Screening of Chelidonium majus isoquinoline alkaloids reveals berberine and chelidonine as selective ligands for the nuclear receptors RORß and HNF4α, respectively.

The nuclear receptors hepatocyte nuclear factor 4α (HNF4α) and retinoic acid receptor-related orphan receptor-ß (RORß) are ligand-regulated transcription factors and potential drug targets for metabolic disorders. However, there is a lack of small molecular, selective ligands to explore the therapeutic potential in further detail. Here, we report the discovery of greater celandine (Chelidonium majus) isoquinoline alkaloids as nuclear receptor modulators: Berberine is a selective RORß inverse agonist and modulated target genes involved in the circadian clock, photoreceptor cell development, and neuronal function. The structurally related chelidonine was identified as a ligand for the constitutively active HNF4α receptor, with nanomolar potency in a cellular reporter gene assay. In human liver cancer cells naturally expressing high levels of HNF4α, chelidonine acted as an inverse agonist and downregulated genes associated with gluconeogenesis and drug metabolism. Both berberine and chelidonine are promising tool compounds to further investigate their target nuclear receptors and for drug discovery.

Biosynthesis of prostaglandin 15dPGJ2 -glutathione and 15dPGJ2-cysteine conjugates in macrophages and mast cells via MGST3.

Inhibition of microsomal prostaglandin E synthase-1 (mPGES-1) results in decreased production of proinflammatory PGE2 and can lead to shunting of PGH2 into the prostaglandin D2 (PGD2)/15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2) pathway. 15dPGJ2 forms Michael adducts with thiol-containing biomolecules such as GSH or cysteine residues on target proteins and is thought to promote resolution of inflammation. We aimed to elucidate the biosynthesis and metabolism of 15dPGJ2 via conjugation with GSH, to form 15dPGJ2-glutathione (15dPGJ2-GS) and 15dPGJ2-cysteine (15dPGJ2-Cys) conjugates and to characterize the effects of mPGES-1 inhibition on the PGD2/15dPGJ2 pathway in mouse and human immune cells. Our results demonstrate the formation of PGD2, 15dPGJ2, 15dPGJ2-GS, and 15dPGJ2-Cys in RAW264.7 cells after lipopolysaccharide stimulation. Moreover, 15dPGJ2-Cys was found in lipopolysaccharide-activated primary murine macrophages as well as in human mast cells following stimulation of the IgE-receptor. Our results also suggest that the microsomal glutathione S-transferase 3 is essential for the formation of 15dPGJ2 conjugates. In contrast to inhibition of cyclooxygenase, which leads to blockage of the PGD2/15dPGJ2 pathway, we found that inhibition of mPGES-1 preserves PGD2 and its metabolites. Collectively, this study highlights the formation of 15dPGJ2-GS and 15dPGJ2-Cys in mouse and human immune cells, the involvement of microsomal glutathione S-transferase 3 in their biosynthesis, and their unchanged formation following inhibition of mPGES-1. The results encourage further research on their roles as bioactive lipid mediators.

Controlling Intramolecular Interactions in the Design of Selective, High-Affinity Ligands for the CREBBP Bromodomain.

CREBBP (CBP/KAT3A) and its paralogue EP300 (KAT3B) are lysine acetyltransferases (KATs) that are essential for human development. They each comprise 10 domains through which they interact with >400 proteins, making them important transcriptional co-activators and key nodes in the human protein-protein interactome. The bromodomains of CREBBP and EP300 enable the binding of acetylated lysine residues from histones and a number of other important proteins, including p53, p73, E2F, and GATA1. Here, we report a work to develop a high-affinity, small-molecule ligand for the CREBBP and EP300 bromodomains [(-)-OXFBD05] that shows >100-fold selectivity over a representative member of the BET bromodomains, BRD4(1). Cellular studies using this ligand demonstrate that the inhibition of the CREBBP/EP300 bromodomain in HCT116 colon cancer cells results in lowered levels of c-Myc and a reduction in H3K18 and H3K27 acetylation. In hypoxia (<0.1% O2), the inhibition of the CREBBP/EP300 bromodomain results in the enhanced stabilization of HIF-1α.

A triple farnesoid X receptor and peroxisome proliferator-activated receptor α/δ activator reverses hepatic fibrosis in diet-induced NASH in mice.

Non-alcoholic steatohepatitis (NASH) - a hepatic manifestation of the metabolic syndrome - is a multifactorial disease with alarming global prevalence. It involves steatosis, inflammation and fibrosis in the liver, thus demanding multiple modes of action for robust therapeutic efficacy. Aiming to fuse complementary validated anti-NASH strategies in a single molecule, we have designed and systematically optimized a scaffold for triple activation of farnesoid X receptor (FXR), peroxisome proliferator-activated receptor (PPAR) α and PPARδ. Pilot profiling of the resulting triple modulator demonstrated target engagement in native cellular settings and in mice, rendering it a suitable tool to probe the triple modulator concept in vivo. In DIO NASH in mice, the triple agonist counteracted hepatic inflammation and reversed hepatic fibrosis highlighting the potential of designed polypharmacology in NASH.

Dual Farnesoid X Receptor/Soluble Epoxide Hydrolase Modulators Derived from Zafirlukast.

The nuclear farnesoid X receptor (FXR) and the enzyme soluble epoxide hydrolase (sEH) are validated molecular targets to treat metabolic disorders such as non-alcoholic steatohepatitis (NASH). Their simultaneous modulation in vivo has demonstrated a triad of anti-NASH effects and thus may generate synergistic efficacy. Here we report dual FXR activators/sEH inhibitors derived from the anti-asthma drug Zafirlukast. Systematic structural optimization of the scaffold has produced favorable dual potency on FXR and sEH while depleting the original cysteinyl leukotriene receptor antagonism of the lead drug. The resulting polypharmacological activity profile holds promise in the treatment of liver-related metabolic diseases.

Urate transporter inhibitor lesinurad is a selective peroxisome proliferator-activated receptor gamma modulator (sPPARγM) in vitro.

Gout is the most common arthritic disease in human but was long neglected and therapeutic options are not satisfying. However, with the recent approval of the urate transporter inhibitor lesinurad, gout treatment has experienced a major innovation. Here we show that lesinurad possesses considerable modulatory potency on peroxisome proliferator-activated receptor γ (PPARγ). Since gout has a strong association with metabolic diseases such as type 2 diabetes, this side-activity appears as very valuable contributing factor to the clinical efficacy profile of lesinurad. Importantly, despite robustly activating PPARγ in vitro, lesinurad lacked adipogenic activity, which seems due to differential coactivator recruitment and is characterized as selective PPARγ modulator (sPPARγM).

Boosting Anti-Inflammatory Potency of Zafirlukast by Designed Polypharmacology.

Multitarget design offers access to bioactive small molecules with potentially superior efficacy and safety. Particularly multifactorial chronic inflammatory diseases demand multiple pharmacological interventions for stable treatment. By minor structural changes, we have developed a close analogue of the cysteinyl-leukotriene receptor antagonist zafirlukast that simultaneously inhibits soluble epoxide hydrolase and activates peroxisome proliferator-activated receptor γ. The triple modulator exhibits robust anti-inflammatory activity in vivo and highlights the therapeutic potential of designed multitarget agents.

Nonacidic Farnesoid X Receptor Modulators.

As a cellular bile acid sensor, farnesoid X receptor (FXR) participates in regulation of bile acid, lipid and glucose homeostasis, and liver protection. Clinical results have validated FXR as therapeutic target in hepatic and metabolic diseases. To date, potent FXR agonists share a negatively ionizable function that might compromise their pharmacokinetic distribution and behavior. Here we report the development and characterization of a high-affinity FXR modulator not comprising an acidic residue.