thiostrepton and nosiheptide

Topics: Alanine; Anti-Bacterial Agents; Boronic Acids; Catalysis; Copper; Enterococcus faecalis; Microbial Sensitivity Tests; Nisin; Protein Processing, Post-Translational; Ribosomes; Solubility; Staphylococcus aureus; Thiazoles; Thiostrepton

Thiopeptides are a family of highly modified peptide metabolites, characterized by a macrocycle bearing a central piperidine/dehydropiperidine/pyridine ring, multiple thiazole rings, and several dehydrated amino acid residues. Thiopeptides have useful antibacterial, antimalarial, and anticancer properties but have not been adapted for human clinical applications, owing in part to their poor water solubility. In 2009, it was revealed that the thiopeptide scaffold is derived from a ribosomally synthesized precursor peptide subjected to extensive posttranslational modifications. Shortly thereafter, three groups developed two types of in vivo strategies to generate thiopeptide variants: precursor peptide mutagenesis and gene inactivation. The thiopeptide analogs and biosynthetic intermediates obtained from these studies provide much-needed insight into the biosynthetic process for these complicated metabolites. Furthermore, the in vivo production of variants can be employed to interrogate thiopeptide structure-activity relationships and may be useful to address the bioavailability issues plaguing these otherwise promising lead molecules. This chapter discusses the in vivo systems developed to generate thiopeptide variants.

Topics: Anti-Bacterial Agents; Genes, Bacterial; Genetic Engineering; Multigene Family; Peptide Biosynthesis; Peptides; Plasmids; Protein Precursors; Ribosomes; Streptomyces; Structure-Activity Relationship; Thiazoles; Thiostrepton

Most thiopeptide antibiotics target the translational machinery: thiostrepton (ThS) and nosiheptide (NoS) target the ribosome and inhibit translation factor function, whereas GE2270A/T binds to the elongation factor EF-Tu and prevents ternary complex formation. We have used several in vitro translational machinery assays to screen a library of thiopeptide antibiotic precursor compounds and identified four families of precursor compounds that are either themselves inhibitory or are able to relieve the inhibitory effects of ThS, NoS, or GE2270T. Some of these precursors represent distinct compounds with respect to their ability to bind to ribosomes. The results not only provide insight into the mechanism of action of thiopeptide compounds but also demonstrate the potential of such assays for identifying lead compounds that might be missed using conventional inhibitory screening protocols.

Topics: Anti-Bacterial Agents; Binding Sites; GTP Phosphohydrolases; Peptide Elongation Factor Tu; Peptides, Cyclic; Prodrugs; Protein Biosynthesis; Ribosomes; Thiazoles; Thiostrepton

The thiopeptide class of antibiotics targets the GTPase-associated center (GAC) of the ribosome to inhibit translation factor function. Using X-ray crystallography, we have determined the binding sites of thiostrepton (Thio), nosiheptide (Nosi), and micrococcin (Micro), on the Deinococcus radiodurans large ribosomal subunit. The thiopeptides, by binding within a cleft located between the ribosomal protein L11 and helices 43 and 44 of the 23S rRNA, overlap with the position of domain V of EF-G, thus explaining how this class of drugs perturbs translation factor binding to the ribosome. The presence of Micro leads to additional density for the C-terminal domain (CTD) of L7, adjacent to and interacting with L11. The results suggest that L11 acts as a molecular switch to control L7 binding and plays a pivotal role in positioning one L7-CTD monomer on the G' subdomain of EF-G to regulate EF-G turnover during protein synthesis.

Topics: Anti-Bacterial Agents; Bacterial Proteins; Bacteriocins; Binding Sites; Crystallography, X-Ray; Deinococcus; Gene Expression Regulation; Models, Molecular; Molecular Sequence Data; Molecular Structure; Peptides; Protein Biosynthesis; Protein Structure, Tertiary; Ribosomal Proteins; Ribosomes; Thiazoles; Thiostrepton

Thiostrepton and micrococcin inhibit protein synthesis by binding to the L11 binding domain (L11BD) of 23S ribosomal RNA. The two compounds are structurally related, yet they produce different effects on ribosomal RNA in footprinting experiments and on elongation factor-G (EF-G)-dependent GTP hydrolysis. Using NMR and an assay based on A1067 methylation by thiostrepton-resistance methyltransferase, we show that the related thiazoles, nosiheptide and siomycin, also bind to this region. The effect of all four antibiotics on EF-G-dependent GTP hydrolysis and EF-G-GDP-ribosome complex formation was studied. Our NMR and biochemical data demonstrate that thiostrepton, nosiheptide, and siomycin share a common profile, which differs from that of micrococcin. We have generated a three-dimensional (3D) model for the interaction of thiostrepton with L11BD RNA. The model rationalizes the differences between micrococcin and the thiostrepton-like antibiotics interacting with L11BD.

Topics: Anti-Bacterial Agents; Bacteriocins; Base Sequence; Binding Sites; Guanosine Triphosphate; Hydrolysis; Magnetic Resonance Spectroscopy; Methylation; Methyltransferases; Models, Molecular; Molecular Sequence Data; Peptides; Protein Binding; Ribosomes; RNA, Ribosomal, 23S; Thiazoles; Thiostrepton

The mode of action of nosiheptide (multhiomycin) on bacterial protein synthesis is closely similar to that of thiostrepton. Both antibiotics inhibit functions of elongation factors Tu and G and greatly reduce the synthesis of guanosine penta- and tetraphosphates in response to stringent factor. Furthermore, the actinomycetes which produce these antibiotics defend themselves against their products in similar fashion. This involves specific pentose-methylation of 23S ribosomal RNA.

Topics: Anti-Bacterial Agents; Bacterial Proteins; Drug Resistance, Microbial; Escherichia coli; Methylation; Pentoses; Peptide Elongation Factors; Ribosomes; Streptomyces; Thiazoles; Thiostrepton