Fluorine NMR is a topic that has come up several times on Practical Fragments (see here, here, and
here). As readers will recall, the 19F nucleus can be readily
detected with a properly equipped NMR spectrometer. The isotope has a wide
range of chemical shifts, and sensitivity to the local environment makes it
easy to detect whether fluorine-containing fragments bind to a protein. But you
don’t need a dedicated fluorine-containing library: in a new paper in ChemBioChem, Claudio Dalvit and
coworkers at Fondazione Istituto Italioano di Tecnologia describe using
fluorinated substrates to screen a membrane enzyme.
The researchers use an approach they call n-fluorine atoms for biochemical screening
(n-FABS). A substrate or cofactor is
labeled with fluorine, and when this is processed by an enzyme, the resulting
change in chemical structure affects the 19F chemical shift, which
is easily detected by NMR. Either substrate or product (or both) can be
observed, and a decrease in product can be attributed to inhibition of the
enzyme.
The researchers were interested in the protein fatty acid
amide hydrolase (FAAH), a membrane-bound enzyme that hydrolyzes lipids such as
endocannabinoids. Of course, membrane-proteins are tough to screen using
fragment-based approaches, and the fact that this enzyme processes lipophilic
substrates makes things even more challenging. The researchers synthesized several
fluorine-containing substrates, but most of these turned out to be insoluble or
formed aggregates, even at low micromolar concentration and even in the presence of detergent.
Ultimately they were able to make one substrate that was soluble at 30
micromolar, sufficient for screening.
Next, the researchers assembled a library of fragments.
Although these did not need to contain fluorine for the n-FABS assay, the researchers chose to focus on fluorine-containing
fragments anyway, perhaps so they could use other NMR methods to confirm
binding. Of 160 commercial fluorine-containing fragments purchased, 113 showed
solubility ≥ 0.1 mM, purity ≥ 75%, and no aggregation. These were combined into
23 pools of 5 and screened for inhibition in the n-FABS assay at 200 micromolar of each fragment. Pools that showed
>15% inhibition were deconvoluted to find the active fragments; some contained
more than one hit. This process led to a remarkably high hit rate of 16.5%. The
IC50 values of all 19 of these hits were then determined using n-FABS and they showed quite a range,
from quite potent (3 micromolar) to low millimolar.
These are nice results and there are clear opportunities for
advancing some of the fragments, but I must admit I was left wanting more. The n-FABS assay is essentially an
inhibition assay, and of course there are all kinds of things that can show
inhibition without proper binding. However, since all the fragments do contain
fluorine, it would be straightforward to actually measure direct binding using NMR; it would be very interesting to see how
many fragments show up in both assays. Perhaps we will see this in a follow-up
study.
1 comment:
I am the 19F-fetishist here and I will be honest, this is a ho-hum paper to me outside of the target. I, like Dan, would like to see more, and quite frankly, expect better from Dr. Dalvit.
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