WO2004011627A2

WO2004011627A2 - A recombinant host cell line screening system for drugs with potential anti-cancer and anti-(retro-) viral activity

Info

Publication number: WO2004011627A2
Application number: PCT/IB2003/002966
Authority: WO
Inventors: David-Nicolas Chaperon
Original assignee: Individual
Current assignee: Individual
Priority date: 2002-07-25
Filing date: 2003-07-24
Publication date: 2004-02-05
Anticipated expiration: 2005-01-25
Also published as: WO2004011627A3; AU2003251079A1

Abstract

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. In addition, 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.

Description

A RECOMBINANT HOST CELL LINE SCREENING SYSTEM FOR DRUGS WITH POTENTIAL ANTI -

CANCER AND ANTI- (RETRO-) VIRAL ACTIVITY.

FIELD OF THE INVENTION

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. In addition, 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.

BACKGROUND OF THE INVENTION

Discovering and developing potential drugs against cancer and (retro-) viruses is very costly and enormously time-consuming. The screening of chemical or natural libraries for potential drugs with existing in vivo or in vitro systems is so far not satisfactory for an efficient development of drugs. Screening pharmacologically interesting drugs such as nucleoside analogues includes in vivo cell culture systems and in vitro direct assays with pure or partially purified target enzymes such as kinases, catabolic enzymes, and DNA/RNA polymerases. Nevertheless, it is well known that the differences between species and cell types and the variability and cost involved in cell culture studies cause enormous problems. The purification of metabolic enzymes involved in nucleoside pathways is costly and complicated, and, in addition, in vitro assays are subject to criticism of their in vivo relevance.

Since bacteria are very fast growing organisms, simple to handle, low in cost and with reliable results, the use of bacterial screening systems would be of interest for the design of anti-cancer and anti- (retro-) viral drugs. This, by expressing human or viral enzymes in Esche chia coli (E. coli). Such in vivo systems could simulate the human or viral conditions of the nucleotide metabolism, especially those involved in the thymidine triphosphate biosynthesis pathways. Some of the enzymes involved in these pathways are the human dTMP kinase dTYMK (Su and Sclafani, 1991, Nucleic Acids Res 25:823- 7; Lavie et al., 1998, Nat Med 3:922-4; Brundiers et al., 1999, J Biol Chem 274:35289- 92; Ostermann et al., 2000, J Mol Biol 304:43-53), the human thymidylate synthase TS (Kaneda et al., 1990, J Biol Chem 265:20277-84; Landis and Loeb, 1998, J Biol Chem 273:25809-17; Rose et al., 2002; Clin Colorectal Cancer 1 :220-9) or the thymidylate synthases of the ThyX family (Myllykallio et al., 2002, Science 297:105-7), the human cytosolic thymidine kinase Tk1 (Arcot and Deininger, 1992, Gene 111 :249-54; Wang et al., 1998, Antimicrob Agents and Chemother. 42:2620-25) and the human mitochondrial thymidine kinase Tk2 (Wang et al., 2000, Biochem Pharmacol 59:1583-8), as well as the HSV TK (Black et al., 1996, Proc Natl Acad Sci USA 93:3525-9; Guettari et al., 1997, Virology 235:398-405; Cazaux et al., 1998, Cancer Gene Ther 5:83-91; Christians et: al., 1999, Nat Biotechnol 17:259-64).

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. coli strains with partial or complete deletions of the relevant genes exist, of which growth or nucleoside/nucleotide activation is dependent on externally provided protein activity (Cazaux et al., 1998, Cancer Gene Ther 5:83-91; Landis and Loeb, 1998, J Biol Chem 273:25809-17; Wang et al., 1998, Antimicrob Agents and Chemother 42:2620-25; Wang et al., 2000, Biochem Pharmacol 59:1583-8; Myllykallio et al., 2002, Science 297:105- 7). The use of more complex organisms for human drug screening is often problematic. A system using a yeast strain with mutations in the 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).

It is known that in human cell, nucleoside analogues of thymidine (pyrimidines) such as AZT, d4T and FUdR are first activated by the human proteins Tkϊ 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). However, the human dTMP kinase dTYMK does not efficiently phosphorylate the monophosphate form of AZT to the diphosphate. This has severe consequences in AIDS therapies (St. Clair et al., 1991 , Science 253:1557-9; Lavie et al., 1997, Nat Med 3:922-4). In contrast, the enzymes of E. coli do phosphorylate AZT to the triphosphate form, integrate it into DNA, and thus inhibit DNA replication (Elwell et al., 1987 Antimicrob Agents Chemother 31:274-80; Shepherd et al., 1992, J Pharm Pharmacol 44:704-6) (Figure 4a-e, Figure 7).

Up to now, the human dTYMK could never be functionally expressed in bacteria such as E. coli (Huang et al., 1994, DNA Cell Biol 13:461-471), which are deficient in their host dTMP kinase activity under all conditions used (Daws and Fuchs, 1984, J Bacteriol 157:440-4). Therefore, 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.

This is the first time that key genes of the human thymidine phosphorylation pathway do completely replace their bacterial homologues for the phosphorylation of thymidine in a recombinant host cell line.

In addition, 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. SUMMARY OF THE INVENTION

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. This is the first time that the key genes of the human thymidine phosphorylation pathway can functionally replace their bacterial homologues for growth in E. coli. The presented invention allows a screening system of the enzymes of the human thymidine phosphorylation pathway combining the most important human kinases in a bacterial background deficient in the bacterial homologues.

One advantage of the present invention is that, contrary to the existing human cell line, bacteria like E. coli are very fast growing organisms, simple to handle, low in cost and with reliable results. In addition, 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.

Further on, 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. BRIEF DESCRIPTION OF THE FIGURES

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.

All these 4 vectors are isopropylthiogalactoside (IPTG) inducible high copy vectors.

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 B178Δfm f strain growing in the presence of an IPTG-inducible αTY -expressing plasmid.

Figure 4b shows that the growth of a B"\78Δtmk strain depends on the expression of the human gene dTYM B" 78Δtmk shows reduced growth in the absence of IPTG when complemented with the human dTYMK.

Figure 4c shows that B178ιιrt 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'\78Δtmk strains with different plasmids in the presence of 500 nM of AZT.

Figure 4e shows the absence of growth of B178wtf and B178Δtmk strains in the presence of 2000 nM of AZT.

Figure 5 describes a schema of the Δtdk deletion strain.

Figure 6 describes a schema of the ΔthyA deletion strain. (Bell-Pedersen et al., 1991 , J Bacteriol 160:371-8)

Figure 7 shows £. coli wt, Δtmk, Δtdk or ΔtmkΔtdk 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. DETAILED DESCRIPTION OF THE INVENTION 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.

These deletions, in the case the recombinant host cell line is £. coli, are designated Δtdk, Δtmk or ΔthyA, 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. In the preferred embodiment of the present invention, 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.

In a preferred embodiment, deletions (Δtdk, Δtmk or ΔthyA) in £. coli have been functionally complemented by both the human dTMP kinase gene dTYMK and the human cytoplasmic thymidine kinase gene tk1.

Also other human genes able to complement the said £. coli Δtmk, Δtdk and/or ΔthyA deletions can be used. In addition to human cytosolic tk1 and dTYMK, the human mitochondrial thymidine kinase tk2 (Wang et al., 2000, Biochem Pharmacol 59:1583-8) as well as the human gene TS (Landis and Loeb, 1998, J Biol Chem 273:25809-17), which is coding for the thymidylate synthase activity can be used alone or in combination with the dTYMK gene and/or the tk1 gene.

In another embodiment, 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.

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. Optionally, 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.

In yet another embodiment, this step e) can replace step d).

Examples of 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 cdcδ.

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 τκ1 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.

Additionally, 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. As an example, a gene library coding for θf 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 Δtmk background.

In a preferred embodiment, 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).

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).

In a preferred embodiment, 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.

Alternatively, 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.

Alternatively, 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.

Yet another concern of the present invention is to provide a kit 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. Alternatively, 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 genome in the presence of a plasmid encoding the E. coli tmk gene (Daws and Fuchs, 1984 J Bacteriol 157:440-4; Reynes et al., 1996, J Bacteriol 178:2804-12) (Figure 1). Further on, applicant functionally replaced the £. coli gene by the bacteriophage T4 gene 1, by the yeast gene cdc8 (Jong and Campbell, 1984, J Biol Chem 259:14394-8) as well as by the human gene dTYMK (Su and Sclafani, 1991 , Nucleic Acids Res 25:823-7; Huang et al., 1994, DNA Cell Biol 13:461-471), which are all homologues of the £. coli thymidylate kinase.

This shows that it is possible to replace the £. coli TMK by further viral, fungal or mammalian thymidylate kinases.

In a second step, the gene thyA (Belfort and Pedersen-Lane, 1984, J Bacteriol 160:371-8), (Figure 6) and tdk (Hiraga et al., 1967, Biochim Biophys Acta 145:41-51; Summers and Raksin, 1993, J Bacteriol 175:6049-51), (Figure 5) of £. coli have been removed by almost complete deletions. 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 £. coli, have been complemented by a human gene, the human cytoplasmic thymidine kinase tk1 (Wang et al., 1998, Antimicrob Agents and Chemother 42:2620-25), this gene product phosphorylates thymidine in the human salvage pathway (Arner and Eriksson, 1995, Pharmacol Ther 67:155-66) (Figure 2).

The system presented here has been tested for its efficiency by demonstrating the known differential behaviour of £. coli and human thymidylate kinase in the presence of AZT, 3'-azido-3'-deoxythymidine (Elwell et al., 1987, Antimicrob Agents Chemother 31 :274-80; Balzarini et al., 1989, J Biol Chem 264:6127-33; Lavie et al., 1997, Nat Med 3:922-4).

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. 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). As an example, 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 Δtmk 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).

According to a preferred best mode of the invention, the triple deleted ΔtdkΔtmkΔthyA E.coli strain, functionally complemented by the human genes dTYMK and tk1, is used for carrying out the invention.

According to another best mode of the invention, the triple deleted ΔtdkΔtmkΔthyA E.coli strain, functionally complemented by the human genes dTYMK , tk1 and tk2 , is used for carrying out the invention.

According to a best mode of the invention, the triple deleted ΔtdkΔtmkΔthyA E.coli strain, functionally complemented by the human genes dTYMK tk1 and TS, is used for carrying out the invention. Further on, 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.

The foregoing description will be more fully understood with reference to the following examples. Such examples are, however, exemplary of methods of practising the present invention and are not intended to limit the scope of the invention.

EXAMPLES

Amplification

Amplification of all genomic DNA by the polymerase chain reaction (PCR) has been performed on the £. coli strain B178, a galE derivative of W3110 (Georgopoulos et al., 1972, Nature New Biol. 239:38-41) and with the following primers: P1 5'->3' upstream of £. coli tmk S'-gtagtggcgggcgagg-S' P2 3'->5' (N-term) of £. coli tmk

5'-atctgcatgcttccagcccctcaatg-3' P3 5'->3' (C-term) of £. coli tmk 5'-atctgcatgctgggtgaaggagttgg-3'

P4 3'->5' downstream of £. coli tmk

5'-gccagaacgtcatccacgtc-3' P5 5'->3' (N-term) of kkal (KanR) of pACYCI 77 5'-atctgcatgctaagttatgagccatattcaac-3' P6 3^*->5' (C-term) of kkal (KanR) of pACYCI 77

5'-atctgcatgccatttagaaaaactcatcgagca-3' P7 5'->3' (N-term) of E. coli tmk 5'-ggaggaattcaccatgcgcagtaagtatatcgt-3'

P8 3'->5' (C-term) of £. coli tmk

5'-acgcgcatgctcatgcgtccaactccttc-3' P9 5' upstream of £. coli tdk

5'-ggagctcgagccttaaattcagttgtg-3' P 10 3'->5' (N-term) of £. coli tdk

5'-actaggatccatatgaaataaagttggctgg-3' P11 5'->3' (C-term) of £. coli tdk

5'-actaggatccagggtgagcagggcac-3' P12 3' downstream of £. coli tdk 5'-ggagtctagacagaagggttgtaaccgc-3

P13 5'->3' (N-term) of human dTMP kinase dTYMK

5'-ggaggcatgctaaggagaattcatatggcggcccggcgc-3' P14 3'->5' (C-term) of human dTMP kinase dTYMK

5'-actatctagaggccggcctcacttccatagctcccc-3' P15 5'->3' (N-term) of human thymidine kinase tk1

5'-ggagtctagagaggaattcatatgagctgcattaacctgc-3' P16 3'->5' (C-term) of human thymidine kinase tk1

5'-actaagatctcagttggcagggctgca -3' The PCR amplification products or plasmids were digested with restriction enzymes indicated in parentheses. The constructed strains have been confirmed by the standard laboratory techniques of PCR and Southern (Sambrook and Russell, 2001 , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA). Tet^R marker has been introduced near to the deletions for the constructions of certain multiple deletion strains. Combinations of the following £. coli deletions have been constructed by bacteriophage P1 transductions as described in Sambrook and Russell (2001 , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA). For expression of human genes, cDNA constructs have been used. Tmk deletion strain

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 Λ-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,

Sphl), and cloned into the vector pMPM-AΩ6 (EcoRI, Sphl) (Mayer, 1995, Gene 163:41-6), using this plasmid or similar constructs as a second copy during the construction of the genomic £. coli Δtmk deletion and kkal (KanR) substitution strain (Figure 1 ).

Tdk deletion strain

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 pHP45ΩCm (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 thyA deletion strain carrying a kanamycin-resistance marker was a gift from M. Belfort (Belfort et al., 1983, Proc Natl Acad Sci USA 80:1858-61 ; Bell-Pedersen et al., 1991 , J Bacteriol 173:1193-200) (Figure 6).

Tmk, Tdk and /or ThyA deletion strain

Construction of multiple deletions strains (ΔtdkΔtmkΔthyA or ΔtmkΔthyA or ΔtdkΔtmk) has been achieved according to standard techniques known from the person skilled in the art.

Human dTYMK kinase expression vector (Figure 3b)

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.

Human tk1 kinase expression vector (Figure 3c)

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).

Human dTYMK and tk1 kinase expression vector (Figure 3d)

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 μg/ml), tetracycline (10 μg/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.

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT rule 13bis)

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.

INDUSTRIAL APPLICABILITY

As described above, 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.

Furthermore, a kit integrating the recombinant host cell line will be highly useful in the pharmaceutical industry as well as in the research laboratories.

Claims

1. A recombinant host cell line characterized in that its 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.

2. A recombinant host cell line characterized in that its thymidylate kinase gene, thymidine kinase gene and thymidylate synthase gene have been functionally complemented by at least one functional homologue of another organism.

3. The recombinant host cell line of claims 1 or 2, characterized in that said host cell is a fungal cell.

4. The recombinant host cell line of claim 3, characterized in that said host cell is a yeast cell.

5. The recombinant host cell line of claims 1 or 2, characterized in that said host cell is a eukaryotic cell.

6. The recombinant host cell line of claim 5, characterized in that said host cell is a mammalian cell.

7. The recombinant host cell line of claims 1 or 2, characterized in that said host cell is a bacterium.

8. The recombinant host cell line of claim 7, characterized in that said host cell is a Gram (-) bacterium.

9. The recombinant host cell line of claim 8, characterized in that said host cell is Escherichia coli.

10. The recombinant host cell line of claim 9, characterized in that the thymidylate kinase is the tmk gene, the thymidine kinase gene is the tdk gene and the thymidylate synthase gene is the thyA gene.

11. The recombinant host cell line of any of claims 1 to 10, characterized in that the functional homologue is a procarytic or a eukarytic gene.

12. The recombinant host cell line of claim 10, characterized in that the functional homologue is a fungal gene, in particular a yeast gene.

13. The recombinant host cell line of any of claims 1 to 10, characterized in that the functional homologue is a viral gene.

14. The recombinant host cell line of any of claims 1 to 10, characterized in that the functional homologue is a bacteriophage gene.

15. The recombinant host cell line of claim 14, characterized in that the bacteriophage gene is the T4 gene 1.

16. The recombinant host cell line of any of claims 1 to 10, characterized in that the functional homologue is a human gene.

17. The recombinant host cell line of claim 16, characterized in that the functional homologue is the human dTMP kinase gene dTYMK.

18. The recombinant host cell line of claim 16, characterized in that the functional homologue is the human cytoplasmic or mitochondrial thymidine kinase gene tk1 or tk2.

19. The recombinant host cell line of claim 16, characterized in that the functional homologue is the human thymidylate synthase TS gene.

20. The recombinant host cell line of any of claims 17 to 19, characterized in that the functional homologues are used as a combination of the human dTMP kinase dTYMK gene, the human thymidylate kinase TS gene and/or the cytoplasmic or mitochondrial thymidine kinase tk1 or tk2 genes.

21. 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.

22. The method of claim 21 characterized in that it further comprises step: e) detecting chemical modifications of candidate compounds using recombinant host cell line or using extracts of the recombinant host cell line.

23. 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 others 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 chemical modifications of candidate compounds using recombinant host cell line or using extracts of the recombinant host cell line.

24. The method according to claims 21 to 23, characterized in that the genome depletion for the tdk, tmk or thyA genes is achieved by a complete deletion of these genes.

25. Use of the method according to claims 21 to 24 for the screening of nucleoside analogues for their activation by said human kinases involved in the dTTP phosphorylation.

26. Use of the method according to claims 21 to 24 for testing chemical variants of nucleoside analogues for improved phosphorylation of the nucleoside analogues.

27. Use of the method according to claims 21 to 24 for testing mutations in the human dTYMK or tk1 genes.

28. Use of the method according to claims 21 to 24 to simplify the screening of chemical libraries of nucleoside analogues for their activation by the human dTYMK or tk1 genes or for inhibition of the complementing nucleoside or nucleotide kinases in £ coli.

29. A kit for the screening of compounds characterized in that it comprises: a) the recombinant host cell line according to any of claims 1 to 20, b) a substrate enabling the growth of said recombinant host cell line, c) and means for measuring cell densities or cell culture growth.

30. A plasmid characterized in that it expresses the human dTYMK and tk1 kinase genes wherein the distance between said two genes has been established for regulating their desired over-production.

31. A process for making the plasmid of claim 30, characterized in that it comprises the steps of:

-cloning the PCR product of primers P15 and P16, -cloning the PCR product of primers P13 and P14, -cloning the PCR product of primers of P14 and P15, or variants thereof.

32. Use of a recombinant host cell line of any of claims 1 to 20, for activity testing, characterisation and purification of any enzyme activity able to complement the absence of the corresponding activity of the recombinant host cell line.

33. Use according to claim 32, characterized in that said enzyme activity is a thymidylate kinase with altered N-terminal amino acid sequence in its first 30 amino acids.

34. Use according to claim 33, characterized in that said thymidylate kinase is the human dTMP kinase dTYMK.

35. Use of a recombinant host cell line of any of claims 1 to 20, in combination with a target of an activated compound.

36. Use according to claim 35, characterized in that the target is a Reverse Transcriptase.

37. Use according to claim 36, characterized in that the target is a HIV Reverse Transcriptase.

38. Use according to claim 35, characterized in that the target is a DNA polymerase.

39. Use according to claim 38, characterized in that the target is a bacterial DNA polymerase.

40. Use according to claim 38, characterized in that the target is a mammalian DNA polymerase.

41. Use according to claim 40, characterized in that the target is a human DNA polymerase.