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The leukotriene B4 receptor 2 (BLT2) is a G-protein coupled receptor activated by 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT), which has been proposed as a promising therapeutic target for diabetic wound healing and gastrointestinal lesions. In this study, the rational design of a fluorescent probe based on the synthetic BLT2 agonist CAY10583 is described. The synthesis of several derivatives of CAY10583 coupled to fluorescein resulted in a traceable ligand suitable for different fluorescence-based techniques. An HTRF-based displacement assay (Tag-lite) on stably transfected CHO-K1 cells was developed to characterize binding properties of diverse BLT2 ligands. Highly specific binding to the BLT2 receptor was demonstrated in staining experiments on mouse skin tissue, and specific modulation of BLT2-induced cAMP signaling provided further evidence for receptor binding and ligand functionality. In conclusion, the fluorescent ligands developed in this study are suitable to investigate the pharmacology of BLT2 receptor ligands in a variety of assay systems.

As a master regulator of neurogenesis, the orphan nuclear receptor tailless homologue (TLX, NR2E1) maintains neuronal stem cell homeostasis by acting as a transcriptional repressor of tumor suppressor genes. It is hence considered as an appealing target for the treatment of neurodegenerative diseases, but a lack of potent TLX modulators as tools to probe pharmacological TLX control hinders further validation of its promising potential. Here, we report the development of a potent TLX agonist based on fragment screening, pharmacophore modeling, and fragment fusion. Pharmacophore similarity of a fragment screening hit and the TLX ligand ccrp2 provided a rational basis for fragment linkage, which resulted in several TLX activator scaffolds. Among them, the fused compound 10 evolved as a valuable TLX agonist tool with submicromolar potency and high selectivity over related nuclear receptors, rendering it suitable for functional studies on TLX.

The transcription factor nerve growth factor-induced clone B (NGFI-B, Nur77, NR4A1) is an orphan nuclear receptor playing a role in cell survival and apoptosis regulation. Pharmacological Nur77 modulation holds promise for cancer and (neuro-)inflammatory disease treatment. The available Nur77 ligand scaffolds based on highly lipophilic natural products cytosporone B, celastrol and isoalantolactone are inadequate for the development of potent Nur77 modulators with favorable properties as chemical tools and future drugs. By fragment library screening and subsequent modeling for fragment extension, we have obtained a set of new Nur77 ligands offering alternative chemotypes for the development of Nur77 agonists and inverse agonists. Computer-aided fragment extension in a second stage screening yielded a Nur77 agonist with significant activation efficacy and preference over the related NR4A receptors.

The ligand-sensing transcription factor tailless homologue (TLX, NR2E1) is an essential regulator of neuronal stem cell homeostasis with appealing therapeutic potential in neurodegenerative diseases and central nervous system tumors. However, knowledge on TLX ligands is scarce, providing an obstacle to target validation and medicinal chemistry. To discover TLX ligands, we have profiled a drug fragment collection for TLX modulation and identified several structurally diverse agonists and inverse agonists of the nuclear receptor. Propranolol evolved as the strongest TLX agonist and promoted TLX-regulated gene expression in human glioblastoma cells. Structure-activity relationship elucidation of propranolol as a TLX ligand yielded a structurally related negative control compound. In functional cellular experiments, we observed an ability of propranolol to counteract glioblastoma cell proliferation and migration, while the negative control had no effect. Our results provide a collection of TLX modulators as initial chemical tools and set of lead compounds and support therapeutic potential of TLX modulation in glioblastoma.

Autophagy is the common name for a number of lysosome-based degradation pathways of cytosolic cargos. The key components of autophagy are members of Atg8 family proteins involved in almost all steps of the process, from autophagosome formation to their selective fusion with lysosomes. In this study, we show that the homologous members of the human Atg8 family proteins, LC3A and LC3B, are druggable by a small molecule inhibitor novobiocin. Structure-activity relationship (SAR) studies of the 4-hydroxy coumarin core scaffold were performed, supported by a crystal structure of the LC3A dihydronovobiocin complex. The study reports the first nonpeptide inhibitors for these protein interaction targets and will lay the foundation for the development of more potent chemical probes for the Atg8 protein family which may also find applications for the development of autophagy-mediated degraders (AUTACs).

The first potent leukotriene B4 (LTB4) receptor type 2 (BLT2) agonists, endogenous 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT), and synthetic CAY10583 (CAY) have been recently described to accelerate wound healing by enhanced keratinocyte migration and indirect stimulation of fibroblast activity in diabetic rats. CAY represents a very valuable starting point for the development of novel wound-healing promoters. In this work, the first structure-activity relationship study for CAY scaffold-based BLT2 agonists is presented. The newly prepared derivatives showed promising in vitro wound-healing activity.

The ligand-sensing transcription factor nuclear receptor related 1 (Nurr1) evolves as an appealing target to treat neurodegenerative diseases. Despite its therapeutic potential observed in various rodent models, potent modulators for Nurr1 are lacking as pharmacological tools. Here, we report the structure-activity relationship and systematic optimization of indole-based inverse Nurr1 agonists. Optimized analogues decreased the receptor's intrinsic transcriptional activity by up to more than 90% and revealed preference for inhibiting Nurr1 monomer activity. In orthogonal cell-free settings, we detected displacement of NCoRs and disruption of the Nurr1 homodimer as molecular modes of action. The inverse Nurr1 agonists reduced the expression of Nurr1-regulated genes in T98G cells, and treatment with an inverse Nurr1 agonist mimicked the effect of Nurr1 silencing on interleukin-6 release from LPS-stimulated human astrocytes. The indole-based inverse Nurr1 agonists valuably extend the toolbox of Nurr1 modulators to further probe the role of Nurr1 in neuroinflammation, cancer, and beyond.

International audience; The emerging pharmacological target soluble epoxide hydrolase (sEH) is a bifunctional enzyme exhibiting two different catalytic activities that are located in two distinct domains. Although the physiological role of the C-terminal hydrolase domain is well-investigated, little is known about its phosphatase activity, located in the N-terminal phosphatase domain of sEH (sEH-P). Herein we report the discovery and optimization of the first inhibitor of human and rat sEH-P that is applicable in vivo. X-ray structure analysis of the sEH phosphatase domain complexed with an inhibitor provides insights in the molecular basis of small-molecule sEH-P inhibition and helps to rationalize the structure−activity relationships. 4-(4-(3,4-Dichlorophenyl)-5-phenyloxazol-2-yl)butanoic acid (22b, SWE101) has an excellent pharmacokinetic and pharmacodynamic profile in rats and enables the investigation of the physiological and pathophysiological role of sEH-P in vivo.

The ligand-activated transcription factor nuclear receptor related-1 (Nurr1) exhibits great potential for neurodegenerative disease treatment, but potent Nurr1 modulators to further probe and validate the nuclear receptor as a therapeutic target are lacking. We have systematically studied the structure-activity relationship of the 4-amino-7-chloroquinoline scaffold contained in Nurr1 activators amodiaquine and chloroquine and discovered fragment-like analogues that activated Nurr1 in several cellular settings. The most active descendants promoted the transcriptional activity of Nurr1 on human response elements as monomer, homodimer, and heterodimer and markedly enhanced Nurr1-dependent gene expression in human astrocytes. As a tool to elucidate mechanisms involving in Nurr1 activation, these Nurr1 agonists induced robust recruitment of NCoR1 and NCoR2 co-regulators to the Nurr1 ligand binding domain and promoted Nurr1 dimerization. These findings provide important insights in Nurr1 regulation. The fragment-sized Nurr1 agonists are appealing starting points for medicinal chemistry and valuable early Nurr1 agonist tools for pharmacology and chemical biology.

The metabolic syndrome (MetS) is a constellation of cardiovascular and metabolic symptoms involving insulin resistance, steatohepatitis, obesity, hypertension, and heart disease, and patients suffering from MetS often require polypharmaceutical treatment. PPARγ agonists are highly effective oral antidiabetics with great potential in MetS, which promote adipocyte browning and insulin sensitization. However, the application of PPARγ agonists in clinics is restricted by potential cardiovascular adverse events. We have previously demonstrated that the racemic dual sEH/PPARγ modulator RB394 (3) simultaneously improves all risk factors of MetS in vivo. In this study, we identify and characterize the eutomer of 3. We provide structural rationale for molecular recognition of the eutomer. Furthermore, we could show that the dual sEH/PPARγ modulator is able to promote adipocyte browning and simultaneously exhibits cardioprotective activity which underlines its exciting potential in treatment of MetS.

Polypharmaceutical regimens often impair treatment of patients with metabolic syndrome (MetS), a complex disease cluster, including obesity, hypertension, heart disease, and type II diabetes. Simultaneous targeting of soluble epoxide hydrolase (sEH) and peroxisome proliferator-activated receptor γ (PPARγ) synergistically counteracted MetS in various in vivo models, and dual sEH inhibitors/PPARγ agonists hold great potential to reduce the problems associated with polypharmacy in the context of MetS. However, full activation of PPARγ leads to fluid retention associated with edema and weight gain, while partial PPARγ agonists do not have these drawbacks. In this study, we designed a dual partial PPARγ agonist/sEH inhibitor using a structure-guided approach. Exhaustive structure-activity relationship studies lead to the successful optimization of the designed lead. Crystal structures of one representative compound with both targets revealed potential points for optimization. The optimized compounds exhibited favorable metabolic stability, toxicity, selectivity, and desirable activity in adipocytes and macrophages.

Designed multitarget ligands are a popular approach to generating efficient and safe drugs, and fragment‐based strategies have been postulated as a versatile avenue to discover multitarget ligand leads. To systematically probe the potential of fragment‐based multiple ligand discovery, we have employed a large fragment library for comprehensive screening on five targets chosen from proteins for which multitarget ligands have been successfully developed previously (soluble epoxide hydrolase, leukotriene A4 hydrolase, 5‐lipoxygenase, retinoid X receptor, farnesoid X receptor). Differential scanning fluorimetry served as primary screening method before fragments hitting at least two targets were validated in orthogonal assays. Thereby, we obtained valuable fragment leads with dual‐target engagement for six out of ten target combinations. Our results demonstrate the applicability of fragment‐based approaches to identify starting points for polypharmacological compound development with certain limitations., Thinking outside the pharmacophore: This study systematically investigates the potential of fragment‐based multiple ligand discovery. We employed a large fragment library for comprehensive screening on five targets and obtained valuable fragment leads with dual‐target engagement for six out of ten target combinations.

Inhibition of multiple enzymes of the arachidonic acid cascade leads to synergistic anti-inflammatory effects. Merging of 5-lipoxygenase (5-LOX) and soluble epoxide hydrolase (sEH) pharmacophores led to the discovery of a dual 5-LOX/sEH inhibitor, which was subsequently optimized in terms of potency toward both targets and metabolic stability. The optimized lead structure displayed cellular activity in human polymorphonuclear leukocytes, oral bioavailability, and target engagement in vivo and demonstrated profound anti-inflammatory and anti-fibrotic efficiency in a kidney injury model caused by unilateral ureteral obstruction in mice. These results pave the way for investigating the therapeutic potential of dual 5-LOX/sEH inhibitors in other inflammation- and fibrosis-related disease models.

Nuclear receptors (NRs) activate transcription of target genes in response to binding of ligands to their ligand-binding domains (LBDs). Typically, in vitro assays use either gene expression or the recruitment of coactivators to the isolated LBD of the NR of interest to measure NR activation. However, this approach ignores that NRs function as homo- as well as heterodimers and that the LBD harbors the main dimerization interface. Cofactor recruitment is thereby interconnected with oligomerization status as well as ligand occupation of the partnering LBD through allosteric cross talk. Here we present a modular set of homogeneous time-resolved FRET–based assays through which we investigated the activation of PPARγ in response to ligands and the formation of heterodimers with its obligatory partner RXRα. We introduced mutations into the RXRα LBD that prevent coactivator binding but do not interfere with LBD dimerization or ligand binding. This enabled us to specifically detect PPARγ coactivator recruitment to PPARγ:RXRα heterodimers. We found that the RXRα agonist SR11237 destabilized the RXRα homodimer but promoted formation of the PPARγ:RXRα heterodimer, while being inactive on PPARγ itself. Of interest, incorporation of PPARγ into the heterodimer resulted in a substantial gain in affinity for coactivator CBP-1, even in the absence of ligands. Consequently, SR11237 indirectly promoted coactivator binding to PPARγ by shifting the oligomerization preference of RXRα toward PPARγ:RXRα heterodimer formation. These results emphasize that investigation of ligand-dependent NR activation should take NR dimerization into account. We envision these assays as the necessary assay tool kit for investigating NRs that partner with RXRα.

Endocannabinoids are important lipid-signaling mediators. Both protective and deleterious effects of endocannabinoids in the cardiovascular system have been reported but the mechanistic basis for these contradicting observations is unclear. We set out to identify anti-inflammatory mechanisms of endocannabinoids in the murine aorta and in human vascular smooth muscle cells (hVSMC). In response to combined stimulation with cytokines, IL-1β and TNFα, the murine aorta released several endocannabinoids, with anandamide (AEA) levels being the most significantly increased. AEA pretreatment had profound effects on cytokine-induced gene expression in hVSMC and murine aorta. As revealed by RNA-Seq analysis, the induction of a subset of 21 inflammatory target genes, including the important cytokine CCL2 was blocked by AEA. This effect was not mediated through AEA-dependent interference of the AP-1 or NF-κB pathways but rather through an epigenetic mechanism. In the presence of AEA, ATAC-Seq analysis and chromatin-immunoprecipitations revealed that CCL2 induction was blocked due to increased levels of H3K27me3 and a decrease of H3K27ac leading to compacted chromatin structure in the CCL2 promoter. These effects were mediated by recruitment of HDAC4 and the nuclear corepressor NCoR1 to the CCL2 promoter. This study therefore establishes a novel anti-inflammatory mechanism for the endogenous endocannabinoid AEA in vascular smooth muscle cells. Furthermore, this work provides a link between endogenous endocannabinoid signaling and epigenetic regulation. Electronic supplementary material The online version of this article (10.1007/s00395-020-0793-3) contains supplementary material, which is available to authorized users.

The nuclear peroxisome proliferator-activated receptor γ has well-validated therapeutic potential in metabolic, inflammatory, and neurodegenerative pathologies, but its activation is also associated with marked adverse effects and novel modes of PPARγ modulation are required. Here, we report the discovery and profiling of a new PPARγ modulator chemotype endowed with remarkable potency and a distinct binding mode in the orthosteric PPARγ ligand-binding site. Its R-enantiomer evolved as a eutomer regarding PPARγ activation with a high eudysmic ratio. The new PPARγ modulator revealed outstanding selectivity over the PPARα and PPARδ subtypes and did not promote adipogenesis in primary human fibroblasts, discriminating it from established agonists.

Hepatocyte nuclear factor 4α (HNF4α) is a ligand-sensing transcription factor and presents as a potential drug target in metabolic diseases and cancer. In humans, mutations in the HNF4α gene cause maturity-onset diabetes of the young (MODY), and the elevated activity of this protein has been associated with gastrointestinal cancers. Despite the high therapeutic potential, available ligands and structure–activity relationship knowledge for this nuclear receptor are scarce. Here, we disclose a chemically diverse collection of orthogonally validated fragment-like activators as well as inverse agonists, which modulate HNF4α activity in a low micromolar range. These compounds demonstrate the druggability of HNF4α and thus provide a starting point for medicinal chemistry as well as an early tool for chemogenomics.

Vitamin E exhibits pharmacological effects beyond established antioxidant activity suggesting involvement of unidentified mechanisms. Here, we characterize endogenously formed tocopherol carboxylates and the vitamin E mimetic garcinoic acid (GA) as activators of the peroxisome proliferator-activated receptor gamma (PPARγ). Co-stimulation of PPARγ with GA and the orthosteric agonist pioglitazone resulted in additive transcriptional activity. In line with this, the PPARγ-GA complex adopted a fully active conformation and interestingly contained two bound GA molecules with one at an allosteric site. A co-regulator interaction scan demonstrated an unanticipated co-factor recruitment profile for GA-bound PPARγ compared with canonical PPARγ agonists and gene expression analysis revealed different effects of GA and pioglitazone on PPAR signaling in hepatocytes. These observations reveal allosteric mechanisms of PPARγ modulation as an alternative avenue to PPARγ targeting and suggest contributions of PPARγ activation by α-13-tocopherolcarboxylate to the pharmacological effects of vitamin E.

Peroxisome proliferator-activated receptor gamma (PPARγ) modulators have found wide application for the treatment of cancers, metabolic disorders and inflammatory diseases. Contrary to PPARγ agonists, PPARγ antagonists have been much less studied and although they have shown immunomodulatory effects, there is still no therapeutically useful PPARγ antagonist on the market. In contrast to non-competitive, irreversible inhibition caused by 2-chloro-5-nitrobenzanilide (GW9662), the recently described (E)-2-(5-((4-methoxy-2-(trifluoromethyl)quinolin-6-yl)methoxy)-2-((4-(trifluoromethyl)benzyl)oxy)-benzylidene)-hexanoic acid (MTTB, T-10017) is a promising prototype for a new class of PPARγ antagonists. It exhibits competitive antagonism against rosiglitazone mediated activation of PPARγ ligand binding domain (PPARγLBD) in a transactivation assay in HEK293T cells with an IC50 of 4.3 µM against 1 µM rosiglitazone. The aim of this study was to investigate the structure-activity relationships (SAR) of the MTTB scaffold focusing on improving its physicochemical properties. Through this optimization, 34 new derivatives were prepared and characterized. Two new potent compounds (T-10075 and T-10106) with much improved drug-like properties and promising pharmacokinetic profile were identified.

Nuclear receptors act as ligand-activated transcription factors translating ligand signals into changes in gene expression. The 48 members of the superfamily of nuclear receptors identified so far are involved amongst others in maintenance of metabolic balance, inflammation, and cancer. Thus, they hold enormous potential for drug discovery and some nuclear receptors are experiencing considerable academic and industrial interest. Nuclear receptor modulator discovery requires reliable, robust, and economic test systems that allow for high throughput. In this chapter, we discuss the principle, strengths, and advantages of hybrid reporter gene assays for nuclear receptor focused drug discovery and describe how they can be developed, established, and validated.

The fatty acid sensing nuclear receptor families retinoid X receptors (RXRs) and peroxisome proliferator-activated receptors (PPARs) hold therapeutic potential in neurodegeneration. Valuable pleiotropic activities of Wy14,643 in models of such conditions exceed its known PPAR agonistic profile. Here, we characterize the compound as an RXR agonist explaining the pleiotropic effects and report its systematic structure-activity relationship analysis with the discovery of specific molecular determinants driving activity on PPARs and RXRs. We have designed close analogues of the drug comprising selective and dual agonism on RXRs and PPARs that may serve as superior pharmacological tools to study the role and interplay of the nuclear receptors in various pathologies. A systematically optimized high potency RXR agonist revealed activity in vivo and active concentrations in brain. With its lack of RXR/liver X receptor-mediated side effects and superior profile compared to classical rexinoids, it establishes a new class of innovative RXR modulators to overcome key challenges in RXR targeting drug discovery.

The protein arginine N-methyltransferase 6 (PRMT6) is overexpressed in a variety of different cancer types and plays a role in human immunodeficiency virus (HIV) infections. Furthermore, the PRMT6 activity might also influence the pathogenesis of neurodegenerative, inflammatory, and cardiovascular diseases, whereby it becomes an interesting target for drug development. Previously reported activity assays for PRMT6 activity are either expensive, time-consuming or use radioactive substrates. To overcome these challenges, we developed a coupled fluorescence-based activity assay using recombinant PRMT6 expressed in E. coli. In the first step of the assay, the fluorogenic substrate Nα-Benzoyl-L-arginine-7-amido-4-methylcoumarin (Bz-Arg-AMC) is methylated by PRMT6, while in a second step the remaining un-methylated substrate is cleaved by trypsin, producing the fluorescent 7-amino-4-methylcoumarin.

Metabolic syndrome (MetS) is a multifactorial disease cluster that consists of dyslipidemia, cardiovascular disease, type 2 diabetes mellitus, and obesity. MetS patients are strongly exposed to polypharmacy; however, the number of pharmacological compounds required for MetS treatment can be reduced by the application of multitarget compounds. This study describes the design of dual-target ligands that target soluble epoxide hydrolase (sEH) and the peroxisome proliferator-activated receptor type γ (PPARγ). Simultaneous modulation of sEH and PPARγ can improve diabetic conditions and hypertension at once. N-Benzylbenzamide derivatives were determined to fit a merged sEH/PPARγ pharmacophore, and structure-activity relationship studies were performed on both targets, resulting in a submicromolar (sEH IC50 = 0.3 μM/PPARγ EC50 = 0.3 μM) modulator 14c. In vitro and in vivo evaluations revealed good ADME properties qualifying 14c as a pharmacological tool compound for long-term animal models of MetS.

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.

Nonalcoholic steatohepatitis arising from Western diet and lifestyle is characterized by accumulation of fat in liver causing inflammation and fibrosis. It evolves as serious health burden with alarming incidence, but there is no satisfying pharmacological therapy to date. Considering the disease's multifactorial nature, modulation of multiple targets might provide superior therapeutic efficacy. In particular, farnesoid X receptor (FXR) activation that revealed antisteatotic and antifibrotic effects in clinical trials combined with inhibition of soluble epoxide hydrolase (sEH) as anti-inflammatory strategy promises synergies. To exploit this dual concept, we developed agents exerting partial FXR agonism and sEH inhibitory activity. Merging known pharmacophores and systematic exploration of the structure-activity relationship on both targets produced dual modulators with low nanomolar potency. Extensive in vitro characterization confirmed high dual efficacy in cellular context combined with low toxicity, and pilot in vivo data revealed favorable pharmacokinetics as well as engagement on both targets in vivo.

Bacterial pathogens modulate host cell apoptosis to establish a successful infection. Pore-forming toxins (PFTs) secreted by pathogenic bacteria are major virulence factors and have been shown to induce various forms of cell death in infected cells. Here we demonstrate that the highly conserved caspase-2 is required for PFT-mediated apoptosis. Despite being the second mammalian caspase to be identified, the role of caspase-2 during apoptosis remains enigmatic. We show that caspase-2 functions as an initiator caspase during Staphylococcus aureus α-toxin- and Aeromonas aerolysin-mediated apoptosis in epithelial cells. Downregulation of caspase-2 leads to a strong inhibition of PFT-mediated apoptosis. Activation of caspase-2 is PIDDosome-independent, and endogenous caspase-2 is recruited to a high-molecular-weight complex in α-toxin-treated cells. Interestingly, prevention of PFT-induced potassium efflux inhibits the formation of caspase-2 complex, leading to its inactivation, thus resisting apoptosis. These results revealed a thus far unknown, obligatory role for caspase-2 as an initiator caspase during PFT-mediated apoptosis.

Mutations in sterile alpha motif (SAM) domain of TP63 have been reported to be associated with ankyloblepharon-ectodermal dysplasia-cleft lip/palate syndrome and Rapp-Hodgkin syndrome. SAM domain, a protein-protein interaction module, is found in cytoplasmic signaling proteins and several transcriptional regulatory proteins which are involved in development and differentiation. Here, we report on a SAM domain mutation (p.Asp564His) in TP63 that predisposed the patients to have nonsyndromic cleft palate and nonsyndromic cleft lip and palate. © 2011 Wiley-Liss, Inc.