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Jun 10, 2013 - Inhibitors of Bacterial Transcription Initiation Complex Formation ... School of Medical Sciences, Department of Pharmacology, The Univ...
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Inhibitors of Bacterial Transcription Initiation Complex Formation Cong Ma,† Xiao Yang,† Hakan Kandemir,‡ Marcin Mielczarek,‡ Elecia B Johnston,†,∥ Renate Griffith,§ Naresh Kumar,‡ and Peter J. Lewis*,† †

School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW 2308, Australia School of Chemistry and §School of Medical Sciences, Department of Pharmacology, The University of New South Wales, Sydney, NSW 2052, Australia

‡

S Supporting Information *

ABSTRACT: Antibiotic resistance is a growing global problem, with very few new compounds in development. Bacterial transcription is an underutilized target for antibiotics, which has been attributed to the similarity of the active site of RNA polymerases (RNAPs) across all domains of life and the ease with which resistance can arise through point mutation at multiple sites within this conserved region. In this study we have taken a rational approach to design a novel set of compounds that specifically target the formation of transcription initiation complexes by preventing the unique bacterial σ initiation factor from binding to RNAP. We have identified the region of RNAP to which these compounds bind and demonstrate that one compound, GKL003, has an inhibition constant in the low nanomolar range. This compound has activity against both Gram-positive and -negative organisms, including a community acquired methicillin-resistant strain of the major pathogen Staphylococcus aureus.

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transcription may be of limited use. However, the SB series of compounds may not bind within the active site cleft of RNAP and have been shown to prevent the formation of RNAP holoenzyme (HE; RNAP core in complex with a σ initiation factor).7 Despite these encouraging results, the binding site for SB compounds remains unknown. Since the first publication of its structure in 1999,9 many Xray diffraction and electron microscopy-derived structures of bacterial RNAP and drug/nucleic acid/transcription factor complexes have been published, providing a wealth of information for detailed analysis of intermolecular interactions that are essential for transcription. σ factors are unique to bacteria, bind to RNAP core (subunit composition, α2ββ′ω) to form HE, and are required for the correct initiation of transcription at promoter sites (e.g., ref 12). Although there is an enormous range of σ factors distributed across the eubacteria, transcription of :housekeeping: genes is dependent on a highly conserved σ70/σA factor. A high-resolution X-ray crystal structure of RNAP in complex with σA from Thermus thermophilus13 has enabled us to examine the interaction between RNAP and σA in great detail. In previous work, using the model Gram-positive organism Bacillus subtilis, we used this information to identify key amino acid residues in σA required for interaction with

ince the development of penicillin before and during WWII, we have become highly dependent on an arsenal of antimicrobial compounds to treat or prevent infections that traditionally had high levels of morbidity and mortality. Antibiotic discovery reached a peak in the 1950s and 1960s and has been in decline ever since. Between 1962 and 2000 no new classes of antibiotic were released to market, and since then only oxazolidinones, lipopeptides, and mutilins have been identified. Resistance to these compounds is rapidly developing,1−5 and there is less commercial interest in discovering and developing new antibiotics than there ever has been. Most antibiotics target three functions: cell wall/cell surface integrity (e.g., β-lactams, glycopeptides, lipopeptides), translation (e.g., aminoglycosides, tetracyclines), or DNA replication/segregation (e.g., quinolones).6 Despite being essential for viability, transcription has rarely proven to be a fertile area for new antibiotic discovery, and to this day, despite many antitranscription compounds being discovered, only one class, the rifamycins, has been available for clinical use (see ref 7), with fidaxomicin (lipiarmycin) recently approved by the FDA in 2011 for use in Clostridium dif f icile infections.8 The vast majority of antitranscription drugs bind within the active site of RNAP, which is a large cleft wide enough to accommodate double stranded DNA,9 that offers multiple sites where point mutations are able to confer resistance to the antibiotic. At least 12 different mutations in the β and β′ subunit confer resistance to rifamycins, and despite its recent approval, mutations conferring resistance to lipiarmycin were reported as far back as 1977.10,11 Therefore, schemes to identify new inhibitors of © XXXX American Chemical Society

Received: April 5, 2013 Accepted: June 10, 2013

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Figure 1. Interaction between the CH region of β′ and the N-terminal domain of σ. (A) Space-filled model of RNA polymerase holoenzyme adapted from ref 14 with the RNA polymerase core colored in gray and σA in blue. The portion corresponding to the interaction between the CH region of the β′ subunit and the N-terminal domain of σA is boxed. (B) Magnified view of the boxed region from panel A, with the β′-CH region colored gray and σA N-terminal domain blue. (C) Sequence alignments of interacting regions of RNAP and σ. From top to bottom: Bacillus subtilis, Staphylococcus aureus, Streptococcus pneumoniae, Clostridium dif f icile, Acinetobacter baumannii, Escherichia coli, and Mycobacterium tuberculosis. Black dots indicate amino acids modeled as being involved in interactions.14 Arrows below indicate α-helices. (D) Location of amino acids on σA2.2 determined to be involved in the interaction with the β′-CH region.14,15 The N-terminal domain of σA has been rotated 90° into the page from the orientation shown in panel B (arrow). The relative importance of the labeled amino acids for their interaction with the β′-CH region is color-coded from yellow to red for least to most important.

Figure 2. Mutational analysis of amino acids in the β′-CH region involved in interaction with σA. (A) Results of ELISAs to determine the role of selected alanine substitutions on the binding of the β′-CH region with σA. Amino acid substitutions are shown below the respective columns, and binding efficiency is plotted relative to wild-type binding. Standard deviations are shown. (B) Amino acids that were mutated on the β′-CH region color coded according to the binding efficiency of the mutant fragment. Binding efficiency 50%, green. (C, D) 180° rotations of close-up views of the intermolecular interactions between the β′-CH region (gray) and σA2.2 (blue). Amino acids are labeled for clarity. Carbon atoms, gray; hydrogen, white; nitrogen, blue; oxygen, red; sulfur, yellow. Hydrogen bonds are represented with dotted green lines.

RNAP.14,15 Several residues in an N-terminal α-helix in a region called σA2.2 were shown to be required for interaction with

RNAP, with a single amino acid, E166, being particularly important.15 σA2.2 interacts with a portion of the β’ subunit B

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σD162, which is important but not essential for intermolecular interaction (Figure 2C14,15). β′R270 was not predicted to be involved in hydrogen bonding in our homology model but does form part of a hydrophobic pocket along with β′L271 and β′I280, which may be important for hydrophobic interactions with σAM169 (Figure 2C). β′I280 is also predicted to form a hydrogen bond with σAQ165 via its main chain oxygen (Figure 2D). β′M287 and β′N283 (colored yellow in Figure 2B) also have important roles in the interaction with σA as mutation of these residues resulted in σ165/σM169 > σD162/σM172 (Figure 1D).14,15 These residues, along with those in the β′-CH region that were mutated to alanine in this study, are indicated by dots in Figure 1C. Amino acids that were mutated in the β′-CH region were chosen on the basis of their predicted interactions with residues in σA2.2 that have been shown to be required for efficient RNAP HE formation.14,15 To assess the contribution of these amino acids to their interaction with σ, ELISA was performed (see Methods), and results are shown in Figure 2A. These studies used an Nterminal fragment of σA containing region 2.2 (σNTD) along with a soluble fragment of the β′-CH region fused to glutathione-S-transferase (see Supporting Information). We categorized binding activities of mutants relative to wild-type into groups using ≤20% (red), 20−30% (orange), 30−50% (yellow), and >50% (green) and mapped them onto the structure of the β′-CH region (Figure 2B). Mutation of β′R267 had the most dramatic effect on σNTD binding (Figure 2A, colored red in Figure 2B), with β′R264 and β′R270 also very important for the interaction (Figure 2A, orange in 2B). We expected β′R267 to be important as it is predicted to form charge-reinforced hydrogen bonds with σAE166, which has previously been shown to be essential for RNAP HE formation,15 as well as with the main chain carbonyl group of σAD162 (Figure 2C). β′R264 is predicted to hydrogen bond with

Figure 3. Pharmacophore model based on the amino acids of σA responsible for HE formation. (A) Features mapped onto the interface between β′ (gray) and σA (blue). (B) The final pharmacophore model with important σA amino acids overlaid with chemical features. In both panels: green spheres, H-bond acceptors; pink spheres, H-bond donor; cyan spheres, hydrophobic groups; gray spheres, exclusion zones. Hbond features are vector-based so each bond has two spheres. One sphere marks the zone of the donor atom, and the second sphere the acceptor atom, with direction being an important factor.

amino acids between σA (blue) and β′ (gray) and Figure 3B shows how the three σA amino acids form the basis of the pharmacophore model. σAE166 contains a hydrogen bond acceptor (green spheres) on an oxygen atom (red), σAQ165 contains a hydrogen bond donor (pink spheres) on a nitrogen atom (blue) and a small hydrophobe point (cyan sphere) on the side chain, and σAM169 contains a large hydrophobe (cyan sphere) toward the end of the side chain. Exclusion zones (gray) helped frame pockets and areas where a small molecule would not fit. C

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Figure 4. Determination of the binding specificity of compounds that inhibit the interaction between σA and RNA polymerase. (A) Structures of GKL001−3 along with their calculated fits to the pharmacophore model. Following fitting to the pharmacophore model, regions identified as hydrogen bond acceptors were circled in green, and hydrophobes in cyan. (B) Composite image of native polyacrylamide gels. Lanes were loaded from left to right with free σA, RNAP HE, RNAP core + σAD162K, RNAP core + σAE166K, RNAP core + σA + GKL001, RNAP core + σA + GKL002, and RNAP core + σA + GKL003. (C) Results of isothermal titration calorimetry experiments to determine the affinity of the interaction between the β′-CH fragment and the σNTD. Top to bottom, titration of σNTD against GST, and σNTD against β′-CH region. (D) Results, top to bottom, of titration of GKL003 against free GST, σNTD, and β′-CH. (E) GKL003 binding to β′-CHK267. (F) GKL003 binding to β′-CHI280. D

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Figure 5. Determination of GKL003 activity using in vitro transcription assays. In all panels, the band shown on the autoradiograph corresponds to the 220 nt transcription runoff product. (A) Results of titration of σA against RNAP core in the presence and absence of GKL003. Above the lanes, the presence of RNAP core, ratio of σA to core, and concentration of GKL003 (μM) present in reactions is shown. Below the lanes, the % inhibition of transcription relative to the respective core/σA reaction in the absence of GKL003 is shown. (B) Results of the titration of GKL003 as an inhibitor of transcription using a level of σA:core of 0.5:1. x-axis, GKL003 concentration (nM); y-axis, % inhibition. Error bars are shown. The inset shows an expanded view of the titration curve at low levels of GKL003. (C) Results of an experiment to ascertain whether GKL003 is capable of competitively inhibiting RNAP HE formation. The details above the lanes indicate the level and presence of RNAP core and GKL003 and whether σA was added to reactions before (1) or after (2) GKL003. See text for further details. Below the lanes, the level of transcription inhibition relative to RNAP HE in the absence of GKL003 is shown. (D) Results of a transcription assay using E. coli σ70 rather than B. subtilis σA. The details above the lanes indicate the presence/absence of RNAP core and σ70 and the level of GKL003. Below, the level of transcription inhibition relative to RNAPHE in the absence of GKL003 is shown. The image has been cropped so that the negative control lane (RNAP core only) lies adjacent to the positive control lane (RNAP HE, no GKL003).

in RNAP HE formation. Free σA ran near the bottom of the gel, whereas RNAP core and HE ran as smears near the top of the gel (Figure 4B). From previous studies14,15 we predicted that σAD162 was of minor importance in the interaction with β′-CH, whereas σAE166 was expected to be essential. As can be seen, only small amounts of free σA could be detected when forming RNAP HE with the σAD162K mutant, but a substantial amount of free σA is present when using σAE166K (Figure 4B), validating our original studies with σA. These results are consistent with the intermolecular interaction predictions14,15 and the mutagenesis experiments with the β′-CH region outlined above, where β′R264 was predicted to form a single hydrogen bond with σAD162 and β′R267 was predicted to form two hydrogen bonds, one with σAE166 and one with σAD162. Next, GKL001, -002, and -003 were tested to determine whether they were capable of preventing the formation of RNAP HE. Although all of the compounds had similar fit values (∼1.6−1.8) and all displayed the ability to prevent the interaction between σA and RNAP, GKL001 and -003 inhibited RNAP HE formation much more efficiently than GKL002 (Figure 4B). Although all of the three compounds furnish two carbonyl groups into the hydrogen acceptor binding site (green spheres, Figure 4A), and at least one indole ring provides a

Identification and Synthesis of Potential Small Molecule Inhibitors. We used our pharmacophore model to screen an in-house library of peptidomimetic compounds based on linked indole rings (see Methods and Supporting Information for details). The top three hits, named GKL001− 003, were synthesized for further investigation and are shown in Figure 4A, along with their fit scores, and predicted fit to the pharmacophore features. In GKL001 and -003, the two indoles are bonded via a 7,7′-glyoxylamide and 7,7′-amide linker, respectively, compared to GKL002, which has a 2,2′-amide linker. Although the compounds all contained hydrogen bond acceptor and hydrophobic features (green and cyan circles, respectively, Figure 4A), they did not contain a hydrogen bond donor in the correct place to fit the original pharmacophore model (pink sphere, Figure 3B). Peptidomimetic Compounds Can Inhibit Initiation Complex Formation. Having synthesized new compounds designed to target the interaction between the β′-CH region and σA2.2, native gel electrophoresis was used to determine whether they were capable of inhibiting RNAP HE formation in vitro. σA mutants D162K and E166K were also used to assess whether amino acids determined to be important for interaction with β′-CH in far-Western blots14,15 were important E

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(Figure 5A). At both σA levels GKL003 caused a significant reduction in transcript levels, even at a concentration of 0.5 μM (96% and 22%, respectively, lanes 4 and 7, Figure 5A). However, the effect of GKL003 on transcription was most apparent when using subsaturating levels of σA (98% and 96%, respectively, lanes 3 and 4, Figure 5A). Next, we determined the inhibition constant (Ki) of GKL003 through titration of increasing amounts of compound against RNAP core in transcription assays using a σA:RNAP core ratio of 0.5:1 (Figure 5B). The transcriptional activity was reduced dramatically even at very low levels of GKL003 and the Ki was determined to be 5.79 ± 0.09 nM. In order to obtain further information on the mechanism of transcription inhibition by GKL003, competition assays were performed to determine whether it was capable of competitively inhibiting transcription. Assays were set up where GKL003 was added to RNAP core before the addition of σA and where it was added after σA had equilibrated with RNAP core. When either 10 or 500 nM of GKL003 was added to assay mixtures before σA ,transcription was reduced 71% and 91%, respectively (lanes 3 and 4, Figure 5C). When similar levels of GKL003 were added following equilibration of σA with RNAP core, transcription was reduced 62% and 91%, respectively (lanes 5 and 6, Figure 5C). These results show that GKL003 is capable of competitively inhibiting the interaction between RNAP core and σA in vitro. The above transcription assays were performed with E. coli RNAP core and B. subtilis σA. Although it is well established that σ-factors are capable of binding to a noncognate RNAP core and correctly initiating transcription, and the regions of RNAP and σ targeted in this study are almost identical (Figure 1C), we repeated experiments using E. coli RNAP core and E. coli σ70 (Figure 5D). On addition of GKL003 to 10 nM and 500 nM, transcription was inhibited 65% and 88%, respectively (lanes 3 and 4, Figure 5D), which is very similar to the levels observed using B. subtilis σA (lanes 3 and 4, Figure 5C). This data shows that our rationally designed compounds are capable of inhibition of transcription, through the targeted antagonism of transcription initiation complex formation at low nanomolar concentrations, which represents a remarkable achievement for a first round screen of new molecules. GKL003 Inhibits Bacterial Growth in Culture. Having established target specificity and the ability of GKL003 to inhibit transcription in vitro, we wished to determine its efficacy on live cells. Since our initial molecules are relatively large (MW 660−804, GKL001−GKL003) and hydrophobic, it was possible that they would not be able to effectively cross membranes and cell walls to have their desired activity on inhibition of transcription within the cytoplasm. E. coli DH5α was used to test the efficacy of GKL003 against Gram-negative cells, and S. aureus USA300, which is a community acquired methicillin-resistant strain (CA-MRSA 20), was used as the Gram-positive organism. Gram-negative cells have an outer membrane that represents a significant physical barrier for the penetration of hydrophobic molecules and is considered one of the major factors behind the lack of efficacy of many antimicrobials against these organisms.21 Therefore, these assays would allow us to determine whether GKL003 had antimicrobial activity and whether it was active against Gramnegative, Gram-positive, or both types of cells. GKL003 was added to exponentially growing starter cultures at a range of concentrations from 125 μM to 2 mM, and cell growth was monitored (Figure 6). GKL003 was able to inhibit growth of

hydrophobic interaction site (bigger cyan sphere, Figure 4A), the lack of activity of GKL002 suggests the 7,7′ linkage to the indole rings in GKL001 and -002 may be important for activity. Therefore, the compounds we had specifically identified as being likely to inhibit RNAP HE formation by targeting the interaction between the σA2.2 and β′-CH regions were able to do this in vitro. Since the effect of GKL001 and -003 in preventing formation of RNAP HE was similar, the remainder of the data presented in this work are based on investigations using GKL003. GKL003 Binds to the β′-CH Region. In order to ascertain whether GKL003 was binding to σNTD or β′-CH, isothermal titration calorimetry experiments were performed. Initially we determined the strength of the interaction between σNTD and β′-CH (Figure 4C). Data were fit to a one -site binding model giving 0.943 ± 0.013 sites, consistent with the known 1:1 interaction of RNAP with σA. A Kd of 1.16 ± 0.17 μM was obtained. Previous studies using whole RNAP core and σ factors and a wide array of techniques have provided Kd’s of between 0.3 and 190 nM, 17 suggesting that interactions between regions 3 and 4 of σ factors and other regions of RNAP contribute to the stability of the interaction despite not being able to compensate for the loss of the σA2.2 interaction with β′-CH (see Figure 4B). The negative enthalpy of −1.8 kcal/mol obtained is consistent with charged residues (e.g., σAE166, β′R264,267) playing a significant role in the formation of intermolecular bonds. Since the β′-CH fragment used in these studies was fused to GST (see Supporting Information Table 1), a control expriment, showing no intermolecular interaction, was performed where σNTD was titrated against pure GST (top trace, Figure 4C). Next, experiments were performed where GKL003 was added to σNTD, β′-CH, or GST (Figure 4D). Since GKL003 was initially dissolved in DMSO prior to dilution in buffer, DMSO was added to all solutions and samples at a final concentration of 1% (v/v) to minimize heat of reaction effects due to solvent mismatch. The data in Figure 4D clearly shows that GKL003 binding is specific for β′-CH, even though heats of reaction remain high at saturation. No binding to σNTD or GST was observed (top 2 thermograms, Figure 4D). In order to obtain higher quality titrations, unsuccessful attempts were made to increase the concentration of β′-CH or change the temperature of the reaction vessel (not shown). The high heats of reaction at saturation are probably related to the heat of intermolecular interactions between dimeric chlorophenyl indoles with β′-CH amino acids, as titration between GKL003 and buffer, σNTD, or free GST did not show any significant heat change. Next we tested whether GKL003 was able to bind to mutant forms of β′-CH in order to confirm that GKL003 is specifically targeting the σA-binding interface. Results for β′R267 and β′I280 are shown in Figure 4E and F, respectively, and clearly show there is little or no interaction of GKL003 with either mutant form. Therefore, the compounds we designed are specifically binding to the same region of β′-CH that is required for interaction with σA2.2. GKL003 Is Capable of Competitive Inhibition of Transcription. To determine the mechanism of action of GKL003, we performed transcription assays using B. subtilis σA with E. coli RNAP core (see Methods). Since the level of σfactors within the cell is known to be less than that of RNAP core,18,19 we initially performed transcription assays with both saturating and subsaturating levels of σA relative to RNAP core F

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synthesis and testing of only nine initial compounds, for which we have presented data on three. We further characterized compound GKL003, to establish which component (σ or RNAP) of the transcription complex was the target for binding and determined the mechanism of inhibition. Remarkably for an initial hit, GKL003 had a Ki of ∼6 nM in in vitro transcription assays, demonstrating that our rational approach is capable of generating information that allows the synthesis of highly effective inhibitory compounds without the need for costly and time-consuming high-throughput screening. Antibiotics that target transcription nearly always target the active site.7 While these compounds can be highly effective, point mutations that confer resistance to the drug rapidly arise, and the active site is highly conserved across all domains of life. We have targeted the interaction between RNAP and the σ factor essential for correct initiation of transcription. This is a nontraditional target as it does not involve any clefts into which a small molecule can tightly bind. Nevertheless, previous investigations have indicated transcription initiation is a valid target for future antibiotic development.7,22−25 Although the binding site of inhibitor activity was not established in all of those studies, antisense peptide nucleic acids have been used to target σ production,24 and of particular relevance to this work, small peptides have been identified that bind to the β′-CH region preventing initiation complex formation.25 While peptides show promise for development as therapeutic compounds, they are relatively unstable, expensive to mass produce, and unsuitable for oral administration. We have shown that compounds can be specifically designed to target a protein interaction surface and effectively inhibit the essential protein−protein interaction at low nanomolar concentrations in vitro. GKL003 was shown to have the ability to inhibit bacterial growth and transcription in culture, but this was at substantially higher concentrations (≥1 mM) than were required for inhibition in vitro, suggesting that cell permeability may be poor for this compound. Nevertheless, GKL003 was broad spectrum in activity, being able to inhibit growth of Grampositive and -negative organisms, including the CA-MRSA strain S. aureus USA300, indicating that there is the potential to refine our compounds to target transcription initiation for antibiotic development, including in bacterial strains carrying multiple resistance determinants to existing compounds.

both Gram-negative (Figure 6A) and Gram-positive bacteria (Figure 6B), with significant inhibition of growth observed at levels of ≥1 mM.

Figure 6. Determination of the activity of GKL003 against bacteria growing in culture. (A) Results of a growth inhibition assay of increasing concentrations of GKL003 against E. coli and (B) results against S. aureus USA300. Black line, positive control (growth in media); dotted black line, DMSO control; light gray line, 0.125 mM; midgray line, 0.5 mM; dark gray line, 2 mM GKL003. All curves are the average of triplicates. (C) Rate of incorporation of 3H-uridine into exponentially growing S. aureus USA300. Solid black line, positive control (no inhibitors added); dark gray line, 0.5 mM GKL003; light gray line, 2 mM GKL003; black dashed line 0.0625 μg/mL rifampicin.

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METHODS

Plasmid Vector Construction, Protein Overproduction and Purification. All fragments of B. subtilis RNAP and σA were cloned, overproduced, and purified as detailed in Supporting Information. Native Polyacrylamide Gel Electrophoresis. Native 6% polyacrylamide gels containing 0.25x Tris-borate EDTA buffer (TBE) and 5% (v/v) glycerol were run in 0.5x TBE on ice at 100 V for 100 min. Reactions were carried out in 10 mM Tris-HCl, 150 mM NaCl, 10 mM MgCl2, 5% (v/v) glycerol, 0.1 mM DTT, pH 7.5. For holoenzyme formation, 1 μL of 18 μM RNAP core was mixed in a 1:1 ratio with 18 μM σA or σA mutant at 37 °C for 10 min. For compound screening, 1 μL of 0.5 μM compound was preincubated with σA at 37 °C for 15 min prior to the addition of RNAP core, followed by incubation at 37 °C for 15 min. Two microliters of 0.025% (w/v) bromophenol blue in 50% glycerol was added to aid sample loading. ELISA-Based Assays. σA was diluted to 250 nM in phosphate buffered saline (PBS), and 100 μL of the solution added into NUNC Maxisorp microtiter plate wells. Following overnight incubation at 4 °C the wells were washed 3 times with 300 μL of PBS and blocked with 300 μL of 1% (w/v) BSA in PBS at RT for 2 h. After blocking, plates were washed three times with wash buffer (PBS, 0.05% (v/v) Tween-20). Then 200 nM wild-type or mutant GST tagged β′ CH in

In order to confirm that the activity of GKL003 on live cells was consistent with the observed in vitro activity, we monitored the incorporation of 3H-uridine into exponentially growing cultures of S. aureus USA300 at different concentrations. As a positive control, cells were also treated with an inhibitory level of the transcription inhibitor rifampicin (dashed lines, Figure 6C). The dose-dependent effect of GKL003 on 3H-uridine incorporation (Figure 6C) closely followed the reduction of growth rate (Figure 6B), suggesting that it is able to inhibit transcription activity in live cells. In this study we describe the rational design, synthesis, and efficacy of a new class of compounds that specifically target the formation of transcription initiation complexes. Identification of compounds with significant and specific activity required the G

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100 μL PBS was added to the wells followed by incubation for 1 h. Wells were washed 3 times in 300 μL of PBS/Tween-20 wash buffer before the addition of 100 μL of rabbit anti-GST primary antibody (1:2000 in PBS) for 1 h. After washing, HRP-conjugated goat antirabbit secondary antibody (1:2000 in PBS) was added to each well and incubated for 1 h. Interactions were detected by the addition of 100 μL of TMB substrate system (3,3′,5,5′-tetramethylbenzidine liquid substrate system for ELISA, Sigma-Aldrich) to each well. The A600 was measured following incubation with shaking at 600 rpm for 6 min in a FLUOstar Optima plate reader (BMG Labtech). Isothermal Calorimetric Titration. Protein solutions (40 μM) in 50 mM KH2PO4, 150 mM NaCl, pH 7.4 were loaded into the reaction cell of an auto-ITC200 isothermal titration calorimeter (GE Healthcare). This was titrated with a 400 μM ligand solution in identical buffer and with the same concentration of DMSO (1% v/v), where appropriate. Experiments were carried out at 25 °C, with 19 injections at 150 s intervals, and a stirring speed of 1000 rpm. Titrations were repeated twice. Transcription Assays. Template DNA comprised a 360 bp fragment encompassing the Pxyl promoter region amplified from pSG115426 using primers 5′-CAAAGCCTGTCGGAATTGG-3′ (forward) and 5′-CCCATTAACATCACCATC-3′ (reverse) and was purified in DEPC treated water. In vitro transcription runoff assays were performed as described previously.27 In noncompetitive inhibition assays, 1 μL of 1 μM core enzyme (Epicenter) was mixed with GKL003 and incubated on ice for 10 min. One microliter of 0.5 μM or 1.0 μM purified σA or σ70 was added into the reaction, followed by 5 μL (0.4 μg) of purified template DNA. The reaction was then made up to 50 μL containing 40 mM Tris-HCl (pH 7.9), 160 mM KCl, 10 mM MgCl2, 1 mM DTT, 5% glycerol, 10 mM each ATP, GTP, and CTP, 2.5 mM UTP, and 10 μCi of α-32P UTP (3000 Ci/ mmol). The reaction was allowed to proceed at 37 °C for 5 min. In competitive inhibition assays RNAP core and σA were premixed and equilibrated on ice for 10 min prior to the addition of GKL003. All other steps were the same. Ten microliters of the reaction mixture was transferred into 5 μL of RNA gel loading buffer (95% formamide, 0.05% bromophenol blue and 0.05% xylene cyanol). The samples were heated at 95 °C for 3 min and run on 6% denaturing polyacrylamide gels with a RNA molecular weight standard (Perfect RNA Marker Template Mix 0.1−1 kb, Novagen). The gel was imaged using a phosphorimager (BioRad) after drying at 60 °C for 1.5 h, and relative band intensity was determined using ImageJ software (Version 1.46; NIH). Pharmacophore Construction and Compound Screening. Interaction characteristics were mapped directly onto σA residues using the “add query feature” in Discovery Studio (DS; Accelrys Software Inc.), version 2.5. Features added included hydrogen bond acceptors, hydrogen bond donors, and hydrophobes. Each feature was defined by a central point, as well as by the radius of a location constraint sphere. Hydrogen bond acceptors and donors were further defined by addition of a second sphere representing the interacting group on β′. Exclusion volumes were added to label regions of space not available to σA. Exclusion volumes were defined by a central point, as well as a spherical location constraint. Conformations were generated for each compound using DS, versions 3.0 or 3.5. Default parameter settings were applied, with the following exceptions: Conformation Method BEST, Maximum Conformations 255, Energy Threshold 20.0. Generated conformations were mapped onto pharmacophores using DS 3.0 or 3.5. Default parameter settings were applied, with the following exceptions: Conformation Generation NONE, Best Mapping Only TRUE, Maximum Omitted Features 1, Fitting Method RIGID. Inhibitor Synthesis. Compounds were synthesized as outlined in the Supporting Information. In Vivo Activity Test. Compounds (50 mM in DMSO) were serially diluted in 100 μL of LB medium in the range 0.125−2 mM into individual wells in a 96-well plate. E. coli DH5α or S. aureus USA300 cells were grown at 37 °C in 5 mL of LB with shaking to OD600 0.6−0.7, and 5 μL of the culture was added to each well. The

plate was incubated in a FLUOstar Optima plate reader (BMG Labtech) at 37 °C shaking at 600 rpm. The OD600 was taken every 10 min over a 16 h period using LB as the blank. Samples were tested in triplicate and the growth of each sample was compared to cells exposed to equal amounts of DMSO. In Vivo Inhibition of Transcription. 3H-uridine uptake assays were performed as detailed in ref 28, except that S. aureus USA300 growing exponentially in LB medium were used.

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ASSOCIATED CONTENT

S Supporting Information *

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]. Present Address

∥ School of Pharmacy and Molecular Sciences, James Cook University, Townsville, QLD 4811, Australia.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by NHMRC grant APP1008014 to P.J.L., R.G., and N.K. We thank E. Harry (UTS) for the kind gift of Staphylococcus aureus USA300.

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REFERENCES

(1) Cui, L., Tominaga, E., Neoh, H. M., and Hiramatsu, K. (2006) Correlation between reduced daptomycin susceptibility and vancomycin resistance in vancomycin-intermediate Staphylococcus aureus. Antimicrob. Agents Chemother. 50, 1079−1082. (2) Migliori, G. B., De Iaco, G., Besozzi, G., Centis, R., and Cirillo, D. M. (2007) First tuberculosis cases in Italy resistant to all tested drugs. Eurosurveillance 12, E070517−070511. (3) Witte, W., and Cuny, C. (2011) Emergence and spread of cfrmediated multiresistance in staphylococci: an interdisciplinary challenge. Future Microbiol. 6, 925−931. (4) Ward, P. B., Johnson, P. D., Grabsch, E. A., Mayall, B. C., and Grayson, M. L. (2001) Treatment failure due to methicillin-resistant Staphylococcus aureus (MRSA) with reduced susceptibility to vancomycin. Med. J. Aust. 175, 480−483. (5) Valencia, R., Arroyo, L. A., Conde, M., Aldana, J. M., Torres, M. J., Fernandez-Cuenca, F., Garnacho-Montero, J., Cisneros, J. M., Ortiz, C., Pachon, J., and Aznar, J. (2009) Nosocomial outbreak of infection with pan-drug-resistant Acinetobacter baumannii in a tertiary care university hospital. Infect. Control Hosp. Epidemiol. 30, 257−263. (6) Walsh, C. (2003) Where will new antibiotics come from? Nat. Rev. Microbiol. 1, 65−70. (7) Villain-Guillot, P., Bastide, L., Gualtieri, M., and Leonetti, J. P. (2007) Progress in targeting bacterial transcription. Drug Discovery Today 12, 200−208. (8) FDA. (2011). FDA approves treatment for Clostridiu, dif f icile infections. http://www.fda.gov/NewsEvents/Newsroom/ PressAnnouncements/2011/ucm257024.htm. (9) Zhang, G., Campbell, E. A., Minakhin, L., Richter, C., Severinov, K., and Darst, S. A. (1999) Crystal structure of Thermus aquaticus core RNA polymerase at 3.3 A resolution. Cell 98, 811−824. (10) Sonenshein, A. L., Alexander, H. B., Rothstein, D. M., and Fisher, S. H. (1977) Lipiarmycin-resistant ribonucleic acid polymerase mutants of Bacillus subtilis. J. Bacteriol. 132, 73−79. (11) Tupin, A., Gualtieri, M., Roquet-Baneres, F., Morichaud, Z., Brodolin, K., and Leonetti, J. P. (2010) Resistance to rifampicin: at the crossroads between ecological, genomic and medical concerns. Int. J. Antimicrob. Agents 35, 519−523.

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ACS Chemical Biology

Articles

(12) Murakami, K. S., and Darst, S. A. (2003) Bacterial RNA polymerases: the wholo story. Curr. Opin. Struct. Biol. 13, 31−39. (13) Vassylyev, D. G., Sekine, S., Laptenko, O., Lee, J., Vassylyeva, M. N., Borukhov, S., and Yokoyama, S. (2002) Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6 A resolution. Nature 417, 712−719. (14) Johnston, E. B., Lewis, P. J., and Griffith, R. (2009) The interaction of Bacillus subtilis sigmaA with RNA polymerase. Protein Sci. 18, 2287−2297. (15) Johnston, E. B. (2010) The RNA Polymerase-sigma interaction as a target for potential novel antimicrobials, in School of Environmental and Life Sciences, University of Newcastle, Newcastle, Australia. (16) Arthur, T. M., Anthony, L. C., and Burgess, R. R. (2000) Mutational analysis of beta ‘260−309, a sigma 70 binding site located on Escherichia coli core RNA polymerase. J. Biol. Chem. 275, 23113− 23119. (17) Ferguson, A. L., Hughes, A. D., Tufail, U., Baumann, C. G., Scott, D. J., and Hoggett, J. G. (2000) Interaction of sigma 70 with Escherichia coli RNA polymerase core enzyme studied by surface plasmon resonance. FEBS Lett. 481, 281−284. (18) Maeda, H., Fujita, N., and Ishihama, A. (2000) Competition among seven Escherichia coli sigma subunits: relative binding affinities to the core RNA polymerase. Nucleic Acids Res. 28, 3497−3503. (19) Delumeau, O., Lecointe, F., Muntel, J., Guillot, A., Guedon, E., Monnet, V., Hecker, M., Becher, D., Polard, P., and Noirot, P. (2011) The dynamic protein partnership of RNA polymerase in Bacillus subtilis. Proteomics 11, 2992−3001. (20) Maree, C. L., Daum, R. S., Boyle-Vavra, S., Matayoshi, K., and Miller, L. G. (2007) Community-associated methicillin-resistant Staphylococcus aureus isolates causing healthcare-associated infections. Emerging Infect. Dis. 13, 236−242. (21) Nikaido, H. (2003) Molecular basis of bacterial outer membrane permeability revisited. Microbiol. Mol. Biol. Rev. 67, 593−656. (22) Andre, E., Bastide, L., Villain-Guillot, P., Latouche, J., Rouby, J., and Leonetti, J. P. (2004) A multiwell assay to isolate compounds inhibiting the assembly of the prokaryotic RNA polymerase. Assay Drug Dev. Technol. 2, 629−635. (23) Andre, E., Bastide, L., Michaux-Charachon, S., Gouby, A., Villain-Guillot, P., Latouche, J., Bouchet, A., Gualtieri, M., and Leonetti, J. P. (2006) Novel synthetic molecules targeting the bacterial RNA polymerase assembly. J. Antimicrob. Chemother. 57, 245−251. (24) Bai, H., Sang, G., You, Y., Xue, X., Zhou, Y., Hou, Z., Meng, J., and Luo, X. (2012) Targeting RNA polymerase primary sigma70 as a therapeutic strategy against methicillin-resistant Staphylococcus aureus by antisense peptide nucleic acid. PLoS One 7, e29886. (25) Husecken, K., Negri, M., Fruth, M., Boettcher, S., Hartmann, R. W., and Haupenthal, J. (2013) Peptide-based investigation of the Escherichia coli RNA polymerase sigma(70):core interface as target site. ACS Chem. Biol. 8, 758−766. (26) Lewis, P. J., and Marston, A. L. (1999) GFP vectors for controlled expression and dual labelling of protein fusions in Bacillus subtilis. Gene 227, 101−110. (27) Yang, X., and Lewis, P. J. (2008) Overproduction and purification of recombinant Bacillus subtilis RNA polymerase. Protein Expression Purif. 59, 86−93. (28) Lewis, P. J., Thaker, S. D., and Errington, J. (2000) Compartmentalization of transcription and translation in Bacillus subtilis. EMBO J. 19, 710−718.

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