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CAYMANCURRENTS ISSUE 31 | WINTER 2019

FATTY LIVER DISEASE (NAFLD/NASH)

Targeting Resistance for the Metabolic Homeostasis Targets Treatment of NASH Page 6 Page 1 Special Feature Inside: Tools to Study NAFLD/NASH A guide to PPAR function and structure Page 3

Pathophysiology of NAFLD Infographic Oxidative Stress, Inflammation, and Apoptosis Targets Page 5 Page 11

1180 EAST ELLSWORTH ROAD · ANN ARBOR, MI 48108 · (800) 364-9897 · WWW.CAYMANCHEM.COM Targeting for the Treatment of NASH Kyle S. McCommis, Ph.D. Washington University School of Medicine, St. Louis, MO The obesity epidemic has resulted in a dramatic escalation Defects in fat secretion do not appear to be a driver of in the number of individuals with hepatic fat accumulation hepatic steatosis, as NAFLD subjects display greater VLDL or steatosis. When not combined with excessive alcohol secretion both basally and after “suppression” by insulin.5 consumption, the broad term for this spectrum of disease Fatty acid β-oxidation is decreased in animal models and is referred to as non-alcoholic fatty liver disease (NAFLD). humans with NAFLD/NASH.6-8 A significant proportion of individuals with simple steatosis will progress to the severe form of the disease known as ↑ Hyperinsulinemia Insulin resistance non-alcoholic steatohepatitis (NASH), involving hepatocyte Hyperglycemia damage, inflammation, and fibrosis. If left untreated, Adipose lipolysis NASH can lead to more severe forms of liver disease such Steatosis as cirrhosis, hepatocellular carcinoma, liver failure, and De novo O ↑ lipogenesis HO eventually necessitate liver transplantation. Due to this large clinical burden, research efforts have greatly expanded Fatty acids to better understand NAFLD pathogenesis and the mechanisms underlying the transition to NASH. Likewise, there are currently no approved agents for treating NAFLD/ NASH, thus efforts to identify therapeutic targets and progress drug development have intensified in recent years. Figure 1. Insulin resistance drives hepatic steatosis. Metabolic syndrome and insulin resistance are the most significant risk factors for the development of NAFLD and PPARs as Drug Targets NASH. Indeed, 60-75% of type 2 diabetic subjects have Because insulin resistance is a direct driver of hepatic lipid NAFLD.1,2 Many aspects of the pathophysiology of insulin accumulation and NASH pathology, the insulin sensitizing resistance are directly causal to NAFLD development compounds (TZDs) were one of the (Figure 1). Insulin-resistant adipose tissue is unable to earliest classes of drugs to be tested in NASH. appropriately inhibit lipolysis, and therefore, plasma free and have both been used in animal models fatty acid delivery to the liver is increased. In exquisite and human trials of NASH and improve almost all aspects labeling experiments, plasma free fatty acids were shown to of NASH pathology.9-15 These TZD compounds are comprise the source of ~60% of liver (TAG) in agonists of the nuclear transcription factor peroxisome NAFLD subjects.3 Inadequate insulin signaling also results proliferator-activated receptor γ (PPARγ).16 PPARγ regulates in increased insulin secretion from pancreatic β-cells and a gene expression program for adipocyte differentiation hyperinsulinemia, which can stimulate hepatic de novo and fatty acid storage. While lipid “sequestration” into lipogenesis.4 Whether derived from free fatty acids or adipose tissue could be protective against hyperlipidemia, de novo lipogenesis, these lipids are largely converted to this PPARγ activation with TZDs is associated with a acyl-CoAs by acyl-CoA synthetases, complexed to glycerol- number of side effects such as weight gain, edema, and 3-phosphate to create diacylglyceride, and lastly, a third bone mineral density loss and fracture risk,17 which have acyl-CoA is added by diacylglycerol acyltransferase (DGAT) greatly diminished the clinical use of TZDs. Interestingly, to form TAG. This TAG, as well as other lipids and sterols, pioglitazone is a much weaker agonist of PPARγ compared are then stored inside lipid droplets. These lipid-bound to rosiglitazone,16 but provides superior improvements organelles are thought to safely store and control the in NASH histology, particularly with regards to fibrosis hydrolysis of these lipids. While somewhat controversial, improvement.18 These results have opened the door to accumulation of other lipid intermediate species such as the possibility that TZDs could have additional target(s) diacylglycerols, ceramides, and acyl-CoAs in the liver has responsible for their pharmacology. Indeed, it was recently been linked to worsening insulin resistance. Other potential observed that TZDs can bind and inhibit the mitochondrial mechanisms for fat accumulation in hepatocytes include pyruvate carrier (MPC), which regulates pyruvate transport decreased fat oxidation and decreased fat secretion in across the inner mitochondrial membrane into the the form of very low-density lipoprotein (VLDL) particles. mitochondrial matrix.19-21 PPARγ-sparing TZD compounds

Cayman Currents 1 Issue 31, Winter 2019 that retain full ability to bind the MPC (MSDC-0602K and inhibition of DGAT-1 or -2 in animal models has been shown MSDC-0160) have been developed.19,22 MSDC-0602K was to improve hepatic steatosis and fibrosis, as well as hepatic recently shown to improve NASH pathology in a mouse and global insulin sensitivity.31-35 Intriguingly, DGAT-2 model23 and is currently in a Phase 2B clinical trial for inhibition was reported to improve steatosis but exacerbate NASH. Inhibiting the MPC may improve NASH pathology hepatic injury in the methionine-choline deficient mouse by improving insulin sensitivity, enhancing fatty acid model of NASH.36 Nevertheless, reducing DGAT expression oxidation, attenuating the upregulated flux of carbon into or activity is still actively being explored to treat NASH, the tricarboxylic acid (TCA) cycle,6,24-26 and reducing hepatic with both small molecule inhibitors and antisense stellate cell activation.23 oligonucleotides in clinical development. Lastly, analogs of Non-TZD compounds have also been developed, which the small secreted peptide encoded in the mitochondrial agonize other PPAR isoforms such as PPARα and PPARδ, genome known as MOTS-c have been developed, which with or without PPARγ activation. In theory, these produce effects similar to insulin action, namely inhibition compounds may combine the insulin-sensitizing effects of of adipose tissue lipolysis. These MOTS-c analogs (CB4209 37 targeting PPARγ with the increased fat oxidation effects of and CB4211) improved NASH histology in a mouse model PPARα and the anti-inflammatory and fatty acid oxidation and will likely progress to clinical trials in the near future. effects of PPARδ. (PPARα/δ agonist) and IVA337/lanifibranor (pan-PPAR agonist) are currently in Phase 2, while (PPARα/δ agonist) is in Phase 3 trials for NASH.

Non-PPAR Drug Targets A number of compounds for other targets that improve insulin sensitivity are currently being developed for NASH. Adipose (TAG) Specific Agonists of the (FXR) have been MPC MPC inhibitors Improved insulin Citrate observed to improve insulin sensitivity and provide anti- sensitivity Mitochondria inflammatory and antifibrotic effects in diabetic patients DNL Acyl-CoA Plasma FFA with NASH.27 is a synthetic analog, which is the predominant compound for FXR DAG Multi- or pan- DGAT inhibitor PPARγ DGAT-1/2 agonism. However, a trial for obeticholic acid in NASH was PPAR agonists or antisense oligonucleotide terminated before completion due to low treatment efficacy PPARα PPARδ TAG and side effects such as pruritis and modest LDL Bile acids, FXR 28 synthetic FXR agonists, elevations. FXR agonism for NASH will likely depend on and FGFs Nucleus Lipid droplets whether newer synthetic agonists lacking the bile acid structure display benefits without similar side effects. Synthetic derivatives of fibroblast growth factors FGF19 and FGF21 can also regulate hepatic FXR and PPARγ Figure 2. Hepatic targets of insulin sensitizing therapies in development for NASH. coactivator 1 (PGC-1) activity and lead to improved insulin sensitivity and improvements in NAFLD/NASH29 but require Conclusions and Future Outlook larger, longer term trials. These pharmacologic agents that likely have hepatocyte-specific actions in addition to their As insulin resistance and derangements in lipid metabolism overall improved insulin sensitivity are depicted in Figure 2. are so intricately linked to the development of NAFLD, The glucagon-like peptide-1 (GLP-1) receptor agonist it is likely that targeting insulin sensitivity will provide predominantly promotes insulin secretion from the broadest spectrum of pharmacology for treating this pancreatic β-cells but can also improve insulin sensitivity.30 disease. While research efforts and compound development GLP-1 receptor agonists provide additional metabolic are active for downstream pathology such as cell stress/ effects, such as weight loss, that could be beneficial in death, inflammation, or antifibrotic compounds, it is NASH. However, these require further clinical testing. hard to envision successful therapeutics that do not also Inhibitors of DGAT-1 or DGAT-2 could potentially improve address the core mechanisms of hepatic lipid accumulation. NASH by preventing TAG formation and forcing enhanced fatty acid oxidation. Indeed, pharmacologic or genetic Continued on Page 3

(800) 364-9897 www.caymanchem.com 2 However, the presence and degree of hepatic fibrosis is the number one predictor of adverse outcomes in NASH,38 and therefore treatment strategies will need to prevent About the Author or reverse fibrosis. Deciphering how altered metabolism Kyle S. McCommis, Ph.D. results in hepatocellular stress and death, inflammation, Dr. McCommis is a research scientist and increased fibrogenesis requires greater research effort. KM Better understanding this transition from steatosis to NASH, who integrates molecular biology as well as how therapeutics reverse this pathology, remain and physiology to study metabolic disease. the ultimate goals for this area of research. In addition to better understanding disease

Article References pathophysiology, his lab investigates the 1. Bazick, J., Donithan, M., Neuschwander-Tetri, B.A., et al. Clinical model for NASH and advanced fibrosis in adult patients with diabetes and NAFLD: Guidelines for referral in NAFLD. Diabetes mechanisms and efficacy of novel therapeutics. Care 38(7), 1347-1355 (2015). 2. Portillo-Sanchez, P., Bril, F., Maximos, M., et al. High prevalence of nonalcoholic fatty liver disease His research training began in cardiovascular in patients with mellitus and normal plasma aminotransferase levels. J. Clin. Endocrinol. Metab. 100(6), 2231-2238 (2015). physiology, and his projects in the lab still involve 3. Donnelly, K.L., Smith, C.I., Schwarzenberg, S.J., et al. Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease. J. Clin. Invest. 115(5), mitochondrial metabolism in heart failure. 1343-1351 (2005). 4. Ferré, P. and Foufelle, F. Hepatic steatosis: A role for de novo lipogenesis and the transcription Since joining Washington University in 2013, factor SREBP-1c. Diabetes Obes. Metab. 12(Suppl. 2), 83-92 (2010). 5. Poulsen, M.K., Nellemann, B., Stødkilde-Jørgensen, H., et al. Impaired insulin suppression of Kyle has also focused on hepatic mitochondrial VLDL-triglyceride kinetics in nonalcoholic fatty liver disease. J. Clin. Endocrinol. Metab. 101(4), 1637-1646 (2016). metabolism in relation to both the pathology and 6. Satapati, S., Sunny, N.E., Kucejova, B., et al. Elevated TCA cycle function in the pathology of diet-induced hepatic insulin resistance and fatty liver. J. Lipid Res. 53(6), 1080-1092 (2012). treatment of diabetes and fatty liver disease. He 7. Rector, R.S., Thyfault, J.P., Uptergrove, G.M., et al. Mitochondrial dysfunction precedes insulin resistance and hepatic steatosis and contributes to the natural history of non-alcoholic fatty liver is currently an Assistant Professor of Medicine disease in an obese rodent model. J. Hepatol. 52(5), 727-736 (2010). 8. Koliaki, C., Szendroedi, J., Kaul, K., et al. Adaptation of hepatic mitochondrial function in humans and will be opening a lab in the Saint Louis with non-alcoholic fatty liver is lost in steatohepatitis. Cell Metab. 21(5), 739-746 (2015). University Department of Biochemistry and Molecular Biology in May 2019. View continued article references on page 12

Tools to Study NAFLD/NASH FEATURED NAFLD ASSAY KIT Alanine Transaminase Colorimetric Activity Assay Kit 1.0 Item No. 700260 0.9 Measure ALT activity in serum, plasma, tissue samples, 0.8 and cell lysates 0.7

Assay 47 samples in duplicate 0.6

Measure ALT activity down to 0.006 U/ml Absorbance (340 nm ) 0.5 Plate-based colorimetric measurement (340 nm) 0.4 0 1 2345 Time (minutes)

Metabolism Assays Item No. Product Name Measure Sample Type(s)

10009381 Adipolysis Assay Kit Adipolysis of Preadipocytes (e.g., differentiated 3T3-L1 cells)

10007640 Cholesterol Fluorometric Assay Kit Total cholesterol Plasma, serum

700310 Free Fatty Acid Fluorometric Assay Kit Free fatty acids Plasma, serum, urine Extracellular glycerol (triglyceride/fatty acid 10011725 Glycerol Cell-Based Assay Kit Cultured cells cycling rate) Insulin (human) ELISA Kit - 26619 Insulin Cell culture supernatant, plasma, serum Manufactured by Bertin Bioreagent

Cayman Currents 3 Issue 31, Winter 2019 Metabolism Assays (continued) Item No. Product Name Measure Sample Type(s) α-Ketoglutarate Fluorometric Intracellular and extracellular 701350 Cultured cells Detection Assay Kit α-ketoglutarate concentrations 10010303 Triglyceride Colorimetric Assay Kit Triglyceride levels Cell lysates, tissue homogenates, plasma, serum Uric acid (end product of human purine 700320 Uric Acid Assay Kit Plasma, serum, urine metabolism)

Cell-Based Imaging Assays Item No. Product Name Measure Sample Type(s)

10006908 Adipogenesis Assay Kit Lipid droplets Preadipocytes (e.g., differentiated 3T3-L1 cells) Cholesterol Cell-Based Detection 10009779 Cholesterol uptake and localization Cultured cells Assay Kit 500001 Lipid Droplets Fluorescence Assay Kit Lipid droplets Cultured cells

10011125 LDL Uptake Cell-Based Assay Kit LDL uptake Cultured cells

10012643 Steatosis Colorimetric Assay Kit Excessive lipid accumulation Cultured cells

Lipid Droplets Fluorescence Assay Kit LDL Uptake Cell-Based Assay Kit A

B Fluorescent Probes Item No. Product Name Localization Live Cell/Tissue Compatible Fixed Cell Compatible

25892 BODIPY 493/503 Lipid droplets

25893 BODIPY 505/515 Lipid droplets

70440 CFilipin III DFree cholesterol 25073 LDLFilipin uptak Complexe in Huh7 human hepatVariousocyt sterolses. Huh7 cells were plated at 1 x 104 cells/well in a 96 well plate and allowed to attach and growLipid for droplets 24 hour ands. LDL-DylightTM 550 was added in serum-free medium at a 25911 IraZolve-L1™ dilution of 1:100 and incubatedendoplasmic overnight. reticulum Nuclei were stained by addition of Hoechst 33342 (available as Item No. 15547) directly to the mediumPolar lipids andto a lipid-rich final concentration of 5 µM and incubation for 30 min at 37°C. 25907 ReZolve-L1™ LDLR staining was completed compartmentsas described in the kit booklet. Fluorescent images (200X) were captured using a Nikon Eclipse microscope equipped with a Roper Scientific camera and MetaMorph software and Standards merged in ImageJ. Panel A: LDLR staining in green, Panel B: LDL uptake in red, Panel C: Hoechst in blue, Panel D: a merged image. Item No. Product Name Purity Item No. Product Name Purity

® 24919 -d11 MaxSpec Standard ≥95% 25546 Cholesterol-d6 ≥99% (d1-d6)

22787 C16 Ceramide-d7 (d18:1-d7/16:0) ≥99% (d1-d7) 25265 Cholesterol-d7 ≥99% (d1-d7)

22788 C18 Ceramide-d7 (d18:1-d7/18:0) ≥99% (d1-d7) 10005057 Docosahexaenoic Acid-d5 ≥99% (d1-d5)

24379 C18 dihydro Ceramide (d18:0/18:0) ≥98% 10005056 Eicosapentaenoic Acid-d5 ≥99% (d1-d5)

24428 C18 dihydro Ceramide-d3 (d18:0/18:0-d3) ≥99% (d1-d3) 9002193 Linoleic Acid-d11 ≥99% (d1-d11)

22789 C24 Ceramide-d7 (d18:1-d7/24:0) ≥99% (d1-d7) 10008650 1-Stearoyl-2-Arachidonoyl-sn-Glycerol ≥95%

22790 C24:1 Ceramide-d7 (d18:1-d7/24:1(15Z)) ≥99% (d1-d7) 10009872 1-Stearoyl-2-Arachidonoyl-sn-Glycerol-d8 ≥99% (d1-d8)

(800) 364-9897 www.caymanchem.com 4 Therapeutically Targeting the Pathophysiology of NAFLD An ideal drug candidate for NAFLD/NASH should 1) positively regulate abnormal lipid metabolism to reduce steatosis, hepatic inflammation, and injury; 2) improve underlying insulin resistance; and 3) induce antifibrotic responses. To aid in the pre-clinical discovery and optimization of novel compounds for the treatment of these diseases, Cayman has curated a large set of tools targeting the nuclear transcription factors, nuclear receptors, G protein-coupled receptors, and proteins of interest to this field.

CCR2-CCR5 antagonists

Steatosis Hepatitis ibrosis Cirrhosis

etasm nammatn Caen CC rutn Ce Stress Atss

· LOXL2 inhibitors · Galectin-3 inhibitors · Vitamin E · Hsp47 inhibitors · Other antioxidants · PPAR agonists · MPC inhibitors · ASK1 inhibitors · PGC-1α activators · Caspase inhibitors Dey a · DGAT inhibitors SGLT2 · GLP-1 receptor inibitors Antibiotics agonists Probiotics atera ruts · FXR ligands entns · DPP-4 inhibitors Det aryrateat · AMPK activators Digestive lipase · ACC inhibitors inibitors use eretn · SCD1 inhibitors · Leptin · FASN inhibitors · SREBP antagonists

EXPLORE PRODUCTS TARGETING NAFLD PATHOPHYSIOLOGY Find biochemicals, assay kits, proteins, and antibodies on the following pages: HNF4α PPAR DGAT-1/2 ACC FFAR1 (GPR40) TRβ 6 PGC-1α 7 GLP-1 8 SCD1 9 10 FFAR4 (GPR120) LXR MPC DPP-4 FASN Antioxidants TGR5 & FXR AMPK Leptin 11 Apoptosis Inhibitors SREBP Chemokine Receptor SGLT2 Antagonists

Cayman Currents 5 Issue 31, Winter 2019 Metabolic Homeostasis Targets

PPAR PGC-1α Peroxisome proliferator-activated receptors (PPARs) Peroxisome proliferator-activated receptor-γ have a critical role as master regulators of lipid and coactivator-1α (PGC-1α) is a metabolic sensor glucose metabolism and demonstrate anti-inflammatory involved in the expression of genes important to properties in the liver. PPAR agonist-targeted therapies gluconeogenesis, fatty acid oxidation, lipid transport, may be beneficial for NASH, but their effectiveness is and mitochondrial biogenesis. Activators of PGC-1α still emerging. may help to regulate energy homeostasis during the development of NAFLD/NASH. Agonists Item No. Product Name Target Inhibitor and Activator 21106 Amorfrutin A PPARγ Item No. Product Name Action 19618 Amorfrutin B PPARγ 22084 SR 18292 Inhibitor 71730 PPARγ 14121 ZLN005 Agonist 23508 Elafibranor PPARα/δ 11908 GQ-16 PPARγ PGC-1α Gene Expression Target 13689 GW 1929 PPARγ Item No. Product Name 25572 Lanifibranor pan-PPAR 11451 Irisin (human recombinant) 18293 MHY908 PPARα/γ 14625 Irisin Polyclonal Antibody 10007853 PPARα/γ 71745 Pioglitazone PPARγ 71740 Rosiglitazone PPARγ MPC 11086 SR 1664 PPARγ The mitochondrial pyruvate carrier (MPC) plays a role via 71750 PPARγ the mTOR pathway in sensing and directing the effects of overnutrition on liver pathology that drive NAFLD. TZDs Single-Step Screening Assays can directly interact with MPC and attenuate its activity. Mitochondrial targets of TZD insulin sensitizers (mTOTs) PPARγ Ligand Screening Assay Kit are being developed as inhibitors of the MPC. Item No. 10007685 mTOT Insulin Sensitizer PPARγ-LBD Ligand Screening Assay Kit Item No. Product Name Item No. 600616 71748 MSDC-0160 MPC Inhibitors Discover our Guide to PPAR Function & Item No. Product Name Structure at the end of this issue! 23445 7ACC2 16980 UK 5099

Targeting Mitochondrial Substrate Oxidation Pathways UK 5099 (+)- (sodium salt) BPTES Item No. 16980 Item No. 11969 Item No. 19284 Mitochondrial pyruvate carrier Carnitine palmitoyltransferase 1 Glutaminase 1 (GLS1) inhibitor (MPC) inhibitor (CPT1) inhibitor Prevents conversion of glutamine Prevents pyruvate transport into Prevents fatty acyl-CoA transport into glutamate the mitochondria into the mitochondria Shifts energy substrate utilization Shifts energy substrate utilization Shifts energy substrate utilization from glutamine oxidation to fatty from glucose to amino acid and from fatty acid oxidation to glucose acids and pyruvate fatty acid oxidation and amino acids

(800) 364-9897 www.caymanchem.com 6 DGAT-1/2 DPP-4 Diacylglycerol acyltransferase-1 and -2 (DGAT-1/2), Dipeptidyl peptidase 4 (DPP-4) degrades incretins such which catalyze the final step in hepatocyte triglyceride as GLP-1. DPP-4 inhibitors can reduce glucagon and biosynthesis, enable triglyceride accumulation in lipid glucose levels and increase insulin secretion. They also droplets during early stages of NAFLD. Inhibitors of improve hepatic steatosis by downregulating SREBP-1c, these enzymes may help to improve hepatic steatosis. SCD1, and FASN and upregulating PPARα and AMPK in Inhibitors the liver. Item No. Product Name Target Inhibitors 10012708 A-922500 DAGT-1 Item No. Product Name 17774 Amidepsine A DAGT 23768 (benzoate salt) 17775 Amidepsine D DAGT 21454 MK-3102 19440 Cochlioquinone A DAGT 23697 (hydrochloride) 16425 PF-04620110 DAGT-1 13252 (–)- (phosphate) 17680 PF-06424439 DAGT-2 18504 25807 T-863 DAGT-1 14705

Convenient Screening Assay GLP-1 Glucagon-like peptide 1 (GLP-1)—an intestinal peptide DPP (IV) Inhibitor Screening Assay Kit that exerts its effects through the GLP-1 receptor—can Item No. 700210 increase glucose-dependent insulin secretion, stimulate Screen for DPP (IV) inhibitors β-cell proliferation, inhibit glucagon secretion, and Includes sitagliptin as a positive control inhibitor increase satiety. GLP-1 receptor agonists may decrease Plate-based fluorometric measurement hepatic lipogenesis in incidences of NAFLD. (ex 530-540 nm, em 585-595 nm) Agonists Item No. Product Name 11096 Exendin-4 (acetate) AMPK 24727 Liraglutide AMP-activated protein kinase (AMPK) maintains energy homeostasis by decreasing ATP-consuming GLP Peptides pathways and increasing ATP-producing pathways. Item No. Product Name AMPK activators inhibit de novo lipogenesis in the liver, 24755 GLP-1 (1-36) amide (human, rat) (trifluoroacetate salt) increase fatty acid oxidation in the liver, and promote GLP-1 (1-37) (human, rat, mouse, bovine) 24460 mitochondrial function in adipose tissue. (trifluoroacetate salt) 15069 GLP-1 (7-36) amide (trifluoroacetate salt) Activators 24414 GLP-2 (human) (trifluoroacetate salt) Item No. Product Name 24756 GLP-2 (1-34) (human) (trifluoroacetate salt) 11900 A-769662 24757 GLP-2 (rat) (trifluoroacetate salt) 10010241 AICAR 14741 AMPK activator 10631 Ampkinone CUSTOM ORGANIC SYNTHESIS 19155 GSK621 13118 (hydrochloride) 17118 Niclosamide (ethanolamine salt) · Process scale-up (mg to 0.5 kg) 10006726 PD 98059 · Stable isotopic labeling 21335 PT1 · Conjugation chemistry 70970 U-0126 19475 YLF-466D Learn more at www.caymanchem.com/services 21644 ZLN024

Cayman Currents 7 Issue 31, Winter 2019 ACC Leptin Acetyl-CoA carboxylase (ACC) converts acetyl-CoA to Leptin—a cytokine-type hormone mainly produced by malonyl-CoA, which contributes to de novo lipogenesis adipocytes—plays a role in modulating lipid deposition in and helps to regulate fatty acid oxidation. AMPK the liver. Because some patients with NASH demonstrate phosphorylation of ACC or pharmacological inhibitors leptin deficiency, recombinant leptin therapy has been of ACC prevents de novo lipogenesis and increases explored in clinical trials. mitochondrial fatty acid oxidation. Assay Kits Inhibitors Manufactured by Bertin Bioreagent Item No. Product Name Targets Item No. Product Name 17691 CP 640,186 ACC1 and ACC2 500010 Leptin (human) EIA Kit 23961 ND-630 ACC1 and ACC2 10007609 Leptin (mouse/rat) EIA Kit 21778 PF-05175157 ACC1 and ACC2 10007608 Leptin Receptor (human) EIA Kit 10005263 TOFA ACC and fatty acid synthesis

SREBP SCD1 Sterol regulatory element‐binding protein (SREBP) Stearoyl-CoA desaturase 1 (SCD1) is the rate-limiting regulates intracellular cholesterol and lipid levels by enzyme in the biosynthesis of oleic acid and palmitoleic promoting the transcription of genes required for acid. Inhibitors of SCD1 may increase fatty acid their synthesis. Inhibitors of SREBP may control the oxidation by activating AMPK in the liver, preventing potentially lipotoxic role of hepatic cholesterol in NASH. triglyceride accumulation in the liver during NASH. Inhibitors and Agonist Inhibitors Item No. Product Name Action Item No. Product Name 11041 Betulin Inhibitor 19123 A-939572 13562 Fatostatin (hydrobromide) Inhibitor 22478 Aramchol 22206 MBX-2982 Agonist 10012562 CAY10566 16161 PluriSln 1 Assay Kits and Antibodies 19379 SW203668 Item No. Product Name 10007417 10-Thiastearic Acid 10010854 SREBP-1 Transcription Factor Assay Kit 10007663 SREBP-2 Polyclonal Antibody 22728 SREBP-2 Polyclonal Antibody - Biotinylated FASN 10007819 SREBP-2 Transcription Factor Assay Kit Fatty acid synthase (FASN) catalyzes the last step in fatty acid biosynthesis and is thought to be a major determinant of the maximal hepatic capacity to generate SGLT2 fatty acids by de novo lipogenesis. Inhibiting FASN may Sodium-glucose cotransporter 2 (SGLT2) inhibitors improve hepatic steatosis and inflammation. decrease glucose reabsorption in renal proximal tubules, which has dual effects of insulin-independent glycemic Inhibitors control and caloric loss that may help to delay the onset Item No. Product Name of NASH. 10005270 (±)-C75 10005647 Cerulenin Inhibitors 19957 Fasnall (benzenesulfonate) Item No. Product Name 19098 GSK837149A 11574 20236 Icaritin 17375 20940 ML-356 23509 (hydrate) 10005263 TOFA 19090 UCM05

(800) 364-9897 www.caymanchem.com 8 HNF4α TGR5 & FXR Hepatocyte nuclear factor 4α (HNF4α), a nuclear G protein-coupled bile acid receptor (TGR5 or GP-BAR1) hormone receptor expressed in liver, controls the basal and farnesoid X receptor (FXR) have roles as master expression of genes involved in bile acid, lipid, glucose, regulators of carbohydrate and lipid metabolism, glucose and drug metabolism. HNF4α antagonists can be used homeostasis, bile acid homeostasis, inflammation, and modulate the expression of known HNF4α target genes. fibrosis. Activators of these receptors may remediate steatosis pathogenesis. Antagonists Item No. Product Name Agonists 12032 BI-6015 Item No. Product Name Target 12031 BIM5078 11686 Betulinic Acid TGR5 20756 Deoxycholic Acid TGR5 9003230 Glycine-β-muricholic Acid TGR5 TRβ 20274 Glycodeoxycholic Acid TGR5 The thyroid hormone receptor β (TRβ) is expressed 21723 Glycolithocholic Acid TGR5 10006611 GW 4064 FXR predominantly in the liver and the brain and functions to 20294 Hyodeoxycholic Acid TGR5 modulate cholesterol and liver triglycerides levels. TRβ 17678 INT-777 TGR5 agonists have shown sustained reduction in liver fat and 20253 Lithocholic Acid TGR5 other markers of NASH in clinical trials. Taurochenodeoxycholic Acid 20275 TGR5 (sodium salt) FEATURED PRODUCT 15935 Taurodeoxycholic Acid (sodium salt) TGR5 22669 Taurohyocholate TGR5 MGL-3196 21956 Taurohyodeoxycholic Acid TGR5 Item No. 23845 20277 Tauroursodeoxycholic Acid TGR5 20289 Tauro-β-muricholic Acid (sodium salt) TGR5 TRβ agonist that lowers non-HDL cholesterol and liver 16291 TGR5 Receptor Agonist TGR5 triglycerides without affecting thyroid stimulating hormone 15121 Ursodeoxycholic Acid (sodium salt) TGR5 levels in rat and rabbit models of hypercholesterolemia 17369 FXR CN 25748 FXR

O 23350 XL335 FXR H N

O N Cl N N N H Scalable Screening Assay O

O TGR5 (GP-BAR1) Reporter Assay Kit

Cl Item No. 601440 No stable cell line required, simply add regular HEK293 or HEK293T cells to the plate the day LXR before assaying Liver X receptors (LXRs) are closely related to Positive control agonist included intrahepatic inflammation and fibrosis. Liver-specific Obtain results within 24-30 hours after adding cells inverse agonists and antagonists are being studied for Amenable to high-throughput screening antifibrosis effects in NASH. 2.0 x 107

Antagonist and Inverse Agonist ) Agonist Positive Control 1.5 x 107 Lithocholic Acid Item No. Product Name Action Betulinic Acid 25443 GSK2033 LXRα and LXRβ antagonist Ursolic Acid 1.0 x 107 Oleanolic Acid INT-777 18771 SR9238 LXRα and LXRβ inverse agonist Deoxycholic Acid CDCA 5.0 x 106 CRE-SEAP Activity (RLU

0 -9 -8 -7 -6 -5 -4 Log[Agonist], M

Cayman Currents 9 Issue 31, Winter 2019 FFAR1 (GPR40) FFAR4 (GPR120) Free fatty acid receptor 1 (GPR40) is activated by Free fatty acid receptor 4 (GPR120) is activated by long-chain fatty acids and several unsaturated fatty both medium-chain and long-chain fatty acids. It is acids such as palmitoleic and docosahexaenoic acid. It important for the differentiation of adipocytes and induces the release of incretins and demonstrates direct plays roles in metabolic balance and the suppression insulinotropic and anti-inflammatory effects. of inflammation. Agonists and Antagonists Agonists and Antagonists Item No. Product Name Action Item No. Product Name Action 21815 AMG 837 Partial agonist 17713 AH 7614 Antagonist 13143 CAY10587 Agonist 20602 Docosadienoic Acid methyl ester Agonist 19388 DC260126 Antagonist 20749 13Z,16Z-Docosadienoic Acid Agonist 10008908 GW 1100 Antagonist 16624 GPR120 Compound A Agonist 10008907 GW 9508 Agonist 17398 Grifolic Acid Partial agonist 24586 2-hydroxy Lauric Acid Partial agonist 17712 GSK137647A Agonist 17335 TAK-875 Agonist 17035 TUG-891 Agonist Assay Kit and Antibodies Item No. Product Name Assay Kit and Antibody 14265 FFAR4 (GPR120) (C-Term) Polyclonal Antibody Item No. Product Name 14904 FFAR4 (GPR120) (Internal) Polyclonal Antibody 10007205 FFAR1 (GPR40) Polyclonal Antibody 15130 FFAR4 (GPR120) (N-Term) Polyclonal Antibody 601190 FFAR1 (GPR40) Reporter Assay Kit 601200 FFAR4 (GPR120) Reporter Assay Kit

H

FEATURED PRODUCT H N Long-lasting gastrointestinal lipase inhibitor — O O O blocks intestinal absorption of dietary fat O O Orlistat Item No. 10005426

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(800) 364-9897 www.caymanchem.com 10 Oxidative Stress, Inflammation, and Apoptosis Targets Antioxidants Item No. Product Name Summary

25091 Calycosin An isoflavone that scavenges 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals

24960 Chicoric Acid A dicaffeoyl ester that reduces blood glucose levels and upregulates antioxidant enzymes A flavonoid that reduces hydrogen peroxide-induced production of reactive oxygen species 25197 Didymin and increases superoxide dismutase, glutathione peroxidase, and catalase activity 70935 (−)-Epigallocatechin Gallate A phenol that decreases oxidation of deoxyguanosine and tyrosine nitration An antihyperuricemic nonpurine inhibitor of both the oxidized and reduced forms of 14127 Febuxostat xanthine oxidase 10005167 An isoflavinoid shown to attenuate hepatic steatosis and inflammation

10739 Isoliquiritigenin A flavonoid that induces nitric oxide synthase and reduces hepatic fat and triglyceride accumulation

19131 NK 252 An Nrf2 activator that activates the NQO1-antioxidant response element A hepatoprotective antioxidant that reduces free radical production and reverses hepatocyte 23720 Tiopronin degeneration 25985 (±)-α-Tocopherol A biologically active form of vitamin E that protects cellular membranes from oxidative damage A pentacyclic triterpenoid with antioxidant properties that reverses increases in hepatic 10072 Ursolic Acid steatosis and levels of hepatic triglycerides and free fatty acids

Anti-Inflammatories Item No. Product Name Summary

14181 Amlexanox An inhibitor of TANK-binding kinase 1 An orally bioavailable anti-inflammatory agent that inhibits 5-lipoxygenase and phosphodiesterases 11948 MN-001 3 and 4 11726 Oleanolic Acid A triterpenoid with anti-inflammatory, anti-hyperlipidemic, and hepatoprotective in vivo effects

18720 Pentoxifylline A methylxanthine derivative shown to have anti-inflammatory activity

70675 trans- A polyphenolic phytoalexin with anti-inflammatory effects A steroid sapogenin that reduces proteinuria and renal macrophage infiltration and decreases 25217 Ruscogenin gene expression of inflammatory cytokines

Apoptosis Inhibitors Item No. Product Name Summary

22204 Emricasan A pan-caspase inhibitor

16226 NQDI-1 A specific inhibitor of apoptosis signal-regulating kinase 1

16209 p-nitro-Pifithrin-α An inactivator of p53

20972 Selonsertib An orally bioavailable inhibitor of apoptosis signal-regulating kinase 1

22083 XMU-MP-1 Inhibits pro-apoptotic, sterile 20-like kinases MST1 and MST2

Chemokine Receptor Antagonists Item No. Product Name Summary

23927 An orally bioavailable antagonist of C-C chemokine receptor type 5

17330 RS 504393 A selective antagonist of the MCP-1 receptor CCR2

Cayman Currents 11 Issue 31, Winter 2019 Additional Inhibitors and Compounds of Interest Item No. Product Name Summary

25990 APC 366 (trifluoroacetate salt) Reduces collagen content and expression of PAR-2 and α-SMA A phytochemical that reduces hepatic collagen expression, serum levels of ALT, 25214 Decursin AST, and ALP, and production of ROS Diethyl 1,4-dihydro-2,4,6-trimethyl-3, An inhibitor of ferrochelatase that induces Mallory-Denk body formation in animal 23230 5-pyridinedicarboxylate models of alcoholic hepatitis and NASH 14173 Naringenin A flavonoid that directly down-regulates TGF-β1 An orally bioavailable pyridone derivative that inhibits fibrosis and increases in 13986 Pirfenidone hepatocyte apoptosis, lobular inflammation, and hepatic expression of fibrosis- related genes 21681 Solithromycin A macrolide antibiotic that may improve histological parameters of NASH An orally bioavailable inhibitor of plasminogen activator inhibitor 1 that protects 22965 TM5441 against high-fat diet-induced NAFLD

Convenient Monitoring Assays Item No. Product Name Summary

501030 Interleukin-6 (human) ELISA Kit Measure IL-6 from human plasma, serum, synovial fluid, and other sample matrices

705002 Lipid Hydroperoxide (LPO) Assay Kit Measure hydroperoxides directly utilizing the redox reactions with ferrous ions

701600 Mitochondrial ROS Detection Assay Kit Fluorometrically assess mitochondrial ROS directly in living cells

601290 ROS Detection Cell-Based Assay Kit (DHE) Fluorometrically measure ROS directly in living cells Measure MDA, a byproduct of lipid peroxidation, in plasma, serum, urine, tissue 10009055 TBARS Assay Kit homogenates, and cell lysates 589201 TNF-α (human) ELISA Kit Measure TNF-α from human plasma, serum, and other sample matrices

500850 TNF-α (mouse) ELISA Kit Measure TNF-α from mouse plasma, serum, and other sample matrices

Article References (continued) 9. Neuschwander-Tetri, B.A., Brunt, E.M., Wehmeier, K.R., et al. Improved nonalcoholic steatohepatitis 25. Satapati, S., Kucejova, B., Duarte, J.A., et al. Mitochondrial metabolism mediates oxidative stress after 48 weeks of treatment with the PPAR-γ ligand rosiglitazone. Hepatology 38(4), 1008-1017 (2003). and inflammation in fatty liver. J. Clin. Invest. 126(4), 1605 (2016). 10. Neuschwander-Tetri, B.A., Brunt, E.M., Wehmeier, K.R., et al. Interim results of a pilot study 26. Kalavalapalli, S., Bril, F., Koelmel, J.P., et al. Pioglitazone improves hepatic mitochondrial function in demonstrating the early effects of the PPAR-γ ligand rosiglitazone on insulin sensitivity, a mouse model of nonalcoholic steatohepatitis. Am. J. Physiol. Endocrinol. Metab. 315(2), aminotransferases, hepatic steatosis and body weight in patients with non-alcoholic steatohepatitis. E163–E173 (2018). J. Hepatol. 38(4), 434-440 (2003). 27. 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Lancet 385(9972), 956-965 (2015). results of the randomized placebo-controlled fatty liver improvement with rosiglitazone therapy 29. Harrison, S.A., Rinella, M.E., Abdelmalek, M.F., et al. NGM282 for treatment of non-alcoholic (FLIRT) trial. Gastroenterology 135(1), 100-110 (2008). steatohepatitis: A multicentre, randomised, double-blind, placebo-controlled, phase 2 trial. Lancet 14. Ratziu, V., Charlotte, F., Bernhardt, C., et al. Long-term efficacy of rosiglitazone in nonalcoholic 391(10126), 1174-1185 (2018). steatohepatitis: Results of the fatty liver improvement by rosiglitazone therapy (FLIRT 2) extension 30. Jinnouchi, H., Sugiyama, S., Yoshida, A., et al. Liraglutide, a glucagon-like peptide-1 analog, trial. Hepatology 51(2), 445-453 (2010). increased insulin sensitivity assessed by hyperinsulinemic-euglycemic clamp examination in 15. Aithal, G.P., Thomas, J.A., Kaye, P.V., et al. Randomized, placebo-controlled trial of pioglitazone in patients with uncontrolled type 2 diabetes mellitus. J. Diabetes Res. 706416 (2015). nondiabetic subjects with nonalcoholic steatohepatitis. Gastroenterology 135(4), 1176-1184 (2008). 31. Yamaguchi, K., Yang, L., McCall, S., et al. Diacylglycerol acyltranferase 1 anti-sense oligonucleotides 16. Lehmann, J.M., Moore, L.B., Smith-Oliver, T.A., et al. An antidiabetic thiazolidinedione is a high reduce hepatic fibrosis in mice with nonalcoholic steatohepatitis. Hepatology 47(2), 625-635 (2008). affinity ligand for peroxisome proliferator-activated receptor γ (PPARγ). J. Biol. Chem. 270(22), 32. Yamamoto, T., Yamaguchi, H., Miki, H., et al. Coenzyme A: Diacylglycerol acyltransferase 1 inhibitor 12953-12956 (1995). ameliorates obesity, liver steatosis, and lipid metabolism abnormality in KKAy mice fed high-fat or 17. 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Targeting acyl-CoA: Diacylglycerol acyltransferase 1 (DGAT1) with thiazolidinedione insulin sensitizers (mTOT)—relationship to newly identified mitochondrial small molecule inhibitors for the treatment of metabolic diseases J. Biol. Chem. 286(48), pyruvate carrier proteins. PLoS One 8(5), e61551 (2013). 41838-41851 (2011). 20. Divakaruni, A.S., Wiley, S.E., Rogers, G.W., et al. Thiazolidinediones are acute, specific inhibitors of 35. Yu, X.X., Murray, S.F., Pandey, S.K., et al. Antisense oligonucleotide reduction of DGAT2 expression the mitochondrial pyruvate carrier. Proc. Natl. Acad. Sci. USA 110(14), 5422-5427 (2013). improves hepatic steatosis and hyperlipidemia in obese mice. Hepatology 42(2), 362-371 (2005). 21. McCommis, K.S., Chen, Z., Fu, X., et al. Loss of mitochondrial pyruvate carrier 2 in the liver leads to 36. Yamaguchi, K., Yang, L., McCall, S., et al. Inhibiting triglyceride synthesis improves hepatic steatosis defects in gluconeogenesis and compensation via pyruvate-alanine cycling. Cell Metab. 22(4), but exacerbates liver damage and fibrosis in obese mice with nonalcoholic steatohepatitis. 682-694 (2015). Hepatology 45(6), 1366-1374 (2007). 22. Chen, Z., Vigueira, P.A., Chambers, K.T., et al. Insulin resistance and metabolic derangements in 37. Cundy, K.C., Grindstaff, K., Magnan, R., et al. AASLD Liver Meeting Abstract 2010: CB4209 and obese mice are ameliorated by a novel peroxisome proliferator-activated receptor γ-sparing CB4211 reduce the NAFLD activity score in the STAM model of NASH, reduce triglyceride levels, thiazolidinedione. J. Biol. Chem. 287(28), 23537-23548 (2012). and induce selective fat mass loss in DIO mice. Hepatology 66(S1), 1064A (2017). 23. McCommis, K.S., Hodges, W.T., Brunt, E.M., et al. Targeting the mitochondrial pyruvate carrier 38. Ekstedt, M., Hagström, H., Nasr, P., et al. Fibrosis stage is the strongest predictor for disease- attenuates fibrosis in a mouse model of nonalcoholic steatohepatitis. Hepatology 65(5), specific mortality in NAFLD after up to 33 years of follow-up. Hepatology 61(5), 1547-1554 (2015). 1543-1556 (2017). 24. Sunny, N.E., Parks, E.J., Browning, J.D., et al. Excessive hepatic mitochondrial TCA cycle and gluconeogenesis in humans with nonalcoholic fatty liver disease. Cell Metab. 14(6), 804-810 (2011). (800) 364-9897 www.caymanchem.com 12 Targeting PPARs: A guide to function and structure While PPARs display a high degree of homology at the protein level, each subtype exhibits distinct, noninter- changeable roles in energy metabolism that range from energy burning to energy storage. Learn more about their functions as fatty acid sensors and regulators of energy homeostasis at www.caymanchem.com/PPARs. www.caymanchem.com

MUSCLE PPARδ PPARα · ↑ Fatty acid oxidation · ↑ Fatty acid oxidation · ↑ Oxidative muscle fibers · ↑ Obesity resistance PPARγ · ↑ Insulin-mediated · ↑ Insulin sensitivity glucose uptake · ↑ Energy uncoupling

LIVER PPARα PPARγ · ↑ Fatty acid oxidation · ↑ Fatty acid storage · ↑ Ketogenesis · ↑ Lipogenesis · ↓ Plasma triglycerides · ↑ Plasma HDL

VESSEL WALL PPARα & PPARγ · ↓ Inflammation · ↑ Reverse cholesterol transport PPARδ · ↓ Inflammation

WHITE ADIPOSE TISSUE PPARγ · ↑ Adipogenesis · Regulates adipokine production · ↑ Lipid repartitioning to fat · ↑ Insulin sensitivity · ↑ Lipogenesis

BROWN ADIPOSE TISSUE PPARδ · ↑ Fatty acid oxidation · ↑ Energy uncoupling PPARγ · ↑ Adipogenesis atura Ansts Catatrs armaeuta Ansts

Creressrs Antanstsarta Ansts PPAR α, δ, γ PPRE DNA DA nn ne man ren an nn man aret ene ransrtn

A1 A NH2 COOH A C D ste

PPARα* PPARδ PPARγ* 1 1 8 1 1 1 11 1 1 1 1 8

A C D A C D A C D eresents srm 1 te anna seuene n nrt eresents srm te anna seuene n nrt

FAAR NR1C2 Synonyms NR1C1 NR1C3 NUC1 PPARβ

CREBBP EP300 CITED2 FAM120B CREBBP MED1 (PBP/DRIP205/TRAP220) EP300 EP300 NCOA1 (RIP160/SRC-1) NCOA1 (RIP160/SRC-1) MED1 (PBP/DRIP205/TRAP220) NCOA2 (SRC-2) NCOA2 (SRC-2) Coactivators NCOA1 (RIP160/SRC-1) NCOA3 (SRC-3) NCOA3 (SRC-3) NCOA2 (SRC-2) NCOA4 PGC-1α NCOA3 (SRC-3) NCOA6 PGC-1α NCOA7 PGC-1β PGC-1α PRIC295 PGC-1β PGC-2

NCOR1 GPS2 NCOR1 NR0B2 PER2 NCOR1 Corepressors RIP140 (NRIP1) RIP140 (NRIP1) RIP140 (NRIP1) NR0B1 SMRT (NCOR2) SMRT (NCOR2) SMRT (NCOR2) NR0B2 SPEN

AM3102 Amorfrutin A Amorfrutin B (+)-Etomoxir (sodium salt) Azelaoyl PAF CAY10599 Selective GW 590735 CAY10592 CDDO Agonists GW 7647 GW 0742 Ciglitazone GW 9578 GW 501516 GW 1929 8(S)-HETE Pioglitazone 12,14 15-deoxy-Δ -Prostaglandin J2 N-Octadecyl-N’-propyl-sulfamide Rosiglitazone Oleoyl Ethanolamide Troglitazone

Bavachinin (±)13-HODE Bavachinin Lanifibranor Lanifibranor Bezafibrate Bavachinin MHY908 Non-Selective and CAY10514 MHY908 Elafibranor CAY10514 Bezafibrate Muraglitazar Dual Agonists CAY10573 Muraglitazar Lanifibranor CAY10573 CAY10573 Elafibranor Tesaglitazar (±)9-HODE Wy 14643 (±)9-HODE Wy 14643 (±)13-HODE

BADGE GW 9662 Partial Agonists GSK3787 Diclofenac MCC-555 GW 6471 and Antagonists Sulindac FMOC-L-Leucine SR 202 G3335 T0070907 CONTACT US PHONE: (800) 364-9897 (USA and Canada only) (734) 971-3335 FAX: (734) 971-3640 EMAIL: Sales: [email protected] Customer Service: [email protected] Technical Support: [email protected] Contract Services: [email protected]

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