EP1203098A2 - Induzierbarer screening zur auffindung von arzneimitteln - Google Patents

Induzierbarer screening zur auffindung von arzneimitteln

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Publication number
EP1203098A2
EP1203098A2 EP00951686A EP00951686A EP1203098A2 EP 1203098 A2 EP1203098 A2 EP 1203098A2 EP 00951686 A EP00951686 A EP 00951686A EP 00951686 A EP00951686 A EP 00951686A EP 1203098 A2 EP1203098 A2 EP 1203098A2
Authority
EP
European Patent Office
Prior art keywords
dna
inhibitor
nos
kinase
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00951686A
Other languages
English (en)
French (fr)
Inventor
Ian George The Wolfson Inst for Biom Rs CHARLES
Weiming The Wolfson Inst for Biomedical Res XU
Lizhi The Wolfson Inst for Biomedical Res LIU
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University College London
Original Assignee
University College London
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GBGB9918077.0A external-priority patent/GB9918077D0/en
Priority claimed from GB0016171A external-priority patent/GB0016171D0/en
Application filed by University College London filed Critical University College London
Publication of EP1203098A2 publication Critical patent/EP1203098A2/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters

Definitions

  • This invention relates to methods for identifying genes which are differentially expressed in the presence of nitric oxide. It also relates to cancer therapy.
  • NO may play a pathophysiological role in promoting tumor growth and in protecting tumors from agents that cause DNA damage. NO production in cancer cells may thus confer resistance to chemotherapeutic drugs, such as bleomycin and cisplatin, and to radiotherapy on those cells.
  • chemotherapeutic drugs such as bleomycin and cisplatin
  • FIG. 2(a) shows generation of NO by EcR293 clone 11, following treatment with muristerone A.
  • EcR293 clone 11 cells were grown with varying concentrations of muristerone A and at different time intervals supernatants were taken and the
  • Figure 6 shows DNA-PK pulldown peptide assays.
  • Figure 6(a) shows samples prepared from control EcR293 clone 11 cells, and cells expressing NO after treatment with 1 ⁇ M or lO ⁇ M muristerone A in the presence of 20 ⁇ M L-NIO. Peptides derived from wild type and mutant p53 peptide were used as substrate.
  • Figure 6(b) shows DNA-PK activity in nuclear extracts prepared from cells treated with muristerone A as described above. Averages and S.D values from three independent experiments are shown.
  • Figure 8(d) shows UV-C irradiation of human EcR293 clone 11 cells: UV-C irradiation of EcR293 clone 11 cells (120mJ/cm 2 ) following treatment with l ⁇ M, lO ⁇ M and lO ⁇ M muristerone A in the presence of 20 ⁇ M L-NIO, lO ⁇ M muristerone A in the presence of 20 ⁇ M wortmannin (WM).
  • the ecdysonse responsive constructs comprise a promoter operably linked to a coding sequence, wherein the promoter is responsive to ecdysone or an analog thereof and the coding sequence codes for a NOS or a functional variant or fragment thereof.
  • An ecdysone-responsive promoter may comprise more than one EcRE, for example 2 to 10 elements or more preferably 4 to 6 elements.
  • the sequence of an EcRE will depend on the exact functional ecdysone receptor used. If a modified functional ecdysone receptor is used (see below) it may be appropriate to use a modified EcRE (see No et al, Proc. Natl. Acad. Sci. USA, 93: 3346-3351). The EcRE(s) and minimal promoter sequences do not have to be immediately adjacent.
  • promoter will depend on the host cell to be used for expression of the coding sequence. Typically, expression in mammalian cells, for example human cells will be required and thus a mammlian promoter will be preferred. Mammalian promoters, such as ⁇ -actin promoters, may be used. Tissue- specific promoters may be used. Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR), the rous sarcoma virus (RSV) LTR promoter, the SV40 promoter, the human cytomegalovirus (CMV) LE promoter, adenovirus, HSV promoters (such as the HSV IE promoters), or
  • TetO 2 sequences may be used. Typically, if more than one TetO 2 site is used those sites will be arranged in the form of an array. However, other intervening nucleotide sequences may be situated between individual TetO 2 sites. For example one, two, three, four, five, up to ten or up to 15 nucleotides may intervene between any two TetO 2 sites.
  • the TetO 2 sequence is 5'-TCCCTATCAGTGATAGAGA-3' (Hillen and Berens, 1994, Annu. Rev. Microbiol. 48, 345-369; Hillen et al, 1983, J. Mol. Biol. 169, 707-721) or a functional variant thereof.
  • the TetO 2 sequence or a functional valiant thereof is capable of being bound by a homodimer of tetR or a functional variant thereof.
  • tetR may have a binding affinity for a functional variant of the TetO 2 sequence which is greater or less than that of tetR for the TetO 2 sequence.
  • a functional variant of the TetO 2 sequence may be a modified version of that sequence obtained by, for example, nucleotide substitution or deletion. Up to 1, up to 2, up to 3, up to 4, up to 5, up to 6 or more nucleotide substitutions or deletions or combinations thereof may be made to the TetO 2 sequence to produce a functional variant of that sequence .
  • Constructs of the invention may be responsive to the antibiotic tetracycline or an analog thereof.
  • Tetracycline binds to tetR homodimers, such that the tetR:tetracylcine complex dissociates from the TetO 2 sequence.
  • the association constant of tetracycline to tetR is 3 x 10 9 M '1 .
  • Preferred analogs of tetracycline will have an association constant substantially similar to or greater than that of tetracycline for tetR. Suitable analogs of tetracycline include doxycycline.
  • the coding sequence may be modified by nucleotide substitutions or deletions. For example up to 1, 2 or 3 to 10, 25, 50, 75 or 100 substitutions or deletions or combinations thereof may be made to produce a functional variant of a NOS.
  • a polynucleotide encoding a NOS may alternatively or additionally be modified by one or more insertions and/or deletions and/or by an extension at either or both ends.
  • the modified polynucleotide generally encodes for a polypeptide which has NOS activity. Degenerate substitutions may be made and/or substitutions may be made which would result in a conservative amino acid substitution when the modified sequence is translated, for example as shown in the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other.
  • Sequence identity may be calculated as follows.
  • the UWGCG Package provides the BESTFIT program which can be used to calculate identity (for example used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, p387- 395) .
  • the PILEUP and BLAST algorithms can be used to calculate identity or line up sequences (typically on their default settings), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol 215:403-10.
  • Software for performing BLAST analyses is publicly available through the National Centre for Biotechnology Information (http://www.ncb i.nlm.nih.gov
  • EcR and USP/RXR are polypeptides which can heterodimerise with their partner and can, when heterodimerised, allow ecdysone- responsive dimerisation to occur. In some cases functional variants may bind to non- wild type EcREs. Examples of functional variants and modified EcREs are described in No et al. Proc. Natl. Acad. Sci. USA, 93: 3346-3351.
  • a functional variant of tetR is a polypeptide which is similar to tetR and which remains capable of binding, as a homodimer, the TetO 2 site or a functional variant thereof and tetracycline or an analog thereof.
  • the binding affinity of a functional variant sequence of tetR for the TetO 2 site or a functional variant thereof or tetracycline or an analog thereof may be substantially the same as the binding affinity of the tetR polypeptide for the TetO 2 site or a functional variant thereof or tetracycline or an analog thereof.
  • a functional variant sequence may have a binding affinity which may be greater or less than that of the tetR polypetide.
  • Oligonucleotides comprising stretches of, for example, 8 to 10 deoxythymidines may be used as primers for reverse trans criptase.
  • random primed cDNA synthesis may be carried out. In that technique short oligonucleotide fragment of, for example, 6 to 10 nucleotides in length made up of many possible sequences are used as primers for the cDNA synthesis reaction. This technique may be suitable for isolating the 5' end of long messages.
  • the product of both dT primed and random primed cDNA synthesis is an RNA-DNA hybrid. From that point several prodedures may be used to convert the RNA-DNA hybrid to double stranded cDNA molecules suitable for cloning into appropriate vectors.
  • Serial analysis of gene expression is a sequence-based approach to the identification of differentially expressed genes through comparative analyses. It allows the simultaneous analysis of sequences that derive from different cell populations or tissues. Three steps form the molecular basis for SAGE: (1) generation of a sequence tag (from 10 to 14 bp) to identify expressed transcripts; (2) ligation of sequence tags to obtain concatemers that can be cloned and sequenced; and (3) comparison of the sequence data to determine differences in expression of genes that have been identified by the tags.
  • a particular substance may stimulate and/or inhibit transcription and/or translation and/or activity.
  • the cumulative overall effect will be important, as in the majority of cases a polypeptide will be the active species.
  • a substance inhibits transcription or translation the effective "activity" of the corresponding gene will be inhibited.
  • the activity per mole of polypeptide will be unaltered; the amount of polypeptide will be simply diminished.
  • Stimulators and inhibitors may be isolated using any suitable method. Typically, however, it will be convenient to use cells of the invention. Cells may be contacted with a test substance and ecdysone or an analog thereof or tetracycline or an analog thereof under conditions in which in the absence of the test substance the expression of the polynucleotide or activity of the polypeptide is inhibited or stimulated in the presence of NO. The cells may then be assayed for the effect that the test substance has on expression of the differentially expressed polynucleotide or activity of the polypeptide. That is, the effect of the test substance on transcription, translation and polypeptide activity may be assayed.
  • a stimulator or inhibitor of a differentially expressed polynucleotide or polypeptide is one which produces a measurable increase or reduction respectively in transcription and or translation of the differentially expressed polynucleotide or activity of the polypeptide encoded by the polynucleotide in the assays described above.
  • Preferred stimulators are those which stimulate expression of a polynucleotide and or activity of a polypeptide by at least 10%, at least 25%, at least 50%, at least 100%, at least, 200%, at least 500% or at least 1000% at a concentration of the activator 1 ⁇ g ml "1 , lO ⁇ g ml "1 , lOO ⁇ g ml “1 , 500 ⁇ g ml "1 , lmg ml “1 ' lOmg ml "1 , lOOmg ml "1 .
  • the percentage inhibition or stimulation represents the percentage increase or decrease in expression/activity in a comparison of assays in the presence and absence of the test substance. Any combination of the above mentioned degrees of percentage inhibition and concentration of stimulator or inhibitor may be used to define a stimulator or inhibitor of the invention, with greater stimulation or inhibition at lower concentrations being preferred.
  • DNA-PKcs One of the genes isolated in screens carried out to identify polynucleotides which are differentially expressed in response to NO is the gene encoding DNA-PKcs.
  • DNA-PK plays an important role in DNA repair and/or DNA damage signalling and DNA-PKcs is a member of the PI 3-kinase family, ie. it is a PI 3- kinase like kinase.
  • Other polypeptides containing a PI 3-kinase-like domain are also implicated in DNA repair.
  • NOS inhibitors used in the present invention typically achieve at least 50% NOS inhibition, more preferably at least 80% NOS inhibition. Ideally, they achieve substantially complete NOS inhibition.
  • a DNA repair enzyme inhibitor is a substance which inhibits transcription and or translation of a gene encoding a DNA repair enzyme and/or inhibits activity of a DNA repair enzyme itself. Any pharmaceutically acceptable inhibitor of DNA repair enzyme can be used in the present invention.
  • the inhibitor may be a pharmaceutically acceptable salt of one the above compounds.
  • Suitable salts include salts with pharmaceutically acceptable acids, both inorganic acids such as hydrochloric, sulphuric, phosphoric, diphosphoric, hydrobromic or nitric acid and organic acids such as citric, fumaric, maleic, malic, ascorbic, succininc, tartaric, benzoic, acetic, methanesulphonic, ethanesulphonic, benzenesulphonic or p-toluenesulphonic acid.
  • inorganic acids such as hydrochloric, sulphuric, phosphoric, diphosphoric, hydrobromic or nitric acid
  • organic acids such as citric, fumaric, maleic, malic, ascorbic, succininc, tartaric, benzoic, acetic, methanesulphonic, ethanesulphonic, benzenesulphonic or p-toluenesulphonic acid.
  • the above assay may alternatively be carried out using a cell line which expresses NOS under the control of an tetracycline-responsive promoter.
  • the invention also provides a method of treatment of a host suffering from retrovirus infection, for example HTV infection, comprising administration of a therapeutically effective amount of an NOS inhibitor.
  • an effective amount of a PI-3 kinase like kinase for example DNA-PKcs, may be co- administered.
  • the invention also provides products containing an NOS inhibitor and a PI-3 kinase like kinase as a combined preparation for simultaneous, separate or sequential use in the treatment of retroviral infection.
  • the condition of a patient suffering from retroviral infection can be improved by administration of products of the invention.
  • a therapeutically effective amount of products of the invention may be given to a patient in need thereof.
  • the invention also provides use of an NOS inhibitor in the manufacture of a medicament for use in the treatment of retroviral infection.
  • the invention additionally provides use of an NOS inhibitor in the manufacture of a medicament for use with a PI-3 kinase like kinase in the treatment of retroviral infection.
  • DNA damaging agents and NOS or DNA repair enzyme inhibitors may be administered in a variety of dosage forms. Thus, they can be administered orally, for example as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules.
  • the DNA alkylating and or cross-linking agents and NOS or DNA repair enzyme inhibitors may also be administered parenterally, either subcutaneously, intravenously, intramuscularly, intrasternally, transdermally or by infusion techniques.
  • lOO ⁇ g of lysate was mixed with lOO ⁇ l reaction reagent from the NOS detect system (Stratagene NOS detect kit, Cat. No. 204500).
  • the kit measures the conversion of [ 14 C]arginine to [ 14 C] citrulline, and is specific for the NOS pathway.
  • Three plasmids were generated, each of which expressed one of the human NOS isofoims under the control of an ecdysone-responsive promoter.
  • An additional plasmid was constructed placing the iNOS cDNA under the control of a tetracycline- regulated promoter.
  • the wild type human eNOS (GenBank accession number: M95296, Coding sequence 21-3632) was cut from its original vector (Bluescript KS) using the restriction endonucleases, Hind ⁇ . and Notl and cloned into pL D (Invitrogen, San Diego, CA; Catalogue No: V705-20) which had been cut with Hindttl and Notl.
  • the resulting plasmid was sequenced to confirm that the cD ⁇ A had inserted in the correct orientation.
  • tetracycline repressor protein tetR
  • human iNOS cDNA tetracycline repressor protein
  • DNA-PKcs protein levels were examined using western blotting ( Figure 5a). For whole-cell extracts, 20 ⁇ g of protein from untreated cells (track 1), and cells generating NO following treatment with l ⁇ M muristerone A (track 2), 5 ⁇ M muristerone A (track 3), lO ⁇ M muristerone A (track 4) and lO ⁇ M muristerone A in the presence of 1 O ⁇ M L-NIO (track 5) were loaded in each well.
  • EcR293 clone- 11 cells were extremely sensitive to treatment with cisplatin. 12.5 ⁇ g/ml causes massive apoptotic cell death within 24hr (see Figure 8b). The amount of cell death was dose dependent: 30 ⁇ g/ml causes 90% cell death; and 150 ⁇ g/ml kills over 95% of cells in 24hr. Remarkably, the NO-induced cells have over 85% protection against such toxicity even at the highest concentration (150 ⁇ g/ml) of cisplatin. To confirm the role of DNA-PK in this protection, 20 ⁇ M wortmannin (a specific inhibitor of PI-3- type kinases) was added in the lO ⁇ M muristerone A induced culture medium. Wortmannin (WM) completely abolished the protective role of NO-generation against the damaging effect of cisplatin (see Figure 8b).
  • wortmannin a specific inhibitor of PI-3- type kinases
  • PKcs increased enzyme activity requires associated DNA damage within the cell. It is possible that the NO-mediated increase in both mRNA and protein for DNA-PKcs acts as a "priming mechanism" enabling the cell to respond rapidly to NO-associated DNA damage. Previous reports have shown that NO triggers DNA damage (Wink et al, 1991, Science 254, 1001-1003; Nguyen et al, 1992, Proc. Natl. Acad. Sci. USA

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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
EP00951686A 1999-07-30 2000-07-28 Induzierbarer screening zur auffindung von arzneimitteln Withdrawn EP1203098A2 (de)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
GBGB9918077.0A GB9918077D0 (en) 1999-07-30 1999-07-30 Inducible screen for drug discovery
GB9918077 1999-07-30
GB0016171A GB0016171D0 (en) 2000-06-30 2000-06-30 Inducible screen for drug discovery
GB0016171 2000-06-30
PCT/GB2000/002932 WO2001009375A2 (en) 1999-07-30 2000-07-28 Inducible screen for drug discovery

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EP1203098A2 true EP1203098A2 (de) 2002-05-08

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CA (1) CA2380323A1 (de)
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US7603758B2 (en) 1998-12-07 2009-10-20 Shell Oil Company Method of coupling a tubular member
WO2004017950A2 (en) * 2002-08-22 2004-03-04 Piramed Limited Phosphadidylinositol 3,5-biphosphate inhibitors as anti-viral agents

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GB2253852B (en) * 1991-02-26 1995-03-22 Ici Plc Vector for expression of heterologous polypeptide comprising inducible selection system
JPH09500526A (ja) * 1993-06-14 1997-01-21 ベーアーエスエフ アクツィエンゲゼルシャフト テトラサイクリン反応性プロモーターによる真核細胞の遺伝子発現の厳密な制御
US5654168A (en) * 1994-07-01 1997-08-05 Basf Aktiengesellschaft Tetracycline-inducible transcriptional activator and tetracycline-regulated transcription units
US5759836A (en) * 1995-03-27 1998-06-02 Hospital For Joint Diseases Osteoarthritis-associated inducable isoform of nitric oxide synthetase
US5908756A (en) * 1996-08-30 1999-06-01 Johns Hopkins University Protein inhibitor of neuronal nitric oxide synthase
EP0948619A1 (de) * 1996-12-09 1999-10-13 Ariad Gene Therapeutics, Inc. Expression der proteine zur behandlung von asthma durch ligand-vermittelte aktivierung der ihren enkodierenden genen
HN1998000125A (es) * 1997-08-28 1999-02-09 Pfizer Prod Inc 2-aminopiridinas con sustituyentes alcoxi ramificados
US6441158B1 (en) * 1997-12-31 2002-08-27 Medical College Of Georgia Research Institute, Inc. Oligomers that bind to ku protein

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AU6454600A (en) 2001-02-19
GB2371547A (en) 2002-07-31
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WO2001009375A3 (en) 2001-05-25
WO2001009375A2 (en) 2001-02-08
GB0204762D0 (en) 2002-04-17

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