Agnieszka Grabowiecka

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.

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.

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.

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.