Systems Biology of Aromatic Compound Catabolism in Facultative Anaerobic Aromatoleum aromaticum EbN1T.

Members of the genus Aromatoleum thrive in diverse habitats and use a broad range of recalcitrant organic molecules coupled to denitrification or O2 respiration. To gain a holistic understanding of the model organism A. aromaticum EbN1T, we studied its catabolic network dynamics in response to 3-(4-hydroxyphenyl)propanoate, phenylalanine, 3-hydroxybenzoate, benzoate, and acetate utilized under nitrate-reducing versus oxic conditions. Integrated multi-omics (transcriptome, proteome, and metabolome) covered most of the catabolic network (199 genes) and allowed for the refining of knowledge of the degradation modules studied. Their substrate-dependent regulation showed differing degrees of specificity, ranging from high with 3-(4-hydroxyphenyl)propanoate to mostly relaxed with benzoate. For benzoate, the transcript and protein formation were essentially constitutive, contrasted by that of anoxia-specific versus oxia-specific metabolite profiles. The matrix factorization of transcriptomic data revealed that the anaerobic modules accounted for most of the variance across the degradation network. The respiration network appeared to be constitutive, both on the transcript and protein levels, except for nitrate reductase (with narGHI expression occurring only under nitrate-reducing conditions). The anoxia/nitrate-dependent transcription of denitrification genes is apparently controlled by three FNR-type regulators as well as by NarXL (all constitutively formed). The resequencing and functional reannotation of the genome fostered a genome-scale metabolic model, which is comprised of 655 enzyme-catalyzed reactions and 731 distinct metabolites. The model predictions for growth rates and biomass yields agreed well with experimental stoichiometric data, except for 3-(4-hydroxyphenyl)propanoate, with which 4-hydroxybenzoate was exported. Taken together, the combination of multi-omics, growth physiology, and a metabolic model advanced our knowledge of an environmentally relevant microorganism that differs significantly from other bacterial model strains. IMPORTANCE Aromatic compounds are abundant constituents not only of natural organic matter but also of bulk industrial chemicals and fuel components of environmental concern. Considering the widespread occurrence of redox gradients in the biosphere, facultative anaerobic degradation specialists can be assumed to play a prominent role in the natural mineralization of organic matter and in bioremediation at contaminated sites. Surprisingly, differential multi-omics profiling of the A. aromaticum EbN1T studied here revealed relaxed regulatory stringency across its four main physiological modi operandi (i.e., O2-independent and O2-dependent degradation reactions versus denitrification and O2 respiration). Combining multi-omics analyses with a genome-scale metabolic model aligned with measured growth performances establishes A. aromaticum EbN1T as a systems-biology model organism and provides unprecedented insights into how this bacterium functions on a holistic level. Moreover, this experimental platform invites future studies on eco-systems and synthetic biology of the environmentally relevant betaproteobacterial Aromatoleum/Azoarcus/Thauera cluster.

Evaluation of Homobivalent Carbolines as Designed Multiple Ligands for the Treatment of Neurodegenerative Disorders.

Neurodegenerative diseases represent a challenge for biomedical research due to their high prevalence and lack of mechanism-based treatments. Because of the complex pathology of neurodegenerative disorders, multifunctional drugs have been increasingly recognized as potential treatments. We identified homobivalent γ-carbolinium salts as potent inihitors of both cholinesterases, N-methyl-D-aspartate receptors, and monoamine oxidases. Homobivalent γ-carbolines displayed similar structure-activity relationships on all tested targets and may present promising designed multiple ligands for the treatment of neurodegenerative disorders.

Selective microemulsion liquid chromatography analysis of dopamine receptor antagonist LE300 and its N-methyl metabolite in mouse sera by using a monolithic silica column.

A highly selective, sensitive, and rapid microemulsion liquid chromatography (MELC) method was developed and validated for the simultaneous determination of a novel type of dopamine receptor antagonist LE300 and its N-methyl metabolite in mouse sera. LE300, its N-methyl metabolite, and pindolol (an internal standard) were detected using excitation and emission wavelengths of 275 and 340 nm, respectively. HPLC analysis by using a monolithic column was performed by directly injecting the sample after appropriate dilution with the microemulsion mobile phase. The chromatographic behaviour of these compounds was studied to demonstrate their chromatographic efficiency, retention, and peak symmetry. The MELC method was validated for its specificity, linearity, accuracy, precision, robustness and stability. An experimental design was used during validation to evaluate method robustness. The calibration curves in serum showed excellent linearity (r=0.997) over concentrations ranging from 10 to 400 ngmL(-1) for LE300 and 15 to 500 ngmL(-1) for its N-methyl metabolite. The mean relative standard deviation (RSD) of the results of inter- and intra-day precision and accuracy of LE300 and its N-methyl metabolite were ≤5%. The overall recoveries of LE300 and its N-methyl metabolite from mouse sera were in the range 97.9-101.5% with %RSD ranging from 0.98% to 3.63%, which were in line with ICH guidelines. The assay was successfully applied in a pharmacokinetic study.

Beta and gamma carboline derivatives as potential anti-Alzheimer agents: A comparison.

Nine novel ß- and γ-carboline derivatives bearing either methyl-, propargyl- or phenethyl-residues at the indole nitrogen were synthesized and tested as potential anti-Alzheimer drugs. Antagonism of recombinantly expressed NMDA receptors, inhibition of cholinesterases, and radical scavenging properties were determined for all compounds. Some were additionally tested in vivo for their ability to reverse scopolamine-induced cognitive impairment in an 8-arm radial maze experiment with rats. For the most promising candidates, the interaction with muscarinic M1 receptors was also investigated. With this set of compounds assays the influence of the scaffold itself and the substituents can be investigated separately. 5-Methyl-γ-carboline (6) was the most potent (0.25 µmol/100 g b.w.) compound in the in vivo test and might be a good starting point for the development of novel anti-Alzheimer drugs.

An interactive SAR approach to discover novel hybrid thieno probes as ligands for D2-like receptors with affinities in the subnanomolar range.

A series of [(phenylpiperazinyl)alkyl]-isoindole-1,3-dione derivatives was synthesized to serve as probes for dopaminergic receptors. Among this series, compound 6a showed the highest affinity towards D4 and D3 receptors with Ki values in the low nanomolar range, and D2/D4- and D2/D3-selectivity indices of 72 and 20, respectively. Optimization rounds were adopted and led to the D4-selective ligand thiophene-2-carboxamide 9a with a Ki (D4) value of 0.62 nM, and to its butyl analog, 10a, with Ki (D4) and Ki (D3) values of 0.03 and 0.26 nM, respectively. Docking experiments revealed the importance of the unique D4 residue Arg186 in manipulating the ligands' D4-subtype-receptor selectivity.

Tacrine-flurbiprofen hybrids as multifunctional drug candidates for the treatment of Alzheimer's disease.

Five tacrine-flurbiprofen hybrid compounds (3a-e) were synthesized as multi-target-directed compounds for the treatment of Alzheimer's disease. Compared to tacrine, two compounds (3d and 3e) showed better acetylcholinesterase (AChE) inhibitory activity and others (3b-e) better or the same butyrylcholinesterase (BuChE) inhibitory activity. Notably, 3d showed a mixed-type inhibitory action for AChE, indicating a "dual-binding site action" of both toward the catalytic active site (CAS) and the peripheral anionic site (PAS), whereas for BuChE, a competitive inhibitory action was observed. Furthermore, a cell-based assay on amyloid-ß inhibition demonstrated that the selected target compound 3d effectively inhibits the formation of amyloid-ß in vitro.

Discovery of highly potent and selective D4 ligands by interactive SAR study.

A series of thienylmethylphenylpiperazins was synthesized and tested for affinity towards the five subtypes of dopaminergic receptors. Compound 5f showed more than 1000 folds selectivity to D4 receptors; analogue 5e showed the highest affinity to D4 receptors with Ki 3.9 nM. An interactive SAR approach was adopted and lead to compound 14a with Ki (D4) as low as 0.03 nM. Molecular docking studies showed a potential, first to report arene cation interaction between the D4 unique residue Arg-186 and the ligands' arene moiety, explaining the importance of having a strong negative electrostatic potential at this area of the compound structure.

NO-donating tacrine derivatives as potential butyrylcholinesterase inhibitors with vasorelaxation activity.

To search for potent anti-Alzheimer's disease (AD) agents with multifunctional effects, 12 NO-donating tacrine-flurbiprofen hybrid compounds (2a-l) were synthesized and biologically evaluated. It was found that all the new target compounds showed selective butyrylcholinesterase (BuChE) inhibitory activity in vitro comparable or higher than tacrine and the tacrine-flurbiprofen hybrid compounds 1a-c, and released moderate amount of NO in vitro. The kinetic study suggests that one of the most active and highest BuChE selective compounds 2d may not only compete with the substrate for the same catalytic active site (CAS) but also interact with a second binding site. Furthermore, 2d and 2l exhibited significant vascular relaxation effect, which is beneficial for the treatment of AD. All the results suggest that 2d and 2l might be promising lead compounds for further research.

Design, synthesis and evaluation of tacrine-flurbiprofen-nitrate trihybrids as novel anti-Alzheimer's disease agents.

To search for multifunctional anti-Alzheimer's disease (AD) agents with good safety, the previously synthesized tacrine-flurbiprofen hybrids 1a and 1b were modified into tacrine-flurbiprofen-nitrate trihybrids 3a-h. These compounds displayed comparable or higher cholinesterase inhibitory activity relative to the bivalent hybrids. Compound 3a was the most potent, which released moderate NO, exerted blood vessel relaxative activity, and showed significant Aß inhibitory effects whereas tacrine and flurbiprofen did not exhibit any Aß inhibitory activity at the same dose. In addition, 3a was active in improving memory impairment in vivo. More importantly, the hepatotoxicity study showed that 3a was much safer than tacrine, suggesting it might be a promising anti-AD agent for further investigation.

Tacrine-ferulic acid-nitric oxide (NO) donor trihybrids as potent, multifunctional acetyl- and butyrylcholinesterase inhibitors.

In search of multifunctional cholinesterase inhibitors as potential anti-Alzheimer drug candidates, tacrine-ferulic acid-NO donor trihybrids were synthesized and tested for their cholinesterase inhibitory activities, release of nitric oxide, vasodilator properties, cognition improving potency, and hepatotoxicity. All of the novel target compounds show higher in vitro cholinesterase inhibitory activity than tacrine. Three selected compounds (3a, 3f, and 3k) produce moderate vasorelaxation in vitro, which correlates with the release of nitric oxide. Compared to its non-nitrate dihybrid analogue (3u), the trihybrid 3f exhibits better performance in improving the scopolamine-induced cognition impairment (mice) and, furthermore, less hepatotoxicity than tacrine.

Discovery of a novel acetylcholinesterase inhibitor by structure-based virtual screening techniques.

Acetylcholinesterase (AChE) is considered to be one of the most important targets for the treatment of Alzheimer's disease (AD). Previously our group has reported a series of tacrine-based hybrids as potent AChE inhibitors (AChEI). To discover more novel scaffolds, molecular docking and dynamics stimulation were applied to acquire the binding models of AChE with the most prominent compounds from our work. A structure-based pharmacophore model plus shape constraints was generated from the binding models and it was then employed to virtually screen commercial databases, giving a focused hit list of candidates. Subsequently, we scored the hit compounds by their molecular binding energies, which were calculated by MM/PBSA method. Fifteen compounds were selected and purchased for testing their anti-AChE effects, while seven of them showed inhibitory effects with IC(50) values ranging from 1.5 to 9.8 µM. The drug-like properties of these compounds, including LogD, AlogP, molecular volume and Lipinski rule of five, were also calculated. Compounds 12 and 16 (IC(50)=2.5 and 1.5 µM, respectively) exhibited potent activity and acceptable drug-like properties, thus might serve as leads for further modification. The data suggest that the presented model might be a valid approach for identification and development of new AChEIs.

Chiral indolo[3,2-f][3]benzazecine-type dopamine receptor antagonists: synthesis and activity of racemic and enantiopure derivatives.

Racemic and enantiopure 8-substituted derivatives of the lead dopamine receptor antagonist LE 300 (1) were prepared, and their affinities for the dopamine receptors (D(1)-D(5)) were tested. The separate enantiomers showed significantly different affinities; the (8S)-methyl and (8R)-hyroxymethyl derivatives where the substituents point below the reference plane of the indolo[3,2-f][3]benzazecine scaffold were markedly more active than their enantiomeric counterparts. The racemic 8-carboxy derivative was shown to be selective for the D(5)-receptor, even against D(1).

Interaction of ionic liquids ions with natural cyclodextrins.

The interaction of natural α-, ß-, and γ-cyclodextrins (CDs) with 14 hydrophobic ionic moieties of ionic liquids (ILs) was systematically examined in dilute aqueous solutions using isothermal titration microcalorimetry (ITC) and NMR spectroscopy. The studied cationic and anionic moieties involved some recently developed heavily fluorinated structures, as well as some others of common use. To isolate the effect of a given ion, the measurements were performed on salts containing the hydrophobic IL ion in question and a complexation-inactive counterion. Additional ITC experiments on ILs whose both cation and anion can interact appreciably with the CD cavity demonstrated that to resolve the effect of individual ions from such data is generally a tricky task and confirmed the superiority of the isolation strategy adopted for the purpose throughout this work. The binding constant, enthalpy and entropy determined at 298.15 K for the 1:1 (ion:CD) inclusion complex formation range in broad limits, being 0 < K < 2 × 10(5), 0 < -Δ(r)H°/(kJ·mol(-1)) < 44, and -28 < TΔ(r)S°/(kJ·mol(-1)) < 14, respectively. The stabilities of complexes of perfluorohexyl bearing ions with ß-CD belong to the highest ever observed with natural CDs in water. The established binding affinity scales were discussed in both thermodynamic and molecular terms. The concepts of hydrophobic interaction and guest-host size matching supported by simple molecular modeling proved useful to rationalize the observed widely different binding affinities and suggest possible binding modes. Enthalpy and entropy contributions to the stability of the ion-CD complexes were found to compensate each other considerably obeying more or less the linear compensation relationship marked by existing literature data on binding other guests to natural CDs. As outliers to this pattern, the most stable complexes of -C(6)F(13) bearing ions with ß-CD were found to receive an enhanced inherent entropy stabilization due to extraordinarily high extent of desolvation occurring in the course of binding.

The influence of hydrogen bonding on the physical properties of ionic liquids.

Potential applications of ionic liquids depend on the properties of this class of liquid material. To a large extent the structure and properties of these Coulomb systems are determined by the intermolecular interactions among anions and cations. In particular the subtle balance between Coulomb forces, hydrogen bonds and dispersion forces is of great importance for the understanding of ionic liquids. The purpose of the present paper is to answer three questions: Do hydrogen bonds exist in these Coulomb fluids? To what extent do hydrogen bonds contribute to the overall interaction between anions and cations? And finally, are hydrogen bonds important for the physical properties of ionic liquids? All these questions are addressed by using a suitable combination of experimental and theoretical methods including newly synthesized imidazolium-based ionic liquids, far infrared spectroscopy, terahertz spectroscopy, DFT calculations, differential scanning calorimetry (DSC), viscometry and quartz-crystal-microbalance measurements. The key statement is that although ionic liquids consist solely of anions and cations and Coulomb forces are the dominating interaction, local and directional interaction such as hydrogen bonding has significant influence on the structure and properties of ionic liquids. This is demonstrated for the case of melting points, viscosities and enthalpies of vaporization. As a consequence, a variety of important properties can be tuned towards a larger working temperature range, finally expanding the range of potential applications.

A novel non-phenolic dibenzazecine derivative with nanomolar affinities for dopamine receptors.

Dibenzazecines are a novel class of dopamine receptor antagonists, characterized by their high affinities as well as their tendency for D(1) selectivity. Hitherto, the most active dibenzazecines were phenolic in nature; a 3-OH substituent was found to result in the highest affinities. However, the phenolic nature of these compounds mostly renders them unsuitable for in vivo application, due to the poor pharmacokinetic profile, imparted by the phenolic group. A novel dibenzazecine derivative was prepared, with methylenedioxy moiety, connecting C(2) amd C(3), instead of the 3-OH group. The newly synthesized derivative 3 showed high affinities similar to the lead LE404, displaying nanomolar affinities for all dopamine receptor subtypes. Its dibrominated derivative 4, though exhibiting almost a fivefold decrease in affinities, still displayed nanomolar ones for all dopamine receptors, except for D(4) . In a functional Ca(2+) assay, both compounds 3 and 4 were found to possess antagonistic properties towards the dopamine receptors.

Effects of ginger constituents on the gastrointestinal tract: role of cholinergic M3 and serotonergic 5-HT3 and 5-HT4 receptors.

The herbal drug ginger (Zingiber officinale Roscoe) may be effective for treating nausea, vomiting, and gastric hypomotility. In these conditions, cholinergic M (3) receptors and serotonergic 5-HT (3) and 5-HT (4) receptors are involved. The major chemical constituents of ginger are [6]-gingerol, [8]-gingerol, [10]-gingerol, and [6]-shogaol. We studied the interaction of [6]-gingerol, [8]-gingerol, [10]-gingerol (racemates), and [6]-shogaol with guinea pig M (3) receptors, guinea pig 5-HT (3) receptors, and rat 5-HT (4) receptors. In whole segments of guinea pig ileum (bioassay for contractile M (3) receptors), [6]-gingerol, [8]-gingerol, [10]-gingerol, and [6]-shogaol slightly but significantly depressed the maximal carbachol response at an antagonist concentration of 10 µM. In the guinea pig myenteric plexus preparation (bioassay for contractile 5-HT (3) receptors), 5-HT maximal responses were depressed by [10]-gingerol from 93 ± 3 % to 65 ± 6 % at an antagonist concentration of 3 µM and to 48 ± 3 % at an antagonist concentration of 5 µM following desensitization of 5-HT (4) receptors and blockade of 5-HT (1) and 5-HT (2) receptors. [6]-Shogaol (3 µM) induced depression to 61 ± 3 %. In rat esophageal tunica muscularis mucosae (bioassay for relaxant 5-HT (4) receptors), [6]-gingerol, [8]-gingerol, [10]-gingerol, and [6]-shogaol (2-6.3 µM) showed no agonist effects. The maximal 5-HT response remained unaffected in the presence of the compounds. It is concluded that the efficiency of ginger in reducing nausea and vomiting may be based on a weak inhibitory effect of gingerols and shogaols at M (3) and 5-HT (3) receptors. 5-HT (4) receptors, which play a role in gastroduodenal motility, appear not to be involved in the action of these compounds.

Residues at the indole-NH of LE300 modulate affinities and selectivities for dopamine receptors.

To further investigate SAR in the class of azecine-type dopamine receptor antagonists, we synthesized a series of derivatives, substituted at the indole-NH of the lead compound LE300 by different alkyl chains in addition to phenylpropyl, allyl, propargyl, and acetyl residues. The affinities of the target compounds for all human dopamine receptors (D(1) -D(5) ) were investigated by radioligand binding assay and their functionality by a calcium assay. Both the affinities and selectivities for the dopamine receptors were found to be affected by the nature of the substituent. The N14-methylated derivative displayed the highest affinities for all D-receptors. In general, the affinities decreased with increasing chain length of the N-alkyl. Different substituents, partly led to altered affinity, and selectivity profile when compared with our lead LE300.

Synthesis, Molecular Modeling, and Biological Evaluation of Novel Tetrahydro-ß-Carboline Hydantoin and Tetrahydro-ß-Carboline Thiohydantoin Derivatives as Phosphodiesterase 5 Inhibitors.

Two series of fused tetrahydro-ß-carboline hydantoin and tetrahydro-ß-carboline thiohydantoin derivatives with a pendant 2,4-dimethoxyphenyl at position 5 were synthesized, and chiral carbons at positions 5 and 11a swing from R,R to R,S, S,R, and S,S. The prepared analogues were evaluated for their capacity to inhibit phosphodiesterase 5 (PDE5) isozyme. The R absolute configuration of C-5 in the ß-carboline hydantoin derivatives was found to be essential for the PDE5 inhibition. Chiral carbon derived from amino acid even if of the S configuration (L-tryptophan) may lead to equiactive or more active isomers than those derived from amino acid with the R configuration (D-tryptophan). This expands the horizon from which efficient PDE5 inhibitors can be derived and may offer an economic advantage. The thiohydantoin derivatives were less active than their hydantoin congeners.

Arylalkylamine-, beta-carboline-, quinolizine- and azecine-derived compounds and their in vitro interaction with the ionotropic 5-HT3 receptor: search for new lead structures.

Specific serotonin receptor agonists and antagonists are marketed with respect to various diseases, most prominently severe emesis. To identify new chemical classes with affinity for the serotonin 5-HT3 channel, several compounds were synthesized which can be structurally classified as arylalkylamines, azecines, quinolizines and beta-carbolines. These were tested in three models: (1) direct effect on ileum (overall model for contracting or relaxant effect), (2) antiserotoninergic effects on rat ileum (crude serotonin model), (3) inhibitory effect on the 5-HT, receptor channel complex expressed in N1E-115 cells (serotonin-induced [14C]guanidinium influx (specific model)). Key findings and conclusion: Several azecine-type compounds exhibit 5-HT3 receptor channel antagonistic properties at concentrations close to that of tropisetron (used as a positive control) and might serve as potential lead structures for the development of further 5-HT3 channel receptor antagonists.

A novel class of nitrovasodilators: potency and in vitro tolerance of organic aminoalkylnitrates.

Experimental studies on organic alkylnitrates have shown that both vasodilator potency and development of in vitro tolerance (tachyphylaxis) correlate with the number of nitrate groups in the molecule. However, introduction of an amino group to ethylnitrate yielded 2-nitrooxyethylammoniumnitrate (1), which is highly active but without inducing in vitro tolerance. Therefore, we prepared a series of aminoalkylnitrates and investigated vasorelaxation and tachyphylaxis on isolated prostaglandin F2alpha-precontracted porcine pulmonary arteries with intact endothelium. Tachyphylaxis was studied by incubating the arteries with EC100 of the respective aminoalkylnitrate and, after a 45-minute washout phase, measuring vasorelaxation again. All of the aminoalkylnitrates caused vasodilation, but the effect did not correlate with the number of nitrate moieties in the molecule. None of the substances was able to outperform compound 1, not even oligonitrates. Generally, structure­activity relationships found for alkylnitrates are obviously not valid for aminoalkylnitrates. Whereas the most potent aminomononitrate 1 evokes no in vitro tolerance, others exhibit tachyphylaxis independently of their vasodilator potency. We have shown that vasorelaxant activity and induction of tachyphylaxis are modulated significantly by introduction of an amino group to the alkylnitrate scaffold. Our results indicate that aminoalkylnitrates have to be considered as an individual class of nitrovasodilators with different structure­activity relationships and vasodilator properties.