Screening methods

Abstract

The present invention provides materials and methods relating to screening for compounds useful in the treatment of Alzheimer's disease and related conditions. In particular, screening methods using tyrosine kinases are provided, as are methods relating to the role of tyrosine kinases as therapeutic targets.

1 .- 51 . (canceled) 
     
     
         52 . An in vitro method of screening for substances which are candidate therapeutic agents for the treatment of tauopathies, said substance being effective to inhibit the phosphorylation of a tau protein by a tyrosine kinase, wherein the tau protein comprises at least one phosphorylation site, the method comprising:
 (a) contacting at least one said substance, the tau protein and tyrosine kinase under conditions in which the tyrosine kinase is capable of phosphorylating the site(s) of the tau protein in the absence of the substance;   (b) detecting whether, and optionally the extent to which, the substance inhibits the phosphorylation of the tau protein at one or more sites of the tau protein by the tyrosine kinase; and,   (c) selecting the substance which inhibits phosphorylation of the tau protein at one or more of the sites;   wherein the tyrosine kinase is selected from the group consisting of Fyn or Syk.   
     
     
         53 . The method of  claim 52 , wherein the tau protein is paired helical filament tau. 
     
     
         54 . The method of  52 , wherein the tau protein is a fragment or derivative of a tau protein having the amino acid sequence set out in  FIG. 1 . 
     
     
         55 . The method of  claim 52 , wherein the tau protein has greater than 80% sequence identity with a tau protein having the amino acid sequence set out in  FIG. 1 . 
     
     
         56 . The method of  claim 52 , wherein the tyrosine kinase phosphorylates tau protein at one or more sites selected from the group consisting of Y18, Y29, Y197, Y310 and Y394 of tau protein. 
     
     
         57 . The method of  claim 52 , wherein the tyrosine kinase is Fyn. 
     
     
         58 . The method of  claim 57 , wherein Fyn phosphorylates tau protein at one or more sites selected from the group consisting of Y18 and Y310 of tau protein. 
     
     
         59 . The method of  claim 52 , wherein the tyrosine kinase is Syk. 
     
     
         60 . The method of  claim 59 , wherein Syk phosphorylates tau protein at Y18, Y29, Y197 and Y394 of tau protein. 
     
     
         61 . The method of  claim 52 , wherein the method comprises detecting in step (b) whether, and optionally the extent to which, the substance undergoing screening inhibits the phosphorylation of a substrate by the tyrosine kinase. 
     
     
         62 . The method of  claim 61 , wherein the substrate of the tyrosine kinase is not a tau protein or a fragment thereof. 
     
     
         63 . The method of  claim 52  wherein the method comprises determining in step (b) whether, and optionally the extent to which, the substance undergoing screening inhibits the phosphorylation of a substrate by the casein kinase 1. 
     
     
         64 . The method of  claim 63 , wherein the substrate of the tyrosine kinase is not a tau protein or a fragment thereof. 
     
     
         65 . The method of  claim 63 , wherein the method further comprises confirming whether a substance selected in an initial screen has the property of inhibiting the phosphorylation of the tau protein under conditions in which the tyrosine kinase is capable of phosphorylating the site(s) of the tau protein in the absence of the substance. 
     
     
         66 . The method of  claim 52  wherein the step of determining the presence, absence or extent of phosphorylation at one or more sites of the tau protein employs mass spectroscopy or a site specific recognition agent which is capable of distinguishing between a phosphorylated and a non-phosphorylated site. 
     
     
         67 . The method of  claim 66 , wherein the site specific recognition agent is a monoclonal antibody. 
     
     
         68 . The method of  claim 52 , wherein the screening is carried out in a multiplex assay employing a solid phase on which a plurality of substrates are immobilised. 
     
     
         69 . The method of  claim 68 , wherein the substrates correspond to phosphorylation sites of tau protein. 
     
     
         70 . The method of  claim 69 , wherein the phosphorylation sites are one or more of the sites selected from the group consisting of Y18, Y29, Y197, Y310 and Y394 of tau protein. 
     
     
         71 . The method of  claim 52 , the method comprising having identified a substance as an inhibitor of said tyrosine kinase, the further step of optimizing the structure of the substance. 
     
     
         72 . A method which comprises having identified a substance by the method of  claim 52 , the further step of manufacturing the substance and/or formulating it in pharmaceutical composition.