Structure Guided Discovery and Optimization of the Pyrimidoindole Inhibitor Scaffold

Crystallographic fragment screening was employed to identify starting scaffolds. Similar to the “needle” screen conducted by scientists at Roche [14], low molecular weight fragments with adjacent hydrogen-bond donor/acceptor moieties that engage the ATP adenine-binding aspartate and structural water in the active-site pocket were selected for screening [15]. Based on an analysis of the binding modes of fragment hits on Enterococcus faecalis GyrB, a pyrrolopyrimidine scaffold was deemed an appealing candidate for optimization because it projected synthetic vectors towards all the highly conserved sub-pockets of the GyrB and ParE active-sites including a site for the introduction of charged functionality [15] (Figure 1).

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Figure 1. Optimization of inhibitor scaffolds.

For the fragment hit and inhibitor candidates C1, C2, C3 and C4, identical cutaway views of solvent accessible surface representations of the active-site pockets of E. faecalis GyrB from the crystal structures of complexes of the inhibitors with the 24 kDa N-terminal fragment of GyrB from E. faecalis GyrB are shown. The bound inhibitors are drawn with green bonds, the conserved ATP-binding aspartate is drawn with blue bonds and the structural water molecule that plays a key role in substrate binding in GyrB and ParE is shown as a red sphere. Potential hydrogen-bonds between the inhibitors, aspartate and water molecule are depicted as dotted lines. Optimization of the pyrrolopyrimidine scaffold led to inhibitors like C1 with good enzyme potency but only moderate Gram-negative antibacterial activity [16]. Expansion of the bicyclic pyrrolopyrimidine scaffold to a tricyclic pyrimidoindole scaffold (C2) fills an interior lipophilic pocket and offers superior optimization vectors to improve enzyme potency. Subsequent elaboration of the tricyclic scaffold with a fluorine atom at R₆ and an aminomethyl moiety at R₈ dramatically improved inhibitor potency and ligand efficiency. The 6-fluoro-N-methyl-9H-pyrimido[4,5-b]indol-8-amine scaffold quantitatively fills the lipophilic interior sub-pockets of the GyrB/ParE active-sites and adds a new hydrogen-bond. C3 and C4 demonstrate sub-nanomolar enzyme potency versus GyrB and ParE enzymes from a broad range of Gram-positive and Gram-negative pathogens; inhibition constants (K_i values) are shown for a representative enzyme panel that includes the full length GyrB and ParE enzymes from E. faecalis, Francisella tularensis, and E. coli.

https://doi.org/10.1371/journal.pone.0084409.g001

Initial SBDD efforts were successful in converting the fragment hit into a pyrrolopyrimidine inhibitor series (e.g. C1) that had both the desired broad enzymatic spectrum and dual-targeting enzymatic profile by expanding the scaffold into the lipophilic interior of the GyrB and ParE active-sites [15,16] (Figure 1). However, activity against Gram-negative pathogens was limited and we ultimately exhausted avenues for improving potency at the enzyme level without elevating compound molecular weight and lipophilicity to a degree that further compromised Gram-negative antibacterial activity and spectrum. Consequently, we examined multiple alternative inhibitor scaffolds that could exploit the structure-activity relationships (SAR) from this initial series while providing greater potency and antibacterial spectrum. Dramatic improvements in inhibitor potency, properties and antibacterial activity were achieved by switching to a pyrimidoindole scaffold (C2, Figure 1). The phenyl (A) ring of the pyrimidoindole scaffold filled the interior pocket and presented superior optimization vectors that allowed for the design of inhibitors that structurally and electronically complement the active-site. An aminomethyl substituent was added to engage the conserved adenine-binding Asp residue with an additional hydrogen-bond while occupying a recessed lipophilic pocket. Shape and charge complementarity to the conserved regions of both GyrB and ParE was further improved by adding a fluorine substituent at the R₆ position. Analysis of representative GyrB and ParE structures from multiple bacterial species revealed subtle differences in the shapes and volumes of the active-site pockets in the vicinity of the R₆ substituent. A fluorine substituent at R₆ was accommodated well in all enzymes, and was optimal for broad enzymatic spectrum and dual-targeting (Figure S1). The binding mode of the pyrimidoindole scaffold oriented the R₂ and R₄ vectors on the (C) ring in directions that were exploited to incorporate substituents that interact with two highly conserved binding pockets at the receptor solvent interface in GyrB and ParE (Figure 2). As a result, we were able to create a highly potent, ligand efficient tricyclic inhibitor series (e.g. C3 and C4) with broad enzymatic spectrum. Since GyrB and ParE are structurally unique and belong to a class of ATP-binding proteins called the GHKL superfamily of ATPases/kinases with an unconventional fold [18], the potent TriBE lead compounds are highly selective for their bacterial targets when compared to eukaryotic ATP-binding proteins like protein kinases and human topoisomerase II (Figure 3). This suggests that TriBE inhibitors will not suffer from those potential sources of off-target toxicity in the host.

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Figure 2. Summary of inhibitor optimization strategies.

A) Side view of the “salt-bridge” pocket from the crystal structure of the complex of E. coli GyrB with C3, with key interactions highlighted. C3 is drawn with green bonds. Potential hydrogen-bonds are depicted as dotted lines. The residues comprising the salt-bridge pocket are drawn with blue bonds and a semi-transparent surface representation of the pocket is shown. The salt-bridge pocket residues curl around the R₂ pyrimidine, forming a U-shaped pocket. R₂ substituents were designed to address the complex structural and electronic features of the salt-bridge pocket. Extensive ab initio and binding free energy calculations of the R₂ methyl pyrimidine of C3 show significant binding energy from a π–cation interaction with the salt-bridge Arg. The methyl pyrimidine also engages the Arg on the outer rim of the salt-bridge pocket through a water-mediated hydrogen-bond. Van der Waals interactions are observed between a conserved proline that defines the face of the salt-bridge pocket opposite the Glu-Arg salt-bridge pair.

B) Alternate view of the E. coli GyrB complex structure with C3, highlighting key polar interactions between the R₄ diamine of C3, active-site residues and an ordered solvent network. The Asn residue shown in the figure (N46 from the E. coli structure) and salt-bridge residues are conserved in all bacterial topoisomerases, while the residues comprising the “pocket floor” (blue for the residues in E. coli GyrB, tan for the residues from the overlaid F. tularensis ParE/C3 complex structure differ between GyrB and ParE enzymes. The R₄ diamine sits at the protein-solvent interface at the outer rim of the lipophilic interior pocket that binds the (A) ring of the inhibitor. The upper face of the inhibitor occupies a highly conserved polar pocket while the lower face occludes a lipophilic shelf (the pocket floor) that is structurally heterogeneous between GyrB and ParE due to sequence differences in the enzymes, as highlighted. The R₄ diamine adopts a low energy conformation that does not impinge on the structurally diverse pocket floor and directs a basic amine out of the pyrimidoindole plane to interact via hydrogen-bonds with the conserved Asn at the mouth of the interior pocket (N46). The basic amine complements the negative electrostatic potential in this region of the active-site; the same anionic pocket captures the terminal amine from a conserved lysine residue involved in phosphate binding in the dimeric complex of E. coli ParE with ADPNP [17]. The R₄ diamine also hydrogen-bonds with an ordered solvent network above the pocket floor. The water molecules from this network shown in the figure were observed in similar positions in GyrB and ParE crystal structures from multiple orthologs (as shown in C), and molecular dynamics simulations showed this water network remained throughout all simulations, and each water molecule had significantly long residence times. Thus, these water molecules were treated as conserved structural elements during inhibitor optimization.

C) Electron density maps showing the positions of C3 and the water network that was conserved across all GyrB and ParE enzymes that were structurally characterized in this study. Final 2|fo-fc| electron density maps contoured at 1.3σ for: i) the 1.6 Å E. coli GyrB complex with C3, ii) the 1.3 Å E. faecalis GyrB complex with C3, and iii) the 2.4 Å F. tularensis ParE complex with C3. The water network that interacts with the R₄ diamine of the inhibitor is conserved in the three protein orthologs. R₄ groups were designed to position an amine that simultaneously hydrogen-bonds with the water network and a conserved Asn residue in the active-site pocket.

https://doi.org/10.1371/journal.pone.0084409.g002

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Figure 3. Selectivity of TriBE inhibitors versus eukaryotic ATP-binding proteins.

Inhibitory activities of C3 and C4 against a panel of divergent human kinases, and human topoisomerase II. All compounds were assayed at 10 μM concentration. Level of inhibition is color-coded as indicated in the inset.

https://doi.org/10.1371/journal.pone.0084409.g003

Optimization of Antibacterial Activity and Spectrum

Parallel optimization of enzyme inhibitor potency and antibacterial activity revealed that modulation of the charge state of the pyrimidoindole inhibitors strongly influenced antimicrobial activity, particularly against Gram-negative organisms. Diamine substituents at R₄ like the (3aR,6aR)-octahydropyrrolo[3,4-b]pyrrole used in C3 were observed to significantly enhance Gram-negative antibacterial activity and spectrum by improving both enzyme inhibitor potency and imparting physicochemical characteristics to inhibitors that addressed the unique characteristics of the Gram-negative cell envelope. Drug efflux pumps and the orthogonal sieving properties of the inner and outer membranes of the Gram-negative cell envelope pose significant barriers to the development of antibacterial agents that need access to cytoplasmic targets; the outer membrane is selectively permeable to small charged or polar molecules via porins, while the inner membrane lipid bilayer is essentially impermeable to charged ions, but contains efflux pumps that actively transport lipophilic molecules out of the cell [19,20]. Fluoroquinolones and tetracyclines, which are active against Gram-negative bacteria, equilibrate between neutral (minor) and charged (predominant) states at physiological pH [20]. The charged species traverse the outer membrane and are poor efflux pump substrates, while the neutral species are able to diffuse across the inner membrane lipid bilayer [20]. The ionizable R₄ diamines used in our pyrimidoindole inhibitors are similar to the diamine substituents used in many fluoroquinolone antibiotics [21]. The relative distributions of charged/neutral species in C3 and C4, calculated using the pKa calculation algorithm [22] in the Advanced Chemistry Development, Inc. software package, are estimated to be in the 95:5 to 90:10 range at pH 7.4. These ratios of charged to neutral species at physiological pH are similar to those calculated for norfloxacin, amifloxacin and perfloxacin [20], and are likely responsible for enabling efficient penetration of the Gram-negative cell envelope.

The in vitro antibacterial activities of C3 and C4 have been evaluated against a diverse array of clinical pathogens, including those with important resistance phenotypes. A summary of the results using representative pathogens is shown in Tables 1 and 2. As expected, cross-resistance with existing antibiotic drug classes was not observed with any resistance phenotype tested. To confirm that the pyrimidoindole lead compounds kill bacterial cells via inhibition of GyrB and ParE, macromolecular synthesis inhibition assays were conducted (Figure 4), and demonstrated that C3 and C4 specifically inhibit DNA (primary) and RNA (secondary) synthesis. This pattern of macromolecular synthesis inhibition has been observed for other validated GyrB/ParE inhibitors including novobiocin [16,23].

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Figure 4. The effects for C3 (A) and C4 (B) on macromolecular synthesis in E. coli (BAS849) imp.

Incorporation of [³H]-precursors of DNA (●), RNA (○), protein (▲) and cell wall (▽) was examined. The MIC value for each compound is indicated by a vertical dashed line. Both compounds exert a primary effect on DNA synthesis and a secondary effect on RNA synthesis.

https://doi.org/10.1371/journal.pone.0084409.g004

Both inhibitors were exceptionally potent against all Gram-positive and fastidious Gram-negative pathogens screened, with MIC₉₀ values below 0.1 μg/mL in nearly all cases. C3 and C4 retained good antimicrobial activity against clinically problematic Gram-negative pathogens, including the highly drug resistant K. pneumoniae carbapenemase positive (KPC) and New Delhi metallo-β-lactamase positive (NDM-1) strains (Table 3). The dual-targeting activities of C3 and C4 effectively suppressed emergence of resistance; both compounds demonstrate spontaneous resistance frequencies in E. coli below 2 x 10^-11 at concentrations 4-fold above the MIC (Table 4). The moderately elevated MIC values observed for some Gram-negative pathogens are likely due to efflux pump activity; this hypothesis is consistent with differences in MIC values observed between isogenic wild-type and pump knockout strains of Escherichia coli (Table 3). The MIC values were not affected by the addition of 20% mouse serum (Table 3), improving the prospects for the effectiveness of this inhibitor in in vivo infection models.

In Vivo Efficacy

The in vitro antimicrobial activities of C3 and C4 translated well in in vivo infection models. Both compounds demonstrated efficacy in several intravenous Gram-negative mouse infection models, including an E. coli neutropenic mouse tissue infection model (Figure 5). Single doses of 5-15 mg/kg reduced the tissue bioburden in mouse thigh by 3 log₁₀ CFU within 24 hours, as compared with a vehicle control. Notably, a single 15 mg/kg dose of C3 reduced tissue bioburden by > 1 log below the stasis level, while the same dose of C4 reduced bioburden to approximately the stasis level.

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Figure 5. Reduction in bioburden after 24 hrs in a neutropenic mouse thigh infection model.

The MIC values for C3, C4 and levofloxacin against the E. coli strain used in the study are 0.13, 0.13 and 0.03 μg/mL, respectively.

https://doi.org/10.1371/journal.pone.0084409.g005

Conclusions

The failure by industry and academia to deliver new antibiotic classes with Gram-negative spectrum over the last several decades highlights the difficulty of the problem. The technical challenges to realizing this goal are becoming addressable due to advances in SBDD combined with a growing understanding of the sieving properties of the Gram-negative cell envelope. The discovery of the TriBE inhibitors demonstrates an approach with general applicability. The selection of suitable targets combined with an optimization strategy that placed equal emphasis on enzymatic potency, spectrum and drug-properties has led to the discovery of an exciting new class of broad-spectrum antibacterial agents with potent activity against dangerous multi-drug resistant Gram-negative pathogens. The TriBE inhibitor series is undergoing further optimization and advancement to preclinical evaluation for utilization in the clinic.

Synthesis of C1

The synthetic methods used to make C1, or (R)-7-((4-(3-aminopyrrolidin-1-yl)-5-chloro-6-ethyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)thio)-1,5-naphthyridine are described elsewhere [15].

Synthesis of C2, C3 and C4

The synthetic methods used to make C2, or (S)-1-(2-(pyrido[2,3-b]pyrazin-7-ylthio)-9H-pyrimido[4,5-b]indol-4-yl)pyrrolidin-3-amine, C3, or 6-fluoro-4-((3aR,6aR)-hexahydropyrrolo[3,4-b]pyrrol-5(1H)-yl)-N-methyl-2-((2-methylpyrimidin-5-yl)oxy)-9H-pyrimido[4,5-b]indol-8-amine, and C4, or 4-((1S,5R,6R)-6-amino-1-methyl-3-azabicyclo[3.2.0]heptan-3-yl)-6-fluoro-N-methyl-2-((2-methylpyrimidin-5-yl)oxy)-9H-pyrimido[4,5-b]indol-8-amine are described elsewhere [24].

Generation, expression and purification of full length E. faecalis GyrB, E. coli GyrB, F. tularensis GyrB, E. faecalis ParE, E. coli ParE and F. tularensis ParE constructs for enzymological studies

Full length ORFs were PCR amplified and cloned into the NcoI and XhoI sites of pET28a (Novagen). The resulting clones contained a C-terminal hexahistadine tag. Sequence verified clones were transformed to the expression strain, E. coli BL21(DE3). Fermentation and purification conditions for all full-length proteins were as follows: Cells were grown at 37°C in 1 liter of Terrific Broth and induced with 1 mM IPTG once an OD₆₀₀ of 0.8 was reached. The cells were harvested after an additional 12 hours of growth at 18°C. The cell pellets were resuspended in 50 mM Tris pH 8.0, 200 mM NaCl. Sonication was used to lyse the cells and the lysate was cleared by centrifugation. The supernatant was loaded onto a bed of Ni-NTA agarose and eluted with a gradient of imidazole in 50 mM Tris pH 8.0, 200 mM NaCl. The final chromatographic step was anion exchange (HiTrap Q HP from GE Healthcare) using a linear gradient of 25 mM Tris pH 8.0, 25-500 mM NaCl, while collecting fractions over 10 column volumes. The full-length GyrB and ParE proteins were soluble when overexpressed and were purified to > 98% purity (as determined by SDS-PAGE).

Generation, expression and purification of E. faecalis GyrB constructs, E. coli GyrB constructs and F. tularensis ParE constructs for crystallization studies

A uniform cloning method was employed for the generation of C-terminal hexahistadine tagged crystallization constructs. All PCR primers pairs were designed to introduce a BsaI recognition sequence, which when cut would produce compatible ends that could be ligated into pET28a treated with NcoI and XhoI. The primers used to generate the F. tularensis ParE construct were FtParE-fwd 5’- AATAATGGTCTCCCATGCAAAACTATAATGCTAAATCT-3’ and FtParE-rev 5’-AATAATGGTCTCCTCGAGTGCATTAATTCTTTTTTGTGC-3’, which encode amino acids 1-382 of the wild type protein. The E. faecalis GyrB construct was generated using the following primer pair: EfGyrB-fwd 5’-CAACACGGTCTCCCATGGGCTTAGAAGCTGTCCGGAAACGTC-3’ and EfGyrB-rev 5’-CCAACAGGTCTCCTCGAGCCGCCTTCATAGTGATACTCTTTTTTA-3’, resulting in a construct comprising residues 18-225 of the wild type sequence. The E. coli GyrB crystallization construct was amplified using the following primer pair:  EcGyrB-fwd 5’-AATAATGGTCTCCCATGG GGCTGGATGCGGTGCGTAA-3’

EcGyrB-rev 5’-AATAATGGTCTCCTCGAG GCCTTCATAGTGGAAGTGG-3’.  This clone contains residues 1-220 of the wild type protein. The expression and purification of this set of proteins was performed as described for the full-length proteins. E. faecalis GyrB and E. coli GyrB required further purification using a size-exclusion column (Superdex 75 from GE Healthcare) previously equilibrated with buffer consisting of 25 mM Tris pH 8.0, 100 mM NaCl.

ATPase assay

GyrB and ParE activity were evaluated using the coupled spectrophotometric Enzchek™ assay in which the enzyme-dependent release of inorganic phosphate from ATP hydrolysis was measured. The assay contained between 20-100 nM GyrB or ParE (active site concentrations) in 50 µM Tris-HCl buffer (pH 7.6), 2 µM MgCl₂, 125 µM NaCl, 0.2 µM 7-methyl-6-thioguanosine, 1U/mL purine nucleoside phosphorylase. The reaction was initiated by addition of 3 µM ATP and monitored at 360 nm for 30 min at 27°C. Inhibitor potency was determined by incubating the target enzyme in the presence of various concentrations of inhibitor ranging between 1.5 nM and 50 µM for 10 minutes prior to addition of ATP substrate. The final concentration of DMSO was kept constant at 2.5% (v/v). Enzyme activity in the presence of inhibitor was expressed relative to the no-inhibitor control and K_i values determined using Morrison tight-binding equation [25] to account for ligand depletion. All analysis was carried out using GraphPad Prism 4.0.

Mechanism of action determination

The mechanism of action of GyrB/ParE targeting compounds was examined by monitoring macromolecular synthesis in S. aureus ATCC 29213 and a permeabilized E. coli strain, E. coli (imp) (BAS849). Macromolecular synthesis in the presence of increasing doses of compound was monitored by measuring the incorporation of radiolabeled precursors of DNA, RNA, protein and cell wall synthesis ([³H]-Thymidine, [³H]-Uridine, [³H]-Leucine, and [³H]-N-acetylglucosamine, respectively). A range of test compound, spanning the established minimal inhibitory concentration, was examined for each precursor. The extent by which synthesis of each pathway was inhibited was determined by calculating the difference in label incorporation relative to untreated controls. The effects were compared to of 5 control antibiotics specific for selected metabolic pathways: tetracycline (protein synthesis), ciprofloxacin and novobiocin (deoxyribonucleic acid [DNA] synthesis), rifampicin (ribonucleic acid [RNA] synthesis), and vancomycin (cell wall synthesis). A detailed description of the macromolecular synthesis assays used is provided elsewhere [26].

Minimum inhibitory concentration assays

Assays for minimum inhibitory concentration (MIC) values reported in Tables 1 and 2 were performed as follows: Test compounds were solubilized in 100% dimethylsulfoxide (DMSO). Sterile deionized water was added to achieve a 50% DMSO stock solution and additional dilutions were made from the stock solutions to achieve a broader testing range. Mueller Hinton II broth (MHB) was used for testing with the exception of H. influenzae, which was tested in Haemophilus Test Medium Broth (HTM), and S. pneumoniae, which was tested in MHB supplemented with 2.5% lysed horse blood (LHB). Assay plates were loaded with 85 µL of test media appropriate for the test organism, 5 µL of drug solution, and 10 µL of bacterial inoculum. For TriBE inhibitors, the final concentration of DMSO was 2.5% with the exception of H. influenzae where the final concentration of DMSO was 0.6%. Final cell concentration in assay plates was approximately 5 x 10⁵ CFU/mL. Plates were incubated at 35°C for 18 to 24 hrs. and microplates were viewed from the bottom using a plate viewer. The MIC was read and recorded as the lowest concentration of drug that inhibited visible growth of the organism. Assays for MIC values reported in Table 3 were performed as follows: Test compounds were solubilized in 100% DMSO. Two-fold dilutions were prepared in 100% DMSO at 50X final MIC assay concentrations (final DMSO concentration of 2% v/v). MHB was used for testing with the exception of H. influenzae, which was tested in HTM, and S. pneumoniae, which was tested in MHB with 3% LHB. A subset of strains was also tested in the presence of 20% heat inactivated mouse serum. Assay plates were loaded with 98 µL of inoculated test media appropriate for the test organism (final cell concentration of approximately 5 x 10⁵ CFU/mL) and 2 µL of drug solution. Plates were incubated at 35°C for 18 to 24 hr and alamarBlue® was used to visualize cell viability. The MIC was read and recorded as the lowest concentration of drug that inhibited visible growth of the organism and color change of alamarBlue®.

Spontaneous incidence of resistance

Spontaneous mutations frequencies were assessed in E. coli (ATCC 25922). Large format assay dishes (245 x 245 mm) were prepared with 200 mL of Mueller Hinton II cation-adjusted agar containing C3, C4 or ciprofloxacin (CIP) at a concentration 4X the MIC value. A suspension of ~1x10¹⁰ CFU in PBS was spread on each plate with glass beads. Plates were incubated at 37°C for 48 h. Putative mutant colonies were cultured on media containing an equivalent concentration of drug and run in MIC assays to confirm resistance phenotypes. Mutation frequencies were determined by dividing the number of resistant colonies by the actual CFU plated. Frequencies listed in Table 5 represent the average of values obtained for 3 plates, each derived from an independent culture.

Mouse efficacy studies

Female CD-1 mice weighing 18 to 20 grams were pre-treated with cyclophosphamide to render the mice neutropenic. Mice were infected with E. coli (ATCC 25922) via injection into the right thigh muscle of 0.1 mL per mouse. One and a half hours post infection mice were treated IV with C3 or C4 at doses ranging from 1 to 15 mg/kg. Levofloxacin was used as a control, dosed once at 10 mg/kg. Four mice were treated with each drug concentration. Twenty-four hours post treatment, mice were euthanized by CO₂ inhalation. The right thighs of the mice were aseptically removed, weighed, homogenized, serially diluted, and plated on TSA medium. The plates were incubated overnight at 37°C in 5% CO₂. CFU per gram of thigh was calculated by enumerating the plated colonies then adjusting for serial dilutions and the weight of the thigh.

Crystallization and structure determination of E. faecalis GyrB C1, E. faecalis GyrB C2, E. faecalis GyrB C3 and E. faecalis GyrB C4 complexes

All complex crystal structures were determined from the C-terminal 6xHis tagged N-terminal 24 kDa ATP-binding domain of E. faecalis GyrB. Complexes with inhibitors were crystallized by hanging drop vapor diffusion, from 10-20 mg/mL protein solutions (concentrated in in 20 mM Tris, pH 8.0, 100 mM NaCl) combined with the appropriate small molecule ligand from a 10 mg/mL DMSO stock solution to a final concentration of 1 mM. Crystals were grown from drops using 1:1 ratios of the concentrated protein-ligand complex solution with the following reservoir/mother liquor solution at 20°C: 25% (w/v) PEG 1500, 3% (v/v) t-butanol, 20% (v/v) glycerol, 20 mM citrate pH 5.6. Crystals were harvested in nylon loops and frozen in liquid nitrogen without any additional cryoprotectant. Data on crystals of the E. faecalis GyrB complexes with C1, C2 and C4 were collected on a Rigaku RU 200 rotating anode generator equipped with a MAR345 image plate detector. Data were processed with Mosflm [27] and merged with programs from the CCP4 suite [28]. Data on crystals of the E. faecalis GyrB complex with C3 were collected on the MBC 4.2.2 beamline at the Advanced Light Source, Lawrence Berkeley National Laboratory using a wavelength of 1 Å, a sample to detector distance of 120 mm and an oscillation angle of 0.5°. A complete data set was recorded on a NOIR-1 CCD detector. Diffraction data were processed, scaled and merged using D*TREK [29]. All the structures were determined by molecular replacement using Phaser [30] in the CCP4 program suite [28] with the coordinates of E. faecalis GyrB, PDB code 4GEE as a model [15]. Refinement was carried out using REFMAC5.5 [31]. A total of 5% of the data were kept aside for R_free calculations. After initial rounds of restrained refinement, clear electron density was observed in the active site for inhibitors and ordered solvent, as well as t-butanol molecule. The model building, incorporation of solvent and ligands were performed with the program Coot [32]. The crystallographic asymmetric unit contains a single protein-ligand complex. Due to dynamic disorder, no electron density was observed for the last 5 histidine residues of the C-terminal histidine tag and residues between 101 (His) and 119 (Gly) belonging to a mobile surface loop. These residues were not included in the final models. The final refinement and geometry statistics are provided in Table 5. All models were validated using PROCHECK [33] and RAMPAGE [34] prior to submission to the Protein Data Bank.

Crystallization and structure determination of the E. coli GyrB C3 complex

The E. coli GyrB C3 complex crystal structure was determined from the C-terminal 6xHis tagged N-terminal 24 kDa ATP-binding domain of E. coli GyrB. The complex was crystallized by sitting drop vapor diffusion, from a 15 mg/mL protein solution (concentrated in in 20 mM Tris, pH 8.0, 100 mM NaCl) combined with C3 from a 10 mg/mL DMSO stock solution to a final concentration of 1 mM. Crystals were grown from drops using 1:1 ratios of the concentrated protein-ligand complex solution with the following reservoir/mother liquor solution at 4°C: 15% (w/v) PEG 3350, 200 mM AmSO₄, 100 mM Tris pH 7.5. Crystals were cryoprotected with mother liquor solution supplemented with 25% (v/v) ethylene glycol and harvested in nylon loops and frozen in liquid nitrogen. Data on a single crystal of E. coli GyrB complexed with C3 were collected on the MBC 4.2.2 beamline at the Advanced Light Source, Lawrence Berkeley National Laboratory using a wavelength of 1 Å, a sample to detector distance of 120 mm and an oscillation angle of 0.5°. A complete data set was recorded on a NOIR-1 CCD detector. Diffraction data were processed, scaled and merged using D*TREK [29]. All the structures were determined by molecular replacement using Phaser [30] in the CCP4 program suite [28] with the coordinates of E. coli GyrB, PDB code 4KZN as a model [35]. Refinement was carried out using REFMAC5.5 [31]. A total of 5% of the data were kept aside for R_free calculations. After initial rounds of restrained refinement, clear electron density was observed in the active site for inhibitors and ordered solvent. The model building, incorporation of solvent and ligands were performed with the program Coot [32]. The crystallographic asymmetric unit contains two protein-ligand complexes. Due to dynamic disorder, no electron density was observed for the last 5 histidine residues of the C-terminal histidine tag and residues between 94 (Leu) and 110 (Lys) belonging to a mobile surface loop. These residues were not included in the final models. The final refinement and geometry statistics are provided in Table 5. The final model was validated using PROCHECK [33] and RAMPAGE [34] prior to submission to the Protein Data Bank.

Crystallization and structure determination of the F. tularensis ParE C3 complex

The F. tularensis ParE C3 complex crystal structure was determined from the C-terminal 6xHis tagged N-terminal 39 kDa ATP-binding domain of F. tularensis ParE. The complex was crystallized by sitting drop vapor diffusion, from a 20 mg/mL protein solution (concentrated in in 20 mM Tris, pH 8.0, 100 mM NaCl) combined with C3 from a 10 mg/mL DMSO stock solution to a final concentration of 1 mM. Crystals were grown from drops using 1:1 ratios of the concentrated protein-ligand complex solution with the following reservoir/mother liquor solution at 4°C: 10% (w/v) PEG 4000, 10% (v/v) isopropanol, 100 mM citrate pH 5.4. Crystals were cryoprotected with mother liquor solution supplemented with 30% (v/v) glycerol and harvested in nylon loops and frozen in liquid nitrogen. Data on a single crystal of F. tularensis ParE complexed with C3 were collected on the MBC 4.2.2 beamline at the Advanced Light Source, Lawrence Berkeley National Laboratory using a wavelength of 1 Å, a sample to detector distance of 120 mm and an oscillation angle of 0.5°. A complete data set was recorded on a NOIR-1 CCD detector. Diffraction data were processed, scaled and merged using D*TREK [29]. All the structures were determined by molecular replacement using Phaser [30] in the CCP4 program suite [28] with the coordinates of F. tularensis ParE, PDB code 4HY1 as a model [15]. Refinement was carried out using REFMAC5.5 [31]. A total of 5% of the data were kept aside for R_free calculations. After initial rounds of restrained refinement, clear electron density was observed in the active site for inhibitors and ordered solvent. The model building, incorporation of solvent and ligands were performed with the program Coot [32]. The crystallographic asymmetric unit contains two protein-ligand complexes. Due to dynamic disorder, no electron density was observed for residues 1-7, 103-111, 227-231, 258-260, 299-302, and 381 and the C-terminal histidine tag of the A chain. The B chain contains similar disorder, in addition to residues 218-381. Disordered residues were not included in the final model. The final refinement and geometry statistics are provided in Table 5. The final model was validated using PROCHECK [33] and RAMPAGE [34] prior to submission to the Protein Data Bank.

Computational design methods

Computational work in support of this project benefited from supercomputing resources at Lawrence Livermore National Laboratory amounting to ~ 135K cpu-hours/week. Virtual high throughput screening using molecular docking and MM-GB/SA [36] rescoring were performed using Schrodinger’s Glide [37] and Prime [38] programs, and Amber10 [39] and Autodock [40]. Molecular dynamics calculations and thermodynamic integrations were performed during the optimization phase using Amber10 [39]. Ab initio ligand strain, polarizability and chemical stability calculations were performed using Gaussian03 at the B3LYP/6-31++G** level of theory). The QSAR and cheminformatics were performed using the Accelrys [41] suite of programs.

Ethics Statement

The mouse efficacy studies were conducted under IACUC protocol # VVS11-008, which was reviewed and approved by Vivisource IACUC. All animal work was conducted at Vivisource which is a PHS-assured facility (PHS/OLAW assurance # A4543-01). All work complied with the animal care and use guidelines from Guide for the Care and Use of Laboratory Animals and that they comply with the applicable regulations (9 CFR, Subchapter A) issued by the U.S. Department of Agriculture (USDA) under the Animal Welfare Act.