Anti-hepatitis C virus activity and toxicity of type III phosphatidylinositol-4-kinase beta inhibitors.

Type III phosphatidylinositol-4-kinase beta (PI4KIIIß) was previously implicated in hepatitis C virus (HCV) replication by small interfering RNA (siRNA) depletion and was therefore proposed as a novel cellular target for the treatment of hepatitis C. Medicinal chemistry efforts identified highly selective PI4KIIIß inhibitors that potently inhibited the replication of genotype 1a and 1b HCV replicons and genotype 2a virus in vitro. Replicon cells required more than 5 weeks to reach low levels of 3- to 5-fold resistance, suggesting a high resistance barrier to these cellular targets. Extensive in vitro profiling of the compounds revealed a role of PI4KIIIß in lymphocyte proliferation. Previously proposed functions of PI4KIIIß in insulin secretion and the regulation of several ion channels were not perturbed with these inhibitors. Moreover, PI4KIIIß inhibitors were not generally cytotoxic as demonstrated across hundreds of cell lines and primary cells. However, an unexpected antiproliferative effect in lymphocytes precluded their further development for the treatment of hepatitis C.

Morphology, testes development and behaviour of unusual triploid males in microchromosome-carrying clones of Poecilia formosa.

In a microchromosome-carrying laboratory stock of the normally all-female Amazon molly Poecilia formosa triploid individuals were obtained, all of which spontaneously developed into males. A comparison of morphology of the external and internal insemination apparatus and the gonads, sperm ploidy and behaviour, to laboratory-bred F(1) hybrids revealed that the triploid P. formosa males, though producing mostly aneuploid sperm, are partly functional males that differ mainly in sperm maturation and sexual motivation from gonochoristic P. formosa males.

In silico prediction of receptor-mediated environmental toxic phenomena-application to endocrine disruption.

It is an objective of our institution to establish a virtual laboratory allowing for a reliable in silico estimation of the harmful effects triggered by drugs, chemicals and their metabolites. In the recent past, we have developed the underlying technology (Multi-dimensional QSAR: Quasar and Raptor) and compiled a pilot system including the 3D models of three receptors known to mediate endocrine-disrupting effects (the aryl hydrocarbon receptor, the estrogen receptor and the androgen receptor, respectively) and validated them against 310 compounds (drugs, chemicals, toxins). Within this set up we could demonstrate that our concepts are able to both recognize toxic compounds substantially different from those used in the training set as well as to classify harmless compounds clearly as being non-toxic. This suggests that our approach can be used for the prediction of adverse effects of drug molecules and chemicals.

Species-specific field testing of Entamoeba spp. in an area of high endemicity.

Entamoeba histolytica has been separated in recent years into 2 morphologically identical species: the apathogenic E. dispar and the pathogenic E. histolytica, only the latter being pathogenic. Although various laboratory techniques allow discrimination between the 2 species there is a lack of field data about the suitability of available diagnostic tests for use in epidemiological studies and few epidemiological studies using species-specific diagnosis have been performed at community level in endemic areas, especially in sub-Saharan Africa. We conducted a repeated cross-sectional study of 967 schoolchildren in central Côte d'Ivoire to compare and evaluate light microscopy, 2 different antigen detection assays, and one polymerase chain reaction (PCR) assay. Microscopy and a non-specific antigen capture Entamoeba enzyme-linked immunosorbent assay (ELISA) were used for the primary screening of all children (time t0). The prevalence of the E. histolytica/E. dispar species complex at t0 was 18.8% by single microscopical examination and 31.4% using the non-specific ELISA. Approximately 2 months after the initial screening, fresh stool specimens were collected on 2 consecutive days (t1 and t2) from (i) all the children who were positive by microscopy at t0 (n = 182) and (ii) 155 randomly selected children who were negative at the primary screening. These samples were tested with a second antigen detection ELISA specific for E. histolytica (n = 238) and with a species-specific PCR assay (n = 193). The second and third examinations (t1 and t2) revealed an additional 43 infections with the species complex E. histolytica/E. dispar, so that the cumulative microscopical prevalence for t1 and t2 was 27.7%. The overall prevalence of E. histolytica by species-specific ELISA antigen detection was low (0.83%), while the prevalence of E. dispar was 15%. When analysing only microscopically positive samples by PCR (n = 129), the ratio E. histolytica: E. dispar was very low (1:46), suggesting that the vast majority of Entamoeba infections in this area were apathogenic. Both species-specific tests performed well but the ELISA was easier to use for large-scale field screening.

Internet laboratory for predicting harmful effects triggered by drugs and chemicals. Concept and call for co-operation.

It is our objective to establish a virtual laboratory on the Internet to allow for an in silico estimation of harmful effects triggered by drugs, chemicals and their metabolites. Presently, our database includes validated models for five biological targets -- the Aryl hydrocarbon, serotonin 5HT-2A, cannabinoid, GABA (gamma-amino butter acid), and steroid receptors. It shall be continuously extended to include surrogates for any bioregulator known or presumed to mediate harmful effects. Free access to this virtual laboratory shall allow any interested party to estimate the harmful potential of a given substance prior to its synthesis. This is achieved by generating the three-dimensional structure of the compound and its possible metabolites in the computer, followed by calculating their binding affinity towards each receptor surrogate in the database. Only compounds/metabolites passing through this surrogate battery without displaying a significant affinity towards any member may be cleared for synthesis and preclinical studies. This way, potentially harmful compounds can be withdrawn from the evaluation pipeline before in vivo test are conducted, hence contributing to the reduction of animal testing in chemical and pharmaceutical research and development.

Multi-dimensional QSAR in drug research. Predicting binding affinities, toxicity and pharmacokinetic parameters.

Quantitative structure-activity relationships (QSAR) are an area of computational research which builds atomistic or virtual models to predict the biological activity or the toxicity of known or hypothetical substances. Of particular interest for the biomedical sciences are three-dimensional receptor surrogates (3D-QSAR) because they allow for the simulation of directional forces such as hydrogen bonds or metal-ligand contacts--key interactions for both molecular recognition and stereospecific ligand binding. While more powerful approaches make use of a genetic algorithm or a neural network to evolve a receptor surrogate, its predictive power still critically depends on the spatial alignment of the ligand molecules--mirroring the pharmacophore hypothesis--used to construct it. To avoid this bias, a recent development at our laboratory includes the possibility to represent each ligand molecule by an ensemble of conformations, orientations and protonation states as the fourth dimension (4D-QSAR). In addition, it allows for a potentially flexible receptor site (mimicking local induced fit) and solvent-accessible or shallow binding pockets. In this account, we seek to document the superiority of 4D-QSAR compared to 3D-concepts with simulations on the steroid, the aryl hydrocarbon and the neurokinin-1 receptor system. More complex, future applications of 4D-QSAR--the establishment of a virtual laboratory for the assessment of receptor-mediated toxicity and the prediction of oral bioavailability--are outlined.

Multiple-conformation and protonation-state representation in 4D-QSAR: the neurokinin-1 receptor system.

Using a 4D-QSAR approach (software Quasar) allowing for multiple-conformation, orientation, and protonation-state ligand representation as well as for the simulation of local induced-fit phenomena, we have validated a family of receptor surrogates for the neurokinin-1 (NK-1) receptor system. The evolution was based on a population of 500 receptor models and simulated during 40 000 crossover steps, corresponding to 80 generations. It yielded a cross-validated r(2) of 0.887 for the 50 ligands of the training set (represented by a total of 218 conformers and protomers) and a predictive r(2) of 0.834 for the 15 ligands of the test set (70 conformers and protomers). A series of five "scramble tests" (with an average predictive r(2) of -0.438) demonstrates the sensitivity of the surrogate toward the biological data, for which it should establish a QSAR. On the basis of this model, the activities of 12 new compounds - four of which have been synthesized and tested in the meantime - are predicted. For most of the NK-1 antagonists, the genetic algorithm selected a single entity - out of the up to 12 conformers or protomers - to preferably bind to the receptor surrogate. Moreover, the evolution converged at an identical protonation scheme for all NK-1 antagonists. This indicates that 4D-QSAR techniques may, indeed, reduce the bias associated with the choice of the bioactive conformation as each ligand molecule can be represented by an ensemble of conformations, orientations, and protonation states.

Deletion monitoring in skin tumors by interphase-FISH using band-specific DNA probes.

Thin sections from archival paraffin blocks of various skin tumors (26 melanomas, 15 squamous cell carcinomas, 5 keratoacanthomas, 5 basal cell carcinomas) were subjected to interphase-FISH (I-FISH) with DNA probes which are specific for chromosomal regions often involved in deletions in human cancer. These were probes for chromosome 3p21, the p53 gene on chromosome 17p13, and, in a few selected cases, a probe for chromosome 9p21. It was demonstrated that deletions of these regions could be reliably detected and related to tumor type and histology, i.e. grading. The most common deletion was that of 3p21 which was found in all studied squamous cell carcinomas (SCC) of low differentiation, in 60% of the Bowen carcinomas, in 70% of the metastatic melanomas less than 1.5 mm thick, and in over 55% of those which thickness over 2 mm. In contrast, FISH-detected p53 deletion was a rare finding in the investigated tumors. However, this gene was even found in an increased copy number in 60% of the poorly differentiated SCCs (grade 4) and in 50% of the non-metastatic melanomas less than 1.5 mm thick. Deletion of 9p21 was detected in 13 of the 14 tumors on which pertinent examinations could be performed. I-FISH was shown to be a reliable technique for the rapid detection of chromosome band specific deletions in archival material of human skin tumors.

[Genetic algorithms in 3D-QSAR: The use of multiple ligand orientations for improved predictions of toxicity] / Genetische Algorithmen im 3D-QSAR: Verwendung multipler Wirkstofforientierungen zur verbesserten Voraussage der Toxizität.

In a recent article in this journal, we discussed the application of a 3D-QSAR technique to the prediction of the toxicity of dibenzodioxins, dibenzofurans, and biphenyls (Vedani et al., 1999a). The use of such methods is legitimate because there is strong evidence that the toxicity is mediated by the Aryl hydrocarbon (Ah) receptor, a regulatory element involved in the mammalian metabolism of xenobiotics. In an extention to a concept developed at our laboratory (Vedani et al., 1998), we now show that safer predictions are possible if instead of a single - and, therefore, necessarily biased - assumption about the mutual orientation of the toxins, an ensemble of possible orientations is used for model construction. The contribution of a single entity within this ensemble to the toxin-receptor interaction energy is determined by a Boltzmann criterion. While in the single-orientation model two of the 26 toxins of an external test set were predicted false positive or false negative, all test substances are correctly predicted in the multiple-orientation model - including up to four different orientations per molecule - within a factor 10 of the experimental binding affinity. These results demonstrate that 3D-QSAR techniques based on a genetic algorithm can be used to predict the toxicity of chemical and pharmacological substances "in computo" if a receptor-mediated mechanism can be assumed. Consequently, the method can be used in toxicological screening assays, thereby replacing stressful tests on animals.

[Genetic algorithms in 3D-QSAR: Predicting the toxicity of dibenzodioxins, dibenzofurans and biphenyls] / Genetische Algorithmen im 3D-QSAR: Voraussage der Toxizität von Dibenzodioxinen, Dibenzofuranen und Biphenylen.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related compounds represent serious environmental health hazards, whose effects include dermal toxicity, immunotoxicity, tumor promotion, developmental and reproductive toxicity. There is strong evidence that the toxicity is mediated by the Aryl hydrocarbon (Ah) receptor, a regulatory element involved in the mammalian metabolism of xenobiotics. Using Quasi-atomistic receptor modeling, a 3D-QSAR technique, we have generated a receptor model for the Ah receptor. The model was trained using 76 dibenzodioxins, dibenzofurans, and biphenyls, and tested using 26 compounds different therefrom. 24 of these test compounds (92.3%) were predicted to within a factor of 10 of the experimental binding affinity. One compound was predicted false positive, another false negative (7.7% total). The sensitivity of the model with respect to the biological activity was demonstrated by means of a scramble test with negative outcome. Quasi-atomistic receptor modeling can be used for the testing of chemicals and pharmaca for a toxicological potential in an early development phase and thereby replace stressful tests on animals

Dispensable and indispensable genes in an ameiotic fish, the Amazon molly Poecilia formosa.

All-female vertebrates are excellent model systems for studying many evolutionary problems. One of these is the Amazon molly. In this review, three aspects of its biology are discussed: (1) An important question is how dispensable genes, such as all male coding genes, evolve in this species. A number of studies found that most of these genes remain remarkably stable and functional. (2) The gynogenetic Amazon mollies have to live in sympatry with males of a gonochoristic species, because sperm are needed to trigger embryogenesis. Yet, Amazon mollies cannot replace their sexual competitors, because this would lead to their own extinction. Studies on the behavior of Amazon mollies and their sperm-donor species indicate that a number of behavior patterns stabilize the mating system by providing Amazon mollies with the copulations they need to reproduce. (3) The age of Amazon mollies has been estimated to be approximately 100,000 years. This is older than predicted by some theoretical models. In Amazon mollies two ways to occasionally incorporate fresh genetic material have evolved. One way is to add one complete set of paternal chromosomes, which, in nature, leads to stable triploid lineages. The second way is the incorporation of minute, centromere-containing microchromosomes. The evolutionary impact of these phenomena, however, is not resolved so far and needs further study.

Pharmacological characterization of muscarine receptors of PC12 (rat phaeochromocytoma) cells.

The muscarine receptors of PC12 (rat phaeochromocytoma) cells were studied in functional and binding experiments. The catecholamine stores of PC12 cells were labelled by incubation of the cells with tritiated noradrenaline. Muscarinic agonists elicited concentration-dependent release of tritium which consisted overwhelmingly of unchanged 3H-noradrenaline. The rank order of potency was: oxotremorine greater than acetylcholine greater than muscarine = methacholine greater than carbachol greater than bethanechol. The release evoked by carbachol (0.1 mmol/l) was inhibited with high potency by the M1-selective antagonist telenzepine (pKi = 8.82), with intermediate potency by pirenzepine (pKi = 7.00) and with low potency by the M2-selective antagonist AF-DX 116 (pKi = 5.74). The binding of 3H-N-methylscopolamine to PC12 membranes was inhibited by various non-selective and subtype-selective muscarinic antagonists with the following rank order of potency: telenzepine = atropine greater than 4-DAMP greater than dicyclomine greater than pirenzepine greater than HHSiD much greater than AF-DX 116. A similar rank order was obtained for the inhibition by these compounds of 3H-telenzepine binding to M1-receptors in membranes of the cerebral cortex of the guinea pig. The Hill coefficients for inhibition of 3H-N-methylscopolamine binding (to PC12 membranes) by pirenzepine, telenzepine and AF-DX 116 were below unity. Specific binding of both 3H-telenzepine and 3H-N-methylscopolamine to muscarine receptors of PC12 membranes was saturable and of high affinity; the maximal number of binding sites was higher for 3H-N-methylscopolamine than for 3H-telenzepine (calculated for the active (+)enantiomer). PC12 cells are presumably endowed with more than one subtype of muscarine receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the morphology of human uric acid stones.

Human uric acid acid renal stones are easily distinguished from other urinary calculi by their globular or spherical shape, their colour and their hardness. Investigations of uric acid crystals grown in the presence of a variety of pigments indicate that a disordered layer structure of the uric acid dihydrate is responsible for the colour of such crystals, caused by the inclusion of pigment molecules into the crystal lattice. This in turn may help to explain the other special properties of uric acid stones.