PubMed Health⌕ Search

Biomedical subjects

L Meijer

Publications and source records attributed to L Meijer.

At least 55 records · Page 3Linked to original sources
PubMed1999

Intra-M phase-promoting factor phosphorylation of cyclin B at the prophase/metaphase transition.

Activation of Cdc2-cyclin B (or M phase-promoting factor (MPF)) at the prophase/metaphase transition proceeds in two steps: dephosphorylation of Cdc2 and phosphorylation of cyclin B. We here investigated the regulation of cyclin B phosphorylation using the starfish oocyte model. Cyclin B phosphorylation is not required for Cdc2 kinase activity; both the prophase complex dephosphorylated on Cdc2 with Cdc25 and the metaphase complex dephosphorylated on cyclin B with protein phosphatase 2A display high kinase activities. An in vitro assay of cyclin B kinase activity closely mimics in vivo phosphorylation as shown by phosphopeptide maps of in vivo and in vitro phosphorylated cyclin B. We demonstrate that Cdc2 itself is the cyclin B kinase; cyclin B phosphorylation requires Cdc2 activity both in vivo (sensitivity to vitamin K3, a Cdc25 inhibitor) and in vitro (copurification with Cdc2-cyclin B, requirement of Cdc2 dephosphorylation, and sensitivity to chemical inhibitors of cyclin-dependent kinases). Furthermore, cyclin B phosphorylation occurs as an intra-M phase-promoting factor reaction as shown by the following: 1) active Cdc2 is unable to phosphorylate cyclin B associated to phosphorylated Cdc2, and 2) cyclin B phosphorylation is insensitive to enzyme/substrate dilution. We conclude that, at the prophase/metaphase transition, cyclin B is mostly phosphorylated by its own associated Cdc2 subunit.

Amino Acid Sequence↗
PubMed1999

Synthesis and in vitro evaluation of novel 2,6,9-trisubstituted purines acting as cyclin-dependent kinase inhibitors.

Novel C-2, C-6, N-9 trisubstituted purines derived from the olomoucine/roscovitine lead structure were synthesized and evaluated for their ability to inhibit starfish oocyte CDK1/cyclin B, neuronal CDK5/p35 and erk1 kinases in purified extracts. Structure activity relationship studies showed that increased steric bulk at N-9 reduces the inhibitory potential whereas substitution of the aminoethanol C-2 side chain by various groups of different size (methyl, propyl, butyl, phenyl, benzyl) only slightly decreases the activity when compared to (R)-roscovitine. Optimal inhibitory activity against CDK5, CDK1 and CDK2, with IC50 values of 0.16, 0.45 and 0.65 microM, respectively, was obtained with compound 21 containing a (2R)-pyrrolidin-2-yl-methanol substituent at the C-2 and a 3-iodobenzylamino group at the C-6 of the purine. Compound 21 proved cytotoxic against human tumor HeLa cells (LD50-6.7 microM versus 42.7 microM for olomoucine, 24-h contact). Furthermore, unlike olomoucine, compound 21 was effective upon short exposure (LD50= 25.3 microM, 2-h contact). The available data suggest that the affinity for CDKs and the cytotoxic potential of the drugs are inter-related. However, no straightforward cell cycle phase specificity of the cytotoxic response to 21 was observed in synchronized HeLa cells. With the noticeable exception of pronounced lengthening of the S-phase transit by 21 applied during early-S in synchronized HeLa cells, and in striking contrast with earlier reports on studies using plant or echinoderm cells. olomoucilnc and compound 21 were unable to reversibly arrest cell cycle progression in asynchronous growing HeLa cells. Some irreversible hlock in GI and G2 phase occurred at high olomoucine concentration, correlated with induced cell death. Moreover, chmronic exposure to lethal doses of compound 21 resulted in massive nuclear fragmentation, evocative of mitotic catastrophe with minour amounts of apoptosis only. It was also found that olomoucine and compound 21 reversibly block the intracellular uptake of nuicleosides with high efficiency.

CDC2 Protein Kinase↗
PubMed1999

Synthesis and application of functionally diverse 2,6,9-trisubstituted purine libraries as CDK inhibitors.

BACKGROUND: Purines constitute a structural class of protein ligands involved in mediating an astonishing array of metabolic processes and signal pathways in all living organisms. Synthesis of purine derivatives targeting specific purine-binding proteins in vivo could lead to versatile lead compounds for use as biological probes or drug candidates. RESULTS: We synthesized several libraries of 2,6, 9-trisubstituted purines using both solution- and solid-phase chemistry, and screened the compounds for inhibition of cyclin-dependent kinase (CDK) activity and human leukemic cell growth. Lead compounds were optimized by iterative synthesis based on structure-activity relationships (SARs), as well as analysis of several CDK-inhibitor cocrystal structures, to afford several interesting compounds including one of the most potent CDK inhibitors known to date. Unexpectedly, some compounds with similar CDK inhibitory activity arrested cellular proliferation at distinctly different phases of the cell cycle and another inhibitor directly induced apoptosis, bypassing cell-cycle arrest. Some of these compounds selectively inhibited growth of cells derived from specific tumors. CONCLUSIONS: 2,6,9-Trisubstituted purines have various and potent biological activities, despite high concentrations of competing endogenous purine ligands in living cells. Purine libraries constitute a versatile source of small molecules that affect distinct biochemical pathways mediating different cellular functions.

Adenine↗
PubMed1999

Indirubin, the active constituent of a Chinese antileukaemia medicine, inhibits cyclin-dependent kinases.

Indirubin is the active ingredient of Danggui Longhui Wan, a mixture of plants that is used in traditional Chinese medicine to treat chronic diseases. Here we identify indirubin and its analogues as potent inhibitors of cyclin-dependent kinases (CDKs). The crystal structure of CDK2 in complex with indirubin derivatives shows that indirubin interacts with the kinase's ATP-binding site through van der Waals interactions and three hydrogen bonds. Indirubin-3'-monoxime inhibits the proliferation of a large range of cells, mainly through arresting the cells in the G2/M phase of the cell cycle. These results have implications for therapeutic optimization of indigoids.

Animals↗
PubMed1999

Characterization of a novel cdk1-related kinase.

The p13suc1/p9CKShs proteins bind tightly to the cyclin-dependent kinases cdk1 and cdk2. The distantly related protein, p15cdk-BP, binds cdk4/6, cdk5 and cdk8. We now show that immobilized p15cdk-BP binds both an HMG-I kinase and a 35-kDa protein that cross-reacts with anti-PSTAIRE antibodies (PSTAIRE is a totally conserved motif located in subdomain III of cdk). This 'cdkX' and the HMG-I kinase also bind to an immobilized inhibitor of cdks (HD). Several properties clearly distinguish cdkX, and its associated HMG-I kinase, from known anti-PSTAIRE cross-reactive cdks: (a) cdkX migrates, in SDS/PAGE, in a position intermediate between prophase phosphorylated cdk1 and metaphase dephosphorylated cdk1; (b) in contrast with cdk1, cdkX and associated HMG-I kinase activity do not decrease following successive depletions on p9CKShs1-sepharose; (c) cdkX and associated HMG-I kinase activity, but not cdk1, decrease following depletions on immobilized inhibitor; (d) cdkX is expressed during the early development of sea urchin embryos; in contrast with cdk1/cyclin B kinase, the p15cdk-BP-bound HMG-I kinase is active throughout the cell cycle; compared with cdk1 it is active later in development; (e) p15cdk-BP-bound HMG-I kinase is essentially insensitive to powerful inhibitors of cdk such as purvalanol, roscovitine, olomoucine, p21cip1 and p16INK4A; HD is only moderately inhibitory. Altogether these results suggest the existence of a new cdk1-related kinase, possibly involved in the regulation of early development. The presence of this kinase in all organisms investigated so far, from plants to mammals, calls for its definitive identification.

Animals↗
PubMed1999

ATP-site directed inhibitors of cyclin-dependent kinases.

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).

Adenine↗
PubMed1998

The cyclin-dependent kinase inhibitors olomoucine and roscovitine arrest human fibroblasts in G1 phase by specific inhibition of CDK2 kinase activity.

The specificity and the temporal location of cell cycle arrest induced by the cyclin-dependent kinase (CDK) inhibitors olomoucine and roscovitine were investigated in normal human fibroblasts. Effects on the cell cycle were compared with those induced by the kinase inhibitor staurosporine, which arrests normal cells in early G1 phase by acting upstream of CDK2. Consistent with their in vitro activity, olomoucine and roscovitine, but not the related compound iso-olomoucine, induced a dose-dependent arrest in G1 phase. Following removal of CDK inhibitors, cells resumed cycle progression entering S phase with a kinetics faster than staurosporine-treated samples. Cellular levels of PCNA, cyclin D1, and cyclin E were not affected by the CDK inhibitors. In contrast, staurosporine significantly reduced the levels of these proteins, as determined by immunocytometry and Western blot analysis. Cyclin A was detectable only in some cells remaining in the G2 + M compartment of samples treated with CDK inhibitors, but not in samples treated with staurosporine. Significant reduction in the hyperphosphorylated forms of retinoblastoma protein was found in samples treated with CDK inhibitors, while only hypophosphorylated forms were observed in staurosporine-treated samples. Concomitantly, CDK2, but not CDK4, activity immunoprecipitated from cells treated with olomoucine or roscovitine was markedly inhibited. These results suggest that in normal cells, CDK2 kinase activity is the specific target of olomoucine and roscovitine.

CDC2-CDC28 Kinases↗
PubMed1998

Exploiting chemical libraries, structure, and genomics in the search for kinase inhibitors.

Selective protein kinase inhibitors were developed on the basis of the unexpected binding mode of 2,6,9-trisubstituted purines to the adenosine triphosphate-binding site of the human cyclin-dependent kinase 2 (CDK2). By iterating chemical library synthesis and biological screening, potent inhibitors of the human CDK2-cyclin A kinase complex and of Saccharomyces cerevisiae Cdc28p were identified. The structural basis for the binding affinity and selectivity was determined by analysis of a three-dimensional crystal structure of a CDK2-inhibitor complex. The cellular effects of these compounds were characterized in mammalian cells and yeast. In the latter case the effects were characterized on a genome-wide scale by monitoring changes in messenger RNA levels in treated cells with high-density oligonucleotide probe arrays. Purine libraries could provide useful tools for analyzing a variety of signaling and regulatory pathways and may lead to the development of new therapeutics.

Adenine↗
PubMed1998

Outcome of a controlled trial of the effectiveness of intensive case management for chronic public inebriates.

OBJECTIVE: The objective of this study was to test whether an intensive case management intervention would be effective with a group of homeless chronic public inebriate clients. The primary goals of the case management were to improve the financial and residential stability of the clients and to reduce their use of alcohol. METHOD: Subjects (N = 298, 81% male) were interviewed at baseline, randomly assigned to treatment and control conditions and given follow-up interviews at 6-month intervals for 2 years. Case management services were provided for the duration of the project. Follow-up rates for the first three interviews averaged 82%. RESULTS: Repeated measures MANCOVAs showed significant group differences favoring the case-managed group in all three areas targeted by the intervention: total income from public sources, nights spent in "own place" out of the previous 60 nights and days drinking out of the previous 30 days. The results held whether the three variables were analyzed jointly or separately and for alternative measures of drinking and homelessness. Although statistically significant, the group differences are generally not large. CONCLUSIONS: The results indicate that case management had a beneficial effect on the clients receiving it. This effect may have been the result of an increase in services received by the case-managed clients.

Adult↗
PubMed1997

Direct in vivo inhibition of the nuclear cell cycle cascade in experimental mesangial proliferative glomerulonephritis with Roscovitine, a novel cyclin-dependent kinase antagonist.

Glomerular injury is characterized by mesangial cell (MC) proliferation and matrix formation. We sought to determine if reducing the activity of cyclin-dependent kinase 2 (CDK2) with the purine analogue, Roscovitine, decreased MC proliferation in vitro and in vivo. Roscovitine (25 microM) inhibited FCS-induced proliferation (P < 0.0001) in cultured MC. Rats with experimental mesangial proliferative glomerulonephritis (Thy1 model) were divided into two groups. A prevention group received daily intraperitoneal injections of Roscovitine in DMSO (2.8 mg/kg) starting at day 1. A treatment group received daily Roscovitine starting at day 3, when MC proliferation was established. Control Thy1 rats received DMSO alone. MC proliferation (PCNA +/OX7 + double immunostaining) was reduced by > 50% at days 5 and 10 in the Roscovitine prevention group, and at day 5 in the treatment group (P < 0.0001). Early administration of Roscovitine reduced immunostaining for collagen type IV, laminin, and fibronectin at days 5 and 10 (r = 0.984; P < 0.001), which was associated with improved renal function (urinary protein/creatinine, blood urea nitrogen, P < 0.05). We conclude that reducing the activity of CDK2 with Roscovitine in experimental glomerulonephritis decreases cell proliferation and matrix production, resulting in improved renal function, and may be a useful therapeutic intervention in disease characterized by proliferation.

Animals↗
PubMed1997

The effect of the cyclin-dependent kinase inhibitor olomoucine on cell cycle kinetics.

The effect of the cyclin-dependent (CDK) inhibitors olomoucine and roscovitine on cell kinetics was studied. To this end, nonsmall cell lung cancer (NSCLC) cell line MR65 and neuroblastoma cell line CHP-212 were pulse labeled with bromodeoxyuridine (BrdUrd) and chased in culture medium, to which various concentrations of olomoucine or roscovitine were added. A dose-dependent inhibition of the G1/S-phase and G2/ M-/G1 transitions was observed. Furthermore, S-phase progression was also inhibited in a dose-dependent manner. Similarly, roscovitine, another CDK inhibitor with a 10-fold higher efficiency for both CDK1 and CDK2 as compared to olomoucine, showed the same effects at a 10-fold lower concentration. At the highest tested doses both olomoucine (200 microM) and roscovitine (40 microM) induced a complete cell cycle block in both cell lines, paralleled by the appearance of apoptotic figures. In these cultures a decrease in CDK1 protein level was found as shown by Western blotting. Bivariate CDK1/DNA analysis confirmed these observations and showed that a subpopulation of cells with characteristics of apoptosis became CDK1 negative. The presented data suggest that cyclins and CDKs are involved at an important nodal point shared by pathways regulating cellular proliferation and apoptosis.

Antimetabolites↗
PubMed1997

Cytokinin-derived cyclin-dependent kinase inhibitors: synthesis and cdc2 inhibitory activity of olomoucine and related compounds.

Cyclin-dependent kinases (cdk) have recently raised considerable interest in view of their essential role in the regulation of the cell division cycle. The structure-activity relationships of cdk inhibition showed that the 1, 3; and 7 positions of the purine ring must remain free, probably for a direct interaction, in which it behaves as a hydrogen bond acceptor. Olomoucine (6-(benzylamino)-2-[(2-hydroxyethyl)amino]-9-methylpurine, OC), roscovitine (6-(benzylamino)-2(R)-[[1-(hydroxymethyl)propyl]amino]-9-isopropylpur ine), and other N6,2,9-trisubstituted adenines were found to exert a strong inhibitory effect on the p34cdc2/cyclin B kinase. Removal or change of the side chain at position 2 or the hydrophobic group at position 9 dramatically decreased the inhibitory activity of olomoucine or roscovitine. Inhibition of cdk with OC and related compounds clearly arrests cell proliferation of many tumor cell lines at G1/S and G2/M transitions and also triggers apoptosis in the target tumor cells in vitro and in vivo. Thus, from a pharmacological point of view, OC may represent a model compound for a new class of antimitotic and antitumor drugs.

Animals↗
PubMed1997

Inhibition of cyclin-dependent kinases by purine analogues: crystal structure of human cdk2 complexed with roscovitine.

Cyclin-dependent kinases (cdk) control the cell division cycle (cdc). These kinases and their regulators are frequently deregulated in human tumours. A potent inhibitor of cdks, roscovitine [2-(1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurin e], was identified by screening a series of C2,N6,N9-substituted adenines on purified cdc2/cyclin B. Roscovitine displays high efficiency and high selectivity (Meijer, L., Borgne, A., Mulner, O., Chong, J. P. J., Blow, J. J., Inagaki, N., Inagaki, M., Delcros, J.-G. & Moulinoux, J.-P. (1997) Eur. J. Biochem. 243, 527-536). It behaves as a competitive inhibitor for ATP binding to cdc2. We determined the crystal structure of a complex between cdk2 and roscovitine at 0.24-nm (2.4 A) resolution and refined to an Rfactor of 0.18. The purine portion of the inhibitor binds to the adenine binding pocket of cdk2. The position of the benzyl ring group of the inhibitor enables the inhibitor to make contacts with the enzyme not observed in the ATP-complex structure. Analysis of the position of this benzyl ring explains the specificity of roscovitine in inhibiting cdk2. The structure also reveals that the (R)-stereoisomer of roscovitine is bound to cdk2. The (R)-isomer is about twice as potent in inhibiting cdc2/cyclin B than the (S)-isomer. Results from structure/activity studies and from analysis of the cdk2/roscovitine complex crystal structure should allow the design of even more potent cdk inhibitors.

Adenosine Triphosphate↗
PubMed1997

Biochemical and cellular effects of roscovitine, a potent and selective inhibitor of the cyclin-dependent kinases cdc2, cdk2 and cdk5.

Cyclin-dependent kinases (cdk) play an essential role in the intracellular control of the cell division cycle (cdc). These kinases and their regulators are frequently deregulated in human tumours. Enzymatic screening has recently led to the discovery of specific inhibitors of cyclin-dependent kinases, such as butyrolactone I, flavopiridol and the purine olomoucine. Among a series of C2, N6, N9-substituted adenines tested on purified cdc2/cyclin B, 2-(1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine (roscovitine) displays high efficiency and high selectivity towards some cyclin-dependent kinases. The kinase specificity of roscovitine was investigated with 25 highly purified kinases (including protein kinase A, G and C isoforms, myosin light-chain kinase, casein kinase 2, insulin receptor tyrosine kinase, c-src, v-abl). Most kinases are not significantly inhibited by roscovitine. cdc2/cyclin B, cdk2/cyclin A, cdk2/cyclin E and cdk5/p35 only are substantially inhibited (IC50 values of 0.65, 0.7, 0.7 and 0.2 microM, respectively). cdk4/cyclin D1 and cdk6/cyclin D2 are very poorly inhibited by roscovitine (IC50 > 100 microM). Extracellular regulated kinases erk1 and erk2 are inhibited with an IC50 of 34 microM and 14 microM, respectively. Roscovitine reversibly arrests starfish oocytes and sea urchin embryos in late prophase. Roscovitine inhibits in vitro M-phase-promoting factor activity and in vitro DNA synthesis in Xenopus egg extracts. It blocks progesterone-induced oocyte maturation of Xenopus oocytes and in vivo phosphorylation of the elongation factor eEF-1. Roscovitine inhibits the proliferation of mammalian cell lines with an average IC50 of 16 microM. In the presence of roscovitine L1210 cells arrest in G1 and accumulate in G2. In vivo phosphorylation of vimentin on Ser55 by cdc2/cyclin B is inhibited by roscovitine. Through its unique selectivity for some cyclin-dependent kinases, roscovitine provides a useful antimitotic reagent for cell cycle studies and may prove interesting to control cells with deregulated cdc2, cdk2 or cdk5 kinase activities.

Adenosine Triphosphate↗
PubMed1997

The cyclin-dependent kinase (cdk) inhibitors, olomoucine and roscovitine, alter the expression of a molluscan circadian pacemaker.

1. In this study, we determined the effects of the cyclin-dependent kinase (cdk) inhibitors, olomoucine and roscovitine, on the circadian rhythm of optic nerve impulse activity recorded from the eye of the marine snail Bulla gouldiana. 2. We found that olomoucine lengthened period and altered circadian phase in a dose-dependent manner without appreciably affecting gene transcription or translation. We also found that the more specific cdk inhibitor, roscovitine, was approximately 10-fold more effective in lengthening circadian period, while the inactive analogue, iso-olomoucine, was ineffective. 3. The current results, along with previous results from our laboratory, are consistent with the hypothesis that the biochemical mechanism responsible for generating the ocular circadian rhythm in B. gouldiana is related to the biochemical mechanism that regulates the eukaryotic cell division cycle, i.e., by modulation of the activity of protein kinases belonging to the cdk family.

Animals↗