Thursday, September 5, 2013
The first fluorescence polarization activity-based protein profiling assay compatible with membrane proteins
Rhomboids are a family of intramembrane proteases which are found in all kingdoms of life and have been found to play a role in bacterial survival and host cell infection by parasites. They are functionally diverse so developing an assay to screen for inhibitors of rhomboids would be hindered if it relied on altering a substrate. To alleviate this problem a German group recently published: 'A New Class of Rhomboid Protease Inhibitors Discovered by Activity-Based Fluorescence Polarization' in PLoS One. In this paper they describe the use of fluorescence activity based-probes (ABPs). ABPs are small molecules that covalently and selectively bind to the active form of an enzyme. When a fluorescent dye is added to the ABP the binding of the ABP can be monitored using fluorescence polarization.
To assist in the design and implementation of this screening platform these scientists selected the POLARstar Omega from BMG LABTECH. Once they found the appropriate conditions that allowed them to solubilize their protein of interest without affecting their fluorescence polarization results they were able to obtain Z' values of over 0.9, indicative of an outstanding screening platform! Based on their results they have identified a new class of inhibitor which they hope will be the basis for additional compounds of therapeutic relevance.