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Antimicrobial Agents and Chemotherapy, June 2003, p. 2051-2055, Vol. 47, No. 6
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.6.2051-2055.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Inhibitors of Pantothenate Kinase: Novel Antibiotics for Staphylococcal Infections

Anthony E. Choudhry,1 Tracy L. Mandichak,1,{dagger} John P. Broskey,1 Richard W. Egolf,1,{dagger} Cynthia Kinsland,2 Tadhg P. Begley,2 Mark A. Seefeld,1 Thomas W. Ku,1 James R. Brown,1* Magdalena Zalacain,1 and Kapila Ratnam1*

Microbial, Musculoskeletal and Proliferative Diseases and Bioinformatics, GlaxoSmithKline Pharmaceuticals, Collegeville Pennsylvania 19426,1 Department of Chemistry and Chemical Biology, Cornell University, Ithaca New York 148502

Received 22 November 2002/ Returned for modification 20 January 2003/ Accepted 28 February 2003


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ABSTRACT
 
Pantothenate kinase (CoaA) catalyzes the first step of the coenzyme A biosynthetic pathway. Here we report the identification of the Staphylococcus aureus coaA gene and characterization of the enzyme. We have also identified a series of low-molecular-weight compounds which are effective inhibitors of S. aureus CoaA.


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TEXT
 
Increasing reports of antibiotic resistance involving opportunistic gram-positive pathogens, including methicillin-resistant Staphylococcus aureus, have emphasized the critical need for the development of antimicrobial compounds with novel modes of action. Coenzyme A (CoA), an essential cofactor for maintaining life, is used in a multitude of biochemical reactions. In most bacteria, CoA is synthesized from pantothenic acid (vitamin B5) in 5 steps (5), with the first step being the phosphorylation of pantothenate by pantothenate kinase (CoaA). Although this pathway also exists in eukaryotes, in most cases there is no sequence homology between the prokaryotic and eukaryotic CoA biosynthetic enzymes (7, 9, 12, 18, 24, 27). Thus, there is the potential for developing highly specific inhibitors of bacterial CoA enyzmes.

Unlike the case for other biosynthetic pathways of bacteria, the genes involved in CoA biosynthesis are not organized as operons. This has delayed the identification of the enzymes responsible for CoA synthesis, even though the intermediate chemical steps have been known since the 1960s (1). With the recent identification of the Escherichia coli genes encoding the enzymes CoaBC and CoaE, the entire pathway is now known for this organism (9, 10, 13, 19, 21). Interestingly, the gene coaA, which encodes the first enzyme in the pathway, has no homolog in the complete genome sequences of the S. aureus strains Mu50 and N315 (11).

Cloning and purification of S. aureus CoaA. Initially, the coaA gene sequences in S. aureus strains Mu50 and N15 (GenBank accession numbers BA000017 and BA000018, respectively) were identified through searches of the ERGO comparative genomic database (previously WIT) (http://ergo.integratedgenomics.com/ERGO/) (8). We cloned the S. aureus RN4220 coaA gene and overexpressed it using standard techniques (4, 17). S. aureus RN4220 coaA was amplified by PCR, introducing an NdeI site at the start codon and an XhoI site after the stop codon, and cloned into pSTBlue1 using the Perfectly Blunt Cloning kit. The gene was excised by digestion with NdeI and XhoI and ligated into similarly digested pET-28a. The final construct encoded the N-terminal six-His-tagged S. aureus CoaA.

Tuner (DE3) cells were transformed with this construct and grown at 37°C in Luria-Bertani medium-50-µg/ml kanamycin. Protein expression was induced by 500 µM isopropylthio-ß-D-galactoside, and cells were harvested 3 h postinduction. The cell pellet was resuspended and sonicated, and cell debris was removed by centrifugation. The supernatant was subjected to Ni-chelating column chromatography followed by a HiTrap Q Sepharose ion exchange column. Enzyme identity was confirmed through N-terminal sequencing and matrix-assisted laser desorption ionization mass spectrometry and purity was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass corresponded to the predicted mass of 29,096 Da.

Characterization of S. aureus CoaA. To verify that this protein catalyzed the same reaction as E. coli CoaA, its activity was assayed using ATP and pantothenate as substrates. The assay monitored ADP formation by coupling it to pyruvate kinase and lactate dehydrogenase. The activity was measured as a change in absorbance at 340 nm during monitoring of depletion of NADH. This change was observed only in the presence of enzyme and all other components of the reaction mixture, confirming the activity as being the ATP-dependent phosphorylation of pantothenate.

For further analysis of the kinetic mechanism, initial velocities were determined at various concentrations of substrates. Nonlinear regression analysis of the observed rates suggest that S. aureus CoaA proceeds via a Bi Bi mechanism which involves formation of a ternary complex prior to the chemical step occurring (Table 1). It has been suggested that E. coli CoaA functions via a similar kinetic mechanism (20).


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  		TABLE 1. Kinetic parameters of S. aureus CoaAa

Evolutionary relationships of staphylococcal CoaA. Genes orthologous to S. aureus coaA occur in other gram-positive bacteria: the pathogens Staphylococcus epidermidis and Staphylococcus haemolyticus; Oceanobacillus iheyensis, an alkaliphilic, halophilic bacillus living in deep-sea sediments (25); Bacillus anthracis, a soil-dwelling bacterium and the causative agent of the disease anthrax; and its closest relative, Bacillus cereus (Fig. 1). Orthologs also occur in eukaryotes, including mammals. Phylogenetic analysis (Fig. 2) shows that S. aureus-like CoaA proteins are distantly related to eukaryotic CoaA proteins, including the Drosophila cell division protein fumble, although there are several amino acid insertions and deletions which clearly delineate eukaryotes from the bacterial species (2, 16). Its limited distribution in bacteria and yet widespread occurrence in eukaryotes suggest that staphylococcal coaA was horizontally transferred from eukaryotes to bacteria. The high level of sequence, and likely structural, divergence between bacterial and mammalian CoaA should permit the development of S. aureus-like CoaA-specific inhibitors.



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  		FIG. 1. Multiple sequence alignments of homologous CoaA (PanK) sequences. Species are S. aureus (stau), O. iheyensis (ocih), B. anthracis (baan), PanK1 ß isoforms of Mus musculus (mouse), Homo sapiens (human), and Drosophila melanogaster (fly), where the protein is called fumble. The GenBank accession numbers and phylogenetic relationships of these sequences are given in Fig. 2. For columns of residues, the level of conservation corresponds to the shading: dark (100%), medium (80%), and light (60%). Public databases were searched for putative CoaA homologs using BLAST (3). Multiple sequence alignments were initially done using the program CLUSTALW v1.7 (26) and subsequently were manually refined using the program SEQLAB of the GCG v10.0 software package (Genetics Computer Group, Madison Wis.) and GeneDoc v2.6.002 (http://www.psc.edu/biomed/genedoc).



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  		FIG. 2. Phylogenetic tree of bacterial and eukaryotic CoaA (PanK) proteins. Species names in bold type were included in the alignment in Fig. 1. The Drosophila protein fumble and known vertebrate protein families (PanK1-4) are indicated. GenBank accession numbers are given in parentheses. (The sCoaA homolog in B. anthracis strain A2012 is anotated as a hypothetical protein [15]. Preliminary B. cereus and S. epidermidis sequence data were obtained from The Institute for Genomic Research website. [http://www.tigr.org]. The S. haemolyticus sequence is available upon request.) For human and mouse PanK1, the numbers of the identical {alpha} and ß isoforms are both listed. PanK homologs for Takifugu rubripes and Bacillus cereus were found from separate genome searches at GenBank. Phylogenetic analyses used a multiple sequence alignment of 221 conserved amino acids (available upon request). The neighbor-joining tree shown here was constructed from pairwise distances between amino acid sequences calculated using the Dayhoff matrix (PHYLIP 3.6 package [Phylogeny Inference Package], version 3.6a2 [http://evolution.genetics.washington.edu/phylip.html]). Maximum parsimony trees (PAUP4.0b5 package [version 4; Sinauer Associates, Sunderland, Mass.]) were made from 100 random sequence additions yielding a single minimal length tree of 1,030 steps. Maximum likelihood trees (PUZZLE 4.0 software [23]) were based on the JTT substitution matrix, rate heterogeneity estimates of an eight-category gamma distribution model, and {alpha} parameter estimation from the data set. Numbers along the branches show the greater than 50% percent occurrence of nodes in 1,000 bootstrap replicates of neighbor-joining (plain text) and maximum parsimony (italicized text) analyses or 1,000 maximum likelihood quartet puzzling steps (bold text). An asterisk indicates that the node was supported more than 70% by all three methods. The scale bar represents the estimated number of amino acid substitutions per site.

Identification of novel inhibitors of staphylococcal CoaA. The first step in the conversion of pantothenic acid (compound 1a; Fig. 3) to CoA involves the CoaA-catalyzed production of 4'-phosphopantothenic acid (compound 2a; Fig. 3). It has been reported that N-substituted pantothenamides (compounds 1b to e; Fig. 3), derivatives that are structurally similar to pantothenate, possess activity against E. coli (6). Recent studies have demonstrated that compound 1b (Fig. 3) is not an inhibitor of the E. coli CoA pathway enzymes but rather acts as a substrate for E. coli CoaA (22). The conjecture is that while this compound, and the antimetabolite intermediates subsequently formed, are substrates for these enzymes, the ultimate mechanism of antibacterial action is due to the inability of the CoA derivative (ethyldethia-CoA) to form acyl-CoA esters, resulting in disruption of essential downstream pathways (22).



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  		FIG. 3. CoaA biosynthetic transformation.

Given the differences between the sequences of the E. coli and S. aureus CoaA enzymes, we attempted to determine if compound 1b also acted as a substrate for the S. aureus enzyme. Studies with compound 1b and related analogs (compounds 1c to e and 2b; Fig. 3) revealed that most of these compounds are inhibitors of the S. aureus CoaA, with the exception of the N-benzyl derivative of pantothenic acid (compound 1e; Fig. 3). Indeed, it appears that compound 1e is accommodated in the active site in a manner such that it acts as a substrate for the enzyme with a specific activity equal to that of pantothenate (A. E. Choudhry et al., unpublished data). All the other compounds (1b to d and 2b; Fig. 3) inhibited S. aureus CoaA with 50 percent inhibitory concentrations (IC50s) in the low micromolar range (Table 2), indicating their potential as effective inhibitors.


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  		TABLE 2. Potential inhibitors of S. aureus CoaAa

The activity of these compounds against several S. aureus strains was determined by the broth microdilution method. Encouragingly, the compounds with the best IC50s exhibited very good MICs (Table 2). 1e, which is a substrate rather than an inhibitor of CoaA, exhibited no antibacterial activity. Not surprisingly, compound 2b, the phosphorylated form of compound 1b, did not exhibit any antibacterial activity either.

Further, the two compounds exhibiting the best MICs, 1c and 1d, were assessed for their cytotoxicity potential against human HepG2 liver cells as described previously (14). The lowest concentrations causing a >=50% decrease in cell viability) for compounds 1c and 1d are 64 and 128 µg/ml, respectively, indicating that neither compound strongly inhibits the growth of human hepatocytes.

Here, we have identified lead compounds for the development of staphylococcus-specific drugs against pantothenate kinase. Chemical optimization of these molecules could lead to the development of novel drugs that are not compromised by existing resistance mechanisms.


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ACKNOWLEDGMENTS
 
We thank Erick Strauss for synthesis of compound 1b and Gilbert Scott at the Protein Core Facility, GlaxoSmithKline Pharmaceuticals, for performing the protein analysis experiments.


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FOOTNOTES
 
* Corresponding author. Mailing address: Microbial, Musculoskeletal and Proliferative Diseases and Bioinformatics, GlaxoSmithKline Pharmaceuticals, 1250 S. Collegeville Rd., Collegeville, PA 19426. Phone: (610) 917-6399. Fax: (610) 917-7901. E-mail for James R. Brown: james.r.brown{at}gsk.com. E-mail for Kapila Ratnam: kapila.2.ratnam{at}gsk.com. Back

{dagger} Present address: The Pennsylvania State University, Eberly College of Science, University Park, PA 16802-6004. Back


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Antimicrobial Agents and Chemotherapy, June 2003, p. 2051-2055, Vol. 47, No. 6
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.6.2051-2055.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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