Current Enzyme Inhibition, 2011, 7, ???-??? 1

1573-4080/11 $58.00+.00 © 2011 Bentham Science Publishers

Polyphosphate Synthesis as a Target for Novel Antibiotics

Francisco P. Chávez*,1,2, Carlos F. Lagos3, Miguel Reyes-Parada2,4,5, Nicolás Guiliani6 and Carlos A. Jerez2,7

1Systems Microbiology Laboratory, Department of Biology, Faculty of Sciences, University of Chile, Santiago, Chile;

2Millennium Institute for Cell Dynamics and Biotechnology (ICDB), Santiago, Chile;

3Department of Pharmacy, Faculty

of Chemistry, Pontificia Universidad Católica de Chile, Santiago, Chile; 4School of Medicine, Faculty of Medical

Sciences, University of Santiago de Chile, Santiago, Chile; 5Facultad de Ciencias de la Salud, Universidad Autónoma

de Chile, Santiago, Chile; 6Laboratory of Bacterial Communication, Department of Biology, Faculty of Sciences,

University of Chile, Santiago, Chile; 7Laboratory of Molecular Microbiology and Biotechnology, Department of

Biology, Faculty of Sciences, University of Chile, Santiago, Chile

Abstract: Inorganic polyphosphate (polyP) is a biopolymer of tens or hundreds of phosphate (Pi) residues linked by high-energy phosphoanhydride bonds. PolyP has been studied mainly in prokaryotes but it is present in all species of the three domains of life. In bacteria, polyP and its processing enzymes play important roles in cellular metabolism as well as in pathogenesis. The genomes of many bacterial species, including pathogens, encode orthologs of the main polyP-synthesizing enzyme, PPK1. This enzyme has been studied in E. coli and its metabolic inhibitors have been reported. The high degree of identity between the PPK1 orthologs in some of the major pathogenic species has prompted the knockout of their ppk1 genes to determine the dependence of virulence on polyP. Although viable, mutants lacking the ppk1 gene have reduced levels of polyP and exhibit multiple structural, functional and virulence defects.

The emergence of multi-drug resistant (MDR) bacteria is the result of antibiotic overuse. Therefore, novel approaches are much needed to tackle them. One of these combines the reduction of bacterial virulence while simultaneously increasing susceptibility to host defenses instead of killing the pathogen. Considering that no PPK1 orthologs have been identified in higher-order eukaryotes, PPK1 exhibits an enormous potential as a novel target for antimicrobial drug design.

In this review we focus on the current state of the art regarding polyP deficiency in pathogenic bacteria and attempts to design inhibitors targeting enzymes responsible for the synthesis of polyP in bacteria.

Keywords: Polyphosphate, polyphosphate kinase 1, PPK1 inhibitors, antibiotics, chemical biology, pathogenic bacteria, virulence, in silico drug design.

INTRODUCTION

Pathogenic bacteria are the cause of countless pathologies affecting humankind. Pneumonia, typhoid fever, diarrhea, tuberculosis, cholera, and even cancer are only a few examples. Antibiotic development revolutionized the practice of medicine, with a large number of agents available to treat diseases that arise from colonization by pathogenic bacteria. However, although at present a number of effective antibiotic treatments exist, there is increasing evidence that certain strains of bacteria are becoming resistant to one or more antibiotics [1]. With the exception of a few agents, resistance to most antibiotics has been observed only a few years after their introduction into clinical use [2].

Most antibiotics target essential bacterial cell functions or growth processes. Some of them target bacterial cell wall synthesis (penicillins, cephalosporins), others the cell membrane (polymyxins), and others interfere with essential enzymes (quinolones, sulfonamides). These types of antibiotics are usually bactericidal (kill bacteria) but those

*Address correspondence to this author at the Biology Department, Faculty of Sciences, University of Chile, Las Palmeras 3425, Ñuñoa, Santiago, Chile; Tel: 056-2-9787185; Fax: 2712983; E-mail: fpchavez@uchile.cl

that target protein synthesis, such as the aminoglycosides, macrolides, and tetracyclines, are usually bacteriostatic (stop bacterial growth) [3].

Strategies are more recently used combining the reduction of bacterial virulence with a simultaneous increase of susceptibility to host defenses instead of killing the pathogen [4]. An example of such strategies is the development of antibiotics targeting bacterial quorum sensing [5], a system of cell-to-cell communication that relies upon the production of extracellular signaling molecules by the bacterial cells [2].

The search of the appropriate molecule or system to be targeted in the bacterial cell is essential for the effectiveness of the new drug. What new cellular targets are interesting for the design of novel antimicrobial drugs?

Our review highlights bacterial inorganic polyphosphate synthesis and second messenger regulation as attractive cellular targets for designing novel antibiotics.

Inorganic Polyphosphate and Bacterial Virulence

Polyphosphate (polyP) is a ubiquitous linear polymer of hundreds of orthophosphate residues (Pi) linked by

2 Current Enzyme Inhibition, 2011, Vol. 7, No. 3 Chávez et al.

phosphoanhydride bonds [6]. PolyP has been found in all three domains of life (Archaea, Bacteria and Eukarya) but it has been studied mainly in prokaryotes [7].

PolyP was primarily described as a reservoir of phosphate and, as in ATP, a source of high-energy phosphate bonds. Further genetic and biochemical experiments have indicated additional roles for polyP in many bacteria [8]. These include inhibition of RNA degradation, activation of the Lon protease during stringent response, involvement in membrane channel structure, and contribution to the resistance to stress generated by heat, oxidants, osmotic challenge, antibiotics and UV radiation. Particularly, a ppk1 mutant of Pseudomonas aeruginosa PAO1 was impaired in motility, biofilm development, quorum sensing and virulence [9].

The main enzymes related with polyP metabolism in bacteria are polyphosphate kinases (PPK1 and PPK2), which reversibly catalyze the polymerization of the terminal phosphate of ATP into a polyP chain, and the exopolyphosphatases (PPX), which produce phosphate when degrading polyP [10]. PPK2, in contrast to the ATP-dependent polyP synthetic activity of PPK1, preferentially catalyses the reverse reaction, i.e. the polyP-driven synthesis of GTP from GDP [11].

Orthologs of both, PPK1 and PPK2 enzymes have been found in many bacterial genomes. Curiously, there are many bacteria with orthologs of either PPK1 or PPK2, or both, or neither [12].

PPK1 from many pathogenic bacteria shares some genetic and biochemical features with its ortholog in Escherichia coli, but also has distinct differences [13].

PPK1 homologs have been found in over 100 prokaryotic species, including 20 major pathogens and, to date, only in two eukaryotes, the social slime mold Dictyostelium discoideum and the yeast Candida humicola, have orthologs to PPK1 [14].

In many bacterial pathogens, knockout of the ppk1 gene results in cellular defects, particularly in the context of virulence toward the host they invade. For example, ppk1 mutants of Salmonella sp. showed a drastic reduction in acid tolerance and invasiveness in epithelial cells, and bacterial survival in macrophages was also severely compromised [15]. In Shigella flexneri, the ethiological agent of bacillary dysentery, ppk1 gene deletion leads to a decrease in several factors that affect virulence, including invasiveness for epithelial cells [15]. Mutants of Vibrio cholerae were also acid-sensitive and stationary phase-defective [16] while ppk1 mutants of Neisseria meningitidis were highly sensitive to killing by human serum [17]. Attenuated synthesis of PPK1 in Mycobacterium tuberculosis, by expression of ppk1antisense, results in the bacterium’s decrease of its ability to colonize and survive in macrophages [18]. Similar results have been reported in Helicobacter pylori, where the lack of the ppk1 gene results in bacterial inability to colonize mice [19].

In the case of the MDR bacterium Pseudomonas aeruginosa PAO1, ppk1 null mutants are deficient in motility, quorum sensing, biofilm formation and virulence in ocular and burned-mouse models [9]. Moreover, this mutant

exhibits much reduced viability after exposure to a -lactam antibiotic [20]. Similar results were reported in ppk1 mutants from Salmonella typhimurium and Salmonella dublin towards Polymyxin B15 [15].

In addition, ppk1 mutants retain as much as 20% of the wild-type (WT) levels of polyP, which are produced by the activity of another polyphosphate kinase, PPK2. The activity of PPK2 in P. aeruginosa differs from PPK1 in two major features. 1) While PPK1 synthesizes polyP mainly from ATP, PPK2 utilizes PolyP to make GTP at a rate 75-fold greater than that of PolyP synthesis from GTP; 2) PPK1 is strictly specific for ATP while PPK2 uses GTP and ATP equally well during PolyP synthesis.

Antimicrobial chemicals targeting bacterial virulence should possess a broad spectrum of activity and little toxicity. Since PPK1 has not been found in mammalian cells, drugs targeting polyP synthesis are less likely to produce secondary effects and probably antibiotic resistance.

Interestingly, PPK1 is essential in P. aeruginosa not only for various types of motility and development of biofilms, but also for the production of virulence factors such as elastase and rhamnolipids [21]. All of these defects are likely to be exerted through a failure in quorum sensing responses. As a result, the P. aeruginosa ppk1-deficient strain was significantly less virulent than the WT, in both ocular and burned-mouse pathogenesis models [22]. We have found similar results in novel virulence assays in Caenorhabditis

elegans and zebrafish animal models. In both cases we found that phosphate starvation induces a hyper-virulence phenotype in P. aeruginosa cells [Chávez et al., unpublished results].

The fact that PPK1 is involved in cellular metabolism rather than in an essential functions, makes it an attractive target for virulence control in pathogenic bacteria.

PolyP deficit causes many structural and functional defects. The link between genotypes and phenotypes observed during polyP deficiency can be the result of complex networks of interaction that can be elucidated by using a Systems Biology approach.

Systems Biology Approach to Study polyP Deficiency

New emerging fields like systems biology are showing great potential for the understanding of intricate biological networks underlying physiological and pathological processes. Systems biology employs in silico techniques to integrate and analyze disparate chemical and biochemical data in a parallel, as opposed to a sequential fashion [23]. Fueled by Omics technologies, it applies network principles and mathematical tools to dynamic modeling and simulation of complex biological systems in a more holistic manner.

In this context, we have used different high-throughput screens that operate at different levels, with the aim of assessing the impact of polyP deficiency on a wide scale. Thus, functional genomics, quantitative proteomics and phenotype microarray analyses were employed to compare E. coli mutants affected in ppk1 and/or ppx genes with WT strains.

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Bioinformatic analyses of our Omics data revealed a link between polyP and E. coli central metabolism, particularly the tricarboxylic acid (TCA) cycle [24]. In addition, by using Phenotypic microarray (Biolog Inc.), a platform that can quantify nearly 2,000 bacterial phenotypes, we observed that PPK1 inhibition could increase the susceptibility of bacteria to known antibiotics [Chávez et al., unpublished results]. Finally, our results suggest that the multiple structural and functional defects found during alteration of polyP in bacteria could be due to the regulatory role of polyP as a second messenger during transcriptional initiation. PolyP could be a regulatory polymer employed by bacteria to cope with starvation, stress and virulence by interacting with alternative Sigma Factors to control the initiation of transcription. Summarizing, our systems biology approach supports the notion that inhibition of polyP synthesis is a strategy of great potential for the development of novel antibiotics.

Broad spectrum of action, low toxicity and decreased likelihood of resistance development, are presumable characteristics of these novel antibiotics.

PolyP Synthesis as a Target for Novel Antibiotics

As discussed above, numerous lines of evidence indicate that altered polyP metabolism, particularly that derived from the PPK1 loss associated with ppk1 gene deletion, produces a decrease in several virulence phenotypes and, more broadly, a reduction of the microorganisms’ resilience [7]. These data set PPK1 as a remarkable potential target for novel antibiotic agents. Indeed, since inhibition of this enzyme should not lead to pathogen death but to an enhanced susceptibility to host defense mechanisms, a lower pressure for resistance development could be envisaged. In addition, as PPK1 has not been found in mammalian cells [25], its inhibitors should exhibit low toxicity. Also, taking into account the high degree of identity between PPK1 orthologs in most of the major known bacterial pathogens, polyP synthesis inhibition might be regarded as a target for broad-range spectrum antibiotics.

Considering the well documented key regulatory role that polyP and PPK1 play in the survival and virulence of bacterial pathogens, the small number of PPK inhibitors (small molecules) described and characterized thus far is rather surprising. This scenario is even more unexpected, taking into account that the crystal structure of E. coli PPK1 was described more than five years ago (Fig. 1) [26]. Nevertheless, the evidence reviewed here and elsewhere (see for example [8, 27], as well as the recent development of methods suitable for high throughput screening of PPK1 inhibitors [28], anticipate a new impulse for the rational design of this type of drugs, offering a novel and attractive mechanism of chemotherapy.

Among the few reports containing data about novel (small) PPK1 inhibitors is a patent application by Arthur Kornberg [29]. In this application, compounds from the ICOS’ chemical library were screened in vitro for their inhibitory activity against PPK1 from E. coli, using a previously reported enzymatic assay [30, 31]. Thus, five compounds, whose identities were not disclosed, showed inhibitory potencies (expressed as IC50) in the low

micromolar range. In addition, the ability of these drugs to inhibit E. coli polyP accumulation in vivo was also assessed. Interestingly, despite their similar in vitro PPK1 inhibitory activity, the compounds induced a differential cellular response, with some of them drastically inhibiting polyP accumulation (more than 60% reduction), whereas others showed almost no effects as compared with control samples. Other small molecules that inhibit PPK1 include guanidine, ADP and inorganic pyrophosphate [32] as well as the adenosine-5-phosphate analog AMPPNP [26]. In addition, larger molecules such as DNA-based aptamers have been described as potent PPK2 inhibitors in Mycobacterium tuberculosis [33]. However, the presumably poor drugability of such compounds makes the rational search of novel small PPK1 inhibitors a salient challenge for drug designers.

Fig. (1). PPK1 structure and ATP binding site from Escherichia

coli.

(A) Schematic representation of the PPK1 monomer from E. coli. The four domains contributing to the formation of the ATP binding site are shown in colors. (B) A close up of the substrate-binding site illustrating the main aminoacids residues that interact with the ATP molecule analog AMPPNP. Doted lines represent hydrogen bonds as suggested by the docking programs.

In this sense, the availability of the crystal structure of PPK1 from E. coli provides key molecular and functional information for the identification of selective ligands [26]. Thus, PKK1 forms an interlocked dimer, with each monomer containing a tunnel that has a unique substrate (ATP)

4 Current Enzyme Inhibition, 2011, Vol. 7, No. 3 Chávez et al.

binding site (Fig. 1). Using a non-hydrolysable ATP analogue, it was shown that the adenine moiety binds to a highly hydrophobic cavity of the binding site, whereas the phosphate groups bind to a positively charged pocket, which also accommodates two Mg2+ ions essential for catalysis. Based on these data, the authors proposed that during polyP formation, ATP might enter from one end of the tunnel while the synthesized chain would exit from the other end.

In the light of structural information, several sites can be envisaged as targets for PPK1 inhibitors. Thus, the ATP-binding pocket as well as the whole tunnel inner surface, including the substrate entrance and product exit sites, represents attractive sites for drug design. In addition, since both the dimeric assembly and the self-phosphorylation at His435 have been shown to be critical for enzyme activity [32], compounds aimed to disrupt these structural/functional features could also be promising drugs.

Chemical Biology Approach to Obtain Novel polyP Synthesis Inhibitors

Today, the process of drug discovery has been brought to a new level with the development of genomics, proteomics and bioinformatics, and efficient technologies such as combinatorial chemistry, high throughput screening, virtual screening, de novo design and structure-based drug design .

The chemical biology approach can help in all aspects of drug discovery i.e., to analyze the target structures for possible binding/active sites, generate candidate molecules, check for their druglikeness, dock these molecules into their target, rank them according to their predicted affinities, and further optimize them to improve their binding characteristics [34, 35].

Hence, we have initiated a virtual biological screening of small molecules contained in public chemical libraries such as the Maybridge Diversity Fragment Library (Thermo Fisher Scientific), one of the first commercially available fragment libraries, and the National Cancer Institute Diversity Set II (National Institutes of Health). Both collections contain optimized structures of two different types of ligand candidates, with the former including mainly fragments while the second comprises more global pharmacophores.

Several compounds were docked into the active site of the E. coli PPK1 crystal structure, using the CHARMM-based CDOCKER and LibDock programs (Accelrys). After scoring with the LUDI consensus empirical function, we selected a group of compounds which are currently under biological evaluation (Fig. 2). Preliminary results have shown that some of these chemicals can potently inhibit PPK1 activity in vitro, and therefore warrant evaluation at the cellular level and in animal models of virulence. In addition, Fig. 3 illustrates the lowest energy poses of different fragments within the ATP binding pocket. After performing an in silico fragment-based assembly, a hybrid molecule was designed (Fig. 3), which fits smoothly into the PPK1 active site.

We expect that these chemical and systems biology approaches will allow us to discover new PPK1 inhibitors, affecting both PPK1 activity and polyP content, which might

cause similar phenotypes to arise as those observed in bacterial ppk1 mutants. Finally, virulence-inhibition

Fig. (2). Molecular docking of a compound from the National Cancer Institute (NCI) library into the E. coli PPK1 active site. The NCI diversity set II library was in silico screened against PPK1 from E. coli using the CHARMM-based CDOCKER program (Accelrys) and scored using the LUDI2 empirical scoring function. The figure shows the lowest energy pose of one of the top 10% scored compound, docked into the PPK1 active site.

Fig. (3). Fragment-based approach for the design of novel PPK1 inhibitors. (A) A small chemical fragment from the Maybridge Diversity Fragment Library docked into the E. coli PPK1 active site. (B) Two different small chemical fragments were in silico joined according to their position within the ATP binding site to generate a novel compound using the de novo drug design program LUDI.

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biological assays, such as those in C. elegans and zebrafish that are being developed in our laboratory, should allow the evaluation of both drug actions against the pathogen and toxicity toward the host.

CONCLUDING REMARKS

Several lines of evidence, from molecular to systems microbiology levels, support the notion that modification of polyP metabolism, particularly synthesis inhibition, is a strategy of great potential for the development of novel antibiotics. Broad spectrum of action, low toxicity and decreased likeliness of resistance development are presumable characteristics of these drugs. Nevertheless, it should be noted that a simultaneous effect upon the normal microbiota might also arise. Although this aspect is common to most antibiotics, differential sensitivity of beneficial microorganisms should be thoroughly evaluated.

It has been observed that PPK1 inhibition can increase the susceptibility of pathogens to known antibiotics. The use of lower doses of clinically relevant drugs might be associated not only with lower levels of resistance but also with less side effects.

ACKNOWLEDGEMENTS

We thank Dr. Bruce Cassels for his comments on the manuscript. This research was supported by Grants 11070180 from FONDECYT and ICM P05-001-F from the Chilean Millennium Research Initiative CFL is a CONICYT PhD fellow.

COMPETING INTERESTS

The authors declare that they have no competing interests.

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Received: July 22, 2011 Revised: August 02, 2011 Accepted: August 27, 2011