NMR screening for lead compounds using tryptophan-mutated proteins.

NMR-based drug screening methods provide the most reliable characterization of binding propensities of ligands to their target proteins. They Lire, however, one of the least effective methods in terms of the amount of protein required and the time needed for acquiring an NMR experiment. We show here that the introduction of tryptophan to proteins permits rapid screening by monitoring a simple I D proton NMR signal of file NH side chain (H-N(epsilon)) of the tryptophan. The method could also provide quantitative characterization of the antagonist-protein and antagonist-protein-protein interactions in the form of (K)(D)s and fractions of the released proteins front their mutual binding. We illustrate the method with the lead Compounds that block, the Mdm2-p53 interaction and by Studying inhibitors that bind to cyclin-dependent kinase 2 (CDK2).