Neuregulin variants and methods of screening and using thereof

Abstract

The present invention provides polypeptide variants of neuregulin-1β (NRG-1β) that have enhanced or decreased binding affinity to ErbB3 and/or ErbB4. The invention also provides methods of screening and producing polypeptide variants of NRG-1β and methods of using polypeptide variants of NRG-1β for treating diseases.

1 . A polypeptide variant of neuregulin-1β comprising amino acid sequence shown in SEQ ID NO:1, wherein the polypeptide variant comprises a different amino acid than that in SEQ ID NO:1, wherein the polypeptide variant has an enhanced binding affinity to ErbB3 compared to polypeptide of SEQ ID NO:1, and wherein 
 at residue 25 said different amino acid is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;    at residue 35 said different amino acid is A, C, D, E, F, G, H, I, L, N, M, P, Q, R, S, T, V, W, or Y; or    at residue 46 said different amino acid is A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y.    
     
     
         2 . The polypeptide variant of  claim 1 , wherein the polypeptide variant consists of the amino acid sequence shown in SEQ ID NO:1, and wherein 
 at residue 25 said different amino acid is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;    at residue 35 said different amino acid is A, C, D, E, F, G, H, I, L, N, M, P, Q, R, S, T, V, W, or Y; or    at residue 46 said different amino acid is A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y.    
     
     
         3 . The polypeptide variant of  claim 1 , wherein 
 at residue 25 said different amino acid is A;    at residue 35 said different amino acid is A; or    at residue 46 said different amino acid is A    
     
     
         4 . The polypeptide variant of  claim 2 , wherein 
 at residue 25 said different amino acid is A;    at residue 35 said different amino acid is A; or    at residue 46 said different amino acid is A.    
     
     
         5 . The polypeptide variant of  claim 1 , wherein the polypeptide variant has a decreased or similar binding affinity to an ErbB4 compared to the polypeptide of SEQ ID NO:1.  
     
     
         6 . The polypeptide variant of  claim 5 , wherein at residue 25 said different amino acid is A.  
     
     
         7 . The polypeptide variant of  claim 5 , wherein at residue 35 said different amino acid is A.  
     
     
         8 . The polypeptide variant of  claim 5 , wherein at residue 46 said different amino acid is A.  
     
     
         9 . A polynucleotide comprising a nucleic acid sequence encoding the polypeptide variant of  claim 1 .  
     
     
         10 . A pharmaceutical composition comprising an effective amount of the polypeptide variant of  claim 1  and a pharmaceutically acceptable excipient.  
     
     
         11 . A pharmaceutical composition comprising an effective amount of the polypeptide variant of  claim 5  and a pharmaceutically acceptable excipient.  
     
     
         12 . A pharmaceutical composition comprising an effective amount of the polynucleotide of  claim 9  and a pharmaceutically acceptable excipient.  
     
     
         13 . A kit comprising the pharmaceutical composition of  claim 10  and an instruction for using the pharmaceutical composition in preventing, treating, or delaying a disease in an individual via activating ErbB2/ErbB3 receptors.  
     
     
         14 . A kit comprising the pharmaceutical composition of  claim 11  and an instruction for using the pharmaceutical composition in preventing, treating, or delaying a disease in an individual via activating ErbB2/ErbB3 receptors.  
     
     
         15 . A method for preventing, treating, delaying development of schizophrenia in a mammal, comprising administering to a mammal, to which such prevention, treatment or delay is needed or desirable, a pharmaceutical composition comprising an effective amount of the polypeptide variant of  claim 1  and a pharmaceutically acceptable excipient.  
     
     
         16 . The method of  claim 15 , wherein the mammal is a human.  
     
     
         17 . A polypeptide variant of neuregulin-1β comprising amino acid sequence shown in SEQ ID NO:1, wherein the polypeptide variant comprises a different amino acid than that in SEQ ID NO:1, wherein the polypeptide variant has an enhanced binding affinity to ErbB4 compared to polypeptide of SEQ ID NO:1, and wherein 
 at residue 16 said different amino acid is A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y;    at residue 31 said different amino acid is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; or    at residue 47 said different amino acid is A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y.    
     
     
         18 . The polypeptide variant of  claim 17 , wherein the polypeptide variant consists of the amino acid sequence shown in SEQ ID NO:1, and wherein at residue 16 said different amino acid is A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y.  
     
     
         19 . The polypeptide variant of  claim 17 , wherein at residue 16 said different amino acid is A.  
     
     
         20 . The polypeptide variant of  claim 18 , wherein at residue 16 said different amino acid is A.  
     
     
         21 . The polypeptide variant of  claim 17 , wherein at residue 31 said different amino acid is A.  
     
     
         22 . The polypeptide variant of  claim 18 , wherein at residue 31 said different amino acid is A.  
     
     
         23 . The polypeptide variant of  claim 17 , wherein at residue 47 said different amino acid is A.  
     
     
         24 . The polypeptide variant of  claim 18 , wherein at residue 47 said different amino acid is A.  
     
     
         25 . The polypeptide variant of  claim 17 , wherein the polypeptide variant has a decreased or similar binding affinity to an ErbB3 compared to the polypeptide of SEQ ID NO:1.  
     
     
         26 . A polynucleotide comprising a nucleic acid sequence encoding the polypeptide variant of  claim 17 .  
     
     
         27 . A pharmaceutical composition comprising an effective amount of the polypeptide variant of  claim 17  and a pharmaceutically acceptable excipient.  
     
     
         28 . A pharmaceutical composition comprising an effective amount of the polypeptide variant of  claim 25  and a pharmaceutically acceptable excipient.  
     
     
         29 . A pharmaceutical composition comprising an effective amount of the polynucleotide of  claim 26  and a pharmaceutically acceptable excipient.  
     
     
         30 . A kit comprising the pharmaceutical composition of  claim 27  and an instruction for using the pharmaceutical composition in preventing, treating, or delaying a disease in an individual via activating ErbB2/ErbB4 receptors.  
     
     
         31 . A kit comprising the pharmaceutical composition of  claim 28  and an instruction for using the pharmaceutical composition in preventing, treating, or delaying a disease in an individual via activating ErbB2/ErbB4 receptors.  
     
     
         32 . A method for preventing, treating, delaying development of viral myocarditis, dilated (congestive) cardiomyopathy, cardiac toxicity, or myocardial infarction in an individual, comprising administering to an individual, to which such prevention, treatment or delay is needed or desirable, a pharmaceutical composition comprising an effective amount of the polypeptide variant of  claim 17  and a pharmaceutically acceptable excipient.  
     
     
         33 . The method of  claim 32 , wherein the mammal is a human.  
     
     
         34 . A method for screening a polypeptide variant of neuregulin-1β having enhanced binding affinity selective to ErbB3, which method comprises: 
 (a) establishing a three-dimensional structure of a neuregulin-1β or a fragment thereof, an ErbB3, an ErbB4, a complex of the neuregulin-1β or the fragment thereof and the ErbB3, and a complex of the neuregulin-1β or the fragment thereof and the ErbB4 by homology modeling;    (b) establishing data of conformational changes and stability of the complex of the neuregulin-1β or the fragment thereof and the ErbB3, and the complex of the neuregulin-1β or the fragment thereof and the ErbB4 in solution by molecular dynamics simulation method;    (c) calculating subtotal binding free energy (ΔG subtotal wildtype ) of the neuregulin-1β or the fragment thereof with the ErbB3 or the ErbB4 by Molecular Mechanics Poisson Boltzmann Surface Area (MM-PBSA) method;    (d) calculating subtotal binding free energy (ΔG subtotal alanine substituted variant ) of an alanine substituted variant of the neuregulin-1β or the fragment thereof with the ErbB3 or the ErbB4 by Molecular Mechanics Poisson Boltzmann Surface Area (MM-PBSA) method, wherein the alanine substituted variant comprises an amino acid of the neuregulin-1β or the fragment thereof substituted by an alanine;    (e) calculating ΔΔG subtotal =ΔG subtotal wildtype −ΔG subtotal alanine substituted variant ;    (f) selecting alanine substituted variant that has a positive value of ΔΔG subtotal  for the complex of the neuregulin-1β or the fragment thereof and the ErbB3, and has a negative value or a value of about zero for ΔΔG subtotal  for the complex of the neuregulin-1β or the fragment thereof and the ErbB4;    whereby a polypeptide variant of neuregulin-1β that has enhanced binding affinity selective to ErbB3 is identified.    
     
     
         35 . A polypeptide variant of a neuregulin-1β identified by the method of  claim 34 .  
     
     
         36 . A method for screening a polypeptide variant of neuregulin-1β having enhanced binding affinity selective to ErbB4, which method comprises: 
 (a) establishing a three-dimensional structure of a neuregulin-1β or a fragment thereof, an ErbB3, an ErbB4, a complex of the neuregulin-1β or the fragment thereof and the ErbB3, and a complex of the neuregulin-1β or the fragment thereof and the ErbB4 by homology modeling;    (b) establishing data of conformational changes and stability of the complex of the neuregulin-1β or the fragment thereof and the ErbB3, and the complex of the neuregulin-1β or the fragment thereof and the ErbB4 in solution by molecular dynamics simulation method;    (c) calculating subtotal binding free energy (ΔG subtotal wildtype ) of the neuregulin-1β or the fragment thereof with the ErbB3 or the ErbB4 by Molecular Mechanics Poisson Boltzmann Surface Area (MM-PBSA) method;    (d) calculating subtotal binding free energy (ΔG subtotal alanine substituted variant ) of an alanine substituted variant of the neuregulin-1β or the fragment thereof with the ErbB3 or the ErbB4 by Molecular Mechanics Poisson Boltzmann Surface Area (MM-PBSA) method, wherein the alanine substituted variant comprises an amino acid of the neuregulin-1β or the fragment thereof substituted by an alanine;    (e) calculating ΔΔG subtotal =ΔG subtotal wildtype −ΔG subtotal alanine substituted variant ;    (f) selecting alanine substituted variant that has a positive value of ΔΔG subtotal  for the complex of the neuregulin-1β or the fragment thereof and the ErbB4, and has a negative value or an value of about zero for ΔΔG subtotal  for the complex of the neuregulin-1β or the fragment thereof and the ErbB3;    whereby a polypeptide variant of neuregulin-1β that has enhanced binding affinity selective to ErbB4 is identified    
     
     
         37 . A polypeptide variant of a neuregulin-1β identified by the method of  claim 36.