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JAC Advance Access originally published online on June 19, 2008
Journal of Antimicrobial Chemotherapy 2008 62(4):720-729; doi:10.1093/jac/dkn261
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© The Author 2008. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Original research

Resistance mapping and mode of action of a novel class of antibacterial anthranilic acids: evidence for disruption of cell wall biosynthesis

John E. Mott1,2, Bailin A. Shaw3, James F. Smith3, Paul D. Bonin1,3, Donna L. Romero1,4, Keith R. Marotti1,5 and Alita A. Miller1,3,*

1 Infectious Diseases Biology, Pharmacia Corporation, 301 Henrietta Street, Kalamazoo, MI 49007, USA 2 Pfizer Global Research and Development, 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA 3 Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, USA 4 Pharma-Vation Consulting LLC, 1201 Turnberry Ridge Court, Chesterfield, MO 63005, USA 5 Pfizer Veterinary Medicine, 301 Henrietta Street, Kalamazoo, MI 49007, USA

* Correspondence address. Antibacterials Biology, Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, USA. Tel: +1-860-686-6808; Fax: +1-860-715-4693; E-mail: alita.a.miller{at}pfizer.com

Received 16 March 2008; returned 3 April 2008; revised 29 May 2008; accepted 2 June 2008


   Abstract
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Objectives: The aim of this study was to characterize the mechanism of action of a novel class of bacterial protein synthesis inhibitors identified in a high-throughput coupled transcription–translation assay.

Methods: Evaluation of the cross-resistance to antibiotics with known mechanisms of action, resistance mapping and biochemical characterization of a novel class of antibacterial anthranilic acids was performed.

Results: No cross-resistance to established classes of antibiotics was found. Resistance was mapped to SA1575, an essential, integral membrane protein predicted to be involved in polysaccharide biosynthesis. Biochemical analysis demonstrated the inhibition of cell wall biosynthesis.

Conclusions: This novel class of antibacterial anthranilic acids inhibits cell wall biosynthesis. Resistance mapped to SA1575, which may represent a novel target for antibacterial drug discovery.

Keywords: Staphylococcus aureus , antibiotic discovery , HTS


   Introduction
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 Introduction
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As currently emphasized in both popular and scientific literature, the growing threat of antibiotic-resistant bacteria renders the need for novel antibacterial agents ever more urgent. Bacterial protein synthesis remains an attractive target for antibiotic discovery efforts due to the lack of homology to higher systems and the many examples of marketed antibiotics within this class. Several recent reports have described antibacterial translation inhibitors with novel mechanisms of action found through high-throughput screening (HTS) efforts, which merit additional characterization.1–4 One such report from our labs described a class of anthranilic acid derivatives discovered from an HTS of a coupled transcription–translation (TT) assay.4 This effort arose from the identification of a chemically attractive hit compound with modest activity both in the enzymatic assay and in the inhibition of bacterial growth.4 The presence of easily modified carboxamide and sulfonamide bonds allowed for extensive structure–activity relationship (SAR) assessment through parallel medicinal chemistry. One- and two-dimensional libraries led to improved potency both in the in vitro assay and in bacterial killing (although the spectrum remained limited to Staphylococcus aureus strains). Further structural modification involved exploration of the central aromatic ring and the sulfonamide amine.5,6 Although antibacterial potency was greatly enhanced, with a number of compounds showing activity at ≤1 mg/L, and a number of these compounds retaining some correlation between TT inhibition and antibacterial activity, there were some significant areas of diversion where compounds retained antibacterial activity with a concomitant loss of potency in the TT assay. This discrepancy suggested that additional or alternative mechanisms of action could be the cause of the enhanced antibacterial activity. Biochemical and genetic studies were undertaken to examine this hypothesis.


   Materials and methods
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Strains and vectors

A list of strains and vectors used in this study is presented in Table 1.


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  		Table 1. Summary of strains and vectors used in this study

 
Media and reagents

The medium for cloning and maintenance of cells containing recombinant plasmids was Luria–Bertani (LB) broth (purchased as a pre-formulated powder from Difco Laboratories), supplemented with 10 mg/L erythromycin or 1 mg/L TT inhibitor (either compound C or D). B2 broth is 10 g/L casein hydrolysate, 25 g/L yeast extract, 1.23 g/L K2HPO4·3H2O, 5 g/L glucose and 25 g/L NaCl, pH 7.5, with NaOH. Mueller–Hinton broth (MHB) was purchased as a pre-formulated powder from Difco. All control antibiotics were purchased from Sigma. Linezolid and TT compounds A–E4 (Figure 1) were synthesized at Pharmacia.


Figure 1
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  		Figure 1. Translation inhibitors used in this study and representative SAR (taken from Larsen et al.4). Activities are shown in parentheses [percentage inhibition of the in vitro transcription–translation (TT) assay at 100 µM, S. aureus MIC in mg/L].

 
Transcription–translation assay

The preparation of S30 ribosomal extract and the TT assay were performed as described by Murray et al.9

MIC determination

The MIC of a drug necessary to inhibit bacterial growth was determined by broth microdilution according to the standards of the CLSI (formerly the NCCLS).10

Generation of resistant strains

Either ethane methyl sulfonate (EMS)-mutagenized (prepared according to Miller11) or untreated (for spontaneous resistance) S. aureus cultures were grown at the desired temperature with aeration to saturation and plated on Mueller–Hinton agar plates containing compound C or D at the concentrations indicated in the text. Individual-resistant colonies were confirmed by re-streaking, and subsequent MIC analyses were performed as shown in Tables 2 and 3.


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  		Table 2. MICs of antibiotics with known mechanisms of action for S. aureus compound C-resistant isolates

 


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  		Table 3. Antibacterial activity of compounds C and D against S. aureus wild-type or compound C-resistant strains transformed with vector bearing either the wild-type (wt) or A48T SA1575 gene

 
DNA manipulations

Plasmids were isolated from cultures grown overnight using Qiagen columns according to the manufacturer's instructions. All plasmid digestions were performed with New England BioLabs restriction enzymes.

PCR conditions

All oligos used in these studies were synthesized and phosphorylated by Genosys [listed in Table S1, available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/)]. PCR was performed using the Applied Biosystems Taq Gold DNA polymerase kit and standard methods, except when the final concentration of MgCl2 was 3 mM. Invitrogen platinum Taq was used for the cloning of the SA1575 and SA1575R15 genes. All PCR products were cloned using the PCR2.1 TA cloning kit from Invitrogen, according to the manufacturer's instructions.

Competent cell preparation and electroporation of S. aureus

Competent cells of S. aureus were prepared and electroporated following the method of Schenk and Laddaga.12

Phage transduction

LB broth and plate media were used for the growth of the Staphylococcus transducing phage {varphi}1113 and for transduction experiments. LB medium was supplemented with 50 mM CaCl2, which was required for phage propagation. The growth and transduction of {varphi}11 was performed using the protocol for Escherichia coli P1 with some modifications. Phage lysates grown on donor cells were clarified by filtration using Millipore Millex-HV 0.45 µm filter units. For erm(B) transduction, cultures were grown to mid-log phase. The {phi}11 phage was serially diluted in LB, and 100 µL of the phage sample was added to 10 mL aliquots of the culture. The infected cells were incubated for 15 min at 37°C, and the cells were centrifuged, washed once with an equal volume of LB medium, kept in 1 mL of LB broth that was added to 4 mL of 55°C LB top agar and overlaid on an LB plate supplemented with 10 mg/L erythromycin. Control ‘transduction’ with cells only (no added phage) or phage only (no added cells) was included. Transduction plates were incubated for 2 days at 37°C. The scoring of the co-transduction of the resistance phenotype was accomplished by sequentially patching transduced colonies onto LB plates supplemented with erythromycin, and LB plates containing 1 mg/L compound C. For co-transformation of the TT-resistant mutant to a wild-type strain, the percentage co-transduction was measured as a ratio of the number of resistant transductants to the total number of transductants tested. For co-transduction from a non-resistant strain, the percentage co-transduction was scored as a ratio of susceptible transductants to the total number of transductants tested.

Tn917 resistance mapping

The RN4220 (pID408ts) was streaked on an LB plate containing 10 mg/L erythromycin, and 20 individual colonies were selected and grown in 4 mL of LB broth at 30°C. Aliquots of 0.5 mL of the overnight cultures were used to subculture the same medium at 43°C. The cultures were allowed to grow for 3 days. They were diluted again and allowed to grow until they reached a stationary phase. This process was repeated several times with a decreasing volume of the inoculum, until all 20 cultures grew to saturation in ~2 days. The cultures were pooled into four groups of five individual cultures. One millilitre of culture from each pool was plated on LB plates with 1 mg/L compound D. The plates were incubated and compound D-resistant cells were scraped from the selection plate to make a second pooled culture. This second pool was used to generate a {phi}11 lysate from each of the four pools (I– IV). The {phi}11 phage lysates were used to transduce RN4220. Two series of transductions were performed. The first series consisted of selecting on LB with 10 mg/L erythromycin, and the second series consisted of plating on LB supplemented with 10 mg/L erythromycin and 1 mg/L compound D.

Resistance mapping using insertional suicide vectors

The insertional suicide vectors were constructed using pUC19-ERM. Nine insertional suicide pUC19-ERM vectors were constructed by cloning a PCR amplicon from a predetermined chromosomal site flanking SA1578 [oligos listed in Table S1, available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/)]. The primers used for the chromosomal amplicon contained either an XbaI or a HindIII restriction site, so the amplicon could be cloned just downstream of the erm(B) gene. The orientation of the amplicon was chosen so that the erm(B) promoter would compensate for possible transcriptional interruptions generated by the insertions. When possible, insertions were placed between genes to avoid potential problems of gene essentiality. Insertions between genes were given a designation referring to both the adjacent genes. The amplicons were cloned into pCR-2.1, and then cloned from these vectors into pUC19-ERM. These suicide vectors were used to transform competent RN4220 cells selecting for erythromycin resistance. Insertion of the suicide vectors at the desired genomic site was confirmed by PCR. A {phi}11 stock was prepared for all nine RN4220 insertion strains. The phage was used to transduce the insertion into the compound C-resistant strain. Colonies from each of the transformations were selected and patched onto LB plates, and LB plates containing 1 mg/L compound C. The percentage of susceptible colonies compared with the total number of colonies tested was determined.

Genomic DNA isolation

Aliquots (1.5 mL) of overnight cultures grown at 37°C in LB were centrifuged and resuspended in 300 µL of TE (Tris 10 mM, EDTA 1 mM, pH 8.0). Ten microlitres of lysostaphin stock solution (10 mg/mL lysostaphin in 20 mM Na acetate, pH 4.5) was added, and the cells were incubated for 30 min at 37°C. Following the addition of 55 µL of 10% SDS and 10 µL of 20 mg/mL solution of proteinase K, the cells were incubated again for 30 min at 37°C. Eighty microlitres of 5 M NaCl and 70 µL of CTAB (cetyl trimethyl ammonium bromide)/NaCl (0.7 M NaCl, 10% w/v CTAB) was then added, and the cells were incubated for 10 min at 65°C. The lysed cells were extracted once with phenol/chloroform/isoamyl alcohol before alcohol precipitation of the DNA.

Sequencing

PCR-generated DNAs (using Invitrogen Platinum Taq) were sequenced directly using an ABI3700 fluorescence sequencer (Applied Biosystems Inc., Foster City, CA, USA) and the ABI BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit Version III (Applied Biosystems Inc.) by the Pharmacia Sequencing Core.

Cloning of SA1575 and SA1575R

The SA1575 gene was isolated by PCR amplification of chromosomal DNA preparations from the RN4220 parental and resistant mutant strains, and cloned into the Invitrogen pCR2.1 TA vector. The intergenic region upstream of the SA1575 gene was included in the amplicons to encompass the SA1575's promoter. The clones were verified by sequencing before subcloning into the S. aureus/E. coli shuttle vector pLI50 as an XbaI/HindIII fragment.

Macromolecular synthesis assay

Inhibition of specific macromolecular synthesis pathways was assessed by measuring the incorporation of radiolabelled precursors in a growing culture of S. aureus as follows. An array of pre-mixed precursors and drugs was prepared in a 96-well plate. Antibiotic stocks were added to give a final concentration range of 0–20x the MIC. 14C-radiolabelled precursors (Amersham) were distributed to the wells containing drugs such that each compound tested was mixed with precursors for each macromolecular pathway as follows: DNA, 5 µL of a 1:2 dilution of thymidine (50 µCi/mL, cat # CFA-219); RNA, 5 µL of a 1:25 dilution of uridine (50 µCi/mL, cat # CFB-51); protein, 7.5 µL of undiluted L-leucine (50 µCi/mL, cat # CFB-183); fatty acid, 5 µL of a 1:2 dilution of sodium acetate (200 µCi/mL, cat # CFA-14); and cell wall, 5 µL of a 1:25 dilution of N-acetyl glucosamine (200 µCi/mL, cat # CFA-485). The final volume of the pre-mixture was adjusted to 10 µL, with final DMSO concentration being no greater than 0.5%. MHB was inoculated with an overnight culture of RN4220 and incubated at 200 rpm at 37°C to an OD600 of 0.4–0.5. An aliquot of 100 µL of this culture was added to the drug/precursor pre-mixture plate and incubated for 25 min with gentle shaking at 35°C. Following incubation, 100 µL of 25% TCA was added to each well, and the plates incubated on ice for 1 h. Incorporated counts were harvested with the Packard FilterMate-96 harvester using ‘UniFilter GF/B’ filter plates. The filter plates were pre-washed with 5% TCA, samples were filtered through, then washed twice with ice-cold 5% TCA. A final wash with 10% ETOH was performed, and the plates dried under a heat lamp for 5 min, then allowed to dry for an additional ~10 min at room temperature. The bottoms of the plates were sealed, and 40 µL of MicroScint scintillation fluid was added per well. The plates were then top-sealed with MultiScreen sealing tape and counted on the TopCount, 1 min per well. Experiments were conducted at least in duplicate. Data analysis was performed using GraphPad Prism.

Peptidoglycan extraction and HPLC analysis

This procedure is a modification of the technique described by Billot-Klein et al.14 S. aureus RN4220 was grown in MHB at 37°C with agitation (~200 rpm). After an overnight incubation, the culture was diluted to an OD600 of 0.3, and then re-incubated at 37°C until OD600 reached ~0.8. At that time, the compound of interest as shown in Figure 4 (at a concentration equal to 2x its MIC in a final volume of 1 mL, with MICs of vancomycin, linezolid and compound D of 1, 2 and 0.03 mg/L, respectively) was added to the cells and they were further incubated at 37°C for 30 min. The cells were then centrifuged and the cell pellet was rapidly resuspended in 250 µL of ice-cold 1 M formic acid. The cells were incubated for 30 min on ice and then centrifuged. Peptidoglycan precursors contained in the acid extract (supernatant) were analysed by HPLC with a µ-Bondpack C18 column (3.9 x 300 mm) from Waters Corp. (Milford, MA, USA) equilibrated in 10 mM ammonium acetate, pH 5.0. The column was eluted with a 0% to 2% acetonitrile gradient (prepared in the equilibration buffer; 35 min gradient at 0.5 mL/min). OD was monitored at 262 nm. The identities of UDP-GlcNAc, UDP-MurNAc-l-Ala, UDP-MurNAc-tripeptide and UDP-MurNAc-pentapeptides were confirmed in control experiments (data not shown) by negative ion electrospray, using a Quattro II quadrapole mass spectrometer. Peaks in the present study were identified based on retention time compared with these controls. Experiments were conducted at least in triplicate.


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Antibacterial activity versus inhibition of translation in vitro

Figure 1 shows the compounds derived from the exploration of the SARs4 under investigation in this study. Compound A is the original hit compound with modest inhibitory activity both in the enzymic assay (30% inhibition of the TT assay at 100 µM) and on bacterial growth (MIC of 16 mg/L). Efforts in initial exploratory medicinal chemistry involving several hundred compounds led to a more than doubled potency in vitro and in antibacterial activity as exemplified by compound B and others.4 However, additional efforts began to reveal a trend of disparate activities: although antibacterial potency was greatly enhanced, with a number of compounds being active at ≤1 mg/L, some disparity was observed between this activity and that observed in the TT assay (compare representative compounds C–E). This discrepancy suggested that additional or alternative mechanisms of antibacterial activity may be in play. Genetic and biochemical studies were undertaken to examine this hypothesis.

Characterization of resistant mutants

Compound C was initially chosen as a good representative of this class (due to its potency in the TT activity and its low MICs) for use in the selection and mapping of S. aureus-resistant mutants. RN4220 was plated at various concentrations on selective medium and grown at 37°C for 2 days. EMS-generated resistant colonies appeared at a frequency of ~1 in 105 cells; spontaneous resistance occurred at a frequency of ~1 in 2 x 107 cells, suggesting that a single mutation may be responsible for the resistance phenotype. To assess whether or not resistance was associated with the TT assay, S30 ribosomal extracts were prepared from RN4220 parent and resistant strains, and their activity was measured9 in the presence and absence of compound C: no change was observed in the susceptibility of the S30 extracts to compound C in the resistant strains (data not shown).

MICs for RN4220 and two different compound C-resistant mutants were determined for 14 antibiotics with known mechanisms of action, which are representative of known inhibitors from various drug classes (Table 2), in order to characterize the mechanism and specificity of the antibacterial activity of the class. Cross-resistance between C and other members of the class was evident, while no significant changes in MICs were observed with other classes of antibiotics. For a few antibiotics, a single dilution variation was observed compared with the parental strain, which is considered a normal variation in the assay. The lack of cross-resistance indicated that changes in either a multisubstrate efflux pump or alterations in cell wall integrity were probably not the source of the resistance, since either of these phenotypes would probably have conferred resistance to more than one class of compounds.

Compound C-resistant strains were cross-resistant to other members of compounds in this class (Table 2), suggesting that the compound C-resistant mutants harboured a general mechanism of resistance for this class. Because the mutation frequency suggested that a single mutation may be responsible for the resistance phenotype, and since the mutation did not affect the IC50 in the TT assays (i.e. those performed using S30 ribosomal extracts from resistant mutants), resistance mapping was undertaken to determine the source of the mutation.

Chromosomal resistance mapping

Due to a limited inventory of compound C, compound D was then selected to continue the resistance mapping studies. This compound was selected because it was quite similar in both structure and activity in the TT assay, with more potent antibacterial activity. This also allowed for characterization of class-specific cross-resistance. Classic transposon resistance mapping to compound D was performed by a series of Tn917 transductions as described in the Materials and methods section. The erythromycin transductions produced >100 transductants for each pool. The erythromycin/compound D selection resulted in transductants for only pool II. This suggested that pool II contains Tn917 either in the compound D resistance gene or adjacent to it. Fifty-one erythromycin-resistant transductants from pool II were patched onto an LB plate containing both erythromycin and compound D. Eighteen of the colonies were resistant for a co-transduction frequency of 35%. One RN4220 erythromycin-resistant, compound D-susceptible strain was purified and stored. Four RN4220 erythromycin-resistant, compound D-resistant strains were purified. These strains were used to determine whether the Tn917 transposon was integrated either into the resistant gene or adjacent to the resistant gene. A {phi}11 lysate was prepared from each of the four isolates, and they were used to transduce RN4220 selecting for erythromycin resistance and tested for the frequency of compound D resistance. The number of transductants was low, but the results show that the transposon is adjacent to the resistant gene with an over co-transduction frequency of 32%. It appeared that each mutant isolated contains very similar transposon insertions since the co-transduction frequencies were very similar (ranging from 25% to 40%). The Tn917 was designated atr::Tn917 for adjacent to translation inhibitor resistance.

The next set of transductions were conducted to determine that the compound D resistance gene linked to the atr::Tn917 insertions was linked to the compound C resistance phenotype isolated in other independent selections. A {phi}11 phage lysate was prepared from RN4220 atr::Tn917 compound D-susceptible strains taken from the original transduction and used to transduce a strain resistant to compound C. Thirty-seven colonies from this transduction were tested for compound D susceptibility. Nine of the 37 colonies were susceptible to compound D for a co-transduction frequency of 24%. The similarity of the co-transduction frequency suggests that the atr::Tn917 insertion is also adjacent to the gene conferring resistance to compound C. Sequence analysis of genomic DNA isolated from RN4220 atr::Tn917 compound C- or D-resistant strains showed that the transposons were both located near the SA1578 gene (based on the sequence from the S. aureus strain N31515).

Resistance maps to SA1575

Finer structure mapping with a suicide plasmid was conducted to define the location of the gene conferring resistance, as described in the Materials and methods section. The general approach was to make insertions of DNA with an erythromycin resistance marker at specific sites in the chromosomal region of the SA1578 gene and to determine the co-transduction frequency between the insertion and the TI resistance gene using the {phi}11 phage. A schematic of the region conferring the resistance, showing the genes to scale and the location of the inserts as determined by both co-transduction (shown in percentages) and finer sequence mapping, is shown in Figure 2. The co-transduction frequencies showed that the resistant mutant was in the SA1570 to SA1575 gene region. Genomic DNA sequencing revealed a single G to A transition at position 1758 in the mutant versus wild-type sequence. The base pair change causes an alanine to threonine codon change (GCT to ACT) at position 48 of the SA1575 protein. The SA1575 genes were sequenced from two independently isolated spontaneous resistant mutants of the S. aureus strain SAUR9218 selected against compound D and were also found to contain only the A48T point mutation. The occurrence of this mutation in three independent strains (one EMS-generated and two spontaneous) suggests that this change is the predominant mechanism of resistance to this class of compounds.


Figure 2
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  		Figure 2. Summation of the co-transduction frequencies between suicide vector insertions and the TT resistance gene used to map resistance to a region near SA1578 as described in the Materials and methods section.

 
Overexpression of SA1575 confers resistance

The SA1575 genes from the wild-type and SA1575 A48T mutant RN4220 were cloned into the pLI50 shuttle vector. The MICs of compound C or D for wild-type and resistant strains transformed with these constructs were determined (Table 3). Overexpression of the wild-type SA1575 gene resulted in an increase in the MICs of both C and D in a wild-type background, but had no effect on the MIC for the resistant strain. The similar fold changes of resistance to compounds C and D suggest that they utilize the same mechanism of action. Overexpression of the SA1575 A48T mutant caused an increase in the resistance of both the wild-type and resistant strains. The vector alone had no effect on the MIC of compound C or D in either strain. This indicates that the A48T mutation conferring resistance was a dominant phenotype. These results were confirmed independently by multicopy suppression screening, in which resistance to compound C was found to be conferred by a clone overexpressing SA1575 from a genomic library of S. aureus genes (data not shown).

SA1575 is an essential gene of unknown function

The SA1575 gene is an uncharacterized 553 amino acid protein whose predicted function is the biosynthesis and degradation of surface polysaccharides and lipopolysaccharides (http://cmr.tigr.org/). Multiple attempts to inactivate the SA1575 gene by suicide vector insertion were not successful (data not shown). This finding, along with the fact that a ts mutation of this gene has been reported in the Microcide patent US6187541, suggests that SA1575 is essential to the survival of S. aureus. There are homologues of this protein in Enterococcus faecalis (EF0669), Streptococcus pneumoniae (SP1529) and other Gram-positive bacteria, but no close homologues are present either in the Gram-negative or human genome databases. Protein analysis suggests that the protein is an integral membrane protein with 14 transmembrane helices. It also contains a polysaccharide synthesis motif from residue 7 to 326 and a MatE domain from residue 300 to 425.16

Antibacterial activity is linked to inhibition of cell wall synthesis

The effect of compound D on macromolecular synthesis pathways in S. aureus was determined by measuring the relative incorporation of radiolabelled precursors as a function of drug concentration. As seen in Figure 3(d), treatment with compound D clearly resulted in the inhibition of cell wall biosynthesis as a function of drug concentration: no other pathways were inhibited (control compounds are shown in Figure 3a–c). Treatment with compound D also resulted in a slight increase in both DNA and protein synthesis, which is a phenotype that we have observed with a number of known cell wall inhibitors, such as D-cycloserine (Figure 3c) and others (data not shown). To expand on these results, the effect of this compound on peptidoglycan precursor accumulation in treated cells was analysed. This accumulation phenotype is a hallmark of known cell wall inhibitors14 such as vancomycin (Figure 4a). In contrast, most compounds targeting other pathways in bacteria do not demonstrate this phenotype, as is shown for the protein synthesis inhibitor linezolid (Figure 4b). A significant accumulation of precursors was seen in the presence of compound D (Figure 4c), suggesting that it specifically inhibits cell wall biosynthesis. In addition, treatment of the compound D-resistant RN4220 strain (whose resistance had been mapped to SA1575) with vancomycin still resulted in the accumulation of peptidoglycan precursors (Figure 4d). This suggests that the SA1575 A48T does not confer resistance to vancomycin. Therefore, it appears that the mechanism by which this class of compounds inhibits cell wall biosynthesis is distinct from that of vancomycin.


Figure 3
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  		Figure 3. Inhibition of S. aureus macromolecular synthesis pathways. The ability to inhibit specific macromolecular pathways was assessed by analysis of incorporation of radiolabelled precursors of either fatty acid, DNA, RNA, protein or cell wall synthesis by S. aureus growing in culture in the presence of a given compound (as a function of concentration). Controls are shown in (a) ciprofloxacin, a DNA synthesis inhibitor, (b) tetracycline, a protein synthesis inhibitor and (c) D-cycloserine, a cell wall inhibitor. Activity of compound D is shown in (d).

 


Figure 4
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  		Figure 4. Accumulation of cell wall precursors in drug-treated S. aureus. Treatment of wild-type S. aureus with compound D (shown in c) results in the accumulation of peptidoglycan precursors similar to vancomycin (shown in a), indicating specific inhibition of cell wall biosynthesis. By way of comparison, a protein synthesis inhibitor (linezolid) has no effect on the accumulation of precursors (shown in b). The effect of vancomycin on compound D-resistant S. aureus (shown in d), whose resistance was mapped to SA1575, is unaffected, suggesting distinct mechanisms of inhibition are at play. The identity of individual precursors [such as UDP-MurNAc (single asterisk) and UDP-MurNac-pentapeptide (double asterisk)] as noted in (a) was confirmed as described in the Materials and methods section.

 

   Discussion
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Mechanisms of drug resistance include changes in cellular permeability, increased drug efflux, chemical modification of the drug and/or alteration of the drug target.17,18 The data presented above show that the SA1575 gene confers resistance to a series of novel antibacterial compounds originally isolated as translation inhibitors, either by its overexpression or generation of an A48T point mutation within the coding sequence. Cross-resistance studies suggest that this mutation does not alter the integrity of the bacterial cell membrane or increase overall efflux because the only cross-resistance that was observed was specific to this class of compounds (Table 2); alterations in efflux or membrane integrity would probably lead to non-specific cross-resistance to at least several drug classes. The A48T mutation maps to a putative polysaccharide synthesis motif that is ubiquitous in Gram-positive bacteria and is found in integral membrane proteins. It is therefore tempting to speculate that these compounds act by directly inhibiting the enzymatic activity of this essential protein and that the A58T-mediated resistance results from alteration of the active site in response to these inhibitors. Related proteins with similar motifs are known to be involved in capsular polysaccharide synthesis in Gram-positive and Gram-negative bacteria, and O-antigen and LPS biosynthesis in Gram-negative bacteria.19–21 However, these motifs are not found in eukaryotes. Thus, although the antibacterial activity of the compounds presented here is limited to S. aureus, this compound class may eventually be adaptable to a wide variety of bacterial targets. Biochemical analyses of the mechanisms of action of these compounds provided additional evidence that they inhibit bacterial growth by specifically targeting cell wall biosynthesis (Figures 3 and 4). Indeed, we found no evidence of inhibition of translation through either genetic or biochemical analysis of the mechanism of action of this class of compounds, despite the fact that they were originally isolated as inhibitors of translation.4 Although some divergence between in vitro activity and antibacterial activity began to emerge as the series was modified to enhance antibacterial activity and other properties (such as reduced protein binding), the majority of the compounds in the series retained their activity in the in vitro translation assay. Therefore, the contribution of the observed translation inhibitory activity to the overall antibacterial effect remains unclear. Because ribosomes isolated from the resistant strains were equally susceptible to these inhibitors as those from wild-type strains (data not shown), it is likely that SA1575 does not play a direct role in bacterial translation.

In conclusion, these studies indicate that the major mode of action of this compound class is the disruption of bacterial cell wall biosynthesis. Future studies will be required to establish whether or not there is a secondary mode of action related to the inhibition of translation as observed in the in vitro assay that originally led to the discovery of this novel class of antibacterials.


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These studies were conducted at and funded by Pfizer Global Research and Development.


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All authors are either current or former employees of Pfizer and own shares in the company.


   Supplementary data
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Table S1 is available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).


   Acknowledgements
 
We wish to acknowledge Don Povendo for his contributions to mapping resistance to SA1575, Leanne M. Seaver, Kathleen A. Schellin, Roger F. Drong, Earl G. Adams and Jerry L. Slightom of the DNA sequencing core at the former Pharmacia and Atli Thorarensen for his critical reading of the manuscript.


   References
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