Non-alcoholic fatty liver disease (NAFLD), the most common liver disease worldwide, currently lacks US FDA-approved drugs for its treatment. Life-style modification and off-label use of certain insulin sensitizers (e.g., metformin), hypoglycemic agents (e.g., pioglitazone), antioxidants (e.g., vitamin E), and cholesterol-lowering drugs (e.g., statins) offer some improvement to hepatic lipid profiles and associated metabolic and cardiovascular disease risk, but more targeted, evidence-based therapies remain on the horizon for the treatment of NAFLD and its further complication non-alcoholic steatohepatitis (NASH). To aid in the pre-clinical discovery and optimization of novel compounds for the treatment of these diseases, Cayman has curated a set of tools designed to examine the pharmacokinetics of drugs targeting nuclear transcription factors, G protein-coupled protein receptors, and enzymes of interest to this field.

FXR and TGR5

Current attention lies in modulating the farnesoid X receptor (FXR) and the G protein-coupled bile acid receptor (TGR5 or GP-BAR1), the two main bile acid receptors expressed in the liver, intestine, kidney, and fat. FXR, TGR5, and bile acid transporters have become targets of increasing interest to biotech and pharma companies given their roles as master regulators of carbohydrate and lipid metabolism, bile acid homeostasis, inflammation, and fibrosis—all of which may influence NAFLD development.

The cholesterol regulator ursodeoxycholic acid is a natural activator of FXR that shows potential in remediating steatosis pathogenesis and has been used to treat other liver diseases. However, prolonged exposure creates complications related to interference with xenobiotic drug detoxification. For this reason, semi-synthetic or synthetic non-steroidal FXR agonists are being sought. The semi-synthetic bile acid, obeticholic acid, has been shown clinically to be beneficial for altering lipid metabolism in patients with NASH, and the non-steroidal derivatives (e.g., fexaramine, GW 4064) show high affinity, potency, and selectivity for FXR while offering better tolerability profiles.^1-3 Beyond FXR, selective targeting of TGR5 activation also represents an attractive therapeutic approach for NAFLD given its role in increasing thermogenic energy expenditure and inducing release of glucagon-like peptide to influence glucose homeostasis. INT-777, a semi-synthetic bile acid, and TGR5 Receptor Agonist, a synthetic small molecule, are two recently developed activators that are selective for TGR5.² As the hunt for high quality agonists continues, Cayman offers reporter assays to screen the functional activity of test compounds against FXR and TGR5.

FXR (human) Reporter Assay Kit

FXR (mouse) Reporter Assay Kit

TGR5 (GP-BAR1) Reporter Assay Kit

Validation of TGR5 reporter assay with different classes of agonists.

SREBP

Lipid homeostasis is regulated by three known isoforms of sterol regulatory element binding protein (SREBP) transcription factors: SREBP-1a, SREBP-1c, and SREBP-2. SREBP-1c acts primarily to activate genes required for fatty acid synthesis. SREBP-2 performs a critical role in the transcriptional regulation of genes involved in cholesterol synthesis and uptake. Because toxic accumulation of free cholesterol in the liver is part of the pathogenesis of NAFLD, modulation of SREBP activity has important clinical implications in the treatment of the disease.³ While selective SREBP antagonists are under development, some plant-derived compounds, including betulin, have been shown to inhibit the SREBP-driven pathway of cholesterol and fatty acid biosynthesis. Cayman offers sensitive ELISAs for detecting the specific transcription factor DNA binding activity of SREBP in nuclear extracts or cell lysates and polyclonal antibodies to detect both precursor and active forms of SREBP2 in tissues and cells.

SREBP-1 Transcription Factor Assay Kit

SREBP-2 Transcription Factor Assay Kit

SREBP-2 Polyclonal Antibody

SREBP-2 Polyclonal Antibody - Biotinylated

SREBP-2 transcription factor binding assay with recombinant control lysate.

PPARs

Agonists of the peroxisome proliferator-activated receptors (PPARs) have also shown anti-inflammatory and anti-fibrotic effects in both preclinical and clinical models of NAFLD/NASH. Focus is shifting away from PPARγ agonists (thiazolidinediones) that carry increased cardiovascular or cancer risk, and toward improving potency of selective PPARα and PPARδ modulators as well as complementary PPARα/δ ligands. For example, in multiple animal disease models and now phase III clinical studies of NASH patients, elafibranor (GFT505), a dual PPARα/δ agonist, demonstrates improvement to steatosis, inflammation, and fibrosis without safety concerns.³ PPARα activation in hepatocytes (the main target of fibrate drugs, GW 7647, and GW 590735) is associated with increased ketogenesis, enhanced fatty acid β-oxidation, lipolysis of triglycerides, reduced inflammatory responses, and enhanced H2O2 detoxification.³ PPARδ activation has also been implicated in benefiting lipid metabolism and energy homeostasis by enhancing hepatic lipid oxidation and insulin sensitivity as well as demonstrating anti-inflammatory effects.3 Cayman offers reporter assays to screen the functional activity of test compounds against all three PPAR isotypes as well as a convenient fluorescence polarization-based single step assay for screening PPARγ ligands. Polyclonal antibodies are also available to study the expression and functions of each isotype.

PPAR (human) Reporter Assays Panel

PPARα (human) Reporter Assay Kit

PPARα (mouse) Reporter Assay Kit

PPARα (rat) Reporter Assay Kit

PPARα Polyclonal Antibody

PPARδ (human) Reporter Assay Kit

PPARδ (mouse) Reporter Assay Kit

PPARδ (rat) Reporter Assay Kit

PPARδ Polyclonal Antibody

PPARγ (human) Reporter Assay Kit

PPARγ (mouse/rat) Reporter Assay Kit

PPARγ Ligand Screening Assay Kit

PPARγ Polyclonal Antibody

Agonist dose-response analyses of human PPARα.

DPP (IV)

Glucagon mimetics, including glucagon-like peptide 1 (GLP-1) agonists (e.g., exendin-4) and dipeptidyl peptidase IV (DPP (IV)) inhibitors (e.g., sitagliptin, vildagliptin), have been shown to reduce hepatocyte steatosis and improve hepatocyte survival by enhancing the unfolded protein response and promoting macroautophagy.^4,5 Several experimental and clinical trials exploring the efficacy of incretin-based therapies for NAFLD treatment suggest that DDP (IV) inhibitors could be a potential candidate for directly affecting hepatocyte lipid metabolism and reducing liver inflammation.⁴ Cayman’s DPP (IV) Inhibitor Screening Assay provides a convenient fluorescence-based method for identifying emerging DPP (IV) inhibitors in a 96-well format.

DPP (IV) Inhibitor Screening Assay Kit

Inhibition of DPP (IV) by sitagliptin.

Convenient Monitoring Assays

Cayman also offers a series of assays for certain biomarkers that are routinely used as indicators of NAFLD/NASH disease states.

View a complete list of Cayman’s NAFLD research tools.

Download the Research Tools for Fatty Liver Disease Brochure (PDF).

References

1. Hambruch, E., Kinzel, O., and Kremoser, C. Nuclear Receptor Research Vol. 3 (2016), Article ID 101207.

2. Arab, J.P., Karpen, S.J., Dawson, P.A., et al. Hepatology65(1), 350–362 (2017).

3. Musso, G., Cassader, M., and Gambino, R. Nat. Rev. Drug Discov.15(4), 249-274 (2016).

4. Blaslov, K., Bulum, T., Zibar, K. et al. World J. Gastroenterol.20(23), 7356-7365 (2014).

5. Liu, J., Wang, G., Jia, Y., et al. Diabetes Metab. Res. Rev.31(4), 329-335 (2015).