Methods, compositions and compound assays for inhibiting amyloid-beta protein production

Abstract

A method for identifying compounds that inhibit amyloid-beta precursor protein processing in cells, comprising contacting a test compound with a GPCR polypeptide, or fragment thereof, and measuring a compound-GPCR property related to the production of amyloid-beta peptide. Cellular assays of the method measure indicators including second messenger and/or amyloid beta peptide levels. Therapeutic methods, and pharmaceutical compositions including effective amyloid-beta precursor processing-inhibiting amounts of GPCR expression inhibitors, are useful for treating conditions involving cognitive impairment such as Alzheimers Disease.

1 . A method for identifying a compound that inhibits the processing of amyloid-beta precursor protein in a mammalian cell, comprising
 (a) contacting a compound with a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4-6, 289-333; and   (b) measuring a compound-polypeptide property related to the production of amyloid-beta protein.   
     
     
         2 . The method according to  claim 1 , wherein said polypeptide comprises SEQ ID NO: 289-333 in an in vitro cell-free preparation. 
     
     
         3 . The method according to  claim 1 , wherein said polypeptide is membrane-bound. 
     
     
         4 . The method according to  claim 2 , wherein said polypeptide is present as a transmembrane cell receptor in a mammalian cell. 
     
     
         5 . The method of  claim 1 , wherein said property is a binding affinity of said compound to said polypeptide. 
     
     
         6 . The method of  claim 4 , wherein said property is activation of a biological pathway producing an indicator of the processing of amyloid-beta precursor protein. 
     
     
         7 . The method of  claim 6  wherein said indicator is a second messenger. 
     
     
         8 . The method of  claim 7  wherein said second messenger is cyclic AMP or Ca 2+ . 
     
     
         9 . The method of  claim 6  wherein said indicator is amyloid-beta peptide. 
     
     
         10 . The method of  claim 9  wherein said amyloid-beta protein is selected from the group consisting of one or more of amyloid-beta peptide 1-42, 1-40, 11-42 and 11-40. 
     
     
         11 . The method of  claim 10  wherein said amyloid-beta protein is amyloid-beta peptide 1-42. 
     
     
         12 . The method according to  claim 6  wherein said indicator induces the expression of a reporter in said mammalian cell. 
     
     
         13 . The method according to  claim 12  wherein the reporter is selected from the group consisting of alkaline phosphatase, GFP, eGFP, dGFP, luciferase and B galactosidase. 
     
     
         14 . The method according to  claim 1 , wherein said compound is selected from the group consisting of compounds of a commercially available screening library and compounds that have been demonstrated to have binding affinity for a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4-6, 289-333. 
     
     
         15 . The method according to  claim 2 , wherein said compound is a peptide in a phage display library or an antibody fragment library. 
     
     
         16 . The method according to  claim 1 , wherein said compound is an aryloxydithiourea, its salts, hydrates, or solvates. 
     
     
         17 . An agent for the inhibition of amyloid-beta precursor processing selected from the group consisting of an antisense polynucleotide, a ribozyme, and a small interfering RNA (siRNA), wherein said agent comprises a nucleic acid sequence complementary to, or engineered from, a naturally occurring polynucleotide sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4-6. 
     
     
         18 . The agent according to  claim 17 , wherein a vector in a mammalian cell expresses said agent. 
     
     
         19 . The agent according to  claim 18 , wherein said vector is an adenoviral, retroviral, adeno-associated viral, lentiviral, a herpes simplex viral or a sendaiviral vector. 
     
     
         20 . The agent according to  claim 19 , wherein said antisense polynucleotide and said siRNA comprise an antisense strand of 17-25 nucleotides complementary to a sense strand, wherein said sense strand is selected from 17-25 continuous nucleotides of a naturally occurring nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4-6. 
     
     
         21 . The agent according to  claim 20 , wherein said siRNA further comprises said sense strand. 
     
     
         22 . The agent according to  claim 20 , wherein said sense strand is selected from 17-25 continuous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1-3. 
     
     
         23 . The agent according to  claim 17 , wherein said siRNA further comprises a loop region connecting said sense and said antisense strand. 
     
     
         24 . The agent according to  claim 23  wherein said loop region comprises a nucleic acid sequence defined of SEQ ID NO: 288. 
     
     
         25 . The agent according to  claim 17 , wherein said agent is an antisense polynucleotide, ribozyme, or siRNA comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 7-287. 
     
     
         26 . A cognitive enhancing pharmaceutical composition comprising a therapeutically effective amount of an agent of  claim 17  in admixture with a pharmaceutically acceptable carrier. 
     
     
         27 . The cognitive enhancing pharmaceutical composition according to  claim 26  wherein said agent comprises a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 7-287, a polynucleotide complementary to said nucleic acid sequence, and a combination thereof. 
     
     
         28 . A method of inhibiting the processing of amyloid-beta precursor protein in a subject suffering or susceptible to the abnormal processing of said protein, comprising administering to said subject a pharmaceutical composition according to  claim 26 . 
     
     
         29 . A method according to  claim 28  for treatment or prevention of a condition involving cognitive impairment or a susceptibility to the condition. 
     
     
         30 . The method according to  claim 29  wherein the condition is Alzheimer's disease. 
     
     
         31 . A pharmaceutical composition for the treatment or prevention of a condition involving cognitive impairment or a susceptibility to the condition, comprising an effective amyloid-beta precursor processing-inhibiting amount of a GPCR antagonist or inverse agonist. 
     
     
         32 . A composition according to  claim 31 , wherein said GPCR inverse agonist is an aryloxydithiourea, its pharmaceutically acceptable salts, hydrates, solvates, or prodrugs thereof in admixture with a pharmaceutically acceptable carrier.