Vinyl Sulfone-Based Inhibitors of Nonstructural Protein 2 Block the Replication of Venezuelan Equine Encephalitis Virus

Article abstract of DOI:10.1021/acsmedchemlett.0c00215

Emerging infectious diseases like those caused by arboviruses such as Venezuelan equine encephalitis virus (VEEV) pose a serious threat to public health systems. Development of medical countermeasures against emerging infectious diseases are of utmost importance. In this work, an acrylate and vinyl sulfone-based chemical series was investigated as promising starting scaffolds against VEEV and as inhibitors of the cysteine protease domain of VEEV's nonstructural protein 2 (nsP2). Primary screen and dose response studies were performed to evaluate the potency and cytotoxicity of the compounds. The results provide structural insights into a new class of potent nonpeptidic covalent inhibitors of nsP2 cysteine protease represented by compound 11 (VEEV TrD, EC₅₀= 2.4 μM (HeLa), 1.6 μM (Vero E6)). These results may facilitate the evolution of the compounds into selective and broad-spectrum anti-alphaviral drug leads.

Full text of DOI:10.1021/acsmedchemlett.0c00215

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Letter
Vinyl Sulfone-Based Inhibitors of Non-Structural Protein 2
Block the Replication of Venezuelan Equine Encephalitis Virus
Huaisheng Zhang, Moeshia Harmon, Sheli Radoshitzky, Veronica
Soloveva, Chris Kane, Allen J Duplantier, and Ifedayo Victor Ogungbe
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.0c00215 • Publication Date (Web): 02 Sep 2020
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Vinyl Sulfone-Based Inhibitors of Non-Structural Protein 2 Block the
Replication of Venezuelan Equine Encephalitis Virus
Huaisheng Zhang, Moeshia Harmon, Sheli Radoshitzky, Veronica Soloveva, Chris Kane,σ
‡
‡
ф
ψ,†
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‡
Allen J. Duplantier, Ifedayo Victor Ogungbe *
‡Department of Chemistry, Physics, and Atmospheric Sciences, Jackson State University, Jackson, MS, 39217-0095
фThe Geneva Foundation, Countermeasure Development Division, U.S. Army Medical Research Institute of Infectious
Diseases (USAMRIID), Frederick, MD 21702-5011
ψCherokee Nation Assurance, Countermeasure Development Division, US Army Medical Research Institute of
Infectious Diseases (USAMRIID), Frederick, MD 21702-5011
σResearch Program Office, U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID), Frederick, MD
2
1702-5011
ABSTRACT: Emerging infectious diseases like those caused by arboviruses such as Venezuelan equine encephalitis virus
(VEEV) pose a serious threat to public health systems. Development of medical countermeasures against emerging infectious
diseases are of utmost importance. In this work, an acrylate and vinyl sulfone-based chemical series was investigated as
promising starting scaffolds against VEEV and as inhibitors of the cysteine protease domain of VEEV’s non-structural protein
2
(nsP2). Primary screen and dose response studies were performed to evaluate the potency and cytotoxicity of the
compounds. The results provide structural insights into a new class of potent non-peptidic covalent inhibitors of nsP2
cysteine protease represented by compound 11 (VEEV TrD, EC50 = 2.4 M (HeLa), 1.6 M (Vero E6). These results may
facilitate the evolution of the compounds into selective and broad-spectrum anti-alphaviral drug leads.
KEYWORDS: Alphavirus, Covalent inhibitors, Cysteine protease, nsP2, Dihydroquinoline
Venezuelan equine encephalitis virus (VEEV) is a mosquito-
borne neurotropic virus. VEEV infections induce flu-like
symptoms, and can progress in severe cases to encephalitis and
death. Fourteen antigenic subtypes of VEEV have been
identified to date. Symptomatic disease in equines and humans
is caused by antigenic subtypes IA/B, IC, and IE whereas
subtype ID is typically avirulent in equines but virulent in
precursor nsP1234. Its final products, viral non-structural
proteins (nsP1, nsP2, nsP3 and nsP4), are obtained via
proteolytic hydrolysis of the polyprotein precursor by virus-
encoded cysteine protease (nsP2). Replication of the viral
genome as well as the transcription of its 26S subgenomic viral
RNA relies on the biochemical actions of nsP1-4. The viral
structural proteins, capsid protein (CP), small peptides (E3 and
6K), and the envelope glycoproteins (E1 and E2) are translated
from the 26S subgenomic viral RNA. VEEV nsP2 is an important
drug target due to the crucial roles it plays in the virus lifecycle.
Apart from proteolytic hydrolysis of the non-structural
polypeptide precursor into functional protein units, nsP2
regulates negative strand RNA synthesis via its
methyltransferase activity. In addition, nsP2 facilitates the
1
,2
humans. New World alphaviruses like VEEV, eastern equine
encephalitis virus (EEEV), and western equine encephalitis
virus (WEEV) are widely distributed in North, Central and
South America. Occasional outbreaks of VEEV have been
3,4
reported over the years in the Americas. The use of VEEV as
a bioweapon was evaluated by both the US and Russia during
5
the Cold War. New World alphaviral infections used to be very
1
0
rare in the US (<10 cases per year) which made the feasibility
of clinical trials for anti-alphaviral therapeutics challenging.
But in 2019, a record high number of EEEV cases was reported
packaging of genomic RNA into virus particles.
To date, only a few compounds including CA-074Me and E64-
d have been reported to inhibit both the protease activity of
6
in the US with at least 14 fatalities. Also, acute infections
11,12
caused by the related Old-World alphavirus, Chikungunya virus
VEEV’s nsP2 and the replication of VEEV, albeit marginally.
(
CHIKV), are becoming more common, and to date, over 1
It is important to point out, however, that a series of
benzamidine-based and potent inhibitors of VEEV that inhibit
viral RNA synthesis is currently under pre-clinical
million cases have been reported worldwide. Old-World
alphaviruses present significant risk to healthcare systems and
livelihood of millions of people in the tropics and subtropical
1
3,14
investigation.
In this work, a phenotypic screening-based
7
-9
regions.
approach was used to identify compounds with antiviral
activity against VEEV from a library of potential covalent
1
5,16
VEEV has a single-stranded, positive-sense RNA genome that is
11.4 kb in size. One of the two open reading frames in the
alphavirus genome encodes the non-structural polyprotein
protease inhibitors.
An initial structure-activity
~
relationship (SAR) studies was used to define the
pharmacophore in the hit compound (1) as well as the in-
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hibitory activity of its analogues against the nsP2 protease (10,
1
2
3
4
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
11). Interactions between the protease and the active
compounds (10-13) were investigated via covalent molecular
docking.
ther 3-amino-1,2-propanediol or 2-amino-1-propanol by Boc-
protection followed sequentially by either periodate oxidation
(1-4 and 9-11) or Dess–Martin periodinane oxidation (12-16),
then Horner-Wadsworth-Emmons olefination of the resulting
aldehyde with appropriate phosphonate, removal of the Boc
group by treatment with TFA, and final amide coupling with the
appropriate acid in the presence of HBTU. Compounds 5-8
were readily available by analogous amide coupling starting
with methyl 4-aminobutyrate.
We used a library derived from our previously reported
fragment-based approach to identify protease inhibitors of
15
VEEV IC-SH3. VEEV IC-SH3 is a virulent strain derived from a
human isolate from a VEE outbreak in 1992–1993 in
1
7
Venezuela.
SH3)-infected Vero and HeLa cells identified an acrylate-based
,2-dihydroquinoline derivative, 1, as a potent VEEV inhibitor
EC50’s of 0.55 µM (Vero) and 6.48 µM (HeLa)). Subsequent
Our initial screening efforts against VEEV (IC-
This initial SAR study revealed that i) partial saturation of the
dihydroquinoline ring is tolerated (2), ii) methyl substituents
at the 2- and/or 4-position of the hydroquinoline motif
improve anti-VEEV activity (1 and 2 vs 4), iii) methyl
substitution at carbon 6 of the 1,2-dihydroquinoline ring
decreases antiviral activity, and iv) the α, β-unsaturation of the
acrylate warhead is required for anti-VEEV activity (Table 1).
Subsequent analogs were evaluated against VEEV-TC83 using
neuronal cell lines including human BE(2)-M17 and mouse
Neuro-2a cells which may be more physiologically relevant in
vitro models as they better represent primary targets of VEEV
infection. VEEV-TC-83 is a live-attenuated virus generated by
serial passage of VEEV Trinidad (TrD) strain in guinea pig heart
cells. Work with this virus can be performed in BSL-2 and
1
(
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
studies to develop SAR around compound 1 were initiated to i)
understand which structural motifs are necessary for
inhibition of VEEV’s replication, ii) determine if the α, β-
unsaturation in the acrylate warhead is crucial for inhibition
(compounds 2-8), iii) determine if similar warheads like
methyl vinyl sulfone and phenyl vinyl sulfone are tolerated, iv)
determine the tolerability of substituents at the γ-carbon, and
v) determine if the dihydroquinoline heterocycle can be
replaced by similar bicyclic motifs. Analogues were
synthesized as outlined in Scheme 1. The structures, detailed
description of synthesis and characterization of all analogs (2-
18
4
3) investigated are described in the supporting information.
therefore for most assays hereon this variant was used.
The compounds were prepared from ei-
O
O
O
H
N
H
N
d
d
O
R
R
O
S
O
O
OH
H
O
O
O
O
N
O
c
a, b
1-4, 9
S3
HO
NH2
H
S2
O
O
O
H
N
S1
O
H
S
N
O
10, 11
S4
O
O
H
d
NH2 HCl
N
R
O
O
O
S5
5-8
O
O
O
H
H
O
O
H
S
N
R
NH2
a, e
N
O
c
S
N
O
HO
H
d
O
O
O
12, 13
S6
S7
S8
O
O
O
O
H
H
S
N
O
d
d
S
N
R
O
O
O
NH2
H
S11
a, e
HO
N
O
14, 15
H
c
O
O
O
O
O
S
H
H
S
N
O
N
R
S9
O
O
S10
S12
16
Scheme 1. Synthesis of target compounds 1-16. Reagents and conditions: (a) CH
O, NaIO , RT, 1 h, 64%; (c) NaH, THF, methyl diethylphosphonoacetate, diethyl((methylsulfonyl)methyl) phosphonate or
diethyl((phenylsulfonyl)methyl) phosphonate, 0˚C, 25 min, 40-70%; (d) 33% TFA in DCM, 0˚C, 1.5 h. Then ACN, R-COOH, Et N,
HBTU, RT, 16 h, 30-40%; (e) DMP, H O-DCM, 23˚C, 1 h, 40-60%.
2 2 3 3 2
Cl :CH OH, Et N, Boc O, 23˚C, 2 h, >90%; (b)
H
2
4
3
2
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Table 1. Anti-VEEV activity of compounds 1-8.
1
2
3
4
5
6
7
8
9
O
O
H
H
N
R
N
R
O
O
O
O
Compound
1-4
5-8
HeLa (VEEV IC-SH3)
CC50 (µM)
VeroE6 (VEEV IC-SH3)
R
EC50 (µM)
SI
EC50 (µM)
CC50 (µM)
SI
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
0
1
.6 ± 0.1
>30
>30
>55
6.5 ± 2.0
>30
>5
1
2
HN
.2 ± 0.1
>26
>4
8.2 ± 3.1
8.6± 2.5
>30
>30
>4
HN
HN
3
8.7 ± 0.8
>30
>30
>3.5
4
5
18 ± 1
>30
>2
-
>30
>30
>30
>30
-
-
HN
>30
HN
6
>30
>30
>30
>30
-
-
>30
>30
>30
>30
-
-
HN
7
8
HN
>30
>30
-
>30
>30
-
HN
RIID reference control E
0.21 ± 0.02
>3
>12
0.14 ± 0.03
>3
>18
The compounds were tested at concentrations of 13.72 nM-30 µM (8 doses, 3-fold dilution). HeLa and VeroE6 cells were treated with the indicated
compounds and 2 h later inoculated with VEEV IC-SH3 (MOI=0.1 and 0.01, respectively). After 20 h, cells were fixed, and stained with antibodies
against E2. High-content quantitative image-based analysis was used to measure relative infection rates.
Substitution of the acrylate warhead with methyl sulfone (e.g.,
0-13, 15) provided low micromolar potency in both neuronal
cell lines (Table 2). This result was particularly encouraging
to metabolic hydrolysis. It is also interesting that while
substitution at the γ-carbon of methyl esters was not tolerated,
mono-substitution of either a methyl or benzyl group on the γ-
carbon of sulfone derivatives retained anti-VEEV activity.
1
since the methyl sulfone, as opposed to an ester, is not prone
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Table 2. Anti-VEEV activity of selected analogues. See Table S1 for full list of analogues.
1
2
3
4
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
O
O
O
O
O
O
H
H
H
H
S
N
R
S
N
R
S
N
R
N
R
O
R R
2 O
R R
1
2
O
O
1
O
1
, 2, 9
10, 11
12-13: R1 = CH3 and R2 = H
16: R = Benzyl and R = H
1
2
1
4-15: R = Benzyl and R = H
1
2
Compound
Human BE(2)-M17
Mouse Neuro-2a
CC50 (µM)
R
EC50 (µM)
CC50 (µM)
SI
EC50 (µM)
>25
SI
Virus
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
4.5 ± 3.5
6.7
1.5
18.1
>25
>25
-
TC83
HN
2
9
>25
11
12
-
22 ± 11
11 ± 5
>1
>2
TC83
TC83
HN
NH
Br
3 ± 5
4
2
1
.0 ± 1.3
5 ± 1a
19.6
30a
>9
7a
5 ± 2
1.4 ± 0.1b
-
27
>30b
-
>5
>22b
-
TC83
TrD
10
HN
HN
HN
HN
6
± 1c
>25 c
> 4 c
TC83
.4 ± 1.0
>25
30a
48c
>18
>12a
6c
2 ± 1
1.6 ± 0.1b
-
>25
>30
-
>12
>19b
-
TC83
TrD
11
12
13
2.4 ± 0.5a
± 1c
8
TC83
4
.5 ± 0.2
>25
>5
3.4 ± 0.1
-
>25
-
>7.3
-
TC83
TC83
2.7 ± 0.2c
>25c
> 9.7 c
3.3 ± 0.2
>25
>7.5
3.7 ± 0.2
>25
>7
TC83
14
17 ± 3
>38
>50
>2
13 ± 6
13 ± 9
>50
>50
>4
>4
TC83
TC83
HN
15
9.3 ± 2.8
>5.4
HN
Compounds 1, 2, 9-13 were tested at concentrations of 11.43 nM-25 µM (8 doses, 3-fold dilution) whereas compounds 14-16 were tested from 22.86
nM-50 µM (8 doses, 3-fold dilution). BE(2)-M17 and Neuro-2a cells were treated with the indicated compounds and 2 h later inoculated with VEEV
TC83 (MOI = 1.2 and 3.5, respectively). After 7 h, cells were fixed, and stained with antibodies against E2. High-content quantitative image-based
analysis was used to measure relative infection rates. Compounds were assayed in VEEV Trinidad-infected Vero cells (MOI = 0.01). Compounds
were assayed in VEEV Trinidad-infected HeLa cells (MOI = 0.1). ). Compounds were assayed in VEEV TC83-infected mouse cortical neurons (MOI
a
b
c
=
0.04) Cells were fixed 20 h after virus inoculation and stained and analyzed as above.
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Table 2 Continued. Anti-VEEV activity of selected analogues. See Table S1 for full list of analogues.
1
2
3
4
5
6
7
8
9
O
O
O
O
O
O
O
H
H
H
H
S
N
R
S
N
R
S
N
R
N
R
O
R R
2 O
12-13: R1 = CH3 and R2 = H
4-15: R = Benzyl and R = H
R R
1
2
O
O
1
O
1, 2, 9
10, 11
16: R = Benzyl and R = H
1
2
1
1
2
Compound
Human BE(2)-M17
Mouse Neuro-2a
R
EC50 (µM)
CC50 (µM)
SI
EC50 (µM)
CC50 (µM)
SI
Virus
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
16
17 ± 5
16
1
19 ± 15
46
>2
TC83
N
ML336
Ref. 13
0.01 ± 0.00
>2.5
>250
0.02 ± 0.01
>2.5
>125
TC83
Compounds 1, 2, 9-13 were tested at concentrations of 11.43 nM-25 µM (8 doses, 3-fold dilution) whereas compounds 14-16 were tested from 22.86
nM-50 µM (8 doses, 3-fold dilution). BE(2)-M17 and Neuro-2a cells were treated with the indicated compounds and 2 h later inoculated with VEEV
TC83 (MOI = 1.2 and 3.5, respectively). After 7 h, cells were fixed, and stained with antibodies against E2. High-content quantitative image-based
analysis was used to measure relative infection rates.
Compounds 10-12 were also evaluated against VEEV-TC83
using mouse primary cortical neurons, and the compounds
displayed anti-VEEV activity (Table 2). Finally, we conclude
that the di- and tetra-hydroquinoline of sulfone derivatives
retained anti-VEEV activity since replacement with the fully
unsaturated quinoline (16) did not result in anti-VEEV activity.
Additional replacements of the unsaturated quinoline ring that
led to inactivity are listed in Table S1 (supporting information).
It is worth pointing out that different MOIs and infection times
were used for the assays in the different cell lines (to achieve
50-80% infection rates at the study endpoint). These variations
might have contributed to the observed EC50 difference. Also,
infection kinetics (such as the length of one virus lifecycle)
might be different between the cells used. Finally, these
differences might be also due to non-specific effects of the
compounds on the cells. Overall, compounds 10-13 were the
most active analogues in neuronal cells in this initial SAR study.
co-workers.12 As shown in Figure 1 and S1, compounds 11-13
inhibited the proteolytic activity of VEEV nsP2 in a dose-
dependent manner. At 20 µM, compounds 11-13 completely
inactivated VEEV nsP2 (1 µM) in the presence of V12 substrate
(a CFP-YFP FRET Substrate, 10 µM; data not shown). At 0.9 µM,
the estimated percentage inhibition after 24 h incubation was
39%, 59% and 71% for 11, 12 and 13, respectively. Covalent
molecular docking was used to predict favored binding
orientations and specific binding interactions of the
compounds with the nsP2 protein. As shown in Figure 2 and 3,
compound 11, which was the most selective in VEEV-infected
BE(2)-M17 and Neuro-2a cells, was predicted to have
significant Van der Waals interactions with Try 547, Asn 545,
Ala 509 and His 510, and complex-stabilizing hydrogen
bonding interactions with Asn 545, His 510 and a key water
molecule within the active site.
The methyl sulfone motif as well as methyl substituents of the
tetrahydroquinoline ring are predicted to be partially exposed
to solvent, and thus these moieties may provide an avenue for
physicochemical property modifications to reduce microsomal
clearance. Co-crystallization of nsP2 with selected inhibitors,
in order to determine the preferred binding modes of the
compounds, is under active investigation.
To confirm the mechanism of action for this series of VEEV
inhibitors, representative compounds were tested for their
ability to inhibit the proteolytic activity of nsP2 in vitro. The
protease was recombinantly expressed, purified, and assayed
using the gel discontinuous assay previously reported by
Legler and
Figure 1. Inhibition of VEEV nsP2 protease by compounds 12 and 13 in a gel discontinuous assay. V12: CFP-YFP FRET substrate;
nsP2: tag-free non-structural protease 2. For both gels, Lane 1 is V12 alone, Lane 2 is V12 + nsP2, Lanes 3-10 are V12 + nsP2 + 12
or 13 at 3.6, 1.8, 0.9, 0.45, 0.22, 0.11, 0.06 and 0.03 µM, respectively. The molecular weight (MW) of uncut V12, the two V12 fragments
cut v12) and tag-free nsP2 are 58.3, 30.9, 27.4, and 38.29 kDa, respectively. The reactions were carried out in 50 mM HEPES buffer
pH 7.4 for 24 h at room temperature. The concentrations of VEEV nsP2 and V12 were 1 µM and 10 µM, respectively.
(
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Figure 2. Modelled complex of VEEV nsP2 and compound 11. The highlighted residues are predicted to have significant Van der
Waals interactions (piecewise linear potential) with compound 11. The blue dash depicts H-bond interaction. The figure was
prepared using Molecular Operating Environment (MOE).
Figure 3. Modelled complex of VEEV nsP2 and compound 11. The left panel is a stick model representation of the predicted
network of H-bond interactions (depicted as the blue dash lines) stabilizing the nsP2-11 complex. Asn 545, His 510 and His 546
residues are predicted to have direct H-bond interactions with compound 11. Carbon atoms are depicted as yellow in 11. The right
panel is a 2D depiction of the proximity (gray contour) of active site residues to compound 11 as well as the extent of exposure
(purple hue) of the compound’s atoms to solvent. The figure was prepared using Molecular Operating Environment (MOE).
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In summary, a new series of non-peptidic dihydroquinoline
(2) Sharma, A; Knollmann-Ritschel, B. Current understanding of
the molecular basis of Venezuelan equine encephalitis virus
pathogenesis and vaccine development. Viruses 2019, 11, 164.
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and tetrahydroquinoline-based covalent inactivators of VEEV’s
nsP2 cysteine protease was identified in this work. The
preliminary SAR study showed that the conformationally more
flexible dihydroquinoline and tetrahydroquinoline rings
provides an appropriate shape for effective binding at the
active site. Ongoing and future work will focus on improving
microsomal stability and potency, determination of in vivo
pharmacokinetics and tolerability, and subsequent evaluation
in an animal model of VEEV infection
(
3) Carrera, J.P; Bagamian, K.H.; Travassos da Rosa, A.P.; Wang,
E.; Beltran, D.; Gundaker, N.D.; Armien, B.; Arroyo, G.; Sosa, N.;
Pascale, J.M.; Valderrama, A.; Tesh, R.B.; Vittor, A.Y.; Weaver, S.C.
Human and equine infection with alphaviruses and flaviviruses in
Panamá during 2010: a cross-sectional study of household contacts
during an encephalitis outbreak. Am. J. Trop. Med. Hyg. 2018, 98,
1
798-1804.
4) Zacks, M.A.; Paessler, S. Encephalitic alphaviruses. Vet.
(
Microbiol. 2011, 140, 281-286.
ASSOCIATED CONTENT
Supporting Information
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(5) Reichert, E.; Clase, A.; Bacetty, A.; Larsen, J. Alphavirus
antiviral drug development: scientific gap analysis and prospective
research areas. Biosecur. Bioterror. 2009, 7, 413-427.
The Supporting Information is available free of charge on the
ACS Publications website. Supplementary data and details of
assays, synthesis and compound characterization are provided
as supporting information (PDF).
(
6) Morens, D.M.; Folkers, G.K.; Fauci, A. S. Eastern equine
encephalitis virus — another emergent arbovirus in the United
States. N. Engl. J. Med. 2019, 381, 1989-1992.
(7) Kosasih H.; de Mast, Q.; Widjaja, S.; Sudjana, P.; Antonjaya,
U.; Ma’roef, C.; Riswari, S.F.; Porter, K.R.; Burgess, T.H.; Alisjahbana,
B., van der Ven, A. Evidence for Endemic Chikungunya Virus
Infections in Bandung, Indonesia. PLoS Negl. Trop. Dis. 2013, 7,
e2483.
AUTHOR INFORMATION
Corresponding Author
(
8) Lemant, J.; Boisson, V.; Winer, A.; Thibault, L.; André, H.;
*ifedayo.v.ogungbe@jsums.edu; +1 601-979-3719
Tixier, F.; Lemercier, M.; Antok, E.; Cresta, M.P.; Grivard, P.;
Besnard, M.; Rollot, O.; Favier, F.; Huerre, M.; Campinos. J.L.;
Michault, A. Serious acute chikungunya virus infection requiring
intensive care during the reunion island outbreak in 2005–2006.
Crit. Care Med. 2008, 36, 2536–2541.
Present Addresses
†
Merck Research Laboratories, Cambridge, MA, USA.
Author Contributions
(
9) Angelini, P.; Macini, P.; Finarelli, A.C.; Pol, C.; Venturelli, C.;
The manuscript was written through contributions of all
authors.
Bellini, R.; Dottori, M. Chikungunya epidemic outbreak in Emilia-
Romagna (Italy) during summer 2007. Parasitologia 2008, 50, 97–
9
8.
Funding Sources
(
10) Kim, D. Y.; Atasheva, S.; Frolova, E. I.; Frolov, I. Venezuelan
This work was carried out in part by resources made available
by the US National Institutes of Health (SC3GM122629 and
G12MD007581). MH was supported by NIH-RISE
equine encephalitis virus nsP2 protein regulates packaging of the
viral genome into infectious virions. J. Virol. 2013, 87, 4202–4213.
(11) Campos-Gomez, J.; Ahmad, F.; Rodriguez, E.; Saeed, M.F. A
novel cell-based assay to measure activity of Venezuelan equine
encephalitis virus nsP2 protease. Virology 2016, 496, 77-89.
(12) Hu, X.; Compton, J.R.; Leary, D.H.; Olson, M.A.; Lee, M.S.;
Cheung, J.; Ye, W.; Ferrer, M.; Southall, N.; Jadhav, A.; Morazzani,
E.M. Glass, P.J; Marugan, J. and Legler, P.M. Kinetic, mutational, and
structural studies of the Venezuelan equine encephalitis virus
nonstructural protein 2 cysteine protease. Biochemistry 2016, 55,
(
5R25GM067122) and was a participant in the Naval Research
Laboratory (NRL) HBCU/MI Summer Internship Program
sponsored by the Office of Naval Research (N0017315RMV04).
Cell-based VEEV screening at USAMRIID was funded by The
Joint Science and Technology Office for Chemical and Biological
Defense (JSTO-CBD) of the Defense Threat Reduction Agency
(DTRA) under plan number CB10217. The funding agency had
no involvement in any aspect of the study including design,
data collection, analysis, interpretation, or composition of this
manuscript. The opinions, interpretations, conclusions, and
recommendations expressed here are those of the authors and
do not represent those of the U.S. Army, U. S. Department of
Defense or the U. S. government.
3
007-3019.
13) Skidmore, A.M.; Adcock, R.S.; Jonsson, C.B.; Golden, J.E.;
(
Chung, D.H. Benzamidine ML336 inhibits plus and minus strand
RNA synthesis of Venezuelan equine encephalitis virus without
affecting host RNA production. Antiviral Res. 2020, 174, 104674.
(
14) Jonsson, C.B.; Cao, X.; Lee, J.; Gabbard, J.D.; Chu, Y.K.;
Fitzpatrick, E.A.; Julander, J.; Chung, D.H.; Stabenow, J.; Golden, J.E.
Efficacy of a ML336 derivative against Venezuelan and eastern
equine encephalitis viruses. Antiviral Res. 2019, 167, 25-34.
(15) McShan, D.; Kathman, S.; Lowe, B.; Xu, Z.; Zhan, J.; Statsyuk,
A.; Ogungbe, I.V. Identification of non-peptidic cysteine reactive
fragments as inhibitors of cysteine protease rhodesain. Bioorg.
Med. Chem. Lett. 2015, 25, 4509-4512.
ACKNOWLEDGMENT
We thank Dr. Pat. Legler (NRL, Washington DC) for very helpful
discussions about nsP2, and for providing the nsP2 and V12
plasmids.
(
16) Kathman, S.G.; Xu, Z.; Statsyuk, A.V. A fragment-based
ABBREVIATIONS
DCM, dichloromethane; ACN, acetonitrile; DMP, Dess–Martin
method to discover irreversible covalent inhibitors of cysteine
proteases. J. Med. Chem. 2014, 57, 4969-4974.
(17) Rico-Hesse, R.; Weaver, S.C.; De Siger, J.; Medina, G.; Salas,
R.A. Emergence of a new epidemic/epizootic Venezuelan equine
encephalitis virus in South America. Proc. Natl. Acad. Sci. U.S.A.
1995, 92, 5278-5281.
periodinane;
HBTU,
N,N,N′,N′-Tetramethyl-O-(1H-
benzotriazol-1-yl)uronium hexafluorophosphate.
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C.; Haddow, A.D.; Weaver, S.C. Endemic Venezuelan equine
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Future Virol. 2011, 6, 721-740.
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