Anti-angiogenic effects of novel cyclin-dependent kinase inhibitors with a pyrazolo[4,3-d]pyrimidine scaffold.

BACKGROUND AND PURPOSE: Cyclin-dependent kinase 5 (CDK5) has recently emerged as an attractive target in several tumour entities. Inhibition of CDK5 has been shown to have anti-angiogenic effects in vitro and in vivo. However, potent inhibitors of CDK5, which can be applied in vivo, are still scarce. We have recently developed a new series of 5-substituted 3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyrimidines that show a preference for inhibiting CDK5 and tested them in vitro and in vivo in a murine model of hepatocellular carcinoma. EXPERIMENTAL APPROACH: All compounds were initially examined for effects on proliferation of HUVECs. The most potent compounds were then tested on migration, and one of them, LGR2674, was selected for assessing effects on nuclear fragmentation, cell cycle, cell viability and metabolic activity. Furthermore, LGR2674 was tested in a tube formation assay and in vivo in a murine model of hepatocellular carcinoma, induced by s.c. injection of HUH7 cells (measurement of in vivo toxicity, tumour vascularization, tumour cell proliferation and tumour size). KEY RESULTS: LGR2674 showed an EC50 in the low nanomolar range in the proliferation and migration assays. Cytotoxic effects started at 50 nM, a concentration that did not influence the cell cycle. In vivo, LGR2674 was well tolerated and caused a clear reduction in vessel density in the tumours; also tumour cell proliferation was inhibited and tumour growth retarded. CONCLUSIONS AND IMPLICATIONS: Pyrazolo[4,3-d]pyrimidine is a novel scaffold for the development of potent CDK inhibitors with in vivo potential. Such structures are good candidates for broadening our pharmacological arsenal against various tumours.

Purification and determination of plant hormones auxin and abscisic acid using solid phase extraction and two-dimensional high performance liquid chromatography.

A method for separation and purification of plant hormones auxin and abscisic acid based on mixed mode reversed-phase anion-exchange solid phase extraction and two-dimensional HPLC was developed. Two-dimensional HPLC in "heart cutting" mode was very efficient in the purification of these two hormones. Its purification power is high enough to allow reliable on-line quantification of both hormones even with non-selective detectors.

Antiproliferative effect of plant cytokinin analogues with an inhibitory activity on cyclin-dependent kinases.

In this study, analogues of olomoucine, a previously described plant cytokinin analogue with cyclin-dependent kinase (CDK) inhibitory activity, were investigated for effect on CDK1 and CDK2 and for effect on cell proliferation. Eight new compounds exhibit stronger inhibitory activity on CDK1 and CDK2 and on cell proliferation than olomoucine. Some active compounds showed low inhibition of proliferation of normal myeloid growth. Improvement of inhibitory activity of known compounds with a C6-benzylamino group was brought about by substitution with one hydroxyl. Also, new C2 substituents associated with inhibitory activity on CDK and on cell proliferation are described. There was a significant correlation between effect on CDK and antiproliferative effect on the KG1 and Molt3 cell lines and on primary human lymphocytes, strongly suggesting that at least part of the antiproliferative effect of cytokinin analogues was due to inhibition of CDK activity. Cytokinin analogues induced apoptosis in a time- and concentration-dependent manner and changes in cell cycle distribution. The antiproliferative and pro-apoptotic effects of plant cytokinin analogues suggest that they are a new class of cytostatic agents and that they may find an application in the chemotherapy of cancer.

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.

Docking-based development of purine-like inhibitors of cyclin-dependent kinase-2.

The cell division cycle is controlled by cyclin-dependent kinases (cdk), which consist of a catalytic subunit (cdk1-cdk8) and a regulatory subunit (cyclin A-H). Purine-like inhibitors of cyclin-dependent kinases have recently been found to be of potential use as anticancer drugs. Rigid and flexible docking techniques were used for analysis of binding mode and design of new inhibitors. X-ray structures of three (ATP, olomoucine, roscovitine) cdk2 complexes were available at the beginning of the study and were used to optimize the docking parameters. The new potential inhibitors were then docked into the cdk2 enzyme, and the enzyme/inhibitor interaction energies were calculated and tested against the assayed activities of cdk1 (37 compounds) and cdk2 (9 compounds). A significant rank correlation between the activity and the rigid docking interaction energy has been found. This implies that (i) the rigid docking can be used as a tool for qualitative prediction of activity and (ii) values obtained by the rigid docking technique into the cdk2 active site can also be used for the prediction of cdk1 activity. While the resulting geometries obtained by the rigid docking are in good agreement with the X-ray data, the flexible docking did not always produce the same inhibitor conformation as that found in the crystal.

In vitro glycosidation potential towards olomoucine-type cyclin-dependent kinase inhibitors in rodent and primate microsomes.

Interspecies differences in glycosidation potential in mammalian tissues represent a factor contributing to ambiguity when endobiotic and/or xenobiotic metabolic pathways are extrapolated from animals to man. Using the TLC/autoradiographic technique, we conducted an in vitro investigation involving mouse, rat, monkey, as well as human liver and kidney microsomes to evaluate their glycoconjugation potential towards (3)H-labeled, purine-derived selective inhibitors of cyclin-dependent kinases such as olomoucine, bohemine, roscovitine, 6-(2-hydroxybenzyl)amino-2-(1-hydroxymethyl-2-methylpropyl)amino-9-isopropylpurine (compound A-4), and 6-(3-hydroxybenzyl)amino-2-[(1(R/S)-hydroxymethyl)propyl]amino-9-isopropylpurine (compound A-5) as aglycones. Principally, this study confirmed the aliphatic hydroxyl group of olomoucine-type inhibitors as a relatively suitable target for glucuronide, glucoside, xyloside, galactoside, and/or N-acetylaminoglucoside conjugation. Of the tissues examined, only the mouse microsomes were able to perform glucosidation and galactosidation reactions with the aglycones. On the other hand, monkey microsomes were superior to the mouse microsomes in a variety of glucuronide conjugates produced with compounds A-4 and A-5.

Academic misconduct: results of faculty and undergraduate student surveys.

Educators in health-related fields are particularly sensitive to academic misconduct because undergraduate students who falsify academic work in such fields can go on to endanger the health and well being of the very people they are meant to assist. This paper presents the results of a survey of 104 faculty and 314 undergraduate students regarding their experience with academic misconduct. Faculty and student definitions of misconduct are compared, the incidence of cheating within each category is reported, and the projected efficacy of methods for controlling misconduct are examined. Major findings include the following: faculty and students differed significantly in their definitions of 24 of the 36 described behaviors, 82% of the surveyed undergraduate students admitted to engaging in some form of academic misconduct during their college careers, few differences in cheating patterns were related to year in school (class) or gender, and faculty and students differed on the impact that changes in environment and procedure were expected to have on cheating.

Antiproliferative activity of olomoucine II, a novel 2,6,9-trisubstituted purine cyclin-dependent kinase inhibitor.

The study describes the protein kinase selectivity profile, as well as the binding mode of olomoucine II in the catalytic cleft of CDK2, as determined from cocrystal analysis. Apart from the main cell cycle-regulating kinase CDK2, olomoucine II exerts specificity for CDK7 and CDK9, with important functions in the regulation of RNA transcription. In vitro anticancer activity of the inhibitor in a panel of tumor cell lines shows a wide potency range with a slight preference for cells harboring a wild-type p53 gene. Cell-based assays confirmed activation of p53 protein levels and events leading to accumulation of p21(WAF1). Additionally, in olomoucine II-treated cells, Mdm2 was found to form a complex with the ribosomal protein L11, which inhibits Mdm2 ubiquitin ligase function. We conclude that perturbations in RNA synthesis may lead to activation of p53 and that this contributes to the antiproliferative potency of cyclindependent kinase inhibitors.

Diverse effects of the cyclin-dependent kinase inhibitor bohemine: Concentration- and time-dependent suppression or stimulation of hybridoma culture.

An analog of aromatic cytokinins, the 2,6,9-trisubstituted purine derivative bohemine, was applied to cultures of mouse hybridoma cells in order to analyze its capacity of suppressing cell growth and maintaining or enhancing the production of monoclonal antibody. Addition of bohemine at concentrations in the range of1-10 muM resulted in a short-term arrest of growth and of monoclonal antibody production. The short-term suppression of cell functions was followed by a significant temporary increase of specific growth rate and of specific production rate. The steady-state viable cell density values, found in semicontinuous cultures, showed a certain stimulation of cell growth in the range of micromolar concentrations of bohemine, and inhibition of growth at 10 and 30 muM concentrations. The profiles of cell cycle phases indicated that hybridoma cells are retarded both at the G(1)/S boundary and at the G(2)/M boundary, depending on bohemine concentration. The existence of the sequence of events,from suppression to stimulation, suggests that bohemine probably modulates more than one regulatory pathway in the cell.

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.

In vitro biotransformation of 2,6,9-trisubstituted purine-derived cyclin-dependent kinase inhibitor bohemine by mouse liver microsomes.

1. Biotransformation pathways of the cyclin-dependent kinase inhibitor 6-benzylamino-2-(3-hydroxypropylamino)-9-isopropylpurine (bohemine) by mouse liver microsomes in vitro were investigated. 2. Metabolite profiles of [8-(3)H]-labelled bohemine were established by TLC/(3)H-autoradiography and enzymatic and MS analyses were used to elucidate the chemical structures of the metabolites. The structures of the main primary metabolites were confirmed by synthesis of authentic compounds. 3. A schema of the primary NADPH-dependent pathways has been proposed involving N(2)- and N9-dealkylation, N(6)-debenzylation, aromatic hydroxylation, and C2 side chain oxidation of bohemine. Three of the primary metabolites detected, 6-(benzylamino)-2-(3-hydroxypropylamino)purine (M4), 6-amino-2-(3-hydroxypropylamino)-9-isopropylpurine (M5) and 6-(4-hydroxybenzylamino)-2-(3-hydroxypropylamino)-9-isopropylpurine (M6), all retaining their parent primary hydroxyl group, were subsequently shown to be converted, by a liver cytosolic NAD(+)-dependent system, into their corresponding carboxylic acids. M6 was subject to microsomal glycosidations requiring UDP-sugar donors. NADPH-dependent conversion of M6 into M5 by microsomes was also demonstrated. 4. Cytochrome P450 (CYP) enzymes-selective inhibitors were used to identify CYPs involved in bohemine biotransformation. The findings suggested that CYP2a and CYP3a substantially contributed to the NADPH-dependent bohemine transformation in vitro. 5. The findings will facilitate experiments designed to dissect enzymatic systems catalysing clearance of C2,C6,N9-trisubstituted purine compounds from mammalian tissues.

In vivo metabolism of 2,6,9-trisubstituted purine-derived cyclin-dependent kinase inhibitor bohemine in mice: glucosidation as the principal metabolic route.

Synthetic cyclin-dependent kinase inhibitors have recently been referred to as effective antiproliferative agents. This study was conducted to characterize clearance of a 3H-labeled, trisubstituted purine-type inhibitor, 8-[3H]bohemine [6-benzylamino-2-(3-hydroxypropylamino)-9-isopropylpurine], in mice. Radioactivity profiles were analyzed by liquid scintillation counting and by thin layer chromatography followed by autoradiography. Metabolite structures were elucidated by mass spectrometry, NMR, and enzymatic analyses. Bohemine was rapidly and completely metabolized in vivo and disappeared from circulation during the first 60 min following intravenous administration. The metabolites were partly eliminated by the hepatobiliary tract and partly by renal excretion. The terminal hydroxyl group located at the C2 side chain of bohemine made the compound susceptible to main metabolic attacks, i.e., distinct types of conjugation reactions with glycosyl donors as well as an oxidative reaction. Other pathways were of relatively minor significance. Bohemine O-beta-D-glucoside was the most abundant metabolite to be excreted. The enzymatic mechanism responsible for bohemine glucosidation in vitro required the presence of a UDP-glucoside donor. Additional glycosidation products were observed after inclusion of UDP-glucuronide, UDP-xylose, UDP-galactose, or UDP-N-acetylglucosamine into microsomal incubates. Glycosidations occurred faster in the kidney incubates than in hepatic ones. The second principal bohemine metabolite was a carboxylic acid, 6-benzylamino-2-(2-carboxyethylamino)-9-isopropylpurine. A cytosolic, 4-methylpyrazole-sensitive alcohol dehydrogenase class I was shown to mediate oxidation of the terminal hydroxyl group of bohemine into this acid, which was the only metabolite found in the blood in significant amounts. However, it displayed only weak cyclin-dependent kinase-1-inhibitory activity (IC(50) > 100 microM) when compared with that of bohemine (IC(50) approximately 1 microM).

Inhibition of cyclin-dependent kinases by purine analogues.

While testing purines related to the non-specific protein kinase inhibitors N6-dimethylaminopurine and N6-(delta 2-isopentenyl)adenine as potential inhibitors of the p34cdc2/cyclin B kinase, we discovered a compound with high specificity, 2-(2-hydroxyethylamino)-6- benzylamino-9-methylpurine (olomoucine). Kinetic analysis of kinase inhibition reveals that olomoucine behaves as a competitive inhibitor for ATP and as a non-competitive inhibitor for histone H1 (linear inhibition for both substrates). The kinase specificity of this inhibition was investigated for 35 highly purified kinases (including p34cdk4/cyclin D1, p40cdk6/cyclin D3, cAMP-dependent and cGMP-dependent kinases, eight protein kinase C isoforms, calmodulin-dependent kinase II, myosin light-chain kinase, mitogen-activated S6 kinase, casein kinase 2, double-stranded RNA-activated protein kinase, AMP-stimulated kinase, eight tyrosine kinases). Most kinases are not significantly inhibited. Only the cell-cycle regulating p34cdc2/cyclin B, p33cdk2/cyclin A and p33cdk2/cyclin E kinases, the brain p33cdk5/p35 kinase and the ERK1/MAP-kinase (and its starfish homologue p44mpk) are substantially inhibited by olomoucine (IC50 values are 7, 7, 7, 3 and 25 microM, respectively). The cdk4/cyclin D1 and cdk6/cyclin D3 kinases are not significantly sensitive to olomoucine (IC50 values greater than 1 mM and 150 microM, respectively). N6-(delta 2-Isopentenyl)adenine is confirmed as a general kinase inhibitor with IC50 values of 50-100 microM for many kinases. The purine specificity of cyclin-dependent kinase inhibition was investigated: among 81 purine derivatives tested, only C2, N6 and N9-substituted purines exert a strong inhibitory effect on the p34cdc2/cyclin B kinase. An essentially similar sensitivity to this olomoucine family of compounds was observed for the brain-specific cdk5/p35 kinase. Structure/activity relationship studies allow speculation on the interactions of olomoucine and its analogues with the kinase catalytic subunit. Olomoucine inhibits in vitro M-phase-promoting factor activity in metaphase-arrested Xenopus egg extracts, inhibits in vitro DNA synthesis in Xenopus interphase egg extracts and inhibits the licensing factor, an essential replication factor ensuring that DNA is replicated only once in each cell cycle. Olomoucine inhibits the starfish oocyte G2/M transition in vivo. Through its unique selectivity olomoucine provides an anti-mitotic reagent that may preferentially inhibit certain steps of the cell cycle.