piperidines and olomoucine

Cyclin-dependent kinases trigger and coordinate transitions between different phases the cell division cycle (CDK1, 2, 3, 4, 6, 7). They also play a role in apoptosis (CDK2), in neuronal cells (CDK5) and in the control of transcription (CDK 7, 8, 9). Intensive screening has lead to the recent identification of a series of chemical inhibitors of CDKs: olomoucine, roscovitine, purvalanol, CVT-313, flavopiridol, g-butyrolactone, indirubins, paullones and staurosporine. Some of these compounds display remarkable selectivities and efficiencies (IC50 < 25 nM). Many have been co-crystallised with CDK2 and their interactions with the kinase have been analysed in atomic detail. These inhibitors all act by competing with ATP for binding at the catalytic site. Most inhibitors present a flat heterocyclic ring system that occupies the purine binding pocket as well as form 2 or 3 hydrogen bonds with Glu-81 and Leu-83. The binding modes of these inhibitors are reviewed in this article. Knowledge of the CDK/inhibitor interactions will be of great help to design inhibitors with improved selectivity our potency as well as to generate affinity chromatography matrices for the purification and identification of their cellular targets. The potential use of CDK inhibitors is being extensively evaluated in cancer chemotherapy and other fields such as the cardiovascular domain (restenosis), dermatology (psoriasis), nephrology (glomerulonephritis) parasitology (unicellular parasites such as Plasmodium, Trypanosomes, Toxoplasm,.etc.), neurology (Alzheimer's disease) and viral infections (cytomegalovirus, H.I.V., herpes).

Topics: Adenine; Adenosine Triphosphate; Amino Acid Sequence; Antineoplastic Agents; Binding Sites; Cell Cycle; Cyclin-Dependent Kinases; Drug Design; Enzyme Inhibitors; Flavonoids; Humans; Isopentenyladenosine; Kinetin; Molecular Sequence Data; Piperidines; Purines; Roscovitine; Sequence Alignment; Sequence Homology, Amino Acid; Staurosporine; Suramin

The eukaryotic cell division cycle is regulated by a family of protein kinases, the cyclin-dependent kinases (cdk's), constituted of at least two subunits, a catalytic subunit (cdk1-7) associated with a regulatory subunit (cyclin A-H). Transient activation of cdk's is responsible for transition through the different phases of the cell cycle. Major abnormalities of cdk's expression and regulation have been described in human tumours. Enzymatic screening is starting to uncover chemical inhibitors of cdk's with anti-mitotic activities. This review summarizes our knowledge of these first inhibitors, their mechanism of action, their effects on the cell cycle, and discusses the potential of such type of inhibitors as anti-tumour agents.

Topics: 4-Butyrolactone; Animals; Cell Cycle; Cyclin-Dependent Kinases; Enzyme Inhibitors; Flavonoids; Humans; Kinetin; Piperidines; Purines; Staurosporine; Suramin

Recent evidence suggests that apoptosis in post-mitotic neurons involves an aborted attempt of cells to re-enter the cell cycle which is characterized by increased expression of cyclins, such as cyclin D1, prior to death. However, such cyclins activation prior to apoptotic cell death remains controversial. Many neurological disorders are characterized by neuronal loss, particularly amyotrophic lateral sclerosis (ALS). ALS is a motoneuronal degenerative condition in which motoneuron loss could be due to an inappropriate return of these cells in the cell cycle. In the present study, we observed that deprivation of neurotrophic factor in purified motoneuron cultures induces an apoptotic pathway. After neurotrophic factor withdrawal, DAPI (4,6-diamidin-2-phenylindol dichlorohydrate) staining revealed the presence of nuclear condensation, DNA fragmentation, and perinuclear apoptotic body. Similarly, release of apoptotic microparticles and activation of caspases-3 and -9 were observed within the first hours following neurotrophic factor withdrawal. Next, we tested whether inhibition of cell cycle-related cyclin-dependent kinases (cdks) can prevent motoneuronal cell death. We showed that three cdk inhibitors, olomoucine, roscovitine and flavopiridol, suppress the death of motoneurons. Finally, we observed early increases in cyclin D1 and cyclin E expression after withdrawal of neurotrophic factors. These findings support the hypothesis that after removal of trophic support, post-mitotic neuronal cells die due to an attempt to re-enter the cell cycle in an uncoordinated and inappropriate manner.

Topics: Animals; Apoptosis; Cell Survival; Cells, Cultured; Cyclin D1; Cyclin E; Cyclin-Dependent Kinases; Flavonoids; Kinetin; Mice; Mitosis; Motor Neurons; Nerve Growth Factors; Piperidines; Protein Kinase Inhibitors; Purines; Roscovitine

Upregulation of cell cycle proteins occurs in both mitotic and post-mitotic neural cells after central nervous system (CNS) injury in adult animals. In mitotic cells, such as astroglia and microglia, they induce proliferation, whereas in post-mitotic cells such as neurons they initiate caspase-related apoptosis. We recently reported that early central administration of the cell cycle inhibitor flavopiridol after experimental traumatic brain injury (TBI) significantly reduced lesion volume, scar formation and neuronal cell death, while promoting near complete behavioral recovery. Here we show that in primary neuronal or astrocyte cultures structurally different cell cycle inhibitors (flavopiridol, roscovitine, and olomoucine) significantly reduce upregulation of cell cycle proteins, attenuate neuronal cell death induced by etoposide, and decrease astrocyte proliferation. Flavopiridol, in a concentration dependent manner, also attenuates proliferation/activation of microglia. In addition, we demonstrate that central administration of flavopiridol improves functional outcome in dose-dependent manner after fluid percussion induced brain injury in rats. Moreover, delayed systemic administration of flavopiridol significantly reduces brain lesion volume and edema development after TBI. These data provide further support for the therapeutic potential of cell cycle inhibitors for the treatment of clinical CNS injury and that protective mechanisms likely include reduction of neuronal cell death, inhibition of glial proliferation and attenuation of microglial activation.

Topics: Animals; Antineoplastic Agents; Astrocytes; Brain; Cell Cycle; Cell Proliferation; Cell Survival; Cells, Cultured; Central Nervous System; Dose-Response Relationship, Drug; Edema; Enzyme Inhibitors; Etoposide; Flavonoids; Kinetin; Magnetic Resonance Spectroscopy; Microglia; Mitosis; Neurons; Piperidines; Purines; Rats; Roscovitine; Up-Regulation

Several Cdc2p-related protein kinases (CRKs) have been described in trypanosomatids but their role in the control of the cell cycle nor their biological functions have been addressed. In Trypanosoma cruzi two CRKs have been identified, TzCRK1 and TzCRK3. In this work we further characterize T. cruzi CRK1 and report the identification of three novel associating cyclins. We demonstrate that CRK1 levels and localization do not vary during the cell cycle, and show that it is localized in the cytoplasm, discrete regions of the nucleus, and is highly concentrated in the mitochondrion DNA (kinetoplast), suggesting a putative control function in this organelle. Using purified anti-CRK1 IgGs, we immunoprecipitated from the soluble fraction of T. cruzi epimastigote forms a protein kinase activity which is not inhibited by CDK inhibitors. In addition, we co-precipitated with p13Suc1p beads a kinase activity that was inhibited by the CDK inhibitor flavopiridol and olomoucine. Lastly, using the yeast two-hybrid system we identified three novel cyclin-like proteins able to associate with TzCRK1, and demonstrate that two of these cyclins also bind the T. cruzi CRK3 protein, indicating that these two CRKs are cyclin-dependent kinases.

Topics: Amino Acid Sequence; Animals; CDC2 Protein Kinase; CDC2-CDC28 Kinases; Cyclin-Dependent Kinases; Cyclins; Cytoplasm; Enzyme Inhibitors; Flavonoids; Histones; Immunoglobulin G; Immunohistochemistry; Kinetin; Mitochondria; Molecular Sequence Data; Piperidines; Precipitin Tests; Protein Kinases; Protozoan Proteins; Purines; Retinoblastoma Protein; Sequence Alignment; Trypanosoma cruzi

Cell cycle regulatory components are interesting targets for cancer therapy. Expression of pRb, cyclin D1, cdk4, cyclin E, cdk2, E2F1 and DP-1 was determined in MCF-7 and MDA-MB-468 breast carcinoma cells, H460 and Calu-6 non-small cell lung carcinoma cells, H82 and SW2 small cell lung carcinoma cells, HCT116 and HT29 colon carcinoma cells and LNCaP and DU-145 prostate carcinoma cells.. For Western blotting, the ratio with actin expression was used to normalize the data; all lines were run on the same gels.. In cell culture, pRb was not detected in MB-468 and H82 was low in SW2 and DU-145 and highest in HCT116; in tumors, pRb was not detected in MB-468, H82, SW2, and DU-145 and was highest in LNCaP and Calu-6. Cyclin D1 was not detected in SW2 cells in culture, was low in MB-468 and H82, and was highest in LNCaP and H460; in tumors, cyclin D1 was low in MB-468, H460, SW2 and DU 145, and was highest in LNCaP. In cell culture, cdk4 was lowest in Calu-6, HCT116, HT29 and DU-145 and highest in H82 and SW2; in tumors, cdk4 was low in MCF-7, MB-468, H460, Calu-6 and HCT116 and was very high in the SW2. Expression of cyclin E was very low in MCF-7 and HT29 and high in H460 in culture and was very low in MCF-7, H460, Calu-6, H82, HT29 and DU-145 in tumors and high in HCT116 and LNCaP. In cell culture, E2F1 was lowest in MB-468, Calu-6, HT29 and DU-145 cells and highest in LNCaP cells; in tumors, E2F1 was lowest in MCF-7, MB-468 and Calu-6 and highest in LNCaP. In cell culture, DP-1 was lowest in MB-468, HCT116 and HT29 and highest in SW2. The MCF-7 and MB-468 lines were most resistant to flavopiridol and olmoucine and the H460 and Calu-6 lines were most resistant to genistein. The SW2 tumor was most responsive to flavopiridol and olomoucine.. There is a high degree of variability in the expression of cell cycle components in human tumor cell lines, resulting in complexity in predicting response to cell cycle directed agents.

Topics: Aged; Animals; Antineoplastic Agents; Blotting, Western; Calmodulin-Binding Proteins; Cell Cycle; Cell Cycle Proteins; Cell Survival; Cyclin-Dependent Kinases; Cyclins; Enzyme Inhibitors; Female; Flavonoids; Genistein; Humans; Kinetin; Male; Mice; Mice, Nude; Middle Aged; Neoplasm Transplantation; Piperidines; Plant Proteins; Purines; Transcription Factor DP1; Transcription Factors; Transplantation, Heterologous; Tumor Cells, Cultured

Here, we compare the pathways by which DNA-damaging agents, NGF deprivation, and superoxide dismutase 1 (SOD1) depletion evoke apoptosis of sympathetic neurons. Previous work raised the hypothesis that cell cycle signaling plays a required role in neuronal apoptosis elicited by NGF deprivation and the DNA-damaging agent camptothecin. To test this hypothesis, we extended our investigation of DNA-damaging agents to cytosine arabinoside (AraC) and UV irradiation. As with NGF deprivation and camptothecin treatment, the cyclin-dependent kinase inhibitors flavopiridol and olomoucine protected neurons from apoptosis induced by AraC and UV treatment. These observations support the model that camptothecin, AraC, and UV treatment cause DNA damage, which leads to apoptosis by a mechanism that, as in the case of NGF deprivation, includes activation of cell cycle components. Flavopiridol and olomoucine, however, had no effect on death induced by SOD1 depletion, suggesting that CDKs do not play a role in this paradigm of neuronal death. To compare further the mechanisms of death evoked by NGF withdrawal, SOD1 depletion, and DNA-damaging agents, we investigated their responses to inhibitors of cysteine aspartases, elements of apoptotic pathways. The V-ICEinh and BAF, two peptide inhibitors of cysteine aspartases, protected neurons in all three death paradigms. In contrast, the cysteine aspartase inhibitory peptide zVAD-fmk conferred protection from NGF withdrawal and SOD1 depletion, but not DNA-damaging agents, whereas acYVAD-cmk protected only from SOD1 depletion. Taken together, these findings indicate that three different apoptotic stimuli activate separate pathways of death in the same neuron type.

Topics: Animals; Antimetabolites, Antineoplastic; Aphidicolin; Apoptosis; Aspartic Acid; Cell Division; Cell Survival; Cyclin-Dependent Kinases; Cysteine Endopeptidases; Cytarabine; DNA Damage; Enzyme Inhibitors; Flavonoids; Humans; Kinetin; Nerve Growth Factors; Neurons; Oxidative Stress; PC12 Cells; Piperidines; Purines; Rats; Rats, Sprague-Dawley; S Phase; Superior Cervical Ganglion; Superoxide Dismutase; Ultraviolet Rays

Previous studies have demonstrated that G1/S cell cycle blockers and inhibitors of cyclin-dependent kinases (CDKs) prevent the death of nerve growth factor (NGF)-deprived PC12 cells and sympathetic neurons, suggesting that proteins normally involved in the cell cycle may also serve to regulate neuronal apoptosis. Past findings additionally demonstrate that DNA-damaging agents, such as the DNA topoisomerase (topo-I) inhibitor camptothecin, also induce neuronal apoptosis. In the present study, we show that camptothecin-induced apoptosis of PC12 cells, sympathetic neurons, and cerebral cortical neurons is suppressed by the G1/S blockers deferoxamine and mimosine, as well as by the CDK-inhibitors flavopiridol and olomoucine. In each case, the IC50 values were similar to those reported for inhibition of death induced by NGF-deprivation. In contrast, other agents that arrest DNA synthesis, such as aphidicolin and N-acetylcysteine, failed to block death. This suggests that the inhibition of DNA synthesis per se is insufficient to provide protection from camptothecin. We find additionally that the cysteine aspartase family protease inhibitor zVAD-fmk inhibits apoptosis evoked by NGF-deprivation but not camptothecin treatment. Thus, despite their shared sensitivity to G1/S blockers and CDK inhibitors, the apoptotic pathways triggered by these two causes of death diverge at the level of the cysteine aspartase. In summary, neuronal apoptosis induced by the DNA-damaging agent camptothecin appears to involve signaling pathways that normally control the cell cycle. The consequent death signals of such deregulation, however, are different from those that result from trophic factor deprivation.

Topics: Animals; Apoptosis; Camptothecin; Cell Differentiation; Cell Division; Cyclin-Dependent Kinases; Enzyme Inhibitors; Flavonoids; G1 Phase; Growth Inhibitors; Humans; Kinetin; Neurons; PC12 Cells; Piperidines; Purines; Rats; S Phase; Sympathetic Nervous System

Previous studies have demonstrated that multiple agents that promote survival of PC12 cells and sympathetic neurons deprived of trophic support also block cell cycle progression. Presently, we address whether inhibition of cell cycle-related cyclin-dependent kinases (CDKs) prevents neuronal cell death. We show that two distinct CDK inhibitors, flavopiridol and olomoucine, suppress the death of neuronal PC12 cells and sympathetic neurons. In addition, we demonstrate that inhibitor concentrations required to promote survival correlate with their ability to inhibit proliferation. Promotion of survival, however, does not correlate with inhibition of extracellular signal-regulated kinase or c-Jun kinase activities or with interference with the activation of c-Jun kinase that accompanies serum/nerve growth factor deprivation. In contrast to their actions on nerve growth factor-differentiated PC12 cells, the CDK inhibitors do not prevent the death of proliferation-competent PC12 cells and, in fact, promote their cell death. These findings support the hypothesis that post-mitotic neuronal cells die after removal of trophic support due to an attempt to re-enter the cell cycle in an uncoordinated and inappropriate manner. We speculate that cycling PC12 cells are not saved by these agents due to a signaling conflict between an inherent oncogenic signal and the inhibition of CDK activity.

Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cell Cycle; Cell Differentiation; Cell Survival; Cells, Cultured; Culture Media; Cyclin-Dependent Kinases; Enzyme Activation; Enzyme Inhibitors; Flavonoids; JNK Mitogen-Activated Protein Kinases; Kinetin; Mitogen-Activated Protein Kinases; Nerve Growth Factors; Nerve Tissue Proteins; PC12 Cells; Piperidines; Purines; Rats; Sympathetic Nervous System

In the course of our studies on cell cycle regulation mechanisms of Plasmodium falciparum, we investigated expression pattern, kinase activity, and localization of PfPK5, a putative malarial member of the family of cyclin-dependent protein kinase (cdks). The kinase was immunoprecipitated from parasites of selected stages and from parasites blocked with the cell-cycle inhibitor aphidicolin. An elevated kinase activity of PfPK5 from aphidicolin-blocked cells suggested that the enzyme might be implicated in the regulation of the parasite's S-phase. To further investigate this hypothetical function, parasite cultures were treated with the specific cdk inhibitors flavopiridol and olomoucine, which act on PfPK5 in vitro at similar concentrations as on other cdks. When applied during the nuclear division cycles of the parasite, both drugs markedly inhibited the DNA synthesis, as predicted from our proposition that PfPK5 is necessary to activate or maintain the parasite S-phase. Immunolocalization studies provide further evidence for this potential role of PfPK5.

Topics: Animals; Aphidicolin; CDC2 Protein Kinase; Cell Nucleus; DNA, Protozoan; Enzyme Inhibitors; Flavonoids; Kinetin; Piperidines; Plasmodium falciparum; Protozoan Proteins; Purines; RNA, Protozoan; S Phase

The central role of cyclin-dependent kinases (CDKs) in cell cycle regulation makes them a promising target for discovering small inhibitory molecules that can modify the degree of cell proliferation. The three-dimensional structure of CDK2 provides a structural foundation for understanding the mechanisms of activation and inhibition of CDK2 and for the discovery of inhibitors. In this article five structures of human CDK2 are summarised: apoprotein, ATP complex, olomoucine complex, isopentenyladenine complex, and des-chloro-flavopiridol complex.

Topics: Adenine; Adenosine Triphosphate; Animals; Binding Sites; CDC2-CDC28 Kinases; Cell Cycle; Crystallography, X-Ray; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Humans; Isopentenyladenosine; Kinetin; Ligands; Macromolecular Substances; Models, Molecular; Piperidines; Protein Conformation; Protein Serine-Threonine Kinases; Purines; Recombinant Proteins