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This version was published on June 1, 2007
Journal of Biomolecular Screening, Vol. 12, No. 4, 457-463 (2007)
DOI: 10.1177/1087057107299717

A Nuclear Magnetic Resonance–Based Functional Assay for Nicotinamide Adenine Dinucleotide Synthetase

Brian J. Stockman

Pfizer Global Research and Development, Groton, Connecticut, brian.j.stockman{at}pfizer.com

Ian J. Lodovice

Pfizer Global Research and Development, Groton, Connecticut

Douglas A. Fisher

Pfizer Global Research and Development, Groton, Connecticut

Alexander S. Mccoll

Pfizer Global Research and Development, Groton, Connecticut

Zhi Xie

Pfizer Global Research and Development, Groton, Connecticut

Nicotinamide adenine dinucleotide synthetase (NadE) is an essential enzyme for bacterial pathogens and is thus a promising antibacterial target. It catalyzes the conversion of nicotinic acid adenine dinucleotide to nicotinamide adenine dinucleotide. Changes in chemical shifts that occur in the nicotinic acid ring as it is converted to nicotinamide can be used for monitoring the reaction. A robust nuclear magnetic resonance—based activity assay was developed using robotically controlled reaction initiation and quenching. The single-enzyme assay has less potential for false positives compared to a coupled activity assay and is especially well suited to the high concentration of compounds in fragment screens. The assay has been used to screen fragment libraries for NadE inhibitors. ( Journal of Biomolecular Screening 2007:457-463)

Key Words: antibacterial • fragment screening • NAD synthetase • NMR screening


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