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WO2002044408A2 - Methode d'identification d'inhibiteurs d'enzymes - Google Patents

Methode d'identification d'inhibiteurs d'enzymes Download PDF

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Publication number
WO2002044408A2
WO2002044408A2 PCT/GB2001/005329 GB0105329W WO0244408A2 WO 2002044408 A2 WO2002044408 A2 WO 2002044408A2 GB 0105329 W GB0105329 W GB 0105329W WO 0244408 A2 WO0244408 A2 WO 0244408A2
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WO
WIPO (PCT)
Prior art keywords
dam
stranded dna
target site
double stranded
colour
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Ceased
Application number
PCT/GB2001/005329
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English (en)
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WO2002044408A3 (fr
Inventor
Ian George Charles
Jamie Richards
Duncan John Maskell
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Arrow Therapeutics Ltd
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Arrow Therapeutics Ltd
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Publication date
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Priority to AU2002223099A priority Critical patent/AU2002223099A1/en
Publication of WO2002044408A2 publication Critical patent/WO2002044408A2/fr
Anticipated expiration legal-status Critical
Publication of WO2002044408A3 publication Critical patent/WO2002044408A3/fr
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/25—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • This invention relates to a method for identifying substances capable of inhibiting the enzyme DNA adenine methylase (Dam). It further relates to assays for identifying Dam activity in a sample, to test kits for identifying substances capable of inhibiting Dam, to antimicrobial inhibitors identified using said method and the use of said inhibitors in medicine.
  • Dam DNA adenine methylase
  • a method for screening a compound for its ability to inhibit D ⁇ A adenine methylase (Dam) characterised in that the ability of the compound to inhibit Dam is determined in an assay comprising: a) a double stranded D ⁇ A comprising at least one Dam target site; b) a Dam and a substrate therefor; c) an enzyme that selectively cleaves the double stranded D ⁇ A at the Dam target site but which will not cleave the double stranded D ⁇ A at a methylated Dam target site (or vice versa); and d) a means for detecting whether the double stranded DNA comprising the Dam target site has been cleaved.
  • Dam adenine methylase
  • the target site is: 5'-GATC-3' 3'-CTAG-5' (SEQ ID NO 1) and the enzyme is Mb ⁇ l or an isoschizomer thereof such as, for example, Dpn ⁇ l.
  • the double stranded DNA comprising the target site is formed by annealing two oligonucleotides.
  • the oligonucleotides are of from 4-100 base pairs in length, more preferably 20-60 base pairs in length.
  • the oligonucleotides are 24-'mers, and when annealed they form a double stranded DNA.
  • the sequence of one such example is given below: 5'-GAGTACGACTGATCAGTCGGCTGA-3 ' 3 ' -CTC ATGCTGACT AGTC AGCCGACT-5 ' . (SEQ ID NO 2)
  • the Dam target site is shown in bold.
  • the oligonucleotides could however have a plurality of such Dam sites.
  • the assay is conducted on a solid support.
  • a solid support for example, a well plate.
  • the reference to "ends” refers to a position, and not necessarily the terminal end, relative to the Dam site.
  • Methods of covalently attaching oligonucleotides to solid supports are known in the art. This can be achieved by, for example, biotinylating one end of the at least one of the two oligonucleotides forming the double stranded DNA.
  • DNA can then be attached to, for example, a streptavidin coated well plate.
  • suitable methods for attaching oligonucleotides to solid supports include: covalently linking an SH-oligonucleotide via an alkylating reagent such as an iodoacetamide or maleimide (Kumar A et al, Nucleic Acids Res 1991 19: 4561); covalently linking an amine-oligonucleotide via an activated carboxylate group (Ganachaud F et al J.
  • Suitable solid supports include microarrays, microtitre plates (for example, 96 or 384-well plates) or magnetic beads.
  • the other end of the double stranded DNA is modified to provide a first component of a colour generating indicator means which indicator means additionally comprises one or more further components for generating the colour indicator.
  • the first component of the colour generating indicator means is fluorescein and the further components of the colour generating indicator means are an alkaline phosphotase anti-fluorescein antibody and p-Nitrophenyl phosphate (pNPP).
  • the principle behind the assay can be summarised as follows:
  • the method utilizes a double stranded DNA comprising at least one Dam target site.
  • One end of the DNA is bound to a solid support such as the well of a microtitre plate.
  • the other end is provided with a means for detecting whether the double stranded DNA comprising the Dam target site has been cleaved.
  • a compound to be tested is added to, for example, the well of the microtitre plate. Dam and a substrate therefor are added and if the compound is not an inhibitor of Dam the Dam will methylate the adenine residue of the Dam target site.
  • the assay relies on the fact an adenine residue in a Dam target site e.g. 5'-GATC-3' 3'-CTAG-5' (SEQ ID NO 1) of a double stranded DNA is ordinarily methylated by Dam in the presence of a substrate e.g. S -adenosyl methionine (SAM). If a compound under test acts as inl ibitor of Dam no, or reduced methylation occurs when Dam and a substrate are added in the presence of the compound.
  • SAM S -adenosyl methionine
  • an enzyme such as, for example, Mb ⁇ l or an isoschizomer thereof e.g. Dpn ⁇ (which only cleaves double stranded DNA at a non-methylated site) it is possible to selectively cleave the double stranded DNA, thereby removing the first component (e.g. fluorescein) of an indicator only if inhibition of Dam has taken place.
  • This selective cleaving can be used to identify those compounds which act as inhibitors.
  • the adenine will have been methylated (the enzyme will be unable to cleave the DNA) and consequently the first component of the colour generating indicator means will remain attached to the DNA and solid support (i.e.
  • the colour generating indicator means e.g. alkaline phosphotase anti-fluorescein antibody and a substrate e.g. pNPP substrate tablets
  • the first component will react with the first component to generate a colour reaction.
  • this can be detected, for example, colourmetrically at 405 nm.
  • a plurality of compounds are simultaneously screened for the purpose of identifying candidate antimicrobials.
  • test kit suitable for use in identifying an inhibitor of Dam, which kit comprises a double stranded DNA comprising at least one Dam target site.
  • the double stranded DNA is bound to a microtitre plate or another solid support used in screening.
  • the double stranded DNA is provided with a means for detecting whether the double stranded DNA comprising the Dam target site has been cleaved in the assay.
  • a preferred means for detecting whether the double stranded DNA comprising the Dam target site has been cleaved in the assay comprises a first component of a colour generating indicator means which colour generating indicator means further comprises one or more further components for generating a colour.
  • the test kit further comprises one or more of the following: a) a Dam and a substrate therefor; b) an enzyme that selectively cleaves the Dam target site but which will not cleave a methylated Dam target site (or vice versa); and c) the one or more further components of the colour generating indicator means.
  • the first component of the colour generating indicator means is fluorescein and the one or more further components of the colour generating indicator means are an alkaline phosphotase anti-fluorescein antibody and pNPP.
  • the invention also relates to inhibitors of Dam which have been identified by a method according to the invention or by using a kit according to the invention and to such inhibitors for use in a method of treatment of the human or animal body, for use in the treatment of a microbial infection or for use in the manufacture of a medicament for use in the treatment of a microbial infection.
  • Any bacterial infection may be treated with an inhibitor of the invention.
  • infections of Gram-positive or Gram-negative bacteria may be treated.
  • an infection of a pathogenic bacterium will be treated with an inhibitor of the invention.
  • An inhibitor of the invention may be used to treat an infection of bacteria, for example, from the genera Escherichia, Salmonella, Nibrio, Haemophilus, Neisseria, Yersinia, Bordetella, Brucella, Shigella, Klebsiella, Enterobacter, Serracia, Proteus, Nibrio, Aeromonas, Pseudomonas, Acinetobacter, Moraxella, Flavobacterium, Actinobacillus, Staphylococcus, Streptococcus, Mycobacterium, Listeria,
  • Examples of some of the above mentioned genera, infections of which may be treated with an inhibitor of the invention, are Escherichia coli - a cause of diarrhoea in humans; Salmonella typhimurium - the cause of salmonellosis in several animal species; Salmonella typhi - the cause of human typhoid fever; Salmonella enteritidis - a cause of food poisoning in humans; Salmonella choleraesuis - a cause of salmonellosis in pigs; Salmonella dublin - a cause of both a systemic and diarrhoeal disease in cattle, especially of new-born calves; Haemophilus i ⁇ fluenzae - a cause of meningitis; Neisseria gonorrhoeae - a cause of gonorrhoea; Yersinia enterocolitica - the cause of a spectrum of diseases in humans ranging from gastroenteritis to fatal septicemic disease; Borde
  • a method of treating a host suffering from a bacterial infection comprises administering to the host a therapeutically effective amount of an inhibitor.
  • the host may be a human or an animal.
  • the assay for Dam can be used to identify inhibitors of Dam.
  • the fact that the assay for inhibitors can be carried out on a solid support has the advantage that cheap plastic microtitre plates can be used to detect the effects of specific compounds on Dam activity.
  • the screen for inhibitor substances is a colourmetric test radioactive isotopes can be avoided making handling safer.
  • the assay is suitable for adaptation to multiple well plate technologies and can be automated using liquid handling robots, allowing modern high tl ⁇ oughput screening techniques to be applied. The invention therefore permits high through-put, flexible and inexpensive screening for substances which inhibit Dam. This will increase the likelihood of potent bioavailable drugs being identified.
  • Oligonucleotides comprising a Dam target site were prepared.
  • the oligonucleotides were: 5 ' -GAGTACGACTGATCAGTCGGCTGA-3 ' (SEQ ID NO 3) and
  • the first ohgonucleotide (SEQ ID NO 3) was modified by biotinylating it at the 5' end to enable the annealed DNA to be bound to a solid support via, for example, streptavidin coated wells.
  • the second ohgonucleotide (SEQ ID NO 4) was modified by introducing a first component of a detection means which detection means enables the user of the assay to determine whether the double stranded DNA (SEQ ID NO 2) formed by annealing the respective strands has been cleaved in the assay.
  • the preferred first component is the first component of a colour generating indicator means, in this example fluorescein. This modification is on the other side of the Dam target site to the modification allowing the DNA to be bound to the solid support and this allows selective detection following cleavage at the site.
  • the oligonucleotides were annealed and the DNA bound to the wellplate.
  • the assay uses a solid support having a double stranded DNA comprising at least one Dam target site bound thereto and is automated using a Beckman robot.
  • Oligonucleotides (SEQ ID NO 3 AND NO 4) were supplied by Sigma Genosys. Each was labelled at the 5 '-position, one with biotin, the other with fluorescein.
  • Oligonucleotides were resuspended in a buffer of 50 mM Tris-HCl pH 7.5 to a concentration of 1 ⁇ g/ ⁇ l.
  • Buffers Streptavidin coating buffer 8 mM sodium carbonate, 17 mM Sodium bicarbonate, pH 9.6.
  • Blocking solution 66 mM Na PO 4 , 138 mM NaCl, 3% (w/v) Bovine serum albumin, 15 M NaN 3 (pH 7.4).
  • Ohgonucleotide coating buffer 50 mM Tris pH 7.4, 500 ⁇ g/ml Bovine serum albumin, 15 mM NaN 3 (pH 7.5).
  • annealed oligonucleotides were diluted to 35ng/ml in the ohgonucleotide coating buffer, and 100 ⁇ l of this solution added to each well of the streptavidin- coated 96-well plate. The plate was incubated for a further 16-20 hr. at 18-25°C in the dark.
  • Dam methylase buffer 50 mM Tris-HCl, 10 mM EDTA, 1 mM DTT (pH 7.5).
  • Dpnll buffer 50 mM BisTris/HCl, 100 mM NaCl, 10 mM MgCl 2 , 1 mM DTT (pH 6.0).
  • Tris-buffered saline (TBS) 50 mM Tris-HCl, 138mM NaCl (pH 8.0).
  • Anti-fluorescein-alkaline phosphatase conjugate was diluted to 1/1000 in Dpnll buffer, and 120 ⁇ l added to the four remaining wells. This group constitutes the Dam-/ Dpnll- controls.
  • pNPP p-nitrophenyl phosphate
  • a substance which inhibits the activity of Dam may do so by binding to the enzyme.
  • Such enzyme inhibition may be reversible or irreversible.
  • An irreversible inhibitor dissociates very slowly from its target enzyme because it becomes very tightly bound to the enzyme, either covalently or non-covalently.
  • Reversible inhibition in contrast with irreversible inhibition, is characterised by a rapid dissociation of the enzyme- inhibitor complex.
  • the test substance may be a competitive inhibitor.
  • the enzyme can bind substrate (forming an enzyme-substrate complex) or inhibitor (enzyme-inhibitor complex) but not both. Many competitive inhibitors resemble the substrate and bind the active site of the enzyme. The substrate is therefore prevented from binding to the same active site.
  • a competitive inhibitor diminishes the rate of catalysis by reducing the proportion of enzyme molecules bound to a substrate.
  • the inhibitor may also be a non-competitive inhibitor. In non-competitive inhibition, which is also reversible, the inhibitor and substrate can bind simultaneously to an enzyme molecule. This means that their binding sites do not overlap.
  • a non- competitive inhibitor acts by decreasing the turnover number of an enzyme rather than by diminishing the proportion of enzyme molecules that are bound to substrate.
  • the inhibitor can also be a mixed inhibitor. Mixed inhibition occurs when an inhibitor both affects the binding of substrate and alters the turnover number of the enzyme.
  • a substance which inhibits the activity of Dam may also do so by binding to the substrate.
  • the substance may itself catalyse a reaction of the substrate, so that the substrate is not available to the enzyme.
  • the inhibitor may simply prevent the substrate binding to the enzyme.
  • Suitable candidate substances include antibody products (for example, monoclonal and polyclonal antibodies, single chain antibodies, chimaeric antibodies and CDR-grafted antibodies) which are specific for Dam.
  • combinatorial libraries, defined chemical identities, peptide and peptide mimetics, oligonucleotides and natural product libraries may be screened for activity as inhibitors of Dam in an assay such as that described above.
  • the candidate substances may be used in an initial screen of, for example, multiple substances per reaction, and the substance of those batches which show inhibition tested individually.
  • Candidate substances which show activity in assays such as those described below can then be tested in in vivo systems, such as an animal model.
  • Candidate inhibitors could be tested for their ability to attenuate microbial infection in mice.
  • the present invention can enable a substance to be identified which is capable of inhibiting the activity of Dam.
  • a substance may be used in a method of treating a microbial, especially bacterial, infection.
  • Such substances may also be used for the manufacture of a medicament for use in the treatment of a microbial infection.
  • a substance identified according to the invention will depend upon the nature of the substance identified.
  • a substance is formulated for clinical use with a pharmaceutically acceptable carrier or diluent.
  • a pharmaceutically acceptable carrier or diluent may be formulated for typical parenteral, intravenous, intramuscular, subcutaneous, intraocular, transdermal or oral administration.
  • a physician will be able to determine the required route of administration for a particular patient and the condition.
  • the pharmaceutical carrier or diluent may be, for example, an isotonic solution.
  • the dose of substance used may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required clinical regimen.
  • a physician will be able to determine the required route of administration and dosage for any particular patient and condition.
  • the test kit of the invention comprises a double stranded DNA comprising at least one Dam target site.
  • This double-stranded DNA is preferably bound to a solid support such as a microtitre plate and comprises a component of a detection means. It may also comprise Dam and a substrate for Dam; an enzyme that selectively cleaves non-methylated GATC motifs such as, for example, Mb ⁇ l or Dpnll; and the reagents of the chosen detection means.

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Abstract

La présente invention concerne une méthode de criblage d'un composé sur la base de sa capacité à inhiber l'ADN adénine méthylase (Dam). Ladite méthode fait appel à un ADN bicaténaire comprenant au moins un site cible Dam. Une extrémité de l'ADN est liée à un support solide tel que le puits d'une plaque de microtitration. L'autre extrémité est dotée d'un moyen permettant de détecter si l'ADN bicaténaire comprenant le site cible Dam a été clivé. Dans le titrage, un composé à tester est ajouté au puits de la plaque de microtitration. La Dam et un substrat associé sont ajoutés et si le composé n'est pas un inhibiteur de Dam, la Dam méthylera le reste d'adénine du site cible Dam. En ajoutant une enzyme qui clive de façon sélective l'ADN bicaténaire sur le site cible Dam mais qui ne coupera pas un site cible Dam méthylé (ou inversement), il est possible de cliver de façon sélective le moyen de détection présent dans l'ADN. Si l'ADN est clivé, le moyen de détection sera perdu dans une étape de lavage subséquente alors que si l'ADN n'est pas clivé, le moyen de détection restera lié à la plaque de microtitration et peut être utilisé afin de générer, par exemple, une réaction colorée qui peut à son tour être utilisée afin de déterminer si le composé à l'essai est un inhibiteur de Dam. L'invention concerne également un matériel permettant de réaliser un tel titrage.
PCT/GB2001/005329 2000-12-01 2001-12-03 Methode d'identification d'inhibiteurs d'enzymes Ceased WO2002044408A2 (fr)

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AU2002223099A AU2002223099A1 (en) 2000-12-01 2001-12-03 A method for identifying enzyme inhibitors

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GB0029379A GB0029379D0 (en) 2000-12-01 2000-12-01 A method for identifying enzyme inhibitors
GB0029379.5 2000-12-01

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008138572A1 (fr) * 2007-05-09 2008-11-20 Jacobs University Bremen Gmbh Procédé et réactifs pour l'étude des réactions de méthylation d'acide nucléique

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU4486097A (en) * 1996-09-19 1998-04-14 Board Of Trustees Of The Leland Stanford Junior University Dna adenine methyltransferases and uses thereof
GB9822067D0 (en) * 1998-10-12 1998-12-02 Harbron Stuart Column-supported hybridisation assay in which excess probe is destroyed
AU776864B2 (en) * 1999-02-02 2004-09-23 Regents Of The University Of California, The Compositions and methods for treating and preventing pathogenic bacterial infection based on the essential role of DNA methylation in bacterial virulence

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008138572A1 (fr) * 2007-05-09 2008-11-20 Jacobs University Bremen Gmbh Procédé et réactifs pour l'étude des réactions de méthylation d'acide nucléique

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AU2002223099A1 (en) 2002-06-11
GB0029379D0 (en) 2001-01-17
WO2002044408A3 (fr) 2003-10-02

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