Orthoformimycin, a Selective Inhibitor of Bacterial Translation

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Orthoformimycin, a Selective Inhibitor of Bacterial Translation Elongation from Streptomyces Containing an Unusual Orthoformate Sonia I. Maffioli,*,† Attilio Fabbretti,‡ Letizia Brandi,‡ Andreas Savelsbergh,§ Paolo Monciardini,† Monica Abbondi,∥,○ Rossana Rossi,⊥ Stefano Donadio,† and Claudio O. Gualerzi*,‡ †

NAICONS, 20138 Milano, Italy Laboratory of Genetics, Department of Biosciences and Biotechnology, University of Camerino, 62032 Camerino, Italy § Institut für Medizinische Biochemie, Universität Witten/Herdecke, 58453 Witten, Germany ∥ Vicuron Pharmaceuticals, 21040 Gerenzano, Italy ⊥ ITB-CNR, 20090 Segrate, Italy ‡

S Supporting Information *

ABSTRACT: Upon high throughput screening of 6700 microbial fermentation extracts, we discovered a compound, designated orthoformimycin, capable of inhibiting protein synthesis in vitro with high efficiency. The molecule, whose structure was elucidated by chemical, spectrometric, and spectroscopic methods, contains an unusual orthoformate moiety (hence the name) and belongs to a novel class of translation inhibitors. This antibiotic does not affect any function of the 30S ribosomal subunit but binds to the 50S subunit causing inhibition of translation elongation and yielding polypeptide products of reduced length. Analysis by fluorescence stopped flow kinetics revealed that EF-Gdependent mRNA translocation is inhibited by orthoformimycin, whereas, surprisingly, translocation of the aminoacyl-tRNA seems to be unaffected.

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Here, we report the discovery, isolation, structure elucidation, and investigation of the mechanism of action of orthoformimycin, a structurally unique compound that inhibits bacterial protein synthesis by an unprecedented mechanism.

ature has designed a remarkable number of antimicrobial compounds that target specific processes in living cells. Among them, translation of mRNA into proteins is targeted by several classes of compounds of microbial origin, many of which have become important antibiotics in the fight against bacterial diseases.1−3 Most of the known antibiotics bind to the ribosome, which thus constitutes a validated target for many synthetic and naturally occurring classes of drugs.4 Although many antibiotics inhibit translation, several components and individual steps of translation remain untargeted by small molecules and thus offer an opportunity for identifying novel structural classes of ribosome inhibitors. The use of a target-based approach to discover translation inhibitors from microbial product extracts, making use of a bacterial cell-free extract programmed with a model mRNA, has previously been reported.5,6 Additional tests in which translation of the same mRNA in a eukaryotic cell-free extract led to the establishment of the selectivity of the inhibition, whereas the effect on poly(U)-directed poly-Phe synthesis allowed the discrimination of elongation versus nonelongation inhibitors thereby increasing the probability of chemical novelty.6 This approach led to the identification of two new classes of ribosome inhibitors, represented by the tetrapeptide GE81112 7,8 and by the dityrosine-like cyclic peptide GE82832.9,10 © XXXX American Chemical Society

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RESULTS AND DISCUSSION Identification of Orthoformimycin. Orthoformimycin was initially identified in a high throughput screen (HTS) of about 6700 microbial fermentation extracts, derived from ca. 4400 actinomycetes and from 450 filamentous fungi. The HTS test was aimed at identifying molecules capable of inhibiting in vitro translation of the universal 027IF2Cp(A) mRNA.5 One of the extracts fulfilling the selection criteria (inhibition of the Escherichia coli but not of the Saccharomyces cerevisiae system) was further characterized. Upon HPLC fractionation, the active fraction showed an m/z of 540 [M + H]+, which did not apparently correspond to any known microbial product. The producer strain ID107558, characterized on the basis of its morphology and 16S rRNA gene sequence, was found to be closely related to the ubiquitous group of species related to Streptomyces griseus,11 with >99.9% identity to S. griseus subsp. Received: June 8, 2013 Accepted: July 29, 2013

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dx.doi.org/10.1021/cb4004095 | ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology

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Figure 1. In vitro and in vivo translation inhibition by orthoformimycin. (a) Effect of the increasing concentrations of orthoformimycin indicated in the abscissa on Renilla Luciferase mRNA (green ▼), poly(U) (red ▲), and 027IF2Cp(A) mRNA (black ●) translation in a partially purified system derived from E. coli and 027IF2Cp(A) mRNA translation in a S. cerevisiae cell-free extract (Δ). (b) In vivo incorporation of [3H] thymidine (green ■), [3H] uridine (red ●), [35S] methionine (black ▲), and [3H] N-acetylglucosamine (blue ▼) by B. subtilis cells exposed to orthoformimycin (130 μg mL−1) at time 10′; the levels of incorporation of each radioactive precursor at the times indicated in the abscissa are normalized point-by-point and expressed as % of the incorporation of each precursor by the antibiotic-treated cells with respect to the nontreated controls taken as 100%. (c) Time-course of [35S] methionine incorporation by untreated (black ●) and orthoformimycin (130 μg mL−1)-treated (red ■) B. subtilis cells; the arrow indicates the time (10′) of the addition of the antibiotic.

deduced from an HR-ESIMS peak at m/z 540.2582 [M + H]+. HPLC analysis indicated that 1 actually consists of two compounds, designated 1A and 1B, present in a 55:45 ratio. Extensive NMR analyses revealed that most signals are isochrones for the two isomers, with just a few differences. The full NMR assignments are reported in Table 1, whereas the 1 H NMR, COSY, TOCSY, HSQC, HMBC, and NOESY spectra can be found in the Supporting Information (Supplementary Figures S1−S6).

griseus and several other members of the S. griseus clade. From a 10 L culture of the strain, we recovered 50 mg of a pure product 1 (see Methods), designated orthoformimycin, as explained below. Secondary screening tests demonstrated that, in an E. colibased in vitro system, orthoformimycin inhibits >80% the translation of different templates, with estimated IC50s of 0.1 μM for poly(U), 0.8 μM for Renilla luciferase mRNA, and 4 μM for 027IF2Cp(A) mRNA (Figure 1A), an inhibitory activity consistent with that displayed by the original fermentation extract. Furthermore, like the crude extract, orthoformimycin confirmed to be incapable of inhibiting translation in a Saccharomyces cerevisiae cell-free system programmed with 027IF2Cp(A) (Figure 1A). Structure Determination. The structure of orthoformimycin is reported in Figure 2A. It exhibits a characteristic UV maximum at 310 nm and a molecular formula C25H37N3O10

Table 1. 1H and 13C NMR Spectroscopic Data for 1 in CD3CN isomer 1A δH (multiplicity; J in Hz)

Figure 2. Chemical structure of orthoformimycin and derivatives. (a) Structure of 1 (A/B). (b) Selected NMR 2D-correlations with proposed cyclitol stereochemistry. (c) Related compounds 2−6. B

1 2 3 3-NMe2 4

3.99 4.95 3.86 2.79 4.64

(m) (app t; 3.4) (m) (bs) (dd; 10.4; 4.7)

5 6 1′ 2′ 2′-Me 3′ 4′ 5′ and 9′ 6′ and 8′ 7′ 7′-NMe2 1″ 2″ 2″-COOH 3″ 4″

4.43 (m) 3.97 (m)

2.04 (d; 1.25) 7.07 (bs) 7.33 (d; 8.8) 6.74 (d; 8.8) 2.887 (s) 5.88 (s) 4.03 (m) 3.89 (m) 3.50 (m)

isomer 1B δC 70.3 48.6 64.0 41.3 72.3 79.2 69.0 175 128 15.8 136 124.6 132 113 151.6 41 118 79 176.3 73 63

δH (multiplicity, J in Hz) 4.01 (m) 4.88 (app t; 3.9) 3.64 (m) 2.69 (bs) 4.55 (dd; 9.44; 5.20) 4.43 (m) 3.90 (m)

2.03 (d; 1.25) 7.07 (bs) 7.33 (d; 8.8) 6.74 (d; 8.8) 2.883 (s) 5.86 (s) 4.09 (m) 3.89 (m) 3.50 (m)

δC 69.9 50.1 65.3 43.0 73.6 79.2 68.6 175 128 15 136 124.6 132 113 151.6 41 116.8 79 176.3 73 63



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Figure 3. MSn fragmentation pattern of orthoformimycin (m/z 540 with R1 = R2 = H) and its acetylated derivative (m/z 680 with R1 = Ac, R2 = Me).

imply that the likely difference between 1A and 1B should be located on one of the three remaining stereocenters, namely, the orthoformate and the α and β positions on the trihydroxybutyric acid. Further investigation will be necessary to establish the absolute configurations of 1A and 1B. The NMR-derived structure is consistent with the molecular formula C25H37N3O10 and with the calculated nine double bond equivalents. HR-MSn studies provided additional support for the proposed structure, with the three portions of 1 clearly discerned by MS fragmentation experiments, as shown in Figure 3 and Supplementary Table S1. The m/z 540 peak yielded a m/z 404 signal, coherent with the loss of the 136 amu neutral fragment, corresponding to the 2,3,4-trihydroxybutyric moiety, and an ion at m/z 394 corresponding to 2, which also represents the main degradation product of 1 (see below). A fragment at m/z 188, corresponding to the aminocinnamoyl portion, was also detected. When the MSn experiment was carried out in negative ionization mode, the only observed species was m/z 392, in agreement with the interpretation illustrated in Figure 3 and Supplementary Table S1. Consistent with the 1A and 1B being two stereoisomers, no differences in the fragmentation pattern were observed after analytical HPLC separation of 1A and 1B (data not shown). Selective chemical reactions, carried out on analytical scale and monitored by LC−MS, further confirmed the structure of 1. The presence of a single carboxylic acid is supported by reaction with TMSCH2N2, which led to a 14-amu heavier product. When left in moderately acidic solutions at RT, 1 slowly degraded within a few days eventually forming 2, after a first transient hydrolysis of the orthoformate ester to form 3 (Figure 2C). When the same incubation was performed in aqueous methanol, the transient formation of 4 was also observed. Consistently, direct treatment of 1 in MeOH, EtOH, or BuOH with a catalytic amount of p-toluensulfonic acic resulted in the quantitative formation, within a few minutes at RT, of 4, 5, and 6, respectively, as expected from the characteristic chemical behavior of the orthoformate ester functionality.14 When 1 was submitted to acetylation in pyridine, a product having m/z 648 was formed, coherent with the addition of three acetyl groups together with a dehydration, probably due to lactonization at 4″-OH of the butyric moiety, consistent with the behavior of aldonic acids.15 Indeed, dilution of the mixture with methanol resulted in the

Although the following discussion refers to isomer 1A, the same observations are applicable to 1B. From HSQC and HMBC experiments, 25 carbon resonances were observed: five quaternary carbons, two of them matching carboxyls (δC 175 and 176.3) and the others corresponding to aromatic and olefinic signals (δC 128, 124.6, 151.6); a single oxygenated methylene (δC 63); fourteen methine groups, one of them corresponding to an olefinic double bond (δC 136), four belonging to an aromatic ring (δC 113 and 132), plus one peculiar signal at δC 118 ppm; and five methyl groups, one of them on a double bond (δC 15.8), while the others on nitrogen atoms (δC 41 and 41.3). COSY and TOCSY correlations highlighted the presence of different spin systems belonging to an aromatic and olefinic portion; a central diamino-cyclitol in which the COSY cross-peak between H2 and H3 indicates a 1,2-diamino substitution; and a 2,3,4-trihydroxybutyric acid appendage. In the HSQC experiment, the proton signal at 5.88 ppm (H-1″) correlates with a carbon at 118 ppm having a long relaxation time; this carbon, in turn, shows an HMBC correlation with both the diaminocyclitol and the trihydroxybutyric acid. These data suggest that an orthoformate group connects these two moieties. The HMBC correlation (Figure 2B) between H-1″ and H-4 indicates that one of the hydroxyls on the cyclitol participates in the orthoformate group. Moreover, the C-1″ chemical shift (δC″ 118) is in agreement with literature data for pentacyclic orthoformates,12 which suggests that two vicinal hydroxyl groups of the cyclitol (HOC4 and HO-C5) are involved in the orthoester. A HMBC correlation between H-2″ and C-1″ confirms the linkage of the trihydroxy-acid to the cyclitol through an orthoester. Finally, the HMBC correlation between H-2 and C-1′ indicated that the cyclitol is amidated to the 4-aminocinnamic moiety. For both the 1A and 1B isomers, NOESY experiments allowed the establishment of the trans configuration for the double bond of the cinnamic moiety due to the absence of a NOE contact between H-3′ and Me-2′. A diagnostic trans diaxial vicinal coupling constant of 10.4 Hz between H-4 and H-3 was clearly observed for both 1A and 1B, suggesting that the cyclitol has a stereochemistry and conformation equivalent to those of hygromycin A,13 which contains a 5,6-fused aminocyclitol Nlinked to a substituted cinnamic acid. NOE contacts between H-2 and Me2N-3 and between H-4 and Me2N-3 (for both 1A and 1B) also confirmed this stereochemistry. These results C



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Figure 4. Target identification and activity of orthoformimycin on the elongation phase of protein synthesis. (a) Residual translation inhibition activity of the supernatants of increasing amounts of 30S (green ▲) and 50S (red ●) subunits and 70S (black ■) ribosomes subjected to ultracentrifugation after preincubation with orthoformimycin. The residual inhibition activity of the supernatant was determined in Renilla luciferase translation tests. (b) translation inhibition (black ▲) and reduction of the product size (red ■) as a function of the increasing concentrations of orthoformimycin; the product size was measured by determining the [3H]Phe/[35S]Met ratio of the synthesized polypeptide. (c) Effect of increasing concentrations of orthoformimycin on the level of fMet-Phe-puromycin formed by ribosomes programmed with 027 mRNA in the presence (red ▲) or absence (black ■) of elongation factor EF-G. Further experimental details are given in Methods.

subtilis (MIC = 70 μg mL−1), Micrococcus luteus and Streptococcus pneumoniae (MIC = 256 μg mL−1), and Haemophilus inf luenzae, Staphylococcus aureus, and an E. coli ΔtolC mutant (MIC = 512 μg mL−1). Despite its weak antibacterial activity, orthoformimycin produced a substantial inhibition of protein synthesis in B. subtilis in vivo, without affecting the incorporation of radiolabeled thymidine, uracil, and N-acetylglucosamine, a clear indication that DNA, RNA, and peptidoglycan (cell wall) syntheses are not inhibited (Figure 1B). As seen from the results presented in Figure 1C, incorporation of [35S] methionine in acid-insoluble protein product continues to increase linearly for at least 2.5 h in the untreated controls, whereas it is strongly inhibited upon the addition of the antibiotic to the culture. The arrest of protein synthesis can be regarded as almost immediate if one takes into the account the time necessary for the hydrophilic inhibitor to enter the cells and reach a sufficiently high internal concentration. Thus, the in vivo behavior of orthoformimycin is typical of a translation inhibitor. Furthermore, since similar results were obtained also with E. coli ΔtolC cells (not shown), it can be concluded that the translational apparatus is the in vivo target of orthoformimycin inhibition in both Gram positive and Gram negative bacteria. In light of the low concentrations required to inhibit in vitro mRNA translation and specific translational functions, it is likely that either the cells are poorly permeable to the rather hydrophilic antibiotic or the molecule is inactivated inside the cells or efficiently pumped out of the cell. Mechanism of Translation Inhibition. The molecular target of orthoformimycin action was investigated by testing its residual activity in the supernatants of 30S and 50S ribosomal subunits or 70S ribosomes subjected to centrifugation after preincubation with increasing amounts of antibiotic. As seen in Figure 4A, the supernatant of 30S ribosomal subunits contains all the expected orthoformimycin inhibitory activity, indicating that the antibiotic was not bound to the small ribosomal subunit. On the contrary, the amount of orthoformimycin remaining in the supernatant decreases upon incubation with increasing concentrations of 70S ribosomes and, even more so, of 50S ribosomal subunits indicating that orthoformimycin

slow formation of a m/z 680 peak, as expected from the methanolysis of the proposed lactone. HR−MS and MSn fragmentation pattern of m/z 680 (Figure 3) confirmed the addition of two acetyl groups on the diamino-cyclitol central unit and of the third acetyl group on the 2,3,4-trihydroxybutyric moiety with concomitant dehydration/methanolysis. All together, these data are consistent with the presence of an orthoformate linking the trihydroxy-butyric and the cyclitol. Thus, in light of its chemical structure and the uniqueness of an orthoformate moiety, we named 1 “orthoformimycin”. Structural Features of Orthoformimycin. Orthoformimycin consists of three portions: a cinnamic aromatic moiety, a central 1,2-diamino cyclitol, and a 2,3,4-trihydroxybutyric acid. Although each portion has precedents in natural products, unique structural features render 1 very intriguing. The bacterial protein synthesis inhibitors puromycin,16 A201A,17 and hygromycin A,13 all produced by Streptomyces and related genera, do contain a cinnamic moiety, but none a 4-amino aromatic substituent as in 1. The latter is instead encountered in trichostatin A18 and vernamycin B19 from Streptomyces spp., which, however, are not reported as translation inhibitors. Similarly, aminocyclitols are frequently present in microbial products (e.g., 1,3-diaminocyclitols in aminoglycosides20 and 1,4-diaminocyclitols in fortimicin21), but 1 contains a previously unreported 1,2-diaminocyclitol, whose amidated nitrogen is in the same position as monoaminocyclitol of hygromycin A. Finally, the 2,3,4-trihydroxybutyric moiety has only been described as part of an antimicrobial metabolite from Streptomyces.22 Most of all, 1 represents the first example of a natural product containing an orthoformate ester, which is thus the hallmark of orthoformimycin. Other orthoesters are encountered in secondary metabolites from fungi, for example, austalides23 and spiromentin,24 or from plants, for example, phragmalin25 and daphnetoxin.26 Only occasionally has their presence been observed in bacterial metabolites, such as orthosomycins27 and hygromycin B.28 Antibacterial Activity. Orthoformimycin shows modest activity against some bacterial strains: measurable effects were seen against Moraxella catarrhalis (MIC = 32 μg mL−1), Bacillus D



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Figure 5. Effect of orthoformimycin on the EF-G-dependent translocation of aminoacyl-tRNA and mRNA. Fluorescence stopped-flow kinetic analysis of the (a) mRNA and (b) aminoacyl-tRNA (Phe-tRNA) EF-G-dependent translocation in the absence of antibiotics (black trace) or in the presence of 20 μM of orthoformimycin (red trace) or 20 μM GE82832/dityromycin 9 (green trace); negative controls in which EF-G was omitted are also shown (orange tracings). Further experimental details can be found in Methods and in ref 9.

ribosomal binding of the elongation factor but perhaps with its subsequent activity on the ribosome. Indeed, analysis by fluorescence stopped-flow kinetics indicated that orthoformimycin strongly inhibits the EF-G-dependent movement of the mRNA on the ribosome (Figure 5A) as well as π-release from EF-G, albeit to a somewhat lesser extent than GE82832/ dityromycin (Supplementary Figure S8C); π-release normally occurs after EF-G-dependent GTP hydrolysis and upon tRNA/ mRNA translocation.30,31 However, orthoformimycin does not affect EF-G-dependent aminoacyl-tRNA translocation (Figure 5B). This is a quite surprising result, at variance with what happens with other translocation inhibitors, which inhibit both mRNA (Figure 5A) and tRNA (Figure 5B) movement as in the case of GE82832/dityromycin9,10 This is a puzzling finding because during translocation mRNA and aminoacyl-tRNA movements are coupled.30,31 Thus, orthoformimycin is either capable of uncoupling by some unknown mechanism the movement of the two ligands on the ribosome or its mechanism of inhibition is more complex than what would superficially appear from its effect on EF-G functions.

strongly binds to free 50S ribosomal subunits and, albeit with a somewhat lower affinity, to 50S associated with the 30S subunits within 70S monomers. The effect of orthoformimycin was subsequently tested on the individual steps of the translation pathway. Unlike the inhibitor of P-site decoding GE81112,7,8 orthoformimycin did not affect mRNA-dependent binding of fMet-tRNA to 30S or 70S ribosomes (Supplementary Figure S7A); furthermore, this antibiotic displayed a modest inhibition (∼40%) of fMetpuromycin formation only at very high concentration (100 μM) indicating that the initiator tRNA bound in its presence is properly placed in the ribosomal P-site (Supplementary Figure S7B). Likewise, binding of aminoacyl-tRNA (Phe-tRNA) to poly(U)-programmed 30S subunit in the presence or absence of elongation factor EF-Tu was not inhibited by orthoformimycin, while it was inhibited, under the same experimental conditions, by the A-site inhibitor tetracycline (Supplementary Figure S7C). Thus, taken together, these data indicate that orthoformimycin does not interfere with either P- or A-site decoding in the small subunit and does not prevent peptide bond formation between the aminoacyl-tRNA analogue puromycin and P-sitebound fMet-tRNA. The lack of inhibition by orthoformimycin of the initiation phase of protein synthesis and the evidence that the antibiotic targets the 50S ribosomal subunit prompted us to investigate possible effects of the inhibitor on the elongation phase of translation. Thus, when the nature of the translational product synthesized in the presence of increasing concentrations of orthoformimycin was analyzed, the size of the peptides produced was found to become progressively smaller with a dose−response curve roughly corresponding to that obtained for translation inhibition (Figure 4B). This finding suggests that the antibiotic might interfere with translocation, as previously observed in other cases;29 consistent with this hypothesis is the evidence that orthoformimycin, at concentrations ranging from 1 to 10 μM, inhibits 30−45% of the EF-G-dependent translocation of fMetPhe-tRNA from the A-site to the P-site, which is necessary to yield fMet-Phe-Puromycin (Figure 4C). Further analyses of the translocation steps showed that orthoformimycin, like the recently characterized translocation inhibitor GE82832/dityromycin,9,10 does not inhibit ribosome- and EF-G-dependent GTPase (Supplementary Figure S8A and B); this finding suggests that the antibiotic does not interfere with the

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CONCLUSIONS Despite many decades of intensive screening, nature can still provide novel, unique scaffolds that target biosynthetic steps essential for the viability of microbial cells. The unique and original chemical structure of orthoformimycin represents one further example of compounds that are unlikely to be encountered in synthetic or combinatorial libraries. We have also demonstrated that orthoformimycin is an effective antibiotic as expected from a selective inhibitor of bacterial translation. An interesting and intriguing finding is the apparent uncoupling between mRNA and aa-tRNA translocation caused by orthoformimycin. To the best of our knowledge, a similar uncoupling was observed only in another case, namely, ribosomes whose 16S rRNA had been cleaved by Colicin E3 displayed an increased tRNA translocation independent from ribosome unlocking.32 Regardless of the precise mechanism by which orthoformimycin interferes with elongation of protein synthesis, our data indicate that in addition to having a novel chemical structure this antibiotic is also endowed with a novel mechanism of action, different from that of other 50S inhibitors.33 Additional work is clearly needed to clarify better the molecular nature of this mechanism. E



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were performed using Bruker standard pulse sequences. Data were processed with WIN NMR software (Bruker). Accurate mass determinations were performed on a Surveyor Accela HPLC system (Thermo Fisher Scientific) connected to the Exactive benchtop mass spectrometer (Thermo Fisher Scientific), equipped with a NSI−ESI ion source. Samples were injected on a C8 reversed phase column (BioBasic C8, 100 × 0.18 mm, 5 μm, 300A, Thermo Fisher Scientific) and were eluted using a gradient (eluent A, 0.1% formic acid in water; eluent B, 0.1% formic acid in acetonitrile) consisting of 5% eluent B for 3 min, 5 to 65% B in 17 min, and 65 to 95% B in 5 min. The flow rate was 100 μL min−1; the eluent was split to achieve a flux of 2 μL min−1 for MS analyses. Samples were dissolved in water. In Vivo Activity. MICs were determined in cation adjusted Müller Hinton medium using 104 CFU mL−1 inocula and 24 h incubation time. Relevant strains used were from the NAICONS collection. Assessment of in vivo orthoformimycin activity was performed as previously described:5,8 B. subtilis ATCC 6633 cells were grown at 37 °C in Spizizen’s medium (1.4% K2HPO4, 0.6% KH2PO4, 0.2% (NH4)2SO4, 0.1% trisodium citrate dehydrate, 0.02% MgSO4(H2O)7, and 0.5% D-glucose supplemented with 0.1% casamino acids) to an OD600 of 0.2, when the culture was divided into four identical aliquots (time 0); each aliquot received one of the following precursors: [3H] thymidine, [3H] uridine, [35S] methionine, or [3H] N-acetylglucosamine. After 10 min, each culture was split into two aliquots, one of which was exposed to 130 μg mL−1 orthoformimycin and the other to 1.5% DMSO. At 10 min intervals, 50 μL samples were withdrawn and mixed with 50 μL of 2% SDS, and the acid (TCA)-insoluble radioactivity present in 50 μL of the resulting extracts was determined by liquid scintillation counting. In Vitro Tests. In vitro mRNA translation (driven by 027IF2Cp(A) mRNA, poly(U), or Renilla luciferase mRNA) and tests of individual translational steps (e.g., fMet-tRNA binding to 30S and 70S ribosomes, Phe-tRNA binding to the ribosomal A-site, and fMetpuromycin formation and fMet-Phe-puromycin formation) were carried out as described.5,6 Rapid Kinetics Analyses. Pretranslocation complexes programmed with MF-mRNA (Dharmacon Res. Inc.) (or fluoresceinlabeled MFmRNA) and carrying deacylated tRNAfMet in the P site and fMetPhe-tRNAPhe in the A site were prepared as previously described.44 Fluorescence stopped-flow measurements were made and the data evaluated as described.10 Experiments were carried out at 37 °C, by rapidly mixing equal volumes each of the pretranslocation complex (0.2 μM in syringe 1) with (20 μM) or without orthoformimycin and EF-G·GTP (4 μM in syringe 2). π-Release from EF-G after GTP hydrolysis was measured by the fluorescence change of MDCC-labeled phosphate-binding protein (PBP-MDCC) as previously described.36

In this connection, a large body of structural information on the ribosomal location of EF-G during various intermediates of translocation is available nowadays;34−41 it seems therefore possible that structural biology techniques may detect a particular conformation in which orthoformimycin traps the EF-G-ribosome complex, thereby shedding light on the molecular mechanism of inhibition of this antibiotic.

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METHODS

High Throughput Screening. Microbial fermentation extracts, prepared as previously described,42 were screened essentially as reported.5 In particular, a 5 μL sample was assayed in 50 μL of translation reaction consisting of an optimized amount of E. coli MRE600 S30 extract driven by the natural-like 027IF2Cp(A) mRNA.6 Bacterial selectivity of the inhibitors found in the primary screening was established by cell-free translation of the same 027IF2Cp(A) mRNA using an optimized amount of Saccharomyces cervisiae SKQ 2M. Preparation of the reagents, cell-free protein synthesis (either in the prokaryotic or the eukaryotic system), and the conditions for product detection and quantification by ELISA were as described.5 Strain Characterization and Cultivation. Strain ID107558, part of the NAICONS strain collection, was isolated from aquatic plants collected from the Sile river, Italy. The strain presents morphological features and 16S rRNA gene sequence, determined as described43 (Accession No. KC008580), typical of the Streptomyces genus. The strain was maintained at 30 °C on S1 plates, as described.43 Streptomyces sp. ID107558 was grown in 500 mL Erlenmeyer flasks containing 100 mL of VSGM medium (24 g L−1 maize dextrin, 10 g L−1 glucose monohydrate, 5 g L−1 yeast extract, and 5 g L−1 soy peptone, at pH 7.2). The cultures were incubated at 30 °C, under stirring (200 rpm), for 48 h, then a 4% inoculum was transferred into a new 500 mL Erlenmeyer flask containing 100 mL of fresh medium and incubated under the same conditions. After 48 h, 0.5 L of the culture were transferred into a 20 L BioFlo 415 bioreactor (New Brunswick Scientific) containing 10 L of G1/0 medium (10 g L−1 glucose monohydrate, 10 g L−1 maltose, 8 g L−1 soybean meal, 2 g L−1 yeast extract, 4 g L−1 calcium carbonate, and 0.15 g L−1 polypropylene glycol, at pH 7.2) and incubated at 30 °C, 500 rpm stirring, and 0.5 vvm aeration. The medium pH was maintained between 6.8 and 7.8 by addition of 0.5 N NaOH or 5% H2SO4, as appropriate. The culture was harvested after 72 h. Recovery and Purification. The 10 L culture was centrifuged (3000 rpm for 10 min) and 500 mL of HP20 resin were added to the supernatant. After leaving the suspension overnight under shaking at 60 rpm, the resin was recovered by filtration (paper filter Scienceware cat. no. 146320010) and treated with 2 × 500 mL of 80% MeOH. The collected methanolic extracts were evaporated to dryness, and the resulting solid was further purified by reverse-phase medium pressure chromatography (C18 RediSep RF column 86g: 40−63 μm particle size, 60 Å pore size, 230−400 mesh) by using a CombiFlash RF Teledyne Isco. Phase A was water and phase B was MeOH, eluting with a 20 min linear gradient from 5 to 70% phase B. Orthoformimycin-containing fractions were pooled and concentrated under vacuum to dryness yielding 50 mg of pure compound. Analytical Methods. LC−MS analyses employed an Agilent 1100 series liquid chromatograph equipped with an 2.7 μm Ascentis express Supelco RP18 column (50 × 4.6 mm) eluted at 1 mL min−1 and at 40 °C. Samples were resolved with a 6 min linear gradient from 5 to 100% phase B, using 0.05% (v/v) TFA in water and acetonitrile as phases A and B, respectively. The effluent from the column was split between a photodiode array detector and an ESI interface of a Bruker Esquire3000 plus ion trap mass spectrometer operating in the positive-ion mode. 1H and 13C 1D- and 2D-NMR spectra (COSY, TOCSY, NOESY, HSQC, and HMBC) were measured in CD3CN at 25 °C using a Bruker AMX 400 MHz spectrometer. Proton and carbon chemical shifts were referenced to the residual solvent signal (CD3CN) at 1.94 and 1.32 ppm, respectively. Two-dimensional experiments including TOCSY, COSY, NOESY, HMQC, and HMBC

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ASSOCIATED CONTENT

S Supporting Information *

NMR spectral data for orthoformimycin and additional results on translation inhibition. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (C.O.G.); smaffioli@ naicons.com (S.I.M.). Present Address ○

FIIRV, Gerenzano 21040, Italy.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to E. Selva and the screening team at Vicuron Pharmaceuticals for preliminary characterization of orthoformiF



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(18) Tsuji, N., Kobayashi, M., Nagashima, K., Wasisaka, Y., and Koizumi, K. (1976) A new antifungal antibiotic, trichostatin. J. Antibiot. 29, 1−6. (19) Bodanszky, M., and Ondetti, M. A. (1964) Structure of the vernamycin B group of antibiotics. Antimicrob. Agents Chemother. 161, 360−365. (20) Houghton, J. L., Green, K. D., Chen, W., and GarneauTsodikova, S. (2010) The future of aminoglycosides: the end or renaissance? ChemBioChem 11, 880−902. (21) Egan, R. S., Stanaszek, R. S., Cirovic, M., Mueller, S. L., Tadanier, J., Martin, J. R., Collum, P., Goldstein, A. W., Larry De Vault, R., Sinclair, A. C., Fager, E. E., and Mitscher, L. A. (1977) Fortimicins A and B, new aminoglycoside antibiotics III. Structural identification. J. Antibiot. 30, 552−563. (22) Saha, M., Ghosh, D., Jr., Ghosh, D., Garai, D., Jaisankar, P., Sarkar, K. K., Dutta, P. K., Das, S., Jha, T., and Mukherjee, J. (2005) Studies on the production and purification of an antimicrobial compound and taxonomy of the producer isolated from the marine environment of the Sundarbans. Appl. Microbiol. Biotechnol. 66, 497− 505. (23) Horak, R. M., Steyn, P. S., and Vleggaar, R. (1985) Metabolites of Aspergillus ustus. Part 1. Application of the heteronuclear selective population inversion (SPI) NMR technique to the structure elucidation of the Austalides A-F, novel ortho ester meroterpenoids. J. Chem. Soc., Perkin Trans. 1, 345−356. (24) Buchanan, M. S., Hashimoto, T., Takaoka, S., and Asakawa, Y. (1995) (+)-Osmundalactone, γ-lactones and spiromentins from the fungus Paxillus atrotomentosus. Phytochemistry 40, 1251−1257. (25) Wu, J., Xiao, Q., Huang, J., Xiao, Z., Qi, S., Li, Q., and Zhang, S. (2004) Xyloccensins O and P, unique 8,9,30-Phragmalin ortho esters from Xylocarpus granatum. Org. Lett. 6, 1841−1844. (26) Stout, G. H., Balkenhol, W. G., Poling, M., and Hickernell, G. L. (1970) The isolation ans structure of Daphnetoxin, the poisonous principle of Daphne species. J. Am. Chem. Soc. 92, 1070−1071. (27) Ollis, W. D., Smith, C., and Wright, D. E. (1979) The orthosomycin family of antibiotics: The constitution of flambamycin. Tetrahedron 35, 105−127. (28) Neuss, N., Koch, K. F., Molloy, B. B., Day, W., Huckstep, L. L., Dorman, D. E., and Roberts, J. D. (1970) Structure of hygromycin B, an antibiotic from Streptomyces hygroscopicus; the use of CMR spectra in structure determination. Helv. Chim. Acta 53, 2314−2319. (29) Matassova, N. B., Rodnina, M. V., Endermann, R., Kroll, H. P., Pleiss, U., Wild, H., and Wintermeyer, W. (1999) Ribosomal RNA is the target for oxazolidinones, a novel class of translational inhibitors. RNA 5, 939−946. (30) Frank, J., Gao, H., Sengupta, J., Gao, N., and Taylor, D. J. (2007) The process of mRNA−tRNA translocation. Proc. Natl. Acad. Sci. U.S.A. 104, 19671−19678. (31) Rodnina, M. V., and Wintermeyer, W. (2011) The ribosome as a molecular machine: the mechanism of tRNA−mRNA movement in translocation. Biochem. Soc. Trans. 39, 658−662. (32) Lancaster, L. E., Savelsbergh, A., Kleanthous, C., Wintermeyer, W., and Rodnina, M. V. (2008) Colicin E3 cleavage of 16S rRNA impairs decoding and accelerates tRNA translocation on Escherichia coli ribosomes. Mol. Microbiol. 69, 390−401. (33) Wilson, D. N. (2011) On the specificity of antibiotics targeting the large ribosomal subunit. Ann. N.Y. Acad. Sci. 1241, 1−16. (34) Stark, H., Rodnina, M. V., Wieden, H. J., Van Heel, M., and Wintermeyer, W. (2000) Large-scale movement of elongation factor G and extensive conformational change of the ribosome during translocation. Cell 100, 301−309. (35) Gao, Y. G., Selmer, M., Dunham, C. M., Kelley, A. C., and Ramakrishnan, V. (2009) The structure of the ribosome with elongation factor G trapped in the posttranslocational state. Science 326, 694−699. (36) Ratje, A. H., Loerke, J., Mikolajka, A., Brunner, M., Hildebrand, P. W., Starosta, A. L., Donhofer, A., Connell, S. R., Fucini, P., Mielke, T., Whitford, P. C., Onuchic, J. N., Yu, Y., Sanbonmatsu, K. Y., Hartmann, R. K., Penczek, P. A., Wilson, D. N., and Spahn, C. M. T.

mycin, to P. Guglierame for antibacterial activity determinations, and to C. L. Pon (Camerino) for suggestions and discussions. This work was partially supported by grants from Regione Lombardia. R.R. was directly supported by a fellowship from Regione Lombardia.

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REFERENCES

(1) Wilson, D. N. (2004) The antibiotics and the inhibition of ribosome function. In Protein Synthesis and Ribosome Structure: Translating the Genome (Nierhaus, K. H., and Wilson, D. N., Eds.), pp 449−527, Wiley-VCH Verlag GmbH & Co, Berlin, Germany. (2) Wilson, D. N. (2009) The A−Z of bacterial translation inhibitors. Crit. Rev. Biochem. Mol. Biol. 44, 393−433. (3) Fabbretti, A., Gualerzi, C. O., and Brandi, L. (2011) How to cope with the quest for new antibiotics. FEBS Lett. 585, 1673−1681. (4) Maffioli, S. (2013) A Chemist’s Survey of Different Antibiotic Classes. In Antibiotics: Targets, Mechanism and Resistance (Gualerzi, C. O., Brandi, L., Fabbretti, A., and Pon, C. L., Eds.), Wiley−VCH Verlag GmbH & Co. KGaA, Berlin, Germany, in press. (5) Brandi, L., Fabbretti, A., Milon, P., Carotti, M., Pon, C. L., and Gualerzi, C. O (2007) Methods for identifying compounds that specifically target translation. Methods Enzymol. 431, 229−267. (6) Brandi, L., Dresios, J., and Gualerzi, C. O (2008) Assays for the identification of inhibitors targeting specific translational steps. Methods Mol. Med. 142, 87−105. (7) Brandi, L., Fabbretti, A., La Teana, A., Abbondi, M., Losi, D., Donadio, S., and Gualerzi, C. O. (2006) Specific, efficient, and selective inhibition of prokaryotic translation initiation by a novel peptide antibiotic. Proc. Natl. Acad. Sci. U.S.A. 103, 39−44. (8) Brandi, L., Lazzarini, A., Cavaletti, L., Abbondi, M., Corti, E., Ciciliato, I., Gastaldo, L., Marazzi, A., Feroggio, M., Fabbretti, A., Maio, A., Colombo, L., Donadio, S., Marinelli, F., Losi, D., Gualerzi, C. O., and Selva, E. (2006) Novel tetrapeptide inhibitors of bacterial protein synthesis produced by a Streptomyces sp. Biochemistry 45, 3692−3702. (9) Brandi, L., Fabbretti, A., Di Stefano, M., Lazzarini, A., Abbondi, M., and Gualerzi, C. O. (2006) Characterization of GE82832, a peptide inhibitor of translocation interacting with bacterial 30S ribosomal subunits. RNA 12, 1262−1270. (10) Brandi, L., Maffioli, S., Donadio, S., Quaglia, F., Sette, M., Milón, P., Gualerzi, C. O., and Fabbretti, A. (2012) Structural and functional characterization of the bacterial translocation inhibitor GE82832. FEBS Lett. 586, 3373−3378. (11) Rong, X., and Huang, Y. (2010) Taxonomic evaluation of the Streptomyces griseus clade using multilocus sequence analysis and DNA−DNA hybridization, with proposal to combine 29 species and three subspecies as 11 genomic species. Int. J. Syst. Evol. Microbiol. 60, 696−703. (12) Bruyère, H., Westwell, A. D., and Jones, A. T. (2010) Tuning the pH sensitivities of orthoester based compounds for drug delivery applications by simple chemical modification. Bioorg. Med. Chem. Lett. 20, 2200−2203. (13) Pittenger, R. C., Wolfe, R. N., Hoehn, M. M., Marks, P. N., Daily, W. A., and McGuire, J. M. (1953) Hygromycin. I. Preliminary studies in the production and biological activity on a new antibiotic. Antibiot. Chemother. 3, 1268−1278. (14) Chalova, O. B., Nasyrov, I. M., Chistoedova, G. P., Kiladze, T. K., Kantor, E. A., and Rakhmankulov, D. L. (1984) Reaction of 2ethoxy-1,3-dioxacycloalkanes with alcohols and thiols. Dokl. Akad. Nauk Tadzh. SSR 27, 724−727. (15) Miller, F. P., Vandome, A. F., and McBrewster, J. (2010) Aldonic Acid, VDM Publishing House, Germany. (16) Suhadolnik, R. J. (1979) Nucleoside Antibiotics, pp 3−50, WileyInterscience, New York. (17) Kirst, H. A., Dorman, D. E., Occolowitz, J. L., Jones, N. D., Paschal, J. W., Hamill, R. L., and Szymansky, E. F. (1985) The structure of A201A, a novel nucleoside antibiotic. J. Antibiot. 37, 575− 586. G



Articles

(2010) Head swivel on the ribosome facilitates translocation by means of intra-subunit tRNA hybrid sites. Nature 468, 713−416. (37) Frank, J., Gao, H., Sengupta, J., Gao, N., and Taylor, D. J. (2007) The process of mRNA−tRNA translocation. Proc. Nat. Acad. Sci. 104, 19671−19678. (38) Zhou, J., Lancaster, L., Donohue, J. P., and Noller, H. F. (2013) Crystal structures of EF-G-ribosome complexes trapped in intermediate states of translocation. Science 340, 1236086−1236086. (39) Pulka, A., and Cate, J. H. D. (2013) Control of ribosomal subunit rotation by elongation factor G. Science 340, 1235970. (40) Tourigny, D. S., Fernandez, I. S., Kelley, A. C., and Ramakrishnan, V. (2013) Elongation factor G bound to the ribosome in an intermediate state of translocation. Science 340, 1235490. (41) Feng, S., Chen, Y., and Gao, Y.-G. (2013) Crystal structure of 70S ribosome with both cognate tRNAs in the E and P sites representing an authentic elongation complex. PloS One 8, e58829. (42) Donadio, S., Monciardini, P., and Sosio, M. (2009) Approaches to discovering novel antibacterial and antifungal agents. Methods Enzymol. 458, 3−28. (43) Mazza, P., Monciardini, P., Cavaletti, L., Sosio, M., and Donadio, S. (2003) Diversity of actinoplanes and related genera isolated from an Italian soil. Microb. Ecol. 45, 362−372. (44) Savelsbergh, A., Katunin, V. I., Mohr, D., Peske, F., Rodnina, M. V., and Wintermeyer, W. (2003) An elongation factor G-induced ribosome rearrangement precedes tRNA−mRNA translocation. Mol. Cell 11, 1517−1523.

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Orthoformimycin, a Selective Inhibitor of Bacterial Translation

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