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Targeting both “Half‐Reactions” of a Glycokinase Enzyme for Drug Discovery
Author(s) -
Olson Matthew W.,
Lanter James,
Sun Weimei,
Struble Geoff,
Gaul Michael,
Lawrence Kuo,
Todd Matthew
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.lb210
Subject(s) - glucokinase , hexokinase , analyte , chemistry , enzyme , drug discovery , drug , biochemistry , fructose , computational biology , glycolysis , pharmacology , chromatography , biology
With regard to drug discovery, two glycokinases are of particular interest based upon clinical data; ketohexokinase ( EC 2.7.1.3 , 1‐3) and glucokinase/hexokinase 4 ( EC 2.7.1.2 , 4‐8). Yet, modulators for either target have had limited success in pharmaceutical development. Despite, decades of basic and applied research, only a few small molecule activators for glucokinase have made it to phase 1 clinical trial. This could in fact, be due to the basic assay designs used for and the initial steps in drug discovery. Traditionally, glycokinase activities have been monitored by radioactive, enzyme‐linked, fluorescence polarization detection of ATP turnover. Described herein is a novel label‐free means of directly monitoring the bi‐bi reaction for a ketohexokinase where detection of enzyme activity utilizes mass spectroscopy. Here, turnover of ATP and fructose to ADP and fructose 1‐P, respectively, can be monitored directly and simultaneously. The selectivity of analyte detection is maximized by MRM, allowing for detection of each analyte by LCMSMS from both cells and tissues. Basic enzyme characterization is demonstrated using HTMSMS. In addition, the mechanisms of action for two unrelated compounds and the implications of target engagement of pharmaceutically active compounds from “test tube” to tissue samples are discussed.