Biological Analysis of Synthetic alpha-helix mimetics

Protein-protein interactions govern many of the processes that occur in living organisms. The ability to disrupt these interactions selectively is a major challenge in chemical biology. They are hard to disrupt with easily made molecules, as proteins are large and the important non-covalent contacts can be distant from each other. We have developed an inhibitor for one class of these interactions where an alpha-helix of one protein binds in the cleft of another. Such interactions occur through specific amino-acid residues projected along one face of the alpha-helix. Our inhibitor is built in a modular fashion, allowing for easy variation of the side chains and rapid formation of a large library of compounds. Current techniques for the study of these interactions rely on a complex assay, where the displacement of a pre-bound ligand is monitored rather than the actual binding event. This proposal seeks to develop a new assay to measure these interactions that will use Fluorescent Resonant Energy Transfer between the inhibitor and the protein, both of which would be fluorescently labelled. Once the inhibitor is bound, the fluorescent labels are brought into close proximity. Excitation of one of the fluorophores will cause the energy to transfer to the other fluorophore over a small distance. This results in a shift in the wavelength of the emitted light, only when binding has occurred. This allows for direct measurement of the binding event, and is also amenable to in vivo measurements using both bulk techniques as well as confocal microscopy. This assay will enable other researchers to visualize the locations of proteins in cells as well as allow for a more rapid screen of active compounds. This could eventually result in a new class of pharmaceuticals able to tackle challenging problems in medicinal chemistry.