1H-imidazole-2,5-dicarboxamides as ns4a peptidomimetics: Identification of a new approach to inhibit hcv-ns3 protease

Article abstract of DOI:10.3390/biom10030479

The nonstructural (NS) protein NS3/4A protease is a critical factor for hepatitis C virus (HCV) maturation that requires activation by NS4A. Synthetic peptide mutants of NS4A were found to inhibit NS3 function. The bridging from peptide inhibitors to heterocyclic peptidomimetics of NS4A has not been considered in the literature and, therefore, we decided to explore this strategy for developing a new class of NS3 inhibitors. In this report, a structure-based design approach was used to convert the bound form of NS4A into 1H-imidazole-2,5-dicarboxamide derivatives as first generation peptidomimetics. This scaffold mimics the buried amino acid sequence Ile-25‘ to Arg-28‘ at the core of NS4A_{21‘–33‘} needed to activate the NS3 protease. Some of the synthesized compounds (Coded MOC) were able to compete with and displace NS4A_{21‘–33‘} for binding to NS3. For instance, N⁵-(4-guanidinobutyl)-N²-(n-hexyl)-1H-imidazole-2,5-dicarboxamide (MOC-24) inhibited the binding of NS4A_{21‘–33‘} with a competition half maximal inhibitory concentration (IC₅₀) of 1.9 ± 0.12 μM in a fluorescence anisotropy assay and stabilized the denaturation of NS3 by increasing the aggregation temperature (40% compared to NS4A_{21‘–33‘}). MOC-24 also inhibited NS3 protease activity in a fluorometric assay. Molecular dynamics simulations were conducted to rationalize the differences in structure–activity relationship (SAR) between the active MOC-24 and the inactive MOC-26. Our data show that MOC compounds are possibly the first examples of NS4A peptidomimetics that have demonstrated promising activities against NS3 proteins.

Full text of DOI:10.3390/biom10030479

biomolecules
Article
1H-Imidazole-2,5-Dicarboxamides as NS4A
Peptidomimetics: Identification of a New Approach to
Inhibit HCV-NS3 Protease
Abdelsattar M. Omar 1,2 , Mahmoud A. Elfaky 3 , Stefan T. Arold 4, Sameh H. Soror 5,6
,
Maan T. Khayat 1, Hani Z. Asfour 7, Faida H. Bamane 8 and Moustafa E. El-Araby 1,
*
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz University, Alsulaymanyah,
Jeddah 21589, Saudi Arabia; asmansour@kau.edu.sa (A.M.O.); mkhayat@kau.edu.sa (M.T.K.)
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Al-Azhar University, Nasr City,
Cairo 11884, Egypt
Department of Natural Products and Alternative Medicine, Faculty of Pharmacy, King Abdulaziz University,
Alsulaymanyah, Jeddah 21589, Saudi Arabia; melfaky@kau.edu.sa
Computational Bioscience Research Center, Division of Biological and Environmental Sciences and
Engineering, King Abdullah University of Science and Technology, Thuwal 23955, Saudi Arabia;
stefan.arold@kaust.edu.sa
2
3
4
5
6
7
8
Center for Scientific Excellence Helwan Structural Biology Research (HSBR), Faculty of Pharmacy, Helwan
University, Ain Helwan, Cairo 11795, Egypt; sameh_soror@pharm.helwan.edu.eg
Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, Helwan University, Ain Helwan,
Cairo 11795, Egypt
Department of Medical Microbiology and Parasitology, Faculty of Medicine, King Abdulaziz University,
Jeddah 21589, Saudi Arabia; hasfour@kau.edu.sa
Department of Biochemistry, Faculty of Medicine, King Abdulaziz University, Jeddah 21589, Saudi Arabia;
fbamanea@kau.edu.sa
*
Correspondence: madaoud@kau.edu.sa
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 6 February 2020; Accepted: 18 March 2020; Published: 21 March 2020
Abstract: The nonstructural (NS) protein NS3/4A protease is a critical factor for hepatitis C virus
(HCV) maturation that requires activation by NS4A. Synthetic peptide mutants of NS4A were found
to inhibit NS3 function. The bridging from peptide inhibitors to heterocyclic peptidomimetics of
NS4A has not been considered in the literature and, therefore, we decided to explore this strategy
for developing a new class of NS3 inhibitors. In this report, a structure-based design approach
was used to convert the bound form of NS4A into 1H-imidazole-2,5-dicarboxamide derivatives as
first generation peptidomimetics. This scaffold mimics the buried amino acid sequence Ile-25‘ to
Arg-28‘ at the core of NS4A21‘–33‘ needed to activate the NS3 protease. Some of the synthesized
compounds (Coded MOC) were able to compete with and displace NS4A21‘–33‘ for binding to
NS3. For instance, N5-(4-guanidinobutyl)-N2-(n-hexyl)-1H-imidazole-2,5-dicarboxamide (MOC-24)
inhibited the binding of NS4A21‘–33‘ with a competition half maximal inhibitory concentration
(IC50) of 1.9 ± 0.12 µM in a fluorescence anisotropy assay and stabilized the denaturation of NS3
by increasing the aggregation temperature (40% compared to NS4A21‘–33‘). MOC-24 also inhibited
NS3 protease activity in a fluorometric assay. Molecular dynamics simulations were conducted to
rationalize the differences in structure–activity relationship (SAR) between the active MOC-24 and
the inactive MOC-26. Our data show that MOC compounds are possibly the first examples of NS4A
peptidomimetics that have demonstrated promising activities against NS3 proteins.
Keywords: NS3 inhibitors; allosteric inhibitors; NS4A; peptidomimetics; imidazole; hepatitis C virus;
molecular dynamics; Flaviviridae; DSLS; binding assay
Biomolecules 2020, 10, 479; doi:10.3390/biom10030479
www.mdpi.com/journal/biomolecules

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1. Introduction
Hepatitis C is a life-threatening, widespread viral infection [
1]. The virus initially infects liver
cells though remains asymptomatic for extended periods. This invisibility constitutes epidemiological
challenges as patients diagnosed with hepatitis C come from two pools: recently infected and late-stage
asymptomatic. The initial symptoms, such as fever, fatigue, nausea, and liver tenderness, can be
misleading, as they are tolerated by most patients. After several years of this blood-borne infection, the
virus activates, replicates, and causes complications that start with liver scarring, fibrosis followed by
cirrhosis and, eventually, liver failure and carcinoma [2,3]. Awareness of hepatitis C health problems
and forceful efforts to combat the spread of hepatitis C virus (HCV) in healthcare settings have led to a
significant decline in new hepatitis C cases. However, the annual death toll from complications of this
disease remains as high as 400,000 due to the above described unique clinical vs. epidemic profile
of the chronic hepatitis C. An estimated 70 million people globally are currently infected with HCV,
constituting a major health problem [
that of HIV. In 2015, hepatitis C-related deaths exceeded that of tuberculosis and malaria combined [
Up until 2013, interferon (or pegylated interferon) and ribavirin were the most effective available
therapies, but their long-term efficacies were as low as 3% [ ]. Identification of the hepatitis C
1
]. In 2013, the death rate from HCV complications surpassed
4].
5
genome–proteome in the early 1990s prompted intensive research that led to the introduction of the
new class of direct antiviral agents (DAAs). DAAs revolutionized the treatment of hepatitis C and
succeeded to control the global crisis of this epidemic that was once described as the “Third Millennium
Challenge” [
infection of different genotypes [
are several remaining challenges that must be met to eradicate HCV [
economically challenged areas, which must be increased [ ]; (2) emerging drug resistance, which is an
expected problem even with combination therapy [ 10]; and (3) efficacy in broader sectors of HCV
2
]. Recently, combinations of DAAs have become an effective way to cover hepatitis C
]. Nonetheless, according to the World Health Organization, there
]: (1) access to the treatment in
6,7
1
8
9,
patients, such as the elderly and impaired kidney and liver patients, which must be improved [11].
Thus, there is a clear need to identify new approaches to inhibit viral targets and to increase the DAA
arsenal [12].
The HCV genome is a positive-sense, single-stranded RNA virus belonging to Flaviviridae [13].
The translated virus genome mainly corresponds to structural and nonstructural proteins. The
nonstructural (NS) proteins include NS3, NS4A, NS4B, NS5A, and NS5B, which comprise factors
essential for the maturation and replication of the virus [14]. NS3 dually functions as a protease
(N-terminal domain) and an RNA helicase (C-terminal domain) [15]. When activated as a protease,
it catalyzes the processing of the viral proteome to functional proteins by cleaving NS3–NS4A,
NS4A–NS4B, NS4B–NS5A, and NS5A–NS5B junctions. The proper folding of NS3 is indispensable
for protease activity and occurs only when it binds to NS4A, a 54 amino acid peptide with multiple
functions. NS4A is not only required for NS3 activation [16
of NS3 into the host cell endoplasmic reticulum [18] and for neutralizing the host cell immune
responses [19 20]. The NS3/4A protease was the first and foremost targeted viral protein with DAAs
,17] but is also important for the integration
,
that bind to its substrate site [21]. Interference with the NS4A binding site, on the other hand, has
not been evaluated thoroughly as a mechanism to allosterically inhibit the NS3 protein, especially by
drug-like peptidomimetics. In this article, we present compounds that were designed to compete with
and replace NS4A on its NS3 binding site, leading to NS3 inhibition [22].
2. Results
2.1. Rationale and Design
The NS4A N-terminal intercalates between the
N-terminal) of NS3. The association with NS4A induces proper conformation of the apoenzyme and
increases the proteolytic activity of NS3 by ~950 times [14 16 23]. Accumulated evidence established
that the central region of NS4A (Gly21‘-Leu32‘) is sufficient for activation of the NS3 protease [24 27].
β-strands A0 and A1 (the first 28 residues of the
,
,
–

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Throughout this paper, the prime (‘) mark is applied to differentiate the residues of NS4A from that
of the NS3 apoenzyme. It has also been confirmed by our laboratory [28 29] and others [26 30] that
,
,
certain mutants can bind to the NS4A site and inhibit the protease function of NS3. In their reporting
of the first crystal structure of NS3/4A, Kim and coworkers stated that “the contact surface between
NS3 and NS4A is quite extensive and provides another possible site for the design of anti-HCV
chemotherapeutic agents” [14]. However, subsequent research focused merely on the discovery of
NS3/4A substrate site inhibitors, while mention of the NS4A site inhibitors is rare, with only a few
reports describing a hypothetical approach [14,31,32]. Accordingly, we decided to pursue this concept
by designing peptidomimetics that bind and replace NS4A on the protease domain of NS3.
The first step in de novo design of NS4A peptidomimetics was to inspect the crystal structures of
NS3–NS4A complexes (Protein Data Bank (PDB) Code: 1NS3) [23]. We noticed that NS4A forms a
β
-stand that is mostly extended, except in a turn (kink) featuring a nearly planar area. This planar
kink is composed of one eclipsed cis bond with a dihedral angle of
−
12◦ (the negative sign denotes
a deviation to the opposite side of the Val-26‘ side chain) and four near perfect planar trans bonds
spanning through Val-26‘ to Arg-28‘ (Figure 1A, torsion table in Supplementary Material Table S1A).
This cis bond largely depends on the presence of Gly-27‘ as it allows lessening the steric conflict with
the Val-26‘side chain.
(A)
(B)
Figure 1.
(
A) The planar kink region of nonstructural (NS)4A extracted from crystal structure 1NS3
(Protein Data Bank (PDB) Code: 1NS3) is shadowed. (
derivatives that mimic this planar area.
B) De novo design of 1H-imidazole-2,5-diamide
Interestingly, this glycine turn is conserved in all crystal structures of HCV NS3 protease in the
Protein Data Bank (PDB) deposited to date, regardless of their genotype [33 35] or of the sequence
constructs used (NS4A fused with NS3 or non-fused) [14 23 35] (see Supplementary Material, Figure
–
,
,
S1A). Accordingly, we postulated that an imidazole nucleus appending two amide groups should
mimic this planar region. Docking experiments by manual visually guided methods suggest that the
1H-imidazole-2,5-dicarboxamide nucleus can occupy the planar region while keeping key interactions
of NS4A in place without posing significant steric conflicts (Figure 1B). In particular, the N1 of the
imidazole acted as a hydrogen bond donor with the backbone carbonyl of Ile-35 (located in the A1
β-sheet), which also made a hydrogen bond with CO of the 5-carboxamide group of the designed
scaffold. The carbonyl of the C2-carboxamide group occupied a planar area in which Val-26‘ rests.
In previous studies, the Val-26‘ side chain was deemed unnecessary for NS4A function and,
therefore, could be safely replaced by the intended amidic carbonyl [26]. The pocket accommodating the
Ile-25‘ hydrophobic side chain has been sought as an opportunity to increase the binding potency of our
compounds. In the context of probing this hydrophobic pocket, we decided to synthesize compounds
of different hydrophobic substituents at the C2 amide group. The imidazole’s C5 carboxamide occupied
the same position of the amide bond between Gly-27‘ and Arg-28‘, conserving a hydrogen bond with
Gln-8 (located within the A0 sheet). Its agmantyl substituents occupied the same position of the Arg-28‘
side chain. In addition, the bifurcate hydrogen bonding between the backbone carbonyl of Val-26‘ and
NH groups of Thr-10 and Arg-11 were compensated by hydrogen bonding accepted by the N3 of the
1H-imidazole core (Figure 2).

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Figure 2. The conservation of important hydrogen bonding in the structure-based design.
) Interactions of NS4A (light green) with some NS3 residues (yellow). ( ) Designed imidazole-2,
(
A
B
5-dicarboxamide (light blue).
2.2. Synthesis of 1H-Imidazole-2,5-Dicarboxamide Derivatives
The final MOC compounds were synthesized using reported chemistry [36
in synthetic Scheme 1. MOC refers to the project code and, therefore, final compound coding is
not necessarily sequential not starting from MOC-1. Starting from cyanoformic acid ethyl ester ( ),
the nitrile group was converted to the N-hydroxyamidino derivative by nucleophilic addition of
hydroxylamine [38]. The pivotal imidazole intermediate was prepared by thermal microwave
–39] as illustrated
1
2
3
irradiation (MWI)-induced cyclocondensation of 2 with tert-butyl propiolate [36,37]. This compound
features the desired imidazole nucleus containing two different ester groups: ethyl (alkali labile) and
t-butyl (acid labile). Therefore, trifluoroacetic acid (TFA) treatment could selectively hydrolyze the
tert-butyl ester to imidazole-5-carboxylic acid 4.
The carboxylic acid was reacted with selected amines of R2 substituents under catalysis of
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI)/1-hydroxybenzotriazole hydrate (HOBt) to
give the intermediates 5a
–
c. The ethyl esters 5a
–
c
were hydrolyzed into the lithium salts 6a
–c
using LiOH. The final compounds were obtained from 6a
–
c
by coupling with the desired amines
(R1-NH2) under the effect of propylphosphonic anhydride (T3P). The previous amide coupling
step of 5a (R2 = 2-(methylamino)-2-oxoethyl) and 5b (R2 = 2-pyridylmethyl) provided MOC-11
(
R1 = n-pentyl) and MOC-33 (R1 = n-hexyl), respectively. The intermediate 5c (R2 = diBoc-agmantyl,
Boc = tert-butoxycarbonyl) [39] resulted in intermediates 7a
–i, which were directly subjected to acidic
conditions for Boc deprotection to obtain the MOC final compounds 23, 24, 26, 27, 28, 29, 30, 31, and 32.

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O
O
N
N
N
O
c
R2NH2
H2N
HO
a
EtO2C
EtO2C
O
O
O
CO2tBu
CO2H
N
N
O
H
H
d
N
b
1
4
3
2
R1
N
O
N
HN
R1
N
HN
N
H
N
H
R1NH2
LiOH
O
HCl
N
R2
N
N
N
H
HN
O
EtO2C
N
O
H
g
H
e
f
H
HN
O
R2
LiO
O
NH
5a-c
NH
NH2
6a-c
Boc
N
N
O
R1NH2
HN
6a-i
H
a, R2
=
Boc
f
N
MOC-23, R1 = n-pentyl
MOC-24, R1 = n-hexyl
6a, R1 = n-pentyl
6b, R1 = n-hexyl
6c, R1 = 2-(cyclohexyloxy)ethyl
6d, R1 = 3-(cyclohexyloxy)propyl
6e, R1 = 2-phenoxyethyl
H
2-(methylamino)-2-oxoethyl
H
MOC-26, R1 = 2-(cyclohexyloxy)ethyl
MOC-27, R1 = 3-(cyclohexyloxy)propyl
MOC-28, R1 = 2-phenoxyethyl
MOC-29, R1 = 3-phenoxypropyl
MOC-30, R1 = 4-isopropoxybutyl
MOC-31, R1 = 6-methylheptyl
N
R1
N
O
N
H
b, R2
=
O
N
R2
N
6f, R1 = 3-phenoxypropyl
6g, R1 = 4-isopropoxybutyl
6h, R1 = 6-methylheptyl
H
MOC-11, R1 = n-pentyl,
pyridin-2-ylmethyl
R2 = 2-(methylamino)-2-oxoethyl
MOC-33, R1 = n-hexyl,
MOC-32, R1 = 3-methylpentyl
Boc
N
6i, R1 = 3-methylpentyl
R2 = pyridin-2-ylmethyl
c, R2
=
HN
Boc
di-Boc-agmantyl
N
H
Scheme 1. Synthesis of NS4A peptidomimetics and the structures of screened compounds.
Reagents and conditions: ( ) NH2OH HCl, Na2CO3, EtOH, H2O ( ) Et3N, toluene, microwave
(MW) (120 ◦C, 300 W, 13 min.
) Trifluoroacetic acid (TFA), dichloromethane (DCM), 12 h.
a
·
b
(c
(d) 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), 1-hydroxybenzotriazole hydrate (HOBt),
DIPEA, THF, room temperature to 60 ◦C. (
e) THF, H2O (f) Propylphosphonic anhydride (T3P), THF,
24–48 h. (g) Dioxane, DCM.
2.3. Comparative Binding Evaluation of MOC Compounds Using Differential Static Light Scattering (DSLS)
We studied the binding of MOC compounds with NS3 by differential static light scattering
(DSLS) [40]. The NS3 domain stability upon binding to NS4A was measured by monitoring denatured
protein aggregation upon increasing the temperature gradually from 25.0 to 85.0 ◦C (0.5 ◦C increments)
at 600 nm. If a ligand binds to the protein, this increases a protein’s aggregation temperature
(Tagg) [40
liganded protein
the agg values that occurred upon binding of the MOC compounds were measured and compared to
that of NS4A21‘–33‘ as a positive control. MOC-11 (
agg 0.82 ◦C 54.6% of the positive control) MOC-24
Tagg 0.60 ◦C 39.8%), and MOC-23 ( Tagg 0.32 ◦C 19.5%) demonstrated significant affinities towards
NS3 (Figure 3).
,
41]. DSLS provides an advantage of being a label-free binding assay technique and
∆Tagg (of
−
free protein) can be used to assess relative binding affinities of ligands [42]. For NS3,
∆
T
∆
T
(
∆
∆

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150
100
50
0
-50
(A)
(B)
(C)
Figure 3. Cont.

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(D)
(E)
) Bar graph representation of changes in thermal stability (∆Tagg) of NS3 when mixed
Figure 3.
(A
with MOC derivatives or NS4A (n = 3). ( ) Plot graphs of protein aggregation (% light intensity)
B–E
increase by increasing temperature (◦C). The aggregation curve of NS3 domain alone is shown in the
right bundles. The left bundles are for NS3 domain mixed with peptides as follows: (B) NS4A21‘–33‘,
(C) MOC-11, (D) MOC-23 and (E) MOC-24.
2.4. Fluorescence Anisotropy (FA) Competition Assay of MOC Compounds with NS4A
This assay measures the potency by which synthetic peptidomimetics affect the binding of labeled
NS4A21‘–33‘. Before testing the MOC compounds, the binding affinity of fluorescein isothiocyanate
NS4A21‘–33‘ (FITC-NS4A21‘–33‘) was first determined (see Supplementary Material Figure S3) based
on our previous work [29]. For this particular protein batch, the optimal ratio to be used in the FA
affinity test was calculated to be 0.5 µM NS3 and 0.1 µM FITC-NS4A21‘–33‘. Compounds that efficiently
compete with NS4A21‘–33‘ are expected to decrease the fluorescence emitted from NS3/FITC-4A21-34‘
mixture. The results showed that MOC-24 had the strongest competition potency in this assay as
it showed a half maximal inhibitory concentration (IC50) of 1.9
MOC-23 (4.7 M). MOC-32 also showed moderate inhibition of NS4A binding (competition IC50
7.7 M). Compounds MOC-11, MOC-30, MOC-31, and MOC-33 demonstrated a weaker affinity at
competition IC50 values between 12 and 40 M, while MOC-26, MOC-27, MOC-28, and MOC-29
exerted no inhibition of NS4A binding up to 100 µM.
µM (Table 1, Figure 4), followed by
µ
µ
µ

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Table 1. Inhibition of NS4A21‘–33‘ binding with NS3 by MOC compounds and NS3 inhibition. Standard
error of mean (SEM), half maximal inhibitory concentration (IC50).
Compound
Competition IC50 ± SEM (µM)
MOC-11
MOC-23
MOC-24
MOC-26
MOC-27
MOC-28
MOC-29
MOC-30
MOC-31
MOC-32
MOC-33
17.3 ± 1.22
4.70 ± 0.327
1.91 ± 0.119
>100
>100
>100
>100
28.5 ± 8.55
12.1 ± 1.69
7.74 ± 0.240
40.3 ± 24.0
Figure 4. Fluorescence anisotropy competition assay for MOC-24. The emitted fluorescence was
measured (480/520 nm) proportional to the amount of bound FITC-NS4A21‘–33‘ (Y axis).
2.5. Enzyme Inhibition Assay
MOC-24 was selected for a qualitative testing for its inhibition of NS3 protease activity. Experiments
were carried out using the SensoLyte® 520 HCV Protease Assay Kit. This assay measures the recovered
fluorescence emitted upon cleavage of the labeled peptide substrate representing the NS4A/NS4B
junction [43]. Considering that NS3 without NS4A is almost a thousand-times less catalytic [23], the
method was modified to suit the purpose of our allosteric inhibition assay. For instance, we used
a large amount of free NS3 protein (2.00
catalytic activity in the absence of cofactors. The addition of 4.00
complete loss of NS3 catalytic activity. As a positive control, an equimolar mixture of NS4A with NS3
(equimolar, 2.00 M each) caused only a 9-fold increase in the catalytic activity of the enzyme (Figure 5),
which was far less than the reported value [23], possibly due to the large amount of protein used.
µM) as a smaller concentration did not show measurable
µM amounts of MOC-24 resulted in a
µ

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Figure 5. The effect of MOC-24 on the NS3 activity (n = 5). Neg_Ctrl = NS3 alone, MOC-24 = NS3
mixed with MOC-24 and Pos_Ctrl = NS3 mixed with NS4A21‘–33‘. The emitted fluorescence (Y axis) are
proportional to the catalytic activity of the enzyme (n = 3).
2.6. Molecular Dynamics Simulation for MOC-24 and MOC-26
In order to rationalize the differences in the binding affinities of MOC-24 and MOC-26 (as
representatives of active and inactive compounds, respectively), we performed a molecular dynamics
(MD) simulation for 20.0 ns at 300 ◦K after manual docking of both compounds to place them in the
intended pocket followed by energy relaxation. The MD simulation tries to disrupt non-bonding
interactions between the ligand and the binding site residues by applying energy snapshots that result
in conformational excitations followed by relaxations until equilibration occurs [44]. Equilibration of
the system can be determined by plotting the protein’s backbone–ligand root mean square deviation
(PL-RMSD) vs. the simulation time. If certain protein–ligand interactions stayed uninterrupted after
the equilibrium was reached, this indicates a higher possibility of occurrence of the postulated binding
mode [45].
First, an MD control experiment was performed on the NS3/4A21‘–32‘ crystal structure (PDB Code
1NS3). As expected, this viral peptide–protein complex maintained a high degree of stability and low
residue fluctuations during most of the simulation period (see Supplementary Material, Figure S4A).
The MD simulation model of MOC-24 demonstrated three important features: (a) the ligand resided
during the entire simulation period (20.0 ns) within the boundaries of the presumed binding region of
NS4A25‘–28‘ located between the A0 and A1 β-strands; (b) the imidazole nucleus had low interaction
fluctuations; and (c) the n-hexyl side chain stayed deep in the hydrophobic pocket accommodating the
Ile-25‘ side chain (a video illustration is available as part of the Supplementary Material). Monitoring
the PL-RMSD of the NS3/MOC-24 simulation indicated that the conformational fluctuations of the NS3
protein (backbone atoms) equilibrated after ~10.0 ns and this status was maintained towards the end
of the simulation period (Figure 6). Meanwhile, the ligand (MOC-24) equilibrated at ~13.4 ns. Both
the protein and the ligand clearly showed the least fluctuations after 17.5 ns and until the simulation
ended at 20 ns, indicating that the complex reached stability at this binding mode.

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Figure 6. Plot of the MOC-24 root mean square deviation (RMSD) (red, right Y scale) and NS3 protein
backbone RMSD (green, left Y scale) against simulation time. The dashed lines indicate distinct phases
of protein and ligand conformation evolving over the simulation time.
Accordingly, we looked carefully at the conformational behavior of the crucial
imidazole-2,5-dicarboxamide core at the three phases of the ligand conformational dynamics. In
general, the imidazole nucleus and especially its NH at position 1 showed low fluctuation (Figure 7).
During the first phase (0–10.0 ns), we noticed that the imidazole-2,5-dicarboxamide core maintained
two hydrogen bonds with Val-35 of the A1
hypothesis described above (Figure 8A). Around 10 ns, the imidazole ring started to detect and interact
with Gln-8, a residue within the opposite A0 -strand located at the periphery of the NS3 protein
β-strand, the presumed situation according to the design
β
(Figure 8B). At ~13.3 ns, the imidazole-2,5-dicarboxamide settled to embrace the Gln-8 backbone via
two hydrogen bonding interactions and, in the process, lost contact with Val-35. After this major flip,
the imidazole committed to Gln-8 and showed low fluctuation towards the end of the simulation
(Figure 8C,D).
Figure 7. Root mean square fluctuations (RMSF) of MOC-24 heavy atoms. Note the highest
conformational instability of the guanidine terminal and low motility of both the imidazole diamide
core and the n-hexyl side chain.

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(A)
(B)
(C)
(D)
Figure 8. Interactions of MOC-24 (yellow) with the most important residues (green) at the significant
time frames. ( ) At the start of the simulations, ( ) at 10 ns when protein stability started, ( ) at 13.4 ns
when imidazole-2,5-diamide completely flipped, and ( ) at the end of the molecular dynamics (MD)
A
B
C
D
simulation. Hydrogen bonds are dashed lines in orange, valid van der Waals (VdW) interaction distances
are shown as dashed lines, and numerical values are displayed in angstroms with a magenta color.
The behavior of the hydrophobic substituent attached to the C2 carboxamide of MOC-24 was a
critical issue in our model as this moiety of the compound is the only difference between the active
MOC-24 and the inactive MOC-26. The N2-(n-hexyl) group of MOC-24 stayed in the deep hydrophobic
pocket although it did not show strong commitments to a certain amino acid in the active site. In
this regard, Leu-64 and Pro-88 were the most interacting residues with the n-hexyl side chain. This
hydrophobic n-hexyl showed a low RMSF compared to the other polar N5-agmantyl substituent
(Figure 7). The MD simulation results are consistent with lack of binding of the inactive MOC-26,
which contains a bulkier hydrophobic substituent that ends with a cyclohexyl group.
As the simulation time progressed, this cyclohexyl group was forced outside the hydrophobic
pocket. In fact, the whole MOC-26 molecule left the NS4A binding region located in between the
A0 and A1 β-sheets. The imidazole made only minor contacts with Gln-8, while most of the ligand
clearly popped outside to settle near the protein surface (Figure 9). This movement indicates a lack
of affinity of MOC-26 for the NS4A binding pocket. The last point obtained from the MD study was
about the role of the polar agmantyl group in MOC-24. This group served as a polar head suitable
for the microenvironment around the Arg-28‘ of the bound NS4A. The group alternated interactions

Biomolecules 2020, 10, 479
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at the periphery during the simulation between Glu-30, Gln-28, and the solvent. This is not an odd
observation as Arg-28‘ showed similar behavior in reported NS3/4A crystal structures [46–48] (see
Supplementary Material, Figure S4B).
Figure 9. Change in the positioning of MOC-24 (upper, ligand colored golden yellow) and MOC-26
(lower, ligand colored magenta). The right images are for protein-ligand (PL) complexes before the
start of the MD simulations and the left two images are for PL complexes after completion of the
MD simulations.
3. Discussion and Conclusions
The introduction of DAA combinations allows for broadening the efficacies of HCV treatment to
the pan-genotypic orbit. These combinations are made possible by the identification of more targets in
the viral proteome. For instance, Vosevi® is a combination of sofosbuvir (NS5B inhibitor), velpatasvir
(NS5A inhibitor), and voxilaprevir (NS3/4A protease inhibitor) that cover HCV genotypes 1–6. We
were truly motivated by the inclusion of NS4A competitors as a less-studied target toward potential
further emboldening of the arsenal of DAA combinations. However, in our extensive literature search,
we could not find a precedent for designing and/or screening small-molecule peptide mimics of NS4A.
An additional advantage of targeting NS4A is that this vital component is a versatile small peptide
commonly expressed in the entire Flaviviridae family of viruses, which, besides HCV, includes other
dangerous pathogenic viruses such as the Zika, West Nile, and dengue viruses. Therefore, effective
NS4A peptidomimetics may constitute a possible general approach to combat Flaviviridae.
Our approach of the peptidomimetic design depends on simple observations: the conformation
of the bound NS4A and the relevance of the residues involved in the design. The virtually designed
1H-imidazole-2,5-dicarboxamide appeared to satisfactorily reproduce the stereochemistry of these

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NS4A features. Indeed, we could experimentally confirm that such compounds display binding
affinity towards NS3 genotype-4, the most abundant type in the Middle East [49 50]. Our work
allowed quantifying the binding affinity of our designed MOC compounds. The FA competition assay
confirmed their binding to the NS4A binding site between the A0 and A1 -strands. Both DSLS and
,
β
FA assays were generally in agreement, showing that MOC-11, MOC-23, and MOC-24 exerted good
affinities towards NS3.
Structurally, the three compounds contain n-pentyl or its homologue n-hexyl in the R1 position.
Larger hydrophobic groups at this position (MOC-26 to MOC-31) had lower binding affinities in
both DSLS and FA tests, indicating that the pocket has limited bulk tolerance. Regarding the R2
position, all screened compounds contained an agmantyl group, except for MOC-11 and MOC-33.
The agmantyl group was chosen as a mimic of the NS4A Arg-28‘. In addition, the agmantyl moiety
was hypothesized to act as a hooking group and direct the imidazole scaffold to its intended position.
MOC-11 and MOC-33 were synthesized appending two different polar groups at the R2 position.
MOC-11 encompassed a glycinyl moiety (backbone mimic).
Compared to the agmantyl analogue MOC-23, MOC-11 produced a higher increase in Tagg, but
showed lower competition potency in the FA assay. This indicates that the presence of the Arg-28‘ side
chain may be replaced with another polar group. This structure–activity relationship (SAR) feature
is important for future research towards optimization of the NS4A peptidomimetics by removing
the guanidinyl moiety—a less desirable group in drug discovery, due to high ionizability and poor
pharmacokinetics. MOC-33 presented a more radically different variant from the Arg-28‘ side chain
group. This compound showed much weaker binding in both tests, indicating that a successful peptide
mimic should include close mimics of those amino acid features (glycinyl or agmantyl).
MOC-24, a homologue of MOC-23, exerted confirmed binding activities as revealed in both DSLS
and FA tests. It showed ~1/3 of the thermal stabilization (∆Tagg) of NS3 when compared to the natural
cofactor NS4A21‘–33‘. In the FA assay, it demonstrated the highest ability to displace NS4A21‘–33‘ from
NS3 binding among the tested compounds. Moreover, our in vitro protease assay showed that MOC-24
can inhibit NS3 by forming an inactive complex. However, this test could not be further developed to
a quantitative application due to the very week catalytic activity of the NS3 in in vitro conditions.
The MD simulation, conducted in the course of this study, was useful in validating the design
hypothesis and in explaining the binding potency differences between MOC-24 and MOC-26 (as
representatives for active and inactive compounds, respectively). It was remarkable that MOC-24
stayed in the region between A0 and A1 β-sheets and the n-hexyl group completely buried deep
in a hydrophobic pocket for the entire MD simulation time. A question that frequently comes to
mind is what conformational effects occurred consequently to binding of NS4A peptidomimetics (e.g.,
MOC-24). This question should be of secondary importance, anyway, as prevention of NS4A binding
by non-peptide mimics should lead to the inability of NS3 to perform its catalytic function.
Collectively, our work introduces a new strategy to interfere with NS3 protease activity using
compounds that represent non-peptide mimics of an NS4A fragment (Ile-25‘ to Arg-28‘). We must
acknowledge that our compounds represented only four amino acids at the core of NS4A and covered
a limited region of the binding site. Considering their promising activities and limited molecular
weight (MOC-24, MW 351.5), the results with imidazole-2,5-carboxamides demonstrate that NS4A
binding is a good target for the discovery of a newer generation of DAAs. Our work can be considered
as a bridge between the peptide and non-peptide mimics of NS4A. Further expansion upon this lead
can pave the way for the future discovery of NS4A competitors as clinical candidates.
4. Experimental
4.1. Chemical Synthesis
Solvents and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA), VWR (Radnor,
PA, USA), or Alfa Aesar (Heffrey, MA, UK). When needed, solvents were dried according the

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procedures described in the literature. Unless otherwise stated, the reactions were performed under
an inert atmosphere of nitrogen. Microwave (MW) reactions were performed using Milestone
StartSynth™ reactor (Milestone Inc., Sorisole, Italy). Melting points (mp) were determined in open
capillary tubes using electrothermal apparatus (Stuart, Staffordshire, UK) and are uncorrected. NMR
results were recorded on Bruker DPX-300 MHz (Bruker, Fällanden, Switzerland). HPLC–MS was
performed on an Agilent 1100/ZQ MSD including a C18 column and a diode-array UV detector. The
mobile phase (containing 0.01 M ammonium acetate) was a gradient starting from 20% acetonitrile/80%
water to 80% acetonitrile/20% water. Purities are reported according to percentage of peak areas at
wavelength 254 nm. According to LC–MS analyses, all compounds in this study were confirmed
to have 95% purity or higher. Infrared spectra were recorded on a Thermo Scientific Nicolet iS10
Fourier transform (FT)-IR Spectrometer. In this report, we only listed the important IR stretching
bands, including NH, OH, CH, C=O, C=N, and/or C=C. In FT-IR, all samples were measured neat.
The synthesis of intermediates 1,2-bis(tert-butoxycarbonyl)-3-(4-aminobutyl)guanidine
ethyl (Z)-3-amino-3-(hydroxyimino)propanoate 38], and 5-(tert-butyl) 2-ethyl
1H-imidazole-2,5-dicarboxylate ( ) [36 37] are described below and their characterization data were
[39],
(
2)
[
3
,
found to be in agreement with the literature data.
4.1.1. Synthesis of 5-(Tert-Butyl) 2-Ethyl 1H-Imidazole-2,5-Dicarboxylate (3)
Water (0.6 mL/mmol) was added dropwise over a period of 2 h to a stirred mixture of ethyl
cyanoformate
1 (1.0 equiv., 3 g, 30.3 mmol), hydroxylamine hydrochloride (1.5 equiv., 3.16 g, 45.4
mmol), and sodium carbonate (0.77 equiv., 2.470 g, 23.3 mmol) in ethanol (24.0 mL) at room temperature
(rt). Upon completion, the reaction was quenched, and the solvent was removed under a vacuum. The
resulting residue was extracted with dichloromethane, and the combined organic layers were washed
with brine, dried over MgSO4, filtered, and concentrated to afford a white solid of the intermediate
(Z)-2-amino-2-(hydroxyimino) acetate (2). The intermediate 2 was recrystallized from chloroform
and n-heptane to afford white crystals (1.881 g, 47%), mp 59–61 ◦C, which were used without further
purification for the next step. This intermediate (1.0 equiv., 1.320 g, 10 mmol) and tert-butyl propiolate
(1.0 equiv., 1.262 g, 10 mmol) and Et3N (1.0 equiv., 1.40 mL, 1 mmol) were added to toluene (5.50 mL).
The resulting solution was microwave-irradiated at 120 ◦C for 10 min at 300 W, after a heating ramp,
for 3 min. The solvent was evaporated, and the final product was purified by crystallization from ethyl
acetate (EtOAc)/hexane to obtain 128 mg (53.3%) of
DMSO-d6) δH ppm 13.72 (br., 1 H), 7.87 (s, 1 H), 4.34 (q, J = 7.2 Hz, 2 H), 1.51 (s, 9 H), 1.32 (t, J = 7.0
3
, mp 160–165 ◦C. 1H NMR (300 MHz, DMSO
Hz, 3 H); 13C NMR (75 MHz, CDCl3)
δ 169.7, 136.5, 129.5, 129.1, 128.9, 127.8, 123.8, 29.7; IR (FT-IR,
c
cm−1): 3025.3, 2916.8, 2843.6, 1695.0, 1619.3, 1594.0, 1571.3, 1505.7; LC–MS (ESI), RT = 1.41 min, m/z
241.3 [M + H]+.
4.1.2. Synthesis of 2-(Ethoxycarbonyl)-1H-Imidazole-4-Carboxylic Acid (4)
A solution of 5-tert-butyl 2-ethyl 1H-imidazole-2,5-dicarboxylate (1 equiv., 4.29 g, 17.6 mmol) and
TFA (21.1 mL) in dichloromethane (DCM, 14.0 mL) was stirred for 12 h at room temperature (rt). The
solvent was evaporated under a vacuum and the remaining residue was treated with MeOH (70.0
1
mL) and filtered to give
4
as a yellow solid (2.841 g, 86.29%), mp > 230 ◦C (decomposed). H NMR
(300 MHz, DMSO–DMSO-d6) δH ppm 13.72 (br. s, 1 H); 12.59 (br. s, 1 H), 7.91 (s, 1H), 4.33 (q, J = 6.9
Hz, 2H), 1.32 (t, J = 7.0 Hz, 3H); LC–MS (ESI), RT = 1.55 min, m/z 185.3 [M + H]+.
4.1.3. General Procedure for the Synthesis of Ethyl 4-(N-Substituted Carbamoyl)-1H-Imidazole-2-
Carboxylate (5a-5c)
Under an inert atmosphere, carboxylic acid (1.0 equiv., 5.21 mmol) was dissolved in dry THF (26 mL)
then EDCI (1.0 equiv., 0.998 g, 5.21 mmol), HOBt (1.0 equiv., 0.704 mg, 5.21 mmol), diisopropylethylamine
(DIPEA) (1.5 equiv., 1.35 mL, 7.80 mmol), and the amine R2NH2 (1.2 equiv., 6.25 mmol) were added in
the indicated order. The mixture was stirred at rt for 1 h then heated to 60 ◦C. After 3 h, EDCI (0.5 equiv.,

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0.500 g, 2.60 mmol), HOBt (0.5 equiv., 0.352 g, 2.60 mmol), and DIPEA (0.75 equiv., 0.677 mL, 3.90 mmol)
were added to the mixture. After completion, the mixture was purified by column chromatography
using gradient elution (50–100% EtOAc in cyclohexane).
Ethyl 4-((2-(methylamino)-2-oxoethyl)carbamoyl)-1H-imidazole-2-carboxylate (5a)
The yield was 77%, mp 74–76 ◦C. 1H NMR (300 MHz, DMSO-d6) δH ppm 8.19 (br. s., 1H), 7.81 (s,
2H), 4.35 (q, J = 7.1 Hz, 2H), 3.81 (d, J = 5.6 Hz, 2H), 2.59 (d, J = 4.5 Hz, 3H), 1.33 (t, J = 7.1 Hz, 3H);
LC–MS (ESI), RT = 1.57 min, m/z 255.3 [M + H]+.
Ethyl 5-((pyridin-2-ylmethyl)carbamoyl)-1H-imidazole-2-carboxylate (5b)
1
The intermediate 5b was obtained as an oily substance and its yield was 94%. H NMR (300 MHz,
DMSO-d6) δH ppm 13.24 (br. s. 1 H), 8.69 (t, J = 5.6 Hz, 1H), 8.51 (d, J = 4.1 Hz, 1H), 8.41 (s, 1H), 7.81 (s,
1H), 7.74 (t, J = 7.0 Hz, 1H), 7.19-7.35 (m, 2H), 4.53 (d, J = 6.0 Hz, 2H), 4.34 (q, J = 7.1 Hz, 2H), 1.32 (t,
J = 7.0 Hz, 3H); LC–MS (ESI), RT = 1.14 min, m/z 275.3 [M + H]+.
Ethyl (E)-4-((4-(2,3-bis(tert-butoxycarbonyl)guanidino)butyl)carbamoyl)-1H-imidazole-2-carboxylate (5c)
To a solution of 1,4-diaminobutane (2.0 equiv., 0.303 g, 3.44 mmol) in THF (2.3 mL) was
added a solution of 1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea (1.0 equiv., 500 mg,
1.72 mmol) in THF (1.7 mL/mmol) within 0.5 h. The solution was stirred at room temperature
for 1 h. After completion, the solvent was removed under vacuum and the product was
purified by column chromatography on a silica gel using a mixture of DCM/MeOH to give
[[(4-aminobutyl)amino](carboxyamino)methylene]carbamic acid di-tert-butyl ester (0.250 g, 52.9%); 1H
NMR (300 MHz, CDCl3) δH ppm 11.51 (br. s., 1 H), 8.36 (br. s., 1 H), 3.40-3.52 (m, 2 H), 2.75 (t, J = 6.7
Hz, 2 H), 1.51-1.71 (m, 4 H), 1.52 (s, 9 H), 1.51 (s, 9 H); LC–MS (ESI), RT = 2.44 min, m/z 331.8 [M + H]+
.
The produced amine [[(4-aminobutyl)amino](carboxyamino)methylene]carbamic acid di-tert-butyl
ester was reacted with the carboxylic acid
4 according to the general procedure for amide coupling as
described above (Section 4.1.3). The intermediate 5c was collected as an oily substance; the yield was
0.097 g, 36.2%. The compound was used in the next step, the same day, without further characterization.
4.1.4. General Procedure for Synthesis of 6a–6c
The ethyl esters (1.0 equiv.) were dissolved in THF/water 4:1 (4 mL/mmol) then LiOH (3.0 equiv.)
was added. The mixture was stirred at rt until completion. Afterward, the solvent was evaporated
under a vacuum. The remaining residues was resuspended in EtOH/toluene and evaporated. No
further purification or characterization was performed, and the salts were used for the next step.
N5-(2-(Methylamino)-2-oxoethyl)-N2-(n-pentyl)-1H-imidazole-2,5-dicarboxamide (MOC-11)
Under an inert atmosphere, the lithium salt of 6a (180 mg, 0.78 mmol) was dissolved in dry THF
(2.3 mL, 3 mL/mmol) then propanephosphonic acid anhydride (1.17 mL of 50% solution in THF, 3.0
equiv.) and the n-pentylamine (0.11 mL, 1.2 equiv.) were added. The mixture was stirred at room
temperature for 72 h. After 48 h, additional 1.0 equiv. of T3P was added to the mixture. Upon reaction
completion, the solvent was evaporated under a vacuum, and the residue was purified on a silica gel
column with gradient elution 0%–20% MeOH in CHCl3 to give 0.107 g (46.0%) of MOC-11 as a white
solid, mp 87 ◦C. 1H NMR (300 MHz, methanol-d4) δH ppm 7.76 (s, 1H), 4.04 (s, 1H), 3.74 (t, J = 6.4 Hz,
2H), 3.38-3.51 (m, 1H), 2.78 (s, 2H), 1.80-2.00 (m, 2H), 1.53-1.80 (m, 2H), 1.20-1.53 (m, 3H), 0.86-1.07 (m,
3H); 13C NMR (75 MHz, methanol-d4) δC ppm 170.8, 48.5, 48.2, 47.9, 47.6, 47.4, 47.1, 46.8, 41.8, 38.9,
29.0, 28.9, 25.0, 22.1, 13.0; LC–MS (ESI), RT = 1.46 min, m/z 296.4 [M + H]+.
N2-(n-Hexyl)-N5-(pyridin-2-ylmethyl)-1H-imidazole-2,5-dicarboxamide (MOC-33)
This compound was prepared according to the procedure described above for the preparation of
MOC-11 starting from 6b and n-hexylamine to obtain MOC-33 as an oily substance. The yield was

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1
33%. H NMR (300 MHz, methanol-d4) δH ppm 8.52 (d, J = 4.5 Hz, 1H), 7.70-7.93 (m, 2H), 7.45 (d, J =
7.9 Hz, 1H), 7.19-7.40 (m, 1H), 4.71 (s, 2H), 3.39 (t, J = 7.0 Hz, 2H), 1.51-1.76 (m, 2H), 1.37 (br. s., 6H),
0.93 (t, J = 6.4 Hz, 3H); 13C NMR (75 MHz, methanol-d4) δC ppm 158.6, 157.8, 148.5, 137.5, 122.5, 121.9,
121.6, 48.5, 48.2, 47.9, 47.7, 47.4, 47.1, 46.8, 43.7, 39.0, 31.3, 29.2, 26.3, 22.3; LC–MS (ESI), RT = 2.74 min,
m/z 330.3 [M + H]+.
N5-(4-Guanidinobutyl)-N2-(n-pentyl)-1H-imidazole-2,5-dicarboxamide (MOC-23)
The lithium carboxylate salt of 6c was reacted with n-pentylamine according the
procedure described above for the preparation of MOC-11.
N5-[[4-(2,3-bis(tert-butoxycarbonyl)guanidino)butyl]carbamoyl]-N2-(n-pentyl)-1H-imidazole-2,5-dicarboxamide
7a) was subjected to a deprotection reaction for the removal of Boc groups. Thus, it was dissolved in
The collected intermediate
(
dry DCM (5 mL/mmol), hydrochloric acid (in 1,4-dioxane solution (5 mL/mmol) was added, and the
reaction mixture was stirred at rt for 2 h. After completion, the reaction mixture was concentrated and
dried under a vacuum and purified using preparative HPLC to furnish MOC-23 (27.3%), mp 106 ◦C.
1H NMR (300 MHz, methanol-d4) δH ppm 7.83 (s, 1H), 3.36-3.60 (m, 5H), 3.25 (br. s., 3H), 1.69 (br. s.,
7H), 1.17-1.50 (m, 6H), 0.95 (br. s., 4H); 13C NMR (75 MHz, methanol-d4) δC ppm 48.5, 48.3, 48.0, 47.7,
47.4, 47.1, 46.8, 40.8, 39.3, 28.9, 28.7, 25.9, 22.1, 13.0; LC–MS (ESI), RT = 1.90 min, m/z 338.4 [M + H]+.
N5-(4-Guanidinobutyl)-N2-(n-hexyl)-1H-imidazole-2,5-dicarboxamide (MOC-24)
This compound was prepared according to the procedure described above for the synthesis
1
of MOC-23. The product was obtained as a yellowish oily substance (37.1%). H NMR (300 MHz,
methanol-d4) δH ppm 7.81 (br. s., 1H), 3.39-3.57 (m, 4H), 3.07-3.27 (m, 3H), 1.61-1.84 (m, 8H), 1.55 (t, J =
7.1 Hz, 1H), 1.18-1.48 (m, 9H), 0.93 (br. s., 4H); 13C NMR (75 MHz, methanol-d4) δC ppm 48.5, 48.2,
48.0, 47.7, 47.4, 47.1, 46.8, 40.8, 39.2, 31.3, 26.4, 25.9, 22.3, 13.0; LC–MS (ESI), RT = 1.78 min, m/z 352.3 [M
+ H]+.
N2-(2-(Cyclohexyloxy)ethyl)-N5-(4-guanidinobutyl)-1H-imidazole-2,5-dicarboxamide (MOC-26)
This compound was prepared according to the procedure described above for the synthesis
1
of MOC-23. The product was obtained as a yellowish oily substance (19.4%). H NMR (300 MHz,
methanol-d4) δH ppm 7.76 (br. s., 1H), 3.50-3.75 (m, 7H), 3.44 (br. s., 3H), 3.25 (br. s., 3H), 1.81-2.09 (m,
5H), 1.63-1.81 (m, 9H), 1.45-1.63 (m, 2H), 1.11-1.44 (m, 7H); 13C NMR (75 MHz, methanol-d4) δC ppm
77.5, 65.6, 48.5, 48.2, 47.9, 47.6, 47.4, 47.1, 46.8, 40.7, 37.0, 31.9, 25.8, 25.6, 23.7; LC–MS (ESI), RT = 2.46
min, m/z 213.2 [M + H]+
N2-(3-(Cyclohexyloxy)propyl)-N5-(4-guanidinobutyl)-1H-imidazole-2,5-dicarboxamide (MOC-27)
This compound was prepared according to the procedure described above for the synthesis
1
of MOC-23. The product was obtained as a yellowish oily substance (41.8%). H NMR (300 MHz,
methanol-d4) δH ppm 7.73 (s, 1H), 3.38-3.76 (m, 7H), 3.13-3.30 (m, 3H), 1.79-2.07 (m, 5H), 1.63-1.79 (m,
7H), 1.56 (d, J = 7.5 Hz, 1H), 1.12-1.42 (m, 5H); 13C NMR (75 MHz, methanol-d4) δC ppm 77.5, 65.6,
48.5, 48.2, 47.9, 47.6, 47.4, 47.1, 46.8, 40.7, 37.0, 31.9, 25.8, 25.6, 23.7; LC–MS (ESI), RT = 1.98 min, m/z
408.3 [M + H]+.
N5-(4-Guanidinobutyl)-N2-(2-phenoxyethyl)-1H-imidazole-2,5-dicarboxamide (MOC-28)
This compound was prepared according to the procedure described above for the synthesis of
1
MOC-23. The product was obtained as an off-white solid; mp 137–139 ◦C (47.8%). H NMR (300 MHz,
methanol-d4) δH ppm 8.01 (s, 1H), 7.28 (t, J = 7.9 Hz, 2H), 6.82-7.10 (m, 3H), 4.19 (t, J = 5.1 Hz, 2H), 3.84
(t, J = 5.1 Hz, 2H), 3.44 (br. s., 2H), 3.25 (br. s., 2H), 1.69 (br. s., 5H); 13C NMR (75 MHz, methanol-d4) δC
ppm 129.2, 114.2, 48.5, 48.2, 47.9, 47.6, 47.4, 47.1, 46.8; LC–MS (ESI), RT = 1.86 min, m/z 388.4 [M + H]+.

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N5-(4-Guanidinobutyl)-N2-(3-phenoxypropyl)-1H-imidazole-2,5-dicarboxamide (MOC-29)
This compound was prepared according to the procedure described above for the synthesis of
1
MOC-23. The product was obtained as an off-white solid mp 135–138 ◦C (37.1%). H NMR (300 MHz,
methanol-d4) δH ppm 7.73 (s, 1H), 7.13-7.38 (m, 2H), 6.80-7.05 (m, 3H), 4.10 (t, J = 5.9 Hz, 2H), 3.61 (t, J
= 6.7 Hz, 2H), 3.44 (br. s., 2H), 3.14-3.30 (m, 2H), 2.11 (quin, J = 6.3 Hz, 2H), 1.52-1.84 (m, 5H); 13
C
NMR (75 MHz, methanol-d4) δC ppm 129.1, 120.4, 114.2, 65.3, 48.5, 48.2, 47.9, 47.6, 47.4, 47.1, 46.8, 40.8,
37.9, 36.4, 29.0, 26.6, 25.8; LC–MS (ESI), RT = 1.51 min, m/z 402.3 [M + H]+
N5-(4-Guanidinobutyl)-N2-(4-isopropoxybutyl)-1H-imidazole-2,5-dicarboxamide (MOC-30)
This compound was prepared according to the procedure described above for the synthesis of
1
MOC-23. The product was obtained as an oily substance (38.4%). H NMR (300 MHz, methanol-d4) δH
ppm 7.73 (s, 1H), 3.35-3.74 (m, 8H), 3.14-3.28 (m, 2H), 1.69 (br. s., 9H), 1.16 (d, J = 6.2 Hz, 6H); 13C NMR
(75 MHz, methanol-d4) δC ppm 71.5, 67.4, 48.5, 48.2, 47.9, 47.6, 47.4, 47.1, 46.8, 40.8, 38.7, 37.9, 27.1, 26.6,
26.1, 25.8, 21.0; LC–MS (ESI), RT = 2.08 min, m/z 382.3 [M + H]+.
N5-(4-Guanidinobutyl)-N2-(6-methylheptyl)-1H-imidazole-2,5-dicarboxamide (MOC-31)
This compound was prepared according to the procedure described above for the synthesis of
1
MOC-23. The product was obtained as a solid, mp 83–84 ◦C (23.7%). H NMR (300 MHz, methanol-d4)
δH ppm 7.74 (s, 1H), 3.38-3.57 (m, 2H), 3.14-3.30 (m, 2H), 1.54-1.83 (m, 6H), 1.22-1.54 (m, 9H), 0.81-1.09
(m, 7H); 13C NMR (75 MHz, methanol-d4) δC ppm 48.5, 48.2, 47.9, 47.6, 47.3, 47.1, 46.8, 40.8, 39.5, 30.6,
28.6, 26.7, 25.8, 23.8, 22.7, 13.0, 9.8; LC–MS (ESI), RT = 1.19 min, m/z 380.3 [M + H]+.
N5-(4-guanidinobutyl)-N2-(3-methylpentyl)-1H-imidazole-2,5-dicarboxamide (MOC-32)
This compound was prepared according to the procedure described above for the synthesis of
MOC-23. The product was obtained as an off-white oily substance (41.2%). 1H NMR (300 MHz,
methanol-d4) δH ppm 7.73 (s, 1H), 3.35-3.58 (m, 4H), 3.14-3.30 (m, 2H), 1.69 (br. s., 6H), 1.35-1.59 (m,
3H), 1.24 (td, J = 7.0, 13.7 Hz, 1H), 0.77-1.09 (m, 6H); 13C NMR (75 MHz, methanol-d4) δC ppm 48.5,
48.2, 47.9, 47.6, 47.4, 47.1, 46.8, 40.8, 37.1, 35.9, 32.1, 29.1, 26.6, 25.8, 18.0, 10.2; LC–MS (ESI), RT = 1.79
min, m/z 352.3 [M + H]+.
4.2. Biological Screening
All reagents used in the biological screenings were purchased from Merck KGaA (Darmstadt,
Germany) in molecular biology grade unless stated otherwise.
4.2.1. NS3 Protein
NS3 Constructs
A synthetic gene coding for the HCV NS3 domain of genotype 4a, the most abundant HCV in Saudi
Arabia and Egypt [51], was synthesized by GenScript (Hong Kong, China), the nucleotide sequence
was optimized for Escherichia. coli codon usage. The synthetic gene was cloned as a NdeI–BamHI
fragment into the expression vector pET-3a Novagen®. The obtained construct was sequenced to
confirm the identity and frame of the expression construct.
NS3 Protein Information
Accession GU085486.1
HCV genotype 4a (The most common genotype in Saudi Arabia) [52]
NS3 from 4 to 182 aa (L/E, F/E, I/Q, V/E, L/Q, C/S)
NS4A 632 to 685 aa (i/n) are:

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G SVVIVGRVNL SGDTAYAQQT RGEESTQETS QTGRDTNENC GEVQVLSTAT QSFLGTAVNG
VMWTVYHGAG SKTISGPKGP VNQMYTNVDQ DLVGWPSPPG VKSLTPCTCG ASDLYLVTRH
ADVVPVRRRG DTRGALLSPR PISTLKGSSG GPLLCPMGHA AGLFRAAVST RGVAKAVDFV
PVESLETT MRSP
NS4A/NS3 fusion protein expression in pET-28a
NS3 protease domain 1–181 aa plus an N-terminal T7 tag and C-terminal His tag (marked
in uppercase)
M ASMTGGQQMG APITAYAQQT RGLFSTIVTS LTGRDTNENC GEVQVLSTAT QSFLGTAVNG
VMWTVYHGAG SKTISGPKGP VNQMYTNVDQ DLVGWPSPPG VKSLTPCTCG ASDLYLVTRH
ADVVPVRRRG DTRGALLSPR PISTLKGSSG GPLLCPMGHA AGLFRAAVCT RGVAKAVDFV
PVESLETTMR sGSHHHHHH
These sequences were expressed in pET-3a
Protein Expression
The sequence of NS3 domain for genotype 4A, was expressed in E. coli Rosette (DE3) pLysS
according to a standard protocol [14]. Therefore, a synthetic gene for NS3 domain was subcloned in
the expression vector pET-3a. A 100 mL bacterial culture in Luria Broth (LB) medium grown overnight
at 37 ◦C was used for inoculation of 10 L LB in a 14 L fermentation flask (New Brunswick Scientific Co.,
Enfield, CT, USA). The media was supplemented with ampicillin at 50 µg/mL. The culture was grown
until the OD600 reached 0.5–0.6; it was then cooled to 25 ◦C and 1 mM isopropyl-
(IPTG) was added for expression overnight, followed by harvesting of cells.
β-thiogalactoside
Protein Purification
The produced protein was purified using equilibrated nickel nitrilotriacetic acid (Ni-NTA) beads
and without removal of the poly-histidine tag. For this process, cells were first resuspended (1.00
g/5 mL) in buffer (50 mM N-2-hydroxyethyl-piperazine-N0-2-ethanesulfonic acid (HEPES), 0.300 M
NaCl, 10.0% glycerol, 2.00 mM β-mercaptoethanol, pH 8.0). Lysozyme was added (1.00 mg/mL)
followed by a protease inhibitor cocktail tablet and the suspension was sonicated. The cell lysate was
centrifuged to collect the clear supernatant that contained the desired NS3 protein. The protein was
purified using pre-equilibrated Ni-NTA beads (Qiagen, Germantown, MD, USA). The beads were
washed with buffer (50.0 mM HEPES, 0.300 M NaCl, 10.0% glycerol, 2.00 mM
20.0 mM imidazole, pH 8.0) and eluted with another buffer (50.0 mM HEPES, 0.300 M NaCl, 10.0%
glycerol, 2.00 mM -mercaptoethanol, 350 mM imidazole, pH 8.0). The fractions were collected and
β-mercaptoethanol,
β
concentrated using an Amicon Ultra-4 3000 MWCO centrifugal device (Merck KGaA (Darmstadt,
Germany). The protein purity after Ni-affinity purification step was not less than 70.0%. The
purity, as estimated by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), was
sufficient to perform all investigations of this study and the protein was stable for several hours at test
conditions [51]. The concentration of NS3 in the final concentrate was measured using a Nanodrop
nanoscale spectrophotometer.
™
When needed, further purification of the protein was accomplished on a Superdex 75 16/90 column
(GE Healthcare, Chicago, IL, USA) equilibrated in 20.0 mM HEPES, 10 mM DDT, 200 mM NaCl, pH
7.60 run at rate of 1 mL/min followed by SDS-PAGE for purity estimation.
4.2.2. NS4A
The cofactor NS4A and the fluorescent fluorescein isothiocyanate NS4A (FITC-NS4A) were
purchased from GenScript (Hong Kong, China). The NS4A structure was identical to that of the HCV
genotype 4a with two lysine residues added at both the N- and C-termini. Thus, the structure of NS4A
used in this study was LL-G21SVVIVGRIVLSG33-LL.
We studied the binding of NS4A and its mutants with NS3 by DSLS using Stargazer-2
™ (Harbinger
Biotechnology and Engineering Corporation, Toronto, Canada). This method assesses protein stability

Biomolecules 2020, 10, 479
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by monitoring aggregate formation using controlled, gradually elevated temperatures [40]. The NS3
domain stability upon binding to NS4A was measured by monitoring denatured protein aggregation
upon increasing the temperature from 25.0 to 85.0 ◦C (0.5 ◦C increments) at 600 nm. The Tagg values
were calculated automatically from raw data using Stargazer-AIR® software. All experiments were
repeated three times using the same batch of NS3 protein.
4.2.3. DSLS Binding Test
The NS3 domain (15.0
was added to a binding buffer (20.0 mM HEPES, 10.0 mM DTT, 200 mM NaCl, pH 7.60) to a final volume
100 L. The mixture was incubated at room temperature with gentle shaking for 2 h. Afterwards, 10
L of the mixture was transferred into a clear bottomed Nunc 384-well plate and covered by 10 µL
µM), alone or mixed with an equimolar equivalent of tested MOC derivative,
µ
µ
paraffin oil to minimize evaporation. Protein aggregation was monitored by tracking the change in
scattered light that was detected by a charged coupled device (CCD) camera. Snapshot images of the
plate were taken every 0.5 ◦C. The pixel intensities in a preselected region of each well were integrated
using image analysis software to generate a value representative of the total amount of scattered light
in that region. These intensities were then plotted against temperature for each sample well and fitted
to obtain the aggregation temperature (Tagg). Aggregation was monitored and analyzed to assess the
effect of NS4A and its synthetic analogues on the stability of the NS3 as an indicator of binding. Each
experiment was repeated three times. Statistical analysis was performed using GraphPad Prism v. 8.0®
and Instat® v. 3.10 software.
4.2.4. Binding and Competition Assay by Fluorescence Anisotropy
In a 96-well plate, we added a place binding buffer (20 mM HEPES, 10 mM DTT, 200 mM NaCl,
pH 7.60) and a mixture of NS3 (0.40
concentration. A serial dilution of MOC compounds (dissolved in 1.50% DMSO in binding buffer)
(1/2 dilution starting from 150 to 0.292 M) were mixed gently for 60 min in a dark place and the
µM) and FITC-NS4A (0.10 µM) at the calculated affinity constant
µ
fluorescence was measured at excitation/emission wavelengths of 480/520 nm. The competition IC50
was calculated according to the recommended equation embedded in GraphPad Prism v. 8.0® and
Instat® software.
4.2.5. Enzyme Inhibition Assay
The assay was performed using SensoLyte-520® HCV protease assay kit fluorometric* (Anaspec,
Fremont, CA, USA) according to a modified procedure to suit the purpose of determination of
allosteric inhibition. NS3 (2.00
5-FAM/QXL 520 fluorescence resonance energy transfer (FRET) peptide was added as instructed by
the assay kit manual. The sequence of this FRET peptide (5-FAM-SLGRKIQIQ) was derived from the
cleavage site of NS4A/NS4B. In the FRET peptide, the fluorescence of 5-FAM is quenched by QXL 520.
µM) was mixed with MOC-24 (4.00 µM) for 15.0 min. Afterwards,
™
™
Upon cleavage into two separate fragments by HCV NS3/4A protease, the fluorescence of 5-FAM is
recovered, and can be monitored at 490 nm/520 nm (excitation/emission). Controls included buffer,
compound, compound + NS3, NS3 + NS4A, and the FRET peptide separately. All test wells and
controls were repeated in triplicate on the same 96-well plate.
4.3. Molecular Modeling
4.3.1. Hardware and Software
The molecular modeling was performed on HP OMEN-X PC (Processor: Intel® Core
™ i9-7920x
CPU @ 2.9 GHz, Installed Memory (RAM): 32.0 GHz, Operating Systems: Windows 10 Enterprise and
Enterprise Linux 7 (Distribution CentOS-7). The molecular computational works were performed
using Schrödinger Maestro™ Suite (Schrödinger, New York, NY, USA).

Biomolecules 2020, 10, 479
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4.3.2. Protein and Ligand Preparation
The NS3/4A (non-fusion protein) crystal structure (PDB Code: 1NS3) was downloaded from the
Protein Data Bank website (rcsb.org). The protein was prepared according to the standard procedure
in the Protein Preparation module of Maestro [53]. Briefly, the first stage included Preprocess followed
by simplification of the protein to a monomer, removal of unused ligands and water. Finally an energy
minimization was performed using an OPLS3e force field [54]. MOC compounds were sketched using
ACD/ChemSketch freeware (Advanced Chemistry Development, Inc., Toronto, ON, Canada), saved
as an sdf file, and imported to Maestro. Ligands were optimized using LigPrep standard procedure
embedded in the Maestro suite. The ligands selected for molecular dynamics were merged (by 3D
visual guidance) onto the prepared NS3 protein after the removal of the co-crystallized NS4A21‘-32‘
peptide to form the complexes that entered in the next step.
4.3.3. Molecular Dynamics
Using Desmond’s System Builder module, the protein–ligand complex was prepared for MD
simulation by applying an OPLS3e molecular mechanics force field after solvation in a water box
(shape, orthorhombic; size calculation method, buffer; distance from protein, 10
volume, minimize volume) that contained 0.15 M NaCl as added ions. Other parameters were used as
default in the System Builder.
×
10
×
10 Å; box
The resulting solvated ligand–protein complex was subjected to Desmond molecular dynamics
simulation after setting the following parameters: simulation time, 20 ns; recording internal: trajectory,
10 ps; approximate number of frames, 1000; ensemble class, normal pressure and temperature (NPT);
temperature, 300 K, pressure, 1.013 bar. Before starting the MD job, the system was allowed to relax
using the default relaxation protocol. Other parameters were used as default.
5. Patents
El-Araby, M. E.; Omar, A. M.; El-Faky, M. A.; Soror, S. H.; Khayat, M. T.; Asfour, H. Z.; Bamane, F. H.
Imidazole-based Compounds as hepatitis C virus inhibitors, US Application 16/384,472, 16 April 2019.
Supplementary Materials: The following are available online at http://www.mdpi.com/2218-273X/10/3/479/s1,
Figure S1-A: Figure S1-A: Conserved conformation of NS4A peptide in bound form with NS3 protease domain.
Figure S1-B. Dihedral angles
θ of the bound NS4A’s planar region (PDB Code: 1NS3). Figure S3: Fluorescence
n
assay for binding of NS4A21‘-34‘ to NS3 (Genotype 4). Figure S4-A: MD simulation of NS3/4A complex. Figure
S4-B. Interactions of Arg-28‘ terminal guanidine at four different reported NS3/4A crystal structures (PDB code are
printed under each corresponding image). Table S1: Dihedral angles of core part of bound NS4A (PDB Code:
1NS3). Video S1: 20 ns MD simulation of MOC-24 binding with NS3.
Author Contributions: Conceptualization, M.E.E.-A. and S.H.S.; Data curation, M.A.E., S.H.S., M.T.K., and
M.E.E.-A.; Formal analysis, S.T.A., S.H.S., M.T.K., H.Z.A., F.H.B., and M.E.E.-A.; Funding acquisition, A.M.O.
and M.E.E.-A.; Investigation, A.M.O., M.T.K., F.H.B., and M.E.E.-A.; Methodology, A.M.O., M.A.E., S.T.A.,
S.H.S., H.Z.A., F.H.B., and M.E.E.-A.; Project administration, A.M.O., H.Z.A., and M.E.E.-A.; Resources, A.M.O.,
S.T.A., H.Z.A., and F.H.B.; Software, A.M.O.; Supervision, S.T.A., S.H.S., and M.E.E.-A.; Validation, M.E.E.-A.;
Visualization, M.E.E.-A.; Writing—original draft, M.A.E., S.H.S., M.T.K., and M.E.E.-A.; Writing—review & editing,
A.M.O., S.T.A., and H.Z.A. All authors have read and agreed to the published version of the manuscript.
Funding: This project was funded by the National Plan for Science, Technology and Innovation (MAARIFAH),
King Abdulaziz City for Science and Technology, the Kingdom of Saudi Arabia; Award number 12-BIO3193-03.
The research reported in this publication was also supported by funding from King Abdullah University of Science
and Technology (KAUST).
Acknowledgments: The authors acknowledge, with thanks, the Science and Technology Unit, King Abdulaziz
University for technical support. Authors are exceptionally grateful to MDPI, the Editor Ms. Sophia Lin, and the
respected reviewers for being very cooperative to publish this manuscript, considering the current circumstances
of the COVID-19 pandemic that hindered us from covering some reviewers’ comments.
Conflicts of Interest: The authors declare no conflict of interest.

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