Production and use of antimicrobial agents

Details Production and use of antimicrobial agents Production and use of antimicrobial agents

2001-03-16

2004-02-24

Swartz, Rodney P (Department: 1645)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving viable micro-organism

C435S069300, C435S325000, C435S320100, C536S023700

Reexamination Certificate

active

06696268

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to the production of antimicrobial agents for use in therapy and to methods for identifying suitable antimicrobial agents.
BACKGROUND TO THE INVENTION
It is now widely recognised that conventional antibiotics are becoming less effective in treating microbial infections due to the spread of resistant microbial strains. It is therefore important to develop new antimicrobial agents to treat infection.
An approach to identifying potential antimicrobial agents is to select a suitable target, the inhibition of which will result in the eradication of the microbe with minimum effects on the host.
An important requirement is that the target should be essential to the viability of the microbe, as often a microbe will have an alternative mechanism for carrying out the function of the target. For example, the microbe may utilise a different enzyme or a different pathway to by-pass the inhibition of the target.
In considering novel single targets (e.g. those not part of large assemblies such as ribosomes) an important consideration is whether the target, usually an enzyme, is unique to bacteria. If so, its properties and suitability for the design or discovery of useful inhibitors can be exploited without detrimental effects on the host. Other targets, such as dihydrofolate reductase (DHFR), are common to both bacterial and animal cells and therefore selective inhibition of the microorganism is essential if dose-dependent toxicity to the host is to be avoided. The antimicrobial drug trimethoprim is a useful example of a clinically-valuable DHFR inhibitor which competes with substrate (dihydrofolate) and is bound 50,000-fold more tightly by bacterial than mammalian forms of DHFR.
There are therefore several considerations that must be made when selecting a suitable target, and these considerations make the selection of a suitable target difficult.
Therefore, there is a recognised need for identifying and characterising suitable targets that may be useful in antimicrobial therapy. In particular, there is a need for identifying targets that may be used to screen compounds for antimicrobial activity which can be used to prevent, reduce or eradicate infections.
SUMMARY OF THE INVENTION
The present invention is based on the realisation that enzymes involved in co-enzyme A (CoA) synthesis may be suitable targets for antimicrobial or antiparasitic agents. In particular, it has been appreciated that the enzyme phosphopantetheine adenylyltransferase (PPAT) is a suitable target to inhibit the Coenzyme A synthetic pathway, thereby preventing bacterial growth. The suitability of PPAT as an antimicrobial or antiparasitic target is based, at least in part, on an appreciation that the differences in the PPAT enzyme in mammalian and bacterial systems may be exploited to provide selective inhibition of the bacterial form. In particular, the mammalian form exists as a bifunctional enzyme complex with dephospho-CoA kinase (dCoAk), whereas the bacterial form contains separate PPAT and dCoAk. Similar structural differences are found in yeast, fungi and parasitic forms of PPAT.
Therefore, the present invention relates to the use of bacterial or similar PPAT enzyme to select for antimicrobial agents. According to the present invention, a method for identifying antimicrobial agents, comprises the steps of:
(i) contacting a sample containing non-mammalian PPAT enzyme with a suitable substrate and a target compound under suitable conditions;
(ii) measuring the activity of the PPAT enzyme;
(iii) comparing the activity of the enzyme to that of a reference sample lacking the target compound; and
(iv) selecting those targets compounds that reduce the activity of the PPAT enzyme.
In a further bodiment, the present invention proposes the use of compounds capable of inhibiting the PPAT enzyme at a cellular or tissue concentration of less than 10 &mgr;M in antimicrobial therapy. In particular, the compounds may be used in the treatment of infection by
E. coli, Pseudomonas aeruginosa, Streptococcus pneumoniae, Haemophilus influenzae, Mycobacterium tuberculosis, Neisseria meningitidis
and
Staphylococcus aureus
, or any other pathogenic bacteria where PPAT activity is required for CoA synthesis.
According to a further embodiment, a substantially pure non-mammalian PPAT enzyme, or active fragment thereof, is used in a method for the identification of an antimicrobial or antiparasitic agent. Similarly, a gene encoding a non-mammalian PPAT enzyme may be used in a method for the identification of an antimicrobial or antiparasitic agent.
DESCRIPTION OF THE INVENTION
According to the present invention, methods for treating bacterial infection comprise selectively inhibiting the non-mammalian form of phosphopantetheine adenylyltransferase (PPAT). It has been recognised that this enzyme, also known as dephospho-CoA pyrophosphorylase, has particular characteristics which make it suitable as a target for inhibiting bacterial growth.
The term “PPAT enzyme” refers to the complete catalytically-active PPAT enzyme, or to fragments of the enzyme that retain catalytic activity. The enzyme may be used in the methods of the present invention either immobilised on a solid support or in solution.
The term “microbial” relates to any microorganism that is capable of causing infection, e.g. bacterial, fungal and yeast. In particular the term relates to bacterial organisms, especially those implicated in infectious disease, for example,
E. coli, Pseudomonas aeruginosa, Streptococcus pneumoniae, Haemophilus influenzae, Mycobacterium tuberculosis, Neisseria meningitidis
and
Staphylococcus aureus.
PPAT enzymes may also be derived and purified from parasitic organisms which require the enzyme activity for CoA synthesis.
The term “target compound” relates to any suitable agent that may be used as an inhibitor of PPAT activity. The compound may be biological, i.e. a protein or peptide that binds to and inhibits PPAT activity or alters the conformation of the enzyme in such a way as to block the substrate from the catalytic region, or may be a chemical compound. In either instance, the compound may be found naturally or may be synthesised. Suitable synthesis techniques will be apparent to those skilled in the art.
Possible target compounds may be chemically related to the natural substrates of PPAT, i.e. phosphopantetheine and ATP. Such substrates are converted to products at the catalytic centre of the enzyme, and so it is reasonable to assume that derivatives or analogues of such substrates may be identified which lead to selective inhibition of non-mammalian PPAT. In addition, compounds unrelated to the natural substrates may also be useful inhibitors. It is also envisaged that the inhibitors of PPAT activity may not act directly on the PPAT enzyme, but may instead act at the genetic level to block further production of PPAT.
The term “activity” refers to the natural biological activity of the PPAT enzyme. Usually this activity takes the form of the transfer of the reactive adenylyl group from ATP to phosphopantetheine, yielding dephospho-CoA. However, enzyme reactions seldom proceed only in the forward direction, and the reverse direction may also be utilised for determining the activity of PPAT. Use of the reverse reaction may be beneficial as it is linked to the formation of ATP which may then be utilised in fluorescence or luminescence detection techniques, for example the ATP-dependent oxidation of luciferin by luciferase with the concomitant production of light. Other ways of measuring PPAT activity will be apparent to those skilled in the art and will be useful in the methods of the present invention.
The term “therapy” refers to the treatment of diseases arising as a result of infection, not only by bacteria but also by fungi or parasites for which PPAT is required for the synthesis of CoA.
The term “medicament” refers to any suitable pharmaceutical composition. Specifically, it refers to a composition comprising the anti-infective agent in any suitable excipient or diluent, and also to differ

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