RESUMO
Abelson kinase (c-Abl) is a ubiquitously expressed, nonreceptor tyrosine kinase which plays a key role in cell differentiation and survival. It was hypothesized that transient activation of c-Abl kinase via displacement of the N-terminal autoinhibitory "myristoyl latch", may lead to an increased hematopoietic stem cell differentiation. This would increase the numbers of circulating neutrophils and so be an effective treatment for chemotherapy-induced neutropenia. This paper describes the discovery and optimization of a thiazole series of novel small molecule c-Abl activators, initially identified by a high throughput screening. Subsequently, a scaffold-hop, which exploited the improved physicochemical properties of a dihydropyrazole analogue, identified through fragment screening, delivered potent, soluble, cell-active c-Abl activators, which demonstrated the intracellular activation of c-Abl in vivo.
Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Pirazóis/farmacologia , Tiazóis/farmacologia , Animais , Sítios de Ligação , Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Estrutura Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-abl/química , Proteínas Proto-Oncogênicas c-abl/metabolismo , Pirazóis/química , Pirazóis/metabolismo , Relação Estrutura-Atividade , Tiazóis/química , Tiazóis/metabolismoRESUMO
Polypeptide deformylase (PDF) catalyzes the deformylation of polypeptide chains in bacteria. It is essential for bacterial cell viability and is a potential antibacterial drug target. Here, we report the crystal structures of polypeptide deformylase from four different species of bacteria: Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae, and Escherichia coli. Comparison of these four structures reveals significant overall differences between the two Gram-negative species (E. coli and H. influenzae) and the two Gram-positive species (S. pneumoniae and S. aureus). Despite these differences and low overall sequence identity, the S1' pocket of PDF is well conserved among the four enzymes studied. We also describe the binding of nonpeptidic inhibitor molecules SB-485345, SB-543668, and SB-505684 to both S. pneumoniae and E. coli PDF. Comparison of these structures shows similar binding interactions with both Gram-negative and Gram-positive species. Understanding the similarities and subtle differences in active site structure between species will help to design broad-spectrum polypeptide deformylase inhibitor molecules.
Assuntos
Amidoidrolases , Aminopeptidases/antagonistas & inibidores , Aminopeptidases/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Inibidores Enzimáticos/metabolismo , Sequência de Aminoácidos , Aminopeptidases/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Sítios de Ligação , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Haemophilus influenzae/efeitos dos fármacos , Haemophilus influenzae/enzimologia , Cinética , Testes de Sensibilidade Microbiana , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos , Ligação Proteica , Conformação Proteica , Homologia de Sequência de Aminoácidos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/enzimologia , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/enzimologiaRESUMO
Polypeptide deformylase (PDF) is an essential bacterial metalloenzyme responsible for the removal of the N-formyl group from the N-terminal methionine of nascent polypeptides. Inhibition of bacterial PDF enzymes by actinonin, a naturally occurring antibacterial agent, has been characterized using steady-state and transient kinetic methods. Slow binding of actinonin to these enzymes is observed under steady-state conditions. Progress curve analysis is consistent with a two-step binding mechanism, in which tightening of the initial encounter complex (EI) results in a final complex (EI*) with an extremely slow, but observable, off-rate (t(1/2) for inhibitor dissociation >or=0.77 days). Stopped-flow measurement of PDF fluorescence confirms formation of EI and provides a direct measurement of the association rate. Rapid dilution studies establish that the potency of actinonin is enhanced by more than 2000-fold upon tightening of EI to form EI*, from K(i) = 530 nM (EI) to Ki*Assuntos
Amidoidrolases/antagonistas & inibidores
, Ácidos Hidroxâmicos/farmacologia
, Amidoidrolases/química
, Antibacterianos/farmacologia
, Ácidos Hidroxâmicos/química
, Cinética
, Ligação Proteica
, Staphylococcus aureus/enzimologia