JMX0207, a Niclosamide Derivative with Improved Pharmacokinetics, Suppresses Zika Virus Infection Both in Vitro and in Vivo

Article abstract of DOI:10.1021/acsinfecdis.0c00217

Flaviviruses causes significant human disease. Recent outbreaks of the Zika virus highlight the need to develop effective therapies for this class of viruses. Previously we identified niclosamide as a broad-spectrum inhibitor for flaviviruses by targeting

Full text of DOI:10.1021/acsinfecdis.0c00217

Subscriber access provided by CORNELL UNIVERSITY LIBRARY
Article
JMX0207, a niclosamide derivative with improved pharmacokinetics,
suppresses Zika virus infection both in vitro and in vivo
Zhong Li, Jimin Xu, Yuekun Lang, Xiaoyu Fan, Lili Kuo, Lianna D'Brant, Saiyang Hu, Subodh
Samrat, Nicole Trudeau, Anil Tharappel, Natasha Rugenstein, Cheri Koetzner, Jing Zhang,
Haiying Chen, Laura Kramer, David Butler, Qing-Yu Zhang, Jia Zhou, and Hongmin Li
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.0c00217 • Publication Date (Web): 31 Aug 2020
Downloaded from pubs.acs.org on September 6, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 51
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
JMX0207, a niclosamide derivative with improved pharmacokinetics, suppresses Zika
virus infection both in vitro and in vivo
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
#
#
Zhong Li , Jimin Xu , Yuekun Lang, Xiaoyu Fan, Lili Kuo, Lianna D'Brant, Saiyang Hu,
Subodh Kumar Samrat, Nicole Trudeau, Anil M. Tharappel, Natasha Rugenstein, Cheri A
Koetzner, Jing Zhang, Haiying Chen, Laura D Kramer, David Butler, Qing-Yu Zhang, Jia
Zhou*, Hongmin Li*
1
Wadsworth Center, New York State Department of Health, 120 New Scotland Ave, Albany,
NY 12208, USA
2
Chemical Biology Program, Department of Pharmacology and Toxicology, University of Texas
Medical Branch, Galveston, TX 77555, USA
3
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona,
Tucson, AZ 85721, USA
4
The Neural Stem Cell Institute, 1 Discovery Drive, Rensselaer, NY 12144, USA
5
Department of Biomedical Sciences, School of Public Health, University at Albany, Albany,
NY 12201, USA
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 2 of 51
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
Abstract
Flaviviruses causes significant human disease. Recent outbreaks of the Zika virus highlight
the need to develop effective therapies for this class of viruses. Previously we identified
niclosamide as a broad-spectrum inhibitor for flaviviruses by targeting the interface between viral
protease NS3 and its cofactor NS2B. Here, we screened a small library of niclosamide derivatives
and identified a new analogue with improved pharmacokinetic properties. Compound JMX0207
showed improved efficacy in inhibition of the molecular interaction between NS3 and NS2B,
better inhibition of viral protease function, and enhanced antiviral efficacy in cell-based antiviral
assay. The derivative also significantly reduced Zika virus infection on 3D mini-brain organoids
derived from pluripotent neural stem cells. Intriguingly, the compound significantly reduced
viremia in a ZIKV animal model. In summary, a niclosamide derivative JMX0207 was identified
which shows improved pharmacokinetics and efficacy against Zika virus both in vitro and in vivo.
Keywords: Flavivirus, Zika virus, Dengue virus, antiviral, protease inhibitor
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
ACS Paragon Plus Environment

Page 3 of 51
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
The genus Flavivirus is composed of more than 70 viruses. Many flaviviruses, including
the four serotypes of the Dengue virus (DENV1-4), the West Nile virus, and the Zika virus (ZIKV
or ZK) cause serious human diseases. Recently, significant ZIKV outbreaks have occurred
worldwide.1-5 ZIKV is transmitted to humans primarily through bites from infected Aedes species
mosquitoes, but may also be transferred prenatally or through sexual activities or blood
transfusions.6-9 ZIKV infection is associated with devastating diseases, including Guillain-Barré
syndrome, a rare neurological syndrome, and congenital Zika syndrome, which is manifested by
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
1
-5
microcephaly, brain abnormalities, and other central nervous system malformations. In addition,
ZIKV persists in neuronal and/or reproductive tissues of animals for up to 60 days after viral
infection, leading to severe damage.10-12 Currently there is neither a safe and effective vaccine nor
a specific therapy for ZIKV. Although a DENV vaccine is recently approved, it is effective in
children between 9 and 16-year-old, and posts increased risk for naïve children due to antibody-
1
3
dependent enhancement. Therefore, there is an unmet medical need to develop direct antivirals
against flaviviruses.
The flavivirus genome encodes a single open reading frame, from which a polyprotein
precursor is expressed.14-16 The polyprotein precursor is post-translationally processed into 10
mature proteins, including three structural and seven non-structural proteins, by viral and host
proteases in a sequential manner.14-15 The viral protease is a heterocomplex composed of viral NS3
1
4
protein and its viral co-factor NS2B. The viral protease has been considered a highly promising
drug target.14, 17-19 Various strategies have been used to develop protease inhibitors. The majority
of efforts have been focused on the viral protease active site,13,16-18 but with limited success in
2
0
finding inhibitors with in vivo efficacy. Recently, alternative strategies to target the NS2B-NS3
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 4 of 51
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
interaction21-22 and allosteric sites23-25 have led to identification of potent protease inhibitors with
in vivo efficacy against Zika virus.
Previously, we developed a split luciferase complementation (SLC)-based high throughput
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
screening strategy to identify inhibitors targeting the NS2B-NS3 interface of DENV2 (DN2).21
Niclosamide was found to be a broad-spectrum inhibitor for flaviviruses by targeting the interface
between viral protease NS3 and its co-factor NS2B, leading to accumulation of non-functional
2
1
26
viral polyprotein precursor. In addition, niclosamide was found to interfere with virus entry and
2
7
endosomal acidification. Moreover, niclosamide was found as a broad-spectrum antiviral against
multiple viruses including SARS Cov-2, the causing agent of current COVID-19 pandemic.28
Unfortunately, niclosamide did not show appreciable in vivo antiviral efficacy towards ZIKV (data
not shown), possibly due to its poor pharmacokinetic properties.26, 29
In this work, we screened a small library of niclosamide derivatives. We found that one
derivative, JMX0207, showed improved efficacy in inhibition of the NS2B-NS3 interaction, better
inhibition of viral protease function, and enhanced antiviral efficacy in cell-based antiviral assay,
compared to niclosamide. JMX0207 was also found to significantly reduce ZIKV infection on a
3
D mini-brain organoid model derived from pluripotent neural stem cells. Intriguingly, JMX0207
significantly improved the pharmacokinetic properties and markedly reduced viremia in a ZIKV
animal model.
Results
Synthesis and screening of Niclosamide derivatives.
Niclosamide was originally used as an anthelmintic drug. It was also found to have anti-
cancer and antibacterial activities.30-34 We previously developed a small library composed of 62
niclosamide derivatives for other purposes.33-38 These niclosamide analogs have been
ACS Paragon Plus Environment

Page 5 of 51
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
resynthesized in-house following our published procedures for antiviral evaluation. To identify
niclosamide derivatives that have equal or better antiviral efficacy and improved pharmacokinetic
properties, we used our SLC-based NS2B-NS3 interaction assay to screen this library, with
niclosamide as a control.
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
a
Table 1. IC , EC , CC (all in µM), and therapeutic index (TI) of niclosamide derivatives.
5
0
50
50
IC50-SLC-
IC50-pro
(DN2)
EC50-DN2
(A549)
EC50-ZK
(A549) (A549)
CC50
TIc
DN2)
TI
(ZK)
Compoundb
Structure
(
(DN2)
NO2
OH
O
N
Niclosamide
HJC0114
H
2.0
21.6
33.6
0.55
>30
0.48
>30
4.8
8.7
10.0
Cl
Cl
O
N
NO2
O
O
18.4
>200
N
H
Cl
Cl
HN
NO2
O
O
3
35
HJC0125
HJC0308
HJC0365
N
16.3
4.9
44.5
39.7
>60
0.21
1.4
0.15
6.2
50.2
17.7
177
239
12.6
27.6
H
Cl
Cl
HCl
O
NO2
H2N
O
N
H
2.8
Cl
Cl
N
N
NO2
O
O
29.5
N
13.2
6.4
6.0
H
Cl
Cl
N
NO2
O
O
O
2
3.3
HJC0381
N
H
10.8
25.9
1.2
3.9
91.1
76
Cl
Cl
Cl
O
NO2
NH2
N
H
HJC0390
HJC0431
HJC0129
6.0
3.1
9.6
46.5
34.0
11.3
0.43
2.2
<0.1
0.36
>30
30.1
28.4
176
70
13
>300
79
Cl
H2N
NO2
O
O
N
H
Cl
Cl
NH2
OH
O
N
H
>30
Cl
Cl
H
N
OH
O
O
N
HJC0140
9.4
25.5
>30
>30
>200
H
Cl
Cl
OH
O
N
S
O
O
HJC0149
JMX0207
H
18.6
1.3
20.3
8.2
0.9
1.1
241
268
103
219
106
Cl
NO2
OH
O
O2N
N
0.31
0.30
31.9
H
Cl
a
IC50-SLC/IC50-pro/EC50/CC50: Compound concentrations required to reduce 50% of the split luciferase
b
complementation (SLC) signal, protease activity, virus production, and cell viability, respectively. Original
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 6 of 51
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
compound names from literature and this work. According to their structures, these compounds can be classified
into three groups, colored as red, magenta, and blue, respectively. TI, therapeutic index (TI) defined as CC50/EC50
All values are in micromolar (µM) except TI. DN2, Dengue virus serotype 2; ZK, Zika virus.
c
.
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 1. Inhibition of the NS2B-NS3 interactions and protease activity. (A) Dose-dependent
inhibition of SLC upon binding of NLuc-NS2B49-66 to GST-CLuc-NS3 by JMX0207. N=3. (B)
Dose-response inhibitions of the DENV2 His-NS2B/His-MBP-NS3 protease activity by
JMX0207. N=3. In both (A) and (B), DMSO control was set as 100%. The values represent means
±
standard deviation (S.D.) in all panels.
Eleven out of 62 niclosamide derivatives showed dose-responsive inhibition of the
interaction between viral NS3 protease and its cofactor NS2B, with IC50-SLC values lower than 30
µM, the highest concentration tested (IC50-SLC/IC50-pro/EC /CC : compound concentrations
50
50
required to reduce 50% of the split luciferase complementation signal, protease activity, virus
production, and cell viability, respectively) (Table 1, Figure 1). The derivatives identified
included seven compounds with O-alkylamino chains, one compound with 3-NO instead of 5-Cl
2
on the salicylic ring, and three compounds with modifications on the nitro group of the aniline
moiety. Of these active derivatives, compounds HJC0308 with 3-aminopropoxy moiety,
HJC0431 with 4-aminobutoxy moiety, and JMX0207 with 3-NO group showed comparable or
2
ACS Paragon Plus Environment

Page 7 of 51
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
better inhibition of the NS2B-NS3 SLC with IC50-SLC values of 4.9 µM, 3.1 µM and 1.3 µM,
respectively, compared to niclosamide.
Inhibition of the NS2B-NS3 protease activity.
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
We next evaluated the inhibition efficacy of these positive derivatives against the viral
2
1
NS2B-NS3 protease function as previously described. Previously, we generated two versions of
the NS3 protease, the NS3 protease domain refolded from inclusion bodies and a soluble version
of the NS3 protease domain fused to maltose-binding protein (MBP).21-22 Our data showed that
the refolded and MBP-tagged NS3 proteins have indistinguishable protease activities when
transactivated by co-factor NS2B. In this work, we used both versions to characterize the inhibitors
and found that the IC values determined from both versions were similar. Therefore, only values
5
0
for the refolded NS3 were shown. As shown in Table 1, most active compounds in SLC assay
except HJC0365 still showed inhibition against the viral NS2B-NS3 protease activity. However,
we found that there seemed to be no strict correlation between IC50-SLC and IC50-pro values. This is
39
not surprising because niclosamide is a known luciferase inhibitor. The SLC inhibition is an
indirect readout of the NS2B-NS3 interactions. Direct inhibition of luciferase activity and/or direct
inhibition of luciferase complementation in addition to inhibition of NS2B-NS3 interaction may
account for the poor correlation between IC50-SLC and IC50-pro values. While IC50-SLC is a poor
indicator of inhibition efficacy of the NS2B-NS3 interactions, it remains a valuable indicator to
eliminate compounds if they failed to inhibit SLC.
Among these derivatives, compounds HJC0140 with 4´-acetamido group (IC50-pro = 25.5
µM), HJC0149 with benzo[b]thiophene 1,1-dioxide moiety (IC50-pro = 20.3 µM) and HJC0381
with (1-methylpiperidin-4-yl)oxy moiety (IC50-pro = 25.9 µM) maintained the same level of
potency, while compounds HJC0129 with 4´-NH group (IC
= 11.3 µM) and JMX0207 with
50-pro
2
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 8 of 51
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
3
-NO group (IC
= 8.2 µM) showed nearly or more than 2-fold improvement in inhibition of
50-pro
2
the viral NS2B-NS3 protease activity, compared to niclosamide (IC50-pro = 21.6 µM) (Table 1 ,
Figure 1). Taken together, the three compounds HJC0129 (IC50-SLC = 9.6 µM and IC50-pro = 11.3
µM), HJC0140 (IC50-SLC = 9.4 µM and IC50-pro = 25.5 µM), and JMX0207 (IC50-SLC = 1.3 µM and
IC50-pro = 8.2 µM) showed potent inhibitory activity against the NS2B-NS3 interactions in both
NS2B-NS3 SLC and NS2B-NS3 protease assays.
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 2. JMX0207 inhibits DENV. (A) Dose-dependent inhibition of DENV2 infectivity by
JMX0207. N=3. (B) Cell viability assay. A549 cells were incubated with various concentrations
of JMX0207 and then assayed for viability at 48 hrs post-incubation. N=3. Error bars in both
panels represent the standard deviations at each concentration.
Inhibition of DENV2 viral replication.
A viral plaque reduction assay was performed next to evaluate the antiviral efficacy of
these derivatives. Human lung carcinoma A549 cells were infected with DENV2 in the presence
of a concentration series of derivatives or a dimethyl sulfoxide (DMSO) control, and viral titers
were measured at 48 hrs post-infection, as described previously.21-22, 40 Our results indicated that
eight out 11 active derivatives showed appreciable antiviral efficacy with EC50-DN2 less than 30
µM (Table 1). The best derivative, JMX0207, showed slightly improved antiviral efficacy (EC50-
of 0.31 µM), compared to niclosamide (EC50-DN2 of 0.55 µM) (Figure 2A).
DN2
ACS Paragon Plus Environment

Page 9 of 51
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
The observed antiviral activity could result from compound’s cytotoxicity. To address this
concern, we next measured the compound cytotoxicity CC using a WST-8 cell proliferation
5
0
2
1
assay, as we described previously. As shown in Table 1 and Fig. 2B, the majority of these
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
derivatives showed improved CC and therapeutic index (defined as CC /EC ), compared to
5
0
50
50
niclosamide.
Figure 3. JMX0207 inhibits ZIKV. (A) Dose-dependent inhibition of ZIKV infectivity. N=3. (B)
qRT-PCR analysis of inhibition of viral RNA from ZIKV-infected A549 cells by JMX0207. N=3.
Error bars in panels (A) and (B) represent the standard deviations at each concentration. (C)
Immunofluorescence assay of inhibition of viral protein production by JMX0207, using pan-
flavivirus anti-E 4G2 antibody (green) (ATCC).
Inhibition of ZIKV replication.
Our previous studies indicated that niclosamide is a broad-spectrum inhibitor against
2
1
multiple flaviviruses . To determine the antiviral spectrum of the derivatives, we evaluated the
antiviral potency of the derivatives against ZIKV. Our results showed that the derivatives were
also potent inhibitors against ZIKV, with EC50-ZK values comparable to those for DENV2 (Table
1
, Figure 3A). Similarly, JMX0207 showed better inhibition efficacy towards ZIKV than
niclosamide (Figure 3A).
JMX0207 treatment leads to reductions in viral RNA yield and protein production.
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 10 of 51
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
Among the derivatives, JMX0207 showed improved efficacy in almost all categories
Table 1), compared to niclosamide. Therefore, we chose JMX0207 to further characterize
(
whether JMX0207 treatment led to inhibition of viral RNA synthesis and viral protein production,
using qRT-PCR and immunofluorescence assay (IFA), respectively. Our results indicated that
JMX0207 significantly reduced the ZIKV RNA copy number in a dose-dependent manner (Figure
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
3
B). Using a pan flavivirus anti-E antibody 4G2, we conducted an IFA to demonstrate that
JMX0207 greatly reduced ZIKV antigen production in A549 cells in a dose-dependent manner
(Figure 3C), presumably because of inhibition of viral replication.
Overall, these experimental results indicated that JMX0207 showed improved anti-
protease, antiviral, and cytotoxicity properties, compared to niclosamide. JMX0207 treatment
results in inhibition of viral infectivity, viral RNA replication, and viral protein production, and is
a broad-spectrum antiviral for flaviviruses.
JMX0207 inhibits viral production in cells relevant to ZIKV pathogenesis.
We next used human induced pluripotent stem cell (iPSC)-derived neural progenitor cell
(
HNPC) 21, 41 to investigate whether JMX0207 is an effective inhibitor in human primary cells
demonstrated to be relevant to ZIKV pathogenesis.42-46 As shown (Figure 4A & 4B), JMX0207
effectively inhibited ZIKV protein expression and RNA synthesis in HNPC in dose-dependent
manner. Overall, these experiments demonstrate that JMX0207 is an effective antiviral in neural
progenitor cells relevant to ZIKV infection.
ACS Paragon Plus Environment

Page 11 of 51
ACS Infectious Diseases
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
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. Inhibition of ZIKV in cells relevant to ZIKV. (A) Immunofluorescence assay (IFA)
of inhibition of viral protein expression in human neural progenitor cells by JMX0207, using pan-
flavivirus anti-E 4G2 antibody (green) (ATCC). Nuclei (blue) was stained in all IFA assays by the
Hoechst stain solution. (B) qRT-PCR analyses of inhibition of viral RNA from ZIKV-infected
human neural progenitor cell (HNPC) by JMX0207. N=3 (C) ZIKV organoid infected with ZIKV-
Venus. The 3D organoids were infected with PBS (Mock), or ZIKV untreated (DMSO), or ZIKV
treated with JMX0207, or Mock treated with JMX0207. Upper panel, bright field image of intact
organoids. Lower panel, Venus fluorescence image (excitation 515 nm, emission 528 nm) of the
intact 3D organoids. (D) Forebrain reginal specification of organoids. Organoids were stained
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 12 of 51
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
positive for forebrain identity markers PAX6 (green, upper panel), FOXG1 (green, lower panel)
and SOX2 (Red (Magenta after merge with DAPI (Blue)), upper panel) at 20 days. The sections
were stained positive for general neuronal marker TUJI (cyan, upper and lower panels) and were
negative for SOX10 (Red, lower panel). DAPI-merged data were shown. Nuclei (DAPI, Blue);
scale bar 200μm. (E) Slices of organoid infected with ZIKV PRVABC59. The 3D organoids were
infected with PBS (Mock), or ZIKV untreated (DMSO), or ZIKV treated with JMX0207. Upper
panel, IFA using anti-E 4G2 antibody (green); blue, DAPI. Lower panel, details of signature
rosette region of the 3D organoids (Mock) or infected region (DMSO/ZIKV and
JMX0207/ZIKV). Red: Pax6. (F) ZIKV production from the 3D organoids at 5 dpi. Culture
supernatants were collected, and virus production was quantified by PFU assay. N=3. Error bars
in panels (B) and (F) represent the standard deviations at each concentration.
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
JMX0207 protects 3D mini-brain organoid from ZIKV infection.
Newborns from mothers with ZIKV infection during pregnancy have significantly
increased risk of developing microcephaly, a birth defect where a baby has smaller than normal
head circumference. Babies born with microcephaly may develop many brain-related or other
neurological problems. Recently, neural stem cell-derived 3D cerebral organoids were used to
dissect ZIKV pathogenesis and anti-viral development.45, 47-49 Compared to the cultured 2D
monolayer cells, the 3D brain organoids can better represent the composition, diversity and
organization of cell types found in the developing human brain.
Therefore, we used the 3D mini-brain organoid model to further investigate whether
JMX0207 can protect against ZIKV-associated neurological damage. Induced pluripotent stem
cells (iPSC) derived from a healthy control (Male, Caucasian) were differentiated using an
ACS Paragon Plus Environment

Page 13 of 51
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
established protocol to generate region-specific organoids patterned to resemble the dorsal
forebrain (Figure 4C).50 Organoids were stained positive for forebrain identity markers
PAX6 (dorsal forebrain progenitors; upper panel), FOXG1 (lower panel) and SOX2 (neural
ectoderm marker, upper panel) at 20 days. The sections were stained positive for general neuronal
marker TUJI (neuron-specific class III β-tubulin; upper and lower panels) and were negative for
SOX10 (Neural crest, Red, lower panel).
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
To evaluate if JMX0207 protects organoids from ZIKV infections, we first generated a
full-length infectious ZIKV clone expressing Venus fluorescent protein (ZIKV-Venus) (details to
be published elsewhere). At day 20, the organoids displayed signature features of forebrain,
including neural rosettes (Figure 4C-E), and were pre-treated with JMX0207 (1.5 µM) or DMSO
control. At day 20, the organoids were infected with either Mock or ZIKV strain PRVABC59 or
ZIKV-Venus, at an estimated multiplicity of infection (MOI) of 1 in the presence of 1.5 µM
JMX0207 or DMSO control. ZIKV infection was quantified by a plaque forming unit (PFU) assay
at 5 dpi and by fluorescence imaging at 7 dpi, respectively. Our results showed that JMX0207 at
1
.5 µM concentration did not have any toxic effect on the 3D organoids, which remained intact
and unchanged in morphology (Figure 4D). Compared to DMSO control, JMX0207 treatment
completely abolished ZIKV infection in the 3D organoid (Figure 4D).
The 3D mini-brain organoids were sliced for immunostaining to investigate the antiviral
effects of JMX0207 (Figure 4E). As shown, untreated organoids (DMSO) were infected with
ZIKV throughout all layers (Figure 4E). In contrast, JMX0207 treatment nearly completely
protected the organoids from ZIKV infection (Figure 4E). Moreover, ZIKV production was also
significantly inhibited by JMX0207 treatment (Figure 4F). Collectively, the results indicated that
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 14 of 51
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
JMX0207 is an effective inhibitor to protect developing human cortical tissue from ZIKV
infection.
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 5. In vivo antiviral activity of JMX0207 against ZIKV. (A) Pharmacokinetic study of
niclosamide and JMX0207. Adult female B6 mice were given a single oral dose of either
niclosamide or JMX0207 at 40 mg/kg. Plasma was obtained at various times after dosing. Both
niclosamide and JMX0207 were extracted from the plasma and then analyzed by LC-MS/MS as
described in the Methods. N=4. (B) Pharmacokinetic study of JMX0207 at different doses. N=4-
5
. The values in panels (A) and (B) represent means ± S.D. (C) Viremia was detected by a plaque
5
forming unit (PFU) assay on day 3 post-infection of ZIKV with a dose of 1.7 × 10 PFU/mouse in
four-week-old A129 mice, which were treated with vehicle or JMX0207 though oral gavage.
Difference between JMX0207 (N=10) or vehicle (N=10) treatment was analyzed by using the
unpaired, two-tailed t-test. *, P=0.0081. Error bars represent data range of median with 95%
confidence interval.
JMX0207 shows improved pharmacokinetic properties.
We next evaluated whether JMX0207 has favorable pharmacokinetic properties using a
mouse model. As shown in Table 2 and Figure 5A, JMX0207 displayed much better
pharmacokinetic properties than the lead compound niclosamide. In contrast to niclosamide, which
had a short T and low C (0.6 µM), when given to B6 mice orally at 40 mg/kg JMX0207 had
1
/2
max
ACS Paragon Plus Environment

Page 15 of 51
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
an excellent pharmacokinetic profile with Cmax of 145 μM (Table 2), which was much higher than
the EC (0.3 μM) required to inhibit ZIKV. The longer T (~11 h) was also more favorable to
5
0
1/2
ensure less frequent drug administration to infected animals. Further pharmacokinetic study with
different doses of JMX0207 indicated that the plasma drug concentration remained at the same
level after the dose was reduced to 20 mg/kg (Figure 5B). More importantly, mice with repeated
oral dosing at 40 mg/kg/day for 7 days did not display any sign of toxicity (N=6). These results
indicate good bioavailability and low toxicity for the derivative JMX0207.
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. Pharmacokinetic properties of niclosamide and JMX0207
a
Tmax ±S.D. (h) C ±S.D. (µM) T ± S.D. (h) AUC
± S.D.
max
1/2
0→∞
Niclosamide 0.7±0.4
JMX0207 1.2±0.5
S.D., standard deviation
0.6±0.1
145±18
4.6±1.0
11±6.8
3.5±0.8
2719±1018
a
JMX0207 reduces viremia in a ZIKV mouse model.
Finally, we evaluated the in vivo antiviral efficacy using a viremia mouse model, as we
described previously.51-52 Our data showed that JMX0207 treatment at 20 mg/kg/day resulted in a
5
significant reduction in ZIKV-induced viremia in the A129 mice inoculated with 1.7x10 PFU
PRVABC59 ZIKV/mouse compared to the vehicle control (Figure 5C). Overall, the results
indicated that JMX0207 not only inhibited viral replication in vitro but also significantly reduced
viremia in an in vivo animal model.
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 16 of 51
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
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 6. Mechanism of action studies. (A) Time of addition. JMX0207 (0.75 µM) was added at
indicated time points post-infection. Viral titers were quantified using plaque forming assay 48 hrs
post-infection. DMSO was added to control. N=3. ***, p<0.001. (B) Dose-dependent inhibition
of DENV2 replicon by niclosamide and JMX0207. N=3. (C) WB analysis of dose response
inhibition of ZIKV NS3 production by JMX0207 treatment (left). Right, Band intensities of PP
and NS3 normalized to the GAPDH control (N=3). Pre-seeded A549 cells in 6-well plate were
treated with JMX0207 or DMSO control and infected with ZIKV with MOI of 0.1, as described
21
previously. At 48 hrs post-infection, cells were washed, harvested, and protected by protease
inhibitor cocktail prior to lysis by SDS-PAGE loading buffer. Upon incubation at 95 ºC for 10
min, sample was subjected to Western blot analysis with anti-ZIKV NS3 (GTX133309, GeneTex,
Inc.) and anti-GAPDH (CB1001, EMD Millipore) as primary antibodies. Error bars in all panels
represent the standard deviations at each condition.
JMX0207 is effective post-infection.
ACS Paragon Plus Environment

Page 17 of 51
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
Antiviral inhibitors are generally divided into two categories, entry and post-entry
replication), respectively. To determine the mode of action, we first performed a time of addition
(
experiment by adding JMX0207 at different time points post-infection (Figure 6A). Our results
showed that JMX0207 was equally effective in reducing ZIKV titer even at 24 hrs post-infection.
The results indicated that JMX0207 inhibited viral replication instead of viral entry, which agrees
with our hypothesis that JMX0207, as a protease inhibitor, attenuates viral production in the post-
entry replication stage.
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
JMX0207 inhibits viral replication using a DENV2 replicon cell line.
To further investigate the mechanism of action, we next used a DENV2 replicon cell line53
to investigate if JMX0207 inhibited flavivirus replication without an entry step. Our results
indicated that both niclosamide and JMX0207 showed dose-responsive inhibition of DENV2
replication using the DENV2 replicon cell line (Figure 6B). JMX0207 has slightly improved
antiviral efficacy (EC50-DN2 of 1.1 µM) compared to niclosamide (EC50-DN2 of 2.0 µM) (Figure
6
B). Overall, the results indicate that JMX0207 is an effective antiviral to inhibit viral replication
instead of entry.
JMX0207 treatment inhibits viral polyprotein precursor (PP) processing
We carried out Western blot (WB) analysis using an antibody recognizing ZIKV NS3
protein (Figure 6C). Our results showed that JMX0207 treatment led to dose-dependent reduction
of viral NS3 protein production, presumably due to inhibition of viral production by JMX0207.
In addition to reduced NS3 protein production, we observed dose-dependent increase of a protein
with high molecular weight (MW) (Figure 6C). It was known from our previous studies that the
high MW protein is the unprocessed viral polyprotein precursor (PP) which can be recognized by
the anti-NS3 antibody21, 23. The results are consistent with the mechanism by which JMX0207
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 18 of 51
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
inhibits viral protease function, leading to inhibition of viral PP processing, accumulation of non-
functional viral PP, and finally virus reduction.
JMX0207 directly binds to the NS2B-NS3 interface.
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
As a niclosamide derivative, JMX0207 is expected to directly bind to the viral NS3
2
1
protease, as did niclosamide. To demonstrate binding, we first carried out a protein thermal shift
assay (PTSA). Our data indicated that the binding of JMX0207 stabilized the viral NS3 protease,
leading to a 0.75 ºC increase in T of the viral protein. The data suggests that JMX0207 directly
m
binds the viral NS3 protease.
Figure 7: Docking and mutagenesis. (A) Predicted docking pose of JMX0207 (magenta) docking
into NS3pro of DENV2 (PDB Code: 2FOM). NS3pro is in blue ribbon representation and binding
site key interaction residues are highlighted in stick presentation. π cation and π stacking are shown
as cyan dotted lines, H-bond in purple and salt bridge in blue. (B) Predicted binding pose of
Niclosamide (green) docked into NS3pro of DENV2. H-bond is shown as purple dotted lines. (C)
JMX0207 and Niclosamide superimposed at the predicted binding site, in surface representation.
ACS Paragon Plus Environment

Page 19 of 51
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
JMX0207 is shown as magenta sticks and Niclosamide in green. (D) Representative SPR analysis
of JMX0207 binding to the MBP-NS3 protease wild-type and mutant L58A.
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
To investigate the molecular interaction between JMX0207 and viral NS3 protease, we
docked JMX0207 to the NS3 protease structure of DENV2 (PDB ID: 2FOM) using Induced Fit
Docking (IFD) protocol, as we describe previously21-22. The docked pose (Figure 7A) depicted
2
1
JMX0207 can be well docked into the same 2B53 pocket identified from previous study . One
NO group of JMX0207 interacts with Y23 and F46 through π cations, while another NO group
2
2
forms H-bonds with Y33 and K61 and a salt bridge with K26. The phenyl ring on one side of
JMX0207 interacts with L58 through hydrophobic interaction, and the other phenyl ring engages
with Y33 with π- π interaction. The phenol forms an H-bond with H60. The O atom on carbonyl
group of JMX02027 forms an additional H-bond with K26 to further stabilize the binding. We
superimposed the IFD docking pose of JMX0207 and the IFD docking pose of niclosamide. The
overlay analysis demonstrates that JMX0207 binds at the 2B53 pockets on NS3 protease of ZIKV
in a similar manner to that of niclosamide (Figure 7B,C), while an additional nitro group of
JMX0207 also forms critical binding interactions with the protease.
To further investigate JMX0207 binding to viral NS3 protein, we used surface plasmon
resonance (SPR) to measure the binding affinity of JMX0207 to the viral NS3 protease. Because
2
2
we previously generated several MBP-NS3 mutants, we used MBP-NS3 fusion protein in the
SPR analyses for better comparison. As shown in Figure 7D, JMX0207 bound the viral NS3
protease with a binding affinity of 1.1 µM. Collectively, our data demonstrated that JMX0207
directly binds to the viral NS3 protease, with an affinity better than that for niclosamide (6.4 µM).21
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 20 of 51
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
In a previous study, we generated five NS3 mutants, some of which had significant impact
2
2
on binding of small molecule inhibitors to the NS2B-NS3 interface and on viral protease activity .
We measured the binding affinity of JMX0207 to these mutants as well. Our data showed that
L58A abolished the binding of JMX0207 (Table 3, Figure 7D). Two other mutations, I25A and
H60A reduced the binding affinity 2 to 3 folds, whereas mutations Y23A and F46A do not have
any effect.
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 3. Binding affinity of JMX0207 to MBP-NS3 wild-type (WT) or mutant
WT
1.1
Y23A
1.2
I25A
2.3
F46A
1.2
L58A
NDa
H60A
2.9
K (µM)
D
a
ND , no detectable binding.
Discussion
Dengue and Zika viruses cause significant human disease, for which there are no specific
vaccines or therapies. Therefore, development of effective antivirals is urgent. Previously we
identified an existing FDA-approved drug, niclosamide, as a potent antiviral against DENV and
ZIKV. However, the poor pharmacokinetic properties of niclosamide prevent its clinical use to
treat DENV and ZIKV infections.
In this study, we screened a small in-house compound library of niclosamide derivatives
and identified JMX0207 as a candidate inhibitor with improved properties. JMX0207 inhibited
the viral NS2B-NS3 protease activity of DENV2 with better efficacy in vitro than niclosamide. In
cell culture, JMX0207 significantly inhibited the growth of representative flaviviruses, including
DENV2 and ZIKV, with EC in nanomolar range. By using IFA, WB, and qRT-PCR analyses,
5
0
we showed that JMX0207 also significantly inhibited viral RNA synthesis and protein production.
More importantly, compared to niclosamide, JMX0207 has reduced cytotoxicity profile towards
ACS Paragon Plus Environment

Page 21 of 51
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
human cells, indicating a larger therapeutic window towards these flaviviruses. In addition, we
showed that JMX0207 could rescue ZIKV-relevant neural progenitor cells from viral infection in
a dose-dependent manner. Moreover, JMX0207 could almost completely protect 3D cortical
organoids originated from neural stem cells from ZIKV infection. The findings establish the
efficiency of JMX0207 to eliminate infection of ZIKV from human neural progenitor cells, and
provide a path forward to minimize the risk of fetal acquired microcephaly resulting from ZIKV
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
5
4
infection of pregnant women. Furthermore, we showed that JMX0207 displayed significantly
improved pharmacokinetic properties, compared to the parent compound niclosamide. The
improved pharmacokinetics allowed us to carry out in vivo antiviral efficacy studies using an
animal model for ZIKV infection. Our data showed that JMX0207 treatment markedly reduced
viral viremia in the A129 ZIKV mouse model infected with clinical strain ZIKV PRVABC59.
Mechanistically, we showed that JMX0207 directly binds to the viral NS3 protease with improved
binding affinity, compared to its lead drug niclosamide. We also showed that JMX0207 treatment
led to dose-dependent inhibition of viral polyprotein precursor processing, a direct consequence
of inhibition of viral protease activity.
Conclusion
We conclude that the niclosamide derivative JMX0207 is a valuable candidate for further
study, due to its demonstrated ability to inhibit several activities required for ZIKV propagation.
When paired with its reduced cytotoxicity profile and superior pharmacokinetic properties, these
positive attributes indicate that JMX0207 might effectively be used to combat viral progression
following initial infection with ZIKV. Successful development of this compound would be a
valuable tool in the prevention of negative ZIKV-related outcomes, including Guillain-Barre
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 22 of 51
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
syndrome, neuropathy and myelitis, as well as pregnancy-related outcomes including miscarriage,
preterm birth, and congenital Zika syndrome.
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
Methods
Synthesis of niclosamide derivatives.
The synthetic route of compound JMX0207 was described as below (Scheme 1), and the synthesis
of other niclosamide derivatives was reported in our previous publications.34-36, 38 The structures
and purity of all synthesized compounds were confirmed by 1H and 13C NMR, HRMS and HPLC
analysis, and all biologically evaluated compounds are >95% pure.
JMX0207 Synthesis.
a
b
O N
2
COOH
O N
2
CO Me
O2N
COOH
2
OH
O
O
1
2
3
Cl
NO2
Cl
NO2
O
O
O
c
d
N
N
H
H
OH
NO2
NO2
4
JMX0207
Scheme 1. JMX0207 synthesis route.
Reagents and conditions: (a) CH I, K CO , DMF, 50 °C, 24 h, 95%. (b) NaOH, H O/MeOH, r.t.,
3
2
3
2
1
h, 93%. (c) 2-chloro-4-nitroaniline, POCl , pyridine, DCM, 0 °C to r.t., 8 h, 67%. (d) BBr ,
3 3
DCM, 0 °C to r.t., 12 h, 96%.
Methyl 2-methoxy-3-nitrobenzoate (2).
CH I (3.9 g, 27.3 mmol) was added to a solution of 3-nitrosalicylic acid (1.0 g, 5.5 mmol) and
3
K CO (2.3 g, 16.4 mmol) in 20 mL of DMF. The mixture was stirred at 50 °C for 24 hrs and then
2
3
diluted with 300 mL of AcOEt. The resulting mixture was washed with water (3 × 70 mL) and
ACS Paragon Plus Environment

Page 23 of 51
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
brine (50 mL), dried over Na SO , and concentrated in vacuo. The residue was purified by silica
2
4
1
gel column (Hexane/EtOAc = 2/1) to afford compound 2 (1.1 g, 95%) as a yellow oil. H NMR
(300 MHz, CDCl ) δ 8.01 – 7.89 (m, 1H), 7.85 – 7.78 (m, 1H), 7.23 – 7.09 (m, 1H), 3.91 (s, 3H),
3
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
13
3
5
2
.87 (s, 3H). C NMR (75 MHz, CDCl ) δ 164.6, 153.1, 145.4, 135.5, 128.3, 127.2, 123.7, 64.1,
3
2.6.
-Methoxy-3-nitrobenzoic acid (3).
NaOH (1.1 g, 27.3 mmol, in 6 mL of H O) was added to a solution of compound 2 (1.1 g, 5.5
2
mmol) in 10 mL of MeOH. The mixture was stirred at r.t. for 1 h, and then the pH value was
adjusted to 5~6 with 1 M HCl (aq.). The mixture was extracted with EtOAc (2 × 120 mL) and
washed with water (80 mL) and brine (60 mL), dried over Na SO , and concentrated to give acid
2
4
3
(1.0 g, 93%) as a light yellow solid for direct use in the next step.
N-(2-Chloro-4-nitrophenyl)-2-methoxy-3-nitrobenzamide (4).
POCl (3.9 g, 25.4 mmol) was added slowly at 0 °C to a solution of compound 3 (1.0 g, 5.1 mmol),
3
2
-chloro-4-nitroaniline (876 mg, 5.1 mmol) and pyridine (8.0 g, 101.4 mmol) in 50 mL of DCM.
After addition, the mixture was stirred at r.t. for 8 h and then poured into 200 mL of ice-water.
1
Compound 4 (1.2 g, 67%) was isolated by filtration as a yellow solid. H NMR (300 MHz, CDCl )
3
δ 10.58 (s, 1H), 8.93 (d, J = 9.3 Hz, 1H), 8.46 (dd, J = 7.8, 1.8 Hz, 1H), 8.37 (d, J = 2.7 Hz, 1H),
8
3
.24 (dd, J = 9.3, 2.4 Hz, 1H), 8.06 (dd, J = 8.1, 1.8 Hz, 1H), 7.46 (t, J = 8.1 Hz, 1H), 4.12 (s,
1
3
H). C NMR (75 MHz, CDCl ) δ 161.9, 151.7, 144.4, 143.6, 140.7, 137.0, 129.9, 128.2, 125.3,
3
125.1, 123.8, 123.1, 121.1, 64.9.
N-(2-Chloro-4-nitrophenyl)-2-hydroxy-3-nitrobenzamide (JMX0207).
BBr (7.4 ml, 7.40 mmol, 1M in DCM) was added dropwise at 0 °C to a solution of compound 4
3
(1.3 g, 3.70 mmol) in 250 mL of DCM. The mixture was stirred at r.t. for 2 h. Then the mixture
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 24 of 51
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
was poured into 200 mL of ice water. The yellow precipitate JMX0207 was isolated by filtration.
The organic layer was separated and concentrated. Then 50 mL of MeOH was added and the
mixture was stirred at r.t for 20 min. The yellow solid was isolated by filtration. The two parts of
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
yellow solids were combined to afford 1.2 g (96%) of compound JMX0207 in total. HPLC purity
1
9
8
9.9% (t = 18.75 min). H NMR (300 MHz, DMSO-d ) δ 12.10 (s, 1H), 8.65 (d, J = 9.3 Hz, 1H),
R
6
.42 (d, J = 2.4 Hz, 1H), 8.34 – 8.24 (m, 2H), 8.13 (d, J = 8.1 Hz, 1H), 7.03 (t, J = 7.8 Hz, 1H).
1
3
C NMR (75 MHz, DMSO-d ) δ 164.2, 154.1, 143.1, 141.3, 139.1, 136.3, 129.8, 124.8, 124.1,
6
+
1
3
23.6, 122.5, 122.4, 117.3. HRMS (ESI) calcd for C H ClN O 338.0180 (M + H) , found
1
3
9
3
6
38.0172.
Cloning, Expression and Purification.
All clones and proteins were generated as previously described.21-22
Split Luciferase Complementation (SLC) Assay.
The SLC assay was performed as previously described.21-22, 55
Protease Inhibition Assay.
The protease inhibition assay was performed using the refolded DENV2 NS3 fusion
protein (50 nM) and a fluorescence resonance energy transfer (FRET) peptide substrate (100 µM)
(
Abz-RRRRSAG-nTyr (NeoBiolab)), as described previously.21-23, 55 Substrate cleavage was
monitored at excitation/emission wavelengths of 360 nm/420 nm (Abz substrate) by a BioTek
Flx800. The rate of increase in relative fluorescence unit (RFU) over time was calculated in the
linear range and normalized as a percent of the DMSO control. The IC /CC /EC was
5
0
50
50
determined by fitting the dose-responsive curve with a non-linear regression function using the
GraphPad Prism 8 (San Diego, CA). All experiments were performed in triplicates.
Cytotoxicity Assay.
ACS Paragon Plus Environment

Page 25 of 51
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
Cytotoxicity was measured by a WST-8 cell proliferation assay kit (Dojindo Molecular
2
1
Technologies, Inc.) as previously described. All experiments were performed in triplicates.
Viral Titer Reduction Assay.
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
A viral titer reduction assay was used to determine the compounds’ effect on DENV2 and
ZIKV strains, as described previously21-22. Human A549 lung carcinoma cells and human primary
neural progenitor cells were used, as described previously.21-22 All experiments were performed in
triplicates.
Immunofluorescence Assay.
The immunofluorescence assay was performed using ZIKV-infected cells treated with
DMSO or JMX0207, as described previously.21-22 A mouse monoclonal pan anti-E antibody 4G2
(
ATCC) and a DyLight® 488 goat anti-mouse IgG (ImmunoReagents, Inc.) were used to monitor
viral E protein expression, as described previously.21-22
Quantitative qRT-PCR.
Quantitative qRT-PCR was performed as described previously.21-22 ZIKV primers
CCGCTGCCCAACACAAG and CCACTAAYGTTCTTTTGCAGACAT with ZIKV probe Cy5-
AGCCTACCT/TAO/TGACAAGCAGTCAGACACTCAA-IAbRQSp were used. The 2−ΔΔCT
(
“delta-delta Ct”) method was used to quantify samples. All experiments were performed in
triplicates.
Western Blot.
Western blot was performed using anti-ZIKV NS3 (GTX133309, GeneTex, Inc.) and anti-
GAPDH (CB1001, EMD Millipore) antibodies, as described previously.21, 23 All experiments were
performed in triplicates.
DENV2 Replicon Assay.
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 26 of 51
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
BHK-21 cells stably expressing DENV2 replicon with a Renilla luciferase (Rluc) reporter
5
3
5
gene were seeded into white 96-well plate at a density of 2 x 10 cells per well. The cells were
o
incubated at 37 C in a humidified incubator with 5% CO for hrs in a 100 µl medium containing
2
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
Minimal Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS), 100 I.U./ml
penicillin, 100 μg/ml streptomycin, and 300 μg/ml G418. Upon 24 hrs incubation, culture medium
was discarded. Fresh culture medium of 100 µl with a concentration series of compounds or
DMSO control was added to the cells in triplicates. The culture was further incubated for 48 hours.
The cells were then washed twice with 100 µl phosphate-buffered saline (PBS), followed by
addition of 25 µl of a lysis buffer containing 1X PBS and 1% Triton X-100. The mixture was
incubated at room temperature with gentle shaking (50 rpm) for 30 minutes. Assay buffer (125 µl)
containing
1X
PBS,
0.05%
(3-((3-cholamidopropyl)
dimethylammonio)-1-
propanesulfonate)(CHAPS), and 0.1% BSA was then added to each well. In the meantime, a fresh
4X working substrate coelenterazine was prepared by diluting 1 µl of stock coelenterazine (2.4
mM) dissolved in ethanol into 10 ml assay buffer (10,000-fold dilution). Finally, 50 µl 4X substrate
coelenterazine was added to each well with a final substrate concentration of 0.06 µM, using a
Veritas luminometer. The luminescence was recorded immediately using the Veritas luminometer.
The luminescence data was normalized to DMSO control. The EC was determined by nonlinear
5
0
regression fitting of normalized experimental data in GraphPad Prism 8.0 (San Diego, CA). All
experiments were performed in triplicates.
Docking.
The crystal structure of NS3pro of DENV2 (PDB Code: 2FOM) was downloaded from RCSB
PDB bank. After excluding the cofactor NS2B peptide, the structure was preprocessed and
optimized with Schrödinger Protein Preparation Wizard using default settings. The 3D structure
ACS Paragon Plus Environment

Page 27 of 51
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
of JMX0207 and niclosamide were created using Schrödinger Maestro and prepared with LigPrep
to generate a low energy conformation suitable for docking. The Induced Fit Docking (IFD)
protocol of Schrödinger Small-Molecule Drug Discovery Suite was employed in this docking
study. The grid box for docking was centered on the 2B53 binding site. The box size was set to 20
Å on each side. Selected side chains of R24, K26, and M59 were temporarily trimmed (the
equivalent of being mutated to alanine) during the initial IFD process and were restored later. The
receptor-ligand complexes structures generated from IFD docking were imported into Schrödinger
Maestro for visualization and analysis of binding site interactions.
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
Surface Plasmon Resonance.
Surface plasmon resonance (SPR) was used to determine the affinity and kinetic analyses
of the interactions between JMX0207 and the MBP-NS3 proteins at 25 °C using a ProteOn XPR36
SPR instrument (Bio-Rad). The MBP-NS3 wild-type or mutant proteins were immobilized onto a
ProteOnTM GLH sensor chip (~9,000 RU) (Bio-Rad). A 2-fold dilution series of compounds were
injected as the analytes. A blank surface blocked by ethanolamine was used as the control surface.
The experiment was carried out at a flow rate of 100 µl/min using a PBSTD buffer containing 1x
PBS, 0.005% surfactant P20, and 5% DMSO. Association (k ) and dissociation (k ) rates, as well
a
d
as the dissociation constant (K ), were obtained by global fitting of the SPR data from multiple
D
concentrations to a simple 1:1 Langmuir binding model, using the ProteOn Manager software suite
(Bio-Rad).
Culturing of human iPSCs.
Human iPSC F11350.1 culture was maintained in feed-free conditions using mTeSR1
medium (StemCell Technologies #85851) with 5X supplement (StemCell Technologies #85852).
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 28 of 51
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
Cells were maintained on six-well plates coated with human embryonic stem cell-qualified
Matrigel at a 1:60 dilution (Corning Catalog #354277) and passaged with ReLeSR (StemCell
Technologies #05872). Cortical 3D organoids were generated based on the protocol described by
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
Yoon et al. Briefly, iPSCs were dissociated with accutase (ThermoFisher #A1110501) and then
6
plated at a density of 3x10 cells per well in AggreWell 800 24-well plate (StemCell Technologies
#
34811) in Essential 8™ medium (ThermoFisher #A1517001) supplemented with 10µM rock
inhibitor (Tocris #Y27632). The plate was centrifuged at 100 x g for 3 min and incubated at 37°C
overnight. The next day (culture day 0) the organoids were transferred to a low attachment 10cm
plate (Corning #3262) and maintained in 10mL of neural induction medium (Essential 6™
medium, Life Technologies, #A1516401) supplemented with two SMAD pathway inhibitors, 10
µM SB431541 (R&D #1614/50) and 2.5 µM dorsomorphin (Tocris #3093), and Wnt pathway
inhibitor 2.5 µM XAV-939 (Tocris #XAV-939). Media were exchanged daily. On day 6, the
medium was changed to neural expansion medium (Neuralbasal A medium, ThermoFisher
#
10888) supplemented with 2% B27 (without Vit A; ThermoFisher #12587001), 1% antibiotic-
antimycotic (ThermoFisher #15240062), 1% glutaMAX (ThermoFisher # 35050061), 20 ng/mL
FGF2 (R&D #233-FB-500) and 20 ng/mL EGF2 (PeproTech #AF-100-15). Media were
exchanged daily.
Cryopreservation.
Organoids were fixed in 4% paraformaldehyde (Santa Cruz) overnight at 4°C. They were
then washed three times in PBS and transferred to 30% sucrose solution for 72 h. Organoids were
then transferred into plastic cryomold (10 X 10 X 5mm; Tissue Tek cat. no. 4565) with embedding
medium (Tissue-Tek OCT compound, Sakura Finetek 4583) and stored at −80°C. For
ACS Paragon Plus Environment

Page 29 of 51
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
immunohistochemistry, 20 μm thick sections were cut with a Leica cryostat (model CM3050S).
Immunohistochemistry.
Cryosections were blocked in 3% bovine serum albumin (BSA), 10% normal goat serum
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
(NGS), and 0.3% Triton X–100 diluted in PBS for 1 hour at room temperature. The sections were
incubated overnight at 4°C with primary antibodies diluted in blocking solution. Sections were
washed three times with PBS and then incubated with appropriate secondary antibodies diluted in
blocking solution for 1 hour. The following primary antibodies were used for
immunohistochemistry: anti-Pax-6 (1:200; Biolegend #901301), anti-Sox2 (1:100; Santa Cruz
#
sc-365823), anti-FoxG1 (1:500; Takara #M227), Sox10 (1: 100; Santa Cruz # sc369692), and
anti-tubulin III (1:1000; Sigma #T8660).
Pharmacokinetics.
Two- to three-month-old female B6 mice were used for the study. Four to five mice per
group were given niclosamide at 40 mg/kg (in 1% DMSO and 0.5% carboxymethylcellulose) or
JMX0207 in the same vehicle at the indicated dose by oral gavage. Sample preparation and liquid
chromatography with tandem mass spectrometry (LC-MS/MS) detection of niclosamide and
JMX0207 were essentially the same as described in a previous study and were performed using a
Sciex 4000 Q-Trap mass spectrometer (AB SCIEX, Framingham, MA) with the Agilent 1200
high-performance liquid chromatography system (Agilent Technologies, Santa Clara, CA).29
JMX0207 was monitored at m/z 336/171. Declustering potential, entrance potential, collision
energy, and collision cell exit potential were optimized for detection and quantification of
JMX0207 at -45, -10, -35, and -14V, respectively. Data from 4 or 5 mice at each time point were
averaged and used to calculate pharmacokinetic parameters, using a pharmacokinetic solver
(Microsoft, Redmond, WA) by assuming a noncompartmental model. Statistical significance of
ACS Paragon Plus Environment