SOS1 over-expression in genital skin fibroblasts from hirsute women: a putative role of the SOS1/RAS pathway in the pathogenesis of hirsutism.

Hirsutism is the development of androgen-dependent terminal body hair in women in places in which terminal hair are normally not found. It is often associated with hyperandrogenemia and/or polycystic ovary syndrome (PCOS), but the existence of uncommom hirsutism forms that are not related to altered androgen plasma levels lead also to the definition of - idiopathic hirsutism. Although the pathophysiology of hirsutism has been linked to increasing 5-alpha reductase (SRD5A) activity and to an alteration of the androgen receptor (AR) transcriptional machinery, many aspects remain unclear. In particular, the relationships between androgens and local factors are poorly understood. In the present paper, we selected for a genital skin biopsy, 8 women affected with severe hirsutism (Ferriman-Gallway score greater than 25) but with normal plasma androgen levels, with the exception of slightly higher serum 3alpha-diol-glucuronide levels, and 6 healthy controls and analyzed their androgen- and insulin-specific transcriptional profile using a specific custom low density microarray (AndroChip 2, GPL9164). We identified the over-expression of the Son of Sevenless-1 (SOS1) gene in all of the hirsute skin fibroblast primary cell cultures compared to control healthy women. Since SOS1 is a guanine nucleotide exchange factor that couples receptor tyrosine kinases to the RAS signaling pathway that controls cell proliferation and differentiation, we further analyzed SOS1 expression, protein level and RAS signaling activation pathway in an in vitro model (NHDF, normal human dermal fibroblast cell line). NHDF treated for 24 h with different concentrations of DHT and T showed an increase in SOS1 levels (both mRNA and protein) and also an activation of the RAS pathway. Our in vivo and in vitro data represent a novel preliminary observation that factors activating SOS1 could act as local proliferative modulators linked to the androgen pathway in the pilosebaceous unit. SOS1 over-expression may play a role in the regulation of the RAS/mitogen-activated protein kinase pathway in the skin, in the hair follicle proliferation and cell cycle, suggesting new perspectives in understanding the pathogenesis of idiopathic hirsutism.

A pilot study on the transcriptional response of androgen- and insulin-related genes in peripheral blood mononuclear cells induced by testosterone administration in hypogonadal men.

The aim of the present study is to determine whether testosterone (T) administration changes the expression profile of androgen- and insulin-related genes in peripheral blood mononuclear cells (PBMC). To this end, we evaluated the gene expression profile of 19 genes (AKT2, CCND1, GSK3ALPHA, IGF1, GSK3BETA, FOXO3, IL6, IGFBP2, UGT2B17, ARA55, CREBBP, CYP11A, HSD17B1, HSD17B7, UGT2B7, SELADIN 1, CLU, PGC1, AKR1C1) selected according their function in the androgen pathways, in a series of 11 hypogonadal men pharmacologically treated with T. We noted that 7 genes were differentially expressed, five of them were up-regulated (AKT2 FC=2.39, CREBBP FC=11.2, GSK3beta FC=5.6, UGT2B7 FC=4.49, UGT2B17 FC=2.88) and two were down-regulated (ARA55 FC= -2.0, CYP11A FC= -2.47). This experience suggests that androgen- and insulin-related genes can be considered useful blood genomic biomarkers for specific steroid drugs.

Androgen- and insulin-related gene signature using a specific low density oligoarray AndroChip 2 in peripheral blood mononuclear cells in agonists, recreational athletes and sedentary subjects.

The early detection of genomic biomarkers (e.g. RNAs) through analysis of circulating blood cells could have a substantial impact on biomedicine, particularly in monitoring clinical trials, drug toxicity and doping in athletes. To achieve this goal, it is essential to develop methods that are sufficiently sensitive to detect biomarker alterations during normal biologic processes, pathogenic processes, and or in response to therapeutic or other intervention. Using a low density microarray (AndroChip 2) we detected a transcriptional profiling signature of 190 genes related to androgen and insulin metabolism pathway, in peripheral blood mononuclear cell (PBMC) in subjects with different intensities of sports activities. We demonstrated that androgen and insulin gene transcriptional levels are independent to sports activity and therefore potentially suitable for drug monitoring and/or drug doping (such as anabolic androgen steroid AAS abuse) and or gene doping.

In silico pharmacology for drug discovery: applications to targets and beyond.

Computational (in silico) methods have been developed and widely applied to pharmacology hypothesis development and testing. These in silico methods include databases, quantitative structure-activity relationships, similarity searching, pharmacophores, homology models and other molecular modeling, machine learning, data mining, network analysis tools and data analysis tools that use a computer. Such methods have seen frequent use in the discovery and optimization of novel molecules with affinity to a target, the clarification of absorption, distribution, metabolism, excretion and toxicity properties as well as physicochemical characterization. The first part of this review discussed the methods that have been used for virtual ligand and target-based screening and profiling to predict biological activity. The aim of this second part of the review is to illustrate some of the varied applications of in silico methods for pharmacology in terms of the targets addressed. We will also discuss some of the advantages and disadvantages of in silico methods with respect to in vitro and in vivo methods for pharmacology research. Our conclusion is that the in silico pharmacology paradigm is ongoing and presents a rich array of opportunities that will assist in expediting the discovery of new targets, and ultimately lead to compounds with predicted biological activity for these novel targets.

In silico pharmacology for drug discovery: methods for virtual ligand screening and profiling.

Pharmacology over the past 100 years has had a rich tradition of scientists with the ability to form qualitative or semi-quantitative relations between molecular structure and activity in cerebro. To test these hypotheses they have consistently used traditional pharmacology tools such as in vivo and in vitro models. Increasingly over the last decade however we have seen that computational (in silico) methods have been developed and applied to pharmacology hypothesis development and testing. These in silico methods include databases, quantitative structure-activity relationships, pharmacophores, homology models and other molecular modeling approaches, machine learning, data mining, network analysis tools and data analysis tools that use a computer. In silico methods are primarily used alongside the generation of in vitro data both to create the model and to test it. Such models have seen frequent use in the discovery and optimization of novel molecules with affinity to a target, the clarification of absorption, distribution, metabolism, excretion and toxicity properties as well as physicochemical characterization. The aim of this review is to illustrate some of the in silico methods for pharmacology that are used in drug discovery. Further applications of these methods to specific targets and their limitations will be discussed in the second accompanying part of this review.

Outcome assessment in patients with chronic obstructive rhinitis CO2 laser treated.

Surgical lasers have been used to restore nasal flow in chronic obstructive rhinitis, with a significant improvement in symptoms having been reported in almost all cases. However, evidence supporting the efficacy at long-term, and studies on the assessment of quality of life remain limited. In the present study, efficacy at long term and improvement in the quality of life were assessed in patients with chronic obstructive rhinitis, treated with CO2 laser. A total of 308 patients with chronic obstructive rhinitis were enrolled. The primary outcome measure assessed was the change in score regarding specific and general symptoms, between baseline to 2-4.5 and 7.8 mean years follow-up. Laser turbinotomy restored nasal flow and induced a change in total score which was statistically significant, for specific and general symptoms at the first, second and third follow-up, p < 0.01. CO2 laser turbinate surgery improved symptoms and quality of life in patients with chronic obstructive rhinitis as observed at 2-4.5 and 7.8 mean years follow-up.

Signs of the times: the need for a stereochemically informative generic name system.

'Research and clinical pharmacologists frequently present data on impure drugs.' Because generic drug names often hide the fact that different stereoisomers (possibly with different pharmacological properties) may be present in the 'pure' preparation, this statement is all too frequently true. However, the problem may be overcome by pharmacologists and publishers adopting the user-friendly SIGNS nomenclature devised and explained here by Miklòs Simonyi, Joseph Gal and Bernard Testa. The acronym stands for 'stereochemically informative generic name system'. Seven prefixes are offered to describe the stereochemical nature of any drug. The appropriate prefix would be attached to the generic name. A generic name without prefix would indicate a single agent with no stereoisomers.

Structure-property relationships of trimetazidine derivatives and model compounds as potential antioxidants.

Twenty-five compounds (trimetazidine derivatives and other compounds, mostly having a free phenolic group) were examined for their radical scavenging and antioxidant properties. Their reaction with DPPH (2,2-diphenyl-1-picrylhydrazyl) as a measure of radical scavenging capacity was assessed by two parameters, namely EC50 (the concentration of antioxidant decreasing DPPH by 50%), and log Z, a kinetic parameter proposed here and derived from initial second-order rate constants and antioxidant/DPPH ratios. Antioxidant activities were determined by the inhibition of lipid peroxidation and albumin oxidation. The most active compounds were derivatives having a trolox or hydroquinone moiety. Physicochemical and structural properties were determined by molecular modeling as lipophilicity (virtual log P calculations) and H-Surf (solvent-accessible surface of hydroxyl hydrogen) and by quantum mechanical calculations (deltaH(ox) = oxidation enthalpy; deltaH(abs) = enthalpy of hydrogen abstraction). QSAR models were derived to identify molecular mechanisms responsible for the reactivity toward the DPPH radical and for the inhibition of lipid peroxidation. A useful prediction of antioxidant capacity could be achieved from calculated molecular properties and the kinetic parameter developed here.

Behavioural and pharmacokinetic studies on nicotine, cytisine and lobeline.

Previous work has suggested that cytisine and lobeline are of low potency in producing nicotine-like behavioural effects, despite having some nicotine-like peripheral effects and potently inhibiting the binding of tritiated nicotine to the brain of the rat. Rats were trained to discriminate nicotine from saline in a two-bar operant conditioning procedure with food reinforcement. It was confirmed that cytisine had a nicotine-like discriminative effect, but it was much less potent than nicotine itself. Lobeline failed to produce a nicotine-like discriminative effect, even at doses that greatly reduced overall rates of responding. Neither drug attenuated discriminative responses to nicotine. The concentrations of drugs in plasma and brain were determined by HPLC in rats of the same sex, strain and age as those used in the behavioural experiments. The rank order of the ratios of concentrations in brain to plasma was lobeline greater than nicotine greater than cytisine, which was directly proportional to their lipophilicity determined by reversed-phase HPLC. Based on the concentrations in brain and known affinities for high-affinity nicotine binding sites, in vivo tests should show cytisine to be slightly more potent than nicotine and lobeline to have nicotine effects in the doses used. These predictions were not fulfilled and thus, the behavioural effects of cytisine and lobeline cannot be correlated with their effects at the binding site for tritiated nicotine. Since pharmacokinetic factors do not account for this discrepancy, a pharmacodynamic explanation will be necessary.

An ab initio study of electronic factors in metabolic hydroxylation of aliphatic carbon atoms.

The monooxygenase-mediated hydroxylations of aliphatic carbon atoms are known to be regioselective for positions alpha to heteroatoms or to pi systems (aromatic rings, carbon-carbon double bonds, carbonyl groups). Ab initio calculations (STO-3G and in some cases 4-31G) were performed on model molecules, indicating that the Mulliken overlap populations (taken as indices of electron bond densities) of Calpha-H bonds being regioselectively hydroxylated are larger than Cbeta-H and Cgamma-H overlap populations. These results support the hypothesis that metabolic C-hydroxylations occur by insertion of an activated oxygen species of electrophilic nature, probably oxene.

Theoretical conformational studies on some dopamine antagonistic benzamide drugs: 3-pyrrolidyl- and 4-piperidyl derivatives.

Model derivatives of 3-pyrrolidyl- and 4-piperidyl-o-methoxybenzamides, as representatives of neuroleptic substituted benzamide drugs, have been investigated by theoretical conformational analysis. Folded conformers of 2-methoxy-N-(1-methyl-3-pyrrolidyl)benzamide have the lowest energy, but extended conformers are only a few kilocalories per mole less stable. As regards to piperidyl derivative, it has been found that folded conformers are of much higher energy than extended ones. These and previous results are discussed in terms of the pharmacologically active conformers of substituted benzamide drugs and of possible modes of interaction with the dopamine receptor.

Predicting blood-brain barrier permeation from three-dimensional molecular structure.

Predicting blood-brain barrier (BBB) permeation remains a challenge in drug design. Since it is impossible to determine experimentally the BBB partitioning of large numbers of preclinical candidates, alternative evaluation methods based on computerized models are desirable. The present study was conducted to demonstrate the value of descriptors derived from 3D molecular fields in estimating the BBB permeation of a large set of compounds and to produce a simple mathematical model suitable for external prediction. The method used (VolSurf) transforms 3D fields into descriptors and correlates them to the experimental permeation by a discriminant partial least squares procedure. The model obtained here correctly predicts more than 90% of the BBB permeation data. By quantifying the favorable and unfavorable contributions of physicochemical and structural properties, it also offers valuable insights for drug design, pharmacological profiling, and screening. The computational procedure is fully automated and quite fast. The method thus appears as a valuable new tool in virtual screening where selection or prioritization of candidates is required from large collections of compounds.

Binding of arylpiperazines, (aryloxy)propanolamines, and tetrahydropyridylindoles to the 5-HT1A receptor: contribution of the molecular lipophilicity potential to three-dimensional quantitative structure-affinity relationship models.

A set of 280 5-HT1A receptor ligands were selected from available literature data according to predefined criteria and subjected to three-dimensional quantitative structure-affinity relationship analysis using comparative molecular field analysis. No model was obtained for serotonin analogues (19 compounds) and aminotetralins (60 compounds), despite a variety of alignment hypotheses being tried. In contrast, the steric, electrostatic, and lipophilicity fields alone and in combination yielded informative models for arylpiperazines (101 training compounds and 12 test compounds), (aryloxy)propanolamines (30 training compounds and four test compounds), and tetrahydropyridylindoles (54 training compounds) taken separately (models A, B, and C). Arylpiperazines and (aryloxy)propanolamines were then combined successfully to yield reasonably good models for 131 compounds (model D). In a last step, the three chemical classes (185 compounds) were combined, again successfully (model E). This stepwise procedure not only ascertains self-consistency in alignments but it also allows statistical signals (i.e., favorable or unfavorable regions around molecules) to emerge which cannot exist in a single chemical class. The models so obtained reveal a number of interaction sites between ligands and the 5-HT1A receptor, and extend the information gathered from a model based on homology modeling.

Molecular electrostatic potential of orthopramides: implications for their interaction with the D-2 dopamine receptor.

The electronic properties of orthopramides, a group of selective D-2 dopamine receptor antagonists, were investigated by calculating molecular electrostatic potentials (MEP) of model compounds with the ab initio STO-3G MO method. The various substitution patterns of the aromatic ring are characterized by a positive region comprising the H-bonded 2-methoxy group and ring positions 2, 3, and 4 and a negative region comprising the CONH group, 5-substituent, and ring positions 5 and 6. The regions of positive and negative potential are separated by a "curtain" running along the longitudinal axis of the molecule. At shorter distances from the plane of the aromatic ring (1.75 and 2.0 A), this "curtain" is quite sinuous, but at greater distances (2.5 and 3.0 A) it tends toward rectilinearity. We postulate that this longitudinal separation, together with the single positive maximum and the three negative minima perceptible at 3.0 A, constitute a distance pharmacophore responsible for the recognition and proper alignment of the ligand. The more complex MEP at 1.75 and 2.0 A are equated with a contact pharmacophore. Comparison of the MEP of orthopramides and dopamine reveals some analogies and suggests a possible mode of binding of these antagonists to the D-2 receptor.

Modeling of beta-adrenoceptors based on molecular electrostatic potential studies of agonists and antagonists.

The molecular electrostatic potential (MEP) of 32 beta-adrenoceptor ligands, mainly antagonists, was calculated by the STO-3G ab initio quantum mechanical method. The MEP of phenylethanolamines (PEAs) features a negative minimum in the meta region (designated M1) which is topographically equivalent to a minimum (designated M2) found in the vicinity of the aromatic ring in all (aryloxy)propanolamines (AOPAs). In these compounds, a second negative zone located beyond the meta position and designated M3 is found in all beta 1-selective antagonists and in some nonselective and beta 2-selective antagonists. The beta 1-selective antagonists feature in the para position an additional zone which is positive (P4) in the full antagonists and negative (M4) in the antagonists displaying intrinsic sympathomimetic activity (ISA). The MEP-based pharmacophoric models of PEAs, AOPAs, and oxime ethers show common elements and lead to a proposed general model for beta-adrenoceptor ligands.

Quantitative structure-activity relationships and eudismic analyses of the presynaptic dopaminergic activity and dopamine D2 and sigma receptor affinities of 3-(3-hydroxyphenyl)piperidines and octahydrobenzo[f]quinolines.

Data from the preceding paper were examined by QSAR and eudismic analyses. A fair parabolic relationship was found between the lipophilicity (measured by a RP-HPLC method) and the sigma-receptor affinity of 3-(3-hydroxyphenyl)piperidines (3HPP derivatives) and octahydrobenzo[f]quinolines (OHBQ derivatives). As far as the dopamine D2 receptor is concerned, the trans-7-hydroxy-OHBQ derivatives show a 10-fold higher affinity than the eutomeric S enantiomers of 3HPP derivatives, once lipophilicity has been accounted for. This difference in affinity is suggested to correspond to the energy necessary for the 3HPP derivatives to adopt the receptor-bound conformation. The R enantiomers of 3HPP derivatives display no apparent increase in D2 affinity with increasing lipophilicity, and indeed the eudismic index in this series increases with affinity (eudismic affinity quotient = 0.70), in agreement with a recent model of the binding of N-propyl-3HPP (3PPP) enantiomers to the D2 receptor. The selectivity in sigma/D2 affinities was found to depend on both lipophilicity and configuration of the ligands; thus, the selectivity is maximal for log kw values of ca. 1.7-2.1 and is much larger for the R than for the S enantiomers of 3HPP derivatives.

Morphine 6-glucuronide and morphine 3-glucuronide as molecular chameleons with unexpected lipophilicity.

Morphine 6-glucuronide, but not morphine 3-glucuronide, is a highly potent opiate receptor agonist. In fact, there is converging evidence that much of the analgesic effect occurring after morphine treatment in humans is due to this metabolite rather than to the parent drug. Yet glucuronides as a rule are considered as highly polar metabolites unable to cross the blood-brain barrier and rapidly excreted by the urinary and/or biliary routes. Here, we report that morphine 6-glucuronide, and to a lesser extent morphine 3-glucuronide, are far more lipophilic than predicted, and in fact not much less lipophilic than morphine itself. Force-field and quantum mechanical calculations indicate that the two glucuronides can exist in conformational equilibrium between extended and folded forms. The extended conformers, because they efficiently expose their polar groups, must be highly hydrophilic forms predominating in polar media such as water; in contrast, the folded conformers mask part of their polar groups, thus being more lipophilic and likely to predominate in media of low polarity such as biological membranes.

Effects of solvation on the ionization and conformation of raclopride and other antidopaminergic 6-methoxysalicylamides: insight into the pharmacophore.

Previous work has shown that raclopride in water at neutral pH exists in a zwitterionic form, suggesting a stereoelectronic structure largely different from that of other benzamides. In the present study, the acid-base behavior of other 6-methoxysalicylamides is shown to be comparable to that of raclopride. An extensive investigation by high-temperature molecular dynamics gave insight into the conformational behavior of neutral and zwitterionic raclopride in vacuum and in water. Partitioning of raclopride and a more rigid analogue with characterization (by first-derivative UV spectroscopy) of the predominant forms in the organic phase indicated that only neutral, internally H-bonded forms partition into the organic solvent. Thus, the predominant forms of 6-methoxysalicylamides will be very different in the aqueous and organic phases. In the latter phase, and hence presumably also in the receptor phase, the drugs exist with a neutral, internally H-bonded phenolic group and are therefore stereoelectronically similar to other substituted benzamides.

Inhibition of monoamine oxidase-B by 5H-indeno[1,2-c]pyridazines: biological activities, quantitative structure-activity relationships (QSARs) and 3D-QSARs.

A large series (66 compounds) of indeno[1,2-c]pyridazin-5-ones (IPs) were synthesized and tested on their monoamine oxidase-A (MAO-A) and MAO-B inhibitory activity. All of the tested compounds acted preferentially on MAO-B displaying weak (nonmeasurable IC50 values) to high (submicromolar IC50 values) activities. The most active compound was p-CF3-3-phenyl-IP (IC50 = 90 nM). Multiple linear regression analysis of the substituted 3-phenyl-IPs yielded good statistical results (q2 = 0.74; r2 = 0.86) and showed the importance of lipophilic, electronic, and steric properties of the substituents in determining inhibitory potency. Various comparative molecular field analysis studies were performed with different alignments and including the molecular lipophilicity potential. This led to a model including the steric, electrostatic and lipophilicity fields and having a good predictive value (q2 = 0.75; r2 = 0.93).

X-ray crystal structure, partitioning behavior, and molecular modeling study of piracetam-type nootropics: insights into the pharmacophore.

To detect possible molecular determinants of amnesia-reverting activity, the conformational properties of a number of rigid and flexible piracetam-type cognition enhancers have been assessed by X-ray diffraction, NMR spectroscopy, and ab initio and high-temperature-quenched molecular dynamics (QMD) calculations. The structures of the preferred conformers in solution derived from 1H-NMR spectral analysis were in good agreement with those found by QMD calculations. Interestingly, the calculation of the average molecular lipophilicity potential on the water-accessible surface of the selected conformers was helpful in interpreting the partitioning behavior observed by measuring octanol-water partition coefficients and capacity factors in reversed-phase high-performance liquid chromatography. While lipophilicity does not play a relevant role, the distance between polar groups, accounted for by the distance between carbonyl oxygens, emerges as a factor, among others, which should influence the amnesia-reversal activity of piracetam-type nootropics.