Method of detection of influenza virus and compounds for use therein

• US 20020055094A1
• Filed: 04/03/2001
• Published: 05/09/2002
• Est. Priority Date: 03/01/1996
• Status: Abandoned Application

• Current Assignee: Betty Jin, Guy Y. Krippner, Keith Geoffrey Watson, Phillip A. Reece, Wen-Yang Wu
• Original Assignee: Betty Jin, Guy Y. Krippner, Keith Geoffrey Watson, Phillip A. Reece, Wen-Yang Wu
• Inventors: Watson, Keith Geoffrey, Reece, Phillip A., Wu, Wen-Yang, Jin, Betty, Krippner, Guy Y.
• Application Number:
  US09/823,766
• Publication Number:
  US 20020055094A1
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: C07D 309/28 with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
• CPC Class Codes: C07D 407/06 linked by a carbon chain containing only aliphatic carbon atoms
• CPC Class Codes: C07D 495/04 Ortho-condensed systems
• CPC Class Codes: G01N 33/56983 Viruses
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes

Abstract

The invention provides a method of detection of influenza virus which utilises compounds able to bind specifically to the active site of influenza virus neuraminidase, and novel compounds for use in the method.

Claims

1. A method of detection of influenza virus, comprising the step of exposing a sample suspected to comprise said virus to a compound (neuraminidase binder) able to bind specifically to the active site of influenza virus neuraminidase.

2. A method according to claim 1, in which the neuraminidase binder is attached to a support material such that virus particles will be selectively captured and concentrated when a sample is passed over or through the support.

3. A method according to claim 1 or claim 2, in which the neuraminidase binder is linked via a spacer group to a surface.

4. A method according to claim 3, in which the spacer group terminates in a functionality able to bind to a surface.

5. A method according to claim 4, in which the terminal functionality is a biotinyl group and the surface is coated with avidin, streptavidin, or an antibody directed against biotin.

6. A method according to claim 4, in which the terminal functionality is an amino group and the surface comprises carboxy groups.

7. A method according to claim 1, in which the neuraminidase binder is linked to a detectable label.

8. A method according to claim 7, in which the detectable label is covalently coupled to the neuraminidase binder.

9. A method according to claim 7 or claim 8 in which the virus particles in a sample are exposed to a neuraminidase binder coupled to a detectable label, under conditions such that the binder binds selectively to the viral neuraminidase on the surface of the viral particle.

10. A method according to claim 7 or claim 8, which comprises the steps of selective capture and concentration of the virus.

11. A method according to claim 1, comprising the step of selective capture and selective detection of influenza virus.

12. A method according to claim 11, comprising the steps of:

a) exposing the sample to a neuraminidase binder bound to a support, and

b) exposing influenza virus particles retained on the support to a neuraminidase binder linked to a detectable label.

13. A method according to any one of the preceding claims, in which the neuraminidase binder has an IC₅₀ for binding of less than 10⁻

6 M.

15. A neuraminidase binder according to claim 14, in which the analogue of neuraminic acid is a compound of formula (II):

16. A neuraminidase binder according to claim 15, in which the X-W-Y group attached at the 7-position is a terminally functionalized N-substituted-alkyl carbamate.

17. A neuraminidase binder according to claim 14, in which the analogue of neuraminic acid is a compound of formula (IIA) or (IIB)

18. A neuraminidase binder according to claim 17, in which the spacer group W is selected from the group consisting of linear peptides;

oligosaccharides;

polyols;

polyethylene glycol groups; and

hydrocarbon groups linked together with oxygen or sulphur atoms or with carbonyl, amido, urea or hydrazide functionalities, or combinations of these groups.

19. A neuraminidase binder according to any one of claims 15 to 18, in which the protecting group for the end functionality Y is selected from the group consisting of esters of the OH, SH and CO₂H groups, carbamates of the NH₂ and CONHNH₂ groups, and acetals of the CH=O group.

20. A neuraminidase binder according to claim 15, in which R is guanidino and R² is acetyl.

21. A neuraminidase binder according to claim 15 or claim 20, in which X is O and Y is NH₂ or NH-biotinyl.

22. A neuraminidase binder according to any one of claims 15, 20 and 21, in which W is a spacer group selected from the group consisting of CONH(CH₂)_n, CONH(CH₂)_nNHCONH(CH₂)_m, CONH(CH₂)_n[NHCO(CH₂)₅]_m, CO(CH₂)_n, CO(CH₂)_nNHCONH(CH₂)_m, CO(CH₂)_nNHCO(CH₂)_q, CONH(CH₂)_nNH(COCH₂NH)_qCOCH₂;

CONH(CH₂CH₂O)_nCH₂CH₂NHCO(CH₂)_q, and CONH(CH₂)_nNHCO(CH₂)_q, wherein n amd m are integers between 2 and 12, and q is 0 or an integer between 1 and 12.

24. A neuraminidase binder according to claim 23, which has an IC₅₀ of less than 10⁻

8 M.

33. A neuraminidase binder according to claim 14 which is linked to a detectable label, wherein the neuraminidase binder is a compound disclosed in International Patent Applications No. WO/92/06691, No. WO/91/16320 or No. WO/96/26933, or in U.S. Pat. No. 5,453,533.

35. A method of synthesis of a neuraminidase binder of formula (II) according to claim 15, in which X is oxygen, comprising the steps of:

a) converting a sialic acid derivative of formula (III) to an 8,9-protected compound of formula (IV),

in which R and R² are as defined for formula (II), R¹ is an alkyl group, and A is a protecting group;

b) acylating the 7-hydroxy group;

c) optionally extending or functionalising the spacer group W′

, and

d) removing the protecting groups.

36. A method of synthesis of a neuraminidase binder of formula (II) according to claim 15, in which X is NH, comprising the steps of

a) reacting a compound of formula (IV) as defined in claim 35 under Mitsonobu reaction conditions to give a leaving group ester with inverted stereochemistry at C7;

and

b) displacing the C7 ester with a nucleophile.

37. A compound of general formula (V) or (VI)

38. A method according to claim 37, in which A is C=O or CMe₂.

39. A method according to claim 35, in which A in formulae (IV) to (VI) is C=O or CMe₂.

40. A method according to claim 35 or claim 36, in which the 7-hydroxy group is acylated with an isocyanate group OCN-W′

-Y, and where W′

is a spacer group as defined for W in claim 15 or claim 18 and Y is as defined in claim 15 or claim 19.

41. A method according to claim 35 or claim 36, in which the 7-hydroxy group is acylated with an acyl chloride of formula ClCOL, in which L is a leaving group, and L is then displaced by an amine of formula H₂N-W′

-Y, where W′

is as defined for W in claim 15 or claim 18, and Y is as defined in claim 15 or claim 19.

14. A neuraminidase binder comprsiing an analogue of neuraminic acid (sialic acid) substituted at the C₇ or equivalent position with a spacer group suitable for coupling to a detectable label or to a surface, in which said analogue does not comprise a detectable label or spacer group which is cleaved by the neuraminidase.

23. A neuraminidase binder according to any one of claims 14 to 18, which has an IC₅₀ of less than 10⁻

6 M.

25. A neuraminidase binder according to any one of claims 14 to 17, selected from the compounds of Table 1, other than Compound 11.

30. A neuramindase binder according to any one of claims 14 to 29, linked to a detectable label.

31. A neuraminidase binder according to claim 30, in which the link is covalent.

32. A neuraminidase binder according to claim 30 or claim 31, in which the detectable label is an epitope suitable for use in an antibody detection kit, in an optical assay device having an active receptive surface, or in an agglutination detection system.

34. A diagnostic composition comprising a compound according to any one of claims 14 to 33, together with a diagnostically-suitable carrier.

26. 5-acetamido-7-(6′

-biotinylamino-triglycinamido-hexyl) -carbamoyloxy-4-guanidino-2,3,4,5-tetradeoxy-D-glycero-D-galacto-non-2-enopyranosonic acid (21).

27. 5-acetamido-7-(6′

-(6″

-(6′

″

-aminocaproyl) -triaminocaproyl)-aminohexyl)-carbamoyloxy-4-guanidino -2,3,4,5-tetradeoxy-D-glycero-D-galacto-non-2-enopyranosonic acid (25).

28. 5-acetamido-7-{6′

-[6″

-(6′

″

-biotinylaminocaproyl) -tri-aminocaproyl]-aminohexyl}-carbamoyloxy-4-guanidino -2,3,4,5-tetradeoxy-D-glycero-D-galacto-non-2-enopyranosonic acid (26).

29. 5-acetamido 7-{6′

-[6″

-(6′

″

-hydrazidosuccinylaminocaproyl)-triaginocaproyl]-aminohexyl}-carbamoyloxy-4-guanidino-2,3,4,5-tetradeoxy-D-glycero-D-galacto-non-2-enopyranosonic acid (30).