Benzenesulfonamide Derivatives as Calcium/Calmodulin-Dependent Protein Kinase Inhibitors and Antiviral Agents against Dengue and Zika Virus Infections

Article abstract of DOI:10.1021/acs.jmedchem.9b01779

Emerging and resurging mosquito-borne flaviviruses are an important public health challenge. The increased prevalence of dengue virus (DENV) infection has had a significant socioeconomic impact on epidemic countries. The recent outbreak of Zika virus (ZIK

Full text of DOI:10.1021/acs.jmedchem.9b01779

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Article
Benzenesulfonamide Derivatives as Calcium/Calmodulin-
Dependent Protein Kinase Inhibitors and Antiviral
Agents Against Dengue and Zika Virus Infections
Wei-Chia Chen, Yogy Simanjuntak, Li-Wei Chu, Yueh-Hsin Ping, Yi-Ling Lee, Yi-Ling Lin, and Wen-Shan Li
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01779 • Publication Date (Web): 23 Jan 2020
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Benzenesulfonamide Derivatives as Calcium/Calmodulin-Dependent
Protein Kinase Inhibitors and Antiviral Agents against Dengue and
Zika Virus Infections
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Wei-Chia Chen,1,2,# Yogy Simanjuntak, * Li-Wei Chu , Yueh-Hsin Ping , Yi-Ling
3,#,
4,5
4,6
Lee, Yi-Ling Lin, * Wen-Shan Li2,8,9,10*
3
3,7,
1Department of Chemistry, National Taiwan Normal University, Taipei 11677, Taiwan.
2Institute of Chemistry, Academia Sinica, Taipei 11529, Taiwan.
3Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan.
4Institute of Biophotonics, National Yang-Ming University, Taipei 11221, Taiwan.
5Reseach Center for Applied Sciences, Academia Sinica, Taipei 11529, Taiwan.
6Department and Institute of Pharmacology, National Yang-Ming University, Taipei
1
1221, Taiwan.
7Genomic Research Center, Academia Sinica, Taipei 11529, Taiwan.
8Doctoral Degree Program in Marine Biotechnology, National Sun Yat-Sen University,
Kaohsiung 80424, Taiwan
9Ph.D Program in Biotechnology Research and Development, Taipei Medical University,
Taipei 11031, Taiwan
10Department of Medicinal and Applied Chemistry, Kaohsiung Medical University,
Kaohsiung 80708, Taiwan
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ABSTRACT
Emerging and resurging mosquito-borne flaviviruses are an important public health
challenge. The increased prevalence of dengue virus (DENV) infection has had a
significant socio-economic impact on epidemic countries. The recent outbreak of Zika
virus (ZIKV) has created an international public health emergency because ZIKV infection
has been linked to congenital defects and Guillain-Barré syndrome. To develop potentially
prophylactic antiviral drugs for combatting these acute infectious diseases, we have
targeted the host calcium/calmodulin-dependent kinase II (CaMKII) for inhibition. By
using CaMKII structure-guided inhibitor design, we generated four families of
benzenesulfonamide (BSA) derivatives for SAR analysis. Among these substances, N-(4-
cycloheptyl-4-oxobutyl)-4-methoxy-N-phenylbenzenesulfonamide (9) showed superior
properties as a lead CaMKII inhibitor and antiviral agent. BSA 9 inhibited CaMKII activity
with an IC value of 0.79 µM and displayed EC values of 1.52 µM and 1.91 µM against
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DENV and ZIKV infections of human neuronal BE(2)C cells, respectively. Notably, 9
significantly reduced the viremia level and increased animal survival time in mouse-
challenge models.
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INTRODUCTION
Dengue virus (DENV) and Zika virus (ZIKV) are mosquito-borne flaviviruses that
pose a threat to global public health. DENV infection in humans is associated with a wide
spectrum of clinical manifestations, which range from mild dengue fever to severe dengue
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hemorrhagic fever and life-threatening dengue shock syndrome . A recent study estimated
2
that DENV infection will reach 390 million cases per year . Although the mortality rate
has been controlled, recurrent dengue outbreaks have a significant socio-economic impact
3
in epidemic countries . Likewise, although the number of fatalities caused by ZIKV
infection is low, ZIKV infection has been linked to onset of Guillain-Barré syndrome and
4
, 5
to birth defects which include microcephaly . ZIKV infection has created an international
public health emergency. It was reported that approximately 4300 infants were born with
microcephaly during the recent outbreak in Brazil4.
Both members of the Flaviviridae family, DENV and ZIKA possess envelopes and
contain a positive-sense RNA genome encoding a polyprotein that is processed by cellular
and viral proteases to form 3 structural (C, capsid; prM, precursor membrane; and E,
envelope) and 7 nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins6,
7.
Viral infection begins with binding of the major structural envelope protein to the host
cell receptor protein followed by entry of the virion into the cell by endocytosis. The
internalized virions undergo acid-induced conformational changes and membrane fusion
to release the viral genome. Translation of viral RNA produces proteins required for viral
RNA replication through RNA-dependent RNA polymerization. The assembly of viral
RNA and viral proteins generates the mature viral particle, which is then released through
6
the cellular secretory pathway . Flaviviruses exhibit a broad tissue tropism. Although
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dendritic cells, monocytes/macrophages, and lymphocytes are considered the major sites
of viral replication, some other cells can be permissive to flavivirus during natural infection
including endothelial cells, neurons and microglia6, 8, 9
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Antiviral drugs and vaccines are two major approaches to control viral diseases. Since
most of the flavivirus infections result in acute diseases, the therapeutic window is narrow
and short. Vaccine development for preventing flavivirus infection has been hampered in
part by antibody-dependent enhancement. Preexisting antibodies that bind to E or prM of
DENV or ZIKV have been shown to enhance viral infection of monocytes through Fc
gamma receptors10, 11. For antiviral development, both viral and cellular proteins essential
for viral replication serve as potential targets. Members of Flaviviridae may share
12
similarities in their life cycles and host–pathogen interactions . Our recent study indicated
the role of calcium/calmodulin-dependent protein kinase II (CaMKII) in mediating the
1
3
binding/entry of Japanese encephalitis virus (JEV), also a member of Flaviviridae . JEV
activated CaMKII via dopamine D2 receptor-phospholipase C signaling to increase the
surface expression of JEV binding/entry molecule in neuronal cells. Therefore, CaMKII
may serve as a suitable target for the development of prophylactic drug against flavivirus
infection.
CaMKII is a serine/threonine-specific protein kinase that is regulated by the calcium
1
4
and calmodulin complex . Ubiquitously expressed in various tissues, CaMKII plays
distinct roles in neurons, cardiomyocytes, endothelial and immune cells14-16. Owing to the
elevated CaMKII activity associated with cardiovascular and brain disease17, 18,
pharmaceutical companies have initiated efforts to develop safe and effective therapeutics
1
7
targeting CaMKII . The goal of our studies in this area is to develop CaMKII inhibitors
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that can be utilized to determine if this kinase is a viable antiviral drug target and if it can
be used as a platform for antiviral drug design. The studies reported below demonstrated
that N-(4-cycloheptyl-4-oxobutyl)-4-methoxy-N-phenylbenzenesulfonamide (9), is a
novel CaMKII inhibitor that displays anti-DENV and anti-ZIKV activity in both cell and
animal studies.
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RESULTS AND DISCUSSION
Design and Synthesis of Benzenesulfonamides (BSAs) 1−16.
Several CaMKII inhibitors including KN-93 and KN-62 were developed previously
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to study the function of CaMKII . In those investigations, it was observed that the presence
of aromatic and sulfonamide groups in these substances is important for their inhibitory
1
9
activities (Chart 1) . Guided by these earlier findings, we designed several new BSAs
derivatives that we proposed would be CaMKII inhibitors. These substances were prepared
and their antiviral activities against DENV serotype 2 (DENV-2) and ZIKV infections were
assessed.
Chart 1. Commercially available and widely used CaMKII inhibitors.
The final lead compounds, 1 and 2, bearing sulfonamide and fluorobenzene moieties,
were prepared by amide bond forming HBTU/DIPEA promoted condensation of keto-acid
1
7 with aniline (Scheme 1). Reduction of the formed amide 18 by using lithium aluminum
2
0
hydride produced amine 19, which was utilized to form the target BSA 2 through reaction
with 4-methoxybenzenesulfonyl chloride. BSA 1 was generated by oxidation of 2 with
pyridinium dichromate in the presence of 4 Å molecular sieves21.
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O
O
H
OH
H
OH
NH2
N
N
(a)
(b)
O
O
F
F
F
19
17
18
O
O
(c)
O
O
O
O
(d)
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S
N
O
S
N
OH
F
F
2
1
Scheme 1. Reagents, conditions and yields: (a) HBTU, DIPEA, THF, 8 h, rt, 92%; (b)
o
o
LiAlH , THF, 4 h, 90 C, 90%; (c) 4-methoxybenzenesulfonyl chloride, Et N, THF, 60 C,
4
3
4 h, 75%; (d) PDC, DCM, 8 h, rt, 70%
BSAs 3 and 4, analogs in which the N-phenyl moiety in 1 is replaced by H and CH3,
respectively, were synthesized by the routes shown in Scheme 2. In both sequences, amino-
ketone 22 was prepared by reaction of chloride 20 with excess sodium azide and a catalytic
amount of potassium iodide to form the intermediate azide 21, followed by selective
reduction using palladium on activated charcoal and hydrogen gas. Reaction of 22 with 4-
methoxybenzenesulfonyl chloride furnished BSA 3. Preparation of BSA 4 began with
2
2
conversion of amine 22 to its N-methyl analog 23 through reductive methylation , and
subsequent methoxybenzenesulfonamide formation.
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O
O
HN
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F
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(
c)
O
O
O
Cl
N3
H N
(a)
(b)
2
F
F
F
20
21
22
O
(d)
O
O
O
S
N
O
(e)
H
N
4
F
F
2
3
o
Scheme 2. Reagents, conditions and yields: (a) KI, NaN , DMF, 4 h, 90 C; (b) Pd/C, H ,
3
2
o
MeOH, 8 h, rt; (c) 4-Methoxybenzenesulfonyl Chloride, Et N, THF, 60 C, 4 h, 48%; (d)
3
o
(
1) HCHO, NaOMe, MeOH, 90 C, 4 h, (2) NaBH , MeOH, rt, 4 h, 63%; (c) 4-
4
o
Methoxybenzenesulfonyl Chloride, Et N, THF, 60 C, 4 h, 78%
3
For structure–activity relationship studies, we designed and synthesized (Scheme 3)
BSA derivatives 5-16, which contain a variety of para-aryl and nitrogen substituents on
the benzenesulfonamide moiety, and several different groups on the acyl carbon of the
terminal ketone. The pathways for forming these BSAs began with reactions of the
2
3
appropriate primary amines 24-27 with γ-butyrolactone in the presence of sodium hydride
o
at 90 C to generate the corresponding amides 28-31, which were reduced using LiAlH to
4
form the corresponding secondary amines 32-35. Condensation reactions of these amines
with selected aryl ring substituted benzenesulfonyl chlorides followed by oxidation with
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pyridinium dichromate (PDC) afforded the respective aldehydes 43-49. Finally, addition
of excess amounts of the appropriate Grignard reagents24, 25 to 43-49 gave secondary
alcohols 50-56, which were oxidized by using PDC to produce the respective BSAs 5-16.
1
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H
H
NH2
(a)
N
(b)
N
R1
R1
OH
R1
OH
O
2
4 : R =phenyl
28-31
32-35
1
2
5 : R =cyclohexyl
1
2
6 : R =tetrahydronaphthalenyl
1
(c)
2
7 : R =4-cyclohexylphenyl
1
R1
37 : phenyl
R2
R2
R2
R2
trifluoromethoxy
phenoxy
methoxy
3
3
4
8 : phenyl
9 : cyclohexyl
0 : phenyl
methyl
O
O
(d)
(e)
O
O
S
N
OH
R3
41 : tetrahydronaphthalenyl methoxy
S
N
O
O
S
N
42 : 4-cyclohexylphenyl
methoxy
R1
R1
O
R1
OH
50-56
37-42
43-49
R1
R2
R3
(
f)
5
6
7
: phenyl
: phenyl
: phenyl
8 : phenyl
: phenyl
0 : phenyl
11 : phenyl
2 : phenyl
13 : phenyl
14 : cyclohexyl
methoxy
methoxy
methoxy
methoxy
methoxy
methyl
cyclohexyl
cyclobutyl
cyclopentyl
cycloheptyl
R2
9
1
trifluoromethoxy cyclopentyl
phenoxy
methoxy
methyl
cycloheptyl
adamantyl
cycloheptyl
cycloheptyl
cycloheptyl
cycloheptyl
1
O
O
S
N
O
methoxy
15 : tetrahydronaphthalenyl methoxy
6 : 4-cyclohexylphenyl
R1
R3
1
methoxy
5-16
o
Scheme 3. Reagents, conditions and yields: (a) (1) NaH, THF, 30 min, 90 C, (2)
o
o
Butyrolactone, THF, 8 h, 90 C, 90%; (b) LiAlH , THF, 8h, 90 C; (c) Sulfonyl Chloride,
4
o
Et N, THF, 60 C, 4 h, 76%; (d) PDC, DCM, 8 h, rt, 65%; (e) (1) R Br, Mg, THF, rt, 2 h,
3
2
o
(
2) Aldehyde, THF, 60 C, 2 h, 60%; (f) PDC, DCM, 8 h, rt.
Structure–Activity Relationship Screening
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BSAs 1-16 utilized in the SAR study contain four substructural units that correspond
to the phenyl, terminal, linker and benzenesulfonamide parts depicted generally in A in
Figure 1. The BSAs can be classified into four families (B-E in Figure 1) based on
differences in these parts.
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B
O
Benzenesulfonamide part
O
O
O
O
Linker part
O
O
O
S
N
O
O
O
O
O
O
O
O
S
N
O
S
N
OH
S
O
S
N
O
HN
F
F
F
F
Phenyl part
Terminal part
1
2
3
4
O
O
O
O
O
O
C
O
O
O
O
O
O
O
O
O
O
O
O
S
N
O
S
N
O
S
N
O
S
N
O
S
N
O
S
N
O
5
6
7
8
9
10
F3C
O
Ph
O
D
O
O
E
O
O
O
O
O
O
O
O
O
O
O
O
O
S
N
O
S
N
O
S
N
O
S
N
O
S
N
O
S
N
O
12
13
14
15
16
11
Figure 1. BSA 1 (A) and families of synthesized BSAs (B, C, D, E) used in the SAR study.
To assess the antiviral activities of the prepared BSAs, human neuroblastoma BE(2)C
cells were infected with the DENV-2 reporter virus DENV-2-eGFP that expresses a green
fluorescence protein. Specifically, BE(2)C cells in a 96-well plate were infected with
DENV-2-eGFP and then incubated in the absence or presence of 1.56 and 3.13 µM of the
BSAs for 24 h. DMSO was used as the solvent control. The antiviral activities of the
compounds were determined by measuring the intensities of eGFP emission from the cells
and are given in Table 1 as percentages of cells infected relative to that of the solvent
control. In addition, the effects of 6.25 µM of each BSA on hindrance of cell proliferation
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(
cytotoxicity) were determined by using the XTT assay and given in Table 1 as a
percentage of cell proliferation relative to that of the solvent control.
In studies with the BSAs in family B (Figure 1), we observed that treatment with BSA
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
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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
1
resulted in only 39.3% of the BE(2)C cells being infected by DENV-2 and a 56.1%
reduction of cell proliferation. In contrast, BSA 2 promoted only 48.5% cellular infection
and had an improved effect (76.3%) on cell proliferation BSAs 3 and 4 did not cause a
reduction in proliferation and had no antiviral potency against DENV-2 infection (3 =
1
22.6%; 4 = 100.9%, Table 1). These data indicate that the benzene ring is required for
anti-dengue activity of the BSA.
Table 1. Antiviral activities of the BSA, KN-62, and KN-93 against DENV-2-eGFP
reporter virus and effect of BSA on cell proliferation.
Relative eGFP intensity of
Cell proliferation of BSA treated-
a,b
a,c
the infected-cells
cells
BSA
BSA Concentration
[1.56 µM] [3.13 µM]
1
2
3
4
5
6
7
8
9
10
55.9
59.4
110.1
100
39.3
48.5
122.6
100.9
100.8
63.3
33.0
25.1
9.6
56.1
76.3
105.7
105.9
105.9
106.6
93.9
90.0
90.0
96.7
92.4
95.5
99.5
97.5
103.6
92.8
68.9
42.8
34.6
31.8
57.3
102.6
74.7
93.9
85.7
87.2
51.1
118.2
73.4
76.9
65.7
21.5
1
1
1
1
1
1
2
3
4
5
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9
1
1
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1
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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
1
6
36.1
33.5
30.5
13.8
22.3
14.5
100.4
65.7
84.3
KN-62
KN-93
a
Values are means of 3 experiments (standard deviations were less than 10%).
b
Normalized eGFP intensity of virus infected-cells treated with BSA to the solvent-treated
0
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9
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9
0
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0
cells (%).
c
Normalized proliferation of non-infected cells treated with 6.25 µM of the BSA to solvent
control (%).
Building on these findings were observations made in studies of BSAs in family C
where the terminal part is modified (Figure 1). The methylketone 5 displayed no effect on
DENV infection (100.8% infection, Table 1), while its cyclohexyl analog 6 has an
observable antiviral potency that is less than that of 1 (1 = 39.3% vs 6 = 63.3, Table 1).
Moreover, the terminal cyclobutyl (7) and cyclopentyl (8) substituted BSAs had antiviral
activities that are similar to those of 1 (and 7 = 33% and 8 = 25.1%, Table 1). Notably, 7
and 8 had greatly improved cytotoxicity as compared with that of 1 (Table 1). In contrast,
the cycloheptyl substituted BSA 9 displayed a remarkable effect in reducing DENV-2
infection (9.6%) and a minor effect on cell proliferation (Table 1), while the adamantyl
substituted analog 10 displayed 51.1% activity against DENV-2 infection and only minor
effect on cell proliferation (Table 1).
In investigations with BSAs in family D where the methoxy group on the
benzenesulfoamide group in 1 is modified, we found that the trifluoromethoxy (11),
phenoxy (12) and methyl (13) derivatives have either no (11 = 118.2%), or little (12 =
73.4%, and 13 = 76.9%) effect on DENV-2 infection and only minor effects on cell
proliferation (Table 1). Likewise, other changes made in the substituents in the BSAs
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9
brought about only minor changes in the effect against DENV-2 infection (14 = 65.7%, 15
=
21.5% and 16 = 13.8%). Notably, the known allosteric inhibitors KN-62 and KN-93
showed a relatively comparable anti-DENV effect with 8 and 9 (Table 1). However, KN-
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
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9
0
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9
0
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0
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9
0
1
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4
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6
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8
9
0
6
2 and KN-93 reduced cell proliferation to ~16-35%, greater than that of 8 or 9 (Table 1).
Taken together, the highest levels of anti-DENV activity occur when the BSAs contain
a N-phenyl and a para-methoxy group on the benzenesulfonamide group. Significantly, the
cycloheptyl-ketone 9, which contains these features, has the highest antiviral activity and
lowest cytotoxicity of the BSA derivatives examined.
BSA 9 Inhibits DENV-2 and ZIKV Infection In Vitro
Next, we evaluated the potency of 9 against wild-type DENV-2 infection of human
neuronal BE(2)C cells. We first carried out a comprehensive evaluation of 9 for cell
toxicity by employing 3 different assays including the LDH cytotoxicity assay, XTT cell
proliferation assay and trypan blue viable cell counting assay. Tested at a concentration up
to 12.5 M against BE(2)C cells, 9 had no significant effect on cytotoxicity, cell
proliferation, or cell viability (Figure 2A). Importantly, in the case of DENV-2 infection,
treatment of cells with 9 significantly reduced the expression of DENV-2 NS3 protein, as
well as viral progeny production, in a dose-dependent manner (Figure 2B-D), with an EC50
of 1.52 µM. In the case of ZIKV infection, cell treatment with 9 significantly inhibited
ZIKV-E protein expression and viral progeny production (Figure 2E-G), with an EC of
5
0
1
2
.91 µM. Thus, at noncytotoxic concentrations, 9 showed antiviral effects against DENV-
and ZIKV infections in BE(2)C cells. To evaluate whether 9 also displays antiviral effect
in other cell type, we tested the antiviral activity of 9 against DENV-2 and ZIKV in human
microglial HMC3 cells. Treatment with noncytotoxic doses of 9 significantly reduced the
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9
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6
expression of viral NS3 protein and the level of progeny production in HMC3 cells (Figure
S1). Furthermore, 9 might display antiviral effect against other viruses that also employ
2
6
endocytosis-mediated viral entry including influenza A H1N1 and sindbis virus (SINV) .
Treatments with 9 greatly reduced the expression of nucleoprotein (NP) in H1N1-infected
cells and the GFP reporter of SINV-infected cells (Figure S2).
0
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9
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6
Figure 2. Determination of the cytotoxicity and antiviral activity of 9 against DENV-2 and
ZIKV infection (multiplicity of infection [MOI], 0.1). (A) The percentage ratio of the
fraction of viable BE(2)C cells treated with 9 relative to that of the solvent control
determined by using the LDH cytotoxicity assay (left panel), XTT cell proliferation assay
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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9
0
1
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9
0
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9
0
1
2
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4
5
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8
9
0
(center panel), and trypan blue viable cell counting assay (right panel). (B)
Immunofluorescence microscope imaging (200x magnification) of DENV-2 NS3 (green)
in DENV-2-infected BE(2)C cells treated with 9 for 48 h at increasing concentration.
Nuclei are stained blue. (C) Western blot of DENV-2 NS3 in DENV-2-infected BE(2)C
cells treated with 9 at increasing concentration. Actin served as the loading control. The
reported values of the ratios of DENV-2 NS3:actin band densities are listed as percentages
of the ratio determined for the solvent control. (D). Virus titer determined for DENV-2-
infected BE(2)C cells treated with 9 at increasing concentration. (E) Immunofluorescence
microscopic imaging (200x magnification) of ZIKV E (green) in ZIKV-infected BE(2)C
cells treated for 48 h with 9 at increasing concentration. Nuclei are stained blue. (F)
Western blot of ZIKV E in ZIKV-infected BE(2)C cells treated with 9 at increasing
concentration. Actin served as the loading control. The reported values of the ratios of
ZIKV E: actin band densities are listed as percentages of the ratio determined for the
solvent control. (G) Virus titer determined for ZIKV-infected BE(2)C cells treated with 9
at increasing concentration. Values are reported as the mean ± standard deviation of 3
independent experiments. *P < 0.05 and **P < 0.01, compared with solvent control.
Compound 9 Blocks Cellular Entry of DENV-2 and ZIKV
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9
To determine the mechanism for the antiviral action of 9, we investigated whether 9
inhibits the early (0-3 hpi) or late (3-24 hpi) stages of viral infection. As revealed by the
results shown in Figure 3A, within early but not late stage of infection, 9 promoted a
significant reduction in DENV-2 NS3 expression in a dose dependent manner. Therefore,
to further examine the mechanism of action, we focused on the early steps of viral infection
by performing virus-binding and virus-entry assays (see “Experimental Section” for
details). Treatment of BE(2)C cells with 9 had no effect on DENV-2 and ZIKV binding
1
1
1
1
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0
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0
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(Figure 3B). However, the levels of DENV-2 and ZIKV entry were significantly reduced
by 9 in a concentration-dependent manner up to 70% and 80% reduction, respectively
(Figure 3C). 9 also greatly reduced the signal from fluorescently labeled DENV-2 and
ZIKV within the cells as compared with that from control cells treated with solvent only
(Figure 3D and 3E). Thus, the antiviral effect of 9 against DENV-2 and ZIKV infection
derives from inhibition of viral entry into the host cell.
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0
Figure 3. The effect of 9 on the DENV-2 and ZIKV cell binding and entry. (A) Western
blot of DENV-2 NS3, ZIKV E, and the loading control actin. BE(2)C cells were infected
with DENV-2 or ZIKV (MOI, 1) in the absence or presence of 3.125 or 6.25 M 9 during
(
0-3 hpi) or after (3-24 hpi) virus adsorption. The reported ratios of the viral protein: actin
band densities are reported as percentages of the ratio determined for the solvent control.
B) The level of DENV-2 or ZIKV binding to exposed BE(2)C cells determined in the
(
absence or presence of 3.125 or 6.25 M 9. Data are mean ± standard deviation of 3
independent experiments. (C) The level of DENV-2 or ZIKV entry to exposed BE(2)C
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cells determined in the absence or presence of 3.125 or 6.25 M 9, using the plaque-
forming assay. Values are mean ± standard deviation of 3 independent experiments. **P
<0.01, compared with solvent control. (D) and (E) Representative confocal microscope
images of fluorescently-labeled DENV-2-infected (D, scale: 20 μm) and ZIKV-infected
1
1
1
1
1
1
1
1
1
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9
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1
2
3
4
5
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7
8
9
0
(E, scale: 15 μm) BE(2)C cells treated with 3.125 M 9 or solvent (control). The labeled
viruses are red and the stained cell membranes are green.
BSA 9 Increases the Survival Time of DENV-2- and ZIKV-Infected Mice
For the purpose of testing the impact of 9 on the survival of mice infected with
-/-
DENV-2 or ZIKV, we used Stat1 mice which exhibit paralysis, hemorrhage, vascular
2
7
leakage and death after being challenged with a mouse-adapted DENV-2 NGC-N strain .
STAT1 is a transcriptional factor activated by various cytokines, and disruption of the
28
mouse Stat1 gene results in compromised innate immunity against viral diseases . Oral
treatment with 9 at doses of 4 or 8 mg/kg body weight immediately following DENV-2
infection, and then daily for a 6 d period, increased the overall mouse survival number from
0
% to 60% (Figure 4A). Notably, immediate treatment with a lower dose of 9 (2 mg/kg
body weight/day) significantly delayed animal mortality (T , 15 days vs 9 days for the
5
0
vehicle control), with a minor effect on overall animal survival (Figure 4A).
Oral treatment with 9 at a dose of 8 mg/kg body weight immediately following
ZIKV infection, and then daily for a 6 d period, significantly delayed ZIKV-induced
lethality as reflected in the increase in the median survival time T from 13 d to 18 d
5
0
(Figure 4B). However when used at the lower dose of 4 mg/kg body weight, 9 did not
improve the survival time (Figure 4B).
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6
Consistent with the improvements observed in mouse survival rate as a result of
immediate treatment with 9, we found that the viral load of DENV-2 or ZIKV-infected
mice was significantly reduced as a result of treatment with 9 (Figure 4C). Taken together,
these findings provide strong evidence that 9 displays antiviral activity against DENV-2
and ZIKV infections.
0
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9
Figure 4. The impact of 9 on viral load and survival time for DENV-2 and ZIKV-infected
-/-
Stat1 mice. (A) Percent of mice surviving during the course of 36 d after infection with
DENV-2 and immediate initiation of a 6 d oral treatment with phosphate-buffered saline
1
1
1
1
1
1
1
1
1
1
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9
0
(PBS; n  5) (control) or 9 at doses of 2, 4 or 8 mg/kg body weight (n = 5). (B) Percent of
mice surviving during the course of 22 d after infection with ZIKV and immediate initiation
of a 6 d oral treatment with PBS (n  5) or 9 at doses of 4 or 8 mg/kg body weight (n = 5).
(C) Viremia levels determined for serum samples assayed on day 3 following the infection
-/-
of Stat1 mice with DENV-2 or ZIKV and immediate treatment with 9. The control and
the doses of 9 used in these experiments are the same as those stated in (A) and (B). The
viremia levels are reported as the mean (black bar) and individual data (circle, n  5). The
*
P < 0.05 and **P < 0.01, compared with solvent control. (D) Percent of mice surviving
during the course of 36 d after infection with DENV-2 and an 8 h delay in the initiation of
the 6 d daily treatment with 9. The control and doses of 9 used in these experiments are the
same are the same as those listed in (A) with the exception of the 8 mg/kg dose. (E) Percent
of mice surviving during the course of 12 d after infection with ZIKV and an 8 h delay in
the initiation of the 6 d daily treatment with 9. The control and doses of 9 used in these
experiments are the same as those listed in (B). The median survival times (T ) reported
5
0
in the figures were determined using the log-rank test.
Using a therapeutic mode, which involves starting administration of the agent 8 h
after infection and ending at day 6 after infection (8-h-delay), we found that treatment with
2 mg of 9/kg body weight/day was sufficient to significantly improve the median survival
time of DENV-2-infected mice from 10 d to 14 d (Figure 4D). Moreover, treatment with
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a higher dose of 9 (4 mg/kg body weight/day) increased the overall survival to 40% and
the median survival time from 10 to 22 d (Figure 4D). However, using a 8-h-delay
treatment of ZIKV-infected mice with 9 resulted in only a minor improvement in the
median survival time from 8 to 10 d, and had no impact on overall survival (Figure 4E).
The difference in virulence and/or viral pathogenesis between DENV-2 and ZIKV in Stat1-
deficient mice may contribute to the differences observed in the effectiveness of 9 towards
delaying or preventing death. Intriguingly, the inhibitory effect of 9 on the early stage of
DENV-2 and ZIKV infection may explain the greater efficacy of compound 9 observed
when administered immediately following infection rather than 8 h after infection.
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BSA 9 inhibits CaMKIIα activity
Previous studies have demonstrated that JEV, enterovirus 71 (EV71), and rabies
virus infection activates CaMKII13, 29, 30. Activation of CaMKII enhances actin assembly
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and the formation of surface focal adhesion including αvβ3 integrin . In addition,
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activation of CaMKII further phosphorylates vimentin . Notably, actin rearrangement,
integrin β3, and vimentin play important roles in flavivirus entry and/or binding13, 32-34
.
CaMKII is activated by the binding of the calcium/calmodulin complex to the C-terminal
region of the regulatory domain. This, in turn, displaces the regulatory domain to facilitate
access of substrates to the active site of the kinase domain. In addition, the binding of
calcium/calmodulin to the regulatory domain mediates the autophosphorylation of Thr286,
which impairs the rebinding of regulatory domain to the active site of the kinase domain.
Thr286 autophosphorylation is greatly enhanced by binding of ATP to the ATP-binding
site within the active site of the kinase domain14, 17, 35
.
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Molecular docking studies were carried out with 9 and CaMKIIα to identify the
most probable binding site. As illustrated in Figure 5A, consistent with our expectation,
we found that 9 is predicted to bind to the active site located at the interface between the
N-lobe and C-lobe of the CaMKIIα kinase domain (Figure 5A). Here, the sulfonamide
group of 9 is positioned to form hydrogen bonds with the backbone amide NH of Ala23
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(2.21 Å) and of Phe24 (2.31 Å) (Figure 5B; green line). Hydrophobic interactions with 9
involve the nonpolar regions of Lys21, Gly22, Glu139, Gly175, and Gly158 (Figure 5B;
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red line). Notably, Lys21, Gly22, and Phe24 are located within the ATP-binding site and
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Glu139 is required for binding the protein substrate .
Intriguingly, cycloheptyl feature in the terminal part of BSA may improve the
antiviral activity. To support this notion, we further performed docking analysis of 8, 7,
and 6 with CaMKIIα. BSA 8, 7, and 6 showed less and less interactions with CaMKIIα
than 9 (Figure S3A-D). The binding free energy (MM/PBSA) of 9-, 8-, 7-, and 6-CaMKIIα
was -65.545, -61.257, -62.294, and -52.295 KJ/mol, respectively (Figure S3A-D). We
further evaluated the probable interactions of 9 with other kinases that play a role in early
steps of DENV-2 and/or ZIKV infection including tyrosine kinase Axl, tyro3, and p38
mitogen-activated protein kinase (p38MAPK)38, 39. There were hydrophobic interactions
between 9 and the kinase domain of Axl, tyro3 or p38MAPK. However, the interactions
were poor and required a relatively higher free energy binding as compared with that of
CaMKIIα (Figure S3E-G).
BSA 9 was tested in parallel with the known allosteric inhibitor KN-62, to
determine its ability to inhibit the activity of human CaMKIIα. The results obtained
2
+
(Figure 5C) confirm the requirement for Ca for activation and show that 9 has an IC =
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.79 µM and KN-62 IC = 1.03 µM. Moreover, 8 and 16 that displayed slightly lower
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potency of anti-DENV effect than 9 also inhibited CaMKIIα activity with IC = 0.84 µM
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and 0.81 µM, respectively (Figure S4). Notably, 2 and 3 that showed intermediate or no
anti-DENV effect also could reduce CaMKIIα activity but it required a relatively higher
concentration as compared with 9, 8, or 16 (Figure S4). Thus, the antiviral property of
BSAs may correlate with their potency to inhibit CaMKIIα activity.
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Figure 5. CaMKII-9 Molecular docking and inhibition. (A) Predicted docking
conformation of 9 in complex with human CaMKIIα (2VZ6.pdb). (B) Hydrogen-bonding
(green line) and hydrophobic interactions (red line) between 9 and the CaMKIIα binding
site residues (black font). (C) Inhibition of CaMKIIα activity with 9 and KN-62. Data are
mean ± standard deviation (n  3). *P < 0.05 and **P < 0.01, compared with the solvent
control.
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9
The antiviral effect of BSA 9 is associated with CaMKIIα expression
Lastly, we evaluated whether the expression of CaMKIIα is essential for the
antiviral property of 9. The mode of antiviral action of 9 was by inhibiting viral entry, so
we monitored the activation of CaMKIIα at the early time of DENV-2 and ZIKV infection.
DENV-2 and ZIKV infection for 4 hours significantly increased the level of
phosphorylated-CaMKIIα (Figure 6A and 6B). Treatment with 3.125 M 9 greatly
repressed the viral induced-CaMKIIα activation (Figure 6A and 6B). Next, we established
CaMKIIα-deficient BE(2)C cells by transduction with a lentivirus expressing shRNA-
targeting CaMKIIα (shCaMKIIα) (Figure 6C). The level of DENV-2 and ZIKV NS3
protein and progeny production in shCaMKIIα-BE(2)C cells was greatly lower than that
of BE(2)C and shLacZ-BE(2)C control cells (Figure 6D and 6E). Importantly, treatments
with 9 significant repressed the infection of DENV-2 and ZIKV in shLacZ-BE(2)C control
cells, whereas 9 displayed minor antiviral effects in shCaMKIIα-BE(2)C cells (Figure 6F-
I). This data suggested that antiviral property of 9 against DENV-2 and ZIKV was
associated with CaMKIIα expression.
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Figure 6. The antiviral activity of BSA9 is associated with CaMKIIα expression. (A-B)
BE(2)C cells were infected with DENV-2 or ZIKV (MOI, 10) in the absence or presence
of 3.125 µM 9 for 4 h. Representative confocal microscope images of phosphorylated-
CaMKIIα (green) and nuclei (blue) of the cells (A, scale: 50 μm). Relative fluorescence
intensity of phosphorylated-CaMKIIα per cell based on high content image analysis (B).
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(
C) Western blot analysis of knockdown efficiency of CaMKIIα. (D-E) Cells were infected
with DENV-2 or ZIKV (MOI, 5) for 24 h. Western blot of DENV-2 and ZIKV NS3. Actin
served as the loading control (D). Virus titer for DENV-2- or ZIKV-infected cells (E). (F-
G) Cells were infected with DENV-2 (MOI, 5) in the absence or presence of 3.125 or 6.25
µM 9 for 24 h. Western blot of DENV-2 NS3 and the loading control actin (F). Virus titer
for the respective DENV-2-infected cells (G). (H-I) Cells were infected with ZIKV (MOI,
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) in the absence or presence of 3.125 or 6.25 µM 9 for 24 h. Western blot of ZIKV NS3
and the loading control actin (H). Virus titer for the respective ZIKV-infected cells (I). The
reported ratios of the CaMKII or viral NS3 protein:actin band densities are reported as
percentages of the ratio determined for the BE(2)C (C), BE(2)C-infected control (D), or
respective solvent control (F and H). Data are mean ± standard deviation of 3 independent
experiments. *P < 0.05 and **P < 0.01, compared with control. ns: not significant.
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CONCLUSION
We developed the novel benzenesulfonamide derivative 9 to inhibit CaMKII
activity. BSA 9 was shown to display anti-DENV-2 and -ZIKV effects during the early
step of viral infection with an EC of 1.52 µM and 1.91 µM, respectively. Importantly, 9
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reduces the viremia level and improves the survival of both DENV-2 and ZIKV-infected
mice. In vitro inhibition studies demonstrated that 9 inhibits the catalytic activity of human
CaMKIIα with an EC of 0.79 µM. Molecular docking results indicate that 9 might exert
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its inhibitory effect by binding to the CaMKIIα active site.
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9
EXPERIMENTAL SECTION
Chemistry: All reagents and solvents were obtained from commercial sources and
used as purchased unless otherwise stated. THF was freshly distilled from
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Na/benzophenone ketyl under a nitrogen atmosphere. H NMR and C NMR spectra were
recorded on Bruker AVIII-400, AV-400 or AV-500. Proton chemical shifts are reported in
parts per million (ppm) relative to the singlet at 7.24 ppm for residual CHCl in the
3
deuteriochloroform. Carbon chemical shifts were reported in parts per million relative to
1
3
the internal C signals in the CDCl (77.0 ppm) and acetone-d (29.1 ppm) resonances.
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High resolution mass spectra were obtained with Waters LCT Premier XE (Waters Corp.,
Manchester, UK) and Bruker, New ultrafleXtremeTM, Bremen, D.E. Analytical thin layer
chromatography (TLC) was performed on silica gel 60 F254 (Merck). Column
chromatography was performed with silica gel 60 (230400 mesh) (Merck). The purity of
compounds was established as >95% by using normal phase HPLC, Waters 1525 Binary
System with a Waters 2489 dual λ absorbance detector at 254 nm for all compounds; eluent,
ethyl acetate and hexane; column Purospher STAR Si (5 μm), Merk and Venusil XBP
Silica, Agela Technologies.
N-(4-(4-Fluorophenyl)-4-oxobutyl)-4-methoxy-N-phenylbenzenesulfonamide (1)
A solution of pyridinium dichromate (0.91 g, 2.43 mmol) and 2 (0.8 g, 1.87 mmol) in DCM
(30 mL) containing 4 Å molecular sieves (1.0 g) was stirred at room temperature for 8 h.
The mixture was diluted with ethyl acetate and water, and separated. The combined organic
layers were dried over magnesium sulfate and concentrated in vacuum. The residue was
subjected to flash column chromatography, followed by normal-phase HPLC to afford the
1
pure 1 as a white solid (70%): mp 93−94 °C. H NMR (400 MHz, CDCl ) δ 1.82-1.89 (m,
3
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