WO2004111261A1

WO2004111261A1 - Carbon-phosphorus or carbon-arsenic bond-containing moieties suitable for linking to antimicrobial agent

Info

Publication number: WO2004111261A1
Application number: PCT/AU2004/000814
Authority: WO
Inventors: George L. Mendz
Original assignee: Unisearch Limited
Priority date: 2003-06-19
Filing date: 2004-06-21
Publication date: 2004-12-23
Also published as: AU2003903113A0

Abstract

A method for identifying a carbon-phosphorus (C-P) or carbon-phosphorus (C-As) bond-containing moiety suitable for linking to an antimicrobial agent in order to generate a pro-drug for administration to an animal for the treatment or prevention of microbial infection in said animal, comprising the steps of: (1) providing a compound comprising the C-P or C-As bond-containing moiety; (2) introducing said compound to a microorganism capable of infecting an animal, or to a lysate thereof; (3) establishing whether the C-P or C-As bond is hydrolysed; and (4) identifying a C-P or C-As bond-containing moiety as suitable for linkage to an antimicrobial agent if the C-P bond is hydrolysed.

Description

CARBON-PHOSPHORUS OR CARBON-ARSENIC BOND-CONTAINING MOIETIES SUITABLE FOR LINKING TO ANTIMICROBIAL AGENT Technical Field

The present invention is concerned with the delivery of antimicrobials to pathogenic microorganisms, and, more particularly, to the design of pro-drugs for administration to an animal for the treatment or prevention of microbial infection in that animal. Background Art Infectious diseases claimed 18 million lives in

2002, with most of those deaths occurring in developing countries. These same regions of the world serve as incubators for emerging strains of bacteria, fungi and other pathogens that are resistant to current antimicrobial therapy. In addition, the agricultural and medical overuse of hitherto potent antibiotics in both developed and developing countries also contributes significantly to the pool of resistant microorganisms. The challenge is to stay one step ahead of these emergent strains.

Currently, several high throughput techniques are employed to achieve the most important objective of developing new types of antibiotics. For example, genomics and proteomics are used to find suitable targets, and combinatorial chemistry to create millions of new molecules never seen before to be tested against those new targets. However, the potentially important strategy of utilising the physiology of infectious microbes to produce therapeutic agents that will act against gene products essential to the survival of the microorganisms has received only limited attention. This type of strategy makes moderate use of genomics, and requires detailed and in-depth knowledge of specific and unique elements of the metabolic machinery of microorganisms that will be employed against them.

Phosphorus has a central role in cell physiology both in the make up of biomolecules and in catalytic processes. Most commonly it is utilised by organisms in the form of phosphate esters derived from orthophosphates, but it can be obtained also from other types of organophosphorus compounds, including phosphonates (Phn) which are characterised by the presence of carbon- phosphorus (C-P) bonds. Phosphonates occur in biogenic and man-made compounds, and are resistant to chemical hydrolysis, thermal decomposition, photolysis and to the action of phosphatases [1] . The C-P bond is much more stable than the oxygen-phosphorus, nitrogen-phosphorus, or sulphur-phosphorus bonds, but can be cleaved by a wide range of microorganisms including Gram-positive and Gram- negative bacteria, yeasts and fungi [2] . In the family Enterobacteriaceae, Phn catabolism has been observed in the genera Campylobacter, Escherichia, Enterobacter, Helicobacter, Klebsiella, Kluyvera and Salmonella . In addition, pathogens of the genera Bacillus, Burkholderia, Candida, Corynebacterium, Pseudomonas also have the ability to degrade phosphonates. Proteus vulgaris, Yersinia enterocolitica and Mycobacterium phlei also have at least some of these activities.

Four pathways for Phn biodegradation are known in bacteria: the phosphonatases, phosphonoacetate hydrolase, phosphonopyruvate hydrolase, and C-P lyase, which differ both in regard to their substrate specificity and their cleavage mechanisms [1] . Phosphonatases have α-amino- alkylphosphonates as specific substrates; phosphonoacetate hydrolase catalyses phosphonoalkyl carboxylic acids; and the only substrate known of phosphonopyruvate hydrolase is phosphonopyruvate. C-P lyases degrade a wide range of phosphonates including phenylphosphonate, which is not catabolised by the other enzymes. Bacteria able to cleave C-P bonds ordinarily have one or two of these pathways. The phosphonatase and C-P lyase pathways have been found in E. aerogenes [3] , whereas only the phosphonatase pathway is present in S. Typhimurium, and degradation of Phn solely by the C-P lyase pathway occurs in E. coli [4] , Klebsiella aerogenes [5] , Klebsiella oxytoca and Klebsiella pneumoniae [6] , and Kluyvera ascorbata and Kluyvera cryorescens [6] . Burkholderia cepacia Palβ is able to utilise phosphonoalanine as a phosphorus source using a phosphonopyruvate hydrolase [7] . Summary of the Invention

The present invention is based on the observation that many mammalian cells do not transport compounds containing carbon-phosphorus (C-P) bonds such those bearing phosphonate groups, and almost all human tissues lack enzyme activities able to cleave the C-P bond. On the other hand, a number of infectious agents including some bacteria and parasites are able to transport phosphonate and have C-P bond cleaving activities. The metabolic differences between human and such microorganisms can be exploited to design compounds which will be transported into the infectious agent and not the cells of host and/or in which the C-P bond only be cleaved by the metabolic machinery of the microorganism. The carbon-arsenic (C-As) bond is believed to behave similarly to the C-P bond. Accordingly, an investigation of the transport and metabolism of C-P or C-As bond-containing compounds in microorganisms will provide unique opportunities for the development of novel antimicrobials targeted against infectious diseases in animals, including humans .

In a first aspect of the present invention provides a method for identifying a carbon-phosphorus (C- P) or carbon-arsenic (C-As) bond-containing moiety suitable for linking to an antimicrobial agent in order to generate a pro-drug for administration to an animal for the treatment or prevention of microbial infection in said animal. The method comprises the steps of:

(1) providing a compound comprising the C-P or C-As bond-containing moiety;

(2) introducing said compound to a microorganism capable of infecting an animal, or to a lysate thereof;

(3) establishing whether the C-P or C-As bond is hydrolysed; and

(4) identifying a C-P or C-As bond-containing moiety as suitable for linkage to an antimicrobial agent if the C-P or C-As bond is hydrolysed.

In the method of the invention, biophysical and biochemical properties of the C-P or C-As bond-containing moiety may be identified which suggest themselves as suitable for exploitation in order to deliver an antimicrobial drug to the causative microorganism. In an embodiment, one may establish transport characteristics of the compound across the cytoplasmic membrane of the microorganism. In particular, parameters such as transport rates, kinetic parameters and temperature and pH dependence of phosphonate transport that are particularly favourable may be identified. In addition, the effect on phosphonate transport systems or the effects of mono and divalent cations and inhibitors of energy transducing systems and other phosphonates on transport rates may also be investigated in order to establish favourable properties .

Similarly, in an embodiment one may establish the C-P or C-As bond cleaving activities in the causative microorganism, for example, it may be desirable to ascertain which phosphonate degradation pathways are operative and the specificity of the relevant enzymes for different phosphonates. Other factors which may be investigated are the location of enzyme activities e.g. in the cytosolic or cell wall fraction, etc, kinetic parameters, substrate specificity and effects of inhibitors of C-P or C-As bond cleavage enzymes.

Advantageously the studies should be made in vivo and in situ, or in systems as close as possible to the intact cell.

It is envisaged that many C-P or C-As bond- containing moieties may be investigated according to the method of the invention, indeed any C-P or C-As bond- containing molecule may be investigated. In an embodiment, the C-P bond-containing compound is a phosphonate or a phosphinate, most commonly a phosphonate, and the C-As compound is an arsonate.

In' an embodiment, the microorganism is selected from the group consisting of Campylobacter spp. , Helicobacter ssp. , Bacillus ssp. , Escherichia ssp. , Enterobacter ssp. , Klebsiella ssp. , Kluyvera ssp. ,

Pseudomonas ssp. , Burkholderia ssp. , Candida ssp. , and Corynebacteria, Mycobacteria, Proteus spp. and Yersinia spp.

Optionally, the genes encoding phosphonate transport and metabolising proteins may be isolated and characterised. The isolated enzymes may be studied, for example, to determine whether they are subjected in the cell to special regulation of their activity. Knock out mutants of the causative microorganism may also be produced in order to ascertain the viability of these mutants in vitro and therefore the essentiality of the relevant genes. With an understanding of the organisation of the genes coding for the proteins of phosphonate pathways and the role of phosphonate uptake and degradation in the physiology of the causative microorganism, the ability to carry out rational design of compounds with antimicrobial activity is enhanced.

According to a second aspect, the present invention provides a method of designing a candidate pro- drug for administration to an animal for the treatment or prevention of microbial infection in said animal. In the method a C-P or C-As bond-containing moiety by the method described above. Then, an antimicrobial agent lethal to the causative microorganism is selected and feasible chemistry for linking the antimicrobial agent to the C-P or C-As bond-containing moiety is established. In a third aspect the present invention provides a method of preparing a candidate pro-drug for administration to an animal for the treatment or prevention of microbial infection in said animal. The method comprises: providing a first reactant comprising a suitable C-P or C-As bond-containing moiety identified as described above; providing a second reactant comprising an antimicrobial agent lethal to the causative microorganism; and reacting the first reactant with the second reactant so as to link the C-P or C-As bond-containing moiety to the antimicrobial agent. In a fourth aspect of the present invention provides a candidate pro-drug for administration to an animal for the treatment or prevention of microbial infection in said animal comprising a C-P or C-As bond- containing moiety linked to an antimicrobial agent lethal to the causative microorganism.

The antimicrobial agent may be any antimicrobial agent, as would be understood by the person skilled in the art. The purpose of the present invention is to synthesize novel pro-drugs, not to identify new antimicrobial agents, and therefore the antimicrobial agent will generally be known for the selected purpose. Furthermore, antimicrobials which are not in common use because they are too toxic to the host for safe use to treat infection, or because there are other practical difficulties associated with their use, may be useable in this invention. For example, if the active group in a compound toxic to the host is blocked by the C-P or C-As bond-containing moiety the compound will become near harmless to the host but have its potency restored when it is internalized and then cleaved into active form by a microorganism infecting the host. It will be appreciated that the candidate prodrugs described above may or may not prove to be active, Nevertheless, rational drug design in this fashion serves a useful purpose in providing candidates with greatly enhanced prospects of activity.

In a fifth aspect, the present invention provides a method for the identification of a pro-drug for administration to an animal for the treatment or prevention of microbial infection. The method comprises providing a candidate pro-drug, and screening the candidate pro-drug for antimicrobial activity.

Conventional methods of screening for activity, as would be well understood by the person skilled in the art, may be used. In a sixth aspect of the present invention there is provided an enzyme which catabolises phosphonate in Campylobacter species and Helicobacter species. The present inventors have found that Campylobacter species and Helicobacter species are also able to catabolise phosphonates despite the fact that genes orthologous to those encoding C-P bond hydrolyzing enzymes in other bacteria have not been identified in the annotated genome of Campylobacter jejuni strain 11168 or Helicobacter pylori strains 26695 and J99. Phosphonate catabolism in Campylobacter species and Helicobacter species is associated with at least two different C-P bond cleavage activities. One is exclusively associated with the cell wall of the bacterium and enables it to hydrolyse phosphonoalkyl carboxylates and phenyl phosphinate, and is believed to be a phosphonatase or a phosphonoacetate hydrolase. The other activity was found in both the cell wall and cytosolic fractions and was able to cleave phenyl phosphonate. It is believed that this activity is a result of the presence of a C-P lyase. Both activities hydrolysed α-amino- methyl-phosphonate, but at different rates.

In most bacteria the catabolism of phosphonate is under phosphate starvation control and the corresponding enzymes are part of the Pho regulon, but this was not the case for either of the activities observed in Campylobacter species or Helicobacter species. Throughout this specification and the claims, the words "comprise", "comprises" and "comprising" are used in a non-exclusive sense, except where the context requires otherwise.

It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country. Brief Description of the Drawings

Figure 1 is a plot which shows rates of C-P bond cleavage of α-amino-methyl phosphonate (AmePhn) , phenyl phosphonate (PhePhn) , and phosphonoacetate (PhnAce) by C. jejuni 81116 cells (A) and whole lysates (B) . Bacterial samples were suspended in NaCl/KCl (150 mM) mixtures, initial substrate concentration was 50 mM. Measurements were carried out at 37°C employing one-dimensional ¹H-NMR _t spectroscopy. Similar data are obtained for all tested strains of Helicobacter pylori. Figure 2 is a plot which shows growth after 24 h of C. jejuni isolate 1125 in defined liquid media with YNB, SC-URA, 10 mM KCl, supplemented with BSA (0.5%) and catalase (0.1%). (A) Without phosphate (") , with added 10 mM of either sodium phosphate (•) , PhnAce (♦) , AmePhn (A) or PhePhn (T) . (B) With PhePhn added as sole phosphorus source at various concentrations. Similar growth curves are obtained for all tested strains of Helicobacter pylori . Modes for Performing the Invention Example 1

Nuclear magnetic resonance (NMR) methods were employed in this study to investigate Phn catabolism in living, metabolically competent bacteria, whole-cell lysates and cell fractions of several Campylobacter spp . In vitro culture techniques employing defined media were used to determine whether C. coli and C. jejuni can survive by utilising phosphorus from phosphonate-group bearing compounds.

Materials and Methods

Materials . Blood Agar Base No. 2, defibrinated horse blood and Brain Heart Infusion (BHI) were from Oxoid Australia (Heidelberg West, VIC, Australia) ; yeast nitrogen base without phosphate 4027-812 (YNB) and synthetic complete supplement mixture 4410-622 (SC-URA) were from Qbiogene (Carlsbad, CA, USA) . Vancomycin was from Eli Lilly (North Ryde, NSW, Australia) . Bovine serum albumin (BSA) , catalase, polymyxin B, trimethoprim, amphotericin, phosphonoacetic acid (PhnAce) , α-amino- methyl phosphonic acid (AmePhn) , α-amino-ethyl phosphonic acid, α-amino-propyl phosphonic acid, α-amino-butyl phosphonic acid, phenyl phosphonic acid (PhePhn) , and phenyl phosphinic acid (PhePhp) were from Sigma (St Louis, MO, USA) . Phosphonoformic acid, 2-phosphonopropionic acid, and 2-phosphonobutyric acid were obtained from Biochemika (Eastgate, Morecambe, UK) . Phenyl arsonic acid (PheAsn) was kindly donated by S. B. Wild (Australian National University, Canberra) . All other reagents were of analytical grade.

Bacterial strains and growth conditions .

Campylobacter coli strain ATCC 33559, C. coli isolate 1041 (CHU Pellegrin, Bordeaux) , C. fetus subspecies fetus strain ATCC 33246, C. hyoilei strain ATCC 51729, C. hyointestinalis strain D1790, C. jejuni strains 11168 and 81116, and C. jejuni isolate 1125 (CHU Pellegrin, Bordeaux) were grown on Campylobacter Selective Agar consisting of Blood Agar Base No. 2, supplemented with 5% (v/v) defibrinated horse blood, 5 mg ml^" vancomycin, 2.5 μg ml^"1 polymyxin B, 2.5 mg ml^" trimethoprim and 2 μg ml^"1 amphoterin, by incubating for 16 h at 37°C under the microaerobic conditions of an atmosphere of 10% CO_{2 ,} in air and 95% relative humidity. Bacteria were harvested with 150 mM NaCl and washed twice by centrifugation in the same buffer. Cells were lysed by thrice freezing in liquid nitrogen and thawing. Cytosolic and cell-wall fractions were obtained by centrifuging whole-cell lysates at 12,000 x g for 45 min at 4⁰C, and collecting the supernatants and pellets .

Measurement of C-P bond hydrolysis . ¹H-NMR and ³¹P- NMR spectroscopy methods were employed to determine rates of phosphonate bond hydrolysis measured in suspensions of bacterial cells, whole-cell lysates and cell fractions. Enzyn activities were measured at 37⁰C in cells or lysates suspends in 9:1 or 8:2 (v/v) NaCl/KCl (150 mM) mixtures, respectively. One-dimensional ¹H-NMR spectra were acquired at 600.13 MHz, using a Bruker DMX-600 spectrometer operating in the Fourier transform mode with quadrature detection. Instrument parameters were: spectral width of 6009.615 Hz, memory size 1 K, acquisition time 1.36 s, number of transients 64 - 88, anc pulse angle 50° (3.0 μs) , and relaxation delay with solvent presaturation 1.75 s. A Gaussian window function with broadening parameter of 0.19 and line width -0.5 to -1.0 Hz was applied prior to Fourier transformation. One-dimensional ³¹P-NMR spectra were acquired at 202.457 MHz using a Bruker DMX-500 spectrometer operating in the Fourier transform mode with quadrature detection. Instrument parameters were: spectral width of 12135.922 Hz, memory size 32 K, acquisitior time 2.70 s, number of transients 80, and pulse angle 50° (3. μs) . Exponential filtering of 1.0 Hz was applied prior to Fourier transformation. Bacterial samples were placed in 5 or 10 mm tubes (Wilmad, Buena, NJ, USA) , for ¹H-NMR or ³¹P-NMR measurements, respectively, and substrates were added to star the reactions. The time-evolution of the utilization of substrates and appearance of products was followed by automatically acquiring sequential spectra. The integrals of the resonances corresponding to the substrates were measured at each time point using standard Bruker programmes. Calibrations of the peaks arising from the substrates were performed by extrapolating the resonance intensity data to zero time and assigning to this intensity the appropriate concentration. Rates of phosphonate hydrolysis were calculate from good fits (correlation coefficients > 0.98) of the data to straight lines.

Kinetic analyses. Michaelis constants (K_m) and maximal velocities (V_max) of phosphonate bond cleavage by suspensions of bacterial cells or lysates were determined frc the maximal rates of enzyme activity of eleven time courses. The values of the kinetic parameters were calculated by nonlinear regression analysis employing the program Enzyme Kinetics (Trinity Software; Campton, NH, USA) .

Statistical analyses. Normality tests, Kruskal- Wallis nonparametric tests, and student's tests of the data were performed employing the STATA 5.0 software (Stata Corporation; College Station, TX, USA) .

Enzyme activities involved in C-P bond hydrolysis . The presence of different enzyme activities involved in the hydrolysis for PhnAce, AmePhn and PhePhn was investigated by measuring their hydrolysis in cytosolic and cell-wall fractions. Cytosols were fractionated by molecular sizes usir ultrafiltration membranes with cut-offs of 500 kDa, 100 kDa and 50 kDa and the activities of each of the tree substrates measured in each fraction. The various enzyme activities were studied also in competition experiments by determining in ce] suspensions and lysates the rates of degradation of each substrate in the presence of the same concentration of one oi the other compounds •

Specificity of phosphonate bond hydrolysis . The specificity of the phosphonate hydrolase activity for phosphono alkyl carboxylates was investigated by measuring tl rates hydrolysis of C-P bonds of compounds with alkyl chains with one to four carbons. The specificity of the C-P lyase activity for α-amino-alkyl phosphonates was studied by measuring the rates of enzyme activity of compounds with alk} chains with one to four carbons, with phenyl phosphonite, anc with phenyl phosphonates with substituents in the phenyl moiety.

Protein determinations . Estimation of the protein content of samples was made by the bicinchoninic acid method employing a microtitre protocol (Pierce, Rockford, ILL, USA) , Absorbances were measured on a Beckman Du 7500 spectrophotometer .

Results

Measurement of phosphonate and arsenate bond hydrolysis . C-P bond hydrolysis in Campylobacter spp. was established by observing the decrease in the levels of PhnAce in suspensions of living cells or whole-cell lysates employii ¹H-NMR and ³¹P-MMR spectroscopy. Time-courses of the substrata incubated with bacterial cells showed a decline in the levels of phosphonate and the appearance of resonances corresponding to acetate (¹H-NMR) and inorganic phosphate Pi (³¹P-NMR) . Phosphonate degradation was observed also with AmePhn, PhePhi PhePhp, and other C-P compounds. In the absence of bacterial cells or lysates no hydrolysis was detected for any of the compounds. Cleavage of C-P bonds was observed for the five species tested C. coli , C. fetus subspecies fetus, C. hyoile: C. hyointestinalis and C. jejuni .

Kinetics of phosphonate cleavage . The kinetic parameters for the hydrolysis of PhnAce, AmePhn and PhePhn were measured in live cells and whole lysate suspensions of ( coli ATCC 33559 (Table 1) . Different K_m and V_max were obtained for the three phosphonates. For live cells and lysates the values determined for the K_m were similar, but the V_1J13x values were significantly different, suggesting that the observed maximum rate of intracellular phosphonate hydrolysis of each compound may be modulated by its rate of transport across the cell wall; the latter being saturated at lower concentrations than the maximum capacity of the cell to degrade these phosphonates .

Differences in phosphonate catabolism between bacterial strains and species. The rates of hydrolysis of 30 inM PhnAce by lysates of C. coli strains NCTC 11366, JC14,

CSIRO 40, A293 and 445, and of C. hyoilei strains CCUG 33450 486, 182, 74 and 58 were compared to ascertain whether there are intraspecific or interspecific differences in the maximuα capacities to hydrolyse this phosphonate (data not shown) . Kruskal-Wallis nonparametric tests were applied to the data obtained for these strains after verifying the normality of the values. The results indicated that there were no significant differences between the rates determined for the various strains of each of the species. In contrast, for 30 i PhnAce average rates of 2.2 ± 0.5 and 0.74 ± 0.22 nmole/min/i were obtained for C. coli and C. hyoilei lysates, respectively; the respective average rates for 30 mM PhePhn were 5.8 ± 1.4 and 2.0 ± 0.6 nmole/min/mg . Comparison of the rates of phosphonate hydrolysis for C. coli and C. hyoilei b] means of a Student's test indicated a significant difference between their average rates (p < 0.001) .

Specificity of C-P bond hydrolysis . The capability of Campylobacter spp. to cleave C-P bonds of compounds of different alkyl chain length was measured for phosphono alky! carboxylates and α-amino-alkyl phosphonates with chains with to 4 carbon atoms. The rates of hydrolysis for the former followed the sequence acetate > formate > propionate and no activity was observed with phosphonobutyrate • For the α-amine alkyl phosphonates the rates followed the sequence methyl > ethyl > propyl > butyl.

The activity observed with phenyl phosphonate was completely abolished by the presence of a nitro substituent : the para position. On the other hand, C-P bond cleavage was observed for phenyl phosphinate; and Campylobacter spp. cells and lysates catabolised the As-C bond of PheAsn.

Enzyme activities involved in C-P bond hydrolysis . The enzyme activities involved in C-P bond cleavage were investigated by measuring the rates of PhnAce, AmePhn and PhePhn hydrolysis in fractions obtained by centrifugation of whole-cell lysates, and in cytosolic fractions with several molecular sizes. Also by measuring the rates of the three phosphonates in competition experiments in the presence of a second substrate.

In whole-cell lysates and lysate fractions of C. coli , C. hyoilei and C. jejuni, PhnAce hydrolysis was detects in the cell-wall fraction, and none could be detected in the cytosolic fraction. Phenyl phosphonate was catabolised at similar rates by whole-cell lysates and the cell-wall fractions, and at rates 60-70% lower in the cytosolic fractions. Separation of whole lysates into fractions showed similar rates of AmePhn hydrolysis in both the resulting fractions. Degradation of AmePhn was observed in cytosolic fractions of molecular sizes 500, 100 and kDa.

In lysate suspensions of each of the three

Campylobacter species the rates of hydrolysis of 50 mM PhnAce were not affected by equimolar concentrations of PhePhn; and were reduced by 30 to 60% in the presence of equimolar concentrations of AmePhn or orthophosphate . Conversely, the rates of hydrolysis of 50 mM PhePhn were not affected by the presence of equimolar concentrations of PhnAce but were reduced by 30 to 40% by the presence of AmePhn or orthophosphate. The rates measured for each of the phosphonates PhnAce, PhePhn and AmePhn in competition experiments with the other two phosphonates are summarised ii Table 2. Similarly, the presence of equimolar concentrations of methyl phosphonate reduced the rates of PhnAce hydrolysis by approximately 60%, but did not affect the hydrolysis of

PhePhn .

Measurements of the effects of phenyl phosphinate c phenyl arsonate on rates of phosphonate hydrolysis served as an indirect verification of these results. PhnAce hydrolysis was reduced by about 50% in the presence of equimolar concentrations of PhePhp or PheAsn, and no hydrolysis of the phosphinate or arsonate were observed under these conditions On the other hand, rates of hydrolysis of PhePhn were not affected by the presence of equimolar concentrations of PheP] or PheAsn, and these compounds were hydrolysed in the present of PhePhn at rates comparable to those observed in when they were the only substrates.

Biological role of C-P bond hydrolysis . The effect! of PhnAce, AmePhn and PhePhn on C. coli and C. jejuni growth were studied by supplementing the BHI broth with each of the phosphonates, one at a time, at concentrations between 0.5 a. 10 mM. The presence of up to 10 mM concentrations of either c the three phosphonates did not affect cell growth, suggesting that the compounds were not cytotoxic at these concentration! In contrast, phenyl arsonate inhibited bacterial growth in a dose dependent manner, and at 5 mM PheAsn the number of coloi forming units was reduced by three orders of magnitude. To determine whether PhnAce, AmePhn or PhePhn could be employed as a phosphorus source to support bacterial growth in vitro, bacteria, including the recent isolates C. coli 1040 and C. jejuni 1125, were grown in defined media with either 10 mM phosphate, or the same concentration of one of the three phosphonates and without any phosphate or phosphonate, as negative control. Growth curves for both species indicated that PhePhn served as a phosphorus source able to support bacterial growth, but PhnAce or AmePhn did not (Fig. 2) .

Discussion

Analyses of the sequenced and annotated genome of C jejuni strain 11168 have guided genetic and metabolic investigations of this bacterium, and have served to understand various aspects of the physiology of C. jejuni anc of other Campylobacter spp . too. However, this approach is limited to the genes which have been identified previously ai by the biochemical data about the bacterium. The catabolism c Phn by Campylobacter spp. described here for the first time demonstrated the presence of a function previously unknown ii the genus, notwithstanding the absence in the C. jejuni genor [8] of genes orthologous to those encoding C-P bond cleaving enzymes in other organisms.

It was demonstrated that the Campylobacter spp. studied hydrolysed Phn compounds, including α-amino-methyl phosphonate, phosphonoacetate and phenylphosphonate, which cε be catabolised by the phosphonatase, phosphonoacetate hydrolase, or C-P lyase pathways. The kinetic parameters of these activities were determined in intact cells and whole- cell lysates. The bacteria also were able to hydrolyse pheny] phosphinate and the C-As bond of phenyl arsonate. There are very few reports of cleavage of carbon-arsenic bonds by bacteria [9] .

A simple interpretation of the data on Phn hydrolysis by cell-wall and cytosolic fractions, and of the effects of competing substrates on the rates of Phn cataboli. is that there were at least two different C-P bond cleavage activities in Campylobacter spp. One was exclusively associated with the cell wall of the bacterium and able to hydrolyse phosphono alkyl carboxylates and phenyl phosphinate The other activity was found in both the cell-wall and cytosolic fractions and able to cleave phenyl phosphonate. Ii addition, both activities hydrolysed α-amino-methyl- phosphonate. The hydrolysis of alkyl- and α-amino-alkyl- phosphonates showed specificity with respect to the length oi the alkyl chains; and the cleavage of phenyl phosphonate was modulated by the type of substituents of the phenyl moiety.

These results suggested that the enzymes responsible for eacl of these activities have only a limited range of substrates. The catabolism of PhePhn indicated the presence of a C-P lyas able to cleave also the C-P bond of α-amino-methy1- phosphonate, similar to those found in other Gram-negative bacteria such as Agrobacterium radiobacter [6] , Alcaligenes eutrophus [10] , E. coli [11] , Rhodobacter capsulatus [10] , ai various species of Klebsiella [6, 12], Kluyvera [6], Pseudomonas [10, 13] and Rhizobium [14, 15] . C-P lyases actii on these substrates have been observed also in the Gram- positive bacteria Arthrobacter atrocyanus [16] , Arthrobacter sp. GLP-I [10] , and Bacillus megaterium [17] . The other Phn cleaving activity could be a phosphonatase or a phosphonoacetate hydrolase, since both substrates are catabolised by it. Commonly, but not always, the expression c the former is regulated by phosphate [3, 16] . For example, th expression of Pseudomonas florescens phosphonatase is regulated by phosphate, but that of its phosphonoacetate hydrolase is not [18, 19] . Thus, the Campylobacter spp. enzyn which hydrolysed phosphonoacetate is likely to be a hydrolase In most bacteria the catabolism of phosphonate is under phosphate starvation control and the corresponding enzymes are part of the Pho regulon [1, 2, 13, 20] . This was not the case for either of the activities observed in Campylobacter spp., suggesting that the corresponding enzymes are different from known C-P cleaving enzymes. This interpretation is supported by the fact that no gene orthologous to those coding for any of the four types of enzymes catabolising Phn in other organisms have been identified in the genome of C. jejuni . The expression of the. enzyme activities in media abundant in phosphate suggested that also they may have other physiological roles.

The results of cell culture experiments in the presence of various phosphorus sources indicated that Campylobacter spp., similarly to E. coli [11] could survive 1 using PhePhn under conditions of phosphorus stress. At variance with E. coli and many other bacteria, the two phosphonate hydrolysis activities were expressed in Campylobacter spp. even when there is abundant orthophosphat€ in the culture medium, suggesting that phosphonate degradatic by Campylobacter spp. also could occur in environments with substantial backgrounds of phosphate as it is the case with I cepacia Palβ [21] , Pseudomonas paucimobilis strain MMMlOl [10] , Pseudomonas putida NG2 [22] , and Rhizobium huakii PMYl [23] . This ability to utilise phosphonate may be important fc the survival of the bacteria as in their transmission from host to host they find environments with different phosphate concentrations . Table 1

Kinetic parameters of C. coli ATCC 33559 and lysat€ for phosphonoacetate (PhnAce) , α-amino-methyl phosphonate (AmePhn) , and phenylphosphonate (PhePhn) . Measurements were carried at 37°C, in NaCl/KCl suspensions.

Table 2 Rates (nmole min^"1 mg^"1) of C-P bond hydrolysis by C coli ATCC 33559 lysates for PhnAce, AmePhn and PhePhn are indicated by rows. The values for a single substrate are give in the diagonal, and in competition experiments with each one of the other substrates in the off-diagonals . Substrate concentrations were 50 mM. Measurements (n = 3) were carried at 37°C, in NaCl/KCl suspensions. Errors determined as averages of the three measurements were estimated at ± 15% .

Example 2

The degradation of phosphonates by the bacterium Helicobacter pylori was investigated employing similar materials and nuclear magnetic resonance spectroscopy and cell culture techniques as in Example 1. The bacteria were capable of cleaving the phosphonate (C-P) bonds of different compounds including _α-amino-methyl-phosphonate, phosphonoacetate and phenyl phosphonate. The kinetic parameters of these activities were determined in vivo in intact cells and in situ in whole-cell lysates. Cleavage of phosphonate-bearing compounds was associated with both the cell-wall and σytosolic fractions. Measurement of the activities of different fractions and results from substrate competition experiments suggested that at least two enzyme activities appeared to be involved in the hydrolysis of C-P bonds. In most bacteria studied to date, activities for phosphonate catabolism are induced under conditions of phosphate limitation; however in H. pylori these activities were expressed in cells grown in media rich in phosphate. In chemically defined media phenyl phosphonate supported bacterial growth and proliferation in the absence of phosphate. Thus, phosphonate utilisation may be a survival mechanism of H. pylori in milieux lacking sufficient phosphate. The expression of these enzyme activities in media abundant in phosphate suggested that also they may have other physiological roles.

References

The disclosure of the following documents is hereir incorporated by reference:

1. Kononova, S. V., and Nesmenayanova, M. A. (2002) Biochemistry (Moscow) 67, 184-195.

2. Wanner, B. L. (1994) Biodegradation 5, 175-184.

3. Lee, K-S., Metcalf, W. W., and Wanner, B. L. (1992) J. Bacteriol. 174, 2501-2510.

4. Jiang, W., Metcalf, W. W., Lee, K. -S., and Wanner, B. L. (1995) J. Bacteriol. 177, 6411-6421.

5. Imazu, K., Tanaka, S., Kuroda, A., Anbe, Y., Kato, J., and Ohtake, H. (1998) Appl . Env. Microbiol. 64, 3754- 3758.

6. Wackett, L. P., Shames, S. L., Venditi, C. P., and Wals]< C. T. (1987) J. Bacteriol. 169, 710-717.

7. Ternan, N. G, Quinn, J. P. (1998) Biochem. Biophys. Res. Commun. 248, 378-381.

8. Parkhill, J., Wren, B. W., Mungall, K., Ketley, J. M., Churcher, C, Basham, D., Chillingworth, T., Davies, R. M., Feltwell, T., Holroyd, S., Jagels, K., Karlyshev, A. V., Moule, S., Pallen, M. J., Penn, C. W., Quail, M. A., Rajandream, M. A., Rutherford, K. M., van Vliet, A. H., Whitehead, S., Barrell, B. G. (2000) Nature 403, 665-66£

9. Quinn J. P., and McMullan G. (1995) Microbiology 141, 721-725.

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11. Metcalf, W. W., and Wanner, B. L. (1993) J. Bacteriol. 175, 3430-3442. 12. Ohtake, H., Wu, H., Imazu, K., Anbe, Y., Kato, J., and Kuroda, A. (1994) Resour. Conserv. Recycl. 18, 125-134. 13. Stover, C. K., Pham, X. Q., Erwin, A. L., Mizoguchi, S. D., Warrener, P., Hickey, M.J., Brinkman, F. S., Hufnagle, W. 0., Kowalik, D.J., Lagrou, M., Garber, R. L., Goltry, L., Tolentino, E., Westbroσk-Wadman, S., Yuan, Y, Brody, L. L., Coulter, S. N., Folger, K. R., Kas, A., Larbig, K., Lim R₇, Smith, K., Spencer, D., Wong, G. K., Wu, Z., Paulsen, I. T., Reizer, J., Saier, M. H., Hancock, R. E., Lory, S., Olson, M. V. (2000) Nature 406, 959-64.

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Claims

Claims :

1. A method for identifying a carbon-phosphorus (C- P) or carbon-arsenic (C-As) bond-containing moiety- suitable for linking to an antimicrobial agent in order to generate a pro-drug for administration to an animal for the treatment or prevention of microbial infection in said animal, comprising the steps of:

(1) providing a compound comprising the C-P or C-As bond-containing moiety; (2) introducing said compound to a microorganism capable of infecting an animal, or to a lysate thereof;

(3) establishing whether the C-P or C-As bond is hydrolysed; and (4) identifying a C-P or C-As bond-containing moiety as suitable for linkage to an antimicrobial agent if the C-P or C-As bond is hydrolysed.

2. A method as claimed in claim 1 comprising observing the decrease in levels of the compound and/or the increase in levels of at least one metabolite of the compound in a suspension of living cells or whole cell lysates .

3. A method as claimed in claim 2 wherein the or each observation employs ¹H-MMR spectroscopy or ³¹P-NMR spectroscopy.

4. A method as claimed in any one of claims 1 to 3 further comprising establishing kinetic parameters for the hydrolysis reaction.

5. A method as claimed in claim 4 wherein the kinetic parameters are the Michaelis constant (K_M) and maximal velocity (V_max) for C-P bond cleavage.

6. A method as claimed in any one of claims 1 to 5 further comprising identifying the enzyme activities involved in C-P or C-As bond cleavage in the microorganism.

7. A method as claimed in any of claims 1 to 6 further comprising establishing transport characteristics of the compound across the cell wall and/or cytoplasmic membrane of the microorganism.

8. A method as claimed in any one of claims 1 to 7 wherein the microorganism is selected from the group consisting of Campylobacter spp. , Helicobacter ssp. , Bacillus ssp., Escherichia ssp., Enterobacter ssp., Klebsiella ssp. , Kluyvera ssp. , Pseudomonas ssp. , Burkholderia ssp. , Candida ssp. , and Corynebacteria, Mycobacteria, Proteus spp. and Yersinia spp.

9. A method for the design of a candidate pro-drug for administration to an animal for the treatment or prevention of microbial infection in said animal, comprising the steps of:

(1) identifying a C-P or C-As bond-containing moiety by the method claimed in any one of claims 1 to 8;

(2) selecting an antimicrobial agent lethal to the causative microorganism;

(3) establishing feasible chemistry for linking them.

10. A method of preparing a candidate pro-drug for administration to an animal for the treatment or prevention of microbial infection in said animal, comprising: providing a first reactant comprising a C-P or C- As bond-containing moiety identified by the method claimed in any one of claims 1 to 8; providing a second reactant comprising an antimicrobial agent lethal to the causative microorganism; and reacting the first reactant with the second reactant so as to link the C-P or C-As bond-containing moiety to the antimicrobial agent.

11. A candidate pro-drug for administration to an animal for the treatment or prevention of microbial infection in said animal comprising a C-P or C-As bond- containing moiety identified by the method claimed in any one of claims 1 to 8 linked to an antimicrobial agent lethal to the causative microorganism.

12. A method for the identification of a pro-drug for administration to an animal for the treatment or prevention of microbial infection, comprising the steps of: providing a candidate pro-drug as claimed in claim 11; and screening said candidate pro-drug for antimicrobial activity.

13. An enzyme which catabolises phosphonate in Campylobacter spp. and Helicobacter ssp. , putatively a phosphonotase or a phosphonoacetate hydrolase, said enzyme characterised in that its activity is associated exclusively with the cell wall and in that it hydrolyses phosphonoalkyl carboxylates, phenyl phosphonate and α- aminomethyl-phosphonate, wherein phosphonate catabolism is not under phosphate starvation control.

14. An enzyme which catabolises phosphonate in Campylobacter spp. and Helicobacter ssp. , putatively a C-P lyase, said enzyme characterised in that its activity is associated with both cell wall and cytosolic fraction and in that it hydrolyses phenyl phosphonate and α- aminomethyl-phosphonate, wherein phosphonate catabolism is not under phosphate starvation control.