WO1995022622A1 - Procedes et compositions de criblage de medicaments anti-sida - Google Patents

Procedes et compositions de criblage de medicaments anti-sida Download PDF

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WO1995022622A1
WO1995022622A1 PCT/US1995/002074 US9502074W WO9522622A1 WO 1995022622 A1 WO1995022622 A1 WO 1995022622A1 US 9502074 W US9502074 W US 9502074W WO 9522622 A1 WO9522622 A1 WO 9522622A1
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hiv
cells
vector
growth
gene
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PCT/US1995/002074
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English (en)
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Lawrence A. Loeb
Baek Kim
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University Of Washington
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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.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1058—Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase

Definitions

  • the present invention is generally directed toward assays and compositions for identifying compounds for AIDS therapy. This invention is more particularly related to screening candidate compounds for the ability to inhibit reverse transcriptase of human immunodeficiency virus.
  • AIDS Acquired Immune Deficiency Syndrome
  • AIDS is caused by an infectious agent that is transmitted by exposure to blood or blood products.
  • Groups reported to be at greatest risk of contacting AIDS include homosexual or bisexual males and intravenous drug users. Hemophiliacs who receive blood products pooled from donors and recipients of multiple blood transfusions are also at risk.
  • AIDS is a disease that damages the body's immune system, leaving victims susceptible to opportunistic infections, malignancies or other pathological conditions against which a normal immune system would have protected the subject. After patients develop symptoms of AIDS, death generally occurs within 2-3 years of diagnosis. Clinical manifestations of the disease in its final stage include a collapse of a patient's immune defenses (which generally involves a depletion of helper T cells) accompanied by the appearance of a Kaposi sarcoma and/or various opportunistic infections. The pronounced depression of cellular immunity that occurs in patients with AIDS and the quantitative modifications of subpopulations of their T lymphocytes suggests that T cells or a subset of T cells are a central target for the infectious agent.
  • HIV human immunodeficiency virus
  • body fluids such as semen, vaginal fluids or blood
  • AIDS is characterized by a disorder associated with an impaired cell-mediated immunity and lymphopenia, in particular, depletion of those T cells that express the T4 (CD4) glycoprotein. This is due to the fact that HIV preferentially infects the CD4 lymphocyte population (CD4 cells).
  • HIV contains two heavily glycosylated external envelope proteins, gpl20 and gp41, which mediate attachment of virions to glycosylated cell surface receptor molecules. These glycoproteins are encoded by the env gene and translated as a precursor, gpl ⁇ O, which is subsequently cleaved into gpl20 and gp41. Gpl20 binds to the CD4 protein present on the surface of helper T lymphocytes, macrophages, and other cells, thus determining the tissue selectivity of viral infection.
  • the CD4 protein is a glycoprotein of approximately 60,000 molecular weight and is expressed on the cell membrane of mature, thymus- derived (T) lymphocytes, and to a lesser extent on cells of the monocyte/macrophage lineage.
  • CD4 cells appear normally to function by providing an activating signal to B cells, by inducing T lymphocytes bearing the reciprocal CD8 marker to become cytotoxic/supressor cells, and/or by interacting with targets bearing major histocompatibility complex (MHC) class II molecules.
  • MHC major histocompatibility complex
  • the CD4 glycoprotein in addition to playing an important role in mediating cellular immunity also serves as the receptor for HIV.
  • a variety of proposed therapeutic approaches have been based upon an attempt to disrupt the interaction of HIV gpl20 with T cell CD4.
  • HIV RT reverse transcriptase
  • integrase integrase
  • host cell-encoded DNA polymerases and RNA polymerase a virus-encoded enzymes
  • HIV RT polymerizes deoxyribonucleotides by using viral RNA as a template and also acts as a DNA polymerase by using the newly synthesized minus strand DNA as a template to produce a double-stranded DNA. Because of the essential role of HIV RT in the invasion of a host organism by the virus, therapeutic approaches have been based upon an attempt to inhibit HIV RT.
  • the most useful drugs for the treatment of AIDS such as azidothymidine (“AZT”)
  • AZT azidothymidine
  • nucleoside analogs directed against HIV RT are nucleoside analogs directed against HIV RT.
  • these inhibitors of HIV RT have had limited success because of the extensive genetic variation and high mutation rate of HIV. Therefore, by rapid evolution of HIV, mutations in HIV RT arise frequently in infected individuals and render the virus resistant to nucleoside analogs and other antiviral therapies.
  • the present invention provides a variety of methods and compositions related to screening compounds for the ability to inhibit reverse transcriptase of human immunodeficiency virus and to screening for active reverse transcriptase mutants.
  • the present invention provides methods of screening for compounds that inhibit reverse transcriptase of human immunodeficiency virus (HIV RT), comprising the steps of: (a) introducing a vector that expresses the gene encoding an HIV RT into a bacterial cell or eucaryotic cell in culture, the host cell containing a conditional mutant in the host cell gene encoding a DNA polymerase; (b) incubating the host cell harboring the vector under conditions that are limiting for the growth of a host cell in the absence of active HIV RT, the conditions further including a candidate compound which may be capable of inhibiting the HIV RT; and (c) detecting the presence or absence of growth, thereby determining whether the candidate compound inhibited the HIV RT.
  • HIV RT human immunodeficiency virus
  • the method comprises the steps of: (a) introducing a vector that expresses the gene encoding an HIV RT into temperature sensitive E. coli polA ⁇ 2recAl ⁇ cells, the host cells containing a conditional mutant in the host cell gene encoding a DNA polymerase such that growth of the host cells at a non-permissive temperature is dependent on expression of active HIV RT; (b) plating dilutions of the host cells harboring the vector onto a plate containing media sufficient for growth and containing a candidate compound which may be capable of inhibiting the HIV RT; (c) incubating the host cells harboring the vector at a temperature not permissive for growth of E.
  • the present invention provides in another aspect E. coli pol Urec m cells that harbor an HIV RT plasmid (pHIV RT).
  • the present invention provides methods of screening for active HIV RT mutants, comprising the steps of: (a) introducing a vector that expresses the gene encoding an HIV RT mutant into a bacterial cell or eucaryotic cell in culture, the host cell containing a conditional mutant in the host cell gene encoding a DNA polymerase; (b) incubating the host cell harboring the vector under conditions that are limiting for the growth of a host cell in the absence of active HIV RT; and (c) detecting the presence or absence of growth, thereby determining whether the HIV RT mutant is active.
  • the method comprises the steps of:
  • coli polAYlrecAl ⁇ W cells that lack a DNA-dependent DNA polymerase activity at the non-permissive temperature; and (d) detecting the presence or absence of growth at low cell density, thereby determining whether the HIV RT mutant is active.
  • the present invention provides methods of screening for compounds that inhibit HIV RT obtained from a patient, comprising the steps of: (a) amplifying the gene for HIV RT from a sample from an individual infected with HIV; (b) introducing the HIV RT gene into a vector that expresses the HIV RT gene; (c) introducing the vector into a bacterial cell or eucaryotic cell in culture, the host cell containing a conditional mutant in the host cell gene encoding a DNA polymerase; (d) incubating the host cell harboring the vector under conditions that are limiting for the growth of a host cell in the absence of active HIV RT, the conditions further including a candidate compound which may be capable of inhibiting the HIV RT; and (e) detecting the presence or absence of growth, thereby determining whether the candidate compound inhibited the HIV RT from the individual.
  • the method comprises the steps of: (a) amplifying the gene for HIV RT from a sample from an individual infected with HIV; (b) introducing the HIV RT gene into a vector that expresses the HIV RT gene; (c) introducing the vector into temperature sensitive E.
  • coli polA ⁇ 2recAl ⁇ cells the host cells containing a conditional mutant in the host cell gene encoding a DNA polymerase such that growth of the host cells at a non-permissive temperature is dependent on expression of active HIV RT; (d) plating dilutions of the host cells harboring the vector onto a plate containing media sufficient for growth and containing a candidate compound which may be capable of inhibiting the HIV RT; (e) incubating the host cells harboring the vector at a temperature not permissive for growth of E.
  • a method for testing the biological effectiveness of candidate compounds for the inhibition of human immunodeficiency virus reverse transcriptase (HIV RT) in vivo comprises: (a) introducing cells containing a vector that expresses the gene encoding an HIV RT into a test animal, the cells containing a conditional mutant in the cell gene encoding a DNA polymerase such that survival of the cells in the animal is dependent on expression of active HIV RT; (b) administering to the animal a compound in a pharmaceutically acceptable form, the compound a candidate for the inhibition of HIV RT; and (c) assessing the ability of the candidate compound to clear the cells from the animal, thereby determining whether the candidate compound inhibits HIV RT in vivo.
  • HIV RT human immunodeficiency virus reverse transcriptase
  • a method for detecting in a blood sample from a warm-blooded animal the presence of an active compound that inhibits human immunodeficiency virus reverse transcriptase comprises: (a) administering to a warm-blooded animal a compound that inhibits HIV RT, the compound in a pharmaceutically acceptable form; (b) isolating a blood sample from the animal; and (c) detecting the presence or absence of the compound by testing the blood sample in the method according to claim 1 wherein the blood sample replaces the candidate compound of the method, thereby determining whether the compound is present in active form in the blood of the animal.
  • Figure 1 demonstrates the functional complementation of E. coli DNA polymerase I by HIV reverse transcriptase.
  • A Growth of the wild-type E. coli polA + recA + with the parent plasmid, pHSG576, at 30°C and 37°C.
  • B Growth of/?o/A12recA718 with pHSG576 at 30°C and 37°C.
  • C Growth of polA ⁇ 2recAl ⁇ with pHIV RT at 30°C and 37°C.
  • D Growth of /?o/A12recA718 with pHIV RT-DN at 30°C and 37°C.
  • coli polA + recA + (JS295) and /?o/A12recA718 (JS200) were transformed with pHSG576, a low copy number plasmid containing a Pol I independent pSClOl replication origin and a chloramphenicol resistance gene (Takeshida et al., Gene 77:63-74, 1987).
  • the /?o/A12recA718 strain (tet R ) was transformed with pHIV RT-2 and pHIV RT-DN. pHIV RT-DN expresses D186N HIV RT mutant protein that is not functional (Larder et al., Proc. Natl. Acad. Sci. USA ⁇ St5:4803-4807, 1989).
  • Transformed cells were grown to log phase in nutrient broth containing tetracycline (7.5 mg/ml), chloramphenicol (34 mg/ml) and IPTG (1 mM) and then 2 x 10 6 cells were deposited and diluted by rotation with a 10 ⁇ l inoculation loop on a nutrient agar plate containing the same concentrations of tetracycline, chloramphenicol and IPTG. Duplicate plates were incubated at 30°C and 37°C for 48 hrs.
  • Figure 2 graphically illustrates the efficiency of substitution of
  • E. coli polA ⁇ 2recAl ⁇ containing either pHSG576 or pHIV-RT was grown to 2 x 10 8 cells per ml at 30°C.
  • the indicated number of cells per plate in progressive dilutions was plated on nutrient agar containing tetracycline, chloramphenicol and IPTG as described in Figure 1.
  • Duplicate plates were incubated for 48 hrs. at 30°C (black bar) or 37°C (open bar) and colonies were scored. The results given are averages from three different experiments.
  • Figure 3 shows the inhibition of HIV RT complementation by AZT.
  • A polA + recA + with pHSG576, grown on nutrient agar with 100 nM AZT (AZT-NA) at 37°C.
  • B polA ⁇ 2recA7 ⁇ S expressing HIV RT on nutrient agar without AZT at 37°C.
  • C /?o/A12recA718 expressing HIV RT on AZT- NA at 37°C.
  • D /?o/A12recA718 expressing HIV RT on AZT-NA at 30°C.
  • E /?o/A12recA718 expressing rat DNA polymerase ⁇ on AZT-NA at 37°C.
  • E Approximately 200 E.
  • coli cells harboring the designated plasmids were plated in duplicate on NA plates containing tetracycline, chloramphenicol and IPTG (as described in Figure 1) with or without AZT (85 nM) and were incubated at 30°C or 37°C for 38 hours.
  • the relative magnification of the colonies is 25X.
  • Figure 4 illustrates inhibition of complementation of HIV reverse transcriptase by AZT.
  • A /? ⁇ /A12recA718 expressing rat DNA polymerase ⁇ .
  • B /?o/A12rec718 expressing HIV RT from pHIV RT-2.
  • C /?o/A12recA718 expressing T215Y HIV RT mutant protein that shows resistance to AZT (St. Clair et al., Science 253:1557-1559, 1991).
  • coli cells expressing the designated proteins were plated in duplicate on nutrient agar containing tetracycline, chloramphenicol and IPTG (as described in Figure 1) and AZT (0, 50, 150, 200 and 250 nM) and were incubated at 30°C (black bar) or 37°C (open bar) for 48 hr.
  • the percent survival is the ratio of colonies formed in the presence and absence of AZT.
  • T215Y HIV RT mutant from pHIVRT-TY is able to complement the growth defect of Pol I ts mutant at non-permissive temperature to the same extent as the wild-type HIV RT.
  • Figure 5 illustrates induction of ddC and AZT sensitivity of E.
  • coli by co-expression of HSV TK and HIV RT.
  • Approximately 250 Pol I ts cells harboring either pHIV RT-2 or pRT-TK were plated to NA plates containing different concentrations of ddC or AZT. Cells plated were incubated at 30°C or 37°C for 48 hrs. Survival of cells was determined by ratios of survived cells from NA plates with or without drugs.
  • B ddC effect on growth of Pol I ts cells containing pRT-TK at 30°C (closed circle) or 37°C (open circle).
  • HIV RT human immunodeficiency virus
  • the present invention is directed toward methods and compositions useful for screening compounds for the ability to inhibit HIV RT, for screening for active HIV RT mutants, for screening a patient's HIV RT to tailor the individual's therapeutic regime, and for assessing the biological effectiveness of anti-HIV RT compounds.
  • a wide variety of cells may be used as the host cell in the methods of the present invention, and include bacterial cells and eucaryotic cells.
  • suitable host cells include bacteria such as E. coli, Salmonella ⁇ e.g., t phimuriur ), Bacillus (e.g., subtilis), Thermus (e.g., aquaticus), yeast such as Saccharomyces (e.g., cerevisiae), and mammalian cells such as hamster cells, human cells, and rat cells, which have been engineered to contain a conditional mutant in the gene encoding a DNA polymerase.
  • bacteria such as E. coli, Salmonella ⁇ e.g., t phimuriur ), Bacillus (e.g., subtilis), Thermus (e.g., aquaticus), yeast such as Saccharomyces (e.g., cerevisiae), and mammalian cells such as hamster cells, human cells, and rat cells, which have been engineered to
  • cells are exposed to mutagenic agents, screened for the mutants defective in DNA synthesis at non-permissive conditions and then analyzed for mutations in DNA polymerases (e.g., Liu et al., Proc. Natl. Acad. Set USA 5(9:797-801, 1983).
  • a DNA polymerase gene may be cloned, the gene altered to produce mutations (e.g., using site-specific mutagenesis) and then transferred to wild type cells for gene displacement or disruption by transposons (e.g., Sweasy et al., Proc. Natl. Acad. Sci. USA 90:4626-4630, 1993).
  • a preferred host cell containing a conditional mutant in the gene encoding a DNA polymerase is E. coli polAYlrecAlXW.
  • This mutant contains a temperature sensitive (ts) DNA polymerase I (Pol I ts ) mutation (Witkin et al., J. Bacteriol. 774:4166-4168, 1992). It is unable to grow at 42°C in rich media at low density due to a failure to join Okazaki fragments during lagging strand DNA synthesis (Sweasy et al., J. Biol. Chem. 267:1407-1409, 1992).
  • HIV RT gene As disclosed within the present invention, introduction of a HIV RT gene into a host cell containing such a conditional mutant ("host cell") is able to genetically complement the host cell such that HIV RT substitutes for the deficient DNA polymerase in promoting cell growth and in plasmid replication.
  • HIV RT gene includes an intact gene as well as portions thereof which encode a polypeptide capable of exhibiting DNA-dependent DNA polymerase activity. For example, Skalka et al. (Reverse Transcriptase, Cold Spring Harbor Laboratory Press, pp. 144-150, 1993) describe deletions of portions of HIV RT without loss of polymerase activity.
  • an HIV RT gene may be prepared from a patient's blood sample (Saiki et al., Science 230: 1350-1354, 1985), from recombinant HIV RT (Kim et al., Proc. Natl. Acad. Sci. USA 92:684-688, 1995), or by homologous recombination of virus (Kellam et al., Proc. Natl. Acad. Sci. USA 89: 1934- 1938, 1992).
  • An HIV RT gene is generally introduced into a host cell via a vector.
  • An HIV RT gene may be introduced into a vector by a variety of means well known to those in the art (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989). For example, a vector may be cut with restriction enzyme(s) and the HIV RT gene ligated into the vector. After a vector is prepared that expresses a gene encoding an HIV RT, the vector is introduced into a host cell by a variety of means well known to those in the art (Sambrook et al., ibid.). In a preferred embodiment, an HIV RT gene is introduced into E.
  • (pHIV RT) HIV RT expressing plasmid
  • Particularly preferred r>o/A12recA718 strains are those harboring a (pHIV RT) plasmid designated as ⁇ HIV RT-1, pHIV RT-2, and pHIV RT-3, having American Type Culture Collection (ATCC) accession numbers of ATCC 69568, ATCC 69569, and ATCC 69570, respectively.
  • ATCC American Type Culture Collection
  • a host cell may contain one or more additional genes encoding an enzyme (such as a kinase) able to phosphorylate nucleosides and/or nucleotides in order to increase the likelihood of finding, through the use of the methods of the present invention, a compound that inhibits HIV RT.
  • an enzyme such as a kinase
  • human or viral genes encoding enzymes with such capabilities may be introduced into a host cell by means such as those described above.
  • a gene(s) encoding an enzyme(s) for phosphorylation is conveniently introduced into a host cell via a single vector that expresses both the gene(s) and an HIV RT gene.
  • a kinase gene may be inserted into the genome of a host cell.
  • a gene need not be intact but may be a portion thereof (e.g., truncated version of intact molecule), provided that the polypeptide expressed is capable of phosphorylation activity.
  • a portion of the intact thymidine kinase gene e.g., as described in Munir et al., Proc. Natl. Acad. Sci. USA 90:4012-4016, 1993 may be used.
  • host cells harboring a vector that expresses the gene encoding an HIV RT are incubated under conditions that are limiting for the growth of a host cell in the absence of active HIV RT.
  • a variety of limiting conditions may be utilized within the methods of the present invention.
  • the conditions may be limiting for growth in the absence of replication of the plasmid where such replication requires a DNA polymerase for replication.
  • host cells are susceptible to an inhibitor (such as an antibiotic) in the absence of replication of the plasmid, which bears a gene conferring upon the host cells resistance to the inhibitor.
  • a gene, such as a chloramphenicol resistance gene, which confers resistance may be introduced into a vector (and then into host cells via the vector) by means such as those described above.
  • Active HIV RT which has been introduced into the host cells via a vector, provides the DNA polymerase activity required for replication of the plasmid bearing the gene which confers resistance to an inhibitor of host cell growth.
  • host cells possessing active HIV RT will be able to grow in the presence of an inhibitor of host cell growth (by replication of a plasmid bearing a gene which confers resistance to the inhibitor).
  • a condition that is limiting for the growth of a host cell is a temperature which is not permissive for growth of cells that lack a DNA-dependent DNA polymerase activity.
  • growth of host cells at a non-permissive temperature is dependent on expression of active HIV RT.
  • host cells may grow at a lower temperature (such as 30° C) even in the absence of a DNA-dependent DNA polymerase activity.
  • a higher temperature e.g., 42°C
  • the host cells either do not grow or may require high cell density to grow.
  • Whether the lack of growth of particular host cells at a non-permissive temperature is host cell density independent (i.e., no growth regardless of cell density) or density dependent (e.g., no growth provided cell density is low) may be readily ascertained by examining the host cells under both low and high cell density conditions. Alternatively, if one does not wish to determine whether lack of growth of host cells at a non-permissive temperature is density dependent or density independent, one may simply always grow the host cells at low cell density since even those host cells whose growth at a non-permissive temperature is density dependent do not grow at a low cell density. It will be evident to those of ordinary skill in the art that other means are suitable for limiting the growth of a host cell in the methods of the present invention.
  • a compound which is a candidate for the inhibition of HIV RT (hereinafter referred to as "candidate compound") is tested in the methods of the present invention using a host cell described above.
  • a candidate compound is included in the incubation conditions which are limiting for the growth of a host cell in the absence of active HIV RT. It may be desirable to test a variety of different (e.g., graded) amounts of a candidate compound. Alternatively, more than one candidate compound may be included. In addition, one or more candidate compounds may be tested in combination with one or more traditional anti-AIDS compounds, such as azidothymidine (“AZT"). Any compound is a candidate compound. Nucleosides and nucleotides, which herein include analogs of either, are examples of candidate compounds.
  • a candidate compound may be isolated from a natural source or prepared synthetically, including by recombinant, enzymatic and/or synthetic chemical means.
  • Candidate compounds may be designed (e.g., based on structure-function studies), generated randomly, or produced by a combination of these approaches.
  • inhibition of HIV RT by a candidate compound refers to reducing or eliminating the DNA synthesis catalyzed by HIV RT.
  • a candidate compound may inhibit HIV RT in a variety of ways, including by binding to HIV RT or by terminating DNA synthesis catalyzed by HIV RT. Whether a candidate compound inhibits the HIV RT expressed in host cells is readily ascertained by detecting the presence or absence of cell growth when the compound is present.
  • An absence of cell growth indicates that the host cells were limited by the inhibition of HIV RT by the candidate compound. It may be desirable to confirm that the lack of host cell growth is due to the inhibition of HIV RT, rather than an indirect affect on the host cell. Such confirmation may be readily accomplished by the testing of one or more appropriate controls. For example, one may show that the candidate compound does not inhibit the growth of a host cell that does not contain a conditional mutant in the host cell gene for which HIV RT provides functional complementation of the defect. A convenient method for ascertaining growth is to plate concentric dilutions of host cells in solid selection plates.
  • a variety of other means of plating or growing cells in liquid culture such as tritiated thymidine incorporation, PCR of sequences and FACS sorting, may be used as well as other methods known to those of ordinary skill in the art. Growth of cells may be assessed visually.
  • the methods of the present invention may be used to screen for active HIV RT mutants.
  • a gene encoding a HIV RT mutant may be an intact gene, or portion thereof, which contains one or more mutations. Ways to prepare such a gene include isolation or amplification from a biological sample, or recombinant methodology, or synthetic production (e.g., synthetic assembly or in vitro mutagenesis). Methodologies for the preparation of mutant genes are well known to those of ordinary skill in the art (e.g., Ausubel et al., Short Protocols in Molecular Biology, 2d ed., Greene Publishing Associates and John Wiley & Son, 1993; Munir et al., Proc. Natl. Acad. Sci.
  • Active HIV RT mutants are those mutants which possess DNA polymerase activity.
  • a HIV RT mutant that is active functionally complements the DNA polymerase defect of a host cell. The complementation permits growth of the host cell under conditions that are limiting for its growth in the absence of active HIV RT.
  • An example of an active HIV RT mutant is that contained on a plasmid designated as pHIV RT-4.
  • E. coli that express this HIV RT mutant are resistant to AZT.
  • pHIV RT-4 /?o/A12recA718 strain harboring (pHIV RT-4), ATCC accession number ATCC 69571, is resistant to AZT.
  • the genetic selection assays disclosed herein permit the collection of a large number of mutant HIV RTs. Extensive mutagenesis of HIV RT enables the development of structure-function relationships that are essential to understanding evolution of HIV RT and to designing compounds which avoid evasion by mutations in HIV RT.
  • the complementation system disclosed herein may be used to predict the likelihood that mutations would arise that render HIV RT resistant to specific compounds or combinations.
  • a library of active HIV RT mutants may be used in the methods of the present invention to screen for a compound or combination of compounds that inhibit all or most of the mutants.
  • a library of active HIV RT mutants may permit design of a compound or combination of compounds as inhibitors to the class of mutants.
  • a combination of compounds with different spectrums of resistant mutations may be used together (combination chemotherapy) to mitigate against the emergence of resistant strains.
  • the present invention provides methods of screening for compounds that inhibit HIV RT obtained from an individual infected with HIV. This permits a therapeutic regime to be tailored to an individual patient.
  • the methods disclosed herein may be used prior to commencing therapy with an anti-HIV RT compound to determine whether the compound is effective against the HIV RT residing in the particular patient.
  • the methods of the present invention may be used to evaluate HIV RT in a patient with AIDS for the presence of mutations that render the virus resistant to specific compounds used in the treatment of the individual patient, and to evaluate the therapeutic effectiveness of candidate compounds.
  • Sources of a sample containing a gene encoding HIV RT from an individual infected with HIV include biological fluids (e.g., blood) and tissue.
  • the gene for HIV RT may be amplified from a sample using a variety of methods well known to those in the art.
  • an HIV RT gene may be amplified by a polymerase chain reaction ("PCR" (see Mullis et al., U.S. Patent Nos. 4,683,195; 4,683,202 and 4,800,159)).
  • PCR polymerase chain reaction
  • a patient's HIV RT gene is introduced into a host cell via a vector as described above. Such a gene may be an intact gene or portion thereof.
  • Host cells harboring such a vector are incubated under limiting conditions, as described above, and in the presence of one or more candidate compounds, as also described above, which may be capable of inhibiting the patient's HIV RT.
  • the ability of a compound (or combination of compounds) to inhibit a patient's HIV RT is determined based on whether the presence or absence of growth of the host cells is detected.
  • the present invention provides methods for testing the biological effectiveness of candidate compounds for the inhibition of HIV RT in vivo.
  • Cells containing a vector that expresses a gene encoding an HIV RT and a conditional mutant in the cell gene encoding a DNA polymerase, are prepared as described above.
  • Such cells e.g., bacterial cells or yeast cells
  • the cells may be introduced by a variety of means including injection and ingestion. Examples of test animals include mice, rats, guinea pigs, rabbits, cats, dogs, swine and non-human primates such as monkeys.
  • conditional mutant in the cell gene encoding a DNA polymerase renders the survival of the cells in the test animal dependent on expression of active HIV RT.
  • One or more candidate compounds are administered to the test animal to determine whether the compound or combination of compounds inhibits HIV RT in vivo.
  • a compound may be administered by a variety of routes including injection, orally or transdermally.
  • a compound is generally formulated in a pharmaceutically acceptable form for administration, and it will be evident to those in the art that the form may depend on the physical-chemical properties of the compound and its route of adsorption into the bloodstream. Following the administration of a compound or combination of compounds to a test animal, the ability of the compound(s) to clear the introduced cells from the test animal is assessed.
  • Such assessment may be performed once after an appropriate time to permit clearance, or may be performed at two or more time intervals until the cells have been cleared or the maximum appropriate time for clearance has been reached. It will be evident to those of ordinary skill in the art that an assessment of cell clearance from a test animal may be performed in a variety of ways.
  • assays typically used for such purposes include removal from the test animal of a blood sample which is then tested for the presence of HIV RT-dependent cells by a variety of tests that include microbiological culture, colony formation on appropriate selective microbiological media, PCR analysis of DNA-specific gene sequences for the introduced cells, fluorescent-activated cell sorting (FACS) analysis using fluorescently-tagged monoclonal antibodies specific for the introduced cells, or reverse transcriptase activity.
  • FACS fluorescent-activated cell sorting
  • the ability of a compound(s) to clear the cells may be assessed based upon the presence or absence of the pathogenic condition. Further, a pathogenic condition may be fatal, so that survival of the test animal is dependent on clearance of the introduced cells. Therefore, under such circumstances, simply assessing the enhanced survival of the test animal is a direct assay of the effectiveness of a candidate compound in blocking test cell growth, i.e., in inhibiting the activity of HIV RT itself.
  • the test animal is a mouse and the cells introduced are derived from Salmonella typhimurium. Because S. typhimurium is fatal to mice, the survival of the test animal is dependent upon the clearance of the bacterial cells. Confirmation that survival of the test animal is dependent on the administration of a compound or combination of compounds is readily demonstrated by the inability of test animals, who are inoculated with bacterial cells, to survive in the absence of administration of the compound(s).
  • the introduced cells are used indirectly to assay the level of candidate compound(s) circulating in the blood of a test animal. This is done by removing blood samples from test animals at a single or multiple times following administration of the compound, or mixture of compounds, and then assessing the anti-HIV RT activity of the compounds present in the blood by their ability to inhibit cell growth in vitro, in an assay as described above.
  • This test may be used to assess directly the bioavailability of candidate compounds and their pharmacokinetic parameters.
  • subsequent metabolism of candidate compounds and their inactivation (or activation) is monitored speedily and safely without the need for exposing either test animals or laboratory and medical personnel to active virus.
  • Application of this particular embodiment to human clinical trials of an anti-HIV RT compound makes possible the direct determination of the bioavailability of the compound and its subsequent pharmacokinetics, both in healthy volunteers as well as HIV-positive individuals.
  • Escherichia coli strain E. coli
  • This E. coli double mutant polA ⁇ 2recAl 18 contains a temperature sensitive (ts) DNA polymerase I (Pol I ts ) mutation. It is unable to grow at 42°C in rich media at low cell density due to a failure of Pol I to join Okazaki fragments during lagging strand DNA synthesis.
  • wild-type E. coli cells MM300 are mutagenized by nitrosoguanidine (Monk and Kinross, J. Bad. 709:971-978, 1972).
  • Mutagenized cells are screened for sensitivity to MMS (methyl methane sulfonate) at 32 (or 30) and 42 (or 37)°C in replica plates.
  • the polAll mutant shows MMS sensitivity only at 42°C.
  • polA12 single mutant is combined with recA718 mutant SCI 8 for polA12 recA718 double mutant (Witkin and Roegner-Maniscalco, 1992).
  • pHSG576 is a low copy number plasmid that is replicated in DNA polymerase I (Pol I) independent manner (Takeshida et al., Gene 77:63-74, 1987). This plasmid contains lacP/O, a lacZ gene fragment that can be scored as a target for mutagenesis by alpha complementation, and also contains a segment with a multiple cloning site. pHSG576 is constructed by combining pSClOl replication origin, the multiple cloning site with lac? 10 from pUC8/9 for a-complementation and a chloramphenicol resistant gene.
  • Haell fragment of pSClOl replication origin of pHSG415r (Brady et al., Gene 27:223-232, 1984), Hhal (Alton and Vapnek, Nature 252:864-869, 1979)-HaeII Tn9-Cm R fragment in pHSG439 (Brady et al., 1984) and Haell lacZ' fragment of pUC8 or pUC9 (Vieira and Messing, Gene 79:259, 1982) are combined for pHSG576 construction.
  • pHIV RT plasmids are pHSG576 derivatives expressing different level of HIV RT.
  • the HIV RT gene fused to a ribosomal binding site (RBS) for expression, was obtained from S. Wilson. This fused HIV RT gene was cloned into pHSG576 between the Hind III and Eco RI restriction sites, generating (pHIV RT-1).
  • pHIV RT-2 is the same as pHIV RT- 1, except that it contains a 61 bp insert between lacP/O and HIV RT gene.
  • pHIV RT-3 is the same as pHIV RT-1, except that it contains a 431 bp insert encoding a 94 amino acid long amino terminal (N-T) of mouse DNA polymerase beta (pol beta) from pBL (Sweasy and Loeb, Proc. Natl. Acad. Sci. USA 90:4626- 4630, 1993) at Hind III site of pHIV RT-1.
  • pHIV RT-3 expresses HIV RT fused to N-terminal 94 amino acids of the DNA polymerase beta gene.
  • pHIV RT-1, -2 and -3 were prepared as follows: a. pHIV RT- 1 : HIV RT gene was amplified from pRT with 5' RT primer and 3' RT primer. The 5' primer is: 5' GAA GAT CTA AGC TTA
  • the 3' primer is: 5* TTT TGA ATT CGC ATG CCT GCA G 3 * .
  • the 5* RT primer contains the sequence that is complementary to 5' end of RT gene, ribosomal binding sequence for protein expression and Hind III.
  • the 3' RT primer contains the sequence that is complementary to 3' end sequence of RT gene in pRT and Eco RI. Amplified DNA was subjected to Hind III and Eco RI enzymes and ligated to pHSG576.
  • pHIV RT-2 61 bp non-coding sequence was inserted to Hind III site of pHIV RT-1.
  • pHIV RT-3 431 bp Hind III fragment of pBL encoding N- terminal 94 amino acid sequence was inserted to Hind III site of pHIV RT-1. Insertion of this fragment produces two different HIV RT proteins. One is a fusion protein (M.W. about 77 Kdal) and the other is wild-type HIV RT protein. Expression level of both protein about 1/6 of the protein expressed from pHIV RT-1.
  • the parent plasmid ( ⁇ HSC576) or pHIV RT was introduced into /?o/A12recA718 cells by electroporation.
  • Pol I ts cells are grown in nutrient broth media (NB, 8 g/1 Difco nutrient broth and 4 g/1 NaCl) until the optical density of the cell culture is at 600 nm (O.D.600) is 0.5. Cells are harvested and washed by autoclaved water three times.
  • Plasmid DNAs are transformed into the washed cell by electroporation as described (Ausubel et al., Short Protocols in Molecular Biology, 2d ed., Greene Publishing Associates and John Wiley & Son, 1-26 - 1-27, 1993). DNA should be prepared in E. coli B strain to get high transformation efficiency (1 x 10 9 cells/ug). Transformed cells are plated onto NA selection plates that contain Difco nutrient agar (11.5 g/1), NaCl (5 g/1) tetracycline (12.5 mg/1), chloramphenicol (34 mg/1) and IPTG (1 mM). Plates are incubated at 30°C for 24 hours.
  • E. coli pol A ⁇ 2 single mutant can form single colonies in rich media and low cell density at 42°C, whereas /?o/A12recA718 double mutant cannot.
  • polAYl single mutant single strand gaps produced by polA ⁇ 2 mutation can be filled by RecA with its recombination repair function, which allows this pol A ⁇ 2 single mutant to grow at 42°C (Witkin and Roegner- Maniscalco, J. Bacteriol. 774:4166-4168, 1992).
  • recA718 mutant the level of RecA function was decreased. Therefore, in/?o/A12recA718 double mutant, single strand gaps produced by pol A ⁇ 2 mutation cannot be repaired, which results in failure of the double mutant to grow as single colonies at high temperatures.
  • the cell density-dependent temperature-sensitive phenotype is demonstrated in Figure 1(A) by plating concentric dilutions of E. coli pol A ⁇ 2recA718 in NA selection plates (Witkin and Roegner-Maniscalco, J. Bacteriol. 774:4166-4168, 1992).
  • a variety of other methods of plating or growing the cells in liquid culture known to those in the art can be utilized.
  • about 2 x 10 6 E. coli cells were introduced at the center of a plate using a 10 ⁇ l inoculation loop. The plate was rotated and the loop was gradually moved to the outside of the plate to display the bacteria in a diverging spiral of increasing dilution. Wild type E.
  • coli, polA + recA + (Pol I + ), containing the parent plasmid (pHSG576) that lacks the HIV RT gene is able to grow both at 30°C and 37°C ( Figure 1A)
  • the Pol I ts mutant containing the parent plasmid (pHSG576) lacking the HIV RT gene grows at 30°C at all dilutions tested, but can only grow at 37°C at the high density in the center of the plate ( Figure IB).
  • This growth deficit can be complemented in the Pol I ts mutant by a plasmid expressing HIV RT (pHIVRT- 2); the infected E.
  • HIV RT DN186 contains a mutation at the substrate binding site and expresses a non-functional reverse transcriptase.
  • the E. coli double mutant harboring the plasmid that expresses DN186 HIV RT (pHIVRT-DN) is unable to grow at 37°C, further indicating that genetic complementation of DNA polymerase I by HIV RT requires that HIV RT is active.
  • Duplicate plates are incubated for 48 hours at 30°C (black bar) or 37°C (open bar) and the number of colonies were scored.
  • the ts phenotype of /?o/A12recA718 mutant is observed at 37°C and HIV RT complements ts the Pol I ts defect.
  • E. coli with pHSG576 are progressively unable to grow at 37°C (compare the number of colonies at 30°C and 37°C in Figure 2).
  • E. coli expressing HIV RT form an equal number of colonies at 30°C and 37°C ( Figure 2).
  • This experiment provides an example that expression of HIV RT is able to fully complements the growth defect exhibited by the /?o/A12recA718 strain.
  • Pol I + mutant harboring pHSG576 also gives high plating efficiency close to 1 at all dilutions and 37°C.
  • pHIV RT-1 and pHIV RT-2 plasmids gave about 60% and 90% plating efficiency at 37°C at low cell density plate (500 cell per plate).
  • the same type of experiment was done with the/ o/A12recA718 strain expressing rat DNA polymerase ⁇ (Sweasy et al., J. Biol. Chem. 267:1407-1409, 1992).
  • the plating efficiency at dilution 5 (about 150 cells per plate) of the Pol I ts strain expressing rat DNA pol ⁇ at 42°C was about 60% to 80% of that obtained at 30°C.
  • the plating efficiency of the Pol I ts cell expressing rat pol ⁇ drastically decreased at low cell density, as was observed for Pol I ts cells containing pHSG576.
  • a number of other assays can be utilized for the quantitation of the ability of HIV RT or different mutants of HIV RT to complement the temperature-sensitive phenotype of bacteria that have mutations in one or more of the genes that code for DNA polymerase. These include but are not limited to: turbidity of culture, plaque formation, and incorporation of radioactive precursors such as 3 H-thymidine into DNA.
  • the assay to measure inhibition of complementation consists of growing the HIV RT expressing cells at elevated temperature that is restrictive for a temperature-sensitive mutant of DNA polymerase I and then determining the extent of inhibition of cell growth by compounds that inhibit HIV RT.
  • Approximately 200 E. coli cells are diluted from the fresh culture, harboring plasmids that either lack or contain the gene for HIV RT and plated in duplicate on NA plates containing tetracycline, chloramphenicol and IPTG (as described in Figure 1).
  • the plasmid harboring E. coli are grown with or without AZT (100 nM) at 30°C or 37°C for 36 hours. pHIV RT-1 and pHIV RT-2 gave about the same sensitivities as pHIV RT-3.
  • the wild type strain forms large smooth colonies on nutrient agar containing 85 nM AZT at 37°C ( Figure 3(A)) and also at 30°C.
  • the Pol I ts strain harboring pHIV RT forms visually similar colonies at 37°C in the absence of AZT ( Figure 3(B)). However, in the presence of AZT, Pol I ts cells expressing HIV RT form only miniature colonies at 37°C ( Figure 3(C)). At 30°C, where Pol I is fully active, the Pol I ts strain containing pHIV RT forms large colonies even in the presence of AZT ( Figure 3(D)).
  • the range of AZT concentrations in nutrient agar that differentiates between strains expressing HIV RT and Pol ⁇ is 40-120 nM.
  • the effectiveness of different nucleotide analogs is ascertained by incubating the cells on nutrient agar containing graded concentrations of the chemicals to be tested as potential drugs for the treatment ofAIDS.
  • Another way to assay inhibitory effect of AZT on complementation system is to determine survival of colonies in the plates with or without AZT.
  • E. coli cells expressing the designated proteins A: rat DNA polymerase ⁇ , B: wild-type HIV RT (pHIV RT-2), C: TY215 HIV RT
  • A rat DNA polymerase ⁇
  • B wild-type HIV RT (pHIV RT-2)
  • C TY215 HIV RT
  • AZT (0, 50, 150, 200 and 250 nM) and were incubated at 30°C (black bar) or 37°C (open bar) for 48 hr.
  • the percent survival is the ratio of colonies formed in the presence and absence of AZT.
  • Pol I ts expressing rat DNA polymerase ⁇ is able to grow even in high concentrations of AZT whereas the Pol I ts strain expressing HIV RT does not grow at high AZT concentrations ( Figure 4B).
  • TY215 an HIV mutant obtained from a patient treated with AZT, contains a mutation in the RT gene that renders HIV RT resistant to AZT in vivo.
  • cells expressing TY215 AZT resistant mutant are able to grow at high concentrations of AZT.
  • the growth of the host cell is, alternatively, dependent on resistance to an antibiotic by the use of a plasmid that both contains an antibiotic resistance gene and requires DNA polymerase I for plasmid replication. This requirement can be substituted by HIV RT, thus rendering growth dependent on the expression of HIV RT.
  • Plasmids An example of this is illustrated by the replication of pBR322 plasmid derivatives (e.g., pBS-SK from Stratagene, San Diego, CA), a plasmid that requires DNA polymerase I for replication initiation (Witkin and Roegner-Maniscalco, J. Bacteriol. 774:4166-4168, 1992; and Sweasy and Loeb, Proc. Natl. Acad. Sci. USA 90:4626-4630, 1993). This class of plasmids cannot be replicated in Pol I ts mutant at non-permissive temperature (37°C-42°C).
  • plasmid into host cells transformation of ⁇ BR322 to Pol I ts mutants containing pHIV RT and pHSG576 plasmids: 1 ⁇ g of pBR322 (obtained from Promega, Madison, WI) is transformed into the Pol I ts mutant cell containing pHSG576 or pHIV RT plasmids as described in section I.A.4., above.
  • the transformed cells are plated to NA selection plates containing carbenicillin (50 mg/1), which is hereto forth designated as the pBR322 NA plate.
  • Pol I ts cells containing pBR322 and either pHSG576 (lacking HIV RT) or pHIV RT plasmids are grown in NB media containing carbenicillin (50 mg/1) for 24 hours at 30 °C. Grown cells are diluted and plated to pBR322 NA plates. Transformed cells were grown to 2 x 10 8 cells per ml in nutrient broth containing tetracycline (12.5 mg/1), chloramphenicol (34 mg/1), IPTG (1 mM) and carbenicillin (50 mg/1).
  • E. coli polA+recA+ pol Al2recA7 IS strain wild-type mutant plasmid pHSG576 pHSG576 pHIV RT-2 pHIV RT-3 plating efficiency 91% 0.5% 28% 11% at 37°C
  • E. Alternative Complementation Systems 1. Other vectors and phages a. E. coli plasmids: This complementation assay is used with any E. coli plasmid that requires Pol I for replication (e.g., pGB2, 6). b. A vector that contains any E. coli promoter is used for transcription of HIV RT gene. c. E. coli phages (e.g., phage lambda) are used as expression vehicles for expression of HIV RT. d. Bacteria other than E. coli (e.g., Salmonella, Thermus aquaticus and Bacillus subtilis) are used in conjunction with host specific phage
  • DNAs e.
  • Other eucaryotic cells containing mutant DNA polymerases are adopted for these assays including mammalian cells with conditional mutants in one of the DNA polymerases.
  • mammalian cells such as a hamster cell line (Liu et al., Proc. Natl. Acad. Sci. USA 80:797, 1983), a human cell line (Dong et al., J. Biol. Chem. 268:1, 1993), or a rat cell line (Chen et al., Proc. Natl. Acad. Sci. USA 97:3054-3057, 1994; Ezzeddine et al., The New Biologist 3:608-614, 1991) are used.
  • yeast may be used.
  • the expression of HIV RT in yeast is carried out with an appropriate yeast vector (e.g., pBKl).
  • pBKl is pYES2 (Invitrogen, San Diego, CA) containing the HIV RT gene (fused to the gal promoter).
  • Expression of HIV RT in yeast is verified by western analysis.
  • the ability of HIV RT to complement mutants with any of the known defects in DNA polymerase activity (vide infra) can be established. • f.
  • Eucaryotic cells a. Production of eucaryotic cells (e.g.. Saccharomyces cerevisiae) with DNA polymerase conditional mutations
  • Saccharomyces cerevisiae The CDC2 gene encodes yeast DNA polymerase III.
  • Several temperature sensitive mutants of this allele e.g., cdc2-l, cdc2-2
  • cdc2 mutants have been shown to exhibit a growth defect at elevated temperatures and are also sensitive to alkylating agents (e.g., MMS) that cause DNA damage.
  • yeast mutants are available for complementation by HIV RT.
  • HIV RT gene is cloned into any one of a variety of yeast expression vectors (e.g., pYES2 from Invitrogen) that contain yeast specific promoters (e.g., gal P of pYES2). HIV RT is expressed in a form of fusion protein with yeast nuclear localization signal to facilitate delivery of HIV RT to the nucleus.
  • yeast expression vectors e.g., pYES2 from Invitrogen
  • yeast specific promoters e.g., gal P of pYES2
  • HIV RT is expressed in a form of fusion protein with yeast nuclear localization signal to facilitate delivery of HIV RT to the nucleus.
  • HIV RT gene was amplified with 5' Hind III RT primer and 3' Eco RI RT primer.
  • 5' Hind III RT primer contains Hind III site and the sequence complementary with 5' end of HIV RT gene of pRT.
  • 3' Eco RI primer contains Eco RI site and the sequence of 3' end of RT gene in pR.
  • Amplified DNA was cloned to pYES2 vector at Hind III and Eco RI sites. Expression of HIV RT in yeast strain (CDC2 + ) was observed by western analysis.
  • c Introduction of vectors to eucaryotic cells: Vectors expressing HIV RT or its fusion protein are transformed into yeast as described in section I.E.l.b., above.
  • Detection of growth and presentation of inhibitor The approach is analogous to that described above the E. coli. The medium and conditions are well known to those familiar with yeast genetics.
  • cdc2-2 Cells harboring pYES2 or pBKl (pYES2 with HIV RT gene) are grown in C-ura media (complete media) at 20°C.
  • HIV RT inhibitors Nucleoside analogs may have to be phosphorylated in yeast prior to inhibiting HIV RT or to be incorporated in yeast DNA and act as chain terminators.
  • nucleoside kinase that can phosphorylate the inhibitors that are to be evaluated.
  • Either the genes can be directly introduced on the yeast chromosome or can be carried on the same plasmid as HIV RT.
  • Genes expressing viral or human kinase are introduced to Pol I ts cells expressing HIV RT by chromosomal integration (e.g., a phage lambda lysogen, bacterial transposons).
  • E. coli contain an endogenous thymidine kinase that can phosphorylate thymidine and analogs of thymidine such as AZT (Munir et al., Proc. Natl. Acad. Sci. USA 90:4012-4016, 1993).
  • nucleoside triphosphate analogs that are able to terminate DNA synthesis.
  • Herpes simplex thymidine kinase was expressed as a vehicle to phosphorylate various types of nucleosides. HSV TK is able to phosphorylate dG, dC, dG as well as T analogs (Cheng, Y-C, Biochem. Biophys. Acta 452:370-381, 1976).
  • a dual plasmid that contains the genes for both HIV RT and herpes thymidine kinase was constructed.
  • the dual plasmid (pRT-TK) was assembled by introducing the wild type HSV TK gene into pHIVRT, the plasmid used in the basic complementation system.
  • pHIVRT is a low copy number plasmid derived from pHSG576 that contains the gene for HIV RT under control of the lac P/O promoter.
  • the inserted HSV TK gene contains a ribosome binding site for initiation of translation.
  • HSV TK gene fused for ribosomal binding site was obtained from pET ⁇ c-HSVTK.
  • the 1.1 kb Xbal/BamHI fragment of pET8c-HSVTK containing HSV TK gene was blunt-ended, and this fragment was inserted into blunt-ended EcoRI site of pHIVRT, generating pRT- TK.
  • pTK is pHSG576 containing 1.1 Kb HSV TK at Smal site.
  • pHIVRT, pRT- TK and pTK contain DNA polymerase I-independent pSClOl replication origin from pHSG576 which is a low copy number plasmid. Both the HIV RT and HSV TK genes are under control of lac P/O and are transcribed within a single mRNA.
  • ddC DNA polymerase I-independent pSClOl replication origin from pHSG576 which is a low copy number plasmid.
  • HSV TK DNA polymerase I-independent pSClOl replication origin from pHSG576 which is a low copy number plasmid.
  • Both the HIV RT and HSV TK genes are under control of lac P/O and are transcribed within a single mRNA.
  • herpes thymidine kinase and HIV RT render the E. coli sensitive to dideoxycytidine ( Figure 5B).
  • both the single and dual plasmids render the cells equally sensitive to AZT ( Figures 5C and 5D).
  • herpes thymidine phosphorylates dideoxycytidine and that herpes thymidine kinase and HIV RT are both expressed in these cells.
  • the well-documented ability of herpes thymidine kinase to catalyze the phosphorylation of a variety of nucleoside analogs significantly expands the utility of this genetic complementation system.
  • Phage DNAs and plasmid can be transformed into E. coli and yeast as described in section I. ⁇ .2.b., above.
  • the assay is used for the simultaneous testing of multiple compounds or for screening nucleoside libraries containing different substituents.
  • This assay is used to carry out structure function relationships with respect to the design of new inhibitors for HIV RT. Mutants of the enzyme can be established by site specific mutagenesis or by selection from random sequence libraries.
  • HIV RT mutant protein e.g., TY215.
  • a plasmid containing a HIV RT gene encoding a HIV RT that is resistant to AZT was prepared based upon known HIV RT mutants (e.g., St. Clair et al., Science 256:1557-1559, 1991; Emini et al., Nature 364:679, 1993).
  • HIV RT containing multiple mutations are used for introducing multiple mutations and selecting HIV RT mutants that function as wild-type protein (e.g., genetic selection). Strategy for designing oligomers and detailed procedures have been previously described (Munir et al., J. Biol. Chem. 2(57:6584-6589, 1992). A segment of the HIV RT gene was replaced with an oligonucleotide that contains 10% random substitution at amino acid positions 67 to 78. This segment encodes a portion of the putative nucleotide binding site.
  • Example III The same protocols described in Example I are utilized. Example III
  • the effectiveness of current nucleoside analogs used in the treatment of AIDS is limited by the rapid emergence of mutations in HIV RT, some of which reduce the affinity of the enzyme for the specific inhibitor (Larder et al., Science 243:1731, 1989).
  • the new mutants are not only resistant to the analog used in the therapy, but frequently are also resistant to related analogs.
  • the present complementation assay offers the evaluation of HIV RT from individual patients for susceptibility to inhibition by a panel of potential inhibitors, e.g., nucleoside analogs.
  • the vector used for the expression of the patient's HIV RT gene is the same as those used for expression of the wild type HIV RT.
  • the complementation assays described are used for testing a panel of known and approved nucleoside analogs and candidate inhibitors of HIV RT. Any putative mutations in HIV RT that evolve during the course of the disease that render the virus resistant to the current therapy being used to treat an individual patient may be susceptible to other HIV RT inhibitors or a combination of inhibitors.

Abstract

La présente invention concerne des procédés et des compositions ayant trait au SIDA, procédés à l'aide desquels on peut cribler les composés potentiels aptes à inhiber la transcriptase inverse du virus de l'immunodéficience humaine ('TI VIH'). Les procédés selon l'invention permettent de détecter des mutants actifs de la transcriptase inverse du VIH. L'invention porte aussi sur des procédés de criblage de composés inhibiteurs de la transcriptase inverse du VIH prélevée sur un patient ainsi que sur des procédés d'essai de l'efficacité biologique des composés potentiellement inhibiteurs de la transcriptase inverse in vivo.
PCT/US1995/002074 1994-02-18 1995-02-16 Procedes et compositions de criblage de medicaments anti-sida WO1995022622A1 (fr)

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WO1996022385A1 (fr) * 1995-01-20 1996-07-25 University Of Washington Procedes et compositions de criblage de medicaments contre l'hepatite
US6942969B2 (en) 1996-01-29 2005-09-13 Virologic, Inc. Compositions and methods for determining anti-viral drug susceptibility and resistance and anti-viral drug screening
EP0852626A4 (fr) * 1996-01-29 1999-08-25 Virologic Inc Compositions et procedes pour determiner la sensibilite et la resistance vis-a-vis de medicaments antiviraux et criblage de medicaments antiviraux
US6242187B1 (en) 1996-01-29 2001-06-05 Virologic, Inc. Compositions and methods for determining anti-viral drug susceptibility and resistance and anti-viral drug screening
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EP1170380A3 (fr) * 1996-01-29 2003-05-02 Virologic, Inc. Compositions et méthodes déterminant une susceptibilité et résistance anti-virale et screening pour des agents anti-viraux
EP0852626A1 (fr) * 1996-01-29 1998-07-15 Virologic, Inc. Compositions et procedes pour determiner la sensibilite et la resistance vis-a-vis de medicaments antiviraux et criblage de medicaments antiviraux
US7279279B2 (en) 1996-01-29 2007-10-09 Monogram Biosciences, Inc. Compositions and methods for determining anti-viral drug susceptibility and resistance and anti-viral drug screening
EP1012334A1 (fr) * 1997-07-30 2000-06-28 Virologic, Inc. Compositions et procedes permettant de determiner la sensibilite et la resistance vis-a-vis de medicaments antiviraux, et criblage de medicaments antiviraux
EP1012334A4 (fr) * 1997-07-30 2004-12-29 Virologic Inc Compositions et procedes permettant de determiner la sensibilite et la resistance vis-a-vis de medicaments antiviraux, et criblage de medicaments antiviraux
EP1383890B1 (fr) * 2001-04-10 2008-05-14 Institut Pasteur Mutants de la desoxycytidine kinase possedant une activite enzymatique elargie
EP1921143A3 (fr) * 2001-04-10 2009-06-03 Institut Pasteur Mutants de la désoxycytidine kinase possédant une activité enzymatique élargie
US7547513B2 (en) 2001-04-10 2009-06-16 Institut Pasteur Mutants of deoxycytidine kinase having extended enzymatic activity

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