RESUMO
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
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
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.