Kelly A. Mercier, Matthew D. Shortridge and Robert Powers Pages 285 - 295 ( 11 )
A multi-step NMR based screening assay is described for identifying and evaluating chemical leads for their ability to bind a target protein. The multi-step NMR assay provides structure-related information while being an integral part of a structure based drug discovery and design program. The fundamental principle of the multi-step NMR assay is to combine distinct 1D and 2D NMR techniques, in such a manner, that the inherent strengths and weakness associated with each technique is complementary to each other in the screen. By taking advantage of the combined strengths of 1D and 2D NMR experiments, it is possible to minimize protein requirements and experiment time and differentiate between nonspecific and stoichiometric binders while being able to verify ligand binding, determine a semi-quantitative dissociation constant, identify the ligand binding site and rapidly determine a protein-ligand co-structure. Furthermore, the quality and physical behavior of the ligand is readily evaluated to determine its appropriateness as a chemical lead. The utility of the multi-step NMR assay is demonstrated with the use of PrgI from Salmonella typhimurium and human serum albumin (HSA) as target proteins.
Multi-step NMR screen, protein-ligand binding, line broadening, saturation transfer difference, 2D 1H-15N HSQC, Salmonella typhimurium, protein PrgI, human serum albumin
University of Nebraska-Lincoln, Department of Chemistry, 722 Hamilton Hall, Lincoln, NE 68588-0304, USA.