A Cy5-labeled S100A10 tracer used to identify inhibitors of the protein interaction with annexin A2.

Protein-protein interactions are increasingly of interest as targets in small-molecule drug discovery. The interaction between the Ca2+- and phospholipid-binding protein Annexin A2 and its binding partner S100A10 has been implicated in angiogenesis and cancer metastasis. Here, we present a methodology to screen for inhibitors of this protein interaction. We developed a Cy5-labeled S100A10 tracer and showed by circular dichroism spectroscopy that the secondary structure is indistinguishable from that of non-labeled S100A10. This tracer was used to develop a binding assay based upon fluorescence resonance energy transfer to a Cy3-labeled Annexin A2 peptide ligand. The binding parameters matched those for unlabeled components as observed by equilibrium dialysis, which we determined separately, as well as those determined by isothermal titration calorimetry. Binding of labeled and unlabeled peptide was specific and mutually competitive. We used this assay for screening a small compound library derived by computational interrogation of the S100A10-binding pocket. Hits were obtained with IC(50) values in range of the IC(50) of the cognate Annexin A2 peptide ligand. Hits were subjected to an exact parallel assay measuring an unrelated protein-protein interaction (antigen-antibody). In this way, we identified genuine hits that inhibited the interaction between S100A10 and Annexin A2 but do not affect the fluorescence readout. These compounds are potentially of interest as candidates for further analysis and medical chemistry exploration. The simple assay format described here can be employed in early-stage exploration of other protein-protein interaction targets.