WO2003013488A2 - Procedes et compositions visant a inhiber rad51 - Google Patents

Procedes et compositions visant a inhiber rad51 Download PDF

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WO2003013488A2
WO2003013488A2 PCT/US2001/024986 US0124986W WO03013488A2 WO 2003013488 A2 WO2003013488 A2 WO 2003013488A2 US 0124986 W US0124986 W US 0124986W WO 03013488 A2 WO03013488 A2 WO 03013488A2
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pnp
rad51
dna
group
fragment
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PCT/US2001/024986
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WO2003013488A3 (fr
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Gurucharan Reddy
David A. Zarling
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Pangene Corporation
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Publication of WO2003013488A3 publication Critical patent/WO2003013488A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention relates to methods and compositions including small molecules for inhibiting RAD51.
  • RecA protein plays a central role in homologous recombination.
  • RecA protein promotes homologous pairing of double-stranded DNA with single-stranded DNA or partially single-stranded DNA molecules (Radding, Genetic Recombination. Washington, American Society for Microbiology, pp 193-230 (1988); Radding, J. Biol. Chem. 266:5355-5358 (1991); Kowalczykowski & Eggleston, Annu. Rev. Biochem. 63:991-1043 (1994)).
  • Radding Genetic Recombination. Washington, American Society for Microbiology, pp 193-230 (1988); Radding, J. Biol. Chem. 266:5355-5358 (1991); Kowalczykowski & Eggleston, Annu. Rev. Biochem. 63:991-1043 (1994)
  • coli recA and yeast rad51 were isolated from all groups of eukaryotes, including mammals (Morita, et al., Proc. Natl. Acad. Sci. USA 90:6577-6580 (1993); Shinohara, et al., Nature Genet. 4:239-243 (1993); Ueye ⁇ ,Experentia 50:223-233 (1994); Maeshima, et al., Gene 160: 195-200 (1995)).
  • Phylogenetic analysis by Ogawa and co-workers suggest there are two sub-families within the eukaryotic RecA homologous: the Rad51-like genes (Rad51 of human, mouse, chicken, S. cerevisiae, S.
  • yeast rad51 gene was cloned and sequenced (Basile et al., Mol. Cell. Biol. 12:3235-3246 (1992); Aboussekhara, et ⁇ .,MOI. Cell. Biol. 12:3224-3234 (1992)) several years ago, it was not initially thought to be a homologue of RecA, because the sequence homology between RecA and Rad51 is limited. The true extent of structural conservation between RecA protein and Rad51 protein became apparent only after the discovery that yeast Rad51 protein forms nucleoprotein filaments nearly identical to the nucleoprotein filaments formed by RecA protein (Ogawa, et al., CSH Symp. Quant. Biol. 58:567-576 (1993); Story et al., Science 259:1892-1896 (1993)).
  • yeast and human Rad51 proteins have been purified and characterized biochemically. Like E. coli RecA protein, yeast and human Rad51 protein polymerizes on single-stranded DNA to form a right-handed helical nucleoprotein filament which extends DNA by 1.5 times its original length (Story, et al., Science 259:1892-1896 (1993); Benson, et al., EMBO J. 13:5764-5771 (1994)).
  • Rad51 protein promotes homologous pairing and strand exchange in an ATP dependent reaction (Sung, Science 265: 1241-1243 (1994); Baumann, et al., Cell 87:757-766 (1996); Gupta, et al., Proc. Natl. Acad. Sci. USA 94:463-468 (1997)).
  • Rad51 protein is important for the repair of double-strand breaks in damaged cells.
  • genes with homology to recA include Rad51, Rad57 and Dmcl.
  • Rad51 is a member of the Rad52 epistasis group, which includes Rad50, Rad51, Rad52, Rad54, Rad55 and Rad57. All these genes were initially identified as being defective in the repair of damaged DNA caused by ionizing radiation and dysfunctional mutants in these genes were subsequently shown to be deficient in both genetic recombination and the recombinational repair of DNA lesions (Game, Radiation sensitive mutants and repair in yeast. Yeast Genetics: Fundamental and applied aspects. J.F.T. Spencer and A.R.W.
  • Rad51 RNA transcripts and protein are present in all cell types, the highest transcript levels are in tissue active in recombination, including spleen, thymus, ovary and testis (Morita, et al., Proc. Natl. Acad. Sci. USA 90:6577-6580 (1993)).
  • Rad51 is specifically induced in murine B cell nuclei undergoing Ig class switch recombination (Li, et al., Proc. Natl. Acad. Sci.
  • Rad51 is enriched in the synaptonemal complexes which join paired homologous chromosomes in spermatocytes undergoing meiosis (Haaf, et al., Proc. Natl. Acad. Sci. USA 92:2298-2302 (1995); Ashley, et al., Chromosoma 104:19-28 (1995); Plug, et al., Proc. Natl. Acad. Sci.
  • Rad51 nuclear localization changes dramatically in response to DNA damage in cultured cell lines, when multiple discreet foci are redistributed in the nucleus and stain vividly with anti-Rad51 antibodies (Haaf, et al., Proc. Natl. Acad. Sci. USA 92:2298-2302 (1995).
  • Rad51 Targeted disruption of Rad51 leads to an embryonic lethal phenotype in mouse and the dying embryo cells are very sensitive to radiation (Tsuzuki, et al., Proc. Natl. Acad. Sci. USA 93:6236-6240 (1996); Lim & Hasty, Mol. Cell. Biol. 16:7133-7143 (1996)). Attempts to generate viable homozygous rad51 "A embryonic stem cells have not been successful. These results show that Rad51 plays an essential role in cell proliferation, a surprise in view of the viability of S. cerevisiae carrying Rad51 deletions.
  • Rad51 is associated with RNA polymerase II transcription complexes (Maldonado, et al., Nature 381:86-89 (1996). Although the specificity and functional nature of these interactions are not clear, these observations taken together point to a pleiotropic role for human Rad51 in DNA metabolism (repair, recombination, transcription), and maintenance of genomic integrity. Human Rad51 protein interacts directly with wild type-p53 protein, and the regions necessary for this interaction have been mapped (Sturzbecher et, al., EMBO 15: 1992- 2002 (1996); Buchhop, et al., Nucleic Acids Res 25:3868-3874 (1997)).
  • Rad51 interacts with two different regions of p53 (amino acids 94-160 and 264-315), and p53 interacts with the region between amino acids 125 and 220 of Rad51. This latter region is necessary for homo-oligomerization of Rad51. Therefore, p53 may inhibit Rad51 activity by blocking the formation of active Rad51 oligomers. Furthermore, p53 inhibits Rad51 ATPase and DNA strand exchange activities. Interestingly, p53 mutants often found in cancer cells, are reported to bind Rad51 less efficiently than wild type 53 and fail to inhibit its biochemical activities.
  • Rad51 and p53 Taken together, known interactions between Rad51 and p53 suggest that 1) in normal cells p53 interacts with and downregulates Rad51 and 2) in tumor cells with p53 mutations, unregulated Rad51 could possibly lead to uncontrolled recombination, genetic instability, and radiation resistance by upregulating DNA recombination and DNA repair (Sturzbecher, et al., EMBO 15:1992-2002 (1996); Ohnishi, et al., Biochem. Biophys. Res. Comm. 245:319-324 (1998).
  • Rad51 also interacts with BRCAl and BRCA2 (Scully, et al., Cell 88:265-275 (1997); Sharan, et al., Nature 386:804-810 (1997)).
  • Inherited mutations inBRCAl cause familial breast and ovarian cancer, and inherited mutations in BRCA2 case familial breast cancer (Wooster, et al., Science 265:2088-2090 (1994); Smith, et al., Nature Genet. 2: 128-131 (1992); Easton, et al., Am. J. Hum. Genet. 52:678-701 (1993); Gayther, et al., Nature Genet. 15: 103-105 (1997)).
  • Rad51B Albala, et al., Genomics 46:476-479 (1997)
  • Rad51C Rad51C
  • XRCC2 Cartwright et al., Nucleic Acids Res 26:3084- 3089-793 (1998)
  • XRCC3 Liu, et al., Mol Cell 1:783 (1998). While these genes are homologous to human Rad51, it is also possible that they are related to certain other members of the Rad52 epistasis group such as Rad55 and Rad57. The chromosomal locations of all these genes have been mapped. XRCC2 maps to chromosome 7q36.1, a region associated with radiation resistance in human glial tumors.
  • the c-Abl tyrosine kinase is activated by ionizing radiation and other DNA damaging agents. It was recently reported that cAbl constitutively interacts with Rad51 and phosphorylates Rad51 on Tyr-54. Thus, ionizing radiation induces c-Abl-dependent phosphorylation of Rad51. Furthermore, phosphorylation of Rad51 by c-Abl inhibits Rad51 binding to DNA and its function in ATP-dependent DNA strand exchange (Yuan, et al., J. Biol. Chem. 273:3799-3802 (1998)).
  • Rad51 for treatment of Rad51 related disorders, and to potentiate radiation therapy by inhibiting double-strand break repair. It is further desirable to downregulate Rad51 to sensitize human tumor cells to chemotherapeutic agents like cisplatin.
  • compositions for inhibiting Rad51 and human Rad51 homologues are provided herein.
  • the methods can be performed in vitro or in vivo and have a number of applications as further described below including the treatment of cancer.
  • compositions and related methods of using Rad51 inhibitors are also provided.
  • a small molecule is used to inhibit Rad51 or a human Rad51 homologue.
  • the biological or biochemical activity of Rad51 or a human Rad51 homologue is inhibited wherein the biological or biochemical activity is selected from the group of DNA dependent ATPase activity, formation of Rad51 foci, nucleic acid strand exchange, DNA binding, filament formation, DNA pairing and DNA repair, wherein said DNA repair is double stranded break repair, single stranded annealing or post replication recombinational repair.
  • the small molecule can be any one of or a combination of small molecules including but not limited to a nucleotide diphosphate, a nucleotide analogue, a DNA minor groove binding drug, a xanthine or a xanthine derivative such as caffeine.
  • the nucleotide diphosphate is selected from the group consisting of ADP, GDP, CDP, UDP and TDP.
  • the nucleotide analogue is a nucleotide diphosphate complexed with aluminum fluoride.
  • the nucleotide diphosphate complexed with aluminum fluoride can be selected from, for example, the group consisting of ADP. A1F4, GDP.A1F4, CDP.A1F4, UDP.A1F4 and TDP.A1F4.
  • the nucleotide analogue is a non-hydrolyzable nucleotide.
  • the non-hydrolyzable nucleotide can be ATP ⁇ S, GTP ⁇ S, UTP ⁇ S, CTP ⁇ S, TTP ⁇ S, ADP ⁇ S, GDP ⁇ S, UDP ⁇ S, CDP ⁇ S, TDP ⁇ S, AMP ⁇ S, GMP ⁇ S, UMP ⁇ S, CMP ⁇ S, TMP ⁇ S, ATP-PNP, GTP-PNP, UTP-PNP, CTP-PNP, TTP-PNP, ADP-PNP, GDP-PNP, UDP-PNP, CDP-PNP, TDP-PNP, AMP-PNP, GMP-PNP, UMP-PNP, CMP- PNP, or TMP-PNP
  • the Rad51 or human Rad51 homologue is a DNA minor groove binding drug which can be selected from the group consisting of distamycin, netropsin, bis-benzimidazole and actinomycin.
  • the inhibitor is a peptide.
  • Inhibiting Rad51 and/or human Rad51 homologues has a number of applications.
  • a Rad51 ,or human Rad51 homologue, inhibitor is combined with a composition comprising Rad51 and components required for Rad51 activity such cell such as DNA and ATP.
  • the composition can be a solution in a test tube for an in vitro assay.
  • the Rad51 and human Rad51 homologue inhibitors are administered to a cell.
  • the cell can be a cancerous cell. In one embodiment, the cell is of an individual with cancer.
  • Also provided herein is a method for inducing sensitivity to radiation and DNA damaging chemotherapeutics in an individual comprising administering to said individual a composition comprising a Rad51, or human Rad51 homologue, inhibitor, wherein said inhibitor is a small molecule.
  • the individual has cancer.
  • a method for inducing apoptosis comprises administering a Rad51, or human Rad51 homologue, inhibitor to a cell.
  • the cell can be cancerous or of an individual with cancer.
  • the small molecule Rad51 or human Rad51 homologue inhibitors provided herein can be administered in a pharmaceutical acceptable carrier.
  • the inhibitors are further administered in conjunction with a ligand which targets the cells in need thereof.
  • the ligand targets cancerous cells.
  • the ligand can be an antibody which targets cell surface ligands which are specific to diseased cells such as cancer cells or cells infected with HIV.
  • a fragment of Rad51 or a human Rad51 homologue wherein said fragment consists essentially of a binding site for a small molecule, wherein said small molecule regulates the biological or biochemical activity of Rad51.
  • the small molecule inhibits Rad51 activity.
  • the site is the p53 binding site on Rad51 or the human Rad51 homologue.
  • the Rad51 homologue can be selected from the group consisting of Rad5 IB, Rad51 C, Rad5 ID, XRCC2 and XRCC3.
  • the present invention is directed to a series of discoveries relating to the pivotal role that Rad51 plays in a number of cellular functions, including those involved in disease states.
  • compositions and methods for inhibiting Rad51 and methods of treatment for disease states associated with Rad51 activity as further defined below using Rad51 inhibitors.
  • Other compositions and methods related to Rad51 inhibition are also described.
  • Rad51 biological or biochemical activity as used herein can be selected from the group consisting of DNA dependent ATPase activity, formation of Rad51 foci, nucleic acid strand exchange, DNA binding, nucleoprotein filament formation, DNA pairing and DNA repair.
  • DNA repair and recombination are generally considered biological activities.
  • DNA repair can be double stranded break repair, single stranded annealing or post replication recombinational repair.
  • Rad51 inhibitors can include inhibitors of Rad51 homologues such as RecA and/or inhibitors that sensitize cells to radiation and also affect aspects of recombination in vivo, which were not previously known to inhibit Rad51.
  • Rad51 as used herein refers to Rad51 and its homologues, preferably human homologues.
  • Rad51 excludes non-human homologues.
  • Rad51 homologues include RecA and Rad51 homologues in yeast and in mammals. Genes homologous to E. coli RecA and yeast Rad51 have been isolated from all groups of eukaryotes, including mammals.
  • Human Rad51 homologues include Rad51B, Rad51C, Rad51D, XRCC2 and XRCC3.
  • Rad51 inhibitors provided herein were not previously known to inhibit RecA or other Rad51 homologues and were not known to induce sensitizing of cells to radiation.
  • Rad51 as used herein excludes homologues thereof.
  • the small molecule is preferably 4 kilodaltons (kd) or less. In another embodiment, the small molecule is less than 3 kd, 2kd or lkd. In another embodiment the small molecule is less than 800 daltons (D), 500 D, 300 D, 200 D or 100 D.
  • Nucleotides as used herein refer to XYP, wherein X can be U, T, G, C or A (base being uracil, thymine, guanine, cytosine or adenine, respectively), and Y can be M, D or T (mono, di or tri, respectively).
  • nucleotides can include xathanine, hypoxathanine, isocytosine, isoguanine, etc.
  • Analogues as used herein includes derivatives of and chemically modified nucleotides and nucleosides. In one embodiment, methyl methanesulfonate is excluded.
  • the Rad51 inhibitor is a nucleotide diphosphate.
  • the Rad51 inhibitor is selected from the group consisting of ADP, GDP, CDP, UDP and TDP. In preferred embodiments, ADP is excluded.
  • the Rad51 inhibitor is a nucleotide analogue.
  • the Rad51 inhibitor is a nucleotide diphosphate complexed with aluminum fluoride.
  • the Rad51 inhibitor is selected from the group consisting of ADP.A1F4, GDP.A1F4, CDP.A1F4, UDP.A1F4 and TDP.A1F4.
  • the Rad51 inhibitor is a non-hydrolyzable nucleotide.
  • the Rad51 inhibitor is selected from the group consisting of ATP ⁇ S, GTP ⁇ S, UTP ⁇ S, CTP ⁇ S, TTP ⁇ S, ADP ⁇ S, GDP ⁇ S, UDP ⁇ S, CDP ⁇ S, TDP ⁇ S, AMP ⁇ S, GMP ⁇ S, UMP ⁇ S, CMP ⁇ S, TMP ⁇ S, ATP-PNP, GTP-PNP, UTP-PNP, CTP-PNP, TTP-PNP, ADP-PNP, GDP-PNP, UDP-PNP, CDP-PNP, TDP- PNP, AMP-PNP, GMP-PNP, UMP-PNP, CMP-PNP, and TMP-PNP
  • ADP ⁇ S is excluded.
  • the Rad51 inhibitor is a DNA minor groove binding drug.
  • the Rad51 inhibitor is selected from the group consisting of distamycin, netropsin, bis-benzimidazole and actinomycin.
  • the Rad51 inhibitor is a peptide.
  • peptide herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides.
  • the protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures.
  • amino acid or “peptide residue”, as used herein means both naturally occurring and synthetic amino acids.
  • nucleic acid analogs are described in Rawls, C & E News p. 35 (June 2, 1997). All of these references are hereby expressly incorporated by reference. These modifications of the ribose-phosphate backbone may be done to facilitate the addition of additional moieties such as labels, or to increase the stability and half-life of such molecules in physiological environments.
  • mixtures of naturally occurring nucleic acids and analogs including PNA can be made.
  • mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made.
  • the nucleic acids may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence.
  • the nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ribo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xathanine hypoxathanine, isocytosine, isoguanine, etc.
  • Rad51 inhibitors may bind to Rad51 , but exclude agents which generally activate Rad51 such as DNA on which Rad51 normally binds to in the process of recombinational activity, ATP, etc.
  • Rad51 inhibitors are obtained from a wide variety of sources, as will be appreciated by those in the art, including libraries of synthetic or natural compounds. Any number of techniques are available for the random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications to produce structural analogs. In a preferred embodiment, the methods include both in vitro and in vivo applications, preferably in vivo. Accordingly, in a preferred embodiment, the methods comprise the steps of administering a Rad51 inhibitor to a sample comprising Rad51 under physiological conditions, preferably to a cell.
  • the cell that the Rad51 inhibitor is administered to may be a variety of cells.
  • the cell is mammalian, and preferably human.
  • the cell may be any cell in a site in need of Rad51 inhibition such as diseased cells including cancerous cells and cells infected with viruses such as FUN as further discussed below.
  • ⁇ LS nuclear localization signal
  • NLS amino acid sequences have been reported including single basic NLS's such as that of the SV40 (monkey virus) large T Antigen (Pro Lys Lys Lys Arg Lys Nal), Kalderon (1984), et al., Cell 39:499-509; the human retinoic acid receptor- ⁇ nuclear localization signal (ARRRRP); ⁇ FKB p50 (EEVQRKRQKL; Ghosh et al., Cell 62: 1019 (1990); ⁇ FKB p65 (EEKRKRTYE; Nolan et al., Cell 64:961 (1991)); and others (see for example Boulikas, J. Cell. Biochem.
  • NLSs incorporated in synthetic peptides or grafted onto reporter proteins or other molecules not normally targeted to the cell nucleus cause these molecules to be concentrated in the nucleus. See, for example, Dingwall, and Laskey, Ann, Rev. Cell Biol. 2:367-390, 1986; Bonnerot, et al., Proc. Natl. Acad. Sci. USA 84:6795-6799, 1987; Galileo, et al., Proc. Natl. Acad. Sci. USA 87:458-462, 1990.
  • a Rad51 inhibitor there are a variety of techniques available for introducing a Rad51 inhibitor into cells.
  • the techniques vary depending upon whether the inhibitor is transferred into cultured cells in vitro, or in vivo in the cells of the intended host.
  • Techniques suitable for the transfer of inhibitors into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc.
  • the currently preferred in vivo transfer techniques include transfection with viral (typically retroviral) vectors and viral coat protein-liposome mediated transfection(Dzau et al., Trends in Biotechnology 11:205-210 (1993)). Special or other liposomes, modified electroporation, chemical treatment or Piezo injection techniques are particularly preferred.
  • the inhibitory agents may be administered in a variety of ways, orally, systemically, topically, parenterally e.g., subcutaneously, intraperitoneally, intravascularly, etc.
  • the inhibitors are applied to the site of a tumor (or a removed tumor) intra- operatively during surgery.
  • the compounds may be formulated in a variety of ways.
  • the concentration of therapeutically active compound in the formulation may vary from about 0.1-100 wt.%.
  • a therapeutic amount for the need is used, for example, to achieve inhibition of cellular proliferation, radiation or chemotherapeutic sensitization or inducing apoptosis.
  • the Rad51 inhibitory molecules can be combined in admixture with a pharmaceutically or physiologically acceptable carrier vehicle.
  • Therapeutic formulations are prepared for storage by mixing the active ingredient having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
  • Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween, Pluronics or PEG.
  • buffers such as phosphate, citrate and other organic acids
  • antioxidants including ascorbic acid
  • compositions can be prepared in various forms, such as granules, aerosols, tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like.
  • Pharmaceutical grade organic or inorganic carriers and/or diluents suitable for oral and topical use can be used to make up compositions containing the therapeutically-active compounds.
  • Diluents known to the art include aqueous media, vegetable and animal oils and fats. Stabilizing agents, wetting and emulsifying agents, salts for varying the osmotic pressure or buffers for securing an adequate pH value, and skin penetration enhancers can be used as auxiliary agents.
  • the inhibitor in some situations it is desirable to provide the inhibitor with an agent that targets the target cells, such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc.
  • an agent that targets the target cells such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc.
  • proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g. capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half-life.
  • the technique of receptor-mediated endocytosis is described, for example, by Wu et al., J. Biol. Chem. 262:4429-4432 (1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87:3410-3414 (1990).
  • Dosages and desired drug concentrations of pharmaceutical compositions of the present invention may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary physician. Animal experiments provide reliable guidance for the determination of effective doses for human therapy. Interspecies scaling of effective doses can be performed following the principles laid down by Mordenti, j. and Chappell, W. "The use of interspecies scaling in toxicokinetics" In Toxicokinetics and New Drug Development, Yacobi et al, Eds., Pergamon Press, New York pp. 42-96 (1989).
  • the methods comprise identifying the inhibitory effect of the Rad51 inhibitor. For example, determining the effect on double strand break repair, homologous recombination, sensitivity to ionizing radiation, class switch recombination, cellular inhibition, induction of apoptosis, etc. Assays are detailed in Park, J. Biol. Chem. 270(26): 15467 (1995) and Li et al., PNAS USA 93: 10222 (1996), Shinohara et al., supra, (1992), all of which are hereby incorporated by reference. Further assays are discussed below in the examples.
  • the invention provides methods of treating disease states requiring inhibition of cellular proliferation.
  • the disease state requires inhibition of at least one of Rad51 expression, translation or the biological activity of Rad51 as described herein.
  • a disease state means either that an individual has the disease, or is at risk to develop the disease.
  • Disease states which can be treated by the methods and compositions provided herein include, but are not limited to hyperproliferative disorders. More particular, the methods can be used to treat, but are not limited to treating, cancer (further discussed below), premature aging, autoimmune disease, arthritis, graft rejection, inflammatory bowel disease, proliferation induced after medical procedures, including, but not limited to, surgery, angioplasty, and the like. Thus, in one embodiment, the invention herein includes application to cells or individuals afflicted or impending affliction with any one of these disorders.
  • compositions and methods provided herein are particularly deemed useful for the treatment of cancer including solid tumors such as skin, breast, brain, cervical carcinomas, pancreas, testicular carcinomas, etc. More particularly, cancers that may be treated by the compositions and methods of the invention include, but are not limited to: Cardiac: sarcoma (angio sarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; Gastrointestinal: esophagus (squamous cell carcinoma,
  • the individual, or patient is generally a human subject, although as will be appreciated by those in the art, the patient may be animal as well. Thus other animals, including mammals such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, etc., and primates (including monkeys, chimpanzees, orangutans and gorillas) are included within the definition of patient.
  • the individual requires inhibition of cell proliferation. More preferably, the individual has cancer or a hyperproliferative cell condition.
  • the methods comprising administering the Rad51 inhibitors provided herein further comprise administering an alkylating agent or radiation.
  • ionizing radiation shall mean all forms of radiation, including but not limited to alpha, beta and gamma radiation and ultra violet light, which are capable of directly or indirectly damaging the genetic material of a cell or virus.
  • the term irradiation shall mean the exposure of a sample of interest to ionizing radiation, and term radiosensitive shall refer to cells or individuals which display unusual adverse consequences after receiving moderate, or medically acceptable (i.e., nonlethal diagnostic or therapeutic doses), exposure to ionizing irradiation.
  • Alkylating agents include BCNU and CCNU. Additionally, radiation sensitizers (e.g., xanthine and xanthine derivatives including caffeine) can be applied with, before or after the Rad51 inhibitors.
  • the Rad51 inhibitors provided herein are administered to prolong the survival time of an individual suffering from a disease state requiring the inhibition of the proliferation of cells.
  • the individual is further administered radiation or an alkylating agent.
  • the binding site is identified by combining the inhibitor with fragments of Rad51.
  • the fragments are from between amino acids 125 and 220.
  • Rad51 125-220 is fragmented to fragments of 5-25 amino acids and then tested separately or in random recombinations to determine the binding site by standard binding techniques.
  • Rad51 protein carries out strand exchange in three distinct phases: I) presynapsis, during which Rad51 protein binds cooperatively and stoichiometrically to single-stranded DNA and forms a right handed helical nucleoprotein filament; II) synapsis, in which duplex DNA is taken up into the nucleoprotein filament and homologously aligns; and III) DNA strand displacement, which produces a recombinant (heteroduplex) double-stranded DNA molecule and a displaced single-stranded DNA .
  • joint molecules also referred to as D-loops
  • final products are monitored either by filter assays, fluormeters, or by gel electrophoresis.
  • hybrid DNA intermediates of the DNA strand exchange reaction contain single-stranded DNA tails, and one of the products of strand exchange is single-stranded DNA; both of which are trapped on nitrocellulose filters.
  • the unreacted linear double- stranded DNA cannot bind to the membrane and is washed away in the filtrate. Since the initial single-stranded DNA used to make the nucleoprotein filament is not labeled, it is not detected.
  • Fluorescence spectroscopy-based assays for monitoring the DNA strand exchange activity of human Rad51 are highly specific and compatible with high throughput screening. Assays based on fluorescence to measure DNA pairing and DNA strand exchange by human Rad51 protein have been developed. This approach enables one to distinguish homologous DNA pairing from subsequent DNA strand exchange. Homologous pairing of a single-stranded oligonucleotide with a duplex oligonucleotide is measured by fluorescence resonance energy transfer (FRET). Energy transfer between two fluorescent dyes indicates their proximity. In the case of DNA, the proximity of two complementary strands labeled with dyes are determined by FRET.
  • FRET fluorescence resonance energy transfer
  • Homologous DNA pairing assay by FRET A test oligonucleotide labeled at its 3' end with fluorescein is used to form the nucleoprotein filament with Rad51. Rhodamine is attached to the 5' end of the complementary strand in duplex DNA. Homologous pairing between the two DNA molecules juxtaposes the two fluorescent molecules, resulting in nonradiative energy transfer from fluorescein to rhodamine when fluorescein is excited at 493 nm, near its excitation peak. As a result of the energy transfer, the fluorescence emission from fluorescein is quenched and that from rhodamine is enhanced.
  • an 83-mer oligonucleotide (minus strand) labeled at its 3' end with fluroescein is preincubated with 1.2 ⁇ M Rad51 protein in a reaction mixture containing 1 mM MgCl 2 , 25 mM HEPES (pH 7.4), 1 mM DTT, 2 mM ATP and 100 ⁇ g of BSA per ml for 4 minutes at 37°C.
  • concentration of MgCl 2 is increased to 30 mM
  • 3 ⁇ M duplex DNA (labeled with rhodamine at the 5' end of the plus strand) is added.
  • DNA strand exchange assay by FRET DNA strand exchange assay by FRET.
  • fluorescein and rhodamine are juxtaposed by 20 A on opposite complementary strands, the emission from the fluorescein is quenched and that from rhodamine is enhanced as a result of energy transfer.
  • both fluorophores are present in the duplex where they are juxtaposed.
  • strand exchange is completed, the two labeled strands are separated from each other as monitored by the enhanced emission from fluorescein.
  • Rad51 protein is added to unlabeled single- strand oligonucleotide for 4 min. at 37°C followed by the addition of the filament to a reaction mixture containing 30 mM MgCl 2 and 3 ⁇ M duplex oligonucleotide (labeled on the 3' end of the minus strand with fluorescein and on the 5' end of the plus strand with rhodamine).
  • the final concentrations of sDNA and protein are 3 ⁇ M and 1.2 ⁇ M, respectively.
  • Fluorescence emission spectra are recorded from 502 to 620 nm upon excitation at 493 nm on an SLM 8000C (SLM Aminco, Urbana, IL) or similar spectrofluorimeter.
  • SLM 8000C SLM Aminco, Urbana, IL
  • inhibitors for Rad51 biological activity are confirmed in a number of ways.
  • the following assays are used to assay for a change in biological activity to initially identify inhibitors, or to determine the specificity of identified inhibitors: D-loop assay, DNA dependent ATPase assay, nucleoprotein filament assay, and complementary single-strand hybridization assay. These assays are unique features of the Rad51 protein and determine the specificity of, for example, small molecules that inhibit Rad51 protein activity.
  • D-loop assay The non-enzymatic uptake of a homologous single-stranded DNA by a negatively supercoiled DNA leads to the formation of a DNA displacement loop (D-loop, Figure 12) (Holloman et al., PNAS, USA 76:1638-1642 (1975)).
  • Rad51 enzymes both catalyze D-loop formation under physiological conditions. Negative superhelicity is not required in these reactions catalyzed by RecA or Rad51. Only members of RecA and Rad51 protein families can catalyze the formation of D-loops under physiological conditions.
  • ATP hydrolysis To monitor ATP hydrolysis, labeled ATP is incubated with single-stranded DNA and Rad51. The reaction mixture is incubated at 37°C for 30 min. and an aliquot of the reaction is applied directly onto CEL 300 PEI/UV 254 thin layer chromatography plates to separate the product of hydrolysis (ADP) from the substrates. Non-DNA dependent ATPases are used as controls for these reactions. Small molecule compounds that inhibit the ATPase activity of Rad51 would not be expected to affect the activity of other ATPase enzymes.
  • nucleoprotein filament formation Rad51 protein binds cooperatively to DNA to form a right-handed helix.
  • the resulting protein-DNA complex is an active nucleoprotein filament which catalyzes DNA pairing and DNA strand exchange reactions .
  • the DNA helix inside the filament is extended 1.5 times the size of B-form duplex length.
  • This structure of the nucleoprotein filament is a hallmark feature of RecA and Rad51 proteins and is DNA sequence independent.
  • the DNA inside the filament is completely protected from phosphodiesterases, as RecA and Rad51 proteins bind to and protect the phosphate backbone from cleavage. Formation of nucleoprotein filaments is easily monitored by protein-based filter binding assays and fluorescent dye displacement assays. Another DNA binding protein is used as a control.
  • Rad51 protein promotes the hybridization of complementary strands of DNA under specific conditions in which the spontaneous renaturation of complementary strands does not occur. Hybridization activity is easy to monitor in a DNA micro array format or by filter binding assays. Other single- strand annealing proteins, such as SSB, are used as controls.
  • SSB single- strand annealing proteins
  • a sulforhodamine B-based optical density assay of protein in cultured cells is used as a cell-based high throughput drug screen for inhibitors of Rad51 activity.
  • the phenotypes screened for are cytotoxicity and growth inhibition in target tumor (breast, brain and prostate) and control (non tumorigenic) cell lines. Cells are placed in 96-well microtiter plates. Next, these drugs are introduced after one day' of culture, and treat for an additional 96 hours. Assays begin at the beginning of drug treatment, at 48 hours and at 96 hours. Qualitative changes are monitored by comparing the amount of cellular protein present at the beginning of the drug incubation period with the amount of protein present in control and test cultures at day 3 and day 5 of growth.
  • Rad51 activity in these samples using methods outlined above to determine whether the biological effects measured are specific to alterations in Rad51 activity. This is done by comparing the results to antisense oligodeoxynucleotides that specifically down-regulate Rad51 protein.
  • Rad51 down-regulation sensitizes cells to DNA damage is determined by assaying Rad51 down-regulation shifts on the dose response curves for DNA damaging agents (radiation, BCNU, cyclophosphamide, cis-platin) in systems that measure growth, survival and death.
  • DNA damaging agents radiation, BCNU, cyclophosphamide, cis-platin
  • the effect of inhibition on the cell cycle is monitored by looking for cell cycle arrest in Gl or G2 phase by FACS technology.
  • the effects on cell growth are screened using the sulforhodamine B assay. Assayed is the effect on cell survival using a clonigenic assay to determine the surviving fraction of clonigenic cells.
  • Apoptosis is assayed using either a flow cytometric assay for subdiploid fractions or by using the TUNEL method, which utilizes terminal deoxynucleotide transferase to incorporate fluorescein-conjugated deoxyuridine triphosphate into DNA nicks formed in apoptotic cells.
  • TUNEL method utilizes terminal deoxynucleotide transferase to incorporate fluorescein-conjugated deoxyuridine triphosphate into DNA nicks formed in apoptotic cells.
  • the experimental blueprint for these studies in human tumor xenografts is: a) define growth curves for the untreated xenograft and for xenografts treated with inhibitors of Rad51. This aim also determines how growth is related to Rad51 down-regulation; b) choose appropriate doses of DNA damaging agents that allow us to compare the responses of the control and Rad51 down-regulated cell lines; c) quantitate the amount of sensitization to DNA damage.
  • the target tumor cell lines chosen herein can all form tumors in nude mice.
  • 1 x 10 5 , 1 x 10 6 , and 1 x 10 7 cells taken from cell culture in the log phase of growth are injected into the flanks of nude mice, and the tumor measured daily for 50 days, until the tumor reaches 2000mm 3 , or until the animal becomes debilitated.
  • Each group has 5 mice.
  • tumors are treated when they reach a volume of 50-100mm 3 . Determination of lethal dose. This is a simple determination of lethality of the lead compound found by escalating single doses in mice which are indexed to intracellular concentrations that down-regulate Rad51 in culture. Toxicity is investigated by physical exam and organ histopathology.
  • tumors Treatment of tumors with lead compounds. Once tumors reach a treatable size, animals are injected with escalating doses of the lead compound. Both systemic and local routes of administration are tested. The tumors are examined for Rad51 activity and for volume. Toxicity of the compound in these animals are assayed by weight gain, serum chemistries and organ histopathology (including liver, lung, kidneys, heart, gastrointestinal tract and brain).
  • Dose finding for DNA damage The growth characteristics of the cell lines are established and then various doses of DNA damaging agents (for example, 0, 5, 10 and 15 Gy for radiation) are used to define a series of growth curves that describe response. This information allows selection of doses to compare control cell lines to the Rad51 down-regulated cell lines. Each group has at least 5 animals and appropriately matched controls.
  • DNA damaging agents for example, 0, 5, 10 and 15 Gy for radiation

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Abstract

L'invention concerne des procédés visant à inhiber l'activité de Rad51 par l'administration d'un inhibiteur de Rad51. Les inhibiteurs de Rad51 décrits constituent de petites molécules, de préférence de moins de 4 kDa.
PCT/US2001/024986 2001-08-09 2001-08-09 Procedes et compositions visant a inhiber rad51 WO2003013488A2 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8597949B2 (en) 2007-07-28 2013-12-03 The University Of Chicago Methods and compositions for modulating RAD51 and homologous recombination
WO2015034038A1 (fr) * 2013-09-06 2015-03-12 学校法人早稲田大学 Composé ayant une activité inhibitrice de la prolifération cellulaire, composition pharmaceutique et procédé de criblage
US9393250B2 (en) 2012-04-12 2016-07-19 University Of Saskatchewan Phthalocyanine compounds useful as RecA inhibitors and methods of using same
US10188691B2 (en) 2016-05-27 2019-01-29 Synthex, Inc. Protein interfaces
US10912761B2 (en) 2012-11-30 2021-02-09 The University Of Chicago Methods and compositions involving RAD51 inhibitors
US11780881B2 (en) 2017-11-22 2023-10-10 Synthex, Inc. Compounds for selective disruption of protein-protein interactions

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WO1998034118A1 (fr) * 1997-01-30 1998-08-06 Yale University Methodes diagnostiques et compositions basees sur la repartition de rad51
EP1006185A1 (fr) * 1998-10-06 2000-06-07 Vlaams Interuniversitair Instituut voor Biotechnologie vzw. Variant tissu-spécifiques d'épissure du gène de recombinaison-réparation rad51b associé aux leiomyomas t(12;14) utérins
WO2000047231A2 (fr) * 1999-02-10 2000-08-17 Pangene Corporation Nouvelle inhibition antisens de rad51
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8597949B2 (en) 2007-07-28 2013-12-03 The University Of Chicago Methods and compositions for modulating RAD51 and homologous recombination
US9198914B2 (en) 2007-07-28 2015-12-01 The University Of Chicago Methods and compositions for modulating RAD51 and homologous recombination
US9393250B2 (en) 2012-04-12 2016-07-19 University Of Saskatchewan Phthalocyanine compounds useful as RecA inhibitors and methods of using same
US10912761B2 (en) 2012-11-30 2021-02-09 The University Of Chicago Methods and compositions involving RAD51 inhibitors
WO2015034038A1 (fr) * 2013-09-06 2015-03-12 学校法人早稲田大学 Composé ayant une activité inhibitrice de la prolifération cellulaire, composition pharmaceutique et procédé de criblage
JP2015051946A (ja) * 2013-09-06 2015-03-19 学校法人早稲田大学 細胞増殖抑制活性を有する化合物、医薬組成物及びスクリーニング方法
US10188691B2 (en) 2016-05-27 2019-01-29 Synthex, Inc. Protein interfaces
US11780881B2 (en) 2017-11-22 2023-10-10 Synthex, Inc. Compounds for selective disruption of protein-protein interactions

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