DDX3X Helicase Inhibitors as a New Strategy to Fight the West Nile Virus Infection

Article abstract of DOI:10.1021/acs.jmedchem.8b01403

Increased frequency of arbovirus outbreaks in the last 10 years represents an important emergence for global health. Climate warming, extensive urbanization of tropical regions, and human migration flows facilitate the expansion of anthropophilic mosquitos and the emerging or re-emerging of new viral infections. Only recently the human adenosinetriphosphatase/RNA helicase X-linked DEAD-box polypeptide 3 (DDX3X) emerged as a novel therapeutic target in the fight against infectious diseases. Herein, starting from our previous studies, a new family of DDX3X inhibitors was designed, synthesized, validated on the target enzyme, and evaluated against the West Nile virus (WNV) infection. Time of addition experiments after virus infection indicated that the compounds exerted their antiviral activities after the entry process, likely at the protein translation step of WNV replication. Finally, the most interesting compounds were then analyzed for their in vitro pharmacokinetic parameters, revealing favorable absorption, distribution, metabolism, and excretion values. The good safety profile together with a good activity against WNV for which no treatments are currently available, make this new class of molecules a good starting point for further in vivo studies.

Full text of DOI:10.1021/acs.jmedchem.8b01403

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Article
DDX3X helicase inhibitors as new strategy to fight West Nile Virus infection
Annalaura Brai, Francesco Martelli, Valentina Riva, Anna Garbelli, Roberta
Fazi, Claudio Zamperini, Alessandro Pollutri, Lucia Falsitta, Stefania Ronzini,
laura maccari, Giovanni Maga, Simone Giannecchini, and Maurizio Botta
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01403 • Publication Date (Web): 05 Feb 2019
Downloaded from http://pubs.acs.org on February 6, 2019
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DDX3X helicase inhibitors as new strategy to fight
West Nile Virus infection
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Annalaura Brai,1,2 Francesco Martelli, Valentina Riva, Anna Garbelli, Roberta Fazi,
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Claudio Zamperini,1,2 Alessandro Pollutri, Lucia Falsitta, Stefania Ronzini, Laura
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1,2,5,*
Maccari, Giovanni Maga, Simone Giannecchini and Maurizio Botta
1Dipartimento Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Via A.
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De Gasperi 2, I-53100 Siena, Italy. Lead Discovery Siena S.r.l., Castelnuovo
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Berardenga, I-53019 Siena, Italy. Department of Experimental and Clinical Medicine,
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University of Florence, I-50134 Florence, Italy. Istituto di Genetica Molecolare, IGM-
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CNR, Via Abbiategrasso 207, I-27100 Pavia, Italy. Biotechnology College of Science
and Technology, Temple University, Biolife Science Building, Suite 333, 1900 N 12th
Street, Philadelphia, Pennsylvania 19122.
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KEYWORDS: DDX3X • antivirals • West Nile Virus• arbovirus • host factors
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ABSTRACT: Increased frequency of arbovirus outbreaks in the last ten years
represents an important emergence for global health. Climate warming, extensive
urbanization of tropical regions and human migrations flows facilitate the expansion of
anthropophilic mosquitos and the emerging or re-emerging of new viral infections. Only
recently the human ATPase/RNA helicase X-linked DEAD-box polypeptide 3 (DDX3X)
emerged as a novel therapeutic target in the fight against infectious diseases. Herein,
starting from our previous studies a new family of DDX3X inhibitors was designed,
synthesized, validated on the target enzyme and evaluated against West Nile Virus
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(WNV) infection. Time of addition experiments after virus infection indicated that the
compounds exerted their antiviral activities after the entry process, likely at the protein
translation step of WNV replication. Finally, the most interesting compounds were then
analyzed for their in vitro pharmacokinetic parameters, revealing favorable ADME
values. The good safety profile together with a good activity against WNV for which no
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treatments are currently available, make this new class of molecules a good starting
point for further in vivo studies.
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INTRODUCTION
West Nile Virus (WNV) is a neurotropic arbovirus member of Flaviviridae family1.
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Since its introduction in Europe and America the number of novel infections is
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increasing, counting during 2017, 204 novel reported cases in Europe and 2,002 in
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America . The number is impressive considering that about 80% of WNV infections are
usually asymptomatic and only the remaining 20% may cause meningitis, encephalitis
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or rarely in death 1% . In addition, probably due to climate warming, the number of
novel human cases sharply increased in EU member States during the current year,
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reaching 710 infections and 63 deaths as of 30 August. The rapid diffusion of WNV is
dependent on its zoonotic life cycle, that involves migratory birds, Culex and Aedes
mosquitos and mammals including horses and humans. In this context inadequate
vector control, globalization and human travel networks are only a few factors that are
contributing to this and other arboviruses spread. Despite continuous efforts have been
made to identify new drugs (Figure 1) or vaccines, no specific therapy or prophylaxis is
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actually available on the market and the treatment remains symptomatic . In this
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context, drug repurposing strategy provided new drug candidates to fight emerging and
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re-emerging viruses , but often the antiviral compounds are poorly active or are direct
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on viral enzymes and are thus generally characterized by a low genetic barrier to
resistance (Figure 1).
Figure 1. 2D chemical structures of the known WNV inhibitors and their potential
inhibitory target
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In the last years the human ATPase/RNA helicase X-linked DEAD-box polypeptide 3
(
DDX3X) protein has been identified as a component of critical host-cell pathways,
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hijacked by several pathogenic human viruses . DDX3X is an ATP-dependent RNA
helicase involved in many aspects of RNA metabolism such as transcription, translation
and RNA decay. It localizes to P-bodies, cytoplasmic foci related to mRNA turnover
which are disrupted in response to the infection of viruses such as WNV or Dengue
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virus (DENV) . Furthermore, after WNV infection DDX3X was released from P-bodies
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and co-localized at a perinuclear region with viral NS3 . It was found, moreover, that
DDX3X knockdown strongly decreased WNV replication, suggesting an important
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implication not yet completely understood . Although its role in the life cycle of different
viruses was extensively studied11-16, only few DDX3X inhibitors have been published as
antiviral agents17-21, and the mechanism of action is clearly described only for HIV-122.
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In a recent work we identified the first inhibitor of the helicase binding site of DDX3X
also active against WNV. Such compound belonged to a family of DDX3X RNA-
competitive molecules characterized by high selectivity and no cytotoxic effects in both
in vitro and in vivo models. Among them, compound 16d (compound 1 of the
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manuscript), belonging to the urea series, was the first DDX3X inhibitor endowed with a
broad spectrum antiviral activity against HIV-1 (both wild-type and drug-resistant
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strains), HCV, and new emerging viruses (WNV and DENV) . Despite 1 represented
the proof of concept that DDX3X inhibitors could be used as novel antivirals drugs, its
poor aqueous solubility limited its bioavailability in preclinical models causing its
accumulation in fat tissues (LogS=-7.05).
With the attempt to enlarge our library of DDX3X inhibitors we performed a homology
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model-based virtual screening that led us to identify another small molecule inhibitor of
DDX3X helicase activity (compound 2 of the present paper) characterized by a
sulfonamide moiety. Despite its low passive permeability, compound 2 was
characterized by a very good aqueous solubility and at the same time by a promising
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anti-enzymatic activity value of 0.36 µM . Pursuing our research of novel drug-like
DDX3X inhibitors, we performed a docking analysis of the two hit compounds 1 and 2.
On the basis of the 3D overlap of the two molecules within the biding site, we designed
a new series of hybrid compounds by merging the key structural moieties of 1 and 2
able to interact with the main residues involved in the binding of both of them (Arg276,
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Pro274 and Arg480). Novel compounds (8-20 and 31-36 ) were then synthesized and
validated against DDX3X enzyme and evaluated for their ability to inhibit WNV
replication, leading to a small library of derivatives endowed with antiviral activities
comparable or higher than the broad spectrum antiviral compound ribavirin. Moreover,
time of addiction experiments highlighted that compounds likely act in the first phases of
WNV replication. Finally, ADME analysis of the most potent derivatives revealed 100-
times improved aqueous solubility with respect to the hit 1, thus allowing to overcome
an important limitation of our compound.
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RESULTS AND DISCUSSION
Molecular modelling
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With the purpose of identifying novel small molecules inhibitors of the DDX3X helicase
binding site, we analyzed the docking poses of our hit compounds 1 and 2 within the
DDX3X RNA binding pocket. The 3D structure of DDX3X in its RNA-bound closed
conformation previously generated by homology modeling20 was used to perform our
studies. As shown in Figure 2A, the docking analysis of compound 2 highlighted
favorable hydrogen bond interactions between the nitrobenzene oxygen atom and
Arg276 and Gly325, as well as between the sulfonamide group and Pro274 and Arg480,
while the hydroxyl substituent is engaged in profitable H-bond interaction with the
residues Pro274 and Arg503. Furthermore, hydrophobic interactions were found with
Phe357, Val500, and Arg503. The predicted binding mode of compound 1 (Figure 2A)
confirmed some key interactions as seen for compound 2, more in detail the triazole
ring interacted with the guanidine group of Arg276, whereas the urea NH groups and
carbonyl moiety were involved in hydrogen bonds with Pro274 and Arg480 respectively.
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Moreover, the butyl terminus established beneficial interactions with Arg326 and
Gly302. Finally, the tolyl ring established hydrophobic contacts with Arg503 and Pro274
side chains, and the phenyl ring made hydrophobic interactions with Phe357.
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Figure 2: A) Overlapped binding mode of compound 1 (pink) and 2 (blue marine) in
DDX3X helicase pocket. Main interactions of both compounds with key residues of the
active site are highlighted. Remarkably, Arg276, Arg480 and Gly302 take important
contacts also with RNA phosphate backbone while Gly325, Pro274 and Arg503 interact
directly with the nucleobases. B) 2D structure representation of schematic idea behind
the merged series design. C) Proposed binding mode for merged derivative 13.
It is worthy to note that two compounds were both able to interact with Arg276 and
Arg480 and to take profitable contacts also with Gly325, Pro274 and Arg503 that our
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computational model20 suggested to be important in RNA strand interactions into the
helicase binding site (Figure 3).
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Figure 3. Surface and cartoon representation of RNA’s main interactions of within the
helicase binding site of our homology model.
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20
Starting from this analysis, we merged the key structural units of 1 and 2 to provide a
new series of compounds able to interact at the same time with most of the residues
involved in the binding of both of them, with a particular attention to Arg276, Pro274 and
Arg480 since their suggested role in RNA strand binding. To this aim, as shown in
Figure 2B, the n-alkyl substituted triazole group was selected from 1 structure, while the
phenyl-substituted sulfonamide moiety was chosen from the structure of compound 2;
the ‘hybrid‘ structure was finally composed by using the common aromatic ring (B) as
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linker and a small library of compounds were designed around this starting structure to
explore the chemical space in relation to the activity against DDX3X.
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Chemistry
The synthesis of compounds was accomplished following the synthetic route reported
in Scheme 1. Aminothiophenol 3 was first converted into azide 4 by diazotization,
subsequent click reaction with the opportune terminal alkynes 5a-c provides triazoles
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6a-c. Oxidative chlorination of thiols 6a-c led to the corresponding sulfonyl chlorides
intermediates that were immediately converted into final compounds 8-20 by
nucleophilic reaction with the opportune aromatic amines.
Scheme 1. Synthesis of sulfonamides 8-20.
i.
ii.
+
N
R1
H N
2
SH
N3
SH
N
SH
N
R1
3
4
5a-c
6a-c
iii.
H
N
H
N
N
N
N
S
R2
iv.
N
N
S
O O
R2
N
O O
R1
R1
8
-20
7a-c
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8
9
Reagents and conditions: i. a) t-BuONO, ACN, 20 min. 0°C; b) TMSN , ACN, 2h r.t.; ii.
3
CuSO ·5 H O, sodium ascorbate, H O t-BuOH (1:1), MW 10 min, 120°C; iii. H O ,
4
2
2
2
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
SOCl ACN, from 0 °C to rt, 15 min; iv. opportune amine, Pyridine (dry), 5h r.t.
2,
Scheme 2. Synthesis of ethers 5a-b.
i.
( )n
OH
( )n
OR1
21 n=1
22 n=2
5a n=1; R =CH CH
1 2 3
5b n=2; R =CH3
1
Reagents and conditions: i. a) NaOH (aq) (6M), 20 min., r.t; b) dimethylsulfate or
diethylsulfate, 60 min, 50-55°C.
Terminal alkynes 5a and 5b were respectively synthesized from propargyl alcohol and
but-3-yn-1-ol by Haworth alkylation, and purified by distillation (Scheme 2). As shown in
Scheme 3, aniline 25 was synthesized by nucleophilic displacement of 1-fluoro-3-
nitrobenzene 23 with morpholine in presence of K CO and subsequent reduction Pd on
2
3
charcoal catalysed.
Scheme 3. Synthesis of aniline 25.
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2
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
N
O
F
i.
ii.
N
NO2
NO2
NH2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
23
24
25
Reagents and conditions: i. a) morpholine, K CO , DMSO (dry), 3h, 200° C, ii. H , Pd/C,
2
3
2
MeOH, 1h.
As shown in Scheme 4, the synthetic route to the final compounds 31-34 first entailed
the preparation of the common azide intermediate 30, using synthetic procedures
described in Scheme 1. Derivatives 31-34 were then obtained by click reaction with the
opportune terminal alkyne. Para-substituted compounds 35 and 36 were synthesized
analogously.
Scheme 4. Synthesis of sulfonamides 31-34.
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CF3
CF3
CF3
H
H
H N
2
i.
N
ii.
N
Cl
O N
2
S
+
O N
2
S
H N
2
S
O O
O O
O O
26
27
28
29
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
iii.
CF3
iv.
CF3
H
N
H
N
N
S
O O
N3
S
O O
R1
N
N
31-34
30
Reagents and conditions: i. Pyr, 5h r.t. ii. H , Pd/C, MeOH; iii a)t-BuONO,CH CN, 20
2
3
min. 0°C; b) TMSN , CH CN, 2h r.t.; iv. Opportune terminal alkyne, CuSO ·5 H O,
3
3
4
2
sodium ascorbate, H O tBuOH (1:1), MW 10 min, 120°C.
2
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Biological evaluation
Derivatives 8-20 and 31-36 were first evaluated for their ability to inhibit the helicase
activity of DDX3X, results were reported in Table 1. Seven compounds showed
promising inhibitory values higher than of 80% (compounds 8, 9, 10, 14, 15, 19 and 31).
The meta substitution of triazole on ring B was more favorite with respect to the para
position, being 36 about 26% less active than the corresponding meta derivative 8. This
is also confirmed by the docking studies, due to the higher shape complementarity of
the binding site and 8 with respect to 36. Three dimensional conformation of the latter
results to be much more constrained within the pocket, and this is likely to be
responsible for the lower activity.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Replacement of triazole butyl chain with isopentyl was well tolerated (compound 31),
when the length was reduced to 2 carbons, the activity decreased of about 25% in
compound 33, in line with a lower occupancy of the binding site and poorer
complementarity between ligand and protein cavity as also provided by the docking
model. Yet according with this observation, the introduction of bulkier substituents such
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as cyclohexyl and phenyl was detrimental for the activity which decreased to 41% and
52% for compounds 34 and 32, respectively.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Alkyl chain of the triazole ring was also explored by checking the effect of introducing
a polar atom. As a result, methyl-ethyl ether derivatives 10 and 15 retained their
activities; in contrast ethyl-methyl ether 11 was found 31% less active than the
corresponding isomer 10. According to our docking studies, the ether oxygen atom can
establish a hydrogen bond interaction with Arg326. In fact, in the ethyl-methyl ether
derivatives, distance between the ether oxygen atom and its counterpart is less
favorable to the hydrogen bond interaction. On the whole, the pocket hosting the
substituted triazole moiety requires a precise spatial disposition (meta substitution
preferred over the para one) while preferring more flexible-less constrained triazole
substituents (butyl and isopentyl preferred over phenyl and cyclohexyl rings) occupying
as much space as possible (lower activity in the presence of ethyl substituent). Polar
atoms are tolerated, and a proper distance to efficiently interact with Arg326 appears to
favor compound activity.
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Analogously, the pocket that houses the other terminal side of our compound series,
i.e. ring A, also presents distinctive hallmarks. In particular, a proper combination of
both polar and hydrophobic interactions appears to be beneficial to compounds’ activity.
Noteworthy, the presence of a hydrogen bond acceptor on ring A leads to high % of
DDX3X inhibition across this chemical series, being hydrogen bond interactions feasible
with Pro274 and Arg503 of the binding pocket as revealed by our docking studies. On
confirmation of that, the introduction of an isoquinolyl- ring (compounds 14 and 15) on
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ring A was well tolerated as well as the -OH and the –OCH ortho substitution
3
(compounds 8 and 19), all bearing hydrogen-bonding moieties. Also compounds 9 and
10, bearing the –CF moiety at the ortho position of ring A, satisfy such requirements,
3
being the trifluoromethyl group reported to have both hydrophobic and polar (hydrogen-
bonding) properties.23 Substitution of the trifluoromethyl or hydroxyl groups of
compounds 8, 9, 10 with a methyl group (compound 13), which shows only hydrophobic
properties, leads to a lower DDX3X % of inhibition in the sulfonamide series. Activity
further decreases in the presence of a fluorine or hydrogen substituents (compounds 12
and 20) which have similar % of inhibition values. Lower activity of compound 12 is
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9
likely to be due to the lower occupancy combined with a less effective hydrogen
bonding interaction, both exerted by the fluorine atom, the unsubstituted derivative 20
likewise showed a reduced activity value of 31%. The abolishment of the activity for
compound 16, where a morpholine moiety was introduced on ring A, is explained by
docking studies to be due to the spatial constraints of region of the binding site, being
able to accomodate the planar isoquinoline system but unfavorable to host the
morpholine ring.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Also, the ortho position of the substituents on ring A results to be preferred, although
the para- methoxy substitution of derivative 18 is tolerated with a slight loss of activity.
Unfortunately, the low solubility of 17and 35 limited their enzymatic evaluation.
Table 1. DDX3X anti-enzymatic activity.
[b]
[b]
Cmpd.ID[a]
Inhibition
(%)
Cmpd.ID[a]
Inhibition
(%)
Structure
Structure
H
H
N
N
H
N
O
N
S
N
N
N
N
N
O
O
1
98±1
17
O
nd
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
OH
H
H
N
N
S
N
N
N
S
NO2
O
O
O
2
8
O
O
98±2
83±3
18
19
80±1
OH
H
O
N
N
N
H
S
N
N
N
O
O
S
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
O
O
98±0.5
CF3
H
H
N
N
N
N
N
S
N
N
N
S
N
N
O
O
O
O
9
98±1
20
31
31±2
96±1
CF3
CF3
H
H
N
N
N
N
N
S
S
N
N
O
O
O
O
10
98±0.5
O
CF3
H
CF3
H
N
N
N
N
N
S
N
S
N
O
O
O
O
11
67±2
24±2
32
33
52±2
71±2
O
F
H
N
N
CF3
H
S
N
N
N
N
O
O
S
N
N
1
2
O
O
CF3
H
H
N
N
N
N
S
N
S
N
N
N
O
O
O
O
13
59±2
34
41±2.1
N
N
N
H
N
N
N
S
N
CF3
H
N
O
O
N
S
1
1
4
5
99±0.5
98±1
35
36
nd
O
O
N
N
N
H
N
N
N
S
N
OH
N
H
O
O
N
S
57±0.5
O
O
O
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9
O
H
N
N
N
N
S
N
O
O
1
6
O
na
[
a] Data represent mean two values of the least two experiments. [b] % inhibition at a fixed concentration of 20 µM.
na: not active. nd: not determined, compound precipitated from medium
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The docking studies suggested that compounds could act as competitive inhibitors in
the helicase binding site. In order to prove our hypothesis, we generated dose-response
curves titrating compound 8 in DDX3X helicase assays, in the presence of increasing
fixed concentrations of the RNA substrate. As shown in the new Figure 4, the inhibitory
potency (ID50) of the compound decreased (higher absolute values) as the RNA
concentrations increased. This is consistent with the hypothesized competitive
mechanism of inhibition, with respect to the RNA substrate.
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Increasing concentrations of the inhibitor 8 were titrated in helicase assays,
in the presence of 30 pmols of DDX3X and increasing fixed concentrations of the
dsRNA substrate. Values are the mean of three independent measurements ± S.D. The
residual enzymatic activity values were plotted as a function of the inhibitor
concentrations and the corresponding ID50 values calculated for each dsRNA
concentration are indicated on the right side of the panel.
The compounds described here have been rationally designed to specifically target
the helicase activity of DDX3X. In order to confirm the selectivity of our compounds, we
tested the ability of 1, 2, 9 and 14 to inhibit the ATPase activity of DDX3X and the
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helicase activity of the related human DEAD-box helicase DDX1. As shown in Table 2,
no inhibition was found with any compound concentration.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. Enzymatic data on selected compounds
ATPase DDX3
IC50, µM
DDX1
IC50, µM
Cpd ID
1
2
9
>200[a]
>200[a]
>200[a]
>200[a]
>200[a]
>200[a]
>200[a]
>200[a]
1
4
[
a] The value >200 indicates that less than 20% of inhibition was observed at 200 µM,
the highest concentration tested.
Then, we quantified the cellular concentrations of the protein DDX3X, in order to
confirm the presence of our target before carrying out anti-WNV and cytotoxicity assays.
As reported in Table 3, African green monkey kidney epithelial (VERO) cells are
endowed by a DDX3X concentration of 421 nM, higher than adenocarcinomic human
alveolar basal epithelial cells (A549) but lower than human hepatocellular carcinoma
cell line Huh-7 (respectively valued 103 nM and 755 nM).
[a]
Table 3. DDX3X cellular expression in Huh-7, A549 and VERO .
Huh-7[b]
VERO[c]
A549[d]
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
DDX3X (nM)
755±170
421±140
103±20
[a] Values represent mean±S.D of three independent experiments. For details see Methods [b] Evaluated in
Huh7: Hepato cellular carcinoma cells. [c] Evaluated in VERO: African green monkey kidney cells. [d]
Evaluated in A549: adenocarcinomic human alveolar basal epithelial cells.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The three cell lines were infected with WNV at a MOI of 0.1, and the antiviral activity
of compound 9 was assayed. As reported in Table 4 its activities seemed proportional to
the DDX3X concentrations. Probably due to the DDX3X implications in pulmonary
25
cancer , 9 shown a cytotoxic activity of 21.3 μM. For these reasons, in order to avoid
interferences due to anticancer related cytotoxic effects, the human Huh-7 cells
characterized by a higher DDX3X concentration of 755 nM were chosen to perform our
studies.
[
a]
Table 4. Comparison of antiviral activities of 9 in different cell lines infected with WNV .
Huh-7[d]
VERO[e]
A549[f]
Cpd. ID
EC50[b]
CC50[c]
EC50[b]
CC50[c]
EC50[b]
CC50[c]
(μM)
(μM)
(μM)
(μM)
(μM)
(μM)
9
23.1 + 0.4
90 + 2.5
>200
>200
12 + 1.1
95 + 5.1
>200
nt
3.4 + 0.8 21.3 +_1.1
46 + 2.3 nt
ribavirin
[
a] Data represent mean two values of the least two experiments. [b] EC : half
50
maximal effective concentration or needed concentration to inhibit 50% viral induced cell
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9
death [c] CC Cytotoxic concentration 50 or needed concentration to induce 50% death
50
of non-infected cells [d] Evaluated in Huh7: Hepato cellular carcinoma cells. [e]
Evaluated in VERO : African green monkey kidney cells. [f] Evaluated in A549:
adenocarcinomic human alveolar basal epithelial cells. Ribavirin was used as a
reference compound. Nt= not tested
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Subsequently, Huh-7 cells were infected with WNV, and compounds endowed with
the best IC s against DDX3X were assayed for their antiviral properties. As reported in
50
Table 5, compounds showed interesting antiviral activities, ranging from 2.3 to 65.9 μM,
comparable or even higher than the broad spectrum antiviral ribavirin, which was used
as a reference compound. Compound 8 showed the highest antiviral activity value of
2.3 μM, followed by 15, 9, 13, 14 and 10. All these compounds had comparable anti-
enzymatic profiles; the overall trend of their cellular activities could be ascribed to their
different physico-chemical parameters, which may influence the cellular penetration,
and consequently the antiviral effects. Compound 15 resulted to be particularly
promising, since it showed a good antiviral activity together with favorable aqueous
solubility, 100 times higher than hit compound 1. Compound 2 showed the lowest
activity, probably due to the limited cellular permeability (see ADME Section Table 6).
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Journal of Medicinal Chemistry
Page 28 of 69
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The mild antiviral activity of compound 13 is in line with its % inhibition of about 60%
against DDX3X and was considered not worthy of further investigation Finally,
compound 12 endowed with the worst DDX3X inhibitory activity was found to be
inactive in the cell-based assay.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 5. Antiviral activities and cytotoxicity of selected compounds against WNV infected Huh-
[
a]
7
cells .
WNV[d]
Huh-7
Cpd. ID
EC50[b]
CC50[c]
(μM)
(μM)
1
2
8
9
8.8 + 0.2
120 + 5.2
2.3 + 0.5
23.1 + 0.4
65.9 + 2.3
>200
>200
175 + 11
>200
>200
1
1
0
2
>200
>
>
200
200
13
14
39.9 + 1.1
55 + 1.1
13 + 0.8
95.5 + 3.2
>200
>200
>200
15
ribavirin
[a] Data represent mean + standard deviation of three experiments. [b] EC50: half maximal effective concentration or needed
concentration to inhibit 50% viral induced cell death [c] CC50 Cytotoxic concentration 50 or needed concentration to induce 50%
death of non-infected cells [d] evaluated in Huh7 cells. Nt= not tested. Ribavirin was used as a reference compound.
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9
In order to better evaluate the antiviral activities of our compounds, we selected 9 as
representative of the series, to investigate the mechanism of action (Figure 5). Time of
addition studies, performed at compound inhibitory concentration near and under the
IC values, clearly demonstrated that 9 acted in the first two hours of WNV life cycle,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
50
since the addition of 9 after 3 hours post infection did not affect viral replication (Figure
5
A). In particular, although an antiviral effect was visible when added before viral
infection, and cell-entry occurred, the substantial activity of the compound 9 also after
two hours of infection could be indicative that its main target activity was in a
subsequent steps of the viral replication. Consequently, we evaluated the effect of 9 in
the production of both genomic and complementary viral RNA. Importantly, our inhibitor
was able to interfere with the concentration of genomic RNA in the early time of
infection (from 2 h to 12 h post infection) only when added 1 h post infection without
significant changes when added 3 h post infection. Moreover, although the
complementary RNA was not revealed up to 12 hours post infection, 24 h post infection
the quantity of complementary RNA was slit lower after the addition of compound 9 1 h
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