An AlphaLISA assay adaptable to HTS in 384 well plates was designed to measure the TG2-FN interaction. The assay measures the interaction between His6-TG2 bound to Ni-chelated acceptor beads and biotinylated FN42 bound to streptavidin coated donor beads. Protein concentrations were titrated to optimize the assay, as the alpha beads have specific binding capacity and become progressively saturated at increasing protein concentrations. At hook point, both beads are saturated generating the maximum signal. Excess protein above these levels inhibits the association between target molecules and beads causing a decrease in signal. The hook point was reached at 10 nM of His6-TG2 and 3 nM of FN42. To further evaluate the performance of the assay in HTS mode, the Z9 value was calculated by taking into account the means and the standard deviations of the positive and the negative controls. In this manuscript, we describe a new strategy to measure the interaction between TG2 and FN through proximity based binding AlphaLISA assay. Application of this assay to the ChemDiv library of chemical compounds led to the discovery of several potent TG2-FN inhibitors, which were 649735-46-6 subsequently shown to block cell adhesion and migration in OC cell lines. Our findings have several implications. First, we demonstrate that the TG2-FN interaction is druggable. Generally PPIs represent a daunting target for disruption by small molecules due to their large interfacial areas and their often noncontiguous contact points. However, the TG2-FN interaction is characterized by a TG2 b-hairpin inserting into a deep pocket of FN, rendering it an attractive complex to be disrupted by SMIs. Resolution of the three-dimensional structure of TG2 has provided important insight into the complex regulation of its functions. In addition to the catalytic triad consisting of Cys- 277, 911710-03-7 His-335, and Asp-358, the protein has the FN-binding within its N-terminal. The binding site to FN is located around the b-hairpin loop, and mutations within this sequence significantly alter formation of a complex with FN. On the surface of various cells, the complex between TG2 and FN is further supported and stabilized by direct interactions of both proteins with integrins, the major adhesion rece
