In situations where FRET-based

substrate Selleck BTK inhibitor is inaccessible, separation approaches, such as the “LabChip” microfluidic system from Caliper and others, might be the best alternative. Another, less frequently used form of a FP-based protease assay is the application of a fluorescein/biotin dual-labeled substrate. In this format, the precise distance between fluorescent label and biotin is irrelevant as there is no FRET phenomenon. Upon cleavage, the fluorescent label is separated from the biotin tag. Addition of streptavidin to the reaction mixture will lead to an increase in FP proportional to the amount of remaining substrate. While there are numerous ways to assay endoproteases, assays for exoproteases that recognize carboxy or amino-terminal residues are far less available. A HTRF assay for carboxypeptidase

B (EC 3.4.17.2) has been developed for HTS where cleavage of a peptide unmasks an epitope which is then recognized by an antibody (Ferrer et al., 2005). HDACs (EC 3.5.1.98) have been assayed for a number of years by radiometric measurements, after extraction of the released acetic acid from hyperacetylated tritiated histone substrate. In a surrogate Rucaparib assay, Schreiber׳s group (Kwon et al., 1998) attached a coumarin label to a known HDAC inhibitor, K-trap, and used the HDAC-labeled K-trap complex to search for novel inhibitors, essentially converting the enzymatic deacetylation reaction into a binding/displacement type of assay. More recently, a commercial fluorogenic assay has become available. In the Fluor-de-Lys system from Biomol, the lysine residue in the substrate is exposed upon deacetylation and, during

a development reaction, is converted via proprietary reagent to a fluorescent product. As with any assay, interpretation of the results requires careful consideration of potential artifacts. The identification of activators for the HDAC known as SIRT1 ( Howitz et al., 2003 and Milne et al., 2007), that is compounds which appear to increase the affinity of SIRT1 for an acetylated p53-derived peptide, was confounded by the fluorescent tag used in the Fluor-de-Lys system. The putative SIRT1 activators were subsequently found to be inactive when a different label was used in the assay or unlabeled peptides were employed and products detected by either Reverse transcriptase HPLC or release of [14C]-nicotinamide ( Kaeberlein et al., 2005 and Pacholec et al., 2010). This again illustrates the necessity to perform an orthogonal assay ( Thorne et al., 2010) – in this case the same enzyme assay but with a different detection readout, before interpreting results. Another suitable assay for SIRT1 which could serve as an orthogonal assay for the Fluor-de-Lys assay employs pro-luciferin substrates and these assays can be miniaturized to a 10 μL assay volume ( Halley et al., 2011). “Label-free” assays have been developed for HDACs using LC/MS for detection of peptides of acetyl-CoA products ( Rye et al., 2011).

T-score was calculated based upon the database from nationwide survey [13]. A central facility performed quality assurance of the Crizotinib purchase longitudinal adjustment, by calibrating each machine with standardized phantoms. All DXA measurements were analyzed at a central site by a radiologist blinded to treatment group assignment. Serum and postprandial urine samples were collected at baseline, 0.5, 1, and 2 months, and every second month thereafter until 36 months for routine analyses, including Ca concentrations. At baseline, 6, 12, 24, and 36 months, we determined serum bone-specific alkaline phosphatase (BSAP) (Metra-BAP EIA; Quidel, San Diego, CA; reference range 7.9 to 29.0 U/L)

and urinary type I collagen N-telopeptide (NTX) (Osteomark; Inverness Medical

Innovations, Waltham, MA; reference range 9.3 to 54.3 nmol BCE/mol Cr) as bone turnover markers, and 25(OH)D (HPLC-competitive protein binding assay), 1,25(OH)2D (HPLC radioreceptor assay) and intact parathyroid hormone (PTH) (Eclusys PTH, Roche Diagnostics, Penzberg, Germany) as calcium-regulating hormones. Nichols Allegro Lite was used for the measurement of 25(OH)D only at enrollment, because manufacturing of the kit was discontinued thereafter. Regression analysis between the two measurements revealed that there was a linear relationship between the 25(OH)D values from HPLC-competitive binding assay (y) and Nichols Allegro Lite assay (x): y = 1.016x + 4.555. DAPT If increase in serum Ca over 11.0 mg/dL (2.75 mmol/L) developed, or if increase in serum Ca over 10.4 mg/dL (2.6 mmol/L) along with urinary Ca over 0.4 mg/dL GF (0.1 mmol/L GF) developed, treatment was discontinued. If serum Ca in these patients subsequently decreased to below 10.4 mg/dL (2.6 mmol/L) and urinary Ca decreased to below 0.4 mg/dL GF (0.1 mmol/L GF), treatment was resumed with reduced doses 3-mercaptopyruvate sulfurtransferase (0.5 μg eldecalcitol and alfacalcidol). Fifteen patients in eldecalcitol group, and 12 patients in alfacalcidol group discontinued treatment. Among them, all 15 patients in eldecalcitol group and 9 patients in alfacalcidol

group resumed treatment with reduced doses. Compliance with the study treatment was assessed with the use of medication diaries and counts of residual medication supplies. All patients were questioned about adverse events at each visit, and all adverse events were analyzed regardless of the investigators’ assessments of causality. The Medical Dictionary for Regulatory Activities (MedDRA, Version 8) was used to categorize reported adverse events. All randomized patients who took any dose of a study drug were included in the safety analysis, and all randomized patients with drug administration who had a baseline assessment and at least one post-randomization assessment were included in the efficacy analysis (Fig. 1). Analysis of vertebral fracture incidence included patients who underwent radiography at baseline and at least once during the study period.

For relative quantification of gene expression, we used the comparative CT method, also known as the 2− ΔΔCT method [35]. Adenomatous polyp counts were analyzed by the Kruskal-Wallis one-way analysis of variance and Dunn’s post-test. Histomorphometry, relative gene expression, and protein quantification data were compared between groups using Mann-Whitney U analysis. selleck compound Statistical significance

was set at P < .05. All analyses were performed with the GraphPad Prism version 5.0 for windows (GraphPad Software, San Diego, CA). On necropsy, 7 months after the last episode of experimentally induced colitis, the only difference observed between experimental groups was that DSS-treated mice had prominently larger MLN compared to the untreated controls. When the intestines were cut open, however, in 5 of the 11 mice, 7 grossly visible, well-sized polyps were found (Figure W1A). The colonic mucosa exophytic tumors, which had the typical cornflower-like appearance of colonic polypoid adenomas ( Figure 1A), had sizes ranging from 2 to 10 mm in diameter and were located either in the descending colon (five of seven) or in the rectum (two of seven). The surface of the largest four polyps (four of seven) had erosions and microhemorrhages. No grossly detectable polyps were found in the intestines of uPA−/−, WT, and WT

+ DSS experimental groups (uPA−/− + DSS polyps = 7 vs WT + DSS polyps = 0, P < .05; Figure 1A). This finding suggested that uPA−/− + DSS mice Bay 11-7085 could model sporadic Ganetespib colorectal polypoid adenomas of humans. To confirm this, we next characterized the histopathologic and selected immunohistochemical

features of inflammation-induced polyps. The DSS-induced colorectal polyps of uPA−/− mice had the typical histopathologic features of colorectal polypoid adenomas that arise spontaneously in humans or after chemically induced carcinogenesis in mouse models (Figure 1B). All of them were tubular adenomas. Four of them were broad-based (four of seven) and three were pedunculated (three of seven). The tumors composed of elongated, branching, tortuous abnormal crypts, separated by small amounts of intervening stroma ( Figure 1B). Neoplastic gland profiles were densely packed, with back-to-back positioning and had irregular shape, which was often angular. They also showed marked variability in shape and size, slit-shaped lumen, and cystic dilatation ( Figures 1, B and C, and S1B). Occasional dilated crypts were filled with mucin and exfoliated cells. The neoplastic glands were lined by highly dysplastic epithelium showing moderate to marked pseudostratification, loss of nuclear polarity, cellular pleomorphism, and atypia ( Figures 1C and W1, B and C). Mitotic figures, including abnormal ones ( Figure W1C), were abundant ( Figures 1, C and D, and W1, B and C), whereas the most advanced lesions contained increased apoptotic cells ( Figure 1D).

A test substance is considered to be sensitiser if it increases the expression (compared to the solvent control) of at least 7 genes measured by qPCR in either the “SENS-IS” or the “ARE” gene sets. To take into account non-specific gene over-expression due to cell stress, the induction of more than 20 genes in the irritation gene check details set, classifies a result as inconclusive and the test substance is re-analysed at a lower concentration. Similarly to the LLNA, potency

is classified according to the concentration of test material needed to induce a positive response: positive at 0.1%: extreme; positive at 1%: strong; positive at 10%: moderate; positive at 50%: weak. Sens-IS is considered to mainly address key event 2 from the skin sensitisation AOP, but may, as ARE-activated genes are included, also provide information on protein reactivity of a test chemical. The SenCeeTox method is a test battery of three independent

assays addressing several key events to provide information on the skin sensitisation potential of substances and to assign them to GW-572016 research buy a certain subset of potency categories (McKim et al., 2012). Protein reactivity is evaluated in a cell-free manner by measurement of the concentration of free glutathione (GSH) after incubation with the test substance for 24 h at room temperature. The amount of free GSH is determined by a colorimetric assay with 5,5′-Dithio-bis(2-nitrobenzoic acid (DTNB) in relation to the vehicle control. An epidermal

skin equivalent (EpiDerm™, MatTek, MA) is used for gene expression analysis and cytotoxicity determination. Viability of skin tissues is measured by assaying for lactose dehydrogenase (LDH) activity. Expression of four housekeeping and seven target genes (NADPH-quinone oxidoreductase 1, Aldoketoreductase 1C2, Interleukin 8, Cytochrome P450 1A1, Aldehyde dehydrogenase 3A, Heme-oxygenase 1, Glutamate cysteine ligase catalytic subunit C) is monitored after topical exposure of the model skin tissues to the test substances at a range of six concentrations (0.1, 5, 100, 250, 500, and 2500 μM) for 24 h. Concentrations, which result in cell viability before of less than 50% compared to the vehicle control, are disregarded for the determination of the sensitising potential/potency. Finally, a gated algorithm is used to transform the viability, gene induction and glutathione reactivity data into a toxicity index for each substance. This method covers key event 1 (in terms of protein reactivity) and 2 (in terms of keratinocyte activation) in the skin sensitisation AOP. The GARD assay uses proliferating MUTZ-3 cells (a human myeloid leukemia-derived cell line) to measure gene expression induced by test substances.

In the EVEROTAC 6-month prospective, open-label pharmacokinetic study, 35 renal transplant patients were randomized to receive EVR 0.75-mg bid or 1.5-mg bid in combination with standard-dose TAC (0.075-mg/kg bid adjusted

to achieve target C0 of 10–15 ng/mL from days 1–14 posttransplant, and then 5–10 ng/mL thereafter to month 6). EVR C0 levels were maintained between 3 and 8 ng/mL from day 42. From day 4 onward, exposure to TAC was similar with both doses of EVR (AUC: 162 ± 61 vs 171 ± 75 ng·h/mL). Significant differences in AUC were not seen, despite the EVR dose, because TAC dosing was adjusted to achieve target levels. Although the pharmacokinetic data suggest that neither EVR dose resulted in statistically significant differences in TAC exposure, the doses of TAC required to maintain target concentrations were buy Lumacaftor higher when administered with EVR 1.5 mg bid than with EVR 0.75-mg bid (12.5 mg vs 9.5 mg at day 14, and 9 mg vs 6 mg at day 42; p < 0.05 for both comparisons). Further, EVR appeared to decrease TAC exposure in a concentration-dependent manner. The data suggest that concomitant treatment with EVR 1.5-mg bid was effective in minimizing BYL719 exposure to TAC. However, further minimization of TAC exposure would likely require doses

of EVR greater than 3 mg/day because this dose was not enough to achieve EVR levels > 3 ng/mL during the first 2 weeks. From the limited

data discussed above, the findings suggest that co-administration with TAC does not influence exposure to EVR. The reported effects of EVR on TAC exposure, however, are, inconsistent. Bay 11-7085 There are only limited published data evaluating the interaction between SRL and TAC. In a recent pharmacokinetic study, both time- and concentration-dependent increases in TAC and SRL were reported. The study assessed drug exposure in 25 de novo kidney transplant patients, who, within 24 h of the transplant surgery were randomized to receive either SRL (15-mg loading dose, 5 mg for 7 days, and 2 mg thereafter) or MMF (2 g/day) for 6 months [37]. Both groups received TAC (0.10–0.15 mg/kg/dose) and corticosteroids. TAC doses were adjusted to keep blood concentration between 10 and 20 ng/mL for the first 30 days, 8–15 ng/mL during months 2 and 3, and 5–10 ng/mL thereafter. From day 7 to month 6, dose-normalized AUC0–12 for TAC increased by 59% in patients receiving SRL and 65% in patients receiving MMF. Over the same period, the dose-normalized AUC0–24 for SRL increased by 65%. Direct concentration-dependent correlations occurred between TAC and SRL blood levels. Increasing TAC or SRL doses were associated with parallel increases in exposure of SRL (p = 0.016) and TAC (p = 0.012), respectively (Fig. 2A and B).

PBMCs were incubated with magnetic microbeads (130-091-153, Monocyte Isolation Kit II, Miltenyi Biotec, Bergisch Gladbach, Germany) in accordance with the

manufacturer’s protocol, and final isolation of monocytes was achieved using a magnetic cell sorter (AutoMACS, Miltenyi Biotec, Germany). PBMCs were differentiated into dendritic cells using an established protocol (Rogler et al., 1998); monocytes were cultivated in flasks for 1 week under optimal conditions (37 °C, 5% CO2, 95% relative humidity [RH]) with 5 ng/mL IL-4 and 50 ng/mL granulocyte–macrophage colony-stimulating factor (GM-CSF). As described above for ivDCs, peripheral blood monocytes were differentiated into macrophages based on the BKM120 solubility dmso established protocol cited, with monocytes being cultivated in Teflon bags for 1 week under optimal conditions (37 °C, 5% CO2, 95% RH). Differentiated macrophages were detached from the Teflon bags by incubation at 4 °C. The monocytic/macrophage-like THP-1 cell line was cultivated in Roswell Park Memorial Institute medium containing 10% fetal calf serum (FCS), supplemented with penicillin (100 U/mL) learn more and streptomycin (100 μg/mL) under standard conditions (37 °C, 5% CO2, 95% RH). Human epithelial colorectal adenocarcinoma (Caco-2) cells were

grown in high glucose Dulbecco’s Modified Eagle’s Medium containing 10% FCS, supplemented with penicillin (100 U/mL) and streptomycin (100 μg/mL) under recommended conditions (37 °C, 10% CO2, 95% RH). For both cell lines, passaging was carried out according to the guidelines

of the American Type Culture Collection (ATCC, 2012). The influence of retinoids on the LPS-induced cytokine response of ivDCs, ivMACs and THP-1 cells was evaluated in each cell type using the same experimental methodology. Primary cells were adjusted to a density of 1 × 106 cells/mL and plated onto 96-well plates (100 μL/well). THP-1 PtdIns(3,4)P2 cells were incubated in six-well plates at a density of 7 × 105 cells/mL. Retinoids were added to the medium (0.01, 0.1, 1.0 and 5.0 μg/mL) for ivDCs, ivMACs and THP-1 cells, and pre-incubated for 1 h prior to stimulation with LPS (to a final concentration of 100 ng/ml) for a further 48 h at 37 °C. Incubation medium was collected and processed for cytokine analysis (Rules-Based Medicine, Austin, Texas, USA) using Human Cytokine MAP A 1.0® array. Levels of IL-15 were below the detection limit of the assay and were excluded from the analysis. For studies in ivDCs, cytokine response data shown are based on at least six (LPS-induced) and at least four (no LPS) independently performed experiments, each corresponding to a different donor. In ivMACs, these data (both LPS-induced and no LPS) were each based on at least four independently performed experiments, each corresponding to a different donor. Data shown for cytokine response in THP-1 cells are based on three independent experiments.

APC activation is therefore a necessary prerequisite for an efficient adaptive immune response. DCs not only provide antigen and co-stimulation to naïve T cells, but also contribute to the initial commitment of naïve T helper cells into Th1, Th2 or other subsets. This directs the efficient induction of T helper cells PD-166866 with appropriate cytokine profiles early during infections, without the need for direct contact between antigen-specific T cells and pathogens. Undigested pathogen-derived antigens are also drained by the lymph and transported to the B cell-rich area of the lymph node, where they are exposed to BCR-expressing cells. An

adaptive immune response is therefore initiated in a draining lymph node by the concerted action of innate immune cells and free antigens. These activate T and B lymphocytes, respectively, to proliferate and differentiate into effector and memory cells. The type of communication employed by the immune system represents a unique approach to multi-system signalling and communication over distances. As well as employing the soluble mediators – proinflammatory messengers, chemokines and soluble danger signals – the immune system uses migratory APCs to physically transport messages from the periphery to the induction sites of adaptive immune response, eg in lymph nodes. Notably, by selectively migrating in response to infectious/cell-damaging events, DCs act as filters

for the adaptive immune response, helping T and B cells to ignore innocuous foreign antigens. Thus, the innate immune response plays an important role in selecting antigens that represent a real selleck chemical threat to the organism that requires an adequate adaptive response. The response to pathogens in humans takes place over a large anatomical distance and in distinct phases, which are summarised in Figure 2.9. The innate immune response is initiated at the site of challenge when a foreign entity triggers a defensive response, which is mediated by chemical signals. These signals attract responding innate immune cells (monocytes, DCs etc) which travel to the site and engulf fragments of the pathogen. The monocytes and DCs then leave

the site via lymphatic vessels and begin to mature and Thiamet G differentiate, while travelling to the local draining lymph nodes. Differentiation gives rise to APCs that interact with naïve T cells at the lymph nodes and bear receptors for the antigenic peptides expressed on the surface of the APC. Molecular, antigenic and cytokine signals combine to direct the differentiation and activation of CD4+ T cells into distinct effector subtypes. This is the induction phase of the adaptive immune response. A sub-population of CD4+ T cells differentiates into memory cells, which are capable of responding rapidly on repeat exposure to the same antigen. CD8+ T cells also receive antigenic and cytokine stimulation from APCs and undergo differentiation either into memory-type cells or armed effector cytotoxic cells.

Special focus was given to the estimation of urinary excretion rates and the direct determination of major conjugation products. Methanol (LC gradient grade) and glacial acetic acid (p.a.) were purchased from find more Merck (Darmstadt, Germany), acetonitrile (ACN, LC gradient grade) from VWR (Leuven, Belgium), creatinine from Sigma

(Schnelldorf, Germany). Deoxynivalenol-3-O-glucuronide and zearalenone-14-O-glucuronide were synthesized by optimized procedures as described elsewhere ( Fruhmann et al., 2012 and Mikula et al., 2012). Other standards were purchased from Sigma (ZEN, α- and β-ZEL) and Romer Labs Diagnostic GmbH Tulln, Austria (DON, 13C15-DON, deepoxy-DON, nivalenol, T-2 toxin, HT-2 toxin, ochratoxin A, aflatoxin M1, fumonisins B1 and B2). Solid standard substances were dissolved in pure methanol (DON-3-GlcA, nivalenol) or ACN (DON, ZEN-14-GlcA, ZEN, α- and β-ZEL). All other standards were delivered in ACN or ACN/H2O (fumonisins B1 and B2) and stored at −20 °C. A combined multi standard working solution for preparation of calibrants and spiking experiments was prepared in ACN containing 10.0 mg/L DON, IDH inhibitor DON-3-GlcA, deepoxy-DON, nivalenol and HT-2, 5.0 mg/L fumonisin B1 and B2, 2.5 mg/L ZEN-14-GlcA, α-ZEL, β-ZEL and T-2, 1.0 mg/L ZEN and 13C15-DON and 0.125 mg/L aflatoxin M1 and ochratoxin A. DON-15-GlcA was separated

and subsequently fractionated from a highly contaminated human urine

sample which contained both, DON-3-GlcA and DON-15-GlcA (Warth et al., 2012a). Sulfate conjugates were not included in the study FER due to a lack of reference standard (DON-sulfate) and poor chromatographic behavior on the used chromatographic column (ZEN-sulfate). Enzymatic hydrolysis of the samples was done using β-glucuronidase from Escherichia coli (Type IX-A, Sigma). 500 μL urine were mixed with 500 μL PBS buffer (75 mM, pH 7.4) containing 3000 units of β-glucuronidase and incubated at 37 °C for 18 h. Digested samples were centrifuged and 200 μL of the supernatant was diluted with 800 μL dilution solvent to result in a total dilution factor of ten like the untreated samples. The study was conducted on a 27 year old, healthy male volunteer who consumed a special diet over a period of eight days as displayed in Fig. 1. On the first and the last two days the person ingested a mycotoxin reduced diet, which was based on rice, vegetables, fruits and milk products to reach Fusarium mycotoxin blank levels in excreted urine. During the four days in between, a diet naturally contaminated with high levels of DON was consumed, with exactly the same servings each day at the same times. This DON intervention diet consisted of cereals with wheat bran for breakfast, maize porridge (including maize flour) for lunch and bread, beer and pop-corn in the evening.

003). Finally, the Vco2/ V˙o2 ratio remained below 0.9 throughout both sessions and did not differ between exercises. NVP-BGJ398 When compared with rest, the heart rate remained unchanged during the ECC exercise, while it increased progressively and significantly (P<.001) in the CON exercise from the beginning of the exercise onwards ( fig 3A). CO increased during both exercises (P<.008) ( fig 3B), but remained lower during ECC exercise (P<.008). SV increased in both exercises, and this increase was greater in ECC exercise than in CON exercise after 11 minutes of exercise (P<.008) ( fig 3C).

ECC cycling exercise was well tolerated when it was tailored to RPE from a prior CON effort test. It is possible to define mechanical work on the basis of perceived exertion, without the need for a more complex evaluation that includes V˙o2 measurement. To date, there are no consensual

criteria to define the intensity of ECC exercise. As for any exercise intervention, the aim is to ensure efficient training while avoiding muscle injury. However it seems necessary to define levels of intermediate exercise intensity for ECC preconditioning. In this study, in order to avoid maximal CON exercise tests with V˙o2 measurement, and thus to simplify the usual strategy, we chose to use the RPE17 during CON exercise to establish the resistance force to apply to the pedals of the ECC ergocycle. Indeed, this RPE can be used in daily clinical practice to determine levels of perceived exertion, corresponding to different workloads PD0325901 solubility dmso during CON exercise,26 with a good reliability.18 The RPE level chosen was validated to establish a stable level of moderate-intensity CON exercise in healthy subjects27 and patients with cardiovascular disease,28 close to 50% of Vo2Vo2peak.20 The use of RPE to adapt an exercise program has been shown to be more Thalidomide effective than the conventional method based on heart rate at the ventilatory threshold in patients with coronary heart disease.29 This led us to choose an RPE

of 12, which corresponds to the ventilatory threshold in healthy subjects17 and 20 and in patients with chronic heart failure30 and coronary artery disease.29 This study confirmed that the perception of exertion is only very slightly modified during low-intensity ECC cycling exercise compared with the resting state and is therefore not an identified way to tailor ECC training to the individual. Plantar pressure induces a different intrinsic feedback, and its cerebral integration was certainly different.31 Indeed, even though the visual and mechanical feedback were the same in the CON and ECC bout, intrinsic feedback processing was certainly different between the 2 modes of cycling.

Such conformational and rotational flexibility has been

verified, for example, through solution – NMR techniques for M2WJ 332 binding to an artificial 13-base pair construct ( Wang et al., PI3K inhibition 2013). In earlier accounts (Vedani et al., 2000, Vedani et al., 2005 and Vedani and Dobler, 2002) we have demonstrated that a 4D representation including all (Boltzmann weighted) feasible poses can provide more accurate estimations of the associated binding affinities. Fig. 8 shows the corresponding 4D ensembles for the very compounds: diethylstilbestrol bound to the estrogen receptor α, genistein bound to the estrogen receptor β, dexamethasone bound to the glucocorticoid receptor and progesterone bound to the progesterone receptor. The individual poses are Boltzmann-weighted, i.e., only the energetically most favorable binding modes contribute significantly to the binding energy. Using the VirtualToxLab, we have estimated the toxic potential (endocrine and metabolic disruption, some aspects of carcinogenicity and cardiotoxicity) selleck compound for over 2500 compounds—drugs, chemicals and natural products—and posted the results on http://www.virtualtoxlab.org.

The aim of the technology is to generate toxicity alerts, i.e., ranking the tested compounds in three groups: toxic potential (TP) ≤ 0.3 (low), 0.3 < TP ≤ 0.6 (moderate) TP > 0.6 (high). Fig. 9 shows the toxic potential for a selection of compounds. More informative than the toxic potential itself is the underlying binding-energy profile (cf. Table 1 for bisphenol A), as it provides specific information at which target protein an elevated binding affinity—potentially triggering an adverse effect—might be expected (cf. also the fingerprinting display GABA Receptor mode in Fig. 5). The VirtualToxLab interface allows exporting the individual binding affinities into a csv file and, hence, to compute a customized toxicity alert. Most important, our technology allows rationalizing a given binding affinity

by inspection of the associated protein–ligand complexes in real-time 3D using the embedded 3D/4D viewer or, after exporting the coordinates in PDB format, with any other software of choice. Fig. 10 shows the computed binding mode of the anabolic steroid tetrahydrogestrinone to the androgen receptor. The associated binding affinity of 32 nM compares reasonably well with the experimental value of 8.5 nM. As the docking and scoring algorithms within the VirtualToxLab are based solely on thermodynamic considerations, it is suggested to probe the kinetic stability of the protein–ligand complex of interest by means of molecular-dynamical simulations. If the key interactions (hydrogen bonds, salt bridges, binding to metal ions, hydrophobic contacts) remain stable throughout a decent simulation time (t ≥ 5.