Method of detection of influenza virus and compounds for use therein

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.

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 IC 50  for binding of less than 10 −6  M.  
     
     
         14 . A neuraminidase binder comprsiing an analogue of neuraminic acid (sialic acid) substituted at the C 7  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.  
     
     
         15 . A neuraminidase binder according to  claim 14 , in which the analogue of neuraminic acid is a compound of formula (II):  
       
         
           
           
               
               
           
         
         wherein  
         R represents an azido group, an unsubstituted or substituted guanidino group, or an unsubstituted or substituted amino group;  
         R 2  represents COCH 3 , COCF 3  or SO 2 CH 3 ;  
         X represents O or NH;  
         W represents a spacer group made up of a chain of atoms having a total length of between four and one hundred atoms, and optionally also comprising substituted carbon and/or nitrogen atoms and optionally including oxygen and/or sulphur atoms;  
         Y represents OH, SH, NH 2,  CH=O, CH=CH 2,  CO 2 H, CONHNH 2  or NH-biotinyl, or a protected form of one of these end functionalities;  
         and wherein the substituent on the amino or guanidino group, when present, is a substituted or unsubstituted C 1-6  alkyl group, or an amino, hydroxy, cyano or alkoxycarbonyl group;  
         with the proviso that X-W-Y does not include the group OC(=Z)NR 5 R 6 , wherein Z represents O or S, and R 5  and R 6  independently represents H or a hydrocarbon group optionally substituted by NH 2,  OH or SR.  
       
     
     
         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)  
       
         
           
           
               
               
           
         
         wherein R, R 2 , W and Y are as defined for formula (II) in  claim 14 , A and B are oxygen or CH 2  or may represent a single bond, and R 1  is a lipophilic C 1 -C 12  alkyl group which is optionally substituted by one or more halogen atoms, cycloalkyl, alkoxy, haloalkoxy or optionally substituted aryl groups.  
       
     
     
         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 CO 2 H groups, carbamates of the NH 2  and CONHNH 2  groups, and acetals of the CH=O group.  
     
     
         20 . A neuraminidase binder according to  claim 15 , in which R is guanidino and R 2  is acetyl.  
     
     
         21 . A neuraminidase binder according to  claim 15  or  claim 20 , in which X is O and Y is NH 2  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(CH 2 ) n , CONH(CH 2 ) n NHCONH(CH 2 ) m , CONH(CH 2 ) n [NHCO(CH 2 ) 5 ] m , CO(CH 2 ) n , CO(CH 2 ) n NHCONH(CH 2 ) m , CO(CH 2 ) n NHCO(CH 2 ) q , CONH(CH 2 ) n NH(COCH 2 NH) q COCH 2 ; CONH(CH 2 CH 2 O) n CH 2 CH 2 NHCO(CH 2 ) q , and CONH(CH 2 ) n NHCO(CH 2 ) q , wherein n amd m are integers between 2 and 12, and q is 0 or an integer between 1 and 12.  
     
     
         23 . A neuraminidase binder according to any one of  claims 14  to  18 , which has an IC 50  of less than 10 −6  M.  
     
     
         24 . A neuraminidase binder according to  claim 23 , which has an IC 50  of less than 10 −8  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.  
     
     
         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).  
     
     
         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.  
     
     
         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.  
     
     
         34 . A diagnostic composition comprising a compound according to any one of  claims 14  to  33 , together with a diagnostically-suitable carrier.  
     
     
         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 R 2  are as defined for formula (II), R 1  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)  
       
         
           
           
               
               
           
         
         in which A is a protecting group;  
         W and W′ are spacer groups as defined for W in  claim 15  or claim  18 ;  
         R and R 2  are as defined in claim  15 ;  
         R 1  is an alkyl group; and  
         Y′ is as defined in  claim 15  or claim  19 ;  
       
     
     
         38 . A method according to  claim 37 , in which A is C=O or CMe 2 .  
     
     
         39 . A method according to  claim 35 , in which A in formulae (IV) to (VI) is C=O or CMe 2 .  
     
     
         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 H 2 N-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 .