WO2004011627A2 - Systeme de criblage d'une lignee de cellules hotes recombinantes destine aux medicaments ayant un potentiel anti-cancer et une activite anti-(retro-) virale - Google Patents

Systeme de criblage d'une lignee de cellules hotes recombinantes destine aux medicaments ayant un potentiel anti-cancer et une activite anti-(retro-) virale Download PDF

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WO2004011627A2
WO2004011627A2 PCT/IB2003/002966 IB0302966W WO2004011627A2 WO 2004011627 A2 WO2004011627 A2 WO 2004011627A2 IB 0302966 W IB0302966 W IB 0302966W WO 2004011627 A2 WO2004011627 A2 WO 2004011627A2
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host cell
cell line
recombinant host
gene
human
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WO2004011627A3 (fr
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David-Nicolas Chaperon
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David-Nicolas Chaperon
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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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • 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
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases
    • G01N2333/91215—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases with a definite EC number (2.7.1.-)
    • 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
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates to a recombinant host cell line and the use thereof in a recombinant host cell line-based screening system in which the host cell genes of the deoxythymidine triphosphate synthesis pathway are functionally complemented by viral, fungal or mammalian homologues.
  • the present invention also relates to the use of a recombinant host cell line for testing inhibitors of enzymes of the thymidine phosphorylation pathway(s), testing improved phosphorylation of nucleoside analogues, their incorporation into DNA as well as mutants of the involved kinases.
  • the present invention relates to the use of the recombinant host cell line for the expression, characterisation and purification of any enzyme activity able to complement the said recombinant host cell line for growth.
  • E. coli Esche chia coli
  • the existing bacterial screening systems have several limitations: they overproduce human enzymes or enzymes of pathogens in the presence of the E. coli wild type or mutant enzymes. Therefore it is difficult to distinguish if the observed effect is specific for E. coli, humans or a combination of both. Only a few E.
  • thymidylate kinase gene cdc8 which is complemented by the human thymidylate kinase (also called thymidine monophosphate kinase) has been shown to present severe deficiencies in growth (Su and Sclafani, 1991 , Nucleic Acids Res 25:823-7).
  • nucleoside analogues of thymidine such as AZT, d4T and FUdR are first activated by the human proteins Tk ⁇ or Tk2 in the salvage pathway and can be later phosphorylated by the human thymidylate kinase dTYMK.
  • the step from the diphosphate to the triphosphate is a less nucleotide-specific reaction. This reaction is carried out by different human nucleoside diphosphate kinases (Van Rompay et al., 2000, Pharmacol Ther 87:189-98; Lacombe et al., 2000, J Bioenerg Biomembr 32:247-58).
  • the thymidine (pyrimidine)-like molecules can either inhibit enzymes of the dTTP biosynthesis pathways, other enzymes of the nucleotide biosynthesis or they inhibit DNA polymerases of virus or the one of fast growing cells as well as Reverse Transcriptases. They often block DNA replication by a chain termination reaction (Balzarini, 2000, Pharmacol Ther 87:175-87).
  • the human dTMP kinase dTYMK is of special interest. This kinase is one of the most important enzymes in the cascade of dTTP biosynthesis, since it is the bottleneck in the activation of drugs such as 3'-azido-3'-deoxythymidine (AZT) (Balzarini et al., 1989, J Biol Chem 264:6127-33; Lavie et al., 1997, Nat Med 3:922-4). AZT is one of the most important drugs used against HIV (Mitsuya et al., 1990, Science 249:1533-44) and is active against certain bacteria (Elwell et al., 1987, Antimicrob Agents Chemother 31:274-80).
  • AZT 3'-azido-3'-deoxythymidine
  • the object of the present invention is to provide improved recombinant host cell lines and the use thereof in a recombinant host cell line-based screening system in which genes of the thymidine phosphorylation pathway are functionally complemented by viral, fungal or mammalian homologues.
  • the present invention allows the expression, characterisation and purification of any thymidylate kinase activity able to complement the absence of the host kinase activity in said host cell line.
  • the object of the present invention is to provide improved recombinant host cell lines for use in a screening system for testing inhibitors of viral, bacterial, fungal or eukaryotic such as mammalian or especially human kinases involved in nucleotide biosynthesis or inhibitors of viral, fungal or eukaryotic such as mammalian or especially human or viral enzymes involved in DNA replication, such as DNA-polymerases or Reverse Transcriptases.
  • This object has been achieved by complementing the host cell thymidylate kinase gene, thymidine kinase gene and thymidylate synthase gene, such as respectively the tmk, tdk and thyA genes in E. coli, which are involved in the dTTP biosynthesis by functional homologues such as the human genes dTYMK and tk1, or related fungal and viral kinases.
  • functional homologues such as the human genes dTYMK and tk1, or related fungal and viral kinases.
  • bacteria like E. coli are very fast growing organisms, simple to handle, low in cost and with reliable results.
  • bacteria like E. coli can be automatized for large-scale screenings alone or in combination with the targets of nucleoside analogues such as human or viral DNA polymerases or Reverse Transcriptases, and do not require the safety standards as do human and viral screening systems.
  • the described screening system can be used as a kit in combination with systems that do separate chemical compounds.
  • Another advantage of the present invention is that it is useful in the design of potential anti- (retro-) viral and anti-cancer drugs based on thymidine (pyrimidine)-like compounds as well as for the creation of structural analogues of existing drugs such as the drugs AZT, d4T, FUdR and others.
  • the invention further allows the screening or selection for mutant enzymes in the described recombinant host cell background for new technologies such as (suicide) gene therapy. Therefore, such recombinant host cell line can also be used for the production of phosphorylated forms of nucleoside analogues.
  • the present invention allows a simplified characterisation and purification of any kinase activity of viral, bacterial, fungal or mammalian origin able to complement the absence of the corresponding protein activity of the recombinant host cell line.
  • the presented invention also includes the use of a human dTMP kinase dTYMK gene which is mutated in the bases coding for its first 30 amino acids of the N-terminus, since the described mutations increase the protein production as well as they change the cellular trafficking/sorting of the protein in a specific host cell background.
  • Figure 1 describes a schema with tmk deletion strain.
  • E. coli tmk to kanR substitution strain on 24.9 min on the £. coli chromosome the expression of the kanamycin resistance gene is under the control of the putative five- gene operon.
  • Figure 2 illustrates the dTTP biosynthesis pathways.
  • Figure 3 shows the plasmid constructions of a) pGP189, control plasmid b) pGP189 with human cDNA of the dTMP kinase dTYMK c) pGP189 with human cDNA of the cytoplasmic thymidine kinase tk1 d) pGP189 with human cDNA of the dTMP kinase dTYMK and the cytosolic thymidine kinase tk1.
  • IPTG isopropylthiogalactoside
  • Figure 4 shows E. coli wild type and E. coli tmk deletion strains growing on plates with various concentrations of AZT and the complementation of E. coli tmk by the human cDNA gene homologue dTYMK.
  • Figure 4a shows the B178 ⁇ fm f strain growing in the presence of an IPTG-inducible ⁇ TY -expressing plasmid.
  • Figure 4b shows that the growth of a B" ⁇ 78 ⁇ tmk strain depends on the expression of the human gene dTYM B" 78 ⁇ tmk shows reduced growth in the absence of IPTG when complemented with the human dTYMK.
  • Figure 4c shows that B178 ⁇ rt strains present growth reduction in the presence of 50 nM AZT whereas the human dTYMK complemented B" 78 mk strain shows normal growth.
  • the overexpression of £. coli tmk strengthens the growth reducing effect.
  • Figure 4d shows the growth of B178wt and B' ⁇ 78 ⁇ tmk strains with different plasmids in the presence of 500 nM of AZT.
  • Figure 4e shows the absence of growth of B178wtf and B178 ⁇ tmk strains in the presence of 2000 nM of AZT.
  • Figure 5 describes a schema of the ⁇ tdk deletion strain.
  • Figure 6 describes a schema of the ⁇ thyA deletion strain. (Bell-Pedersen et al., 1991 , J Bacteriol 160:371-8)
  • Figure 7 shows £. coli wt, ⁇ tmk, ⁇ tdk or ⁇ tmk ⁇ tdk strains on minimal plates with complementing plasmids in the presence of AZT.
  • Figure 8 shows wild type and examples of changed N-terminal residues of the human dTMP kinase dTYMK (dTYMK-1 to dTYMK-3) with increased kinase production and activity in the bacterium £. coli.
  • the present invention relates to recombinant host cell line characterized in that their thymidylate kinase gene alone or in combination with the thymidine kinase gene and/or thymidylate synthase gene have been functionally complemented by at least one functional homologue of another organism.
  • Another organism means for example a procaryotic organism or an eukaryotic organism such as mammalian and in particular humans.
  • Such functional homologues are, for example, the human dTMP kinase dTYMK and the human cytoplasmic thymidine kinase Tk1 , viral or bacteriophage kinases like the T4 gene product or fungal dTMP kinase like the yeast kinase CDC8.
  • deletions in the case the recombinant host cell line is £. coli, are designated ⁇ tdk, ⁇ tmk or ⁇ thyA, and can be used alone or in combination so that their specific function or combined functions are complemented by any homologue gene/genes from other organisms.
  • the terms "complete deletion” as used herein refer to a deletion of about 80-100% of the complete gene.
  • Examples of recombinant host cell lines include cells like yeast, bacterial cells, eukaryotic cells such as mammalian cells.
  • the Gram (-) bacterium Escherichia coli (E. coli) has been used as a model system.
  • the concerned deletions resulting from a genetic procedure, have been functionally complemented by the human dTMP kinase gene dTYMK, and/or by the human cytoplasmic thymidine kinase gene tk1, by the bacteriophage T4 gene 7 or by the yeast thymidylate kinase gene cdc8.
  • deletions ( ⁇ tdk, ⁇ tmk or ⁇ thyA) in £. coli have been functionally complemented by both the human dTMP kinase gene dTYMK and the human cytoplasmic thymidine kinase gene tk1.
  • deletions of the £. coli genes for nucleoside diphosphate kinase activity are considered, as long as they do not code for essential £. coli genes (Lu and Inouye, 1996, Proc Natl Acad Sci USA 93:5720-5).
  • the corresponding human nucleotide diphosphate kinases of the Nm23-NDP kinase family (Lacombe et al., 2000, J Bioenerg Biomembr 32:247-58) is then co-expressed with the said complementing human genes presenting the whole human dTTP phosphorylation synthesis pathway with deletions in the main genes of the thymidine phosphorylation pathway of £. coli.
  • Another object of the invention relates to the use of the recombinant host cell line for testing nucleoside analogues of thymidine (pyrimidines) for their activation and/or inhibition by said kinases involved in thymidine phosphorylation.
  • pyrimidines nucleoside analogues of thymidine
  • a further object of the invention relates to the use of the recombinant host cell line for simplifying the screening of chemical or natural libraries of nucleoside analogues such as thymidine (pyrimidine)-like analogues for their activation by the human kinases Tk1 and human dTMP kinase and/or for inhibition of these complementing nucleoside/nucleotide kinases in £. coli.
  • Chemical modifications such as phosphorylation or others can be detected using the said recombinant host cell line in vivo or in the form of cell extracts or fractions.
  • the reduced growth or cell death indicates activation or inhibition of the used targets in the recombinant host cell line in vivo whereas chemical modifications using cell extracts of recombinant host cell line can be detected using standard detection procedures.
  • a further object of this invention relates to a recombinant host cell line based screening method characterized in that it comprises the following steps: a) introducing a vector that expresses the human dTMP kinase gene dTYMK alone or in combination with the human cytoplasmic thymidine kinase gene tk1 or other viral, fungal or mammalian kinase able to complement for said host cell having its genome deleted for the tdk, tmk or thyA genes, b) incubating said host cell harbouring said vector under conditions that permits its growth, c) exposing the host cell to a compound to be screened, d) detecting the presence or absence of growth or death, thereby determining whether said candidate compound is activated, inhibited and / or integrated into the host DNA.
  • preceding steps a), b), c), and d), can be supplemented with a step e) detecting chemical modifications such as phosphorylation of candidate compounds using recombinant host cell line or using extracts of the recombinant host cell line.
  • this step e) can replace step d).
  • kinases used in step a) of the described recombinant host cell line based screening method are the human mitochondrial thymidine kinase tk.2, the human gene TS, the bacteriophage T4 gene 1 and the yeast thymidylate kinase gene cdc ⁇ .
  • the described recombinant host cell line based screening method can be used in the screening of compounds acting as a) inhibitors of the mammalian, fungal or viral proteins which are coded by complementing genes of the deleted genes tdk, tmk or thyA of the recombinant host cell line, b) inhibitors of other human, viral or bacterial enzymes involved in other enzymatic steps of the nucleoside metabolism other than that of the genes mentioned in a), c) inhibitors of eukaryotic, prokaryotic or viral DNA or RNA polymerases, as well as of Reverse Transcriptases, d) antibiotic molecules, which integrate into host DNA.
  • Another object of the present invention is to provide vectors expressing the human dTYMK and/or tk1 genes. These genes have been cloned in high copy vectors expressing sufficient amounts of enzymatic activity for complementing the deleted recombinant host cell line genes. For polycistronic expression of the human dTYMK and tk1 genes, a system based on changing the distance between the co-expressed genes has been established for regulating the desired over-production of the human dTYMK and ⁇ 1 proteins.
  • the vector is able to combine a sufficient production of a essential protein with a non-toxic production of a second essential or non essential protein (Figure 3d).
  • the distance between the first and second gene can be changed resulting in that the strength of the translation of the second gene product can be controlled.
  • Increasing the distance between two genes in a polycistronic expression system reduces the relative expression of the second gene of the operon.
  • Using a weak translation initiation signal (ribosomal binding site) does further reduce the translation of the second gene.
  • the described expression vectors can be used in studies for testing growth or sensitivity mutants coding in the corresponding complementing genes in combination, especially for the human dTYMK and tk1 genes.
  • the described invention simplifies the screening or selection of a specific function of a protein(s) used for the complementation of said recombinant host cell lines.
  • a gene library coding for ⁇ f the human dTYMK can be screened or selected for increased or decreased phosphorylation of drugs such as AZT.
  • the mutated human protein must nevertheless be able to complement the essential function of the TMK protein in a £. coli ⁇ tmk background.
  • recombinant host cell line, cell extracts or fractions can be used in combination or consecutively with the targets of activated thymidine (pyrimidine)-like nucleoside analogues such as mammalian, in particular human or viral DNA/RNA polymerases as well as Reverse Transcriptases such as the HIV Reverse Transcriptase (Farmerie et al., 1987, Science 236:305-8; Hizi et al., 1988, Proc Natl Acad Sci USA 85:1218-22).
  • activated thymidine (pyrimidine)-like nucleoside analogues such as mammalian, in particular human or viral DNA/RNA polymerases as well as Reverse Transcriptases such as the HIV Reverse Transcriptase (Farmerie et al., 1987, Science 236:305-8; Hizi et al., 1988, Proc Natl Acad Sci USA 85:1218-22).
  • the described bacterial gene deletions can also be combined with existing screening systems as described in Kim and Loeb (1995, J Virol 69:6563-6) and Sweasy and Loeb, (1992, J Biol Chem 267:1407-10).
  • the bacterial strains used in the screening method can be tested in liquid cultures such as M9 minimal or LB media.
  • the bacterial strains can be tested in liquid cultures containing different concentrations of compounds of the separated chemical or natural libraries and/or with different concentrations of the bacterial strains used.
  • the described screening system functions also on solid media such as M9 minimal agar or LB agar media. Dilutions of the described bacterial strains can be tested on solid media containing different concentrations of the fractions of the separated chemical or natural libraries similar as shown in Figures 4 and 7. All these steps can be automatized.
  • the described screening system also allows the use of cell extracts or fractions of the described recombinant host cell line.
  • a further object of the invention relates to the use of the recombinant host cell line for the expression, activity testing, characterisation or purification of any enzyme activity able to complement the absence of the corresponding activity of the recombinant host cell line.
  • the growth of the used recombinant host cell line is depending on externally provided enzymatic activity. Therefore, any complementing activity from other organism can be over-produced, tested, characterised or purified in the used recombinant host cell line as long as the provided gene product is able to complement for the missing essential function(s) of the recombinant host cell line. This is of special interest in the case of the example of the bacterium £. coli, since this organism is a preferred production vessel for recombinant proteins.
  • kits characterized in that it comprises: a) the recombinant host cell line according to the present invention, b) a substrate enabling the growth of said recombinant host cell line, c) means for measuring cell densities and/or cell culture growth.
  • the described kit contains solid culture medium, such as M9 minimal agar or LB agar, or liquid cultures such as M9 minimal or LB media or contains cell extracts of the described recombinant host cell line according to the present invention.
  • the described invention is based on the creation of a clean deletion of the essential £. coli thymidylate kinase gene tmk at 24.9 min in the £.
  • coli gene tdk product is responsible for the phosphorylation of thymidine in the salvage pathway, whereas THYA modifies dUMP to dTMP in the de novo pathway (Belfort et al., 1983, Proc Natl Acad Sci USA 80:1858- 61). Again, these two genes, which are essential in combination for the growth of £.
  • the invention demonstrates that £. coli shows an increased resistance towards AZT when its tmk gene has been replaced by its human dTYMK counterpart ( Figure 4 and 7).
  • Mutants of human dTYMK gene product with changed functionality can be generated and combined with the above-described system.
  • the described recombinant host cell line are of out-standing use for the screening or selection of any thymidylate kinase activity with changed substrate specificity.
  • the described system can be used for the development of modified human dTYMK kinase activity especially for (suicide) gene therapy.
  • Traditional systems try to modify the HSV Tk (Christians et al., 1999, Nat Biotechnol 17:259-64) for increasing the activation of nucleoside analogues in target cells.
  • the described system can use modified human protein(s) such as the one of the human dTMP kinase dTYMK in combination with wild type or modified Tk1.
  • modified human protein(s) such as the one of the human dTMP kinase dTYMK in combination with wild type or modified Tk1.
  • the above-described £. coli deletion strains which are deficient in the described bacterial genes and are complemented by functional homologues coding for changed substrate specificity, can also be used for the production of phosphorylated forms of nucleoside analogues of thymidine (pyrimidine).
  • pyrimidine thymidine
  • the human dTMP kinase dTYMK inefficiently activates the nucleoside analogues AZT (Balzarini et al., 1989, J Biol Chem 264:6127-33) and a recombinant £.
  • coli ⁇ tmk strain which is complemented with the human dTYMK gene is accumulating the monophosphate form of AZT. Therefore modified genes coding for enzymes with changed substrate specificity can be screened or selected in recombinant host cell line and can this way be used for the production of phosphorylated forms of nucleoside analogues of thymidine (pyrimidines).
  • the triple deleted ⁇ tdk ⁇ tmk ⁇ thyA E.coli strain functionally complemented by the human genes dTYMK and tk1, is used for carrying out the invention.
  • the triple deleted ⁇ tdk ⁇ tmk ⁇ thyA E.coli strain functionally complemented by the human genes dTYMK , tk1 and tk2 , is used for carrying out the invention.
  • the triple deleted ⁇ tdk ⁇ tmk ⁇ thyA E.coli strain functionally complemented by the human genes dTYMK tk1 and TS, is used for carrying out the invention.
  • the applicant has surprisingly shown that mutations in the first 30 N- terminal amino acids of the wild-type human dTMP kinase dTYMK change the production and activity levels of the protein in the bacterial background of £. coli. (Huang et al., 1994, DNA Cell Biol 13:461-471) (Figure 8). Alleles of dTYMK coding for changed N-terminal amino acid sequences and which change the cellular location or trafficking of dTYMK can be used for the expression of the human dTYMK gene in different backgrounds.
  • This deletion strain was constructed using the system of Link et al., (1997, J Bacteriol 179:6228-37) by replacing the majority of the £. coli gene tmk with a kanamycin-resistance marker, which is under the control of the promoter(s) of a putative five-gene operon at 24.9 min in the £. coli genome.
  • the kanamycin-resistance gene kkal was PCR-amplified from the vector pACYC177 (Chang and Cohen, 1978, J Bacteriol 134:1141-56) using primers P5 and P6 (Sphl).
  • the genomic PCR products of primers P1 and P2 (Aatll, Sphl), P3 and P4 (Sail, Sphl), and the kanamycin-resistance gene were cloned into a plasmid (Aatll, Sail) carrying a Bglll/Hindlll fragment in the region of tmk.
  • This Bglll/Hindlll fragment was originally subcloned from a ⁇ -transducing- phage (Kohara et al., 1987, Cell 50:495-508) containing £. coli DNA at 24.9 min of the £. coli genome (S. Raina, personal communication).
  • a BgllllSall fragment from the plasmid carrying the replacement of tmk by the kanamycin-resistance marker was cloned into the vector pKO3 (BamHI, Sail).
  • the minimal £. coli gene tmk was amplified with primers P7 and P8 (EcoRI,
  • the tdk deletion strain was constructed using the system of Yu et al., (2000, Proc Natl Acad Sci USA 97:5978-83). Again, an almost complete deletion of the tdk gene was created (Figure 5).
  • the PCR amplification products of primers P9 and P10 (Xhol, BamHI), primers P11 and P12 (BamHI, Xbal), and the chloramphenicol-resistance marker of the vector pHP45 ⁇ Cm (BamHI) (Fellay et al., 1987, Gene 52:147-52) were cloned into the vector pGP189 (Xhol, Xbal) (Genevaux, 2000, unpublished results). The resulting plasmid was used as the basis for linear transformation into £. coli as described ( Figure 5). ThyA deletion strain
  • the dTMP kinase gene has been cloned from mRNA of human embryonic kidney
  • TM cells 293T using standard RT-PCR DNA amplification techniques (Superscript one- step RT-PCR with Platinum Taq, Gibco BRL, Life Technologies) with the primers P13 and P14 (EcoRI, Xbal).
  • the PCR cDNA product was cloned into the vector pGP189 (EcoRI, Xbal) (Genevaux, 2000, unpublished results) ( Figure 3a), which is the vector pSE380 (Invitrogen Corporation, San Diego CA) with a 39 bp deletion of the base pairs 268 to 306.
  • the human cDNA tk1 gene was PCR amplified from the plasmid pTrcHUMtkl (Wang et al., 1998, Antimicrob Agents and Chemother 42:2620-25) using primers P15 and P16 (EcoRI, Bglll). The amplification product has been cloned into the vector pGP189 (EcoRI, Bglll).
  • a plasmid for the expression of both the dTYMK and the tk 1 kinases was created by cloning the PCR product of primers P15 and P16 (Xbal, Bglll) of the tk1 gene (Wang et al., 1998, Antimicrob Agents and Chemother 42:2620-25) downstream of the human dTYMK kinase gene in the human dTYMK kinase expression vector ( Figure 3b).
  • Use of the recombinant host cell line in a screening test Figures 4 and 7
  • the recombinant £. coli strains were inoculated over-night in LB liquid cultures with the necessary antibiotics (ampicillin (100 ⁇ g/ml), tetracycline (10 ⁇ g/ml)) and 0.1 to 1 mM IPTG. The following day, the optical density (OD) of the cultures was measured and equal cell densities were diluted in 10 times steps in minimal M9 or VB medium (Sambrook and Russell, 2001 , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA; Vogel and Bonner, 1956, J Biol Chem 218:97-106) containing antibiotics and IPTG as described for the overnight cultures. These dilutions were spotted on minimal M9 or VB agar plates in the absence or in the presence of different concentrations of IPTG and/or a compound to be tested. Afterwards they were incubated overnight for 12 hours or more at 37°C.
  • a recombinant £. coli microorganism, according to the present invention, has been deposited with the Institut Pasteur, Collection Nationale de Cultures de Microorganismes in Paris, under accession number of deposit CNCM I-3072 on July 21 , 2003.
  • the recombinant host cell line is useful in a recombinant host cell line-based screening system for testing inhibitors of enzymes of the thymidine phosphorylation pathway(s), testing improved phosphorylation of nucleoside analogues, their incorporation into DNA as well as mutants of the involved kinases and for the expression, characterisation and purification of any enzyme activity able to complement the said recombinant host cell line for growth.
  • kit integrating the recombinant host cell line will be highly useful in the pharmaceutical industry as well as in the research laboratories.

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Abstract

La présente invention concerne une lignée de cellules hôtes recombinantes et son utilisation dans un système de criblage de lignée de cellules hôtes recombinantes dans lequel les gènes de cellule hôte de la voie de synthèse de la déoxythymidine triphosphate sont fonctionnellement complétés par des homologues viraux, fongiques ou mammaliennes. La présente invention concerne également l'utilisation d'une lignée de cellules hôtes recombinantes pour tester les inhibiteurs des enzymes de la ou des voie(s) de la thymidine phosphorylation et pour tester la phosphorylation améliorée d'analogues nucléosides, leur incorporation dans l'ADN ainsi que les mutants des kinases impliquées. Enfin, l'invention concerne l'utilisation de la lignée de cellules hôtes recombinantes pour l'expression, la caractérisation et la purification de n'importe quelle activité enzymatique capable de compléter cette lignée de cellules hôtes recombinantes pour la croissance.
PCT/IB2003/002966 2002-07-25 2003-07-24 Systeme de criblage d'une lignee de cellules hotes recombinantes destine aux medicaments ayant un potentiel anti-cancer et une activite anti-(retro-) virale WO2004011627A2 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006091158A1 (fr) * 2005-02-25 2006-08-31 Rönnerbol Holding Ab Methode et trousse permettant de determiner l'activite de la thymidine kinase, et leur mode d'utilisation
JP2008531012A (ja) * 2005-02-25 2008-08-14 レンネルボル・ホールディング・アクチボラゲット チミジンキナーゼ活性の測定のための方法およびキットならびにその使用
EA012438B1 (ru) * 2005-02-25 2009-10-30 Рённербол Интернешнл Аб Способ и набор для определения тимидинкиназной активности и их применение
AU2006217129B2 (en) * 2005-02-25 2011-06-30 Ronnerbol International Ab A method and kit for determination of thymidine kinase activity and use thereof
US8765378B2 (en) 2005-02-25 2014-07-01 Biovica International Ab Method and kit for determination of thymidine kinase activity and use thereof
US9376707B2 (en) 2005-02-25 2016-06-28 Biovica International Ab Kit for determination of thymidine kinase activity and use thereof

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