RESUMO
Twenty-nine basidiomycetes were screened in surface and liquid cultures for their capability to biotransform the chloroacetamide herbicide Dimethenamid-P (DMTA-P). The basidiomycete Irpex consors converted 70% of the herbicide (0.5 g L-1 DMTA-P) in liquid cultures within 6 days, applying a minimal medium under non-ligninolytic conditions. Nine transformation products of DMTA-P were identified by liquid chromatography-mass spectrometry analysis of the culture supernatants. The four main metabolites were isolated and subjected to GC-MS analysis and NMR spectroscopy. The analyses revealed that the thiophene ring was oxidized at three different positions. Metabolite M1 was identified as the S-oxide, which was isolable and relatively stable at room temperature. In metabolite M2, one methyl substituent of the thiophene ring was hydroxylated. The two metabolites M3A and M3B were diastereomers, but fully separated by HPLC. Here, oxidation of the aromatic CH carbon resulted in prototropic rearrangement to an αß-unsaturated thiolactone. None of the three major metabolites of DMTA-P has been described before.
Assuntos
Acetanilidas/metabolismo , Basidiomycota/metabolismo , Biotransformação , Radioisótopos de Carbono/análise , Basidiomycota/crescimento & desenvolvimento , Cromatografia Líquida de Alta Pressão , Cromatografia Gasosa-Espectrometria de Massas , Espectrometria de Massas , Oxirredução , Tiofenos/análiseRESUMO
The bacterial ribosome is a major target of naturally occurring thiopeptides antibiotics. Studying thiopeptide (e.g. thiostrepton) binding to the GAR's 23S·L11 ribosomal subunit using docking methods is challenging. Regarding the target, the binding site is composed of a flexible protein-RNA nonbonded interface whose available crystal structure is of medium resolution. Regarding the ligands, the thiopeptides are chemically complex, flexible, and contain macrocycles. In this study we developed a combined MD-docking-MD workflow that allows us to study thiopeptide-23S·L11 binding. It is shown that docking thiostrepton-like ligands to an MD-refined receptor structure instead of the medium resolution crystal leads to better convergence to the native-like docking pose and a better reproduction of experimental binding affinities. By applying an energy decomposition analysis, we identify key structural binding elements within GAR's rRNA-protein binding site and within the ligand structures.
Assuntos
Desenho de Fármacos , RNA Ribossômico/química , Relação Estrutura-Atividade , Tioestreptona/química , Sítios de Ligação , Cristalografia por Raios X , Modelos Moleculares , Ligação Proteica , RNA Ribossômico/metabolismo , Ribossomos/química , Ribossomos/metabolismo , Tioestreptona/uso terapêuticoRESUMO
Ribosome-targeting antibiotics exert their antimalarial activity on the apicoplast of the malaria parasite, an organelle of prokaryote origin having essential metabolic functions. These antibiotics typically cause a delayed-death phenotype, which manifests in parasite killing during the second replication cycle following administration. As an exception, treatment with the antibiotic thiostrepton results in an immediate killing. We recently demonstrated that thiostrepton and its derivatives interfere with the eukaryotic proteasome, a multimeric protease complex that is important for the degradation of ubiquitinated proteins. Here, we report that the thiostrepton-based compounds are active against chloroquine-sensitive and -resistant Plasmodium falciparum, where they rapidly eliminate parasites before DNA replication. The minor parasite fraction that escapes the fast killing of the first replication cycle is arrested in the schizont stage of the following cycle, displaying a delayed-death phenotype. Thiostrepton further exhibits gametocytocidal activity by eliminating gametocytes, the sexual precursor cells that are crucial for parasite transmission to the mosquito. Compound treatment results in an accumulation of ubiquitinated proteins in the blood stages, indicating an effect on the parasite proteasome. In accordance with these findings, expression profiling revealed that the proteasome is present in the nucleus and cytoplasm of trophozoites, schizonts, and gametocytes. In conclusion, thiostrepton derivatives represent promising candidates for malaria therapy by dually acting on two independent targets, the parasite proteasome and the apicoplast, with the capacity to eliminate both intraerythrocytic asexual and transmission stages of the parasite.
Assuntos
Antimaláricos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/metabolismo , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Tioestreptona/farmacologia , Animais , Western Blotting , Cloroquina/farmacologia , Resistência a Medicamentos , Técnica Indireta de Fluorescência para Anticorpo , Células HeLa , Humanos , Microscopia Imunoeletrônica , Plasmodium falciparum/patogenicidade , Complexo de Endopeptidases do Proteassoma/metabolismo , Esquizontes/efeitos dos fármacos , Esquizontes/metabolismo , Trofozoítos/efeitos dos fármacos , Trofozoítos/metabolismoRESUMO
Ribosomally produced thiopeptide antibiotics are highly promising lead compounds targeting the GTPase-associated region (GAR) of the bacterial ribosome. A representative panel of GAR mutants suspected to confer resistance against thiopeptide antibiotics was reconstituted in vitro and quantitatively studied with fluorescent probes. It was found that single-site mutations of the ribosomal 23S rRNA binding site region directly affect thiopeptide affinity. Quantitative equilibrium binding data clearly identified A1067 as the base contributing most strongly to the binding environment. The P25 residue on the ribosomal protein L11 was essential for binding of the monocyclic thiopeptides micrococcin and promothiocin B, confirming that the mutation of this residue in the producer organism confers self-resistance. For the bicyclic thiopeptides thiostrepton and nosiheptide, all studied single-site resistance mutations on the L11 protein were still fully capable of ligand binding in the upper pM range, both in the RNA-protein complex and in isolated 70S ribosomes. These single-site mutants were then specifically reconstituted in Bacillus subtilis, confirming their efficacy as resistance-conferring. It is thus reasoned that, in contrast to modifications of the 23S rRNA in the GAR, mutations of the L11 protein do not counteract binding of bicyclic thiopeptides, but allow the ribosome to bypass the protein biosynthesis blockade enforced by these antibiotics in the wild type.
Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Farmacorresistência Bacteriana , Peptídeos/farmacologia , Sequência de Aminoácidos , Antibacterianos/química , Antibacterianos/metabolismo , Bactérias/citologia , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Modelos Moleculares , Mutação , Peptídeos/química , Peptídeos/metabolismo , Conformação Proteica , RNA Bacteriano/antagonistas & inibidores , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico/antagonistas & inibidores , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/efeitos dos fármacos , Subunidades Ribossômicas Maiores de Bactérias/genéticaRESUMO
The natural product thiopeptide antibiotic thiostrepton is shown to undergo facile epimerization at its thiazoline residue in favor of the naturally observed D-configuration, suggesting that a classical epimerase enzyme may not be involved in its biosynthesis.
Assuntos
Cisteína , Racemases e Epimerases/metabolismo , Tioestreptona/biossíntese , Antibacterianos/biossíntese , Tioestreptona/químicaAssuntos
Antibacterianos/biossíntese , Peptídeos Cíclicos/biossíntese , Compostos de Sulfidrila/química , Sequência de Aminoácidos , Antibacterianos/química , Genes Bacterianos , Dados de Sequência Molecular , Família Multigênica , Biossíntese Peptídica , Peptídeos Cíclicos/química , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Tioestreptona/biossínteseAssuntos
Antibacterianos/química , Antibacterianos/metabolismo , Corantes Fluorescentes/análise , GTP Fosfo-Hidrolases/metabolismo , Ribossomos/enzimologia , Tioestreptona/análogos & derivados , Tioestreptona/metabolismo , Antibacterianos/farmacologia , Corantes Fluorescentes/química , RNA Ribossômico 23S/genética , RNA Ribossômico 23S/metabolismo , Ribossomos/efeitos dos fármacos , Ribossomos/genética , Staphylococcus aureus/citologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo , Tioestreptona/farmacologiaRESUMO
Proximity-induced covalent capture (PICC) has been established for the investigation of ligand binding to composite protein/oligonucleotide target complexes. The RNA-induced attachment of the thiopeptides Thiostrepton and Nosiheptide to engineered Cys mutants of the ribosomal protein L11 was highly position selective and allowed mapping of their binding site at amino acid resolution.