RESUMO
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.
Assuntos
Janus Quinase 3/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Pirazinas/química , Pirróis/química , Desenho de Fármacos , Humanos , Janus Quinase 3/metabolismo , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Fosforilação , Piperidinas/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Técnicas de Silenciamento de Genes , Niacina/farmacologia , Placa Aterosclerótica/tratamento farmacológico , Placa Aterosclerótica/metabolismo , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/genética , Receptores de Prostaglandina/antagonistas & inibidores , Receptores de Prostaglandina/genética , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Apolipoproteínas E/deficiência , Colesterol/metabolismo , Interações Medicamentosas , Determinação de Ponto Final , Feminino , Humanos , Masculino , Camundongos , Niacina/uso terapêutico , Placa Aterosclerótica/genética , Receptores Imunológicos/deficiência , Receptores de LDL/deficiência , Receptores de Prostaglandina/deficiência , Receptores de Tromboxano A2 e Prostaglandina H2/metabolismoRESUMO
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.
Assuntos
Janus Quinase 3/antagonistas & inibidores , Éteres Fenílicos/química , Inibidores de Proteínas Quinases/química , Piridazinas/química , Pirróis/química , Sítios de Ligação , Domínio Catalítico , Avaliação Pré-Clínica de Medicamentos , Janus Quinase 3/metabolismo , Simulação de Acoplamento Molecular , Éteres Fenílicos/síntese química , Éteres Fenílicos/metabolismo , Ligação Proteica , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/metabolismo , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Desenho de Fármacos , Janus Quinase 3/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Sítios de Ligação , Cristalografia por Raios X , Interações Hidrofóbicas e Hidrofílicas , Indazóis/química , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 1/metabolismo , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/metabolismo , Janus Quinase 3/metabolismo , Simulação de Acoplamento Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Pirazinas/síntese química , Pirazinas/química , Pirazinas/metabolismo , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Inibidores da Proteína Ativadora de 5-Lipoxigenase/síntese química , Proteínas Ativadoras de 5-Lipoxigenase/química , Alcanos/síntese química , Antialérgicos/síntese química , Derivados de Benzeno/síntese química , Inibidores da Proteína Ativadora de 5-Lipoxigenase/farmacocinética , Inibidores da Proteína Ativadora de 5-Lipoxigenase/farmacologia , Proteínas Ativadoras de 5-Lipoxigenase/metabolismo , Alcanos/farmacocinética , Alcanos/farmacologia , Animais , Antialérgicos/farmacocinética , Antialérgicos/farmacologia , Derivados de Benzeno/farmacocinética , Derivados de Benzeno/farmacologia , Humanos , Concentração Inibidora 50 , Injeções Intravenosas , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Ratos , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Proteínas de Transporte/metabolismo , Leucotrienos/biossíntese , Proteínas de Membrana/metabolismo , Complexos Multiproteicos/análise , Membrana Nuclear/metabolismo , Proteínas Ativadoras de 5-Lipoxigenase , Animais , Artrite/enzimologia , Artrite/metabolismo , Proteínas de Membrana/análise , Camundongos , Células Mieloides/química , Células Mieloides/metabolismo , Neutrófilos/química , Neutrófilos/metabolismo , Membrana Nuclear/químicaRESUMO
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.
Assuntos
Proteínas de Ligação a DNA/agonistas , Metabolismo dos Lipídeos/fisiologia , Receptores Citoplasmáticos e Nucleares/agonistas , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Administração Oral , Animais , Colesterol/metabolismo , HDL-Colesterol/sangue , HDL-Colesterol/metabolismo , AMP Cíclico/farmacologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Isoxazóis/farmacologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Receptores X do Fígado , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estrutura Molecular , Receptores Nucleares Órfãos , Compostos de Fenilureia/farmacologia , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Pirazinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Triglicerídeos/sangue , Triglicerídeos/metabolismoRESUMO
The recent identification of liver X receptors (LXR) as regulators of the cholesterol and phospholipid export pump ABCA1 has raised the possibility that LXR agonists could be developed as HDL-raising agents, possibly also acting on the artery wall to stimulate cholesterol efflux from lipid-laden macrophages. Presently several pharmaceutical companies are working to develop such compounds, which will require finding a path for separating these beneficial effects from the detrimental stimulation of triglyceride synthesis also inherent to LXR agonists. Other challenges to the drug development process include species differences, which makes prediction of in vivo effects of LXR agonists in humans difficult. This review summarizes the present state of knowledge on LXR as a drug target and discusses possible solutions for dissociating the favorable effects of LXR agonists from their unwanted effects.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Arteriosclerose/tratamento farmacológico , HDL-Colesterol/efeitos dos fármacos , Hiperlipidemias/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/agonistas , Fatores de Transcrição , Transportador 1 de Cassete de Ligação de ATP , Animais , Artérias/efeitos dos fármacos , Artérias/metabolismo , Arteriosclerose/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Colesterol/metabolismo , HDL-Colesterol/sangue , Proteínas de Ligação a DNA/metabolismo , Homeostase , Humanos , Hiperlipidemias/metabolismo , Hipertrigliceridemia/prevenção & controle , Receptores X do Fígado , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Nucleares Órfãos , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Especificidade da Espécie , Proteína de Ligação a Elemento Regulador de Esterol 1RESUMO
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.
Assuntos
Proteínas de Ligação a DNA/agonistas , Fungos/metabolismo , Receptores Citoplasmáticos e Nucleares/agonistas , Esteroides/isolamento & purificação , Triterpenos/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Fungos/química , Concentração Inibidora 50 , Ligantes , Fígado/metabolismo , Receptores X do Fígado , Receptores Nucleares Órfãos , Receptores Citoplasmáticos e Nucleares/metabolismo , Esteroides/química , Esteroides/farmacologia , Triterpenos/química , Triterpenos/farmacologiaRESUMO
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.
Assuntos
Antivirais/análise , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/análise , Vírus da Influenza A/enzimologia , Ribonucleoproteínas/análise , Antivirais/farmacologia , RNA Polimerases Dirigidas por DNA/genética , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Vírus da Influenza A/efeitos dos fármacos , Ribonucleoproteínas/genética , Ribonucleoproteínas/farmacologia , Replicação Viral/efeitos dos fármacos , Replicação Viral/fisiologiaRESUMO
The liver X receptors, LXRalpha and LXRbeta, are members of the nuclear receptor superfamily. Originally identified as orphans, both receptor subtypes have since been shown to be activated by naturally occurring oxysterols. LXRalpha knockout mice fail to regulate cyp7a mRNA levels upon cholesterol feeding, implicating the role of this receptor in cholesterol homeostasis. LXR activation also induces the expression of the lipid pump involved in cholesterol efflux, the gene encoding ATP binding cassette protein A1 (ABCA1). Therefore, LXR is believed to be a sensor of cholesterol levels and a potential therapeutic target for atherosclerosis. Here we describe a synthetic molecule named F(3)MethylAA [3-chloro-4-(3-(7-propyl-3-trifluoromethyl-6-(4,5)-isoxazolyl)propylthio)-phenyl acetic acid] that is more potent than 22(R)-hydroxycholesterol in LXR in vitro assays. F(3)MethylAA is capable not only of inducing ABCA1 mRNA levels, but also increasing cholesterol efflux from THP-1 macrophages. In rat hepatocytes, F(3)MethylAA induced cyp7a mRNA, confirming conclusions from the knockout mouse studies. Furthermore, in rat in vivo studies, F(3)MethylAA induced liver cyp7a mRNA and enzyme activity. A critical species difference is also reported in that neither F(3)MethylAA nor 22(R)-hydroxycholesterol induced cyp7a in human primary hepatocytes. However, other LXR target genes, ABCA1, ABCG1, and SREBP1, were regulated.
Assuntos
Colesterol 7-alfa-Hidroxilase/biossíntese , Colesterol 7-alfa-Hidroxilase/genética , Regulação Enzimológica da Expressão Gênica/genética , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores do Ácido Retinoico/agonistas , Receptores dos Hormônios Tireóideos/agonistas , Animais , Apolipoproteína A-I/metabolismo , Northern Blotting , Colesterol/metabolismo , Proteínas de Ligação a DNA , Indução Enzimática/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Humanos , Isoxazóis/farmacologia , Receptores X do Fígado , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Receptores Nucleares Órfãos , Fenilacetatos/farmacologia , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade da Espécie , Ativação Transcricional/genética , Triglicerídeos/metabolismoRESUMO
Activation of liver X receptors (LXRs) induces reverse cholesterol transport and increases high-density lipoprotein cholesterol in vivo. Here, we describe novel, functional, homogeneous cell-based fluorescence resonance energy transfer assays for identifying agonists of LXRs using beta-lactamase as the reporter gene. Stable Chinese hamster ovary cell lines expressing LXRalpha-GAL4 or LXRbeta-GAL4 fusion proteins that regulate beta-lactamase transcription from upstream 7 x UAS GAL4 DNA binding sequences were generated and characterized. Synthetic and natural ligands of LXR dose-dependently activated the expression of beta-lactamase in a subtype-specific manner. These assays were used to demonstrate that a 1-pyridyl hydantoin small molecule LXR synthetic ligand specifically activates LXRalpha receptors. The beta-lactamase assays were optimized for cell density, dimethyl sulfoxide sensitivity, and time of agonist stimulation. Clonal LXRbeta-GAL4-beta-lactamase cells were miniaturized into an ultra high throughput (3456-well nanoplates) screening format.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Fígado/metabolismo , Miniaturização/métodos , Receptores Citoplasmáticos e Nucleares/agonistas , beta-Lactamases/química , Animais , Benzoatos/química , Benzoatos/farmacologia , Benzilaminas/química , Benzilaminas/farmacologia , Células CHO , Cricetinae , Proteínas de Ligação a DNA , Relação Dose-Resposta a Droga , Humanos , Receptores X do Fígado , Receptores Nucleares Órfãos , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
Effects of drought and varying plant density on the competitive coexistence of two winter annual Erodium species were studied using multiple regression analysis. Significant indications of resource partitioning were detected for interspecific mixtures under spring drought. Competitive superiority also was environment-dependent with E. botrys dominating with drought in autumn, while E. brachycarpum dominated with drought in spring. The results suggest that competitive coexistence in Erodium is promoted by processes both equilibrial (e.g., resource partitioning) and nonequilibrial (e.g. competitive reversals).
RESUMO
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.
Assuntos
Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Piridazinas/síntese química , Piridazinas/farmacologia , Baço/enzimologia , Amidas/síntese química , Amidas/farmacologia , Animais , Biologia Computacional , Simulação por Computador , Desenho de Fármacos , Canais de Potássio Éter-A-Go-Go/efeitos dos fármacos , Humanos , Técnicas In Vitro , Camundongos , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Conformação Molecular , Mutagênese/efeitos dos fármacos , Testes de Mutagenicidade , Inibidores de Proteínas Quinases/farmacocinética , Piridazinas/farmacocinética , Ratos , Baço/efeitos dos fármacos , Relação Estrutura-Atividade , Difração de Raios XRESUMO
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.
RESUMO
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.
Assuntos
Imunidade Adaptativa/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Administração Oral , Aminopiridinas/química , Aminopiridinas/farmacologia , Animais , Artrite Experimental/metabolismo , Artrite Experimental/prevenção & controle , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/metabolismo , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Citometria de Fluxo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células Jurkat , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Modelos Moleculares , Estrutura Molecular , Osteoclastos/citologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Ligação Proteica , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Piridazinas/química , Piridazinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/administração & dosagem , Bibliotecas de Moléculas Pequenas/química , Quinase SykRESUMO
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.
Assuntos
Anti-Inflamatórios não Esteroides/síntese química , Ciclopropanos/síntese química , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 3/antagonistas & inibidores , Pirazinas/síntese química , Pirróis/síntese química , Administração Oral , Animais , Anti-Inflamatórios não Esteroides/farmacocinética , Anti-Inflamatórios não Esteroides/farmacologia , Células CACO-2 , Cristalografia por Raios X , Ciclopropanos/farmacocinética , Ciclopropanos/farmacologia , Técnicas de Silenciamento de Genes , Ensaios de Triagem em Larga Escala , Humanos , Interleucina-2/fisiologia , Janus Quinase 1/genética , Janus Quinase 1/metabolismo , Janus Quinase 3/genética , Janus Quinase 3/metabolismo , Camundongos , Modelos Moleculares , Pirazinas/farmacocinética , Pirazinas/farmacologia , Pirróis/farmacocinética , Pirróis/farmacologia , RNA Interferente Pequeno/genética , Ratos , Receptores de Interleucina-6/fisiologia , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismoRESUMO
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.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirazinas/síntese química , Pirróis/síntese química , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/enzimologia , Proteínas Sanguíneas/metabolismo , Cristalografia por Raios X , Humanos , Camundongos , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Pirazinas/farmacologia , Pirazinas/toxicidade , Pirróis/farmacologia , Pirróis/toxicidade , Relação Estrutura-Atividade , Quinase SykRESUMO
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.
Assuntos
Perfilação da Expressão Gênica , Placa Aterosclerótica/genética , Placa Aterosclerótica/imunologia , Biomarcadores , Feminino , Humanos , Macrófagos/imunologia , Masculino , Dados de Sequência Molecular , Proteínas/genética , Proteínas/imunologiaRESUMO
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.