Studies of antibacterial activity (in vitro and in vivo) and mode of action for des-acyl tridecaptins (DATs).

Tridecaptins comprise a class of linear cationic lipopeptides with an N-terminal fatty acyl moiety. These 13-mer antimicrobial peptides consist of a combination of d- and l-amino acids, conferring increased proteolytic stability. Intriguingly, they are biosynthesized by non-ribosomal peptide synthetases in the same bacterial species that also produce the cyclic polymyxins displaying similar fatty acid tails. Previously, the des-acyl analog of TriA1 (termed H-TriA1) was found to possess very weak antibacterial activity, albeit it potentiated the effect of several antibiotics. In the present study, two series of des-acyl tridecaptins were explored with the aim of improving the direct antibacterial effect. At the same time, overall physico-chemical properties were modulated by amino acid substitution(s) to diminish the risk of undesired levels of hemolysis and to avoid an impairment of mammalian cell viability, since these properties are typically associated with highly hydrophobic cationic peptides. Microbiology and biophysics tools were used to determine bacterial uptake, while circular dichroism and isothermal calorimetry were used to probe the mode of action. Several analogs had improved antibacterial activity (as compared to that of H-TriA1) against Enterobacteriaceae. Optimization enabled identification of the lead compound 29 that showed a good ADMET profile as well as in vivo efficacy in a variety of mouse models of infection.

Zebrafish embryo neonicotinoid developmental neurotoxicity in the FET test and behavioral assays.

The need for reliable, sensitive (developmental) neurotoxicity testing of chemicals has steadily increased. Given the limited capacities for routine testing according to accepted regulatory guidelines, there is potential risk to human health and the environment. Most toxicity studies are based on mammalian test systems, which have been questioned for low sensitivity, limited relevance for humans, and animal welfare considerations. This increased the need for alternative models, one of which is the zebrafish (Danio rerio) embryo. This study assessed selected neonicotinoids at sub-lethal concentrations for their effects on embryonic development and behavior. The fish embryo acute toxicity test (OECD TG 236) determined the lowest observable effective concentrations, which were used as the highest test concentrations in subsequent behavioral assays. In the FET test, no severe compound-induced sublethal effects were seen at < 100 µM. In the coiling assay, exposure to ≥ 1.25 µM nicotine (positive control) affected both the burst duration and burst count per minute, whereas ≥ 50 µM thiacloprid affected the mean burst duration. Exposure to ≥ 50 µM acetamiprid and imidacloprid induced significant alterations in both mean burst duration and burst count per minute. In the swimming assay, 100 µM acetamiprid induced alterations in the frequency and extent of movements, whilst nicotine exposure only induced non-significant changes. All behavioral changes could be correlated to findings in mammalian studies. Given the quest for alternative test methods of (developmental) neurotoxicity, zebrafish embryo behavior testing could be integrated into a future tiered testing scheme.

Acute effects of the imidacloprid metabolite desnitro-imidacloprid on human nACh receptors relevant for neuronal signaling.

Several neonicotinoids have recently been shown to activate the nicotinic acetylcholine receptor (nAChR) on human neurons. Moreover, imidacloprid (IMI) and other members of this pesticide family form a set of diverse metabolites within crops. Among these, desnitro-imidacloprid (DN-IMI) is of special toxicological interest, as there is evidence (i) for human dietary exposure to this metabolite, (ii) and that DN-IMI is a strong trigger of mammalian nicotinic responses. We set out here to quantify responses of human nAChRs to DN-IMI and an alternative metabolite, IMI-olefin. To evaluate toxicological hazards, these data were then compared to those of IMI and nicotine. Ca2+-imaging experiments on human neurons showed that DN-IMI exhibits an agonistic effect on nAChRs at sub-micromolar concentrations (equipotent with nicotine) while IMI-olefin activated the receptors less potently (in a similar range as IMI). Direct experimental data on the interaction with defined receptor subtypes were obtained by heterologous expression of various human nAChR subtypes in Xenopus laevis oocytes and measurement of the transmembrane currents evoked by exposure to putative ligands. DN-IMI acted on the physiologically important human nAChR subtypes α7, α3ß4, and α4ß2 (high-sensitivity variant) with similar potency as nicotine. IMI and IMI-olefin were confirmed as nAChR agonists, although with 2-3 orders of magnitude lower potency. Molecular docking studies, using receptor models for the α7 and α4ß2 nAChR subtypes supported an activity of DN-IMI similar to that of nicotine. In summary, these data suggest that DN-IMI functionally affects human neurons similar to the well-established neurotoxicant nicotine by triggering α7 and several non-α7 nAChRs.

Dynamic Modeling of Mitochondrial Membrane Potential Upon Exposure to Mitochondrial Inhibitors.

Mitochondria are the main bioenergetic organelles of cells. Exposure to chemicals targeting mitochondria therefore generally results in the development of toxicity. The cellular response to perturbations in cellular energy production is a balance between adaptation, by reorganisation and organelle biogenesis, and sacrifice, in the form of cell death. In homeostatic conditions, aerobic mitochondrial energy production requires the maintenance of a mitochondrial membrane potential (MMP). Chemicals can perturb this MMP, and the extent of this perturbation depends both on the pharmacokinetics of the chemicals and on downstream MMP dynamics. Here we obtain a quantitative understanding of mitochondrial adaptation upon exposure to various mitochondrial respiration inhibitors by applying mathematical modeling to partially published high-content imaging time-lapse confocal imaging data, focusing on MMP dynamics in HepG2 cells over a period of 24 h. The MMP was perturbed using a set of 24 compounds, either acting as uncoupler or as mitochondrial complex inhibitor targeting complex I, II, III or V. To characterize the effect of chemical exposure on MMP dynamics, we adapted an existing differential equation model and fitted this model to the observed MMP dynamics. Complex III inhibitor data were better described by the model than complex I data. Incorporation of pharmacokinetic decay into the model was required to obtain a proper fit for the uncoupler FCCP. Furthermore, oligomycin (complex V inhibitor) model fits were improved by either combining pharmacokinetic (PK) decay and ion leakage or a concentration-dependent decay. Subsequent mass spectrometry measurements showed that FCCP had a significant decay in its PK profile as predicted by the model. Moreover, the measured oligomycin PK profile exhibited only a limited decay at high concentration, whereas at low concentrations the compound remained below the detection limit within cells. This is consistent with the hypothesis that oligomycin exhibits a concentration-dependent decay, yet awaits further experimental verification with more sensitive detection methods. Overall, we show that there is a complex interplay between PK and MMP dynamics within mitochondria and that data-driven modeling is a powerful combination to unravel such complexity.

A combined in vitro approach to improve the prediction of mitochondrial toxicants.

Drug induced mitochondrial dysfunction has been implicated in organ toxicity and the withdrawal of drugs or black box warnings limiting their use. The development of highly specific and sensitive in vitro assays in early drug development would assist in detecting compounds which affect mitochondrial function. Here we report the combination of two in vitro assays for the detection of drug induced mitochondrial toxicity. The first assay measures cytotoxicity after 24h incubation of test compound in either glucose or galactose conditioned media (Glu/Gal assay). Compounds with a greater than 3-fold toxicity in galactose media compared to glucose media imply mitochondrial toxicity. The second assay measures mitochondrial respiration, glycolysis and a reserve capacity with mechanistic responses observed within one hour following exposure to test compound. In order to assess these assays a total of 72 known drugs and chemicals were used. Dose-response data was normalised to 100× Cmax giving a specificity, sensitivity and accuracy of 100%, 81% and 92% respectively for this combined approach.

Evaluation of an optimized protocol using human peripheral blood monocyte derived dendritic cells for the in vitro detection of sensitizers: Results of a ring study in five laboratories.

Allergic contact dermatitis is a delayed T-cell mediated allergic response associated with relevant social and economic impacts. Animal experiments (e.g. the local lymph node assay) are still supplying most of the data used to assess the sensitization potential of new chemicals. However, the 7th amendment to the EU Cosmetic Directive have introduced a testing ban for cosmetic ingredients after March 2013. We have developed and optimized a stable and reproducible in vitro protocol based on human peripheral blood monocyte derived dendritic cells to assess the sensitization potential of chemicals. To evaluate the transferability and the predictivity of this PBMDCs based test protocol, a ring study was organized with five laboratories using seven chemicals with a known sensitization potential (one none-sensitizer and six sensitizers, including one pro-hapten). The results indicated that this optimized test protocol could be successfully transferred to all participating laboratories and allowed a correct assessment of the sensitization potential of the tested set of chemicals. This should allow a wider acceptance of PBMDCs as a reliable test system for the detection of human skin sensitizers and the inclusion of this protocol in the toolbox of in vitro methods for the evaluation of the skin sensitization potential of chemicals.

Predicting in vivo phospholipidosis-inducing potential of drugs by a combined high content screening and in silico modelling approach.

Drug induced phospholipidosis (PLD) is an adverse side effect which can affect registration of new drug entities. Phospholipids can accumulate in lysosomes, organelles essential in cellular biogenesis and if compromised can lead to cellular toxicity. Drug accumulation in lysosomes (lysosomotropism) is a known mechanism leading to PLD, however phospholipidosis can also occur indirectly by altering synthesis and processing of phospholipids. Drug induced PLD can be measured in vitro using High Content Screening (HCS) approaches, by either determining accumulation of phospholipids conjugated to dyes in cells or by determining accumulation of drugs within lysosomes, by competitive loss of lysosomal dye uptake. In this study we validate two in vitro assays using HepG2 and H9c2 cells in conjunction with in silico models based on physico-chemical properties using 56 compounds (28 phospholipidogenic, 25 non-phospholipidogenic and three kidney specific). Using HCS to determine PLD and lysosomal trapping in HepG2 cells in combination with in silico modelling increase the overall prediction of PLD in vivo with a sensitivity of 96%, specificity of 92% and overall accuracy of 94%. The findings of this study demonstrate the applicability of in vitro and in silico approaches to understand the mechanism underlying PLD and the utility of these approaches as a screening strategy in the pharmaceutical industry to select drug candidates with a low in vivo PLD liability.

Putting the parts together: combining in vitro methods to test for skin sensitizing potentials.

Allergic contact dermatitis is a common skin disease and is elicited by repeated skin contact with an allergen. In the regulatory context, currently only data from animal experiments are acceptable to assess the skin sensitizing potential of substances. Animal welfare and EU Cosmetic Directive/Regulation call for the implementation of animal-free alternatives for safety assessments. The mechanisms that trigger skin sensitization are complex and various steps are involved. Therefore, a single in vitro method may not be able to accurately assess this endpoint. Non-animal methods are being developed and validated and can be used for testing strategies that ensure a reliable prediction of skin sensitization potentials. In this study, the predictivities of four in vitro assays, one in chemico and one in silico method addressing three different steps in the development of skin sensitization were assessed using 54 test substances of known sensitizing potential. The predictivity of single tests and combinations of these assays were compared. These data were used to develop an in vitro testing scheme and prediction model for the detection of skin sensitizers based on protein reactivity, activation of the Keap-1/Nrf2 signaling pathway and dendritic cell activation.

Intralaboratory validation of four in vitro assays for the prediction of the skin sensitizing potential of chemicals.

Allergic contact dermatitis is induced by repeated skin contact with an allergen. Assessment of the skin sensitizing potential of chemicals, agrochemicals, and especially cosmetic ingredients is currently performed with the use of animals. Animal welfare and EU legislation demand animal-free alternatives reflected in a testing and marketing ban for cosmetic ingredients beginning in 2013. The underlying mechanisms of induction and elicitation of skin sensitization are complex and a chemical needs to comply several properties being skin sensitizing. To account for the multitude of events in the induction of skin sensitization an in vitro test system will consist of a battery of various tests. Currently, we performed intralaboratory validations of four assays addressing three different events during induction of skin sensitization. (1) The Direct Peptide Reactivity Assay (DPRA) according to Gerberick and co-workers (Gerberick et al., 2004) using synthetic peptides and HPLC analysis. (2) Two dendritic cell activation assays based on the dendritic cell like cell lines U-937 and THP-1 and flow cytometric detection of the maturation markers CD54 and/or CD86 (Ashikaga et al., 2006; Python et al., 2007; Sakaguchi et al., 2006). (3) Antioxidant response element (ARE)-dependent gene activity in a HaCaT reporter gene cell line (Emter et al., 2010). We present the results of our intralaboratory validation of these assays with 23 substances of known sensitizing potential. The sensitivity, specificity, and accuracy of the individual tests were obtained by comparison to human epidemiological data as well as to data from animal tests such as the local lymph node assay.