Fluorine NMR-Based Screening on Cell Membrane Extracts

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 con-ditions, 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 19 F NMR spectroscopy, to the detection of high- and low-affinity inhibitors of a membrane enzyme in cell extracts and determination of their IC 50 values. Our approach can allow the discovery of novel binding fragments against targets known to be difficult to purify or where mem-brane-association is required for activity. These results pave the way for future applications of the methodology to these relevant and complex biological systems. MBP), overall might explain the differences in terms of IC 50 values. In addition, it should be pointed out that in the FAAH-enriched membrane preparations, the presence of other proteins and metabolites, and thus possible off-target interactions, can result in a weaker affinity of the compound for the desired target. This effect is expected to be more pronounced for fragments that, due to their small size, can promiscuously bind to several proteins. Therefore, some of the very-low-affinity inhibitors could escape detection resulting in “false negatives”. In conclusion, our findings demonstrate that n -FABS using cell extracts is feasible. This approach is particularly useful for the discovery of inhibitors against enzymes that cannot be easily overexpressed or purified as recombinant proteins in a functional and pure form, or that require membrane components for their activity. Both potent inhibitors and weakly binding fragments could be identified, and their IC 50 values reliably measured. Of note, the use of membrane preparations allowed us to run the screening against hFAAH, which is known to have unfavorable biochemical properties and very low-expres-sion yields in recombinant systems. [28] The successful imple-mentation of the methodology in cell extracts opens novel perspectives for its application in the drug discovery process for human diseases. Finally, this work encourages us to explore the possibility to develop n -FABS directly on living cells.

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 19 F NMR spectroscopy, to the detection of high-and low-affinity inhibitors of a membrane enzyme in cell extracts and determination of their IC 50 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.
Monitoring enzyme-mediated substrate metabolism in intact cells, cell lysates, or membrane preparations provides a more physiological environment for analyses and can lead to relevant advances in lead discovery. However, despite several examples of enzyme assays reported in literature, only a small number of methodologies are compatible with such biological samples. [1] These are generally restricted to fluorescence-based detection methods, and the technologies upon which these methods are based include the need for reporter assays. Importantly, the use of cell lysates or membrane preparations to monitor the enzyme activities is poorly applicable to highly sensitive label-free spectroscopic methods or biosensor-based technologies, which mainly rely on isolated biochemical systems containing purified enzyme targets.
n-Fluorine atoms for biochemical screening (n-FABS) [2][3][4] is an NMR functional assay based on fluorine spectroscopy that requires the labeling of the substrate (or cofactor) of the enzyme with a fluorine-containing group and allows the direct measurement of the conversion of the substrate (S) into the product (P). n-FABS is a homogeneous assay and does not need radioactive labeling, secondary coupled chemical or enzymatic reactions, or labeled antibodies. n-FABS has been successfully applied to several pharmaceutically relevant targets resulting in the identification of novel inhibitors. [5][6][7][8][9][10][11] In a recent work, we reported its application to the screening of chemical fragments against a purified recombinant membrane enzyme. [12] Indeed, membrane proteins are challenging targets for biochemical assays. Often, modification of membrane enzymes with fusion partners, such as maltose binding protein (MBP), or point mutations are required to enhance the level of expression of these proteins, their solubility and stability in solution. [13][14][15][16][17][18] Here, we show that n-FABS method can be extended to cell extracts, therefore using the enzyme of interest in more physiological conditions. As a model protein, we used fatty acid amide hydrolase (FAAH), a membrane-bound serine hydrolase responsible for the catabolism of a class of endogenous bioactive lipids called fatty acid ethanolamides (FAEs). FAEs, including N-arachidonylethanolamine (AEA, anandamide) and N-palmitoylethanolamine (PEA), [19] are involved in the regulation of a wide range of physiopathological conditions, such as pain, inflammation, and cognitive/emotional states. AEA is an endogenous agonist of the cannabinoid receptors, CB 1 and CB 2 , and FAAH inhibition in vivo leads to the increase of AEA levels in neural synaptic space, inducing analgesic effects comparable to those of marijuana. [20] Therefore, FAAH has been proposed as a relevant therapeutic target for the treatment of pain and central nervous system (CNS) disorders. [21][22][23][24][25] Recently, we identified and synthetized a soluble fluorinated substrate analogue (ARN1203) [12] of AEA. Using ARN1203, we successfully performed n-FABS against FAAH resulting in the identification of inhibitors with novel chemical scaffolds. [12] This study was carried out using a purified recombinant truncated form of rat FAAH (lacking the first 32 residues of the N-terminal transmembrane a-helix) fused to MBP as proposed by Labar et al. [26] Herein, we demonstrate the feasibility of performing an efficient functional screening by n-FABS method using cell membrane preparations of human FAAH (hFAAH)-overexpressing HEK293 cells, and the subsequent IC 50 determination of the identified hits.
First, we checked whether the hFAAH-enriched membrane preparation (for details, see the Materials and Methods Section in the Supporting Information) was able to cleave the fluorinated substrate ARN1203 (Figure 1 a) and whether the known FAAH inhibitor URB597 [23] completely quenched the reaction. The enzymatic activity of the hFAAH preparation on ARN1203 (60 mm) was monitored over 24 h by recording the 19 F NMR spectra with 1 H decoupling. The same sample was also analyzed in the presence of URB597 (40 mm). Changes in the 19 F NMR signals of the substrate (S) and product (P) over time are shown in Figure 1 b. The 19 F NMR signal of the substrate (S) decreases in intensity, whereas the 19 F NMR signal of the fluorinated product (P) increases in intensity with time. In the presence of URB597, no product signal is observable even after 24 hours, indicating that URB597 completely inhibits the enzyme (Figure 1 c).
In order to rule out the cleavage of the substrate by other en-dogenous enzymes, we compared the activity of the preparation using cell extracts from untransfected (control) and transfected HEK293 cells. The enzymatic reactions were quenched with URB597 (40 mm) after 2, 4, 6 and 24 hours, and 19 F NMR spectra were recorded for the eight samples (see Figure S2 in the Supporting Information). No modification of the substrate occurred in the presence of the control (Figure 2 a), while the reaction in the presence of hFAAH membrane preparations went almost to completion after 24 h (Figure 2 b). The 1D 1 H NMR spectra of this last preparation after 2 and 24 hours of incubation were also recorded (Figure 2 c).
Although the 19 F NMR spectra were different for the two samples at different time points (Figure 2 b), the corresponding 1 H NMR spectra were indistinguishable. The presence of large signals from detergent and many signals from endogenous components present in the membrane preparations hinders Figure 1. a) Enzymatic degradation of the substrate (ARN1203) by hFAAH membrane preparation (of the two products of the enzymatic reaction, only the fluorinated product is shown). b) 19 F NMR spectra of ARN1203 (60 mm) in the presence of total protein extract (0.008 mg mL À1 ) from FAAH-enriched HEK293 membrane preparation. c) The same sample as shown in panel b) but in the presence of URB597 (40 mm). S and P indicate the 19 F NMR signals of substrate and product, respectively. The 19 F NMR spectra were recorded at different time intervals over 24 h, as indicated below the spectra. the observation of the substrate and product signals thus preventing their quantification. This evidence underlines the enormous advantage of 19 F NMR spectroscopy compared with 1 H NMR spectroscopy when working with cellular extracts.
n-FABS was then applied to hFAAH membranes preparation for inhibitor screening and IC 50 measurements. Two known FAAH inhibitors (1 and 2, Figure 3 b) [27] and two fragments previously identified (3 and 4, Figure 3 b) [12] were used for validation. The experiments were performed in an end-point format, and the integrals of the product signal in the presence and absence of the test molecules were measured and compared. This format of the assay, using the fluorinated substrate of Figure 1 a, allows for the screening of approximately 150-200 samples per day. The compounds can be tested as single compounds or in mixtures for increased throughput. As expected, all four compounds were found to be active in our assay. Determination of the IC 50 values was then performed at increasing inhibitor concentrations. The plot of the integrals of the 19 F NMR product signal as a function of the concentration of the test molecules, as shown for entry 1 in Figure 3 a, allowed the calculation of the IC 50 values for the four molecules (Figure 3 b).
Remarkably, the IC 50 values obtained for entries 1 and 2 in Figure 3 b (0.189 mm and 0.292 mm, respectively) showed a rank of potency similar to that obtained previously using samples of recombinant purified MBP-FAAH, [12] and were in line with those obtained by radiometric assays. [27] Moreover, our approach allowed us to measure accurately the IC 50 values of the two fragments 3 and 4, despite their relatively weak potencies (29 mm and 31 mm, respectively). Figure 3 b shows that the IC 50 values obtained with the membrane preparations were generally higher than those measured with the recombinant enzyme. The differences between the two protein samples with respect to the source (human versus rat), length (full versus truncated), and construct type (native versus fused with MBP), overall might explain the differences in terms of IC 50 values. In addition, it should be pointed out that in the FAAHenriched membrane preparations, the presence of other proteins and metabolites, and thus possible off-target interactions, can result in a weaker affinity of the compound for the desired target. This effect is expected to be more pronounced for fragments that, due to their small size, can promiscuously bind to several proteins. Therefore, some of the very-low-affinity inhibitors could escape detection resulting in "false negatives".
In conclusion, our findings demonstrate that n-FABS using cell extracts is feasible. This approach is particularly useful for the discovery of inhibitors against enzymes that cannot be easily overexpressed or purified as recombinant proteins in a functional and pure form, or that require membrane components for their activity. Both potent inhibitors and weakly binding fragments could be identified, and their IC 50 values reliably measured. Of note, the use of membrane preparations allowed us to run the screening against hFAAH, which is known to have unfavorable biochemical properties and very low-expression yields in recombinant systems. [28] The successful implementation of the methodology in cell extracts opens novel perspectives for its application in the drug discovery process for human diseases. Finally, this work encourages us to explore the possibility to develop n-FABS directly on living cells.

Experimental Section
Detailed descriptions of the h-FAAH-enriched HEK293 membrane preparation and of the NMR experiments are provided in the Supporting Information.