WO2013038183A1

WO2013038183A1 - Methods of screening for antimicrobial compounds

Info

Publication number: WO2013038183A1
Application number: PCT/GB2012/052259
Authority: WO
Inventors: Adrian Lloyd
Original assignee: University Of Warwick
Priority date: 2011-09-13
Filing date: 2012-09-12
Publication date: 2013-03-21
Also published as: GB201115793D0

Abstract

The invention provides methods of screening for antimicrobial compounds in which an enzyme which is capable of catalysing a reaction between a nucleotide diphosphate and a substrate to produce a nucleotidylate species is incubated with a test compound and a nucleotide diphosphate. The rate and/or extent of the reaction is then compared to a control reaction carried out in the absence of the test compound. Where the nucleotide diphosphate is AP₄A, the rate or extent of the reaction may be assessed by following ATP production using a hexokinase/glucose dehydrogenase couple and monitoring the change in absorbance (A₃₄₀) of the reaction mixture. These methods are particularly suitable for screening for potential antibiotic agents which target bacterial aminoacyl t RNA synthetase enzymes. The methods are also amenable to use in high- throughput screening.

Description

Methods of screening for antimicrobial compounds

The present invention relates to methods of screening for compounds having

antimicrobial activity. In particular, methods of the invention are useful for screening for inhibitors of synthetase enzymes, such as aminoacyl transfer-RNA synthetases, where such inhibitors may possess antimicrobial or other desirable activities. The invention also relates to methods for measuring the levels of mononucleotide phosphates, especially adenosine triphosphate, in the presence of dinucleotide phosphates or analogues thereof, as well as methods of screening for test compounds using said methods.

The search for new and improved antimicrobial agents is key to the fight against pathogenic microbes such as bacteria, viruses and parasites. The emergence of widespread resistance to clinical antimicrobial agents, perhaps most notably methicillin- resistant Staphylococcus aureus (MRSA), means that it is essential to continue to develop new and improved antimicrobial drugs. One essential stage in many drug development strategies is the large-scale screening of compounds or compound fragments which interact with a known biological target (e.g. a protein target) for their ability to modulate the effect of the target (e.g. to inhibit the catalytic activity of the protein). Many strategies exist for the screening of potentially antimicrobial compounds based on the evaluation of their modulating effects on several important microbial targets.

An important strategy in the development of antibacterial agents is linked to the inhibition of protein synthesis, as exemplified by the successful use of antibiotics such as chloramphenicol, tetracycline and erythromycin. The bacterial aminoacyl transfer- RNA (tRNA) synthetase which supplies protein synthesis with aminoacyl-tRNA substrates is a clinically-proven protein target, as exemplified by mupirocin

(bactroban™). However the continual development of resistance in clinical infection to these antibiotics makes development of new antibiotics essential in targeting protein synthesis, as well as other functions essential for pathogen viability. Many drugs in clinical use can differentiate between the bacterial and mammalian aminoacyl tRNA synthetase (aaRS) enzymes and so represent a particularly important class of microbial targets for the development of new and improved drugs. For example, the antibacterial agent mupirocin (produced naturally by bacteria such as Pseudomonas fluorescens) is a potent inhibitor of isoleucyl-tRNA^lle synthetase (NeRS) and is clinically exploited as an antibiotic targeting Staphylococcal infections. However, the chemical lability of the drug does not allow it to be applied systemically. Furthermore, mupirocin is inactive against the causative agent of tuberculosis and it can become subject to resistance.

All of the natural aaRSs required to direct protein synthesis in microbes from the 20 "natural" amino acids represent potential antibiotic targets in screening for new compounds or compound fragments. Other microbial pathways which may be targeted by drug development strategies include fatty-acid biosynthesis, e.g. by modulation of fatty acyl-CoA synthetase (FAS) enzymes; synthesis of "non-natural" peptides by certain microbes, e.g. by modulation of non-ribosomal peptide synthetase (NRPS) enzymes; as well as coenzyme A (CoA) biosynthesis, e.g. by modulation of 4- phosphopantetheine adenylyl transferase (4-PAT) enzymes, and nucleic acid synthesis and repair, e.g. by modulation of RNA ligase enzymes. All of the enzymes listed above share a common feature, namely that each catalyses a reaction which proceeds via an adenylate intermediate or, in the case of 4-PAT, generates an adenylate product.

For example, aaRSs share a common mechanism involving activation of the cognate amino acid by reaction with adenosine triphosphate (ATP) to form a central aminoacyl adenylate (Figure 1 ). Consistent with their function, aaRSs then catalyse the attack of this central intermediate by tRNA to form the correct aminoacyl-tRNA product (Figure 1 , reaction 1 ). However, in the absence of tRNA, these enzymes can catalyse the cleavage of this central aminoacyl adenylate by pyrophosphate or by a second molecule of ATP (Figure 1 , reactions 2 and 3, respectively). These side-reactions generate ATP and adenosine tetraphosphoadenosine (AP₄A - also known as diadenosine

tetraphosphate and bis(5'-adenosyl) tetraphosphate), respectively (exemplified in Figure 1 , reactions 2 and 3). AP₄A is a molecule known to be produced in many organisms and is believed to be associated with regulation of vasodilation, platelet aggregation, synaptic

neurotransmission and cell cycle control in mammals, as well as in regulation of the stress response in bacteria. Known methods for the detection of AP₄A include radiolabelling (e.g. with tritium) and mass spectroscopic methods.

For example, one method for assaying aaRS activity takes advantage of the ability of trichloroacetic acid to precipitate tRNA onto disks of filter paper (see e.g. Bollum, J. Biol. Chem. (1959) 234 (10);2733-2740). Essentially, aaRS is incubated with [¹⁴C] or [³H] amino acid, ATP and tRNA. The incubation mixture is pipetted onto a disk of filter paper which is immediately immersed in 10% (w/v) cold trichloroacetic acid. The excess radioactive material is washed off with repeated washes of trichloroacetic acid. Disks are finally washed in ethanol, dried and the radioactivity is counted by liquid scintillation counting. This radioactive method can sensitively detect activity (although not necessarily detection of an aaRS inhibitor) but suffers from a number of drawbacks including the high cost of purchasing radiolabeled compounds and the creation of radioactive waste with its associated high disposal costs; the use of corrosive

substances (e.g. trichloroacetic acid); the large number of wash steps involved which make automation for high/medium throughput work virtually impossible; a compromised sensitivity when performing kinetic analysis; and, perhaps most significantly, an inability to monitor product accumulation continuously without needing to carry out product purification steps.

There exists an acute need for new methods of screening for antimicrobial compounds or compound fragments which can modulate biological targets, especially targets involved in reactions that proceed via adenylate intermediates such as aaRSs. It would be particularly desirable to develop new screening methodologies which have the sensitivity to test the properties of compounds which bind weakly to the biological target, e.g. to provide for sensitive screening of compound fragments which can be further developed into highly specific drugs.

The present inventor has surprisingly discovered that some enzymes which catalyse reactions proceeding via an adenylate intermediate (e.g. aaRSs) can utilise AP₄A as a substrate (exemplified in Figure 2). The extent and/or rate of the enzyme-catalysed reaction of AP₄A with a nucleophile can be modulated by the presence of test compounds and measurement of the extent and/or rate of this reaction can report directly on whether and/or to what extent the test compound can act as a modulator of the enzyme. In particular, because of the relatively weak binding affinity of AP₄A to these enzymes, the binding of compounds with weak inhibition constants (having K, values of the order of mM) can be measured accurately. This makes the methods of the invention especially suitable for use in drug development using fragment-based drug design techniques and for incorporation into high-throughput screening methods.

The present inventor has also surprisingly discovered that ATP can sensitively and accurately be measured in the presence of dinucleotide phosphates (such as AP₄A; see Figure 3). This finding provides the possibility of methods for detecting and/or quantifying ATP in a reaction mixture which is contaminated by levels of dinucleotide phosphates that would otherwise render the mixture incapable of measurement using known enzymatic methodologies. The detection methods of the invention are simpler and cheaper than the methods known in the art for discriminating between ATP and dinucleotide phosphates in samples (e.g. using mass spectrometry). Furthermore, the methods of the invention are amenable to miniaturised assays and/or may be used for high-throughput screening. In a first aspect, the invention provides a method of screening for compounds having an antimicrobial activity, the method comprising the steps:

(i) incubating an enzyme which is capable of catalysing a reaction between a

dinucleotide phosphate, or analogue thereof, and a substrate to produce a

nucleotidylate species with:

(a) a dinucleotide phosphate, or analogue thereof,

(b) a substrate which is capable of reacting with the dinucleotide phosphate or analogue thereof to form a nucleotidylate species, and

(c) a test compound,

and

(ii) determining the rate and/or extent of the reaction,

wherein a reaction having a lower rate and/or a lower extent when determined in the presence of the test compound compared to the rate or extent of a control reaction in the absence of the test compound is indicative of the test compound having

antimicrobial activity, and

either wherein the substrate is covalently attached to the enzyme or wherein the substrate is not covalently attached to the enzyme.

In one embodiment, the invention excludes methods in which carboxylic acid-containing substrates which are not covalently attached to the enzyme are used. In another embodiment, the invention excludes methods in which phosphoric acid-containing substrates which are not covalently attached to the enzyme are used. By "carboxylic acid-containing" and "phosphoric acid-containing" substrates is meant substrates in which said carboxylic or phosphoric acid groups are capable of reacting as nudeophiles in the reaction with the dinucleotide phosphate.

Preferably, the nucleotidylate species is an acyl nucleotidylate. In this embodiment, the substrate comprises a carboxylic acid group, which substrate is preferably not covalently attached to the enzyme. Accordingly, the invention provides a method of screening for compounds having an antimicrobial activity, the method comprising the steps:

(i) incubating an enzyme which is capable of catalysing a reaction between a

dinucleotide phosphate, or analogue thereof, and a substrate comprising a carboxylic acid group to produce an acyl nucleotidylate species with:

(a) a dinucleotide phosphate, or analogue thereof,

(b) a substrate comprising a carboxylic acid group, and

(c) a test compound,

and

(ii) determining the rate and/or extent of the reaction,

wherein a reaction having a lower rate and/or a lower extent when determined in the presence of the test compound compared to the rate or extent of a control reaction in the absence of the test compound is indicative or suggestive of the test compound having antimicrobial activity.

Alternatively, and also preferably, the nucleotidylate species is a phosphoryl

nucleotidylate. In this embodiment, the substrate comprises a phosphoric acid group, which substrate is preferably not covalently attached to the enzyme. Accordingly, the invention provides a method of screening for compounds having an antimicrobial activity, the method comprising the steps:

(i) incubating an enzyme which is capable of catalysing a reaction between a

dinucleotide phosphate, or analogue thereof, and a substrate comprising a phosphoric acid group to produce a nucleotidylate species with:

(a) a dinucleotide phosphate, or analogue thereof,

(b) a substrate comprising a phosphoric acid group, and

(c) a test compound,

and

(ii) determining the rate and/or extent of the reaction,

The invention relates to a method of screening for compounds having an antimicrobial activity. As used herein, the term "antimicrobial" is used in its general sense to refer to a property of the test compound to prevent or reduce the growth, spread, formation or other activity of organisms such as bacteria, viruses, protozoa, moulds, fungi, algae or other micro-organisms, including parasites. The bacteria may be Gram-positive bacteria and/or Gram-negative bacteria. The bacteria may be anaerobic and/or aerobic.

The bacteria may, for example, be Staphylococcus aureus including methicillin-resistant Staphylococcus aureus, (1V1RSAJ, Streptococci, vancomycin-resistant enterococci (VRE), Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Fusarium species, Salmonela species, Shigella species, Yersinia species, Bacillus species,

Campylobacteria, Clostridium botulinum, C. perfringes or Listeria monocytogenes. Particular fungi of interest in the present specification are clinically-significant fungi, e.g. fungi which grow in or on mammals. Examples of clinically significant fungi include Cryptococcus species, Canidida albicans, Rhizopus species, Aspergillus fumigatus, Peniciiiium species, Absidia species, Scedosporium apiospermum, Phialophora verrucosa, Cunninghamella species, Tricothecium species, Ulocladium species, and Fonsecae species. In one embodiment the fungus is a fungus of the genus Peniciiiium, e.g. Peniciiiium marneffei. In some embodiments, the fungus comprises Candidas or Aspergillus or Magnaporthe or Fusarium infections. More specifically, in some embodiments, the infection can be caused by Candidas or Aspergillus or Magnaporthe or Fusarium, for example, Candida albicans, Candida tropicalis, Candida parapsilokis, Candida krusei, Candida dubliniensis, Cryptococcus neoformans, A. fumigatus, A.

flavus, A. niger, Magnaporthe grisea or Fusarium moniforme.

Clinically-significant protozoa are of particular interest, especially those which are causative agents of disease in humans. Examples of such protozoa include

trypanosomatids, especially those causing sleeping sickness, Chagas disease or leishmaniasis. Exemplary protozoa include Trypanosoma brucei (e.g. subspecies T b. brucei, T b. gambiense or T. b. rhodesiense), Trypanosoma cruzi and those of the genus Leishmania (e.g. L. major, L. tropica, L aethiopica or L. mexicana).

Preferably, the antimicrobial activity is an antibacterial activity, most preferably one which prevents or reduces the growth of at least one bacteria, a bacteriostatic activity or a bactericidal activity. Most preferably, the antimicrobial activity is an antibiotic activity. Alternatively, the antimicrobial activity is an antitrypanosomal activity, especially one which prevents or reduces the growth of at least one trypanosome, e.g. T. brucei.

The enzyme is one which is capable of catalysing a reaction between a dinucleotide phosphate, or analogue thereof, and a nucleophilic substrate (e.g. comprising a carboxylic or phosphoric acid group) in order to produce a nucleotidylate species (e.g. an acyl nucleotidylate). Where the substrate is covalently attached to the enzyme, e.g. the substrate is an amino acid side-chain of the enzyme, the nucleotidylate species formed is an enzyme-bound nucleotidylate. ln a preferred embodiment, the enzyme is capable of catalysing the conversion of the nudeotidylate species (e.g. the acyl nudeotidylate) into a nucleotide triphosphate (NTP), e.g. by reaction with inorganic pyrophosphate. Most preferably, it is an enzyme which is capable of catalysing the conversion of an adenylate intermediate into ATP, preferably an enzyme which is capable of catalysing the conversion of an acyl adenylate

intermediate into ATP.

The enzyme is preferably a prokaryotic enzyme or a non-mammalian enzyme. It may be a microbial enzyme, e.g. a bacterial, fungal, viral or parasitic eukaryotic enzyme, especially an enzyme from a pathogenic microbe, e.g. a pathogen of an animal species, e.g. a mammal, especially a human pathogen. Some examples of human pathogens are listed above.

The term "pathogen" denotes a micro-organism which can temporarily or permanently colonise a host organism in such a way as to be detrimental to the health of the host, i.e. which causes disease in the host. In a preferred embodiment, the enzyme is an enzyme wherein there are differences between the source organism and a homologous enzyme in the host organism which the source organism is a pathogen, i.e. such that there exists the possibility for developing test compounds that preferentially target the enzyme of the pathogenic organism over any homologous enzymes in the host.

In an alternative embodiment, the enzyme is a eukaryotic enzyme, e.g. a mammalian enzyme. Preferred mammalian enzymes include those from humans, farm animals (e.g. sheep, cows, pigs and horses) and laboratory animals (e.g. rats, mice and rabbits).

Preferably, the enzyme is a synthetase, a ligase or a transferase, e.g. a synthetase or a transferase.

Preferred enzymes are selected from an aminoacyl tRNA synthetase (aaRS), a fatty- acyl CoA synthetase (FAS), a non-ribosomal peptide synthetase (NRPS), a 4- phosphopantetheine adenylyl transferase (4-PAT) and an RNA or DNA ligase, e.g.

selected from an aaRS, an NRPS or a 4-PAT. The enzyme may be a functional part or fragment of a native or recombinant enzyme, e.g. a fragment of the enzyme comprising its catalytic site, and having catalytic activity.

In some embodiments, the enzyme is an amino acid tRNA synthetase (aaRS). Such enzymes are preferably classified by the International Union of Biochemistry and Molecular Biology classification EC 6.1 .1 (ligases forming aminoacyl-tRNA and related compounds), especially selected from EC 6.1 .1.1 to 6.1.1.27.

The aaRS may, for example, be an alanyl, arginyl, asparaginyl, aspartyl, cysteinyl, glutaminyl, glutamyl, glycyl, histidyl, isoleucyl, leucyl, lysyl, methionyl, phenylalanyl, prolyl, seryl, threonyl, tryptophanyl, tyrosyl or valyl tRNA synthetase.

Preferred enzymes include isoleucyl-tRNA^lle synthetase (e.g. EC 6.1 .1 .5), alanyl- tRNA^Ala synthetase (e.g. EC 6.1 .1.7) and valyl-tRNA^Val synthetase (e.g. EC 6.1.1 .9).

Other preferred enzymes include leucyl-tRNA^Leu synthetase (e.g. EC 6.1 .1.4) and asparaginyl -tRNA^Asn synthetase (e.g. EC 6.1 .1 .22). A further preferred enzyme is Seryl- tRNA^Ser synthetase (e.g. EC 6.1 .1 .1 1 ). Preferably the aaRS is obtained from a bacterial species, e.g. a bacterium of the genus Escherichia, Staphylococcus, Streptococcus, Mycobacterium, Haemophilus, Chlamydia, Pseudomonas, Shigella, Campylobacter, Acinetobacter or Salmonella.

Bacterial species of particular interest include Escherichia coli; Staphylococcus aureus (especially MRSA and VRSA strains), Staphylococcus epidermidis and Staphylococcus saprophytics; Streptococcus pneumoniae (especially those strains with high level penicillin resistance), Streptococcus pyogenes, Streptococcus agalactiae and

Streptococcus faecalis (also called Enterococcus faecalis and Enterococcus faecicum - especially those strains resistant to vancomycin); Mycobacterium tuberculosis and Mycobacterium leprae; Pseudomonas aeruginosa, Pseudomonas oryzihabitans and Pseudomonas plecoglossicida; Shigella boydii, Shigella dysenteriae, Shigella flexneri and Shigella sonnei; Campylobacter jejuni, Campylobacter coli, Campylobacter upsaliensis and Campylobacter lari; and Salmonella enterica and Salmonella bongori; Haemophilus influenzae; Chlamydia pneumoniae and Chlamydia trachomotis; and Acientobacter baumanii - especially strains of this and other Gram-negative pathogens with extended spectrum β-lactamase mediated β-lactam resistance. In preferred embodiments, the aaRS is derived from a bacterial species which has resistance to one or more aaRS inhibitors, e.g. antibiotics such as β-lactams,

vancomycin, sulphonamides, mupirocin, and/or which is resistant to agents which target the ribosomal protein synthesis, e.g. chloramphenicol, tetracycline erythromycin. The aaRS is especially derived from Staphylococcal species such as S. aureus (e.g. MRSA or MSSA) or other Staphylococci that carry the mupA resistance gene encoding the mupirocin-resistant isoleucyl-tRNA^lle synthetase.

In other embodiments of the invention, the enzyme is a fatty acyl CoA synthetase (FAS). This enzyme is preferably classified under EC 6.2.1 (acid-thiol ligases), especially selected from EC 6.2.1.3, 6.2.1.19 and 6.2.1.20. Preferred enzymes include long-chain fatty-acid-CoA ligases (e.g. EC 6.2.1.3).

In especially preferred embodiments, the FAS is derived from a pathogenic

microorganism, e.g. a bacterium of the genus Mycobacterium, especially derived from M. tuberculosis or M. leprae.

In yet other embodiments, the enzyme is a non-ribosomal peptide synthetase (NRPS). This enzyme is preferably classified under EC 2.7 (enzymes transferring phosphorus- containing groups), especially EC 2.7.8.7 (holo-ACP synthase).

In preferred embodiments, the NRPS is derived from a microorganism that produces a non-ribosomal peptide toxin, e.g. from a cyanobacterium of the genus Nostoc. In a further preferred embodiment the NRPS is derived from an organism that produces a non-ribosomal peptide siderophore, e.g. a Gram-negative organism selected from E. coli and P. aeruginosa or from Salmonella, Klebsiella, Shigella and Yersinia species. In further embodiments, the enzyme is a ligase. This enzyme is preferably an ATP- dependent DNA or RNA ligase classified under EC 6.5.1 , especially EC 6.5.1.1 (DNA ligases) or EC 6.5.1.3 (RNA ligases). RNA ligases are particularly preferred. In preferred embodiments, the ligase is derived from a pathogenic microorganism, especially a virus or a parasitic organism such as a protozoan. Especially preferably, the ligase is an RNA-editing ligase from a trypanosomatid, e.g. RNA-editing ligase 1 or 2 from T. brucei. In yet other embodiments, the enzyme is a 4-phosphopantetheine adenylyl transferase (4-PAT). This enzyme is preferably classified under EC 2.7.7 (nucleotidyltransferase enzymes transferring phosphorus-containing groups), especially EC 2.7.7.3

(pantetheine-phosphate adenylyltransferase). In preferred embodiments, the 4-PAT is derived from a pathogenic microorganism, e.g. from a pathogenic Gram-negative bacterium of the genus Escherichia, Pseudomonas, Haemophilus or Salmonella, or from a pathogenic Gram-positive bacterium of the genus Streptococcus, Staphylococcus, Enterococcus or Mycobacterium. Most preferably, the enzyme is an aminoacyl tRNA synthetase (aaRS).

Enzymes for use in methods according to the invention may be obtained from

commercial sources or may be prepared according to protocols known in the art. It is preferred that the enzymes of the invention are used in a form that is substantially free of contaminating substances (e.g. other enzymes) that can affect the reaction rates measured, especially in a form substantially free of contaminating pyrophosphatase. Known methods may be employed for the removal of contaminating substances such as size exclusion and affinity chromatography. Removal of contaminating substances from an enzyme preparation may be achieved by binding the enzyme to a solid phase via an enzyme-binding moiety attached to the solid phase, which enzyme-binding moiety binds to the test enzyme but not to the contaminating substance. The bound enzyme may then be washed to remove contaminating substance and optionally eluted from the solid phase. For example, removal of contaminating inorganic pyrophosphatase from a valyl- tRNA^Val synthetase preparation may be achieved by binding the synthetase to a Procion Green HE4BDA dye (e.g. from ICI Americas Inc.) covalently attached to Sepharose 4B (e.g. from GE Healthcare), washing the beads and eluting the purified synthetase from the solid support. Alternatively, pyrophosphatase activity could be eliminated by the addition of potassium fluoride to the assays, e.g. at a concentration of about 50 mM.

The substrate is a substrate for or on the enzyme. The substrate is a nucleophile capable of reacting with the dinucleotide phosphate to produce a nucleotidylate species, e.g. an acyl nucleotidylate species.

Where the substrate is covalently attached to the enzyme, the product of the reaction is an enzyme-bound nucleotidylate species. In this case, the substrate is preferably a nucleophilic side-chain of an amino acid making up the primary sequence of the enzyme. Examples of such substrates include the side-chains of tyrosine, cysteine, serine, threonine, lysine, arginine, aspartic acid, glutamic acid, asparagine or glutamine residues, especially lysine residues.

Where the substrate is not covalently bound to the enzyme, the substrate preferably comprises a nucleophilic carboxylic or phosphoric acid group which is capable of attacking the dinucleotide phosphate.

The nature of the substrate will depend on the reaction being catalysed and will, in turn, determine the nature of the nucleotidylate intermediate or product formed in the reaction.

In some embodiments, the carboxylic acid or phosphoric acid group may be replaced by an equivalent group thereof.

The term "equivalent" used in the context of carboxylic acid-containing compounds denotes a functional group having the capacity to participate in chemical reactions by way of nucleophilic substitution in substantially the same way as carboxylic acids.

Examples of carboxylic acid equivalents include esters, thioesters, amides, acid anhydrides and acid chlorides. In the context of phosphoric acid-containing compounds, the term "equivalent" is used to denote phosphorothioates and other phosphate derivatives with one or more O atoms replaced by S, inter alia.

Where the enzyme is an aaRS or an NRPS, the carboxylic acid-containing substrate is preferably an amino acid (or an equivalent thereof) and the intermediate formed is an aminoacyl adenylate intermediate. Preferred amino acids include natural amino acids (e.g. isoleucine, alanine, leucine, asparagine, lysine, aspartic acid, methionine, cysteine, phenylalanine, glutamic acid, threonine, glutamine, tryptophan, glycine, valine, proline, serine, tyrosine, arginine, histidine) as well as non-natural amino acids, which include amino acids with non- natural side-chains (e.g. hydroxyproline, ornithine and taurine) and/or D-amino acid stereochemistry.

Especially preferred for use in reactions catalysed by amino acid tRNA synthetase enzymes and NRPS enzymes are the cognate amino acids, i.e. the amino acid (or acids) which is (or are) the natural substrate for the said enzyme. For example, in a screening method for modulators of the isoleucyl-tRNA^lle synthetase, the substrate is preferably L- isoleucine.

Hence the invention particularly relates to methods wherein the enzyme is a synthetase, preferably an aminoacyl tRNA synthetase and the substrate is the cognate amino acid (e.g. the enzyme is valyl-tRNA^Val synthetase and the substrate is valine).

Where the enzyme is a FAS, the substrate is preferably a fatty acid (or an equivalent thereof) and the intermediate formed is a fatty-acyl adenylate intermediate. Preferred fatty acids include medium and long-chain fatty acids, e.g. fatty acids of formula R-C(0)OH wherein R is a saturated or unsaturated aliphatic group comprising at least 6 carbon atoms in a linear chain, especially at least 8, 10, 12, 14 or 16 carbon atoms in a linear chain. Especially preferably, R is a saturated or unsaturated aliphatic group comprising between 18 and 60 carbon atoms in the longest hydrocarbon chain, especially between 20 and 30 carbon atoms or between 40 and 60 carbon atoms in the longest chain. In a preferred embodiment the fatty acid is a mycolic acid, especially a cyclopropane-containing fatty acid, e.g. selected from alpha-mycolic acid, methoxy- mycolic acid and keto-mycolic acid.

Where the enzyme is a 4-PAT, the nucleophile is preferably a phosphoric acid- containing nucleophile, e.g. 4'-phosphopantetheine (or an equivalent thereof). In this case, the species formed is a nucleotidyl-phosphate species, e.g. a 3'-dephospho-CoA species.

Where the enzyme is a ligase, the nucleophile is preferably the side-chain of an amino acid of the enzyme, e.g. a lysine residue. In this case, the species formed is an enzyme-bound nucleotidylate species, e.g. an enzyme lysyl-adenylate species.

The screening methods of the invention involve an enzyme-catalysed reaction between a substrate comprising a nucleophilic group (e.g. a carboxylic acid group or a phosphoric acid group) and a dinucleotide phosphate or an analogue thereof. Preferably the dinucleotide phosphate is a compound of formula (I):

X¹-Q-R_n-X² (I) wherein

X¹ and X² independently denote nucleosides comprising a nucleobase and a 5-carbon sugar;

Q and R independently denote phosphate groups, wherein the attachment of X¹ and X² to the adjacent phosphate group is via the 5' oxygen on the 5-carbon sugar; and n denotes 0, 1 , 2, 3, 4 or 5. Preferred nucleobases are adenine, cytosine, guanine, thymine, uracil and inosine, especially adenine, guanine and uracil, most preferably adenine.

Preferred 5-carbon sugars are ribose (e.g. D-ribose) and deoxyribose (e.g. 2-deoxy-D- ribose) groups.

Preferably the phosphate group is an -0-P(0)(OH)- group or an ionic derivative thereof. Preferably n denotes the integer 1 , 2 or 3, especially 2 or 3. Most preferably n is 3.

In an especially preferred embodiment, X¹ and X² denote the same nucleoside.

Preferably, the nucleosides are selected from adenosine, cytidine, guanosine, thymidine and uridine, most preferably adenosine.

Most preferably, the dinucleotide phosphate is X¹-Q-R3-X¹, particularly preferably AP₄A (adenosine tetraphospho adenosine). Other preferred dinucleotide phosphates include AP₄G (adenosine tetraphospho guanosine), AP₄U (adenosine tetraphospho uridine) and AP₃A (adenosine triphospho adenosine).

By "analogue" of a dinucleotide phosphate is meant a compound which is chemically related to compounds of formula (I) but which does not fall under the definition of formula (I). Dinucleotide phosphate analogues are compounds which have substantially the same characteristics as a closely-related dinucleotide phosphate, e.g. in terms of binding affinity and reactivity values.

Examples of dinucleotide phosphate analogues include analogues of compounds of formula (I) wherein one or both nucleobases and/or 5-carbon sugars is (or are) substituted by one or more amino, halogen (e.g. -F, -CI, -Br or -I) or methyl groups; wherein one or more amino groups thereon is replaced by a methylene or

halomethylene groups; and/or wherein one or more methyl groups thereon is are replaced by hydrogen or halogen atoms. Other examples of analogues include analogues of compounds of formula (I) wherein one or more heteroatoms are independently replaced by a different heteroatom selected from O, N and S. Dinucleotide phosphate analogues according to the invention include compounds which comprise one of more methylene and/or halomethylene groups in the phosphate chain and/or include one or more phosphorothioate groups in the backbone.

According to this embodiment, the dinucleotide phosphate analogue comprises a group "R_n" of formula (II):

[-(CH₂)_p-(PY₃-)-(CH₂)_q-]_n (II) wherein n is as defined herein and, for each integer value of n, each Y is independently selected from O and S, preferably O, and p and q in each case are independently 0 or 1 , with the proviso that at least one "p" or at least one "q" must be 0. Each methylene group may independently be substituted by 1 or 2 halogen atoms, especially by 2 fluorine atoms. In a preferred embodiment, the group Q denotes (PY₃ ^"), wherein each Y is independently selected from O and S and is preferably O.

Dinucleotide phosphates and dinucleotide phosphate analogues for use in methods according to the invention may be obtained from commercial sources (e.g. from Sigma- Aldrich) or may be synthesised using known methodology, either with or without enzyme-catalysed steps (see e.g. Guranowski A., Acta. Biochim. Pol. 2003; 50(4):947- 72 and the documents cited therein, and also Eliahu et al. J. Med. Chem. (2010) 53 (24):8485-8697, the disclosures of which and particularly the dinucleotide phosphates and dinucleotide phosphate analogues disclosed therein are incorporated herein by reference). In a related aspect, the invention provides the use of a dinucleotide phosphate as defined herein (e.g. AP₄A), or an analogue thereof, in a method of screening one or more test compounds for antimicrobial activity, e.g. by determining the extent to which said test compounds act as inhibitors of an aminoacyl tRNA synthetase enzyme. The enzyme-catalysed reaction is one which is capable of producing an acyl

nudeotidylate species or intermediate. By "nudeotidylate" is meant a nucleoside monophosphate moiety wherein the nucleoside is as herein defined with regard to X¹ and/or X².

A preferred nudeotidylate species is adenylate. Examples of acyl nudeotidylate species are aminoacyl nucleotidylates, preferably aminoacyl adenylate, and fatty acyl nucleotidylates, preferably fatty acyl adenylate.

Hence in one embodiment, step (i) comprises

(i) incubating an aminoacyl tRNA synthetase which is capable of catalysing a reaction between a dinudeotide phosphate, or analogue thereof, and a cognate amino acid to produce an aminoacyl nudeotidylate species with:

(a) a dinudeotide phosphate, or analogue thereof,

(b) a cognate amino acid, and

(c) a test compound.

Preferably, the dinudeotide phosphate is AP₄A and the aminoacyl nudeotidylate is aminoacyl adenylate.

Hence in another embodiment, step (i) comprises:

(i) incubating a fatty acyl CoA synthetase which is capable of catalysing a reaction between a dinudeotide phosphate, or analogue thereof, and a fatty acid group to produce a fatty acyl nudeotidylate species with:

(a) a dinudeotide phosphate, or analogue thereof,

(b) a fatty acid group, and

(c) a test compound.

Preferably, the dinudeotide phosphate is AP₄A and the fatty acyl nudeotidylate is a fatty acyl adenylate. ln yet a further embodiment, step (i) comprises

(i) incubating a non-ribosomal peptide synthetase which is capable of catalysing a reaction between a dinucleotide phosphate, or analogue thereof, and an amino acid to produce an aminoacyl nucleotidylate species with:

(a) a dinucleotide phosphate, or analogue thereof,

(b) a cognate amino acid, and

(c) a test compound. Preferably, the dinucleotide phosphate is AP₄A and the aminoacyl nucleotidylate is aminoacyl adenylate.

In a further embodiment, step (i) comprises

(i) incubating a 4-phosphopantetheine adenylyl transferase which is capable of catalysing a reaction between a dinucleotide phosphate, or analogue thereof, and 4'- phosphopantetheine to produce a dephospho-CoA species, or a nucleobase analogue thereof, with:

(a) a dinucleotide phosphate, or analogue thereof,

(b) 4'-phosphopantetheine, and

(c) a test compound.

Preferably, the dinucleotide phosphate is AP₄A and the dephospho-CoA species is 3- dephospho-CoA. In a still further embodiment, step (i) comprises

(i) incubating a DNA or RNA ligase which is capable of catalysing a reaction between a dinucleotide phosphate, or analogue thereof, and an amino acid side-chain on said ligase to produce a nucleotidylate species with:

(a) a dinucleotide phosphate, or analogue thereof, and

(b) a test compound.

Preferably, the dinucleotide phosphate is AP₄A and the nucleotidylate species is an enzyme-bound adenylate. The test compound or compounds for screening according to the present invention may be small organic molecules prepared by combinatorial synthesis techniques and the like. However, the term "test compound" is not limited to such organic compounds and may include biomolecules such as nucleic acids (e.g. DNA or RNA), polypeptides (e.g.

functional enzymes and enzyme domains, structural proteins and protein domains, and oligopeptides having between 2 and 40 amino acids, especially between 5 and 10 amino acids), antibodies and antigen binding fragments thereof, as well as viruses, phage, bacteria, liposomes and the like.

Preferred test compounds are carbon-containing molecules having a molecular weight between 15 and 10,000 Da. In one embodiment, the test compounds have a molecular weight between 50 and 5,000 Da, especially between 100 and 2,000 Da, e.g. between 150 and 1 ,000 Da, between 200 and 750 or between 350 and 500 Da. In another embodiment, the test compounds have a molecular weight between 15 and 500 Da, especially between 25 and 300 Da, e.g. between 30 and 200 or between 40 and 150 Da. In one embodiment, the test compound is a molecule coupled to a siderophore, in which case the molecular weight would be that described above with an additional 150 to 1 ,500 Da to take account of the siderophore, e.g. between 200 and 6,500 Da, between 250 and 3,500 Da or between 156 and 2,000 Da. In a further embodiment the test compound (i.e. having a molecular weight as described above) is coupled to one or more sugar moieties, e.g. to a monosaccharide, an oligosaccharide or a polysaccharide. Preferably, the siderophore is a hydroxamate or a catecholate.

As described herein, the screening methods of the invention are particularly suitable for screening for compounds which exhibit weak binding (in the mM range) to the

nucleotidylate-forming enzyme and this makes the said methods particularly suitable to screen for low molecular weight compounds, e.g. as defined above. Screening methods of the invention may include other components in the reaction mixture wherein the enzyme is incubated with the substrate and the dinucleotide phosphate, i.e. in step (i) and optionally in step (ii). These optional further components may include buffers, e.g. HEPES, Tris and the like; salts such as MgC , NaCI, KCI and the like; reducing agents such as dithiothreitol and the like; and other components typically included in enzyme reactions.

In embodiments of the screening methods defined herein wherein the enzyme is capable of catalysing the conversion of the nucleotidylate intermediate into the corresponding NTP (e.g. by reaction with inorganic pyrophosphate) the enzyme may be incubated with inorganic pyrophosphate in step (i) and optionally in step (ii).

It is preferred that the reaction is carried out in the absence of the natural acceptor substrate of the nucleotidylate intermediate.

For example, where the enzyme is an aaRS, step (i) of the screening method (and optionally also step (ii)) is preferably carried out in the absence or substantially in the absence of tRNA, preferably in the absence of the cognate tRNA (i.e. the tRNA which carries the amino acid which is acted upon by the aaRS).

Where the enzyme is a FAS or an NRPS, step (i) of the screening method (and optionally also step (ii)) is preferably carried out in the absence or substantially in the absence of Coenzyme A and/or an acyl carrier protein (especially a holo-acy\ carrier protein).

Where the enzyme is a DNA or RNA ligase, step (i) of the screening method (and optionally also step (ii)) is preferably carried out in the absence or substantially in the absence of nucleotide or ribonucleotide, respectively.

In certain embodiments, the reaction is free or substantially free of enzymes having pyrophosphatase activity. In some embodiments of the invention, one or more of the reagents (e.g. the enzyme, the substrate, the dinucleotide phosphate, etc.) may be bound to or otherwise

immobilised on a solid phase or solid support. This may facilitate the separation of components from the reaction mixture, e.g. to facilitate measurement of the rate and/or extent of the reaction, and may also enable the components to be efficiently recycled.

The enzyme is incubated with the test compound and the dinucleotide phosphate or analogue thereof under conditions which are suitable for the nucleotidylate species (e.g. the acyl nucleotidylate) to be formed.

Typically, the incubation of the enzyme with the other components of the reaction mixture will be done under conditions at which the enzyme adopts an essentially native structure. Preferably, the incubation is carried out at a pH between 5 and 10, especially between 6.8 and 8.6, e.g. between 7 and 8, such as about 7.5. Preferred temperatures are generally in the range of 15 to 50 °C, especially in the range of 20 to 42 °C, e.g. between 25 and 37 °C, such as about 32 °C.

The enzyme is preferably incubated with the other components of the reaction mixture for a time sufficient to allow the extent or rate of the reaction to be measured, e.g. step (i) and optionally also step (ii) of the methods of the invention have a sufficient duration to allow the generation of a detectable species which can be used to determine the rate and/or extent of the reaction.

Preferred lengths of time for incubation include periods between 10 second and 2 hours, preferably between 60 seconds and 1 hour, especially between 5 and 30 minutes.

Preferably the incubation is carried out for a period of time of at least 10 seconds, e.g. at least 20, 30 or 60 seconds, or at least 1 minute, e.g. at least 2, 5, 10, 20 or 30 minutes. In some embodiments of the invention, the incubation is carried out in the presence of or subsequently in the presence of a second nucleophile which is capable of initiating the cleavage of the nucleotidylate species, e.g. the acyl nucleotidylate. Examples of such second nucleophiles include pyrophosphate. Generally, the reaction taking place in the methods of the invention comprises a first and, optionally, a second step. In the first step, the dinucleotide phosphate and nudeophile (e.g. the carboxylic acid group-containing substrate) react to yield the nucleotidylate species, e.g. the acyl nucleotidylate species. This first step liberates a first nucleoside-containing product (exemplified in Figure 2). Where the dinucleotide phosphate is a compound of formula (I), the first nucleoside-containing product may be described as X₂-R_n-H (or a salt or ionic form thereof), wherein X₂, R and n are as herein defined. Thus, where n=0 the first nucleoside-containing product is a nucleoside (or analogue thereof) and where n>0 the first nucleoside-containing product is a nucleotide or nucleotide polyphosphate (or an analogue thereof). In the second step, the nucleotidylate intermediate species is typically cleaved by a further nudeophile (e.g. pyrophosphate) to reform the nudeophile reactant from step one (e.g. amino acid) and to yield a second nucleoside-containing product (exemplified in Figure 2). In the case where the dinucleotide phosphate is a compound of formula (I) and the further nudeophile is pyrophosphate, the second nucleoside-containing product may be described as X Q-OP(0)(OH)-OP(0)(OH)₂, wherein X! and Q are as herein defined. Generally, the second nucleoside-containing product is a nucleotide

triphosphate (NTP), e.g. ATP.

In step (ii) of the screening methods described herein, the rate and/or extent of the reaction is determined. This may be done by any suitable means.

For example, the rate and/or extent of the reaction may be determined by determining the rate and/or extent of the consumption of dinucleotide phosphate; the rate and/or extent of the consumption of the nudeophile (i.e. the substrate comprising a carboxylic acid group); the rate and/or extent of the production of the first nucleoside-containing product; the rate and/or extent of the production of the acyl nucleotidylate species; and/or the rate and/or extent of the production the second nucleoside-containing product. Preferably, the rate and/or extent of production of a nucleotide phosphate moiety or analogue thereof derived from the dinucleotide phosphate of formula (I): X¹-Q-R_n-X²

is determined.

Preferably, in embodiments wherein n is 3, the rate and/or extent of production of X¹- triphosphate and/or X² -triphosphate is determined. In especially preferred

embodiments, e.g. where the dinucleotide phosphate is AP₄A, the rate and/or extent of production of ATP is determined

Hence, in one embodiment, the invention provides a method of screening for

compounds having an antimicrobial activity, the method comprising the steps:

(i) incubating an enzyme which is capable of catalysing a reaction between AP^, or analogue thereof, and an amino acid to produce an acyl adenylate species with:

(a) AP₄A, or an analogue thereof,

(b) a test compound, and

(c) optionally an amino acid,

and

(ii) determining the rate and/or extent of the production of ATP,

wherein a reaction having a lower rate and/or a lower extent of production of ATP when determined in the presence of the test compound compared to the rate or extent of production of ATP in a control reaction in the absence of the test compound is indicative of the test compound having antimicrobial activity.

In a preferred embodiment, the enzyme is an amino acyl tRNA synthetase and the amino acid incubated with the enzyme and test compound is the cognate amino acid for the enzyme. In another preferred embodiment, the enzyme is an RNA ligase and the amino acid is part of the enzyme.

In preferred embodiments, the rate and/or extent of the reaction is determined by measuring the rate and/or extent of the production of ATP. Methods for measuring the rate or extent of the production of ATP are known in the art. In the context of the current invention, the methods utilised should preferably be able to measure specifically the rate or extent of the production of ATP in a reaction mixture which comprises ATP and at least one dinucleotide phosphate or analogue thereof as herein defined.

Known methods for discriminating between ATP and nucleotide diphosphates in a reaction mixture include chromatographic methods and mass spectroscopic methods which may utilise radiolabeled species. Such methods allow for the sensitive and accurate detection of ATP in the presence of dinucleotide phosphates which might otherwise interfere with the results obtained. However, these methods are time- consuming, expensive and typically require multiple steps for sample preparation and analysis. In contrast, the enzymatic methods of the present invention are simple, cheap and can be carried out in situ, e.g. with detection being effected by photometric means.

Hence, in one embodiment, methods for the detection and/or quantification of ATP are preferably light-based methods, e.g. using fluorescent, photometric and/or luminescent means to determine the presence and/or level of ATP in the reaction mixture.

Preferably the methods are not radioactive methods, e.g. the molecular components of the reaction mixture are not labelled with radioisotopes, e.g. with radioisotopes such as ³H, ³²P and ³³P.

The preferred ATP detection methods operate by action of an ATP-utilising enzyme on ATP in the reaction mixture. Preferably, the ATP-utilising enzyme has a low propensity to catalyse a reaction utilising the dinucleotide phosphate or analogue thereof as a substrate to generate the detectable species, e.g. the enzyme substantially does not catalyse said reaction. Preferably, the enzyme has no detectable rate of catalysis of the reaction utilising the dinucleotide phosphate.

The amount of ATP-utilising enzyme used in the methods of the invention will depend on the relative concentrations of ATP and dinucleotide phosphate and also on the kinetic parameters of the enzyme and could readily be determined by the skilled person. Typically, the enzyme concentration will be chosen so as to provide a signal for detection and/or quantification of ATP that is measurable over a period of seconds, minutes or hours, especially over a period of between 1 second and 12 hours, e.g. 10 seconds to 4 hours, 30 seconds to 1 hour, or 1 minute to 30 minutes. Typical enzyme concentrations include 0.01 μΜ to 1 mM, e.g. 0.05 to 10 μΜ, especially 0.1 to 1 μΜ, e.g. around 0.5 μΜ.

In one embodiment, the ATP-utilising enzyme generates a detectable species which may be directly detected or quantified. Preferably the directly-detectable species is light (i.e. photons) or a chemical species which has an absorption, emission or fluorescence profile which allows for that species to be detected by spectrophotometric means (e.g. NADPH).

Preferably, the ATP-utilising enzyme is a light-producing enzyme which utilises ATP as an energy source, for example luciferase. Luciferase enzymes for use according to the invention may be obtained from commercial sources (e.g. Sigma-Aldrich) or can be purified from organisms which naturally produce luciferase (e.g. Photinus pyralis) or from recombinant organisms (e.g. recombinant E. coli) using techniques known in the art.

In an alternative embodiment, the ATP-utilising enzyme generates a detectable species which may be indirectly detected or quantified. By "indirectly" is meant that the detectable species is involved in a further interaction or reaction to generate the signal that permits the detection and/or quantification in step (ii) of the methods of the invention.

One example of an indirectly-detectable species would be a chemical species which itself is a substrate for one or more further reactions that generate a directly-detectable species as defined above. Preferably, the detectable species is a substrate for a reaction which generates a chemical species having an absorption, emission or fluorescence profile which allows for the species to be detected by photometric means. The coupling reaction which utilises the indirectly-detectable species to generate a directly-detectable species is preferably an enzyme-catalysed reaction which is not significantly affected by the presence of dinucleotide phosphate (or analogues thereof) so as to allow the coupling reaction agents and the ATP-utilising reaction agents to be present in the same reaction mixture.

In a particularly preferred embodiment, the ATP-utilising enzyme is a

phosphotransferase enzyme (e.g. having EC classification 2.7.1 ), especially a hexokinase (e.g. having EC classification 2.7.1.1 ). Hexokinase enzymes catalyse the conversion of ATP and a hexose to ADP and hexose 6-phosphate.

Hexose substrates for hexokinases include D-Glucose, D-mannose, D-fructose, sorbitol and D-glucosamine, especially D-glucose.

Suitable enzymes can be obtained commercially (e.g. from Roche or Sigma-Aldrich) or purified from natural (e.g. Leuconostoc mesenteroides) or recombinant organisms using methods known in the art. An especially preferred hexokinase is Leuconostoc mesenteroides hexokinase (e.g. available from Roche).

Thus, in one embodiment, the ATP detection and/or quantification methods of the invention utilise a hexokinase as the ATP-utilising enzyme (e.g. see Figure 3).

Preferably, at least one dinucleotide phosphate is AP₄A and the reaction mixture further comprises a hexose substrate as defined herein.

Hence, in some embodiments, step (ii) comprises determining the rate and/or extent of the production of ATP using an hexokinase.

Where the ATP-utilising enzyme generates an indirectly-detectable species, it is preferred that this species is converted to an oxidised (or reduced) species which is either itself directly detectable or involved in the concomitant reduction (or oxidation) of another molecule which is directly detectable. Preferably the indirectly-detectable species is the substrate for a dehydrogenase which catalyses the oxidation of the indirectly-detectable species and the concomitant reduction of NAD⁺ (or NADP⁺) to NADH (or NADPH). Coupling of the reaction to NADH (or NADPH) production allows for the determination of the rate and/or extent of the reaction by measuring a change in absorbance, e.g. at 340 nm, or fluorescence, e.g. excitation at 340 nm and emission at 420 nm.

Preferred dehydrogenases are categorised under EC 1.1.1 , especially as glucose-6- phosphate dehydrogenases (e.g. EC 1.1.1.49). An especially-preferred dehydrogenase is Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase (e.g. available from Roche).

Especially preferred dehydrogenases may be obtained from commercial sources (e.g. from Roche or Sigma-Aldrich) or purified from natural (e.g. Leuconostoc mesenteroides) or recombinant organisms using methods known in the art.

Thus, in a preferred embodiment, the invention provides methods of detecting and/or quantifying ATP wherein the detectable species is glucose-6-phosphate which is detected and/or quantified by subsequent conversion to 6-phosphogluconate with concomitant production of NADPH (e.g. see Figure 3). Measurement of a change in absorbance at 340 nm (or a change in fluorescence) can detect and/or quantify the amount of glucose-6-phosphate in the reaction mixture, which in turn can determine the presence and/or level and/or rate of production of ATP in the reaction mixture.

Typically, supplementation of a reaction mixture containing hexokinase with glucose 6- phosphate dehydrogenase does not limit the activity of the ATP-utilising enzyme and so measurement of NADPH levels is in direct proportion to the activity of the enzyme, i.e. the rate of NADPH production is directly proportional to the rate of ATP production at a steady state. Preferably, the invention provides a method of screening for compounds having an antimicrobial activity as described herein wherein the rate and/or extent of the reaction is determined using a method of detecting and/or quantifying ATP as defined herein. Accordingly, in a preferred embodiment, step (ii) of the method of the invention comprises

(ii) determining the rate and/or extent of the production of ATP by:

(a) incubating said reaction mixture, or a part thereof, with hexokinase;

(b) detecting and/or quantifying glucose-6-phosphate generated by utilisation of ATP by the hexokinase;

wherein the production of glucose-6-phosphate at a lower rate and/or a lower extent when determined in the presence of the test compound compared to the rate or extent produced in a control reaction in the absence of the test compound is indicative of the test compound having an antimicrobial activity.

In yet another preferred embodiment, step (ii) of the method of the invention comprises (ii) determining the rate and/or extent of the production of ATP by:

(a) incubating said reaction mixture, or a part thereof, with hexokinase;

(b) simultaneously or subsequently incubating said reaction mixture or a part thereof with a dehydrogenase, preferably glucose-6-phosphate dehydrogenase, and determining the rate and/or extent of the change in absorption of the reaction mixture at 340 nm,

wherein an increase in the absorption of the reaction mixture when determined in the presence of the test compound compared to the absorption seen in a control reaction in the absence of the test compound is indicative of the test compound having

antimicrobial activity.

In some embodiments of the invention, step (i) may be carried out separately and/or at different times from step (ii). For example, incubating step (i) may be carried out and the reaction mixture stored (e.g. the incubation reaction (i) may be terminated, chilled, frozen or suspended) before determining step (ii) is carried out. The suspension of the reaction may be performed by a significant drop in temperature, e.g. to below 0 °C, and/or by acidification, e.g. to a pH of between 2 and 4. The methods for detecting ATP according to the invention enable the determination of the presence and/or level of ATP in a reaction mixture comprising at least one dinucleotide phosphate or analogue thereof. The methods allow the detection of ATP in the presence of varying amounts of dinucleotide phosphate, even in the presence of a large excess of dinucleotide phosphate. Because the ATP-utilising enzyme does not utilise dinucleotide phosphates or analogues thereof, or only utilises it to a small extent, high specificity and sensitivity is maintained. In one embodiment, the method is used to detect and/or quantify ATP in the reaction mixture at a level above about 0.5 μΜ, especially above about 1 or 2 μΜ. Preferably, the method is used to detect ATP at a level below about 1 mM, especially below 0.5 or 0.3 mM. For example, the method is used to detect and/or quantify ATP in the reaction mixture at a level of between 1 and 200 μΜ, especially about 10, 50 or 100 μΜ.

Levels of dinucleotide phosphate (or analogues thereof) which can be accommodated in the ATP-detection methods of the invention will depend on the nature of the enzyme, especially the relative strength of binding between the enzyme and the dinucleotide phosphate and the rate of formation of detectable species from the dinucleotide phosphate as compared to the corresponding values with ATP. Typically, high levels of dinucleotide phosphate or analogues thereof can be accommodated.

In these embodiments, the invention may be used to detect and/or quantify ATP, wherein dinucleotide phosphate (e.g. AP₄A) or an analogue thereof is present in the reaction mixture at a level above about 1 μΜ, especially above about 0.1 mM, 1 mM or 10 mM. Preferably, the dinucleotide phosphate or analogue thereof is present at a level below about 100 mM, especially below about 50 mM, 20 mM, 10 mM, 5 mM or 2 mM, e.g. between 50 μΜ and 4 mM or between 0.2 mM and 0.8 mM, especially about 0.4 mM or 0.7 mM. The above values may relate to the levels of a single dinucleotide phosphate species (or analogue) or to the total amount of all dinucleotide phosphate species (or analogues) in the reaction mixture. In the latter case, the effective molarity is calculated as the sum of the molarities of each individual dinucleotide phosphate species (or analogue). Preferably, where the reaction mixture comprises magnesium cations (Mg²⁺), the dinucleotide phosphate is present in a molar concentration of less than that of the magnesium cations. In particular embodiments, the ATP detection and/or quantification methods of the invention are carried out on reaction mixtures comprising ATP and at least one dinucleotide phosphate or analogue thereof in a molar ratio of between 1 : 1 and 1000:1 ATP:nucleotide phosphate. Especially, the molar ratio is between 4: 1 and 10: 1 , e.g. around 6: 1 ATP:nucleotide phosphate. In alternative embodiments, the ATP detection and/or quantification methods of the invention are carried out on reaction mixtures with an excess of dinucleotide phosphate, i.e. comprising ATP and at least one dinucleotide phosphate or analogue thereof in a molar ratio of between 1 : 1 and 1 : 1000

ATP:nucleotide phosphate. Especially, the molar ratio is between 1 : 10 and 1 : 100, or between 1 :4 and 1 : 10, e.g. around 1 :6 ATP:nucleotide phosphate. By molar ratio is meant the number of moles of ATP per mole of dinucleotide phosphate or analogue thereof. Assuming that the reaction mixture maintains a constant volume, the molar ratio is equal to the ratio of the molarities of each component.

In a particularly preferred aspect of the invention, screening methods as described herein are performed wherein the step of measuring the extent and/or rate of the reaction (e.g. step (ii)) utilises a method of detecting and/or quantifying ATP in the reaction mixture according to the corresponding methods as also described herein.

Seen from this aspect, the invention provides methods of screening as described wherein in step (i) the adenylate-forming enzyme is incubated under conditions where ATP is capable of being formed, and wherein step (ii) comprises the method of detecting and/or quantifying ATP in the reaction mixture as also described herein.

Methods of screening for compounds having potential antimicrobial activity utilising the ATP detecting methods of the invention are particularly advantageous because all of the reagents (including the adenylate-forming enzyme, ATP-utilising enzyme(s) and their substrates) may be included in a single reaction mixture, thus considerably simplifying the process. Furthermore, because the extent and/or rate of the reactions can be monitored in real-time using spectrophotometric means, the method is well suited to automation and scaling up to high-throughput screening.

Such screening methods are quicker, cheaper, simpler to perform and simpler to analyse. In addition, for the reasons given hereinbefore, these methods have the potential to screen for compounds that modulate clinically-relevant targets (e.g.

Streptococcal aaRS enzymes, mycobacterial FAS enzymes and trypanosomal RNA ligases) and which bind with high or low affinity, facilitating the development of fragment-based drug design strategies.

The methods outlined herein are also suitable for assaying the presence and/or level of a dinucleotide phosphate or an analogue thereof in a reaction mixture. Generally speaking, the enzymes described herein catalyse a first reaction between a dinucleotide phosphate and a substrate comprising a nucleophilic group (e.g. a carboxylic acid group or phosphoric acid group) to form a nucleotidylate species (e.g. an acyl adenylate) with concomitant release of a nucleotide phosphate. The enzyme may catalyse a further reaction of nucleophilic attack by a nucleophile (e.g. pyrophosphate) on the

nucleotidylate species to liberate the substrate and a further nucleotide species (e.g. a nucleotide diphosphate). This reaction scheme allows for the detection and/or quantification of dinucleotide phosphate in a reaction mixture by direct or indirect detection of the nucleotide species produced by the first and second steps of the reaction, e.g. using methods as described herein.

Thus in a further aspect, the invention provides a method for detecting and/or quantifying the level or amount of dinucleotide phosphate or analogue thereof in a sample, said method comprising:

(i) incubating an enzyme which is capable of catalysing a reaction between the dinucleotide phosphate or analogue thereof, and a substrate to produce a nucleotidylate species, with:

(a) the sample, and

(b) the substrate,

and

(ii) determining the rate and/or extent of the reaction, wherein the rate and/or extent of the reaction is indicative of the level or amount of dinucleotide phosphate or analogue thereof in the sample.

In one embodiment, the invention provides a method for detecting and/or quantifying the level or amount of dinucleotide phosphate or analogue thereof in a sample, said method comprising:

(i) incubating an enzyme which is capable of catalysing a reaction between the dinucleotide phosphate or analogue thereof, and a substrate comprising a carboxylic acid group to produce an acyl nucleotidylate species, with:

(a) the sample, and

(b) a substrate comprising a carboxylic acid group,

and

(ii) determining the rate and/or extent of the reaction,

wherein the rate and/or extent of the reaction is indicative of the level or amount of dinucleotide phosphate or analogue thereof in the sample.

In a further embodiment, the invention provides a method for detecting and/or quantifying the level or amount of dinucleotide phosphate or analogue thereof in a sample,

said method comprising:

(i) incubating an enzyme which is capable of catalysing a reaction between the dinucleotide phosphate or analogue thereof, and a substrate comprising a phosphoric acid group to produce an acyl nucleotidylate species, with:

(a) the sample, and

(b) a substrate comprising a phosphoric acid group,

and

(ii) determining the rate and/or extent of the reaction,

Preferably, the reaction is carried out under conditions wherein the enzyme and substrate are present in excess. Typically, the reaction mixture will first be incubated in the absence of at least one key component of the enzyme reaction, e.g. the enzyme, the enzyme substrate or the nucleophile, under conditions whereby the reaction could take place if the missing components were included in order to remove any contaminating molecules in the reaction mixture which could give a false reading. For example, where step (ii) of the method relies upon detection and/or quantification of nucleotide triphosphate (e.g. ATP) produced in step (i), the reaction mixture is preferably incubated under conditions whereby the nucleotide triphosphate in the reaction mixture is removed (e.g. by conversion to the corresponding diphosphate) before the missing component (e.g. pyrophosphate) is added to allow the desired reaction to proceed. Preferably the contaminating molecule(s) are removed from the reaction mixture by an enzymatic reaction (e.g. an enzyme couple) which is responsible for the detection and/or quantification in step (ii). For example, where the method detects and/or quantifies the level or amount of AP₄A in the reaction mixture, the enzyme may be alanyl-tRNA^Ala synthetase, the substrate may be alanine and the nucleophile may be pyrophosphate. The amounts of substrate and nucleophile are typically chosen so as not to limit the absolute response of the assay, i.e. to allow essentially all of the AP₄A in the reaction mixture to be converted to ATP. Where the assay is performed in the presence of hexokinase, glucose-6-phosphate dehydrogenase and NADP⁺ (as described herein), the absorbance change due to NADPH production is directly proportional to AP^ concentration (i.e. in a 1 :2 ratio of AP^ consumption to NADPH production) and therefore provides a convenient and quantitative assay for AP₄A.

In an especially preferred embodiment, step (ii) comprises detecting and/or quantifying ATP as described herein, especially a method utilising the hexokinase/glucose-6- phosphate dehydrogenase enzyme couple described herein. In another embodiment of the invention, there is provided a method of detection and/or quantification of adenosine triphosphate (ATP) in a reaction mixture comprising ATP and at least one dinucleotide phosphate or analogue thereof, the method comprising: i) incubating with said reaction mixture an enzyme that utilises ATP to generate a detectable substrate at a substantially faster rate than it utilises said at least one dinucleotide phosphate or analogue thereof to generate the detectable substrate;

ii) detecting and/or quantifying said detectable substrate; and

iii) determining the presence and/or level of ATP in the reaction mixture based on the detection and/or quantification of step (ii).

In yet another embodiment, there is provided a method of detection and/or

quantification of adenosine triphosphate (ATP) in a reaction mixture comprising ATP and AP₄A, the method comprising:

i) incubating said reaction mixture with hexokinase; and

ii) detecting and/or quantifying the rate and/or extent of glucose-6-phosphate

generated;

wherein the rate and/or level of glucose-6-phosphate which is generated by utilisation of ATP by the hexokinase is indicative of the rate of production and/or level of ATP in the reaction mixture.

In such a method, glucose-6-phosphate is generated by utilisation of ATP by the hexokinase.

In a related aspect, the invention provides the use of hexokinase in a method of discriminating between ATP and a dinucleotide phosphate (e.g. AP₄A) or an analogue thereof. The detection and/or quantification of the rate and/or extent of glucose-6-phosphate generated may be carried out by the methods described herein.

Preferably, the detection and/or quantification of the rate and/or extent of glucose-6- phosphate generated is carried out by converting the glucose-6-phosphate to 6- phosphogluconate and detecting and/or quantifying the concomitant production of NADPH (e.g. by measuring absorbance at 340 nm). Preferably, glucose-6-phosphate is converted to 6-phosphogluconate using a

dehydrogenase as herein defined, e.g. glucose-6-phosphate dehydrogenase.

The screening methods of the invention are particularly suited to the high-throughput screening of compounds. High-throughput methods of screening according to the invention refer to methods of screening as defined herein wherein a plurality of compounds are screened substantially simultaneously. Preferably at least 10 compounds are screened, especially at least 100, at least 1000 or at least 10000 compounds are screened substantially simultaneously. By "substantially

simultaneously" is meant that each incubation step (i) and/or measurement step (ii) of each screening assay is carried out within a 24 hour period, preferably within a 4 hour period and especially within a 1 hour period. Particularly preferably, each incubation step (i) and/or measurement step (ii) of each screening assay is carried out

simultaneously, i.e. the periods in which said steps are carried out overlap, at least in part.

The screening methods of the invention are also particularly suitable for use in methods of fragment-based drug design, in particular in methods of designing a drug to act as a modulator of an adenylate-forming enzyme as defined herein, e.g. a drug having an antimicrobial activity. Preferred methods of fragment-based drug design are methods of designing a drug to act as an inhibitor of a microbial enzyme, especially an inhibitor of a bacterial aaRS, a bacterial FAS or a trypanosomal RNA ligase. Especially preferred methods include methods of designing an antimicrobial drug, e.g. an antibiotic or antitrypanosomal agent.

Fragment-based drug design typically starts with detection of a very weak interaction between a protein target and a chemically simple ligand (a drug 'fragment'). At this point, a crystal structure of this fragment:target complex may be used to guide chemical elaboration of the small molecule, to increase its potency. Then, further cycles of crystallography and compound redesign increase the potency and specificity of the elaborated fragment for its target, until the fragment assumes the parameters required for therapeutic utility. The whole approach is predicated on the ability in the first instance to detect very weak interactions in the region of multi-millimolar potency. The kinetics inherent in the screening and ATP detection assays described herein allow these assays to monitor weakly-binding compounds, because of the very favourable competitive environment they present to a weakly-interacting modulator. Thus, the methods of the invention are particularly useful for fragment-based drug discovery.

The methods of fragment-based drug design of the invention preferably comprise identifying at least one compound by a method of screening as defined herein, where this compound is a modulator of the nucleotidylate-forming enzyme, and combining all or a part (e.g. the part of the molecule which is responsible for the modulator properties) of said at least one compound with another compound that is known to bind to said nucleotidylate-forming enzyme. The other compound may be also a compound identified using a screening method as described herein. Thus, the method of drug design may comprise identifying two or more compounds which modulate the

adenylate-forming enzyme and combining all or a part of said compounds to form a compound having improved modulatory properties, e.g. improved binding, enhancing or inhibitory properties. When "combining all" of a compound with another compound, as would readily be understood by the skilled person, this means that substantially all of the compound identified appears in the combined product. Typically, a functional group on the compound is used to make the connection between compounds and this would generally involve the replacement of at least one atom, e.g. a hydrogen atom, with a bond between the two compounds. For example, a first modulatory compound comprising a carboxylate group might be combined with a second enzyme-binding compound comprising an amine group by the formation of an amide bond. The combined compound would be considered, for the purposes of this application, as a compound derived from the combination of "all" of the two substituent compounds, despite the fact that amide formation is a reaction in which three atoms (in the form of water) are lost.

The combination of compounds identified using screening methods of the invention may be done using conventional chemical reactions carried out on the individual compounds, e.g. using linking groups such as bifunctional linking groups, or may be the result of de novo chemical synthesis of the "combined" product. Libraries of combined products may be also generated using combinatorial methods known in the art. In a preferred embodiment, the methods of drug design include at least two steps of compound identification and combination. In this embodiment, stages of the method are carried in an iterative fashion to produce combined products having an even higher modulatory activity.

The screening methods according to the invention may be used in processes for preparing the modulatory compounds. Such processes comprise screening for one or more modulatory compounds using a method as defined herein, selecting said one or more compounds and optionally preparing the one or more compounds for

administration by admixture with one or more pharmaceutically acceptable agents, e.g. excipients, carriers or diluents. The admixture of said compounds with one or more pharmaceutically acceptable agents provides a pharmaceutical composition which may be adapted for administration, for example by enteral (e.g. oral or rectal), parenteral (e.g. by injection or infusion, especially intravenous injection), inhalation, nasal, buccal or sublingual routes.

In particular, the invention provides a process for the preparation of a pharmaceutical composition having an antimicrobial activity, the process comprising the steps:

(i) identifying a compound having an antimicrobial activity by a screening method as as described herein,

(ii) producing a pharmaceutical composition by combining the compound with one or more pharmaceutically acceptable excipients, diluents or carriers.

A related aspect of the invention provides modulatory or test compounds which are identified by one or more methods of screening as herein defined. Especially provided are antimicrobial compounds, e.g. those which inhibit a microbial aaRS, identified by one or more screening methods defined herein.

Hence the invention further provides a compound having an antimicrobial activity which has been identified by a screening method of the invention.

The invention further provides a pharmaceutical composition comprising a compound having an antimicrobial activity which has been identified by a screening method of the invention, optionally together with one or more pharmaceutically acceptable excipients, diluents or carriers.

Conventional pharmaceutical agents may be used in the preparation of these compositions such as binding agents, lubricants, fillers, sweeteners, taste-masking agents, solvating agents, pH modifiers, buffers, isotonic agents, carriers, etc. Such pharmaceutical agents are generally known in the art.

Also provided are libraries of said modulatory compounds, especially libraries comprising (e.g. consisting essentially of) at least 10, at least 100, at least 1000 or at least 10000 said compounds. These libraries preferably contain between 50 and 5000 compounds, especially between 200 and 2000 compounds. The library of compounds may be contained within one or more multi-well plates, e.g. one or more 96-well plates. The present invention also provides kits for use in methods as herein described.

Preferably the kits comprise one or more dinucleotide phosphates (e.g. AP₄A) or analogues thereof and one or more of the enzymes referred to herein, optionally with one or more substrates comprising a nucleophilic group, e.g. a carboxylic acid or phosphoric acid group.

Especially preferably, the kits comprise AP₄A, one or more aaRS enzymes and one or more amino acids, preferably amino acids which are the cognate amino acids of said one or more aaRS enzymes.

In another embodiment, the kits comprise an ATP-utilising enzyme (e.g. hexokinase) and at least one dinucleotide phosphate or analogue thereof.

Optionally, the kit further comprises means for converting a detectable substrate into a signal, e.g. one or more enzymes for converting the detectable substrate into a luminescent, fluorescent or photometric signal. Optionally, the kits comprise one or more test compounds as defined herein and further optionally comprise instructions for the use of the kit in the performance of the said methods, e.g. including the method steps set out herein. In a preferred embodiment, the kits of the invention comprise, separately or in combination:

(i) AP₄A,

(ii) one or more aaRS enzymes,

(iii) hexokinase, and

(iv) glucose-6-phosphate dehydrogenase,

wherein the components of the kit are as hereinbefore defined, optionally together with instructions for use in a method of the invention.

Preferably the kits of the invention further comprise one or more of:

(v) one or more amino acids which are the cognate amino acids of said one or more aaRS enzymes,

(vi) pyrophosphate (e.g. sodium pyrophosphate)

(vii) NADP⁺,

(viii) D-glucose,

(ix) a buffer (e.g. HEPES),

(x) MgCI₂ and/or KCI, and

(xi) a reducing agent (e.g. dithiothreitol).

In a further preferred embodiment, the kits of the invention may comprise, separately or in combination:

(i) AP4A,

(ii) one or more FAS enzymes,

(iii) hexokinase, and

(iv) glucose-6-phosphate dehydrogenase,

Preferably said kits further comprise one or more of: (v) one or more fatty acids which are substrates for said one or more FAS enzymes,

(vi) pyrophosphate (e.g. sodium pyrophosphate)

(vii) NADP⁺,

(viii) D-glucose,

(ix) a buffer (e.g. HEPES),

(x) MgCI₂ and/or KCI, and

(xi) a reducing agent (e.g. dithiothreitol). In another preferred embodiment, the kits of the invention may comprise, separately or in combination:

(i) AP4A,

(ii) one or more NRPS enzymes,

(iii) hexokinase, and

(iv) glucose-6-phosphate dehydrogenase,

Preferably said kits further comprise one or more of:

(v) one or more amino acids which are the cognate amino acids of said one or more NRPS enzymes,

(vi) pyrophosphate (e.g. sodium pyrophosphate)

(vii) NADP⁺,

(viii) D-glucose,

(ix) a buffer (e.g. HEPES),

(x) MgCI₂ and/or KCI, and

(xi) a reducing agent (e.g. dithiothreitol).

In yet another preferred embodiment, the kits of the invention may comprise, separately or in combination:

(i) AP4A,

(ii) one or more 4-PAT enzymes,

(iii) hexokinase, and (iv) glucose-6-phosphate dehydrogenase,

wherein the components of the kit are as hereinbefore defined, optionally together with instructions for use in a method of the invention. Preferably said kits further comprise one or more of:

(v) 4-phosphopantetheine,

(vi) NADP⁺,

(vii) D-glucose,

(viii) a buffer (e.g. HEPES),

(ix) MgCI₂ and/or KCI, and

(x) a reducing agent (e.g. dithiothreitol).

In a yet further preferred embodiment, the kits of the invention may comprise, separately or in combination:

(i) AP₄A,

(ii) one or more RNA and/or DNA ligase enzymes,

(iii) hexokinase, and

(iv) glucose-6-phosphate dehydrogenase,

Preferably said kits further comprise one or more of:

(v) pyrophosphate (e.g. sodium pyrophosphate)

(vi) NADP⁺,

(vii) D-glucose,

(viii) a buffer (e.g. HEPES),

(ix) MgCI₂ and/or KCI, and

(x) a reducing agent (e.g. dithiothreitol). BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the reaction scheme for processes catalysed by aaRSs. In the conventional reaction, an amino acid and ATP react to form the central aminoacyl intermediate which is attacked by a tRNA in step 2 to yield the aminoacyl tRNA and AMP. In the absence of the tRNA, attack on the central intermediate by pyrophosphate (step 1 ) or ATP (step 3) yields ATP or AP₄A respectively.

Figure 2 shows the reaction between AP₄A and an amino acid catalysed by aaRS (with valine as a specific example of the amino acid). The first step of the reaction liberates ATP which can be quantified to report on the rate of the reaction. In the second step, in the presence of pyrophosphate, the aminoacyl adenylate intermediate is released from the enzyme to regenerate the amino acid and liberate a further molecule of ATP. Figure 3 shows a reaction scheme for detection of ATP using an enzyme (hexokinase) which converts ATP to a detectable substrate (ADP) without acting significantly on dinucleotide phosphate present in the reaction mixture. In the example illustrated in Figure 3, the ADP produced by the hexokinase may be detected using

spectrophotometric, fluorescent or bioluminescent means, whilst the glucose-6- phosphate produced by action of the hexokinase in the presence of ATP is coupled to the reduction of NADP⁺ by glucose-6-phosphate dehydrogenase in order to provide NADPH and a characteristic change in absorbance at 340 nm.

Figure 4 shows the time-courses of AP₄A assays using valyl-tRNA^Val synthetase (Fig. 4A), isoleucyl-tRNA^lle synthetase (Fig. 4B) and alanyl-tRNA^Ala synthetase (Fig 4C) enzymes all from Escherichia coli acting on AP₄A in the presence and absence of the standard inhibitors TSA (5'-0-(A/-(L-threonyl)-sulphamoyl)-adenosine), mupirocin and ASA (5'-0-(A/-(L-alanyl)-sulphamoyl)-adenosine), respectively. Figure 5 shows an example of kinetic analysis (using double reciprocal plots) comparing enzyme activity with AP₄A and isoleucine concentrations at various concentrations of test compound 1 (a putative enzyme inhibitor) using the AP₄A assay with Escherichia coli isoleucyl-tRNA^lle synthetase. Figure 5A shows that the inhibition is competitive with respect to AP₄A (K, = 0.140 mM) and Figure 5B shows that the inhibition is competitive with respect to isoleucine (K, = 0.184 mM).

Figure 6 shows the quantitative analysis of AP₄A concentration in a reaction mixture using Streptococcus pneumoniae alanyl-tRNA^Ala synthetase, pyrophosphate and alanine to cleave AP₄A into two equivalents of ATP which are quantified using a hexokinase and glucose-6-phosphate dehydrogenase enzyme couple. The change in absorbance observed is that expected from the stoichiometry of the reaction. Figure 7 shows the quantitative analysis of AP₄A concentration in a reaction mixture using an absorbance-based Type 1 T4 RNA ligase activity assay as described in

Example 5.

Figure 8 shows, schematically, the principle of an AP₄A-based RNA ligase detection assay, e.g. as described in Example 5, in which the net reaction is AP^ + PP, = 2x ATP.

EXAMPLES

The present invention is further defined in the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. The disclosure of each reference set forth herein is incorporated herein by reference in its entirety. Example 1 - Non-radiometric AP₄A substrate-based in vitro aaRS assays

Preparation of the target enzyme

The alanyl-tRNA^Ala synthetase gene was amplified by polymerase chain reaction from the chromosomal DNA of Escherichia coii or Streptococcus pneumoniae. The PCR fragment was cloned as described between the Nde1 and Xho1 restriction sites of the Novagen expression vector pET26a, yielding the alaRS gene fused in frame with a 3' sequence encoding a hexa-histidine C-terminus. The target protein was then over- expressed in soluble form in Escherichia coii BL21 DE3* Rosetta, and purified by immobilised metal affinity chromatography on immobilised cobalt resin (ClonTech Corp.)

The other aaRS enzymes that were used (E. coii isoleucyl-tRNA^lle synthetase and E. coii valyl-tRNA^Val synthetase were provided by Professors C. Fishwick and I. Chopra (Leeds University)) were cloned into pET26a over-expression vectors and were purified as described for alanyl-tRNA^Ala synthetase, except that the valyl enzyme required further purification on Sepharose 4B-immobilised Procion Green HE4BD (an

immobilised triazine dye), from which it was eluted by 2 M NaCI, in order to free it of contaminating inorganic pyrophosphatase. Assay conditions

Assay were conducted at 37°C in a final volume of 0.2 ml of buffer (50 mM HEPES, 10 mM MgCI₂, pH 7.6, 1 mM dithiothreitol, 50 mM KCI) containing 10 mM D-glucose, 0.5 mM NADP⁺, 1 .7 mM/min Leuconostoc mesenteroides hexokinase (Roche, i.e. an amount of enzyme which, under manufacturer's standard conditions, causes an increase in product concentration of 1.7x10^"3 moles per litre of glucose-6-phosphate per minute), 0.85 mM/min Leuconostoc mesenteroides glucose-6-phosphate

dehydrogenase (Roche) and 10% (v/v) DMSO. Addition of further components depended on the exact assay being carried out. Assays carried out using Escherichia coii Valine aaRS (VaIRS) included 4 mM L-valine, 0.39 mM AP₄A and 2.4 μΜ ValRS. Assays carried out using Escherichia coii Isoleucine aaRS (NeRS) included 10 μΜ L-isoleucine, 0.7 mM AP₄A and 2.4 μΜ HeRS. Assays carried out using Escherichia coli alanine aaRS (AlaRS) included 2 mM L-alanine, 0.41 mM AP₄A and 0.93 μΜ AlaRS. These components and the components listed in the paragraph above (i.e. in the absence of pyrophosphate) define the "reaction mixture" as used in this Example.

Once the components of the reaction mixture were combined, a background rate was measured by determining the change in absorbance at 340 nm over time. The assay was then initiated by the addition of 20 μΜ pyrophosphate (VaIRS, lleRS, and AlaRS) and the initial rate served as a positive control (i.e. a no-inhibitor control).

Known inhibitory compounds were added to the reaction mixture to test the

responsiveness of the assay. The inhibitor TSA (Integrated DNA Technologies) was used at 50 μΜ in the case of VaIRS; mupirocin (Sigma) at 5 μΜ was used in the case of lleRS; and ASA (Integrated DNA Technologies) was used at 5 μΜ in the case of AlaRS. Reactions were initiated 5 minutes after mixing by the addition of sodium pyrophosphate to a final concentration of 20 μΜ.

Results

Typical time courses are illustrated in Figures 4A, 4B and 4C for activity measurements of lleRS, VaIRS and AlaRS in the presence of DMSO alone or in the presence of known inhibitory compounds. The vertical lines indicates the addition of pyrophosphate at 5 minutes after mixing.

Inspection of the measured rate before and after addition of pyrophosphate for the assays with and without inhibitors clearly shows that a significantly different rate was measured and in each case illustrates the effective 0% (no inhibitor) and 100% (added inhibitor) inhibition that could be measured under these experimental conditions. The length of the linear phase of the initiated reaction (around 5 to 12 minutes) is sufficient to accurately and reproducibly determine the steady state activity of the target enzyme and impact of test compounds upon it. Example 2 - Use of the AP₄A assay to screen for potential aaRS inhibitors

Methods

An assay reaction mixture was prepared according to Example 1 using the NeRS enzyme and further comprising mupirocin (as a known inhibitor). The rate of ATP production observed in the presence of 5 μΜ mupirocin was used to define 100% inhibition.

Potential inhibitor test compounds were added to the reaction mixture from stock solutions made up in DMSO such that the final concentration of DMSO remained at 10% (v/v) in the reaction mixture. Concentrations of test compounds were between 0.1 and 1 mM. Within this assay, selectivity of an inhibitor for the enzyme target, as opposed to the coupling enzyme detection system, can easily be established by the addition of ATP, the assayed product of the target synthetase and the substrate of the coupling enzyme detection system and by the determination of any change in rate.

One of the test compounds (compound 1 ) was further investigated in assays which varied the concentration of AP₄A (between about 1 and 6 mM), isoleucine (between about 0.04 and 0.2 mM) and compound 1 (between 0 and 3 mM) to obtain kinetic parameters.

Results

From a library of 33 test compounds (small organic compounds), percent inhibition of between 0 and 82.2% was observed across the library. Two of the compounds (compounds 1 and 2) displayed greater than 75% inhibition when compared with mupirocin. Inhibition of as low as 1.3% was recorded for the other test compounds.

By the above assay, mupirocin was calculated to have an IC₅o value of 0.35±0.06 μΜ. The IC₅₀ values calculated for test compounds 1 and 2 were 0.22±0.04 mM and 0.90±0.06 mM respectively. The analysis of enzyme inhibition relative to AP₄A and isoleucine binding indicate that compound 1 has a K, of 0.140 mM with respect to AP₄A (see Figure 5A) and a K, of 0.184 mM with respect to isoleucine (see Figure 5B). The intersecting pattern of lines on the y axis of both plots in Figure 5 indicates that compound 1 competes for the binding site of isoleucine and AP₄A. This observation is entirely consistent with the conclusion that compound 1 targets the isoleucyl-adenylate binding pocket of HeRS. Similar data were obtained for compound 2 (not shown). The results of this experiment demonstrate that the assay is an efficient and highly sensitive assay for screening potential modulators of nucleotidylate-forming enzymes.

Example 3 - Assay sensitivity to weakly-interacting inhibitors The interaction of AP₄A with HeRS, VaIRS and AlaRS was compared using the methodology set out in Example 1.

Comparison of the behaviour of these three enzymes with respect to binding of AP₄A with literature regarding ATP utilization (the nucleotide used in all other reported synthetase assays) suggests that AP₄A binds considerably more weakly than ATP (a factor of between 17 and 66 times more weakly). Similarly, the K_m values for the cognate amino acids in these AP₄A assays compared to literature values indicate that the cognate amino acids bind to the enzyme in the presence of AP₄A significantly more weakly than they do in other ATP-dependent assays (a factor of between 28 and 341 times more weakly). The following values were obtained experimentally or were obtained from the literature ("[amino acid]" refers to the cognate amino acid for the aaRS):

¹ Yanagisawa T, Lee JT, Wu HC and Kawakami M, (1994). J. Biol. Chem. 269, 24304-24309;

2 Myers G, Blank U and Soil D, (1971 ). J. Biol. Chem. 246, 4955-4964;

³ Davis MW, Buetchter DD and Schimmel P, (1994). Biochemistry 33, 9904-991 1 ;

⁴ Xu B. Trawick B. Krudy GA, Philips RM, Zhou L and Rosevear PR, (1994).

Biochemistry 33, 398-402;

⁵ Wang P, Fichera A. Kumar K, and Tirrell DA (2004). Angewandte Chemie 116, 3750-3752;

⁶ Guo M, Chong YE, Shapiro R, Beebe K, Yang X-L and Schimmel P, (2009). Nature 462, 808-812.

The markedly weaker binding of AP₄A suggests a much more favourable competitive environment for a weakly binding inhibitor to register a signal in the assays of the invention. It can be calculated, for instance, that inhibitors binding weakly enough to possess Kj values of up to at least 2.7 mM should be detectable by these assays. This is the level of sensitivity that is required for fragment based drug discovery (see e.g. Murray and Blundell, Curr. Op. Struct. Biol. (2010) 20 (4):497-507). This demonstrates the utility of the AP₄A assay in the detection of inhibitors with an extremely wide range of affinities for the target enzymes. Example 4 - Assay to determine the presence of dinucleotide phosphate

The quantitative analysis of AP₄A concentration in a reaction mixture was performed using the methods described hereinbefore. Assay conditions were as described in Example 1 and the reaction included the following components: S. pneumoniae alanyl- tRNA^Ala synthetase (1.8 μΜ)), sodium pyrophosphate (0.471 mM), L-alanine (25 mM) and AP₄A (27.2 μΜ). ATP generation was assayed using the hexokinase/glucose-6- phosphate dehydrogenase couple as described in Example 1 with the course of the reaction being followed over time by the change in absorbance at 340nm.

The reaction catalysed involves the cleavage of AP₄A into two equivalents of ATP which were quantified spectrophotometrically. The net reaction is AP₄A + PP,→ 2 ATP.

The results are shown in Figure 6, in which the change in absorbance shows on the y- axis converted to ATP concentration by comparison to a standard curve. The expected value to be obtained by generation of 2 molecules ATP from each molecule of AP₄A would yield an ATP concentration of 54.4 μΜ. The experimental value observed after 1 10 minutes was 59.2 μΜ, i.e. a factor of 2.18 times the concentration of AP₄A. This example demonstrates that methods of the invention can sensitively and accurately detect and quantify dinucleotide phosphates in reaction mixtures.

Example 5 - Assay to determine the activity of a Type 1 T4 RNA ligase The AP₄A assay described in the previous Examples was used to evaluate the ability of a Type 1 T4 RNA ligase (EC 6.5.1 .3 obtained from New England Biolabs) to utilise AP₄A.

Assays were carried out at 37 °C in a final volume of 0.2 ml of buffer (50 mM HEPES, 10 mM MgCI₂, pH 7.6, 1 mM dithiothreitol, 50 mM KCI) containing 10 mM D-glucose; 0.5 mM NADP⁺; 1 .7 mM/min Leuconostoc mesenteroides hexokinase (Roche), i.e. an amount of enzyme which, under manufacturer's standard conditions, causes an increase in product concentration of 1.7x10^"3 moles per litre of glucose-6-phosphate per minute; and 0.85 mM/min Leuconostoc mesenteroides glucose-6-phosphate

dehydrogenase (Roche). For the results presented in Figure 7, AP₄A and T4 RNA ligase were added to final concentrations of 2.5 mM, and 1.26 μΜ respectively. After establishment of a background rate, RNA ligase activity was initiated by addition of pyrophosphate to a final concentration of 0.5 mM.

ATP generation was assayed using the hexokinase/glucose-6-phosphate

dehydrogenase couple as described above with the course of the reaction being followed over time by the change in absorbance at 340 nm. The reaction catalysed involved the cleavage of AP₄A into two equivalents of ATP which were quantified spectrophotometrically. The net reaction is AP₄A + PP,→ 2 ATP (illustrated in Figure 8).

The results are shown in Figure 7, in which the change in absorbance is shown on the y-axis. It can clearly be seen that addition of pyrophosphate after 2 minutes led to a steady increase in the absorbance, which can be used to determine the rate of ATP generation and hence the turnover of the RNA ligase.

This example demonstrates that ATP-utilising RNA ligases are amenable for use methods of screening for modulatory compounds as described herein.

Claims

1. A method of screening for compounds having an antimicrobial activity, the method comprising the steps:

(i) incubating an aminoacyl tRNA synthetase (aaRS) enzyme with:

(a) a dinucleotide phosphate, or analogue thereof,

(b) an amino acid which is the natural substrate for said aaRS enzyme, and

(c) a test compound,

and

(ii) determining the rate and/or extent of the reaction,

antimicrobial activity.

2. A method of screening for compounds having an antimicrobial activity, the method comprising the steps:

(i) incubating an enzyme which is capable of catalysing a reaction between a dinucleotide phosphate, or analogue thereof, and a substrate to produce a nucleotidylate species with:

(a) a dinucleotide phosphate, or analogue thereof,

(b) a substrate which is capable of reacting with the dinucleotide phosphate or analogue thereof to form a nucleotidylate species, either wherein the substrate is covalently attached to the enzyme or wherein the substrate is not covalently attached to the enzyme, and

(c) a test compound,

and

(ii) determining the rate and/or extent of the reaction,

antimicrobial activity.

3. A method as claimed in claim 2, wherein the substrate comprises a carboxylic acid group or a phosphoric acid group.

4. A method as claimed in claim 3 wherein the nucleotidylate species is an acyl nucleotidylate or a phosphoryl nucleotidylate.

5. A method of screening for compounds having an antimicrobial activity, the method comprising the steps:

(i) incubating an enzyme which is capable of catalysing a reaction between a dinucleotide phosphate, or analogue thereof, and a substrate comprising a carboxylic acid group to produce an acyl nucleotidylate species with:

(a) a dinucleotide phosphate, or analogue thereof,

(b) a substrate comprising a carboxylic acid group, and

(c) a test compound,

and

(ii) determining the rate and/or extent of the reaction,

antimicrobial activity.

6. A method as claimed in any one of claims 2 to 5, wherein the enzyme is a synthetase, preferably selected from the group consisting of an aminoacyl tRNA synthetase (aaRS), a fatty-acyl CoA synthetase (FAS) and a non-ribosomal peptide synthetase (NRPS).

7. A method as claimed in any one of claims 2 to 5, wherein the enzyme is a ligase, preferably an RNA ligase.

8. A method of screening for compounds having an antimicrobial activity, the method comprising the steps: (i) incubating an enzyme which is capable of catalysing a reaction between a dinucleotide phosphate, or analogue thereof, and a substrate comprising a phosphoric acid group to produce a nucleotidylate species with:

(a) a dinucleotide phosphate, or analogue thereof,

(b) a substrate comprising a phosphoric acid group, and

(c) a test compound,

and

(ii) determining the rate and/or extent of the reaction,

antimicrobial activity.

9. A method as claimed in any one of claims 2 to 4 or 8, wherein the enzyme is a transferase, preferably a 4-phosphopantetheine adenylyl transferase (4-PAT).

10. A method as claimed in any one of the preceding claims, wherein the dinucleotide phosphate is AP₄A.

11. A method as claimed in any one of the preceding claims, wherein step (ii) comprises determining the rate and/or extent of the production of ATP.

12. A method as claimed in any one of the preceding claims, wherein step (ii) comprises

(ii) determining the rate and/or extent of the production of ATP by:

(a) incubating said reaction mixture, or a part thereof, with hexokinase;

13. A method as claimed in any one of claims 1 to 10, wherein step (ii) comprises: (ii) determining the rate and/or extent of the production of ATP by:

(a) incubating said reaction mixture, or a part thereof, with hexokinase;

antimicrobial activity.

14. A method for detecting and/or quantifying the level or amount of dinucleotide phosphate or analogue thereof in a sample, said method comprising:

(a) the sample, and

(b) a substrate, preferably wherein the substrate is capable of reacting with any dinucleotide phosphate or analogue thereof in the sample to form a nucleotidylate species, either wherein the substrate is covalently attached to the enzyme or wherein the substrate is not covalently attached to the enzyme, and

(ii) determining the rate and/or extent of the reaction,

15. A method as claimed in claim 14, either wherein the substrate comprises a carboxylic acid group and the nucleotidylate species is an acyl nucleotidylate, or wherein the substrate comprises a phosphoric acid group and the nucleotidylate species is a phosphoryl nucleotidylate.

16. A method as claimed in claim 14 or 15, wherein the enzyme is as defined in claim 6, claim 7 or claim 9.

17. A method as claimed in any one of claims 14 to 16, wherein the dinucleotide phosphate is AP₄A.

18. A method of detection and/or quantification of adenosine triphosphate (ATP) in a reaction mixture comprising ATP and at least one dinucleotide phosphate or analogue thereof, the method comprising:

i) incubating with said reaction mixture an enzyme that utilises ATP to generate a detectable substrate at a substantially faster rate than it utilises said at least one dinucleotide phosphate or analogue thereof to generate the detectable substrate;

ii) detecting and/or quantifying said detectable substrate; and

19. A method of detection and/or quantification of adenosine triphosphate (ATP) in a reaction mixture comprising ATP and AP₄A, the method comprising:

i) incubating said reaction mixture with a hexokinase; and

ii) detecting and/or quantifying the rate and/or extent of glucose-6-phosphate generated;

20. A kit comprising, separately or in combination:

(i) AP

(ii) one or more aaRS enzymes,

(iii) hexokinase, and

(iv) glucose-6-phosphate dehydrogenase,

optionally together with instructions for use in a method as defined in any one of claims 1 to 19.

21. A kit as claimed in claim 20, wherein the kit additionally comprises one or more components selected from the group consisting of:

(vi) pyrophosphate (e.g. sodium pyrophosphate)

(vii) NADP⁺,

(viii) D-glucose,

(ix) a buffer (e.g. HEPES),

(x) MgCI₂ and/or KCI, and

(xi) a reducing agent (e.g. dithiothreitol).

22. A kit comprising, separately or in combination:

(i) AP4A,

(ii) one or more FAS enzymes,

(iii) hexokinase, and

(iv) glucose-6-phosphate dehydrogenase,

23. The kit of claim 22, wherein the kit further comprises one or more components selected from the group consisting of:

(v) one or more fatty acids which are substrates for said one or more FAS enzymes,

(vi) pyrophosphate (e.g. sodium pyrophosphate)

(vii) NADP⁺,

(viii) D-glucose,

(ix) a buffer (e.g. HEPES),

(x) MgCI₂ and/or KCI, and

(xi) a reducing agent (e.g. dithiothreitol).

24. A kit comprising, separately or in combination:

(i) AP4A, (ii) one or more NRPS enzymes,

(iii) hexokinase, and

(iv) glucose-6-phosphate dehydrogenase,

25. The kit of claim 24, wherein the kit further comprises one or more components selected from the group consisting of:

(vi) pyrophosphate (e.g. sodium pyrophosphate)

(vii) NADP⁺,

(viii) D-glucose,

(ix) a buffer (e.g. HEPES),

(x) MgCI₂ and/or KCI, and

(xi) a reducing agent (e.g. dithiothreitol).

26. A kit comprising, separately or in combination:

(i) AP4A,

(ii) one or more 4-PAT enzymes,

(iii) hexokinase, and

(iv) glucose-6-phosphate dehydrogenase,

27. The kit of claim 26, wherein the kit further comprises one or more components selected from the group consisting of:

(v) 4-phosphopantetheine,

(vi) NADP⁺,

(vii) D-glucose,

(viii) a buffer (e.g. HEPES),

(ix) MgCI₂ and/or KCI, and

(x) a reducing agent (e.g. dithiothreitol).

28. A kit comprising, separately or in combination:

(i) AP4A,

(ii) one or more RNA and/or DNA ligase enzymes,

(iii) hexokinase, and

(iv) glucose-6-phosphate dehydrogenase,

29. The kit of claim 28, wherein the kit further comprises one or more components selected from the group consisting of:

(v) pyrophosphate (e.g. sodium pyrophosphate)

(vi) NADP⁺,

(vii) D-glucose,

(viii) a buffer (e.g. HEPES),

(ix) MgCI₂ and/or KCI, and

(x) a reducing agent (e.g. dithiothreitol).