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P2028
nanoCF™ Assay system  A Novel Tool for Mechanistic Studies
Part Two  Compound Profiling & Mechanism of Action
Presenter Christopher Hoyle, Eksigent Technologies, USA
Additional Authors: David Wyrick, Don Arnold
Eksigent Technologies has developed a novel assay platform, the principles of operation and process of which are detailed in an accompanying poster, Part One. The use and versatility of nanoCF assay system for characterization biochemical reactions at the nanoliter scale is demonstrated. The compatibility of nanoCF with standard fluorescence-based detection modes (FLINT, TR-FRET) and industry standard biochemical reagents is demonstrated with examples provided for proteases and multi-substrate enzymes.  In addition, nanoCF is suitable for determining % inhibition and IC50 of test compounds of unknown potency. The quality of data is superior to that generated by conventional microtiter plate methods and enables a novel streamlined approach to the generation of mechanistic data for determining the mode of action of inhibitors. A model enzyme system is used to determine, in a single run, both the inhibition mechanism and potency of a known inhibitor. It is apparent that nanoCF is suitable for use in a variety of mechanistic experiments - enzyme reaction kinetic mechanism determination; steady state kinetic constants (Km and kcat); effects of reagents components on reagent kinetics; kinetic isotope effects; pH effects on reaction kinetics; assessment of synergism or antagonism between two small molecule effectors.