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Antimicrobial Agents and Chemotherapy, October 2007, p. 3688-3698, Vol. 51, No. 10
0066-4804/07/$08.00+0     doi:10.1128/AAC.00392-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Discovery of Novel DNA Gyrase Inhibitors by High-Throughput Virtual Screening

David A. Ostrov,^1* José A. Hernández Prada,¹ Patrick E. Corsino,² Kathryn A. Finton,² Nhan Le,² and Thomas C. Rowe^2*

Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine, 1600 SW Archer Road, Gainesville, Florida 32610,¹ Department of Pharmacology and Therapeutics, University of Florida College of Medicine, 1600 SW Archer Road, Gainesville, Florida 32610-0267²

Received 22 March 2007/ Returned for modification 21 May 2007/ Accepted 27 July 2007

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The bacterial type II topoisomerases DNA gyrase and topoisomerase IV are validated targets for clinically useful quinolone antimicrobial drugs. A significant limitation to widely utilized quinolone inhibitors is the emergence of drug-resistant bacteria due to an altered DNA gyrase. To address this problem, we have used structure-based molecular docking to identify novel drug-like small molecules that target sites distinct from those targeted by quinolone inhibitors. A chemical ligand database containing approximately 140,000 small molecules (molecular weight, <500) was molecularly docked onto two sites of Escherichia coli DNA gyrase targeting (i) a previously unexplored structural pocket formed at the dimer interface of subunit A and (ii) a small region of the ATP binding pocket on subunit B overlapping the site targeted by coumarin and cyclothialidine drugs. This approach identified several small-molecule compounds that inhibited the DNA supercoiling activity of purified E. coli DNA gyrase. These compounds are structurally unrelated to previously identified gyrase inhibitors and represent potential scaffolds for the optimization of novel antibacterial agents that act on fluoroquinolone-resistant strains.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Fluoroquinolones (see Fig. 1) are a clinically important class of antibacterial drugs that target the type IIA topoisomerases DNA gyrase and topoisomerase IV, two highly homologous enzymes that play essential roles in bacterial DNA replication (reviewed in references 5, 12, 15, 29, and 46). DNA gyrase is a heterotetrameric protein consisting of two GyrA subunits and two GyrB subunits (A₂B₂) encoded by the gyrA and gyrB genes, respectively. The GyrA subunit mediates the enzyme-catalyzed DNA breakage-reunion reaction and contains the active-site tyrosine that forms a covalent complex with the 5'-labeled ends of the transiently cleaved DNA duplex. The GyrB subunit contains an ATPase activity which facilitates the DNA strand-passing reaction of DNA gyrase. Topoisomerase IV, a paralogue of DNA gyrase, is also a heterotetramer, consisting of two ParC and two ParE subunits which are homologues of the GyrA and GyrB subunits of DNA gyrase, respectively. Fluoroquinolones interact with the DNA breakage-reunion subunit of DNA gyrase and topoisomerase IV, leading to the stabilization of the covalent topoisomerase/DNA cleavable complex which blocks DNA replication.

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FIG. 1. Chemical structures of known inhibitors that target the A (the fluoroquinolone ciprofloxacin) and B (novobiocin and cyclothialidine) subunits of bacterial DNA gyrase.

Resistance to fluoroquinolones is associated primarily with mutations in the quinolone resistance-determining region (QRDR) of the genes encoding GyrA and/or ParC (8, 14, 50; reviewed in references 12 and 20). This region encodes a 50- to 60-amino-acid stretch in the N-terminal regions of these polypeptides. Resistance to fluoroquinolones in Escherichia coli is most commonly associated with amino acid substitutions at S83 and D87 in GyrA, which map to the putative DNA binding surface of -helix 4 (see Fig. 2) (39).

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FIG. 2. Strategy for the identification of small-molecule inhibitors targeted to the dimer interface of E. coli DNA GyrA by using DOCK v5.1.0. (A) The dimeric form of E. coli DNA GyrA is depicted, showing one subunit with red helices and the other subunit with teal helices. The scoring grid used in the docking analysis is depicted by the blue box. The positions of -helix 3 (3) and -helix 4 (4) are identified by arrows. (B) The site for molecular docking was selected based on spheres (not shown) positioned at the dimer interface in close proximity to the key catalytic residue Y122. The S83 and D87 residues are shown by the red and blue spheres, respectively, in -helix 4. Compound NSC 103003 is shown in the orientation posed by DOCK v5.1.0. The positions of Y122 and S83 are indicated. The figure was made with PYMOL.

DNA gyrase is also the target of coumarin and cyclothialidine drugs (Fig. 1), which inhibit GyrB-associated ATPase activity (reviewed in reference 35). Crystallographic analysis indicates that both drugs form key hydrogen bonds with D73 and a conserved water molecule in the ATP binding site of GyrB (30). Resistance to coumarin drugs in E. coli occurs primarily by a mutation of R136 to L, H, C, S, or A (7, 10). Interestingly, topoisomerase IV is 5- to 10-fold more resistant to coumarin antibiotics than DNA gyrase, and recent studies indicate that this may be due to a single amino acid substitution of a methionine for isoleucine at position 74 in the ParE subunit of topoisomerase IV (2).

In an effort to discover novel inhibitors that would act on microbial topoisomerases resistant to the known DNA gyrase inhibitors, we utilized a molecular docking screening strategy to identify structural elements outside the QRDR of bacterial GyrA that could potentially be targeted with small molecules. Molecular docking has led to the successful discovery of novel ligands for more than 30 targets (reviewed in reference 43). This strategy has been successfully applied primarily to a large number of enzymatic target proteins, such as aldose reductase, Bcl-2, matriptase, adenovirus protease, AmpC ß-lactamase, carbonic anhydrase, hypoxanthine phosphoribosyltransferase, dihydro-dipicolinate, and cyclin-dependent kinase 4. The generation of a new class of potent cyclin-dependent kinase 4 inhibitors is a prototypic example of the "scaffold-based approach" to integrate molecular docking with inhibitor design using virtual libraries of small molecules (19). With the increasing number and accuracy of crystal structures in recent years, molecular docking has become an important tool for the synthetic elaboration of novel therapeutics based on chemical scaffolds. In this study, we utilized solved crystal structures of the A and B subunits of DNA gyrase for molecular docking of small molecules onto both established and novel drug-targeting sites in this enzyme. Several of the top-scoring compounds were found to inhibit the DNA supercoiling activity of purified E. coli DNA gyrase.

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Database preparation. The National Cancer Institute/Developmental Therapeutics Program (NCI/DTP) maintains a repository of 139,644 samples (plated compound set) (38). The three-dimensional coordinates for the NCI/DTP plated compound set in the MDL sd format were converted to the mol2 format by the program SDF2MOL2 (UCSF). Partial atomic charges for ligand atoms were calculated using SYBDB (UCSF) (13) and added to the mol2 file representing the NCI/DTP plated compound set (approximately 140,000 small molecules).

Molecular docking. The procedure for molecular docking involves (i) the selection of structural pockets in DNA gyrase suitable for interactions with drug-like small molecules and (ii) molecular docking simulations where each one of approximately 140,000 small molecules (molecular weight, <500) is positioned in the selected structural pocket and scored based on predicted polar (e.g., H bond) and nonpolar (e.g., van der Waals) interactions. The 10 highest-scoring compounds for each selected structural pocket were obtained for use in DNA gyrase inhibition assays. Docking calculations were performed with the 15 October 2002 development version of DOCK v5.1.0 (13, 41).

The coordinates for the crystal structure of a 59-kDa fragment of gyrase A from E. coli, Protein Data Bank (PDB) code 1AB4, were used in the molecular docking calculations (39). The biologically relevant dimeric form of DNA gyrase A was generated by applying the crystallographic symmetry operation (–1.0, 0.0, 0.0, 119.6 Å, 0.0, –1.0, 0.0, 119.6 Å, 0.0, 0.0, 1.0, 0.0) to the coordinates in PDB 1AB4 using CCP4 (23). The molecular surface of the structure was explored using sets of spheres to describe potential binding pockets. The spheres literally fill in the available pocket spaces where a ligand might be able to form a complex. DOCK uses the spheres as a guide to search for orientations of each molecule that fit into the selected sites. The sites selected for molecular docking were defined using the SPHGEN program and filtered through the CLUSTER program (13). The SPHGEN program generates an unbiased grid of points that reflect the actual shape of the selected site. The CLUSTER program groups the selected spheres to define the points that are used by DOCK to match (superimpose) potential ligand atoms with spheres. Seventy-two spheres were used to define the gyrase A site for molecular docking. Each compound in the NCI/DTP database was positioned in the selected site in 100 different orientations. Intermolecular AMBER energy scoring (van der Waals + columbic), contact scoring, and bump filtering were implemented in DOCK v5.1.0 (13). PYMOL (9) was used to generate molecular graphic images.

The ATP binding site in DNA gyrase subunit B was targeted with small molecules by screening approximately 140,000 compounds, using a molecular docking protocol similar to that used for DNA gyrase subunit A, described above. For molecular docking into a structural pocket of DNA gyrase subunit B, the site selection criteria were based on the position of novobiocin in the ATP binding site (27). The coordinates were from the crystal complex of novobiocin and Thermus thermophilus, PDB code 1KIJ (26). PDB 2SPH (UCSF) was used to place spheres at the positions of novobiocin atoms. The novobiocin-bound structure of E. coli gyrase B is not available, but conserved residues in the ATP binding site were identified by aligning DNA gyrase subunit B sequences from E. coli and T. thermophilus by using ClustalX (6). Sequence variability was plotted on the molecular surface of T. thermophilus by using PYMOL to demonstrate the high degree of sequence and structural similarities. Forty-four spheres were used to define the site on gyrase B for molecular docking. Scoring was calculated in a 5-Å grid surrounding the spheres (GRID; UCSF). All molecular docking jobs were performed on SGI Octane workstations running DOCK (UCSF) in IRIX6.5. The 10 highest-scoring compounds for each selected structural pocket were obtained for use in DNA gyrase inhibition assays. Analysis of docked compounds was performed with HBPLUS (36) and plotted using LIGPLOT (45).

Chemicals and enzymes. The small-molecule inhibitors used in this study were obtained from the Drug Synthesis and Chemistry Branch of the National Cancer Institute and are listed in Tables 1 and 3. Plasmids containing the A (pPH11) or B (pAG111) subunit of E. coli DNA gyrase (kindly provided by Tony Maxwell, Norwich Research Park, United Kingdom) were overexpressed in E. coli XL1-Blue (Stratagene), and the resulting polypeptides were purified as described previously by Maxwell and Howells (34). DNA gyrase activity was then reconstituted by mixing the two subunits together at a ratio of 1:1.4 (GyrA/GyrB).

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TABLE 1. GyrA compoundsa

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TABLE 3. GyrB compoundsa

Preparation of relaxed DNA substrate. Relaxed plasmid DNA substrate was prepared by incubating supercoiled pRSET A DNA (Invitrogen) with wheat germ topoisomerase I (Promega) according to the manufacturer's specifications. The relaxed pRSET A DNA was then extracted with phenol-chloroform before ethanol precipitation and resuspension in 10 mM Tris-HCl, pH 7.5, 1 mM EDTA.

DNA supercoiling assay. Supercoiling by DNA gyrase was determined using a standard agarose gel assay as described previously (18). Reaction mixtures (20 µl) containing 25 mM Tris-acetate, pH 7.9, 20 mM potassium acetate, 10 mM magnesium acetate, 2 mM dithiothreitol, 1.5 mM ATP, 5 mM spermidine-HCl, 50 µg/ml bovine serum albumin, 0.5 µg relaxed pRSET A DNA (Invitrogen), and various concentrations of drug were incubated on ice for 10 min. Reactions were then initiated by adding DNA gyrase (2.5 nM GyrA and 3.5 nM GyrB) and incubating the mixture at 30°C for 30 min. Reactions were terminated by adding 4 µl of stop solution (3% sodium dodecyl sulfate, 30% Ficoll, 0.6 mg/ml bromophenol blue, 60 mM EDTA), and the samples were loaded onto a 1% agarose gel in 40 mM Tris-acetate, pH 8.1, 2 mM EDTA. Following electrophoresis at 12 V for 20 h, gels were stained with ethidium bromide (1 µg/ml) and the DNA was visualized under UV light. The gel image was captured using a Bio-Rad Gel Doc apparatus, and the DNA bands were quantitated using Scion Image Beta software version 4.0.2 (Scion Corporation). The percentage of supercoiled DNA in each drug-treated sample was calculated relative to that in non-drug-treated gyrase controls.

ATPase assay. The DNA-independent ATPase activity of E. coli GyrB was measured by a spectrophotometric method that couples the hydrolysis of ATP with the oxidation of NADH (31). The methodology and reaction conditions were the same as those previously reported using 40 nM of purified E. coli GyrB and 2 mM ATP (23). The apparent K_i [K_i(app)] value for Cancer Chemotherapy National Service Center (NSC) compound 20116 was determined as follows: 1/slope of a plot of inhibitor concentration versus (V₀/V_i) – 1, where V₀ and V_i represent the rates of ATP hydrolysis in the absence and presence of inhibitor, respectively (23). The K_i value was then calculated using the equation K_i = K_i(app)/(1 + [ATP]/K_m), using a K_m of 0.3 mM for E. coli gyrase ATPase (44).

DNA unwinding assay. Drug-induced DNA unwinding was measured as described previously (28), using pGEM2 plasmid DNA (Promega, Madison, WI) and wheat germ DNA topoisomerase I (Promega, Madison, WI).

Bacterial growth studies. To determine the effects of drugs on E. coli XL1-Blue cell growth, overnight cultures grown in LB media at 37°C were diluted to an optical density at 595 nm (OD₅₉₅) of 0.05 in 50 ml fresh LB media and incubated with shaking at 37°C until cells entered log-phase growth. Cells (10 ml) were then transferred into flasks containing various concentrations of filter-sterilized drug, and cell growth continued with shaking at 37°C. Cell growth was monitored at OD₅₉₅ using a Pharmacia LKB Ultrospec III spectrophotometer (Cambridge, England).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Discovery of a novel structural pocket on E. coli DNA gyrase subunit A for structure-based drug design. The goal of this approach was to develop a strategy to inhibit DNA gyrase activity in E. coli by interfering with a functionally important structural pocket that is not affected by common mutations that cause resistance. Resistance to fluoroquinolones is associated primarily with mutations that occur in a small region (QRDR) of the A subunits of the DNA gyrase and topoisomerase IV genes, which encode a 50- to 60-amino-acid stretch in the N-terminal regions of the GyrA and ParC polypeptides, respectively (8, 14, 50; reviewed in references 12 and 20). In the case of E. coli GyrA, fluoroquinolone resistance is most often associated with amino acid substitutions at S83 and D87, which reside within the putative DNA binding surface of -helix 4 (Fig. 2). Since more than 80% of the mutations that confer fluoroquinolone resistance occur in -helix 4 of E. coli DNA GyrA, we targeted small molecules to a structural pocket that is not in contact with S83 or D87. The targeted region includes -helix 3, which forms part of the GyrA dimer interface, and is in close proximity to Y122 (3.9 Å), the key catalytic residue involved in the DNA breakage-reunion reaction of DNA gyrase (Fig. 2) (39). This structural pocket is in close proximity to the catalytic residue Y122 but distant (18 Å) from S83 and D87, which are commonly altered in fluoroquinolone-resistant bacteria.

We used molecular docking to position each of approximately 140,000 compounds in 100 different orientations at the GyrA dimer interface. The 10 highest-scoring compounds based on van der Waals and electrostatic potential were then obtained from the NCI/DTP (38) and tested for inhibition of DNA gyrase supercoiling activity in vitro by using drug concentrations of up to 100 µg/ml (Table 1 and Fig. 3). Compounds (defined by NSC identification numbers) NSC 103003, NSC 130847, and NSC 20115 were found to significantly inhibit DNA gyrase supercoiling activity with 50% inhibitory concentrations (IC₅₀s) of 50, 72, and 737 µM, respectively. The most potent compound, NSC 103003, also had the highest overall energy score, –15.68 kcal per mol. Inhibition of DNA gyrase by NSC 103003 was further tested in the presence and absence of Triton X-100 to ascertain whether inhibition was an artifact due to sequestration of DNA gyrase by drug aggregates. McGovern et al. has shown that low concentrations of Triton X-100 (0.01%) can prevent or reverse this type of promiscuous enzyme inhibition by promoting the dissociation of enzyme from drug aggregates (37). Inhibition of DNA gyrase activity by NSC 103003 was not significantly affected by Triton X-100 at either 0.01% or 0.025% (Table 2), indicating that inhibition was not due to nonspecific drug aggregation effects. To determine whether the inhibition of DNA gyrase might involve drug intercalation into DNA, a DNA unwinding assay was done. NSC 130847 and NSC 20115 did not cause DNA unwinding at 100 and 1,600 µM, respectively (data not shown). However, NSC 103003 did cause significant DNA unwinding at concentrations of 50 µM, indicating that this compound may inhibit gyrase by altering DNA structure.

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FIG. 3. Chemical structures of the highest-scoring GyrA targeted compounds identified by DOCK v5.1.0. The NSC and Chemical Abstracts Service (CAS) numbers are given below each compound.

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TABLE 2. Effect of Triton X-100 on drug inhibition of DNA gyrase

In silico docking and in vitro functional testing of compounds targeting the ATP binding pocket of DNA gyrase subunit B. The ATP binding pocket of DNA gyrase subunit B is highly conserved between species, and crystallographic and biochemical evidence indicates that coumarin and cyclothialidine drugs act by competing with ATP for binding to the structural pocket defined by N46, E50, D73, R76, I78, K103, V118, and T165 in T. thermophilus and E. coli GyrB (Fig. 4) (25, 27, 30; reviewed in reference 35). There are currently no solved structures of wild-type E. coli DNA gyrase B bound to novobiocin. Therefore, we used the crystal structure of novobiocin bound to the highly conserved ATP binding pocket of T. thermophilus (Fig. 4) for our docking analysis. Similar to the strategy used for the A subunit of DNA gyrase, we screened approximately 140,000 molecules in the structural pocket defined by novobiocin bound to GyrB. The 10 highest-scoring compounds (Table 3 and Fig. 5) were then tested for inhibition of E. coli DNA gyrase supercoiling activity. One advantage of this approach is that it should selectively identify inhibitors that act through conserved binding motifs present in both T. thermophilus and E. coli enzymes. Such inhibitors would be expected to have a broader spectrum of antibacterial activity than that of inhibitors that target species-specific motifs.

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FIG. 4. Strategy for identification of ATPase inhibitors targeting the ATP binding site of T. thermophilus DNA GyrB by using DOCK v5.1.0. (A) The dimeric form of T. thermophilus DNA GyrB is depicted, showing one subunit with red helices and yellow strands and the other subunit with teal helices and magenta strands. The site for molecular docking was selected based on spheres (shown in green), and the scoring grid used in the docking analysis is depicted by the blue box. The ATP binding site is identified by a black arrow. (B and C) The positions of 5'adenylyl-ß, -imidodiphosphate (ADP-PNP) and novobiocin, respectively, are shown within the ATP binding pocket of GyrB. (D) NSC 20116 is shown in the orientation posed by DOCK v5.1.0 in the ATP binding pocket of GyrB. The molecular surface is colored based on the sequence similarity between E. coli and T. thermophilus GyrB, with red depicting the most highly conserved residues. The figure was generated with PYMOL. (E) Inhibition of E. coli DNA gyrase ATPase by NSC 20116. Gyrase ATPase activity was measured in the presence of various concentrations of NSC 20116 and plotted as (V0/Vi) – 1 versus the inhibitor concentration as described in Materials and Methods. The Ki value (0.149 mM) for the plot was calculated as described in Materials and Methods.

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FIG. 5. Chemical structures of the highest-scoring GyrB targeted compounds identified by DOCK v5.1.0. The NSC and Chemical Abstracts Service (CAS) numbers are given below each compound.

NSC compounds 20116 and 7784 inhibited DNA gyrase activity, with IC₅₀ values of 338 and 814 µM, respectively (Table 3). The inclusion of Triton X-100 in the assay did not reverse inhibition by NSC 20116, suggesting that inhibition was not due to enzyme adsorption to drug aggregates (Table 2). Inhibition is unlikely to involve DNA intercalation since neither NSC 20116 nor NSC 7784 caused DNA unwinding at concentrations of up to 640 µM and 1.6 mM, respectively (data not shown).

Both NSC 20116 and NSC 7784 were also tested for inhibition of gyrase ATPase activity. Although NSC 7784 had no effect on ATPase activity at concentrations of up to 1.5 mM, NSC 20116 significantly inhibited ATPase activity, with a K_i of 149 µM (Fig. 4E), supporting the hypothesis that this latter compound targets the ATP binding pocket of GyrB.

The chemical structure of NSC 20116 differs significantly from those of known DNA gyrase inhibitors, suggesting a different mode of interaction with the ATP binding pocket (Fig. 4B to D). Cyclothialidine and novobiocin share a common binding motif involving an H bond donor to D73 and an H bond acceptor from a conserved water molecule (25, 27, 30, 48). These drugs also interact with residue R136, and an alteration in this residue to L, H, C, S, or A confers drug resistance (reviewed in reference 35). Although NSC 20116 is predicted to form a hydrogen bond with D73, it is not predicted to interact with R136 (Fig. 6). Therefore, mutations at R136 that confer resistance to coumarin and cyclothialidine drugs are not likely to cause cross-resistance to NSC 20116.

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FIG. 6. Comparison of the binding modes of novobiocin and NSC 20116 in the ATP binding pocket of T. thermophilus GyrB. (A) Molecular interactions between novobiocin and GyrB based on the crystal PDB code 1KIJ. Hydrogen bonds are depicted by dashed lines. Residues involved in van der Waals interactions are shown with red arcs. (B) NSC 20116 in the orientation posed by molecular docking by using the T. thermophilus coordinates from PDB code 1KIJ. Residues are numbered according to the corresponding residues in E. coli GyrB. The figure was made using the programs DOCK v5.1.0 (13), HBPLUS (36), and LIGPLOT (45).

Inhibition of E. coli growth by NSC 103003 and NSC 20116. Logarithmically growing E. coli XL1-Blue cells were incubated with various concentrations of either NSC 103003 or NSC 20116, and growth was monitored by following the absorbance of cell cultures at OD₅₉₅ (Fig. 7). NSC 103003 did not significantly inhibit growth in the micromolar range, although some inhibition was seen at 1.2 mM. This concentration was approximately 20-fold higher than concentrations required to inhibit purified DNA gyrase activity in vitro, suggesting that NSC 103003 does not effectively accumulate inside E. coli. Alternatively, the lack of growth inhibition by NSC 103003 could be due to drug instability or metabolic inactivation by bacterial enzymes. In contrast, NSC 20116 completely inhibited growth within 30 min at 192 µM, a concentration that inhibits purified DNA gyrase activity by >30%. There is an apparent dose-related delay in growth inhibition by NSC 20116 in the low micromolar range since growth inhibition was delayed for 30 or 60 min following the treatment of cells with 64 µM or 21 µM of drug, respectively (Fig. 7).

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FIG. 7. Inhibition of E. coli XL1-Blue growth by NSC 103003 and NSC 20116. Growth of E. coli XL1-Blue was monitored in the presence of various concentrations of either NSC 103003 (A) or NSC 20116 (B) as described in Materials and Methods. DMSO, dimethyl sulfoxide.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Fluoroquinolones are highly effective antimicrobial drugs that target the bacterial type II topoisomerases DNA gyrase and topoisomerase IV. Unfortunately, resistance to this class of drugs has become increasingly prevalent and is usually due to alterations in DNA gyrase/topoisomerase IV or to a decrease in intracellular drug levels caused by changes in membrane permeability or overexpression of drug efflux pumps. Alterations in DNA gyrase or topoisomerase IV are usually localized within the QRDR in the N terminus of the GyrA or ParC subunit of DNA gyrase or topoisomerase IV, respectively. In the case of E. coli DNA gyrase, clinical resistance is most frequently associated with chromosomal point mutations resulting in amino acid substitutions at codons 83 and 87 of GyrA. Quinolone resistance in other gram-negative as well as gram-positive organisms is also typically associated with similar substitutions at the equivalent conserved residues in the A subunits of DNA gyrase and/or topoisomerase IV. One potential approach to circumvent quinolone resistance would be to identify novel inhibitors that target a different region of GyrA. To this end, we have used a virtual screening method (DOCK v5.1.0) to search for inhibitors that might interact with an essential GyrA dimerization domain near Y122, a key catalytic residue involved in the DNA breakage-reunion reaction of DNA gyrase. Our preliminary findings have identified several compounds that inhibit DNA gyrase supercoiling activity at concentrations in the micromolar range. One of these compounds, NSC 103003, was found to be a DNA intercalator, suggesting that inhibition was due to compound-induced perturbation of DNA structure and not an effect on GyrA dimerization. In contrast, NSC 130847 and NSC 20115 are not DNA intercalators, and whether these drugs inhibit gyrase by interfering with dimerization or by steric hindrance of Y122 remains to be tested. The distinct chemical structures of NSC 130847 and NSC 20115 compared with those of fluoroquinolone derivatives (Fig. 1 and 3) and their predicted orientations in binding at the GyrA dimer interface (outside the QRDR) (Fig. 2) suggest that optimized derivatives might potentially be active against fluoroquinolone-resistant strains of E. coli.

In addition to targeting the A subunit of DNA gyrase, we used molecular docking in an attempt to identify several small-molecule inhibitors that putatively target the ATP binding pocket of the B subunit of DNA gyrase. This region is a well-established target site for coumarin and cyclothialidine drugs. Structural analyses have shown that the binding of these two drug classes to GyrB is stabilized by several critical hydrogen bonds involving D73 and a conserved water molecule in the ATP binding site (reviewed in reference 35). Previous in silico screening studies have focused on the identification of small-molecule inhibitors that utilize these two key hydrogen bonds (3, 32, 40). This contrasts with our study, which did not require small-molecule inhibitors to form these two hydrogen bonds. Since we did not constrain the molecular docking search by imposing interactions with D73 and an ordered water molecule that participates in binding to known GyrB inhibitors, this study differs significantly from all previous studies that employ molecular docking on DNA gyrase (3, 32, 40). Interestingly, our top-scoring compound (NSC 20116) is predicted to be positioned in the adenosine binding site and to interact with D73 (Fig. 6), validating our current approach as an alternative method for identifying novel inhibitors. However, in contrast to coumarin and cyclothialidine inhibitors, NSC 20116 is not predicted to interact with R136, which is the most frequently mutated residue conferring coumarin resistance in DNA gyrase (7, 10, 35). This suggests that resistance due to R136 mutations is not likely to confer cross-resistance to NSC 20116. The molecular docking results also suggest that the top-scoring compound, NSC 20116, is positioned in the ATP binding pocket such that there is space available for lead derivatization and development of potent, novel, specific DNA gyrase inhibitors (Fig. 4D).

Interestingly, although the ATP binding pockets of DNA gyrase and topoisomerase IV are highly conserved, coumarin drugs are typically 5- to 50-fold less active against topoisomerase IV. Recent studies suggest that coumarin resistance of E. coli topoisomerase IV is due primarily to a methionine residue at position 74 (equivalent to I78 in E. coli DNA gyrase) (2). When isoleucine is exchanged for methionine in the ParE subunit of topoisomerase IV, the IC₅₀ of novobiocin decreases from 210 to 12 nM (an 18-fold decrease), which is less than the IC₅₀ of novobiocin for E. coli DNA gyrase (46 nM). X-ray analysis of the gyrase/novobiocin complex indicates that I78 has a hydrophobic interaction with the novobiose sugar moiety of novobiocin (Fig. 5A) (30). Although M74 occupies a nearly identical position in the ATP binding site of topoisomerase IV ParE, the M74 side chain is no longer close enough to interact with and stabilize the binding of novobiocin, providing an explanation for the difference in potency of novobiocin against gyrase and topoisomerase IV (2). Docking analysis of NSC 20116 in the active site of DNA gyrase also shows a potential interaction with isoleucine 78, suggesting that this compound, like novobiocin, may have decreased activity against topoisomerase IV. Additional docking analysis that takes this parameter into consideration may allow the identification of drugs that can effectively target both DNA gyrase and topoisomerase IV. A drug that dually targets both enzymes is likely to be a more potent inhibitor of bacterial growth and less susceptible to drug resistance that arises due to mutations in DNA gyrase. This type of an approach has recently led to the identification of several aminobenzimidazoles, VRT-125853 and VRT-752586, that dually inhibit the ATPase activities of both DNA gyrase and topoisomerase IV (17, 33).

NSC 20116 completely blocked E. coli growth within 30 min at 192 µM, a concentration that inhibits purified DNA gyrase activity by approximately 30%. There was a significant dose-dependent delay in growth inhibition by NSC 20116 at concentrations of <192 µM. At 64 and 21 µM, growth inhibition was not observed until 30 to 60 min following drug treatment. The DNA gyrase inhibitor novobiocin has also been reported to cause a delayed inhibition of bacterial growth (4). The mechanism underlying the delay in growth inhibition at lower drug concentrations is presently unclear. However, in addition to its role in DNA replication, DNA gyrase also functions to modulate gene transcription by regulating DNA topology/supercoiling (reviewed in reference 47). Recent microarray analyses indicate that novobiocin changes the expression of many genes (16, 42). Possibly, the delayed growth inhibition at lower drug concentrations is due to decreased expression of some essential function that eventually becomes growth limiting following one or more cell divisions.

This study demonstrates a novel application of virtual screening methods, which are now traditionally used in drug discovery. The availability of large libraries of compounds with known structures permits the sampling of an ever-growing number of candidates for lead compounds (more than 3 million are currently available). The accuracy of molecular docking methods is continuously improving due to the increased quality of structural information, primarily from solved crystal structures, available on drug targets. A major goal of structural genomics efforts is to define all protein folds to permit accurate structural models of all proteins. As knowledge of drug target structures and of the ligand-receptor interaction becomes increasingly available, a new level of specificity can be achieved by in silico screening using large ligand databases. The potential identification of small-molecule inhibitors by using this approach is further enhanced by the availability of more than 3 million structures in databases such as ZINC (21), which now includes the compounds in the NCI/DTP repository used in this study.

Significant limitations to existing virtual screening methods include long computation demands and false-positive hits resulting from inevitable inaccuracies in the scoring functions. In addition, small molecules have aggregation effects on functional assays, which can result in false-positive results. In this study, we addressed the limitations of virtual screening methods by relaxing search parameters, thus reducing computational time, by using a combination of scoring algorithms, and by testing the selected compounds in activity assays in the presence and absence of detergent. The search parameters for molecular docking typically position each potential ligand in tens of thousands of orientations in the selected site (1). In comparison to other molecular docking studies, this study found that far fewer orientations were needed to identify active compounds. In addition, the scoring functions in this study were established with a priority on the van der Waals contact score (nonpolar interactions), with electrostatic potential (polar interactions) having a secondary priority. The oriented compounds were ranked by the overall energy score, which is the sum of van der Waals and electrostatic contributions. Using the latest development version of DOCK v5.1.0, this set of parameters yielded several active inhibitors in the 10 highest-scoring compounds out of approximately 140,000. In contrast, the identification of novel inhibitors in other virtual screening studies required the testing of 100 to 1,000 compounds (43). Molecular docking, using the parameters described in this study, resulted in hit rates comparable to those previously observed (20 to 80%), which are significantly higher than those obtained by random screening (0.2 to 2.4%) (11, 49). These data demonstrate that this virtual screening method is a more rapid, economical, and accurate approach than previously considered.

In this study, we identified novel targets for drug interaction that differ from existing drug binding structural pockets in DNA gyrase. Compounds selected by high-throughput molecular docking, and their derivatives, may be able to overcome resistance to available DNA gyrase inhibitors (i.e., quinolone drugs). Furthermore, in addition to identifying compounds that may circumvent known mechanisms of antimicrobial drug resistance, this type of virtual screening method is likely to become an essential tool for identifying novel lead compounds which modulate the activity of important therapeutic targets for a broad range of human diseases.

 
We thank Tony Maxwell for providing the DNA gyrase expression constructs used in these studies.

D.A.O. is currently supported by NIH R21 HL080222 and Cure Autism Now Foundation 2908051-12.

 
* Corresponding author. Mailing address for David A. Ostrov: Department of Pathology, Immunology and Laboratory Medicine, University of Florida, College of Medicine, Gainesville, FL 32610. Phone: (352) 273-8166. Fax: (352) 273-8285. E-mail: ostroda{at}pathology.ufl.edu. Mailing address for Thomas C. Rowe: Department of Pharmacology and Therapeutics, P.O. Box 100267, Gainesville, FL 32610. Phone: (352) 392 3530. Fax: (352) 392-9696. E-mail: tomrowe{at}ufl.edu

Published ahead of print on 6 August 2007.

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Antimicrobial Agents and Chemotherapy, October 2007, p. 3688-3698, Vol. 51, No. 10
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