RESUMO
Rhomboids are relatively recently discovered intramembrane serine proteases that are conserved throughout evolution. They have a wide range of biological functions, and there is also much speculation about their potential medical relevance. Although rhomboids are weakly inhibited by some broad-spectrum serine protease inhibitors, no potent and specific inhibitors have been identified for these enzymes, which are mechanistically distinct from and evolutionarily unrelated to the classical soluble serine proteases. Here we report a new biochemical assay for rhomboid function based on the use of quenched fluorescent substrate peptides. We have developed this assay into a high-throughput format and have undertaken an inhibitor and activator screen of approximately 58,000 small molecules. This has led to the identification of a new class of rhomboid inhibitors, a series of monocyclic ß-lactams, which are more potent than any previous inhibitor. They show selectivity, both for rhomboids over the soluble serine protease chymotrypsin and also, importantly, between different rhomboids; they can inhibit mammalian as well as bacterial rhomboids; and they are effective both in vitro and in vivo. These compounds represent important templates for further inhibitor development, which could have an impact both on biological understanding of rhomboid function and potential future drug development.
Assuntos
Ensaios de Triagem em Larga Escala , Proteínas de Membrana/metabolismo , Monobactamas , Proteínas Recombinantes de Fusão/metabolismo , Serina Proteases/metabolismo , Inibidores de Serina Proteinase , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Escherichia coli , Corantes Fluorescentes/análise , Expressão Gênica , Cinética , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Monobactamas/síntese química , Monobactamas/farmacologia , Relação Quantitativa Estrutura-Atividade , Proteínas Recombinantes de Fusão/genética , Serina Proteases/genética , Inibidores de Serina Proteinase/síntese química , Inibidores de Serina Proteinase/farmacologia , Bibliotecas de Moléculas Pequenas/análise , Especificidade da Espécie , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por SubstratoRESUMO
A high-throughput screening campaign identified a number of imidazopyridazines as novel inhibitors of the malarial kinase PfPK7. Further synthetic chemistry efforts enabled the preparation of a number of analogues with promising in vitro potencies. Although these compounds show likely broad spectrum inhibitory activity, they represent a useful starting point for further chemical optimisation.
