Roscovitine is a proteostasis regulator that corrects the trafficking defect of F508del-CFTR by a CDK-independent mechanism.

BACKGROUND AND PURPOSE: The most common mutation in cystic fibrosis (CF), F508del, causes defects in trafficking, channel gating and endocytosis of the CF transmembrane conductance regulator (CFTR) protein. Because CF is an orphan disease, therapeutic strategies aimed at improving mutant CFTR functions are needed to target the root cause of CF. EXPERIMENTAL APPROACH: Human CF airway epithelial cells were treated with roscovitine 100 µM for 2 h before CFTR maturation, expression and activity were examined. The mechanism of action of roscovitine was explored by recording the effect of depleting endoplasmic reticulum (ER) Ca(2+) on the F508del-CFTR/calnexin interaction and by measuring proteasome activity. KEY RESULTS: Of the cyclin-dependent kinase (CDK) inhibitors investigated, roscovitine was found to restore the cell surface expression and defective channel function of F508del-CFTR in human CF airway epithelial cells. Neither olomoucine nor (S)-CR8, two very efficient CDK inhibitors, corrected F508del-CFTR trafficking demonstrating that the correcting effect of roscovitine was independent of CDK inhibition. Competition studies with inhibitors of the ER quality control (ERQC) indicated that roscovitine acts on the calnexin pathway and on the degradation machinery. Roscovitine was shown (i) to partially inhibit the interaction between F508del-CFTR and calnexin by depleting ER Ca(2+) and (ii) to directly inhibit the proteasome activity in a Ca(2+) -independent manner. CONCLUSIONS AND IMPLICATIONS: Roscovitine is able to correct the defective function of F508del-CFTR by preventing the ability of the ERQC to interact with and degrade F508del-CFTR via two synergistic but CDK-independent mechanisms. Roscovitine has potential as a pharmacological therapy for CF.

CDK/CK1 inhibitors roscovitine and CR8 downregulate amplified MYCN in neuroblastoma cells.

To understand the mechanisms of action of (R)-roscovitine and (S)-CR8, two related pharmacological inhibitors of cyclin-dependent kinases (CDKs), we applied a variety of '-omics' techniques to the human neuroblastoma SH-SY5Y and IMR32 cell lines: (1) kinase interaction assays, (2) affinity competition on immobilized broad-spectrum kinase inhibitors, (3) affinity chromatography on immobilized (R)-roscovitine and (S)-CR8, (4) whole genome transcriptomics analysis and specific quantitative PCR studies, (5) global quantitative proteomics approach and western blot analysis of selected proteins. Altogether, the results show that the major direct targets of these two molecules belong to the CDKs (1,2,5,7,9,12), DYRKs, CLKs and CK1s families. By inhibiting CDK7, CDK9 and CDK12, these inhibitors transiently reduce RNA polymerase 2 activity, which results in downregulation of a large set of genes. Global transcriptomics and proteomics analysis converge to a central role of MYC transcription factors downregulation. Indeed, CDK inhibitors trigger rapid and massive downregulation of MYCN expression in MYCN-amplified neuroblastoma cells as well as in nude mice xenografted IMR32 cells. Inhibition of casein kinase 1 may also contribute to the antitumoral activity of (R)-roscovitine and (S)-CR8. This dual mechanism of action may be crucial in the use of these kinase inhibitors for the treatment of MYC-dependent cancers, in particular neuroblastoma where MYCN amplification is a strong predictor factor for high-risk disease.

CR8, a potent and selective, roscovitine-derived inhibitor of cyclin-dependent kinases.

Among the ten pharmacological inhibitors of cyclin-dependent kinases (CDKs) currently in clinical trials, the purine roscovitine (CYC202, Seliciclib) is undergoing phase 2 trials against non-small-cell lung and nasopharyngeal cancers. An extensive medicinal chemistry study, designed to generate more potent analogues of roscovitine, led to the identification of an optimal substitution at the N6 position (compound CR8). An extensive selectivity study (108 kinases) highlights the exquisite selectivity of CR8 for CDK1/2/3/5/7/9. CR8 was 2- to 4-fold more potent than (R)-roscovitine at inhibiting these kinases. Cocrystal structures of (R)-CR8 and (R)-roscovitine with pCDK2/cyclin A showed that both inhibitors adopt essentially identical positions. The cellular effects of CR8 and (R)-roscovitine were investigated in human neuroblastoma SH-SY5Y cells. CR8 inhibited the phosphorylation of CDK1 and 9 substrates, with a 25-50 times higher potency compared to (R)-roscovitine. CR8 was consistently more potent than (R)-roscovitine at inducing apoptotic cell death parameters: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium reduction (40-fold), lactate dehydrogenase release (35-fold), caspases activation (68-fold) and poly-(ADP-ribose)polymerase cleavage (50-fold). This improved cell death-inducing activity of CR8 over (R)-roscovitine was observed in 25 different cell lines. Altogether these results show that second-generation analogues of (R)-roscovitine can be designed with improved antitumor potential.

[Search for new anti-angiogenic agents with structural analogy with thalidomide]. / Recherche de nouveaux agents anti-angiogéniques: analogues structuraux de la thalidomide.

Despite its teratogenic effects, thalidomide has been reintroduced in human anticancer treatment for its anti-angiogenic activity, especially observed in patients with multiple myeloma. Here, we report the synthesis of new analogues designed to increase the thalidomide anti-tumour properties. The anti-angiogenic activity of the compounds was tested on EA.hy 926 endothelial cell lines. In this model, that is easier to manipulate than HUVEC cells, thalidomide is active in a similar dose range as reported on HUVEC cells and one of our compounds is more efficient.

Development of a sensitive and specific new plasma 4-androstene-3,17- dione time-resolved fluoroimmunoassay (TR-FIA).

We describe, for the first time to our knowledge, the development of a new, non-isotopic time resolved-fluoroimmunoassay of 4-androstene-3,17-dione in plasma or serum. This steroid exhibits a key role in steroid metabolism and is often assayed in the investigation of various pathologic endocrine states. Most of the 4-androstene-3,17-dione immunoassays are performed using a radioactive tracer. We synthesized a biotinylated 4-androstene-3,17-dione tracer from 4-androstene-3,17-dione-3-carboxymethyloxime by acylation of biotinylaminopropylammonium trifluoroacetate. A specific rabbit anti 6-hemisuccinate-4-androstene-3,17-dione/BSA was indirectly bound via an anti-rabbit sheep antibody immobilized on microtiter plate wells. The amount of biotinylated-4-androstene-3,17-dione tracer was then measured by adding streptavidin-europium, and the europium fluorescence was quantified by time resolved-fluorescence (TR-FIA, Delfia System). The plasma 4-androstene-3,17-dione-levels measured with this non-isotopic assay were compared to those measured with a radioimmunoassay previously published. In both cases, the same anti-4-androstene-3,17-dione antibody was used, and the assays were performed after an extraction step and a chromatographic step. The results obtained by the two methods were virtually the same. However, the main advantages of the new plasma 4-androstenedione-3,17-dione time-resolved-fluorescence immunoassay were its greater sensitivity than radioimmunoassay and its higher precision.

A new specific and sensitive time resolved-fluoroimmunoassay of 11-deoxycortisol in serum.

A biotinylated 11-deoxycortisol tracer was synthesized from 11-deoxycortisol-3-carboxymethyloxime and the conjugate obtained by acylation of biotinylaminopropylammonium trifluoroacetate. This biotinylated tracer was used to develop an 11-deoxycortisol time-resolved-fluoroimmunoassay (TR-FIA). The tracer was quantified after adding streptavidine-Europium. A TR-FIA sensitive standard curve, with displacement of 20, 50, and 80% of tracer was obtained with 12.4, 70.7, and 512.8 pg of 11-deoxycortisol, respectively. After extraction followed by Celite chromatography, purified serum samples were simultaneously assayed by RIA and TR-FIA. The results obtained by the two methods were practically identical, however, this new specific, non-isotopic 11-deoxycortisol assay has the advantage of being more sensitive than RIA, thus well-suited to accurate measurement in endocrinological studies, particularly when serum 11-deoxycortisol levels in patients are just above the highest normal values. Moreover, this non-isotopic assay is cheaper than RIA.

Selective antibodies to methadone enantiomers: synthesis of (R)- and (R,S)-methadone conjugates and determination by an immunoenzymatic method in human serum.

Selective antibodies to (R)-methadone (Mtd) and to its racemate were produced in rabbits by immunization with conjugates of (R)- or (R,S)-hemisuccinyl-methadol-bovine serum albumin, respectively. A hapten was first prepared by reduction of (R)- or (R,S)-Mtd with sodium borohydride, followed by esterification with succinic anhydride. The conjugation of hapten with albumin was achieved by the mixed anhydride method. After immunization of rabbits, the titers and specificity of each antibody were determined by ELISA. The antibodies obtained were tested with (R)-, (S)-, (R,S)-Mtd, its major metabolite (EDDP), and some drugs of abuse (morphine, codeine, cocaine). The sensitivities of antibodies to (R)- and (R,S)-Mtd were about 1 and 2 ng/ml, respectively. Selective (R)-antibodies recognized (R)-Mtd about 40 times more avidly than the (S)-isomer, while an antiserum against (R,S)-Mtd recognized (R)- and (S)-isomers to about the same degree. Both selective antibodies showed little interference (about 0.5%) with EDDP metabolite and no crossreactivity with morphine, codeine, and cocaine. These two selective antibodies were used to develop an immunoenzymatic method (ELISA) for the determination of (R)- and (R,S)-Mtd in serum samples of patients under maintenance treatment for narcotic addiction.

Plasma 17-OH pregnenolone: comparison of a time-resolved fluoroimmunoassay using a new tracer 17-OH pregnenolone-3-oxyacetyl-biotine with a radioimmunoassay using 125I 17-OH pregnenolone-3-hemisuccinate-histamine.

In this article we described, for the first time to our knowledge, the development of a new non isotopic immunoassay (time resolved-fluoroimmunoassay) for determining 17alpha-hydroxypregnenolone levels in plasma or serum. This steroid is indeed the most relevant steroid for the diagnosis of 3beta-hydroxysteroid dehydrogenase deficiency. For the hapten tracer, we synthesized a biotin-oxyacetyl 17-hydroxypregnenolone conjugate. A specific polyclonal rabbit anti-17-hydroxypregnenolone was indirectly bound via an immobilized sheep anti-rabbit antibody on microtiter plate wells. The amount of biotin-17-hydroxypregnenolone conjugate bound was then measured by adding Streptavidin-Europium, and the Europium fluorescence was quantified by Time Resolved -Fluorescence (TR-FIA, Delfia System). The plasma 17-hydroxypregnenolone levels of this non isotopic assay were comparatively measured with a radioimmunoassay previously published and using the same anti 17-hydroxypregnenolone antibody. In both cases, the assays were performed after a extraction step and a chromatographic step. The sensitivity of this 17-hydroxypregnenolone time resolved-fluoroimmunoassay was higher than that of 17-hydroxypregnenolone radioimmunoassay. The compared results of plasma 17-hydroxypregnenolone, performed with these two methods were not significantly different. A practical advantage is the stability of the biotine tracer, comparatively to the radioactive 125I 17-hydroxypregnenolone tracer which requires a new labeling every two months.

Plasma 21-deoxycortisol: comparison of a time-resolved fluoroimmunoassay using a biotinylated tracer with a radioimmunossay using (125)iodine.

Plasma 21-deoxycortisol (21DF) is an excellent marker of 21-hydroxylase deficiency. Currently, it is the only marker able to detect heterozygous carriers with 21-hydroxylase deficiency after ACTH stimulation. We have already developed radioimmunoassays for 21DF using first tritiated, then 125I-21DF which had a ten-fold higher sensitivity. However, because the lifespan of 125I-21DF is short, the tracer needs to be reprepared every two months and this multiplies the risk of contamination by radioactive 125I vapours. We therefore developed a non-isotopic 21DF assay that uses a 21DF-biotin conjugate with a original bridge, a diaminopropyl arm, linking the steroid to biotin. The 21DF-biotin conjugate was measured by time-resolved fluorescence after adding streptavidin-europium to the microtitration wells. The analytical qualities of this assay were very similar to those of the radioimmunoassay using 125I-21DF as tracer. The results obtained by the two methods, in either normal subjects or patients with 21-hydroxylase deficiency, were virtually the same.

Development of a plasma 17alpha-hydroxyprogesterone time resolved-fluorescence immunoassay involving a new biotinylated tracer.

A biotinylated 17alpha-hydroxyprogesterone probe (3) was prepared from 17alpha-hydroxyprogesterone-3-carboxymethyloxime and conjugate obtained by acylation of biotinylaminopropylammonium trifluroacetate. This new tracer was used in the development of a 17alpha-hydroxyprogesterone time-resolved fluoroimmunoassay using streptavidin-europium. The new method was compared to a long-standing radioimmunoassay method and found to be more sensitive and economical.

Highly sensitive and specific time-resolved fluoroimmunoassay (TR-FIA) of cyproterone acetate and free cyproterone.

We have developed a non-isotopic TR-FIA for Cyproterone acetate and Cyproterone in plasma. Synthesis of the biotinylated tracers, biotinylated Cyproterone acetate, and Cyproterone, as well as the preparation of anti-Cyproterone acetate and anti-Cyproterone antisera are reported. The specificity of anti-Cyproterone acetate antiserum resulting from the coupling of link bridge (link bridge between steroid and BSA), on the 3-position on the steroid skeleton, allowed to carry out the Cyproterone acetate assay directly on extracted plasma (without chromatography). On the other hand Cyproterone assays require a purification step, including extraction plus chromatography, because the plasma Cyproterone acetate concentrations in Cyproterone acetate-treated women are 200 times higher than for Cyproterone. Theses plasma TR-FIA of Cyproterone acetate and Cyproterone presented the advantage of needing only small doses of radioactivity for recovery controls, and better practicability related to the only existing RIA described to date.

Genetic, biochemical, and clinical studies of patients with A328V or R213C mutations in 11betaHSD2 causing apparent mineralocorticoid excess.

Apparent mineralocorticoid excess is a recessively inherited hypertensive syndrome caused by mutations in the 11beta-hydroxysteroid dehydrogenase type 2 gene, which encodes the enzyme normally responsible for converting cortisol to inactive cortisone. Failure to convert cortisol to cortisone in mineralocorticoid-sensitive tissues permits cortisol to bind to and activate mineralocorticoid receptors, causing hypervolemic hypertension. Typically, these patients have increased ratios of cortisol to cortisone and of 5alpha- to 5beta-cortisol metabolites in serum and urine. We have studied 3 patients in 2 families with severe, apparent mineralocorticoid excess and other family members in terms of their genetic, biochemical, and clinical parameters, as well as normal controls. Two brothers were homozygous for an A328V mutation and the third patient was homozygous for an R213C mutation in the 11beta-hydroxysteroid dehydrogenase type 2 gene; both mutations caused a marked reduction in the activity of the encoded enzymes in transfection assays. The steroid profiles of the 7 heterozygotes and 2 other family members studied were completely normal. The results of a novel assay used to distinguish 5alpha- and 5beta-tetrahydrometabolites suggest that 5beta-reductase activity is reduced or inhibited in apparent mineralocorticoid excess. In 1 patient undergoing renal dialysis for chronic renal insufficiency, direct control of salt and water balance completely corrected the hypertension, emphasizing the importance of mineralocorticoid action in this syndrome.

Rapid determination of methadone and its major metabolite in biological fluids by gas-liquid chromatography with thermionic detection for maintenance treatment of opiate addicts.

A rapid gas-liquid chromatographic assay is developed for the quantification of methadone (Mtd) and its major metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), in biological fluids of opiate addicts. After alkaline extraction from samples with lidocaine hydrochloride as internal standard, Mtd and EDDP are separated on SP-2250 column at 220 degrees C and detected with a thermionic detector. The chromatographic time is about 6 min. The relative standard deviations (R.S.D.) of Mtd and EDDP standards are between 1.5 and 5.5%. Most drugs of abuse (morphine, codeine, narcotine, cocaine, benzoylecgonine, cocaethylene, dextropropoxyphene etc) are shown not to interfere with this technique. The method has been applied to study the levels of Mtd and EDDP metabolite in serum, saliva and urine of patients under maintenance treatment for opiate dependence. EDDP levels were found higher than those of Mtd in urine samples from four treated patients, but lower in serum and undetectable in saliva. However, Mtd concentrations were higher in saliva than in serum.

Crystal structure of 11,18-oxidosteroids obtained by hypoiodination of cortisol acetonide.

The photochemical hypoiodination of cortisol acetonide, without neutralization of the excess of acidity during the work-up of the reaction, led to a mixture of 11 beta,18-oxido-17 alpha,21-dihydroxy-4-pregnen-3,20-dione and 11 beta,19-oxido-17 alpha,21-dihydroxy-4-pregnen-3,20-dione. Side chain cleavage of the former compound gave 11 beta,18-oxido-4-androsten-3,17-dione. The crystal structures of both of these 11 beta,18-oxidosteroids were determined by X-ray. The ring conformations are discussed and compared with those of aldosterone.

Enantioselective high-performance liquid chromatography determination of methadone enantiomers and its major metabolite in human biological fluids using a new derivatized cyclodextrin-bonded phase.

The simultaneous determination of methadone (Mtd) enantiomers and its major metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), in human urine and serum by enantioselective HPLC using a new Cyclobond 1-2000 RSP column is described. After alkaline extraction from urine or serum with estazolam as an internal standard, Mtd enantiomers and its metabolite (EDDP) are separated on the previous column with reversed-mobile phase and detected at 210 nm. Peak resolutions are about 2.0 for Mtd enantiomers. The relative standard deviations (R.S.D.) of Mtd and EDDP standards are between 0.5 and 4.5%. Most drugs of abuse are shown not to interfere with this technique. The method has been applied to study the levels of each Mtd enantiomer and of its racemic metabolite in urine and serum of patients under maintenance treatment for opiate dependence. In urine, R-(-)-Mtd levels are always higher (about 2+/-0.5-fold) than those of S-(+)-Mtd and in most cases, metabolite concentrations are greater than those of global Mtd enantiomers. However, the R-(-) enantiomer levels of residual drug in serum of some patients were lower than those of its antipode. This method is suitable for pharmacokinetic and toxicological studies of Mtd enantiomers and its major metabolite in biological fluids.

Radioimmunoassay of cortisone in serum, urine, and saliva to assess the status of the cortisol-cortisone shuttle.

We have developed a new assay for cortisone (E) in serum, saliva, and urine involving Celite chromatography followed by RIA with 125I-labeled E and scintillation proximity assay. The chromatography step separates cortisol (F) from E, and in combination with their RIAs, permits assessment of the status of the F-E shuttle. We report the results of basal, postcorticotropin (ACTH), and postdexamethasone E and F concentrations and their circadian fluctuations in the serum, saliva, and urine of healthy volunteers. The serum and urine F/E ratios were increased in patients with ectopic ACTH secretion, whereas in adrenal adenoma and Cushing disease only the urinary ratio was increased. In chronic renal insufficiency this ratio was increased in serum (23.5 +/- 3.9) but diminished in saliva (0.38 +/- 0.11), and in apparent mineralocorticoid excess the ratios were high in serum (44.3 +/- 9.3) and urine (5.35 +/- 0.85) compared with those of healthy subjects (serum 9.8 +/- 3.5, urine 0.52 +/- 0.29, saliva 0.52 +/- 0.29).

Use of a zwitterionic cyclodextrin as a chiral agent for the separation of enantiomers by capillary electrophoresis.

The purity and enantioselectivity of a novel chiral agent, the zwitterionic mono-(6-delta-glutamylamino-6-deoxy)-beta-cyclodextrin (beta-CD-Glu), were studied by capillary electrophoresis. Chiral separation of the enantiomers of chlorthalidone was obtained at pH 2.3, a pH at which beta-CD-Glu is partially protonated. Comparison with the cationic mono-(6-amino-6-deoxy)-beta-cyclodextrin (beta-CD-NH2) enantioselectivity clearly shows that the greater the difference in mobility between the free analyte and the analyte-cyclodextrin complex, the better the resolution. Hydrobenzoin enantiomers were separated at pH 11.2, a pH at which beta-CD-Glu is anionic. Under these conditions, the migration order was opposite to that observed in the presence of beta-CD-NH2 at pH 2.3. When no separation was obtained directly with beta-CD-Glu, a dual cyclodextrin system was developed. Carprofen enantiomers were resolved at pH 2.3 in the presence of a beta-CD-Glu/trimethyl-beta-cyclodextrin (TM-beta-CD) system in which the charged CD confers a non-zero mobility to the analyte, while the neutral CD allows chiral recognition.

In vitro and in vivo immunosuppressive potential of thalidomide and its derivative, N-hydroxythalidomide, alone and in combination with cyclosporin A.

Thalidomide (Thd) has been shown to have interesting immunosuppressive properties and strong action against TNF-alpha. It is used for treating a variety of immune-mediated pathology and inflammatory diseases. The purpose of this work was to evaluate the in vitro and in vivo immunosuppressive effects of Thd and its derivative, N-Hydroxythalidomide (H-Thd), alone and in combination with cyclosporin A (CsA), upon different in vitro lymphocyte activation pathways and in vivo local graft-versus-host-reaction (GvHR). At different concentrations, both Thd and H-Thd alone inhibited the lymphocyte proliferation induced by alloantigen (MLR), mitogens (Con A, PWM) and superantigen (SEB) with an activity of 50-75% that of CsA, however, in some tests, immunosuppressive potency of H-Thd was shown to be higher than that of Thd. In vivo using GvHR, Thd and H-Thd alone proved as active as CsA. The association in vitro and in vivo of each compound with CsA at different low concentrations, produced an additive effect as strong as CsA used alone at high therapeutic concentrations. In summarizing, this study revealed that: (1) despite its weaker potency in vitro than that of CsA, H-Thd presents interesting immunosuppressive properties similar to, and in some cases, better than Thd, and (2) the combination of H-Thd or Thd with CsA at suboptimal concentrations leads to high activity.

Intrinsic selectivity in capillary electrophoresis for chiral separations with dual cyclodextrin systems.

Defined as the ratio of the affinity factors of the analytes for a complexing agent, the intrinsic selectivity is representative of the very nature of the complexing agent. When more than one complexing agent are present in the background electrolyte, it is possible to define several intrinsic selectivities according to whether complexing agents are considered separately or all together. A theoretical model with respect to selectivity is presented for separations that involve two complexing agents, using the concept of apparent constant for complex formation. When only independent complexation occurs (absence of mixed complexes), then the intrinsic selectivity of a complexing agent X in the presence of a complexing agent Y can be easily related to the intrinsic selectivity of each complexing agent and to complex formation constants. Dual systems of cyclodextrins (CDs), implementing the cationic mono(6-amino-6-deoxy)-ß-cyclodextrin (ß-CD-NH(2)) and a neutral CD (trimethyl-ß-CD (TM-ß-CD) or dimethyl-ß-CD (DM-ß-CD)), were studied to illustrate this model and to offer an alternative to the separation of neutral enantiomers when ß-CD-NH(2) shows no or insufficient stereoselectivity. With a dual ß-CD-NH(2)/TM-ß-CD system at pH 2.3, arylpropionic acid enantiomers were baseline resolved and benzoin derivatives were partially resolved. For the arylpropionic acids, ß-CD-NH(2), which is not stereoselective, confers on them a nonzero mobility, while TM-ß-CD allows the chiral recognition. A study of the respective influence of ΤM-ß-CD and ß-CD-NH(2) concentrations was performed to determine the optimal conditions with respect to resolution. This theoretical approach allowed characterization of the intrinsic selectivity of neutral CDs for pairs of neutral enantiomers and therefore identification of the potential of neutral chiral agents for neutral enantiomers.

A simple synthesis of 11,19-oxidosteroids.

The photochemical hypoiodination of cortisol acetonide gave a mixture of 18-iodocortisol acetonide and of the 11 beta,19-oxidoderivative. The proportion of the two products was slightly modified by the reaction temperature. Deprotection of the acetonide group of the 11 beta,19-oxidoderivative gave 11 beta,19-oxido-17 alpha,21-dihydroxy-4-pregnen-3,20-dione which led to the formation of 11 beta,19-oxido-4-androsten-3,17-dione upon treatment with sodium bismutate.