Method of Forming Fragment Ligated Inhibitors

Abstract

Methods for developing non-peptidic inhibitors that target the polo-box domain of PLK1 proteins are described. Methods include developing structure activity relationships for peptidic inhibitors followed by development of non-peptide fragment alternatives for portions of the peptide inhibitors. The non-peptide fragment can provide similar structure activity relationship as the replaced peptide. Fragment alternatives to key binding determinants are identified in an iterative computational and synthetic process facilitated through understanding of the peptide structure-activity relationships. The approach is informed by peptide structure-activity data obtained through synthesis and testing of truncated and mutated analogs of known PBD binding motifs.

What is claimed is: 
     
         1 . A method for developing a fragment ligated inhibitor, the method comprising iteratively replacing amino acid residues of a peptide inhibitor, the amino acid residues being replaced by a method comprising:
 determining the structure activity relationship of a peptide inhibitor or fragment thereof with a protein and thereby identifying a first peptide fragment of the peptide inhibitor to be replaced, the first peptide fragment including one or more amino acid residues;   computationally and individually docking each of a plurality of non-peptide fragment alternatives into a volume of a binding site of the protein, the binding site being known to interact with the first peptide fragment of the peptide inhibitor;   scoring each of the plurality of non-peptide fragments through energetic and geometric evaluation of docked poses of the non-peptide fragments;   selecting one of the first non-peptide fragments based upon the scores thus obtained;   replacing through chemical synthesis the first fragment of the peptide inhibitor with the selected non-peptide fragment to form a first generation fragment ligated inhibitor;   assaying the first generation fragment ligated inhibitor in vitro to obtain data concerning the first generation fragment ligated inhibitor;   developing a second generation non-peptide fragment derivative of the selected non-peptide fragment;   computationally docking the second generation non-peptide fragment derivative into the volume of the binding site of the protein;   scoring the second generation non-peptide fragment derivative through energetic and geometric evaluation of docked poses of the second generation non-peptide fragment derivative;   replacing through chemical synthesis the selected non-peptide fragment of the first generation fragment ligated inhibitor with the second generation non-peptide fragment derivative to form a second generation fragment ligated inhibitor;   assaying the second generation fragment ligated inhibitor in vitro to obtain data concerning the second generation fragment ligated inhibitor; and   replacing at least one additional fragment of the peptide inhibitor with at least one additional non-peptide fragment, the at least one additional non-peptide fragment of the peptide inhibitor including one or more amino acid residues of the peptide inhibitor, the first fragment and the at least one additional fragment being different fragments of the peptide inhibitor.   
     
     
         2 . The method of  claim 1 , wherein at least one of the non-peptide fragments is a naphthoic acid derivative or a benzoic acid derivative. 
     
     
         3 . The method of  claim 1 , wherein the peptide fragment to be replaced comprises 2, 3, or 4 amino acid residues of the peptide inhibitor. 
     
     
         4 . The method of  claim 1 , wherein the peptide fragment to be replaced comprises one or more of the terminal amino acid residues of the peptide inhibitor. 
     
     
         5 . The method of  claim 1 , wherein the peptide fragment to be replaced comprises one or more of the core amino acid residues of the peptide inhibitor. 
     
     
         6 . The method of  claim 1 , wherein the fragment ligated inhibitor is a kinase selective inhibitor. 
     
     
         7 . The method of  claim 1 , wherein the fragment ligated inhibitor is a non-ATP competitive inhibitor. 
     
     
         8 . The method of  claim 1 , wherein the first generation fragment ligated inhibitor and/or the second generation fragment ligated inhibitor are assayed according to an anti-proliferative process. 
     
     
         9 . The method of  claim 1 , wherein the first generation fragment ligated inhibitor and/or the second generation fragment ligated inhibitor are assayed according to a flow cytometry process. 
     
     
         10 . The method of  claim 1 , wherein the first generation fragment ligated inhibitor and/or the second generation fragment ligated inhibitor are assayed according to a subcellular localization process. 
     
     
         11 . The method of  claim 1 , wherein the fragment ligated inhibitor is capable of inhibiting subcellular localization. 
     
     
         12 . The method of  claim 1 , wherein the fragment ligated inhibitor is capable of inhibiting substrate phosphorylation.