Target Engagement Capabilities in Drug Discovery at the G-INCPM – QR2 Case Study

Quinone reductase 1 (NQO1) and 2 (QR2) are enzymes that reduce harmful quinones and allow their removal from the cell, thus preventing oxidative damage. We have shown recently QR2 involvement in cognitive function in rodent. It is important to identify potent inhibitors of QR2 to better understand its role and effect in the brain. In collaboration with Prof Kobi Rosenblum’s team, a High Throughput Screening (HTS) assay was developed to asses QR2 enzymatic activity. 200,000 compounds were screened, of which a few validated hits were followed by our medicinal chemistry team. The next step in drug discovery is to pursue target engagement. This is defined as the identification of proteins directly binding to small molecules. Our goal is to identify and quantify binding of relevant small molecules to QR2 protein. To achieve this we employed Surface Plasmon Resonance (SPR) and Cellular Thermal Shift Assay (CETSA). In SPR methods, QR2 is immobilized on an SPR crystal and the small molecule solution is injected over the immobilized QR2. This type of experiment run in vitro in purified solution. The CETSA, on the other hand, is performed in the context of a whole cell and reports binding in situ. CETSA results report at which temperature QR2 protein denatures; Small molecules that bind to QR2 and stabilize it will show a thermal shift. Using these methods we were able to validate the binding of a number of new small molecules to QR2, showing quantitative binding data for this protein for the first time.