In vitro synergistic activity of NCL195 in combination with colistin against Gram-negative bacterial pathogens.

In this study, the potential of using the novel antibiotic NCL195 combined with subinhibitory concentrations of colistin against infections caused by Gram-negative bacteria (GNB) was investigated. We showed synergistic activity of the combination NCL195 + colistin against clinical multidrug-resistant GNB pathogens with minimum inhibitory concentrations (MICs) for NCL195 ranging from 0.5-4 µg/mL for Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, whereas NCL195 alone had no activity. Transmission electron microscopy of the membrane morphology of E. coli and P. aeruginosa after single colistin or combination drug treatment showed marked ultrastructural changes most frequently in the cell envelope. Exposure to NCL195 alone did not show any change compared with untreated control cells, whereas treatment with the NCL195 + colistin combination caused more damage than colistin alone. Direct evidence for this interaction was demonstrated by fluorescence-based membrane potential measurements. We conclude that the synergistic antimicrobial activity of the combination NCL195 + colistin against GNB pathogens warrants further exploration for specific treatment of acute GNB infections.

The antibiotic robenidine exhibits guanabenz-like cytoprotective properties by a mechanism independent of protein phosphatase PP1:PPP1R15A.

The aminoguanidine compound robenidine is widely used as an antibiotic for the control of coccidiosis, a protozoal infection in poultry and rabbits. Interestingly, robenidine is structurally similar to guanabenz (analogs), which are currently undergoing clinical trials as cytoprotective agents for the management of neurodegenerative diseases. Here we show that robenidine and guanabenz protect cells from a tunicamycin-induced unfolded protein response to a similar degree. Both compounds also reduced the tumor necrosis factor α-induced activation of NF-κB. The cytoprotective effects of guanabenz (analogs) have been explained previously by their ability to maintain eIF2α phosphorylation by allosterically inhibiting protein phosphatase PP1:PPP1R15A. However, using a novel split-luciferase-based protein-protein interaction assay, we demonstrate here that neither robenidine nor guanabenz disrupt the interaction between PPP1R15A and either PP1 or eIF2α in intact cells. Moreover, both drugs also inhibited the unfolded protein response in cells that expressed a nonphosphorylatable mutant (S51A) of eIF2α. Our results identify robenidine as a PP1:PPP1R15A-independent cytoprotective compound that holds potential for the management of protein misfolding-associated diseases.

Gram-Positive and Gram-Negative Antibiotic Activity of Asymmetric and Monomeric Robenidine Analogues.

Desymmetrisation of robenidine (1: N',2-bis((E)-4-chlorobenzylidene)hydrazine-1-carboximidhydrazide) and the introduction of imine alkyl substituents gave good antibiotic activity. Of note was the increased potency of two analogues against vancomycin-resistant Enterococci (VRE), one of which returned a MIC of 0.5 µg mL-1 . Five analogues were found to be equipotent or more potent than the lead 1. Introduction of an indole moiety resulted in the most active robenidine analogue against methicillin-resistant S. aureus (MRSA), with a MIC of 1.0 µg mL-1 . Imine C=NH isosteres (C=O/C=S) were inactive. Monomeric analogues were 16-64 µg mL-1 active against MRSA and VRE. An analogue that lacks the terminal hydrazide NH moiety showed modest Gram-negative activity at 64 µg mL-1 . A 4-tert-butyl analogue was shown to be active against both Gram-positive and -negative strains at 16-64 µg mL-1 . In general, additional modifications with aromatic moieties was poorly tolerated, except with concomitant introduction of an imine C-alkyl group. The activity of these analogues against MRSA and VRE ranged from 8 µg mL-1 to inactive (MIC>128 µg mL-1 ) with the naphthyl and indole analogues. Gram-negative activity was most promising with two compounds at 16 µg mL-1 against E. coli. Against P. aeruginosa, the highest activity observed was with MIC values of 32 µg mL-1 with another two analogues. Combined, these findings support the further development of the (E)-2-benzylidenehydrazine-1-carboximidamide scaffold as a promising scaffold for the development of antibiotics against Gram-positive and Gram-negative strains.

Evaluation of robenidine analog NCL195 as a novel broad-spectrum antibacterial agent.

The spread of multidrug resistance among bacterial pathogens poses a serious threat to public health worldwide. Recent approaches towards combating antimicrobial resistance include repurposing old compounds with known safety and development pathways as new antibacterial classes with novel mechanisms of action. Here we show that an analog of the anticoccidial drug robenidine (4,6-bis(2-((E)-4-methylbenzylidene)hydrazinyl)pyrimidin-2-amine; NCL195) displays potent bactericidal activity against Streptococcus pneumoniae and Staphylococcus aureus by disrupting the cell membrane potential. NCL195 was less cytotoxic to mammalian cell lines than the parent compound, showed low metabolic degradation rates by human and mouse liver microsomes, and exhibited high plasma concentration and low plasma clearance rates in mice. NCL195 was bactericidal against Acinetobacter spp and Neisseria meningitidis and also demonstrated potent activity against A. baumannii, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae and Enterobacter spp. in the presence of sub-inhibitory concentrations of ethylenediaminetetraacetic acid (EDTA) and polymyxin B. These findings demonstrate that NCL195 represents a new chemical lead for further medicinal chemistry and pharmaceutical development to enhance potency, solubility and selectivity against serious bacterial pathogens.

Robenidine Analogues as Gram-Positive Antibacterial Agents.

Robenidine, 1 (2,2'-bis[(4-chlorophenyl)methylene]carbonimidic dihydrazide), was active against MRSA and VRE with MIC's of 8.1 and 4.7 µM, respectively. SAR revealed tolerance for 4-Cl isosteres with 4-F (8), 3-F (9), 3-CH3 (22), and 4-C(CH3)3 (27) (23.7-71 µM) and with 3-Cl (3), 4-CH3 (21), and 4-CH(CH3)2 (26) (8.1-13.0 µM). Imine carbon alkylation identified a methyl/ethyl binding pocket that also accommodated a CH2OH moiety (75; 2,2'-bis[1-(4-chlorophenyl)-2-hydroxyethylidene]carbonimidic dihydrazide). Analogues 1, 27 (2,2'-bis{[4-(1,1-dimethylethyl)phenyl]methylene}carbonimidic dihydrazide), and 69 (2,2'-bis[1-(4-chlorophenyl)ethylidene]carbonimidic dihydrazide hydrochloride) were active against 24 clinical MRSA and MSSA isolates. No dose-limiting cytotoxicity at ≥2× MIC or hemolysis at ≥8× MIC was observed. Polymyxin B addition engendered Escherichia coli and Pseudomonas aeruginosa Gram-negative activity MIC's of 4.2-21.6 µM. 1 and 75 displayed excellent microsomal stability, intrinsic clearance, and hepatic extraction ratios with T1/2 > 247 min, CLint < 7 µL/min/mg protein, and EH < 0.22 in both human and mouse liposomes for 1 and in human liposomes for 75.

Influence of three coccidiostats on the pharmacokinetics of florfenicol in rabbits.

In-feed Medication has been used for a long time to prevent coccidiosis, a worldwide protozoal disease in rabbits. Florfenicol (FFC) has been widely used in veterinary clinics for bacterial diseases treatment. Therefore, the use of combinations of coccidiostats with FFC in rabbits is common. In the present study, we aimed to evaluate the effect of three coccidiostats, sulfaquinoxaline (SUL), robenidine (ROB), and toltrazuril (TOL), as feed additives on the pharmacokinetic profile of FFC in rabbits. The disposition kinetics of FFC in rabbits were investigated after a single intravenous injection (25 mg/kg) in rabbits fed anticoccidial-free diets or feeds containing SUL (250 ppm), ROB (66 ppm), or TOL (2 ppm), respectively, for 20 days. Plasma FFC concentrations were determined by the high performance liquid chromatography (HPLC) method. The pharmacokinetic parameters of FFC were analyzed using a non-compartmental analysis based on the statistical moment theory. The results demonstrated that ROB feeding resulted in an obvious decrease in plasma FFC level as compared with anticoccidial-free feeding. The terminal elimination half-life (t1/2z), area under the concentration-time curve (AUC), area under the first moment curve (AUMC), and mean residence time (MRT) significantly decreased, whereas the elimination rate constant (λz) and total body clearance (CLz) obviously increased in rabbits pretreated with ROB. However, we did not find that SUL or TOL feeding had any effect on the pharmacokinetic profile of FFC. Our findings suggested that more attention should be paid to the use of FFC in rabbits supplemented with ROB.

In vitro destruction of Eimeria oocysts by essential oils.

This study aims to assess the ability of essential oils (EOs) to destroy Eimeria oocyst in vitro using microscopic counting and 273 nm absorbing material release. A screening for the ability of ten EOs to destroy Eimeria oocyst was carried out in liquid medium. Out of these ten, artemisia, tea tree, thyme and clove EOs were identified as being the most effective. The treatment of Eimeria oocyst with these EOs leads to their lysis as shown by the release of substances absorbing at 273 nm. These results were obtained after approximately three hours contact. Four EOs were proven to destroy Eimeria oocysts in a few hours at low concentration. This destructive effect is a consequence of their lysis. This work is a preliminary contribution aiming to develop a new generation of natural efficient agents for destroying Eimeria oocyst to fight coccidiosis in broiler chicken.

Fate and antibacterial potency of anticoccidial drugs and their main abiotic degradation products.

The antibacterial potency of eight anticoccidial drugs was tested in a soil bacteria bioassay (pour plate method), EC(50)-values between 2.4 and 19.6 microM were obtained; however, one compound, nicarbazin exhibited an EC(50)-value above the maximum tested concentration (21 microM, 9.1 mg L(-1)). The potency of mixtures of two of the compounds, narasin and nicarbazin, was synergistic (more than additive) with 10-fold greater antibacterial potency of the mixture than can be explained by their individual EC(50)-values. The influence of pH, temperature, oxygen concentration and light on the transformation of robenidine and salinomycin was investigated. Robenidine was transformed by photolysis (DT(50) of 4.1 days) and was unstable at low pH (DT(50) of approximately 4 days); salinomycin was merely transformed at low pH, the latter into an unknown number of products. The antibacterial potency of the mixtures of transformation products of robenidine after photolysis and at low pH was comparable with that of the parent compound. Finally five photo-transformation products of robenidine were structural elucidated by accurate mass measurements, i-FIT values (isotopic pattern fit) and MS/MS fragmentation patterns.

Effect of subcutaneously administered diclazuril on the output of Eimeria species oocysts by experimentally infected rabbits.

The effect of subcutaneously injected diclazuril on the output of Eimeria species oocysts was studied in experimentally infected rabbits. Diclazuril was administered either prophylactically at 0.5, 1 or 2 mg/kg bodyweight two days before each rabbit was inoculated with 20,000 oocysts of a mixed-species field isolate of Eimeria or therapeutically at 1, 2 or 4 mg/kg bodyweight five days after they were inoculated. The prophylactic treatments significantly reduced (P<0.05) the output of oocysts in faeces and the numbers in the rabbits' livers at all doses. The therapeutic treatment at 4 mg/kg also caused a significant reduction (P<0.05) in oocyst shedding, but the lower doses resulted in only a moderate reduction. The shedding of the pathogenic species Eimeria stiedae, Eimeria magna, Eimeria irresidua, Eimeria flavescens, Eimeria piriformis and Eimeria intestinalis was significantly reduced (P<0.05) in all the treated groups. The burden of oocysts in the livers of the therapeutic groups (4000 to 9000) were significantly lower (P<0.05) than in the inoculated but untreated control group (23,000), but higher than in the prophylactic groups (around 1000).

Mechanisms of allosteric regulation of Trypanosoma cruzi S-adenosylmethionine decarboxylase.

S-Adenosylmethionine decarboxylase (AdoMetDC) is a pyruvoyl-dependent enzyme that catalyzes an essential step in polyamine biosynthesis. The polyamines are required for cell growth, and the biosynthetic enzymes are targets for antiproliferative drugs. The function of AdoMetDC is regulated by the polyamine-precursor putrescine in a species-specific manner. AdoMetDC from the protozoal parasite Trypanosoma cruzi requires putrescine for maximal enzyme activity, but not for processing to generate the pyruvoyl cofactor. The putrescine-binding site is distant from the active site, suggesting a mechanism of allosteric regulation. To probe the structural basis by which putrescine stimulates T. cruzi AdoMetDC we generated mutations in both the putrescine-binding site and the enzyme active site. The catalytic efficiency of the mutant enzymes, and the binding of the diamidine inhibitors, CGP 48664A and CGP 40215, were analyzed. Putrescine stimulates the k(cat)/K(m) for wild-type T. cruzi AdoMetDC by 27-fold, and it stimulates the binding of both inhibitors (IC(50)s decrease 10-20-fold with putrescine). Unexpectedly CGP 48664A activated the T. cruzi enzyme at low concentrations (0.1-10 microM), while at higher concentrations (>100 microM), or in the presence of putrescine, inhibition was observed. Analysis of the mutant data suggests that this inhibitor binds both the putrescine-binding site and the active site, providing evidence that the putrescine-binding site of the T. cruzi enzyme has broad ligand specificity. Mutagenesis of the active site identified residues that are important for putrescine stimulation of activity (F7 and T245), while none of the active site mutations altered the apparent putrescine-binding constant. Mutations of residues in the putrescine-binding site that resulted in reduced (S111R) and enhanced (F285H) catalytic efficiency were both identified. These data provide evidence for coupling between residues in the putrescine-binding site and the active site, consistent with a mechanism of allosteric regulation.