| Assay for identifying biological targets of polynucleotide-binding compounds -> Monitor Keywords |
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Assay for identifying biological targets of polynucleotide-binding compoundsRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic AcidAssay for identifying biological targets of polynucleotide-binding compounds description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20080096201, Assay for identifying biological targets of polynucleotide-binding compounds. Brief Patent Description - Full Patent Description - Patent Application Claims
BACKGROUND OF THE INVENTION [0002] The saframycins, isolated in the late 1970s, are a family of microbial fermentation products with significant anti-proliferative activity and anti-microbial activity against gram-positive bacteria (Arai, T.; Kubo, A. In The Alkaloids; Brossi, A., Ed.; Academic Press: New York, 1983; Vol. 21, Chapter 3; Arai, T.; Takahashi, K. the Journal of Antibiotics 1977, 30, 1015-1018). Several saframycin analogues have been isolated and characterized in recent years (see, e.g., DE 2839668; U.S. Pat. No. 4,248,863; U.S. Pat. No. 4,372,947; U.S. Pat. No. 5,023,184; U.S. Pat. No. 4,837,149; and EP 329606; each of which is incorporated herein by reference). For example, saframycins A-H, R, and S have been isolated from the culture broths of Streptomyces lavendulae, and saframycins M.sub.x1 and M.sub.x2, have been isolated from the culture broths of the myxobacterium, Myxococcus xanthus, each of the saframycins varying in the oxidation state of the ring system and in substitution of the core structure (Saito et al. Chem. Pharm. Bull. 43:777, 1995). Certain saframycins, namely A and C, exhibit extreme cytotoxicity toward cultured cells and toward several experimental tumor cell lines including leukemias L1210 and P388 and Ehrlich carcinoma (Arai, T.; Mikami, Y.; Okamoto, K.; Tokita, H.; Teras, K. In Advances in cancer chemotherapy; University Park Press, Baltimore, 1978, 235-251; Arai, T.; Takahashi, K.; Ishiguro, K.; Mikami, Y. Gann 1980, 71, 790-796; Ishiguro, K.; Sakiyami, Takahashi, K.; Arai, T. Biochemistry 1981, 17, 2545-2550; Arai, T.; Takahashi, K.; Nakahara, S.; Kubo, A. Experientia 1980, 36, 1025-1027; Myers, A. G.; Plowright, A. T. J. Am. Chem. Soc. 2001, 123, 5114; Martinez, E.; Owa, T.; Schreiber, S. L.; Corey, E. J. Proc. Natl. Acad. Sci. USA 1999, 96, 3496-3501). TABLE-US-00001 TABLE 1 IC.sub.50s in Tumor Cell Lines Compound A375 melanoma A549 lung carcinoma (-)-Saframycin A 5.3 nM 133 nM QAD 1.2 nM 4.4 nM Ecteinascidin 743 0.15 nM 1.0 nM Saframycin A has been shown to block RNA synthesis in cultured cells, and it has been suggested that saframycins A and C exhibit this potency because of their ability to bind covalently to DNA (for a discussion of the biological activity of the saframycins see, for example, Lown et al. Biochemistry, 1982, 21, 419; Ishiguro et al. Biochemistry, 1978, 17, 2545; Rao et al. Chem. Res. Toxicol., 1990, 3, 262, 1990; Ishiguro et al. J. Biol. Chem., 1981, 256, 2162). [0003] Saframycin A (SafA), the most potent member of this series, exhibits wide spectrum anti-cancer activity against Ehrlich ascites tumor, B16 melanoma, and murine leukemias P388 and L1210 (Arai, T.; Mikami, Y.; Okamoto, K.; Tokita, H.; Teras, K. In Advances in cancer chemotherapy; University Park Press, Baltimore, 1978; pp 235-251; Arai, T.; Takahashi, K.; Ishiguro, K.; Mikami, Y. Gann 1980, 71, 790-796; Ishiguro, K.; Sakiyami, Takahashi, K.; Arai, T. Biochemistry 1981, 17, 2545-2550; Arai, T.; Takahashi, K.; Nakahara, S.; Kubo, A. Experientia 1980, 36, 1025-1027). A structurally related class of non-quinoid natural products, the ecteinascidins, was found to possess an even more potent antiproliferative activity (Rinehart, K. L.; Holt, T. G.; Fregeau, N. L.; Keifer, P. A.; Wilson, G. R.; Perun, T. J.; Sakai, R.; Thompson, A. G.; Stroh, J. G.; Shield, L. S.; Seigler, D. S.; Li, L. H.; Martin, D. G.; Grimmelikhuijzen, C. J. P.; Gade, G. Journal of Natural Products 1990, 53, 771-792; Sakai, R.; Rinehart, K.; Guan, Y.; Wang, A. H. J. Proc. Natl. Acad. Sci. USA 1992, 89, 11456-11460). One member of the class, ecteinascidin 743 (Et-743), has advanced to Phase III clinical trials, and it was found to be particularly active against soft tissue sarcomas without obvious toxicity (Jimeno, J. M.; Faircloth, G.; Cameron, L.; Meely, K.; Vega, E. Gomez, A.; Sousa-Faro, J. M. F.; Drugs Future 1996, 21, 1155-1165; Bowman, A.; Twelves, C.; Hoekman, K.; Simpson, A.; Smyth, J.; Vermorken, J.; Hoppener, F.; Beijnen, J.; Vega, E.; Jimeno, J. Hanauske, A. R. Ann. Oncol. 1998, 9 (Suppl. 2), 119; Taamma, A.; Misset, J. L.; Riofrio, M. Guzman, C.; Brain, E.; LopezLazaro, L.; Rosing, H.; Jimeno, J. M.; Cvitkovi, E. J. Clin. Oncol. 2001, 19, 1256-1265). By employing an efficient synthetic route to SafA, a number of saframycin structural analogues have been prepared (see U.S. Ser. No. 10/011,466, filed Nov. 5, 2001; U.S. Ser. No. 60/245,888, filed Nov. 3, 2000; each of which is incorporated herein by reference). The most active analogue, the quinaldic acid analogue of SafA (QAD), possesses nearly 30-fold greater activity than SafA in a lung carcinoma cell line and 4-fold greater activity in a melanoma cell line. QAD has been reported to show 100-fold greater potency in three human sarcoma cell lines as compared to Et-743 (Myers, A. G.; Kung, D. W., J. Am. Chem. Soc. 1999, 121, 10828-10829; Myers, A. G.; Plowright, A. T. J. Am. Chem. Soc. 2001, 123, 5114-5115). [0004] Due to the potent antiproliferative activity of the ecteiniscidins, saframycins, and related analogues, elucidating their biological mode of action has been an area of active research ever since their discovery. Elucidating the biological target of the saframycins may lead to the development of assays which can be used to identify better pharmaceutical agents with the same mode of action as the saframycins. Also, since many other anti-proliferative agents are known to bind DNA, an activity which is believed to be central to their biological activities, the assays developed for use with saframycins may be applicable to other DNA-binding agents. SUMMARY OF THE INVENTION [0005] The present invention stems from the recognition that some anti-proliferative agents exert their effects on cells by binding to nuclear DNA, thereby forming a binary complex, which is then recognized by a cellular target. This recognition event may take the form of a DNA-repair enzyme that repairs the lesion or it may lead to a signaling event that initiates apoptosis (or both). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) has been found to be the cellular target of the saframycin A/dsDNA binary complex (see Examples below). Saframycin A has been shown to bind in the minor groove of DNA in a sequence-selective manner, and to alkylate the N2 position of guanine through an imminium ion intermediate generated from the elimination of a cyano or hydroxyl group at C21 of saframycin A (FIG. 1) (Arai, T.; Mikami, Y.; Okamoto, K.; Tokita, H.; Teras, K. In Advances in cancer chemotherapy; University Park Press, Baltimore, 1978; pp 235-251; Arai, T.; Takahashi, K.; Ishiguro, K.; Mikami, Y. Gann 1980, 71, 790-796; Ishiguro, K.; Sakiyami, Takahashi, K.; Arai, T. Biochemistry 1981, 17, 2545-2550; Arai, T.; Takahashi, K.; Nakahara, S.; Kubo, A. Experientia 1980, 36, 1025-1027; Lown et al. Biochemistry 1982, 21, 419). The resulting binary complex then interacts with GAPDH to mediate the lethality of saframycin A within the cell. Saframycin A, the quinaldic acid analogue (QAD), and ecteinascidin 743 all are found to target cellular GAPDH and thus are believed to have a common biological mechanism of action. [0006] In one aspect, the present invention provides a novel method of identifying the biological target of a DNA-binding agent. The method includes the formation of a binary complex of the polynucleotide and agent on a solid support such as a resin. The interaction between polynucleotide and agent is preferably covalent; however, the covalent interaction may be reversible. Either the polynucleotide or the agent may be attached directly to the solid support; however, preferably the polynucleotide is directly linked to the solid support through techniques known in the art. Once the binary complex is formed, it can be used to locate by affinity chromatography the biological target in a cell lysate, extracellular fluid, serum, plasma, blood, or any other solution or suspension which may be thought to contain the biological target. Once the ternary complex of agent, polynucleotide, and biological target has been formed on the solid support, the solid support with ternary complex attached is washed to remove non-binding proteins and cellular debris. The biological target can then be isolated and identified using various techniques in the field of protein biochemistry including SDS-polyacrylamide gel electrophoresis, silver staining, antibody binding, and/or protein microsequencing by mass spectroscopy. This novel method was used to successfully identify GAPDH as the cellular target of the saframycin A/dsDNA binary complex. The present invention also includes kits containing the reagents useful in the method of identifying a biological target. These kits may include saframycin A, saframycin analogues, ecteinascidin, ecteinascidin analogues, GAPDH protein, polynucleotides, cell lines, media, buffers, reagents, affinity resins, gels, nitrocellulose filters, vials, instruction manuals, etc., which may be useful in the practice of the inventive method. [0007] In another aspect, the present invention provides a method of identifying compounds with a known mechanism of action. In certain embodiments, the method allows for the identification of compounds with a greater affinity for the biological target as compared to a known agent. For example, having identified the target of saframycin A as GAPDH, the inventive method can be used to identify other compounds which also target GAPDH. A polynucleotide sequence is attached to the wells of a multi-well plate. In each well is then delivered a test compound. The compound is allowed to react and form a binary complex with the attached polynucleotide. A solution of GAPDH or cell lysate is then added to each well, and the amount of GAPDH bound by the test compound/polynucleotide binary complex is quantitated for comparison to other GAPDH-targeting compounds such as saframycin A, QAD, ecteinascidin 743, and phthalascidin. The amount of GAPDH bound by the binary complex may be quantitated by any known method including immunochemistry, SDS-PAGE followed by visualization with silver staining or Coomassie Blue staining, Western blot, fluorescence, chemiluminescence, capillary electrophoresis, etc. Compounds such as saframycin A, QAD, ecteinascidin 743, and phthalascidin may be used as positive controls in determining the amount of GAPDH bound. Compounds known to not target GAPDH, such as mitomycin C and cis-platin, may be used as negative controls. Compounds identified using the inventive method for screening may be further characterized using southwestern blotting, capillary electrophoresis, affinity chromatography, competition studies, siRNA studies, GAPDH translocation studies, in vitro cytotoxicity studies, in vivo animal model studies, etc. [0008] The method of identifying compounds targeting GAPDH is amenable to high-throughput screening techniques including robotic assisted fluid delivery, combinatorial chemistry, microfluidics, and computer analysis of the resulting data. In certain embodiments, a collection of compounds such as a combinatorial library may be provided for screening. In other embodiments, a historical collection of chemical compounds from a pharmaceutical company may screened using the inventive method. All compounds identified using the inventive method are within the scope of the present invention as well as methods of treating a proliferative disease such as cancer and other neoplasms using the identified compounds. [0009] In another aspect, the present invention provides kits with conveniently packaged materials needed to perform the inventive methods. The kits may include polynucleotides, affinity resins, chemical compounds (such as saframycin A, ecteinascidin 743, cis-platin, trans-platin, etc.) with known targets or mechanisms of action, buffers, reagents, purified proteins such as GAPDH, antibodies useful in detecting biological targets, cell lines, cell lysates, polyacrylamide gels, vials, multi-well plates, radioisotopes, software for high-throughput screening and statistical analysis, instruction manuals, etc. For example, a kit for screening chemical compounds or small molecules to identify compounds targeting GAPDH may include polynucleotides optionally bound to a solid support, saframycin A as a positive control, mitomycin C or cis-platin as a negative control, purified GAPDH, antibodies directed to GAPDH, and an instruction manual. The kit may also contain various buffers and reagents useful in the practice of the screening method. Preferably, the materials are conveniently packaged in aliquots useful in the practice of the inventive methods and/or useful in high throughput screening with robotic equipment for fluid delivery. [0010] The inventive methods and systems are not only useful in identifying compounds targeting GAPDH like saframycin A and ecteinascidin 743, but they may also be used to identify the targets of other agents and compounds with similar activity. For example, cis-platinated DNA has previously been found to bind HMG1. Therefore, the inventive methods could be used to identify compounds which bind DNA and the resulting binary complex targets HMG1. Other DNA binding agents useful in the inventive screening methods include topoisomerase poisons, e.g., camptothecin (and its analogs) and quinolone, which are known to target DNA topoisomerase II. BRIEF DESCRIPTION OF THE DRAWING [0011] FIG. 1A shows the structure of (-)-saframycin A (SafA); ecteinascidin 743 (Et 743); the quinaldic acid analogues of (-)-saframycin A, QAD R.dbd.CN, HQ R.dbd.OH; phthalascidin (Pt 650), an analogues of ecteinascidin 743; bispyridal, an inactive analogue of saframycin A. FIG. 1B shows the alkylation of DNA by (-)-saframycin A at a guanine residue. The DNA alkylation process is reversible and the DNA-drug adduct is labile. The alkylation reaction has been found to be sequence selective as the drug prefers to bind to certain three base pair motifs such as 5'-GGG-3',5'-GCC-3', and 5'-GGPy-3', wherein Py is a pyrimidine. [0012] FIG. 2 shows the scheme for constructing a drug-based AffiGel-15 affinity resin. [0013] FIG. 3 shows the use of a DNA-drug binary complex as an affinity ligand to identify the protein target of the complex. [0014] FIG. 4 is a photograph of a silver-stained SDS-PA gel showing the affinity chromatography results. The affinity matrix was incubated in bovine brain lysate at 4.degree. C., washed extensively, and heat denatured to release bound proteins. Lane 1: molecular weight standard; Lane 2: dsDNA resin:control matrix to lane 3, 4, and 5; Lane 3: QAD alkylated dsDNA resin; Lane 4: QAD alkylated dsDNA resin with 40 equivalents of QAD/dsDNA complex free in solution; Lane 5: Pt 650 alkylated dsDNA resin; Lane 6: dsDNA resin: control matrix to lane 7; Lane 7: SafA alkylated dsDNA resin; Lane 8: dsDNA resin control matrix to lane 9; Lane 9: MMC alkylated dsDNA resin; Lane 10: dsDNA resin control matrix to lane 11 and 12; Lane 11: cis-platin alkylated dsDNA resin; Lane 12: trans-platin alkylated dsDNA resin; Lane 13: dsDNA resin control matrix to lane 14; Lane 14: bispyridal alkylated dsDNA. [0015] FIG. 5 depicts the Southwestern blot technique using GAPDH and saframycins. FIG. 5A is a schematic of the Southwestern blot technique. FIG. 5B shows the Southwestern blot results with GAPDH and saframycins. The membranes with GAPDH were incubated with the various .sup.32P-labeled dsDNA-drug complexes. Lane 1: free dsDNA control; Lane 2: HQ alkylated dsDNA; Lane 3: QAD alkylated dsDNA; Lane 4: Pt 650 alkylated dsDNA; Lane 5: SafA alkylated dsDNA. [0016] FIG. 6 shows the nuclear GAPDH translocation after HQ treatment in HeLa-S3 cells. A. Accumulation of GAPDH in nuclear extracts from HeLa cells at 1 day, 2 days, and 3 days after treatment with 17.5 nM HQ. B. Confocal laser scanning micrograph of optical sections of HeLa-S3. Control was treated with 0.5% DMSO for 2 days; drug treated cells were incubated with 17.5 nM HQ in 0.5% DMSO for 2 days. [0017] FIG. 7 is a bar graph showing the results of assays for the effect of the DNA-drug binary complex on GAPDH glycolysis activity. The addition of drug, DNA, or dsDNA-drug complex to the glycolysis mixture showed no observable changes to the glycolytic activity of GAPDH. [0018] FIG. 8 shows GAPDH siRNA silencing experiments. GAPDH siRNA silencing renders A549 tumor cells resistant to QAD not cis-platin. FIG. 8A shows GAPDH expression levels as determined by Western blot analysis. FIG. 8B shows GI.sub.50 of cis-platin in A549 tumor cells unchanged when treated with GAPDH siRNA (left) and GI.sub.50 of QAD in A549 tumor cells increased approximately 9-fold when treated with GAPDH siRNA (right). DEFINITIONS [0019] A chemical compound or compound as used herein can include organometallic compounds, organic compounds, inorganic compounds, metals, transitional metal complexes, and small molecules. In certain preferred embodiments, polynucleotides are excluded from the definition of compounds. In other preferred embodiments, polynucleotides and peptides are excluded from the definition of compounds. In a particularly preferred embodiment, the term compounds refers to small molecules (e.g., preferably, non-peptidic and non-oligomeric) and excludes peptides, polynucleotides, transition metal complexes, metals, and organometallic compounds. [0020] A drug or drug candidate may include any chemical compound used in the clinic or to be used in the clinic for the treatment or prevention of a disease or condition. In certain embodiments, the drug or drug candidate is a small molecule. In other embodiments, the drug or drug candidate is a protein or peptide. In other embodiments, the drug or drug candidate is a polynucleotide. In certain embodiments, a drug or drug candidate is any pharmaceutical agent that has been approved by the Food and Drug Administration (FDA) for the treatment of a disease. A drug candidate may be in pre-clinical or clinical testing, and it may not be approved by the Food and Drug Administration but may be in the process of receiving FDA approval. For example, drugs for human use listed by the FDA under 21 C.F.R. .sctn..sctn. 330.5, 331 through 361; 440-460, and drugs for veterinary use listed by the FDA under 21 C.F.R. .sctn..sctn. 500-582, incorporated herein by reference, are all considered drugs in the present invention. For a more comprehensive discussion of drugs and drug candidates, see Goodman & Gilman's The Pharmacological Basis of Therapeutics, Ninth Ed., McGraw-Hill, 1996; The Merck Index, Eleventh Ed., 1989; The Merck Manual, Seventeenth Ed. 1999; the entire contents of each of which are hereby incorporated by reference. In certain preferred embodiments, a drug is an anti-neoplastic agent. In other embodiments, a drug is an antibiotic. Continue reading about Assay for identifying biological targets of polynucleotide-binding compounds... 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