Bioorganic & Medicinal Chemistry Letters 24 (2014) 3986–3996

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier.com/locate/bmcl

Docking based virtual screening and molecular dynamics study

to identify potential monoacylglycerol lipase inhibitors
Obaid Afzal a, Suresh Kumar a, Rajiv Kumar a, Ahmad Firoz b, Manu Jaggi c, Sandhya Bawa a,⇑
a
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), New Delhi 110062, India
b
Biomedical Informatics Center of ICMR, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India
c
Dabur Research Foundation, Ghaziabad, Uttar Pradesh, India

a r t i c l e i n f o a b s t r a c t

Article history: Monoacylglycerol lipase (MAGL) is one of the key enzymes of the endocannabinoid system (ECS). It
Received 4 March 2014 hydrolyzes one of the major endocannabinoid, 2-arachidonoylglycerol (2-AG), an endogenous full agonist
Revised 20 May 2014 at G protein coupled cannabinoid receptors CB1 and CB2. Numerous studies showed that MGL inhibitors
Accepted 10 June 2014
are potentially useful for the treatment of pain, inflammation, cancer and CNS disorders. These provoc-
Available online 19 June 2014
ative findings suggested that pharmacological inhibition of MAGL function may confer significant thera-
peutic benefits. In this study, we presented hybrid ligand and structure-based approaches to obtain a
Keywords:
novel set of virtual leads as MAGL inhibitors. The constraints used in this study, were Glide score, binding
Monoacylglycerol lipase
E-Pharmacophore mapping
free energy estimates and ADME properties to screen the ZINC database, containing approximately 21
Molecular docking million compounds. A total of seven virtual hits were obtained, which showed significant binding affinity
Virtual screening towards MAGL protein. Ligand, ZINC24092691 was employed in complex form with the protein MAGL,
Binding free energy estimates for molecular dynamics simulation study, because of its excellent glide score, binding free energy and
Molecular dynamics simulation ADME properties. The RMSD of ZINC24092691 was observed to stay at 0.1 nm (1 Å) in most of the trajec-
MAGL inhibition assay tories, which further confirmed its ability to inhibit the protein MAGL. The hits were then evaluated for
their ability to inhibit human MAGL. The compound ZINC24092691 displayed the noteworthy inhibitory
activity reducing MAGL activity to 21.15% at 100 nM concentration, with an IC50 value of 10 nM.
Ó 2014 Elsevier Ltd. All rights reserved.

Monoacylglycerol lipase (MAGL) is a 33 kDa cytosolic serine a/
b-hydrolase, consisting of 303 amino acid residues, that is also
found associated with the cell membrane. In mammals, MAGL
hydrolyzes one of the major endocannabinoid, 2-arachidonoylglyc-
erol (2-AG), an endogenous full agonist at G protein coupled
cannabinoid receptors CB1 and CB2, into free fatty acid (FFAs) and
glycerol through a mechanism that involves a classical Ser122,
Asp239 and His269 catalytic triad.1–3 MAGL activity not only
decreases MAGs along with increase in FFAs, but also leads to an
increase in specific FFA-derived signalling lipids, such as phospha-
tidic acid (PA), lysophosphatidic acid (LPA), sphingosine-1-
phosphate (S1P), prostaglandins PGE2 and PGD2.4,5 In the brain,
MAGL-driven 2-AG hydrolysis was shown to provide the principal
source of the neuroinflammatory prostaglandins. Moreover genetic
or pharmacological MAGL inhibition has exerted anti-inflammatory
and neuroprotective effects in animal models of Parkinson’s and
Alzheimer’s disease.6–8 Additionally, MAGL inhibitors have been
⇑ Corresponding author. Tel.: +91 11 26059688/5644.
E-mail addresses: drsbawa@rediffmail.com, sbawa@jamiahamdard.ac.in
(S. Bawa).
found to increase 2-AG levels in the brain and spinal cord and also
cause weakened mechanical and cold allodynia via CB1 receptors,
establishing their capability to reduce neuropathic pain.9,10 Thus,
MAGL inhibitors are potentially useful for the treatment of pain,
inflammation, and CNS disorders. In cancer cells, MAGL
overexpression has been proposed to act as the key metabolic
switch capable of redirecting lipids from storage sites toward the
synthesis of protumorigenic signaling lipids.11 It has been shown
that levels of MAGL are considerably high in aggressive cancer cells
compared with less aggressive cancer cells and this lipase, through
breakdown of monoacylglycerols (MAGs), controls the level of free
fatty acids (FFA) in cancer cells. The consequential MAGL-FFA
pathway promotes migration, survival, and in vivo tumour
growth.12–14 Collectively these provocative findings suggest
that pharmacological inhibition of MAGL function may confer
significant therapeutic benefits.
MAGL inhibitors, such as JZL184 (IC50 = 10 nM)15 and
Ly218324012 (IC50 = 20 nM)16 act via the catalytic triad Ser122,
Asp239 and His269, to form a covalent bond with the serine resi-
due. However, both these inhibitors lack selectivity17 and in other
studies JZL184 exhibited lower inhibitory activity on hMAGL with

http://dx.doi.org/10.1016/j.bmcl.2014.06.029
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
 O. Afzal et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3986–3996
the selectivity in part due to inability of the bulky lipophilic
bisarylcarbinol moiety to fit into a relatively narrow binding
pocket.18,19 Another inhibitor, SAR629 co-crystallised with hMAGL,
appeared outside the active site, suggesting an alternative
mechanism of action.20 Recently, some MAGL inhibitors have been
reported, which were found to be more potent than earlier
reported inhibitors.21,22
Despite the elucidation of MAGL 3D structure, although potent
and increasingly selective MAGL inhibitors have been described,
their number is still limited. In this study, we presented hybrid
ligand- and structure-based virtual screening approaches to obtain
a novel set of potential inhibitors for MAGL. The validation of the
docking poses was confirmed by 20 ns molecular dynamics study.
Top hits from these approaches are then evaluated for their ADME
profile based on the calculation of physicochemical properties. A
total of six top hits were screened for their potential to inhibit
MAGL by in vitro enzyme assay.
All computations were performed on an Intel(R) Core(TM) i5-
2400 CPU @ 3.10 GHz capacity processor with memory of 8 GB
RAM running with the Window 7 operating system. PHASE 3.5
and Glide 5.9 implemented in the Maestro 9.4 software package
was used for generating E-Pharmacophore model and docking
studies.23 The crystal structure of human MAGL protein (PDB code:
3PE6, resolution-1.35 Å) was retrieved from Protein Data Bank
(PDB) and was utilized for molecular modelling studies. The
human MAGL protein consist a common folding motif of the a/b
hydrolase fold having 8b sheets, which forms a partial b-barrel dec-
orated on both sides with 8a-helices. The catalytic triad of MAGL,
located in the centre of the binding pocket, consisting of amino
acid residues Ser122, Asp239, and His269. The catalytic nucleo-
philic amino acid Ser122, frequently referred to as the ‘nucleophilic
elbow’, resides on a tight turn between strand b5 and helix a3. The
structurally conserved network of hydrogen bond donors, com-
prises of nucleophilic elbow and the loop connecting a1 and b3
(Gly50, Ala51, Met123, and Gly124) is called the oxyanion hole.
This oxyanion hole serves to stabilize the anionic transition state
of the catalytic reaction carried out by the MAGL.24 Protein was
prepared with the Protein Preparation Wizard in Maestro using
options: bond orders were assigned, hydrogen atoms were added,
Figure 1. Crystal structure of ZYH (blue) bound to MGL compared to the predicted binding mode (brown).
3987
formal charges were treated and water molecules were deleted.
Hydrogen bonding network was then optimized using the exhaus-
tive sampling option and the protein was minimized to an RMSD
limit from the starting structure of 0.3 Å using the Impref module
of Impact with the OPLS_2005 force field. Docking grids were gener-
ated with the default settings in Glide using the co-crystallized
ligand (ZYH) to define the centre of the grid box (20  20  20 Å).
Default parameters were used and no constraints were included
during grid generation.
The energy minimized 3D structure was analysed to gain
insight into the binding pattern of ZYH with the MAGL protein.
The amide carbonyl of co-crystallized ligand ZYH projects into
the oxyanion hole and forms hydrogen bonds with amino hydro-
gen of Ala51 and amide nitrogen of Met123, adjacent to the cata-
lytic Ser122. The pyrimidine–piperazine–azetidine part of the
ligand projects into a narrow amphiphilic pocket and fills the avail-
able active site space almost completely. This portion of the ligand
does not participate in hydrogen bond interactions; instead the
pyrimidine ring forms a p–p stacking interaction of with Tyr194,
which provides further interaction energy with the protein. The
benzoxazole and cyclohexane portion of the ligand is positioned
in a spacious hydrophobic environment and forms mostly van
der Waals interactions with the amino acid residues Leu148,
Ala151, Ala156, Phe159, Leu205, Leu213, Leu214 and Val217. For
a validation of the accuracy of the Glide docking program, the
RMS deviation between the co-crystallized structure ZYH and the
most reasonable binding modes of ZYH docked with Glide XP
was calculated. The results of control docking showed that Glide
determined the optimal orientation of the docked inhibitor, ZYH
to be close to that of the original orientation found in the crystal
(Fig. 1). The RMS deviation between the experimental conforma-
tion and the calculated docked conformation for ZYH in MAGL
was 0.67 Å.
Two different approaches were used in this study to identify
potential virtual MAGL inhibitors. In the first approach,
structure-based virtual screening methodology was used, which
provided a scope for search of new scaffolds that could bind in
the active site of protein. Second approach was based on
E-Pharmacophore script, which has been introduced recently in
3988 O. Afzal et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3986–3996
Schrodinger, a method that uses energetic analysis of structure-
based fragment docking to elucidate key features for molecular
recognition. This hybrid ligand- and structure-based methodology
uses an atomic breakdown of the energy terms from the Glide XP
scoring function to locate key pharmacophoric features from the
docked fragments.25
Structure based virtual screening was employed to screen
approximately 21 million compounds available on ZINC data-
base.26 Prior to the virtual screening with docking simulation, they
were filtered on the basis of Lipinski’s ‘Rule of Five’ to adopt only
the compounds with physicochemical properties of potential drug
candidates and without reactive functional group(s).27 To remove
the structural redundancies in the chemical library, the structur-
ally similar compounds with Tanimoto coefficient smaller than
0.1 were clustered into a single representative molecule. The
ligands were prepared for docking using LigPrep module of Schro-
dinger. All the possible stereoisomers were generated at a selected
pH range 7 ± 2 and energy was minimized using the OPLS_2005
Figure 2. Workflow of structure-based and hybrid ligand- and structure-based virtual screening.
force field. As a consequence, a docking library consisting of 13.2
million compounds was constructed. The workflow used for struc-
ture-based virtual screening is presented in Figure 2. The ligands
were first subjected to high throughput virtual screening (HTVS),
which is intended for rapid screening because of more restricted
conformational sampling. The docking criteria in HTVS stage was
set in such a way so that only the top 1% (132,000 in number) of
all the docked states were passed on to the next stage of standard
precision (SP) docking, where a more extensive sampling using
Monte Carlo procedure was carried out. From here, only top 1%
(1320 in number) of all the good scoring state was retained and
passed on to the third stage in which extra precision (XP) docking
was performed. XP docking does a more extensive sampling and
uses anchor-and-grow strategy to weed out false positives. XP
docking employs a more stringent scoring function than the SP
Glidescore, which includes terms for hydrophobic enclosure and
desolvation penalties.28 Top 10% (132 compounds) having good
XP Gscore values were selected and evaluated for binding free
 O. Afzal et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3986–3996
energy estimates using MM-GBSA, employing VSGB 2.0 continuum
dielectric model as solvent model.29 Among them five structurally
diverse hits were selected on the basis of binding free energy esti-
mates, XP Gscore and visual inspection of binding pattern. The
docking scores (XP Gscore) and binding free energy estimates of
the top five compounds docked to MAGL are listed in Table 1.
The 3D complex structures of top five compounds docked to MAGL
in the active site are depicted in Figure 3. These top docked com-
pounds were found to superimpose at the same site as the co-crys-
tallized ligand. The ligand interaction diagram tool of Schrodinger
was used to visualize the 2D representation of protein-ligand con-
tacts with the interacting residues and is represented in Figure 4.
Structural analysis of the top five hits clearly showed the presence
of two hydrophobic domains connected through the linker having
carbonyl functional group in cyclic or acyclic amide form. In all the
top five hits, it was observed that, less hydrophobic part of the
ligand (amphiphilic in nature) goes towards the amphiphilic
pocket of the MAGL protein constituted by both polar and non-
polar amino acids, while more hydrophobic part remains in the
spacious hydrophobic domain of the active site constituted by only
hydrophobic amino acids. Amide carbonyl function of all the hits
interacts and forms hydrogen bond with one or more key amino
acid residues, Ala51, Met123 and Ser122 of the oxyanion hole in
the active site (catalytic triad) of MAGL. Tyr194 and Phe159 are
involved in p–p stacking interaction, while amino acids Val270,
Val191, Leu184, Ile179, Leu205, Val217, Leu241, Leu148, Ala151,
Leu214, Phe159, Leu213, Tyr58 etc., were found to be involved in
hydrophobic interactions.
The other approach used in this study was E-Pharmacophore
mapping and database screening. The literature was extensively
surveyed to collect diverse set of human MAGL inhibitors from
experimental investigations.15,20,21,24,30–39 The three dimensional
coordinates of all the known inhibitors (25 in number) were
Table 1
XP Gscore and binding free energy estimates of the top five hits obtained from structure based virtual screening
S. No. ZINC Id. Structure
1 09531022
2 01459944
3 05313458
4 12863377
5 24092691
3989
generated using Maestro module of Schrodinger. Ligands were
prepared using LigPrep 2.6 with Epik 2.4 to expand protonation
and tautomeric states at 7.0 ± 2.0 pH units and energy was min-
imized using the OPLS 2005 force field. E-Pharmacophore com-
bines the aspects of ligand and structure-based approaches to
enhance enrichments rather than ligand information alone. The
method presented here attempts to take a step beyond the sim-
ple contact scoring since it incorporates structural and energetic
information using the scoring function in Glide XP.25 The work-
flow used for E-Pharmacophore based database screening is pre-
sented in Figure 2. The compiled and prepared 25 inhibitors were
then docked to the protein structure using Glide XP docking.
‘Write XP descriptor information’ was chosen during the docking
run which deduced energy terms such as hydrogen-bond interac-
tions, electrostatic interaction, hydrophobic enclosure, and p–p
stacking interactions and the rest of the parameters were kept
default. Of all the collected 25 MGL inhibitors, 7 were selected
on the basis of Glide XP docking and binding free energy esti-
mates. The 7 MAGL inhibitors selected for E-Pharmacophore
modelling are presented in Table 2. They were redocked using
Glide XP docking and the Xpdes result file was then given as
an input which contained the information about protein ligand
interactions for energy-optimized pharmacophore (E-Pharmaco-
phore) modelling. The pharmacophore sites scored on the basis
of energies and the top scoring sites were selected based on
energetic ranking to generate a pharmacophore hypothesis. Four
pharmacophoric features along with their scores are: A18
(3.97), R32 (2.09), R29 (1.95) and R27 (1.08). The
generated pharmacophore hypothesis along with the intersite
distances and excluded volumes are given in Figure 5. Conform-
ers were generated for all the tautomers in the database and
matched for all the four pharmacophoric features with a distance
matching tolerance of 3.0. The fitness score was taken as:
Binding free energy (Kcal/mol) XP Gscore (Kcal/mol)
123.465 11.624
123.287 13.696
107.014 12.732
93.083 12.288
90.468 9.118
3990 O. Afzal et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3986–3996

Figure 3. Docked conformation of the top 5 hits obtained from structure-based virtual screening, overlapped in the active site of MGL.

 
1:0  align score Where, fitness is a score that measures how well the matching
Fitness ¼ 1:0  þ 1:0  vector score þ 1:0
1:2 conformation in the database superimposes with that of the refer-
 volume score ence ligands conformation. The hits were rejected with align score
greater than 1.2, vector score less than 1.0 and a volume score less

Figure 4. 2D representation of Protein–ligand contacts of human MGL with the top 5 hits obtained from structure-based virtual screening.
 O. Afzal et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3986–3996 3991

Table 2
XP Gscore, binding free energy and IC50 values of top 7 MAGL inhibitors used for E-Pharmacophore mapping

S. No. Ligand Structure XP Gscore Binding free energy (Kcal/mol) IC50 value

1 ZYH 10.831 145.586 NA

2 MAGL24 9.118 104.690 9 nM

3 MAGL20 8.718 103.373 9 nM

4 MAGL21 8.645 97.131 12 nM

5 MAGL19 8.491 87.312 8 nM

6 MAGL23 8.029 104.175 33 nM

7 CAY10499 7.992 85.47 0.5 lM—20 nM

Figure 5. Pharmacophore hypothesis along with the intersite distance (A) and excluded volume. (B) Orange torus indicate aromatic ring feature and pink sphere indicates
acceptor feature.

than 0. Also, receptor-based excluded volumes were integrated to after first HTVS of ZINC database in structure-based virtual screen-
refine screening process, which helped in reducing false positives ing was chosen for the preparation of Phase database. 7850 ligands
by rejecting inactive compounds.28 132,000 ligands screened out obtained from database screening were then docked to crystal
3992 O. Afzal et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3986–3996
structure of human MAGL using HTVS, Glide SP and Glide XP dock-
ing protocol. Seven ligands were then scored on the basis of bind-
ing free energy estimates and top 3 hits were selected. Alignment
of top 3 hits along with the pharmacophoric features are depicted
in Figure 6. The docking scores (XP Gscore) and binding free energy
estimates of the top three compounds docked to MAGL are listed in
Table 3. The 3D complex structure of top three compounds docked
to MAGL in the active site is depicted in Figure 7. These top docked
compounds were found to superimpose at the same site as the
co-crystallized ligand, ZYH. The ligand interaction diagram tool of
Schrodinger was used to visualize the 2D representation of
protein-ligand contacts with the interacting residues and is
represented in Figure 8. One of the top 3 hit, ZINC2409269 is same
as obtained in structure-based virtual screening. Structural analy-
sis of ZINC0940564 and ZINC40391523 showed the presence of
two hydrophobic domains connected through the heterocyclic lin-
ker having carbonyl functional group. This carbonyl functional
group interacts and forms hydrogen bond with one or more key
amino acid residues, Ala51, Met123 and Ser122 of the oxyanion
hole in the active site (catalytic triad) of MAGL. Key amino acid res-
idue Tyr194 in the amphiphilic region of MAGL forms p–p stacking
interaction with the benzene ring in all the 3 top hits. Amino acids
Val270, Val191, Leu184, Ile179, Leu205, Val217, Leu241, Leu148,
Ala151, Leu214, Phe159, Leu213, Tyr58 etc., were found to be
involved in hydrophobic interactions.
It is increasingly becoming apparent that ADME (Absorption,
Distribution, Metabolism and Excretion) properties are crucial
determinants for the successful development of new drugs. Unfa-
vourable ADME properties can lead to rejection of a drug in the
later stages of drug process.40 All the hits obtained, were further
processed for ADME, Lipinski’s ‘Rule of 5’ and Jorgensen’s ‘Rule of
3’ using QikProp tool of Schrodinger which is built using
experimental details of 710 compounds including 500 drugs and
heterocyclic compounds.41 The QikProp properties of top 7 hits
are listed in Table 4. QikProp calculates properties like molecular
weight, molecular volume, no. of H-bond donors, no. of H-bond
acceptors, polar surface area, QP log Po/w (Predicted octanol/water
partition coefficient) and violations related to Lipinski’s ‘Rule of 5’
27
and Jorgensen’s ‘Rule of 3’ 42 to filter out compounds with clear-
cut undesirable properties. Compounds that satisfy Lipinski’s ‘Rule
of 5’ are considered drug-like and compounds with fewer (and
preferably no) violations of Jorgensen’s ‘rule of 3’, are more likely
to be orally available. All the top 7 hits showed excellent ADME
properties and passed Lipinski’s ‘Rule of 5’ having no violations.
In addition, 4 out of 7 hits were shown to be 100% orally
bioavailable. The excellent ADME property of these hits makes
them promising candidates for as MAGL inhibitors.
Figure 6. Alignment of top 3 hits obtained from E-Pharmacophore based screening, with the pharmacophore features.
ZINC24092691 was employed in complex form with the protein
MAGL, for molecular dynamics simulation study. Molecular
dynamics were employed into complex model to assess the stabil-
ity of the protein in the presence of the ligand as well as the suitable
orientation of the inhibitor in the active site of protein-
ZINC24092691 complex. The molecular dynamics study was per-
formed using GROMACS 4.6.3 software.43,44 The ligand parameters
were calculated with a PRODRG server in the framework of the
GROMOS force field. The model was solvated in a cubic box of
1 nm dimension by applying GROMOS96 43a1 force. The SPC water
model was used in order to create the aqueous environment. The
system was neutralized by adding 2 Na+ and 6 Cl counter ions.
All the simulations were carried out at constant temperature
(300 K) and pressure (1 atm). Particles Mesh Ewald (PME) electro-
static and periodic boundary conditions were applied in all direc-
tions.45 Subsequently, it was subjected to a steepest descent
energy minimization until a tolerance of 1000 kJ/mol, so that the
system can get rid of the high energy interactions and steric clashes.
All the bond lengths were constrained with the LINCS method, 46,47
the energy minimized system was treated for 100 ps equilibration
run of MD with position restraints on the protein within a cut-off
radius of 0.9 nm. The pre-equilibrated system was consequently
subjected to 20 ns production MD simulation with a time-step of
2 fs at constant temperature (300 K), pressure (1 atm) and without
any position restraints. Snapshots were collected every 2 ps and all
the analyses of the MD simulation were carried out by GROMACS
4.6 analysis tools. Some of the analyses like Root Mean Square Devi-
ation (RMSD), Potential Energy (PE), and Root Mean Square Fluctu-
ation (RMSF) were used to check the stability of the model in
explicit condition for 20 ns. RMSD of the protein backbone atoms
with reference to crystal were plotted as a function of time to check
the stability of the system throughout the simulation. During the
20 ns of time, the RMSD of the system tends to be converged indi-
cating that the system is stable and well equilibrated. The analysis
of the RMSD plot for backbone showed that after a small rearrange-
ment from the initial conformation, the complex was stable during
entire MD simulations period (Fig. 9(A) and (B)). The RMSD for
backbone before and after simulation remained within the limit
of 0.15 Å. Figure 9A and B showed the overall RMSD analysis of
backbone which explained the protein structure deviation at
atomic level with respect to the function of time. The relative
flexibility of the model was characterized by plotting the RMSF
relative to the average structure obtained from the MD simulation
trajectories. To examine the flexible regions of protein, RMSF plot
was generated with respect to their individual residues (Fig. 10).
The RMSD of ZINC24092691 was observed to stay at 0.1 nm (1 Å)
in most of the trajectories (Fig. 11). MD simulations showed that
 O. Afzal et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3986–3996 3993

Table 3
XP Gscore and binding free energy estimates of the top three hits obtained from E-Pharmacophore based screening

S. No. ZINC Id. Structure Binding free energy (Kcal/mol) XP Gscore (Kcal/mol)

1 24092691 90.468 9.118

2 09405640 87.530 10.805

3 40391523 87.013 11.731

Figure 7. Docked conformation of the top 3 hits obtained from E-Pharmacophore based screening, overlapped in the active site of MGL.

Figure 8. 2D representation of Protein–ligand contacts of human MGL with the top 3 hits obtained from E-Pharmacophore based screening.

the binding mode of ZINC24092691 was maintained. Furthermore, formed additional two H-bonds with the amino acid residues
the investigation of protein-ligand interactions during MD simula- MET123 and GLY52, leading to overall four H-bonds within the
tion suggested that the amide functional group of ZINC24092691 active site of the protein, making it a potential inhibitor (Fig. 12).
3994 O. Afzal et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3986–3996

Table 4
QikProp property obtained for 7 top hits

ZINC Id. Mol_MW (Dalton) SASA (Å2) Volume (Å3) Donor HB Accept HB QP log Po/w % Human oral absorption Rule of five Rule of three
(130–725) (300–1000) (500–2000) (0–6) (2–20) (2.0–6.0) (>80% is high <25% is poor) (max. is 4) (max. is 3)
1459944 478.931 794.149 1428.583 1 7.75 4.495 100 0 1
5313458 390.441 706.718 1247.417 2 7.5 2.263 77.918 0 0
9405640 475.475 747.068 1334.339 2 10 2.583 80.377 0 1
9531022 428.505 755.281 1372.294 3 6 3.714 90.663 0 1
12863377 393.46 744.493 1298.783 2 5.5 4.068 100 0 1
24092691 385.442 691.444 1191.175 1 6 4.207 100 0 1
40391523 480.457 773.499 1387.776 0.25 8 4.363 100 0 1

Mol_MW: molecular weight in Dalton; SASA: total solvent accessible surface area; volume: total solvent-accessible volume; donar HB:
Hydrogen bond donar; accept HB: hydrogen bond acceptor; QP log Po/w: predicted octanol/water partition coefficient.
Values in parentheses depict the recommended values.

Figure 9. (A) RMSD of protein backbone atom with respect to time over the course of the 20 ns MD simulation runs. (B) RMSD of protein backbone atom relative to the crystal
structure.

The in vitro hMAGL inhibition assay was carried out by the
method reported by Muccioli et al., 2008.32 Among the top seven
hits, six were purchased. We were unable to procure or synthe-
size ZINC09405640. So, a total of six compounds were tested
for their ability to inhibit human MAGL in serial dilutions
(1:10) from 100 lM to 1 nM (six concentrations in triplicate)
using dimethyl sulfoxide (DMSO) as solvent. Assays were carried
out in a 96-well plate (180 ll total volumes). Assay buffer 10 mM
Tris–HCl, pH 7.2, containing 1 mM EDTA was used for the assay as
well as for the dilution of pure human MAGL. 4-Nitrophenylace-
tate (NPA) was used as substrate, which by the action of MAGL
produced yellow product 4-nitrophenol. The 96-well plate was
incubated at 37 °C for 15 min prior to absorbance measurement
at 405 nm. Average absorbance of background, 100% MAGL
activity (IA) and inhibitor wells were determined. The average
absorbance of background well, contained buffer and DMSO only
was systematically subtracted from the average absorbance of IA
and inhibitors. The% MAGL activity for the inhibitors was
calculated by the formula: % MAGL activity = Inhibitor/IA  100.
Results are quoted as% MAGL activity in Table 5 using

Figure 10. RMSF of C-alpha atoms averaged over all subunits. Figure 11. Ligand (ZINC24092691) positional RMSD.
 O. Afzal et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3986–3996 3995

Figure 12. Binding mode of ZINC24092691 before and after simulation.

Table 5
Selected six compounds and their inhibitory effects against hMAGL

% MAGL activity IC50

1 nM 10 nM 100 nM 1 lM 10 lM 100 lM
ZINC9531022 95.10 91.51 87.42 75.21 62.72 57.60 >100 lM
ZINC1459945 93.78 89.70 80.55 70.26 57.78 51.09 100 lM
ZINC5313458 92.35 88.26 77.15 69.70 59.28 51.85 100 lM
ZINC12863377 90.36 78.05 42.51 30.45 23.07 18.61 39 nM
ZINC24092691 81.40 51.23 21.15 17.10 15.09 14.28 10 nM
ZINC40391523 97.80 95.66 89.10 83.12 74.50 72.30 >100 lM
CAY10499 97.56 94.17 91.92 18.36 6.66 5.12 424 nM

CAY10499 used as standard control; IC50 value was calculated from Graph pad prism, version 6.01.

Figure 13. 2D and 3D interaction of ZINC24092691 with hFAAH (PDB Id: 3LJ6).

CAY10499 as standard positive control. Out of the six compounds
tested, ZINC24092691 and ZINC12863377 reduced the MAGL
activity less than 50% at 100 nM concentration. The most potent
compound was the benzthiazole–benztriazole analogue, ZINC
24092691 reducing% MAGL activity to 51.23 at 10 nM, 21.15 at
100 nM and 14.28 at 100 lM concentrations. Compound
ZINC12863377 also showed good MAGL inhibitory potential with
an IC50 value of 39 nM. The results for CAY10499 (IC50 = 424 nM)
were comparable to that reported previously (IC50 = 350 nM,
Muccioli et al.32). None of the compound showed complete
inhibition of MAGL, a feature possibly showing the characteristic
of reversible inhibition.
Since ZINC24092691 showed the best hMAGL inhibitory
potential with an IC50 value of 10 nM, it was chosen to study fur-
ther its affinity with human Fatty acid amide hydrolase (hFAAH),
another lipid metabolising enzyme of the endocannabinoid
system. ZINC 24092691 was docked to the prepared hFAAH protein
(PDB Id: 3LJ6) to determine its selectivity. The 2D and 3D image of
the docked structure is depicted in Figure 13. The docked ligand
showed only one p–p interaction with the amino acid residue
3996 O. Afzal et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3986–3996
PHE-192, with a low XP Gscore value of 3.128. Furthermore, no
H-bonding was observed, which showed its minimal interaction
with the protein FAAH and high selectivity towards MAGL.
In conclusion, this study reports novel MAGL inhibitors showing
significant binding affinity towards MAGL protein by structure-
based and hybrid ligand- and structure-based virtual screening
approach. A careful analysis of all the top hits obtained either by
structure-based or hybrid structure- and ligand based virtual
screening showed three key essential features for MAGL inhibition.
A amphiphilic benz-fused heterocyclic ring (to form p–p stacking
interaction with Tyr194 and also interact with the polar as well
as non-polar amino acids) linked with bulky hydrophobic part (to
fill up the hydrophobic space and to interact with the hydrophobic
amino acids) connected through a linker having carbonyl
functional group either in cyclic or acyclic amide form or in a
heterocyclic system (to form H-bond with one or more key amino
acid residues, Ala51, Met123 and Ser122 of the oxyanion hole in
the active site). These novel hits can be taken up for further studies
to investigate their biological relevance as potent inhibitors or to
design novel MAGL inhibitors.
Acknowledgments
Authors are thankful to Department of Pharmaceutical Chemis-
try, Jamia Hamdard, New Delhi for providing necessary facilities.
One of the authors (Obaid Afzal) is grateful to the Council of Scien-
tific and Industrial Research (CSIR), New Delhi, India, for providing
financial assistance.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.
06.029.
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