Bis(aminomethyl)phosphinic Acid, a Highly Promising Scaffold for the Development of Bacterial Urease Inhibitors

Inhibitors of bacterial ureases are considered to be promising compounds in the treatment of infections caused by <i>Helicobacter pylori</i> in the gastric tract and/or by urealytic bacteria (e.g., <i>Proteus</i> species) in the urinary tract. A new, extended transition state scaffold, bis­(aminomethyl)­phosphinic acid, was successfully explored for the construction of effective enzyme inhibitors. A reliable methodology for the synthesis of phosphinate analogues in a three-component Mannich-type reaction was elaborated. The obtained molecules were assayed against ureases purified from <i>Sporosarcina pasteurii</i> and <i>Proteus mirabilis</i>, and aminomethyl­(<i>N</i>-<i>n</i>-hexylaminomethyl)­phosphinic acid was found to be the most potent inhibitor, with a <i>K</i>_i = 108 nM against the <i>S. pasteurii</i> enzyme

Bisphosphonic acids as effective inhibitors of <i>Mycobacterium tuberculosis</i> glutamine synthetase

<p>Inhibition of glutamine synthetase (GS) is one of the most promising strategies for the discovery of novel drugs against tuberculosis. Forty-three bisphosphonic and bis-<i>H</i>-phosphinic acids of various scaffolds, bearing aromatic substituents, were screened against recombinant GS from <i>Mycobacterium tuberculosis.</i> Most of the studied compounds exhibited activities in micromolar range, with <i>N</i>-(3,5-dichlorophenyl)-2-aminoethylidenebisphoshonic acid, <i>N</i>-(3,5-difluorophenyl)-2-aminoethylidene-bisphoshonic acid and <i>N</i>-(3,4-dichlorophenyl)-1-hydroxy-1,1-ethanebisphosphonic acid showing the highest potency with kinetic parameters similar to the reference compound – <i>L</i>-methionine-<i>S</i>-sulfoximine. Moreover, these inhibitors were found to be much more effective against pathogen enzyme than against the human ortholog. Thus, with the bone-targeting properties of the bisphosphonate compounds in mind, this activity/selectivity profile makes these compounds attractive agents for the treatment of bone tuberculosis.</p

1,2-Benzisoselenazol-3(2<i>H</i>)‑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(2<i>H</i>)-one (ebselen), displayed <i>K</i>_i 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 (<i>pGEM::ureOP</i>) 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

1,2-Benzisoselenazol-3(2<i>H</i>)‑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(2<i>H</i>)-one (ebselen), displayed <i>K</i>_i 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 (<i>pGEM::ureOP</i>) 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

Cytotoxicity of nine urease inhibitors expressed as inhibition of proliferation in four normal cell lines.

<p>Cell proliferation was evaluated using a sulforhodamine B assay against mouse fibroblasts BALB/3T3, murine mammary gland epithelium Eph4-Ev, immortalized murine endothelial cell line from peripheral lymph node HEC A10, and human mammary gland MCF-10A.</p

Structures and numbering of phosphinic and phosphonic compounds tested for inhibition of recombinant urease from <i>H</i>. <i>pylori</i>.

<p>Structures and numbering of phosphinic and phosphonic compounds tested for inhibition of recombinant urease from <i>H</i>. <i>pylori</i>.</p

The influence of IPTG and nickel ion concentration on the expression of recombinant <i>H</i>. <i>pylori</i> urease in <i>E</i>. <i>coli</i> Rosetta2 (DE3) strain.

<p>The influence of IPTG and nickel ion concentration on the expression of recombinant <i>H</i>. <i>pylori</i> urease in <i>E</i>. <i>coli</i> Rosetta2 (DE3) strain.</p

SDS-PAGE electrophorograms of native <i>H</i>. <i>pylori</i> J99 urease partially purified from the culture supernatant.

<p>Cells were grown for 72 h in the presence of 15 mM urea. M–molecular weight marker, SE–a soluble protein fraction before purification, PhS–fractions containing active urease after hydrophobic interaction purification using Phenyl Sepharose, CM–original culture medium (control). The red arrows indicate α and β subunits of urease with molecular weights of 61.7 and 29.5 kDa, respectively.</p

Inhibitory effects of compounds (■) 1, (●) 13, (▲) 21 and (▼) 24 towards urease in intact <i>H</i>. <i>pylori</i> J99 (♦).

<p>Cells grown in the presence of 15 mM urea assayed with 0.5 mM inhibitors without preincubation (A) and after 2 h of preincubation with 0.1 mM inhibitors in the growth medium (B); cells grown without urea assayed with 0.5 mM compounds without preincubation (C) and after 2 h of preincubation with 0.1 mM inhibitors in PBS (D).</p

The optimization of the induction conditions for the highly efficient expression of soluble protein.

<p><i>E</i>. <i>coli</i> Rosetta2 (DE3) that contained <i>pGEM</i>::<i>ureOP</i> was grown at 37°C. When the OD₆₀₀ reached 0.6, protein expression was induced under 96 different conditions. The cells were grown for an additional 18 h, harvested, and disrupted via sonication, and analyzed using SDS-PAGE electrophorograms. Lane M: protein molecular weight marker (kDa). Lane M: protein molecular weight marker (kDa). Cell-free extracts obtained from cultures carried out at different steps of optimization. Line 1: 750 μM Ni ²⁺; Line 2: 750 μM IPTG, and 750 μM Ni²⁺; Lane 3: induction temperature 22°C, 750 μM IPTG and 750 μM Ni²⁺; Line 4: time of incubation 24 h, 22°C, 750 μM IPTG and 750 μM Ni²⁺.</p