Hit Validation: Competition Binding

When known ligands (inhibitors) for the target are available, competition binding studies can be readily performed using TINS. Note that the ligands do not have to be small molecules. Competition binding studies can be carried out using antibodies or other protein (or DNA) ligands. The figure shows two examples carried out in the NMR spectrometer.

Here the target has been immobilized and placed in both cells of the dual-cell sample holder. At the top, a substrate for the immobilized enzyme has been injected into one cell and the substrate plus a potent inhibitor into the other. In the absence of the inhibitor, the substrate is quantitatively converted into product, while in the presence of the inhibitor it remains unperturbed. Below, a fragment hit has been injected +/- the same inhibitor. In the presence of the inhibitor it does not bind, as evidenced by the significantly larger NMR signals.