Discovery of novel dengue virus entry inhibitors via a structure-based approach

Article abstract of DOI:10.1016/j.bmcl.2017.06.049

Dengue is a mosquito-borne virus that has become a major public health concern worldwide in recent years. However, the current treatment for dengue disease is only supportive therapy, and no specific antivirals are available to control the infections. Therefore, the need for safe and effective antiviral drugs against this virus is of utmost importance. Entry of the dengue virus (DENV) into a host cell is mediated by its major envelope protein, E. The crystal structure of the E protein reveals a hydrophobic pocket occupied by the detergent n-octyl-β-D-glucoside (β-OG) lying at a hinge region between domains I and II, which is important for the low-pH-triggered conformational rearrangement required for fusion. Thus, the E protein is an attractive target for the development of antiviral agents. In this work, we performed prospective docking-based virtual screening to identify small molecules that likely bind to the β-OG binding site. Twenty-three structurally different compounds were identified and two of them had an EC₅₀ value in the low micromolar range. In particular, compound 2 (EC₅₀?=?3.1?μM) showed marked antiviral activity with a good therapeutic index. Molecular dynamics simulations were used in an attempt to characterize the interaction of 2 with protein E, thus paving the way for future ligand optimization endeavors. These studies highlight the possibility of using a new class of DENV inhibitors against dengue.

Full text of DOI:10.1016/j.bmcl.2017.06.049

Accepted Manuscript
Discovery of novel Dengue virus entry inhibitors via a structure-based approach
Emilse S. Leal, M. Gabriela Aucar, Leopoldo G. Gebhard, Nestor G. Iglesias,
María J. Pascual, Juan J. Casal, Andrea V. Gamarnik, Claudio N. Cavasotto,
Mariela Bollini
PII:
S0960-894X(17)30653-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.06.049
BMCL 25083
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 May 2017
15 June 2017
19 June 2017
Please cite this article as: Leal, E.S., Aucar, M.G., Gebhard, L.G., Iglesias, N.G., Pascual, M.J., Casal, J.J., Gamarnik,
A.V., Cavasotto, C.N., Bollini, M., Discovery of novel Dengue virus entry inhibitors via a structure-based approach,
Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.06.049
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Emilse S. Leal, M. Gabriela Aucar, Leopoldo G. Gebhard, Nestor G. Iglesias, María J. Pascual, Juan J. Casal,
Andrea V. Gamarnik, Claudio N. Cavasottoand Mariela Bollini

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Discovery of novel Dengue virus entry inhibitors via a structure-based approach
Emilse S. Leala, ‡, M. Gabriela Aucarb,c, ‡, Leopoldo G. Gebhardd, Nestor G. Iglesiasd, María J. Pascuale,
Juan J. Casala, Andrea V. Gamarnikd, Claudio N. Cavasottob,* and Mariela Bollinia, ∗
a Laboratorio de Química Medicinal, Centro de Investigaciones en Bionanociencias (CIBION)-CONICET, Godoy Cruz 2390, Ciudad de Buenos Aires,
Argentina.
b Laboratory of Computational Chemistry and Drug Design, Instituto de Investigación en Biomedicina de Buenos Aires (IBioBA) - CONICET - Partner Institute
of the Max Planck Society, Godoy Cruz 2390, Ciudad de Buenos Aires, Argentina.
c Instituto de Modelado e Innovación Tecnológica, CONICET, and Departamento de Fisica, FCENA-UNNE, Avda. Libertad 5460, Corrientes, Argentina.
d Fundación Instituto Leloir-CONICET, Av. Patricias Argentinas 435, Ciudad de Buenos Aires, Argentina.
e Instituto de Investigaciones Biotecnológicas, Universidad Nacional de San Martín, CONICET, Av. 25 de Mayo y Francia, San Martín, Prov. de Buenos Aires,
Argentina.
.
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Dengue is a mosquito-borne virus that has become a major public health concern worldwide in
recent years. However, the current treatment for dengue disease is only supportive therapy, and
no specific antivirals are available to control the infections. Therefore, the need for safe and
effective antiviral drugs against this virus is of utmost importance. Entry of the dengue virus
(DENV) into a host cell is mediated by its major envelope protein, E. The crystal structure of the
E protein reveals a hydrophobic pocket occupied by the detergent n-octyl-β-D-glucoside (β-OG)
lying at a hinge region between domains I and II, which is important for the low-pH-triggered
conformational rearrangement required for fusion. Thus, the E protein is an attractive target for
the development of antiviral agents. In this work, we performed prospective docking-based
virtual screening to identify small molecules that likely bind to the β-OG binding site. Twenty-
three structurally different compounds were identified and two of them had an EC50 value in the
low micromolar range. In particular, compound 2 (EC50 = 3.1 µM) showed marked antiviral
activity with a good therapeutic index. Molecular dynamics simulations were used in an attempt
to characterize the interaction of 2 with protein E, thus paving the way for future ligand
optimization endeavors. These studies highlight the possibility of using a new class of DENV
inhibitors against Dengue
Keywords:
Keyword_1 Envelope protein,
Keyword_2 Structure-based drug design,
Keyword_3 Virtual screening,
Keyword_4 Anti-dengue drugs
Keyword_5 Molecular dynamics
2009 Elsevier Ltd. All rights reserved.
Dengue virus (DENV) is the most prevalent mosquito-borne
viral pathogen and has become a major public health concern
worldwide. Presently, dengue is endemic in 112 countries around
the world.1 Mostly in tropical and subtropical areas, each year
50–100 million individuals are infected with DENV resulting in
nearly 500,000 severe illnesses and 25,000 deaths.2,3 Four distinct
serotypes (DENV1-4) of the virus are the major contributors in
circulation of DENV in the world population.4 Although the
number of dengue cases increases every year, there are no
approved therapeutics available for the treatment of these
infections.
that are present in the infectious virus particle, composed of a
lipid bilayer that anchors envelope and membrane proteins,
surrounding the viral RNA complexed with the capsid protein.
The other seven are nonstructural proteins (i.e.; NS1, NS2A,
NS2B, NS3, NS4A, NS4B, and NS5) involved in amplifying the
viral genome once the virus reaches the cytoplasm of the cell.7
During viral morphogenesis, the capsid protein recruits the viral
RNA genome on the cytosolic face of the endoplasmic reticulum
(ER), and the assembled nucleocapsid buds into the ER lumen
acquiring a coat that contains the Envelope (E) glycoprotein and
the precursor Membrane protein, prM. Then, the particle passes
through the secretory pathway, where a furin-like protease
cleaves prM to M in a late trans-Golgi compartment.8 The
cleavage releases a constraint on the E glycoprotein and primes
the particle for low-pH-triggered membrane fusion. The E
DENV belongs to the flavivirus genus of the Flaviviridae
family of positive-sense single-stranded RNA viruses.5,6 The
RNA genome encodes ten proteins; three of them (the so-called
Capsid, Membrane and Envelope proteins) are structural proteins
———
∗
Corresponding Author mariela.bollini@cibion.conicet.gov.ar; cnc@cavasotto-lab.net, ccavasotto@ibioba-mpsp-conicet.gov.ar.
‡These authors equally contributed to this work.

glycoprotein is a major component of the virion surface and plays
an important role in binding to the host receptor, drives viral
fusion, and induces immunogenic response in host cell.9,10
aminopyridin-2-ol, Cs2CO3, DMF, 100 oC, 24 h; (d) 5-(Pyridin-2-
yl)thiophene-2-carboxylic acid, BOP, DIPEA, rt, overnight.
The identities of the purchased compounds and compound 2
were verified by NMR and mass spectrometry, and the purity
was found to be at least 95% by HPLC
The crystal structure of the DENV E protein reveals a
hydrophobic pocket occupied by the detergent n-octyl-β-D-
glucoside (β-OG) lying at a hinge region between domains I and
II of the protein, which is important for the low-pH-triggered
conformational rearrangement required for membrane fusogenic
activity. Therefore, the β-OG binding site was proposed as an
appropriate target for developing small-molecule inhibitors of
virus-host membrane fusion process. 11–14
Cl
N
S
O
N
N
N
NH
O
O
HN
O
O
N
NH
O
Cl
Cl
S
2
The available structural data of the DENV E opened up the
possibility to identify antiviral agents interfering with the early
steps of DENV infection. In this work, we undertook a multistep
hierarchical prospective structural-based virtual screening
(SBVS) strategy targeting the β-OG hydrophobic binding site of
the solved crystal structure of protein E (PDB code: 1OKE) to
identify novel entry inhibitors with improved potency and
efficacy against E. Docking-based virtual screening was
performed using approximately 110000 small-molecules from the
Maybridge database using the ICM software, which proved
successful for finding hits on several other targets.15–18 The
screening protocol began with an ADME filter,19 in order to
retain only potentially non-toxic and druggable molecules, which
were subjected to two independent high-throughput docking
(HTD) screenings, where the best score per molecule was kept.20
The top ranked 500 molecules were manually inspected using the
following criteria: (i) docking pose overlapping with the co-
crystallized β-OG ligand, (ii) interaction with binding site amino
acids, (iii) scaffold diversity, (iv) commercial availability, and (v)
synthetic tractability for potential modifications. All molecules
containing unwanted structural features were removed such as
those with readily hydrolizable and/or highly electrophilic
functional groups. Finally, 23 compounds were taken into
consideration for in vitro studies (Scheme S1, Supplementary
Information) and were either purchased or synthesized. In
particular, compound 2 was prepared and the synthetic route is
shown in Scheme 1. The quinazoline ring was installed by
3
1
N
Cl
HN
O
H
N
O
O
HO
H
N
NH
OH
S
O
S
N
NH
5
4
O
CF3
Figure 1. Chemical structures of novel anti-dengue inhibitors.
Antiviral activity was next assessed using a reporter DENV
assay based on the measurement of luciferase expression from a
recombinant fully functional virus bearing a luciferase coding
sequence in the context of the viral genome.21,22 After infection of
cells in culture with the reporter DENV, luciferase activity first
peaks at 8 hours as a result of translation of the incoming
genomes, reflecting virus entry into the host cells. At 24 hours
post infection, amplification of the genome by the viral
polymerase results in an increase in luciferase activity that
reflects virus RNA synthesis. Luciferase activity was measured at
8 and 24 hours post infection with the reporter DENV in
untreated or treated cells with each of the 23 compounds
identified in the virtual screening. The results showed that 5 hits
displayed antiviral activity against DENV (Figure 1, Table 1).
Compounds 2, 5 and 4 showed marked antiviral inhibition at 8
hours post infection with a good selectivity index. Compounds 1
and 3 showed moderate inhibitory activities and no cytotoxicity
was detected at 100 µM. In order to examine the selective
antiviral potential of the compounds identified in the virtual
screening, we tested these compounds against bovine viral
diarrhea virus (BVDV), another member of the Flaviviridae
family. None of them demonstrated significant antiviral activity
at 50 µM. Taken together, our results suggested that active
compounds interfere with early steps of the DENV life cycle,
most likely during viral entry.
o
condensing aniline 6 with formamide at 150 C. Heating of
compound 7 in excess of thionyl chloride afforded 4-chloro
quinazoline 8. The nucleophilic displacement of chlorine in
compound 8 by 2-amino-2-pyridinol proceeded in the presence of
o
Cs2CO3 in DMF at 100 C in a good yield, and produced the
intermediate 9. Finally, the target compound 2 was obtained by
amidation with 5-(pyridin-2-yl)thiophene-2-carboxylic acid
mediated
by
BOP
[1-benzotriazolyl-oxytris-
(dimethylamino)phosphonium-hexafluoro-phosphate]
as
a
coupling reagent (Scheme 1).
Table 1. In vitro antiviral effect, cytotoxicity and selectivity of
compounds identified (as hits) from the virtual screening.
EC50 ( µM)a
CC50
SIc
(DENV)
Compound
(µM)b
DENV-2d BVDV
22.7 1.2 >50
1
2
3
4
5
a
>100
>100
>100
>100
>100
>5
3.1 1.1
>50
>32
>4
28.7 1.0 >50
10.8 1.1 >50
>9
5.1 0.9
>50
>20
Scheme 1. Synthetic Route for the Preparation of compound 2. Reagents
and conditions (a) Formamide, 150 oC; (b) SOCl2, reflux, 8 h; (c) 5-
d
EC50: 50 % effective concentration 8 h post infection

b CC50: 50 % cytotoxic concentration
c SI: selectivity index
All the compounds also had a reasonable stability in buffer
(>48 h), so no intrinsic stability issues were expected
To better characterize the likely interaction of the new ligands
with protein E, and to explore the role of water molecules (cf.
Ref. 23) (omitted during the docking stage), that might be
involved in mediating protein-ligand interaction, we performed
100 ns MD simulation on the most active compound (2). The
docked pose of compound 2 was used as the initial conformation.
It can be observed that the protein remains stable throughout the
simulation (Figure S2). The predicted binding pose of 2 within
the E protein is displayed in Figure 2a.
The 4-substituted 7-chloro-quinazoline moiety remains
exposed to the solvent, while the pyridine attached to it makes
contacts with Thr48, Glu49, Leu135, Gln271 and Leu277 (Figure
2b); the system is further stabilized by a dynamic moderate
hydrogen bond between the pyridine N and the HN of Ala50,
exhibiting an interatomic distance below 2.5 Å during half of the
last 50 ns simulation. The O in the amide is intermittently
exposed to the solvent through a narrow channel. Several water
molecules were found throughout the simulation within the
binding site, bridging the interaction of the ligand with the
protein. A water molecule with almost 100% occupancy in the
last 50 ns makes hydrogen bonds with the N of the terminal
pyridine, the hydroxyl group of Tyr137, the carboxylic O of
Thr280, and another water molecule, which belongs to a small
conserved cluster of three buried water molecules that also
interact with the HN of Leu 191 and His282, the carboxylic O of
Thr189 and the side chain of His282. A stable π-π interaction
between the terminal pyridine ring and Phe193 is also observed
throughout the simulation, with an average ring centroid distance
of 3.7 Å and an average planes angle of 14° (Figure 1). The 2-
(thiophen-2-yl) pyridine moiety is stabilized by hydrophobic
contacts with Val130, Leu191, Asp192, Leu207, Ile270, Phe279,
and Thr280. It should be stressed that compound 2 overlaps very
well with co-crystallized ligand β-OG (Figure 2c). The predicted
protein-ligand interaction pattern is in line with recent models.14
Figure 2. Predicted interaction of compound 2 within the E
protein β-OG binding site, extracted from the molecular
dynamics simulation. (A) Ribbon representation of protein E
with compound 2 within the binding site (B). Interaction of 2
with the binding site amino acids and water molecules. For
simplicity, backbone nitrogens, carbonyl oxygens are not
displayed, except when interacting directly with 2. Only polar
hydrogens are shown. Hydrogen bonds are shown as a line of
colored spheres. Color code: 2, yellow carbons; DENV E
carbons, white; oxygens, red; nitrogens, blue; sulphur, green;
polar hydrogens, dark grey. (C). Superposition of 2 with the β-
OG ligand within the binding site. Color code: DENV E protein,
gray ribbon; β-OG, white carbons.
Finally, the pharmacologic characteristics of drug candidates
were predicted using QikProp program19,24 and are summarized
in Table 2. QikProp generates physically relevant descriptors and
uses them to perform ADMET predictions. A compound is
viewed as potentially problematic if it does not satisfy a “rule-of-
three”: predicted log S > -6.0, PCaco > 30 nm/s, and maximum
number of primary metabolites of six. The most potent
compounds reported here compare favorably with all these limits.
In humans, the pH value in stomach is 1-2, while in small
intestine it is 5-7. Because compound solubility often depends on
pH in solution, it is important to consider different pH buffers.
Taking into account the relevance of drug solubility, in vivo
absorption and distribution, we tested the in vitro solubility and
stability in PBS media for active compounds 2, 4, 5 at pH 2.0,
6.4, and 7.4. The solubility data are summarized in Table 3; all
compounds showed good solubility and are well inside the range
normally observed for oral drugs, 4-4000 µg/mL24 corresponding
to an S of 10-2 -10-5 M for compounds with a molecular weight of
400. The ClogP values for the listed compounds are between 3
and 5, which are in the normal range of 0-5 for oral drugs 25. The
aqueous solubility pattern of the assayed compounds was similar
to the predicted solubility.
Compounds 2, 4 and 5 showed low to moderate solubility
ranging from 1-10 µg/mL at pH 7.4. However, these compounds
are more soluble at pH 2.0 and pH 6.4.
Figure 3. Plane angles (left panel) and ring centroid distances
(right panel) for the π-π interaction between Phe193 and the
terminal pyridine moiety of compound 2

Table 3. Calculated pharmacological characteristics, experimental solubility and stability for active compounds.
f
Aqueous Solubility
PBS
EC50
(μM)a
b
c
d
e
stability
Compound
QlogP
QlogS
QPCaco
#met
µg/mL
pH
(h)
2.0
6.4
7.4
2.9
6.1
1.2
2
4
5
3.1
4.2
3.9
3.7
-4.5
-5.2
-5.2
785.8
520.5
1735.6
4
5
3
12.7
48.3
8.2
10.1
32.1
13.1
>48
>48
>48
10.8
5.1
aEC50 (µM) was calculated from dose–response curves by Prism software. The results represent the average of two or more independent experiments.
b octanol/water partition coefficient (log Po/w)
c aqueous solubility
d Caco-2 cell permeability
e number of metabolites
f Experimental solubility at pH 2, 6.4, 7.4. Reference values at pH 6.4 for piroxicam 7.3 µg/mL and efavirenz 68. 3 µg/mL. Values are means of two independent
experiments, each running triplicate
Here, we have described a prospective structure-based drug
discovery approach to identify novel and potent inhibitors of
DENV entry targeting protein E. We performed a multistep
hierarchical high-throughput docking of the 110000 Maybridge
compounds library, leading to the identification of the
quinazoline hit 2 with an EC50 of 3 µM, and compound 5 with an
EC50 of 5 µM, both with a good therapeutic index. Molecular
docking for compound 5 and molecular dynamics approaches for
2 predicted strong interactions of both compounds with the key
amino acids in the β-OG binding pocket of protein E that are
crucial for membrane fusion to deliver the viral genome in the
cytoplasm of the infected cell, in agreement with earlier
works.13,14 In summary, we have provided novel DENV
inhibitors that interfere early during infection, stablishing a
platform for the development of new and more potent antivirals
against this important human pathogen.
Acknowledgments
This work has been supported by the Agencia Nacional de
Promoción Científica y Tecnológica, Argentina (PICT 2013-0078
and PICT 2014-1884 to MB; PICT 2014-3599 to CNC and MB),
CONICET (PIP 2014 11220130100721 to MB and CNC), and
FOCEM-Mercosur (COF 03/11).
MB thanks William Jorgensen for providing an academic license
for QikProp software. CNC thanks Molsoft LLC for providing an
academic license for the ICM program. The authors thank the
National System of High Performance Computing (Sistemas
Nacionales de Computación de Alto Rendimiento, SNCAD) and
the Computational Centre of High Performance Computing
(Centro de Computación de Alto Rendimiento, CeCAR) for
granting use of their computational resources, and Diego Alvarez
for technical help and critical discussion of the manuscript.
Computational methods
High-throughput docking
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