RESUMEN
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.
Asunto(s)
PPAR gamma , Tiazolidinedionas , PPAR gamma/agonistas , PPAR gamma/genética , PPAR gamma/metabolismo , Ligandos , Factores de Transcripción/metabolismo , Tiazolidinedionas/química , Sitios de UniónRESUMEN
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.
Asunto(s)
Proteína de Unión a CREB/metabolismo , PPAR gamma/metabolismo , Multimerización de Proteína , Receptor alfa X Retinoide/metabolismo , Benzoatos/farmacología , Células HEK293 , Humanos , Ligandos , Mutación/genética , Coactivador 1 de Receptor Nuclear/metabolismo , PPAR gamma/agonistas , PPAR gamma/química , Dominios Proteicos , Multimerización de Proteína/efectos de los fármacos , Estabilidad Proteica/efectos de los fármacos , Reproducibilidad de los Resultados , Receptor alfa X Retinoide/química , Receptor alfa X Retinoide/genética , Retinoides/farmacología , Rosiglitazona/farmacología , Activación Transcripcional/genéticaRESUMEN
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.
Asunto(s)
Neoplasias , Receptores de Esteroides , Humanos , Ligandos , Receptores Nucleares Huérfanos/uso terapéutico , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Apoptosis , Neoplasias/tratamiento farmacológicoRESUMEN
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-1ß 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.
Asunto(s)
Ácidos Araquidónicos/metabolismo , Quimiocina CCL2/biosíntesis , Endocannabinoides/metabolismo , Músculo Liso Vascular/metabolismo , Alcamidas Poliinsaturadas/metabolismo , Animales , Antiinflamatorios/farmacología , Ácidos Araquidónicos/farmacología , Quimiocina CCL2/efectos de los fármacos , Endocannabinoides/farmacología , Epigénesis Genética/efectos de los fármacos , Humanos , Ratones , Músculo Liso Vascular/efectos de los fármacos , Alcamidas Poliinsaturadas/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
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.
Asunto(s)
Factor Nuclear 4 del Hepatocito/química , Factor Nuclear 4 del Hepatocito/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Calorimetría , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos , Fructosa-Bifosfatasa/genética , Regulación de la Expresión Génica/efectos de los fármacos , Células Hep G2 , Factor Nuclear 4 del Hepatocito/genética , Humanos , Ligandos , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
The retinoid X receptor (RXR) is a ligand-sensing transcription factor acting mainly as a universal heterodimer partner for other nuclear receptors. Despite presenting as a potential therapeutic target for cancer and neurodegeneration, adverse effects typically observed for RXR agonists, likely due to the lack of isoform selectivity, limit chemotherapeutic application of currently available RXR ligands. The three human RXR isoforms exhibit different expression patterns; however, they share high sequence similarity, presenting a major obstacle toward the development of subtype-selective ligands. Here, we report the discovery of the saturated fatty acid, palmitic acid, as an RXR ligand and disclose a uniform set of crystal structures of all three RXR isoforms in an active conformation induced by palmitic acid. A structural comparison revealed subtle differences among the RXR subtypes. We also observed an ability of palmitic acid as well as myristic acid and stearic acid to induce recruitment of steroid receptor co-activator 1 to the RXR ligand-binding domain with low micromolar potencies. With the high, millimolar endogenous concentrations of these highly abundant lipids, our results suggest their potential involvement in RXR signaling.
Asunto(s)
Ácido Palmítico/metabolismo , Isoformas de Proteínas/metabolismo , Receptores X Retinoide/metabolismo , Línea Celular , Dimerización , Regulación de la Expresión Génica/fisiología , Células HEK293 , Humanos , Ligandos , Ácido Mirístico/metabolismo , Coactivador 1 de Receptor Nuclear/metabolismo , Transducción de Señal/fisiología , Ácidos Esteáricos/metabolismoRESUMEN
Caspases are key enzymes responsible for mediating apoptotic cell death. Across species, caspase-2 is the most conserved caspase and stands out due to unique features. Apart from cell death, caspase-2 also regulates autophagy, genomic stability and ageing. Caspase-2 requires dimerization for its activation which is primarily accomplished by recruitment to high molecular weight protein complexes in cells. Here, we demonstrate that apoptosis inhibitor 5 (API5/AAC11) is an endogenous and direct inhibitor of caspase-2. API5 protein directly binds to the caspase recruitment domain (CARD) of caspase-2 and impedes dimerization and activation of caspase-2. Interestingly, recombinant API5 directly inhibits full length but not processed caspase-2. Depletion of endogenous API5 leads to an increase in caspase-2 dimerization and activation. Consistently, loss of API5 sensitizes cells to caspase-2-dependent apoptotic cell death. These results establish API5/AAC-11 as a direct inhibitor of caspase-2 and shed further light onto mechanisms driving the activation of this poorly understood caspase.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Caspasa 2/metabolismo , Inhibidores de Caspasas/metabolismo , Cisteína Endopeptidasas/metabolismo , Proteínas Nucleares/metabolismo , Apoptosis , Proteínas Reguladoras de la Apoptosis/deficiencia , Proteínas Reguladoras de la Apoptosis/genética , Autofagia , Caspasa 2/química , Dominio de Reclutamiento y Activación de Caspasas , Cisteína Endopeptidasas/química , Activación Enzimática , Células HeLa , Humanos , Espectrometría de Masas , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Unión Proteica , Multimerización de ProteínaRESUMEN
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⯵M against 1⯵M 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.
Asunto(s)
Cinamatos/farmacología , PPAR gamma/antagonistas & inhibidores , Quinolinas/farmacología , Animales , Cinamatos/síntesis química , Cinamatos/farmacocinética , Células HEK293 , Humanos , Masculino , Ratones , Microsomas Hepáticos/metabolismo , Estructura Molecular , Quinolinas/síntesis química , Quinolinas/farmacocinética , Ratas , Rosiglitazona/farmacología , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Proteínas Nucleares/análisis , Péptidos/química , Proteína-Arginina N-Metiltransferasas/análisis , Fluorescencia , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteína-Arginina N-Metiltransferasas/química , Proteína-Arginina N-Metiltransferasas/genética , Proteínas Recombinantes/análisis , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
UNLABELLED: The nucleotide-based second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) is involved in regulating a plethora of processes in bacteria that are typically associated with lifestyle changes. Myxococcus xanthus undergoes major lifestyle changes in response to nutrient availability, with the formation of spreading colonies in the presence of nutrients and spore-filled fruiting bodies in the absence of nutrients. Here, we investigated the function of c-di-GMP in M. xanthus and show that this bacterium synthesizes c-di-GMP during growth. Manipulation of the c-di-GMP level by expression of either an active, heterologous diguanylate cyclase or an active, heterologous phosphodiesterase correlated with defects in type IV pilus (T4P)-dependent motility, whereas gliding motility was unaffected. An increased level of c-di-GMP correlated with reduced transcription of the pilA gene (which encodes the major pilin of T4P), reduced the assembly of T4P, and altered cell agglutination, whereas a decreased c-di-GMP level correlated with altered cell agglutination. The systematic inactivation of the 24 genes in M. xanthus encoding proteins containing GGDEF, EAL, or HD-GYP domains, which are associated with c-di-GMP synthesis, degradation, or binding, identified three genes encoding proteins important for T4P-dependent motility, whereas all mutants had normal gliding motility. Purified DmxA had diguanylate cyclase activity, whereas the hybrid histidine protein kinases TmoK and SgmT, each of which contains a GGDEF domain, did not have diguanylate cyclase activity. These results demonstrate that c-di-GMP is important for T4P-dependent motility in M. xanthus. IMPORTANCE: We provide the first direct evidence that M. xanthus synthesizes c-di-GMP and demonstrate that c-di-GMP is important for T4P-dependent motility, whereas we did not obtain evidence that c-di-GMP regulates gliding motility. The data presented uncovered a novel mechanism for regulation of T4P-dependent motility, in which increased levels of c-di-GMP inhibit transcription of the pilA gene (which encodes the major pilin of T4P), ultimately resulting in the reduced assembly of T4P. Moreover, we identified an enzymatically active diguanylate cyclase that is important for T4P-dependent motility.
Asunto(s)
GMP Cíclico/análogos & derivados , Fimbrias Bacterianas/fisiología , Myxococcus xanthus/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos , GMP Cíclico/genética , GMP Cíclico/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Movimiento , Estructura Terciaria de ProteínaRESUMEN
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.
Asunto(s)
Apoptosis/fisiología , Caspasa 2/metabolismo , Cisteína Endopeptidasas/metabolismo , Apoptosis/efectos de los fármacos , Toxinas Bacterianas/farmacología , Regulación hacia Abajo , Células HeLa , Proteínas Hemolisinas/farmacología , Humanos , Queratinocitos/metabolismo , Proteínas Citotóxicas Formadoras de Poros/farmacología , Potasio/análisis , Potasio/metabolismoRESUMEN
The leukotriene A4 hydrolase (LTA4H) is a bifunctional enzyme, containing a peptidase and a hydrolase activity both activities having opposing functions regulating inflammatory response. The hydrolase activity is responsible for the conversion of leukotriene A4 to pro-inflammatory leukotriene B4, and hence, selective inhibitors of the hydrolase activity are of high pharmacological interest. Here we present the thermodynamic characterization of structurally distinct inhibitors of the LTA4H that occupy different regions of the binding site using different biophysical methods. An in silico method for the determination of stabilized water molecules in the binding site of the apo structure of LTA4H is used to interpret the measured thermodynamic data and provided insights for design of novel LTA4H inhibitors.
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Inhibidores Enzimáticos/farmacología , Epóxido Hidrolasas/antagonistas & inhibidores , Termodinámica , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Epóxido Hidrolasas/metabolismo , Humanos , Modelos Moleculares , Estructura Molecular , Relación Estructura-ActividadRESUMEN
The ability to escape apoptosis is a hallmark of cancer-initiating cells and a key factor of resistance to oncolytic therapy. Here, we identify FAM96A as a ubiquitous, evolutionarily conserved apoptosome-activating protein and investigate its potential pro-apoptotic tumor suppressor function in gastrointestinal stromal tumors (GISTs). Interaction between FAM96A and apoptotic peptidase activating factor 1 (APAF1) was identified in yeast two-hybrid screen and further studied by deletion mutants, glutathione-S-transferase pull-down, co-immunoprecipitation and immunofluorescence. Effects of FAM96A overexpression and knock-down on apoptosis sensitivity were examined in cancer cells and zebrafish embryos. Expression of FAM96A in GISTs and histogenetically related cells including interstitial cells of Cajal (ICCs), "fibroblast-like cells" (FLCs) and ICC stem cells (ICC-SCs) was investigated by Northern blotting, reverse transcription-polymerase chain reaction, immunohistochemistry and Western immunoblotting. Tumorigenicity of GIST cells and transformed murine ICC-SCs stably transduced to re-express FAM96A was studied by xeno- and allografting into immunocompromised mice. FAM96A was found to bind APAF1 and to enhance the induction of mitochondrial apoptosis. FAM96A protein or mRNA was dramatically reduced or lost in 106 of 108 GIST samples representing three independent patient cohorts. Whereas ICCs, ICC-SCs and FLCs, the presumed normal counterparts of GIST, were found to robustly express FAM96A protein and mRNA, FAM96A expression was much reduced in tumorigenic ICC-SCs. Re-expression of FAM96A in GIST cells and transformed ICC-SCs increased apoptosis sensitivity and diminished tumorigenicity. Our data suggest FAM96A is a novel pro-apoptotic tumor suppressor that is lost during GIST tumorigenesis.
Asunto(s)
Apoptosis/genética , Proteínas Portadoras/genética , Tumores del Estroma Gastrointestinal/genética , Proteínas Supresoras de Tumor/genética , Animales , Factor Apoptótico 1 Activador de Proteasas/genética , Línea Celular , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Expresión Génica/genética , Células HEK293 , Humanos , Células Intersticiales de Cajal/metabolismo , Metaloproteínas , Ratones , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Mitocondrias/genética , Pez Cebra/genéticaRESUMEN
The COVID-19 pandemic has highlighted the lack of effective, ready-to-use antivirals for the treatment of viruses with pandemic potential. The development of a diverse drug portfolio is therefore crucial for pandemic preparedness. Viral macrodomains are attractive therapeutic targets as they are suggested to play an important role in evading the innate host immune response, making them critical for viral pathogenesis. Macrodomains function as erasers of mono-ADP-ribosylation (deMARylation), a post-translational modification that is involved in interferon signaling. Herein, we report the development of a modular HTRF-based assay, that can be used to screen for inhibitors of various viral and human macrodomains. We characterized the five most promising small molecule SARS-CoV-2 Mac1 inhibitors recently reported in the literature for potency and selectivity and conducted a pilot screen demonstrating HTS suitability. The ability to directly detect enzymatic activity makes the DeMAR assay a valuable addition to the existing tools for macrodomain drug discovery.
RESUMEN
Noxious stimuli activate nociceptive sensory neurons, causing action potential firing and the release of diverse signaling molecules. Several peptides have already been identified to be released by sensory neurons and shown to modulate inflammatory responses and inflammatory pain. However, it is still unclear whether lipid mediators can be released upon sensory neuron activation to modulate intercellular communication. Here, we analyzed the lipid secretome of capsaicin-stimulated nociceptive neurons with LC-HRMS, revealing that oleic acid is strongly released from sensory neurons by capsaicin. We further demonstrated that oleic acid inhibits capsaicin-induced calcium transients in sensory neurons and reverses bradykinin-induced TRPV1 sensitization by a calcineurin (CaN) and GPR40 (FFAR1) dependent pathway. Additionally, oleic acid alleviated zymosan-mediated thermal hypersensitivity via the GPR40, suggesting that the capsaicin-mediated oleic acid release from sensory neurons acts as a protective and feedback mechanism, preventing sensory neurons from nociceptive overstimulation via the GPR40/CaN/TRPV1-axis.
RESUMEN
Various disorders are accompanied by histamine-independent itching, which is often resistant to the currently available therapies. Here, it is reported that the pharmacological activation of Slack (Kcnt1, KNa1.1), a potassium channel highly expressed in itch-sensitive sensory neurons, has therapeutic potential for the treatment of itching. Based on the Slack-activating antipsychotic drug, loxapine, a series of new derivatives with improved pharmacodynamic and pharmacokinetic profiles is designed that enables to validate Slack as a pharmacological target in vivo. One of these new Slack activators, compound 6, exhibits negligible dopamine D2 and D3 receptor binding, unlike loxapine. Notably, compound 6 displays potent on-target antipruritic activity in multiple mouse models of acute histamine-independent and chronic itch without motor side effects. These properties make compound 6 a lead molecule for the development of new antipruritic therapies targeting Slack.
Asunto(s)
Canales de Potasio , Prurito , Animales , Ratones , Antipruriginosos/uso terapéutico , Histamina/metabolismo , Loxapina/uso terapéutico , Canales de Potasio/metabolismo , Prurito/tratamiento farmacológico , Prurito/metabolismoRESUMEN
G protein-coupled receptor G2A was postulated to be a promising target for the development of new therapeutics in neuropathic pain, acute myeloid leukemia, and inflammation. However, there is still a lack of potent, selective, and drug-like G2A agonists to be used as a chemical tool or as the starting matter for the development of drugs. In this work, we present the discovery and structure-activity relationship elucidation of a new potent and selective G2A agonist scaffold. Systematic optimization resulted in (3-(pyridin-3-ylmethoxy)benzoyl)-d-phenylalanine (T-10418) exhibiting higher potency than the reference and natural ligand 9-HODE and high selectivity among G protein-coupled receptors. With its favorable activity, a clean selectivity profile, excellent solubility, and high metabolic stability, T-10418 qualifies as a pharmacological tool to investigate the effects of G2A activation.
Asunto(s)
Receptores Acoplados a Proteínas G , Humanos , Relación Estructura-Actividad , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Animales , Fenilalanina/farmacología , Fenilalanina/análogos & derivados , Fenilalanina/química , Fenilalanina/síntesis química , Estructura MolecularRESUMEN
The neuron derived orphan receptor (NOR-1, NR4A3) is among the least studied nuclear receptors. Its physiological role and therapeutic potential remain widely elusive which is in part due to the lack of chemical tools that can directly modulate NOR-1 activity. To probe the possibility of pharmacological NOR-1 modulation, we have tested a drug fragment library for NOR-1 activation and repression. Despite low hit-rate (<1 %), we have obtained three NOR-1 ligand chemotypes one of which could be rapidly expanded to an analogue comprising low micromolar inverse NOR-1 agonist potency and altering NOR-1 regulated gene expression in a cellular setting. It confirms druggability of the transcription factor and may serve as an early tool to assess the role and potential of NOR-1.
Asunto(s)
Receptores Citoplasmáticos y Nucleares , Factores de Transcripción , Regulación de la Expresión Génica , Ligandos , NeuronasRESUMEN
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.
Asunto(s)
Diseño de Fármacos , Receptores Nucleares Huérfanos/agonistas , Animales , Supervivencia Celular/efectos de los fármacos , Estabilidad de Medicamentos , Células HEK293 , Humanos , Ligandos , Microsomas Hepáticos/metabolismo , Receptores Nucleares Huérfanos/metabolismo , Piperazina/química , Piperazina/metabolismo , Piperazina/farmacología , Unión Proteica , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
Several lines of evidence suggest the ligand-sensing transcription factor Nurr1 as a promising target to treat neurodegenerative diseases. Nurr1 modulators to validate and exploit this therapeutic potential are rare, however. To identify novel Nurr1 agonist chemotypes, we have employed the Nurr1 activator amodiaquine as template for microscale analogue library synthesis. The first set of analogues was based on the 7-chloroquiolin-4-amine core fragment of amodiaquine and revealed superior N-substituents compared to diethylaminomethylphenol contained in the template. A second library of analogues was subsequently prepared to replace the chloroquinolineamine scaffold. The two sets of analogues enabled a full scaffold hop from amodiaquine to a novel Nurr1 agonist sharing no structural features with the lead but comprising superior potency on Nurr1. Additionally, pharmacophore modeling based on the entire set of active and inactive analogues suggested key features for Nurr1 agonists.