Detection of Ligands from a Dynamic Combinatorial Library by X-ray Crystallography

摘要

Dynamic combinatorial chemistry (DCC) is an approach to molecular recognition in which specific members of a combinatorial library are selected and amplified with the use of a template. The principle difference between DCC and traditional combinatorial chemistry is that the reaction linking the building blocks together in DCC is reversible and there is an ongoing interchange between the different members of the dynamic combinatorial library (DCL) under thermodynamic control. A DCL is therefore able to respond to molecular recognition events owing to the presence of a template, such as a protein, which can stabilize a particular member of the library and induce a shift in the equilibrium, favoring the formation of the selected species. A drawback of the method is that it usually requires excess quantities of protein for an effect on the equilibrium to be observed. Also, the effect can only be detected by comparison of identical libraries, generated with and without the protein component present, using either mass spectrometric analysis or HPLC chromatograms as a fingerprint. We now report a complimentary approach in which ligands are observed directly by X-ray crystallography by interpretation of electron-density maps from crystals exposed to a dynamic combinatorial library mixture. We call this technology dynamic combinatorial X-ray crystallography or DCX. This approach was used to detect rapidly potent inhibitors of the cyclin-dependent kinase 2 protein. DCX has key advantages over previously reported DCC technologies in that direct identification of the ligand is possible fro the mixture of components in the DCL, and its detailed binding mode is defined from the electron-density maps. Furthermore, only very small amounts of protein are required for each individual DCX experiment compared with previously reported DCC protocols.