Desulfurization of 2-thiouracil nucleosides: conformational studies of 4-pyrimidinone nucleosides

4-Pyrimidinone ribofuranoside (H(2)o(4)U) and 4-pyrimidinone 2'-deoxyribofuranoside (dH(2)o(4)U) were synthesized by the oxidative desulfurization of parent 2-thiouracil nucleosides with m-chloroperbenzoic acid. The crystal structures of H(2)o(4)U and dH(2)o(4)U and their conformations in solution were determined and compared with corresponding 2-thiouracil and uracil nucleosides. The absence of a large 2-thiocarbonyl/2-carbonyl group in the nucleobase moiety results in C2'-endo puckering of the ribofuranose ring (S conformer) in the crystal structure of H(2)o(4)U, which is not typical of RNA nucleosides. Interestingly, the hydrogen bonding network in the crystals of dH(2)o(4)U stabilizes the sugar moiety conformation in the C3'-endo form (N conformer), rarely found in DNA nucleosides. In aqueous solution, dH(2)o(4)U reveals a similar population of the C2'-endo conformation (65%) to that of 2'-deoxy-2-thiouridine (62%), while the 62% population of the S conformer for H(2)o(4)U is significantly different from that of the parent 2-thiouridine, for which the N conformer is dominant (71%). Such a difference may be of biological importance, as the desulfurization process of natural tRNA 2-thiouridines may occur under conditions of oxidative stress in the cell and may influence the decoding process.     

Juvenile animal studies and pediatric drug development: a European regulatory perspective

During the workshop organized by ILSI/HESI on May 5-6, 2010 on the value of juvenile animal toxicity studies, the implementation of the European Pediatric Regulation and in particular the review process of the nonclinical part of the Pediatric Investigation Plan (PIP) were described. A PIP is intended to outline the development of a medicinal product in the pediatric population (i.e. quality, safety, efficacy of the medicine and timing of studies); it is reviewed and agreed by the Pediatric Committee (PDCO) of the European Medicines Agency (EMA). The Nonclinical Working Group (NcWG) supports the PDCO in the review process of the nonclinical part of a PIP and is composed of members from the PDCO, the EMA Safety Working Party, additional experts from national competent authorities and the FDA. This article summarizes the NcWG review process and outcomes of 97 approved or ongoing PIPs, from the establishment of the NcWG in November 2008 to May 2010, as presented during the workshop. Juvenile animal studies were proposed by the applicant in 33% or required by the NcWG in 26% of the PIPs. The requirements were mainly motivated by concerns regarding potential developmental toxicities, in view of the young age of the pediatric population to be investigated, the lack of knowledge concerning the maturation of the pharmacological target, the lack of sufficient (non)clinical data, observed toxicities in the adult (non)clinical studies and the long duration of the intended treatments. Most juvenile animal studies were in the therapeutic areas of oncology, infectious diseases and endocrinology. In about 14% of the PIPs submitted, the NcWG requested either justifications of, or amendments to the study designs proposed by the applicants (e.g. justification of endpoints, study duration, species selection and timing with regards to clinical pediatric studies). Generally, only one species was selected or proposed for the juvenile studies, the rat being the most prevalent. The number of juvenile studies initially proposed by the applicant plus those requested by the NcWG was higher than the number of studies included in the "key binding elements" of the PIP opinions. This apparent discrepancy was mainly due to additional information or justifications submitted by the applicant during the clock stop. It was noted that the PIPs initially submitted often lacked information relevant to the nonclinical evaluation. Therefore, during the workshop, the need to provide scientifically based justifications when no juvenile animal studies are proposed in the initial PIP submission was stressed.     

Investigating the effect of emetic compounds on chemotaxis in Dictyostelium identifies a non-sentient model for bitter and hot tastant research

Novel chemical entities (NCEs) may be investigated for emetic liability in a range of unpleasant experiments involving retching, vomiting or conditioned taste aversion/food avoidance in sentient animals. We have used a range of compounds with known emetic /aversive properties to examine the possibility of using the social amoeba, Dictyostelium discoideum, for research into identifying and understanding emetic liability, and hence reduce adverse animal experimentation in this area. Twenty eight emetic or taste aversive compounds were employed to investigate the acute (10 min) effect of compounds on Dictyostelium cell behaviour (shape, speed and direction of movement) in a shallow chemotaxic gradient (Dunn chamber). Compound concentrations were chosen based on those previously reported to be emetic or aversive in in vivo studies and results were recorded and quantified by automated image analysis. Dictyostelium cell motility was rapidly and strongly inhibited by four structurally distinct tastants (three bitter tasting compounds--denatonium benzoate, quinine hydrochloride, phenylthiourea, and the pungent constituent of chilli peppers--capsaicin). In addition, stomach irritants (copper chloride and copper sulphate), and a phosphodiesterase IV inhibitor also rapidly blocked movement. A concentration-dependant relationship was established for five of these compounds, showing potency of inhibition as capsaicin (IC(50) = 11.9 ± 4.0 μM) > quinine hydrochloride (IC(50) = 44.3 ± 6.8 μM) > denatonium benzoate (IC(50) = 129 ± 4 μM) > phenylthiourea (IC(50) = 366 ± 5 μM) > copper sulphate (IC(50) = 1433 ± 3 μM). In contrast, 21 compounds within the cytotoxic and receptor agonist/antagonist classes did not affect cell behaviour. Further analysis of bitter and pungent compounds showed that the effect on cell behaviour was reversible and not cytotoxic, suggesting an uncharacterised molecular mechanism of action for these compounds. These results therefore demonstrate that Dictyostelium has potential as a non-sentient model in the analysis of the molecular effects of tastants, although it has limited utility in identification of emetic agents in general.     

A densitometrical method for the study of pattern formation in a ciliateChilodonella

Summary An easy and sensitive method is reported here for testing the similarities of individual patterns by photographically transforming maps of these patterns to given, deductively chosen conventions involving constant distances between selected reference points. A cumulative map is produced by loading all landmarks from a set of individual maps on to one sheet of paper. The use of various a priori conventions results in variable cumulative maps, which are then optically transformed on an analog digital converter, with additional input for optical picture processing. The densitometrical maps thus obtained may be compared as to the cumulative degree of areas of maximal and minimal density of landmarks. The best conventions are those that yield the map with the most contrast.Maps of spatial patterns of the sites of contractile vacuole pore (CVP) primordia in an early stage of divisional morphogenesis of the ciliateChilodonella steini were compared after four different transformations and adjustments of the same set of individual maps. The best focusing of the sites of CVP differentiation was achieved by use of the postoral axis, defined by the center of the oral apparatus and the posterior end of the cell as the scaling parameter. The composite domain map obtained by optical transformation of this cumulative map could distinguish the specific CVP territories observed in earlier work (Kaczanowska 1981). These results confirm earlier findings that indicated the site of the oral apparatus is an important reference point in CVP primordia positioning. They also strongly suggest the existence of an overriding scaling factor governing the positioning of sites of differentiation in both dimensions of the developmental field. The method of superposition and scaling of pattern maps is generally applicable to situations in which pattern elements appear at discrete points on a flat surface.     

Pre-steady-state kinetic characterization of the DinB homologue DNA polymerase of Sulfolobus solfataricus

Equilibrium as well as pre-steady-state measurements were performed to characterize the molecular basis of DNA binding and nucleotide incorporation by the thermostable archaeal DinB homologue (Dbh) DNA polymerase of Sulfolobus solfataricus. Equilibrium titrations show a DNA binding affinity of about 60 nm, which is approximately 10-fold lower compared with other DNA polymerases. Investigations of the binding kinetics applying stopped-flow and pressure jump techniques confirm this weak binding affinity. Furthermore, these measurements suggest that the DNA binding occurs in a single step, diffusion-controlled manner. Single-turnover, single dNTP incorporation studies reveal maximal pre-steady-state burst rates of 0.64, 2.5, 3.7, and 5.6 s(-1) for dTTP, dATP, dGTP, and dCTP (at 25 degrees C), which is 10-100-fold slower than the corresponding rates of classical DNA polymerases. Another unique feature of the Dbh is the very low nucleotide binding affinity (K(d) approximately 600 mum), which again is 10-20-fold lower compared with classical DNA polymerases as well as other Y-family polymerases. Surprisingly, the rate-limiting step of nucleotide incorporation (correct and incorrect) is the chemical step (phosphoryl transfer) and not a conformational change of the enzyme. Thus, unlike replicative polymerases, an "induced fit" mechanism to select and incorporate nucleotides during DNA polymerization could not be detected for Dbh.     

Real time spectral analysis during phytochrome chromophore and chromoprotein purification

The plant photoreceptor phytochrome senses light quality and quantity in the red region of the spectrum, directing adaptation and development. The functional holo-protein is a dimeric chromoprotein which is formed by an autoassembly reaction between the translation product and the open chain tetrapyrroles phytochromobilin (PPhiB) or phycocyanobilin (PCB). We are interested in structure/function relationships within the phytochrome molecule, in particular chromophore/protein interaction during the assembly and photoactivation, using IR and NMR spectroscopy. For this we use an automated F/HPLC system running in a darkroom to purify large amounts of protein and chromophore separately. To obtain highly pure PCB chromophore we developed improved extraction and purification methods in which the final step is RPC-18 HPLC. As there are many spectrally only slightly different tetrapyrroles in the extract, the triple-wavelength monitoring offered by the F/HPLC detector was inadequate for distinguishing between PCB and impurities. Furthermore, lambda(max) for the phytochrome Pfr signalling state lies between 705 and 730 nm, beyond the range of the detector. Also, as both holo-protein and chromophore are photoactive, we wished to minimize light exposure of the eluate. We therefore implemented a miniature CCD-based flow UV-vis spectrophotometer using a xenon flash and quartz fiber optics enabling us monitor the entire 250-800 nm spectrum of the eluate to an accuracy of +/-3 x 10(-3)A in real time. The instrumentation described can be added to any chromatographic system, thereby allowing the purification of any molecule to be monitored easily and efficiently.     

Study on the influence of cranberry extract ?uravit S·O·S(?) on the properties of uropathogenic Escherichia coli strains, their ability to form biofilm and its antioxidant properties

Consumption of cranberries is known to exert positive health effects, especially against urinary tract infections. For this reason, presumably, they are widely used in folk medicine. Different aspects of cranberry phenolics activity were studied in individual papers but complex study in this matter is missing. The aim of the present study is to provide complex data concerning various aspects of cranberry extract activity. We studied the effects of subinhibitory concentrations of commercially available extract (?uravit S·O·S(?)) against two Escherichia coli strains isolated from urine of patients with pyelonephritis. Additionally the main extract anthocyanins were characterized. The activity of extract against lipid peroxidation and its radical scavenging ability were also assessed. ?uravit S·O·S(?) decreased the hydrophobicity of one of the studied E. coli strains, reduced swimming motility and adhesion to epithelial cells of both studied strains, it also limited the ability of bacteria to form biofilm. Expression of curli was not affected by cranberry extract, the assessment of P fimbriae expression was not reliable due to extract-induced agglutination of erythrocytes. Cranberry extract caused filamentation in both studied E. coli strains. It also showed pronounced antioxidant and radical scavenging properties. The properties of the studied cranberry extract show that it could be effectively used in prevention and/or elimination of urinary tract infections, specially the recurrent ones.     

The EPI bioassay identifies natural compounds with estrogenic activity that are potent inhibitors of androgenic pathways in human prostate stromal and epithelial cells

The reactive stromal phenotype is an important factor for prostate cancer progression and may be a new target for treatment and prevention. A new high efficiency preclinical protocol, the EPI bioassay, reflects the interaction of endocrine, paracrine and immune, (EPI) factors on induced androgen metabolism in human prostate reactive stroma. The bioassay is based on co-culturing human primary prostate stromal cells and LAPC-4 prostatic adenocarcinoma cells in a downscaled format of 96-well-plates for testing multiple doses of multiple target compounds. Metabolism of dehydroepiandrosterone (DHEA) with or without TGFβ1-induced stimulation (D+T) of the reactive stroma phenotype was assessed by increased testosterone in the media and PSA production of the epithelial prostate cancer cells. Using the non-metabolizable androgen R1881, effects from direct androgen action were distinguished from stromal androgen production from DHEA. Stromal cell androgenic bioactivity was confirmed using conditioned media from D+T-treated stromal cell monocultures in an androgen-inducible AR screening assay. We further showed that both agonists to estrogen receptor (ER), DPN (ERβ) and PPT (ERα), as well as estrogenic natural compounds including soy isoflavones attenuated D+T-induced PSA production. Studies with the pure ER agonists showed that activating either ERα or ERβ could inhibit both D+T-mediated and R1881-mediated PSA production with the D+T effect being more pronounced. In conclusion, natural compounds with estrogenic activity and pure ER agonists are very potent inhibitors of stromal conversion of DHEA to androgenic metabolites. More studies are needed to characterize the mechanisms involved in estrogenic modulation of the endocrine-immune-paracrine balance of the prostate microenvironment.     

Synthesis and SAR-study for novel arylpiperazine derivatives of 5-arylidenehydantoin with α₁-adrenoceptor antagonistic properties

The study is focused on a series of 5-arylidenehydantoin derivatives with a phenylpiperazine-hydroxypropyl fragment at N3 of the hydantoin ring. The compounds were assessed on their affinity for α(1)-adrenoceptors and evaluated in functional bioassays for their antagonistic properties. Crystal structures of (Z)-5-(4-chlorobenzylidene)-3-(3-(4-(2-ethoxyphenyl)piperazin-1-yl)-2-hydroxypropyl)imidazolidine-2,4-dione (7) and hydrochloride of (Z)-5-(4-chlorobenzylidene)-3-(2-hydroxy-3-(4-(2-methoxyphenyl)piperazin-1-yl)propyl)imidazolidine-2,4-dione (10a) were solved using the X-ray diffraction method. Classical molecular mechanics (MMFFs force field, MCMM, MacroModel) were used to predict 3D structure of compounds 5a-18a using a crystal structure of 7. SAR analysis was performed on the basis of Barbaro's pharmacophore model and structural properties of previously investigated α(1)-adrenoceptor antagonists possessing a hydantoin fragment. Most of the compounds exhibited significant affinities for α(1)-ARs in nanomolar range (40-290 nM). The highest activities (K(i)<75 nM) were observed for compounds possessing a 2-alkoxyphenylpiperazine fragment and two methoxy substituents at the benzylidene moiety. The results indicated that chemical properties, number and positions of substituents at the 5-arylidene fragment influenced the power of α(1)-affinities as follows: 3,4-di CH(3)O>2,4-di CH(3)O>4-Cl>2,3-di CH(3)O>H>4-N(CH(3))(2).     

Combined surgery and radiotherapy in the treatment of ductal carcinoma in situ of the breast: preliminary results of the Hungarian multicenter prospective randomised study

The aim of this work is to report the preliminary results of the Hungarian multicentric randomised DCIS study. Between 2000 and 2007, 278 patients with ductal carcinoma in situ (DCIS) treated by breast-conserving surgery were randomised according to predetermined risk groups. Low/intermediate-risk patients (n=29) were randomised to 50 Gy whole-breast irradiation (WBI) or observation. High-risk cases (n=235) were allocated to receive 50 Gy WBI vs. 50 Gy WBI plus 16 Gy tumour bed boost. Very high-risk patients (patients with involved surgical margins; n=14) were randomised to 50 Gy WBI plus 16 Gy tumour bed boost or reoperation (reexcision plus radiotherapy or mastectomy alone). Immunohistochemistry (IHC) was performed to detect the expression of potential molecular prognostic markers (ER, PR, Her2, p53, Bcl-2 and Ki-67). At a median follow-up of 36 months no recurrence was observed in the low/intermediate- and very high-risk patient groups. In the high-risk group, 4 (1.7%) local recurrences and 1 (0.4%) distant metastasis occurred. No patient died of breast cancer. In the high-risk group of patients, the 3- and 5-year probability of local recurrence was 1.1% and 3.1%, respectively. The positive immunostaining for Her2 (38%), p53 (37%) and Ki-67 (44%) correlated with a high nuclear grade. Significant inverse correlation was found between the expression of ER (77%), PR (67%), Bcl-2 (64%) and grade. Preliminary results suggest that breast-conserving surgery followed by radiotherapy yields an annual local recurrence rate of less than 1% in patients with DCIS. IHC of molecular prognostic markers can assist to gain insight into the biologic heterogeneity of DCIS.