Cyclin based inhibitors of CDK2 and CDK4
Structural and functional analysis of peptide inhibitor binding to the cyclin D1 groove has been investigated and used to design peptides that provide the basis for structure-activity relationships, have improved binding and have potential for development as chemical biology probes, as potential diagnostics and as therapeutics in the treatment of proliferative diseases including cancer and inflammation.
1. A method for forming a first peptide inhibitor of a CDK/cyclin D complex, the method comprising:
generating an in silico model comprising a second peptide inhibitor bound to a cyclin protein, this cyclin protein differing from the cyclin D of the CDK/cyclin D complex;
superimposing the cyclin D of the CDK/cyclin D complex on the in silico model such that an corresponding alpha carbon atoms of the cyclin D of the CDK/cyclin D complex and an of the cyclin protein are overlayed and the binding sites of the two cyclins are sequence aligned with one another;
deleting the cyclin protein from the in silico model to form a second in silico model of the second peptide inhibitor bound to the cyclin D of the CDK/cyclin D complex, wherein the deletion is carried out in steps such that an energy minimum is converged upon;
determining the location of conformation differences between the model of the cyclin protein bound to the second peptide inhibitor and the model of the cyclin D protein of the CDK/cyclin D complex bound to the second peptide inhibitor;
altering the structure of the second peptide inhibitor to develop an in silico model of the first peptide inhibitor, the structure alteration comprising replacement of one or more residues of the second peptide inhibitor with a different amino acid or a synthetic substituent; and
forming the first peptide inhibitor based upon the in silico model of the first peptide inhibitor, and testing whether the affinity of the cyclin D protein of the CDK/cyclin D complex to the first peptide inhibitor is greater than the affinity of the cyclin D protein of the CDK/cyclin D complex to the second peptide inhibitor.
2. The method according to claim 1 , wherein the cyclin D protein of the CDK/cyclin D complex is a cyclin D1 protein.
3. The method according to claim 1 , wherein the cyclin protein is a cyclin A protein.
4. The method according to claim 3 , wherein the cyclin protein is a cyclin A2 protein.
5. The method according to claim 1 , wherein the CDK/cyclin D complex is a CDK4/cyclin D complex.
6. The method according to claim 1 , wherein the second peptide inhibitor comprises less than about 10 amino acids.
7. The method according to claim 1 , wherein the step of altering the structure of the second peptide inhibitor further comprises adding an amino acid or a synthetic substituent to an end of the second peptide inhibitor.
8. The method according to claim 1 , wherein the second peptide inhibitor is selected from the group consisting of HAKRRLIF (SEQ ID NO: 6), SAKRRLFG (SEQ ID NO: 5), PVKRRLDL (SEQ ID NO: 7), and RRLIF (SEQ ID NO: 4).
9. The method according to claim 1 , wherein the step of altering the structure of the second peptide inhibitor comprises altering the N-terminus of the second peptide inhibitor.
10. The method of claim 1 , wherein the structure alteration of the second peptide inhibitor comprises replacement of one or more residues of the second peptide inhibitor with one of the following:
11. The method of claim 1 , wherein the structure alteration of the second peptide inhibitor comprises replacement of a phenylalanine residue of the second peptide inhibitor.
12. The method of claim 1 , wherein the structure alteration of the second peptide inhibitor comprises replacement of an arginine residue of the second peptide inhibitor.
13. The method of claim 1 , wherein the structure alteration of the second peptide inhibitor comprises replacement of a residue of the second peptide inhibitor with a citrullene residue.
14. The method of claim 9 , wherein the alteration of the N-terminus of the second peptide inhibitor comprises capping the N-terminus with 1-(3,5-dichlorophenyl)-5-methyl-1H-1,2,4-triazole-3-carboxylic acid.
15. The method of claim 9 , wherein the alteration of the N-terminus of the second peptide inhibitor comprises capping the N-terminus with a triazole, pyrazole, furan, pyrrole, or thiazole substituent.
16. The method of claim 9 , wherein the alteration of the N-terminus of the second peptide inhibitor comprises capping the N-terminus with a substituent having the following structure:
17. The method of claim 9 , wherein the alteration of the N-terminus of the second peptide inhibitor comprises capping the N-terminus with a substituent having the following structure: