High-throughput cellular assays to find a small molecule inhibitor of the TrkA receptor (360G-Wellcome-211340_Z_18_Z)

This project aims to identify novel TrkA receptor antagonists that in the future might be developed into drugs to reduce chronic pain symptoms upon the binding of NGF. NGF levels have been shown to be elevated in injury, inflammation and chronic pain states. Sequestering of NGF with anti-NGF antibodies has been shown to reduce hyperalgesia. However, antibody therapies are costly due to manufacturing and purification processes. To find novel TrkA receptor antagonists, a virtual screen against a novel allosteric site within the catalytic domain of the TrkA receptor has identified 125 compounds with potential to dock into the site and inhibit receptor function. I will test these compounds on a cellular assay using NFAT-bla CHO-K1 reporter lines that have had human TrkA, TrkB or TrkC genes stably transfected into them and overexpressed. Using reporter gene b-lactamase, activated upon neurotrophins binding to the Trk receptors, the substrate CCF4-AM (directly loaded onto the cells) is cleaved. This can be measured by changes in the cell FRET value from 530nm to 460nm. This will allow us to test if any compounds identified in the virtual screen inhibit TrkA function, and whether they show selectivity for TrkA over the other two Trk receptors.