Calmodulin in starfish oocytes. I. Calmodulin antagonists inhibit meiosis reinitiation.
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3',5'-Cyclic-AMP Phosphodiesterases↗
Biomedical subjects
L Meijer
Publications and source records attributed to L Meijer.
Properties and potential-applications of chemical inhibitors of cyclin-dependent kinases.
Cyclin-dependent kinases (CDKs) trigger and co-ordinate the cell division cycle phases. They also play a role in neuronal cells and in the control of transcription. Intensive screening has led in the past few years to the identification of a series of chemical inhibitors of CDKs. Some of these compounds display remarkable selectivity and efficiency (IC50 <25 nM). Many have been co-crystallised with CDK2, and their atomic interactions with the kinase have been analysed in detail: all are located in the ATP-binding pocket of the enzyme. These inhibitors are antimitotic, they arrest cells in G1 and, at higher doses, in G2/M. Furthermore, they facilitate or even trigger apoptosis in proliferating cells. In contrast, they protect neuronal cells from apoptosis. The potential use of these inhibitors is being extensively evaluated in cancer chemotherapy (clinical trials, Phase I and II). Possible clinical applications are being investigated in other fields: cardiovascular (restenosis, tumoural angiogenesis, atherosclerosis), nephrology (glomerulonephritis), dermatology (psoriasis), parasitology (unicellular parasites such as Plasmodium, Trypanosoma, Toxoplasma, etc.), neurology (Alzheimer's disease), viral infections (cytomegalovirus, human immunodeficiency virus, herpes). We anticipate the discovery of novel selective and powerful inhibitors in the near future, and hope for their efficient applications in various human diseases.
Cell Division↗
KID, a Kinase Inhibitor Database project.
The Kinase Inhibitor Database is a small specialized database dedicated to the gathering of information on protein kinase inhibitors. The database is accessible through the World Wide Web system and gives access to structural and bibliographic information on protein kinase inhibitors. The data in the database will be collected and submitted by researchers working in the kinase inhibitor field. The submitted data will be checked by the curator of the database before entry.
Databases as Topic↗
Cyclin-dependent kinases: initial approaches to exploit a novel therapeutic target.
Cyclin-dependent kinases (CDKs) have been recognized as key regulators of cell cycle progression. Alteration and deregulation of CDK activity are pathogenic hallmarks of neoplasia. Therefore, inhibitors or modulators would be of interest to explore as novel therapeutic agents in cancer, as well as other hyperproliferative disorders. Flavopiridol is a semisynthetic flavonoid that emerged from an empirical screening program as a potent antiproliferative agent that mechanistic studies demonstrated to directly inhibit CDKs 1, 2, and 4 as a competitive ATP site antagonist. Initial clinical trials have shown that concentrations that inhibit cell proliferation and CDK activity in vitro can be safely achieved in humans, and additional clinical trials will establish its clinical potential. To address the need for additional chemotypes that may serve as lead structures for drugs that would not have the toxicities associated with flavopiridol, compounds with a similar pattern of cell growth inhibitory activity in the National Cancer Institute's in vitro anticancer drug screen have been recognized by the computer-assisted pattern recognition algorithm COMPARE and then screened for anti-CDK activity in a biochemical screen. The benzodiazepine derivative NSC 664704 (7,12-dihydro-indolo[3,2-d][1]benzazepin-6(5H)-one) was revealed by that approach as a moderately potent (IC50 0.4 microM) inhibitor of CDK2, which in initial experiments shows evidence of causing cell cycle redistribution in living cells. NSC 664704 is, therefore, a candidate for further structural optimization, guided in part by understanding of the ATP-binding site in CDK2. This approach represents one way of combining empirical screening information with structure-based design to derive novel candidate therapeutic agents directed against an important cellular target.
Animals↗
Screening for antimitotic compounds using the cdc25 tyrosine phosphatase, an activator of the mitosis-inducing p34cdc2/cyclin Bcdc13 protein kinase.
A universal intracellular factor, the "M phase-promoting factor" (MPF), triggers the G2/M transition of the cell cycle in all organisms. In late G2, it is present as an inactive complex of tyrosine-phosphorylated p34cdc2 and unphosphorylated cyclin Bcdc13. In M phase, its activation as an active MPF displaying histone H1 kinase activity originates from the specific tyrosine dephosphorylation of the p34cdc2 subunit by the tyrosine phosphatase p80cdc25. We describe here a colorimetric assay of recombinant human cdc25A tyrosine phosphatase used as a cell cycle-specific target to screen for antimitotic compounds. The glutathione-S-transferase/cdc25A tyrosine phosphatase fusion protein is produced in large amounts of Escherichia coli and easily purified by affinity chromatography on glutathione-agarose. Optimal purification, storage and assay conditions (concentrations of enzyme, p-nitrophenylphosphate and dithiothreitol; duration of assay) have been determined. Using this system we tested 15 compounds currently used in cancer treatment; none of them displayed any inhibitory activity. However, the assay detected the inhibitory activity of vanadate, a reported tyrosine phosphatase inhibitor. The simplicity, speed and possible extensive automation of this assay using an essential cell cycle-regulating component provide a highly specific mechanism-based screen for antimitotic drugs discovery.
Amino Acid Sequence↗
A new screening test for antimitotic compounds using the universal M phase-specific protein kinase, p34cdc2/cyclin Bcdc13, affinity-immobilized on p13suc1-coated microtitration plates.
A universal intracellular factor, the "M phase-Promoting Factor" (MPF), triggers the G2/M transition of the cell cycle in all organisms. This factor displays an easily assayable histone H1 kinase (H1K) activity and is composed of at least two subunits, p34cdc2 (catalytic) and cyclin Bcdc13 (regulatory). We describe here a microtitration plate assay using affinity-immobilized H1K-MPF as a cell cycle-specific target to screen for antimitotic compounds. First, meiotic starfish oocytes were selected as the most convenient and abundant source of M phase extracts containing high levels of H1K. Second, we used the strong and specific interaction between p34cdc2 and p13suc1 to affinity-immobilize H1K-MPF on p13suc1-coated microtitration plates. p13suc1-coated wells specifically retain the M phase kinase, the activity of which is assayed with histone H1 and gamma-32P-ATP. Among 10 microtitration plates, Maxisorp plates (Nunc) proved to be the most efficient at retaining H1K through p13suc1. Experimental conditions to coat the plates with p13suc1, to immobilize and to measure p34cdc2/cyclin Bcdc13 kinase activity, as well as to store p13suc1-precoated plates, have been optimized. Using this system we tested 18 currently used anticancer drugs and S or G2 inhibitors; none of them displayed any inhibitory activity. The microtitration assay has allowed the detection of two H1K inhibitors, isopentenyladenine (I50: 40 microM) and staurosporine (I50: 3.2 nM). This affinity-immobilized H1K-MPF can thus now be used as a simple screening system to detect inhibitors of a major cell cycle-regulating component. This method may prove useful to screen for antimitotic compounds of potential anticancer interest.
Acetylcholine↗