title: NMR screening for lead compounds using tryptophan-mutated proteins.
abstract: NMR-based drug screening methods provide the most reliable characterization of binding propensities of ligands to their target proteins. They are, 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 1D proton NMR signal of the NH side chain ((N)H(epsilon)) of the tryptophan. The method could also provide quantitative characterization of the antagonist-protein and antagonist-protein-protein interactions in the form of KDs and fractions of the released proteins from 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).
authors.affiliation: Max Planck Institute for Biochemistry, D-82152 Martinsried, Germany.
authors.forename: Ulli
authors.initials: U
authors.lastname: Rothweiler
authors.forename: Anna
authors.initials: A
authors.lastname: Czarna
authors.forename: Lutz
authors.initials: L
authors.lastname: Weber
authors.forename: Grzegorz M
authors.initials: GM
authors.lastname: Popowicz
authors.forename: Kinga
authors.initials: K
authors.lastname: Brongel
authors.forename: Kaja
authors.initials: K
authors.lastname: Kowalska
authors.forename: Michael
authors.initials: M
authors.lastname: Orth
authors.forename: Olaf
authors.initials: O
authors.lastname: Stemmann
authors.forename: Tad A
authors.initials: TA
authors.lastname: Holak
mesh_terms: D051357:Cyclin-Dependent Kinase 2 / Q000737:chemistry / Q000235:genetics; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D006801:Humans; D007854:Lead / Q000032:analysis*; D008024:Ligands; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D009942:Organometallic Compounds / Q000032:analysis*; D017354:Point Mutation; D025941:Protein Interaction Mapping / Q000379:methods; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D051736:Proto-Oncogene Proteins c-mdm2 / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics*; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry
chemical_list: D008024:Ligands; D009942:Organometallic Compounds; D011506:Proteins; D016159:Tumor Suppressor Protein p53; D007854:Lead; D014364:Tryptophan; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D051357:Cyclin-Dependent Kinase 2