Methods and Means For Inhibition of CDK4 Activity

Abstract

p21 WAF1 interacts with cyclin D1 and Cdk4. Peptide fragments of p21 inhibit the interaction and/or affect Cdk4 activity. The peptides, derivative peptides and non-peptidyl mimetics thereof are useful in affecting activity of Cdk4, such as RB phosphorylation and cellular proliferation, indicative of therapeutic usefullness in treatment of tumors and other hyperproliferative disorders. Assay and screening methods allow identification of such modulators, especially inhibitors, of Cdk4 activity.

1 . A method of ameliorating a disorder characterised by abnormal cell proliferation which comprises contacting a cell with, or causing the cell to express, a substance selected from the group consisting of:
 (i) a peptide fragment of p21 consisting of an amino sequence selected from the group consisting of:
 RERWNFDFVTETPLEGDFAW (peptide 4); 
 KACRRLFGPVDSEQLSRDCD (peptide 2); 
 KxxRRyFzP, wherein x may be any amino acid, y and z may be hydrophobic, and each of the underlined residues may be absent or different; 
 KRRQTSMTDFYHSKRRLIFS (peptide 10); 
 KRRQTSATDFYHSKRRLIFS; 
 KRRQTSMTAFYHSKRRLIFS; 
 TSMTDFYHSKRRLIFSKRKP (peptide 11); 
 KRRLIFSK; or 
 xyLzF, wherein y and z are any amino acid and x is preferably R; 
   (ii) the peptide fragment of (i) coupled to a coupling partner or fused to a non-p21 sequence;   (iii) a peptide fragment of 40 amino acids or less of p21, or a derivative of such a fragment, the fragment or derivative including the amino sequence
 KxxRRyFzP, wherein x may be any amino acid, y and z may be hydrophobic, and each of the underlined residues may be absent or different, and 
   
       wherein the derivative has the property of binding to or interacting with cyclin D1 and/or cdk4; and
 (iv) the peptide fragment or derivative of (iii) coupled to a coupling partner or fused to a non-p21 sequence; 
 such that abnormal cell proliferation is ameliorated. 
 
     
     
         2 . The method of  claim 1  wherein said disorder is a hyperproliferative disorder. 
     
     
         3 . The method of  claim 1  wherein the coupling partner is a carrier peptide for delivery to cells. 
     
     
         4 . The method of  claim 3  wherein the carrier peptide has the sequence RQIKIWFQNRRMKWKK. 
     
     
         5 . A method of interfering with interaction between p21 and cyclin D1 and/or Cdk4, comprising contacting p21 and/or Cdk4 with a substance selected from the group consisting of
 (i) a peptide fragment of p21 consisting of an amino sequence selected from the group consisting of:
 RERWNFDFVTETPLEGDFAW (peptide 4); 
 KACRRLFGPVDSEQLSRDCD (peptide 2); 
 KxxRRyFzP, wherein x may be any amino acid, y and z may be hydrophobic, and each of the underlined residues may be absent or different; 
 KRRQTSMTDFYHSKRRLIFS (peptide 10); 
 KRRQTSATDFYHSKRRLIFS; 
 KRRQTSMTAFYHSKRRLIFS; 
 TSMTDFYHSKRRLIFSKRKP (peptide 11); 
 KRRLIFSK; or 
 xyLzF, wherein y and z are any amino acid and x is preferably R; 
   (ii) the peptide fragment of (i) coupled to a coupling partner or fused to a non-p21 sequence;   (iii) a peptide fragment of 40 amino acids or less of p21, or a derivative of such a fragment, the fragment or derivative including the amino sequence
 KxxRRyFzP, wherein x may be any amino acid, y and z may be hydrophobic, and each of the underlined residues may be absent or different, and 
   
       wherein the derivative has the property of binding to or interacting with cyclin D1 and/or cdk4; and
 (iv) the peptide fragment or derivative of (iii) coupled to a coupling partner or fused to a non-p21 sequence. 
 
     
     
         6 . A method of modulating a p21-mediated effect on Cdk4 activity, the method including contacting p21 and/or Cdk4 with a substance selected from the group consisting of
 (i) a peptide fragment of p21 consisting of an amino sequence selected from the group consisting of:
 RERWNFDFVTETPLEGDFAW (peptide 4); 
 KACRRLFGPVDSEQLSRDCD (peptide 2); 
 KxxRRyFzP, wherein x may be any amino acid, y and z may be hydrophobic, and each of the underlined residues may be absent or different; 
 KRRQTSMTDFYHSKRRLIFS (peptide 10); 
 KRRQTSATDFYHSKRRLIFS; 
 KRRQTSMTAFYHSKRRLIFS; 
 TSMTDFYHSKRRLIFSKRKP (peptide 11); 
 KRRLIFSK; or 
 xyLzF, wherein y and z are any amino acid and x is preferably R; 
   (ii) the peptide fragment of (i) coupled to a coupling partner or fused to a non-p21 sequence;   (iii) a peptide fragment of 40 amino acids or less of p21, or a derivative of such a fragment, the fragment or derivative including the amino sequence
 KxxRRyFzP, wherein x may be any amino acid, y and z may be hydrophobic, and each of the underlined residues may be absent or different, and 
   
       wherein the derivative has the property of binding to or interacting with cyclin D1 and/or cdk4; and
 (iv) the peptide fragment or derivative of (iii) coupled to a coupling partner or fused to a non-p21 sequence. 
 
     
     
         7 . The method of  claim 5  or  6  wherein the method is performed in vitro or ex vivo. 
     
     
         8 . The method of  claim 5  or  6  wherein the method is performed in vivo. 
     
     
         9 . The method of  claim 5  or  6  wherein the cell is contacted with the substance. 
     
     
         10 . The method of  claim 5  or  6  wherein the cell is caused to express the substance by expression by the cell of a nucleic acid molecule encoding the substance. 
     
     
         11 . A method of ameliorating a disorder characterised by abnormal cell proliferation, said method comprising expressing in a target cell a nucleic acid molecule encoding a substance selected from the group consisting of
 (i) a peptide fragment of p21 consisting of an amino sequence selected from the group consisting of:
 RERWNFDFVTETPLEGDFAW (peptide 4); 
 KACRRLFGPVDSEQLSRDCD (peptide 2); 
 KxxRRyFzP, wherein x may be any amino acid, y and z may be hydrophobic, and each of the underlined residues may be absent or different; 
 KRRQTSMTDFYHSKRRLIFS (peptide 10); 
 KRRQTSATDFYHSKRRLIFS; 
 KRRQTSMTAFYHSKRRLIFS; 
 TSMTDFYHSKRRLIFSKRKP (peptide 11); 
 KRRLIFSK; or 
 xyLzF, wherein y and z are any amino acid and x is preferably R; 
   (ii) the peptide fragment of (i) coupled to a coupling partner or fused to a non-p21 sequence;   (iii) a peptide fragment of 40 amino acids or less of p21, or a derivative of such a fragment, the fragment or derivative including the amino sequence
 KxxRRyFzP, wherein x may be any amino acid, y and z may be hydrophobic, and each of the underlined residues may be absent or different, and 
   
       wherein the derivative has the property of binding to or interacting with cyclin D1 and/or cdk4; and
 (iv) the peptide fragment or derivative of (iii) coupled to a coupling partner or fused to a non-p21 sequence; 
 such that abnormal cell proliferation is ameliorated. 
 
     
     
         12 . A method of preparing a pharmaceutical composition for use in treating a hyperproliferative disease, said method comprising mixing a pharmaceutically acceptable excipient with a substance selected from the group consisting of:
 (i) a peptide fragment of p21 consisting of an amino sequence selected from the group consisting of:
 RERWNFDFVTETPLEGDFAW (peptide 4); 
 KACRRLFGPVDSEQLSRDCD (peptide 2); 
 KxxRRyFzP, wherein x may be any amino acid, y and z may be hydrophobic, and each of the underlined residues may be absent or different; 
 KRRQTSMTDFYHSKRRLIFS (peptide 10); 
 KRRQTSATDFYHSKRRLIFS; 
 KRRQTSMTAFYHSKRRLIFS; 
 TSMTDFYHSKRRLIFSKRKP (peptide 11); 
 KRRLIFSK; or 
 xyLzF, wherein y and z are any amino acid and x is preferably R; 
   (ii) the peptide fragment of (i) coupled to a coupling partner or fused to a non-p21 sequence;   (iii) a peptide fragment of 40 amino acids or less of p21, or a derivative of such a fragment, the fragment or derivative including the amino sequence
 KxxRRyFzP, wherein x may be any amino acid, y and z may be hydrophobic, and each of the underlined residues may be absent or different, and 
   
       wherein the derivative has the property of binding to or interacting with cyclin D1 and/or cdk4; and
 (iv) the peptide fragment or derivative of (iii) coupled to a coupling partner or fused to a non-p21 sequence. 
 
     
     
         13 . A method of preparing a pharmaceutical composition for use in treating a hyperproliferative disease, said method comprising mixing a pharmaceutically acceptable excipient with a nucleic acid sequence encoding a substance selected from the group consisting of:
 (i) a peptide fragment of p21 consisting of an amino sequence selected from the group consisting of:
 RERWNFDFVTETPLEGDFAW (peptide 4); 
 KACRRLFGPVDSEQLSRDCD (peptide 2); 
 KxxRRyFzP, wherein x may be any amino acid, y and z may be hydrophobic, and each of the underlined residues may be absent or different; 
 KRRQTSMTDFYHSKRRLIFS (peptide 10); 
 KRRQTSATDFYHSKRRLIFS; 
 KRRQTSMTAFYHSKRRLIFS; 
 TSMTDFYHSKRRLIFSKRKP (peptide 11); 
 KRRLIFSK; or 
 xyLzF, wherein y and z are any amino acid and x is preferably R; 
   (ii) the peptide fragment of (i) coupled to a coupling partner or fused to a non-p21 sequence;   (iii) a peptide fragment of 40 amino acids or less of p21, or a derivative of such a fragment, the fragment or derivative including the amino sequence
 KxxRRyFzP, wherein x may be any amino acid, y and z may be hydrophobic, and each of the underlined residues may be absent or different, and 
   
       wherein the derivative has the property of binding to or interacting with cyclin D1 and/or cdk4; and
 (iv) the peptide fragment or derivative of (iii) coupled to a coupling partner or fused to a non-p21 sequence. 
 
     
     
         14 . The method of  claim 13  wherein said nucleic acid sequence is part of a vector. 
     
     
         15 . A method according to  claim 14  wherein said vector is a viral vector. 
     
     
         16 . A method according to  claim 14  wherein said vector is a plasmid vector.