Fluorine NMR‐Based Screening on Cell Membrane Extracts

Abstract

The possibility of measuring the action of inhibitors of specific enzymatic reactions in intact cells, cell lysates or membrane preparations represents a major advance in the lead discovery process. Despite the relevance of assaying in physiological conditions, only a small number of biophysical techniques, often requiring complex set‐up, are applicable to these sample types. Here, we demonstrate the first application of n‐fluorine atoms for biochemical screening (n‐FABS), a homogeneous and versatile assay based on 19F NMR spectroscopy, to the detection of high‐ and low‐affinity inhibitors of a membrane enzyme in cell extracts and determination of their IC50 values. Our approach can allow the discovery of novel binding fragments against targets known to be difficult to purify or where membrane‐association is required for activity. These results pave the way for future applications of the methodology to these relevant and complex biological systems.

19F NMR as a tool in chemical biology

“…The results clearly showed that the proposed n-FABS method was successful in detecting strong inhibitors of FAAH activity in cells. Also, it allowed the accurate quantification of the corresponding IC 50 values, all of which were consistent with those obtained in previous work but measured in cell membrane extracts [46].…”

Fluorine nuclear magnetic resonance-based assay in living mammalian cells

“…hFAAH overexpressing HEK293 cells hFAAH-overexpressing HEK293 (hFAAHeHEK293) cells were obtained as described previously [30]. The cells were cultured at 37 C in a humidified incubator (5% CO 2 ) using Dulbecco's modified Eagle's medium (DMEM; EuroClone, code ECB7501L) containing 10% fetal bovine serum, 1% L-glutamine, and 1% penicillinestreptomycin (complete DMEM).…”

“…where [30]. The selected fragments were tested at 200 mM and displayed different levels of inhibition (see Fig.…”

“…Recently, we successfully applied n-FABS to identify inhibitors of the membrane-bound serine amidase, fatty acid amide hydrolase (FAAH), as a purified protein [28,29] or in enriched membrane extracts [30]. FAAH is responsible for the catabolism of a class of endogenous bioactive lipids (anandamide and other fatty acid amides) [31] and represents a potentially important pharmaceutical target for the treatment of inflammation, pain, and central nervous system disorders [32e39].…”

19F-Modified Proteins and 19F-Containing Ligands as Tools in Solution NMR Studies of Protein Interactions

“…In these experiments, titration with an inhibitor of the enzyme permits one to determine the IC 50 value (the inhibitor concentration at which 50% inhibition of the reaction is observed), based on the difference in the 19 F resonance intensity in the initial state and the enzyme-modified state. Such experiments can be modified for other purposes, including simultaneous screening against multiple proteins, determining the function of a newly sequenced protein, and for screening in living cells or cell extracts (Dalvit, Ardini, Fogliatto, Mongelli, & Veronesi, 2004;Dalvit et al, 2003;Forino et al, 2005;Frutos, Tarrago, & Giralt, 2006;Lambruschini et al, 2013;Veronesi et al, 2014).…”