Identification of small molecule binding sites within proteins using phagedisplay technology

Affinity selection of peptides displayed on phage particles was used as the basis for mapping molecular contacts between small molecule ligands and th eir protein targets. Analysis of the crystal structures of complexes betwee n proteins and small molecule ligands revealed that virtually all ligands o f molecular weight 300 Da or greater have a continuous binding epitope of 5 residues or more. This observation led to the development of a technique f or binding site identification which involves statistical analysis of an af finity-selected set of peptides obtained by screening of libraries of rando m, phage-displayed peptides against small molecules attached to solid surfa ces. A random sample of the selected peptides is sequenced and used as inpu t for a similarity scanning program which calculates cumulative similarity scores along the length of the putative receptor. Regions of the protein se quence exhibiting the highest similarity with the selected peptides proved to have a high probability of being involved in ligand binding. This techni que has been employed successfully to map the contact residues in multiple known targets of the anticancer drugs paclitaxel (Taxol(TM)), docetaxel (Ta xotere(TM)) and 2-methoxestradiol and the glycosaminoglycan hyaluronan, and to identify a novel paclitaxel receptor [1]. These data corroborate the ob servation that the binding properties of peptides displayed on the surface of phage particles can mimic the binding properties of peptides in naturall y occurring proteins. It follows directly that structural context is relati vely unimportant for determining the binding properties of these disordered peptides. This technique represents a novel, rapid, high resolution method for identifying potential ligand binding sites in the absence of three-dim ensional information and has the potential to greatly enhance the speed of development of novel small molecule pharmaceuticals.