ge-2270-a and pulvomycin

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

Pulvomycin inhibits protein synthesis by preventing the formation of the ternary complex between elongation factor Tu (EF-Tu) x GTP and aa-tRNA. In this work, the crystal structure of Thermus thermophilus EF-Tu x pulvomycin in complex with the GTP analogue guanylyl imino diphosphate (GDPNP) at 1.4 A resolution reveals an antibiotic binding site extending from the domain 1-3 interface to domain 2, overlapping the domain 1-2-3 junction. Pulvomycin binding interferes with the binding of the 3'-aminoacyl group, the acceptor stem, and 5' end of tRNA. Only part of pulvomycin overlaps the binding site of GE2270 A, a domain 2-bound antibiotic of a structure unrelated to pulvomycin, which also hinders aa-tRNA binding. The structure of the T. thermophilus EF-Tu x GDPNP x GE2270 A complex at 1.6 A resolution shows that GE2270 A interferes with the binding of the 3'-aminoacyl group and part of the acceptor stem of aa-tRNA but not with the 5' end. Both compounds, pulvomycin more markedly, hinder the correct positioning of domain 1 over domains 2 and 3 that characterizes the active form of EF-Tu, while they affect the domain 1 switch regions that control the EF-Tu x GDP/GTP transitions in different ways. This work reveals how two antibiotics with different structures and binding modes can employ a similar mechanism of action.

Topics: Aminoglycosides; Anti-Bacterial Agents; Crystallography; Peptide Elongation Factor Tu; Peptides, Cyclic; Phenylalanine-tRNA Ligase; Protein Conformation; Thermus thermophilus; Thiazoles

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

The antibiotic GE2270A prevents stable complex formation between elongation factor Tu (EF-Tu) and aminoacyl-tRNA (aatRNA). In Escherichia coli we characterized two mutant EF-Tu species with either G257S or G275A that lead to high GE2270A resistance in poly(Phe) synthesis, which at least partially explains the high resistance of EF-Tu1 from GE2270A producer Planobispora rosea to its own antibiotic. Both E. coli mutants were unexpectedly found to bind GE2270A nearly as well as wild-type (wt) EF-Tu in their GTP-bound conformations. Both G257S and G275A are in or near the binding site for the 3' end of aatRNA. The G257S mutation causes a 2.5-fold increase in affinity for aatRNA, whereas G275A causes a 40-fold decrease. In the presence of GE2270A, wt EF-Tu shows a drop in aatRNA affinity of at least four orders of magnitude. EF-Tu[G275S] and EF-Tu[G275A] curtail this drop to about two or one order, respectively. It thus appears that the resistance mutations do not prevent GE2270A from binding to EF-Tu.GTP and that the mutant EF-Tus may accommodate GE2270A and aatRNA simultaneously. Interestingly, in their GDP-bound conformations the mutant EF-Tus have much less affinity for GE2270A than wt EF-Tu. The latter is explained by a recent crystal structure of the EF-Tu.GDP.GE2270A complex, which predicts direct steric problems between GE2270A and the mutated G257S or G275A. These mutations may cause a dislocation of GE2270A in complex with GTP-bound EF-Tu, which then no longer prevents aatRNA binding as in the wt situation. Altogether, the data lead to the following novel resistance scenario. Upon arrival of the mutant EF-Tu.GTP.GE2270.aatRNA complex at the ribosomal A-site, the GTPase centre is triggered. The affinities of aatRNA and GE2270A for the GDP-bound EF-Tu are negligible; the former stays at the A-site for subsequent interaction with the peptidyltransferase centre and the latter two dissociate from the ribosome.

Topics: Actinomycetales; Adenine; Amino Acid Substitution; Aminoglycosides; Anti-Bacterial Agents; Drug Resistance, Microbial; Escherichia coli; Guanosine Diphosphate; Guanosine Triphosphate; Models, Molecular; Mutation; Peptide Elongation Factor Tu; Peptides; Peptides, Cyclic; Poly U; Protein Binding; Protein Biosynthesis; Protein Conformation; RNA, Bacterial; RNA, Transfer, Amino Acyl; Thermodynamics; Thermus; 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

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

The sensitivity of elongation factor Tu (EF-Tu) from different species of bacteria to the EF-Tu-binding antibiotics efrotomycin, pulvomycin and MDL 62879 was tested by measuring the effect of these antibiotics on cell-free protein synthesis systems. EF-Tu from four different Gram-negative species was sensitive to all three antibiotics. Among Gram-positive bacteria, EF-Tu of Bacillus subtilis, Staphylococcus aureus and Enterococcus faecalis was resistant to efrotomycin and less sensitive to pulvomycin than EF-Tu of Gram-negative bacteria. EF-Tus from streptococci were significantly less sensitive than EF-Tus from Gram-negative bacteria to both efrotomycin and pulvomycin. All of the EF-Tus were sensitive to MDL 62879. The same sensitivity pattern emerged from GDP exchange assays, performed with partially purified EF-Tu from different bacterial species and pure Escherichia coli EF-Ts. These results suggest that the site of action of MDL 62879 is more conserved among bacterial species than those of efrotomycin and pulvomycin. Heterogeneity of EF-Tus from different bacterial species was also reflected in differences in their apparent molecular masses estimated by SDS-PAGE. EF-Tus from the Gram-positive species had higher molecular masses than those from all but one of the Gram-negative species.

Topics: Aminoglycosides; Anti-Bacterial Agents; Bacterial Proteins; Drug Resistance, Microbial; Gram-Negative Bacteria; Gram-Positive Bacteria; Guanosine Diphosphate; Peptide Elongation Factor Tu; Peptides; Peptides, Cyclic; Pyridones; Species Specificity; Thiazoles

The new thiazolyl peptide antibiotic GE2270 A, isolated from Planobispora rosea strain ATCC 53773, is shown to inhibit bacterial protein biosynthesis in vitro by affecting specifically the GTP-bound form of elongation factor Tu (EF-Tu). The 'off' rate of EF-Tu.GTP is slowed down 400-fold, locking GTP on EF-Tu, whereas EF-Tu.GDP is unaffected. Therefore, on the EF-Tu.guanine nucleotide interaction, GE2270 A mimicks the effect of aa-tRNA. In line with this, the binding of aa-tRNA to EF-Tu.GTP is hindered by the antibiotic, as shown by the absence of a stable ternary complex and the inhibition of the enzymatic binding of aa-tRNA to the ribosome. This blocks the elongation cycle. GE2270 A does not essentially modify the intrinsic GTPase activity of EF-Tu, but impairs the stimulation by ribosomes of this reaction. The negative effect of GE2270 A on the EF-Tu.GTP interaction with aa-tRNA bears similarities with that of the structurally unrelated pulvomycin, whereas marked differences were found by comparing the effects of these two antibiotics on EF-Tu.GDP. This work emphasizes the varieties of the transitional conformations which tune the EF-Tu interaction with GTP and GDP.

Topics: Aminoglycosides; Anti-Bacterial Agents; Escherichia coli; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Molecular Structure; Peptide Chain Elongation, Translational; Peptide Elongation Factor Tu; Peptides; Peptides, Cyclic; Protein Binding; Ribosomes; Thiazoles