title: Applications of NMR screening techniques to the pharmaceutical target Checkpoint kinase 1.
abstract: Ligand screening techniques based on NMR spectroscopy are not as sensitive as other commonly used methods like fluorescence, radiolabeling and surface plasmon resonance. However, using modern NMR instrumentation, they can achieve reliable screening under near physiological condition using as little as 4.6 nmol of receptor and 100 nmol of ligand. Additionally, these NMR methods can also provide valuable and specific information on the ligand under investigation such as the dissociation constant KD, the binding epitope and most importantly some structural information on the actual conformation in the bound state. In this manuscript, we describe the use of NMR based screening techniques ("Saturation Transfer Difference" (STD) and "Water Ligand Observed via Gradient SpectroscopY" (WaterLOGSY)) to detect small therapeutic molecules that interact with the DNA damage checkpoint enzyme Checkpoint kinase 1 (Chk1). After the identification of the most potent ligand, we used specific NMR experiments to perform the epitope mapping of this ligand ("Group epitope mapping-STD" (GEM-STD), "Difference of Inversion REcovery rate with and without Target IrradiatiON" (DIRECTION)) and to characterize its bound conformation ("Transferred-Nuclear Overhauser Effect SpectroscopY" (tr-NOESY), "Transferred-Rotating frame Overhauser Effect SpectroscopY" (tr-ROESY)). Finally, we used molecular docking procedures to position the ligand within the active site of Chk1. On the experimental level, a comparison between NMR studies performed in a 90%H2O/10%D2O buffer and a 100% D2O buffer is also presented and discussed.
authors.affiliation: Institut de Recherches Servier, Analytical and Physical Chemistry Department, 11 rue des Moulineaux, 92150 Suresnes, France. Electronic address: nathalie.lancelot@fr.netgrs.com.
authors.forename: N
authors.initials: N
authors.lastname: Lancelot
authors.affiliation: Bruker BioSpin, Laboratoire d'applications RMN, 34 rue de l'industrie, 67166 Wissembourg, France. Electronic address: martial.piotto@bruker.fr.
authors.forename: M
authors.initials: M
authors.lastname: Piotto
authors.affiliation: Institut de Recherches Servier, Chimie Partenariats et Modélisation Moléculaire, 125 Chemin de Ronde, 78290 Croissy-Sur-Seine, France.
authors.forename: I
authors.initials: I
authors.lastname: Theret
authors.affiliation: Institut de Recherches Servier, Chimie Partenariats et Modélisation Moléculaire, 125 Chemin de Ronde, 78290 Croissy-Sur-Seine, France.
authors.forename: B
authors.initials: B
authors.lastname: Lesur
authors.affiliation: Institut de Recherches Servier, Analytical and Physical Chemistry Department, 11 rue des Moulineaux, 92150 Suresnes, France.
authors.forename: P
authors.initials: P
authors.lastname: Hennig
mesh_terms: D000818:Animals; D000071877:Checkpoint Kinase 1; D004249:DNA Damage / Q000502:physiology; D015195:Drug Design; D018604:Epitope Mapping; D000939:Epitopes; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D062105:Molecular Docking Simulation / Q000379:methods*; D058990:Molecular Targeted Therapy; D004364:Pharmaceutical Preparations / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D061987:Sf9 Cells; D018411:Spodoptera
chemical_list: D000939:Epitopes; D008024:Ligands; D004364:Pharmaceutical Preparations; D011494:Protein Kinases; D000071877:Checkpoint Kinase 1
keywords: Checkpoint kinase 1; Group epitope mapping; NMR screening; Saturation Transfer Difference; Transferred NOESY