E2 displacement assay for identifying inhibitors of HPV

An assay for the identification of inhibitors of HPV, comprising:

• a) contacting a HPV E2 transactivation domain with a probe to form a E2:probe complex and measuring a signal from said probe to establish a base line level;
• b) incubating the E2:probe complex with a test compound and measuring the signal from said probe;
• c) comparing the signal from step b) with the signal from step a);
• wherein said probe is a compound of formula (I) or its enantiomers or diastereoisomers thereof:
• wherein:
• A is a 5- or 6-membered homocyclic ring, or a 5- or 6-membered heterocyclic ring containing 1 or more heteroatoms selected from N, O and S;
• X is H and W is OH;
• or X and W together form a carbonyl group or an epoxide;
• R1 is H;
• or one or two substituents independently selected from the group consisting of:
• hydroxy, halo, lower alkyl, lower alkoxy, lower thioalkyl, haloalkyl, or —C(O)R2 wherein R2 lower alkyl, alyloxy or benzyloxy;
• Y is phenyl optionally mono- or di-substituted with R5 or C(O)R6, wherein R5 is lower alkyl, lower cycloalkyl, lower alkoxy, halo, hydroxy, nitrile or trifluoromethyl, and R6 is lower alkyl, lower cycloalkyl, lower alkoxy, hydroxy or trifluoromethyl;
• said phenyl ring being optionally fused with a saturated or unsaturated 4 to 6-membered ring optionally containing a heteroatom selected from N, O and S;
• or Y is a heterocycle (Het) containing one or more heteroatom selected from N, O or S, said Het optionally mono- or di-substituted with R5 or C(O)R6, wherein R5 and R6. are as defined above;
• said Het being optionally fused with a saturated or unsaturated 4 to 6-membered ring optionally containing a heteroatom selected from N, O and S;
• or Y is ethylene-phenyl, said ethylene moiety being optionally mono-substituted with lower alkyl, wherein said phenyl ring is optionally mono- or di-substituted with R5 or C(O)R6, wherein R5 and R6 are as defined above;
• said phenyl ring being optionally fused with a saturated or unsaturated 4- to 6-membered ring optionally containing a heteroatom selected from N, O and S;
• or Y is ethylene-Het, said ethylene moiety being optionally mono-substituted with lower alkyl, wherein Het is optionally mono- or di-substituted with R5 or C(O)R6, wherein R5 and R6 are as defined above;
• said Het being optionally fused with a saturated or unsaturated 4 to 6-membered ring optionally containing a heteroatom selected from N, O and S;
• R3 is selected from the group consisting of:
• lower alkyl, lower cycloalkyl, lower alkylene, aryl or lower aralkyl, all of which optionally mono- or di-substituted with:
• lower alkyl, lower cycloalkyl, haloalkyl, halo, CN, azido, lower alkoxy, (lower alkyl)acyl, C1-6 thioalkyl, C1-6 alkylsulfonyl, NHC(O)-lower alkyl, NHC(O)-aryl, NHC(O)—O-lower alkyl, NHC(O)O-aryl, aryl, aryloxy, hydroxy, nitro, amino, or Het, said Het optionally mono- or di-substituted with lower alkyl, lower cycloalkyl, lower alkoxy, halo, hydroxy, nitrile, trifluoromethyl, C(O)R6 wherein R6 is as defined above;
• said lower cycloalkyl, aryl, lower aralkyl or Het being optionally fused with a saturated or unsaturated 4 to 6-membered ring optionally containing a heteroatom selected from N, O and S;
• and
• R4 is a carboxylic acid, a salt or an ester thereof;
• and wherein said;
• probe of formula (I) is labeled with a detectable label or an affinity tag, wherein wavy lines represent bonds of unspecified stereochemistry;
• and wherein said signal is selected from:
• fluorescence, resonance energy transfer, time resolved fluorescence, radioactivity, fluorescence polarization, change in the intrinsic spectral
• properties, luminescence and plasma-resonance;
• whereby a modulation in said signal is an indication that said test compound binds to said transactivation domain.