Ras inhibitory peptides and uses thereof

Abstract

The present invention provides peptides comprising a sequence of X-6X-5X-4X-3X-2X-1X1PX3X4PX6X7PGX10X11AX13X14X15X16LX18X19X20X21X22X23LX25X26YLX29X30X31X32 wherein the amino acids X-6, X-5, X-4, X-3, X-2, X-1, X1, X3, X4, X6, X7, X10, X11, X13, X14, X15, X16, X18, X19, X20, X21, X22, X25, X26, X29, X30, X31, and X32 are as defined herein. The present invention further provides pharmaceutical compositions comprising the peptides and methods of using the peptides for treating proliferative diseases such as cancer which are associated with Ras. Also provided are methods of screening a library of peptide dimers using a peptide dimer display technology.

1 . A peptide comprising a sequence of: 
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 1) 
                 
                     
                   X 1 PX 3 X 4 PX 6 X 7 PGX 10 X 11 AX 13 X 14 X 15 DLX 18 X 19 YX 21 X 22 RLX 25   
                 
                     
                     
                 
                     
                   X 26 YLKX 29 X 30 VA 
                 
             
                
                
                
                
               
            
           
         
       
       wherein:
 X 1  is Gly, Arg, or an amino acid capable of cross-linking the peptide with another peptide; 
 X 3 , X 4 , and X 6  are each independently a charged amino acid; 
 X 10  is a charged amino acid; 
 X 7  is Tyr or an amino acid capable of cross-linking the peptide with another peptide; 
 X 11  is a charged amino acid or an amino acid capable of cross-linking the peptide with another peptide; 
 X 13  is Ser or an amino acid capable of cross-linking the peptide with another peptide; 
 X 14  is Ile or Glu; 
 X 15  is Glu, Asp, Gln, or Gly; 
 X 18  is an aromatic or hydrophobic amino acid; 
 X 19  is Glu, Asp, Gln, Ala, or Trp; 
 X 21 , X 25 , X 29  are each independently a Tyr, Phe, Trp, His, or an amino acid with a cyclohexyl side chain, wherein Tyr, Phe, Trp, cylohexyl side chain can be substituted with one or more fluorines; 
 X 22  is Ala, Gly, Ser, or Val; 
 X 26  is Asn, Leu, Ile, or His; 
 X 30  is Ala or Arg; 
 wherein the peptide comprises one to two amino acids capable of cross-linking the peptide with another peptide; or 
 a peptide comprising a sequence of: 
 
       X- 6 X- 5 X- 4 X- 3 X- 2 X-iX 1 PX 3 X 4 PX 6 X 7 PGX 10 X 11 AX 13 X 14 X 15 X 16 LX 18 X 19 X 20 X 21 X 22 X 23 LX 25 X 26  YLX 29 X 30 X 31 X 32  (SEQ ID NO: 13), wherein
 X 3  and X 4  are each independently a neutral or charged amino acid; 
 X 6  is a charged amino acid or an amino acid capable of cross-linking the peptide with another peptide; 
 X 7  is Tyr, His, or an amino acid capable of cross-linking the peptide with another peptide; 
 X 10  is Pro, charged amino acid, or an amino acid capable of cross-linking the peptide with another peptide; 
 X 11  is Ala, Ser, neutral or charged amino acid or an amino acid capable of cross-linking the peptide with another peptide; 
 X 13  is Ser, Pro, Thr, or an amino acid capable of cross-linking the peptide with another peptide; 
 X 14  is Ile, Glu, Val, Leu, or an amino acid capable of cross-linking the peptide with another peptide; 
 X 15  is Glu, Lys, Arg, Ala, Ser, Asp, Gln, or Gly; 
 X 16  is Asp, Glu, Gln, Ala, or Ser; 
 X 18  is an aromatic or hydrophobic amino acid; 
 X 19  is Glu, Lys, Leu, Met, His, Asp, Gln, Ala, Ser, Trp, or Arg; 
 X 20  is Tyr or Phe; 
 X 21 is Gln, Tyr, Phe, Trp, His, or an amino acid with a cyclohexyl side chain, wherein Tyr, Phe, Trp, cylohexyl side chain can be substituted with one or more fluorines; 
 X 22  is Ala, Gln, Trp, Leu, Tyr, Gly, Ser, Val, or Asn; 
 X 23  is Arg, Asp, Leu, or Ala; 
 X 25  is Gln, Tyr, Phe, Trp, His, Asp, or an amino acid with a cyclohexyl side chain, wherein Tyr, Phe, Trp, cylohexyl side chain can be substituted with one or more fluorines; 
 X 26  is Asn, Ala, Leu, Arg, Phe, Ile, His, or Gln; 
 X 29  is Ala, Leu, Glu, Asn, Gln, Tyr, Phe, Trp, His, or an amino acid with a cyclohexyl side chain, wherein Tyr, Phe, Trp, cylohexyl side chain can be substituted with one or more fluorines; 
 X 3 ( )is Ala, Arg or Val; 
 X 31  is V or an amino acid capable of cross-linking the peptide with another peptide; 
 X 32  is V, Ala, Arg, Ser, or an amino acid capable of cross-linking the peptide with another peptide; 
 X- 6 , X- 5 , X- 4 , X- 3 , X- 2 , X- 1 , and X 1  are optional; and 
 wherein the peptide comprises one to two amino acids capable of cross-linking the peptide with another peptide; or 
 a peptide comprising a sequence of: 
 X 1 PX 3 X 4 PX 6 X 7 PGX 10 AAX 13 X 14 AALHAYX 21 AX 23 LX 25 NYLX 29 X 30 VX 32  (SEQ ID NO: 48, wherein 
 X 1  is optionally present and when present, X 1  is Gly, Arg, or an amino acid capable of cross-linking the peptide with another peptide; 
 X 3 , X 4  and X 6  are each independently a neutral or positively charged amino acid; 
 X 7  is Tyr or an amino acid capable of cross-linking the peptide with another peptide; 
 X 10  is Asp or a neutral amino acid; 
 X 13  is Ser, Thr or an amino acid capable of cross-linking the peptide with another peptide; 
 X 14  is Ile or Leu; 
 X 21 , X 25 , X 29  are each independently a Tyr, Phe, Trp, His, or an amino acid with a cyclohexyl side chain, wherein Tyr, Phe, Trp, cylohexyl side chain can be substituted with one or more fluorines; 
 X 23  is Arg, Leu or Ala; 
 X 30  is Ala or Arg; 
 X 32  is Ala, Ser, or Arg; and 
 wherein the peptides comprise one to two amino acids capable of cross-linking the peptide with another peptide; or 
 a peptide dimer comprising a first peptide associated with a second peptide, wherein the first and second peptides each independently comprise a peptide sequence of: 
 
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 1) 
                 
                     
                   
                     X 1 PX 3 X 4 PX 6 X 7 PGX 10 X 11 AX 13 X 14 X 15 DLX 18 X 19 YX 21 X 22 RLX 25   
                   
                 
                     
                     
                 
                     
                   
                     X 26 YLKX 29 X 30 VA 
                   
                 
             
                
                
                
                
               
            
           
         
       
       wherein:
 X 1  is Gly, Arg, or an amino acid capable of cross-linking the peptide with another peptide; 
 X 3 , X 4 , and X 6  are each independently a charged amino acid; 
 X 10  is a charged amino acid; 
 X 7  is Tyr or an amino acid capable of cross-linking the peptide with another peptide; 
 X 11  is a charged amino acid or an amino acid capable of cross-linking the peptide with another peptide; 
 X 13  is Ser or an amino acid capable of cross-linking the peptide with another peptide; 
 X 14  is Ile or Glu; 
 X 15  is Glu, Asp, Gln, or Gly; 
 X 18  is an aromatic or hydrophobic amino acid; 
 X 19  is Glu, Asp, Gln, Ala, or Trp; 
 X 21 , X 25 , X 29  are each independently a Tyr, Phe, Trp, His, or an amino acid with a cyclohexyl side chain, wherein Tyr, Phe, Trp, cylohexyl side chain can be substituted with one or more fluorines; 
 X 22  is Ala, Gly, Ser, or Val; 
 X 26  is Asn, Leu, Ile, or His; 
 X 30  is Ala or Arg; 
 wherein the first and second peptides each comprises one to two amino acids cross-linking the first peptide with the second peptide. 
 
     
     
         2 . (canceled) 
     
     
         3 . The peptide of  claim 1 , wherein
 if X- 6  is present, then X- 6  is Gly and X- 5  to X 1  are present;   if X- 5  is present, then X- 5  is Cys or Sec and X- 4  to X 1  are present;   if X- 4  is present, then X- 4  is Gly and X- 3  to X 1  are present;   if X- 3  is present, then X- 3  is Gly and X- 2  to X 1  are present;   if X- 2  is present, then X- 2  is Pro, Cys, Sec, or Gly and X-i and X 1  are present;   if X- 1  is present, then X- 1  is Arg or Gly and X 1  is present; and   if X 1  is present, X 1  is Gly, Arg, or an amino acid capable of cross-linking the peptide with another peptide.   
     
     
         4 . The peptide of  claim 3 , wherein
 X- 6  to X 1  are not present; X 11 , X 15 , X 16 , X 19  are each Ala or Ser; X 18  is His; X 20  is Tyr, and X 26  is Asn.   
     
     
         5 - 6 . (canceled) 
     
     
         7 . A peptide dimer comprising a first peptide associated with a second peptide, wherein the first and second peptides each independently comprise a peptide sequence of SEQ ID NO: 13 of  claim 1 . 
     
     
         8 . A peptide dimer comprising a first peptide associated with a second peptide, wherein the first and second peptides each independently comprise a peptide sequence of SEQ ID NO: 48 of claim  5 . 
     
     
         9 . A peptide dimer comprising a first peptide associated with a second peptide, wherein the first and second peptides each independently comprise a peptide sequence selected from SEQ ID NO: 1, 13, or 48 of  claim 1 . 
     
     
         10 . The peptide dimer of  claim 1 , wherein the dimer is a homodimer. 
     
     
         11 . The peptide dimer of  claim 1 , wherein the dimer is a heterodimer. 
     
     
         12 - 58 . (canceled) 
     
     
         59 . The peptide of  claim 1 , wherein the peptide is a first peptide that associates with a second peptide to form a peptide dimer that binds to a target protein, wherein the target protein is Ras, a Ras mutant, Myc/Max, RalA, Beta catenin, YAP/TEAD, and NEMO/IκB kinase, 
     
     
         60 - 78 . (canceled) 
     
     
         79 . A peptide comprising a oligomerization domain comprising a sequence of:
 PX a X b PX c X d P (SEQ ID NO: 2), wherein X a , X b , X c  is any non-proline amino acid, and X d  is an amino acid capable of cross-linking the peptide with a second peptide, or
 a peptide comprising a oligomerization domain comprising a sequence of: 
   PX a X b PX c X d P (SEQ ID NO: 2), wherein X a , X b  is any non-proline amino acid; and X c  and X d  is an amino acid capable of cross-linking the peptide with a second peptide.   
     
     
         80 . (canceled) 
     
     
         81 . The peptide of  claims 79 , wherein the second peptide comprises a sequence of: PX a X b PX c X d P (SEQ ID NO: 2). 
     
     
         82 . The peptide of  claim 79 , further comprising an alpha-helical domain comprising a sequence of:
 X 13 X 14 X 15 DLX 18 X 19 YX 21 X 22 RLX 25 X 26 YLX 29 X 30 VA (SEQ ID NO: 3), wherein X 13 , X 14 , X 15 , X 18 , X 19 , X 21 , X 22 , X 25 , X 26 , X 29 , and X 30  are as defined in any one of the preceding claims.   
     
     
         83 - 100 . (canceled) 
     
     
         101 . A method of treating a disease or condition associated with Ras in a subject in need thereof comprising administering a peptide of  claim 1  to the subject. 
     
     
         102 . The method of  claim 101 , wherein the disease associated with Ras is a proliferative disease. 
     
     
         103 . The method of  claim 101 , wherein the proliferative disease is cancer. 
     
     
         104 . The method of  claim 101 , wherein the disease associated with Ras is CFC syndrome, capillary malformation-arteriovenous malformation syndrome, Costello syndrome, Legius syndrome, Neurofibromatosis type 1, Noonan syndrome, or Noonan syndrome with multiple lentigines (formerly LEOPARD syndrome). 
     
     
         105 . A method of screening a library of peptide dimers, comprising the steps of:
 transforming display cells with a vector encoding a first peptide and a second peptide, wherein the first and second peptides associate to form a peptide dimer fused to a cell wall protein,   contacting the display cells with a first label, wherein the first label comprises a target protein and associates with a cell expressing the peptide dimer having enhanced binding to the target and does not associate with a cell which does not express the peptide dimer having enhanced binding to the target;   isolating the display cells with which the first label is associated; and   identifying the first and second peptides which exhibit enhanced binding to the target.   
     
     
         106 . The method of  claim 105 , wherein the peptide dimer is a peptide homodimer or a peptide heterodimer of two peptides each comprising an alpha-helical structure. 
     
     
         107 - 114 . (canceled) 
     
     
         115 . A nucleic acid encoding a peptide of  claim 1 . 
     
     
         116 . A host cell comprising a nucleic acid of  claim 115 . 
     
     
         117 . A host cell comprising a peptide of  claim 1 .