If you read this blog you know I love 19F NMR. I am a big fan of it for ligand based screening or as a secondary screen in target-based mode. Well, this paper is the first to use target-based NMR to screen small molecules. Using 3-fluorotyrosine-labeled protein and using what they call Protein observed fluorine NMR (PrOF-NMR), the interrogate a PPI (CBP/p300-KIX) and determine this interaction's ligandability.
Starting from the Maybridge Ro3 Library, they found 508 19F containing fragments. These were put into 85 mixtures (5 or 6 compounds each) and screened at 833uM and 2.5 % DMSO (Figure 1, KG-501 is a control compound). Each experiment was performed in 5 minutes (with a quick reference spectrum) which is faster than SOFAST HSQC (for 15N labeled proteins). 15 mixtures gave hits which upon deconvolution gave 4 actives (>2 SD in chemical shift), a 0.8% active rate.
|Figure 1. Typical 19F Data|
They then titrated the four confirmed fragments to determine the Kd, which for 3 of them was in the mM range. They followed this up with Analog by Catalog and developed some SAR. Lastly, they used H-N HSQC to verify that the compounds do bind and where they data shows. They do.
Some thoughts on this. The use of 3FY is only one amino acid that can be used. Fluorotryptophan can also be used, so this method can easily be applied to other systems. Secondly, 19F can accomodate much larger pool sizes (within reason). And there is no reason why both could not be used. Of course, one of the things not noted here is that they produce single mutants in order to ID each individual residue. I think you could live without residue specific assignments and still get tremendous value out of this method. I would be curious what others think. We bring up impractical tools all the time, so I really want to applaud this paper. Here is a very practical new method for screening.