Development of Fragment-Based n-FABS NMR Screening Applied to the Membrane Enzyme FAAH

Article abstract of DOI:10.1002/cbic.201300347

Despite the recognized importance of membrane proteins as pharmaceutical targets, the reliable identification of fragment hits that are able to bind these proteins is still a major challenge. Among different ¹⁹F NMR spectroscopic methods, n-fluorine atoms for biochemical screening (n-FABS) is a highly sensitive technique that has been used efficiently for fragment screening, but its application for membrane enzymes has not been reported yet. Herein, we present the first successful application of n-FABS to the discovery of novel fragment hits, targeting the membrane-bound enzyme fatty acid amide hydrolase (FAAH), using a library of fluorinated fragments generated based on the different local environment of fluorine concept. The use of the recombinant fusion protein MBP-FAAH and the design of compound 11 as a suitable novel fluorinated substrate analogue allowed n-FABS screening to be efficiently performed using a very small amount of enzyme. Notably, we have identified 19 novel fragment hits that inhibit FAAH with a median effective concentration (IC₅₀) in the low mM-μM range. To the best of our knowledge, these results represent the first application of a ¹⁹F NMR fragment-based functional assay to a membrane protein.

Full text of DOI:10.1002/cbic.201300347

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DOI: 10.1002/cbic.201300347
Development of Fragment-Based n-FABS NMR Screening
Applied to the Membrane Enzyme FAAH
Chiara Lambruschini,[a] Marina Veronesi,[a] Elisa Romeo,[a] Gianpiero Garau,[a]
Tiziano Bandiera,[a] Daniele Piomelli,[a, b] Rita Scarpelli,[a] and Claudio Dalvit*[a, c]
Despite the recognized importance of membrane proteins as
pharmaceutical targets, the reliable identification of fragment
hits that are able to bind these proteins is still a major chal-
lenge. Among different 19F NMR spectroscopic methods, n-fluo-
rine atoms for biochemical screening (n-FABS) is a highly sensi-
tive technique that has been used efficiently for fragment
screening, but its application for membrane enzymes has not
been reported yet. Herein, we present the first successful appli-
cation of n-FABS to the discovery of novel fragment hits, tar-
geting the membrane-bound enzyme fatty acid amide hydro-
lase (FAAH), using a library of fluorinated fragments generated
based on the different local environment of fluorine concept.
The use of the recombinant fusion protein MBP-FAAH and the
design of compound 11 as a suitable novel fluorinated sub-
strate analogue allowed n-FABS screening to be efficiently per-
formed using a very small amount of enzyme. Notably, we
have identified 19 novel fragment hits that inhibit FAAH with
a median effective concentration (IC50) in the low mm–mm
range. To the best of our knowledge, these results represent
the first application of a 19F NMR fragment-based functional
assay to a membrane protein.
Introduction
Over the last decade, the fragment-based approach (FBA) has
become an established and efficient method for hit identifica-
tion and optimization as an appealing alternative to high-
throughput screening (HTS).[1] The central idea in FBA is to
identify fragments that bind to target proteins from a relatively
small library of compounds (100–1000) complying with the
rule of three.[2] Such libraries are able to cover the chemical
space more thoroughly than libraries of larger molecules and
are more likely to contain cores that fit into the target binding
site.[3] Due to their low molecular weight, fragments usually
bind weakly to the target macromolecules (mm–mm range). In
order to detect such weak binding, sensitive biophysical tech-
niques such as NMR, fluorescence spectroscopy, surface plas-
mon resonance, and X-ray crystallography[4] have been applied.
Although FBAs have been successfully applied against solu-
ble protein targets, and several compounds have advanced to
clinical trials,[5] the use of this approach on membrane-bound
targets is still in its infancy. These proteins constitute more
than 40% of drug targets, demonstrating their notable rele-
vance for the treatment of different diseases. The development
of drugs targeting membrane proteins has been based pre-
dominantly on the screening of large compound libraries.[6]
In general, major challenges in targeting membrane proteins
include: 1) production and purification of sufficient quantity of
functional target; 2) stabilization of the protein in a homogene-
ous system suitable for the assay; and 3) high levels of false
positives due to nonspecific partitioning of fragments into
detergent systems. Although different new reagents and tech-
niques have enabled the production and purification of a varie-
ty of membrane proteins,[7] continuous efforts are dedicated to
the development of efficient and reliable assays for the investi-
gation of this class of challenging targets.
n-Fluorine atoms for biochemical screening (n-FABS)[8] has
been demonstrated to be a powerful and sensitive method for
performing biochemical assays in an FBA.[9] n-FABS is a bio-
chemical methodology which requires the labeling of the
enzyme substrate (or cofactor) with a fluorine-containing
group. It is based on the extreme sensitivity of 19F isotropic
chemical shift to small perturbations, resulting in different
19F NMR signals for the starting substrate and the enzymatically
modified substrate (or cofactor), even when the modification
occurs far from the fluorine moiety. Its robustness results in
the detection of even weak inhibitors and in reliable IC50 deter-
minations of the hits.
[a] C. Lambruschini,+ M. Veronesi,+ Dr. E. Romeo, Dr. G. Garau, Dr. T. Bandiera,
Prof. Dr. D. Piomelli, Dr. R. Scarpelli, Dr. C. Dalvit
Department of Drug Discovery and Development
Fondazione Istituto Italiano di Tecnologia
Via Morego 30, 16163 Genova (Italy)
[b] Prof. Dr. D. Piomelli
Department of Anatomy and Neurobiology
3216 Gillespie Neuroscience Facility, University of California
Irvine, CA 92697-1275 (USA)
[c] Dr. C. Dalvit
University of Neuchꢀtel
Avenue de Bellevaux 51, 2000 Neuchꢀtel (Switzerland)
E-mail: claudio.dalvit@unine.ch
[+] These authors contributed equally to this work.
Recently, attention has been also dedicated to the develop-
ment of more sophisticated approaches to build efficient frag-
ment libraries. In this context, a strategy based on the local en-
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201300347.
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Results and Discussion
vironment of fluorine concept has been recently proposed by
Vulpetti et al.[10] The component fragments of such libraries are
selected based on their different fluorine local environmental
fingerprints, which allows for better detection of putative fluo-
rophilic hot spots on the desired biological target.[11]
The aim of this work was the development of a fast, efficient,
and reliable biochemical fragment screening method applica-
ble to FAAH, combining n-FABS methodology with the use of
a fragment library. In order to apply this approach, three main
requirements had to be properly fulfilled: 1) availability of a
stable and active form of FAAH in aqueous/detergent solution;
2) identification of a suitable fluorinated substrate of FAAH;
and 3) design and construction of a library of fragments.
Our main interest was focused on the development of an n-
FABS assay for fragment screening against membrane proteins.
For this purpose, we selected the fatty acid amide hydrolase
(FAAH) enzyme[12] and screened a small fluorinated library built
in-house. FAAH is a membrane-bound serine hydrolase, mainly
expressed in brain, testis, and liver, responsible for the catabo-
lism of a class of endogenous bioactive lipids called fatty acid
ethanolamides (FAEs), such as N-arachidonylethanolamine
(AEA, anandamide) and N-palmitoylethanolamine (PEA;
Scheme 1). FAEs are involved in several physiological functions
Expression of the recombinant membrane enzyme FAAH
FAAH is a 63 kDa protein whose structure was first solved by
Bracey et al. in 2002,[16] expressed in E. coli as a truncated func-
tional form of the enzyme (DTM-FAAH) which lacks 29 residues
at the N terminus. The crystal structure showed that this
enzyme anchors cell membranes through the elongated trans-
membrane a-helix of the N terminus and two highly hydro-
phobic a-helices (a18 and a19). The enzyme presents two in-
ternal channels: the membrane access channel, which allows
the substrate to access the active site catalytic triad (Lys142,
Ser217, and Ser241), and the acyl chain binding pocket, which
accommodates the fatty acid chain of the substrate during
hydrolysis.
The insertion of an N-terminal fusion protein increases the
amount of membrane protein that is heterologously ex-
pressed, and maltose-binding protein (MBP) is particularly ef-
fective at promoting overexpression of soluble well-folded pro-
teins.[17] Hence, we generated a construct of DTM-rFAAH (rat
FAAH residues 33–574) bearing an MBP at the protein N termi-
nus as a fusion protein, and a hexahistidine (His6) tag at the
C terminus to optimize the performance of the recombinant
protein (MBP-rFAAH) purification. This system allowed us to
produce this membrane protein with a yield of ~5 mgLÀ1 of
culture medium and purity exceeding 80% after a single affini-
ty purification step using Ni–NTA agarose. Importantly, activity
of recombinant MBP-rFAAH was stable over time (see the Sup-
porting Information). Moreover, WaterLOGSY[18] experiments on
MBP-rFAAH in the presence of a five-fragment mixture from
our fluorinated library demonstrated that the enzyme did not
aggregate (data not shown).
Scheme 1. Selected natural substrates (PEA and AEA) and fluorinated sub-
strate analogues (compounds 1–11).
and in the regulation of a wide range of mammalian behaviors,
including pain, inflammation, and cognitive/emotional states.
Inhibition of FAAH raises the extracellular levels of AEA and
leads to indirect activation of cannabinoid receptors.[13] Over
the last decade, a significant number of reversible and irrever-
sible FAAH inhibitors have been reported.[14] These molecules
might be useful in the treatment of pain, inflammation, drug
addiction, appetite regulation, sleep disorders, anxiety, and de-
pression.[15]
The requirement of a purified stable functional enzyme and
the identification of a suitable fluorinated enzyme substrate
has rendered n-FABS assay development particularly challeng-
ing when applied to FAAH, for two main reasons: 1) FAAH is
a membrane-bound enzyme; and 2) the natural substrates of
FAAH are characterized by high lipophilicity and low solubility
in aqueous solutions.
Identification of fluorinated FAAH substrates
One of the challenges we encountered in the setup of the n-
FABS assay was the identification of an aqueous soluble fluori-
nated FAAH substrate. It has been reported that FAAH catalyz-
es the hydrolysis of several FAEs.[19] We selected AEA and PEA
as our starting point for the design of new fluorinated ana-
logues, which are shown in Scheme 1.
Vandevoorde et al.[20] reported that several PEA analogues
bearing different substituents in the amine moiety (head
chain), including the fluoroethyl group of compound 1, are rec-
ognized by FAAH, demonstrating that the OH group of the
head chain in the natural substrate is not fundamental for
FAAH recognition. On the other hand, the authors also ob-
In the present work, we report on the development of an
NMR-based fragment screening assay that can overcome the
challenges posed by targeting FAAH. We demonstrate the
performance of our methodology through the screening of
our in-house fluorinated fragment library and identification of
novel fragment hits, with a good hit rate (16.5%). To the best
of our knowledge, the present study represents the first appli-
cation of 19F NMR fragment-based biochemical screening for a
membrane protein.
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served a low solubility of those compounds in aqueous buffers.
For this reason, we designed analogues bearing the fluorinated
moiety not only in the head chain, such as in compounds 2
and 3, but also in the acyl portion (tail chain), such as in com-
pounds 4–6, aiming to enhance the solubility of the fluorinat-
ed PEA analogues by maintaining the polar hydroxy group of
the head chain.
porting Information for experimental details). Under these con-
ditions, compounds 1–6 demonstrated low solubility (<5 mm)
and a strong tendency to aggregate. On the other hand, com-
pounds 7–11 showed improved solubility in PBS. Unfortunate-
ly, compounds 8 and 9 aggregate at concentrations higher
than 10 mm (Figure S1 in the Supporting Information).
Therefore, compounds 7, 10, and 11 were selected as possi-
ble substrates of FAAH in the setup of the n-FABS assay. The
stability and Km of the three candidates were then evaluated.
To determine the stability of 7, 10, and 11, we recorded their
19F NMR spectra in PBS at room temperature every 60 min over
14 h. No differences in 19F NMR signals were observed, indicat-
ing that the three compounds are sufficiently stable at room
temperature (Figure S2).
With regard to the fluorinated AEA analogues, we decided
to introduce the fluorinated moiety only in the head chain
(compounds 7–11) for easier synthetic accessibility. Among
these, compounds 7 and 9 are known FAAH substrates[20–21] in
which the CH2OH group is replaced by a CH2F and a CF3
moiety, respectively. Compound 8 is a close analogue of com-
pounds 7 and 9, while compounds 10 and 11, bearing a polar
OH group in the head chain, were designed with the aim of
enhancing the solubility of known FAAH substrates 7 and 9.
To select a suitable fluorinated substrate, we applied three
criteria: 1) solubility and stability; 2) lack of aggregation in
assay buffer; and 3) affinity profile (Km and enzyme efficiency).
Solubility, stability, and aggregation state were determined in
phosphate-buffered saline (PBS, pH 7.4) with 8% D2O and
0.05% Triton X-100 using 19F NMR experiments (see the Sup-
To calculate Km values and enzyme efficiencies of 7, 10, and
11, we determined the concentrations of the enzymatic prod-
ucts by analyzing the integral values of 19F NMR signals of the
products at increasing concentrations of the substrates (in Fig-
ure 1A), data at 40 mm are shown as an example). The reac-
tions were quenched after 50 min for 7 and 70 min for 10 and
11 by adding the known potent FAAH inhibitor URB597.[13] The
rate of the reaction, obtained by the measurement of the inte-
Figure 1. A) 19F NMR spectra of substrate 7 (40 mm) in the presence of 10 nm MBP-rFAAH, and substrates 10 and 11 (40 mm) in the presence of 30 nm MBP-
rFAAH. S and P indicate the 19F NMR signals of substrate and product, respectively. B) Michaelis–Menten plots. The best data fit afforded the Km and Vmax
values.
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gral of the 19F NMR signal of the product divided by the incu-
bation time, as a function of the substrate concentration, was
then plotted. The best fit of these data afforded Km and Vmax
values and, consequently, also the enzyme efficiency (kcat/Km)
values. (Figure 1B). Unfortunately, it was not possible to accu-
rately calculate the Km for compound 7 due to aggregation at
concentrations higher than 40 mm. As can be observed in
Figure 1, there is a large difference in the 19F chemical shift be-
tween the substrate and product signals for both CF3- and
CH2F-containing compounds.
ꢀ100 mm in PBS (pH 7.4) with 8% D2O and 5 mm EDTA, purity
ꢀ75% and lack of aggregation. For experimental details on
compounds characterization, see the Supporting Information.
The final set of 115 compounds was used to generate 23 mix-
tures of five compounds each to screen against FAAH.
n-FABS assay, deconvolution, and IC50 determination
Our n-FABS assay was first validated by determining IC50 values
of known FAAH inhibitors. The derived IC50 values showed the
same rank order of potencies reported in literature, obtained
using different substrate and assay conditions (for experimen-
tal details, see the Supporting Information).
Finally, compound 11 was selected as suitable fluorinated
substrate for the n-FABS assay based on its superior enzyme
efficiency (kcat/Km =1407mÀ1 sÀ1) compared to compound 10
(kcat/Km =542mÀ1 sÀ1). To the best of our knowledge, the ability
of compounds 10 and 11 to behave as FAAH substrates is an
unprecedented result. It is worth noting that compound 7,
which is an efficient substrate according to Figure 1 despite
the uncertainty of its Km value, can also be used for n-FABS.
This would require working at low-mm substrate concentra-
tions, in order to avoid aggregation, which is currently possible
with an NMR spectrometer equipped with 19F cryogenic probe
technology.
The 23 mixtures in our fluorinated library were tested at a
concentration of 200 mm using enzyme and substrate concen-
trations of 15 nm and 30 mm, respectively, as described in the
Experimental Section. This substrate concentration that corre-
sponds to the Km is suitable for a balanced assay, thus allowing
the efficient detection of competitive, uncompetitive, and non-
competitive inhibitors. In this particular case, the inhibition
binding constant, Ki, corresponds to the IC50/2 value for both
competitive and uncompetitive inhibitors and to the IC50 value
for noncompetitive inhibitors. Nine mixtures showed inhibition
>15% and were deconvoluted for hit identification. An exam-
ple of screening and the following deconvolution of an active
mixture are shown in Figure 2. The disappearance of the 19F
signal of the product (P) with tested mixture 17 indicates the
presence of inhibitor(s); on the other hand, no changes in in-
tensities were observed in the presence of mixture 9, which is
therefore an inactive mixture (Figure 2A). The deconvolution
consists in a stepwise process that allows for the identification
of an active fragment(s). Mixture 17 was first split into two sets
of three and two compounds, respectively, and assays were
carried out on these new sets. Only the mixture containing
fragments 4 and 5 was active (Figure 2B). Fragments 4 and 5
were then tested as single compounds to identify the active
one: fragment 5 (Figure 2C). Through this process, of the nine
mixtures showing inhibition >15%, we identified 19 active flu-
orinated fragments. In some mixtures, more than one hit could
be detected.
Compounds 10 and 11 were synthesized using standard re-
action conditions (Scheme 2A) by treatment of arachidonoyl
Scheme 2. Synthesis of fluorinated AEA analogues 10 and 11. Reagents and
conditions: a) i: DMF (cat.), (COCl)2, CH2Cl2, 08C to RT, 3 h; ii: Et3N, RCH2NH2,
CH2Cl2, 08C to RT, 12 h; b) potassium phthalimide, DMF, 808C, 4 h; 61%;
c) 13% HCl, 1008C, 12 h, 92%.
Determination of the IC50 values of our fragment hits was
performed with n-FABS experiments at different concentrations
of the inhibitors in triplicate samples by measuring the integral
values of the 19F NMR signal of enzymatic product (P). IC50
values were then calculated by plotting these values as a func-
tion of the inhibitor concentration, as shown for hit 1 and hit 6
in Figure 3. It is worth noting that the IC50 value can also be
determined by the 19F NMR signal of the substrate. This is pos-
sible because the n-FABS assay allows for simultaneous detec-
tion of the substrate and product, which is not feasible with
many biophysical methods applied to biochemical assays.
Based on the conditions of the assay, our methodology al-
lowed for the measurement of IC50 values ranging from low
nm (limit determined by protein concentration) to mm values
(limit determined by compound solubility). The structures of
the fragment hits, the corresponding rFAAH IC50 values, and
the binding efficiency index (BEI)[23] are reported in Table 1.
chloride with the appropriate fluorinated amine, which was
either commercially available or obtained from the correspond-
ing chloride by Gabriel synthesis, as reported in Scheme 2B).
See the Supporting Information for experimental details of the
synthesis of all substrates.
Construction of the fluorinated fragment library
We built our fluorinated fragment library according to the
methodology developed by Vulpetti et al.[10] We analyzed
about 7000 commercial fluorinated compounds containing CF
or CF3 moieties and clustered them as a function of the fluo-
rine environments. Out of the 160 ordered compounds,
115 compounds passed the SPAM filter,[22] showing solubility
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Figure 2. n-FABS screening and deconvolution. S and P indicate the 19F NMR signal of substrate 11 and product, respectively. The asterisk indicates the ab-
sence of product and, consequently, the presence of an active compound. A) 19F NMR spectra of an inactive (mixture 9) and active (mixture 17). B) Only the
set of mixture 17 containing fragments 4 and 5 was active. C) Fragment 5 was identified as active compound.
Notably, structural analysis of the identified fragment hits re-
vealed that our scaffolds, except for the trifluoromethyl ketone
(hit 3) and trifluoroacetyl benzotriazole (hit 14), do not contain
evident chemical overlapping substructures with any of the
published FAAH inhibitors.[14a] A common structural leitmotif
may be highlighted between hits 2, 15, and 17, bearing a
fused bicyclic 6,5-ring system, and hits 18 and 19 as anilide de-
rivatives of five-membered heteroaromatic rings. Interestingly,
hit 16, a known fluorinated selective COX-2 inhibitor (niflumic
acid), was previously found to inhibit FAAH activity.[24] It is also
worth noting the presence of several CF3 aromatic-containing
molecules. It has been shown that these moieties are often
found in proximity of a-helices (a secondary structure element
abundant in FAAH), with the CF3 group making close interac-
tions with the side chain of hydrophobic residues in positions i
and i+3 or i+4 of the helix.[11b,25] It is also worth noting the
identification of very weak inhibitors with IC50 in the mm
range, as it is for hit 17 and hit 18, despite the low concentra-
tion at which the fragments were tested. At the 200 mm con-
centration used for screening, the two hits showed only a
~15% inhibition. Often, these hits would be discarded in HTS
assays because these small values of inhibition are contained
in the error bars of the measurements. However, the robust-
ness and reliability of the n-FABS allowed their selection for
Figure 3. IC50 determination for two fragment hits: A) hit 1 and B) hit 6. Their
chemical structures are reported in Table 1.
IC50 determination. In general, most of the identified hits are
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Table 1. Structures and IC50 and BEI values of the identified hits.
Table 1. (Continued)
Hit
Structures
FAAH
BEI[a]
Hit
Structures
FAAH
BEI[a]
IC50 [mm]
IC50 [mm]
hit 15
110 Æ27
162Æ44
15
hit 1
4.2Æ1
23
hit 16
13
hit 2
hit 3
hit 4
3Æ1
22
22
22
26Æ6
58Æ21
hit 17
1125Æ230
10
10
hit 5
hit 6
450Æ45
32Æ8
21
20
hit 18
hit 19
1315Æ260
53% at 100 mM
hit 7
147Æ24
20
[a] BEI (binding efficiency index)=pIC50 [m]/MW [kDa].
hit 8
hit 9
270Æ60
465Æ60
20
20
amenable to chemical structural evolution which, in combina-
tion with the novelty of the structural frameworks, may allow
for the identification of new FAAH inhibitors.
Conclusions
hit 10
137Æ31
19
Membrane proteins are challenging targets in drug discovery
and, therefore, the application of an FBA is often not straight-
forward. Herein, we report the use of the n-FABS method for
screening a library of 115 fluorinated fragments, targeting the
membrane-bound enzyme FAAH. The screening was per-
formed by using recombinant MBP-rFAAH and compound 11
as a novel fluorinated and soluble substrate analogue. The
small amount of enzyme required for NMR screening of the
fragment library (15 nm concentration for each mixture) indi-
cates that the method can be extended to efficient screening
against those membrane enzymes that can only be produced
in very small quantities. The low concentration required for n-
FABS allows for better solubility of the membrane protein and
reduces aggregation. In addition, screening with n-FABS ap-
plied to membrane proteins does not require the use of ex-
pensive deuterated detergents, because the 19F NMR spectra
are recorded. We identified 19 novel fragment hits that inhibit
hit 11
hit 12
143Æ47
563Æ36
18
18
hit 13
hit 14
720Æ90
400Æ65
17
16
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Synthesis of (5Z,8Z,11Z,14Z)-N-(3,3,3-trifluoro-2-hydroxy-propyl)-
icosa-5,8,11,14-tetraenamide (10): DMF (1 mL, 0.01 mmol) and
oxalyl chloride (55 mL, 0.66 mmol) were added to a solution of
arachidonic acid (100 mg, 0.33 mmol) in dry CH2Cl2 (2 mL) at 08C
under N2 atmosphere. After stirring for 3 h, the solvent was
removed under reduced pressure, and the crude product was
washed with dry CH2Cl2 (3ꢁ2 mL, evaporation after each step). The
residue was then dissolved in dry CH2Cl2 (2 mL) under
N2 atmosphere and added through a cannula to a solution of dry
Et3N (70 mL, 0.49 mmol) and 3-amino-1,1,1-trifluoropropan-2-ol
(55 mg, 0.43 mmol) in dry CH2Cl2 (2.5 mL) at 08C. After stirring
overnight at room temperature, a 2 n HCl solution (15 mL) was
added. The reaction mixture was extracted with CH2Cl2 (3ꢁ15 mL),
and the organic layer was washed with brine and dried over
Na2SO4. After evaporation of the solvent, the crude product was
purified by preparative HPLC-MS to afford 10 (70 mg, 51%) as a col-
orless oil: Rf =0.56 (CH2Cl2/MeOH, 98:2); 1H NMR (400 MHz,
[D6]DMSO) d=0.85 (t, J=6.9 Hz, 3H), 1.21–1.37 (m, 6H), 1.54 (p,
J=7.6 Hz, 2H), 2.02 (q, J=6.8 Hz, 4H), 2.10 (t, J=7.5 Hz, 2H), 2.72–
2.86 (m, 6H), 3.06 (ddd, J=13.6, 7.6, 5.7 Hz, 1H), 3.37–3.45 (m, 1H),
3.93–4.05 (m, 1H), 5.26–5.41 (m, 8H), 6.39 (d, J=6.4 Hz, 1H),
8.03 ppm (t, J=5.6 Hz, 1H); 13C NMR (101 MHz, [D6]DMSO) d=13.9,
21.9, 25.2, 26.2, 26.6, 28.7, 30.9, 34.6, 39.1, 67.4 (q, J=28.6 Hz),
125.3 (q, J=283.4 Hz), 127.5, 127.6, 127.8, 128.0, 128.1, 129.4,
129.9, 172.4 ppm; MS (ESI): m/z calcd. for C23H36F3NO2: 416 [M+H]+,
found: 416.
the enzyme with IC50 values in the low mm–mm range. The
fragment evolution of selected hits is currently ongoing, and
the results will be described in due course. To the best of our
knowledge, our results represent the first application of
19F NMR fragment-based screening to a membrane protein
using a functional assay. We are confident that these results
will contribute to future application of the n-FABS methodolo-
gy to other membrane proteins.
Experimental Section
FAAH construct generation: the encoding sequence of rat
rFAAHDTM (97–1722 bp) was amplified from cDNA clone 7370226
(Open Biosystem) using the following primer pair: forward 5’-
GGGAAT TCCATA TGGGGC GCCAGA AGGCCC-3’ (NdeI site is under-
lined); reverse 5’-ATAGTT TAGCGG CCGCTC AATGAT GATGAT
GATGAT GAGGGG TCATCA GCT-3’ (NotI site is underlined). A His6
tag was introduced in the reverse primer sequence. To obtain the
construct MBP-rFAAH-His6 pMALc5x, the amplified rFAAHDTM was
cloned in a pMALc5x vector in frame with N-terminal MBP.
Protein expression and purification: Expression of the MBP-
rFAAH-His6 protein was carried out in the E. coli Rosettagami 2
(DE3)pLysS strain (Novagen). At an optical density (OD600) of 0.6,
bacteria were induced by the addition of 0.25 mm IPTG for 16 h at
258C. Cells were then harvested by centrifugation, resuspended in
buffer (50 mm sodium phosphate, 0.3 m NaCl, 10 mm imidazole,
pH 7.4), and lysed by sonication and addition of 1% Triton X-100.
The lysate was incubated for 1 h with benzonase nuclease and
centrifuged at 15000g for 30 min. The clarified supernatant was in-
cubated for 2 h with Ni–NTA agarose (Qiagen) and washed with
buffer containing increasing concentrations of imidazole. Elution
was performed with buffer containing 0.25 m imidazole. The pro-
tein was then concentrated, and the buffer was exchanged with
a solution of 50 mm sodium phosphate, 0.3 m NaCl, and 0.1%
CHAPS, pH 7.4. Protein aliquots were stored at À808C until use.
Synthesis of 2-(3-fluoro-2-hydroxypropyl)isoindoline-1,3-dione
(12): to
a suspension of potassium phthalimide (300 mg,
1.60 mmol) in dry DMF (3.2 mL) was added 1-chloro-3-fluoropro-
pan-2-ol (150 mg, 1.33 mmol) at room temperature. After stirring
at 808C for 4 h, the reaction was cooled to room temperature and
H2O (5 mL) was added. The reaction mixture was extracted with
EtOAc (5ꢁ15 mL), and the organic layer was washed with brine
and dried over Na2SO4. After evaporation of the solvent, the crude
product was purified by automated column chromatography, elut-
ing with cyclohexane/EtOAc (from 100:0 to 75:25) to afford 12
1
(180 mg, 61%) as a white solid: H NMR (400 MHz, CDCl3) d=2.71
Chemistry: For experimental and analytical details, see the Sup-
porting Information.
(s, 1H), 3.90–3.95 (m, 2H), 4.16 (dp, J=20.0, 4.9 Hz, 1H), 4.47 (ddd,
J=47.0, 9.8, 4.7 Hz, 1H), 4.51 (ddd, J=47.2, 9.8, 4.1 Hz, 1H), 7.75
(dd, J=5.5, 3.1 Hz, 2H), 7.88 ppm (dd, J=5.5, 3.1 Hz, 2H); MS (ESI):
m/z calcd. for C11H10FNO3: 224 [M+H]+, found: 224.
Synthesis of (5Z,8Z,11Z,14Z)-N-(2-fluoroethyl)icosa-5,8,11,14-tet-
raenamide (7): DMF (1 mL, 0.01 mmol) and oxalyl chloride (43 mL,
0.45 mmol) was added to a solution of arachidonic acid (75 mg,
0.25 mmol) in dry CH2Cl2 (1.5 mL) at 08C under N2 atmosphere.
After stirring for 3 h, the solvent was removed under reduced pres-
sure, and the crude product was washed with dry CH2Cl2 (3ꢁ2 mL,
evaporation after each step). The residue was then dissolved in dry
CH2Cl2 (1.5 mL) under N2 atmosphere and added through a cannula
to a solution of dry Et3N (80 mL, 0.55 mmol) and 2-fluoroethylamine
hydrochloride (30 mg, 0.31 mmol) in dry CH2Cl2 (2.5 mL) at 08C.
After stirring overnight at room temperature, 2 n HCl solution
(15 mL) was added. The reaction mixture was extracted with
CH2Cl2 (3ꢁ15 mL), and the organic layer was washed with brine
and dried over Na2SO4. After evaporation of the solvent, the crude
product was purified by column chromatography using a SI (5 g)
cartridge, eluting with CH2Cl2 (100%) to afford 7 (26 mg, 31%) as
Synthesis of 1-amino-3-fluoro-propan-2-ol hydrochloride (13): A
suspension of 12 (125 mg, 0.56 mmol) in 13% HCl solution (1 mL)
was stirred at 1008C overnight. The reaction was then cooled to
room temperature, and H2O (2 mL) was added. The mixture was
washed with EtOAc (3ꢁ15 mL), and the aqueous phase was evapo-
rated under reduced pressure to dryness to afford 13 (66 mg,
1
92%) as a white solid: H NMR (400 MHz, [D6]DMSO) d=2.68–3.01
(m, 2H), 3.88–4.01 (m, 1H), 4.37 (ddd, J=47.2, 9.7, 4.8 Hz, 1H), 4.41
(ddd, J=47.2, 9.6, 4.3 Hz, 1H), 5.80 (s, 1H), 7.99 ppm (s, 3H); MS
(ESI): m/z calcd. for C3H8FNO: 94, found: 94.
Synthesis of (5Z,8Z,11Z,14Z)-N-(3-fluoro-2-hydroxy-propyl)icosa-
5,8,11,14-tetraenamide (11): DMF (1 mL, 0.01 mmol) and oxalyl
chloride (55 mL, 0.66 mmol) were added to a solution of arachidon-
ic acid (100 mg, 0.33 mmol) in dry CH2Cl2 (2 mL) at 08C under
N2 atmosphere. After stirring for 3 h, the solvent was removed
under reduced pressure, and the crude product was washed with
dry CH2Cl2 (3ꢁ2 mL, evaporation after each step). The residue was
then dissolved in dry CH2Cl2 (2 mL) under N2 atmosphere and
added through a cannula to a solution of dry Et3N (120 mL,
0.82 mmol) and 13 (55 mg, 0.43 mmol) in dry CH2Cl2 (2.5 mL) at
08C. After stirring overnight at room temperature, a 2 n HCl solu-
1
a colorless oil: Rf =0.28 (CH2Cl2); H NMR (400 MHz, CDCl3) d=0.89
(t, J=6.5 Hz, 3H), 1.23–1.42 (m, 6H), 1.73 (p, J=7.4 Hz, 2H), 2.06
(q, J=6.9 Hz, 2H), 2.12 (q, J=6.8 Hz, 2H), 2.21 (t, J=7.6 Hz, 2H),
2.77–2.87 (m, 6H), 3.57 (dq, J=28.4, 5.1 Hz, 2H), 4.49 (dt, J=47.4,
4.7 Hz, 2H), 5.27–5.48 (m, 8H), 5.76 ppm (s, 1H); 13C NMR
(101 MHz, CDCl3) d=14.2, 22.7, 25.6, 25.8, 26.8, 27.4, 29.5, 31.7,
36.1, 40.0 (d, J=19.5 Hz), 83.0 (d, J=166.0 Hz), 127.7, 128.0, 128.3,
128.4, 128.8, 129.0, 129.2, 130.7, 173.1 ppm.
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2013, 14, 1611 – 1619 1617

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Keywords: FAAH · fluorine NMR screening · fragment-based
approach · membrane proteins
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2013, 14, 1611 – 1619 1618

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Received: May 29, 2013
Published online on August 5, 2013
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2013, 14, 1611 – 1619 1619