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(12) Patent Application Publication (10) Pub. No.: US 2004/0023207 A1 Polansky (43) Pub US 20040023207A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2004/0023207 A1 Polansky (43) Pub. Date: Feb. 5, 2004 (54) ASSAYS FOR DRUG DISCOVERY BASED ON Publication Classification MICROCOMPETITION WITH A FOREIGN POLYNUCLEOTIDE (51) Int. Cl." .............................. C12O 1/70; C12O 1/68; C12N 15/85; C12N 15/86 (52) U.S. Cl. .................. 435/5; 435/6; 435/455; 435/456 (76) Inventor: Hanan Polansky, Rochester, NY (US) (57) ABSTRACT Correspondence Address: A recent discovery showed that microcompetition between a Hanan Polansky foreign polynucleotide and a cellular polynucleotide is a risk 3159 S. Winton Rd. factor for Some of the major chronic diseases. The invention Rochester, NY 14623 (US) uses this novel discovery to present assays for Screening compounds based on their effectiveness in modulating Such microcompetition. The effective compounds can be used in (21) Appl. No.: 10/211,295 treatment of these chronic diseases. The invention also presents assays for Screening compounds that can be used in treatment of chronic diseaseS resulting from other foreign (22) Filed: Aug. 1, 2002 polynucleotide-type disruptions. Patent Application Publication Feb. 5, 2004 Sheet 1 of 6 US 2004/0023207 A1 s e 2 9 s H c 2 4 competitor plasmid/test plasmid Figure 1 1 O 09% 1 2 3 4 competitor plasmid/test plasmid -H Ltk- (pSV2Neo) -A-ML (pSV2neo) . A- - - Ltk- (pA1 Oneo) - - - - - - Mill (pA1 Oneo) Figure 2 Patent Application Publication Feb. 5, 2004 Sheet 2 of 6 US 2004/0023207 A1 e 2 r U H c O s S 2 3. Molar ratio (pSV2NeolhMT-ilA-CAT) Figure 3 Patent Application Publication Feb. 5, 2004 Sheet 3 of 6 US 2004/0023207 A1 2 x e S. 0.2 0.3 0.4 Molar ratio of bgallCAT Figure 5 p Controls o Differen tiated Patent Application Publication Feb. 5, 2004 Sheet 4 of 6 US 2004/0023207 A1 5% FBS S-RPM Serum-free S-RPM 35 p---...----...- - - - - - - How - Humun 3 4 3 4 5 6 Time (days) Time (days } pIRES-AR pcDNA-AR pSG5-AR Figure 8 Patent Application Publication Feb. 5, 2004 Sheet 5 of 6 US 2004/0023207 A1 w s s w2 f Figure 9 -o-Empty vector - - - - - - AntiSense -A-Wild type Figure 10 Patent Application Publication Feb. 5, 2004 Sheet 6 of 6 US 2004/0023207 A1 3x} wn; : KC "h 250 error- or --- E er.E i. m E 2000 --------- --------------- rt w 2 15x -- a-- 1 y & - E s x co-si ? asC- six or . .''.g..." a'aa?h.A ?h - wow"ws.x was aw: I -- k. h as: Stase A 3.38663 occagas:the oes ::::::: 13 30 45 75 90 s 12 13 Days Figure 11 t - ac C k A. d Assass333333333333333.5AAA e.g. gigger pet 0 on 9. r s -G5 5 63 72 9. 18 7 days Figure 12 US 2004/0023207 A1 Feb. 5, 2004 ASSAYS FOR DRUG DSCOVERY BASED ON the cellular Rb gene, a pharmaceutical composition can be MICROCOMPETITION WITH A FOREIGN administered to the Subject that reduces the copy number of POLYNUCLEOTDE the p300/cbp virus by, for instance, reducing viral replica tion, reduces binding of a p300/cbp transcription factor, Such BACKGROUND OF THE INVENTION as GABP, to the p300/cbp virus, increases expression of the p300/cbp transcription factor, increases binding of the p300/ 0001. The cause of many cases of the major chronic cbp transcription factor to the Rb promoter by, for instance, diseases is unknown. Therefore, treatment is focused on Stimulating phosphorylation of the p300/cbp transcription clinical Symptoms associated with the disease rather than the factor, or increases expression of Rb, through, for instance, cause. As a result, in many cases, the treatment shows transfection of an exogenous Rb gene, reduced degradation limited efficacy and Serious negative side effects. of the Rb protein, or administration of exogenous Rb protein 0002 Recently, the National Cancer Institute (NIH Guide (see more examples below). In the case of another foreign 2000") announced a program aimed to "reorganize the polynucleotide-type disruption, for example, the composi "front-end,' or gateway, to drug discovery in cancer. The tion may reverse the effects of Such disruption. For instance, new approach promotes a three Stage discovery process, microcompetition with a p300/cbp virus reduces expression first, discovery of the molecular mechanisms underlying of Rb. A mutation can also reduce the expression of Rb. neoplastic transformations, cancer growth and metastasis, Therefore, Such mutation is a foreign polynucleotide-type Second, Selection of a novel molecular target within the disruption. Microcompetition with a p300/cbp virus can discovered biochemical pathway associated with the disease result in cancer, and, therefore, a mutation in the Rb pro State; finally, design of a new drug that modifies the Selected moter that reduces Rb expression can also result in cancer. target. The program encourages moving away from Screen To ameliorate the Symptoms of cancer resulting from Such ing based on a clinical effects, Such as tumor cell shrinkage, mutation in the Rb gene, a pharmaceutical composition can either in Vivo or in Vitro, to Screening, or drug design, based be administered to the Subject that Stimulates complex on molecular effects. According to the NCI, Screening by a formation between a p300/cbp transcription factor and Rb. desired clinical effect identified drugs that traditionally 0006. In second aspect, the invention provides assays for demonstrated clear limitations in patients, while Screening Screening test compounds to find compounds which modu by a desired molecular effect should produce more effica late microcompetition between a foreign polynucleotide and cious and Specific drugs. a cellular polynucleotide, an effect of Such microcompeti 0003. The best drugs reverse the molecular events that tion, or an effect of another foreign polynucleotide-type cause a disease. Following the discovery of microcompeti disruption. tion between foreign polynucleolitides and cellular genes as 0007 A further aspect of the invention provides methods the cause of many chronic disease cases, the present inven for determining the risk of developing the molecular, cellu tion presents methods for treating chronic diseases, methods lar and clinical Symptoms associated with a chronic disease. for evaluating the effectiveness of a compound for use in The method may include detecting in a biological Sample modulating the progression of chronic diseases, and meth obtained from a Subject at least one of the following: (i) a ods for determining whether a Subject has a chronic disease, foreign polynucleotide, Specifically, a p300/cbp virus (ii) or has an increased risk of developing clinical Symptoms modified expression or bioactivity of a gene Suceptible to asSociated with Such disease. microcompetition with a foreign polynuleotide, Specifially, a p300/cbp regulated gene (iii) presence of a genetic lesion in BRIEF SUMMARY OF THE INVENTION a gene Suceptible to microcompetition with a foreign polyn 0004. In one aspect, the invention presents methods for culetide, Specifically, a gene encoding a p300/cbp factor, a treating chronic diseases. In a preferred embodiment, the p300/cbp regulated gene, p300/cbp factor kinase or p300/ methods feature administration to a Subject a therapeutically cbp phosphatase, or p300/cbp agent (iv) presence of a effective amount of a pharmaceutical or nutraceutical com genetic lesion in a DNA binding box of a p300/cbp tran position that attenuates microcompetition between a foreign Scription factor. polynucleotide and a cellular polynucleotide, attenuates an effect of Such microcompetition, or attenuates an effect of BRIEF DESCRIPTION OF THE FIGURES another foreign polynucleotide-type disruption. A pharma 0008 FIG. 1 shows the observed relative CAT activity as ceutical or nutraceutical composition may include, but not a function of the relative concentration of the competitor limited to, Small molecule (organic or inorganic), polynucle plasmid pX1.0 to the test plasmid pSV2CAT. otide, polypeptide or antibody. 0009 FIG.2 shows the observed relative CAT activity as 0005 For example, to ameliorate a disease symptom a function of the relative concentration of the competitor resulting from microcompetition between a foreign poly plasmid pSV2neo to the test plasmid pSV2CAT, and the nucleotide and a cellular polynucleotide, a pharmaceutical relative concentration of the competitor plasmid pA10neo to composition can be administered to the Subject that reduces the test plasmid pSV2CAT, in either Ltk- or ML fibroblast the cellular copy number of the foreign polynucleotide, cells. reduces complex formation between the foreign polynucle otide and a cellular transcription factor, increases complex 0010 FIG. 3 shows the observed relative CAT activity, formation between the microcompeted cellular transcription expressed as the ratio between CAT activity in the presence factor and the cellular polynucleotide, or reverses an effect of pSV2neo and CAT activity in the absence of pSV2neo, as of microcompetition on the expression or activity of a a function of the molar ratio of pSV2Neo to hMT-IIA-CAT. polypeptide with expression regulated by the cellular poly 0011 FIG. 4 shows accumulation of triglyceride assayed nucleotide. For example, in the case of a p300/cbp virus and by oil red staining in untreated F442A cells or in cells US 2004/0023207 A1 Feb. 5, 2004 transfected with either a vector expressing the SV40 large T 0023 a) Microcompetition Related Elements antigen or the “empty vector p7.IPNeo. 0024 (1) Microcompetition 0012 FIG. 5 shows the observed relative CAT activity as a function of the molar ratio between the competitor plasmid 0.025 Definition CMV-Bgal and the test plasmid PDGF-B-CAT, or between 0026 Assume the DNA sequences DNA and DNA-bind the competitor plasmid SV40-fgal and the test plasmid the transcription complexes C and C, respectively. If C PDGF-B-CAT. and C include the same transcription factor, DNA and DNA, are called “microcompetitors." A special case of 0013 FIG.
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