ge-2270-a and mocimycin

Elfamycins are a relatively understudied group of antibiotics that target the essential process of translation through impairment of EF-Tu function. For the most part, the utility of these compounds has been as laboratory tools for the study of EF-Tu and the ribosome, as their poor pharmacokinetic profile and solubility has prevented implementation as therapeutic agents. However, due to the slowing of the antibiotic pipeline and the rapid emergence of resistance to approved antibiotics, this group is being reconsidered. Some researchers are using screens for novel naturally produced variants, while others are making directed, systematic chemical improvements on publically disclosed compounds. As an example of the latter approach, a GE2270 A derivative, LFF571, has completed phase 2 clinical trials, thus demonstrating the potential for elfamycins to become more prominent antibiotics in the future.

Topics: Actinomycetales; Actinomycetales Infections; Aminoglycosides; Anti-Bacterial Agents; Drug Design; Escherichia coli; Guanosine Triphosphate; Peptide Elongation Factor Tu; Peptides, Cyclic; Polyenes; Pyridones; Ribosomes; Thiazoles

Since the pioneering discovery of the inhibitory effects of kirromycin on bacterial elongation factor Tu (EF-Tu) more than 25 years ago [1], a great wealth of biological data has accumulated concerning protein biosynthesis inhibitors specific for EF-Tu. With the subsequent discovery of over two dozen naturally occurring EF-Tu inhibitors belonging to four different subclasses, EF-Tu has blossomed into an appealing antimicrobial target for rational drug discovery efforts. Very recently, independent crystal structure determinations of EF-Tu in complex with two potent antibiotics, aurodox and GE2270A, have provided structural explanations for the mode of action of these two compounds, and have set the foundation for the design of inhibitors with higher bioavailability, broader spectra, and greater efficacy.

Topics: Aminoglycosides; Anti-Bacterial Agents; Drug Resistance, Bacterial; Macromolecular Substances; Peptide Elongation Factor Tu; Peptides, Cyclic; Polyenes; Protein Synthesis Inhibitors; Pyridones; Thiazoles

The antibiotic kirromycin inhibits prokaryotic protein synthesis by immobilizing elongation factor Tu (EF-Tu) on the elongating ribosome. Streptomyces ramocissimus, the producer of kirromycin, contains three tuf genes. While tuf1 and tuf2 encode kirromycin-sensitive EF-Tu species, the function of tuf3 is unknown. Here we demonstrate that EF-Tu3, in contrast to EF-Tu1 and EF-Tu2, is resistant to three classes of EF-Tu-targeted antibiotics: kirromycin, pulvomycin, and GE2270A. A mixture of EF-Tu1 and EF-Tu3 was sensitive to kirromycin and resistant to GE2270A, in agreement with the described modes of action of these antibiotics. Transcription of tuf3 was observed during exponential growth and ceased upon entry into stationary phase and therefore did not correlate with the appearance of kirromycin in stationary phase; thus, it is unlikely that EF-Tu3 functions as a resistant alternative for EF-Tu1. EF-Tu3 from Streptomyces coelicolor A3(2) was also resistant to kirromycin and GE2270A, suggesting that multiple antibiotic resistance is an intrinsic feature of EF-Tu3 species. The GE2270A-resistant character of EF-Tu3 demonstrated that this divergent elongation factor is capable of substituting for EF-Tu1 in vivo.

Topics: Aminoglycosides; Anti-Bacterial Agents; Drug Resistance, Bacterial; Gene Expression Regulation, Bacterial; Models, Molecular; Peptide Elongation Factor Tu; Peptides, Cyclic; Pyridones; RNA, Bacterial; RNA, Messenger; Streptomyces; Streptomyces coelicolor; Thiazoles; Transcription, Genetic

The antibiotic pulvomycin is an inhibitor of protein synthesis that prevents the formation of the ternary complex between elongation factor (EF-) Tu.GTP and aminoacyl-tRNA. In this report, novel aspects of its action on EF-Tu are described. Pulvomycin markedly affects the equilibrium and kinetics of the EF-Tu-nucleotide interaction, particularly of the EF-Tu.GTP complex. The binding affinity of EF-Tu for GTP is increased 1000 times, mainly as the consequence of a dramatic decrease in the dissociation rate of this complex. In contrast, the affinity for GDP is decreased 10-fold due to a marked increase in the dissociation rate of EF-Tu.GDP (25-fold) that mimics the action of EF-Ts, the GDP/GTP exchange factor of EF-Tu. The effects of pulvomycin and EF-Ts can coexist and are simply additive, supporting the conclusion that these two ligands interact with different sites of EF-Tu. This is further confirmed on native PAGE by the ability of EF-Tu to bind the EF-Ts and the antibiotic simultaneously. Pulvomycin enhances the intrinsic EF-Tu GTPase activity, like kirromycin, though to a much more modest extent. As with kirromycin, this stimulation depends on the concentration and nature of the monovalent cations, Li(+) being the most effective one, followed by Na(+), K(+), and NH(4)(+). In the presence of pulvomycin (in contrast to kirromycin), aa-tRNA and/or ribosomes do not enhance the GTPase activity of EF-Tu. The property of pulvomycin to modify selectively the conformation(s) of EF-Tu is also supported by its effect on heat- and urea-dependent denaturation, and tryptic digestion of the protein. Specific differences and similarities between the action of pulvomycin and the other EF-Tu-specific antibiotics are described and discussed.

Topics: Aminoglycosides; Anti-Bacterial Agents; Binding Sites; Enzyme Inhibitors; Enzyme Stability; GTP Phosphohydrolases; Guanosine Diphosphate; Guanosine Triphosphate; Hydrolysis; Peptide Elongation Factor Tu; Peptide Elongation Factors; Peptides, Cyclic; Protein Denaturation; Pyridones; Thiazoles; Trypsin; Urea

Elongation factor Tu (EF-Tu) is encoded by the tuf gene of the plastid organelle of the malaria parasite Plasmodium falciparum. A range of structurally unrelated inhibitors of this GTP-dependent translation factor was shown to have antimalarial activity in blood cultures. The most active was the cyclic thiazolyl peptide amythiamicin A with an IC50 = 0.01 microM. Demonstrable complexes were formed in vitro between a recombinant version of P. falciparum EF-Tu(pl) and inhibitors that bind to different sites on EF-Tu; these included the antibiotics kirromycin, GE2270A and enacyloxin IIa.

Topics: Animals; Anti-Bacterial Agents; Binding Sites; Genes, Protozoan; Macrocyclic Compounds; Peptide Elongation Factor Tu; Peptides, Cyclic; Plasmodium falciparum; Plastids; Polyenes; Protozoan Proteins; Pyridones; Recombinant Proteins; Thiazoles

Sensitivity of EF-Tu1 of the GE2270A producer Planobispora rosea towards GE2270A, pulvomycin and kirromycin was determined by band-shift assays for EF-Tu1-antibiotic complex formation and by in vitro translation experiments. EF-Tu1 of P. rosea appeared to be not only totally resistant to GE2270A, but also ten times more resistant to kirromycin than EF-Tu1 of Streptomyces coelicolor. In contrast, P. rosea EF-Tu1 was found to be not resistant to pulvomycin, an antibiotic that just like GE2270A blocks EF-Tu x GTP x aminoacyl-tRNA complex formation. Previous in vivo and in vitro experiments with mixed populations of antibiotic resistant and sensitive EF-Tu species had shown that sensitivity to kirromycin and pulvomycin is dominant over resistance. In the case of GE2270A we observed, however, that sensitivity is recessive to resistance, which again points to a different action mechanism than in the case of pulvomycin. Besides the tuf1 gene encoding the regular elongation factor EF-Tu1 a gene similar to S. coelicolor tuf3 for a specialized EF-Tu was located in the P. rosea genome. The tuf1 gene was isolated and sequenced. The amino acid sequence of EF-Tul of P. rosea not only exhibits an unusual Tyr160 substitution (comparable to those described for kirromycin-resistant EF-Tus), but also shows significant changes of conserved amino acids in domain 2 that may be responsible for GE2270A resistance (the latter do not resemble those leading to pulvomycin resistance). P. rosea EF-Tu1 thus is a first example of a bacterial EF-Tu with resistance against two divergently acting antibiotics.

Topics: Actinomycetales; Amino Acid Sequence; Aminoglycosides; Anti-Bacterial Agents; Bacterial Proteins; Cell-Free System; Cloning, Molecular; Drug Resistance, Microbial; Genes, Bacterial; Kinetics; Molecular Sequence Data; Peptide Biosynthesis; Peptide Elongation Factor Tu; Peptides; Peptides, Cyclic; Protein Biosynthesis; Protein Structure, Secondary; Pyridones; Recombinant Proteins; Sequence Homology, Amino Acid; Streptomyces; Thiazoles

Using a cell-free protein-synthesis system, we have established that the elongation factor (EF) Tu (EF-Tu) of the actinomycete Planobispora rosea, the producer of the thiazolyl peptide GE2270, a specific EF-Tu inhibitor, is highly resistant to its own antibiotic, while it is completely inhibited by kirromycin, which is another inhibitor of this factor. P. rosea was found to possess a single tuf gene, located between fus and rpsJ, encoding other components of the protein-synthesis machinery. The P. rosea tuf gene was expressed as a translational fusion to malE in Escherichia coli, and the resulting EF-Tu with an N-terminal Gly-Met extension was able to promote poly(U)-directed poly(Phe) synthesis in cell-free systems. This activity was not affected by GE2270, and the recombinant protein was incapable of binding the antibiotic, indicating that the P. rosea EF-Tu is intrinsically resistant to this inhibitor. Inspection of the translated tuf sequence revealed a number of amino acid substitutions in highly conserved positions. These residues, which are likely to be involved in conferring GE2270 resistance, map in EF-Tu domain II, as do the only two known mutations conferring resistance to this class of thiazolyl peptides in Bacillus subtilis.

Topics: Actinomycetales; Amino Acid Sequence; Anti-Bacterial Agents; ATP-Binding Cassette Transporters; Carrier Proteins; Cloning, Molecular; Conserved Sequence; Dose-Response Relationship, Drug; Drug Resistance, Microbial; Escherichia coli; Escherichia coli Proteins; Genes, Bacterial; Maltose-Binding Proteins; Models, Molecular; Molecular Sequence Data; Monosaccharide Transport Proteins; Mutation; Peptide Elongation Factor Tu; Peptides, Cyclic; Periplasmic Binding Proteins; Pyridones; Recombinant Fusion Proteins; Recombinant Proteins; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Thiazoles

Antibiotic MDL 62,879 inhibits bacterial protein synthesis by acting on elongation factor Tu (EF-Tu). In this study we show that the inhibition of protein synthesis by MDL 62,879 in an Escherichia coli cell-free system was fully reversed by addition of stoichiometric amounts of EF-Tu but not by large excesses of EF-Ts, ribosomes, or aa-tRNA. MDL 62,879 bound tightly to EF-Tu and formed a stable 1:1 MDL 62,879:EF-Tu (M:EF-Tu) complex. We show that binding of MDL 62,879 to EF-Tu strongly affects the interaction of EF-Tu with aa-tRNA and causes rapid dissociation of preformed EF-Tu.aa-tRNA complex, suggesting that inhibition of aa-tRNA binding is due to a conformational change in EF-Tu rather than competition for the aa-tRNA binding site. Indication of a conformational change in EF-Tu induced by MDL 62,879 is further confirmed by proteolytic cleavage experiments: MDL 62,879 binding strongly protects EF-Tu against trypsin cleavage. The observed effects of MDL 62,879 appear to be different from those of the kirromycin class of antibiotics, which also inhibit protein synthesis by binding to EF-Tu, suggesting two distinct binding sites. Indeed, the M:EF-Tu complex was able to bind stoichiometric amounts of kirromycin to form a 1:1:1 M:EF-Tu:kirromycin (M:EF-Tu:K) complex, providing direct evidence that the two antibiotics bind to independent and distinct sites on the EF-Tu molecule. The interaction of the M:EF-Tu:K complex with aa-tRNA and other co-factors suggest that the contemporary binding of the two antibiotics locks EF-Tu into an intermediate conformation in which neither antibiotic exhibits complete dominance.

Topics: Aminoglycosides; Anti-Bacterial Agents; Binding Sites; Chromatography, High Pressure Liquid; Models, Chemical; Peptide Elongation Factor Tu; Peptides; Peptides, Cyclic; Poly U; Pyridones; RNA, Transfer, Amino Acyl; Thiazoles