MS/NMR: A structure-based approach for discovering protein ligands and fordrug design by coupling size exclusion chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy

A protocol is described for rapidly screening small organic molecules for t heir ability to bind a target protein while obtaining structure-related inf ormation as part of a structure-based drug discovery and design program. Th e methodology takes advantage of and combines the inherent strengths of siz e exclusion gel chromatography, mass spectrometry, and NMR to identify boun d complexes in a relatively universal high-throughput screening approach. S ize exclusion gel chromatography in the spin column format provides the hig h-speed separation of a protein-ligand complex from free ligands. The spin column eluent is then analyzed under denaturing conditions by electrospray ionization mass spectrometry (MS) for the presence of small molecular weigh t compounds formerly bound to the protein. Hits identified by MS are then i ndividually assayed by chemical shift perturbations in a 2D H-1-N-15 HSQC N MR spectrum to verify specific interactions of the compound with the protei n and identification of the binding site on the protein. The utility of the MS/NMR assay is demonstrated with the use of the catalytic fragment of hum an fibroblast collagenase (MMP-1) as a target protein and the screening of a library consisting of similar to 32 000 compounds for the identification of molecules that exhibit specific binding to the RGS4 protein.