title: 2-Fluoro-ATP as a versatile tool for 19F NMR-based activity screening.
abstract: 19F NMR-based methods have found utility in activity-based screening assays. However, because enzymes catalyze a diverse set of reactions, a large variety of fluorinated substrates would need to be identified to target each one separately. We have developed a more streamlined approach that is applicable to many enzymes that utilize ATP as a substrate. In this method, a fluorine-containing ATP analogue, 2-fluoro-ATP, is used to monitor the reaction. Applications are described for nicotinamide adenine dinucleotide synthetase and 3-phosphoinositide dependent kinase-1. Fragment screening results for the latter indicate that this technique can identify compounds that inhibit as well as activate reactions. The present results, together with previous biochemical studies from other laboratories, have shown that 2-fluoro-ATP can serve as a substrate for nine enzymes that are representative of three of the six enzyme subclasses, namely the transferases, hydrolases, and ligases. This suggests that 2-fluoro-ATP is suitable as a universal tool for screening ATP-requiring enzymes. Importantly, 2-fluoro-ATP has been determined to be a valid substrate for a variety of kinases, including both small molecule and protein kinases, suggesting that it may be useful for investigating the large number of pharmaceutically relevant kinases.
authors.affiliation: Pfizer Global Research & Development, Groton, Connecticut 06340, USA. brian.j.stockman@pfizer.com
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Stockman
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D004798:Enzymes / Q000032:analysis* / Q000378:metabolism; D005461:Fluorine / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D009703:Nucleoside-Phosphate Kinase / Q000032:analysis / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000032:analysis / Q000378:metabolism; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D013379:Substrate Specificity
chemical_list: D004798:Enzymes; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; C038022:2-fluoro-ATP; D005461:Fluorine; D000255:Adenosine Triphosphate; D017346:Protein Serine-Threonine Kinases; D009703:Nucleoside-Phosphate Kinase; C020093:dTMP kinase