RESUMEN
Stimulation of protease-activated receptor 1 (PAR1) on endothelium by activated protein C (APC) is protective in several animal models of disease, and APC has been used clinically in severe sepsis and wound healing. Clinical use of APC, however, is limited by its immunogenicity and its anticoagulant activity. We show that a class of small molecules termed "parmodulins" that act at the cytosolic face of PAR1 stimulates APC-like cytoprotective signaling in endothelium. Parmodulins block thrombin generation in response to inflammatory mediators and inhibit platelet accumulation on endothelium cultured under flow. Evaluation of the antithrombotic mechanism showed that parmodulins induce cytoprotective signaling through Gßγ, activating a PI3K/Akt pathway and eliciting a genetic program that includes suppression of NF-κB-mediated transcriptional activation and up-regulation of select cytoprotective transcripts. STC1 is among the up-regulated transcripts, and knockdown of stanniocalin-1 blocks the protective effects of both parmodulins and APC. Induction of this signaling pathway in vivo protects against thromboinflammatory injury in blood vessels. Small-molecule activation of endothelial cytoprotection through PAR1 represents an approach for treatment of thromboinflammatory disease and provides proof-of-principle for the strategy of targeting the cytoplasmic surface of GPCRs to achieve pathway selective signaling.
Asunto(s)
Células Endoteliales/metabolismo , Inflamación/metabolismo , Receptor PAR-1/agonistas , Trombosis/metabolismo , Animales , Apoptosis , Factor Xa/metabolismo , Técnicas de Silenciamiento del Gen , Glicoproteínas/genética , Glicoproteínas/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Lipopolisacáridos , Masculino , Ratones , Ratones Endogámicos C57BL , Microcirculación , Péptido Hidrolasas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Transcripción Genética , Regulación hacia ArribaRESUMEN
The enzyme protein disulfide isomerase (PDI) is essential for the correct folding of proteins and the activation of certain cell surface receptors, and is a promising target for the treatment of cancer and thrombotic conditions. A previous high-throughput screen identified the commercial compound STK076545 as a promising PDI inhibitor. To confirm its activity and support further biological studies, a resynthesis was pursued of the reported ß-keto-amide with an N-alkylated pyridone at the α-position. Numerous conventional approaches were complicated by undesired fragmentations or rearrangements. However, a successful 5-step synthetic route was achieved using an aldol reaction with an α-pyridone allyl ester as a key step. An X-ray crystal structure of the final compound confirmed that the reported structure of STK076545 was achieved, however its lack of PDI activity and inconsistent spectral data suggest that the commercial structure was misassigned.
RESUMEN
The Natriuretic Peptide Receptors (NPRs) regulate vascular sodium levels and have been of significant interest for the potential treatment of hypertension and related cardiovascular complications. The peptidomimetic antagonist M372049 is a valuable probe for the study of NPR-C signaling, unfortunately it is presently not commercially available. Described is a detailed protocol for its synthesis that does not require specialized apparatus and builds upon a prior patent from Veale and colleagues. Key steps include a base-mediated lactam formation and a solid-supported peptide synthetic sequence. An X-ray crystal structure of a key lactam intermediate was obtained to confirm the structure and relative stereochemistry of the compound.
RESUMEN
The voltage-sensing domain (VSD) is a conserved structural module that regulates the gating of voltage-dependent ion channels in response to a change in membrane potential. Although the structures of many VSD-containing ion channels are now available, our understanding of the structural dynamics associated with gating transitions remains limited. To probe dynamics with site-specific resolution, we utilized NMR spectroscopy to characterize the VSD derived from Shaker potassium channel in 1-palmitoyl-2-hydroxy-sn-glycero-3-phospho-(1'-rac-glycerol) (LPPG) micelles. The backbone dihedral angles predicted based on secondary chemical shifts using torsion angle likeliness obtained from shift (TALOS+) showed that the Shaker-VSD shares many structural features with the homologous Kv1.2/2.1 chimera, including a transition from α-helix to 310 helix in the C-terminal portion of the fourth transmembrane helix. Nevertheless, there are clear differences between the Shaker-VSD and Kv1.2/2.1 chimera in the S2-S3 linker and S3 transmembrane region, where the organization of secondary structure elements in Shaker-VSD appears to more closely resemble the KvAP-VSD. Comparison of microsecond-long molecular dynamics simulations of Kv 1.2-VSD in LPPG micelles and a 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) bilayer showed that LPPG micelles do not induce significant structural distortion in the isolated voltage sensor. To assess the integrity of the tertiary fold, we directly probed the binding of BrMT analog 2-[2-({[3-(2-amino-ethyl)-6-bromo-1H-indol-2-yl]methoxy}k7methyl)-6-bromo-1H-indol-3-yl]ethan-1-amine (BrET), a gating modifier toxin, and identified the location of the putative binding site. Our results suggest that the Shaker-VSD in LPPG micelles is in a native-like fold and is likely to provide valuable insights into the dynamics of voltage-gating and its regulation.
Asunto(s)
Glicerol/análogos & derivados , Glicerol/química , Micelas , Resonancia Magnética Nuclear Biomolecular , Canales de Potasio de la Superfamilia Shaker/química , Secuencia de Aminoácidos , Dominios Proteicos , Estructura Secundaria de ProteínaRESUMEN
Cytoprotection by activated protein C (aPC) after ischemia-reperfusion injury (IRI) is associated with apoptosis inhibition. However, IRI is hallmarked by inflammation, and hence, cell-death forms disjunct from immunologically silent apoptosis are, in theory, more likely to be relevant. Because pyroptosis (ie, cell death resulting from inflammasome activation) is typically observed in IRI, we speculated that aPC ameliorates IRI by inhibiting inflammasome activation. Here we analyzed the impact of aPC on inflammasome activity in myocardial and renal IRIs. aPC treatment before or after myocardial IRI reduced infarct size and Nlrp3 inflammasome activation in mice. Kinetic in vivo analyses revealed that Nlrp3 inflammasome activation preceded myocardial injury and apoptosis, corroborating a pathogenic role of the Nlrp3 inflammasome. The constitutively active Nlrp3A350V mutation abolished the protective effect of aPC, demonstrating that Nlrp3 suppression is required for aPC-mediated protection from IRI. In vitro aPC inhibited inflammasome activation in macrophages, cardiomyocytes, and cardiac fibroblasts via proteinase-activated receptor 1 (PAR-1) and mammalian target of rapamycin complex 1 (mTORC1) signaling. Accordingly, inhibiting PAR-1 signaling, but not the anticoagulant properties of aPC, abolished the ability of aPC to restrict Nlrp3 inflammasome activity and tissue damage in myocardial IRI. Targeting biased PAR-1 signaling via parmodulin-2 restricted mTORC1 and Nlrp3 inflammasome activation and limited myocardial IRI as efficiently as aPC. The relevance of aPC-mediated Nlrp3 inflammasome suppression after IRI was corroborated in renal IRI, where the tissue protective effect of aPC was likewise dependent on Nlrp3 inflammasome suppression. These studies reveal that aPC protects from IRI by restricting mTORC1-dependent inflammasome activation and that mimicking biased aPC PAR-1 signaling using parmodulins may be a feasible therapeutic approach to combat IRI.
Asunto(s)
Inflamasomas/efectos de los fármacos , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteína C/farmacología , Daño por Reperfusión/prevención & control , Animales , Animales Recién Nacidos , Anticoagulantes/farmacología , Apoptosis/efectos de los fármacos , Células Cultivadas , Citoprotección/efectos de los fármacos , Citoprotección/genética , Immunoblotting , Inflamasomas/metabolismo , Riñón/irrigación sanguínea , Riñón/efectos de los fármacos , Riñón/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/prevención & control , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Sustancias Protectoras/farmacología , Receptor PAR-1/genética , Receptor PAR-1/metabolismo , Daño por Reperfusión/metabolismoRESUMEN
A new series of simplified azasordarin analogs was synthesized using as key steps a Diels-Alder reaction to generate a highly substituted bicyclo[2.2.1]heptane core, followed by a subsequent nitrile alkylation. Several additional strategies were investigated for the generation of the key tertiary nitrile or aldehyde thought to be required for inhibition at the fungal protein eukaryotic elongation factor 2. This new series also features a morpholino glycone previously reported in semisynthetic sordarin derivatives with broad spectrum antifungal activity. Despite a lack of activity against Candida albicans for these early de novo analogs, the synthetic route reported here permits more comprehensive modifications of the bicyclic core and structure-activity relationship studies that were not heretofore possible.
Asunto(s)
Antifúngicos/síntesis química , Antifúngicos/farmacología , Indenos/síntesis química , Indenos/farmacología , Alquenos/química , Antifúngicos/química , Antifúngicos/metabolismo , Técnicas de Química Sintética , Factor 2 Eucariótico de Iniciación/química , Factor 2 Eucariótico de Iniciación/metabolismo , Indenos/química , Indenos/metabolismo , Simulación del Acoplamiento Molecular , Nitrilos/química , Conformación ProteicaRESUMEN
A set of novel Kv7.2/7.3 (KCNQ2/3) channel blockers was synthesized to address several liabilities of the known compounds XE991 (metabolic instability and CYP inhibition) and the clinical compound DMP 543 (acid instability, insolubility, and lipophilicity). Using the anthrone scaffold of the prior channel blockers, alternative heteroarylmethyl substituents were installed via enolate alkylation reactions. Incorporation of a pyridazine and a fluorinated pyridine gave an analog (compound 18, JDP-107) with a promising combination of potency (IC50â¯=â¯0.16⯵M in a Kv7.2 thallium flux assay), efficacy in a Kv7.2/7.3 patch clamp assay, and drug-like properties.
Asunto(s)
Antracenos/farmacología , Canal de Potasio KCNQ2/antagonistas & inhibidores , Canal de Potasio KCNQ3/antagonistas & inhibidores , Trastornos Mentales/tratamiento farmacológico , Enfermedades Neurodegenerativas/tratamiento farmacológico , Bloqueadores de los Canales de Potasio/farmacología , Antracenos/síntesis química , Antracenos/química , Relación Dosis-Respuesta a Droga , Canal de Potasio KCNQ2/metabolismo , Canal de Potasio KCNQ3/metabolismo , Estructura Molecular , Bloqueadores de los Canales de Potasio/síntesis química , Bloqueadores de los Canales de Potasio/química , Relación Estructura-ActividadRESUMEN
Through a structure-based drug design project (SBDD), potent small molecule inhibitors of pyruvate carboxylase (PC) have been discovered. A series of α-keto acids (7) and α-hydroxycinnamic acids (8) were prepared and evaluated for inhibition of PC in two assays. The two most potent inhibitors were 3,3'-(1,4-phenylene)bis[2-hydroxy-2-propenoic acid] (8u) and 2-hydroxy-3-(quinoline-2-yl)propenoic acid (8v) with IC50 values of 3.0⯱â¯1.0⯵M and 4.3⯱â¯1.5⯵M respectively. Compound 8v is a competitive inhibitor with respect to pyruvate (Kiâ¯=â¯0.74⯵M) and a mixed-type inhibitor with respect to ATP, indicating that it targets the unique carboxyltransferase (CT) domain of PC. Furthermore, compound 8v does not significantly inhibit human carbonic anhydrase II, matrix metalloproteinase-2, malate dehydrogenase or lactate dehydrogenase.
Asunto(s)
Ácidos Cumáricos/uso terapéutico , Piruvato Carboxilasa/antagonistas & inhibidores , Ácidos Cumáricos/farmacología , Diseño de Fármacos , HumanosRESUMEN
Novel analogs of the allosteric, biased PAR1 ligand ML161 (parmodulin 2, PM2) were prepared in order to identify potential anti-thrombotic and anti-inflammatory compounds of the parmodulin class with improved properties. Investigations of structure-activity relationships of the western portion of the 1,3-diaminobenzene scaffold were performed using an intracellular calcium mobilization assay with endothelial cells, and several heterocycles were identified that inhibited PAR1 at sub-micromolar concentrations. The oxazole NRD-21 was profiled in additional detail, and it was confirmed to act as a selective, reversible, negative allosteric modulator of PAR1. In addition to inhibiting human platelet aggregation, it showed superior anti-inflammatory activity to ML161 in a qPCR assay measuring the expression of tissue factor in response to the cytokine TNF-alpha in endothelial cells. Additionally, NRD-21 is much more plasma stable than ML161, and is a promising lead compound for the parmodulin class for anti-thrombotic and anti-inflammatory indications.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Oxazoles/farmacología , Receptor PAR-1/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Regulación Alostérica/efectos de los fármacos , Antiinflamatorios no Esteroideos/química , Relación Dosis-Respuesta a Droga , Estabilidad de Medicamentos , Humanos , Ligandos , Estructura Molecular , Oxazoles/síntesis química , Oxazoles/química , Agregación Plaquetaria/efectos de los fármacos , Receptor PAR-1/metabolismo , Relación Estructura-ActividadRESUMEN
An essential coordinator of all DNA metabolic processes is Replication Protein A (RPA). RPA orchestrates these processes by binding to single-stranded DNA (ssDNA) and interacting with several other DNA binding proteins. Determining the real-time kinetics of single players such as RPA in the presence of multiple DNA processors to better understand the associated mechanistic events is technically challenging. To overcome this hurdle, we utilized non-canonical amino acids and bio-orthogonal chemistry to site-specifically incorporate a chemical fluorophore onto a single subunit of heterotrimeric RPA. Upon binding to ssDNA, this fluorescent RPA (RPAf) generates a quantifiable change in fluorescence, thus serving as a reporter of its dynamics on DNA in the presence of multiple other DNA binding proteins. Using RPAf, we describe the kinetics of facilitated self-exchange and exchange by Rad51 and mediator proteins during various stages in homologous recombination. RPAf is widely applicable to investigate its mechanism of action in processes such as DNA replication, repair and telomere maintenance.
Asunto(s)
Recombinación Homóloga , Proteína de Replicación A/química , Proteína de Replicación A/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Azidas/química , ADN de Cadena Simple/metabolismo , Colorantes Fluorescentes/química , Microscopía Fluorescente , Fenilalanina/análogos & derivados , Fenilalanina/química , Recombinasa Rad51/metabolismo , Proteína de Replicación A/genética , Proteínas de Saccharomyces cerevisiae/genética , Triptófano/químicaRESUMEN
Drugs do not act solely by canonical ligand-receptor binding interactions. Amphiphilic drugs partition into membranes, thereby perturbing bulk lipid bilayer properties and possibly altering the function of membrane proteins. Distinguishing membrane perturbation from more direct protein-ligand interactions is an ongoing challenge in chemical biology. Herein, we present one strategy for doing so, using dimeric 6-bromo-2-mercaptotryptamine (BrMT) and synthetic analogues. BrMT is a chemically unstable marine snail toxin that has unique effects on voltage-gated K+ channel proteins, making it an attractive medicinal chemistry lead. BrMT is amphiphilic and perturbs lipid bilayers, raising the question of whether its action against K+ channels is merely a manifestation of membrane perturbation. To determine whether medicinal chemistry approaches to improve BrMT might be viable, we synthesized BrMT and 11 analogues and determined their activities in parallel assays measuring K+ channel activity and lipid bilayer properties. Structure-activity relationships were determined for modulation of the Kv1.4 channel, bilayer partitioning, and bilayer perturbation. Neither membrane partitioning nor bilayer perturbation correlates with K+ channel modulation. We conclude that BrMT's membrane interactions are not critical for its inhibition of Kv1.4 activation. Further, we found that alkyl or ether linkages can replace the chemically labile disulfide bond in the BrMT pharmacophore, and we identified additional regions of the scaffold that are amenable to chemical modification. Our work demonstrates a strategy for determining if drugs act by specific interactions or bilayer-dependent mechanisms, and chemically stable modulators of Kv1 channels are reported.
Asunto(s)
Canal de Potasio Kv1.4/química , Membrana Dobles de Lípidos/química , Caracoles/química , Triptaminas/química , Secuencia de Aminoácidos , Animales , Humanos , Ligandos , Unión Proteica , Relación Estructura-Actividad , Xenopus laevisRESUMEN
Several classes of ligands for Protease-Activated Receptors (PARs) have shown impressive anti-inflammatory and cytoprotective activities, including PAR2 antagonists and the PAR1-targeting parmodulins. In order to support medicinal chemistry studies with hundreds of compounds and to perform detailed mode-of-action studies, it became important to develop a reliable PAR assay that is operational with endothelial cells, which mediate the cytoprotective effects of interest. We report a detailed protocol for an intracellular calcium mobilization assay with adherent endothelial cells in multiwell plates that was used to study a number of known and new PAR1 and PAR2 ligands, including an alkynylated version of the PAR1 antagonist RWJ-58259 that is suitable for the preparation of tagged or conjugate compounds. Using the cell line EA.hy926, it was necessary to perform media exchanges with automated liquid handling equipment in order to obtain optimal and reproducible antagonist concentration-response curves. The assay is also suitable for study of PAR2 ligands; a peptide antagonist reported by Fairlie was synthesized and found to inhibit PAR2 in a manner consistent with reports using epithelial cells. The assay was used to confirm that vorapaxar acts as an irreversible antagonist of PAR1 in endothelium, and parmodulin 2 (ML161) and the related parmodulin RR-90 were found to inhibit PAR1 reversibly, in a manner consistent with negative allosteric modulation.
Asunto(s)
Benzamidas/farmacología , Calcio/metabolismo , Fenilendiaminas/farmacología , Receptor PAR-1/antagonistas & inhibidores , Receptor PAR-2/antagonistas & inhibidores , Tecnología Farmacéutica/métodos , Regulación Alostérica , Benzamidas/síntesis química , Línea Celular , Células Endoteliales/metabolismo , Humanos , Iminas/farmacología , Indazoles/síntesis química , Indazoles/farmacología , Lactonas/farmacología , Ligandos , Oligopéptidos/síntesis química , Oligopéptidos/farmacología , Fenilendiaminas/síntesis química , Piridinas/farmacología , Receptor PAR-1/agonistas , Receptor PAR-2/agonistas , Urea/análogos & derivados , Urea/síntesis química , Urea/farmacologíaRESUMEN
Protease-activated receptor-1 (PAR1) couples the coagulation cascade to platelet activation during myocardial infarction and to endothelial inflammation during sepsis. This receptor demonstrates marked signaling bias. Its activation by thrombin stimulates prothrombotic and proinflammatory signaling, whereas its activation by activated protein C (APC) stimulates cytoprotective and antiinflammatory signaling. A challenge in developing PAR1-targeted therapies is to inhibit detrimental signaling while sparing beneficial pathways. We now characterize a novel class of structurally unrelated small-molecule PAR1 antagonists, termed parmodulins, and compare the activity of these compounds to previously characterized compounds that act at the PAR1 ligand-binding site. We find that parmodulins target the cytoplasmic face of PAR1 without modifying the ligand-binding site, blocking signaling through Gαq but not Gα13 in vitro and thrombus formation in vivo. In endothelium, parmodulins inhibit prothrombotic and proinflammatory signaling without blocking APC-mediated pathways or inducing endothelial injury. In contrast, orthosteric PAR1 antagonists such as vorapaxar inhibit all signaling downstream of PAR1. Furthermore, exposure of endothelial cells to nanomolar concentrations of vorapaxar induces endothelial cell barrier dysfunction and apoptosis. These studies demonstrate how functionally selective antagonism can be achieved by targeting the cytoplasmic face of a G-protein-coupled receptor to selectively block pathologic signaling while preserving cytoprotective pathways.
Asunto(s)
Endotelio Vascular/lesiones , Lactonas/efectos adversos , Piridinas/efectos adversos , Receptor PAR-1/antagonistas & inhibidores , Trombosis/tratamiento farmacológico , Trombosis/prevención & control , Animales , Apoptosis , Sitios de Unión , Células COS , Chlorocebus aethiops , Citoplasma/metabolismo , Endotelio Vascular/efectos de los fármacos , Exocitosis , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inflamación , Ligandos , Inhibidores de Agregación Plaquetaria/química , Proteína C/química , ARN Interferente Pequeño/metabolismo , Transducción de SeñalRESUMEN
A new series of potent inhibitors of cellular lipid uptake from HDL particles mediated by scavenger receptor, class B, type I (SR-BI) was identified. The series was identified via a high-throughput screen of the National Institutes of Health Molecular Libraries Small Molecule Repository (NIH MLSMR) that measured the transfer of the fluorescent lipid DiI from HDL particles to CHO cells overexpressing SR-BI. The series is characterized by a linear peptidomimetic scaffold with two adjacent amide groups, as well as an aryl-substituted heterocycle. Analogs of the initial hit were rapidly prepared via Ugi 4-component reaction, and select enantiopure compounds were prepared via a stepwise sequence. Structure-activity relationship (SAR) studies suggest an oxygenated arene is preferred at the western end of the molecule, as well as highly lipophilic substituents on the central and eastern nitrogens. Compound 5e, with (R)-stereochemistry at the central carbon, was designated as probe ML279. Mechanistic studies indicate that ML279 stabilizes the interaction of HDL particles with SR-BI, and its effect is reversible. It shows good potency (IC50=17 nM), is non-toxic, plasma stable, and has improved solubility over our alternative probe ML278.
Asunto(s)
Alanina/análogos & derivados , Antígenos CD36/antagonistas & inhibidores , Furanos/química , Compuestos Heterocíclicos/química , Tetrazoles/química , Alanina/síntesis química , Alanina/química , Alanina/metabolismo , Animales , Antígenos CD36/genética , Antígenos CD36/metabolismo , Células CHO , Cricetinae , Cricetulus , Evaluación Preclínica de Medicamentos , Lipoproteínas HDL/metabolismo , Unión Proteica , Relación Estructura-Actividad , Tetrazoles/síntesis química , Tetrazoles/metabolismoRESUMEN
We report a new series of 8-membered benzo-fused lactams that inhibit cellular lipid uptake from HDL particles mediated by Scavenger Receptor, Class B, Type I (SR-BI). The series was identified via a high-throughput screen of the National Institutes of Health Molecular Libraries Small Molecule Repository (NIH MLSMR), measuring the transfer of the fluorescent lipid DiI from HDL particles to CHO cells overexpressing SR-BI. The series is part of a previously reported diversity-oriented synthesis (DOS) library prepared via a build-couple-pair approach. Detailed structure-activity relationship (SAR) studies were performed with a selection of the original library, as well as additional analogs prepared via solution phase synthesis. These studies demonstrate that the orientation of the substituents on the aliphatic ring have a critical effect on activity. Additionally, a lipophilic group is required at the western end of the molecule, and a northern hydroxyl group and a southern sulfonamide substituent also proved to be optimal. Compound 2p was found to possess a superior combination of potency (av IC50=0.10µM) and solubility (79µM in PBS), and it was designated as probe ML312.
Asunto(s)
Antígenos CD36/antagonistas & inhibidores , Lactamas/farmacología , Metabolismo de los Lípidos , Animales , Antígenos CD36/fisiología , Humanos , Lactamas/química , Relación Estructura-ActividadRESUMEN
Changes in calcium concentration in cells are rapidly monitored in a high-throughput fashion with the use of intracellular, fluorescent, calcium-binding dyes and imaging instruments that can measure fluorescent emissions from up to 1,536 wells simultaneously. However, these instruments are much more expensive and can be challenging to maintain relative to widely available plate readers that scan wells individually. Described here is an optimized plate reader assay for use with an endothelial cell line (EA.hy926) to measure the protease-activated receptor (PAR)-driven activation of Gαq signaling and subsequent calcium mobilization using the calcium-binding dye Fluo-4. This assay has been used to characterize a range of PAR ligands, including the allosteric PAR1-targeting anti-inflammatory "parmodulin" ligands identified in the Dockendorff lab. This protocol obviates the need for an automated liquid handler and permits the medium-throughput screening of PAR ligands in 96-well plates and should be applicable to the study of other receptors that initiate calcium mobilization.
Asunto(s)
Calcio , Humanos , Calcio/metabolismo , Calcio/análisis , Xantenos/química , Compuestos de Anilina/química , Línea Celular , Colorantes Fluorescentes/química , Ligandos , Receptor PAR-1/metabolismo , Células Endoteliales/metabolismo , Señalización del Calcio/fisiologíaRESUMEN
BACKGROUND AND OBJECTIVE: Protease-activated receptor-1 (PAR1) has emerged as an important link between coagulation and the complications of obesity including metabolic dysfunction-associated steatotic liver disease (MASLD). PAR1 is expressed by various cells and cleaved by different proteases to generate unique tethered agonists that activate distinct signaling pathways. Mice expressing PAR1 with an R41Q mutation have disabled canonical thrombin-mediated signaling, whereas R46Q mice express PAR1 resistant to non-canonical signaling by activated protein C (APC). METHODS: Mice with whole body and hepatocyte-selective PAR1 deficiency, as well as PAR1 R41Q and R46Q mice were fed a high fat diet to induce MASLD. RESULTS AND CONCLUSIONS: High fat diet (HFD)-fed R41Q mice displayed reduced hepatic steatosis and liver/body weight ratio. In contrast, HFD-fed R46Q mice displayed increased relative liver weight and hepatic steatosis alongside increased serum ALT activity. Surprisingly, despite the distinct impact of PAR1 mutations on steatosis, selective deletion of PAR1 in hepatocytes had no impact. To evaluate a viable PAR1-targeted approach, mice with HFD-induced obesity were treated with the allosteric PAR1 modulator NRD-21, which inhibits canonical PAR1 inflammatory signaling but promotes PAR1 protective, non-canonical anti-inflammatory signaling. NRD-21 treatment reduced plasma TNFα, serum ALT activity, hepatic steatosis, and insulin resistance (HOMA-IR), but increased plasma active GLP-1. The results suggest non-hepatocellular canonical PAR1 cleavage drives MASLD in obese mice and provide translational proof-of-concept that selective pharmacological modulation of PAR1 yields multiple metabolic benefits in experimental obesity.
RESUMEN
Continuing efforts to discover novel means of combating fluconazole resistance in Candida albicans have identified an indole derivative that sensitizes strains demonstrating resistance to fluconazole. This tetracycle (3, ML229) does not appear to act through established Hsp90 or calcineurin pathways to chemosensitize C. albicans, as determined in Saccharomyces cerevisiae models, and may be a useful probe to uncover alternative resistance pathways.
Asunto(s)
Candida albicans/efectos de los fármacos , Farmacorresistencia Fúngica/efectos de los fármacos , Fluconazol/farmacología , Indoles/química , Indoles/farmacología , Transducción de SeñalRESUMEN
A major obstacle in diabetes is the metabolic or hyperglycemic memory, which lacks specific therapies. Here we show that glucose-mediated changes in gene expression largely persist in diabetic kidney disease (DKD) despite reversing hyperglycemia. The senescence-associated cyclin-dependent kinase inhibitor p21 (Cdkn1a) was the top hit among genes persistently induced by hyperglycemia and was associated with induction of the p53-p21 pathway. Persistent p21 induction was confirmed in various animal models, human samples and in vitro models. Tubular and urinary p21-levels were associated with DKD severity and remained elevated despite improved blood glucose levels in humans. Mechanistically, sustained tubular p21 expression in DKD is linked to demethylation of its promoter and reduced DNMT1 expression. Two disease resolving agents, protease activated protein C (3K3A-aPC) and parmodulin-2, reversed sustained tubular p21 expression, tubular senescence, and DKD. Thus, p21-dependent tubular senescence is a pathway contributing to the hyperglycemic memory, which can be therapeutically targeted.
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Diabetes Mellitus , Nefropatías Diabéticas , Hiperglucemia , Animales , Senescencia Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Diabetes Mellitus/patología , Nefropatías Diabéticas/patología , Humanos , Hiperglucemia/patología , RiñónRESUMEN
We report the outcome of a high-throughput small-molecule screen to identify novel, nontoxic, inhibitors of Trypansoma cruzi, as potential starting points for therapeutics to treat for both the acute and chronic stages of Chagas disease. Two compounds were identified that displayed nanomolar inhibition of T. cruzi and an absence of activity against host cells at the highest tested dose. These compounds have been registered with NIH Molecular Libraries Program (probes ML157 and ML158).