03 March 2014

FABS-ulous Screening Against Membranes

The blog is running on a 2 hour delay today thanks to the winter storm (we actually got a nothing burger here from it).
If you follow this blog, and actually read what I say, you will know I have a 19F-fetish.  Thus, whenever another paper comes out, I gravitate towards it.  Claudio Dalvit is really one of the primary (if not THE) drivers of 19F NMR screening development.  He has been discussed on this blog often.  Most recently, back in October, when he published an example of n-FABS against a membrane target, FAAH.   Now, he is back with this paper: "Fluorine NMR-based Screening on Cell Membrane Extracts".  I was immediately transported back to my days at Lilly where in our group we came up with the great idea to try to screen (using STD) against crude membrane preps.  I don't remember much but my lab mate being unsuccessful in the end for any number of reasons.  Obviously, the development of a robust, biophysical technique which can be applied to intact cells, cell lysates, or membrane preps would be a significant addition to the entire biophysical toolbox.  Currently, only biochemical assays largely based upon fluorescence can do this.  n-FABS, as decribed previously, relies on the substrate of the target (which is labeled with at least one 19F atom) being converted by target action into product, and thus causing a chemical shift change in the 19F.  This is easily detected by NMR and voila, an assay is born. This work is an extension of the previous work on FAAH and very similar to this work by Brian Stockman.  The 19F chemical shift of substrate and product are easily differentiated and roughly quantitatable:
The proper controls showed that this activity is solely due to the TOI.  What makes this assay so appealing is shown in the next figure:
This figure shows the 1H spectrum of the reaction at 2hr (top) and 24hr (bottom).  There is virtually no difference in this spectrum, indicating that it is impossible to follow the substrate due to large signals from detergents and endogenous protonated signals.  For me this is the key to this.  We all know membrane proteins are hard to do, especially with fragments.  I have always wondered where 19F fits in the biophysical toolbox, especially in light of recent discussions where it presumed that 19F could out perform 1H.  In discussions, I have said that 19F runs circles around 1H when the ligands are highly aliphatic.  Well, this is the converse, and still just as true, when the sample matrix is ugly with "other stuff", in this case the stuff that keeps the target in solution.  One major drawback is that this approach is NOT a binding approach, and thus would be of limited utility against non-enzymatic membrane targets, such as a majority of membrane targets.  In the majority of membrane targets, SPR may be the most robust approach.

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