Synthetic Guaiacol Derivatives as Promising Myeloperoxidase Inhibitors Targeting Atherosclerotic Cardiovascular Disease

Article abstract of DOI:10.1002/cmdc.202000084

Myeloperoxidase (MPO) is known to cause oxidative stress and inflammation leading to cardiovascular disease (CVD) complications. MPO-mediated oxidation of lipoproteins leads to dysfunctional entities altering the landscape of lipoprotein functionality. The specificity of guaiacol derivatives toward preventing MPO-mediated oxidation to limit MPO's harmful effects is unknown. Diligent in silico studies were accomplished for a portfolio of compounds with guaiacol as a building block. The compounds’ activity toward MPO inhibition was also validated. The role of these chemical entities in controlling MPO-mediated oxidation of lipoproteins (LDL and HDL) was shown to agree with our approach of developing powerful MPO inhibitors. The mechanism of MPO inhibition was demonstrated to be reversible in nature. This study reveals that there is great potential for guaiacol derivatives as therapeutics for CVD by modulating lipid profiles, reducing atherosclerotic plaque burden, and subsequently optimizing cardiovascular functions.

Full text of DOI:10.1002/cmdc.202000084

Accepted Article
Title: Discovery of novel synthetic hydroxyanisole derivatives as
promising myeloperoxidase inhibitors (MPOIs) targeting
atherosclerotic CVD
Authors: Premkumar Jayaraj, Sampath Parthasarathy, Sanjay
Rajagopalan, Chandrakala Aluganti, and Desikan Rajagopal
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000084
Link to VoR: https://doi.org/10.1002/cmdc.202000084
01/2020

10.1002/cmdc.202000084
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Discovery of novel synthetic guaiacol derivatives as promising
myeloperoxidase inhibitors (MPOIs) targeting atherosclerotic
CVD
Premkumar Jayaraj [a], Sampath Parthasarathy [b], Sanjay Rajagopalan [c][d], Chandrakala Aluganti *[b],
Rajagopal Desikan *[a, b]
[a]
J. Premkumar, and Dr. D. Rajagopal *,
Department of Chemistry, School of Advanced Science, Vellore Institute of Technology-Vellore, Tamilnadu-632014. India.
E-mail: desikanrajagopal@gmail.com
[b]
Dr. P. Sampath, Dr. D. Rajagopal *, and Dr. A. Chandrakala *,
Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL 32832, USA.
E-mail: chandrakala.alugantinarasimhulu@ucf.edu
[c]
[d]
Dr. R. Sanjay,
Division of Cardiovascular Medicine, Harrington Heart and Vascular Institute, Cleveland, Ohio, USA.
Dr. R. Sanjay,
Cardiovascular Research Institute, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Abstract: Myeloperoxidase (MPO) is known to cause oxidative stress
and inflammation leading to CVD complications. MPO mediated
oxidation of lipoproteins leads to dysfunctional entities altering the
landscape of lipoproteins functionality. The specificity of guaiacol
derivatives toward preventing MPO mediated oxidation to limit MPO’s
harmful effects is unknown. Diligent in silico studies were
accomplished for a portfolio of compounds keeping guaiacol as a
building block. Validation of compounds activity toward MPO inhibition
is also established. The role of these chemical entities on controlling
MPO-mediated oxidation of lipoproteins (LDL and HDL) is shown to
agree with our approach of developing powerful MPO inhibitors. The
mechanism of MPO inhibition is demonstrated to be reversible in
nature. The outcome of this study reveals that there is a great
opportunity available for guaiacol derivatives as potential therapeutics
for CVD by modulating lipid profiles, reducing atherosclerotic plaque
burden and subsequently optimizing cardiovascular functions.
performs a crucial role in the inflammatory network. In the case of
inflammation, ROS also participates in the induction of cell
multiplication, migration of white blood cells, and increases the
extracellular matrix production, oxidation of lipids, tissue damages,
[6][7][8]
oxidative stress markers, and endothelial dysfunctions
.
Other roles of ROS including growth and differentiation, apoptotic
signaling pathway, gene expression, and progression of diabetes
followed atherosclerosis [9][10]. Oxidative stress is the main reason
for organ dysfunction in human subjects and it may lead to
complications such as atherosclerosis, Parkinson’s, heart failure
and stroke.
ROS also acts as a mediator of lipid accumulation and can react
with unsaturated fatty acids (UFA) of lipid membranes and induce
lipid peroxidation. Lipoprotein, LDL (Low-density lipoprotein) and
HDL (High-density lipoprotein), a combination of lipids and
[11][12][13]
proteins, moves through the blood vessel
. Non-
atherogenic LDL can deposit at artery wall until it stays in native
form. Oxidized-LDL (Ox-LDL), also called atherogenic lipid, is
shown to have prompted many CVD events. MPO in presence of
H2O2 can promote unfavorable reactions within the premises of
LDL leading to the formation of Ox-LDL [14].Consuming excessive
food, smoking, poorly controlled diabetes, and stress are some of
the risk factors to increase the level of Ox-LDL [15][14]. This will lead
to excessive deposition of Ox-LDL within the artery wall. This
Introduction
Myeloperoxidase (MPO) is a peroxidase heme protein which is
present in human leukocytes [1]. This hemo protein can oxidize
biomolecules by way of generating hypochlorous acid (HOCl) and
sometimes a minor amount of hypobromous (HOBr) [2]. A specific
chemical reaction between hydrogen peroxide (H2O2) with
Thiocyanate-SCN (Pseudo-halide) and halides (Bromide-Br,
Chloride-Cl,) to generate corresponding hypohalous acid. These
are extremely powerful oxidants which oxidize biomolecules
whatever it encounters. For example, HOCl is a chemically stable
intermediate for a short duration of time. However, due to its
chemically labile nature, HOCl is shown to react with tyrosine
triggers biochemical reactions that will have a cascading effect in
[16]
the formation of atherosclerotic plaque
and subsequently
initiate several cardiovascular complications. High-density
lipoprotein (HDL), another member of the lipid family, has a
contrasting behavior compare to LDL. The main structural
component of HDL is apolipoprotein A-1 (apoA-1) which clears
cholesterols from macrophages foam cell via the kidney. Mainly,
HDL plays a significant part in RTC (Reverse cholesterol
transport) mechanism and modulation of inflammation thereby
preventing atherosclerosis progression. In recent literature
reports suggest that HDL loses its cardioprotective properties
when it is oxidatively damaged (modified) [17]. Literature reports
also suggest the effect of MPO-mediated oxidation of HDL to
present in biomolecules to form 3-chlorotyrosine (3-ClTyr) and
sometimes forms dityrosine (Di-Tyr)
[3][4][5]
via radical C-C bond
formation. These modified amino acid residues are considered as
specific markers for MPO catalyzed chemical transformation that
are taking place within the bio-environment. Due to chemical
modifications within the lipids and protein structures, it causes
extreme damage to host tissues. Apart from MPO-mediated HOCl,
other reactive oxygen species (ROS) react aggressively with
biomolecules and structurally modify them altogether. ROS
transform it as Ox-HDL in the human artery wall. The existence of
[18]
3-ClTyr and 3-NO2Tyr (3-nitro tyrosine)
within the
atherosclerotic plaque, characteristic biomarkers of MPO-
1
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10.1002/cmdc.202000084
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mediated products, was established by high-resolution mass
spectrometry. Modified HDL may involve in the occurrence of pro-
inflammatory processes causing extensive damage to biological
The binding modes of the active site of MPO complex structures
were illustrated in Figure S1 (Supplementary data). These
illustrations demonstrated that the docked poses get docking
scores (Binding energy) of protein-ligand complexes which are
depicted in Table 2. Based on the binding energy of docked
molecules in the MPO active site pocket, we concluded that all
the designed molecules have been shown to possess strong
potential binding capacity within the active site of a protein.
Structure Activity Relationship (SAR) study has provided a
platform for optimized lead structures. These active molecules
were successfully synthesized and validated for protein inhibition
assay.
systems [19][20]
.
MPO is known to participate in the atherosclerotic event.
Atherosclerosis causes plaque formation in a large or medium
artery blood vessel causing vasoconstriction followed by
restricted or decreased blood flow. The constituents of
atherosclerotic plaque are ox-lipids, cell remnants, SMC (smooth
muscle cells), endogenous collagen and deposition of calcium on
the surface of blood vessels [21]. These depositions may lead to
the formation of atherosclerotic lesion.
Last few decades, health care industries have watched
Table 1. Lipinski rules for the proposed derivatives.
tremendous growth in the development of antioxidant therapy.
[22]
Oxidative stress modulators, vitamins (Vitamin-C and E)
,
Compound
Mw
HBD
<5
HBA
<10
Clog
P<5
Log P
carotenes (α-Carotene and β-Carotene) [23][24], and phytonutrients
(cinnamyl, curcumin) are reported in the literature [25][26]. Many
pharmaceutical industries devote their drug discovery effort
toward developing chemical agents utilizing nature-based
supplements without compromising the pharmacological effect of
the parental structure aiming at specific disease targets. Guaiacol
as an important class of organic compounds holding excellent
bioactive properties including modulation of oxidative stress,
which is abundantly available in “Corn” (Zea mays), and “Bamboo
shoot” (Phyllostachys edulis), “Cinnamon spice” (Lauraceae), and
“Turmeric” (Zingiberaceae). These phytonutrients are shown to
have the highest free radical scavenging capacity.
<500
#
##
###
0
1
2
3
4
5
6
7
8
9
435.21
435.21
444.12
443.13
403.17
210.10
209.06
239.07
209.06
3
3
3
2
2
3
3
3
3
5
5
8
7
4
4
4
5
4
+ 4.24
+ 4.24
+ 6.15
+ 7.11
+ 6.99
- 0. 33
- 0. 07
- 0. 29
- 0. 07
3.12
3.12
5.35
5.97
6.03
0.54
0.93
0.81
0.93
3.19
3.19
5.07
5.68
5.73
0.66
1.24
0.98
1.24
0
4.91
5.79
5.63
0.81
0.47
0.60
0.47
Mw - Molecular weight; HBD - Hydrogen Bond Donor; HBA - Hydrogen Bond
Acceptor; Clog P – Calculated log P; LogP - Partition Coefficient for n-
octanol/water; (#) by Crippen's fragmentation; (##) by Viswanathan's
fragmentation; (###) by Broto's method.
A recent report from our collaborative lab has demonstrated the
usefulness of supplements for atherosclerosis treatment, specific
to MPO inhibition [27][28]. Although several scientific reports clearly
indicted MPO as one of the main reasons for the manifestation of
cardiovascular disease (CVD), yet no single drug is available in
the market as an MPO inhibitor (MPOI), though AstraZeneca’s
product, AZD4831, is under clinical trial for heart failure with
preserved ejection fractions [29]. Considering the importance of
developing novel chemical entities as MPOI for CVD treatment,
we have used guaiacol as a basic building block to create novel
scaffolds for MPOI [30]. The advantages of this moiety are
functionalization with other existing anti-inflammatory compounds,
efficacy improvement and safety [31][32]. The outcome of this
approach revealed promising results which firms up developing
active agents as some anti-atherosclerotic compounds by way of
MPOI.
Table 2. In silico binding energy and in vitro IC50 value of compounds 1 to 9 with
MPO protein.
Compound
ΔG Score (Kcal /mol)
IC50 (µM)
11.00
16.00
05.06
09.04
14.00
03.03
00.82
01.04
02.08
1
2
3
4
5
6
7
8
9
-7.52
-8.00
-7.26
-6.80
-6.29
-6.18
-5.90
-6.50
-5.71
ADMET predicted profiles & regressions
ADMET properties were evaluated on the parameters of
absorbance of the drug followed by drug distribution, metabolism
and excretion. All the designed molecules have no violations in
ADMET parameters and data are given in Table S2 (Provided in
supplementary data).
Results
In silico validations
The prediction of Lipinski rules for the proposed derivatives are
given in Table 1. Out of nine compounds, three compounds 3, 4
and 5 violated Clog-p rule, however all other parameters are well
within the acceptable level. It is known in the literature that the
Lipinski rule violated chemical entities have been developed as
drug for various diseases [33][34]. Lipinski prediction was computed
using Chemdraw Professional-15 software.
Chemical synthesis
The designed guaiacol derivatives 1 to 9 contain various chemical
bonding such as amide, ester, hydrazide and hydroxamic acid
functional groups. It is well documented that amide and ester
bonds can exert pertinent biological response and it also offers
[35][36]
avenues for new chemical development
.
Several
commercial drugs [37] hold these two types of chemical functional
groups. Also, hydrazide and hydroxamic acid moieties are shown
Molecular docking studies
The molecular docking studies were carried out with designed
MPOIs. These designed molecules were docked at the possible
active site of MPO protein (5FIW) and the binding energy of the
MPO complexes was found to be between -5.71 to -8.00 kcal/mol.
to possess a crucial role in MPO inhibition [38][39][40]
.
2
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Scheme 2. Synthesis of compound 1 to 9 by method-2.
Scheme 1. Synthesis of compound 1 to 9 by method-1.
To achieve targeted guaiacol derivatives with good yield, high
purity, and scalable methodology, two methods are adapted, In
the Scheme 1, acid is converted into acid chloride followed by
treating it with various amines and alcohols to obtain respective
products. However, the synthesis of acid chloride from acid has a
limitation in terms of product yield and lack of chemical stability at
RT. To overcome this difficulty, another method was adapted. As
depicted in Scheme 2, acid is directly treated with amine /
alcohols via the Steglich condensation process to yield amide /
ester functionalized compounds (compound 1 to 5). Compounds
6 to 9, hydroxamic and hydrazide derivatives were synthesized as
reported in the literature [41]. On comparison, we achieved 80%
yield with a high level of purity and stability of the compounds
using scheme 2 compare to the scheme 1. It is noteworthy that
the scheme 2 can be used for bulk synthesis of proposed
compounds for advanced clinical development of the compounds
as CVD therapeutics. The purity of these compounds stands
above 96% and it was determined by the UPLC method. These
compounds were fully characterized by 1H and 13C NMR
techniques (Bruker Instrument 400 MHz); NMR data were
provided in supplementary data as Figure S4. Molecular mass
was determined by LC-ESI Mass spectrometer; spectral data
were given in supplementary data Figure S5. (The complete
synthetic procedures and spectral data are provided in the
supplementary data).
The IC50 values of all the synthesized compounds are presented
in Table 2. Two lead compounds, 7 (IC50= 0.82 µM) and 8 (IC50=
1.4 µM) showed maximum inhibition with minimal concentration
of inhibitors. Similarly, compounds 6 and 9 have inhibited MPO
under comparable concentration. We used salicylhydroxamic acid
as a positive control.
Antioxidant assay
Antioxidant activity is assessed by observing the decrease in the
DPPH radical absorbance at 517 nm. Results were reported as
percentage inhibition of free radical generated during the course
of reaction in presence of MPOIs. Similarly, ABTS antioxidant
assay was performed without using any substrate. ABTS assay
with an absorption maximum of 734 nm has excellent water
solubility and chemical stability. Figure 1 provides the details of
DPPH and ABTS radicals inhibition percentage by compounds 1
to 9 (50µM). Our in vitro antioxidant study with MPOIs along with
ascorbic acid (50µM) as positive control showed there is a
correlation in antioxidant activity in comparison with a known
antioxidant. Out of nine compounds, compounds 6 to 9 seem to
show moderate radical scavenging reactivity. On the other hand,
compounds 7 and 8 showed maximum inhibition of radical.
LDL oxidation in presence of MPO
In vitro MPO Inhibition assay
LDL was subjected to oxidation in the presence of 0.2 U MPO with
The biological activity of guaiacol derivatives was assessed by the
inhibition of MPO activity in a cell-free system using commercially
available MPO enzyme and 3,3,5,5-tetramethylbenzidine (TMB)
as a substrate [42][43]. The IC50 value of inhibitors was determined
by plotting percentage inhibition by measuring the absorbance at
650 nm against inhibitor concentrations.
and without compounds. The presence of conjugated dienes has
[44]
been examined by monitoring the UV absorption at 234 nm
.
As represented in Figure 2A, MPO produced Ox-LDL attained
maximum increment in absorption at about 3.5 h. In the presence
of compounds, there was a rise in lag time delineating that the
compounds could extend the time of oxidation rate.
Leukomethylene blue (LMB) and thiobarbituric acid reactive
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substance (TBARS) assay values also corroborated the results
Figure 2. Compound 1 to 9 inhibit the oxidation of Lipoproteins
with the oxidation peak as shown in Figure 2B & 2C.
Figure 1. % of DPPH and ABTS Inhibition by Compound 1 to 9 (50 µM).
Reduction of peroxidase by MPOIs
Peroxides offer a major function in inducing inflammation and
further progress of the illness. As shown in Figure 3, except
compounds 1, 4 and 5, all the compounds notably decrease the
peroxide quantity of FFAOOH (free fatty acid peroxides) when
incubated together. Similarly, all the other compounds were able
to reduce H2O2 as shown in Figure 4, which is a substrate for
MPO reactions.
Effect of MPOIs on LPS-mediated pro-inflammatory gene
expression in RAW macrophages
LPS actively influenced the mRNA levels of pro-inflammatory
genes in RAW macrophages. However, as seen in Figure 5 & 6,
in the presence of different MPOIs, the pro-inflammatory genes
tumor necrotic factor-α (TNF-alpha) and interleukin-1β (IL-1β)
were reduced differently in a dose-dependent manner. Only
MPOIs did not cause any inflammatory gene expression.
*Plasma from consented subjects is used to isolate LDL for MPO oxidation.
100μg LDL was treated with compounds (1 to 9) (25μg) and then oxidation was
carried out with 0.2U MPO in 1ml PBS and OD was measured at 234 nm. As
shown in Figure 2A all compound delayed the initiation of LDL oxidation. After
oxidation, the samples were used to determine peroxide content and
thiobarbituric acid substances using LMB (Figure 2B) and TBARS (Figure 2C)
assays. *P < .05.
MTT cell viability assay
MTT assay (cell viability) protocol using RAW macrophage cells
[45]
(RAW 264.7 from the ATCC)
method was adopted. The
outcome of this study indicated that conjugates 7 and 8 are highly
safe for human consumption and non-toxic even at higher
concentrations. Table 3 shows the % of cell viability at various
concentrations of lead compounds.
Purity of the lead MPOIs
For the purpose of biological study, we have used compounds
with more than 96% purity. Out of nine synthesized compounds,
we have selected compounds 7 and 8 based on the docking score
and screening of inhibition assay. Based on the UPLC-DAD study,
purity of the compound 7 and 8 was shown to be 97.37% and
96.18% respectively. Figure S7 (Supplementary Information)
represents the chromatogram of UPLC-DAD purity graph.
Table 3. The cell viability result for RAW Macrophages cell line by different
concentration of lead compound.
Compound
Cell viability %
Concentration (µM)
Compound-7
Compound-8
Control
10
100
94.3
92.0
88.4
86.8
82.2
100
90.8
87.3
85.4
82.9
79.9
20
Mechanism of non-suicidal MPO Inhibition
i) UV-Visible spectroscopic method:
MPO acts on a variety of substrates and oxidizes them, it is
theoretically possible that our inhibitors were acting as substrates
and competitively masking the formation of product(s) from the
30
40
50
4
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Figure 3. Guaiacol derivative in decomposition of HPODE:
*200 nmoles/mL of HPODE (FFAOOH) was carefully incubated with various concentrations of compound 1 to 9 (0-25 µg) for one hour at 37 °C. Lipid peroxide
formed in the reaction mixture was evaluated by LMB assay. All the compounds (1 to 9) revealed remarkable reduction in the peroxide content of FFAOOH in a
concentration dependent manner. *P < 0.05; **P<0.01,
added substrate. However, we concluded that this chemistry was
unlikely as the substrate was added several folds in excess. The
spectroscopic method of evaluating the reaction mixture failed to
reveal any additional products in the presence of MPO. There was
no colored product even when the reaction mixture contained only
the inhibitors and not the TMB substrates. As can be seen from
Figure 7A & 7B, there is no indication of chemical transformation
that could have occurred when our lead inhibitors can covalently
link at the MPO active site since UV-Visible spectrum of lead
inhibitors with and without MPO showed same absorption bands.
compounds 7 and 8 inhibit MPO irreversibly (suicide inhibition),
the nature of resultant product will be chemically modified. The
modified structure can be easily observed in TLC when compare
to parent compound. A similar pattern of reactivity was observed
when MPO is reacted with lead inhibitors 7 and 8 in presence of
protein substrate, tetramethylbenzidine, as given in Figure 7C.
Overall, the presence of hydroxamic acid and hydrazides
functional groups within the basic building blocks has significantly
increased the MPO inhibition potentials and peroxide
decomposition assays. Additionally, LDL and gene expression
studies also correlated the above results. With these observations
in hand, two lead molecules 7 and 8 are considered as
prospective chemical agents for the treatment of MPO-mediated
CVD.
ii) Thin layer chromatography method:
The Formation of any product from inhibitor due to oxidation of
MPO will be reflected on TLC by characteristic new spots when it
is eluted with specific solvent systems (eluant). Based on the TLC
experiment, we observed that the lead inhibitors 7 and 8, when
they were reacted with MPO protein in a suitable buffer, did not
produce any characteristic oxidative products. As can be seen
from TLC in Figure 7C, spots corresponding to before and after
MPO treatment appear at the same Rf-value indicating there was
no specific product formed between inhibitor and MPO delineating
the reversible nature of the inhibitor. Assuming the lead
Structure-activity relationship
Critical in vitro assays have been used to identify two lead
compounds, 7 and 8 wherein functional group modifications are
invoked to improve the efficacy of the final structures. Chemical
linkage of anti-inflammatory drugs with guaiacol moiety yielded
compounds (1 to 5) which have moderately reduced MPO
inhibition concentration up to 7-fold compared to parent structures.
5
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Figure 4. Guaiacol derivative in decomposition of H2O2:
*200 nmoles/ml of H2O2 was incubated with variable concentrations of compound 1 to 9 (0-25 µg) for 1hr at 37°C. Peroxide decomposition was measured by
LMB assay. All the compounds (1 to 9) showed significant reduction in the peroxide content of H2O2 in a concentration dependent manner. *P < 0.05;
**P<0.01.
Conversion of acid functionality of guaiacol derivatives to ester
groups (6a to 9a) has slightly decreased the IC50 value from ~85
µM to ~55 µM. Further, modification of ester functionality (6a - 9a)
into hydroxamic acid and hydrazide functional group, there was a
dramatic decrease in the inhibition concentrations. There was a
30-fold decrease in IC50 value for hydroxamic acid and 22-fold
decrease in IC50 value for hydrazide functionality compare with 6a
- 9a. The substituted guaiacol derivatives (1 to 5) need ~4-fold
excess of a compound concentration (~7 µM) to inhibit MPO
compare to hydroxamic acid substituted guaiacol derivatives (6 to
9) which require ~1.5 µM concentration. With regard to the
substitution of anti-inflammatory agents (1 to 5) in the LDL
oxidation reaction, there was a 10-fold decrease in the inhibition
concentration when hydroxamic acid substituents are used. For
HPODE (13-Hydroperoxylinoleic acid) and H2O2 decomposition
assay, we observed that there was 90% higher efficacy in
peroxide degradation for compounds 6 to 9, when compared to
compounds 1 to 5. This observation is also applicable to multiple
concentrations ranging from 1µg to 25µg. As a representative
sample, Figure 8 provides comparative inhibition (IC50) value of
basic building block and its modified structures. Overall, the
presence of hydroxamic acid and hydrazides functional groups
within the basic building blocks have significantly increased the
MPO inhibition potentials and peroxide decomposition assays.
Additionally, LDL and gene expression studies also corroborated
the above results. With these observations in hand, two lead
molecules 7 and 8 are considered as prospective chemical agents
for the treatment of MPO-mediated CVD.
Discussion
Based on the literature reports, there is a strong correlation exists
between phenolic compounds and MPO inhibition [28]. The
molecule, such as Primaquine (Anti-malarial drug) [46], Niflumic
acid (anti-inflammatory agent) [47], Flufenamic acid (Antipyretic
[48]
and anti-inflammatory agent)
,
Mefenamic acid (anti-
inflammatory agent) [49] have the potential chemical functionalities
with specific medicinal properties to improve the efficacy [50]
The concept of drug repurposing is getting prominence and this
investigation is focused on the chemical combination of the
existing drug with guaiacol based phytonutrients to realize
synergism in bioactivity. A well-known example is curcumin
wherein presence of guaiacol type structures offers special
properties to the parent molecules.
6
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Figure 5. LPS mediated pro-inflammatory gene expression in- RAW 264.7 cells in presence of guaiacol derivatives (Compound 1 to 9). RAW 264.7 cells have been
incubated with LPS and compounds in serum free condition for 24 h. RNA was separated and qPCR analysis was executed for TNF-α gene in RAW cells treated
with LPS in presence and absence of guaiacol derivatives of various concentrations using appropriate primers. Results are given as mean ± SD and significance
considered as *P < 0.05.
and ABTS antioxidant assays were measured with all the
Synthesis of these compounds was designed in such a way that
methods can be adopted to prepare compounds for large scale
production. The docking results, as well as biochemical assays
with our inhibitors, revealed that MPO inactivation could occur
either via active site blockade or rendering MPO inactive by
compound II. This hypothesis is supported by literature reports
that have shown phenols and anilines prevent MPO cascading
reactions from producing hypochlorous acid by scavenging the
enzyme in its dormant compound II form [51]. This type of inhibition
is reversible because superoxide reduces compound II back to its
native form. Therefore, at this point of time, we reasonably believe
that compounds 1-9 may act as reducing substrate and may
promote compound II formation and it could act as a substitute for
hydrogen peroxide in compound I formation. It is also received
our focus that the large binding cavity of MPO can be utilized to
compounds and outcome of this data indicated that all nine
compounds are average to good antioxidants. The degree of
antioxidant potentials slightly varies for compounds 6 to 9, when
compared to other structures. However, difference in antioxidant
activity of 7 and 8 with other compounds seems to be comparable
with positive control.
In view of the implication of MPO in CVD, it is important to
investigate on the effect of MPOI toward lipids oxidation and its
prevention. Involvement of Ox-LDL in atherosclerotic plaque
development and characterization of MPO-mediated biomarker
products within the lesion demonstrate that importance of lipids in
CVD. LDL and HDL on oxidation can generate a characteristic
diene which absorbs at 234 nm. Increase in the absorption at 234
nm indicates the formation of elevated level of diene due to
oxidation of lipids. In vitro oxidation of isolated LDL with and
further optimize the guaiacol derivatives that we have synthesized. without MPOIs has shown that there was an increase in lag time
Additional docking results of MPO with other specific guaiacol
derivatives (data not shown) have provided very weak lead
when oxidation was done in presence of MPOI suggesting that
the proposed compounds can delay the oxidation of LDL thereby
reduce the Ox-LDL level as represented in Figure 2A. The
oxidized samples are further studied by treating them with LMB
and TBARS assays by measuring its characteristic absorption at
660 nm and 532 nm respectively. Once again, based on the data
provided in the Figure 2B and 2C, it is reasonably assumed that
compound 7 and 8 can control the oxidation of LDL.
structures [52]
.
Next, the results of specific MPO inhibition assay namely, TMB
assay revealed that the proposed structures are moderate to high
active inhibitors. Within the nine compounds, compounds 7 and 8
showed inhibition in sub-micromolar to high nanomolar
concentration. The inhibition values of these two compounds
correlate well with binding energy of these molecules with MPO
in docking score. To further substantiate these findings, DPPH
7
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Figure 6. LPS mediated pro-inflammatory gene expression in- RAW 264.7 cells in presence of MPOIs. RAW 264.7 cells have been incubated with LPS and MPOIs
in serum free condition for 24 h. RNA was separated and qPCR analysis was executed for IL-1β gene in RAW cells treated with LPS in presence and absence of
guaiacol derivatives of various concentrations using appropriate primers. Results are given as mean ± SD and significance considered as *P < 0.05.
Figure 7A & 7B. The UV pattern of MPO reaction of lead inhibitors in presence and absence of MPO with compound 7 and 8. Figure 7c. The TLC pattern of MPO
reaction of lead inhibitors in presence and absence of MPO with compound 7 and 8.
This can offer an explanation that these compounds can be
considered as powerful MPOI and efficient modulators of lipid
oxidation thereby it can offer cardio protection. Next, the effect of
MPOI on peroxide decomposition was investigated. Peroxides
can cause damage by inducing inflammation and further
progression of the disease. Hence, the decomposition of H2O2
and HPODE was studied in presence of MPOI and it was
estimated by LMB assay. In line with activity prediction,
compound 7 and 8 seem to decompose peroxides more efficiently
compare to other compounds.
By establishing good activity response under in vitro condition, we
turned our attention to cell line study to mimic physiological
condition. If there is any correlation existing between in vitro and
cell line study, there is a greater chance of identifying lead
compounds within the proposed structures. Several diseases
including cardiovascular, diabetes, inflammatory bowel disease,
cancer, and different types of neurological disorders are
8
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10.1002/cmdc.202000084
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associated with oxidative stress and inflammation. The influence
of these two factors on the disease progression is not yet
completely known. In this methodology, we studied the effect of
MPOIs on LPS induced inflammation.
that most of the compounds used in this study were methoxy
phenols, which seem to have therapeutic potential. Further, it
might provide
a regulatory component in several anti-
inflammatory pathways and possess numerous beneficial effects.
Figure 8. Representative example for comparative inhibition (IC50) value of
Even though several pharmacological as well as nutritional
approaches exist, still the mortality rate is continued to rise due to
oxidative stress mediated CVD and inflammatory disease. This
emphasizes the vital requirement of new therapeutic agents to
address this malady. The current study evaluated the potential
therapeutic nature of several guaiacol derivatives as specific
MPOIs useful for CVD treatment. This will open up new avenues
for clinicians and pharmaceutical companies to explore further in
this line of investigation.
parent and its modified structure to reveal SAR study.
Conclusion
Revelation of chemically modified guaiacol derivatives as MPOIs
is demonstrated to operate exceedingly well based on the
outcome of in vitro and cell line studies. Blockade of MPO active
site is the basis for its inhibition which is substantiated by
computation docking study. Based on the literature reports that
have shown phenols and anilines inhibit MPO from generating
hypochlorous acid by deactivating the enzyme in its dormant form
which can be extended to proposed guaiacol derivatives also. The
reversible mode of inhibition is postulated since superoxide
reduces compound II back to its native form. Compounds 7 and 8
may act as reducing substrates and can support compound II
generation and it could behave as alternate for H2O2 in compound
I formation. Results based on in vitro and cell line-based assay
and effect of compounds 7 and 8 on LDL revealed that compound
7 and 8 are efficient MPO inhibitors and can provide promising
therapeutic properties against CVD.
Indeed, each compound showed different levels of attenuation to
LPS induced inflammation, however all the compounds were
significantly attenuated LPS mediated pro-inflammatory cytokines
TNF-α and IL-1β as shown in Figure 5 and 6. Also, enzymatic
and non-enzymatic LDL oxidation mechanisms are different and
various MPOIs can inhibit or delayed the enzymatic (MPO)
oxidation of LDL. Also, all anti-inflammatory molecules may not
be antioxidant molecules and vice versa; further some MPOIs are
needed in high doses for their beneficial action in oxidative
stress/inflammation pathway.
These compounds sometimes had divergent effects on multiple
measures such as conjugated dienes assay, methylene blue
assay. These effects are congruent with the effects on in vitro
assays suggesting that the effects on measurements of lipid
Future perspective
functionality
can
sometimes
diverge,
necessitating
The discernment of MPO-mediated pathways has improved
dramatically and it is therefore very essential to suitably earmark
interventions and resources, for the scope bringing out an inhibitor
with a minimum risk-benefits ratio. In order to develop MPO
inhibitors as crucial therapeutic agents, it is necessary to include
numerous multiple intricate parameters that are involved to
impact the positive outcome of the study. This is not only for CVD
but other illness of which MPO is implicated. The guaiacol
derivatives as MPOIs are expected to show their effect to a
multiple target, but at the same time offer minimum efficacy on
immune suppression addressing the involvement on the progress
of infections due to lack of MPO. This study undoubtedly would
pave the way for advance pre-clinical development in the area of
cardioprotection that are bio-compatible and non-toxic due to
natural origin of the basic building blocks.
comprehensive evaluation of a number of parameters. However,
the molecules are non-toxic even at high concentrations
demonstrated by viability assays. The purity of the synthesized
compound was determined by UPLC method and it was
established to be above 96%.
Further, the mechanism of inhibition of MPO is important in terms
of understanding the interaction between inhibitor and protein.
Essentially, the proposed inhibitors deactivate the MPO protein
through reversible pathway. The evidence for this reversible
mechanism (non-suicidal inhibition) is ascertained from UV-
Visible study and TLC study. Irreversible inhibitors undergo
chemical modification due to covalent linkage with protein which
can be inferred by observing change in absorption pattern in UV
studies. Similarly, formation of chemically modified product can
be observed in thin layer chromatographic studies. Lack of
characteristic change in the UV spectrum and TLC studies point
out to the fact that proposed lead structures inhibit MPO in a
reversible fashion.
Experimental section
Materials
Existing literature reports suggest that the guaiacol derivatives
are more potent anti-inflammatory agents and can efficiently
inhibit various inflammatory signaling pathways such as TNF
alpha, TLR4 and NFkB [53][54]. In addition, recent studies from our
collaborator laboratory have demonstrated the whole sesame oil
aqueous extract (SOAE: enriched with methoxy phenolic
compounds) efficiently attenuated the NFkB mediated
inflammatory mechanism [55]. All these results strengthen our data
Starting materials were purchased from Sigma Aldrich, Bangalore,
India. All reagents were purchased from SD Fine chemicals, India.
1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI) and
dimethyl aminopyridine (DMAP) were purchased from Alfa Aesar
chemicals (Germany). CDCl3 and DMSO-d6 were purchased from
Merck Pvt. Ltd, India. Proteins and other biological reagents were
purchased from Sigma Aldrich Pvt. Ltd. Solvents were purchased
from SD Fine chemicals India Ltd. Polymerase chain reaction
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10.1002/cmdc.202000084
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(PCR) primers and TrizolTM testing agent were purchased from
MPO co-substrate; H2O2, NaCl, with variable concentration of
guaiacol derivatives were used to prepare a dose response curve
Invitrogen,
Carlsbad,
CA.
Human
leukocyte
MPO
(myeloperoxidase), Lipopolysaccharide (LPS) (E. Coli) and
regular reagents and chemicals were purchased from Sigma, St.
Louis, MO, USA.
and finally determine the IC50 values [42]
.
In vitro antioxidant evaluation
Briefly, 100 µM DPPH solution in methanol was made and 1000
µL of this DPPH solution was mixed to a total volume of 3 mL of
the total reaction mixture containing MPOIs at different
concentration. The solution was shaken thoroughly and kept it
~27 ºC (RT) for 30 minutes. Absorbance was then determined at
517 nm. By spectroscopically monitoring the colour change will
provide a clue on antioxidant properties of the compound. A
similar study was performed using the ABTS method of
antioxidant study to scavenge 2,2’ azinobis (ethylbenzthiozoline
6-sulfonic acid) (ABTS+) radical cation to identify antioxidant
potentials of the compounds. Briefly, by reacting 7 mM ABTS in
water and 2.45 mM K2S2O8 (1:1) in the absence of light (dark) at
~27 ºC (RT) for a period of 16 h, ABTS·+ cation radical was
generated. Appropriate dilution of ABTS·+ species with methanol
is done and finally, absorbance at 734 nm was measured.
Combination of MPOIs at different concentration to a required
volume of diluted ABTS·+ solution, the absorbance was
determined at 30 min after the initial mixing. An applicable
background/solvent blank was determined in each measurement.
To compare efficiency, ascorbic acid was used as a positive
control. Measurement was also done in the absence of an
inhibitor.
Methods
In silico studies
Lipinski rule was invoked to predict the feasibility of the proposed
structures to ensure the success rate of the proposed compound
library is relatively high.
Molecular docking: An AutoDock-1.5.6 docking tool was used
for molecular modelling studies
[56]
using protein-heme complex
from the RCSB-PDB (Protein Data Bank - https://www.rcsb.org)
crystal structure entry by using PDB ID: 5FIW. The original X-ray
crystal structure resolution was 1.7 Å used. This protein was
employed as an input structure for protein preparation. Out of four
chains, A, B, C, and D; chain A and C were considered for docking
while the crystal symmetry-related B, D chains were removed.
Hydrogen atoms were added to the myeloperoxidase heme
followed by the addition of Gasteiger and Kollman charges. It may
be noted that the PDB entry did not contain any crystallographic
water molecules in the protein-heme complex. The ligands were
drawn using Chemdraw Professional-15.0 and the grid box was
prepared to cover active site pocket. Then the grid run was
performed followed by docking protocol was evaluated. After the
completion of docking procedure, critical analysis of various
parameters was performed for the protein-ligand complex. The
analysed complex was visualized using Discovery Studio
Visualizer-2019-R2 and the images were taken in 2D and 3D
format. None of the MPO structures in the PDB contained a
natural ligand and we have created lead entities based on the
guaiacol pharmacophore building block.
Isolation and oxidation of lipoproteins
Subsequent Institutional Review Panel approval, blood sample
was collected in a heparinized vial from healthy volunteers after
receiving their consent and stored on cold condition. Plasma was
separated by centrifuging the blood at 3000 rpm for 20 min. Low-
density lipoprotein was separated from ordinary blood plasma by
consecutive ultra-centrifugation using a Beckman TL-100 tabletop
ultracentrifuge (Beckman, Palo Alto, CA) [59]. The separated
lipoprotein was dialyzed against 0.3mM ethylenediamine
tetraaceticacid (EDTA) in 1x phosphate buffer saline (PBS) of pH
7.4 for 12 h then it was filtered and sterilized. The protein amount
was quantified using the Bio-Rad DC protein assay (Hercules,
CA). LDL sample was made to undergo oxidation rapidly /
immediately after dialysis. Oxidation of LDL (100μg/mL) was
performed with 0.2U MPO and 100μM hydrogen peroxidase
(H2O2) in 1000 µL of phosphate buffer saline at 37°C both in
presence and absence of 25μgm of the MPO inhibitors. The
formation of conjugated dienes has been observed at an optical
ADMET: admetSAR database is used to derive Absorption
Distribution Metabolism Excretion and Toxicity (ADMET) Profile
prediction and regression have been calculated for all designed
compounds [57]
.
Chemical synthesis of MPOI
The synthesis of proposed guaiacol derivatives was achieved by
adapting two different chemical method to synthesize proposed
compounds with high yield and purity. Accordingly, for synthetic
method-1 (Scheme 1), conversion of acid to acid chloride followed
by coupling reaction with substituted guaiacol based amine and
alcohol moieties has been implemented. In method 2 (Scheme 2),
we used substituted acid derivatives directly coupled with amine
and alcohol moieties by Steglich condensation processes
(compound 1 to 5). To synthesize compound 6a to 9a by acid to
ester followed by treatment with amine by condensation reaction
yielded compound 6 to 9. The Scheme-2 is a feasible approach
in terms of yield, cost-effectiveness, with easy process
methodology. The complete details of Scheme-1 and Scheme-2
are provided in the supplementary data file.
density of 234nm for
4
h
using Jenway DB-6500
spectrophotometer furnished with an 8-chamber cuvette changer.
[60]
Peroxide content was quantified using LMB
assays by the degree of LDL oxidation processes.
and TBARS
Preparation of HPODE and Incubation with MPOIs
13-Hydroperoxylinoleic acid, 13-HPODE of 200 nmol/mL was
[61]
prepared as already discussed
and was used to measure the
effect of MPOI on FFAOOH. HPODE was incubated with
increasing concentrations of compounds (0-25μg) for 1 h at 37ºC.
Lipid peroxide produced in this reaction method was tested by
leucomethylene blue (LMB) assay. Compounds also were
incubated with H2O2 to determine whether it has a similar effect
as seen with HPODE.
MPO inhibition assay
Tetramethylbenzidine (TMB) assay
MPO inhibition assay was performed based on the literature
report using TMB as an enzyme-substrate [58]. Salicylhydroxamic
acid, a known MPO inhibitor, was used as a positive control. Other
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10.1002/cmdc.202000084
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FULL PAPER
Cell culture
1 mL with 50 mM sodium acetate (CH3COONa) buffer at pH 5.6.
The reaction was induced by adding MPO. Wavelength scan
between 200 to 800 nm was used to detect any products
formation and it was reflected in the spectrum. Generated
products were identified by comparing it with a spectrum without
MPO. This experiment was carried out with two controls, one
without MPO enzyme and another with an MPO enzyme.
RAW 264.7 macrophages were purchased from American Type
Culture Collection (ATCC). Cells have been grown as a single
layer in dishes and flasks and maintained in DMEM medium
supplemented with 10% fetal bovine serum (Sigma, St Louis, MO,
USA), L-glutamine 2mM, and 1x antibiotic penicillin-streptomycin
solution. Cells have been maintained in a 5% carbon dioxide
(CO2) atmosphere at 37°C. For experimental studies, the cells
were incubated in serum-free conditions.
ii) By thin layer chromatography method
Initially, a suitable mobile phase (eluant) was identified to
determine the precise Rf value for each inhibitor on a pre-coated
UV active silica gel plate (EMD, TLC Silica gel 60 F254). MPO
mediated reaction was carried out by reaction mixture contains
100 mU human MPO (Sigma-Aldrich, St. Louis, MO), 2 µM H2O2,
8 µM of TMB, followed by the addition of lead molecules. The final
volume was made to 1 mL with 50 mM sodium acetate buffer at
pH 5.6. The reaction was induced by adding MPO. The organic
compound present in the mixture was extracted with a minimum
amount of organic solvent and concentrated followed by loaded
on a pre-coated TLC plate and eluted with a known mobile phase.
The appearance of any new products apart from the inhibitors was
determined either by exposing the plate to UV light or to iodine
vapour to get a bright spot on the TLC plate.
i) Incubation of RAW 264.7 cells with LPS and MPOIs
To detect the variation in gene expressions of TNF-α and IL-1β,
RAW 264.7 cells (2x105 cells/well) were pre-incubated in serum-
free DMEM for 3 h. Cells were incubated with 10ng/ml LPS either
in the presence and absence of 5 or 25μg of compounds for 24 h.
At the end of one-day incubation, cells have been collected in
TrizolTM for RNA separation (isolation) [62]
.
ii) cDNA Synthesis and RT-PCR reaction
Total RNA from cells was separated by using the TrizolTM testing
agent. 1μg of RNA was reverse transcribed into cDNA by using
SuperscriptTMIII First Strand Synthesis system (Invitrogen,
Carlsbad, CA). cDNA (50ng) compound used to execute
Quantitative RT-PCR (real-time polymerase chain reaction) by
CFX96 iCycler Multicolor RT-PCR Detective System (Bio-Rad,
Hercules, CA) with SYBR Green (Invitrogen Carlsbad, CA). PCR
was performed with IL-1β and TNF-α particular primers for a
mouse Table S6 (available in supplementary file), consequential
in 200-bp pieces. For the purpose of comparison in gene, we used
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers,
consequential in a 200-bp fragment. PCR was executed with an
initial method of denaturation at 50ºC for 2 minutes, 95ºC for 10
minutes followed by 40 cycles of 95ºC for 20 secs and 60ºC for
20 sec. Melt curves were constituted for the reactions. Normalized
fold aspects were measured by a 2-ΔΔCt methodology.
Statistical analysis
Statistical values are mentioned as mean ± SD, and statistical
calculations have been carried out by way of Student t-test at the
significance of P < 0.05.
Conflicts of interest
The authors declare no conflicts of interest.
Keywords
Myeloperoxidase; Hypochlorous acid; Heme protein; Low-density
lipoprotein (LDL); Chlorotyrosine;
Purity of lead MPOIs
Author contributions
In order to confirm the highest purity level of the synthesized
compound for in vitro testing, we established purity determination
PJ has done in silico studies and synthesis of guaiacol derivatives.
RD and CA have contributed in terms of in vitro studies including
enzyme-based MPO assay, cell line-based assay, and LDL
assays. RD and CA have contributed to manuscript preparation.
SP and SR have provided direction in terms of biochemical
aspects of the study and manuscript revision. RD has supervised
the in silico, synthesis and in vitro studies.
with
UPLC-DAD
method
(Ultra
Performance
Liquid
Chromatography Waters model) and purity of the lead compound
was found to be >96%.
Mechanism of non-suicide Inhibition
Irreversible inhibitors are shown to act as suicidal compounds.
They strongly bind within the active site of the enzyme forming a Acknowledgment
tight binding. These bindings may be formed between the inhibitor
and the heme group or between the inhibitor and the amino acids
located near the heme group. Two families of compounds were
characterized as irreversible inhibitors including aminobenzoic
acid hydrazide and thioxanthenes. Most of the reported
compounds in the literature are reversible competitive inhibitors
of MPO. They react reversibly with the active site of the enzyme.
We established the reversible nature of the MPOI by performing
two relevant experiments as given below viz. UV-Visible
spectroscopic method and thin-layer chromatographic method.
Authors are grateful to VIT-RGEMS for the financial support. We
also thank DST-VIT-FIST for NMR, VIT-SIF for GC-MS and other
instrumentation facilities. RD would also like to acknowledge UCF
for a visiting professorship.
Abbreviations
MPO, myeloperoxidase; LDL, low density lipoprotein; HDL, high
density lipoprotein; HOCl, hypochlorous acid; HOBr, hypobromos;
H2O2, hydrogen peroxide; Br, bromide; Cl, chloride; SCN,
thiocyanate; 3-ClTyr, 3-Chlorotyrosine; Di-Tyr, dityrosine; ROS,
reactive oxygen species; ox-LDL, oxidized LDL; apo A-1,
apolipoprotein A-1; RTC, reverse cholesterol transport; ox-HDL,
oxidized HDL; 3-NO2Tyr, 3-nitro tyrosine; SMC, smooth muscle
cells; CVD, cardiovascular disease; EDCI, 1-ethyl-3-(3-
i) By UV-Visible spectrophotometer
Typically, the reaction mixture contains 100 mU human MPO
(Sigma-Aldrich, St. Louis, MO), 2 µM H2O2, 8 µM of TMB, followed
by the addition of lead molecules. The final volume was made to
dimethylaminopropyl)
carbodiimide;
DMAP,
dimethyl
11
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10.1002/cmdc.202000084
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FULL PAPER
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aminopyridine; PCR, polymerase chain reaction; LPS,
lipopolysaccharide; ADMET, adsorption distribution metabolism
excretion and toxicity; PDB, protein data bank; TLC, thin layer
chromatography; EDTA, ethylenediaminetetraacetic acid; PBS,
phosphate buffer saline; LMB, leucomethylene blue; MPOI, MPO
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