RESUMO
The WalK (a histidine kinase)/WalR (a response regulator, aka YycG/YycF) two-component system is indispensable in the signal transduction pathway for the cell-wall metabolism of Bacillus subtilis and Staphylococcus aureus. The inhibitors directed against WalK would be expected to have a bactericidal effect. After we screened 1368 culture broths of Streptomyces sp. by a differential growth assay, walkmycin A, B and C, which were produced by strain MK632-100F11, were purified using silica-gel column chromatography and HPLC. In this paper, the chemical structure of the major product (walkmycin B) was determined to be di-anthracenone (C(44)H(44)Cl(2)O(14)), which was very similar to BE40665A. MICs of walkmycin B against B. subtilis and S. aureus were 0.39 and 0.20 microg ml(-1), and IC(50) measurements against WalK were 1.6 and 5.7 microM, respectively. To clarify the affinity between WalK and walkmycin B, surface plasmon resonance was measured to obtain the equilibrium dissociation constant, K(D1), of 7.63 microM at the higher affinity site of B. subtilis WalK. These results suggest that walkmycin B inhibits WalK autophosphorylation by binding to the WalK cytoplasmic domain.
Assuntos
Antracenos/farmacologia , Antibacterianos/farmacologia , Proteínas Quinases/metabolismo , Antracenos/química , Antibacterianos/química , Bactérias/efeitos dos fármacos , Estrutura Molecular , Streptomyces/enzimologiaRESUMO
A two-component signal transduction system (TCS) is an attractive target for antibacterial agents. In this chapter, we review the TCS inhibitors developed during the past decade and introduce novel drug discovery systems to isolate the inhibitors of the YycG/YycF system, an essential TCS for bacterial growth, in an effort to develop a new class of antibacterial agents.
Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Bactérias/genética , Fenômenos Fisiológicos Bacterianos/efeitos dos fármacos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Desenho de Fármacos , Farmacorresistência Bacteriana Múltipla/genética , Farmacorresistência Bacteriana Múltipla/fisiologia , Histidina Quinase , Testes de Sensibilidade Microbiana , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/genética , Proteínas Quinases/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Interface Usuário-ComputadorRESUMO
A response regulator YycF and its cognate sensor kinase YycG constitute the two-component signal transduction system essential for growth of Gram-positive bacteria with a low GC content. We have determined the X-ray crystal structure of the effector domain of Bacillus subtilis YycF involved in DNA binding. The structure, containing a winged helix-turn-helix motif, was found to be very similar to that of the response regulator PhoB from Escherichia coli. Specific binding of YycF to the PhoB-regulated alkaline phosphatase promoter was also demonstrated.
Assuntos
Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/química , DNA/química , Regulação Bacteriana da Expressão Gênica , Sequências Hélice-Volta-Hélice , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Cristalografia por Raios X , DNA/metabolismo , Sequências Hélice-Volta-Hélice/genética , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Estrutura Terciária de ProteínaRESUMO
Ascochytatin, a new spirodioxynaphthalene metabolite produced by a marine-derived fungus, was found from a screening program focused on the bacterial two-component regulatory system. The structure of ascochytatin was determined by spectroscopic methods, including NMR and MS. The relative stereochemistry was determined by an X-ray crystallographic analysis, and the absolute stereochemistry was determined by the modified Mosher's method.
Assuntos
Antibacterianos/farmacologia , Ascomicetos/química , Naftalenos/farmacologia , Compostos de Espiro/farmacologia , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antibióticos Antineoplásicos/isolamento & purificação , Antibióticos Antineoplásicos/farmacologia , Ascomicetos/classificação , Bacillus subtilis/efeitos dos fármacos , Bactérias/efeitos dos fármacos , Linhagem Celular Tumoral , Fenômenos Químicos , Físico-Química , Cromatografia em Camada Fina , Cristalografia por Raios X , Meios de Cultura , Humanos , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Modelos Moleculares , Naftalenos/química , Naftalenos/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Espectrofotometria Infravermelho , Espectrofotometria Ultravioleta , Compostos de Espiro/química , Compostos de Espiro/isolamento & purificaçãoRESUMO
Two-component signal-transduction systems (TCSs) of bacteria are considered to form an intricate signal network to cope with various environmental stresses. One example of such a network in Escherichia coli is the signal transduction cascade from the EvgS/EvgA system to the PhoQ/PhoP system, where activation of the EvgS/EvgA system promotes expression of PhoP-activated genes. As a factor connecting this signal transduction cascade, we have identified a small inner membrane protein (65 aa), B1500. Expression of the b1500 gene is directly regulated by the EvgS/EvgA system, and b1500 expression from a heterologous promoter simultaneously activated the expression of mgtA and other PhoP regulon genes. This activation was PhoQ/PhoP-dependent and EvgS/EvgA-independent. Furthermore, deletion of b1500 from an EvgS-activated strain suppressed mgtA expression. B1500 is localized in the inner membrane, and bacterial two-hybrid data showed that B1500 formed a complex with the sensor PhoQ. These results indicate that the small membrane protein, B1500, connected the signal transduction between EvgS/EvgA and PhoQ/PhoP systems by directly interacting with PhoQ, thus activating the PhoQ/PhoP system.
Assuntos
Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Quinases/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Clonagem Molecular , Escherichia coli , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana/genética , Dados de Sequência Molecular , Ligação Proteica , Proteínas Quinases/genética , Transdução de Sinais , Fatores de Transcrição/genéticaRESUMO
We have developed two screening systems for isolating inhibitors that target bacterial two-component signal transduction: (1) a differential growth assay using a temperature-sensitive yycF mutant (CNM2000) of Bacillus subtilis, which is supersensitive to histidine kinase inhibitors, and (2) a high-throughput genetic system for targeting the homodimerization of histidine kinases essential for the bacterial two-component signal transduction. By using these methods, we have been able to identify various types of inhibitors that block the autophosphorylation of histidine kinases with different modes of actions.
