E2 displacement assay for identifying inhibitors of HPV

• US 6,825,176 B2
• Filed: 02/05/2003
• Issued: 11/30/2004
• Est. Priority Date: 02/07/2002
• Status: Expired due to Term

• Current Assignee: Applied Materials Incorporated, Boehringer Ingelheim Limited (C.H. Boehringer Sohn AG & Co. KG)
• Original Assignee: Boehringer Ingelheim Limited (C.H. Boehringer Sohn AG & Co. KG)
• Inventors: Yoakim, Christiane, White, Peter
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US10/358,790
• Publication Number:
  US 20030194698A1
• Time in Patent Office: 664 Days
• Field of Search: 435/6, 514/1, 514/44, 514/351, 514/152, 514/231.5, 536/23.1, 548/127
• US Class Current: 514/44.00R
• CPC Class Codes: C07D 407/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07D 491/10 Spiro-condensed systems
• CPC Class Codes: G01N 2333/025 Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
• CPC Class Codes: G01N 2500/10 involving cells
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: G01N 33/534 with radioactive label
• CPC Class Codes: G01N 33/535 with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/56983 Viruses
• CPC Class Codes: G01N 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/583 with non-fluorescent dye label
• CPC Class Codes: G01N 33/60 involving radioactive labelled substances

Abstract

The present invention generally relates to an assay for identifying inhibitors of Human Papillomavirus (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 R¹, A, X, W, Y, R³ and R⁴ are as defined herein; or a derivative thereof, wherein said derivative is a probe of formula (I) 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.

Claims

1. 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, C^1-6 thioalkyl, C^1-6 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.

2. An assay for the identification of inhibitors of HPV replication, comprising:

a) contacting a transactivation domain of HPV E2 protein with a probe of formula I as defined in claim 1, to form an E2;

probe complex and measuring a signal from said probe to establish a base line level;

b) incubating a E2 protein with a test compound;

b′

) adding a probe of formula (I) to said mixture of E2 and test compound from step b) and measuring the signal from said probe; and

c) comparing the signal from step a) with the signal from step b′

);

whereby a modulation in said signal is an indication that said test compound binds to said transactivation domain.

3. The assay according to claim 1, wherein said detectable label is selected from the list consisting of:

a fluorescent label, a chemiluminescent label, a colorimetric label, an enzymatic marker, and a radioactive isotope.

4. The assay according to claim 1, wherein said detectable label is a fluorescent label selected from the list consisting of:

fluorescein, Oregon green, dansyl, rhodamine, tetra-methyl rhodamine, Texas-red, phycoerythrin BODIPY®

FL, BODIPY®

493/503 and Eu³⁺.

5. The assay according to claim 4, wherein said detectable label is a chemiluminescent label.

6. The assay according to claim 3, wherein said radioactive isotope is selected from the list consisting of:

³H, ¹⁴C, and ¹²⁵I.

7. The assay according to claim 1, wherein said affinity tag comprises a ligand whose strong affinity for a receptor is used to extract from a solution the entity to which said ligand is attached.

8. The assay according to claim 7, wherein said ligand is selected from the list consisting of:

biotin, a poly-histidine peptide and a defined epitope recognizable by a specific antibody.

9. The assay according to claim 1, wherein said probe is a compound of formula (I), present in an “

cis/cis”

relative stereochemistry represented as follows;

wherein R¹ is a lower alkyl group;

A is a 6-membered carbocyclic ring or a 5-membered sulfur-containing heterocycle;

X is H and W is OH;

or X and W form a carbonyl group;

Y is an phenyl group optionally mono- or di-substituted with lower alkyl or halo;

R³ is aryl substituted with a fluorescent label, a chemiluminescent label, or a radioactive label; and

R⁴ is a carboxyl group.

10. The assay according to claim 9, wherein said probe comprises pure enantiomers of compounds of formula (Ia) or (Ib) with the relative stereochemistry “

cis/cis”

;

wherein R¹, A, X, W, Y, R³ and R⁴ are as defined in claim 9.

11. The assay according to claim 10, wherein said probe comprises pure cis/cis enantiomers of compounds of formulae IIa and IIb:

wherein R¹ is a lower alkyl group;

X and W form a carbonyl group;

Y is an phenyl group optionally mono- or di-substituted with lower alkyl or halo;

R³ is aryl substituted with a fluorescent label, a chemiluminescent label, or a radioactive label; and

R⁴ is a carboxyl group.

12. The assay according to claim 11, wherein for said probe, R¹ is methyl, Y is phenyl substituted with R⁵ wherein R⁵ is one or two substituents selected from:

Cl or Br; and

R³ is phenyl substituted with —

CH₂—

NH—

C(O)—

R^3A or —

(CH₂)—

NH—

C(S)—

R^3A wherein R^3A is a fluorescent label, a chemiluminescent label, or a radioactive label.

13. The assay according to claim 12, wherein said probe has the following formula:

wherein R⁵ is di-bromo, and R³ is phenyl substituted with —

CH₂—

NH—

C(O)—

R^3A or —

(CH₂)—

NH—

C(S)—

R^3A wherein R^3A is a tritiated —

CH₃, a fluorescent label or a chemiluminescent label.

14. The assay according to claim 13, wherein R³ is selected from:

wherein the * represents a tritium label.

15. The assay according to claim 14, wherein said R³ is

wherein the * represents a tritium label.

16. The assay according to claim 1, wherein said probe is selected from:

17. The assay according to claim 1, wherein said E2 transactivation domain is selected from the list consisting of:

full length E2 protein, a protein comprising amino acids 1-190 of the full length E2 protein, SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.5.

18. The assay according to claim 17, wherein said E2 transactivation domain is from a low risk type papillomavirus.

19. The assay according to claim 18, wherein said low risk papillomavirus is selected from:

HPV-6 and HPV-11.

20. The assay according to claim 19, wherein said low risk papillomavirus is HPV-11.

27. An assay of claim 1 for identifying inhibitors of HPV using a probe having the formula:

wherein R⁵ is lower alkyl, lower cycloalkyl, lower alkoxy, halo, hydroxy, nitrile or trifluoromethyl, and R³ is aryl substituted with a fluorescent label, a chemiluminescent label, or a radioactive label.

29. A kit for testing compounds that potentially bind to the transactivation domain of HPV E2, said kit comprising a E2:

probe complex as defined in claim 1; and

instructions on how to use said probe for identifying test compounds binding to said transactivation domain.

30. A reagent for testing compounds that potentially bind to the transactivation domain of HPV E2, said reagent comprising a E2:

probe complex as defined in claim 1.

31. The assay according to claim 5 wherein said chemiluminescent label is luciferase.

21. A probe that binds to the transactivation domain of HPV-11 E2 and is capable of being displaced by a potential inhibitor thereof;

said probe having the formula;

wherein R⁵ is lower alkyl, lower cycloalkyl, lower alkoxy, halo, hydroxy, nitrile or trifluoromethyl, and R³ is aryl substituted with a fluorescent label, a chemiluminescent label, or a radioactive label.

22. The probe according to claim 21, wherein R⁵ is one or two halogen substituents.

23. The probe according to claim 22, wherein R³ is phenyl substituted with —

CH₂—

NH—

C(O)—

R^3A or —

(CH₂)—

NH—

C(S)—

R^3A wherein R^3A is a fluorescent label, a chemniluminescent label, or a radioactive label.

24. The probe according to claim 23, wherein R³ is selected from:

wherein the * represents a tritium label.

25. The probe according to claim 24, wherein said R³ is

wherein the * represents a tritium label.

26. The probe according to claim 21, selected from:

invention is selected from the group consisting of;

28. A kit for testing compounds that potentially bind to HPV E2, said kit comprising a probe as defined in claim 21;

and instructions on how to use said probe for identifying test compounds binding to said E2.