Yes! Our latest paper reporting on screening for protein-protein interaction inhibitors with F2H is now online: http://www.ncbi.nlm.nih.gov/pubmed/24476585.
This is also my first first-author paper from my postdoctoral studies at ChromoTek! Together with Big Pharma (Janssen Pharmaceutica NV) we worked on this project, blind-testing compounds on p53/Mdm2 and p53/Mdm4 interactions, and this was a very rewarding experience. Even more exciting was that our independent results aligned extremely well! All right, coming from basic science, one is just used to things not always working that smoothly. The greater was my surprise how easy and fast it was to screen protein interactions with microscopy here, just using GFP- and RFP-fusion proteins.
Fluorescence images show bait and prey interaction in the cellular F2H assay.
Upper row: GFP-bait forms a bright green spot in the nucleus of F2H cells. RFP-prey interacts with the GFP-bait and forms a co-localizing red spot.
In my former lab, cell-based analysis of protein-protein interactions by microscopy would mean trying your luck with FRET. And FRET always came in a package with 4-hour late night slots at the confocal, freezing in that spaghetti-top (own fault) and hoping that the software does not crash and your settings will not go to Nirvana, because you still need 18 more cells to get that statistics. And pray that no one comes up with an idea to try a “couple of inhibitors” of this interaction! In different concentrations!
Anyways, screening for inhibitors of protein-protein interactions went much more efficient with F2H. It takes only half a second to look at a cell to say if it is an interaction or not. You should still check about 50 cells per condition – which all in all takes a few minutes. Thus, for our paper I screened 20 compounds in 8 different concentrations on p53/Mdm2 and p53/Mdm4 interactions. This would have taken me ages with cell-based FRET. Disclosure: to speed things up even more, we let an automated 96-well microscope image the interactions and run high-content analysis.
F2H-Assay proved useful when testing not only small-molecule inhibitors, but also stapled peptides, and for real-time monitoring of protein-protein interaction dynamics in living cells as well. Our collaboration with Sir D. Lane’s group on these topics resulted in a few more papers, all published in 2013: http://www.ncbi.nlm.nih.gov/pubmed/23214419, http://www.ncbi.nlm.nih.gov/pubmed/23653682, http://www.ncbi.nlm.nih.gov/pubmed/24278380.
Being so happy with our F2H success, I really wonder if there are easier ways to see proteins interacting?
Posted by Larisa Yurlova on Thu, Mar 20, 2014