RESUMO
Here, we described the design, by fragment merging and multiparameter optimization, of selective MMP-13 inhibitors that display an appropriate balance of potency and physicochemical properties to qualify as tool compounds suitable for in vivo testing. Optimization of potency was guided by structure-based insights, specifically to replace an ester moiety and introduce polar directional hydrogen bonding interactions in the core of the molecule. By introducing polar enthalpic interactions in this series of inhibitors, the overall beneficial physicochemical properties were maintained. These physicochemical properties translated to excellent drug-like properties beyond potency. In a murine model of rheumatoid arthritis, treatment of mice with selective inhibitors of MMP-13 resulted in a statistically significant reduction in the mean arthritic score vs control when dosed over a 14 day period.
RESUMO
Comprehensive and unbiased detection methods are a prerequisite for high-throughput screening (HTS) campaigns within drug discovery research. Label-free matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has been introduced as an HTS-compatible readout for biochemical test systems to support the drug discovery process. So far, reported HTS applications were based on surface-modified systems or proof-of-concept studies. We present the utilization of a MALDI-TOF-based screening platform to identify inhibitors of human cyclic GMP-AMP synthase (cGAS), a mediator of innate immune response whose aberration has been causally correlated to a number of inflammatory disorders. In this context, the development and validation of a MALDI-TOF-based activity assay is reported to demonstrate fast, robust, and accurate detection of chemical cGAS inhibition by direct quantification of the physiological reaction product cyclic GMP-ATP (cGAMP). Results from a screen of a diverse library of more than 1 million small molecules in 1536-well format against the catalytic cGAS activity are presented with excellent assay performance and data quality. Identified hits were qualified in dose-response experiments and confirmed by RapidFire-MS measurements. Conclusively, the presented data provide the first proof of applicability of direct automated MALDI-TOF MS as a readout strategy for large-scale drug discovery HTS campaigns.
Assuntos
DNA/genética , Ensaios de Triagem em Larga Escala , Nucleotidiltransferases/antagonistas & inibidores , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Citosol/enzimologia , DNA/efeitos dos fármacos , Descoberta de Drogas , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Humanos , Nucleotidiltransferases/genética , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Interferon regulatory factor 5 (IRF5) is a key transcription factor of innate immunity, which plays an important role in host restriction to viral infection and inflammation. Genome-wide association studies have implied the association of IRF5 with several autoimmune diseases, including systemic lupus erythematosus (SLE), Sjogren's syndrome, inflammatory bowel disease and multiple sclerosis. However, the regulation of IRF5-mediated immunity is not well understood. To uncover new regulators in IRF5 pathway, we used two "omics" approaches: affinity purification coupled with mass spectrometry and a high throughput RNAi screen. Proteomics identified 16 new IRF5 interactors while RNAi-mediated knockdown found 43 regulators of the TLR7-dependent IRF5 signaling pathway. NXF1 was identified in both screens. Stimulation with TLR7 ligand enhances formation of IRF5-NXF1 protein complexes. Gain or loss-of-function experiments revealed NXF1 selectively regulates TLR7-driven IRF5 transcriptional activity, suggesting a new role for NXF1 in the IRF5 signaling pathway.
Assuntos
Fatores Reguladores de Interferon/metabolismo , Proteômica , Interferência de RNA , Transdução de Sinais , Genes Reporter , Humanos , Imunidade Inata , Interferons/biossíntese , Proteínas de Transporte Nucleocitoplasmático , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Isoformas de Proteínas , Proteômica/métodos , Proteínas de Ligação a RNA , Reprodutibilidade dos TestesRESUMO
A series of nonsteroidal "dissociated" glucocorticoid receptor agonists was optimized for drug-like properties such as cytochrome P450 inhibition, metabolic stability, aqueous solubility, and hERG ion channel inhibition. This effort culminated in the identification of the clinical candidate compound ( R )-39.
RESUMO
Synthesis and structure-activity relationship (SAR) of a series of alkyl and cycloalkyl containing non-steroidal dissociated glucocorticoid receptor (GR) agonists is reported. This series of compounds was identified as part of an effort to replace the CF3 group in a scaffold represented by 1a. The study culminated in the identification of compound 14, a t-butyl containing derivative, which has shown potent activity for GR, selectivity against the progesterone receptor (PR) and the mineralocorticoid receptor (MR), in vitro anti-inflammatory activity in an IL-6 transrepression assay, and dissociation in a MMTV transactivation counter-screen. In a collagen-induced arthritis mouse model, 14 displayed prednisolone-like efficacy, and lower impact on body fat and free fatty acids than prednisolone at an equivalent anti-inflammatory dose.
Assuntos
Descoberta de Drogas , Glucocorticoides/síntese química , Metanol/química , Receptores de Glucocorticoides/agonistas , Animais , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Artrite/tratamento farmacológico , Sítios de Ligação , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Glucocorticoides/química , Glucocorticoides/farmacologia , Humanos , Concentração Inibidora 50 , Metanol/síntese química , Metanol/farmacologia , Camundongos , Modelos Moleculares , Estrutura Molecular , Prednisolona/química , Prednisolona/farmacologia , Ligação Proteica/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
Synthesis and structure-activity relationship (SAR) of a series of nonsteroidal glucocorticoid receptor (GR) agonists are described. These compounds contain "diazaindole" moieties and display different transcriptional regulatory profiles in vitro and are considered "dissociated" between gene transrepression and transactivation. The lead optimization effort described in this article focused in particular on limiting the transactivation of genes which result in bone side effects and these were assessed in vitro in MG-63 osteosarcoma cells, leading to the identification of (R)-18 and (R)-21. These compounds maintained anti-inflammatory activity in vivo in collagen induced arthritis studies in mouse but had reduced effects on bone relevant parameters compared to the widely used synthetic glucocorticoid prednisolone 2 in vivo. To our knowledge, we are the first to report on selective glucocorticoid ligands with reduced bone loss in a preclinical in vivo model.
Assuntos
Osso e Ossos/efeitos dos fármacos , Receptores de Glucocorticoides/agonistas , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Relação Estrutura-AtividadeRESUMO
The K(+) channel KCa3.1 is required for Ca(2+) influx and the subsequent activation of CD4 T cells. The class II phosphatidylinositol 3 kinase C2ß (PI3KC2ß) is activated by the T-cell receptor (TCR) and is critical for KCa3.1 channel activation. Tripartite motif containing protein 27 (TRIM27) is a member of a large family of proteins that function as Really Interesting New Gene (RING) E3 ubiquitin ligases. We now show that TRIM27 functions as an E3 ligase and mediates lysine 48 polyubiquitination of PI3KC2ß, leading to a decrease in PI3K enzyme activity. By inhibiting PI3KC2ß, TRIM27 also functions to negatively regulate CD4 T cells by inhibiting KCa3.1 channel activity and TCR-stimulated Ca(2+) influx and cytokine production in Jurkat, primary human CD4 T cells, and Th0, Th1, and Th2 CD4 T cells generated from TRIM27(-/-) mice. These findings provide a unique mechanism for regulating class II PI3Ks, and identify TRIM27 as a previously undescribed negative regulator of CD4 T cells.
Assuntos
Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD4-Positivos/imunologia , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Ubiquitinação , Animais , Cálcio/metabolismo , Citocinas/biossíntese , Proteínas de Ligação a DNA/deficiência , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Ativação do Canal Iônico , Células Jurkat , Camundongos , Mucoproteínas/metabolismo , Proteínas Nucleares/deficiência , Fosfatidilinositol 3-Quinases/metabolismo , Poliubiquitina/metabolismo , Ligação Proteica , Proteólise , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/imunologia , Células Th1/imunologia , Células Th2/imunologia , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina-Proteína LigasesRESUMO
We report a SAR of non-steroidal glucocorticoid mimetics that utilize indoles as A-ring mimetics. Detailed SAR is discussed with a focus on improving PR and MR selectivity, GR agonism, and in vitro dissociation profile. SAR analysis led to compound (R)-33 which showed high PR and MR selectivity, potent agonist activity, and reduced transactivation activity in the MMTV and aromatase assays. The compound is equipotent to prednisolone in the LPS-TNF model of inflammation. In mouse CIA, at 30 mg/kg compound (R)-33 inhibited disease progression with an efficacy similar to the 3 mg/kg dose of prednisolone.
Assuntos
Glucocorticoides/química , Glucocorticoides/farmacologia , Indóis/química , Indóis/farmacologia , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/metabolismo , Animais , Células HeLa , Humanos , Camundongos , Modelos Moleculares , Relação Estrutura-AtividadeRESUMO
Matrix metalloproteases (MMPs) play an important role in cartilage homeostasis under both normal and inflamed disease states and, thus, have become attractive targets for the treatment of arthritic diseases. Herein, we describe the identification of a potent, selective MMP-13 inhibitor, developed using fragment-based structure-guided lead identification and optimization techniques. Virtual screening methods identified a novel, indole-based MMP-13 inhibitor that bound into the S1' pocket of the protein exhibiting a novel interaction pattern hitherto not observed in MMP-13 inhibitors. X-ray crystallographic structures were used to guide the elaboration of the fragment, ultimately leading to a potent inhibitor that was >100-fold selective over nine other MMP isoforms tested.
Assuntos
Indóis/síntese química , Inibidores de Metaloproteinases de Matriz , Cristalografia por Raios X , Humanos , Indóis/química , Metaloproteinase 13 da Matriz/química , Modelos Moleculares , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Relação Estrutura-AtividadeRESUMO
Syntheses and structure-activity relationships (SAR) of nonsteroidal glucocorticoid receptor (GR) agonists are described. These compounds contain azaindole moieties as A-ring mimetics and display various degrees of in vitro dissociation between gene transrepression and transactivation. Collagen induced arthritis studies in mouse have demonstrated that in vitro dissociated compounds (R)-16 and (R)-37 have steroid-like anti-inflammatory properties with improved metabolic side effect profiles, such as a reduced increase in body fat and serum insulin levels, compared to steroids.
Assuntos
Anti-Inflamatórios não Esteroides/síntese química , Piridinas/síntese química , Pirróis/síntese química , Receptores de Glucocorticoides/agonistas , Esteroides/química , Tecido Adiposo/efeitos dos fármacos , Animais , Anti-Inflamatórios não Esteroides/efeitos adversos , Anti-Inflamatórios não Esteroides/farmacologia , Aromatase/biossíntese , Aromatase/genética , Inibidores da Aromatase/farmacologia , Artrite Experimental/tratamento farmacológico , Disponibilidade Biológica , Células Cultivadas , Indução Enzimática , Feminino , Humanos , Ligação de Hidrogênio , Insulina/sangue , Interleucina-1/farmacologia , Interleucina-6/antagonistas & inibidores , Interleucina-6/biossíntese , Interleucina-6/genética , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Piridinas/efeitos adversos , Piridinas/farmacologia , Pirróis/efeitos adversos , Pirróis/farmacologia , Ratos , Ratos Sprague-Dawley , Estereoisomerismo , Relação Estrutura-Atividade , Ativação TranscricionalRESUMO
We have recently reported the discovery of a novel class of glucocorticoid receptor (GR) antagonists, exemplified by 3, containing a 1,2-dihydroquinoline molecular scaffold. Further SAR studies of these antagonists uncovered chemical modifications conveying agonist functional activity to this series. These agonists exhibit good GR binding affinity and are selective against other nuclear hormone receptors.
Assuntos
Quinolinas/química , Quinolinas/farmacologia , Receptores de Glucocorticoides/agonistas , Quinolinas/metabolismo , Receptores de Glucocorticoides/metabolismo , Relação Estrutura-AtividadeRESUMO
We report the discovery of a novel class of glucocorticoid receptor (GR) ligands based on 1,2-dihydroquinoline molecular scaffold. The compounds exhibit good GR binding affinity and selectivity profile against other nuclear hormone receptors.
Assuntos
Quinolinas/síntese química , Quinolinas/farmacologia , Receptores de Glucocorticoides/antagonistas & inibidores , Receptores de Glucocorticoides/metabolismo , Anti-Inflamatórios/farmacologia , Dexametasona/farmacologia , Células HeLa , Humanos , Interleucina-1/farmacologia , Interleucina-6/metabolismo , Ligantes , Vírus do Tumor Mamário do Camundongo/genética , Modelos Moleculares , Estrutura Molecular , Regiões Promotoras Genéticas/efeitos dos fármacos , Quinolinas/química , Receptores de Estrogênio/efeitos dos fármacos , Receptores de Estrogênio/metabolismo , Receptores de Mineralocorticoides/efeitos dos fármacos , Receptores de Mineralocorticoides/metabolismo , Receptores de Progesterona/efeitos dos fármacos , Receptores de Progesterona/metabolismo , Relação Estrutura-Atividade , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genéticaRESUMO
Compound 1, a potent glucocorticoid receptor ligand, contains a quaternary carbon bearing trifluoromethyl and hydroxyl groups. This paper describes the effect of replacing the trifluoromethyl group on binding and agonist activity of the GR ligand 1. The results illustrate that replacing the CF3 group with a cyclohexylmethyl or benzyl group maintains the GR binding potency. These substitutions alter the functional behavior of the GR ligands from agonists to antagonists. Docking studies suggest that the benzyl analog 19 binds in a similar fashion as the GR antagonist, RU486. The central benzyl group of 19 and the C-11 dimethylaniline moiety of RU486 overlay. Binding of compound 19 is believed to force helix 12 to adopt an open conformation and this leads to the antagonist properties of the non-CF3 ligands carrying a large group at the center of the molecule.