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.

Targeting the Alternative Vitamin E Metabolite Binding Site Enables Noncanonical PPARγ Modulation.

The lipid-sensing transcription factor PPARγ is the target of antidiabetic thiazolidinediones (TZD). At two sites within its ligand binding domain, it also binds oxidized vitamin E metabolites and the vitamin E mimetic garcinoic acid. While the canonical interaction within the TZD binding site mediates classical PPARγ activation, the effects of the second binding on PPARγ activity remain elusive. Here, we identified an agonist mimicking dual binding of vitamin E metabolites and developed a selective ligand of the second site, unveiling potential noncanonical regulation of PPARγ activities. We found that this alternative binding event can simultaneously occur with orthosteric ligands and it exerted different effects on PPARγ-cofactor interactions compared to both orthosteric PPARγ agonists and antagonists, indicating the diverse roles of the two binding sites. Alternative site binding lacked the pro-adipogenic effect of TZD and mediated no classical PPAR signaling in differential gene expression analysis but markedly diminished FOXO signaling, suggesting potential therapeutic applications.

Emerging TACnology: Heterobifunctional Small Molecule Inducers of Targeted Posttranslational Protein Modifications.

Posttranslational modifications (PTMs) play an important role in cell signaling and they are often deregulated in disease. This review addresses recent advances in the development of heterobifunctional small molecules that enable targeting or hijacking PTMs. This emerging field is spearheaded by proteolysis-targeting chimeras (PROTACs), that induce ubiquitination of their targets and, thus, tag them for degradation by the proteasome. Within the last decade, several improvements have been made to enhance spatiotemporal control of PROTAC-induced degradation as well as cell permeability. Inspired by the success story of PROTACs, additional concepts based on chimeric small molecules have emerged such as phosphatase-recruiting chimeras (PhoRCs). Herein, an overview of strategies causing (de-)phosphorylation, deubiquitination as well as acetylation is provided, and the opportunities and challenges of heterobifunctional molecules for drug discovery are highlighted. Although significant progress has been achieved, a plethora of PTMs have not yet been covered and PTM-inducing chimeras will be helpful tools for chemical biology and could even find application in pharmacotherapy.

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.

The Transcriptional Repressor Orphan Nuclear Receptor TLX Is Responsive to Xanthines.

The orphan nuclear receptor tailless homologue (TLX) is expressed almost exclusively in neural stem cells acting as an essential factor for their survival and is hence considered as a promising drug target in neurodegeneration. However, few studies have characterized the roles of TLX due to the lack of ligands and limited functional understanding. Here, we identify xanthines including caffeine and istradefylline as TLX modulators that counteract the receptor's intrinsic repressor activity. Mutagenesis of residues lining a cavity within the TLX ligand binding domain altered the activity of these ligands, suggesting direct interactions with helix 5. Using xanthines as tool compounds, we observed a ligand-sensitive recruitment of the co-repressor silencing mediator for retinoid or thyroid-hormone receptors, TLX homodimerization, and heterodimerization with the retinoid X receptor. These protein-protein interactions evolve as factors that modulate the TLX function and suggest an unprecedented role of TLX in directly repressing other nuclear receptors.

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.

Design and Structural Optimization of Dual FXR/PPARδ Activators.

Nonalcoholic steatohepatitis (NASH) is considered as severe hepatic manifestation of the metabolic syndrome and has alarming global prevalence. The ligand-activated transcription factors farnesoid X receptor (FXR) and peroxisome proliferator-activated receptor (PPAR) δ have been validated as molecular targets to counter NASH. To achieve robust therapeutic efficacy in this multifactorial pathology, combined peripheral PPARδ-mediated activity and hepatic effects of FXR activation appear as a promising multitarget approach. We have designed a minimal dual FXR/PPARδ activator scaffold by rational fusion of pharmacophores derived from selective agonists. Our dual agonist lead compound exhibited weak agonism on FXR and PPARδ and was structurally refined to a potent and balanced FXR/PPARδ activator in a computer-aided fashion. The resulting dual FXR/PPARδ modulator comprises high selectivity over related nuclear receptors and activates the two target transcription factors in native cellular settings.

l-Thyroxin and the Nonclassical Thyroid Hormone TETRAC Are Potent Activators of PPARγ.

Thyroid hormones (THs) operate numerous physiological processes through modulation of the nuclear thyroid hormone receptors and several other proteins. We report direct activation of the nuclear peroxisome proliferator-activated receptor gamma (PPARγ) and retinoid X receptor (RXR) by classical and nonclassical THs as another molecular activity of THs. The T4 metabolite TETRAC was the most active TH on PPARγ with nanomolar potency and binding affinity. We demonstrate that TETRAC promotes PPARγ/RXR signaling in cell-free, cellular, and in vivo settings. Simultaneous activation of the heterodimer partners PPARγ and RXR resulted in high dimer activation efficacy. Compared to fatty acids as known natural ligands of PPARγ and RXR, TETRAC differs markedly in its molecular structure and the PPARγ-TETRAC complex revealed a distinctive binding mode of the TH. Our observations suggest a potential connection of TH and PPAR signaling through overlapping ligand recognition and may hold implications for TH and PPAR pharmacology.

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.

A Novel Biphenyl-based Chemotype of Retinoid X Receptor Ligands Enables Subtype and Heterodimer Preferences.

The nuclear retinoid X receptors (RXRs) are key ligand sensing transcription factors that serve as universal nuclear receptor heterodimer partners and are thus involved in numerous physiological processes. Therapeutic targeting of RXRs holds high potential but available RXR activators suffer from limited safety. Selectivity for RXR subtypes or for certain RXR heterodimers are promising strategies for safer RXR modulation. Here, we report systematic structure-activity relationship studies on biphenyl carboxylates as new RXR ligand chemotype. We discovered specific structural modifications that enhance potency on RXRs, govern subtype preference, and vary modulation of different RXR heterodimers. Fusion of these structural motifs enabled specific tuning of subtype preferential profiles with markedly improved potency. Our results provide further evidence that RXR subtype selective ligands can be designed and present a novel chemotype of RXR modulators that can be tuned for subtype and heterodimer preferences.

Computer-Assisted Discovery and Structural Optimization of a Novel Retinoid X Receptor Agonist Chemotype.

As universal heterodimer partners of many nuclear receptors, the retinoid X receptors (RXRs) constitute key transcription factors. They regulate cell proliferation, differentiation, inflammation, and metabolic homeostasis and have recently been proposed as potential drug targets for neurodegenerative and inflammatory diseases. Owing to the hydrophobic nature of RXR ligand binding sites, available synthetic RXR ligands are lipophilic, and their structural diversity is limited. Here, we disclose the computer-assisted discovery of a novel RXR agonist chemotype and its systematic optimization toward potent RXR modulators. We have developed a nanomolar RXR agonist with high selectivity among nuclear receptors and superior physicochemical properties compared to classical rexinoids that appears suitable for in vivo applications and as lead for future RXR-targeting medicinal chemistry.

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.

Opportunities and Challenges for Fatty Acid Mimetics in Drug Discovery.

Fatty acids beyond their role as an endogenous energy source and storage are increasingly considered as signaling molecules regulating various physiological effects in metabolism and inflammation. Accordingly, the molecular targets involved in formation and physiological activities of fatty acids hold significant therapeutic potential. A number of these fatty acid targets are addressed by some of the oldest and most widely used drugs such as cyclooxygenase inhibiting NSAIDs, whereas others remain unexploited. Compounds orthosterically binding to proteins that endogenously bind fatty acids are considered as fatty acid mimetics. On the basis of their structural resemblance, fatty acid mimetics constitute a family of bioactive compounds showing specific binding thermodynamics and following similar pharmacokinetic mechanisms. This perspective systematically evaluates targets for fatty acid mimetics, investigates their common structural characteristics, and highlights demands in their discovery and design. In summary, fatty acid mimetics share particularly favorable characteristics justifying the conclusion that their therapeutic potential vastly outweighs the challenges in their design.

DrugBank screening revealed alitretinoin and bexarotene as liver X receptor modulators.

In silico screening of DrugBank database to detect liver X receptor (LXR) agonism of marketed drugs using a self-organizing map and successive LXR-Gal4 hybrid reporter gene assay evaluation in vitro discovered alitretinoin and bexarotene as partial liver X receptor agonists. Dose-response curves demonstrated that plasma concentrations observed in clinical trials are sufficient for LXR activation and thus could account for LXR-mediated side-effects such as hypercholesterolemia and hyperlipidemia. The discovered drugs are the first reported dual LXR/RXR agonists and can serve as lead structures for LXR and dual LXR/RXR modulator development.