18 February 2015

19F Target-based Screening Reduced to Practice

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. 


Jacob said...

With such a technique is the an easy way to check that the Fluorine modification isn't the primary contributor to the interaction?
(or is it obvious that there not and I's just missing it?)

Dr. Teddy Z said...

Are you thinking hydrogen bonding through fluorine? I would expect this NOT to be the case, but there is no reason why it couldn't be contributing.

Unknown said...

One trick we have done, is change the fluorinated amino acid labeling approach you use to monitor the binding event, such as 3-fluorotyrosine, to either 4-fluorophenylalanine or 5-fluortryptophan (or even a different isomer of fluorotyrosine) in the binding site, and compare the Kd. You can also use a complementary method without fluorine to compare. We haven't seen significant effects yet, but it doesn't mean they can't occur.

Jacob said...

Mostly just being concerned that as with any modification its not an artifactual consequence.
I was hoping for an simple check (ex. you can tell from the shift that is or isn't a contact point)-- While its not a major issue if one wants to check it sounds like there is no easy way out, a second assay is needed (NMR or otherwise)

Jacob said...

Dr. Pomerantz, While your here any thoughts on how the results might translate to the hit rates of other Kix domains such as MED15 (PBDID:2GUT) or GAL11 (PDBID:2K0N)?

Unknown said...

We looked at an old sequence alignment of other KIX domains from a nice paper by the Naar group. A lot of the aromatics seem to be conserved, so probably worth a try. In general the protein protein interaction
field has seen a large enrichment of aromatics (particularly Y and W) at PPI interfaces, which is why we decided to label them.

Jacob said...

thanks for taking the time to answer
much obliged

Stephan Zech said...

As usual in NMR, any experiment works well for ubiquitin, lysozyme, or similarly sized proteins (12 kDa in this case). Good luck getting this running for a 'regular' drug target (usually 35 kDa or bigger). The 19F relaxation will kill the signal and sensitivity will be much lower than proton detect experiments on the protein. So this can be a nice technology addition, for fairly small proteins.

Matthew R. Lee said...

The 508 cpds screened appear to be non-fluorinated, as only 1 of the 4 hits contained fluorine atoms.

Shouldn't we consider work from Fesik's groups to also be target-based ligand screening? Perhaps what is first about this paper is the screen against a fluorine-labelled protein?