Fluorinated fragments vs FAAH – functionally

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.