Synthesis of terpenoid oxo derivatives with antiureolytic activity.

Urease is an important virulence factor for a variety of pathogenic bacteria strains such as Helicobacter pylori, which colonizes human gastric mucosa, and Proteus sp., responsible for urinary tract infections. Specific inhibition of urease activity could be a promising adjuvant strategy for eradication of these pathogens. Due to the interesting antiureolytic activity of carvone and the scant information regarding the inhibitory properties of corresponding monoterpenes, we decided to study selected monoterpenic ketones and their oxygen derivatives. Several monoterpenes and their terpenoid oxygen derivatives were evaluated in vitro against Sporosarcina pasteurii urease. The most effective inhibitors-derivatives of ß-cyclocitral (ester 10 and bromolactone 14)-were described with [Formula: see text] of 46.7 µM and 45.8 µM, respectively. Active inhibitors of native urease were tested against H. pylori and Proteus mirabilis whole cells. Here, the most active inhibitor, 14, was characterized with IC50 values of 0.32 mM and 0.61 mM for P. mirabilis and H. pylori, respectively. The antibacterial activity of a few tested inhibitors was also observed. Compound 14 limited the growth of E. coli ([Formula: see text]= 250 µg/mL). Interestingly, 10 was the only compound that was effective against both Gram-negative and Gram-positive bacteria. It had a [Formula: see text] of 150 µg/mL against E. coli and S. aureus. In the presented study a group of novel antiureolytic compounds was characterised. Besides carvone stereoisomers, these are the only terpenoid urease inhibitors described so far.

Novel O-alkyl Derivatives of Naringenin and Their Oximes with Antimicrobial and Anticancer Activity.

In our investigation, we concentrated on naringenin (NG)-a widely studied flavanone that occurs in citrus fruits. As a result of a reaction with a range of alkyl iodides, 7 novel O-alkyl derivatives of naringenin (7a⁻11a, 13a, 17a) were obtained. Another chemical modification led to 9 oximes of O-alkyl naringenin derivatives (7b⁻13b, 16b⁻17b) that were never described before. The obtained compounds were evaluated for their potential antibacterial activity against Escherichia coli, Staphylococcus aureus, and Bacillus subtilis. The results were reported as the standard minimal inhibitory concentration (MIC) values and compared with naringenin and its known O-alkyl derivatives. Compounds 4a, 10a, 12a, 14a, 4b, 10b, 11b, and 14b were described with MIC of 25 µg/mL or lower. The strongest bacteriostatic activity was observed for 7-O-butylnaringenin (12a) against S. aureus (MIC = 6.25 µg/mL). Moreover, the antitumor effect of flavonoids was examined on human colon cancer cell line HT-29. Twenty-six compounds were characterized as possessing an antiproliferative activity stronger than that of naringenin. The replacement of the carbonyl group with an oxime moiety significantly increased the anticancer properties. The IC50 values below 5 µg/mL were demonstrated for four oxime derivatives (8b, 11b, 13b and 16b).

Inhibition of oxytocin and vasopressin neuron activity in rat hypothalamic paraventricular nucleus by relaxin-3-RXFP3 signalling.

KEY POINTS: Relaxin-3 is a stress-responsive neuropeptide that acts at its cognate receptor, RXFP3, to alter behaviours including feeding. In this study, we have demonstrated a direct, RXFP3-dependent, inhibitory action of relaxin-3 on oxytocin and vasopressin paraventricular nucleus (PVN) neuron electrical activity, a putative cellular mechanism of orexigenic actions of relaxin-3. We observed a Gαi/o -protein-dependent inhibitory influence of selective RXFP3 activation on PVN neuronal activity in vitro and demonstrated a direct action of RXFP3 activation on oxytocin and vasopressin PVN neurons, confirmed by their abundant expression of RXFP3 mRNA. Moreover, we demonstrated that RXFP3 activation induces a cadmium-sensitive outward current, which indicates the involvement of a characteristic magnocellular neuron outward potassium current. Furthermore, we identified an abundance of relaxin-3-immunoreactive axons/fibres originating from the nucleus incertus in close proximity to the PVN, but associated with sparse relaxin-3-containing fibres/terminals within the PVN. ABSTRACT: The paraventricular nucleus of the hypothalamus (PVN) plays an essential role in the control of food intake and energy expenditure by integrating multiple neural and humoral inputs. Recent studies have demonstrated that intracerebroventricular and intra-PVN injections of the neuropeptide relaxin-3 or selective relaxin-3 receptor (RXFP3) agonists produce robust feeding in satiated rats, but the cellular and molecular mechanisms of action associated with these orexigenic effects have not been identified. In the present studies, using rat brain slices, we demonstrated that relaxin-3, acting through its cognate G-protein-coupled receptor, RXFP3, hyperpolarized a majority of putative magnocellular PVN neurons (88%, 22/25), including cells producing the anorexigenic neuropeptides, oxytocin and vasopressin. Importantly, the action of relaxin-3 persisted in the presence of tetrodotoxin and glutamate/GABA receptor antagonists, indicating its direct action on PVN neurons. Similar inhibitory effects on PVN oxytocin and vasopressin neurons were produced by the RXFP3 agonist, RXFP3-A2 (82%, 80/98 cells). In situ hybridization histochemistry revealed a strong colocalization of RXFP3 mRNA with oxytocin and vasopressin immunoreactivity in rat PVN neurons. A smaller percentage of putative parvocellular PVN neurons was sensitive to RXFP3-A2 (40%, 16/40 cells). These data, along with a demonstration of abundant peri-PVN and sparse intra-PVN relaxin-3-immunoreactive nerve fibres, originating from the nucleus incertus, the major source of relaxin-3 neurons, identify a strong inhibitory influence of relaxin-3-RXFP3 signalling on the electrical activity of PVN oxytocin and vasopressin neurons, consistent with the orexigenic effect of RXFP3 activation observed in vivo.

Activity of fluconazole and its Cu(II) complex towards Candida species.

Candida species, although they are present as commensal organisms in the digestive tract of healthy individuals, can produce a broad spectrum of serious illnesses in compromised hosts. Fluconazole, a water-soluble triazole with bioavailability greater than 90 %, has been extensively used to treat a wide range of Candida infections. However, a growing resistance of microorganisms in the treatment leads to the discovery of new drugs or modifications of existing ones. The aim of the present study was to investigate whether coordination of Cu(II) ions to fluconazole affects its antifungal activity. The in vitro susceptibility tests and antifungal studies were performed with two Candida spp.: Candidaglabrata and Candida albicans. Overall, 34 strains of the former and 16 strains of the latter were treated with fluconazole, its Cu(II) complex and free Cu(II) ions. The obtained MIC values in 16 cases of the C. glabrata and in 5 cases of the C. albicans were lower for the complex in comparison to the drug. This implies that the complex is more effective against particular strains than the parent drug. The most significant improvement in the complex drug efficacy was observed for fluconazole-resistant species.

Inhibitory activity of catecholic phosphonic and phosphinic acids against Helicobacter pylori ureolysis.

Catechols have been reported to be potent covalent inhibitors of ureases, and they exhibit activity by modifying cysteine residues at the entrance to enzymatic active sites. Following these principles, we designed and synthesized novel catecholic derivatives that contained carboxylate and phosphonic/phosphinic functionalities and assumed expanded specific interactions. When studying the chemical stability of the molecules, we found that their intrinsic acidity catalyzes spontaneous esterification/hydrolysis reactions in methanol or water solutions, respectively. Regarding biological activity, the most promising compound, 2-(3,4-dihydroxyphenyl)-3-phosphonopropionic acid (15), exhibited significant anti-urease potential (Ki = 2.36 µM, Sporosarcinia pasteurii urease), which was reflected in the antiureolytic effect in live Helicobacter pylori cells at a submicromolar concentration (IC50 = 0.75 µM). As illustrated by molecular modeling, this compound was bound in the active site of urease through a set of concerted electrostatic and hydrogen bond interactions. The antiureolytic activity of catecholic phosphonic acids could be specific because these compounds were chemically inert and not cytotoxic to eukaryotic cells.

Exploration of Thiourea-Based Scaffolds for the Construction of Bacterial Ureases Inhibitors.

A series of 32 thiourea-based urease inhibitors were synthesized and evaluated against native bacterial enzyme and whole cells of Sporosarcina pasteurii and Proteus mirabilis strains. The proposed inhibitors represented structurally diverse thiosemicarbazones and thiocarbohydrazones, benzyl-substituted thiazolyl thioureas, 1H-pyrazole-1-carbothioamides, and dihydropirimidine-2(1H)-thiones. Kinetic characteristics with purified S. pasteurii enzyme determined low micromolar inhibitors within each structural group. (E)-2-(1-Phenylethylidene)hydrazine-1-carbothioamide 19 (Ki = 0.39 ± 0.01 µM), (E)-2-(4-methylbenzylidene)hydrazine-1-carbothioamide 16 (Ki = 0.99 ± 0.04 µM), and N'-((1E,2E)-1,3-diphenylallylidene)hydrazinecarbothiohydrazide 29 (Ki = 2.23 ± 0.19 µM) were used in modeling studies that revealed sulfur ion coordination of the active site nickel ion and hydrogen bonds between the amide group and the side chain of Asp363 and Ala366 carbonyl moiety. Whole-cell studies proved the activity of compounds in Gram-positive and Gram-negative microorganisms. Ureolysis control observed in P. mirabilis PCM 543 (e.g., IC50 = 304 ± 14 µM for 1-benzyl-3-(4-(4-hydroxyphenyl)thiazol-2-yl)thiourea 52) is a valuable achievement, as urease is recognized as a major virulence factor of this urinary tract pathogen.

Optimized Ebselen-Based Inhibitors of Bacterial Ureases with Nontypical Mode of Action.

Screening of 25 analogs of Ebselen, diversified at the N-aromatic residue, led to the identification of the most potent inhibitors of Sporosarcina pasteurii urease reported to date. The presence of a dihalogenated phenyl ring caused exceptional activity of these 1,2-benzisoselenazol-3(2H)-ones, with Ki value in a low picomolar range (<20 pM). The affinity was attributed to the increased π-π and π-cation interactions of the dihalogenated phenyl ring with αHis323 and αArg339 during the initial step of binding. Complementary biological studies with selected compounds on the inhibition of ureolysis in whole Proteus mirabilis cells showed a very good potency (IC50 < 25 nM in phosphate-buffered saline (PBS) buffer and IC90 < 50 nM in a urine model) for monosubstituted N-phenyl derivatives. The crystal structure of S. pasteurii urease inhibited by one of the most active analogs revealed the recurrent selenation of the Cys322 thiolate, yielding an unprecedented Cys322-S-Se-Se chemical moiety.

N-substituted aminomethanephosphonic and aminomethane-P-methylphosphinic acids as inhibitors of ureases.

Small unextended molecules based on the diamidophosphate structure with a covalent carbon-to-phosphorus bond to improve hydrolytic stability were developed as a novel group of inhibitors to control microbial urea decomposition. Applying a structure-based inhibitor design approach using available crystal structures of bacterial urease, N-substituted derivatives of aminomethylphosphonic and P-methyl-aminomethylphosphinic acids were designed and synthesized. In inhibition studies using urease from Bacillus pasteurii and Canavalia ensiformis, the N,N-dimethyl derivatives of both lead structures were most effective with dissociation constants in the low micromolar range (Ki=13±0.8 and 0.62±0.09 µM, respectively). Whole-cell studies on a ureolytic strain of Proteus mirabilis showed the high efficiency of N,N-dimethyl and N-methyl derivatives of aminomethane-P-methylphosphinic acids for urease inhibition in pathogenic bacteria. The high hydrolytic stability of selected inhibitors was confirmed over a period of 30 days using NMR technique.

Covalent Inhibition of Bacterial Urease by Bifunctional Catechol-Based Phosphonates and Phosphinates.

In this study, a new class of bifunctional inhibitors of bacterial ureases, important molecular targets for antimicrobial therapies, was developed. The structures of the inhibitors consist of a combination of a phosphonate or (2-carboxyethyl)phosphinate functionality with a catechol-based fragment, which are designed for complexation of the catalytic nickel ions and covalent bonding with the thiol group of Cys322, respectively. Compounds with three types of frameworks, including ß-3,4-dihydroxyphenyl-, α-3,4-dihydroxybenzyl-, and α-3,4-dihydroxybenzylidene-substituted derivatives, exhibited complex and varying structure-dependent kinetics of inhibition. Among irreversible binders, methyl ß-(3,4-dihydroxyphenyl)-ß-(2-carboxyethyl)phosphorylpropionate was observed to be a remarkably reactive inhibitor of Sporosarcina pasteurii urease (kinact/KI = 10 420 s-1 M-1). The high potential of this group of compounds was also confirmed in Proteus mirabilis whole-cell-based inhibition assays. Some compounds followed slow-binding and reversible kinetics, e.g., methyl ß-(3,4-dihydroxyphenyl)-ß-phosphonopropionate, with Ki* = 0.13 µM, and an atypical low dissociation rate (residence time τ = 205 min).

Synthesis of nitrogen-containing monoterpenoids with antibacterial activity.

Incorporation of the Beckmann rearrangement into the presented research resulted in the formation of nitrogen-containing terpenoid derivatives originating from naturally occurring compounds. Both starting monoterpenes and obtained derivatives were subjected to estimation of their antibacterial potential. In the presented study, Staphylococcus aureus was the most sensitive to examined compounds. The Minimal Inhibitory Concentration (MIC) experiments performed on S. aureus demonstrated that the (-)-menthone oxime (-)-8 and (+)-pulegone oxime (+)-13 had the best antibacterial activity among the tested derivatives and starting compounds. Their MIC90 value was 100 µg/mL. The obtained derivatives were also evaluated for their inhibitory activity against bacterial urease. Among the tested compounds, three active inhibitors were found - oxime 14 and lactams (-)-15 and 16 limited the activity of Sporosarcina pasteurii urease with Ki values of 174.3 µM, 43.0 µM and 4.6 µM, respectively. To our knowledge, derivative 16 is the most active antiureolytic lactam described to date.

Current methodology of MTT assay in bacteria - A review.

The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium assay is a popular tool in estimating the metabolic activity of living cells. The test is based on enzymatic reduction of the lightly colored tetrazolium salt to its formazan of intense purple-blue color, which can be quantified spectrophotometrically. Under properly optimized conditions the obtained absorbance value is directly proportional to the number of living cells. Originally, the MTT assay was devised for use in eukaryotic cells lines and later applied for bacteria and fungi. As the mechanism of MTT reduction was studied in detail mostly considering eukaryotic cells, the lack of information resulted in generating a vast variety of MTT based protocols for bacterial enzymatic activity evaluation. In the presented article the main aspects of the MTT assay applicability in bacterial research were summarized, with special emphasis on sources of inaccuracies and misinterpretation of the test results.

Aminophosphinates against Helicobacter pylori ureolysis-Biochemical and whole-cell inhibition characteristics.

Urease is an important virulence factor from Helicobacter pylori that enables bacterial colonization of human gastric mucosa. Specific inhibition of urease activity can be regarded as a promising adjuvant strategy for eradication of this pathogen. A group of organophosphorus inhibitors of urease, namely, aminophosphinic acid and aminophosphonic acid derivatives, were evaluated in vitro against H. pylori urease. The kinetic characteristics of recombinant enzyme activity demonstrated a competitive reversible mode of inhibition with Ki values ranging from 0.294 to 878 µM. N-n-Hexylaminomethyl-P-aminomethylphosphinic acid and N-methylaminomethyl-P-hydroxymethylphosphinic acid were the most effective inhibitors (Ki = 0.294 µM and 1.032 µM, respectively, compared to Ki = 23 µM for the established urease inhibitor acetohydroxamic acid). The biological relevance of the inhibitors was verified in vitro against a ureolytically active Escherichia coli Rosetta host that expressed H. pylori urease and against a reference strain, H. pylori J99 (CagA+/VacA+). The majority of the studied compounds exhibited urease-inhibiting activity in these whole-cell systems. Bis(N-methylaminomethyl)phosphinic acid was found to be the most effective inhibitor in the susceptibility profile studies of H. pylori J99. The cytotoxicity of nine structurally varied inhibitors was evaluated against four normal human cell lines and was found to be negligible.

Whole-cell Proteus mirabilis urease inhibition by aminophosphinates for the control of struvite formation.

The study evaluated the in vitro impact of a series of aminophosphinic urease inhibitors on Proteusmirabilis. The group of compounds comprised structurally diverse analogues of diamidophosphate built on an N-C-P scaffold. The influence of urease inhibition on urea-splitting activity was assessed by whole-cell pH-static kinetic measurements. The potential to prevent struvite formation was determined by monitoring changes in pH and ionic composition of artificial urine medium during P. mirabilis growth. The most active compounds exhibited stronger positive effect on urine stability than the acknowledged inhibitor acetohydroxamic acid. The high anti-ureolytic and pH-stabilizing effect of urease inhibitors 4 and 14 was well correlated with their reported kinetic properties against pure urease from P. mirabilis (Ki values of 0.62±0.09 and 0.202±0.057 µM, respectively, compared to 5.7±0.4 µM for acetohydroxamic acid). The effect of repressed ureolysis upon the viability of Proteus cells was studied using MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] metabolic efficiency assay and LIVE/DEAD fluorescent staining. Most of the compounds caused whole-cell dehydrogenase activity loss; four structures (1, 2, 4 and 14) reduced the culture viability by nearly 70 % at 1 mM concentration. Results of dual fluorescent staining suggested that besides urea-splitting prevention, the structures additionally exerted an outer-membrane-destabilizing effect.

1,2-Benzisoselenazol-3(2H)-one Derivatives As a New Class of Bacterial Urease Inhibitors.

Urease inhibitors are considered promising compounds for the treatment of ureolytic bacterial infections, particularly infections resulting from Helicobacter pylori in the gastric tract. Herein, we present the synthesis and the inhibitory activity of novel and highly effective organoselenium compounds as inhibitors of Sporosarcina pasteurii and Helicobacter pylori ureases. These studied compounds represent a class of competitive reversible urease inhibitors. The most active compound, 2-phenyl-1,2-benzisoselenazol-3(2H)-one (ebselen), displayed Ki values equal to 2.11 and 226 nM against S. pasteurii and H. pylori enzymes, respectively, indicating ebselen as one of the most potent low-molecular-weight inhibitors of bacterial ureases reported to date. Most of these molecules penetrated through the cell membrane of the Gram-negative bacteria Escherichia coli (pGEM::ureOP) in vitro. Furthermore, whole-cell studies on the H. pylori J99 reference strain confirmed the high efficiency of the examined organoselenium compounds as urease inhibitors against pathogenic bacteria.

Interferences in the Optimization of the MTT Assay for Viability Estimation of Proteus mirabilis.

BACKGROUND: The chromogenic assay based on MTT bioreduction was adapted to Proteus mirabilis viability estimations. We primarily intended to use the assay for the evaluation of novel antimicrobial compounds, including structures with possible permeabilizing activity. Therefore, the influence of basic permeabilizing agents like Triton X-100 and EDTA upon the MTT assay was studied. METHODS: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was used as a substrate for the whole-cell dehydrogenase activity estimations. The amount of formazan product was evaluated in the end-point reactions terminated with acidic isopropanol or in the continuous reactions run in the presence of low detergent concentrations. RESULTS: The generally established procedure of the end product dissolution with acidic isopropanol caused absorbance instability which strongly affected the results accuracy. The disadvantage was especially pronounced when the assay was conducted in Mueller-Hinton Broth. PBS with 0.01% Triton X-100 used as the reaction medium allowed to omit the formazan dissolution step and follow the microbial MTT reduction in a continuous mode. It was observed that in Proteus mirabilis with a compromised outer membrane the assay score was artificially increased above the untreated control. CONCLUSION: The dependence of the assay results on the cell integrity might be a major drawback of the MTT assay application for the evaluation of novel antimicrobials against Gram-negative microorganisms. On the other hand, the MTT reduction could be conveniently used to assay the permeabilization degree in biotechnological protocols.

Excitatory orexinergic innervation of rat nucleus incertus--Implications for ascending arousal, motivation and feeding control.

Orexin/hypocretin peptides play a central role in the integrated control of feeding/reward and behavioural activation, principally via interactions with other neural systems. A brainstem area involved in behavioural activation is the nucleus incertus (NI), located in the posterior ventromedial central grey. Several studies have implicated NI in control of arousal/stress and reward/feeding responses. Orexin receptor mRNA expression identifies NI as a putative target of orexin modulation. Therefore, in this study we performed neural tract-tracing and immunofluorescence staining to characterise the orexinergic innervation of NI. Our results indicate a convergent innervation of the NI area by different orexin neuron populations, with an abundance of orexin-A-containing axons making putative synaptic contacts with relaxin-3-positive NI neurons. The influence of orexin-A on NI neuron activity was investigated using patch-clamp recordings. Orexin-A depolarised the majority (64%) of recorded neurons and this effect was maintained in the presence of tetrodotoxin and glutamate and GABA receptor antagonists, indicating a likely postsynaptic action. Voltage-clamp experiments revealed that in 'type I' NI neurons comprising relaxin-3-positive cells, orexin-A acted via L-type calcium channels, whereas in 'type II' relaxin-3-negative neurons, activation of a sodium/calcium exchanger was involved. A majority of the orexin-A sensitive neurons tested for the presence of orexin receptor mRNA, were OX2 mRNA-positive. Immunohistochemical staining for putative orexin receptors on NI neurons, confirmed stronger expression of OX2 than OX1 receptors. Our data demonstrate a strong influence of orexin-A on NI neurons, consistent with an important role for this hypothalamic/tegmental circuit in the regulation of arousal/vigilance and motivated behaviours.

Computer-aided optimization of phosphinic inhibitors of bacterial ureases.

Urease inhibitors can be considered as a tool to control the damaging effect of ureolytic bacteria infections in humans which occur commonly in the developed countries. Computer-aided optimization of the aminomethylphosphinate structures by modifying both their N- and P-termini led to the invention of a novel group of inhibitors of bacterial ureases. Introduction of P-hydroxymethyl group into the molecule resulted in considerable increase of the inhibitory activity against enzymes purified from Bacillus pasteurii and Proteus vulgaris as compared with their P-methyl counterparts described previously. The designed compounds represent a competitive reversible class of urease inhibitors. The most potent, N-methyl-aminomethyl-P-hydroxymethylphosphinic acid, displayed K(i) = 360 nM against P. vulgaris enzyme.

Design, synthesis, and evaluation of novel organophosphorus inhibitors of bacterial ureases.

A new group of organophosphorus inhibitors of urease, P-methyl phosphinic acids was discovered by using the structure based inhibitor design approach. Several derivatives of the lead compound, aminomethyl(P-methyl)phosphinic acid, were synthesized successfully. Their potency was evaluated in vitro against urease from Bacillus pasteurii and Proteus vulgaris. The studied compounds constitute a group of competitive, reversible inhibitors of bacterial ureases. Obtained thiophosphinic analogues of the most effective structures exhibited kinetic characteristics of potent, slow binding urease inhibitors, with Ki = 170 nM (against B. pasteurii enzyme) for the most active N-( N'-benzyloxycarbonylglycyl)aminomethyl(P-methyl)phosphinothioic acid.