THE TOXICOLOGY-METABOLISM LINK. Cell Metabolism Assays. for. Toxicology. Research

Transcription

1 THE TOXICOLOGY-METABOLISM LINK Cell Metabolism Assays for Toxicology Research

2 SCREENING FOR TOXIC & ADVERSE PHENOTYPES USING FUNCTIONAL METABOLISM ASSAYS SCREENING The drug discovery process is evolving to demonstrate increased therapeutic agent efficacy using optimized methodologies, including correlating in vitro toxicity and in vivo outcomes. Researchers are leveraging Seahorse XF technology to identify potential adverse effects early in the drug discovery process. The Seahorse XF Cell Mito Stress Test measures the key parameters of respiration: basal respiration, proton leak, ATP-linked respiration, maximal respiration, and spare respiratory capacity. The Seahorse XF Glycolysis Stress Test measures the key parameters of glycolytic activity: glycolysis, glycolytic capacity, and glycolytic reserve. These XF assays are used to measure desired and adverse drug affects. / Extracellular Acidification Rate (ECAR) (% of DMSO) Ascorbate DMSO Sertraline Bisacodyl Clemastine Vinpocetine Nonoxynol-9 Meclizine Menadione Mefloquine Chlorhexidine Nifuroxazide Thonzonium Clofilium Alexidine Antimycin S glu/gal Treated Treated FCCP uncoupled Uncoupled Rate/Vehicle rate/vehicle Control control Number of Compounds Number of compounds STDV=3 STDV=1 MEAN STDV=-1 STDV= Human fibroblast cells (Gohil VM et al., 21. Nat Biotechnol.) Rabbit renal proximal tubular cells (Beeson CC et al., 21. Anal Biochem.) Seahorse XF assays identifies chemicals that significantly affect metabolism when plotted against cellular growth in galactose -relative to glucose-containing media. Seahorse XF assay predicts nephrotoxicity in kidney cells during a primary screening of the Library of Pharmacologically Active Compounds (LOPAC) HepG2 + K27 4 SH-SY5Y + K27 3 HepG2 + Anti-A 2 1 SH-SY5Y + Anti-A.1.1 Drug (nm) [acute] (% Normalized) Biotin Buspirone Clotrimazole Efavirenz Fluoxetine Fluvastatin Imatinib Nefazodone Nimesulide Phenformin Risperidone Rotenone Tolcapone Troglitazone FCCP Concentration (μm) Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Human liver carcinoma cells (HepG2); Human neuroblastoma cells (SH-SY5Y) (Prado A et al., 215. J Appl Toxicol.) Human liver carcinoma cells (Wang R et al., 215. J Biomol Screen.) Seahorse XF assay reveals drug toxicity in liver and neuronal cells. Seahorse XF assay is consistent and reproducible for evaluating the effect of multiple compounds on HepG2 cells.

3 THE WORLD S MOST ADVANCED METABOLIC ANALYZERS FOR TOXICOLOGY RESEARCH MODELS OF TOXICITY Determining a relevant in vitro model that can accurately recapitulate in vivo toxicity is crucial to preventing late-stage trial loss. By using Seahorse XF technology to assess functional metabolism, greater comprehensive knowledge about the drug effect can be gained, which is essential to a successful drug discovery program. 2 * (Cam) * Chloramphenicol Norfloxacin (Nor) 1 5 None Cam Bactericidal Antibiotics 3 ** (pmol/min/worms) 4 15 Combination 2 1 vehicle Dox. (μg/ml) Dox. (μg/ml) C. elegans E. coli (Moullan N et al., 215. Cell Rep.) (Lobritz MA et al., 215. PNAS.) Seahorse XF assay reveals doxycycline (Dox)-mediated mitochondrial dysfunction in C. elegans. Seahorse XF assay reveals connection between decreased respiration and killing arrest, in E. coli, confirming metabolomics data. XF DATA IN PUBLICATIONS There are over 2, references utilizing Seahorse XF technology published in leading journals such as Nature and Cell. Scientists are embracing Seahorse XF technology to identify metabolic phenotypes and reprogramming to target metabolic pathways for therapeutic purposes. Glucose Pyruvate Lactate IV III GLYCOLYSIS ECAR (Extracellular Acidification Rate) Cells generate ATP via glycolysis independent of II I MITOCHONDRIAL RESPIRATION OCR (Oxygen Consumption Rate) Mitochondria consume oxygen when oxidizing fatty acids or oxygen, producing lactic acid and other substrates to generate ATP. protons.seahorse XF Analyzers Seahorse XF Analyzers measure measure glycolysis by measuring mitochondrial respiration the extracellular acidification by measuring the oxygen rate (ECAR) of cells. consumption rate (OCR) of cells.

4 MEASURING THE KEY PARAMETERS OF FUNCTIONAL CELL METABOLISM DRUG TOXICITY While some therapeutic candidates are widely employed, there can be adverse effects linked to these drugs. Researchers are using Seahorse XF technology to understand the mechanisms associated with adverse effects in clinical situations. (pmol/min/5 x 1 4 cells) Oligomycin FCCP FCCP Human glioblastoma cells Rotenone/ (Funes HA et al., 215. J Antimicrob Chemother.) Vehicle EFV(1µm) EFV (µm) Seahorse XF Cell Mito Stress Test reveals an Efavirenz (EFV) -mediated dose response decline in mitochondrial respiration in human glioblastoma cells. ENVIRONMENTAL TOXICITY Exposure to environmental contaminants is a primary health concern. Mitochondria are a key component of cellular response to environmental pollutants. Seahorse XF technology is providing the tools to explore the toxicity of new chemicals Oligomycin FCCP Rat neuronal cells (Tiernan CT et al., 215. Toxicol Sci.) Control MeHg Rotenone Seahorse XF assay reveals mitochondrial dysfunction induced by methylmercury (MeHg) exposure in neuronal cells. Compound FCCP Oligomycin Rotenone/ (% of pre-dose) DMSO µm 1 1 µm µm Human liver-derived cells (Kenna JG et al., 215. J Pharmacol Exp Ther.) (Treatment % Control) A B C D E Rabbit renal proximal cells (Wills LP et al., 215. Toxicol Sci.) A = 2-methyl-4, 6-dinitrophenol B = perfluorooctanesulfonamide C = tributyltin D = pentachlorophenol E = didecldimethylammonium chlor Seahorse XF Cell Mito Stress Test reveals a dose-dependent reduction in mitochondrial respiration in human liver-derived cells (Huh-7) following treatment with sitaxentan, an endothelial receptor agonist. Seahorse XF assay identifies perfluorooctanesulfonamide, a pesticide and disinfectant, as a mitochondrial uncoupler. (pmol/min/1 x 1 4 cells) METH (mm) 4-MMC (mm) GBR1299 (μm) Seahorse XF Cell Mito Stress Test reveals a potential rescue of the mephedione-mediated reduction in neuronal cell maximal respiration. ** ** * Human neuroblastoma cells (den Hollander B et al., 214. Toxicol Sci.)

5 FUNCTIONAL METABOLIC ASSAYS THE INEVITABLE TOXICOLOGY-METABOLISM LINK The optimal paradigm for drug discovery includes eliminating ineffective drug candidates early in the process, rather than during the most costly and time-consuming phases of pre-clinical and clinical trials. Furthermore, the elimination of drug candidates goes beyond efficacy to identifying adverse effects, reliably predicting toxicity, and using relevant models that closely mimic the target host, is the ideal strategy. Mitochondria are at the nexus of cellular energy generation, intracellular signaling, and cell death and survival regulation. Therefore, compromised mitochondria, which strains the expansive and intricate cellular metabolic network, has been linked to various disease pathologies and etiologies. This valuable organelle presents a sensitive system to test for acute and toxic effects. With traditional cellular toxicology approaches representing only an acute study of drug toxicity, researchers are expanding their focus to include functional metabolism to predict toxicity prior to in vivo and patient studies. Seahorse XF technology is providing researchers the necessary tools to measure the functional, metabolic impact of their compound of interest faster and easier, using live cells. Drug and Environmental Toxicity Screening Functional Metabolism Toxicity Models SUBSTRATES AND METABOLISM GENES & METABOLISM LINK GLUCOSE FATTY ACID GLUCOSE LACTATE PYRUVATE FATTY ACID CITRATE PI3/AKT HIF1 GLYCOLYSIS p53 MYC Acetyl-CoA MYC HIF1 TCA HIF1 PYRUVATE LIPID SYNTHESIS ETC GLUTAMATE TCA PI3/AKT GLYCOLYSIS (ECAR) MITOCHONDRIAL RESPIRATION (OCR) GLUTAMINE MYC GLUTAMINOLYSIS

6 GOLD STANDARD METABOLIC ASSAYS MEASURING THE KEY PARAMETERS OF CELL METABOLISM Seahorse XF Cell Mito Stress Test Profile Mitochondrial Respiration Oligomycin Basal Respiration ATP Production FCCP Rotenone & Maximal Respiration Proton Leak Non-mitochondrial Oxygen Consumption Spare Time (minutes) Seahorse XF Glycolysis Stress Test Profile Extracellular Acidification Rate (ECAR) (mph/min) Glycolytic Function Glucose Glycolysis Oligomycin Glycolytic 2-DG Non-glycolytic Acidification Glycolytic Reserve Time (minutes) Seahorse XF Cell Energy Phenotype Test Metabolic Phenotype & Potential Seahorse XF Mito Fuel Flex Test Profile Mitochondrial Function 1 Mitochondrial Respiration Aerobic Baseline Phenotype Quiescent Metabolic Potential Glycolysis Energetic Stressed Phenotype Glycolytic Extracellular Acidification Rate (ECAR) % GLC+GLN+FA Fuel Oxidation Dependency Flexibility Dependency Glucose Pathway Glutamine Pathway Fatty Acid Flexibility Dependency Corporate Headquarters European Headquarters Asia-Pacific Headquarters Seahorse Bioscience Inc. 16 Esquire Road North Billerica, MA 1862 US Phone: Seahorse Bioscience Europe Fruebjergvej 3 21 Copenhagen DK Phone: Seahorse Bioscience Asia 199 Guo Shou Jing Rd, Suite 27 Pudong, Shanghai 2123 CN Phone: Rev All rights reserved. Seahorse Bioscience and the Seahorse logo are trademarks of Seahorse Bioscience Inc. The content in this brochure is for informational purposes only and may be subject to change without notice.