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1. (WO2002050259) COMPOSITIONS, METHODS AND KITS FOR IDENTIFYING PROTEIN-PROTEIN INTERACTION DISRUPTING AGENTS
Dernières données bibliographiques dont dispose le Bureau international   

N° de publication :    WO/2002/050259    N° de la demande internationale :    PCT/CA2001/001770
Date de publication : 27.06.2002 Date de dépôt international : 13.12.2001
Demande présentée en vertu du Chapitre 2 :    17.07.2002    
CIB :
C12N 15/10 (2006.01)
Déposants : NATIONAL RESEARCH COUNCIL OF CANADA [CA/CA]; Building M 58, Montreal Road, Ottawa, Ontario K1A 0R6 (CA) (Tous Sauf US).
ZHAO, Hui-Fen [CA/CA]; (CA) (US Seulement).
SHEN, Shi-Hsiang [CA/CA]; (CA) (US Seulement)
Inventeurs : ZHAO, Hui-Fen; (CA).
SHEN, Shi-Hsiang; (CA)
Mandataire : CONN, David; Borden Ladner Gervais LLP, World Exchange Plaza, 100 Queen Street, Suite 1100, Ottawa, Ontario K1P 1J9 (CA)
Données relatives à la priorité :
60/255,910 18.12.2000 US
Titre (EN) COMPOSITIONS, METHODS AND KITS FOR IDENTIFYING PROTEIN-PROTEIN INTERACTION DISRUPTING AGENTS
(FR) COMPOSITIONS, METHODS AND KITS FOR IDENTIFYING PROTEIN-PROTEIN INTERACTION DISRUPTING AGENTS
Abrégé : front page image
(EN)The present invention provides compositions, methods, and kits for identifying agents that are capable of disrupting protein-protein interaction in a mammalian reverse two-hybrid system. A tetracycline repressor protein was fused with the inhibitory KRAB domain as a suppressor to tightly regulate the reverse two-hybrid system for mammalian cells. Binding of the chimeric protein to the tetracycline operator sequence provided within a promoter entirely suppressed the expression of two interactive proteins. When both an inducer and a candidate protein-protein interaction disrupting agent such as a drug were added in the cell culture together, the reporter gene was either activated by the interaction of protein-pair if the drug was ineffective or remained silent due to the disruption of the protein-protein interaction by the effective drug. The plasmids for the suppressor and the reporter were integrated into chromosomes. Constructs for the expression of interactive proteins were either stably or transiently transfected into the cells. The utility of this system for screening drugs, particularly for enzyme inhibitor drugs, was demonstrated by using two well characterized interactions of the type I receptor for TGF$g(b) with FKPB12 and the EGF receptor with p85. The interaction between TGF$g(b)RI and FKPB12 or between EGF receptor and p85 were disrupted by FK506 and kinase inhibitor AG1478, respectively. The mammalian reverse two-hybrid system of the present invention can also be used for high throughput screening of compounds that disrupt protein-protein interactions.
(FR)The present invention provides compositions, methods, and kits for identifying agents that are capable of disrupting protein-protein interaction in a mammalian reverse two-hybrid system. A tetracycline repressor protein was fused with the inhibitory KRAB domain as a suppressor to tightly regulate the reverse two-hybrid system for mammalian cells. Binding of the chimeric protein to the tetracycline operator sequence provided within a promoter entirely suppressed the expression of two interactive proteins. When both an inducer and a candidate protein-protein interaction disrupting agent such as a drug were added in the cell culture together, the reporter gene was either activated by the interaction of protein-pair if the drug was ineffective or remained silent due to the disruption of the protein-protein interaction by the effective drug. The plasmids for the suppressor and the reporter were integrated into chromosomes. Constructs for the expression of interactive proteins were either stably or transiently transfected into the cells. The utility of this system for screening drugs, particularly for enzyme inhibitor drugs, was demonstrated by using two well characterized interactions of the type I receptor for TGF$g(b) with FKPB12 and the EGF receptor with p85. The interaction between TGF$g(b)RI and FKPB12 or between EGF receptor and p85 were disrupted by FK506 and kinase inhibitor AG1478, respectively. The mammalian reverse two-hybrid system of the present invention can also be used for high throughput screening of compounds that disrupt protein-protein interactions.
États désignés : AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, OM, PH, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZM, ZW.
Organisation régionale africaine de la propriété intellectuelle (ARIPO) (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZM, ZW)
Office eurasien des brevets (OEAB) (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM)
Office européen des brevets (OEB) (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR)
Organisation africaine de la propriété intellectuelle (OAPI) (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG).
Langue de publication : anglais (EN)
Langue de dépôt : anglais (EN)