WO1999061625A1 - N-acetyltransferases mycobacteriennes - Google Patents

N-acetyltransferases mycobacteriennes Download PDF

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Publication number
WO1999061625A1
WO1999061625A1 PCT/GB1999/001692 GB9901692W WO9961625A1 WO 1999061625 A1 WO1999061625 A1 WO 1999061625A1 GB 9901692 W GB9901692 W GB 9901692W WO 9961625 A1 WO9961625 A1 WO 9961625A1
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acetyltransferase
arylamine
protein
mycobacterial
nat
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PCT/GB1999/001692
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English (en)
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Edith Sim
Mark Payton
John Sinclair
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Isis Innovation Limited
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Priority to EP99925163A priority Critical patent/EP1082441A1/fr
Priority to JP2000551009A priority patent/JP2002516109A/ja
Publication of WO1999061625A1 publication Critical patent/WO1999061625A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)

Definitions

  • Arylamine N-acetyltransferases catalyse the N- acetylation of arylamines, the N- and O-acetylation of arylhydroxylamines, the N-0 transfer of an acetyl group from acetylhydroxylamines and the N- acetylation of aromatic hydrazines (1 ,2).
  • Their substrates include drugs (e.g. isoniazid, a hydrazine) and carcinogens (e.g. aminofluorene).
  • NATs are found in a wide range of organisms and the enzymes form a unique family of proteins on the basis of sequence similarity.
  • the enzymes from bacteria constitute a subgroup of the NAT enzymes on the basis of sequence comparison. All of the enzymes use acetylCoA as a cofactor in the acetylation reactions and the reaction mechanism is thought to involve the transfer of an acetyl group to an active site sulphydryl group in the protein followed by the transfer of the acetyl group from the acetylated intermediate to an aromatic amine or hydrazine substrate.
  • N-acetyltransferase activity in bacteria was confined to Salmonella typhimunum.
  • N- hydroxyarylamine O-acetyltransferase NhoA transferase
  • NAT activity has been demonstrated to be associated with the sensitivity of S. typhimunum strains when they are used as indicators of mutagenicity in the Ames test.
  • the NAT from S. typhimunum when aligned with NATs from eukaryotes for maximum similarity, has a 20 amino acid gap corresponding to a region in eukaryotic NATs predicted to be random coil.
  • the structural prediction suggests that eukaryotic NATs and S. typhimunum NAT each consist of alpha-helix in the N-terminal region and predominantly beta- sheet in the C-terminal half, with a loop on the C-terminal side between the two regions in eukaryotes which is missing in S. typhimunum NAT.
  • Isoniazid is used for the treatment of TB. Although the molecular mechanism which renders M. tuberculosis Vietnamesely sensitive to isoniazid has resulted in potential targets, one of which has been crystallised, it is not yet fully established how isoniazid acts. It has been proposed that isoniazid interferes with the synthesis of mycolic acids which are a characteristic component of the cell walls of mycobacteria, including M. tuberculosis. It is thought to be an oxidation product of isoniazid which is the active agent and isoniazid resistance has been associated with mutation in the katG (catalase-peroxidase gene). Other loci have been associated with isoniazid resistance.
  • Ahpc alkyl hydoperoxide reductase expression is thought to counteract the action of activated isoniazid.
  • a further protein, InhA which catalyses the NADH-specific reduction of enoyl- ACP in long chain fatty acid synthesis has been proposed as another target for isoniazid. The drug does not appear to bind to the InhA protein. Isoniazid resistance can only be partially accounted for by the currently identified potential causes.
  • the inventors propose that the enzyme arylamine
  • N-acetyltransferase has a role in modulating the activity of isoniazid in M. tuberculosis. Mutations which affect either the NAT protein itself or the expression of the NAT protein in M. tuberculosis may modify NAT activity in isoniazid resistant strains.
  • NAT arylamine N-acetyltransferase
  • the invention provides, in one aspect, the Mycobacterium tuberculosis arylamine N-acetyltransferase protein having the sequence of figure 1 or peptide fragments greater than 5, preferably greater than 10, contiguous amino acid residues thereof.
  • the Mycobacterium tuberculosis arylamine N-acetyltransferase protein or peptide fragments thereof may have an altered sequence from that shown in figure 1 , such that the amino acid at position 207 is arginine instead of glycine.
  • Mycobacterium smegmatis arylamine N- acetyltransferase protein having the amino acid sequence of figure 2 or peptide fragments greater than 5, preferably greater than 10, contiguous amino acid residues thereof.
  • the present invention also provides a protein or a fragment thereof or a polypeptide containing a mimetope of an epitope of a Mycobacterium tuberculosis or Mycobacterium smegmatis arylamine N- acetyltransferase protein or fragment thereof.
  • Proteins and fragments thereof and polypeptides of the invention may be recovered from cells of organisms expressing an arylamine N-acetyltransferase gene or generated by expression of an arylamine N-acetyltransferase gene or coding sequence contained in a nucleic acid of the present invention in an appropriate expression system and host, or obtained by de novo synthesis or a combination thereof, by techniques well known in the art of recombinant DNA technology.
  • the proteins, fragments thereof and polypeptides of the invention will contain naturally occurring L- ⁇ -amino acids and may also contain one or more non- naturally occurring ⁇ -amino acids having the D- or L-configuration.
  • variants of the said protein and fragments e.g. variants having conservative substitutions of one residue for another one at one or more locations, having at least 80% sequence identity, with the said protein or fragments, and longer proteins and peptides containing the characteristic protein or fragments here defined, said longer protein or peptides having been artificially obtained.
  • the Mycobacterium smegmatis arylamine N-acetyltransferase gene having the sequence of figure 2 or oligonucleotide fragments greater than 8, preferably at least 20, contiguous nucleotide residues thereof.
  • the gene or oligonucleotide fragments may have sequences differing from the sequences of the arylamine N-acetyltransferase gene or oligonucleotide fragments, due to alternative codon usage and/or encoding alternative amino acids sequences, conservative or otherwise, or mimetopes.
  • Single stranded DNA may be the transcribed strand or the non- transcribed (complementary) strand.
  • RNA's of the invention include unprocessed and processed transcripts of DNA, messenger RNA (mRNA) containing the arylamine N-acetyltransferase gene and anti-sense RNA containing a sequence complementary to the arylamine N- acetyltransferase gene.
  • mRNA messenger RNA
  • Nucleic acids encoding Mycobacterial arylamine N- acetyltransferases are envisaged as useful primers for polymerase chain reactions (PCRs) conducted to ascertain the susceptibility of a particular strain of Mycobacterium to anti-mycobacterial drugs.
  • PCRs polymerase chain reactions
  • PCR methods including the use of fluorescent primers for rapid screening, and restriction fragment length polymorphism (RFLP) analysis, can be used on TB isolates to allow a correct course of treatment to be carried out.
  • variants of the said gene or fragments having at least 80% sequence identity with the said genes and fragments; and longer polynucleotides containing the characteristic genes or fragment here defined, the said longer polynucleotide having been obtained by recombinant nucleic acid technology.
  • mycobacterial strains transformed with one or more synthetic DNA constructs encoding one or more genes or proteins of the invention for example hybrid proteins containing amino acid sequence found in two or more arylamine N-acetyltransferase proteins.
  • mycobacterial strains transformed with a synthetic DNA construct which abolishes endogenous arylamine N-acetyltransferase activity are also provided.
  • the invention provides antibodies raised against the Mycobacterium tuberculosis arylamine N-acetyltransferase protein having the sequence of figure 1 or peptide fragments greater than 5 contiguous amino acid residues thereof.
  • antibodies raised against the Mycobacterium smegmatis arylamine N-acetyltransferase protein having the sequence of figure 2 or peptide fragments greater than 5 contiguous amino acid residues thereof.
  • antibodies raised against the Salmonella typhimurium arylamine N-acetyltransferase protein having the sequence in figure 3 or peptide fragments greater than 5 contiguous amino acid residues thereof.
  • the antibodies provided by the invention may have been raised against a protein or a fragment thereof or a polypeptide containing a mimetope of an epitope of a Mycobacterium tuberculosis, Mycobacterium smegmatis or Salmonella typhimurium arylamine N-acetyltransferase protein or fragment thereof.
  • Antibodies against such proteins and fragments as well as fragments of such antibodies including chemically derived and recombinant fragments of such antibodies, and cells, such as eukaryotic cells, for instance, hybridomas and prokaryotic recombinant cells capable of expressing and, preferably secreting antibodies or fragments thereof against such proteins or fragments, also form part of the present invention.
  • Antibodies may be obtained by immunization of a suitable host animal and recovery of the antibodies, by culture of antibody producing cells obtained from suitably immunized host animals or by in vitro stimulation of B-cells with a suitable arylamine N-acetyltransferase protein, fragment or polypeptide or arylamine N-acetyltransferase mimetope, protein, fragment or polypeptide and culturing of the cells. Such cells may be immortalised as necessary for instance by fusion with myeloma cells.
  • Antibodies may also be produced by 'phage-display' technology known in the art. Antibody fragments may be obtained by well known chemical and biotechnological methods. All these techniques are well known to practitioners of the arts of biotechnology.
  • a sample for example a biological sample, for example from an infected individual.
  • a method of detecting mycobacteria in a sample for example a biological sample, which comprises the steps of providing a biological sample which has been treated so as to allow the release of cytoplasmic components of mycobacteria present in the sample, bringing said treated sample into contact with one or more antibodies raised against a mycobacterial arylamine N-acetyltransferase, and determining the binding of said antibody to one or more cytoplasmic components of the sample.
  • the sample may treated by sonication to release the cytoplasmic components.
  • the invention provides a method of screening for compounds that bind to a Mycobacterial N-acetyltransferase protein by bringing one or more molecules of an N-acetyltransferase protein into contact with one or more compounds to be screened for binding and detecting said binding.
  • Such compounds may be produced by combinatorial or rational design methods as known in the art.
  • One such method provided comprises the steps of immobilising one or more test compounds on a solid support; bringing an aliquot of N-acetyltransferase protein into contact with the said immobilised test compounds; removing unbound protein from the solid support; detecting the bound protein.
  • the N-acetyltransferase protein used in the method may be arylamine N-acetyltransferase from Mycobacterium tuberculosis, Mycobacterium smegmatis or Salmonella typhimurium, or fragments thereof, or N-acetyltransferase proteins or fragments thereof from eukaryotes including humans.
  • a non-mycobacterial NAT protein is used in the compound screen, the compound may also optionally be screened using a mycobacterial NAT.
  • a screened compound may be subsequently tested for binding to Human NAT1 or NAT2 or other eukaryotic NATs, for example, bovine or badger.
  • This may be by the steps of: immobilising one or more screened compounds which bind to mycobacterial N-acetyltransferase on a solid support; bringing an aliquot of a human N-acetyltransferase protein into contact with the said immobilised test compounds; removing unbound protein from the solid support; detecting the bound protein.
  • the detection of the bound protein may be by means of antibodies raised against complete molecules, fragments, or a polypeptide containing a mimetope of an epitope, of a human NAT protein .
  • the detection of bound protein in such a method may be by means of antibodies raised against complete molecules, fragments or a polypeptide containing a mimetope of an epitope, of arylamine N- acetyltransferase from Mycobacterium tuberculosis, Mycobacterium smegmatis or Salmonella typhimurium.
  • Other methods of protein detection known in the art for example, the use of antibody fragments containing at least one epitope binding site and other ELISA and fluorescence techniques, are also envisaged.
  • the compounds being screened for are those that are ligands of NAT, herein defined as including compounds which are substrates of NAT or which modulate the enzymic activity of NAT such as inhibitors, for example inhibitors of the acetylation of aromatic amines and hydrazines by Mycobacterial N-acetyltransferase proteins. More preferably such compounds inhibit the acetylation of arylamines by Mycobacterial N-acetyltransferase proteins. Most preferably such compounds are ligands for Mycobacterial but not mammalian N-acetyltransferase proteins.
  • the invention provides a method of controlling the growth of a Mycobacterium by bringing said Mycobacterium into contact with a compound which is a ligand of a arylamine N- acetyltransferase protein expressed by said Mycobacterium.
  • the ligand maybe a substrate or an inhibitor of the arylamine N-acetyltransferase.
  • the method of controlling the growth of a Mycobacterium may comprise bringing the said Mycobacterium into contact with an aromatic amine or hydrazine and a compound, for example a NAT ligand, capable of diminishing the activity of an arylamine N-acetyltransferase protein expressed by said Mycobacterium for said aromatic amine or hydrazine.
  • a preferred method of according to the invention controls the growth of
  • Mycobacterium tuberculosis Mycobacterium leprae, Mycobacterium bovis or Mycobacterium avum, though the control of other pathogenic mycobacterial species is envisaged.
  • the aromatic amine or hydrazine and the inhibitory compound may be applied to the mycobacterium separately or simultaneously.
  • the invention provides in a further aspect, the use of a compound which is a NAT ligand, in the manufacture of a medicament for the treatment of mycobacterial infections.
  • the ligand may be a substrate or an inhibitor of mycobacterial arylamine N-acetyltransferase.
  • the inhibitor may diminish the activity of a mycobacterial arylamine N-acetyltransferase protein towards a second compound which is an aromatic amine or hydrazine, which may be incorporated in the medicament also.
  • an anti-mycobacterial formulation which contains an effective concentration of a compound which is a ligand of a mycobacterial N-acetyltransferase protein.
  • the ligand may be a substrate or an inhibitor of the mycobacterial arylamine N-acetyltransferase.
  • an anti-mycobacterial formulation is a mixture of an effective concentration of an anti-mycobacterial aromatic amine or hydrazine and an effective concentration of a compound capable of diminishing the activity of a mycobacterial arylamine N-acetyltransferase protein.
  • a preferred anti- mycobacterial hydrazine is isoniazid, though other compounds such as ethionamide, dapsone or p-aminosalicylate are envisaged.
  • NAT genes which modulate the expression of NAT.
  • Such compounds can be screened by methods known in the art using the antibodies and/or gene sequences of the invention to monitor levels of one or more NAT gene expression products upon exposure of a target mycobacterium to candidate molecules.
  • Such compounds may be used in the manufacture of medicaments for the treatment of mycobacterial infections or be contained in anti-mycobacterial formulations in an effective concentration.
  • Multiple isoforms of NAT have been found in many eukaryotes including humans, chickens and mice. In humans, two isoforms exist, NAT1 and NAT2, which are 87% homologous in amino acid sequence but have different tissue distributions and substrate specificities.
  • NAT1 is widely distributed and uses p-aminobenzoic acid (pABA) and suiphamethoxazole as substrates.
  • NAT2 has been shown to metabolise isoniazid (INH) and arylamine sulphonamides.
  • the cytosolic NAT enzymes all share a high sequence identity and appear to have been conserved throughout evolution.
  • a conserved amino acid sequence denoted as PFENL by the single letter code within the N-terminal region may be involved in substrate binding due to its capacity to make hydrophobic interactions with aromatic groups.
  • An RGGdC region, where d is either Y or W, of the N-terminal half on the molecule is also highly conserved.
  • Site directed mutagenesis has been used to show that the conserved cysteine in the RGGdC amino acid sequence found in all characterised NATs (C69 in Salmonella typhimurium) is essential for activity.
  • the more variable C-terminal region may confer substrate specificity.
  • the invention provides, in a further aspect, a method of identifying nucleic acid sequences which at least partially encode, or amino acid sequences which at least partially comprise, putative enzymes which are potential targets for anti-mycobacterial drugs or putative enzymes useful for the synthesis of anti-mycobacterial drugs, by means of identifying within said sequences nucleotides encoding or amino acids comprising, protein sequences with similarity greater than 80% to the following sequence:
  • PFENL(X) n 5 - 5 oRGGdC where d is W or Y.
  • similarity is greater than 90% and most preferably greater than 95%.
  • n is between 20 and 30, and more preferably n is equal to 24 such that the first R residue is 29 residues on the C-terminal side of the P residue.
  • the invention also provides a method of isolating DNA sequences, from any source of genomic or cDNA, which at least partially encode putative enzymes which are potential targets for anti-mycobacterial drugs or putative enzymes useful for the synthesis of anti-mycobacterial drugs, by means of a primer extension reaction, such as the polymerase chain reaction, which uses one or more sense primer which encodes the amino acid sequence PFENL and one or more anti-sense primer which encodes the amino acid sequence RGGdC, where d is either W or Y.
  • the product of the primer extension reaction may be subsequently labelled and used as a probe, by methods known in the art, to screen a DNA library, genomic or cDNA, which may be from the same organism as the source of the template DNA used in the primer extension reaction.
  • a mixture of degenerate sense primers and a mixture of degenerate anti-sense primers, comprising all possible encoding sequences for the said amino acid sequences are used in the primer extension reaction.
  • Possible primer mixtures as described using the IUPAC-IUB code, are: Sense primer: 5' CCNTTYGARAAYYTN 3';
  • the invention also relates to the use of the three dimensional structure of a Mycobacterial arylamine N-acetyltransferase protein for the design of compounds capable of binding to said arylamine N- acetyltransferase protein by methods known in the art.
  • the invention further relates to the use of the three dimensional structure of a Mycobacterial arylamine N-acetyltransferase protein for determining or predicting the structure of a further arylamine N-acetyltransferase protein and using the structural information so obtained for the design of compounds capable of binding to said further arylamine N- acetyltransferase protein.
  • the invention also provides compounds which are substrates of arylamine N-acetyltransferase proteins which have the following formula:
  • R C1-C20 alkyl
  • Such compounds may be used as model substrates in screens for compounds which modify NAT activity, or used in the manufacture of medicaments for treatment of mycobacterial infections, or contained in an anti-mycobacterial formulation in an effective concentration.
  • NATs catalyse a classic ping pong reaction.
  • the first step involves the acetylation of the enzyme.
  • the enzyme starting state is restored by the transfer of the acetyl group to a suitable arylamine substrate, such as 4-aminoveratrole (4-AV), anisidine (ANS), p-aminosalicylic acid (PAS), dapsone, or hydrazine substrates such as isoniazid (INH).
  • Organisms which do not appear to possess NAT may duplicate its activity using other enzymes.
  • the identification of a NAT-like sequence in M. tuberculosis is particularly interesting because the anti-bacterial agent isoniazid is a substrate for one of the human NAT isoenzymes and isoniazid is rendered inactive by acetylation. Humans who are fast acetylators (now identified as carrying one or two fast alleles at the NAT2 locus) inactivate isoniazid more rapidly than slow acetylators.
  • a library from M. tuberculosis was screened. A clone was obtained with the expected sequence. The NAT region has subsequently been cloned into the expression vector pET28b and encodes for a protein of the expected size upon SDS-PAGE analysis (approximately 32 kDa, which includes a hexahistidine fusion tag). A clone from a library of the closely related M. smegmatis has been found and sequenced. It is 65% identical to putative NAT from M. tuberculosis. The M. smegmatis NAT, expressed in E. coli has the expected molecular weight and N-acetyltransferase activity with substrates, including isoniazid.
  • Figure 1 shows the nucleotide and derived protein sequence of the M. tuberculosis NAT gene. Letters below sequence represent the predicted amino acids in the single-letter code. Two number code indicates nucleotide and derived amino acid number, respectively. The stop codon is marked by an asterisk. Amino acids in bold face represent areas of conservation typical of NAT genes. Previously reported conserved arginines t (; Biochem J, (1997) Delomenie et al) and cysteine ⁇ (; J. Biol. Chem, (1992) Dupret & Grant and J. Biol. Chem (1992) Watanabe et al) are shown.
  • Figure 2 shows the nucleotide and derived protein sequence of the M. smegmatis NAT gene. Letters below sequence represent the predicted amino acids in the single-letter code. Two number code indicates nucleotide and derived amino acid number, respectively. The stop codon is marked by an asterisk. Amino acids in bold face represent areas of conservation typical of NAT genes. Previously reported conserved arginines t (; Biochem J, (1997) Delomenie et al) and cysteine ⁇ (; J. Biol. Chem,
  • Figure 3 shows the nucleotide and derived protein sequence of the S.typhimurium NAT gene. Letters below sequence represent the predicted amino acids in the single-letter code. Two number code indicates nucleotide and derived amino acid number, respectively. The stop codon is marked by an asterisk. Amino acids in bold face represent areas of conservation typical of NAT genes. Previously reported conserved arginines t (Biochem J, (1997) Delomenie et al) and cysteine ⁇ (; J. Biol. Chem, (1992) Dupret & Grant and J. Biol. Chem (1992) Watanabe et al) are shown. Figure 4 shows the specific activity of heterologously expressed M.
  • smegmatis NAT with 4-AV, ANS , p-aminobenzoic acid (at 0.2 mM) and isoniazid (at 0.015 mM) as substrates.
  • the y-axis shows the activity in E. coli lysate relative to anisidine (1 equivalent to 14 nmol/min/mg protein).
  • Figure 5 shows the effect of expression of M. tuberculosis nat on the growth of M. smegmatis in the presence of isoniazid (INH).
  • results for cultures of M. smegmatis either transformed with pACE-1 alone (open circles) or with pACE-1 containing M. tuberculosis nat (solid circles) grown in minimal medium containing acetamide and cultures of pACE-1 containing M. tuberculosis nat grown in minimal medium containing glucose (triangles) are shown.
  • Figure 6 shows a sequence comparison between S. typhimurium NAT, M. tuberculosis NAT, M. smegmatis NAT and Human NAT2" '*' represents the active cysteine residue. Areas underlined represent conserved regions found in all NATs used to screen for M. smegmatis NAT. Dark shading shows conserved amino acids.
  • Figure 7 shows a western blot analysis of the recombinant and endogenous NAT gene products of M. smegmatis and S. typhimurium and also endogenous NAT in an M. smegmatis soluble cell extract.
  • Recombinant proteins were expressed in E. coli. Proteins were detected using a polyclonal antibody raised against purified recombinant
  • Lane A 500 ng total protein containing
  • Figure 8 shows SDS-PAGE and Western blot analysis of bacterial lysates. Total lysate concentrations were adjusted to load equivalent amounts of recombinant protein.
  • Figure 9 shows a schematic representation of construct (3.6 kbp) used to ablate the endogenous nat gene of M. smegmatis.
  • Gene knockout was achieved using the suicide vector approach. This was achieved by the electroporation of the construct ligated into a vector, incapable of replicating in mycobacteria, into M. smegmatis. Selection was by kanamycin resistance.
  • Figure 10 shows PCR analysis for potential nat gene knockouts and targeted integration. A representative sample of 40 kanamycin resistant colonies are shown.
  • A. PCR amplification using primers specific for M. smegmatis nat demonstrates a lack of the gene (therefore gene knockout) in sample KO 9, as indicated by the lower arrow (828 bp).
  • the upper arrow represents product obtained with kanamycin gene inserted into nat (2.1 kbp). As expected this is not present in MC2 155 (wild type M. smegmatis), this possesses only the nat gene.
  • Lanes represent, M; (Hind Ill/Bam H 1 digested lambda phage marker), C; No template DNA, C2; vector containing M.
  • smegmatis nat, C knockout construct used to create nat-gene ablation in M. smegmatis, MC2 155; wild type M. smegmatis, KO 3, 4, 8, 16, 21 and KO 27 are single crossover (meioploidy) events, and KO 9; a double crossover or gene knockout event.
  • B PCR amplification of the same samples used in Fig. 2. A using primers representing the 3' terminus of the kanamycin insert running in a 5' direction together with primers designed to amplify outside of knockout cassette. This determines integration to the 3' end and/or a targeted event.
  • C 1 , C 2 , C 3 and MC 2 155 do not possess the required sequence to generate product. All others are at least integrated correctly at the 3' end of nat to yield a product size of 2.1 kbp.
  • Figure 11 shows 'A' a gel electrophoresis and 'B/C Southern blot analysis of selected potential knock out strains of M. smegmatis.
  • Lanes represent, A; vector containing gene knock out construct (M. smegmatis nat gene with kanamycin insert), B; vector containing M. smegmatis nat gene only, C; blank lane, Mc2 155; wild type M. smegmatis, KO 3-27; selected strains of kanamycin resistant M. smegmatis.
  • KO 9 clearly depicts a nat gene knock out event.
  • Panel A demonstrates agarose gel analysis of equal amounts of digested chromosomal DNA using Eco Rl and Hind HI visualised using ethidium bromide.
  • Panel B is a southern blot of Panel A using the nat gene open reading frame as a probe.
  • Panel C is Panel B stripped and reprobed with the kanamycin gene.
  • Figure 12 shows the acetylation capacity of isoniazid by M. smegmatis lysates containing recombinant M. smegmatis NAT, endogenous M. smegmatis NAT and M. smegmatis with the nat gene ablated (KO 9).
  • the y-axis shows acetylated isoniazid per mg total protein per hour. Experiments performed in triplicate.
  • Figure 13 shows sensitivity to increasing concentrations of isoniazid during growth (OD ⁇ oonm) in 7H9 media of M. smegmatis wild type (Mc2 155), M. smegmatis over expressing M. tuberculosis nat (TBNAT) and M. smegmatis with the nat gene knocked out (KO 9).
  • TBNAT demonstrates an increased resistance to isoniazid and the nat gene knock out demonstrates an increased sensitivity to isoniazid.
  • Figure 14 is a plot of the rate of acetylation by NAT on a range of long chain (C2-C12) aminophenolethers of the formula:
  • the synthesised compounds were assayed with S. typhimurium NAT to determine the rate of acetylation.
  • the 200 ⁇ l assay contained 9% DMSO, 91 % 20mM Tris-HCI buffer pH 7.5, 90 ⁇ M compound, 400 ⁇ M Acetyl CoA and 1 ⁇ g NAT.
  • the rates of acetylation were compared with an identical solution without the NAT enzyme and the difference attributed to the enzyme.
  • Figure 15 shows the effect of on a range of long chain (C2- C12) aminophenolether compounds upon the growth of wild-type M. smegmatis.
  • Cultures of M. smegmatis were grown in the presence of approximately 25 mg/ml of each compound and a measurement of growth via an optical density reading at 580nm was taken after 24 hours.
  • the percentage inhibition of growth for the C4, C6, C8, C10 and C12 para and ortho compounds, compared to cultures in which the compounds were absent is shown.
  • Example 1 Mycobacterium tuberculosis arylamine N-acetyltransferase recombinant protein production
  • NAT arylamine N-acetyltransferase
  • This vector contains a T7 polymerase promoter, regulated by IPTG via a lac operon, which transcribes any insert with a T7 polymerase promoter.
  • the nucleotide sequence was 56% homologous to a gene from S. typhimurium with proven NAT activity and was calculated to have an open reading frame (ORF) corresponding to a 32 kDa protein.
  • ORF open reading frame
  • the M. tuberculosis gene encoded an enzyme possessing NAT activity it was expressed and tested for activity using a colourimetric assay (3).
  • Combined supernatants and pellets from cultures of BL21 E. coli containing the cloned M. tuberculosis NAT in pET28b appeared to be enzymatically inactive when assayed colourimetrically with anisidine, an arylamine substrate.
  • NAT soluble M. tuberculosis NAT could be produced further expression studies were performed under a variety of conditions. In case the concentrations of soluble protein were too small to be resolved by SDS page, the colourimetric assay was used to determine NAT activity. Initially, expression of insoluble protein was shown to increase with time after induction. Similarly, expression of insoluble protein over time decreased with temperature down to 12°C whilst no corresponding increase in soluble protein was observed.
  • M. tuberculosis NAT clone produced inactive inclusion bodies of the 32 kDa protein, as visualised by denaturing SDS-PAGE. This is the size expected for NAT, although it showed no NAT activity.
  • the protein was not soluble and cytosolic enzymes of this type only show activity when they are expressed in a soluble form.
  • the formation of inclusion bodies allowed the protein to be purified using simple physical techniques. N-terminal amino acid sequencing of the purified recombinant protein further confirmed its identity.
  • M. tuberculosis NAT was denatured in 6 M urea; a standard denaturant for refolding inclusion bodies.
  • M. tuberculosis NAT at 50 mg/ml is almost entirely soluble in 6M urea.
  • Urea at 6 M did not affect NAT activity but did alter the absorbance of the solution at 450 nm in a linear fashion.
  • the detection limits of the apparatus allowed measurements to be made in up to 60 mM urea, with greatest confidence at urea concentrations of less than 10 mM.
  • M. tuberculosis when over-expressed in M. smegmatis using the inducible mycobacterial expression construct.
  • NAT from M. smegmatis or from M. tuberculosis was expressed in either E. coli or in M. smegmatis.
  • the lysates were centrifuged at 11 ,600 g for 30 min and the amount of protein corresponding to the additional band at 30 000-33 000 mol. weight on SDS- PAGE as determined by Coomassie blue staining was estimated in samples of supernatant and resuspended pellet. The percentage solubility in each host is shown below:
  • M. smegmatis and M. tuberculosis NAT enzymes are expressed in a soluble form in M. smegmatis, the total amount of protein induced from the two constructs is different, with the M. smegmatis construct resulting in expression of approximately five times more protein than the M. tuberculosis construct.
  • M. smegmatis NAT and M. tuberculosis NAT are equally soluble. The optimum conditions for the over-expression of M. tuberculosis and M. smegmatis NAT in M.
  • Example 2 Cloning of a gene from Mycobacterium smegmatis encoding arylamine N-acetyltransferase
  • M. smegmatis is a very closely related non-pathogenic species which is used as a model mycobacterial system; its genetics are understood and plasmid expression systems are being developed (4,5).
  • M. smegmatis also possesses NAT
  • a g ridded library representing the M. smegmatis genome was screened using M. tuberculosis NAT cDNA.
  • a 400 bp probe template from the M. tuberculosis NAT open reading frame was chosen because it encodes the PFENL and RGGYC regions conserved in NATs.
  • the 400 bp probe template was radiolabelled with a conventional kit (Amersham-Pharmacia Biotech) and used to probe a gridded library representing the M. smegmatis whole genome.
  • a low stringency screen was performed to detect clones containing the PFENL and RGGYC regions hybridising to the probe.
  • Six clones which hybridised strongly to the 400 bp probe were obtained from the screen. Restriction digestion with BamHI was performed to test for insert size. Results were confirmed using a Southern blot to check for hybridisation to the original probe. Most clones appeared sufficiently large to contain an entire NAT gene.
  • the gridded genomic library was screened. A Southern blot confirmed that all six clones of the clones obtained appeared to contain DNA encoding for PFENL and RGGdC, where d is W or Y, sequences which are also conserved in NATs.
  • the gridded library manufacturing process which utilises blunt end religation, creates a SatnHI site 1 in every 4 occasions. All of the clones had at least one BamHI site and so were linearised.
  • Clone '20F22' contained two BamHI sites and it was thus completely excised from its vector to give a smaller fragment.
  • Clone 20F22 was selected for sequencing along with a second representative clone from the remainder (5115). Plasmids previously obtained from M. smegmatis clones 5115 and 20F22 were digested with Xho ⁇ to excise the inserts from the pBluescript vectors (Stratagene). The fragments were gel purified and analysed. The clones were also digested with ⁇ /ofl to give a set of overlapping fragments. Clone 20F22 contained an internal ⁇ /o l site which gave two fragments. The excised inserts were ligated into pGEM11Z, (Promega). Constructs were sequenced with M13 forward and reverse primers.
  • Example 3 Mycobacterium smegmatis arylamine N-acetyltransferase recombinant protein production
  • the 825 bp clone (5115) corresponding to the putative 275 residue NAT open reading frame was inserted into a pET28b recombinant protein expression vector (Novagen) and used to transform BL21 E. coli (Promega).
  • the open reading frame is relatively short for a NAT protein and, given that the similar M. tuberculosis NAT ORF contains an extra 24 base pairs a second transformation was performed using a clone of the open reading frame and a further 105 base pairs beyond the termination codon.
  • Recombinant soluble protein with a relative molecular weight of 32,000 was expressed from both transformed bacterial colonies consisting of 30,000 of M. smegmatis NAT protein and 2,100 from the fusion hexahistidine affinity tag.
  • the specific activity of recombinant M. smegmatis with a variety of substrates was measured using a 60 minute assay for soluble NAT expressed from the 875 bp M. smegmatis insert.
  • Polyclonal antibodies raised against M. tuberculosis NAT provide a useful tool for specific protein detection. For instance the detection of native protein expression from lysates isolated from drug resistant or sensitive M. tuberculosis would be essential for understanding NAT'S ability to metabolise isoniazid in vivo.
  • M. tuberculosis NAT inclusion bodies were isolated using sonication.
  • Nonionic detergents were used to remove membrane proteins by the following method:
  • the resuspended pellets were spun down at 10,000 x g for 10 minutes at 4°C.
  • the resuspension buffer 50 mM Tris-HCI, 2 mM EDTA, 4 mM DTT and 1 mM Pefabloc (protease inhibitor) at pH 8.0
  • the pellet was resuspended and spun at 10,000 x g for 10 minutes at 4°C. The supernatant was discarded without disturbing the pellet or the gelatinous strands of material.
  • the pellet was carefully resuspended in Triton-EDTA solution (1% (v/v) Triton X-100, 1 mM EDTA) and spun at 10,000 x g for 10 minutes at 4 °C.
  • This preparation was further purified on a 8% acrylamide gel. Gel fragments containing the protein, were mixed with an equal amount of Freund's incomplete adjuvant and injected into rabbits. Rabbits were injected using the following protocol:
  • Freund's complete adjuvant relies on components derived from mycobacterial cell walls to stimulate a sufficient humoral immune response to produce antibodies. To prevent non-specific recognition of other mycobacterial proteins or cell wall components, Freund's incomplete adjuvant was used. An adequate immune response was achieved by leaving the protein in acrylamide. The resulting antibodies have been shown to strongly bind M. tuberculosis NAT and cross react weakly with S. typhimurium NAT.
  • Antibodies to a peptide of M. tuberculosis NAT were also raised.
  • the hydrophobicity plots generated from the sequences of M. tuberculosis NAT and M. smegmatis NAT reveal differences between the C-terminal regions and a region between residues 160-180.
  • the peptides CDELLARQPGADAP from the C-terminus of M. tuberculosis NAT and CDVQARVAEVLDT from the C-terminus of M. smegmatis NAT were synthesised and coupled to soybean trypsin inhibitor as a carrier protein for immunisation (750 ⁇ g) with Freund's Incomplete Adjuvant in rabbits, with two subsequent boosts (600 ⁇ g).
  • the antisera raised against these peptides are capable of distinguishing between the NAT proteins of M. tuberculosis and M. smegmatis.
  • the C- terminal region of both proteins is an important antigenic epitope of NAT.
  • Antibodies raised against this region are therefore useful in the detection and identification of mycobacteria in samples, for example which have been treated to release proteins or protein fragments from within the mycobacterial cell wall.
  • M. smegmatis NAT inclusion bodies were isolated using sonication. Triton X100 and lgepal-630 detergents were used to remove membrane proteins by the same method outlined in the previous example: This preparation was further purified on a 8% acrylamide gel. Gel fragments containing the protein, were mixed with an equal amount of Freund's incomplete adjuvant and injected into rabbits. Rabbits were injected using the same protocol tabulated in the previous example. 2 ml of blood were removed during bleeds to test antibodies using dot or Western blots or ELISA. 1 ml of M. smegmatis NAT in acrylamide, mixed with Freund's incomplete adjuvant, was injected at each boost and for the priming.
  • Freund's complete adjuvant relies on components derived from mycobacterial cell walls to stimulate a sufficient humoral immune response to produce antibodies. To prevent non-specific recognition of other mycobacterial proteins or cell wall components, Freund's incomplete adjuvant is used. An adequate immune response is achieved by leaving the protein in acrylamide. The antibodies raised against whole M. smegmatis NAT protein were found to cross reactive with the M. smegmatis NAT protein.
  • NAT from S.typhimurium (0.6 mg) was used to immunise rabbits as a primary injection in Freund's complete adjuvant followed by two booster injections (0.5 mg) firstly in adjuvant and then in saline at two week intervals. A weak response was obtained. The animals were boosted with 1 mg of pure protein in Freund's incomplete adjuvant on up to 4 occasions at up to monthly intervals before the final bleed. The antibody reacts extremely strongly at 1 :100,000 dilution and monospecifically with NAT from S. typhimurium and with NAT from M. smegmatis, see figure 7
  • a M. smegmatis strain lacking a functional nat gene was created by use of a DNA construct (shown in figure 9) capable of ablating the endogenous nar gene.
  • Gene knockout was achieved using a suicide vector containing the construct which was placed into M. smegmatis via electroporation. The vector was incapable of replicating in mycobacteria and transformed strains were selected by kanamycin resistance.
  • a representative sample of 40 kanamycin resistant colonies were tested by PCR analysis for potential nat gene knockouts and targeted integration, this is shown in figures 10 and 11.
  • PCR amplification using primers specific for M. smegmatis nat were used to demonstrate a lack of the gene (therefore gene knockout) in sample one sample designated KO 9. PCR amplification of the same samples was to determine integration to the 3' end and/or a targeted event.
  • Example 8 Testing of isoniazid with engineered strains of M. smegmatis with altered NAT expression.
  • Lysates were obtained from cultures of wild M. smegmatis (Mc2 155), a recombinant strain that was over-expressing NAT and strain KO9 in which the nat gene has been ablated. The ability of these lysates to carry out the acetylation of isoniazid was investigated. The results are shown in figure 12. It can be seen from in figure 12 that acetylation is increased four-fold in the NAT over-expressing strain compared to the wild type strain. The K09 strain shows a depletion in acetylation relative to the other two strains.
  • M. smegmatis strains in the presence of isoniazid was also investigated.
  • a wild-type strain Mc2 155
  • TBNAT strain over-expressing M. tuberculosis NAT
  • strain KO9 lacking a NAT function were cultured in 7H9 media supplemented with acetamide, in different concentrations of isoniazid (1 , 2 and 10 ⁇ g/ml).
  • the results are shown in figure 13. It can be seen that TBNAT demonstrates an increased resistance to isoniazid and the nat gene knock out demonstrates an increased sensitivity to isoniazid.
  • Example 9 Compounds shown to be NAT substrates and anti- mycobacterial agents
  • the 4-isomer compounds also show activity with NAT from E. coli, M. smegmatis and M. tuberculosis
  • the para (4-isomer) and ortho (2-isomer) compounds were additionally tested for an inhibitory effect upon the growth of M. smegmatis.
  • Cultures of M. smegmatis were grown in the presence of approximately 25 mg/ml of each compound and a measurement of growth via an optical density reading at 580nm was taken after 24 hours.
  • the percentage inhibition of growth for the C4, C6, C8, C10 and C12 para and ortho compounds, compared to cultures in which the compounds were absent is shown is shown in figure 15. It can be seen that the C8 para-isomer compound that has highest NAT activity of the group also has the highest inhibitory effect upon the growth of M. smegmatis. It is therefore shown that compounds which are good substrates of NAT may be used to inhibit mycobacterial growth.
  • the prevention of NAT from acting upon endogenous substrates as a result the exogenous substrate being present in the culture may be the mechanism behind the impaired growth observed.
  • NAT genes from 16 clinical isolates of M. tuberculosis isoniazid resistant strains were amplified by PCR, cloned and both strands sequenced. Two strains displayed a genuine allelic variant of the wild-type
  • M. tuberculosis NAT sequence open reading frame was amplified from DNA obtained from M. tuberculosis clinical isolates displaying isoniazid resistance using the following primers:
  • the resulting amplification products were digested with
  • the sequence comparison ( Figure 6) of the mycobacterial NATs show that the NAT from mycobacteria are slightly different in size from other known NATs.
  • the mammalian NATs have 290 amino acids, that from S. typhimurium and E. coli have 281 amino acids.
  • the M. tuberculosis NAT has 283 amino acids and the M. smegmatis has 275 amino acids.
  • the active site cysteine and the arginine which has been implicated in catalysis are conserved Comparative hydrophobicity plots show a 12 residue region, 162-174 where M. tuberculosis NAT is significantly more hydrophobic than M. smegmatis NAT.
  • NAT enzyme from M. tuberculosis Whilst the NAT enzyme from M. tuberculosis is not active when expressed in lysates of E coli, it could be refolded to a form which was active with anisidine as substrate.
  • the NAT enzyme from M. tuberculosis is active with isoniazid as substrate when expressed in another mycobacterium (M. smegmatis) ( Figure 5).
  • the stability of the two enzymes may be different. These differences may contribute to the inherent differences of M. tuberculosis and M. smegmatis in sensitivity to isoniazid. Mutations which affect either the NAT protein itself or the expression of the NAT protein in M. tuberculosis may increase NAT activity in isoniazid resistant strains of TB. Administration of ligands of mycobacterial NAT, together with isoniazid may increase its effectiveness against other pathogenic mycobacteria, such as M. leprae which causes leprosy or M. bovis which infects cattle. The current work gives new insights into the mechanism of isoniazid metabolism in mycobacteria and may help tackle the current resurgence in tuberculosis, the largest cause of preventable deaths in the world. References

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Abstract

L'invention concerne des protéines de N-acétyltransférase arylamine provenant de Mycobacterium tuberculosis, ainsi qu'un gène et une protéine de N-acétyltransférase arylamine provenant de Mycobacterium smegmatis et des anticorps produits contre les produits géniques et protéines y relatifs. L'invention concerne également des méthodes permettant de détecter des mycobactéries à l'aide des anticorps et des souches mycobactériennes exprimant ces protéines ou manquant de celles-ci. Cette invention concerne par ailleurs des méthodes destinées à détecter par criblage des composés qui sont des ligands des protéines de N-acétyltransférase arylamine provenant de mycobactéries, par exemple des substrats et des inhibiteurs. Ces ligands, dont des exemples sont donnés, peuvent être utilisés dans des médicaments destinés à traiter les infections mycobactériennes, ou dans des formulations anti-mycobactériennes, et peuvent par ailleurs être conçus au moyen de structures tridimensionnelles des protéines de cette invention. L'invention concerne enfin des méthodes d'identification de séquences d'acide nucléique associées à des enzymes reconnues comme étant utiles pour synthétiser des médicaments anti-mycobactériens, ou pour servir de cibles à ceux-ci.
PCT/GB1999/001692 1998-05-27 1999-05-27 N-acetyltransferases mycobacteriennes WO1999061625A1 (fr)

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Publication number Priority date Publication date Assignee Title
EP2133424A1 (fr) 2004-11-29 2009-12-16 Kirin Beer Kabushiki Kaisha Peptide de transport vers les chromoplastes dans les pétales et procédé de construction de plante dotée de pétales jaunâtres par son utilisation
CN110787164A (zh) * 2019-10-08 2020-02-14 天津国际生物医药联合研究院 奥替尼啶在抑制乙酰基转移酶和抗分枝杆菌感染中的应用

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EP0562547A2 (fr) * 1992-03-23 1993-09-29 Tokyo Metropolitan Institute for Neurosciences Gènes humains d'arylamine-N-acétyl-transferase
WO1994026765A1 (fr) * 1993-05-13 1994-11-24 Albert Einstein College Of Medicine Of Yeshiva University, A Division Of Yeshiva University Utilisation des genes de m. tuberculosis, m. bovis et m. smegmatis conferant une resistance a l'isoniazide

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EP0562547A2 (fr) * 1992-03-23 1993-09-29 Tokyo Metropolitan Institute for Neurosciences Gènes humains d'arylamine-N-acétyl-transferase
WO1994026765A1 (fr) * 1993-05-13 1994-11-24 Albert Einstein College Of Medicine Of Yeshiva University, A Division Of Yeshiva University Utilisation des genes de m. tuberculosis, m. bovis et m. smegmatis conferant une resistance a l'isoniazide

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PAYTON M. ET AL.: "Cloning and characterization of Arylamine N-acetyltransferase genes from Mycobacterium smegmatis and Mycobacterium tuberculosis: increased expression results in Isoniazid resistance", JOURNAL OF BACTERIOLOGY, vol. 181, no. 4, February 1999 (1999-02-01), WASHINGTON, DC, US, pages 1343 - 1347, XP002120996, ISSN: 0021-9193 *
WATANABE M. ET AL.: "Involvement of Cys69 residue in the catalytic mechanism of N-Hydroxyarylamine O-Acetyltransferase of Salmonella typhimurium", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 267, no. 12, 25 April 1992 (1992-04-25), AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE, MD, US, pages 8429 - 8436, XP002120012, ISSN: 0021-9258 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2133424A1 (fr) 2004-11-29 2009-12-16 Kirin Beer Kabushiki Kaisha Peptide de transport vers les chromoplastes dans les pétales et procédé de construction de plante dotée de pétales jaunâtres par son utilisation
CN110787164A (zh) * 2019-10-08 2020-02-14 天津国际生物医药联合研究院 奥替尼啶在抑制乙酰基转移酶和抗分枝杆菌感染中的应用

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