RESUMO
Vancomycin is a potent glycopeptide antibiotic that has evolved to specifically bind to the D-Ala-D-Ala dipeptide termini of nascent peptidoglycans. Although this mode of action is well established, several studies indicate that vancomycin and analogues exploit noncanonical target sites. In order to address all vancomycin targets in clinically relevant Staphylococcus aureus and Enterococcus faecalis strains we developed a series of small-molecule photoaffinity probes based on vancomycin. Proteomic profiling revealed the specific labeling of two previously unknown vancomycin targets that are likely to contribute to its antibiotic activity. The specific inhibition of the major staphylococcal autolysin Atl confirms previous observations that vancomycin alters S. aureus cell morphology by interaction with the autolytic machinery. Moreover, in E. faecalis the vancomycin photoprobe specifically binds to an ABC transporter protein, which likely impedes the uptake of essential nutrients such as sugars and peptides. The labeling of these two prominent membrane targets in living cells reveals a thus far unexplored mode of vancomycin binding and inhibition that could allow a rational design of variants with improved activity.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Antibacterianos/farmacologia , Enterococcus faecalis/efeitos dos fármacos , Marcadores de Fotoafinidade/química , Staphylococcus aureus/efeitos dos fármacos , Vancomicina/farmacologia , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antibacterianos/síntese química , Antibacterianos/química , Sítios de Ligação/efeitos dos fármacos , Relação Dose-Resposta a Droga , Enterococcus faecalis/citologia , Enterococcus faecalis/metabolismo , Testes de Sensibilidade Microbiana , Estrutura Molecular , Proteômica , Staphylococcus aureus/citologia , Staphylococcus aureus/metabolismo , Estereoisomerismo , Relação Estrutura-Atividade , Vancomicina/síntese química , Vancomicina/químicaRESUMO
Target analysis of acivicin derived 3-halodihydroisoxazoles scaffolds in living non-pathogenic and pathogenic bacteria.
Assuntos
Isoxazóis/química , Oxazóis/química , Aldeído Desidrogenase/química , Proteínas de Bactérias/química , Isomerismo , Oxazóis/síntese química , Staphylococcus aureus/enzimologiaRESUMO
Selective release of enriched biomolecules from solid support is a desirable goal in proteomic and metabolomic studies. Here we demonstrate that photocleavage of a light-sensitive phenacyl ester bond is a suitable alternative cleavage strategy for the selective release of enriched biomolecules form avidin beads circumventing the disadvantages of conventional heat denaturation procedures.
Assuntos
Fotoquímica , Cromatografia Líquida de Alta Pressão , Ressonância Magnética Nuclear Biomolecular , Espectrometria de Massas por Ionização por Electrospray , Raios UltravioletaRESUMO
Aberrant PI3K/Akt signaling has been implicated in many human cancers, including prostate carcinomas. Currently different therapeutic strategies target the inhibition of this survival pathway. The nucleoside analog triciribine (TCN), which was initially described as a DNA synthesis inhibitor, has recently been shown to function as an inhibitor of Akt. Here, we demonstrate that TCN inhibits Akt phosphorylation at Thr308 and Ser473 and Akt activity in the human prostate cancer cell line PC-3. In addition, TCN sensitized PC-3 cells to TRAIL- and anti-CD95-induced apoptosis, whereas the cells remained resistant to DNA damaging chemotherapeutics. The observed sensitization essentially depended on the phosphorylation status of Akt. Thus, prostate cancer cell lines displaying constitutively active Akt, e.g. PC-3 or LNCaP, were sensitized to death receptor-induced apoptosis. Most importantly with respect to therapeutic application, derivatives of both TCN and TRAIL are already tested in current clinical trials. Therefore, this combinatorial treatment might open a promising therapeutic approach for the elimination of hormone-refractory prostate cancers, which are largely resistant to conventional DNA damaging anticancer drugs or irradiation.