Inhibition of D-Ala:D-Ala ligase through a phosphorylated form of the antibiotic D-cycloserine.

D-cycloserine is an antibiotic which targets sequential bacterial cell wall peptidoglycan biosynthesis enzymes: alanine racemase and D-alanine:D-alanine ligase. By a combination of structural, chemical and mechanistic studies here we show that the inhibition of D-alanine:D-alanine ligase by the antibiotic D-cycloserine proceeds via a distinct phosphorylated form of the drug. This mechanistic insight reveals a bimodal mechanism of action for a single antibiotic on different enzyme targets and has significance for the design of future inhibitor molecules based on this chemical structure.

Biological evaluation of diazene derivatives as anti-tubercular compounds.

Despite efforts made in chemotherapeutic research in the past and present, Mycobacterium tuberculosis (M.tb), the etiological agent of tuberculosis, still causes more than a million deadly casualties each year, second only to HIV. The rapid generation and spread of drug resistant strains, a problem exacerbated by co-infection with HIV demands further efforts in the investigation of novel classes of anti-tubercular compounds. A library of eight substituted diazenecarboxamides, three carbamoyldiazenecarboxylates and four diazene-1,2-dicarboxamides was synthesized in a straightforward manner followed by a biological evaluation of the compounds. We observed minimal inhibitory concentrations below 10 µg/mL against the H37Rv lab strain of M.tb. Three compounds that showed a potency of 90% growth inhibition of M.tb at a concentration lower than 10 µg/mL were further evaluated and showed potency against other clinically relevant mycobacterial species such as Mycobacterium bovis, Mycobacterium avium and Mycobacterium ulcerans. The selected compounds were examined for acute cell toxicity on a murine macrophage like monocyte cell line J774 A.1 in which the cell viability was reduced by 50% at concentrations ranging from 7.4 µg/mL to 20.7 µg/mL. Neither of the three compounds showed signs of genotoxicity by VITOTOX or by Comet assay. The study was complemented by demonstration of the inhibition of intracellular replication of M.tb H37Rv inside J774 A.1 cells at 2 µg/mL concentration and the susceptibility of a MDR LAM-1 strain at concentrations between 5 and 1 µg/mL of the most active compound.

Crystallization and preliminary X-ray analysis of a UDP-MurNAc-tripeptide D-alanyl-D-alanine-adding enzyme (PaMurF) from Pseudomonas aeruginosa.

The ATP-dependent UDP-MurNAc-tripeptide:D-Ala-D-Ala ligase MurF catalyses the last step in the cytoplasmic phase of peptidoglycan biosynthesis, which is critical in the formation of the bacterial cell wall and in the recycling of peptidoglycan intermediates. In this study, the crystallization of MurF from the Gram-negative pathogen Pseudomonas aeruginosa in the presence of its UDP-MurNAc-tripeptide substrate is reported. The crystals belonged to space group P212121, with unit-cell parameters a = 57.81, b = 87.29, c = 92.61 Å, and data were collected to 1.92 Šresolution, allowing study of the enzyme in the substrate-liganded form for the first time.

Synthesis and biological evaluation of 4-nitro-substituted 1,3-diaryltriazenes as a novel class of potent antitumor agents.

We describe the synthesis and biological activity of a new class of 1,3-diaryltriazenes, namely 4-nitro-substituted 1,3-diaryltriazenes. Structure-activity relationship analysis reveals that 1,3-diaryltriazenes can be modified from inactive to highly cytotoxic compounds by the introduction of two nitro groups at the para positions of benzene rings and two additional electron-withdrawing groups (bromo, chloro, trifluoromethyl or fluoro substituents) at their ortho position. In order to increase the solubility of the modified compounds, we introduced various acyl groups to their triazene nitrogen. The results of LC-MS/MS analysis showed that N-acyltriazenes can be considered as prodrugs of non-acylated triazenes. Selected 3-acetyl-1,3-bis(2-chloro-4-nitrophenyl)-1-triazene (8b) is highly cytotoxic against different tumor cell lines, including cisplatin-resistant laryngeal carcinoma cells. Notably, its antiproliferative activity is significantly higher against tumor cells than against normal cells. DNA binding analysis suggests that neither 8b nor its non-acylated derivative 8a bind into the minor groove of DNA. Instead, 8b induces reactive oxygen species that could provoke endoplasmic reticulum (ER(a)) stress finally leading to apoptosis. Our data suggest that 4-nitro-substituted 1,3-diaryltriazenes are a new class of anticancer molecules which preferentially target malignant cells and may serve as potential antitumor agents.

Design and synthesis of new hydroxyethylamines as inhibitors of D-alanyl-D-lactate ligase (VanA) and D-alanyl-D-alanine ligase (DdlB).

The Van enzymes are ATP-dependant ligases responsible for resistance to vancomycin in Staphylococcus aureus and Enteroccoccus species. The de novo molecular design programme SPROUT was used in conjunction with the X-ray crystal structure of Enterococcus faeciumd-alanyl-d-lactate ligase (VanA) to design new putative inhibitors based on a hydroxyethylamine template. The two best ranked structures were selected and efficient syntheses developed. The inhibitory activities of these molecules were determined on E. faecium VanA, and due to structural similarity and a common reaction mechanism, also on d-Ala-d-Ala ligase (DdlB) from Escherichia coli. The phosphate group attached to the hydroxyl moiety of the hydroxyethylamine isostere within these systems is essential for their inhibitory activity against both VanA and DdlB.

Diazenedicarboxamides as inhibitors of D-alanine-D-alanine ligase (Ddl).

D-Alanine-D-alanine ligase (Ddl) catalyzes the biosynthesis of an essential bacterial peptidoglycan precursor D-alanyl-D-alanine and it represents an important target for development of new antibacterial drugs. A series of semicarbazides, aminocarbonyldiazenecarboxylates, diazenedicarboxamides, and hydrazinedicarboxamides was synthesized and screened for inhibition of DdlB from Escherichia coli. Compounds with good inhibitory activity were identified, enabling us to deduce initial structure-activity relationships. Thirteen diazenedicarboxamides were better inhibitors than D-cycloserine and some of them also possess antibacterial activity, which makes them a promising starting point for further development.