RESUMO
The Structural Genomics Consortium (SGC) and its clinical, industry and disease-foundation partners are launching open-source preclinical translational medicine studies.
Assuntos
Linhagem Celular , Avaliação Pré-Clínica de Medicamentos/métodos , Cultura Primária de Células , Humanos , Pacientes , Parcerias Público-Privadas , Pesquisa Translacional BiomédicaRESUMO
Small-molecule inhibitors that target bromodomains outside of the bromodomain and extra-terminal (BET) sub-family are lacking. Here, we describe highly potent and selective ligands for the bromodomain module of the human lysine acetyl transferase CBP/p300, developed from a series of 5-isoxazolyl-benzimidazoles. Our starting point was a fragment hit, which was optimized into a more potent and selective lead using parallel synthesis employing Suzuki couplings, benzimidazole-forming reactions, and reductive aminations. The selectivity of the lead compound against other bromodomain family members was investigated using a thermal stability assay, which revealed some inhibition of the structurally related BET family members. To address the BET selectivity issue, X-ray crystal structures of the lead compound bound to the CREB binding protein (CBP) and the first bromodomain of BRD4 (BRD4(1)) were used to guide the design of more selective compounds. The crystal structures obtained revealed two distinct binding modes. By varying the aryl substitution pattern and developing conformationally constrained analogues, selectivity for CBP over BRD4(1) was increased. The optimized compound is highly potent (Kd = 21 nM) and selective, displaying 40-fold selectivity over BRD4(1). Cellular activity was demonstrated using fluorescence recovery after photo-bleaching (FRAP) and a p53 reporter assay. The optimized compounds are cell-active and have nanomolar affinity for CBP/p300; therefore, they should be useful in studies investigating the biological roles of CBP and p300 and to validate the CBP and p300 bromodomains as therapeutic targets.
Assuntos
Proteína de Ligação a CREB/química , Proteína p300 Associada a E1A/química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Sítios de Ligação , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Técnicas de Química Sintética , Cristalografia por Raios X , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos/métodos , Proteína p300 Associada a E1A/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Genes p53 , Células HeLa/efeitos dos fármacos , Humanos , Indóis/química , Isoxazóis/química , Ligantes , Microssomos Hepáticos/efeitos dos fármacos , Modelos Moleculares , Estrutura Molecular , Estrutura Terciária de Proteína , Bibliotecas de Moléculas Pequenas/metabolismo , Relação Estrutura-AtividadeAssuntos
Biomarcadores Farmacológicos/química , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Sondas Moleculares/química , Biomarcadores Farmacológicos/metabolismo , Permeabilidade da Membrana Celular , Humanos , Sondas Moleculares/metabolismo , Terapia de Alvo Molecular , Relação Estrutura-Atividade , Estudos de Validação como AssuntoRESUMO
The design and profile of a series of indole containing long acting beta(2)-adrenoceptor agonists is described. Evaluation of these analogues using an in vitro guinea pig trachea tissue model demonstrates that analogues within this series have salmeterol-like duration of action with potential for long duration of action in humans.