RESUMO
Tumor necrosis factor (TNF) is a pleiotropic cytokine belonging to a family of trimeric proteins with both proinflammatory and immunoregulatory functions. TNF is a key mediator in autoimmune diseases and during the last couple of decades several biologic drugs have delivered new therapeutic options for patients suffering from chronic autoimmune diseases such as rheumatoid arthritis and chronic inflammatory bowel disease. Attempts to design small molecule therapies directed to this cytokine have not led to approved products yet. Here we report the discovery and development of a potent small molecule inhibitor of TNF that was recently moved into phase 1 clinical trials. The molecule, SAR441566, stabilizes an asymmetrical form of the soluble TNF trimer, compromises downstream signaling and inhibits the functions of TNF in vitro and in vivo. With SAR441566 being studied in healthy volunteers we hope to deliver a more convenient orally bioavailable and effective treatment option for patients suffering with chronic autoimmune diseases compared to established biologic drugs targeting TNF.
RESUMO
The primary target of a novel series of immunosuppressive 7-piperazin-1-ylthiazolo[5,4- d]pyrimidin-5-amines was identified as the lipid kinase, PI4KIIIß. Evaluation of the series highlighted their poor solubility and unwanted off-target activities. A medicinal chemistry strategy was put in place to optimize physicochemical properties within the series, while maintaining potency and improving selectivity over other lipid kinases. Compound 22 was initially identified and profiled in vivo, before further modifications led to the discovery of 44 (UCB9608), a vastly more soluble, selective compound with improved metabolic stability and excellent pharmacokinetic profile. A co-crystal structure of 44 with PI4KIIIß was solved, confirming the binding mode of this class of inhibitor. The much-improved in vivo profile of 44 positions it as an ideal tool compound to further establish the link between PI4KIIIß inhibition and prolonged allogeneic organ engraftment, and suppression of immune responses in vivo.
Assuntos
Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/farmacocinética , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Piperazinas/farmacologia , Piperazinas/farmacocinética , Piperidinas/farmacologia , Transplante Homólogo , Administração Oral , Animais , Disponibilidade Biológica , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/metabolismo , Humanos , Imunossupressores/administração & dosagem , Imunossupressores/metabolismo , Imunossupressores/farmacocinética , Imunossupressores/farmacologia , Camundongos , Simulação de Acoplamento Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Piperazinas/administração & dosagem , Piperazinas/metabolismo , Piperidinas/administração & dosagem , Piperidinas/metabolismo , Conformação ProteicaRESUMO
Phosphoinositide 3-kinases (PI3K) are key signaling enzymes regulating cellular survival, development, and function. Expression of the PI3Kδ isoform is largely restricted to leukocytes and it plays a key role in immune cell development and function. Seletalisib is a novel small-molecule inhibitor of PI3Kδ that was evaluated in biochemical assays, cellular assays of adaptive and innate immunity, and an in vivo rat model of inflammation. Our findings show that seletalisib is a potent, ATP-competitive, and selective PI3Kδ inhibitor able to block protein kinase B (AKT) phosphorylation following activation of the B-cell receptor in a B-cell line. Moreover, seletalisib inhibited N-formyl peptide-stimulated but not phorbol myristate acetate-stimulated superoxide release from human neutrophils, consistent with a PI3Kδ-specific activity. No indications of cytotoxicity were observed in peripheral blood mononuclear cells (PBMCs) or other cell types treated with seletalisib. Findings from cellular assays of adaptive immunity demonstrated that seletalisib blocks human T-cell production of several cytokines from activated T-cells. Additionally, seletalisib inhibited B-cell proliferation and cytokine release. In human whole blood assays, seletalisib inhibited CD69 expression upon B-cell activation and anti-IgE-mediated basophil degranulation. Seletalisib showed dose-dependent inhibition in an in vivo rat model of anti-CD3-antibody-induced interleukin 2 release. Collectively, these data characterize seletalisib as a selective PI3Kδ inhibitor and potential therapeutic candidate for the treatment of B-cell malignancies and autoimmune diseases driven by dysregulated proinflammatory cytokine secretion.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Piridinas/química , Piridinas/farmacologia , Quinolinas/química , Quinolinas/farmacologia , Animais , Células Cultivadas , Classe I de Fosfatidilinositol 3-Quinases , Relação Dose-Resposta a Droga , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/enzimologia , Masculino , Fosfatidilinositol 3-Quinases/metabolismo , Ratos , Ratos Endogâmicos Lew , Ratos WistarRESUMO
LRP5 and LRP6 are proteins predicted to contain four six-bladed ß-propeller domains and both bind the bone-specific Wnt signaling antagonist sclerostin. Here, we report the crystal structure of the amino-terminal region of LRP6 and using NMR show that the ability of sclerostin to bind to this molecule is mediated by the central core of sclerostin and does not involve the amino- and carboxyl-terminal flexible arm regions. We show that this structured core region interacts with LRP5 and LRP6 via an NXI motif (found in the sequence PNAIG) within a flexible loop region (loop 2) within the central core region. This sequence is related closely to a previously identified motif in laminin that mediates its interaction with the ß-propeller domain of nidogen. However, the NXI motif is not involved in the interaction of sclerostin with LRP4 (another ß-propeller containing protein in the LRP family). A peptide derived from the loop 2 region of sclerostin blocked the interaction of sclerostin with LRP5/6 and also inhibited Wnt1 but not Wnt3A or Wnt9B signaling. This suggests that these Wnts interact with LRP6 in different ways.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteína Wnt1/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Proteínas Morfogenéticas Ósseas/genética , Calorimetria , Linhagem Celular , Cristalografia , DNA Complementar , Marcadores Genéticos/genética , Humanos , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/química , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica , Homologia de Sequência de Aminoácidos , Proteína Wnt1/genéticaRESUMO
Structural biology studies typically require large quantities of pure, soluble protein. Currently the most widely-used method for obtaining such protein involves the use of bioinformatics and experimental methods to design constructs of the target, which are cloned and expressed. Recently an alternative approach has emerged, which involves random fragmentation of the gene of interest and screening for well-expressing fragments. Here we describe the application of one such fragmentation method, combinatorial domain hunting (CDH), to a target which historically was difficult to express, human MEK-1. We show how CDH was used to identify a fragment which covers the kinase domain of MEK-1 and which expresses and crystallizes significantly better than designed expression constructs, and we report the crystal structure of this fragment which explains some of its superior properties. Gene fragmentation methods, such as CDH, thus hold great promise for tackling difficult-to-express target proteins.
Assuntos
MAP Quinase Quinase 1/química , MAP Quinase Quinase 1/genética , Engenharia de Proteínas , Clonagem Molecular , Cristalização , Cristalografia , Escherichia coli , Humanos , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
A number of novel fused thiophene derivatives have been prepared and identified as potent inhibitors of MEK. The SAR data of selected examples and the in vivo profiling of compound 13 h demonstrates the functional activity of this class of compounds in HT-29 PK/PD models.
Assuntos
Química Farmacêutica/métodos , MAP Quinase Quinase Quinases/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Tiofenos/química , Animais , Linhagem Celular Tumoral , Cristalografia por Raios X/métodos , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Concentração Inibidora 50 , Camundongos , Modelos Químicos , Fosforilação , Eletricidade Estática , Relação Estrutura-Atividade , Fatores de TempoRESUMO
4-(1,3-Thiazol-2-yl)morpholine derivatives have been identified as potent and selective inhibitors of phosphoinositide 3-kinase. The SAR data of selected examples are presented and the in vivo profiling of compound 18 is shown to demonstrate the utility of this class of compounds in xenograft models of tumor growth.
Assuntos
1-Fosfatidilinositol 4-Quinase/antagonistas & inibidores , Morfolinas/farmacologia , Tiazóis/farmacologia , Animais , Técnicas de Química Combinatória , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Isoenzimas , Camundongos , Estrutura Molecular , Morfolinas/síntese química , Morfolinas/química , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The synthesis and P2Y2 activities of a novel series of nucleoside triphosphates are described. Many of these compounds were potent agonists of the P2Y2 receptor.
Assuntos
Nucleotídeos/síntese química , Nucleotídeos/farmacologia , Agonistas do Receptor Purinérgico P2 , Linhagem Celular Tumoral , DNA Complementar/biossíntese , DNA Complementar/genética , Humanos , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Receptores Purinérgicos P2Y2 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Relação Estrutura-Atividade , Uridina Trifosfato/farmacologiaRESUMO
A series of analogues of the peptide deformylase (PDF) inhibitor BB-3497 where the P3' amide bond was replaced with a ketone functionality is described. The in vitro antibacterial profiling of these compounds revealed that they demonstrate activity against pathogens associated with respiratory tract infections.