The GPC spin column/ESI-MS screening protocol rapidly analyzes the ability of small organic molecules to bind non-covalently to target protein molecules.
The methodology takes advantage of and combines the inherent strengths of size exclusion gel chromatography in the spin column mode, reversed-phase HPLC and ESI-MS in a nearly universal high throughput screening approach. The methodology has been automated to screen large libraries of chemical compounds with known structures while optimizing each operational step and minimizing analysis time. The methodology has been successfully applied at a number of pharmaceutical institutions, resulting in the identification of a number of new and novel inhibitors/antagonists of proteins of therapeutic interest. The GPC spin column/ESI-MS screening methodology can complement and even supplement the cell-based assays presently in vogue for HTS and surely deserves exploration when HTS fails to identify compounds with desirable biological and chemical properties.
A unique feature of this technology is the uncoupling of the GPC spin column step from the (HPLC)/ESI-MS detection step, thereby permitting the optimization of each of the analytical steps to rapidly produce reliable drug discovery non-covalent binding data. The strength of this GPC spin column screening methodology is the direct identification in mixtures of the small minority of ligands that non-covalently bind to protein targets and the elimination of non-binding ligands from the eluate. A challenge for the GPC spin column/ESI-MS technique is the identification of ligands of low abundances and unknown structures, as found in very complex mixtures, such as natural products extracts, tissue extracts and combinatorial libraries, that bind non-covalently with protein targets. With advanced software and future instrumental developments, screening problems of this complexity will be solvable using the GPC spin column/ESI-MS technology.
The site of non-covalent binding of the ligand to the protein is not directly measurable by GPC spin column/ESI-MS. To directly obtain the binding site, X-ray and NMR techniques are used. Site directed mutagenesis and displacement of known binders coupled with GPC spin column/ESI-MS can be used to identify non-covalent binding sites.
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