ABSTRACT
Inhibition of complex I of the mitochondrial respiratory chain (cI) by rotenone and methyl-phenylpyridinium (MPP +) leads to the degeneration of dopaminergic neurons in man and rodents. To formally describe this mechanism of toxicity, an adverse outcome pathway (AOP:3) has been developed that implies that any inhibitor of cI, or possibly of other parts of the respiratory chain, would have the potential to trigger parkinsonian motor deficits. We used here 21 pesticides, all of which are described in the literature as mitochondrial inhibitors, to study the general applicability of AOP:3 or of in vitro assays that are assessing its activation. Five cI, three complex II (cII), and five complex III (cIII) inhibitors were characterized in detail in human dopaminergic neuronal cell cultures. The NeuriTox assay, examining neurite damage in LUHMES cells, was used as in vitro proxy of the adverse outcome (AO), i.e., of dopaminergic neurodegeneration. This test provided data on whether test compounds were unspecific cytotoxicants or specifically neurotoxic, and it yielded potency data with respect to neurite degeneration. The pesticide panel was also examined in assays for the sequential key events (KE) leading to the AO, i.e., mitochondrial respiratory chain inhibition, mitochondrial dysfunction, and disturbed proteostasis. Data from KE assays were compared to the NeuriTox data (AO). The cII-inhibitory pesticides tested here did not appear to trigger the AOP:3 at all. Some of the cI/cIII inhibitors showed a consistent AOP activation response in all assays, while others did not. In general, there was a clear hierarchy of assay sensitivity: changes of gene expression (biomarker of neuronal stress) correlated well with NeuriTox data; mitochondrial failure (measured both by a mitochondrial membrane potential-sensitive dye and a respirometric assay) was about 10-260 times more sensitive than neurite damage (AO); cI/cIII activity was sometimes affected at > 1000 times lower concentrations than the neurites. These data suggest that the use of AOP:3 for hazard assessment has a number of caveats: (i) specific parkinsonian neurodegeneration cannot be easily predicted from assays of mitochondrial dysfunction; (ii) deriving a point-of-departure for risk assessment from early KE assays may overestimate toxicant potency.
Subject(s)
Electron Transport Chain Complex Proteins/antagonists & inhibitors , Electron Transport/drug effects , Enzyme Inhibitors/toxicity , Mitochondria/drug effects , Mitochondria/metabolism , Pesticides/toxicity , Biomarkers , Cell Line , Cell Line, Tumor , Dopaminergic Neurons/drug effects , Dopaminergic Neurons/metabolism , Drug-Related Side Effects and Adverse Reactions , Electron Transport Complex I/antagonists & inhibitors , Electron Transport Complex II/antagonists & inhibitors , Electron Transport Complex III/antagonists & inhibitors , Humans , Proteostasis/drug effects , Risk Assessment , TranscriptomeABSTRACT
The purpose of this project report is to introduce the European "GOLIATH" project, a new research project which addresses one of the most urgent regulatory needs in the testing of endocrine-disrupting chemicals (EDCs), namely the lack of methods for testing EDCs that disrupt metabolism and metabolic functions. These chemicals collectively referred to as "metabolism disrupting compounds" (MDCs) are natural and anthropogenic chemicals that can promote metabolic changes that can ultimately result in obesity, diabetes, and/or fatty liver in humans. This project report introduces the main approaches of the project and provides a focused review of the evidence of metabolic disruption for selected EDCs. GOLIATH will generate the world's first integrated approach to testing and assessment (IATA) specifically tailored to MDCs. GOLIATH will focus on the main cellular targets of metabolic disruption-hepatocytes, pancreatic endocrine cells, myocytes and adipocytes-and using an adverse outcome pathway (AOP) framework will provide key information on MDC-related mode of action by incorporating multi-omic analyses and translating results from in silico, in vitro, and in vivo models and assays to adverse metabolic health outcomes in humans at real-life exposures. Given the importance of international acceptance of the developed test methods for regulatory use, GOLIATH will link with ongoing initiatives of the Organisation for Economic Development (OECD) for test method (pre-)validation, IATA, and AOP development.
Subject(s)
Diabetes Mellitus/epidemiology , Endocrine Disruptors/adverse effects , Fatty Liver/epidemiology , Obesity/epidemiology , Adipocytes/drug effects , Adipocytes/pathology , Diabetes Mellitus/chemically induced , Diabetes Mellitus/prevention & control , Fatty Liver/chemically induced , Fatty Liver/prevention & control , Humans , Metabolic Networks and Pathways/drug effects , Obesity/chemically induced , Obesity/prevention & control , Risk AssessmentABSTRACT
Drugs that accumulate in lysosomes reach very high tissue concentrations, which is evident in the high volume of distribution and often lower clearance of these compounds. Such a pharmacokinetic profile is beneficial for indications where high tissue penetration and a less frequent dosing regime is required. Here, we show how the level of lysosomotropic accumulation in cells can be predicted solely from molecular structure. To develop quantitative structure-activity relationship (QSAR) models, we used cellular accumulation data for 69 lysosomotropic macrocycles, the pharmaceutical class for which this type of prediction model is extremely valuable due to the importance of cellular accumulation for their anti-infective and anti-inflammatory applications as well as due to the fact that they are extremely difficult to model by computational methods because of their large size (Mw > 500). For the first time, we show that five levels of intracellular lysosomotropic accumulation (as measured by liquid chromatography coupled to tandem mass spectrometry-LC-MS/MS), from low/no to extremely high, can be predicted with 60% balanced accuracy solely from the compound's structure. Although largely built on macrocycles, the eight non-macrocyclic compounds that were added to the set were found to be well incorporated by the models, indicating their possible broader application. By uncovering the link between the molecular structure and cellular accumulation as the key process in tissue distribution of lysosomotropic compounds, these models are applicable for directing the drug discovery process and prioritizing the compounds for synthesis with fine-tuned accumulation properties, according to the desired pharmacokinetic profile.
Subject(s)
Computational Biology/methods , Lysosomes/chemistry , Macrocyclic Compounds/pharmacokinetics , Chromatography, Liquid , Macrocyclic Compounds/chemistry , Molecular Structure , Quantitative Structure-Activity Relationship , Tandem Mass Spectrometry , Tissue DistributionABSTRACT
Certain environmental chemicals affect the body's energy balance and are known as metabolism disrupting chemicals (MDCs). MDCs have been implicated in the development of metabolic diseases, such as obesity and type 2 diabetes. In contrast to their well-known impact on developing adipocytes, MDC effects leading to altered energy balance and development of insulin resistance in mature white adipocytes, constituents of adult adipose tissue, are largely unclear. Here, we investigated the effects of six well-established environmental MDCs (bisphenol A (BPA), perfluorooctanoic acid (PFOA), triclosan (TCS), p,p-dichlorodiphenyl-dichloroethylene (ppDDE), tributyltin chloride (TBT) and triphenyl phosphate (TPP)) on mature human white adipocytes derived from mesenchymal stem cells in vitro. We aimed to identify biomarkers and sensitive endpoints of their metabolism disrupting effects. While most of the tested exposures had no effect on adipocyte glucose consumption, lipid storage and assessed gene expression endpoints, the highest concentration of triclosan affected the total lipid storage and adipocyte size, as well as glucose consumption and mRNA expression of the glucose transporter GLUT1, leptin and adiponectin. Additionally, an increased expression of adiponectin was observed with TPP and the positive control PPARγ agonist rosiglitazone. In contrast, the lipidomic analysis of the cell culture medium after a 3-day exposure was extremely sensitive and revealed concentration-dependent changes in the extracellular lipidome of adipocytes exposed to nearly all studied chemicals. While some of the extracellular lipidome changes were specific for the MDC used, some effects were found common to several tested chemicals and included increases in lysophosphatidylcholines, glycerophospholipids and ceramides and a decrease in fatty acids, with possible implications in inflammation, lipid and glucose uptake. This study points to early signs of metabolic disruption and likely systemic effects of mature adipocyte exposure to environmental chemicals, as well as to the need to include lipidomic endpoints in the assessment of adverse effects of MDCs.
Subject(s)
Diabetes Mellitus, Type 2 , Triclosan , Humans , Adipocytes, White , Lipidomics , Adiponectin , Triclosan/toxicity , Glucose/pharmacologyABSTRACT
Azithromycin is a macrolide antibiotic with well-described anti-inflammatory properties which can be attributed, at least partially, to its action on macrophages. We have previously shown, with 18 different macrolide molecules, that IL-6 and PGE2 inhibition correlates with macrolide accumulation, as well as with their binding to phospholipids in J774A.1 cells. The present study was performed in order to substantiate the hypothesis that biological membranes are a target for macrolide anti-inflammatory activity. By analyzing the effect of azithromycin on overall eicosanoid production, we found that in LPS-stimulated J774A.1 cells, azithromycin, like indomethacin, inhibited the synthesis of all eicosanoids produced downstream of COX. Upstream of COX, azithromycin inhibited arachidonic acid release in the same way as a cPLA2 inhibitor, while indomethacin had no effect. Further comparison revealed that in LPS-stimulated J774A.1 cells, the cPLA2 inhibitor showed the same profile of inhibition as azithromycin in inhibiting PGE2, IL-6, IL-12p40 and arachidonic acid release. Therefore, we propose that the anti-inflammatory activity of azithromycin in this model may be due to interactions with cPLA2, causing inadequate translocation of the enzyme or disturbing physical interactions with its substrates.
Subject(s)
Anti-Bacterial Agents/pharmacology , Anti-Inflammatory Agents/pharmacology , Azithromycin/pharmacology , Lipopolysaccharides/immunology , Macrophages/drug effects , Macrophages/immunology , Animals , Anti-Bacterial Agents/immunology , Anti-Inflammatory Agents/immunology , Anti-Inflammatory Agents, Non-Steroidal/immunology , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Arachidonic Acid/immunology , Azithromycin/immunology , Cell Line , Dinoprostone/immunology , Eicosanoids/immunology , Group IV Phospholipases A2/antagonists & inhibitors , Indomethacin/immunology , Indomethacin/pharmacology , Interleukin-12 Subunit p40/immunology , Interleukin-6/immunology , Macrophages/metabolism , Mice , Prostaglandin-Endoperoxide Synthases/genetics , RNA, Messenger/genetics , Tumor Necrosis Factor-alpha/immunologyABSTRACT
Exceptional therapeutic effects of macrolides in treating various infections and inflammatory conditions can be significantly contributed to their unique pharmacokinetic properties. Macrolides accumulate in cells and tissues, with concentrations usually 10 to more than 100 times higher of those measured in plasma. Intracellular distribution of macrolides has so far been examined using extensive subcellular fractionation techniques, radiolabeled compounds and conventional pharmacokinetic methods. In this study we evaluated four fluorescently labeled macrolides on their applicability to monitor azithromycin distribution in vitro and in vivo. 9-Deoxo-9a-{3-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]propyl}-9a-aza-9a-homoerythromycin A (9a-NBD-azithromycin) was selected as a compound with most similar cellular pharmacokinetics to azithromycin. 9a-NBD-azithromycin demonstrated antimicrobial properties comparable to azithromycin, displayed the same biological activity profile in LPS-stimulated J774A.1 murine macrophage cells and, even though it accumulated in cells almost 50% more than azithromycin, it showed same rate of retention. Identical to azithromycin, 9a-NBD-azithromycin was localized in lysosomes of J774A.1 cells. Two hours after 9a-NBD-azithromycin was administered intraperitonally to mice, a strong fluorescent signal was located in kidneys and liver and slightly weaker in the spleen. In kidneys, the signal was concentrated in tubuli, and glomeruli were negative. Patchy florescence in hepatocytes supports lysosomal cellular localization. Weaker staining of white pulp compared to red pulp of spleen is in agreement with lower accumulation of azithromycin in lymphocytes compared to other cell types present. We conclude that 9a-NBD-azithromycin can be used as a fluorescent analog of azithromycin to visualize its distribution in in vitro systems, and is also suitable for in vivo studies.
Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacokinetics , Azithromycin/chemistry , Azithromycin/pharmacokinetics , Fluorescent Dyes/chemistry , Fluorescent Dyes/pharmacokinetics , Animals , Anti-Bacterial Agents/pharmacology , Azithromycin/pharmacology , Bacteria/drug effects , Bacterial Infections/drug therapy , Cell Line , Cell Line, Tumor , Cell Survival/drug effects , Fluorescent Dyes/pharmacology , Humans , Male , Mice , Mice, Inbred BALB C , Microbial Sensitivity Tests , Structure-Activity Relationship , Tissue DistributionABSTRACT
In vitro models of adipogenesis are phenotypic assays that most closely mimic the increase of adipose tissue in obesity. Current models, however, often lack throughput and sensitivity and even report conflicting data regarding adipogenic potencies of many chemicals. Here, we describe a ten-day long adipogenesis model using high content analysis readouts for adipocyte number, size, and lipid content on primary human mesenchymal stem cells (MSC) sensitive enough to compare bisphenol A derivatives quantitatively in a robust and high throughput manner. The number of adipocytes was the most sensitive endpoint capable of detecting changes of 20% and was used to develop a benchmark concentration model (BMC) to quantitatively compare eight bisphenols (tested at 0.1-100 µM). The model was applied to evaluate mixtures of bisphenols obtaining the first experimental evidence of their additive effect on human MSC adipogenesis. Using the relative potency factors (RPFs), we show how a mixture of bisphenols at their sub-active concentrations induces a significant adipogenic effect due to its additive nature. The final active concentrations of bisphenols in tested mixtures reached below 1 µM, which is within the concentration range observed in humans. These results point to the need to consider the toxicity of chemical mixtures.
Subject(s)
Adipocytes/drug effects , Adipogenesis/drug effects , Benzhydryl Compounds/toxicity , High-Throughput Screening Assays/methods , Mesenchymal Stem Cells/drug effects , Phenols/toxicity , Adaptation, Biological , Adipocytes/physiology , Adipogenesis/physiology , Cell Differentiation , HumansABSTRACT
There is increasing evidence of a role for environmental contaminants in disrupting metabolic health in both humans and animals. Despite a growing need for well-understood models for evaluating adipogenic and potential obesogenic contaminants, there has been a reliance on decades-old in vitro models that have not been appropriately managed by cell line providers. There has been a quick rise in available in vitro models in the last ten years, including commercial availability of human mesenchymal stem cell and preadipocyte models; these models require more comprehensive validation but demonstrate real promise in improved translation to human metabolic health. There is also progress in developing three-dimensional and co-culture techniques that allow for the interrogation of a more physiologically relevant state. While diverse rodent models exist for evaluating putative obesogenic and/or adipogenic chemicals in a physiologically relevant context, increasing capabilities have been identified for alternative model organisms such as Drosophila, C. elegans, zebrafish, and medaka in metabolic health testing. These models have several appreciable advantages, including most notably their size, rapid development, large brood sizes, and ease of high-resolution lipid accumulation imaging throughout the organisms. They are anticipated to expand the capabilities of metabolic health research, particularly when coupled with emerging obesogen evaluation techniques as described herein.
Subject(s)
Adipocytes , Zebrafish , 3T3-L1 Cells , Adipocytes/metabolism , Adipogenesis , Animals , Caenorhabditis elegans , Cell Differentiation , Mice , Obesity/metabolismABSTRACT
Obesity is a multifactorial disease with both genetic and environmental components. The prevailing view is that obesity results from an imbalance between energy intake and expenditure caused by overeating and insufficient exercise. We describe another environmental element that can alter the balance between energy intake and energy expenditure: obesogens. Obesogens are a subset of environmental chemicals that act as endocrine disruptors affecting metabolic endpoints. The obesogen hypothesis posits that exposure to endocrine disruptors and other chemicals can alter the development and function of the adipose tissue, liver, pancreas, gastrointestinal tract, and brain, thus changing the set point for control of metabolism. Obesogens can determine how much food is needed to maintain homeostasis and thereby increase the susceptibility to obesity. The most sensitive time for obesogen action is in utero and early childhood, in part via epigenetic programming that can be transmitted to future generations. This review explores the evidence supporting the obesogen hypothesis and highlights knowledge gaps that have prevented widespread acceptance as a contributor to the obesity pandemic. Critically, the obesogen hypothesis changes the narrative from curing obesity to preventing obesity.
Subject(s)
Endocrine Disruptors , Adipogenesis , Adipose Tissue , Child, Preschool , Endocrine Disruptors/toxicity , Environmental Exposure/adverse effects , Humans , Obesity/etiologyABSTRACT
BACKGROUND AND PURPOSE: Efficacy of current antimalarial treatments is declining as a result of increasing antimalarial drug resistance, so new and potent antimalarial drugs are urgently needed. Azithromycin, an azalide antibiotic, was found useful in malaria therapy, but its efficacy in humans is low. EXPERIMENTAL APPROACH: Four compounds belonging to structurally different azalide classes were tested and their activities compared to azithromycin and chloroquine. in vitro evaluation included testing against sensitive and resistant Plasmodium falciparum, cytotoxicity against HepG2 cells, accumulation and retention in human erythrocytes, antibacterial activity, and mode of action studies (delayed death phenotype and haem polymerization). in vivo assessment enabled determination of pharmacokinetic profiles in mice, rats, dogs, and monkeys and in vivo efficacy in a humanized mouse model. KEY RESULTS: Novel fast-acting azalides were highly active in vitro against P. falciparum strains exhibiting various resistance patterns, including chloroquine-resistant strains. Excellent antimalarial activity was confirmed in a P. falciparum murine model by strong inhibition of haemozoin-containing trophozoites and quick clearance of parasites from the blood. Pharmacokinetic analysis revealed that compounds are metabolically stable and have moderate oral bioavailability, long half-lives, low clearance, and substantial exposures, with blood cells as the preferred compartment, especially infected erythrocytes. Fast anti-plasmodial action is achieved by the high accumulation into infected erythrocytes and interference with parasite haem polymerization, a mode of action different from slow-acting azithromycin. CONCLUSION AND IMPLICATIONS: The hybrid derivatives described here represent excellent antimalarial drug candidates with the potential for clinical use in malaria therapy.
Subject(s)
Antimalarials , Malaria , Animals , Antimalarials/pharmacology , Antimalarials/therapeutic use , Azithromycin/pharmacology , Azithromycin/therapeutic use , Chloroquine/pharmacology , Chloroquine/therapeutic use , Dogs , Malaria/drug therapy , Mice , Plasmodium falciparum , RatsABSTRACT
Given the speed of viral infection spread, repurposing of existing drugs has been given the highest priority in combating the ongoing COVID-19 pandemic. Only drugs that are already registered or close to registration, and therefore have passed lengthy safety assessments, have a chance to be tested in clinical trials and reach patients quickly enough to help in the current disease outbreak. Here, we have reviewed available evidence and possible ways forward to identify already existing pharmaceuticals displaying modest broad-spectrum antiviral activity which is likely linked to their high accumulation in cells. Several well studied examples indicate that these drugs accumulate in lysosomes, endosomes and biological membranes in general, and thereby interfere with endosomal pathway and intracellular membrane trafficking crucial for viral infection. With the aim to identify other lysosomotropic drugs with possible inherent antiviral activity, we have applied a set of clear physicochemical, pharmacokinetic and molecular criteria on 530 existing drugs. In addition to publicly available data, we have also used our in silico model for the prediction of accumulation in lysosomes and endosomes. By this approach we have identified 36 compounds with possible antiviral effects, also against coronaviruses. For 14 of them evidence of broad-spectrum antiviral activity has already been reported, adding support to the value of this approach. Presented pros and cons, knowledge gaps and methods to identify lysosomotropic antivirals, can help in the evaluation of many drugs currently in clinical trials considered for repurposing to target COVID-19, as well as open doors to finding more potent and safer alternatives.
Subject(s)
Antiviral Agents/therapeutic use , Betacoronavirus , Coronavirus Infections/drug therapy , Drug Repositioning , Lysosomes/drug effects , Pandemics , Pneumonia, Viral/drug therapy , Anti-Inflammatory Agents/pharmacokinetics , Antiviral Agents/adverse effects , Antiviral Agents/pharmacokinetics , Arrhythmias, Cardiac/chemically induced , Azithromycin/pharmacokinetics , Azithromycin/therapeutic use , COVID-19 , Chemical and Drug Induced Liver Injury/etiology , Chloroquine/pharmacokinetics , Chloroquine/therapeutic use , Computer Simulation , Drug Evaluation, Preclinical , Endosomes/drug effects , Humans , Hydrogen-Ion Concentration , Hydroxychloroquine/pharmacokinetics , Hydroxychloroquine/therapeutic use , Intracellular Membranes/physiology , Lysosomes/chemistry , Membrane Lipids/metabolism , Models, Biological , Phospholipids/metabolism , SARS-CoV-2 , Surface-Active Agents/pharmacokinetics , Virus Internalization , COVID-19 Drug TreatmentABSTRACT
Many marketed pharmaceuticals reach extremely high tissue concentrations due to accumulation in lysosomes (lysosomotropism). Quantitative prediction of intracellular concentrations of accumulating drugs is challenging, especially for macrocyclic compounds that mainly do not fit in current in silico models. We tested a unique library of 47 compounds (containing 39 macrocycles) specifically designed to cover the entire range of accumulation intensities observed with pharmaceuticals so far. For the first time, we show that intracellular concentration of compounds measured by liquid chromatography with tandem mass spectrometry correlates with the induction of phospholipidosis and inhibition of autophagy, but the highest correlation was observed with the increase of lysosomal volume (R = 0.95), all measured by high-throughput imaging assays. Based only on imaging data, we developed a 5-class in vitro model for the prediction of compound accumulation with the accuracy of 81%. The measured change of total lysosomal volume can thus be used in high-throughput screening for determination of the actual intensity of intracellular accumulation of new macrocyclic compounds. The models are largely based on macrocycles, greatly improving the screening and prediction of intracellular accumulation of this challenging class. However, all tested nonmacrocyclic compounds fitted well in the models, indicating potential use of the models in broader chemical space.
Subject(s)
Lysosomes/chemistry , Macrocyclic Compounds/metabolism , Animals , Cell Line , Cell Line, Tumor , Hep G2 Cells , High-Throughput Screening Assays/methods , Humans , Mice , Phospholipids/metabolism , RAW 264.7 CellsABSTRACT
Some cationic amphiphilic drugs (CADs) have been individually reported to interfere with the differentiation of immune system cells, such as macrophages and dendritic cells. To investigate the possible generic nature of this process, in this study we aimed to see whether these drugs are capable of interfering with the differentiation of adipocytes. Further, we investigated whether this feature might be connected to the lysosomotropic character of these drugs, and their disturbance of intracellular membrane trafficking rather than to the individual pharmacologic properties of each drug. Thus, for the selected set of compounds consisting of seven structurally and pharmacologically diverse CADs and three non-CAD controls we have measured the impact on differentiation of 3T3-L1K murine preadipocytes to adipocytes. We conclude that CADs indeed inhibit adipocyte differentiation, as shown morphologically, at the level of lipid droplet formation and on the expression of genetic markers of adipocytes. Furthermore, the intensity of this inhibitory effect was found to strongly positively correlate with the extent of drug accumulation in adipocytes, with their affinity for phospholipid membranes, as well as with their ability to induce phospholipidosis and inhibit autophagy.
Subject(s)
Adipocytes/drug effects , Autophagy/drug effects , Cell Differentiation/drug effects , Cell Membrane/drug effects , Hydrophobic and Hydrophilic Interactions , Lysosomes/drug effects , Phospholipids/metabolism , 3T3 Cells , Adipocytes/cytology , Adipogenesis/drug effects , Animals , Cell Membrane/metabolism , Gene Expression Regulation/drug effects , Lipid Droplets/drug effects , Lysosomes/metabolism , MiceABSTRACT
Many chemicals accumulate in organisms through a variety of different mechanisms. Cationic amphiphilic drugs (CADs) accumulate in lysosomes and bind to membranes causing phospholipidosis, whereas many lipophilic chemicals target adipose tissue. Perfluoroalkyl substances (PFASs) are widely used as surfactants, but many of them are highly bioaccumulating and persistent in the environment, making them notorious environmental toxicants. Understanding the mechanisms of their bioaccumulation is, therefore, important for their regulation and substitution with new, less harmful chemicals. We compared the highly bioaccumulative perfluorooctanesulfonic acid PFOS to its three less bioaccumulative alternatives perfluorooctanoic acid (PFOA), perfluorohexanoic acid (PFHxA) and perfluorobutane sulfonic acid (PFBS), in their ability to accumulate and remain in lung epithelial cells (NCI-H292) and adipocytes (3T3-L1K) in vitro. As a reference point we tested a set of cationic amphiphilic drugs (CADs), known to highly accumulate in cells and strongly bind to phospholipids, together with their respective non-CAD controls. Finally, all compounds were examined for their ability to bind to neutral lipids and phospholipids in cell-free systems. Cellular accumulation and retention of the test compounds were highly correlated between the lung epithelial cells and adipocytes. Interestingly, although an anion itself, intensities of PFOS accumulation and retention in cells were comparable to those of CAD compounds, but PFOS failed to induce phospholipidosis or alter lysosomal volume. Compared to other lipophilicity measures, phospholipophilicity shows the highest correlation (RË2â¯=â¯0.75) to cellular accumulation data in both cell types and best distinguishes between high and low accumulating compounds. This indicates that binding to phospholipids may be the most important component in driving high cellular accumulation in lung epithelial cells, as well as in adipocytes, and for both CADs and bioaccumulating PFASs. Obtained continuous PLS models based on compound's affinity for phospholipids and neutral lipids can be used as good prediction models of cellular accumulation and retention of PFASs and CADs.
Subject(s)
Alkanesulfonic Acids/metabolism , Fluorocarbons/metabolism , Lysosomes/metabolism , Pharmaceutical Preparations/metabolism , Phospholipids/metabolism , Adipocytes/cytology , Adipocytes/metabolism , Alkanesulfonic Acids/chemistry , Animals , Azithromycin/chemistry , Azithromycin/metabolism , Caproates/chemistry , Caproates/metabolism , Caprylates/chemistry , Caprylates/metabolism , Cations/chemistry , Cell Line , Cell Survival , Epithelial Cells/cytology , Epithelial Cells/metabolism , Fluorocarbons/chemistry , Humans , Least-Squares Analysis , Linear Models , Lipids/chemistry , Mice , Pharmaceutical Preparations/chemistry , Phospholipids/chemistry , Sulfonic Acids/chemistry , Sulfonic Acids/metabolismABSTRACT
The aim of this study was to investigate lipophilicity and cellular accumulation of rationally designed azithromycin and clarithromycin derivatives at the molecular level. The effect of substitution site and substituent properties on a global physico-chemical profile and cellular accumulation of investigated compounds was studied using calculated structural parameters as well as experimentally determined lipophilicity. In silico models based on the 3D structure of molecules were generated to investigate conformational effect on studied properties and to enable prediction of lipophilicity and cellular accumulation for this class of molecules based on non-empirical parameters. The applicability of developed models was explored on a validation and test sets and compared with previously developed empirical models.
Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacokinetics , Azithromycin/analogs & derivatives , Azithromycin/pharmacokinetics , Clarithromycin/analogs & derivatives , Clarithromycin/pharmacokinetics , Humans , Models, Biological , Models, Chemical , Molecular Conformation , Molecular Dynamics SimulationABSTRACT
Macrolones are a new class of antimicrobial compounds consisting of a macrolide scaffold linked to a 4-quinolone-3-carboxylic acid moiety via C(4â³) position of a macrolide. As macrolides are known to possess favorable pharmacokinetic properties by accumulating in inflammatory cells, in this study we determined the intensity of accumulation in human polymorphonuclear leukocytes (PMNs) of 57 compounds of the macrolone class and analyzed the relationship between the molecular structure and this cellular pharmacokinetic property. Accumulation of macrolones ranged from 0 to 5.5-fold higher than the standard macrolide azithromycin. Distinct structural features in all three considered molecule parts: the macrolide scaffold, quinolone moiety and the linker, affect cellular accumulation. Interestingly, while the parent macrolide, azithromycin, accumulates approximately 3-fold more than clarithromycin, among macrolones all clarithromycin derivatives accumulated in PMNs significantly more than their azithromycin counterparts. Modeling cellular accumulation of macrolones with simple molecular descriptors, as well as with the measured octanol-water distribution coefficient, revealed that the number of hydrogen bond donors and secondary amide groups negatively contribute to macrolone accumulation, while lipophilicity makes a positive contribution.
Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Macrolides/chemistry , Macrolides/pharmacology , Neutrophils/metabolism , Cells, Cultured , Humans , Structure-Activity RelationshipABSTRACT
Azithromycin, a macrolide antibacterial, has been shown to modify the phenotype of macrophages. We have investigated whether azithromycin in vitro is able to modulate the differentiation of human blood monocytes to DCs. iA-DCs appear to have a unique phenotype, characterized by increased granularity, adherence, and a surface molecule expression profile similar to that of MDCs, namely, CD1aâ»CD14â»CD71âºCD209(high), as well as high CD86 and HLA-DR expression. The iA-DC phenotype is associated with increased IL-6 and IL-10 release, increased CCL2 and CCL18 expression and release, and M-CSF expression, as well as reduced CCL17 expression and release. Upon maturation with LPS, A-DCs and MDCs exhibit decreased expression of HLA-DR and costimulatory molecules, CD40 and CD83, as well as an increase in IL-10 and a decrease in CCL17 and CXCL11 secretion. These modulated responses of iA-DCs were associated with the ability to reduce a MLR, together with enhanced phagocytic and efferocytotic properties. Azithromycin, added 2 h before activation of iDCs with LPS, enhanced IL-10 release and inhibited IL-6, IL-12p40, CXCL10, CXCL11, and CCL22 release. In conclusion, azithromycin modulates the differentiation of blood monocyte-derived DCs to form iA-DCs with a distinct phenotype similar to that of iMDCs, accompanied by enhanced phagocytic and efferocytic capabilities. It also modifies LPS-induced DC maturation by decreasing surface molecule expression required for T cell activation, increasing IL-10 production, and inducing MLR-reducing properties.