Enzyme-Activated Indicator Displacement Assay for the Detection of Esterases

Friday, September 30, 2016
10:00 a.m.
208 Tory Building

The 2016-17 OCCI Lecture Series presents Dr. Clifford Rossitor, of the Department of Chemistry at SUNY (Potsdam).


High-throughput screening (HTS) of molecules for enzyme inhibition in multi-well plate assays is an integral part of lead generation and optimization in the pharmaceutical industry. The increased reliance on HTS necessitates the development of stable, sensitive and robust detection methods to reduce the time-consuming and expensive impact of false positives and negatives.

Fluorescence-based assays have several benefits in the sensitive detection of enzyme activity over other detection methods (electrochemical or mechanical) including rapid detection, high sensitivity, ease of signal quantification, absence of surface binding events and preservation of sample integrity. Unfortunately, the majority of fluorescent-based systems use modulation of fluorescence intensity for signal transduction, thus limiting assay reproducibility. Assays capable of exploiting additional signaling methodologies, such as fluorescence resonance energy transfer (FRET), without the need of large fluorophores which are known to effect substrate binding (stronger binding), catalytic turnover (reactivity may be lowered or absent compared to non-labeled substrates), and solubility are highly sought after. Herein we present a new enzyme assay format which uses a metallo-protein as a molecular container in our modified indicator displacement assay.