GB2387600A - Pantothenate kinase - Google Patents

Abstract

An isolated pantothenate kinase protein derived from Staphylococcus aureus is shown to be a suitable target for antimicrobial therapy. The protein may also be used to diagnose Staphylococcal infections and also to screen molecules for their ability to inhibit Staphylococcal growth.

Description

GENE AND PROTEIN

Field of the Invention

This invention relates to the production of antimicrobial agents for use in therapy, to methods for identifying suitable antimicrobial agents and to the use of staphylococcal-

5 specific genes and proteins for diagnosis of infections.

Background to the Invention

The staphylococci are a medically important genera of bacteria known to cause a number of diseases in humans, for example, septicemia, endocarditis, toxic shock syndrome, urinary tract infection and osteomyelitis.

10 It is now widely recognised that conventional antibiotics are becoming less effective in treating microbial infections due to the spread of resistant microbial strains.

One particularly prevalent and clinically difficult resistant organism is methicillin-resistant Staphylococcus aureus (MRSA) where treatment options are limited. It is therefore important to develop new antimicrobial agents to treat infection, and in particular, to 15 develop new antimicrobial agents to treat staphylococcal infections.

An approach to identifying potential antimicrobial agents is to select a suitable target, the inhibition of which will result in the eradication of the microbe with minimum effects on the host. Such targets may be broad spectrum in that they are found in most pathogenic bacteria, or they may be narrow spectrum, that is, confined to one organism, 20 or to one class of organisms. Such narrow spectrum or niche targets have the advantage that inhibitors act only against the pathogen containing the target and do not affect the natural microbial population of the host. Thus narrow spectrum inhibitors are less likely to result in the selection of resistance mechanisms.

In considering novel single targets (e.g. those not part of large assemblies such as 25 ribosomes) an important consideration is whether the target, usually an enzyme, is unique to bacteria. If so, its properties and suitability for the design or discovery of useful inhibitors can be exploited without detrimental effects on the host. Other targets, such as dihydrofolate reductase (DHFR), are common to both bacterial and animal cells and therefore selective inhibition of the microorganism is essential if dose-dependent toxicity 30 to the host is to be avoided. The antimicrobial drug trimethoprim is a useful example of a clinically valuable DHFR inhibitor which competes with the substrate (dihydrofolate) and binds 50000-fold more tightly to bacterial than mammalian forms of DEER.

There are therefore several considerations that must be made when selecting a suitable target, and these considerations make the selection of a suitable target difficult.

Therefore, there is a recognised need for identifying and characterizing suitable targets that may be useful in antimicrobial therapy. In particular, there is a need for 5 identifying targets that may be used to screen compounds for antimicrobial activity which can be used to prevent, reduce or eradicate infections.

Pantothenic acid is an essential dietary requirement in mammals and is a necessary precursor for the biosynthesis of coenzyme A (Abiko, 1975, Metabolic Pathways, Vol. 7, ppl-25). Coenzyme A is cofactor In a wide variety of biochemical pathways and it is 10 estimated that about 4% of all enzymes use coenzyme A or a thioester of coenzyme A as substrate. The enzymatic conversion of pantothenic acid to coenzymeA proceeds via five distinct chemical reactions (Fig. 1) which occur in prokaryotes and eukaryotes.

Pantothenate kinase (PK) is the first enzyme in the conversion of pantothenate to coenzyme A and catalyzes the phosphorylation of pantothenate (vitamin B5) to give 4' 15 phosphopantothenate in an ATPdependent reaction. The PK of Escherichia cold has been extensively studied and a high resolution crystal structure has been solved (Yun et al, 2000, J. Biol. Chem., 275:28093-28099).

PK of E. cold is encoded by the coaA gene (Vallari and Rock, 1987, J. Bact., 169:5795-5800). A number of bacteria, including some pathogens, contain genes 20 presumed to encode PK, since these genes show a high identity to the E. cold coaA gene.

Such genes can be identified in published bacterial genomes such as the genomes of Streptococcus pneumonias, Haemophilus influence, Mycobacterium tuberculosis, Enterococcusfaecalis and others using algorithms such as BLAST (Altschul et al 1990, J. Mol. Biol., 215: 403-410).

25 Other genes that encode PK but show little homology to the coax gene of E. cold exist. These include a gene identified from Bacillus subtilis (0 01/21772 A2) which was previously named yacB, and has now been named coax. Another class of genes encoding PK has been found in fungi such as Aspergillus nidulans (Carder et al, 1999, J. Biol. Chem., 274:2014-2020) and Saccharomyces cerevisiae. Homologues ofthe fungal 30 genes can be found in genome sequences from higher eukaryotes including human, mouse (Rock et al, 2000, J. Biol. Chem., 275:1377-1383) and Drosophila melanogaster.

There is little conservation of sequence between these three classes of genes encoding PK. TheA. nidulans gene shows only 10.7% identity with the coaA gene of E. cold (Fig. 2), and the coaX gene of B. subtilis is only 7. 9% identical to the GOaA gene of E. coli(Fig. 3), while theA. nidulansgeneisonly5.5%identicalto the coaXgeneofB.

5 sublilis (Fig. 4).

Summary of the Invention

The present invention is based on the realisation that enzymes involved in coenzyme A biosynthesis may be suitable targets for antibacterial agents.

In particular, it has been appreciated that the enzyme pantothenate kinase is a 10 suitable target to inhibit the coenzyme A synthetic pathway, thereby preventing bacterial growth. Further, it has been realized that PK from Staphylococcus aureus is a suitable target to inhibit to prevent growth of staphylococci. The suitability of staphylococcal PK is based, at least in part, on an appreciation that the differences between the PK of Staphylococcus aureus and the PK enzymes of other organisms may be exploited to 15 provide selective inhibition of the staphylococcal enzyme.

Moreover, the difference between the PK of Staphylococcus aureus and the PK enzymes of other organisms may provide a method of diagnosing staphylococcal infections by using a staphylococcal PK-specific antibody, or by PCR amplification and detection of the staphylococcal PK gene, or by some other technique well known to those skilled in the 20 art.

The present invention relates to the nucleotide sequence of the staphylococcal pantothenate kinase gene and the amino acid sequence of its encoded pantothenate kinase protein, as well as derivatives and analogues thereof, and antibodies thereto. The present invention further relates to the use of staphylococcal pantothenate kinase genes and the 25 encoded proteins or derivatives or analogues thereof, and antibodies thereto, in assays for the detection and in treatment/prevention of disease states associated with staphylococcal infections. It further relates to the use of staphylococcal pantothenate kinase protein or derivatives or analogues thereof in the screening of small molecule inhibitors capable of binding to the pantothenate kinase functional protein and their use in, but not limited to, 30 structure-based drug design.

Detailed Description of the Invention

The PK gene can be from one of many staphylococcal species, including but not limited to Staphylococcus aureus and Staphylococcus epidermidis, in naturally occurring sequence or in variant form, or from any source, whether natural, synthetic or 5 recombinant. In a specific embodiment described herein, the PK gene sequence is a Staphylococcus aureus sequence (Fig. 5). Due to the inherent degeneracy ofthe genetic code, other DNA sequences which encode substantially the same or a functionally equivalent PK, are within the scope ofthe invention. Such DNA sequences include those which are capable of hybridizing to the staphylococcal PK sequence under stringent 10 conditions which are defined as those hybridizing conditions that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCI / 0.0015 M sodium citrate / 0.1% SDS at 50 C; (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin / O. 1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer at pH 6.5 15 with 0.75 M NaCl, 0.075 M sodium citrate at 42 C; or (3) employ 50% formamide, 5 times SSC (0.75 MNaCl, 0. 075 M sodium pyrophosphate, 5 times Denhardt's solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS, and 10% dextran sulfate at 42 C, with washes at 42 C in 0.2 times SSC and 0.1% SDS.

Altered DNA sequences which may be used in accordance with the invention 20 include deletions, insertions, inversions, or substitutions of different nucleotides resulting in a sequence that encodes the same or a functionally equivalent gene product. The gene product itself may contain deletions, additions or substitutions of amino acid residues within the PK sequence, which result in a functionally equivalent PK protein. Amino acid substitutions may be made on the basis of similarity of physical attributes such as polarity, 25 charge, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. The DNA sequence of the invention may be engineered in order to alter the PK coding sequence for a variety of ends including but not limited to alterations which modify processing and expression of the gene product. For example; mutations may be 30 introduced using techniques which are well known in the art, e.g. site-directed mutagenesis, to insert new restriction sites, etc.

-- The PK protein can be from one of many staphylococcal species, including but not limited to Staphylococcus aureus and Staphylococcus epidermidis, in naturally occurring sequence or in variant form, or from any source, whether natural, synthetic or recombinant. In a specific embodiment described herein, the PK protein is a 5 Staphylococcus aureus protein.

As defined herein, a PK derivative may be a fragment or amino acid variant of the PK sequence shown in (Fig. 6) as long as the fragment or amino acid variant is capable of displaying one or more biological activities associated with the full length PK protein.

Such biological activities include, but are not limited to, the pantothenate kinase enzyme 10 function attributed to the polypeptide chain. Nucleic acids encoding such derivatives or analogs are also within the scope of the invention. A preferred PK variant is one sharing at least 70% amino acid homology, a particularly preferred variant is one sharing at least 80% amino acid sequence homology and another particularly preferred PK protein variant is one sharing at least 90% amino acid sequence homology to the naturally occurring PK 15 protein over at least 25, at least 50, at least 75, or at least 100 contiguous amino acids of the PK amino acid sequence. As used herein, amino acid sequence homology refers to amino acid sequences having identical amino acid residues or amino acid sequences containing conservative changes in amino acid residues. In another embodiment, a PK homologous protein is one that shares the foregoing percentages of sequences identical 20 with the naturally occurring PK protein over the recited lengths of amino acids.

In another embodiment ofthe invention, the coding sequence ofthe PK gene can be synthesized in whole or in part using chemical synthesis methods well known in the art.

In addition, the protein itself, in whole or in part, could be produced by chemical synthesis methods using solid phase chemistries well known in the art and the sequence of such 25 synthetic polypeptides confirmed by amino acid sequencing, amino acid analysis or mass spectrometry. Another embodiment of the invention relates to the use of staphylococcal PK to select for antimicrobial agents. According to the present invention, a method for identifying antimicrobial agents, comprises the steps of: 30 (i) contacting a sample containing staphylococcal PK with a suitable substrate and a target compound under suitable conditions; (ii) measuring the activity of the PK enzyme; |

! 6 (iii) comparing the activity ofthe enzyme to that of a reference sample lacking the target compound; and (iv) selecting those target compounds that reduce the activity of the PK enzyme. 5 In a further embodiment, the present invention proposes the use of compounds capable of inhibiting the PK enzyme at a cellular or tissue concentration of less than 10 aM in antimicrobial therapy. In particular, the compounds may be used in the treatment of infection by Staphylococcus aureus or Staphylococcus epidermidis.

The invention also relates to the use of staphylococcal PK polynucleotides and 10 polypeptides as diagnostic reagents. Detection ofthe staphylococcal PK polynucleotides and/or polypeptides in a eukaryote, such as a mammal, and especially a human, will provide a diagnostic method for diagnosis of disease, and/or the response ofthe infectious organism to antibiotic therapy. Eukaryotes, particularly humans and other mammals, suspected to be infected with staphylococci, may be detected at the nucleic acid or amino 15 acid level by a variety of well known techniques such as detection of PK polynucleotide sequences by PCR or some other amplification technique, and detection of the PK polypeptide by radioimmunoassay, antibody detection and ELISA assays, and other methods well known to those skilled in the art.

The PK genomic sequence is provided by the present invention. Also included 20 within the scope of the present invention are polynucleotide sequences of PK consisting of at least 8 nucleotides, at least 15 nucleotides, at least 25 nucleotides, at least 100 nucleotides, at least 500 nucleotides, or at least 801 nucleotides. Sequence analysis ofthe PK gene sequence reveals an open reading frame of 801 nucleotides, potentially encoding a protein of 267 amino acids with a predicted molecular weight of 29025Da.

25 The present invention is illustrated by way of examples disclosing the cloning and sequencing of staphylococcal PK.

Example

Cloning ofthe pantothenate kinase gene from Staphylococcus aureus by functional complementation of an E. cold temperature-sensitive mutant strain.

30 The S. aureus PK gene was cloned using a functional complementation approach.

A sample of a Staphylococcus aureus genomic library containing Staplococcus aureus RN450 genornic DNA inserted into Lambda ZAP 11 vector (Stratagene) was used to

produce a phagemid library, following the manufacturer's instructions. A portion ofthe phagemid library, sufficient to represent the genomic library five times over, was used to transform electrocompetent E. cold DV62. E cold DV62 (Vallari and Rock, 1987, J. Bact., 169:5795-5800) is a strain of E. cold that carries a temperature-sensitive coaA gene 5 such that the strain grows at 30 C (the permissive temperature), but not at 44 C (the non-

permissive temperature). The transformed cells were plated on L-agar plates containing 100,ug/ml ampicillin and samples were incubated at 30 C and 44 C. The incubation at 30 C confirmed that the transformation produced sufficient colonies to represent the entire S. aureus genome. Following incubation at the non-permissive temperature, forty 10 colonies were obtained. Of these, 32 were selected for follow-up and were analysed and characterized by digestion with restriction enzymes. Ofthe selected phagemids,31 proved to be identical. E. cold DV62 transformants containing each ofthese 32 phagemids were re-tested for growth at the non- permissive temperature wherein the 31 identical phagemids were able to support growth, whereas the dissimilar phagemid did not support growth at 15 44 C and was therefore deemed to be a false positive.

Sequence analysis of one of the 31 identical phagemids revealed that the insert sequence spanned 4.2 kb and contained three major open reading frames called ORF 1, ORF 2 and ORF 3, encoding hypothetical proteins of 397, 223 and 267 amino acids respectively. Each of these open reading frames was of unknown function and could 20 plausibly encode a pantothenate kinase protein. In order to identify the PK gene, the 4.2 kb region was dissected by (1) removing a HindIII fragment from the insert which had the effect of deleting ORT 1 encoding the 397 amino acid hypothetical protein and (2) removing an MfeI fragment from the insert which had the effect of deleting ORF 2 encoding the 223 amino acid hypothetical protein together with 299 amino acids from the 25 C-terminus ofthe 397 amino acid hypothetical protein encoded by ORF 1. These deletion constructs were tested for complementation of the mutation in the temperature sensitive strain E. cold DV62, wherein both deletion constructs were able to complement the mutation. Thus the remaining intact open reading frame, ORF 3, encoding a 267 amino acid hypothetical protein, was able to produce functional PK activity when heterologously 30 expressed in E. coli.

Analysis of the sequence of the 267 amino acid protein showed very limited similarity with the coaX gene of B. subtilis and the coaA gene of E. cold (Figs. 7 and 8).

Pairwise alignments were produced using the Blosum62mt2 algorithm (Vector NTi) and the identity between the ORF 3 protein and E. cold coaA was 12.7 % and the identity between the ORF 3 protein and B. subtilis coaXwas 12. 4%. The 267 amino acid protein shows greater similarity with some ofthe PK enzymes from eukaryotes which are encoded 5 for by panK genes (Fig. 9), with 17.7% identity between the ORF 3 protein and PK1 p from mouse and 18.2% identity to human PK1,8.

Thus the staphylococcus PK enzyme is sufficiently different from the PK enzymes produced by other bacteria, and from the PK enzymes produced by eukaryotes, including mammals and particularly humans, that selective inhibition ofthe staphylococcal enzyme 10 should be possible. Furthermore, the sequences of the PK gene (and protein) are sufficiently different from the sequences of the eukaryotic PK genes (and proteins), including those of mammals and particularly of humans, and from the sequences ofthe PK genes (and proteins) of other bacteria, that diagnostic methods based on these differences could be designed to detect staphylococcal infections.

Claims (15)

1. An isolated protein comprising the amino acid sequence shown in Fig. 6; SEQ ID No. 1.

2. An isolated DNA molecule comprising the DNA sequence shown in Fig. 5; SEQ 5 ID No. 2.

3. Use of the product of claim 1 or claim 27 in therapy or diagnosis.

4. A method for identifying an anti-staphylococcal agent, comprising the steps of: (i) contacting a sample containing staphylococcal PK enzyme with a suitable substrate and a target compound; 10 (ii) measuring the activity of the enzyme; (iii) comparing the activity ofthe enzyme to that of a reference sample lacking the target compound; and (iv) selecting a target compound that reduces the activity of the enzyme.

5. A method according to claim 4, wherein the enzyme is obtainable from 15 Staphylococcus aureus.

6. A method according to claim 4 or claim 5, wherein the staphylococcal PK enzyme is defined as in SEQ ID NO. 1 or a functional equivalent thereof.

7. A method according to any of claims 4 to 6, wherein the target compound does not inhibit the mammalian PK enzyme.

20

8. A method according to any of claims 4 to 7, wherein the target compound reduces the activity of the enzyme by at least 20% at a concentration of less than 1 OO'lM..

9. An anti-staphylococcal compound identified using a method according to any of claims 4 to 8.

10. A compound according to claim 9, for use in a method of therapy.

25

11. Use of a compound according to claim 9, for the manufacture of a medicament for the treatment of a staphylococcal infection.

12. The use of a substantially pure staphylococcal PK enzyme, or fragment thereof, in a method for the identification of an anti-staphylococcal agent

13. The use of a gene encoding a staphylococcal PK enzyme, or fragment thereof, in 30 a method for the identification of an antistaphylococcal agent.

14. The use of a gene encoding staphylococcal PK enzyme for the detection of Staphylococcus aureus in human disease.

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15. The use of an antibody against a staphylococcal enzyme for the detection of Staphylococcus aureus in human disease.