ADP Hunter™ Plus: A Rapid, Quantitative HTS Assay for GDP and UDP

ADP Hunter™ Plus: A Rapid, Quantitative HTS Assay for GDP and UDP

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There are numerous assays available to measure the activity of enzymes involved in the transfer of a phosphate from ATP to a substrate macromolecule.  However, there are only a few assays for enzymes that utilize other nucleotide triphosphates, opening opportunities to address pharmacologically relevant targets.
GTPases are a broad family of enzymes with a wide variety of activities such as signal transduction, Ras-mediated cell signaling and endocytosis.  Hydrolysis of GTP to GDP is a critical step for GTPase activity and hence can be used to characterize these enzymes.
UDP-glycotransferases are involved in polysaccharide biosynthesis, post-translational modification and glucuronidation of small molecules.  The latter is particularly relevant for drug discovery since UDP-transferases are important in drug metabolism and removal.  There are over 200 glycotransferases in humans which are increasingly being targeted for therapeutic intervention.
DiscoveRx has developed ADP Hunter Plus and ADP Quest, an homogeneous fluorescent assay for detection of nucleotide diphosphates, including ADP, GDP and UDP.  ADP Quest was specifically developed for real time kinetics of enzymes.  This is a non-antibody, general purpose biochemical assay that does not require a labeled substrate and generates a positive red-shifted fluorescent read-out that is compatible with any fluorescent plate reader.  There are currently limited available approaches for GTPase such as HPLC, ELISA, IP or Pi assays.  The DiscoveRx assay offers much greater ease of use, with an HTS-friendly procedure, excellent sensitivity and reproducibility.  We will present data here highlighting the key features of the assay, the versatility of the technology, as well as results using our assay with an enzyme that produces UDP.