RESUMEN
There is a lack of FDA-approved tocolytics for the management of preterm labor (PL). In prior drug discovery efforts, we identified mundulone and mundulone acetate (MA) as inhibitors of in vitro intracellular Ca2+-regulated myometrial contractility. In this study, we probed the tocolytic potential of these compounds using human myometrial samples and a mouse model of preterm birth. In a phenotypic assay, mundulone displayed greater efficacy, while MA showed greater potency and uterine-selectivity in the inhibition of intracellular-Ca2+ mobilization. Cell viability assays revealed that MA was significantly less cytotoxic. Organ bath and vessel myography studies showed that only mundulone exerted inhibition of myometrial contractions and that neither compounds affected vasoreactivity of ductus arteriosus. A high-throughput combination screen identified that mundulone exhibits synergism with two clinical-tocolytics (atosiban and nifedipine), and MA displayed synergistic efficacy with nifedipine. Of these combinations, mundulone+atosiban demonstrated a significant improvement in the in vitro therapeutic index compared to mundulone alone. The ex vivo and in vivo synergism of mundulone+atosiban was substantiated, yielding greater tocolytic efficacy and potency on myometrial tissue and reduced preterm birth rates in a mouse model of PL compared to each single agent. Treatment with mundulone after mifepristone administration dose-dependently delayed the timing of delivery. Importantly, mundulone+atosiban permitted long-term management of PL, allowing 71% dams to deliver viable pups at term (>day 19, 4-5 days post-mifepristone exposure) without visible maternal and fetal consequences. Collectively, these studies provide a strong foundation for the development of mundulone as a single or combination tocolytic for management of PL.
Asunto(s)
Productos Biológicos , Trabajo de Parto Prematuro , Nacimiento Prematuro , Tocolíticos , Femenino , Recién Nacido , Ratones , Animales , Humanos , Tocolíticos/farmacología , Tocolíticos/uso terapéutico , Nacimiento Prematuro/tratamiento farmacológico , Nifedipino/farmacología , Nifedipino/uso terapéutico , Mifepristona/uso terapéutico , Productos Biológicos/uso terapéutico , Trabajo de Parto Prematuro/tratamiento farmacológicoRESUMEN
The 3 human RAS genes, KRAS, NRAS, and HRAS, encode 4 different RAS proteins which belong to the protein family of small GTPases that function as binary molecular switches involved in cell signaling. Activating mutations in RAS are among the most common oncogenic drivers in human cancers, with KRAS being the most frequently mutated oncogene. Although KRAS is an excellent drug discovery target for many cancers, and despite decades of research, no therapeutic agent directly targeting RAS has been clinically approved. Using structure-based drug design, we have discovered BI-2852 (1), a KRAS inhibitor that binds with nanomolar affinity to a pocket, thus far perceived to be "undruggable," between switch I and II on RAS; 1 is mechanistically distinct from covalent KRASG12C inhibitors because it binds to a different pocket present in both the active and inactive forms of KRAS. In doing so, it blocks all GEF, GAP, and effector interactions with KRAS, leading to inhibition of downstream signaling and an antiproliferative effect in the low micromolar range in KRAS mutant cells. These findings clearly demonstrate that this so-called switch I/II pocket is indeed druggable and provide the scientific community with a chemical probe that simultaneously targets the active and inactive forms of KRAS.
Asunto(s)
Descubrimiento de Drogas , Preparaciones Farmacéuticas/química , Proteínas Proto-Oncogénicas p21(ras)/química , Guanosina Trifosfato/metabolismo , Humanos , Modelos Moleculares , Nanopartículas/químicaRESUMEN
The Receptor for Advanced Glycation End products (RAGE) is a pattern recognition receptor that signals for inflammation via the NF-κB pathway. RAGE has been pursued as a potential target to suppress symptoms of diabetes and is of interest in a number of other diseases associated with chronic inflammation, such as inflammatory bowel disease and bronchopulmonary dysplasia. Screening and optimization have previously produced small molecules that inhibit the activity of RAGE in cell-based assays, but efforts to develop a therapeutically viable direct-binding RAGE inhibitor have yet to be successful. Here, we show that a fragment-based approach can be applied to discover fundamentally new types of RAGE inhibitors that specifically target the ligand-binding surface. A series of systematic assays of structural stability, solubility, and crystallization were performed to select constructs of the RAGE ligand-binding domain and optimize conditions for NMR-based screening and co-crystallization of RAGE with hit fragments. An NMR-based screen of a highly curated ~14 000-member fragment library produced 21 fragment leads. Of these, three were selected for elaboration based on structure-activity relationships generated through cycles of structural analysis by X-ray crystallography, structure-guided design principles, and synthetic chemistry. These results, combined with crystal structures of the first linked fragment compounds, demonstrate the applicability of the fragment-based approach to the discovery of RAGE inhibitors.
Asunto(s)
Benzamidas/química , Diseño de Fármacos/métodos , Imidazoles/química , Receptor para Productos Finales de Glicación Avanzada/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/química , Benzamidas/metabolismo , Benzamidas/farmacología , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Imidazoles/metabolismo , Imidazoles/farmacología , Ligandos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Receptor para Productos Finales de Glicación Avanzada/química , Receptor para Productos Finales de Glicación Avanzada/genética , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Bibliotecas de Moléculas Pequeñas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-ActividadRESUMEN
Staphylococcus aureus is a serious threat to public health due to the rise of antibiotic resistance in this organism, which can prolong or exacerbate skin and soft tissue infections (SSTIs). Methicillin-resistant S. aureus is a Gram-positive bacterium and a leading cause of SSTIs. As such, many efforts are under way to develop therapies that target essential biological processes in S. aureus. Antimicrobial photodynamic therapy is an effective alternative to antibiotics; therefore we developed an approach to simultaneously expose S. aureus to intracellular and extracellular photosensitizers. A near infrared photosensitizer was conjugated to human monoclonal antibodies (MAbs) that target the S. aureus iron-regulated surface determinant (Isd) heme acquisition proteins. In addition, the compound VU0038882 was developed to increase photoactivatable porphyrins within the cell. Combinatorial photodynamic treatment of drug-resistant S. aureus exposed to VU0038882 and conjugated anti-Isd MAbs proved to be an effective antibacterial strategy in vitro and in a murine model of SSTIs.
Asunto(s)
Staphylococcus aureus Resistente a Meticilina , Infecciones de los Tejidos Blandos , Infecciones Estafilocócicas , Animales , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Humanos , Ratones , Fármacos Fotosensibilizantes/farmacología , Infecciones de los Tejidos Blandos/tratamiento farmacológico , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureusRESUMEN
Lactate dehydrogenase (LDH) is a critical enzyme in the glycolytic metabolism pathway that is used by many tumor cells. Inhibitors of LDH may be expected to inhibit the metabolic processes in cancer cells and thus selectively delay or inhibit growth in transformed versus normal cells. We have previously disclosed a pyrazole-based series of potent LDH inhibitors with long residence times on the enzyme. Here, we report the elaboration of a new subseries of LDH inhibitors based on those leads. These new compounds potently inhibit both LDHA and LDHB enzymes, and inhibit lactate production in cancer cell lines.
Asunto(s)
Compuestos de Anilina/farmacología , Antineoplásicos/farmacología , Diseño de Fármacos , Éteres/farmacología , L-Lactato Deshidrogenasa/antagonistas & inhibidores , L-Lactato Deshidrogenasa/metabolismo , Compuestos de Anilina/química , Antineoplásicos/química , Línea Celular Tumoral , Éteres/química , Humanos , L-Lactato Deshidrogenasa/químicaRESUMEN
The mitotic spindle is a microtubule-based machine that segregates a replicated set of chromosomes during cell division. Many cancer drugs alter or disrupt the microtubules that form the mitotic spindle. Microtubule-dependent molecular motors that function during mitosis are logical alternative mitotic targets for drug development. Eg5 (Kinesin-5) and Kif15 (Kinesin-12), in particular, are an attractive pair of motor proteins, as they work in concert to drive centrosome separation and promote spindle bipolarity. Furthermore, we hypothesize that the clinical failure of Eg5 inhibitors may be (in part) due to compensation by Kif15. In order to test this idea, we screened a small library of kinase inhibitors and identified GW108X, an oxindole that inhibits Kif15 in vitro. We show that GW108X has a distinct mechanism of action compared with a commercially available Kif15 inhibitor, Kif15-IN-1 and may serve as a lead with which to further develop Kif15 inhibitors as clinically relevant agents.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Cinesinas/antagonistas & inhibidores , Sondas Moleculares/farmacología , Oxindoles/farmacología , Quinazolinonas/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Cinesinas/metabolismo , Sondas Moleculares/síntesis química , Sondas Moleculares/química , Estructura Molecular , Oxindoles/síntesis química , Oxindoles/química , Quinazolinonas/síntesis química , Quinazolinonas/química , Relación Estructura-ActividadRESUMEN
K-RAS is mutated in approximately 30% of human cancers, resulting in increased RAS signaling and tumor growth. Thus, RAS is a highly validated therapeutic target, especially in tumors of the pancreas, lung and colon. Although directly targeting RAS has proven to be challenging, it may be possible to target other proteins involved in RAS signaling, such as the guanine nucleotide exchange factor Son of Sevenless (SOS). We have previously reported on the discovery of small molecules that bind to SOS1, activate SOS-mediated nucleotide exchange on RAS, and paradoxically inhibit ERK phosphorylation (Burns et al., PNAS, 2014). Here, we describe the discovery of additional, structurally diverse small molecules that also bind to SOS1 in the same pocket and elicit similar biological effects. We tested >160,000 compounds in a fluorescence-based assay to assess their effects on SOS-mediated nucleotide exchange. X-Ray structures revealed that these small molecules bind to the CDC25 domain of SOS1. Compounds that elicited high levels of nucleotide exchange activity in vitro increased RAS-GTP levels in cells, and inhibited phospho ERK levels at higher treatment concentrations. The identification of structurally diverse SOS1 binding ligands may assist in the discovery of new molecules designed to target RAS-driven tumors.
Asunto(s)
Sistema de Señalización de MAP Quinasas , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteína SOS1/metabolismo , Células HeLa , Humanos , Proteínas Proto-Oncogénicas p21(ras)/química , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteína SOS1/química , Proteína SOS1/genéticaRESUMEN
This letter describes the on-going SAR efforts to develop PLD1, PLD2 and dual PLD1/2 inhibitors with improved physiochemical and disposition properties as well as securing intellectual property position. Previous PLD inhibitors, based on a triazaspiro[4.5]decanone core proved to be highly selective PLD2 inhibitors, but with low plasma free fraction (rat, human fuâ¯<â¯0.03), high predicted hepatic clearance (rat CLhepâ¯>â¯65â¯mL/min/kg) and very short half-lives in vivo (t1/2â¯<â¯0.15â¯h). Removal of a nitrogen atom from this core generated a 2,8-diazaspiro[4.5]decanone core, harboring a new chiral center, as well as increased sp3 character. This new core demonstrated enantioselective inhibition of the individual PLD isoforms, enhanced free fraction (rat, human fuâ¯<â¯0.13), engendered moderate predicted hepatic clearance (rat CLhepâ¯â¼â¯43â¯mL/min/kg), improved half-lives in vivo (t1/2â¯>â¯3â¯h), and led to the first issued US patent claiming composition of matter for small molecule PLD inhibitors.
Asunto(s)
Inhibidores Enzimáticos/química , Fosfolipasa D/metabolismo , Compuestos de Espiro/química , Animales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacocinética , Células HEK293 , Semivida , Humanos , Concentración 50 Inhibidora , Fosfolipasa D/antagonistas & inhibidores , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Ratas , Compuestos de Espiro/síntesis química , Compuestos de Espiro/farmacocinética , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Ewing sarcomas are rare mesenchymal-derived bone and soft tissue tumors in children. Afflicted children with distant metastases have poor survival despite aggressive therapeutics. Epithelial-to-mesenchymal transition in epithelial carcinomas is associated with loss of E-cadherin and resistance to apoptosis. ML327 is a novel small molecule that we have previously shown to reverse epithelial-to-mesenchymal transition features in both epithelial and neural crest-derived cancers. Herein, we sought to evaluate the effects of ML327 on mesenchymal-derived Ewing sarcoma cells, hypothesizing that ML327 initiates growth arrest and sensitizes to TNF-related apoptosis-inducing ligand. ML327 induced protein expression changes, increased E-cadherin and decreased vimentin, consistent with partial induction of mesenchymal-to-epithelial transition in multiple Ewing Sarcoma cell lines (SK-N-MC, TC71, and ES-5838). Induction of epithelial features was associated with apoptosis, as demonstrated by PARP and Caspase 3 cleavage by immunoblotting. Cell cycle analysis validated these findings by marked induction of the subG0 cell population. In vitro combination treatment with TRAIL demonstrated additive induction of apoptotic markers. Taken together, these findings establish a rationale for further in vivo trials of ML327 in cells of mesenchymal origin both alone and in combination with TRAIL.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Isoxazoles/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Niacinamida/análogos & derivados , Bibliotecas de Moléculas Pequeñas/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Antígenos CD , Antineoplásicos/química , Cadherinas/genética , Cadherinas/metabolismo , Caspasa 3/genética , Caspasa 3/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Sinergismo Farmacológico , Transición Epitelial-Mesenquimal/efectos de los fármacos , Regulación de la Expresión Génica , Humanos , Isoxazoles/química , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/patología , Niacinamida/química , Niacinamida/farmacología , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patología , Transducción de Señal , Bibliotecas de Moléculas Pequeñas/química , Vimentina/genética , Vimentina/metabolismoRESUMEN
Aberrant activation of the small GTPase Ras by oncogenic mutation or constitutively active upstream receptor tyrosine kinases results in the deregulation of cellular signals governing growth and survival in â¼30% of all human cancers. However, the discovery of potent inhibitors of Ras has been difficult to achieve. Here, we report the identification of small molecules that bind to a unique pocket on the Ras:Son of Sevenless (SOS):Ras complex, increase the rate of SOS-catalyzed nucleotide exchange in vitro, and modulate Ras signaling pathways in cells. X-ray crystallography of Ras:SOS:Ras in complex with these molecules reveals that the compounds bind in a hydrophobic pocket in the CDC25 domain of SOS adjacent to the Switch II region of Ras. The structure-activity relationships exhibited by these compounds can be rationalized on the basis of multiple X-ray cocrystal structures. Mutational analyses confirmed the functional relevance of this binding site and showed it to be essential for compound activity. These molecules increase Ras-GTP levels and disrupt MAPK and PI3K signaling in cells at low micromolar concentrations. These small molecules represent tools to study the acute activation of Ras and highlight a pocket on SOS that may be exploited to modulate Ras signaling.
Asunto(s)
Indoles/metabolismo , Modelos Moleculares , Complejos Multiproteicos/metabolismo , Piperidinas/metabolismo , Conformación Proteica , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Proteína SOS1/metabolismo , Cromatografía Liquida , Cromatografía en Capa Delgada , Cristalografía por Rayos X , Polarización de Fluorescencia , Células HeLa , Humanos , Ligandos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Estructura Molecular , Complejos Multiproteicos/química , Proteínas Proto-Oncogénicas p21(ras)/química , Proteína SOS1/químicaRESUMEN
E-cadherin is a ubiquitous trans-membrane protein that has important functions in cellular contacts and has been shown to play a role in the epithelial mesenchymal transition. We have previously reported the use of an HTS screen to identify compounds that are capable of restoring e-cadherin in cancer cells. Here, we report the additional medicinal chemistry optimization of these molecules, resulting in new molecules that restore e-cadherin expression at low micromolar concentrations. Further, we report preliminary pharmacokinetic data on a compound, ML327, that can be used as a probe of e-cadherin restoration.
Asunto(s)
Cadherinas/biosíntesis , Isoxazoles/farmacología , Animales , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Isoxazoles/síntesis química , Isoxazoles/química , Ratones , Estructura Molecular , Ratas , Relación Estructura-ActividadRESUMEN
The systematic exploration of a series of triazole-based agonists of the cation channel insect odorant receptor is reported. The structure-activity relationships of independent sections of the molecules are examined. Very small changes to the compound structure were found to exert a large impact on compound activity. Optimal substitutions were combined using a 'mix-and-match' strategy to produce best-in-class compounds that are capable of potently agonizing odorant receptor activity and may form the basis for the identification of a new mode of insect behavior modification.
Asunto(s)
Drosophila melanogaster/fisiología , Receptores Odorantes/agonistas , Animales , Indoles/química , Indoles/farmacología , Estructura Molecular , Receptores Odorantes/efectos de los fármacos , Relación Estructura-Actividad , Triazoles/química , Triazoles/farmacologíaRESUMEN
Replication protein A (RPA) is the primary single-stranded DNA (ssDNA) binding protein in eukaryotes. The N-terminal domain of the RPA70 subunit (RPA70N) interacts via a basic cleft with a wide range of DNA processing proteins, including several that regulate DNA damage response and repair. Small molecule inhibitors that disrupt these protein-protein interactions are therefore of interest as chemical probes of these critical DNA processing pathways and as inhibitors to counter the upregulation of DNA damage response and repair associated with treatment of cancer patients with radiation or DNA-damaging agents. Determination of three-dimensional structures of protein-ligand complexes is an important step for elaboration of small molecule inhibitors. However, although crystal structures of free RPA70N and an RPA70N-peptide fusion construct have been reported, RPA70N-inhibitor complexes have been recalcitrant to crystallization. Analysis of the P61 lattice of RPA70N crystals led us to hypothesize that the ligand-binding surface was occluded. Surface reengineering to alter key crystal lattice contacts led to the design of RPA70N E7R, E100R, and E7R/E100R mutants. These mutants crystallized in a P212121 lattice that clearly had significant solvent channels open to the critical basic cleft. Analysis of X-ray crystal structures, target peptide binding affinities, and (15)N-(1)H heteronuclear single-quantum coherence nuclear magnetic resonance spectra showed that the mutations do not result in perturbations of the RPA70N ligand-binding surface. The success of the design was demonstrated by determining the structure of RPA70N E7R soaked with a ligand discovered in a previously reported molecular fragment screen. A fluorescence anisotropy competition binding assay revealed this compound can inhibit the interaction of RPA70N with the peptide binding motif from the DNA damage response protein ATRIP. The implications of the results are discussed in the context of ongoing efforts to design RPA70N inhibitors.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteína de Replicación A/genética , Proteínas Adaptadoras Transductoras de Señales/química , Cristalización , Cristalografía por Rayos X , Proteínas de Unión al ADN/química , Polarización de Fluorescencia , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Dominios y Motivos de Interacción de Proteínas , Proteína de Replicación A/antagonistas & inhibidores , Proteína de Replicación A/química , Electricidad EstáticaRESUMEN
Currently, there is a lack of FDA-approved tocolytics for the management of preterm labor (PL). In prior drug discovery efforts, we identified mundulone and its analog mundulone acetate (MA) as inhibitors of in vitro intracellular Ca 2+ -regulated myometrial contractility. In this study, we probed the tocolytic and therapeutic potential of these small molecules using myometrial cells and tissues obtained from patients receiving cesarean deliveries, as well as a mouse model of PL resulting in preterm birth. In a phenotypic assay, mundulone displayed greater efficacy in the inhibition of intracellular-Ca 2+ from myometrial cells; however, MA showed greater potency and uterine-selectivity, based IC 50 and E max values between myometrial cells compared to aorta vascular smooth muscle cells, a major maternal off-target site of current tocolytics. Cell viability assays revealed that MA was significantly less cytotoxic. Organ bath and vessel myography studies showed that only mundulone exerted concentration-dependent inhibition of ex vivo myometrial contractions and that neither mundulone or MA affected vasoreactivity of ductus arteriosus, a major fetal off-target of current tocolytics. A high-throughput combination screen of in vitro intracellular Ca 2+ -mobilization identified that mundulone exhibits synergism with two clinical-tocolytics (atosiban and nifedipine), and MA displayed synergistic efficacy with nifedipine. Of these synergistic combinations, mundulone + atosiban demonstrated a favorable in vitro therapeutic index (TI)=10, a substantial improvement compared to TI=0.8 for mundulone alone. The ex vivo and in vivo synergism of mundulone and atosiban was substantiated, yielding greater tocolytic efficacy and potency on isolated mouse and human myometrial tissue and reduced preterm birth rates in a mouse model of PL compared to each single agent. Treatment with mundulone 5hrs after mifepristone administration (and PL induction) dose-dependently delayed the timing of delivery. Importantly, mundulone in combination with atosiban (FR 3.7:1, 6.5mg/kg + 1.75mg/kg) permitted long-term management of PL after induction with 30 µg mifepristone, allowing 71% dams to deliver viable pups at term (> day 19, 4-5 days post-mifepristone exposure) without any visible maternal and fetal consequences. Collectively, these studies provide a strong foundation for the future development of mundulone as a stand-alone single- and/or combination-tocolytic therapy for management of PL.
RESUMEN
N-Acyl-phosphatidylethanolamine hydrolyzing phospholipase D (NAPE-PLD) is a zinc metallohydrolase that hydrolyzes N-acyl-phosphatidylethanolamines (NAPEs) to form N-acyl-ethanolamines (NAEs) and phosphatidic acid. Several lines of evidence suggest that reduced NAPE-PLD activity could contribute to cardiometabolic diseases. For instance, NAPEPLD expression is reduced in human coronary arteries with unstable atherosclerotic lesions, defective efferocytosis is implicated in the enlargement of necrotic cores of these lesions, and NAPE-PLD products such as palmitoylethanolamide and oleoylethanolamide have been shown to enhance efferocytosis. Thus, enzyme activation mediated by a small molecule may serve as a therapeutic treatment for cardiometabolic diseases. As a proof-of-concept study, we sought to identify small molecule activators of NAPE-PLD. High-throughput screening followed by hit validation and primary lead optimization studies identified a series of benzothiazole phenylsulfonyl-piperidine carboxamides that variably increased activity of both mouse and human NAPE-PLD. From this set of small molecules, two NAPE-PLD activators (VU534 and VU533) were shown to increase efferocytosis by bone-marrow derived macrophages isolated from wild-type mice, while efferocytosis was significantly reduced in Napepld-/- BMDM or after Nape-pld inhibition. Together, these studies demonstrate an essential role for NAPE-PLD in the regulation of efferocytosis and the potential value of NAPE-PLD activators as a strategy to treat cardiometabolic diseases.
Asunto(s)
Enfermedades Cardiovasculares , Fosfolipasa D , Ratones , Humanos , Animales , Fosfatidiletanolaminas/metabolismo , Encéfalo/metabolismo , Macrófagos/metabolismo , Enfermedades Cardiovasculares/metabolismoRESUMEN
N -acyl-phosphatidylethanolamine hydrolyzing phospholipase D (NAPE-PLD) is a zinc metallohydrolase that hydrolyzes N -acyl-phosphatidylethanolamine (NAPEs) to form N -acyl-ethanolamides (NAEs) and phosphatidic acid. Several lines of evidence suggest that reduced NAPE-PLD activity could contribute to cardiometabolic diseases. For instance, NAPEPLD expression is reduced in human coronary arteries with unstable atherosclerotic lesions, defective efferocytosis is implicated in the enlargement of necrotic cores of these lesions, and NAPE-PLD products such as palmitoylethanolamide and oleoylethanolamide have been shown to enhance efferocytosis. Thus, enzyme activation mediated by a small molecule may serve as a therapeutic treatment for cardiometabolic diseases. As a proof-of-concept study, we sought to identify small molecule activators of NAPE-PLD. High-throughput screening followed by hit validation and primary lead optimization studies identified a series of benzothiazole phenylsulfonyl-piperidine carboxamides that variably increased activity of both mouse and human NAPE-PLD. From this set of small molecules, two NAPE-PLD activators (VU534 and VU533) were shown to increase efferocytosis by bone-marrow derived macrophages isolated from wild-type mice, while efferocytosis was significantly reduced in Napepld -/- BMDM or after Nape-pld inhibition. Together these studies demonstrate an essential role for NAPE-PLD in the regulation of efferocytosis and the potential value of NAPE-PLD activators as a strategy to treat cardiometabolic diseases.
RESUMEN
Wnt/ß-catenin signaling is critically involved in metazoan development, stem cell maintenance and human disease. Using Xenopus laevis egg extract to screen for compounds that both stabilize Axin and promote ß-catenin turnover, we identified an FDA-approved drug, pyrvinium, as a potent inhibitor of Wnt signaling (EC(50) of â¼10 nM). We show pyrvinium binds all casein kinase 1 (CK1) family members in vitro at low nanomolar concentrations and pyrvinium selectively potentiates casein kinase 1α (CK1α) kinase activity. CK1α knockdown abrogates the effects of pyrvinium on the Wnt pathway. In addition to its effects on Axin and ß-catenin levels, pyrvinium promotes degradation of Pygopus, a Wnt transcriptional component. Pyrvinium treatment of colon cancer cells with mutation of the gene for adenomatous polyposis coli (APC) or ß-catenin inhibits both Wnt signaling and proliferation. Our findings reveal allosteric activation of CK1α as an effective mechanism to inhibit Wnt signaling and highlight a new strategy for targeted therapeutics directed against the Wnt pathway.
Asunto(s)
Caseína Quinasa Ialfa/metabolismo , Proliferación Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Compuestos de Pirvinio/farmacología , Transducción de Señal/efectos de los fármacos , Proteínas Wnt/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales , Poliposis Adenomatosa del Colon/genética , Poliposis Adenomatosa del Colon/metabolismo , Animales , Proteína Axina , Quinasa de la Caseína I/genética , Quinasa de la Caseína I/metabolismo , Extractos Celulares , Línea Celular Tumoral , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Oocitos/citología , Oocitos/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteínas Wnt/química , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Proteínas de Xenopus , Xenopus laevis , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Further chemical optimization of the MLSCN/MLPCN probe ML077 (KCC2 IC(50)=537 nM) proved to be challenging as the effort was characterized by steep SAR. However, a multi-dimensional iterative parallel synthesis approach proved productive. Herein we report the discovery and SAR of an improved novel antagonist (VU0463271) of the neuronal-specific potassium-chloride cotransporter 2 (KCC2), with an IC(50) of 61 nM and >100-fold selectivity versus the closely related Na-K-2Cl cotransporter 1 (NKCC1) and no activity in a larger panel of GPCRs, ion channels and transporters.
Asunto(s)
Piridazinas/química , Simportadores/antagonistas & inhibidores , Tiazoles/química , Estructura Molecular , Piridazinas/farmacología , Relación Estructura-Actividad , Tiazoles/farmacología , Cotransportadores de K ClRESUMEN
Plasma membrane organization profoundly impacts cellular functionality. A well-known mechanism underlying this organization is through nanoscopic clustering of distinct lipids and proteins in membrane rafts. Despite their physiological importance, rafts remain a difficult-to-study aspect of membrane organization, in part because of the paucity of chemical tools to experimentally modulate their properties. Methods to selectively target rafts for therapeutic purposes are also currently lacking. To tackle these problems, we developed a high-throughput screen and an accompanying image analysis pipeline to identify small molecules that enhance or inhibit raft formation. Cell-derived giant plasma membrane vesicles were used as the experimental platform. A proof-of-principle screen using a bioactive lipid library demonstrates that this method is robust and capable of validating established raft modulators including C6- and C8-ceramide, miltefosine, and epigallocatechin gallate as well as identifying new ones. The platform we describe here represents a powerful tool to discover new chemical approaches to manipulate rafts and their components.