Set-up and validation of a high throughput screening method for human monoacylglycerol lipase (MAGL) based on a new red fluorescent probe

Article abstract of DOI:10.3390/molecules24122241

Monoacylglycerol lipase (MAGL) is a serine hydrolase that has a key regulatory role in controlling the levels of 2-arachidonoylglycerol (2-AG), the main signaling molecule in the endocannabinoid system. Identification of selective modulators of MAGL enables both to provide new tools for investigating pathophysiological roles of 2-AG, and to discover new lead compounds for drug design. The development of sensitive and reliable methods is crucial to evaluate this modulatory activity. In the current study, we report readily synthesized long-wavelength putative fluorogenic substrates with different acylic side chains to find a new probe for MAGL activity. 7-Hydroxyresorufinyl octanoate proved to be the best substrate thanks to the highest rate of hydrolysis and the best Km and Vmax values. In addition, in silico evaluation of substrates interaction with the active site of MAGL confirms octanoate resorufine derivative as the molecule of choice. The well-known MAGL inhibitors URB602 and methyl arachidonylfluorophosphonate (MAFP) were used for the assay validation. The assay was highly reproducible with an overall average Z⁰ value of 0.86. The fast, sensitive and accurate method described in this study is suitable for low-cost high-throughput screening (HTS) of MAGL modulators and is a powerful new tool for studying MAGL activity.

Full text of DOI:10.3390/molecules24122241

molecules
Article
Set-Up and Validation of a High Throughput
Screening Method for Human Monoacylglycerol
Lipase (MAGL) Based on a New Red
Fluorescent Probe
Matteo Miceli 1, Silvana Casati 1 , Roberta Ottria 1 , Simone Di Leo 2, Ivano Eberini 3,
Luca Palazzolo 3 , Chiara Parravicini 3 and Pierangela Ciuffreda 1,
*
1
Dipartimento di Scienze Biomediche e Cliniche “Luigi Sacco”, Università degli Studi di Milano, Via G.B.
Grassi 74, 20157 Milano, Italy; matteo.miceli@unimi.it (M.M.); silvana.casati@unimi.it (S.C.);
roberta.ottria@unimi.it (R.O.)
Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università degli Studi di Milano,
Via Fratelli Cervi 93, 20090 Segrate (MI), Italy; simone.dileo@unimi.it
2
3
Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Via Balzaretti 9,
20133 Milano, Italy; ivano.eberini@unimi.it (I.E.); luca.palazzolo@unimi.it (L.P.);
chiara.parravicini@unimi.it (C.P.)
*
Correspondence: pierangela.ciuffreda@unimi.it; Tel.: +39-02-50319695
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Anton Simeonov
Received: 7 May 2019; Accepted: 14 June 2019; Published: 15 June 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Monoacylglycerol lipase (MAGL) is a serine hydrolase that has a key regulatory role
in controlling the levels of 2-arachidonoylglycerol (2-AG), the main signaling molecule in the
endocannabinoid system. Identification of selective modulators of MAGL enables both to provide
new tools for investigating pathophysiological roles of 2-AG, and to discover new lead compounds
for drug design. The development of sensitive and reliable methods is crucial to evaluate this
modulatory activity. In the current study, we report readily synthesized long-wavelength putative
fluorogenic substrates with different acylic side chains to find a new probe for MAGL activity.
7-Hydroxyresorufinyl octanoate proved to be the best substrate thanks to the highest rate of
hydrolysis and the best Km and Vmax values. In addition, in silico evaluation of substrates interaction
with the active site of MAGL confirms octanoate resorufine derivative as the molecule of choice.
The well-known MAGL inhibitors URB602 and methyl arachidonylfluorophosphonate (MAFP) were
used for the assay validation. The assay was highly reproducible with an overall average Z0 value
of 0.86. The fast, sensitive and accurate method described in this study is suitable for low-cost
high-throughput screening (HTS) of MAGL modulators and is a powerful new tool for studying
MAGL activity.
Keywords: high-throughput screening; endocannabinoid; monoacylglycerol lipase; assay
development
1. Introduction
The endocannabinoid system (ES) is a modulatory system that plays a critical role in a
variety of neuronal functions including motor activity, nociception, and appetite control as well
as peripheral functions such as energy metabolism and inflammation [
1]. This wide range of effects
makes it an interesting target for drug development although a difficult one to act upon due to
life-threatening psychological adverse effects [
2] linked to the direct antagonist action against the
endocannabinoid receptors.
Molecules 2019, 24, 2241; doi:10.3390/molecules24122241
www.mdpi.com/journal/molecules

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Inhibition or enhancement of the ES hydrolytic regulatory enzymes seems a very promising
strategy to act on the ES in a more fine-tuned way [ ]. Monoacylglycerol lipase (MAGL), a soluble
3
serine hydrolase that associates with cell membranes, is a very promising target for pharmacological
inhibition, since it is the main hydrolase implicated in the degradation of the main signaling molecule
in the ES, 2-AG. The hydrolysis of 2-AG is catalyzed also by two additional serine hydrolases, α-β
hydrolase domain 6 and 12 (ABHD6 and ABHD12). MAGL, however, is responsible for approximately
85% of 2-AG hydrolysis [4].
Identification of new promising lead compounds for MAGL inhibition from large libraries of
molecules needs simple, highly sensitive, specific, inexpensive and reliable assays suitable for a
high-throughput format. The most commonly assay procedures used to screen and characterize
MAGL inhibitors quantify the hydrolysis products of radiolabeled MAGL substrates [
arachidonic acid released upon 2-AG hydrolysis by using high-performance liquid chromatography
(HPLC) with UV [ ] or with mass spectrometric detection [ ]. Another method assesses the
rate of MAGL-catalyzed hydrolysis of p-nitrophenyl alkyl esters by monitoring the liberation
of p-nitrophenol [ ]. Furthermore, a fluorescence-based assay has been developed applying
5,6] or the
7
8
9
7-hydroxycoumarinyl arachidonate (7-HCA) [10], as well as an HPLC method with fluorescence
detection, using as fluorogenic probe 1,3-dihydroxypropan-2-yl-4-pyren-1-ylbutanoate [11]. We have
previously reported an efficient protocol for a MAGL continuous assay, based on a new long-wavelength
fluorogenic substrate, 7-hydroxyresorufinyl arachidonate 1g [12]. Here we report the synthesis and the
evaluation, as MAGL assay substrates, of 10 alkyl esters of fluorescent resorufin with the aim to propose
a high-throughput screening (HTS) method for MAGL activity based on a new red fluorogenic probe.
Essential to the success of any HTS program that seeks to discover modulators of the function of specific
proteins, is the development of a high-quality screening, which requires an accurate, and homogeneous
biochemical readout of protein activity, robust assay reproducibility and appropriate sensitivity [13,14].
Furthermore, possible interferences due to the concentrations of library compounds used as protein
activity modulators in the assay could occur, so shifting the emission wavelength to the red reduces
these interferences [15]. It must also be underlined that a suitable substrate has to be readily accessible,
stable in solution and with low rate of spontaneous hydrolysis.
We have previously reported an efficient protocol for a MAGL continuous assay, based on a
new long-wavelength fluorogenic substrate, 7-hydroxyresorufinyl arachidonate 1g [12]. Here we
report new esters of fluorescent resorufin with different classes of acyl chains such as linear, i.e.,
acetate (1a), butyrate (1b), octanoate (1c), dodecanoate (1d), icosanoate (1e), oleate (1f), branched, i.e.,
2-methylhexanoate (1h), 2-ethylhexanoate (1i) and 2-butyloctanoate (1j), and aromatic, i.e., benzoate
(1k) (Figure 1) and compared them with 1g. Moreover, we investigated the MAGL substrate specificity
depending on the acyl chain, and determined the kinetic constants. The differences of substrate
interaction with MAGL active site were also analyzed using structural in-silico techniques.

Molecules 2019, 24, 2241
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a R= CH3;
N
O
b R= CH2CH2CH3;
c R= CH2(CH2)5CH3;
d R= CH2(CH2)9CH3;
e R= CH2(CH2)17CH3;
O
O
R
O
1
g R=
i R=
k R=
f R=
h R=
j R=
Figure 1. Structures of 7-hydroxyresorufynil esters: acetate (1a), butyrate (1b), octanoate
1c), dodecanoate (1d), icosanoate (1e), oleate (1f), arachidonate (1g), 2-methylhexanoate (1h),
2-ethylhexanoate (1i) 2-butyloctanoate (1j), benzoate (1k).
(
2. Results
2.1. Synthesis of 1a–k
For the synthesis of fluorogenic compounds, two different methods have been followed. While the
synthesis of the esters 1a–g involves passing through the formation of the acyl chloride, the compounds
1h–k required Steglich esterification [16]. In the case of fatty acid with branched acyl chain such as
2-methylhexanoic acid, 2-ethylhexanoic acid and 2-butyloctanoic acid the symmetrical anhydrides
were isolated instead of the esters. Therefore, the use of oxalyl chloride has to be avoided with
sterically hindered carboxylic acids. In the Steglich esterification indeed, the addition of 3 mol %
DMAP accelerates the DCC-activated esterification of carboxylic acids with alcohols to such an extent
that formation of side products is suppressed. Synthesized molecules were completely characterized
by 1D NMR, as well as 2D NMR homocorrelation (COSY) and heterocorrelation (HMQC and HMBC).
The purity of all compounds measured with NMR was greater than 98%.
2.2. Substrates Screening
Screening experiment were performed at 5 µM in Tris-HCl containing EDTA (10% DMSO) in the
presence of 25 ng of human recombinant MAGL (hMAGL). Typical MAGL assays use bovine serum
albumin (BSA) since it assists in the solubility of the substrate and might prevent its non-specific
binding to the plastic labware [10]. We decided not to use BSA because it is not compatible with lipase
assay due to its well-known esterase-like activity [17]. As reported in Figure 2, at 5
shows a considerable spontaneous hydrolysis, compound 1b a smaller one while all the other esters
seem to be stable. During the set-up of the assay conditions also spontaneous hydrolysis of 1a
and 1h at all concentration used for kinetic experiments (data not shown) have been evaluated.
At 25 M, the highest concentration assessed (Supplementary Figure S1), compounds 1a show a
hydrolysis rate increment proportional to the concentration as expected, even if almost negligible for
1c. Compounds 1i seems to be still stable with no significant increment in hydrolysis rate, while
compounds 1d show an atypical behavior, indeed, to a five-fold increase of the ester concentration
correspond a less increment in the rate of hydrolysis. For this reason, the solubility in aqueous buffer
of substrates 1d at 25 M was verified by dynamic light scattering (DLS) analyses (Supplementary
µM compound 1a
–d,
–
k
µ
–c
–
k
–h
–h
µ
Figure S3). DLS analyses revealed the presence of micelles and/or aggregates that are formed at this
concentration, due to the hydrophobic nature of the substrates.

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Figure 2. Time course of 1a
(hMAGL). 25ng/well of hMAGL (circles) or buffer alone (empty squares) were incubated in 96-well black
plate, at room temperature, with 5 M of each compound in total volume of 100 L/well, as described
in the experimental section. Fluorescence was measured at indicated time points with a Jasco FP-8300
–d, 1f, and 1h–k hydrolysis by human recombinant monoacylglycerol lipase
µ
µ
fluorometer (Jasco Europe, Cremella (LC) Italy) using the kinetic mode (
λ
= 571 nm,
λ
em
= 588 nm,
ex
slit = 5.0). Data are mean ± standard error of independent experiments.
2.3. Kinetic Studies
The amount of enzyme used in the assay was optimized to 25 ng/well to maintain linearity
over time and to maintain substrate consumption below 10% to adhere to the assumptions of the
Michaelis–Menten equation [12]. Enzyme kinetics were analyzed by measuring product formation
rates in the presence of different amounts of the substrates. Standard curves, showing fluorescence
response vs. fluorophore concentration, were constructed from a resorufin dilution series, transforming
relative fluorescence units (RFU) into picomoles of resorufin. Figure 3 reports the action of hMAGL
on different concentrations of all synthesized substrates. To evaluate the real enzymatic activity,
the amount of free resorufin released from the spontaneous hydrolysis of the substrate was subtracted.
The initial linear part of the resulting curve and its slope were considered to measure the enzyme
activity [18]. Michaelis–Menten kinetic parameters, Km and Vmax values, were derived with Prism
GraphPad software, Version 6.0c applying a nonlinear regression analysis and Michaelis-Menten fit
(Table 1). As shown in Figure 3, fluorometric determination of the kinetic constants for compounds
1a and 1b was impossible. For all other compounds, Michaelis-Menten curves were constructed
considering hMAGL hydrolysis at five concentrations (0.5, 1.0, 2.5, 5.0, 10.0 µM) and the obtained
results for Km and Vmax are reported in Table 1. Among new compounds, 1j has the best fit with
Michaelis-Menten regression but has a very low rate of hydrolysis.

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Figure 3. Action of hMAGL on different concentrations of 1a
v/v). The reactions were conducted in 50 mM Tris-HCl buffer (pH 7.4, 1 mM EDTA) with 25 ng/well of
hMAGL. Values are the means of triplicates standard error. The kinetic parameters Km and Vmax were
–d, 1f, 1h–j, in the presence of DMSO (10%,
±
determined via computer-assisted nonlinear regression analysis using GraphPad Prism 6.0c (GraphPad
Software, San Diego, CA 92108).
Table 1. List of tested substrates with kinetic and modeling parameters.
Vmax
(nmol/min/mg
LogD at
pH 7.4 a
Docking
Score
Fitting
Model
Compound
Km (µM)
MM-GBSA
r2
protein)
1a
1b
1c
1d
1e
1f
1g
1h
1i
1.69
2.83
4.61
6.39
9.94
8.69
8.50
4.26
4.71
6.49
3.74
n/a
n/a
n/a
n/a
106 ± 5.4
24 ± 1.2
n/a
13 ± 0.64
25.8 ± 0.88
33 ± 2.509
1.3 ± 0.1
0.67 ± 0.03
n/a
−6.7
−7.7
−8.2
−10.5
−9.8
−10.8
−10.7
−8.5
−8.8
−8.4
−8.9
−52.4
−60.5
−72.0
−81.4
−67.8
−73.2
−76.0
−60.3
−62.8
−65.4
−63.2
n/a
n/a
M-M
M-M
n/a
M-M
M-M
M-M
M-M
M-M
n/a
n/a
n/a
0.66 ± 0.14
0.31 ± 0.09
n/a
0.42 ± 0.1
0.87 ± 0.13
1.09 ± 0.02
2.8 ± 0.7
1.1 ± 0.1
n/a
0.8488
0.6731
n/a
0.7391
0.9908
0.8859
0.9236
0.9450
n/a
1j
1k
a
Data generated using the Chemicalize https://chemicalize.com/ (developed by ChemAxon, http://www.chemaxon.
com).
Kinetic parameters reported in Table 1 highlight the substrate preference of hMAGL. Of all the
compounds newly synthesized, the fastest hydrolyzed was 1c. Compound 1e has a very low solubility
in DMSO (<0.5 nM) and could not be assessed as hMAGL substrate. Actually, even LogD value
(Table 1) highlights higher affinity of this compound to non-polar organic solvents. Substrate 1k was
excluded from further investigations due to its inappreciable hydrolysis by hMAGL. All substrates
have Km values ranging from 0.31 to 2.8
µM, in accordance to that of 0.87 µM already calculated for
1g 12]. Maximum velocities are similar as well, ranging from 0.67 to 33 nmol/min/mg protein, with
[
the exception of 1j that displays a very low hydrolysis rate with a Vmax of 0.67 nmol/min/mg protein.
Interestingly, 1c has a Vmax of 106 nmol/min/mg protein. For this reason, compound 1c was selected as
fluorogenic substrate for the set-up of a new method.
2.4. Validation of 1c for Screening Assay
In order to validate compound 1c for HTS two known MAGL inhibitors, URB602 and Methyl
arachidonylfluorophosphonate (MAFP) [7,19] were used. The dose-response curves are shown in

Molecules 2019, 24, 2241
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Figure 4. After incubation of hMAGL with the inhibitor, 1c was rapidly added and fluorescence read
for 30 min. The activity of hMAGL was calculated as described for the kinetic assay. IC50 for URB602
was found to be 8.1 µM, which is in line with literature data [20]. As already known, MAFP had the
lowest IC50 value, our experiments gave an IC50 of 15.3 nM, which is in the range of values indicated
in literature [12,20].
Figure 4. Inhibition of human recombinant MAGL with known inhibitors: methyl arachidonyl
fluorophosphonate (A), URB602 (B). The assay was carried out as described in Experimental procedures
section. Data derived from two independent experiments performed in triplicate and calculated as
non-linear regressions using sigmoid dose-response setting with variable Hill slope by GraphPad Prism
6.0c. In panel B 100% inhibition is not achieved due to the low solubility of URB 602 in assay conditions.
2.5. Docking Studies
The identified binding site corresponds to the catalytic active site identified by Lauria et al. [21
]
Table 1 reports the Glide XP docking score and MM-GBSA binding energies for the top scoring pose of
each tested compound, showing that all of them are able to bind MAGL in its catalytic site. Docking
simulations provided overlapping poses for all the tested compounds and a good superposition of
their common resorufin group, confirming the accuracy of this approach. Moreover, the carbonyl
group for all the tested compounds, except for 1j, overlaps the carbonyl group of the natural substrate
2-AG in proximity to Ser122, according to Lauria et al. [21]. In particular, Ala51, Ser122 and Met123
establish hydrogen bond interactions with the carbonyl and ester oxygen atoms (Figure 5), while
His121 and His269 are involved in π–π interaction with the resorufin group, as reported in Table S2
of Supplementary. As shown in Figure 5, the alkyl chains of the resorufine ester accommodate in a
well-shaped pocket surrounded by hydrophobic/aromatic residues, such as Leu148, Leu213, Leu214,
Ile179, Ala151, Ala156, Phe159, Phe209. Accordingly, in silico results also underline the importance of
the contribution of the hydrophobic interactions in the stability of the complexes.
Figure 5. (A) Docking poses of compound 1c into the MAGL binding site. MAGL is shown in ribbon
representation; tested compound and Ser122 are shown in stick representation and hydrogens are
hidden. (B) Ligand interaction diagram of compound 1c into the MAGL binding site.

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3. Discussion
Here we present 7-hydroxyresorufinyl octanoate (1c) as a new fluorogenic substrate to evaluate
MAGL activity by HTS. Although numerous assay methodologies have been published in the past
decades for measurement of MAGL activity, few of them allow the screening of a large number of
compounds showing high sensibility, high throughput, and low assay cost. In order to overcome these
issues, we investigated the use of an alternative substrate for measuring MAGL activity. The native
substrates for MAGL include 2-AG (C20:4) and 2-oleoyl glycerol (2-OG, C18:1) both containing
long hydrocarbon fatty acid chains with relatively unstable cis olefins. Nevertheless, MAGL is able
to hydrolyze a vast array of esters structurally very different, such as p-nitrophenyl esters [9] or
7-hydroxycoumarinyl esters [22]. We chose resorufine as fluorescent probe because its derivatives
have proven to be an excellent fluorescence marker and have the advantages to be hardly susceptible
to background signals. Often pharmacological and biochemical research need to measure enzymatic
activity in crude cell lysate or turbid solution [23], the shift of emission wavelengths at higher values
give less interference [24]. In the current approach, 10 fluorogenic alkyl esters of resorufin with different
acyl chains are synthesized and evaluated as MAGL assay substrates (Figure 1). Selected compound 1c
ensure lower background signals, thanks to resorufine in comparison with 7-methylumbelliferone,
higher stability and lower costs if compared to arachidonoyl or oleyl derivatives.
MAGL fluorescence assays were carried out in the presence of 5 µM substrate, 10 % DMSO and at
pH 7.4, a value that attempts to mimic the native intracellular conditions. Diverging too far from the
physiological pH could affect the interactions between the enzyme and the substrates, thus giving
an altered view of the potential biochemical and cellular activity of these compounds. Moreover,
this pH value can be maintained by various buffers, such as Tris-HCl, PBS, sodium phosphate, Mops
and Hepes, and allowed us a prompt comparison with previous data [12]. Among the synthesized
compounds, linear acylic chains of up to 12 carbon atoms are substrates for MAGL, as well as the
derivative of oleic acid, as expected, since 2-OG, an oleic acid derivative, is one of the native substrates
of the protein. The addition of just two more carbons to the side chain makes the hydrolysis of this
compound almost non-existent. The same result is obtained when the branching becomes as long
as four carbon atoms. When in aqueous buffer, 1a and 1b show the highest rate of spontaneous
hydrolysis among our compounds (Figure 2), to the point that fluorometric determination of their
kinetic constants proved impossible (Figure 3). For resorufin acetate 1a, this behavior was already
reported by Maeda and colleagues [25], leading to the impossibility of using that compound as a probe.
Even if 1b showed an inferior hydrolysis rate than 1a (Figure 1), this compound is unsuitable for the
achievement of Michaelis-Menten conditions in an aqueous environment (Figure 3) [26]. On the other
hand, compounds with a long linear chain or with a branched side chain, showed low to negligible
hydrolysis. These differences may be explained with the susceptibility of acyl resorufins towards
nucleophilic attack [27]. Short linear side chains (1a
water, while side chains with branches next to the carboxyl group (1h
on water molecules, protecting the ester group from hydrolysis. A similar phenomenon reasonably
happens with medium (1c 1d) and long (1f 1g) alkyl chains. When in an aqueous environment, the
,
1b) leave the
α
carbon exposed to the attack of
,
1i, 1j) exert a steric hindrance
,
,
alkyl chain folds on itself via van der Waals interactions next to the carboxyl group, thus mimicking the
side branch protecting effect. Moreover, the formation of aggregates, as highlighted by DLS analyses
(Supplementary Figure S2), could be a reasonable explanation for the unusual behavior observed
in spontaneous hydrolysis experiments of compounds 1d–h where to a five-fold increase of ester
concentration corresponds a less increase in the rate of the hydrolysis. In Figure 6, hydrolysis of
different substrates by MAGL, normalized against compound 1c, is reported to better underline the
high difference between the hydrolysis rate displayed by 1c and the other proposed MAGL substrates.

Molecules 2019, 24, 2241
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Figure 6. Normalized hydrolysis of different substrates by hMAGL. Release of 7-hydroxyresorufin
upon hydrolysis of different resorufynil esters (each at a concentration of 5 M) by 25 ng hMAGL
µ
was measured in triplicates, the slopes of the regression lines were averaged and then normalized
against the slope of the most efficiently hydrolyzed compound. The error bars represent the standard
deviation of the results. The following compounds were used as drawn below the bars: acetate (1a),
butyrate (1b), octanoate (1c), dodecanoate (1d), oleate (1f), arachidonate (1g), 2-methylhexanoate (1h),
and 2-butyloctanoate (1j). The specific activity of the hMAGL preparation was 241.9 U/mg.
The presence of aggregates in the substrates’ solutions at 25
µM concentration prompted us to
consider only solutions until 10 M concentration for calculations of kinetic parameters. Observing
µ
obtained results (Figure 3), 1f gives a good fit with Michaelis-Menten type kinetics indeed, its side
chain, the same of natural substrate 2-OG, seems to allow a better accommodation in the enzyme
active site. It is possible that the enhanced rigidity of the alkyl chain, conferred by the presence of the
double bond, guides the substrate to a conformation favorable to enzymatic hydrolysis, hindering
disadvantageous folding in the enzyme active site.
The scale of Y axis (Figure 3), and the Vmax and Km values (Table 1), highlight how steric hindrance,
due to the different length of acyl side chain on C2, reduce the rate of substrate hydrolysis. Interestingly,
1j shows a Michaelis-Menten kinetic behavior even if with very low hydrolysis rate. In this case,
the bulkier side chain likely exerts an even stronger effect of steric hindrance, which does not allow
reaching the concentration needed to manifest the inhibitory effect. Compound 1c proved to be a
very interesting substrate for MAGL. Indeed, with respect to the previously described arachidonate
1g [12] and oleate 1f, the two resorufine derivatives of the MAGL natural substrates, 1c shows slightly
higher XP Glide docking score, MM-GBSA binding energy and Km values. In the molecular docking
poses, 1c resorufin perfectly overlaps the 1g one, while the linear side chain of 1c mimics the 2-AG side
chain. Focussing our attention specifically on the difference in activity pointed out for compound 1c
versus 1b, as already reported in the Results section and in Figure S4, a significant relative difference in
the extension of the hydrophobic interactions can be one of the factors contributing to the different
stability of the complexes, as shown by the different of the solvation/desolvation energy reported in
the Prime MM-GBSA energy column in Table 1. The Vmax for 1c, however, is much higher than that of
1g, as indicated also by the direct comparison of the catalytic speed of the two substrates. In addition,
the smaller substrate 1c has superior aqueous solubility properties and contains no potential labile cis
olefins. Therefore, the signal to background ratio of 1c (seven to one) is suitable for the screening for
MAGL inhibitors in high-throughput screening. For all these reasons, 1c was selected and validated as
new red fluorogenic probe for the HTS method.
MAFP and URB602, two well-known serine hydrolase and MAGL inhibitors, were chosen
for validation [
values for both MAGL inhibitors using hMAGL and 1c (5
those reported in literature [12 20]. In order to evaluate and validate the assay for its potential in
7,
19]. After a 60 min pre-incubation of the enzyme with the inhibitor, the IC50
µM) were obtained and compared with
,
high-throughput screening applications, we further determined the Z-factor [28], a dimensionless
statistical parameter that reflects both the assay signal dynamic range and the data variation associated

Molecules 2019, 24, 2241
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with the signal measurements. The assay was highly reproducible with an overall average Z0 value
of 0.86. The objective of this study was to identify a substrate readily accessible, stable in solution
and with low rate of spontaneous hydrolysis for realizing an HTS method for MAGL activity using
human recombinant MAGL. It should be considered that MAGL belongs to the group of serine
hydrolases (SHs), a superfamily of enzymes able to cleavage ester, amide, or thioester bonds of protein,
peptide, and small molecule substrates [29] including esterases, lipases, peptidases, and amidases [30].
Within this family, there are the SHs that regulate the biosynthesis and degradation of two major
endocannabinoids, AEA and 2-AG, as well as other lipid-metabolizing serine hydrolases that are
ubiquitously distributed [31]. Therefore, in the potentiality to apply 1c as a probe for experiments
performed in complex biological systems, as living cells or cell lysates, the hydrolysis of the probe 1c
by other SHs enzymes should be considered. These results demonstrated that our fluorometric method
could be successfully applied to identify compounds that modulate MAGL activity.
4. Materials and Methods
4.1. Chemicals
Arachidonic acid, resorufin, and all other reagents and solvents were purchased from
Sigma-Aldrich. Reactions progress was monitored by analytical thin-layer chromatography (TLC) on
pre-coated aluminum foils. Monoacylglycerol lipase (human recombinant, 50 µg) was purchased from
Cayman Chemical. All steps, which included resorufin, were carried out protecting the compound
from light.
Fluorescence signals were recorded by a Jasco FP-8300 fluorometer using the kinetic mode
(
λ
ex = 571 nm
microtiter plates.
1H-NMR spectra were recorded in CDCl3 (isotopic enrichment 99.95%) solutions at 300 K using a
Bruker AVANCE 500 instrument (Bruker Italia Srl, Milan, Italy) (500.13 MHz for 1H, 125.76 MHz for
13C) using 5 mm inverse detection broadband probes and deuterium lock. Chemical shifts (
) are given
, λem = 588 nm, slit = 2.5 nm in both cases) in black, flat-bottomed, 96-well polystyrene
δ
as parts per million relative to the residual solvent peak (7.26 ppm for 1H) and coupling constants (J)
are in Hertz (Supplementary Table S4).
4.2. Synthesis
4.2.1. Synthesis of 7-Hydroxyresorufinyl-Derivatives 1a–1g
To a solution of the opportune carboxylic acid (0.46 mmol) in dry CH2Cl2 (2 mL), oxalyl
◦
chloride (58
µ
L, 0.69 mmol) in dry CH2Cl2 (0.5 mL) was added dropwise at 0 C under stirring.
N,N-dimethylformamide (DMF, 1 drop) was added next. The reaction mixture was stirred at room
temperature for 3 h and then concentrated under vacuum, giving crude acyl chloride. This residue was
dissolved in dry CH2Cl2 (1 mL) and added dropwise to an ice-cold suspension of resorufin (50 mg,
0.23 mmol) and triethylamine (48 µL, 0.35 mmol) in dry CH2Cl2 (3 mL) and then stirred overnight at
room temperature. After dilution with CH2Cl2 the salts residues were removed by filtration obtaining
a brick-red solution; that was washed with 0.5 M HCl (2 mL) and saturated NaHCO3 (2.5 mL), dried
on anhydrous Na2SO4 and concentrated to give the crude product that was purified with column
chromatography on silica gel (Supplementary Table S1). Yields 62–92%.
4.2.2. Synthesis of Resorufin Esters 1h–1k
To a stirred solution of the opportune carboxylic acid (0.46 mmol) in dry CH2Cl2 (3 mL), were
added DMAP (2.5 mg. 0.02 mmoli) and resorufin (50 mg, 0.23 mmol). DCC (72 mg, 0.35 mmoli) was
added to the reaction mixture at 0 ◦C. Stirring was continued for 5 min at 0 ◦C and for 3 h at 20 ◦C.
Precipitated urea was then filtered off and the filtrate evaporated down in vacuo. The residue was
taken up in CH2Cl2 and, if necessary, filtered free of any further precipitated urea. The CH2Cl2 solution

Molecules 2019, 24, 2241
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was washed twice with 0.5 M HCl (2 mL) and saturated NaHCO3 (2.5 mL), dried on anhydrous Na2SO4
and concentrated to give the crude product that was purified by column chromatography on silica gel,
yields 82–92% (Supplementary Table S1).
4.3. Stability of the Substrates
The stability of all substrates was tested in 50 mM Tris-HCl buffer (pH 7.4, 1 mM EDTA),
as previously reported [12]. Briefly, 10
µL of the substrates dissolved in DMSO (final concentration
5
µ
M) were added to 90 L of the buffer. The fluorescence increase at 588 nm was monitored at intervals
µ
of 1 min over 90 min at rt., using a Jasco FP-8300 fluorometer (λex = 571 nm, λem = 588 nm).
4.4. Substrates Screening
hMAGL activity was monitored following the increase of resorufin fluorescence (
em = 588 nm), at intervals of 1 min. hMAGL (specific activity 241.9 U/mg) was diluted to 250 ng/mL
in 50 mM Tris-HCl buffer (pH 7.4), with 1mM EDTA (reaction buffer). Each reaction well contained
80 L Tris-HCl 50 mM with EDTA 1 mM, 10 L of reaction buffer with 25 ng hMAGL and the putative
substrate dissolved in 10 L of DMSO (final concentrations 5 M or 25 M for all other substrates),
total volume 100 L [12]. In addition, resorufin calibration curve and negative control, containing
90 L of reaction buffer and no hMAGL, were measured. All resorufin ester solutions were freshly
λex = 571 nm,
λ
µ
µ
µ
µ
µ
µ
µ
prepared in DMSO.
4.5. Kinetic Assays of hMAGL
Reagent solutions for each substrate were prepared diluting first 5 mM stock solution with DMSO
(5
experiments 10
reagent solution were added to each well. The final concentrations were for hMAGL 25 ng/100
(7.6 nM), for all substrates 0.5 M, 1 M, 2.5 M, 5 M, 10 M, and 25 M, and DMSO 10%. For negative
controls 10 L of reaction buffer were added instead of enzyme. In addition, blank samples (90 L of
reaction buffer, 10 L DMSO) were analyzed. Fluorescence was recorded at room temperature for 50
cycles with a cycle time of 3 min. A standard curve was generated by plotting fluorescence of five
µM, 1
µM, 25
µM, 50
µM, 100
µM and 250
µM), and then 1:9 in the reaction buffer. For kinetic
µL of the reaction buffer, containing 25 ng of hMAGL, and 90
µL of the appropriate
µL
µ
µ
µ
µ
µ
µ
µ
µ
µ
concentrations of resorufin (0.02 µM, 0.1 µM, 0.5 µM, 1 µM, 5 µM) prepared by diluting DMSO stocks
in reaction buffer (10% DMSO).
4.6. Data Analysis
All experiments were performed in triplicate and independently replicated at least once. The curve
generated from the hydrolysis of the substrates was used to convert raw fluorescence data into
nmol/mL/min of resorufin produced. The values of negative controls were subtracted from the
enzymatic curve. The initial velocities were determined from the linear portion of the resulting curve.
Kinetic data were elaborated using GraphPad Prism 6.0c and Microsoft Excel graphing software; kinetic
parameters Km and Vmax were calculated using GraphPad, applying a nonlinear regression analysis
(Michaelis-Menten). The quantitative data were calculated as means
was calculated using the equation Z0 = 1
(3 h + 3 l)/|
±
standard errors. The Z0-factor
− µl|, where h and l are the standard
h and L are the mean signal
−
σ
σ
µh
σ
σ
deviations of the high and low signal controls, respectively, and
intensities of the high and low signal controls, respectively [28].
µ
µ
4.7. Validation of 1c for Screening Assay
MAFP and URB602 were chosen for the method validation due to their well-known MAGL
inhibitory activity [ 19]. To prepare inhibitors stock solutions, commercial MAFP solution (10 mg/mL
in ethanol) was diluted to 200 M in DMSO and 15 M URB602 solution in DMSO was obtained from
powder. Eight different working solution were then prepared by dilution with DMSO. 10 L of diluted
7
,
µ
µ
µ

Molecules 2019, 24, 2241
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hMAGL solution containing 25 ng of the enzyme and 10
µL of the appropriate MAFP or URB602
solution were added to wells of a 96-well plate and the volume was adjusted to 95
buffer (Tris-HCl 50 mM with EDTA 1 mM). In control wells, 10 L of DMSO were added instead of
inhibitor solution and the black samples containing only reaction buffer and DMSO (10%) also were
prepared. Final concentrations of MAFP were 1.0 M, 500.0 nM, 100.0 nM, 50.0 nM, 10.0 nM, 5.0 nM,
and 0.1 nM; final concentrations of URB602 were 75.0 M, 50.0 M, 25.0 M, 10.0 M, 5.0 M, 1.0 M,
and 100.0 nM. A 100.0 1c working solution was prepared by diluting a 5.0 mM DMSO stock 1:50
in DMSO. After 60 min of incubation at 25 ◦C, 5.0
L of 1c working solution was added to each well
to give a final substrate concentration of 5.0 M (10% DMSO). Fluorescence was recorded at room
µL with reaction
µ
µ
µ
µ
µ
µ
µ
µ
µM
µ
µ
temperature for 30 cycles, with a cycle time of 1 min. All experiments were performed in triplicate
and independently replicated at least once and the mean of the three obtained values was used for
calculation. The mean fluorescence value of a blank was subtracted from the value of each sample and
control well to normalize data at each time point, the mean value of control wells was subtracted to the
mean value of each sample. From the slop of kinetic curves, residual enzymatic activity was calculated
and IC50 values were obtained by non-linear regression analysis of log[concentration]/inhibition curves.
IC50 was determined as the concentration of inhibitor that results in an initial velocity 50% that of the
sample containing no inhibitor. IC50 was used along with previously calculated Km to determine Ki.
4.8. In Silico Molecular Docking Simulations
All the computational procedures were carried out by the Schrödinger Small-Molecule Drug
Discovery Suite 2018-01 (Schrödinger, Cambridge, USA). The crystallographic structure of the catalytic
domain of hMAGL was downloaded from the RCSB PDB (PDB ID: 3PE6, resolution of 1.35 Å) [32].
Since the selected MAGL crystallographic structure presents three engineered mutations for increasing
the quality of the diffracting crystal, the Schrödinger Protein Refinement tool was used to mutate Ala36,
Ser169 and Ser176 in Lys36, Leu169 and Leu176, respectively. The wild-type MAGL structure was
then energy minimized using the Schrödinger Protein Preparation Wizard in order to fix structural
issues in the three-dimensional (3D) structure. Tested ligands were built through the Schrödinger
Maestro Build Toolbar and prepared for docking by the Schrödinger Ligand Preparation, generating
the stereoisomers of 1h, 1i, and 1j. A receptor grid, which defines the MAGL active site, was generated
via the Schrödinger Receptor Grid Generation, centring a cubic box, with an edge of 20 Å, on the
co-crystallized inhibitor. The molecular docking procedure was carried out by the Schrödinger Glide
Docking in the “extra precision (XP)” mode in order to evaluate the ability of the tested ligand to bind
the MAGL catalytic domain, keeping only the 20 top-scoring poses. The top-scoring solution for each
ligand was submitted to the Schrödinger Prime MM-GBSA, which integrates molecular mechanics
energies combined with the generalized Born and surface area continuum solvation [33] in order to
compute ligand binding and ligand strain energies for a set of ligands and a single receptor.
4.9. Dynamic Light Scattering Analyses
Dynamic light scattering experiments were performed in a custom modified setup (Scitech 100).
The wavelength of the excitation light is
λ = 532 nm and the scattered light was collected at a constant
angle
θ
= 90◦. Both the excitation light and the scattered light were introduced and fetched by means
of polarization-maintaining fibers. The sample was put in a cylindrical glass capillary (O.D. = 3 mm
I.D. 2.4 mm) and sealed with a silicon cap. The intensity time autocorrelation was obtained using g2
(τ) a digital correlator (flex-03d Correlator.com). Each g2 (τ) was obtained after averaging 600 s and for
each experiment three g2 (τ) were acquired.
5. Conclusions
We present here the set-up and validation of a new HTS method for MAGL activity based on a
red fluorogenic probe. Starting from the synthesis of a few long-wavelength fluorogenic compounds,
characterized by different acylic side chains, and their application to the fluorometric determination

Molecules 2019, 24, 2241
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of MAGL activity, 7-hydroxyresorufinyl octanoate (1c) was selected as the best substrate for the HTS
method. 1c is the eligible substrate among the compound tested, being the substrate with the higher
rate of hydrolysis and the best Km and Vmax values. in-silico docking studies show the favorable
interactions between 1c and MAGL active site. Moreover, compound 1c can be easily prepared in
milligram and gram scale and is very stable in solution as demonstrated by the low rate of spontaneous
hydrolysis. The probe 1c was validated using the well-known MAGL inhibitors URB602 and MAFP, in
assay condition.
In conclusion, the fast, sensitive, and accurate method described in this study is a powerful new
tool for the high-throughput assay of MAGL activity and screening of MAGL modulators.
Supplementary Materials: The following are available online. Table S1. Mobile phases used for the separation of
synthesis products. Table S2. Ligand: MAGL interactions. Table S3. Decay times and corresponding radius. Table
S4. Assignment of 1H and 13C-NMR Chemical Shift in CDCl3. Figure S1. Comparison between spontaneous
hydrolysis of 1a–d, 1f, and 1h–k. Figure S2: Time correlation functions for the scattered intensity (at θ
= 90◦).
Figure S3. Binding poses of tested compounds into the MAGL binding site. 1H- and 13C-NMR spectra of
compounds 1a–k. Figure S4. MAGL complexes with compounds 1b and 1c.
Author Contributions: Conceptualization P.C.; Investigation, M.M., S.C., R.O., C.P., L.P., S.D.L.; Validation, M.M.;
Formal Analysis, M.M., R.O., L.P., C.P; Resources P.C. and I.E.; Writing—Original Draft Preparation, M.M., R.O.,
S.C., I.E. and P.C.; Writing—Review & Editing, R.O., S.C., and P.C.; Funding Acquisition, P.C. and I.E.
Funding: This work was supported by “Università degli Studi di Milano, Linea 2 – Azione A 2017-2018” funding
to P.C. and MIUR Progetto Eccellenza to I.E.
Acknowledgments: The authors thank the University of Milan for financial support.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds Ola and Olad8 are available from the authors. Samples of
loading compounds are not available.
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).