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1.
J Am Chem Soc ; 126(24): 7462-3, 2004 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-15198592

RESUMO

Ramoplanin is a potent cyclic lipoglycodepsipeptide antibiotic that disrupts bacterial cell wall synthesis by binding to the peptidoglycan intermediate Lipid II and blocking its polymerization to form the carbohydrate chains of peptidoglycan. Although ramoplanin is a promising compound for certain indications, it has limitations that impede IV administration for systemic use. However, it may be possible to overcome these limitations with analogues. In this manuscript, we dissect the effects of structural changes to ramoplanin. The studies described here combine total synthesis with enzyme kinetics, NMR analysis, and MIC measurements to shed light on the roles of key structural features in this antibiotic in Lipid II binding, transglycosylation inhibition, and biological activity. The results should serve as a foundation for the design of synthetically accessible analogues with improved biological properties.


Assuntos
Antibacterianos/síntese química , Antibacterianos/farmacologia , Depsipeptídeos , Peptídeos Cíclicos/síntese química , Antibacterianos/química , Bactérias/citologia , Bactérias/efeitos dos fármacos , Bactérias/metabolismo , Sequência de Carboidratos , Desenho de Drogas , Testes de Sensibilidade Microbiana , Modelos Biológicos , Dados de Sequência Molecular , Estrutura Molecular , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia
2.
Chem Biol ; 11(5): 703-11, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15157881

RESUMO

Nucleotide-glycosyltransferases (NDP-Gtfs) play key roles in a wide range of biological processes. It is difficult to probe the roles of individual glycosyltransferases or their products because, with few exceptions, selective glycosyltransferase inhibitors do not exist. Here, we investigate a high-throughput approach to identify glycosyltransferase inhibitors based on a fluorescent donor displacement assay. We have applied the screen to E. coli MurG, an enzyme that is both a potential antibiotic target and a paradigm for a large family of glycosyltransferases. We show that the compounds identified in the donor-displacement screen of MurG are selective for MurG over other enzymes that use similar or identical substrates, including structurally related enzymes. The donor displacement assay described here should be adaptable to many other NDP-Gtfs and represents a new strategy to identify selective NDP-Gtf inhibitors.


Assuntos
Proteínas da Membrana Bacteriana Externa/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/química , Glicosiltransferases/antagonistas & inibidores , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Animais , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Desenho de Drogas , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Polarização de Fluorescência/métodos , Glicosiltransferases/química , Glicosiltransferases/metabolismo , Cinética , Ligantes , N-Acetilglucosaminiltransferases/química , N-Acetilglucosaminiltransferases/metabolismo
3.
J Am Chem Soc ; 125(29): 8736-7, 2003 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-12862463

RESUMO

Ramoplanin is a lipglycodepsipeptide antibiotic that inhibits peptidoglycan biosynthesis. Its mechanism of action has been the subject of debate. It was originally proposed to inhibit the MurG step of peptidoglycan synthesis by binding Lipid I. In this paper, we report that ramoplanin inhibits bacterial transglycosylases by binding to Lipid II, the substrate for these enzymes. The inhibition curves reveal that the inhibitory species has a stoichiometry of 2:1 ramoplanin:Lipid II. A Job titration confirms that ramoplanin binds as a dimer to Lipid II. The apparent dissociation constant is in the nanomolar range, which is unusually low given the nature of the interacting species. We show that Lipid II binding is coupled to the formation of a higher order species, which may explain the tight binding. We also present a testable model for the binding-competent dimeric conformation of ramoplanin.


Assuntos
Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa , Proteínas de Bactérias , Proteínas de Transporte , Depsipeptídeos , Hexosiltransferases/antagonistas & inibidores , Complexos Multienzimáticos/antagonistas & inibidores , Muramilpentapeptídeo Carboxipeptidase , Peptídeos Cíclicos/farmacologia , Peptidil Transferases/antagonistas & inibidores , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Uridina Difosfato Ácido N-Acetilmurâmico/metabolismo , Antibacterianos/metabolismo , Dimerização , Relação Dose-Resposta a Droga , Cinética , N-Acetilglucosaminiltransferases/metabolismo , Proteínas de Ligação às Penicilinas , Peptídeos Cíclicos/metabolismo , Conformação Proteica
4.
J Am Chem Soc ; 125(37): 11168-9, 2003 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-16220917

RESUMO

MurG is a glycosyltransferase involved in the biosynthesis of bacterial peptidoglycan. It is a potentially important antibiotic target, but no inhibitors of the enzyme have been reported. In general, inhibitors of glycosyltransferases have been difficult to design. Furthermore, no glycosyltransferase inhibitors have been identified through high-throughput screening, perhaps because appropriate screens for glycosyltransferase inhibition have not been developed. In this manuscript, we describe the development of a high-throughput screen for MurG that was used to screen a 50 000 compound library for inhibitors. The screen, which can be generalized to other glycosyltransferases, led to the identification of a family of active-site directed MurG inhibitors. The family of inhibitors contains a five-membered heterocyclic core that appears to function as a diphosphate mimic with respect to the presentation of substituents. We discuss the implications of this result and the utility of the screen for identifying inhibitors of other glycosyltransferases.


Assuntos
Proteínas da Membrana Bacteriana Externa/antagonistas & inibidores , Proteínas da Membrana Bacteriana Externa/química , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Glicosiltransferases/antagonistas & inibidores , Glicosiltransferases/metabolismo , N-Acetilglucosaminiltransferases/antagonistas & inibidores , N-Acetilglucosaminiltransferases/química , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Sítios de Ligação , Inibidores Enzimáticos/química , Concentração Inibidora 50 , Estrutura Molecular , N-Acetilglucosaminiltransferases/metabolismo , Conformação Proteica
5.
J Am Chem Soc ; 124(47): 13970-1, 2002 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-12440876

RESUMO

Ramoplanin is a cyclicdepsipeptide antibiotic that inhibits peptidoglycan biosynthesis. It was proposed in 1990 to block the MurG step of peptidoglycan synthesis by binding to the substrate of MurG, Lipid I. The proposed mechanism of MurG inhibition has become widely accepted even though it was never directly tested. In this paper, we disprove the accepted mechanism for how ramoplanin functions, and we present an alternative mechanism. This work has implications for the design of ramoplanin derivatives and may influence how other proposed substrate binding antibiotics are studied.


Assuntos
Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa , Depsipeptídeos , Inibidores Enzimáticos/farmacologia , Peptídeos Cíclicos/farmacologia , Peptidoglicano/biossíntese , Antibacterianos/química , Inibidores Enzimáticos/química , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Bactérias Gram-Positivas/efeitos dos fármacos , Bactérias Gram-Positivas/enzimologia , Cinética , Monossacarídeos/metabolismo , N-Acetilglucosaminiltransferases/antagonistas & inibidores , N-Acetilglucosaminiltransferases/metabolismo , Ressonância Magnética Nuclear Biomolecular , Oligopeptídeos/metabolismo , Peptídeos Cíclicos/química
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