Arylamide as Potential Selective Inhibitor for Matrix Metalloproteinase 9 (MMP9): Design, Synthesis, Biological Evaluation, and Molecular Modeling

Article abstract of DOI:10.1021/acs.jcim.9b00630

Previous studies have reported that compounds bearing an arylamide linked to a heterocyclic planar ring have successfully inhibited the hemopexin-like domain (PEX9) of matrix metalloproteinase 9 (MMP9). PEX9 has been suggested to be more selectively targe

Full text of DOI:10.1021/acs.jcim.9b00630

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Pharmaceutical Modeling
Arylamide as Potential Selective Inhibitors for Matrix Metalloproteinase 9
(MMP9): Design, Synthesis, Biological Evaluation and Molecular Modeling
maywan hariono, Rina Fajri Nuwarda, Muhammad Yusuf, Rollando Rollando, Riris
Istighfari Jenie, belal Omar Al-najjar, Jeffry Julianus, Kevin Cahaya Putra, Ervan
Setyo Nugroho, Yohanes Krisna Wisnumurti, Sangga Putra Dewa, Benedictus Wisnu
Jati, Reynaldo Tiara, Ratna Dwi Ramadani, Lailatul Qodria, and Habibah Wahab
J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.9b00630 • Publication Date (Web): 11 Dec 2019
Downloaded from pubs.acs.org on December 12, 2019
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Arylamide as Potential Selective Inhibitors for
Matrix Metalloproteinase 9 (MMP9): Design,
Synthesis, Biological Evaluation, and Molecular
Modeling
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Maywan Hariono , Rina F. Nuwarda , Muhammad Yusuf , Rollando Rollando , Riris I.
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Jenie , Belal Al-Najjar , Jeffry Julianus , Kevin C. Putra , Ervan S. Nugroho , Yohanes K.
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Wisnumurti , Sangga P. Dewa , Benedictus W. Jati , Reynaldo Tiara , Ratna D.
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Ramadani , Lailatul Qodria , Habibah A. Wahab
1Faculty of Pharmacy, Sanata Dharma University, Depok, Sleman 55282, Yogyakarta,
Indonesia
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2Faculty of Pharmacy, Padjadjaran University, Jatinangor, Sumedang 45363, West Java,
Indonesia
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3Chemistry Department, Faculty of Mathematics and Natural Sciences, Padjadjaran
University, Jatinangor, Sumedang 45363, West Java, Indonesia
4Pharmacy Program, Faculty of Science and Technology, Ma Chung University, Malang
65151, Indonesia
5Cancer Chemoprevention Research Center, Faculty of Pharmacy, Gadjah Mada
University, Sekip Utara 55281, Yogyakarta, Indonesia
6Faculty of Pharmacy and Medical Sciences, AlAhliyya Amman University, Amman
19328, Jordan
7Pharmaceutical Technology Department, School of Pharmaceutical Sciences and USM-
RIKEN Centre for Ageing Science (URICAS), Universiti Sains Malaysia, 11800 Minden,
Pulau Pinang, Malaysia
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Abstract
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Previous studies have reported that compounds bearing arylamide linked to heterocyclic
planar ring, have successfully inhibited the hemopexin-like domain (PEX9) of matrix
metalloproteinase9 (MMP9). PEX9 has been suggested to be more selectively targeted
than MMP9’s catalytic domain in degrading extracellular matrix in some pathologic
conditions, especially in cancer. In this study, we aim to synthesize and evaluate ten
arylamide compounds as MMP9 inhibitors through enzymatic assay as well as a cellular
assay. The mechanism of inhibition for the most active compounds was investigated by
molecular dynamics simulation (MD). Molecular docking was performed using
AutoDock4.0 with PEX9 as the protein model to predict the binding of the designed
compounds. The synthesis was carried out by reacting aniline derivatives with 3-
bromopropanoyl chloride using pyridine as the catalyst at room temperature. MMP9
assay was conducted using the FRET-based MMP9 kits protocol and gelatin zymography
assay. Cytotoxicity assay was done using the MTT method, and the MD simulation was
performed using AMBER16. Assay on MMP9 demonstrated activities of three compounds
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(2, 7, and 9) with more than 50% inhibition. Further inhibition on MMP9 expressed by 4T1
showed two compounds (7 and 9) inhibited its gelatinolytic activity for more than 50%.
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The cytotoxicity assay against 4T1 cells results in the inhibition of the cell growth with
EC of 125 M and 132 M for 7 and 9, respectively. MD simulation explained a stable
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interaction of 7 and 9 in PEX9 at 100 ns with the free energy of binding: -8.03 kcal/mol
and -6.41 kcal/mol, respectively. Arylamides have potential effects as selective MMP9
inhibitors in inhibiting breast cancer cell progression.
Introduction
Breast cancer, the most common cancer in female, is now the second leading cause of
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death-related cancer in the world . As with any other cancer, the disease is attributed to
complicated pathways, in which cell selectivity becomes the major issue in its
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pharmacotherapy . The development of anti-cancer chemotherapeutic agents by
targeting selective proteins or nucleic acid for breast cancer is challenging and mostly
met with some serious adverse side effects as well as resistance3.
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Breast cancer is categorized into three types according to the most common receptors
known to fuel breast cancer growth, i.e., estrogen, progesterone, and human epidermal
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growth factor receptor 2 (HER-2) . Another type in which those three receptors are not
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present is called a triple-negative type . Several proteins have been reported to be
responsible for cancer cell growth. However, in the triple-negative breast cancer, matrix
metalloproteinase 9 (MMP9) has been identified to be highly expressed during
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angiogenesis as well as cell migration . MMP9 is one of the MMP subfamilies, classified
as gelatinase that degrades extracellular matrix (ECM). It also has gelatin as the substrate
7, 8
for the enzyme activity . The genomic structure of MMP9 is composed of the propeptide
and a catalytic domain, which is bridged by SH-Zn, followed by three fibronectins which
are linked to the terminal hemopexin-like (PEX9) domain9, 10. The enzyme is activated by
disconnecting the SH-Zn bridge (called a cysteine switch) to interact with the substrate11,
12.
Therefore, by interrupting this interaction using peptidomimetics or small molecules,
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the progress of angiogenesis and cell migration could be well controlled . Unfortunately,
many MMP9 inhibitors failed in the clinical trials due to the presence of adverse drug side
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effects (ADS) such as musculoskeletal syndrome . High homology in all MMPs catalytic
sites has been suggested to contribute to a non-selective inhibition leading to ADS15, 16
.
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Dufour et al. (2011) and Alford et al. (2017) have developed MMP9 inhibitors by targeting
the non-catalytic site, i.e., hemopexin-like domain (PEX9)17, 18. This domain has low-
homology amongst all MMPs. Therefore it is an interesting target for disrupting breast
cancer with less adverse side effects19, 20. Dufour et al. studied the pharmacophore of two
of their most active inhibitors (Fig. 1a) via molecular docking into the binding site of PEX9.
They suggested that the enzyme inhibitory activity, on one hand, was due to the six-
member heterocycle ring, which provides a planar conformation that fits into the cavity of
PEX9 domain blades. On the other hand, the arylamide moiety facilitates flexible
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conformation that bound to the surface near the cavity .
Analogs of these inhibitors could be synthesized by modifying the heterocyclic planar ring
using purine, as suggested in Scheme 1. The retrosynthesis identified that the analogs
could be synthesized through the nucleophilic acyl substitution reaction between the
arylamide with bromine attached at the alkyl chain’s terminal as the leaving group and
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purine. Thus, the arylamide has a role as an intermediate compound, which in turn could
be synthesized from aniline derivatives with acetyl chloride derivatives.
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In this present study, we designed and synthesized ten arylamide compounds by
modifications at its para-position. The design was initiated by studying the binding mode
of these compounds in the PEX9 binding site via molecular docking. The compounds
were then evaluated for their potency to inhibit the activity of MMP9 using fluorescence
binding and gelatin zymography assays. Then, cytotoxicity assay was performed on the
selected active compounds to the cell growth of 4T1, a cell model for the triple-negative
breast cancer. Finally, a molecular dynamics study was carried out to gain a deep
understanding of the interaction of the most active compounds with PEX9. This study
found three compounds that are potentially developed as an anti-breast cancer agent.
Methods
Molecular design
21
The protein (PEX9) with PDB id 1ITV was downloaded from www.rcsb.org . The control
docking was carried out by extracting the sulfate ligand from PEX9 using Discovery Studio
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.5 (www.accelrys.com) and then prepared using AutodockTools 1.5.6.
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(www.scripps.edu) . The protein was protonated and assigned with Kollman charges
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while the ligand with Gasteiger charges. The grid box was set with the number of grid
point = 70x70x70; grid point spacing = 0.375Å; and the center of coordinate with x =
42.053, y = -30.855, and z = -1.804. Docking was performed using AutoDock 4.2 with the
Lamarckian Genetic Algorithm (LGA) for 250 iterations. The free energy of binding was
calculated as the sum of final intermolecular energy, van der Waals, H-bond, desolvation,
electrostatic, final total internal energy, torsional free and unbound system energy. The
docking poses were visualized using Discovery Studio 3.5, and the docking parameter
was defined as valid when the RMSD value between initial and post-docking poses was
23
not greater than 2.0 Å . The new ligands were sketched and energetically optimized
using Marvin Sketch with MM+ as the force field (www.chemaxon.com), prepared and
docked with the same parameters of the control docking.
Chemistry
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All reactions were carried out using standard techniques for the exclusion of moisture24
except for those in aqueous media. The progress of the reaction was monitored using
TLC on 0.25 mm silica F254 and detected under UV light. FTIR spectra were determined
using Shimadzu. H-NMR and 13C-NMR spectra were determined using Bruker
spectrometer (700 MHz) with TMS as an internal standard, and the mass spectra were
determined using QP2010S Shimadzu with EI 70 ev. Melting points were obtained using
a STUART SMP electro-thermal apparatus and were uncorrected.
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In a round bottom flask, aniline or its derivatives (3.59 mmol) was mixed with pyridine
(7.18 mmol) and then added with 3-bromopropionyl chloride (8 mmol). The mixture was
stirred for 20-30 minutes at room temperature. The progress of the reaction was
monitored using TLC and color tests using DAB HCl. When the product was formed, the
mixture was then washed with Na CO 10%, followed by filtration. The solid product was
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collected and recrystallized using suitable organic solvents (adapted from25-28). The
characterization and the NMR spectra of the compounds are presented in Supporting
Information.
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In vitro fluorescence binding assay (FBA)
The MMP9 enzyme kit was purchased from Biovision containing lyophilized MMP9,
FRET-based MMP9 substrate, MMP9 assay Buffer, and NNGH (N-isobutyl-N-(4-
methoxyphenylsulfonyl) glycylhydroxamic acid) inhibitor as the positive control. The
lyophilized enzyme was reconstituted with 110 L glycerol 30% in deionized water. The
reconstituted enzyme was diluted into 550 L of buffer and ready to be used in the assay.
Compound’s sample was prepared by dissolving it in DMSO to have a final concentration
of 200 g/ml in the 96-microwell plate. The final concentration of DMSO in each well was
less than 2%. Each sample (1 L) was well mixed with the buffer (44 L), followed by the
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addition of the enzyme (5 L) into each well. The mixture was then incubated at 37 C for
30 minutes. The substrate (50 L; 40 M) was added to the mixture and then re-incubated
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at 37 C for 60 minutes. The fluorescence was read using Tecan Infinite Pro200 Microplate
Reader at 325/393 nm (adapted from29, 30
)
In vitro gelatin zymography assay (GZA)
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T1 metastatic cell line (ATCC CRL­ 2539) was cultured in Dulbecco's Modified Eagle
Medium (DMEM) medium (Gibco) with 10% Fetal Bovine Serum (Gibco), 1%
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Penicillin­Streptomycin (Gibco) and 0.5% fungizone (Gibco). The 4T1 cells (1 × 10 ) were
seeded into each well of a 6-well plate and incubated at 37°C in a CO2 incubator for 24
h. Cells were incubated with 10 M sample, in serum-free medium for 24 h. The medium
was collected and subjected to polyacrylamide gel electrophoresis (PAGE) on a 10%
SDS-PAGE gel containing 0.1% gelatin and run in the SDS running buffer. The gels were
washed in a renaturing solution containing 2.5% Triton X-100 for 30 minutes, then
incubated with buffer (50 mM Tris-HCl, 150 mM NaCl, 10 mM CaCl2) for 20 h at 37°C.
The gels were stained using 0.5% Coomassie Brilliant Blue and incubated for 30 min at
RT and destained with a destaining solution (10% v/v methanol and 5% v/v acetic acid).
Gels were then scanned and analyzed with image-J software31, 32. Pentagamavunone-1
((2E,5E)
-2-
[
(4-hydroxy-3,5-dimethylphenyl)methylidene]-5-[(3-methoxy-4,5-
dimethylphenyl) methylidene]cyclopentanone) was used as the positive control33.
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In vitro MTT cytotoxicity assay
4T1 and Vero cell cytotoxicity were determined using MTT assay: Cells were seeded into
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well with volume 100 L (10 cells) per well and checked under an inverted microscope
to see its distribution. Cells were then incubated under CO atmosphere overnight until
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returned to normal condition and ready to be used. The normal cells in the plate were
then taken from the incubator, and the media was discarded. The concentration series of
the sample in 0.1% DMSO was seeded into the well and then incubated under CO2
atmosphere for 24 hours. The media was then removed, and 100 L of MTT reagent (0.5
g/mL) was applied per well followed by incubation under CO atmosphere for four hours
2
until the formazan being formed. The reaction was then stopped by adding 100 L of 10%
SDS in 0.1 N HCl. The plate was then wrapped using aluminum foil and stored in a dark
room at room temperature overnight. Absorbance was read using the Tecan Infinite
3
4
Pro200 Microplate Reader at 595 nm (adapted from ). Doxorubicin was added to the
cultures at the following concentrations: 0, 2.5, 5, 10, 20, and 40ꢀg in a final volume of
100ꢀμL as a positive control using DMSO 0.1%
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Molecular dynamics
The interactions of three compounds with PEX9 were further investigated by MD
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simulation, which are two active compounds (7 and 9), and the least active compound
(4) were simulated for comparison. AMBER16 was utilized to perform geometry
minimization and MD simulation in an explicit TIP3P water model. First, minimization
was done by using 1000 and 1000 steps of steepest descent and conjugate gradient,
respectively. The MD system was gradually heated to 310 K over 60 ps in the NVT
2
ensemble. A harmonic restraint of 5 kcal/molÅ on the complex was used in the heating
stage, followed by a 1 ns of NPT equilibration. During the equilibration stage, the
2
harmonic restraint was gradually reduced by 1 kcal/molÅ until it reached zero. Finally,
the production run in the NPT ensemble was carried out for 100 ns. The time step for
the production run was 2 fs with the use of the SHAKE algorithm. Langevin thermostat
was used to control the temperature, where the collision frequency parameter was set
-1
to 1 ps . Berendsen barostat was used to control the pressure, where the coupling
constant and target pressure was set to 1 ps and 1 bar, respectively. The cutoff value
for nonbonded interactions was set to 9 Å. Particle Mesh Ewald was activated to treat
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long-range electrostatic interactions. Cpptraj in AmberTools was used to analyze the
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MD trajectories . The binding energy between the compounds and PEX9 was
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computed using the MM/PBSA (Molecular Mechanics / Poisson-Boltzmann Surface
Area) method implemented in the MMPBSA.py program of AMBER16. The calculation
of binding energy was calculated from the addition of molecular mechanical (MM)
energy change in the gas phase and solvation free energy. The MM energy composed
of internal energy, Coulomb electrostatic term, and van der Waals interaction term.
Whereas the solvation free energy composed of electrostatic and non-electrostatic
solvation energy.
Result
Molecular docking and synthesis design
The design was initiated from an arylamide ring that was predicted to contribute to the
17
inhibition of PEX9 activity . Although diverse functional groups have been suggested
could be substituted at the para position of the ring to study the structure-activity
18
relationships, their contribution, however, was not discussed . Therefore, we are
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interested in exploring these contributions in terms of the binding to PEX9 via docking
study. Previously, an active arylamide that had OCHF attached to the p-position
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demonstrated that the electron-withdrawing group (EWG), F and CH (steric group) might
2,
be responsible to PEX9 inhibition (Dufour et al. (2011)). Thus, we hypothesize that
attaching the electron-withdrawing group (EWG) together with a steric functional group at
the p-position will increase the arylamide activity. In this study, we attached EWG, i.e.
nitro (NO = 2), chloro (Cl = 3) and fluoro (F = 4), EWG plus a steric/ hydrophobic group
2
such as ethylaceto (COOC H = 5), sulfonamide (SO NH = 6), sulfonamide-pyrimidine
2
5
2
2
(7), sulfonamide methylpyrimidine (8), sulfonamide dimethylpyrimidine (9), at p-
arylamide. We also designed a compound that has an electron-donating group (EDG)
with a steric/ hydrophobic character (OCH = 10) attached at 3, 4, and 5 positions, to
3
investigate whether EDG plus a steric character could also contribute to the inhibition of
PEX9 activity. As a control, arylamide (1) with no substitution at the p-position was also
synthesized for the study. The reaction is presented in Scheme 2.
Docking of sulfate ion as the control ligand into PEX9 showed an RMSD of 1.69 Å (less
23
than 2.0 Å), indicating that the docking parameters are reproducible . Table 1 shows the
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docking results where the free energy of binding (∆Gbind) of all the compounds studied
are less than -6 kcal/mol (-6.40 to – 11.36 kcal/mol) predicting the ability of the ligands to
bind competitively with the substrate into PEX9’s binding site. Most ligands displayed
hydrogen bond interaction with GLU14, GLU60, ARG106, ASP151, and GLN154 of the
PEX9’s binding site. Thus, these compounds have proceeded for the synthesis.
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Chemistry
The synthetic products showed a negative colorimetric reaction with dimethylamino
benzaldehyde HCl (DAB HCl), indicating that the nucleophilic acyl substitution (S A) has
N
taken place. It has been well documented that free aromatic amine reacts with DAB HCl
by forming an orange color describing a Schiff base reaction to produce an imine (-CH=N-
). The arylamide products were then confirmed for their purity using TLC, showing a pure
single spot. The presence of a carbonyl functional group was confirmed by FTIR, showing
-1
a very strong stretching band near 1500 cm . The proton NMR demonstrates aromatic
proton which varies in chemical shifts depending on the chemical environment, but most
of them are deshielded at 6-8 ppm. The ethylene proton is confirmed by the presence of
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two triplet signals nearby 2.7 ppm and 3.3 ppm, respectively. The confirmation of Br
group, as well as the whole structure, was carried out using mass spectroscopy. For
compounds with a sulfonamide functional group, the mass/ion was not observed in the
parent molecule. However, there is always 65 of mass/ion deduction due to the
rearrangement of the amine group to attach the arylamide ring at p-position when the SO2
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being fragmented . Following the confirmation of the structures of the ten synthesized
compounds, bioactivity against MMP9 in vitro was carried out.
In vitro fluorescence binding assay
In the in vitro binding assay, MMP9 is reacted with its substrate to perform proteolysis.
The substrate is a peptide linked to the fluorophore, cleaved by MMP9, leading to
fluorescence, which can be measured spectrophotometrically as the enzyme activity37, 38
.
The presence of a synthetic compound should reduce the fluorescence, indicating the
inhibition of MMP9-substrate binding. In this study, the tested compounds demonstrated
4 to 68% inhibition at 200 g/mL (Fig. 2). The high percentage of inhibitions are exhibited
by 2 (67%), 7 (66%), and 9 (68%), whereas the other seven compounds showed less
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than 50% inhibition. The moderate to low activities were performed by 8 (49%), 5 (25%),
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(11%), 3 (10%), 6 (9%), 4 and 10 (both were 4%). These results indicate that arylamide
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compounds can inhibit the activity of MMP9 from low to high potency. Interestingly,
compounds with sulfonamide-pyrimidine substitutions at para position perform higher
inhibitory activities with % inhibition = 66 and 68 for non-methylated (6) and
dimethylatedpyrimidine (8), respectively. In addition, the p-nitro substituted compound (2)
also demonstrates a high percentage of inhibition (67%) against MMP9. Sulphonamide
and nitro groups are two strong electron-withdrawing groups, thus strengthening our
hypothesis on the role of EWG in MMP9 inhibition. Compound 9 with the highest
percentage inhibition was then re-run for its IC experiment calculating the microMolar
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concentration of 70 µM. Although the IC of 9 was not as potent as NNGH (47.80 nM) as
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the positive control, however, 9 could be less toxic than NNGH. It was well studied that
MMP9 inhibitor with hydroxamate-like structure fails in clinical study due to its non-
14
selective inhibition towards the catalytic site .
In vitro gelatin zymography assay (GZA)
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4
T1, a triple-negative breast cancer cell line from Mus musculus, was used to test the
gelatinase activity of the samples. Alignment of amino acid sequences between
hemopexin MMP9 expressed by MDA-MB-231 from humans and MMP9 from 4T1 of Mus
musculus demonstrated 61% identity. The high homology between the two MMP9s
suggested that 4T1 could be used as a cell model to express MMP9. The sequence
alignment of PEX9 (PDB 1ITV) with MMP9 from Mus musculus is presented in Fig. S1
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(Supporting Information).
Gelatine zymography is a simple method to determine the inhibitory activity of a
39
compound against MMP9 . In this experiment, 4T1 cells expressing MMP9 can be
detected using electrophoresis. MMP9 will precede gelatinolysis during electrophoresis
and show its existence as a transparent band with a blue background in the absence of
40
gelatinolysis . The band is assigned as a 100% intensity of MMP9 being expressed by a
4T1 cell. In the presence of MMP inhibitor, the intensity should be reduced indicating the
41
inhibition of such inhibitor in MMP9 gelatinolytic activity .
Fig. 2b presents a zymogram of MMP9 expressed by 4T1 cells consisting of five bands;
untreated cell 1, untreated cell 2, and cell treated by 2, 7, and 9 at 10 µM of concentration.
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It is clear that the band intensity of the untreated 4T1 cells is different that the 4T1 cells
treated with those three active compounds indicating that 2, 7 and 9 have inhibited the
activity of MMP9. As presented in Fig. 2b, the average intensities of the untreated cell 1
is 12947.3, whereas, in the cells treated with 2, the band intensities are reduced to 8266.2.
The treated cells showed reducing activity down to 63.8 % compared to the control cell,
indicating that MMP9 was inhibited by 2 up to 36.2%. Interestingly, the activity of MMP9
was reduced by 7 and 9 down to 19.1% and 23.3%, respectively, indicating that these
two most active compounds inhibited 80.9% and 76.7% MMP9 gelatinolytic activity,
respectively. These data support the initial findings using fluorescence assay that 2, 7,
and 9 were able to inhibit the activity of MMP9. PGV-1, as the positive control, showed
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35.39% inhibition towards gelatinolysis at the concentration of 2 µM. In respect to the
concentration used by 2, 7, and 9, the concentration of the positive control was 5x lower
than the tested compounds. Therefore, we could assume when they were at the same
concentration, the positive control could be more potent than the tested compounds.
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In vitro MTT cytotoxicity assay
The toxicity of the three compounds was tested toward 4T1 cells as well as normal Vero
cells to determine the safety and selectivity of the compounds. To assess the cell
metabolic activity, the MTT assay was carried out. The number of viable cells present
was reflected by NAD(P)H-dependent oxidoreductase enzyme under defined conditions.
The reduction of tetrazolium dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide formed purple color by the enzyme into insoluble formazan product. The cell was
then solubilized by an organic solvent such as DMSO while releasing a soluble formazan,
which was measured spectrophotometrically. Since the reduction of MTT can only occur
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in metabolically active cells, the level of activity is a measure of viability cell . Fig. 3
illustrates the drug-dose dependent curves for the three active compounds (2, 7, and 9)
and an inactive compound (4) against 4T1 cells.
The assay system well worked by showing the positive control inhibited 50% of cell growth
at 37.30 µM. All the three compounds have effective concentrations to inhibit the 4T1 cell
growth within a narrow range (125-139 M), with the best EC exhibited by 7 (EC = 125
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M), followed by 9 (EC = 132 M) and 2 (EC = 139 M). Although 4 showed only 4%
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inhibition in MMP9 fluorescence binding assay, the EC of the compound in the cellular
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assay is 229 M.
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Fig. S2 (Supporting Information) illustrates the morphology of the Vero cell before and
after treatments using 7. The cell size is smaller than its normal size and shrank because
of the loss of cytoplasmic fluid due to the membrane disruption, as shown by the detached
cell upon the compound’s treatment. All the three compounds showed high CC on the
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Vero cells. A compound which has a concentration to stop at least 50% of non-cancer
cell growth for more than 10 M is categorized as non-toxic43, 44. Thus, the three most
active compounds against MMP9 are also relatively safe for further development, as
confirmed by the calculated safety index (SI).
The safety index (SI) is a ratio of CC to EC in which the higher the value of the SI, the
50
50
less toxic the compound would be. The results showed that among three compounds, the
SI was calculated as followed: 9>4>2>7. Therefore, the safest compound is 9 with SI =
5.80. Table 2 is the summary of the arylamides’ activities in inhibiting MMP9, MMP9 being
expressed by 4T1 cells, 4T1 cells, and the normal Vero cells. The trend seemed similar
to the assay using either FBA or GZA, that compared to the positive control, the tested
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compounds were still less potent. However, we still hope that in further study, the 9 could
be less toxic than doxorubicin.
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Molecular dynamics study
MD simulation was performed to investigate further the interaction of the active inhibitors,
7
and 9, on the MMP9 binding site. It is noted that 7 and 9 are very similar, only differed
by the two methyl groups on the pyrimidine ring. The RMSD profile of these two
compounds during the simulation showed that 7 is more stable than 9 in the binding site
of PEX9 (Fig. 4).
The simulation result suggested that the high affinity of 7 and 9 in the PEX9 is due to the
conserved H-bond with ARG106 (Fig. 5). Nevertheless, the binding mode between 7 and
9 (Fig. 5a and 5b, respectively) with the ARG106 is different. It is shown that the
sulfonamide group of 9 is more oriented towards ARG106 as compared to that of 7. This
observation is supported by the time-dependent distance between ARG106 and H-bond
acceptor of the ligand (Fig. 5d), indicating that the H-bond between 7 and ARG106 was
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only conserved until 60 ns of simulation. Whereas, that of 9 was stable until the end of
the simulation.
1
1
1
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9
0
The least active compound 4 is also simulated for comparison purposes. It is shown that
the binding mode of 4 is quite different from the two most actives. The bromo group of 4,
unlike the other two, is oriented outside the binding site (Fig. 5c). In this study, the addition
of EWG is expected to improve the inhibition activity by participating in the interaction with
PEX9. Therefore, the orientation of the bromo group outside the binding site of PEX9
could explain the least activity of 4 compared to the other compounds.
The MM/PBSA binding energy of 7 (-8.03 kcal/mol) is lower than 9 (-6.41 kcal/mol),
suggesting that 7 has a stronger binding with PEX9 than 9 (Table 1). This result agrees
with the higher stability of 7 in the enzyme’s binding site, as depicted by the RMSD values
over 100 ns (Fig. 4). Moreover, the least activity of 4 is implied by the highest binding
energy (-3.34 kcal/mol) as compared to 7 and 9.
Table 3 shows the binding energy decomposition of compound 7, 9, and 4 in the binding
site of PEX9. The electrostatic term represents the H-bond occupancy between ligands
and receptor. In agreement with the binding mode and H-bond analysis, 7 has stronger
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7
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9
1
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electrostatic energy than 9. Interestingly, the electrostatic energy of 9 is higher than the
least active compound 4. However, hydrophobic interaction formed by 7 and 9, which
represented by van der Waals and surface area terms (non-polar solvation + dispersion),
was stronger than that of 4. Therefore, it can be suggested that hydrophobic interaction
plays an important role in stabilizing the ligand at the PEX9 binding site, besides the
electrostatic interaction. Nevertheless, it can be suggested that the stabilization of ligand
inside the binding site of PEX9 is also contributed by the H-bond formation with ARG106.
Table 3 also shows that the occupancy of H-bond of 7 and 9 with ARG106 is higher than
0
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4.
Discussion
In this study, we have shown that the arylamide scaffold is the backbone that binds to the
cavity of PEX9 while interacting with surrounded important amino acids. Without any
substitution at para position, 1 could inhibit MMP9, albeit with a low % inhibition. In
contrast, locating EWG, such as ethylaceto (5) and sulfonamide (6), increases back the
activities. The characters of EWG in 7, 8 and 9 are observed by adjoining pyrimidine ring
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to NH-SO . In addition, the steric character is increased by the addition of the methyl (8)
2
and dimethyl group (9) to the pyrimidine ring, thus contributing to more pronounced
inhibition toward MMP9 activity. The attachment of halogen EWG such as chloro (3) and
fluoro (4) did not improve the binding with PEX9 as shown in docking study, where only
one hydrogen bond interaction with GLN154 is stabilizing the interaction between the
ligands and PEX9, thus contributing to the higher free energy of binding. This result is
supported by the in vitro studies where the two halogenated arylamide are found inactive.
Thus we suggested that there is the need for the addition of a steric group at this position.
1
1
1
1
1
1
1
1
1
1
2
2
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0
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0
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Although halogen is EWG, however, the EWG strength is considered moderate . Thus,
it is insufficient to possess the inhibition of the enzyme. This result strengthens the
hypothesis that a strong EWG with steric characters would increase the activity of
arylamide in inhibiting MMP9. Attaching EDG together with a steric character such as
OCH at 4, 5 and 6 positions (10) decreases the activity.
3
Compound 2 was designed with a relatively low free energy of binding (-6.95 kcal/ mol),
implying its potential to interact with PEX9 strongly. In fact, these compounds interact with
ARG106 via H-bond interactions (Fig. 6), which was also seen with the sulfate ion in the
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6
control docking. The in vitro assay using the fluorescence method agrees with the
prediction using in silico method, that compound 2 inhibits the proteolytic activity of MMP9
towards peptide substrate. This result is further supported by the results of gelatin
zymographic assay (GZA), where 2 also inhibits the gelatinolytic activity of MMP9
expressed in the 4T1 cells by as much as 36.2%. Interestingly, 2 is also less cytotoxic to
the Vero cell lines as compared to 7.
0
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0
Because the activity of 2 is lower than 7 and 9 in GZA, we did not study the dynamic
behavior of 2 via MD simulation. However, docking results predicted that 2 interacts with
PEX9 via H-bond interactions with ARG106 and GLU14 by O-nitro and O-carbonyl,
respectively. H-bond between O-nitro with ARG106 showed strong EWG character at the
para position of arylamide as indicated by the distance of H-bond (1.9Å).
Compounds 7 and 9 are expected to bind more selectively in the hemopexin-like domain
(PEX9) than in the catalytic domain. The expected molecular mechanism is by
interrupting the homodimerization of two PEX9 monomers at blade 4 via hydrophobic
interactions. When the homodimerization of PEX9 is interrupted, its binding to lipoprotein-
related protein-1 (LRP-1) receptor is also disrupted, therefore, there is no signal
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transduction to an extracellular-signal-regulated kinase (ERK1/2) and Akt, to lead the cell
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migration . On the other hand, the binding of the inhibitor to the PEX9 will act as an
1
1
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1
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0
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0
allosteric inhibitor to interrupt the proteolysis of ECM which holds the Vascular Endothelial
Growth Factor (VEGF). Hence, there is no VEGF binding into its receptor at the
membrane to proceed the angiogenesis.
MD simulation showed that the ligand-binding with important residues at the active site
plays an important role in stabilizing the interaction. MD simulation has been used for
47
evaluating the stability of ligand binding predicted by docking . It is shown that low
binding energy predicted in docking is not always indicating stable interaction. Thus it
must be further studied by dynamic simulation. Sulfone compounds have been reported
having MMP9 inhibition activity at a nanomolar concentration, which includes bis-
arylsulfonamide hydroxamate , arylsulfone , biaryl ether sulfonamide , and 11C-
4
8
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50
51
radiolabeled sulfonamide . However, there is no further study on the binding selectivity
of these compounds toward MMP9. To the best of our knowledge, the study of arylamide
incorporated with sulfonamide as MMP9 inhibitor and its dynamic behavior toward PEX9
binding site has not been reported elsewhere. However, there is still a need to confirm
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