RESUMEN
Novel antiviral drugs, which are less prone to resistance development, are desirable alternatives to the currently approved drugs for the treatment of potentially serious influenza virus infections. The viral polymerase is highly conserved and serves as an attractive target for antiviral drugs since potent inhibitors would directly stop viral replication at an early stage. Recent structural studies on the functional domains of the heterotrimeric influenza polymerase, which comprises subunits PA, PB1, and PB2, opened the way to a structure-based approach for optimizing inhibitors of viral replication. These strategies, however, are limited by the use of isolated protein fragments instead of employing the entire ribonucleoprotein complex (RNP), which represents the functional form of the influenza polymerase in infected cells. In this study, we have established a screening assay for efficient and reliable analysis of potential influenza polymerase inhibitors of various molecular targets such as monoselective polymerase inhibitors targeting the endonuclease site, the cap-binding domain, and the polymerase active site, respectively. By utilizing whole viral RNPs and a radioactivity-free endpoint detection with the capability for efficient compound screening while offering high-content information on potential inhibitors to drive medicinal chemistry program in a reliable manner, this biochemical assay provides significant advantages over the currently available conventional assays. We propose that this assay can eventually be adapted for coinstantaneous analysis and subsequent optimization of two or more different chemical scaffold classes targeting multiple active sites within the polymerase complex, thus enabling the evaluation of drug combinations and characterization of molecules with dual functionality.
Asunto(s)
Antivirales/análisis , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , ARN Polimerasas Dirigidas por ADN/análisis , Virus de la Influenza A/enzimología , Ribonucleoproteínas/análisis , Antivirales/farmacología , ARN Polimerasas Dirigidas por ADN/genética , Evaluación Preclínica de Medicamentos/métodos , Humanos , Virus de la Influenza A/efectos de los fármacos , Ribonucleoproteínas/genética , Ribonucleoproteínas/farmacología , Replicación Viral/efectos de los fármacos , Replicación Viral/fisiologíaRESUMEN
The discovery of a novel series of pyrrolopyrazines as JAK inhibitors with comparable enzyme and cellular activity to tofacitinib is described. The series was identified using a scaffold hopping approach aided by structure based drug design using principles of intramolecular hydrogen bonding for conformational restriction and targeting specific pockets for modulating kinase activity.
Asunto(s)
Janus Quinasa 3/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Pirazinas/química , Pirroles/química , Diseño de Fármacos , Humanos , Janus Quinasa 3/metabolismo , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Fosforilación , Piperidinas/farmacología , Pirimidinas/farmacología , Pirroles/farmacología , Relación Estructura-ActividadRESUMEN
Inhibition of spleen tyrosine kinase has attracted much attention as a mechanism for the treatment of cancers and autoimmune diseases such as asthma, rheumatoid arthritis, and systemic lupus erythematous. We report the structure-guided optimization of pyridazine amide spleen tyrosine kinase inhibitors. Early representatives of this scaffold were highly potent and selective but mutagenic in an Ames assay. An approach that led to the successful identification of nonmutagenic examples, as well as further optimization to compounds with reduced cardiovascular liabilities is described. Select pharmacokinetic and in vivo efficacy data are presented.
Asunto(s)
Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Piridazinas/síntesis química , Piridazinas/farmacología , Bazo/enzimología , Amidas/síntesis química , Amidas/farmacología , Animales , Biología Computacional , Simulación por Computador , Diseño de Fármacos , Canales de Potasio Éter-A-Go-Go/efectos de los fármacos , Humanos , Técnicas In Vitro , Ratones , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Conformación Molecular , Mutagénesis/efectos de los fármacos , Pruebas de Mutagenicidad , Inhibidores de Proteínas Quinasas/farmacocinética , Piridazinas/farmacocinética , Ratas , Bazo/efectos de los fármacos , Relación Estructura-Actividad , Difracción de Rayos XRESUMEN
INTRODUCTION: Spleen tyrosine kinase (SYK) is a key integrator of intracellular signals triggered by activated immunoreceptors, including Bcell receptors (BCR) and Fc receptors, which are important for the development and function of lymphoid cells. Given the clinical efficacy of Bcell depletion in the treatment of rheumatoid arthritis and multiple sclerosis, pharmacological modulation of Bcells using orally active small molecules that selectively target SYK presents an attractive alternative therapeutic strategy. METHODS: A SYK inhibitor was developed and assayed in various in vitro systems and in the mouse model of collagen-induced arthritis (mCIA). RESULTS: A novel ATP-competitive inhibitor of SYK, 6-[(1R,2S)-2-Amino-cyclohexylamino]-4-(5,6-dimethyl-pyridin-2-ylamino)-pyridazine-3-carboxylic acid amide, designated RO9021, with an adequate kinase selectivity profile and oral bioavailability, was developed. In addition to suppression of BCR signaling in human peripheral blood mononuclear cells (PBMC) and whole blood, FcγR signaling in human monocytes, and FcϵR signaling in human mast cells, RO9021 blocked osteoclastogenesis from mouse bone marrow macrophages in vitro. Interestingly, Toll-like Receptor (TLR) 9 signaling in human Bcells was inhibited by RO9021, resulting in decreased levels of plasmablasts, immunoglobulin (Ig) M and IgG upon B-cell differentiation. RO9021 also potently inhibited type I interferon production by human plasmacytoid dendritic cells (pDC) upon TLR9 activation. This effect is specific to TLR9 as RO9021 did not inhibit TLR4- or JAK-STAT-mediated signaling. Finally, oral administration of RO9021 inhibited arthritis progression in the mCIA model, with observable pharmacokinetics (PK)-pharmacodynamic (PD) correlation. CONCLUSIONS: Inhibition of SYK kinase activity impinges on various innate and adaptive immune responses. RO9021 could serve as a starting point for the development of selective SYK inhibitors for the treatment of inflammation-related and autoimmune-related disorders.
Asunto(s)
Inmunidad Adaptativa/efectos de los fármacos , Inmunidad Innata/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Administración Oral , Aminopiridinas/química , Aminopiridinas/farmacología , Animales , Artritis Experimental/metabolismo , Artritis Experimental/prevención & control , Enfermedades Autoinmunes/tratamiento farmacológico , Enfermedades Autoinmunes/metabolismo , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/metabolismo , Línea Celular Tumoral , Células Cultivadas , Citometría de Flujo , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células Jurkat , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Modelos Moleculares , Estructura Molecular , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Unión Proteica , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/química , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/metabolismo , Piridazinas/química , Piridazinas/farmacología , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Bibliotecas de Moléculas Pequeñas/química , Quinasa SykRESUMEN
We report the discovery of a novel series of ATP-competitive Janus kinase 3 (JAK3) inhibitors based on the 5H-pyrrolo[2,3-b]pyrazine scaffold. The initial leads in this series, compounds 1a and 1h, showed promising potencies, but a lack of selectivity against other isoforms in the JAK family. Computational and crystallographic analysis suggested that the phenyl ether moiety possessed a favorable vector to achieve selectivity. Exploration of this vector resulted in the identification of 12b and 12d, as potent JAK3 inhibitors, demonstrating improved JAK family and kinase selectivity.
Asunto(s)
Janus Quinasa 3/antagonistas & inhibidores , Éteres Fenílicos/química , Inhibidores de Proteínas Quinasas/química , Piridazinas/química , Pirroles/química , Sitios de Unión , Dominio Catalítico , Evaluación Preclínica de Medicamentos , Janus Quinasa 3/metabolismo , Simulación del Acoplamiento Molecular , Éteres Fenílicos/síntesis química , Éteres Fenílicos/metabolismo , Unión Proteica , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/metabolismo , Relación Estructura-ActividadRESUMEN
Using a structure based design approach we have identified a series of indazole substituted pyrrolopyrazines, which are potent inhibitors of JAK3. Intramolecular electronic repulsion was used as a strategy to induce a strong conformational bias within the ligand. Compounds bearing this conformation participated in a favorable hydrophobic interaction with a cysteine residue in the JAK3 binding pocket, which imparted high selectivity versus the kinome and improved selectivity within the JAK family.
Asunto(s)
Diseño de Fármacos , Janus Quinasa 3/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Sitios de Unión , Cristalografía por Rayos X , Interacciones Hidrofóbicas e Hidrofílicas , Indazoles/química , Janus Quinasa 1/antagonistas & inhibidores , Janus Quinasa 1/metabolismo , Janus Quinasa 2/antagonistas & inhibidores , Janus Quinasa 2/metabolismo , Janus Quinasa 3/metabolismo , Simulación del Acoplamiento Molecular , Unión Proteica , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/metabolismo , Estructura Terciaria de Proteína , Pirazinas/síntesis química , Pirazinas/química , Pirazinas/metabolismo , Relación Estructura-ActividadRESUMEN
We describe the discovery of several pyrrolopyrazines as potent and selective Syk inhibitors and the efforts that eventually led to the desired improvements in physicochemical properties and human whole blood potencies. Ultimately, our mouse model revealed unexpected toxicity that precluded us from further advancing this series.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirazinas/síntesis química , Pirroles/síntesis química , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/enzimología , Proteínas Sanguíneas/metabolismo , Cristalografía por Rayos X , Humanos , Ratones , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Estructura Molecular , Unión Proteica , Pirazinas/farmacología , Pirazinas/toxicidad , Pirroles/farmacología , Pirroles/toxicidad , Relación Estructura-Actividad , Quinasa SykRESUMEN
Organic impurities in compound libraries are known to often cause false-positive signals in screening campaigns for new leads, but organic impurities do not fully account for all false-positive results. We discovered inorganic impurities in our screening library that can also cause positive signals for a variety of targets and/or readout systems, including biochemical and biosensor assays. We investigated in depth the example of zinc for a specific project and in retrospect in various HTS screens at Roche and propose a straightforward counter screen using the chelator TPEN to rule out inhibition caused by zinc.
RESUMEN
The Janus kinases (JAKs) are involved in multiple signaling networks relevant to inflammatory diseases, and inhibition of one or more members of this class may modulate disease activity or progression. We optimized a new inhibitor scaffold, 3-amido-5-cyclopropylpyrrolopyrazines, to a potent example with reasonable kinome selectivity, including selectivity for JAK3 versus JAK1, and good biopharmaceutical properties. Evaluation of this analogue in cellular and in vivo models confirmed functional selectivity for modulation of a JAK3/JAK1-dependent IL-2 stimulated pathway over a JAK1/JAK2/Tyk2-dependent IL-6 stimulated pathway.
Asunto(s)
Antiinflamatorios no Esteroideos/síntesis química , Ciclopropanos/síntesis química , Janus Quinasa 1/antagonistas & inhibidores , Janus Quinasa 3/antagonistas & inhibidores , Pirazinas/síntesis química , Pirroles/síntesis química , Administración Oral , Animales , Antiinflamatorios no Esteroideos/farmacocinética , Antiinflamatorios no Esteroideos/farmacología , Células CACO-2 , Cristalografía por Rayos X , Ciclopropanos/farmacocinética , Ciclopropanos/farmacología , Técnicas de Silenciamiento del Gen , Ensayos Analíticos de Alto Rendimiento , Humanos , Interleucina-2/fisiología , Janus Quinasa 1/genética , Janus Quinasa 1/metabolismo , Janus Quinasa 3/genética , Janus Quinasa 3/metabolismo , Ratones , Modelos Moleculares , Pirazinas/farmacocinética , Pirazinas/farmacología , Pirroles/farmacocinética , Pirroles/farmacología , ARN Interferente Pequeño/genética , Ratas , Receptores de Interleucina-6/fisiología , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
The use of nicotinic acid to treat dyslipidemia is limited by induction of a "flushing" response, mediated in part by the interaction of prostaglandin D(2) (PGD(2)) with its G-protein coupled receptor, DP1 (Ptgdr). The impact of DP1 blockade (genetic or pharmacologic) was assessed in experimental murine models of atherosclerosis. In Ptgdr(-/-)ApoE(-/-) mice versus ApoE(-/-) mice, both fed a high-fat diet, aortic cholesterol content was modestly higher (1.3- to 1.5-fold, P < 0.05) in Ptgdr(-/-)ApoE(-/-) mice at 16 and 24 weeks of age, but not at 32 weeks. In multiple ApoE(-/-) mouse studies, a DP1-specific antagonist, L-655, generally had a neutral to beneficial effect on aortic lipids in the presence or absence of nicotinic acid treatment. In a separate study, a modest increase in some atherosclerotic measures was observed with L-655 treatment in Ldlr(-/-) mice fed a high-fat diet for 8 weeks; however, this effect was not sustained for 16 or 24 weeks. In the same study, treatment with nicotinic acid alone generally decreased plasma and/or aortic lipids, and addition of L-655 did not negate those beneficial effects. These studies demonstrate that inhibition of DP1, with or without nicotinic acid treatment, does not lead to consistent or sustained effects on plaque burden in mouse atherosclerotic models.
Asunto(s)
Técnicas de Silenciamiento del Gen , Niacina/farmacología , Placa Aterosclerótica/tratamiento farmacológico , Placa Aterosclerótica/metabolismo , Receptores Inmunológicos/antagonistas & inhibidores , Receptores Inmunológicos/genética , Receptores de Prostaglandina/antagonistas & inhibidores , Receptores de Prostaglandina/genética , Animales , Aorta/efectos de los fármacos , Aorta/metabolismo , Apolipoproteínas E/deficiencia , Colesterol/metabolismo , Interacciones Farmacológicas , Determinación de Punto Final , Femenino , Humanos , Masculino , Ratones , Niacina/uso terapéutico , Placa Aterosclerótica/genética , Receptores Inmunológicos/deficiencia , Receptores de LDL/deficiencia , Receptores de Prostaglandina/deficiencia , Receptores de Tromboxano A2 y Prostaglandina H2/metabolismoRESUMEN
A search for a suitable replacement for the central norbornyl scaffold presented in the recently disclosed novel FLAP inhibitors is herein described, as well as the SAR study performed on the endo and exo-aryl groups.
Asunto(s)
Inhibidores de Proteína Activante de 5-Lipoxigenasa/síntesis química , Proteínas Activadoras de la 5-Lipooxigenasa/química , Alcanos/síntesis química , Antialérgicos/síntesis química , Derivados del Benceno/síntesis química , Inhibidores de Proteína Activante de 5-Lipoxigenasa/farmacocinética , Inhibidores de Proteína Activante de 5-Lipoxigenasa/farmacología , Proteínas Activadoras de la 5-Lipooxigenasa/metabolismo , Alcanos/farmacocinética , Alcanos/farmacología , Animales , Antialérgicos/farmacocinética , Antialérgicos/farmacología , Derivados del Benceno/farmacocinética , Derivados del Benceno/farmacología , Humanos , Concentración 50 Inhibidora , Inyecciones Intravenosas , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Ratas , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
BACKGROUND: Atherosclerosis is a complex disease requiring improvements in diagnostic techniques and therapeutic treatments. Both improvements will be facilitated by greater exploration of the biology of atherosclerotic plaque. To this end, we carried out large-scale gene expression analysis of human atherosclerotic lesions. METHODS AND RESULTS: Whole genome expression analysis of 101 plaques from patients with peripheral artery disease identified a robust gene signature (1514 genes) that is dominated by processes related to Toll-like receptor signaling, T-cell activation, cholesterol efflux, oxidative stress response, inflammatory cytokine production, vasoconstriction, and lysosomal activity. Further analysis of gene expression in microdissected carotid plaque samples revealed that this signature is differentially expressed in macrophage-rich and smooth muscle cell-containing regions. A quantitative PCR gene expression panel and inflammatory composite score were developed on the basis of the atherosclerotic plaque gene signature. When applied to serial sections of carotid plaque, the inflammatory composite score was observed to correlate with histological and morphological features related to plaque vulnerability. CONCLUSIONS: The robust mRNA expression signature identified in the present report is associated with pathological features of vulnerable atherosclerotic plaque and may be useful as a source of biomarkers and targets of novel antiatherosclerotic therapies.
Asunto(s)
Perfilación de la Expresión Génica , Placa Aterosclerótica/genética , Placa Aterosclerótica/inmunología , Biomarcadores , Femenino , Humanos , Macrófagos/inmunología , Masculino , Datos de Secuencia Molecular , Proteínas/genética , Proteínas/inmunologíaRESUMEN
Leukotrienes (LTs) are signaling molecules derived from arachidonic acid that initiate and amplify innate and adaptive immunity. In turn, how their synthesis is organized on the nuclear envelope of myeloid cells in response to extracellular signals is not understood. We define the supramolecular architecture of LT synthesis by identifying the activation-dependent assembly of novel multiprotein complexes on the outer and inner nuclear membranes of mast cells. These complexes are centered on the integral membrane protein 5-Lipoxygenase-Activating Protein, which we identify as a scaffold protein for 5-Lipoxygenase, the initial enzyme of LT synthesis. We also identify these complexes in mouse neutrophils isolated from inflamed joints. Our studies reveal the macromolecular organization of LT synthesis.
Asunto(s)
Araquidonato 5-Lipooxigenasa/metabolismo , Proteínas Portadoras/metabolismo , Leucotrienos/biosíntesis , Proteínas de la Membrana/metabolismo , Complejos Multiproteicos/análisis , Membrana Nuclear/metabolismo , Proteínas Activadoras de la 5-Lipooxigenasa , Animales , Artritis/enzimología , Artritis/metabolismo , Proteínas de la Membrana/análisis , Ratones , Células Mieloides/química , Células Mieloides/metabolismo , Neutrófilos/química , Neutrófilos/metabolismo , Membrana Nuclear/químicaRESUMEN
Current immunoassays for the measurement of leukotriene B(4) (LTB(4)) typically utilize an enzyme-linked immunosorbent assay (ELISA) format that requires multiple incubations and washing steps and often expensive immunoassay kits. We have developed a bead-based, mix and read, indirect fluorescence-linked immunosorbent assay utilizing fluorometric microvolume assay technology (FMAT). The assay employs a monoclonal anti-LTB(4) antibody-coated onto goat antimouse antibody coupled polystyrene beads and an AlexaFluor-647-coupled LTB(4) ligand. Because the FMAT measurement is made only in the portion of the well volume containing the settled beads coated with AF647-LTB(4), the free label in the solution is not measured. Similarly, substances present in plasma that interfere with other immunoassays are largely ignored. The assay is robust (Z=0.8; S/N=250) and can be measured in the presence of relatively high concentrations of dimethyl sulfoxide or serum. It is inexpensive (<0.10 dollars/assay) and amenable to robotics and has a sensitivity comparable to that of the most sensitive ELISA assays; the concentration of LTB(4) giving 50% inhibition (IC(50)) was ca. 55pg/ml. Cross-reactivity in the FMAT assay was comparable to that of the ELISA assay with significant cross-reactivity found only with 20-hydroxy LTB(4) and 12-epi LTB(4). Measurements of LTB(4) determined by FMAT were equivalent to those measured by standard ELISA in samples of ionophore-stimulated human neutrophils or whole blood.
Asunto(s)
Fluorometría , Inmunoensayo , Leucotrieno B4/análisis , Humanos , Inmunoensayo/métodos , Leucotrieno B4/sangre , Neutrófilos/metabolismoRESUMEN
Liver X receptor (LXR) alpha and LXRbeta are closely related nuclear receptors that respond to elevated levels of intracellular cholesterol by enhancing transcription of genes that control cholesterol efflux and fatty acid biosynthesis. The consequences of inactivation of either LXR isoform have been thoroughly studied, as have the effects of simultaneous activation of both LXRalpha and LXRbeta by synthetic compounds. We here describe the effects of selective activation of LXRalpha or LXRbeta on lipid metabolism. This was accomplished by treating mice genetically deficient in either LXRalpha or LXRbeta with an agonist with equal potency for both isoforms (Compound B) or a synthetic agonist selective for LXRalpha (Compound A). We also determined the effect of these agonists on gene expression and cholesterol efflux in peritoneal macrophages derived from wild-type and knockout mice. Both compounds raised HDL-cholesterol and increased liver triglycerides in wild-type mice; in contrast, in mice deficient in LXRalpha, Compound B increased HDL-cholesterol but did not cause hepatic steatosis. Compound B induced ATP-binding cassette transporter (ABC) A1 expression and stimulated cholesterol efflux in macrophages from both LXRalpha and LXRbeta-deficient mice. Our data lend further experimental support to the hypothesis that LXRbeta-selective agonists may raise HDL-cholesterol and stimulate macrophage cholesterol efflux without causing liver triglyceride accumulation.
Asunto(s)
Proteínas de Unión al ADN/agonistas , Metabolismo de los Lípidos/fisiología , Receptores Citoplasmáticos y Nucleares/agonistas , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Administración Oral , Animales , Colesterol/metabolismo , HDL-Colesterol/sangre , HDL-Colesterol/metabolismo , AMP Cíclico/farmacología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Isoxazoles/farmacología , Hígado/efectos de los fármacos , Hígado/metabolismo , Receptores X del Hígado , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Estructura Molecular , Receptores Nucleares Huérfanos , Compuestos de Fenilurea/farmacología , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Pirazinas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Triglicéridos/sangre , Triglicéridos/metabolismoRESUMEN
Cholesterol homeostasis is tightly controlled process that involves a variety of regulators including liver X receptors (LXR). Agonists of LXR are expected to increase cholesterol efflux, lower LDL, and raise HDL levels. Screening of a natural product library of microbial extracts using a LXR-scintillation proximity assay (SPA) binding assay and bioassay-guided fractionation of a number of fungal extracts led to the isolation of five ergostane and a cycloartane derivative. These compounds exhibited IC50 value ranging 0.5 approximately 9 microM in the binding assay for a-receptor and a number of these showed in vitro agonist activity in the coactivator association assays but lacked the cell based LXR activation. The isolation and LXR activity of these compounds are described.
Asunto(s)
Proteínas de Unión al ADN/agonistas , Hongos/metabolismo , Receptores Citoplasmáticos y Nucleares/agonistas , Esteroides/aislamiento & purificación , Triterpenos/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , Hongos/química , Concentración 50 Inhibidora , Ligandos , Hígado/metabolismo , Receptores X del Hígado , Receptores Nucleares Huérfanos , Receptores Citoplasmáticos y Nucleares/metabolismo , Esteroides/química , Esteroides/farmacología , Triterpenos/química , Triterpenos/farmacologíaRESUMEN
Liver X receptors (LXR) are nuclear hormone receptors that play a critical role in cholesterol homeostasis. They regulate the expression of the ABCA1 gene, which mediates the efflux of cholesterol out of cells. LXR agonists are expected to increase cholesterol efflux, lower LDL, and raise HDL levels. Screening of a natural product library of microbial extracts using a LXR-SPA binding assay and bioassay-guided fractionation of an active extract of a Streptomyces sp. (MA6657) led to the discovery of two new hexacyclic aromatic ketones, (-)-anthrabenzoxocinone [(-)-ABX (1)], an enantiomer of BE-24566B, and (-)-bischloroanthrabenzoxocinone [(-)-BABX (2)]. The IC50 values of LXRalpha-SPA binding are 2 microM for (-)-ABX and 10 microM for (-)-BABX. This extract was also found to inhibit type II fatty acid synthesis, and its active component, (-)-BABX, was responsible for the majority of the inhibition. All three compounds showed good Gram-positive antibacterial activity (MIC 0.5-2 microg/mL). Details of the isolation, structure elucidation, LXR ligand binding, antibacterial activity, and selectivity of inhibition of 1 and 2 are described.
Asunto(s)
Antibacterianos/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/aislamiento & purificación , Receptores Citoplasmáticos y Nucleares/metabolismo , Streptomyces/química , Antraquinonas/aislamiento & purificación , Antibacterianos/química , Antibacterianos/farmacología , Compuestos Heterocíclicos de 4 o más Anillos/química , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Concentración 50 Inhibidora , Ligandos , Receptores X del Hígado , Estructura Molecular , Receptores Nucleares Huérfanos , EstereoisomerismoRESUMEN
It has been demonstrated that liver X receptors (LXR) play a significant role in cholesterol homeostasis. Agonists of LXR are expected to increase cellular cholesterol efflux, lower LDL, and raise HDL levels. Screening of a natural product library of plant extracts using a LXR-SPA binding assay and bioassay-guided fractionation of a number of plant and marine gorgonian extracts led to the isolation of a number of active compounds. These included acanthoic acid (1) and alcohol (2), viperidone (3), polycarpol (4), rosacea acid (5), a cycloartane derivative (6), a new cycloartane analogue (7), betulinic acid (8), and gorgostane derivatives (9, 10, and 11). Of these compounds, 1, 4, and 11 exhibited potent binding affinity for alpha-receptor with IC(50) values of 0.25, 0.12, and 0.07 microM, respectively. Functionally they also showed strong coactivator association stimulation for LXRalpha receptor with EC50 values of 0.18, 0.03, and 0.05 microM, respectively. They also exhibited 15-, 8-, and 13-fold induction of the alpha-receptor in a transactivation assay in HEK-293 cells, respectively. In general these compounds were selective for the LXR alpha-receptor over the beta-receptor in all assays and were much better stimulators of the alpha-receptor than the endogenous steroid ligands.
Asunto(s)
Proteínas de Unión al ADN/agonistas , Diterpenos/aislamiento & purificación , Plantas Medicinales/química , Hidrocarburos Policíclicos Aromáticos/aislamiento & purificación , Receptores Citoplasmáticos y Nucleares/agonistas , Esteroides/aislamiento & purificación , Triterpenos/aislamiento & purificación , Animales , Annonaceae/química , Antozoos/química , Bahamas , Cactaceae/química , Campanulaceae/química , Células Cultivadas , Costa Rica , Diterpenos/química , Diterpenos/farmacología , Guyana , Humanos , Concentración 50 Inhibidora , Receptores X del Hígado , Estructura Molecular , Olacaceae/química , Receptores Nucleares Huérfanos , Triterpenos Pentacíclicos , Perú , Pinaceae/química , Hidrocarburos Policíclicos Aromáticos/química , Hidrocarburos Policíclicos Aromáticos/farmacología , Esteroides/química , Esteroides/farmacología , Triterpenos/química , Triterpenos/farmacología , Estados Unidos , Ácido BetulínicoRESUMEN
A series of podocarpic acid amides were identified as potent agonists for Liver X receptor alpha and beta subtypes, which are members of a nuclear hormone receptor superfamily that are involved in the regulation of a variety of metabolic pathways including cholesterol metabolism. We recently reported podocarpic acid anhydride and imide dimers as potent LXR agonists. Through parallel organic synthesis, we rapidly identified a series of new podocarpate leads with stable structures exemplified by adamantyl- and phenylcyclohexylmethyl-podocarpic acid amides (14 and 18). Compound 18 exhibited LXRalpha/beta 50/20 nM (binding affinity) and 33.7/35.3-fold receptor inductions. Synthesis, SAR, and biological activities of new podocarpate analogs are discussed.
Asunto(s)
Abietanos/química , Abietanos/farmacología , Abietanos/uso terapéutico , Amidas/química , Aterosclerosis/tratamiento farmacológico , Proteínas de Unión al ADN/agonistas , Fenantrenos/química , Fenantrenos/farmacología , Fenantrenos/uso terapéutico , Receptores Citoplasmáticos y Nucleares/agonistas , Animales , Área Bajo la Curva , Cromatografía Liquida , Receptores X del Hígado , Masculino , Espectrometría de Masas , Receptores Nucleares Huérfanos , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
Liver X receptors are nuclear receptors that regulate metabolism of cholesterol. They are activated by oxysterols resulting in increased transcription of the ABCA1 gene, promoting cholesterol efflux and HDL formation. We have identified podocarpic acid anhydride as a 1nM agonist of LXRalpha and beta receptors. Functionally this agonist was over 8-10-fold better activator of LXR receptors compared to one of the natural ligands, 22-(R)-hydroxy cholesterol, in HEK-293 cells. An imide analog increased the level of HDL by 26%, decreased LDL by 10.6%, and increased triglyceride by 51% in hamsters. Discovery, synthesis, SAR and details of the activities of dimers have been described.