Wnt signaling agonist molecules

Abstract

The present application relates to novel therapeutic agents, particularly to agents capable of activating (GPR)124/RECK/Frizzled/lipoprotein receptor-related protein (LRP)-mediated Wnt signaling, wherein said agents do not activate Frizzled/LRP -mediated Wnt signaling in the absence of RECK and/or GPR124. The present agents are particularly useful for the prevention or treatment of neurovascular disorders or central nervous system (CNS) disorders comprising neurovascular dysfunction.

What is claimed is: 
     
         1 . An agent capable of activating G-protein coupled receptor (GPR)124/RECK/Frizzled/lipoprotein receptor-related protein (LRP)-mediated Wnt signaling, wherein said agent does not activate Frizzled/LRP-mediated Wnt signaling in the absence of RECK and/or GPR124. 
     
     
         2 . The agent according to  claim 1 , wherein said agent is capable of inducing heteromerization of Frizzled and LRP polypeptides at a cell membrane in the presence of RECK and GPR124, but not in the absence of RECK and/or GPR124. 
     
     
         3 . The agent according to  claim 2 , wherein said agent is capable of concurrently binding to Frizzled and LRP polypeptides at a cell membrane in the presence of RECK and GPR124, but not in the absence of RECK and/or GPR124. 
     
     
         4 . The agent according to  claim 1 , wherein said agent is capable of binding to the GPR124 and/or the RECK polypeptide. 
     
     
         5 . The agent according to  claim 1 , wherein said agent is capable of binding to the RECK polypeptide. 
     
     
         6 . The agent according to  claim 5 , wherein said agent is capable of binding to a cysteine knot 4 (CK4) region, to a CK5 region, or to the CK4 and CK5 regions, of RECK polypeptide. 
     
     
         7 . The agent according to  claim 1 , wherein said agent is capable of binding to a cysteine-rich domain (CRD) of the Frizzled polypeptide. 
     
     
         8 . The agent according to  claim 1 , wherein said agent is capable of binding to a DKK-binding site and/or a Wnt-binding site of the LRP polypeptide. 
     
     
         9 . The agent according to  claim 1 , wherein the agent is selected from the group consisting of a chemical substance, an antibody, an antibody fragment, an antibody-like protein scaffold, a protein, a polypeptide, a peptide, a peptidomimetic, an aptamer, a photoaptamer, a spiegelmer, a nucleic acid and a combination of two or more thereof. 
     
     
         10 . The agent according to  claim 1 , wherein said agent comprises a RECK-binding domain, a Frizzled-binding domain and a LRP-binding domain, wherein said RECK-binding domain, said Frizzled-binding domain, and said LRP- binding domain are derived from a Wnt7polypeptide. 
     
     
         11 . The agent according to  claim 10 , wherein said RECK-binding domain comprises:
 an amino acid sequence having at least 25% sequence identity to the amino acid sequence                HVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID     NO: 17);                   or   an amino acid sequence having at least 25% sequence identity to the amino acid sequence                VEPVRASRNKRPTFLKIKKPLSYRKPMDT (SEQ ID NO: 18);                  or   an amino acid sequence having at least 25% sequence identity to the amino acid sequence                VEVVRASRLRQPTFLRIKQLRSYQKPMET (SEQ ID NO: 19);                  or   an amino acid sequence                XXXVXAXRXXXXXFLXIXXXXXYXKXXXX(SEQ ID NO: 20),                             VXAXRXXXXXFLXIXXXXXYXK (SEQ ID NO:),                             XXXVXAXRXXXXXFLXXXXXXXXXKXXXX(SEQ ID NO: 21),                  or               VXAXRXXXXXFLXXXXXXXXXK (SEQ ID NO: 23)                  wherein Xis any amino acid, preferably wherein the amino acid sequence shows at least 50% sequence identity to any one of SEQ ID NO: 18 or SEQ ID NO: 19.   
     
     
         12 . The agent according to  claim 1 , wherein said agent consists essentially of a fragment of a Wnt7 polypeptide. 
     
     
         13 . The agent according to  claim 12 , wherein said fragment consists essentially of a N-terminal domain (NTD) of the Wnt7 polypeptide. 
     
     
         14 . The agent according to  claim 1 , wherein said agent is a variant of the Wnt7 polypeptide. 
     
     
         15 . The agent according to  claim 14 , wherein
 the glutamine (Q) residue at the position corresponding to position 17 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than glutamine (Q);   the isoleucine (I) residue at the position corresponding to position 20 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than isoleucine (I);   the praline (P) residue at the position corresponding to position 25 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than praline (P);   the alanine (A) residue at the position corresponding to position 27 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than alanine (A);   the isoleucine (I) residue at position corresponding to position 28 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than isoleucine (I);   the glutamate (E) residue at the position corresponding to position 33 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than glutamate (E);   the methionine (M) residue at the position corresponding to position 37 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than methionine (M);   the leucine (L) residue at the position corresponding to position 39 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than leucine (L);   the glutamate (E) residue at the position corresponding to position 41 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than glutamate (E);   the phenylalanine (F) residue at the position corresponding to position 44 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than phenylalanine (F),   the arginine (R) residue at the position corresponding to position 50 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than arginine (R);   the asparagine (N) residue at the position corresponding to position 52 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than asparagine (N);   the valine (V) residue at the position corresponding to position 68 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than valine (V);   the isoleucine (I) residue at the position corresponding to position 129 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than isoleucine (I);   the phenylalanine (F) residue at the position corresponding to position 131 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than phenylalanine (F);   the lysine (K) residue at the position corresponding to position 133 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than lysine (K);   the phenylalanine (F) residue at the position corresponding to position 135 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than phenylalanine (F);   the isoleucine (I) residue at the position corresponding to position 141 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than isoleucine (I);   the arginine (R) residue at the position corresponding to position 146 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than arginine (R);   the arginine (R) residue at the position corresponding to position 158 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than arginine (R);   the lysine (K) residue at the position corresponding to position 159 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than lysine (K);   the lysine (K) residue at the position corresponding to position 181 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than lysine (K);   the arginine (R) residue at the position corresponding to position 191 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than arginine (R);   the lysine (K) residue at the position corresponding to position 198 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than lysine (K);   the lysine (K) residue at the position corresponding to position 200 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than lysine (K);   the lysine (K) residue at the position corresponding to position 205 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than lysine (K);   the valine (V) residue at the position corresponding to position 205 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than valine (V);   the glutamate (E) residue at the position corresponding to position 208 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than glutamate (E);   the arginine (R) residue at the position corresponding to position 214 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than arginine (R);   the lysine (K) residue at the position corresponding to position 216 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than lysine (K);   the praline (P) residue at the position corresponding to position 218 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than praline (P);   the lysine (K) residue at the position corresponding to position 222 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than lysine (K);   the isoleucine (I) residue at the position corresponding to position 223 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than isoleucine (I);   the tyrosine (Y) residue at the position corresponding to position 229 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than tyrosine (Y);   the praline (P) residue at the position corresponding to position 232 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than praline (P);   the threonine (T) residue at the position corresponding to position 235 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than threonine (T);   the glutamate (E) residue at the position corresponding to position 248 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than glutamate (E);   the arginine (R) residue at the position corresponding to position 289 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than arginine (R);   the tryptophan (W) residue at the position corresponding to position 291 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than tryptophan (W), preferably by an alanine (A) residue;   the threonine (T) residue at the position corresponding to position 307 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than threonine (T); and/or   the lysine (K) residue at the position corresponding to position 318 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more amino acid residues other than lysine (K).   
     
     
         16 . A nucleic acid encoding the agent according to  claim 1  wherein said agent is a protein, polypeptide or a peptide. 
     
     
         17 . A nucleic acid expression cassette comprising the nucleic acid according to  claim 16 , operably linked to a promoter and/or transcriptional and translational regulatory signals. 
     
     
         18 . A vector comprising the nucleic acid according to  claim 16 . 
     
     
         19 . A pharmaceutical composition comprising the agent according to  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         20 . A method for treating a neurovascular disorder or a central nervous system (CNS) disorder comprising neurovascular dysfunction comprising administering the agent according to  claim 1  to an individual in need thereof. 
     
     
         21 . The method according to  claim 20 , wherein said neurovascular disorder is selected from the group consisting of ischemic stroke, hemorrhagic stroke, ischemia/reperfusion injury, brain aneurysms, arteriovenous malformations (AVMs), cavernous malformations, vasculitis, cerebral hemorrhage, subarachnoid hemorrhage, spinal vascular malformations, carotid artery stenosis, Moyamoya disease and intracranial atherosclerosis and combinations thereof, or said CNS disorder is selected from the group consisting of multiple sclerosis, ischemic stroke, brain cancer, epilepsy, dementia, vascular dementia, HIV-1-associated dementia, Alzheimer’s disease, Parkinson’s disease, Huntington disease, amyotrophic lateral sclerosis, infectious brain diseases, traumatic brain injuries, migraine and chronic traumatic encephalopathy and combinations thereof. 
     
     
         22 . An in vitro method for identifying an agent useful as a therapeutic, said method comprising determining whether a test agent activates GPR124/RECK/Frizzled/LRP-mediated Wnt signaling but not Frizzled/LRP-mediated Wnt signaling in the absence of RECK and/or GPR124. 
     
     
         23 . The in vitro method according to  claim 22 , comprising:
 contacting the test agent with a cell capable of GPR124/RECK/Frizzled/LRP-mediated Wnt signaling and measuring said Wnt signaling, and   contacting the test agent with a cell capable of Frizzled/LRP-mediated Wnt signaling but not GPR124/RECK/Frizzled/LRP-mediated Wnt signaling, and measuring said Wnt signaling.   
     
     
         24 . The in vitro method according to  claim 23 , wherein the method further comprises determining whether the test agent is capable of binding to the RECK polypeptide. 
     
     
         25 . A pharmaceutical composition comprising the nucleic acid according to  claim 16 . 
     
     
         26 . A method for treating a neurovascular disorder or a central nervous system (CNS) disorder comprising neurovascular dysfunction comprising administering the nucleic acid according to  claim 16  to an individual in need thereof.