|Home | About | Journals | Submit | Contact Us | Français|
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 utilized 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 provide not only 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.
The translational machinery represents one of the major targets within the cell for antibiotics (reviewed by Spahn and Prescott, 1996; Wilson, 2004). Many clinical important classes of antibiotics, such as the tetracyclines, phenylpropanoids (chloramphenicol), macrolides (erythromycin), and aminoglycosides (gentamicin) inhibit translation by binding to the ribosome. Despite the potency of many of these drug classes, antibiotic resistance among clinically relevant pathogens is an increasing problem and thus the need for new antibiotics is more urgent than ever before. One class of antibiotics that has received renewed interest in the recent years is the thiopeptide family (reviewed by Bagley et al., 2005; Nicolaou et al., 2009), because of their effectiveness against Gram-positive bacteria, in particular, methicillin-resistant Staphlococcus aureus (MRSA), as well as against the malarial parasite Plasmodium falciparum (McConkey et al., 1997). Thiopeptide antibiotics are composed of oxazoles and thiazoles, as well as non-natural amino acids that are linked together to form complex macrocyclic frameworks (Figure 1A–D).
Two distinct families of thiopeptide compounds target the translational apparatus, one that targets the ribosome, referred to here as Class I thiopeptides, and the other, referred to as Class II thiopeptides, which targets the elongation factor EF-Tu. The best characterized of the Class I compounds include thiostrepton (ThS) and nosiheptide (NoS) (Figure 1A, B), both of which have been crystallized bound to the large ribosomal subunit (Harms et al., 2008) (see later Figure 6A, B and G). These structures reveal that the Class I thiopeptides bind within a region of the ribosome that is part of the GTPase associated center (GAC), so-named because it is involved in translation G-protein factor binding and stimulation of GTPase activity (Wilson and Nierhaus, 2005). Biochemically, Class I thiopeptides have been shown to inhibit 70S initiation complex (70SIC) formation, by interfering with the initiation G-protein IF2 (Brandi et al., 2004) and references therein; (Grigoriadou et al., 2007) as well as elongation, by interfering with both the G-proteins EF-Tu (Gale et al., 1981; Gonzalez et al., 2007; Modelell et al., 1971) necessary for rapid cognate aminoacyl-tRNA binding to the ribosome, and EF-G, which catalyzes translocation of the tRNA2-mRNA complex from the A and P sites to the P and E-sites (Pan et al., 2007; Pestka, 1970; Rodnina et al., 1999; Seo et al., 2006; Weisblum and Demohn, 1970). In contrast, the structurally similar Class II thiopeptides (Figure 1D) do not bind to the ribosome, but instead interact directly with EF-Tu (reviewed by Parmeggiani and Nissen, 2006). The crystal structure of the Class II thiopeptide GE2270A bound to EF-Tu reveals that the drug binds within a cleft between domains I and II of EF-Tu and directly overlaps with the binding site of the terminal end of the aminoacyl-tRNA (Parmeggiani et al., 2006; Parmeggiani and Nissen, 2006) (see later Figure 7A–C). GE2270A is thought to prevent the closing of domain I and II, which is necessary for the induced-fit binding of EF-Tu to the tRNA, thereby preventing ternary complex formation (Parmeggiani and Nissen, 2006).
Although ThS is already in veterinary usage, its low water solubility and poor bioavailability has so far precluded its use in human medicine. Recent success has been reported in the total synthesis of a number of Class I and II thiopeptides (reviewed by Hughes and Moody, 2007; Nicolaou et al., 2009), including amongst others ThS (Nicolaou et al., 2005a; Nicolaou et al., 2005c), and GE2270A (Nicolaou et al., 2008b; Nicolaou et al., 2006). Such synthetic studies pave the way to generating improved thiopeptide derivatives by identifying synthetic fragments (or derivatives thereof) that display biological activity or can act as new lead compounds.
We have utilized a series of translation machinery assays to screen a library of thiopeptide antibiotic precursor compounds. Unlike the parent antibiotics thiostrepton, nosiheptide and GE2270T, only a few of the precursor compounds display any significant inhibitory properties, even at high concentrations. Instead, however, four structurally distinct families of precursor compounds (Figure 1E–H) were discovered that relieve the inhibitory effect imparted by the parent compounds. The different precursor families exhibit differential effects with respect to the inhibitory antibiotic that is counteracted, as well as to the target whether the ribosome or EF-Tu. Two of the families represent completely new compounds with respect to their ability to bind to ribosomes and thus open the path to the development of novel antimicrobials. The application of such screening strategies will enable the identification of new lead compounds that are not detected using conventional inhibitory screening protocols.
Several assays were used to examine thiopeptide precursor compounds for their abilities to bind to the thiostrepton binding site, either by mimicking ThS inhibition of specific ribosomal functions, or by protecting the ribosome against the inhibitory effects of ThS via a competition effect. These assays, which are discussed in turn below, measure (i) IF2 conformational change during the conversion of 30S initiation complex (30SIC) to 70S initiation complex (70SIC), (ii) ribosome-dependent stimulation of the GTPase activity of EF-G and (iii) the cell-free synthesis of green fluorescent protein (GFP) using an Escherichia coli in vitro coupled transcription-translation (TT) assay.
The initiation factor IF2 is essential for 70SIC formation from 30SIC and 50S subunits (Antoun et al., 2003; Grigoriadou et al., 2007). Elsewhere we have shown that the fluorescence of a Cy3 derivative of IF2 (IF2Cy3) increases on 70SIC formation resulting from the binding of a 50S subunit to a 30SIC containing IF2Cy3 (Qin et al., 2009). This increase is inhibited by both ThS and NoS (Figure 2A), largely as a result of the effect of these antibiotics in inhibiting both the rate and extent of 70SIC formation (Grigoriadou et al., 2007) and data not shown). Measuring the extent of fluorescence change is thus a convenient way of monitoring thiopeptide precursor effects on 70SIC formation. A library of thiopeptide precursor compounds, as well three forms of the EF-Tu inhibitor GE2270 (A, T, and C1) were screened for this activity, along with ThS and NoS as positive controls. Only one precursor, denoted PA1 (Figure 2B), showed any measurable activity in inhibiting the fluorescence change, with an apparent Ki, 25 µM, some 60 – 400-fold higher than for ThS or NoS, respectively. Although PA1 does not bind with very high affinity, it apparently does so with considerable structural specificity, since the inhibitory effect was not seen for compounds PA2-PA3 which have either stereochemical or minor constitutional differences from PA1 (Figure 1E). The thiopeptide precursors were also screened for their abilities to reverse ThS inhibition of the IF2Cy3 fluorescence increase on 70SIC formation. In no case was such reversal observed.
Vacant 70S ribosomes are known to stimulate the GTPase activity of EF-G via formation of a 70S•EF-G•GTP complex. Such stimulation is strongly inhibited by ThS (Pestka, 1970; Weisblum and Demohn, 1970). We used two multiple turnover GTPase assays to screen thiopeptide precursors for inhibitory activity.
The first assay measured EF-G GTPase activity, via formation of a Malachite Green complex (see Experimental Procedures), for hundreds of turnovers. Under conditions for which ThS (1 µM) almost completely abolished such activity, we identified three distinct classes of precursor compounds (PA–PC; Figure 1E–G) that exhibited modest inhibitory effects when added at 50 µM (Figure 3A). However, we note that none of these compounds added at 10 µM showed appreciable inhibition (data not shown). As expected, the negative control, GE2270T, had no effect at a concentration of 50 µM (Figure 3A). In order to determine whether the modest inhibitory effects seen in Figure 3A were specific for EF-G, we next checked whether these compounds could also inhibit the ribosome-dependent stimulation of the Tet(M) GTPase. Tet(M) is a GTPase that binds to the ribosome analogously to EF-G, and confers resistance to the antibiotic tetracycline by weakening its binding to the ribosome (reviewed by Connell et al., 2003). Similar to the results for EF-G, representatives of the PA and PC families exhibited modest inhibitory effects on Tet(M) GTPase at high concentration (100 µM) (Figure 3B). However, in contrast to EF-G, little or no inhibition was observed for the PB family, suggesting that it is specific for EF-G.
The second assay measured EF-G GTPase activity from the fluorescence increase of released Pi binding to the fluorescent phosphate binding protein MDCC-PBP (Brune et al., 1994; Seo et al., 2006). This assay, which can in principle be used for single turnover measurement, was here employed to measure several turnovers, as determined by the stoichiometric ratio (5:1) of MDCC-PBP to ribosome. As performed, this assay could only detect very potent inhibitors of EF-G GTPase, since fluorescence wasn’t measured until 1 min after initiation of reaction, whereas in the absence of inhibition the full fluorescence change is complete within 5 – 10 sec (data not shown). It is thus no surprise, given the results presented in Figure 3A, that although ThS inhibited this assay with an apparent Ki of 1.1 µM, neither any of the precursors tested, nor even NoS or MiC, showed measurable inhibition up to a concentration of 100 µM (Figure 3C). In contrast, both NoS and MiC added at very low concentration protected against inhibition by 1.2 µM ThS, with half-maximal effects seen at 0.04 µM and 0.11 µM, respectively (Figure 3D). However, none of the precursor compounds afforded similar protective effects up to 50 – 100 µM of added precursor.
Although as expected, ThS, NoS and GE2270T were potent inhibitors of GFP synthesis using an in vitro TT assay (Figure 4, lanes 2–4), none of the precursor compounds tested displayed any significant inhibitory activity in this assay (Figure 4, white bars), even at high concentrations (50–100 µM). In contrast, addition of 50 µM of representative precursor compounds from four structural distinct classes PA-PD (Figure 1E–G), could reverse the inhibitory effect of 5 µM ThS (Figure 4, grey bars). The most effective protection was seen with PA2, which restored translation back to levels observed in the absence of antibiotic (Figure 4, lane 6). In comparison, PB1, PC1 and PD1 restored translation to 40–60% of the original levels (Figure 4, lanes 13–28). Additionally, we find that the 5S, 6R stereoisomer of PA2 (PA4) exhibited some protective properties (~35% compared to 100% for PA2). The structural specificity of these effects is clear from the failure of precursors that are chemically related to PA2 to exhibit similar protective effects against ThS inhibition. These include PA3, (Figure 4, lanes 6 and 8), which differs from PA2 by lacking only a double bond within the central dehydropiperidine ring (Figure 1E), and PD2 (Figure 4, lanes 26 and 28) which, with respect to PD1, has an altered side-chain on one of the thiazole rings (Figure 1H).
Interestingly, PA2, PB1, PC1 and PD1 displayed marked differences in their abilities to reverse the inhibitory effects of NoS (5 µM) and GE2270T (25 µM), as compared to the inhibitory effects of ThS. Thus, as shown in Figure 5, PA2 was an omnipotent protector of translation, restoring translation levels in the presence of all three thiopeptide inhibitors, with the following order of efficiency: ThS (100 %) > GE2270T (80 %) > NoS (60 %). PD1 rescued translation in the presence of ThS and GE2270T, but not NoS, and PB1 and PC1 efficiently rescued translation only against ThS. As was true for ThS, neither PA3, PB2, PC2, nor PD2 were able to reverse inhibition by NoS or GE2270T (data not shown).
The specific protective effect of the precursor compounds against ThS suggests that these compounds specifically compete with ThS for binding to the ribosome. Structural (Harms et al., 2008; Jonker et al., 2007) and biochemical data (Spahn and Prescott, 1996; Xing and Draper, 1996) for ThS suggests that the high affinity of this drug for the ribosome results from cooperative interaction between nucleotides in H43/44 of the 23S rRNA and the L11-NTD (Figure 6A, B). Given the structural similarity between PA2 and ThS, it is possible to model the position of this compound bound to the ribosome (Figure 6C; Harms et al., 2008). The substitution of the double-bond in the piperidine ring of PA2 to generate PA3 abolishes the protective effect of the compound (Figure 4). This is likely to result from differences in the planarity of the piperidine ring between PA3 and PA2, which in turn leads to differences in the relative position (by 0.5 Å) of the attached thiazole moiety, which, based on the model, would shift it towards Pro27 of L11-NTD and thus encroach on the thiopeptide binding site (data not shown). Such modest displacements within drug binding sites have been shown to have dramatic effects on the affinity of compounds, and often lead to antibiotic resistance (Blaha et al., 2008; Tu et al., 2005).
Although it is more difficult to model the PB and PC series of compounds based on the available structures, it is clear that the aromatic rings within these families suggest a potential mode of binding that establishes simultaneous stacking interactions with both H43/44 and L11-NTD (Figure 6D, E). Alterations that disrupt these rings, as seen for PC2 (Figure 6F), could explain a reduced binding and corresponding loss in protection (Figure 4). The PD class of precursors is structurally most similar to the pyridine core of NoS (Figure 1). NoS is oriented differently on the ribosome compared to ThS, establishing stacking interactions with Pro22 but not Pro26 (Figure 6G; Harms et al., 2008). Based on our modeling, PD1 can make analogous interactions with Pro22 as NoS (Figure 6H), whereas the inactive PD2 cannot (Figure 6I).
The thiopeptide GE2270A has been crystallized in complex with EF-Tu, revealing that the drug binds within a covered groove in domain II, and spans across the active site cleft of EF-Tu to interact with the domain I, the G domain (Figure 7A; Parmeggiani et al., 2006). GE2270A overlaps the binding site of the terminal A76 and aminoacyl moiety of the tRNA (Figure 7B) and is believed to prevent the closing of domains I and II necessary for the induced fit binding of aa-tRNA (Figure 7C), thereby preventing ternary complex (EF-Tu• GTP•tRNA) formation (reviewed by Parmeggiani and Nissen, 2006). The structural similarities between PA and PD and GE2270A (Figure 1) suggest that these compounds would also bind within the groove of domain II of EF-Tu (Figure 7D) and overlap with the A76 of the tRNA (Figure 7E). However, the truncated nature of these compounds prevents them from establishing additional interactions with domain I, even in the closed tRNA-bound ternary complex state of EF-Tu (Figure 7F).
Development of improved antimicrobial agents will be necessary to combat the prevalence of multi-drug resistant bacteria. A number of biochemical approaches have been taken to identify functionally important hotspots on the ribosome that do not overlap with previously known antibiotic binding sites (Laios et al., 2004; Llano-Sotelo et al., 2009b; Yassin et al., 2005; Yassin and Mankin, 2007). In addition, a recent study has developed an assay using fluorescently labeled ribosomal proteins to monitor binding of small molecules, such as antibiotics, to the ribosome, which is amenable to high-throughput screening (Llano-Sotelo et al., 2009a). Here we present several translation-related assays utilizing high-throughput 96- or 384-microtiter plate formats that have been used to screen a library of thiopeptide precursor compounds for their abilities to inhibit one or more aspects of translation and/or reverse the inhibition of known thiopeptide antibiotics. These screens identified four distinct families of precursor compounds, termed PA-PD, which could act as potential lead compounds for development of novel antimicrobials.
Two of the families identified, PA and PD, contain a six-membered nitrogen heterocycle core (PA, dehydropiperidine; PD, pyridine) analogous to the thiopeptide antibiotics thiostrepton and GE2270A (Figure 1). The crystal structures of thiopeptides bound to the ribosome (Harms et al., 2008) and of GE2270A bound to EF-Tu (Parmeggiani et al., 2006) reveal the importance of the heterocycle core of these compounds for interaction with their respective targets, and allows modeling of how PA and PD members are likely to interact with the ribosome and/or EF-Tu (Figure 5 and and6).6). The resulting models are consistent with the rescue of translation in the presence of ThS and GE2270T by family members, such as PA2 and PD2, probably by direct competition for binding between the precursor compound and the thiopeptide antibiotic. In addition, PA1 and PA2 displayed inhibitory activity against translational GTPases IF2 (Figure 2B), and EF-G and Tet(M) (Figure 3A,B), respectively. However, compared to the parent thiopeptide compounds, much higher concentrations of the precursor compounds were necessary to exhibit similar effects, most likely indicating the much lower binding affinity of the precursors. The ineffectiveness of precursor compound PA2 as a direct inhibitor was surprising, since this compound has been previously reported to exhibit antimicrobial activity against methicillin-resistance Staphlococcus aureus and vancomycin-resistant Enterococcus faecalis with a minimal inhibitory concentration (MIC) of 5 µM (Nicolaou et al., 2005b). Our results suggest therefore that the inhibitory effect of PA2 in vivo may in fact not be related to translation, but verification of this point will require further investigation.
The other two families identified in our screen, PB and PC, have not, to our knowledge, been previously reported to target the translational machinery. PB1 is chemically similar to the thiazolidine precursor compound used to generate the pyridine core of amythiamicins (Nicolaou et al., 2008a), which target EF-Tu analogously to GE2270A (Parmeggiani et al., 2006; Parmeggiani and Nissen, 2006). The PC series of compounds contain a protected β-hydroxy-α-aminoacid, which is a precursor in the synthesis of GE2270A/T/C1. Curiously, the PB and PC families display much higher specificity for the ribosome than for EF-Tu, as evidenced by the ability of PB1 and PC1 to restore translation more efficiently in the presence of ThS, as compared with GE2270A (Figure 5). Although PB1 and PC1 are structurally distinct (Figure 1), we believe the common aromatic/cyclic nature of both these compounds is important for ribosome binding. Accommodation of EF-G on the ribosome involves the insertion of domain V of EF-G into the crevice between H43/44 and L11-NTD. Inhibition by Class I thiopeptides has been proposed to stem in part from their physically linking L11-NTD to H43/44, thereby locking the cleft shut (Harms et al., 2008). We suggest that PB1 and PC1 can also span the L11-rRNA crevice (Figure 6D, E) and perform this locking function, analogous to ThS/PA2 (Figure 6B, C) and NoS/PD1 (Figure 6G, H). Similarly to PA/D, the high concentrations of PB/C required to inhibit the ribosome-dependent GTPase activity of EF-G are indicative of their low binding affinities for the ribosome. Such low affinity may allow facile displacement of precursors from the ribosome, as a result of translation factors (IF2 or EF-G) binding, or from EF-Tu, during ternary complex formation, thus explaining the absence of any direct inhibitory effect of any of the precursors on GFP synthesis. The differential effects of the precursors on the GTPase assays compared to the TT assay is probably related to the ribosome concentrations in the GTPase assays being ~10× – 100× less (30 × 300 nM) compared to the TT assay (~2 µM) and to the putative higher affinity of EF-G for translating rather than empty ribosomes (Sergiev et al., 2005).
The majority of clinically used antibiotics targeting the ribosome bind either to the decoding region on the small subunit or within either the peptidyltransferase center or the adjacent peptide exit tunnel of the large subunit, where they interact almost exclusively with ribosomal RNA (Spahn and Prescott, 1996; Wilson, 2004). The Class I thiopeptide compounds, however, are distinct in that they target a different region of the ribosome, namely the GTPase-associated region or translation-factor binding site, where they interact with both rRNA and ribosomal protein L11. As a consequence, no cross-resistance has been found between thiopeptide antibiotics and other clinically important drugs. The compounds such as PA-PD identified in our study provide lead structures for the development of novel antimicrobial agents that target this region of the ribosome. Furthermore, the ability of some precursor compounds, such as PA1 and PD1, to bind both EF-Tu and the ribosome suggests the feasibility of developing antimicrobials that are dual inhibitors of ribosomes and ternary complex formation.
The translational machinery represents one of the major targets within the cell for antibiotics, with many clinical important classes of antibiotics inhibiting translation by binding to the ribosome. Despite the potency of many of these drug classes, antibiotic resistance among clinically relevant pathogens is an increasing problem and there is an urgent need for improved antibiotics. We present herein a series of translation machinery assays that can be used to screen for lead compounds that not only inhibit specific steps of translation, but also relieve the inhibitory effects of other inhibitory compounds. Using these assays to screen a library of thiopeptide precursor compounds, we have identified four distinct families of compounds that inhibit either IF2, EF-G and/or Tet(M), as well as confer protective effects against thiopeptide translation inhibitors of both the ribosome and EF-Tu. Our findings not only elucidate the mechanism of action of thiopeptide compounds, but also illustrate the potential of such high-throughput assays to identify distinct lead compounds that might be missed using conventional inhibitory screening protocols. Whereas the IF2 and EF-G GTPase assays are specifically useful for screening antibiotics interfering with translation G-factor proteins, the TT assay is generally applicable for screening all classes of translation inhibitors, including those targeting the peptidyltransferase and decoding centers of the ribosome, and other ribosomal sites, in addition to those interfering with translation G-factor proteins.
GE2270A, T and C1 and the library of thiopeptide precursor compounds were synthesized as described previously (Nicolaou et al., 2005b; Nicolaou et al., 2008a; Nicolaou et al., 2008b; Nicolaou et al., 2006). Thiostrepton was purchased from Sigma, while nosiheptide was the kind gift from Prof. Floss and micrococcin P1 was supplied by Dr. Torsten Stachlhaus. The tetM gene (Tn916) cloned into the pET24b vector was the kind gift from Prof. V. Burdett. Tet(M) protein was expressed in BL21 (DE3) pRIL cells in 20 °C with 0.2 mM IPTG. E. coli EF-G cloned into pQE70 vector was expressed in XL1 blue cells. Both proteins were purified using Ni-NTA metal-affinity chromatography (Qiagen), followed by gel-filtration chromatography on a HiLoad 26/60 Superdex 75 prep grade column (Amersham-Pharmacia). For the experiments described in Figures 2 and 3CD, ribosomes, IF2Cy3, IF1, IF3, 30S subunits, MDCC-labeled phosphate-binding protein (PBP), 022AUG mRNA, and fMet-tRNAfMet, were prepared as described previously (Qin et al., 2009) as was EF-G (Pan et al., 2007).
Reactions were carried out in a 384-well assay plate. 50S subunits were preincubated (15 min at 37 °C) with a series of concentrations of the test compounds in DMSO that are transferred from a premade compound plate to the assay plate by a Perkin-Elmer Evolution P3 liquid handler. Reaction was initiated by addition of 30S initiation complex (30SIC) to each well of the plate. Fluorescence (579 nm) was read with a 2103 EnVision Multilabel Plate Reader on excitation at 550 nm. For the reversal experiment, 30SIC was preincubated with ThS (10 min, 37 °C), followed by a second preincubation with test compounds (10 min, 37 °C), and reaction was initiated by 50S addition.
For both assays described below, reactions performed in the absence of ribosomes were used as a background signal to account for the intrinsic GTPase activity of EF-G or Tet(M).
GTPase activity was measured using the Malachite Green Phosphate Kit (BioAssay) that quantifies the green complex formed between Malachite Green, molybdate and free orthophosphate. All reactions contained 30 nM E. coli 70S ribosomes, 20 µM GTP and 60 nM protein in the presence or absence of antibiotics as necessary. Reactions were transferred into 96-well microtiter plates and color formation was measured on Tecan - Infinite M1000 microplate reader at 650 nm.
GTPase activity was measured using the MDCC-labeled PBP complex, which measures free phosphate release as an increase in fluorescence, and utilizes a Pi-MOP system to minimize the background due to phosphate present in the original medium (Brune et al., 1994; Seo et al., 2006). Reactions were carried out in a 384-well assay plate. Ribosomes were preincubated (15 min at 37 °C) with a series of concentrations of the test compounds in DMSO that are transferred from a premade compound plate to the assay plate by a Perkin-Elmer Evolution P3 liquid handler. Reaction was initiated by addition of an ice-cold solution containing EF-G, PBP-MDCC, and GTP to each well of the plate, a process that was completed in under 30 sec. Fluorescence (450 nm) was read within 1 min using a 2103 EnVision Multilabel Plate Reader, on excitation at 405 nm. For the reversal experiment, ribosomes were preincubated with ThS (10 min, 37 °C), followed by a second preincubation with test compounds (10 min, 37 °C), and reaction was initiated by EF-G, PBP-MDCC, and GTP as above.
All coupled transcription-translation experiments were performed using an E. coli lysate-based system in the presence and absence of antibiotics as described previously (Dinos et al., 2004; Szaflarski et al., 2008). Reactions were transferred into 96-well microtiter plates and the GFP fluorescence was measured with a Typhoon Scanner 9400 (Amersham Bioscience) using a Typhoon blue laser module (Amersham Bioscience). Images were then quantified using ImageQuantTL (GE Healthcare) and represented graphical using SigmaPlot (Systat Software, Inc.).
Chemical structures for the precursor compounds were drawn and converted to 3D coordinates using ChemDraw (Advanced Chemistry Development, Inc. Toronto, Canada). PA2 models utilized the thiostrepton binding position on the Deinococcus radiodurans 50S (D50S) subunit (PDB ID 3CF5) (Harms et al., 2008), whereas PD1 and PD2 were based on the D50S-nosiheptide complex (PDB ID 2ZJP) (Harms et al., 2008). PyMol (http://www.pymol.org) was used to model the PB1 and PC compounds, align EF-Tu ●GE2270A (yellow; PDB ID 2C77; (Parmeggiani et al., 2006) and EF-Tu●tRNA (blue; PDB ID 1TTT; (Nissen et al., 1995) on the basis of domain II, as well as to prepare all X-ray structure figures.
We would like to thank Prof. Roland Beckmann and his coworkers for generous support and creating a stimulating work environment. This work was financed by the Deutsche Forschungsgemeinschaft (WI3285/1-1 to D.N.W.) and by the National Institutes of Health (GM071014 to B.S.C.).
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.