RESUMO
A microplate, scintillation proximity assay to measure the coupled transglycosylase-transpeptidase activity of the penicillin binding proteins in Escherichia coli membranes was developed. Membranes were incubated with the two peptidoglycan sugar precursors UDP-N-acetyl muramylpentapeptide (UDP-MurNAc(pp)) and UDP-[(3)H]N-acetylglucosamine in the presence of 40 µM vancomycin to allow in situ accumulation of lipid II. In a second step, vancomycin inhibition was relieved by addition of a tripeptide (Lys-D-ala-D-ala) or UDP-MurNAc(pp), resulting in conversion of lipid II to cross-linked peptidoglycan. Inhibitors of the transglycosylase or transpeptidase were added at step 2. Moenomycin, a transglycosylase inhibitor, had an IC50 of 8 nM. Vancomycin and nisin also inhibited the assay. Surprisingly, the transpeptidase inhibitors penicillin and ampicillin showed no inhibition. In a pathway assay of peptidoglycan synthesis, starting from the UDP linked sugar precursors, inhibition by penicillin was reversed by a 'neutral' combination of vancomycin plus tripeptide, suggesting an interaction thus far unreported.
Assuntos
Escherichia coli/metabolismo , Proteínas de Ligação às Penicilinas/metabolismo , Penicilinas/administração & dosagem , Peptidoglicano Glicosiltransferase/metabolismo , Peptidoglicano/biossíntese , Peptidil Transferases/metabolismo , Vancomicina/administração & dosagem , Bioensaio/instrumentação , Bioensaio/métodos , Avaliação Pré-Clínica de Medicamentos/instrumentação , Interações Medicamentosas , Ativação Enzimática , Desenho de Equipamento , Escherichia coli/efeitos dos fármacos , Miniaturização , Peptídeos/administração & dosagem , Mapeamento de Interação de Proteínas/instrumentaçãoRESUMO
The parasite Toxoplasma gondii can lead to toxoplasmosis in those who are immunocompromised. To combat the infection, the enzyme responsible for nucleotide synthesis thymidylate synthase-dihydrofolate reductase (TS-DHFR) is a suitable drug target. We have used virtual screening to determine novel allosteric inhibitors at the interface between the two TS domains. Selected compounds from virtual screening inhibited TS activity. Thus, these results show that allosteric inhibition by small drug-like molecules can occur in T. gondii TS-DHFR and pave the way for new and potent species-specific inhibitors.