Uncover New Drug Targets Through Cell Metabolism

Uncover New Drug Targets Through Cell Metabolism Drug Target eBook Cellular Metabolism A New Avenue in Drug Discovery Topics Drug target identification is a key step in the discovery and development of safe and effective therapies. Often thought of as simply providing ‘housekeeping’ functions, energy metabolism is now emerging as a critical contributor to many cellular functions. Dysfunctional metabolism is also associated with a growing number of different disease states. Therefore, looking at the genes, proteins, and pathways that modulate energy metabolism is a promising new avenue for developing novel therapeutic strategies for a broad range of diseases. This handy reference eBook provides examples of metabolic targets impacting key disease areas including: - Cancer - Immuno-oncology/Immunotherapy - Neurodegenerative diseases - Diabetes, Cardiovascular and Acquired Metabolic Disorders We hope that this eBook will inspire you to look at your therapeutic strategy in a new way, through the lens of energy metabolism. To learn how you can incorporate Agilent solutions for measuring energy metabolism into your target identification workflows, see more here: www.agilent.com/chem/drugdiscovery-cellmetabolism 3 Cancer Altered energy metabolism is now recognized as a hallmark of cancer1 . Identification of targets affecting metabolic pathways, therefore, provides critical insights into potential effective cancer therapies. 4 Target: Oncogenes, associated molecular targets and metabolic pathways Oncogenes are responsible for rewiring cancer cell energy metabolism towards biomass accumulation and glycolytic energy production, rather than the mitochondrial energy production that occurs in nonproliferating cells. Intermediates in oncogene pathways (including genes, proteins, and enzymes) represent promising targets for cancer therapies. Target: Nutrient transport and utilization To support biomass accumulation, cancer cells increase substrate and nutrient uptake from the tumor microenvironment. Potential metabolic targets may focus on inhibiting or activating genes, proteins, and pathways associated with substrate and nutrient transport or utilization. Phenogram: Simultaneous measurement of oxidative phosphorylation and glycolysis 40 OCR (pmol/min) OCR (pmol/min) ECAR (mpH/min) ECAR (mpH/min) glycolytic glycolytic Oxidative Chemoresistance isogenic counterpart Chemosensitive Oxidative 150 50 40 100 30 30 50 10 0 0 0 20 40 60 80 0 10 20 30 Energetic Energetic A2780 PEO1 C200 Learn more Determine substrate dependency of cancer Learn more cell models Time (min) Inhibitor Oligo FCCP Rot/AA 0 24 48 72 96 120 OCR (pmol/min/1,000 Cells) 1 2 3 4 5 6 7 8 9 10 0 Cancer Significant advances have been made in defining druggable targets in cancer cells, with metabolic intermediates being highlighted as key targets in cancer drug discovery. PEO4 A549 Mitochondrial Respiration Control Etomoxir (LCF As) UX5099 (Glucose/Pyruvate) BPTES (Glutamine) Adapted from Dar, S., et al. Bioenergetic Adaptations in Chemoresistant Ovarian Cancer Cells. Sci Rep. 2017. 7 (1): 8760. 5 Table 1 lists several promising metabolic targets for cancer therapies. The stage of development and effectiveness of these metabolic targets range from preclinical to clinical and postclinical and represent areas of opportunity for drug target identification and validation. Target ID Pathway/Function Description References GLUT1 Glycolysis, glucose transport Glucose transporter 2 xCT Glutamine, glutamate metabolism - cystine acquisition for GSH synthesis Cystine-glutamate antiporter encoded by SLC7A11 gene 3 MCT1 Lactate metabolism Monocarboxylate transporter encoded by SLC16A1 gene 4-6 GAPDH Glycolysis Glycolytic enzyme 7 LDHA Glycolysis, NAD/NADH balance Glycolytic enzyme, catalyses pyruvate-lactate conversion 8-13 GLS Glutamine metabolism Amidohydrolase enzyme, catalyzes glutamine-glutamate conversion 14-18 CPT1 Long-chain fatty acid transport/oxidation Mitochondrial enzyme, catalyzes transfer of an acyl group of a long chain fatty acyl-CoA from coenzyme A to L-carnitine 19 PDK1 Pyruvate metabolism/oxidation Protein kinase that regulates pyruvate dehydrogenase (PDH) activity 20 MGLL Fatty acid synthesis Monoacylglycerol lipase, catalyze hydrolysis of monoacilglycerides into glycerol and long-chain fatty acids 21 HIF1 Regulates the hypoxic response Hypoxia-inducible transcription factor 22 mTOR Regulates cell proliferation, transcription, translation, and autophagy Kinase encoded by MTOR gene 23-24 Table 1. Examples of promising metabolic targets for cancer therapies. Therapeutic targeting of metabolic synthetic lethality in cancer research “The rapid proliferation of cancer cells requires simply more energy, ATP, and macromolecules. Therefore cells have to rewire their metabolic network to meet such demands. That opens many potential opportunities for targeting tumor metabolism in cancer therapy.” – Yuting Sun, PhD Senior Research Scientist, MD Anderson Cancer Center View eSeminar Cancer 6 Immuno-oncology / Immunotherapy Modulating the activity of immune cells for the treatment of a variety of pathologies such as cancer or autoimmune diseases is one of the fasting growing areas in drug discovery. Metabolic reprogramming plays a key role in the immediate response of the immune cell, as well as directing cell fate. Different immune cell types have unique metabolic requirements, to support energetic and biosynthetic demands. * * * * 7 The balance of nutrients and metabolites in the tumor microenvironment can significantly affect metabolic programs, altering immune cell response. Targeting critical metabolic pathways is emerging as a promising strategy to regulate immune function and has been shown to strongly influence the efficacy of immune checkpoint as well as adoptive CAR-T therapies. Monitor real-time immune cells response to checkpoint blockade Bezafibrate enhances the effectiveness of PD-L1 blockade in antitumor immunity by activating mitochondrial respiration of killer T cells to promote survival and proliferation and improve effector function. Adapted from Chowdhury, P. S. et al. PPAR-Induced Fatty Acid Oxidation in T Cells Increases the Number of Tumor-Reactive CD8(+) T Cells and Facilitates Anti-PD-1 Therapy. Cancer Immunol Res. 2018. 6 (11): 1375-1387. Time (min) FCCP Rotenone/ Antimycin A Oligomycin OCR (pmol/min) 200 150 100 50 0 0 20 40 60 80 100 120 anti-PD-L1 Bezafibrate ECAR(mpH/min) OCR (pmol/min) Spare respiratory capacity (pmol/min) 120 80 40 0 + – – – + + 0 0 30 35 40 45 50 60 70 80 Learn more Immuno-oncology / Immunotherapy Ctrl IgG anti-PD-L1 anti-PD-L1 + bezafibrate 8 Monitor immune cell fitness and function Core metabolic pathways dictate potency and persistence in adoptive T cell therapy “There’s a huge interest in how energetic metabolism really regulates the core immune function. Much of that work has really come from our understanding and study of T cells: how metabolism regulates T cell differentiation, and T cell effector function.” – John Connolly, PhD Chief Scientific Officer, Tessa Therapeutics Ltd Research Director, Institute of Molecular and Cellular Biology (IMCB) Cellular bioenergetics plays a critical role in determination of fate of engineered immune cells. Cell Fate Tuning CAR Engineering CAR Engineering CD28, TCR 4-1BB TCR Immune Memory Persistence Central Memory Oxidative Phosphorylation Tumor Killing Effector memory Aerobic glycolysis T cell Cell Fate Tuning Activated Vehicle anti-CD3/CD28 Baseline Fit Oligo FCCA R/A Ovarian Cancer Relative OCR (%) Time (min) Ascites (%) Fitness Exhausted 200 150 100 50 30 60 90 120 0 0 Real-time Activation Injection of anti-CD3 and anti-CD28 activator Fate & Persistence Fitness Learn more View eSeminar Immuno-oncology / Immunotherapy Time (min) Time (min) Oligo FCCA R/A Ovarian Cancer ECAR (mpH/min) Relative OCR (%) Vehicle anti-CD3/CD28 05 0 10 20 200 30 150 100 50 0 0 30 60 90 120 0 1001 50 Ascites (%) Fit Exhausted 9 Mitochondrial dysfunction has been implicated as the most common cause of neurodegenerative disease, which is primarily linked to dysfunctional respiration (OXPHOS), altered homeostasis of metabolic intermediates and trigger of cell death mechanisms25. Identification of specific proteins affecting mitochondrial metabolic function provides insight into potential drug targets for neurodegenerative disease. Neurodegenerative Diseases 10 Mitochondria are cellular powerhouses responsible for the production of energy (ATP) and the regulation of several other processes that are critical to cellular homeostasis. Neuronal cells have high energy demands and limited regeneration capabilities; consequently, these cells are highly dependent on mitochondrial function for survival. Indeed, there is increasing evidence that mitochondrial proteins and mitochondrial dysfunction are responsible for the onset of neurodegenerative diseases, including Alzheimer’s, Parkinson’s, Huntington’s, and amyotrophic lateral sclerosis (ALS). Target: Mitochondrial proteins and intermediates in mitochondrial dysfunction Studies measuring metabolic pathways activity and intermediates indicate that mitochondrial dysfunction is the primary cause of neurodegenerative disease. Metabolic targets and key driving factors are attributed to inherited mutations (mtDNA mutations), mitochondrial membrane permeability and potential disruption, disrupted mitochondrial fusion or fission, impaired protein and ion homeostasis, reactive oxygen species or accumulation of toxic aggregates, and dysfunctional mitophagy. Neurodegenerative Diseases Mitochondrial Spare Respiratory Capacity provides a strong indicator of neurodegenerative disease Adapted from Theurey, P., et al. Systems biology identifies preserved integrity but impaired metabolism of mitochondria due to a glycolytic defect in Alzheimer's disease neurons. Aging Cell. 2019. 18 (3): e12924. Learn more OCR (pmol O/min/µg protein) OCR (pmol O2/min/µg protein) Time (min) Non-mitochondrial OCR Basal Maximal OCR Spare capacity Oligo FCCP Anti A ATP synthesis H leak Oligo FCCP Anti A Basal WT WT ** *** TgAD TgAD H+ lcak ATP synthesis Maximal OCR Non-mito. OCR Spare capacity 0 4 8 12 16 20 25 20 15 10 5 20 40 60 0 0 ** 11 Table 2 lists some mitochondrial proteins that have been studied as potential targets for neurodegenerative disorders. Mitochondrial proteins represent a relatively new target for drug development compared to other drug targets in neurodegenerative diseases. Although few therapies targeting mitochondrial proteins have reached the stage of clinical trials, they represent a promising area of opportunity for novel targets. Target ID Pathway/Function Description Diseases References CypD Mitochondrial function/health Peptidyl prolyl isomerase F, associates with the inner mitochondrial membrane during the mitochondrial membrane permeability transition (mPTP formation) Alzheimer's, Parkinson's, amyotrophic lateral sclerosis (ALS) 26–28 PINK1 PInk1/Parkin pathway, mitophagy Mitochondria-specific kinase PTEN Induced Kinase 1 Parkinson's 29 LRRK2 Cytoplasmic GTPase and kinase activites Member of the leucine-rich repeat kinase family Parkinson's 30–32 Drp-1 Mitophagy GTPase that regulates mitochondrial fission Parkinson's 33–34 DJ-1 Cellular oxidative stress response Redox-sensitive chaperone/sensor for oxidative stress, encoded by PARK7 gene Parkinson's 35 ABAD Oxidation of isoleucine, branched-chain fatty acids, xenobiotics, sex hormones, and neuroactive steroids Mitochondrial enzyme encoded by the HSD17B10 gene Alzheimer's 36–39 MPC Pyruvate oxidation Mitochondrial pyruvate carrier Neurodegeneation due to excitotoxic injury 40 SOD1 Antioxidant enzyme, detoxification of ROS in cells Superoxide dysmutase isoform present in the cytoplasm and mitochondrial intermembrane space, encoded by SOD1 Amyotrophic lateral sclerosis (ALS) 41–42 Table 2. Examples of promising metabolic targets for neurodegenerative disorders. Mitochondrial pyruvate metabolism controls neuronal function and excitotoxic death “There’s quite a bit of history about the limitations that we have had over the years, basically up until the development of high-throughput respirometry, in what we could learn, and certainly the speed in which we could gather data.” –Anne N. Murphy – Ajit S. Divakaruni, PhD Asst. Professor of Molecular and Medical Pharmacology, University of California, Los Angeles – Anne N. Murphy, PhD Professor of Pharmacology, University of California, San Diego Neurodegenerative Diseases View eSeminar Environmental factors relating to excess nutrients and stress are key drivers of obesity, leading to further complications, including type 2 diabetes and cardiovascular disease. Modulation of metabolic targets to restore the balance of energy homeostasis to “normal” represents a promising therapeutic strategy for reversing or preventing these damaging syndromes. 12 Diabetes, Cardiovascular, and Other Acquired Metabolic Disorders 13 Different tissues are designed to use different nutrients to meet their energy demands. Excessive nutrient and substrate availability, or long-term exposure to the wrong balance of substrates have a cascade of effects, including increased adipose tissue, inflammation, and insulin resistance. Identifying those proteins active in energy substrate uptake and utilization, including metabolic pathways and intermediates, are rich sources of potential drug targets. Target: Lipid versus glucose metabolism, and insulin resistance There is a negative feedback between lipid metabolism and glucose metabolism as an energy source. The energy balance between fatty acid oxidation and glucose oxidation (including reducing mitochondrial uncoupling) is affected by the activity of proteins and enzymes involved in substrate uptake and utilization. Diabetes, Cardiovascular, and Other Acquired Metabolic Disorders Natural products that expand and induce Brown adipose tissue using innovative Stem Cell Metabolism Assays. – Shahzad Ali, PhD, Senior Research Scientist, Plasticell Limited Illustrative Discovery Workflow Step 1: Validate Phenotype of in vitro Model of Brown Adipose Tissue (BAT) Step 2: Screen for Metabolic Activators of BAT Step 3: Select and Re-characterise Leads for Mechanism of Action and Development Agilent MitoXpress Xtra Oxygen Consumption Assay: Identify Hits increasing basal OXPHOS Agilent Seahorse XF Cell Mito Stress Test: Higher metabolic activity and proton leak, characteristic of BAT Example Hit compound with increased basal and spare respiratory capacity View eSeminar OCR (pmol/min) Time (min) 1µM Antimycin 0.2% DMSO extract 8 OCR 0 20 40 60 80 100 100 90 80 70 60 50 40 30 20 10 00 0 2 4 6 8 10 12 800 600 400 200 Time (min) ADSC-d BAI Antimycin A Rotenone FCCP Oligomycin ADSC-d WAT OCR (pmol/min) 0 0 50 100 150 200 14 Table 3 lists proteins that have been studied previously as potential targets for metabolic disorders relating to obesity, diabetes, and cardiovascular disease. Target ID Pathway/Function Description References PPARα Controls expression of (hepatic) genes involved in fatty acid uptake and intracellular transport, fatty acid oxidation, lipogenesis, ketogenesis, and lipoprotein/cholesterol metabolism (in adipocytes) Ligand activated nuclear receptor/transcription factor 43 PPARγ Regulation of ADD, function, insulin sensitivity, lipogenesis, lipid storage, and glucose metabolism Ligand activated nuclear receptor/transcription factor 44 AMPK AMPK activation results in stimulation of hepatic / skeletal fatty acid oxidation, ketogenesis, and glucose uptake, inhibition of FA synthesis, lipogenesis, and modulation of insulin secretion ADP-activated protein kinase, a key enzyme in cellular energy homeostasis 45 GLUT4 Glycolysis/glucose oxidation Insulin-regulated glucose transporter 46, 47 CPT1 Long chain fatty acid transport/oxidation Mitochondrial enzyme, catalyzes transfer of an acyl group of a long chain fatty acyl-CoA from coenzyme A to L-carnitine 45 MPC Pyruvate oxidation Mitochondrial pyruvate carrier, therapeutic target for modulating energy balance and the metabolic profile 48 UCPs ETC/OXPHOS Family of mitochondrial proteins present mainly in brown adipose tissue, uncouples ETC from OXPHOS 49 Table 3. Examples of promising metabolic targets for diabetes, cardiovascular, and other aquired metabolic disorders. Impaired mitochondrial ß-oxidation in cardiomyocytes: role of the cardiac renin-angiotensin system and miR-208 “The pathogenic mechanisms of diabetic cardiomyopathy are complex. What is central in the diabetic heart are changes in it’s energy substrate utilization and metabolism.” – Margriet Ouwens Ph.D. German Diabetes Center, DDZ Institute for Clinical Biochemistry and Pathobiochemistry, Düsseldorf Diabetes, Cardiovascular, and Other Acquired Metabolic Disorders View eSeminar 15 Explore how you can incorporate Agilent solutions for measuring energy metabolism into your drug discovery workflows Discover Agilent Cell Analysis Publications Visit the eSeminars web page to view past webinars on-demand Learn how to run our assays Meet with an Expert Useful Resources 16 Citations 1. Hanahan, D., & Weinberg, RA. (2011) Hallmarks of Cancer. The Next Generation. Cell. 144(5), 646-674. 2. Chan, D. A. et al. (2011) Targeting GLUT1 and the Warburg effect in renal cell carcinoma by chemical synthetic lethally. Sci. Transl. Med. 3, 94ra70. 3. Timmerman, L. A. et al. (2013) Glutamine sensitivity analysis identifies the xCT antiporter as a common triple-negative breast tumor therapeutic target. Cancer Cell. 24, 450–465. 4. Polanski, R. et al. (2014) Activity of the monocarboxylate transporter 1 inhibitor AZD3965 in small cell lung cancer. Clin. Cancer Res. 20, 926-937. 5. Birsoy, K et al. (2013) MCT1-mediated transport of a toxic molecule is an effective strategy for targeting glycolytic tumors. Nature Genet. 45, 104–108. 6. Sonveaux, P. et al. (2008) Targeting lactate-fueled respiration selectively kills hypoxic tumor cells in mice. J. Clin. Invest. 118, 3930–3942. 7. Ganapathy-Kanniappan, S. et al. (2013) Anticancer efficacy of the metabolic blocker 3-bromopyruvate: specific molecular targeting. Anticancer Res. 33, 13–20 8. Boudreau, A. et al. (2016) Metabolic plasticity underpins innate and acquired resistance to LDHA inhibition. Nat. Chem. Biol. 12, 779–786. 9. Cui, W. et al. (2016) Discovery of 2-((3-cyanopyridin-2-yl) thio) acetamides as human lactate dehydrogenase A inhibitors to reduce the growth of MG-63 osteosarcoma cells: virtual screening and biological validation. Bloorg. Med. Chem. Lett. 26, 3984–3987. 10. Billiard, J. et al. (2013) Quinoline 3-sulfonamides inhibit lactate dehydrogenase A and reverse aerobic glycolysis in cancer cells. Cancer Metab. 1, 1-19. 11. Rai, G. et al. (2017) Discovery and optimization of potent, cell active pyrazole-based inhibitors of lactate dehydrogenase (LDH). J. Med. Chem. 55, 3285–3306. 12. Ward, R.A. et al. (2012) Design and synthesis of novel lactate dehydrogenase A inhibitors by fragment-based lead generation.J. Med. Chem. 55, 3285–3306. 13. Le, A. et al. (2010) Inhibition of lactate dehydrogenase A induces oxidative stress and inhibits tumor progression. Proc. Natl. Acad. Sci. 107, 2037–2042. 14. Xiang, Y. et al. (2015) Targeted inhibition of tumor-specific glutaminase diminishes cell-autonomous tumorigenesis. J. Clin. Invest. 125, 2293–2306. 15. Shroff, E. H. et al. (2015) MYC oncogene overexpression drives renal cell carcinoma in a mouse model through glutamine metabolism. Proc. Natl. Acad. Sci. 112, 6539–6544. 16. Gross, M.I. et al. (2014) Antitumor activity of the glutaminase inhibitor CB839 in triple-negative breast cancer. Mol. Cancer Ther. 13, 890–901. 17. Wang, J. B. et al. (2010) Targeting mitochondrial glutaminase activity inhibits oncogenic transformation. Cancer Cell 18, 207–219. 18. Seltzer, M. J. et al. (2010) Inhibition of glutaminase preferentially slows growth of glioma cells with mutant IDH1. Cancer Res. 70, 8981–8987. 19. Pike, L. S. et al. (2011) Inhibition of fatty acid oxidation by etomoxir impairs NADPH production and increases reactive oxygen species resulting in ATP depletion and cell death in human glioblastoma cells. Biochim. Biophys. Acta 1807, 726–734. 20. Michelakis, E. D., Webster, L. & Mackey, J. R. (2008) Dichloroacetate (DCA) as a potential metabolic-targeting therapy for cancer Br. J. Cancer 99, 989–994. 21. Nomura, D. K. et al. (2010) Monoacylglycerol lipase regulates a fatty acid network that promotes cancer pathogenesis Cell 140, 49–61. 22. Wilson, W. R. & Hay, M. P. (2011) Targeting hypoxia in cancer therapy. Nature Rev. Cancer 11, 393–410. 23. Sabatini, D. M. (2006) mTOR and cancer, insights into a complex relationship. Nature rev. Cancer 6, 729–734. 24. Benjamin, D. et al. (2011) Rapamycin passes the torch a new generation of mTOR inhibitors. Nature Rev. Drug Discov. 10, 868–880. 25. Lee, J. (2016) Mitochondrial drug targets in neurodegenerative diseases. Bioorganic & Medical Chemistry Letters. 26, 714-720. 17 26. Guo, H. X. et al. (2005) Novel cyclophilin D inhibitors derived from quinoxalineexhibit highly inhibitory activity against rat mitochondrial swelling and Ca2+ uptake/release Acta Pharmacol. Sin. 26, 1201–1211. 27. Valasani, K. R. et al. (2014) Design, synthesis, in silico and in vitro studies of novel 4-methylthiazole-5-carboxylic acid derivatives as potent anti-cancer agents Bioorg. Med. Chem. Lett. 24(18), 4580–4585. 28. Elkamhawy, A. et al. (2014) Novel quinazoline-urea analogues as modulars for Aβ-induced mitochondral dysfunction: design, synthesis, and molecular docking study. Med. Chem. 84, 466–475. 29. Hertz, N. T. et al. (2013) A neo-substrate that amplifies catalytic activity of parkinson's-disease-related kinase PINK1. Cell. 154, 737–747. 30. Li, T. et al. (2015) A Novel GTP-Binding Inhibitor, FX2149, Attenuates LRRK2 Toxicity in Parkinson's Disease Models. PLoS One. 10, e0122461. 31. Li, T. & Yang, D. et al. (2014) Discovery of highly potent, selective, and brainpenetrant aminopyrazole leucine-rich repeat kinase 2 (LRRK2) small molecule inhibitors. Med. Chem. 57, 921–936 32. Estrada, A. A. et al. (2014) Discovery of highly potent, selective, and brainpenetrant aminopyrazole leucine-rich repeat kinase 2 (LRRK2) small molecule inhibitors. Med. Chem. 57, 921–936. 33. Qi, X. et al. (2013) A novel Drp 1 inhibitor diminishes aberrant mitochondrial fission and neurotoxicity. Cell. Sci. 126, 789–802. 34. Lackner, L. L. & Nunnari, J. (2010) Small molecule inhibitors of mitochondrial divisionsn tools that translate basic biological research into medicine. Chem. Biol. 17, 578–583. 35. Kitamura, Y. et al. (2011) Neuroprotective effect of a new DJ-1-binding compound against neurodegeneration in Parkinson's disease and stroke model rats. Mol. Neurodegener. 6(48), 1–19. 36. Kissinger, C. R. et al. (2004) Molecular dynamics simulations of the amyloidbeta binding alcohol dehydrogenase (ABAD) enzyme. Mol. Biol. 16(21), 9511–9518. 37. Lim, Y.T. et al. (2011) Inhibitation of the mitochondrial enzyme ABAD resotres the amyloid-β-mediated deregulation of estradiol. PLoS One.6, e28887. 38. Valaasani, K.R. et al. (2014) Identification of human ABAD inhibitors for rescuing Aβ-mediated mitochondrial dysfunction. Curr. Alzeimer Res.11, 128-136. 39. Ayan, D., Maltais, R, & Prior, D. (2012) Identification of a 17β-hydroxysteroid dehydrogenase type 10 steroidal inhibitor: a tool to investigate the role of type 10 in Alziheimer’s disease and prostate cancer. ChemMedChem.7, 1181-1184. 40. Divakaruni, A. S. et al. (2019) Inhibition of the nitochondrial pyruvate carrier protects from excitotoxic neuronal death. J Cell Biol. 4, 1091-1105. 41. Kalmar, B. et al. (2008) Cellular toxicity of mutant SOD1 protein is linked to an easily soluble, non-aggregated from in vito. Neurobiol Dis. 49, 49-56. 42. Lange, D. J. et al. (2013) Pyrimethamine decreases levels of SOD1 in leukocytes and cerebrospinal fluids of ALS patients: a phase I plot study. Amytroph lateral Scler Frontotemporal Desener. 14(3) 199-204. 43. Han et al. (2017) PPARs: regulators of metabolism and as therapeutic targets in cardiovascular disease. Part I: PPAR-α Future Cardiol. 13(3), 259-278. 44. Han, L. et al. (2017) PPARs: regulators of metabolism and as therapeutic targets in cardiovascular disease. Part Ii: PPAR- β/ δ and PPAR- γ. Future Cardiol. 13(3), 279-296. 45. Fukshima, A. et al. (2015) Myocardial Energy Substrate Metabolism in Heart Failure: from Pathways to Therapeutic Targets. Current Pharmaceutical Design. 21,3654-3664. 46. Schreiber, I, et al. (2017) BMPs as new insulin sensitizers: enhanced glucose uptake in mature 3T3-L1 adipocytes via PPAR gamma and GLUT4 upregulation. Sci Rep.7 (1), 17192. 47. Bhowmik, A. & Banu, S. (2017) Therapeutic targets of type 2 diabetes: an overview. MOJ Drug Des Develop Ther. 1(3), 00011. 48. Divakaruni, A,S. et al. (2013) Thiazolidinediones are acute, specific inhibitors of the mitochondrial pyruvate carrier. PNAS. 110(14), 5422-5427. 49. Samudio, I. et al. (2019) Mitochondrial Uncoupling and the Warbug Effect. Molecular basis for the Reprogrmming of Cancer Cell Metabolism. Cancer Res 69(6), 2163-2166. Learn more www.agilent.com/chem/drugdiscovery-cellmetabolism Live Chat www.agilent.com/chem/discoverXF U.S. and Canada 1 800 227 9770 agilent_inquiries@agilent.com Europe UK 0800 096 Denmark 45 8830 5083 Germany 0800 180 66 78 Netherlands 0800 022 7243 Other EU 45 3136 9878 info_agilent@agilent.com Asia Pacific China 800 820 3278 Singapore 65 6571 0888 inquiry_lsca@agilent.com For Research Use Only. Not for use in diagnostic procedures. DE.6191666667 This information is subject to change without notice. © Agilent Technologies, Inc. 2020 Published in the USA, June 18, 2020 5994-2059EN