology.

[12]  R. Maccioni,et al.  The protein kinase Cdk5. Structural aspects, roles in neurogenesis and involvement in Alzheimer's pathology. , 2001, European journal of biochemistry.

[13]  L Meijer,et al.  Biochemical and cellular effects of roscovitine, a potent and selective inhibitor of the cyclin-dependent kinases cdc2, cdk2 and cdk5. , 1997, European journal of biochemistry.

[14]  A. Bitonti,et al.  Crystal structure of human cyclin-dependent kinase 2 in complex with the adenine-derived inhibitor H717. , 2001, Journal of medicinal chemistry.

[15]  G L Trainor,et al.  Cyclin-dependent kinase inhibitors: useful targets in cell cycle regulation. , 2000, Journal of medicinal chemistry.

[16]  S. Cox,et al.  Indenopyrazoles as novel cyclin dependent kinase (CDK) inhibitors. , 2001, Journal of Medicinal Chemistry.

[17]  J. Adams,et al.  Recent progress towards the identification of selective inhibitors of serine/threonine protein kinases. , 1999, Current opinion in drug discovery & development.

[18]  Doris Marko,et al.  Indirubin, the active constituent of a Chinese antileukaemia medicine, inhibits cyclin-dependent kinases , 1999, Nature Cell Biology.

[19]  P. Jeffrey,et al.  Structural basis of cyclin-dependent kinase activation by phosphorylation , 1996, Nature Structural Biology.

[20]  Li-Huei Tsai,et al.  A decade of CDK5 , 2001, Nature Reviews Molecular Cell Biology.

[21]  P. Toogood.  Cyclin‐dependent kinase inhibitors for treating cancer , 2001, Medicinal research reviews.

[22]  S H Kim,et al.  Exploiting chemical libraries, structure, and genomics in the search for kinase inhibitors. , 1998, Science.

[23]  S H Kim,et al.  Structural basis for specificity and potency of a flavonoid inhibitor of human CDK2, a cell cycle kinase. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[24]  G. Schwartz,et al.  Cyclin-dependent kinase and protein kinase C inhibitors: a novel class of antineoplastic agents in clinical development. , 2000, Cancer journal.

[25]  S. Mani,et al.  Cyclin-dependent kinase inhibitors: novel anticancer agents , 2000, Expert opinion on investigational drugs.

[26]  P. Schultz,et al.  Synthesis and application of functionally diverse 2,6,9-trisubstituted purine libraries as CDK inhibitors. , 1999, Chemistry & biology.

[27]  A. Harwood,et al.  Lithium inhibits glycogen synthase kinase-3 by competition for magnesium. , 2001, Biochemical and biophysical research communications.

[28]  S. Kim,et al.  Purification and crystallization of human cyclin-dependent kinase 2. , 1993, Journal of molecular biology.

[29]  J Mottram,et al.  Intracellular targets of cyclin-dependent kinase inhibitors: identification by affinity chromatography using immobilised inhibitors. , 2000, Chemistry & biology.

[30]  G. Trainor,et al.  Identification of selective inhibitors of cyclin dependent kinase 4. , 2001, Bioorganic & medicinal chemistry letters.

[31]  V S Lamzin,et al.  Automated refinement of protein models. , 1993, Acta crystallographica. Section D, Biological crystallography.

[32]  D. Fabbro,et al.  Selective in vivo and in vitro effects of a small molecule inhibitor of cyclin-dependent kinase 4. , 2001, Journal of the National Cancer Institute.

[33]  S H Kim,et al.  Inhibition of cyclin-dependent kinases by purine analogues: crystal structure of human cdk2 complexed with roscovitine. , 1997, European journal of biochemistry.

[34]  G L Trainor,et al.  Quinazolines as cyclin dependent kinase inhibitors. , 2001, Bioorganic & medicinal chemistry letters.

[35]  D O Morgan,et al.  Cyclin-dependent kinases: engines, clocks, and microprocessors. , 1997, Annual review of cell and developmental biology.

[36]  H. Eldar-Finkelman,et al.  Glycogen synthase kinase 3: an emerging therapeutic target. , 2002, Trends in molecular medicine.

[37]  G. Murshudov,et al.  Refinement of macromolecular structures by the maximum-likelihood method. , 1997, Acta crystallographica. Section D, Biological crystallography.

[38]  G. Mcmahon,et al.  A novel cdk2-selective inhibitor, SU9516, induces apoptosis in colon carcinoma cells. , 2001, Cancer research.

[39]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[40]  I. Dominguez,et al.  Missing links in GSK3 regulation. , 2001, Developmental biology.

[41]  M. Roussel,et al.  Glycogen synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular localization. , 1998, Genes & development.

[42]  J. Wu,et al.  Characterization of novel inhibitors of cyclin-dependent kinases. , 1999, Biochemical and biophysical research communications.

[43]  Seung Ki Lee,et al.  A Specific Inhibitor of Cyclin-dependent Protein Kinases, CDC2 and CDK2 , 1996, Molecules and Cells.

[44]  P. Marin,et al.  Transgenic murine cortical neurons expressing human Bcl-2 exhibit increased resistance to amyloid β-peptide neurotoxicity , 1999, Neuroscience.

[45]  Sung-Hou Kim,et al.  Crystal structure of cyclin-dependent kinase 2 , 1993, Nature.

[46]  P. A. Harris,et al.  Oxindole-based inhibitors of cyclin-dependent kinase 2 (CDK2): design, synthesis, enzymatic activities, and X-ray crystallographic analysis. , 2001, Journal of medicinal chemistry.

[47]  A. Bitonti,et al.  THE DESIGN AND SYNTHESIS OF PURINE INHIBITORS OF CDK2. III , 2001 .

[48]  R. Jope,et al.  The multifaceted roles of glycogen synthase kinase 3β in cellular signaling , 2001, Progress in Neurobiology.

[49]  L. Meijer,et al.  Anti-mitotic properties of indirubin-3′-monoxime, a CDK/GSK-3 inhibitor: induction of endoreplication following prophase arrest , 2001, Oncogene.

[50]  J. Navaza,et al.  AMoRe: an automated package for molecular replacement , 1994 .

[51]  L. Tsai,et al.  Activity and regulation of p35/Cdk5 kinase complex. , 2000, Methods in enzymology.

[52]  G L Snyder,et al.  Paullones are potent inhibitors of glycogen synthase kinase-3beta and cyclin-dependent kinase 5/p25. , 2000, European journal of biochemistry.

[53]  B G Benson,et al.  Prevention of chemotherapy-induced alopecia in rats by CDK inhibitors. , 2001, Science.

[54]  L. Meijer,et al.  Intracellular targets of paullones : identification by affinity chromatography using immobilized inhibitor. , 2002 .

[55]  M. Kitagawa,et al.  Butyrolactone I, a selective inhibitor of cdk2 and cdc2 kinase. , 1993, Oncogene.

[56]  L. Kuyper,et al.  Binding mode of the 4-anilinoquinazoline class of protein kinase inhibitor: X-ray crystallographic studies of 4-anilinoquinazolines bound to cyclin-dependent kinase 2 and p38 kinase. , 2000, Journal of medicinal chemistry.

[57]  R. Maccioni,et al.  The protein kinase Cdk5 , 2001 .

[58]  G L Snyder,et al.  Indirubins inhibit glycogen synthase kinase-3 beta and CDK5/p25, two protein kinases involved in abnormal tau phosphorylation in Alzheimer's disease. A property common to most cyclin-dependent kinase inhibitors? , 2001, The Journal of biological chemistry.

[59]  N. Pavletich.  Mechanisms of cyclin-dependent kinase regulation: structures of Cdks, their cyclin activators, and Cip and INK4 inhibitors. , 1999, Journal of molecular biology.

[60]  K D Watenpaugh,et al.  The Cyclin-dependent Kinases cdk2 and cdk5 Act by a Random, Anticooperative Kinetic Mechanism* , 2001, The Journal of Biological Chemistry.

[61]  L. Meijer,et al.  Cinnamaldehydes inhibit cyclin dependent kinase 4/cyclin D1. , 2000, Bioorganic & medicinal chemistry letters.

[62]  L. Meijer,et al.  ATP-site directed inhibitors of cyclin-dependent kinases. , 1999, Current medicinal chemistry.

[63]  D. Boschelli,et al.  Pyrido[2,3-d]pyrimidin-7-one inhibitors of cyclin-dependent kinases. , 2000, Journal of medicinal chemistry.

[64]  M. Barbacid,et al.  Genetic Analysis of Mammalian Cyclin-Dependent Kinases and Their Inhibitors , 2000, Biological chemistry.

[65]  L. Meijer,et al.  Chemical inhibitors of cyclin-dependent kinases. , 1996, Trends in cell biology.

[66]  A H Calvert,et al.  Identification of novel purine and pyrimidine cyclin-dependent kinase inhibitors with distinct molecular interactions and tumor cell growth inhibition profiles. , 2000, Journal of medicinal chemistry.

[67]  P. Cohen,et al.  The renaissance of GSK3 , 2001, Nature Reviews Molecular Cell Biology.

[68]  Young-Tae Chang,et al.  Targeting hyperproliferative disorders with cyclin dependent kinase inhibitors , 2000 .

[69]  A Joly,et al.  CVT-313, a Specific and Potent Inhibitor of CDK2 That Prevents Neointimal Proliferation* , 1997, The Journal of Biological Chemistry.

[70]  Jacques Dumas,et al.  Protein kinase inhibitors: emerging pharmacophores 1997 - 2000 , 2001 .

[71]  A. Harwood,et al.  Regulation of GSK-3 A Cellular Multiprocessor , 2001, Cell.

[72]  Punit Marathe,et al.  Discovery of aminothiazole inhibitors of cyclin-dependent kinase 2: synthesis, X-ray crystallographic analysis, and biological activities. , 2002, Journal of medicinal chemistry.

[73]  D. Cooper,et al.  Protein Kinases as Therapeutic Targets , 2000, Pharmaceutical Research.

[74]  Philip Cohen,et al.  GSK3 takes centre stage more than 20 years after its discovery. , 2001 .