Inhibition of Flaviviruses by Targeting a Conserved Pocket on the Viral Envelope Protein

Article abstract of DOI:10.1016/j.chembiol.2018.05.011

Viral envelope proteins are required for productive viral entry and initiation of infection. Although the humoral immune system provides ample evidence for targeting envelope proteins as an antiviral strategy, there are few pharmacological interventions that have this mode of action. In contrast to classical antiviral targets such as viral proteases and polymerases, viral envelope proteins as a class do not have a well-conserved active site that can be rationally targeted with small molecules. We previously identified compounds that inhibit dengue virus by binding to its envelope protein, E. Here, we show that these small molecules inhibit dengue virus fusion and map the binding site of these compounds to a specific pocket on E. We further demonstrate inhibition of Zika, West Nile, and Japanese encephalitis viruses by these compounds, providing pharmacological evidence for the pocket as a target for developing broad-spectrum antivirals against multiple, mosquito-borne flavivirus pathogens. Countermeasures against dengue, Zika, and other flaviviruses are a large, unmet medical need. de Wispelaere et al. validate a conserved pocket of the flavivirus envelope protein as a target for small-molecule antivirals with broad-spectrum activity against flaviviruses.

Full text of DOI:10.1016/j.chembiol.2018.05.011

Article
Inhibition of Flaviviruses by Targeting a Conserved
Pocket on the Viral Envelope Protein
Graphical Abstract
Authors
Melissanne de Wispelaere,
Wenlong Lian, Supanee Potisopon, ...,
Jarrod A. Marto, Nathanael S. Gray,
Priscilla L. Yang
cyanohydrazone
O
CN
N
H
N
CF3
N
2
,4-disubstituted
pyrimidine
O
4,6-disubstituted
H
pyrimidine
N
N
O
Correspondence
priscilla_yang@hms.harvard.edu
N
H
N
N
N
H
OCF3
OCF3
In Brief
Dengue, Zika,
West Nile, Japanese
encephalitis virus
envelope proteins
Countermeasures against dengue, Zika,
and other flaviviruses are a large, unmet
medical need. de Wispelaere et al.
validate a conserved pocket of the
flavivirus envelope protein as a target for
small-molecule antivirals with broad-
spectrum activity against flaviviruses.
target
membrane
cytosol
pre-fusion
post-fusion
E trimer
E dimer
viral lipid
membrane
Fusion
pore
Highlights
d
d
d
d
Small molecules targeting the dengue virus E protein inhibit
membrane fusion
The target of these inhibitors is a pocket conserved in other
flavivirus E proteins
Zika and other flaviviruses are also inhibited by compounds
targeting this site
These compounds block infection without the risk of
antibody-dependent enhancement
de Wispelaere et al., 2018, Cell Chemical Biology 25, 1–11
August 16, 2018 ª 2018 Elsevier Ltd.
https://doi.org/10.1016/j.chembiol.2018.05.011

Please cite this article in press as: de Wispelaere et al., Inhibition of Flaviviruses by Targeting a Conserved Pocket on the Viral Envelope Protein, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.05.011
Cell Chemical Biology
Article
Inhibition of Flaviviruses by Targeting a Conserved
Pocket on the Viral Envelope Protein
Melissanne de Wispelaere,1 Wenlong Lian, Supanee Potisopon,
,9
1,9
1,6,9
Pi-Chun Li,2,9 Jaebong Jang, Scott B. Ficarro,
2
3,4
5
1
1,7
1,8
1
5
2
Margaret J. Clark, Xuling Zhu, Jenifer B. Kaplan, Jared D. Pitts, Thomas E. Wales, Jinhua Wang, John R. Engen,
Jarrod A. Marto, Nathanael S. Gray, and Priscilla L. Yang
Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA
2Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School and Department of Cancer Biology, Dana-Farber
Cancer Institute, 360 Longwood Avenue, Boston, MA 02215, USA
3
,4
2
1,10,
*
1
3Department of Cancer Biology, Department of Oncologic Pathology, Blais Proteomics Center, Dana-Farber Cancer Institute, 360 Longwood
Avenue, Boston, MA 02215, USA
4Department of Pathology, Brigham and Women’s Hospital, 45 Francis Street, Boston, MA 02115, USA
5Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, USA
6Present address: Bioaster, Lyon 69007, France
7Present address: Elpidera/Moderna Therapeutics, Cambridge, MA 02139, USA
8Present address: Abbvie Bioresearch Center, Worcester, MA, USA
9These authors contributed equally
10Lead Contact
*Correspondence: priscilla_yang@hms.harvard.edu
https://doi.org/10.1016/j.chembiol.2018.05.011
SUMMARY
bat DENV, ZIKV, and many other flavivirus pathogens.
Geographic spread of the Aedes mosquito species that trans-
Viral envelope proteins are required for productive mits DENV and ZIKV, and ZIKV’s recent explosive emergence
viral entry and initiation of infection. Although the hu- in the Western Hemisphere have heightened the need for coun-
termeasures that can reduce transmission and therapeutic inter-
ventions that can prevent severe disease resulting from infec-
tion. Due to the lack of success to date of efforts aimed at
moral immune system provides ample evidence for
targeting envelope proteins as an antiviral strategy,
there are few pharmacological interventions that
producing antivirals against the viral polymerase and protease,
have this mode of action. In contrast to classical anti-
alternative antiviral strategies to inhibit infection are of growing
viral targets such as viral proteases and polymer-
relevance.
ases, viral envelope proteins as a class do not have
a well-conserved active site that can be rationally tar-
geted with small molecules. We previously identified
Small molecules that target the envelope protein on the sur-
face of virions have the potential to engage their target extracel-
lularly and to block the viral replication cycle at its earliest step.
compounds that inhibit dengue virus by binding to its Although this antiviral strategy is well-validated by the humoral
envelope protein, E. Here, we show that these small immune response to many viruses, there are few antivirals that
molecules inhibit dengue virus fusion and map the act by this mode of action. A major barrier in developing antivirals
targeting envelope proteins is the absence of a conserved active
binding site of these compounds to a specific pocket
on E. We further demonstrate inhibition of Zika, West
Nile, and Japanese encephalitis viruses by these
compounds, providing pharmacological evidence
for the pocket as a target for developing broad-spec-
trum antivirals against multiple, mosquito-borne fla-
vivirus pathogens.
site analogous to those of viral proteases and polymerases. For
DENV, the viral envelope protein, E, exists as 90 prefusion di-
mers on the surface of mature virions. Viral entry is initiated by
engagement of E with attachment factors on the plasma mem-
brane of the host cell, followed by uptake of the virion by a cla-
thrin-dependent process (Acosta et al., 2008; Krishnan et al.,
2007; van der Schaar et al., 2008). Acidification of the endosomal
compartment triggers significant structural changes leading to
reorganization and refolding of E as a postfusion trimer (Allison
et al., 1995; Bressanelli et al., 2004; Modis et al., 2004). This
structural transformation induces fusion of the viral and endoso-
INTRODUCTION
Dengue virus (DENV) is a mosquito-borne virus from the genus mal membranes and creates a pore that allows escape of the
Flavivirus, which includes several other human pathogens viral nucleocapsid into the host cytosol where the viral RNA
including yellow fever, West Nile, Japanese encephalitis, and genome can be expressed.
Zika viruses (YFV, WNV, JEV, and ZIKV, respectively). Over
Over a decade ago, crystallization of the DENV serotype 2
00 million dengue infections occur annually (Bhatt et al., (DENV2) E protein in the presence of millimolar concentrations
013), resulting in disease that can escalate to dengue hemor- of the detergent b-octylglucoside (bOG) enabled Modis et al.
3
2
rhagic fever and dengue shock syndrome. We currently lack a (2003) to detect binding of a detergent molecule in a pocket be-
broadly protective vaccine or specific antiviral treatment to com- tween domains I and II. The location of the detergent-binding site
Cell Chemical Biology 25, 1–11, August 16, 2018 ª 2018 Elsevier Ltd.
1

Please cite this article in press as: de Wispelaere et al., Inhibition of Flaviviruses by Targeting a Conserved Pocket on the Viral Envelope Protein, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.05.011
(
the ‘‘bOG pocket’’) at a hinge region for the major conforma- affected at concentrations of the compounds sufficient to reduce
tional changes that occur during E-mediated membrane fusion infectivity by 10-fold (IC90). Since DENV virions attach to the
led to the proposal that small molecules that bind at this site plasma membrane, but their clathrin-mediated uptake is in-
ꢀ
might inhibit DENV entry. While multiple small molecules that hibited at 4 C (van der Schaar et al., 2007), we quantified the
inhibit DENV entry by binding E and/or by preventing fusion number of viral genomes attached to cells at this temperature
have been reported (Clark et al., 2016; Kampmann et al., 2009; and found it to be unaffected by the inhibitors (Figure 1B). To
Poh et al., 2009; Schmidt et al., 2012; Wang et al., 2009; Zhou determine how the inhibitors might affect E’s catalysis of mem-
et al., 2008), the structural basis for their inhibitory activities brane fusion, we monitored the low pH-triggered fusion of
has not been determined. Furthermore, although residues lining DENV virions with trypsin-loaded liposomes using digestion of
this pocket appear to conserved across other flaviviruses, the viral core protein, located on the interior of the viral lipid
neither inhibitors of other flavivirus E proteins nor co-crystal bilayer, as a marker for successful creation of a fusion pore (Fig-
structures of other flavivirus E proteins with bOG or other ligands ure 1C). Following incubation of DENV2 virions with liposomes,
have been reported. Validation of the bOG pocket as a bona fide we detected pH-dependent digestion of core, which was
pharmacological target mediating the inhibition of DENV and blocked in the presence of compounds 2-12-2, 7-148-6, and
other flaviviruses has thus remained tantalizing but elusive.
other pyrimidines (Figures 1C and S2). Protection of the viral
We report here on 2,4-diamino pyrimidines and 4,6-disubsti- capsid is concentration dependent and correlates well with the
tuted pyrimidines that target the prefusion form of the DENV inhibitor concentrations at which antiviral activity is observed,
E protein (DENV E) and block viral entry by inhibiting membrane thus providing strong evidence that these compounds inhibit
fusion. A resistance mutation, E-M196V, located adjacent to the DENV by interfering with E’s function in the fusion step of
bOG pocket, reduces small-molecule-binding affinity and re- viral entry.
stores virus entry and infectivity in the presence of both types
of pyrimidines, suggesting that this pocket may be the molecular A Mutation Adjacent to the bOG-Binding Pocket Confers
target of these compounds. We confirmed this hypothesis by Antiviral Resistance
demonstrating crosslinking of a photoactivatable inhibitor to a To understand how these compounds interact with DENV E to
region of E located in the bOG pocket and showing that other inhibit its fusogenic activity, we serially passaged DENV2 NGC
substitutions in the bOG pocket introduced by site-directed in the presence of different inhibitors to select for resistance.
mutagenesis affect inhibitor binding to recombinant, soluble E. While in most cases no resistance was observed after >10
Comparison of the effects of site-directed mutations on com- passages (data not shown), we did observe emergence of a
pounds representative of the two different pyrimidine inhibitors resistant quasispecies after six passages in the presence of
series, as well as a previously reported cyanohydrazone inhibitor the 2,4-diamino pyrimidine 7-148-6 (Figure 2A). Sequencing of
of DENV E, suggests differences in how these inhibitors engage the quasispecies at passage 7 revealed an A to G mutation re-
the bOG pocket. Consistent with conservation of residues in sulting in substitution of valine for methionine at residue 196 of
the bOG pocket, we demonstrate inhibition of ZIKV, WNV, and E. This residue lies adjacent to the potential ligand-binding site
JEV by both pyrimidine and cyanohydrazone inhibitors, with ev- occupied by the bOG detergent molecule (PDB: 1OKE) in the
idence for both DENV-specific antiviral activity as well as broad- DENV2 soluble, prefusion E dimer crystal structure (Modis
spectrum activity against multiple flaviviruses. These findings et al., 2003) (Figure 2B).
establish the bOG pocket as a bona fide target for pharmacolog-
Following three rounds of virus plaque purification, we isolated
ical inhibition of flavivirus entry and provide a foundation for and sequence-verified a DENV2 NGC E-M196V clone and
defining structure-activity relationships for developing antivirals confirmed that this virus has reduced sensitivity to 7-148-6.
that target E.
The IC90 of 7-148-6 against DENV2 NGC is 13 ± 2 mM (average
of four independent experiments; representative experiment
shown in Figure 2C). Although 7-148-6’s solubility under the con-
ditions of the antiviral assays limited our ability to saturate inhibi-
tion of the E-M196V mutant in two of three independent experi-
ments, we can estimate the lower bound of the IC90 value to be
RESULTS
Disubstituted Pyrimidines Inhibitors of DENV Inhibit
E-Catalyzed Membrane Fusion
We recently reported the discovery and characterization of 2,4- 30 mM (Figure 2C). The DENV2 E-M196V virus exhibits similar
diamino and 4,6-disubstituted pyrimidines that inhibit DENV en- resistance to related 2,4-diamino pyrimidine 2-12-2 (Figure S3).
try by binding directly to the prefusion form of E (Clark et al., We independently confirmed this by generating DENV2 single-
2
016). For our current studies, we focused on representative cycle reporter virus particles (RVPs) (Ansarah-Sobrinho et al.,
compounds from each of these classes (Figure 1A). We addition- 2008) to show that the E-M196V substitution significantly allevi-
ally included the cyanohydrazone 3-110-22 as a control since we ates the inhibitory effects of 7-148-6 on RVP entry (Figure 2D).
had previously shown it to inhibit DENV fusion, although its bind-
We reasoned that the M196V mutation might confer resistance
ing site on E remains unknown (Schmidt et al., 2012). To investi- by interfering with binding of 7-148-6 and 2-12-2, by enabling E
gate how these compounds affect E’s function during viral entry, to mediate fusion despite binding of the inhibitor, and/or by
we first quantified antiviral activity against the New Guinea C increasing the proportion of non-infectious particles that can
strain of dengue virus serotype 2 (DENV2 NGC) for representative act as a ‘‘molecular sponge’’ to decrease inhibitor binding by in-
compounds using a previously established viral infectivity assay fectious virions. To examine the extent to which the mutation af-
(Clark et al., 2016) (Figures 1A and S1). We then determined how fects inhibitor binding, we expressed soluble, prefusion DENV2
E’s functions in virion attachment and membrane fusion are wild-type E and E-M196V proteins (sE and sE-M196V) and
2
Cell Chemical Biology 25, 1–11, August 16, 2018

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Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.05.011
A
IC90 μM (DENV2 NGC)
non-linear
virus or RVP
inhibitor
preincubation
wash
and
quantify
single-cycle
yield of
[inhibitor]
to reduce
yield by
compound
infection in
IC90
=
empirical*
regression
+
in presence overlay
of inhibitor
with
infectious virus
1 log10 unit
F
3
CO
2
-12-2
N
N
medium in supernatants
5
8 ± 1
13 ± 2
21 ± 1
2
,4-disubstituted
N
H
N
N
N
H
pyrimidine
OCF
3
OR
[inhibitor]
luciferase
IC50 = to reduce
luciferase
activity
7-148-6
4
3
5 min
7°C
60 min
37°C
60 min
37°C
assay
25
15
N
H
N
H
2
,4-disubstituted
(RVPs)
OCF
O
3
pyrimidine
by 50%
F
3
CO
GNF-2
N
N
IC90 8 μM
IC90 14.3 μM
N
H
NH
2
4
3
2
1
×104
×104
×104
4,6-disubstituted
2
pyrimidine
F
3
CO
1-100-1
,6-disubstituted
pyrimidine
N
N
4
7.5
0.7
11 ± 1
×104
1×104
N
O
H
×104
3
-110-22
O
CN
H
N
CF
3
4.0 ± 0.4
N
0
0
cyanohydrazone
O
0
.01 0.1
1
10 100
0.01 0.1
1
10 100
[2-12-2] μM
[7-148-6] μM
pH 8.0
pH 5.5
2-12-2
B
C
03
+ liposomes
Trypsin
2-12-2
1
1
1
virus + inhibitor
preincubation
E
C
02
01
pH 8.0
1
0 min
4
5 min
7°C
3
2
-
6
in
-
ed
ct
pH 5.5
SO 2-
ar
1
-
48
p
1
e
fe
in
DM
2
7
h
n
u
Figure 1. Effects of Inhibitors on the Attachment and Fusion Steps of DENV Entry
A) Infectivity assays using live virus or single-cycle reporter virus were designed to assess inhibition of viral entry when compound treatment is limited to a
(
preincubation with the inoculum and the initial 1 hr infection, after which non-adsorbed virus and compound were washed away. Productive viral entry was
assessed by quantifying the single-cycle yield of infectious virus produced. IC90 values—defined as the concentration of compound required to reduce single-
cycle viral yield by 10-fold—were previously estimated empirically (marked by ‘‘*’’) (Schmidt et al., 2012; Clark, et al., 2016) and were determined here by titration
and non-linear regression analysis. Values in the table are the average and SD from n R 2 independent experiments. Graphs are representative data from one of n
R 2 independent experiments with error bars indicating the standard deviation for experimental replicates within that experiment. Graphs for 2,4-disubstituted
pyrimidines 2-12-2 and 7-148-6 are shown here; graphs showing representative data for GNF2 and 1-100-1 are provided in Figure S1.
ꢀ
(B) Effects of compounds on the attachment step of DENV2 entry were assessed by incubation of cells with virus at 4 C in the presence of representative inhibitors
followed by washes to remove non-specifically bound material and qT-PCR analysis of isolated RNA to quantify the number of attached DENV2 genomes.
Heparin was used as a positive control. Graphs are representative data from one of n R 2 independent experiments with error bars indicating the standard
deviation for experimental replicates within that experiment.
(C) Fusion of DENV2 virions with synthetic liposomes encapsulating trypsin is triggered by low pH. Formation of a fusion pore allows trypsin (blue dots) to access
and digest core protein (green) from the interior of the virion, while envelope protein (E) (red ovals) on the exterior of the virion remains intact. Western blot analysis
of the reactions for (C and E) shows that compound 2-12-2 protects core from digestion upon exposure of the virion-liposome mixture to acidic pH, indicating
concentration-dependent inhibition of viral fusion. Representative data are shown for n R 2 independent experiments. Final concentrations of 2-12-2 were 5, 15,
25, and 40 mM. Analogous data for other compounds are provided in Figure S2.
used biolayer interferometry to measure equilibrium dissociation Disubstituted Pyrimidine Inhibitors of DENV E Bind in
constants (K ) for their interaction with 7-148-6 (Figure 2E) and the bOG Pocket
-12-2 (Figure S3). The wild-type sE binds to both compounds To probe the site of inhibitor binding more directly, we synthe-
D
2
with K
D
values of 6.1 ± 1.8 mM for 7-148-6 and 6.5 ± 2.1 mM for sized JBJ-16-111, an analog of the 4,6-disubstituted pyrimidine
2
-12-2, whereas sE-M196V has reduced affinity with K of 1-100-1 conjugated via linker to a diazirine group suitable for
D
greater than 13 and 12 mM for 7-148-6 and 2-12-2, respectively. photocrosslinking experiments (Figure 3A). A recombinant pro-
This demonstrates that the mutation affects the interactions of tein comprising DENV2 E domains I and II was preincubated
7
-148-6 and 2-12-2 with DENV2 sE through either a direct or with JBJ-16-111 and then irradiated with long-wave UV light to
an allosteric mechanism. Reduced sensitivity of the DENV2 activate the photocrosslinking group. Following trypsin digestion
E-M196V virus to the 4,6-disubstituted pyrimidines 1-100-1 of the crosslinking reaction, capillary electrophoresis-mass
and GNF2 (Figures 2F and S4) further suggests that this mutation spectrometry analysis detected crosslinking to a peptide corre-
may similarly affect the interaction of these inhibitors with E.
sponding to residues 247–284. Fragment ion data further
Cell Chemical Biology 25, 1–11, August 16, 2018
3

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Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.05.011
A
B
C
DENV2 wildtype,
ko
IC90 12 ± ±2 μM
1
00
1
1
1
1
06
04
lo
g
DENV2 E-M196V,
IC90 ≥±30 μM
f
5
0
0
02
DMSO
-148-6
7
00
2.5 5 10 20 40
[
7-148-6] ꢀM
1
2 3 4 5 6 7 8 9
passage
βOG
N
M196
N
H
N
N
H
OCF3
D
E
F
sE wt, Kd 3.2 ± 1.5 μM
sE M196V, Kd ≥ 15μM
DENV2 wildtype,
100
50
0
IC90 11 ± ±1 μM
DMSO
p= 0.03
0.15
.10
1
0 μM 7-148-6
DENV2 E-M196V,
IC90 31± ±17 μM
1
00
0
5
0
0
0.05
0.00
2
.5
5
10 20 40
1-100-1] μM
[
0
5
10 15 20 25
F3CO
DENV2 DENV2
wt E-(M196V)
N
N
[7-148-6] μM
N
H
O
Figure 2. Selection of a Mutation Conferring Resistance to the 2,4-Disubstituted Pyrimidine 7-148-6
A) DENV2 NGC was serially passaged in the presence of 7-148-6 or vehicle control (‘‘DMSO’’) and titered by viral plaque formation assay after each passage.
(
A resistant quasispecies was observed by passage 6. Sequencing of the resistant and vehicle control quasispecies identified a mutation leading to substitution of
valine for methionine at position 196 of the viral E protein.
(
B) Structure of the DENV2 soluble E prefusion dimer structure (PDB: 1OKE) with domains I and II highlighted in red and yellow. The E-M196V substitution at the
base of b strand f, highlighted in magenta, is located adjacent to the molecule of b-octoglucoside (bOG, cyan).
C) IC90 values against DENV2 NGC and the DENV2 NGC E-M196V mutant virus were measured as described in Figure 1A. Single-cycle viral yields were
(
normalized to the titers of the DMSO-treated controls. The IC90 of 7-148-6 against DENV2 NGC is 13 ± 2 mM (average of four independent experiments;
representative data shown). Although 7-148-6’s solubility under the conditions of the antiviral assays limited our ability to saturate inhibition of the E-M196V
mutant in two of three independent experiments, we estimated the lower bound of the IC90 value to be 30 mM; representative data for the experiment in which we
were able to saturate inhibition are shown with error bars indicating the standard deviation for experimental replicates within that experiment. Analogous data for
2-12-2 are presented in Figure S3.
(D) Recombinant, single-cycle reporter viruses for DENV2 wild-type and the E-M196V mutant were generated and used in the infectivity assay, as outlined in
Figure 1A, to confirm that the E-M196V mutation confers resistance to 7-148-6. Luminescence readings were normalized to the DMSO-treated controls.
Representative data are shown for n R 2 independent experiments with error bars indicating the standard deviation for experimental replicates within that
experiment.
(
E) Equilibrium dissociation constants for the interaction of wild-type recombinant, soluble DENV2 E protein (DENV2 sE) or the E-M196V mutant with 7-148-6 were
measured by biolayer interferometry. K values in the figure legend represent the average and standard deviation of two or more independent experiments, with
the graphed data for one such experiment shown. Analogous data for 2-12-2 is presented in Figure S3.
F) IC90 values for 4,6-disubstituted pyrimidine 1-100-1 against DENV2 NGC and the DENV2 NGC E-M196V viruses were measured as described in Figure 1A.
D
(
Single-cycle viral yields were normalized to the titers of the DMSO-treated controls. Graph is of data from a representative experiment. IC90 values presented in
the figure legend are the average and SD of n R 2 independent experiments with error bars indicating the standard deviation for experimental replicates within
that experiment. Analogous data for 4,6-disubstituted pyrimidine GNF2 are presented in Figure S4.
suggest that the peptide is labeled between residues S274 and by targeting E (Schmidt, 2011; Schmidt et al., 2012). To assess
L283, which line one side of the bOG pocket (Figure 3B–3C).
As a complementary approach to probe the interaction of the we measured equilibrium K
the impact of these point mutations on small-molecule-binding,
values for compounds representa-
D
inhibitors with the bOG pocket, we used site-directed mutagen- tive of the 2,4-diamino pyrimidine (2-12-2 and 7-148-6), the 4,6-
esis to introduce substitutions in this region of the protein. disubstituted pyrimidine (GNF-2 and 1-100-1), and the cyanohy-
We successfully generated additional recombinant sE proteins drazone (3-110-22) inhibitor series (Table S1).
bearing substitutions F193L, Q200A, Q200E, Q271A, Q271E,
The T171A substitution had no effect on binding of all three
M272S, and F279S, all of which line the bOG pocket (Figure 4A). structural classes of inhibitors, consistent with the idea that, if
We additionally produced sE-T171A, a substitution that lies this mutation confers resistance it does so via a mechanism
distant from the bOG pocket but that may confer DENV2 resis- that does not affect inhibitor binding. The other mutations had
tance to 3-110-22, a cyanohydrazone that inhibits DENV fusion variable effects on binding of inhibitors to E. To visualize a
4
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Please cite this article in press as: de Wispelaere et al., Inhibition of Flaviviruses by Targeting a Conserved Pocket on the Viral Envelope Protein, Cell
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of 3-110-22 appears more sensitive to changes made in b
A
B
C
F
3
CO
N
N
ko
0
strands f and g and the k loop, with M196V, Q200E/A, Q271E/
l
o
A, and M272S all causing significant loss of binding, whereas
each of the pyrimidines appears to tolerate at least one substitu-
tion on this side of the pocket.
N
H
O
g
f
1
-100-1
F
3
CO
As a third example, the Q200A and Q271A substitutions cause
significant loss of binding to all of the inhibitors. This could sug-
gest participation of these residues in hydrogen bonds that are
important for inhibitor binding. This idea is consistent with the
observation that the Q200E mutant binds to all of the com-
pounds with affinities comparable with wild-type. Likewise, bind-
ing of the Q271E mutant to GNF-2, 7-148-6, or 2-12-2 occurs
with affinity comparable with wild-type and may reflect a role
of hydrogen bonding at this residue that impacts binding of these
inhibitors but not 1-100-1 and 3-110-22. Collectively, these data
support a model in which all three classes of inhibitors bind in
the bOG pocket and suggest that each class makes specific—
and distinct—contacts with the pocket. Since the M196V and
M272S substitutions appeared to affect all of the inhibitors, we
questioned whether they might have more global effects on E’s
structure (e.g., dimerization, structural dynamics). This appears
unlikely to be the case, since hydrogen-deuterium exchange
mass spectrometry of the wild-type sE and sE-M196V and sE-
M272S proteins detected no evidence for global structural
changes (Data S1).
N
N
N N
H
N
N
H
O
peptide
274-283
βOG
Met-196
O
JBJ-16-111
3
5
8
.3e+05
1
00
00
600
.9e+04
*
6
1100
1600
.2e+04
1
100
m/z
2
74-283
KQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLK
Targeting the Ligand-Binding Pocket between Domains
I and II as a General Strategy for Inhibiting Flaviviruses
Although all characterized flavivirus E proteins share common
domain structures and organization, small-molecule inhibitors
of other flavivirus E proteins have not been reported. Alignment
of residues proximal to bOG in the DENV2 sE structure (PDB:
[
+JBJ-16-111]
Figure 3. Photocrosslinking of a 4,6-Disubstituted Inhibitor in the
bOG Pocket
1OKE) with the analogous residues of Zika, West Nile, and Jap-
(
(
A) Structures of inhibitor JBJ-16-111 and parental compound 1-100-1.
B) Higher-energy collisional dissociation tandem mass spectrometry (MS/MS)
anese encephalitis viruses (Figure 5A) showed significant con-
servation. For example, the majority of residues that pack around
the hydrocarbon tail of bOG deep in the pocket are identical
(L135, F193, and F279) or have conservative substitutions
spectrum of the peptide KQDVVVLGSQEGAMHTALTGATEIQMSSGNLLF
TGHLK (residues 247–284) acquired during capillary electrophoresis-mass
spectrometry analysis of tryptic peptides from the UV crosslinking reaction
between domains I and II protein and JBJ-16-111. Unmodified b- and y-type
ions are marked with blue and red circles, respectively, while y-ions
modified by JBJ-16-111 are highlighted in purple. Abundant probe-related
ions at m/z 431.12 and 374.07 (green), along with neutral-loss ion at m/z
(V130 and L198) in DENV2 and ZIKV E proteins, while residues
that interact with the glucoside at the top of the pocket are diver-
gent, especially those in the fg b strands and the kl hairpin (Fig-
ures 5A and 5B). JEV and WNV E proteins exhibit similar patterns
of conservation with DENV and ZIKV at the bottom of the pocket
adjacent to the viral membrane and divergence in solvent-
exposed residues at the top of the pocket (Figure 5A). Based
on this analysis, we questioned whether the DENV E inhibitors
might also inhibit other flaviviruses.
9
93.24 (denoted *), suggest that JBJ-16-111 is readily lost during MS/MS.
Fragment ion evidence suggests that the peptide is labeled between S274
and L283 (see arrows; assumes C-terminal lysine is not modified due to suc-
cessful trypsin cleavage). ++, doubly charged ion.
(
C) DENV2 E structure (PDB: 1OKE) with residues 274–283 highlighted
in green.
To examine this possibility, we tested representative com-
mutational ‘‘footprint’’ for each inhibitor, we compared equilib- pounds from the 2,4-diamino pyrimidine series (2-12-2), the
rium K values for binding of each compound with wild-type 4,6-disubstituted pyrimidine series (GNF-2), and the cyanohy-
and mutant proteins and mapped decreases in affinity onto the drazone series (3-110-22), for activity against ZIKV PF13-
D
DENV2 sE-bOG co-crystal structure (PDB: 1OKE) (Figures 4B– 251013-18 (Aubry et al., 2016) using an infectivity assay analo-
4F). Within this limited analysis, compounds in the same inhibitor gous to that used to measure anti-DENV2 IC90 values as
class appeared to have footprints more similar to each other depicted in Figure 1A. Compound 3-110-22 inhibits ZIKV
than to those of the other two structural classes. For example, PF13-251013-18 with an IC90 of 4.0 ± 0.6 mM (Figure 5C), which
2
,4-diamino pyrimidines 2-12-2 and 7-148-6, which differ is comparable with its activity against DENV2 NGC. Both 2-12-2
only in the substituents at the 4-position are only modestly (IC90 = 46 mM) and GNF-2 (IC90 = 107 mM) have more modest ef-
affected by the M196V mutation, whereas binding of cyanohy- fects on ZIKV PF13-251013 (Figure S5). Consistent with its anti-
drazone 3-110-22 and 4,6-disubstituted pyrimidines GNF-2 ZIKV activity, 3-110-22 interacts with recombinant, soluble ZIKV
and 1-100-1 are more significantly affected. Similarly, binding E (ZIKV sE) in biolayer interferometry experiments with a K
D
of
Cell Chemical Biology 25, 1–11, August 16, 2018
5

Please cite this article in press as: de Wispelaere et al., Inhibition of Flaviviruses by Targeting a Conserved Pocket on the Viral Envelope Protein, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.05.011
A
B
C
F3CO
H
N
H N
N
2
k
0
-l
Q271E/A
F279S
0
loop
2-12-2
GNF2
N
H
N
N
H
M272S
O
N
N
OCF3
OCF
3
βOG
g
T171A
F193L
M196V
Q200E/A
Q271E/A
M272S
F279S
T171A
F193L
T171A
f
M196V
Q200E/A
Q271E/A
M272S
F279S
f
Q200E/A
M196V
F193L
B
C
D
E
N
F
3
-110-22
O
CN
H
H
O
N
N
CF
7-148-6
1-100-1
OCF
3
3
N
N
H
N
N
H
N
N
O
OCF
3
T171A
F193L
T171A
F193L
T171A
F193L
M196V
Q200E/A
Q271E/A
M272S
F279S
M196V
Q200E/A
Q271E/A
M272S
F279S
M196V
Q200E/A
Q271E/A
M272S
F279S
E
F
Figure 4. Loss-of-Affinity Footprints of Representative DENV E Inhibitors
The ligand-binding pocket of the DENV2 E structure (PDB: 1OKE) is shown with bOG (cyan) bound.
A) Substitutions were introduced at seven different residues lining the pocket as shown. Residues 200 and 271 were mutated from glutamine to glutamate (E) and
alanine (A).
B–F) Loss of affinity for each mutation is mapped onto the PDB: 1OKE structure for cyanohydrazone 3-110-22, 2,4-diaminopyrimidines 2-12-2 and 7-148-6, and
,6-disubstituted pyrimidines GNF2 and 1-100-1. Magenta, >5-fold loss of affinity; light pink, 3- to 5-fold loss of affinity; green, <2-fold loss of affinity; gray, data
indeterminate due to poor signal-to-background. Color coding at residue 271 reflects the effects of the Q271E mutation because the Q271A mutant exhibited
significant loss of binding with all inhibitors. K values are presented in Table S1.
(
(
4
D
2
.1 ± 0.7 mM (Figure 5D). Since 3-110-22’s anti-DENV2 activity is drugs that target the fusogenic activities of viral glycoproteins.
correlated with activity in a competitive luminescence proximity Although biomimetic peptides have been shown to inhibit the en-
assay using recombinant DENV2 sE (Clark et al., 2016), we addi- try of diverse enveloped viruses in cell culture by interfering with
tionally confirmed that 3-110-22’s activity against Zika sE in an the conformational changes of the viral glycoprotein required for
analogous competitive luminescence proximity assay has an fusion (Kirschner et al., 2007; Porotto et al., 2006; Rapaport et al.,
IC50 value of approximately 1 mM (Figure 5E). We also observed 1995; Schmidt et al., 2010), inhibition of HIV gp41 by the peptide
significant inhibition of both JEV and single-cycle reporter WNV T20 (Kilby et al., 1998) remains the only example of an approved
(
Pierson et al., 2006) by one or more compounds (Figures 5F and drug that acts by this mechanism. Pursuit of small molecules that
S5), albeit at concentrations generally higher than those required inhibit the membrane-fusing activity of viral glycoproteins has
to inhibit authentic DENV2 infection. Together, these observa- been limited by the absence of obvious drug-binding pockets
tions demonstrate that 3-110-22 and other inhibitors of DENV2 on these proteins, and a paucity of target-based assays that
E can inhibit other flavivirus pathogens and suggest that the can be used to discover and optimize small molecules that inhibit
ligand-binding pocket between domains I and II is an antiviral membrane fusion.
target conserved across this viral genus.
The discovery of the detergent bOG bound in a region of the
DENV2 prefusion E protein (Modis et al., 2003), which had previ-
ously been implicated in regulating the pH at which viral fusion
occurs (Butrapet et al., 2011; Lee et al., 1997), suggested that
this pocket might present a bona fide pharmacological target
on E. Over the past decade, numerous virtual screening efforts
DISCUSSION
The bOG-Binding Pocket of the DENV E Protein as a
Pharmacological Target Mediating Antiviral Activity
Direct-acting antiviral drugs that target virally encoded enzymes, (Kampmann et al., 2009; Poh et al., 2009; Wang et al., 2009; Yen-
such as viral polymerases or proteases, have demonstrated namalli et al., 2009; Zhou et al., 2008) have produced com-
enormous benefit to patients infected with HIV and HCV. While pounds that inhibit DENV entry, but clear evidence for the target
there is precedent for small-molecule inhibitors of viral entry, site(s) of these compounds on E in the form of high-resolution
these have more typically targeted host co-receptors (e.g., structural data, biochemical crosslinking experiments, or anal-
CXCR5) or the capsid proteins of non-enveloped viruses (e.g., ysis of antiviral resistance has been elusive. To date, the best ev-
pleconaril). In contrast, there is a paucity of examples of antiviral idence for binding in the bOG pocket comes from work by Zhou
6
Cell Chemical Biology 25, 1–11, August 16, 2018

Please cite this article in press as: de Wispelaere et al., Inhibition of Flaviviruses by Targeting a Conserved Pocket on the Viral Envelope Protein, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.05.011
f-g strands
f-g hairpin
A
B
4
8
54 127
138 189
212 266
282
DENV2 NGC,16681
DENV2 S2
ZIKV H/PF 2013
JEV RP-9
JEV SA14
TEAKQPA GKVVQPENLEYT TGLDFNEMVLLQMENKAWLVHRQW GATEIQMSSGN-LLFTGHLK
TEAKQPA GKIVQPENLEYT TGLDFNEMVLLQMKDKAWLVHRQW GATEIQMSSGN-LLFTGHLK
TTVSNMA GKSIQPENLEYR TGLDFSDLYYLTMNNKHWLVHKEW GALEAEMDGAKGRLSSGHLK
IEASQLA GRTIQPENIKYE SGLNTEAFYVMTVGSKSFLVHREW GAIVVEYSS-SVKLTSGHLK
IEASQLA GRTIQPENIKYK SGLNTEAFYVMTVGSKSFLVHREW GAIVVEYSS-SVMLTSGHLK
MEAANLA GRTILKENIKYE SGIDTNAYYVMTVGTKTFLVHREW GAIPVEFSSNTVKLTSGHLK
MEAANLA GRTILKENIKYE SGIDTSAYYVMTVGTKTFLVHREW GAIPVEFSSNTVKLTSGHLK
WNV NY99
WNV Kunjin
C
D
IC90 4.0 ± 0.6 μM
K 2.1 ± 0.7 μM
D
ko
6
4
2
×
×
×
105
105
105
0
0.06
0.04
0.02
0.00
Q271
lo
F279
g
D0
f
E0
e
L198
0.01 0.1
1
10 100
0
5
10 15 20
L135
[3-110-22] μM
[3-110-22] μM
V130
F193
E
70000
IC50 1μM
6
5
4
3
0000
0000
0000
0000
West Nile Virus RVP
-12-2, IC 29 μM
Japanese encephalitis virus
2
7-148-6, IC 36 μM
90
50
F
3-110-22, IC 2.4 μM
3-110-22, IC 5.9 μM
50
90
20000
0.1
×105
1
10
2
1
1
00
[
3-110-22] μM
×105
5
0
0
0
[
inhibitor] μM
[inhibitor] μM
Figure 5. Pharmacological Validation of the bOG Pocket as a Target for Inhibition of Zika Virus and Other Flaviviruses
A) Sequence alignment of the residues proximal to bOG in the high-resolution structure of DENV2 S2 sE co-crystallized with bOG (PDB: 1OKE and 1OAN). DENV2
(
NGC is used as a reference sequence. Blue indicates residues that are identical; green residues are conservative substitutions; gray residues are non-conserved
substitutions.
(
B) The bOG-binding pocket of DENV2 sE showing bOG-proximal residues that are identical to ZIKV E (blue), conserved with ZIKV E (green), or non-
conserved (gray).
C) 3-110-22 inhibits ZIKV PF13-251013-18 in the viral infectivity assay (Figure 1A) with IC90 value of 3.8 ± 0.8 mM. Graphs are representative data from one of
n R 2 independent experiments with error bars indicating the standard deviation for experimental replicates within that experiment.
(
(D) 3-110-22 binds to recombinant ZIKV sE with a K
D
value of 2.1 ± 0.7 mM.
(E) 3-110-2 disrupts the interaction of ZIKV sE with GNF2-biotin in a competitive luminescence proximity assay with IC50 of approximately 1 mM. Graphs are
representative data from one of n R 2 independent experiments with error bars indicating the standard deviation for experimental replicates within that
experiment.
(F) Cyanohydrazone and pyrimidine inhibitors of DENV2 E also inhibit West Nile reporter virus and Japanese encephalitis virus. Figure S5 presents data for
additional compounds that inhibit Zika and Japanese encephalitis viruses. IC90 and K values presented in the text are the average and SD for n R 2 independent
D
experiments. Graphs show representative data for an independent experiment with error bars reflecting the SD for 2 or more experimental replicates.
1
et al. (2008), who monitored the H spectrum of bOG in the pres- 1OKE structure. Loss of affinity caused by mutation of residues
ence of DENV E by saturation transfer difference nuclear mag- in the pocket was also observed for compounds from all three in-
netic resonance spectroscopy. They observed loss of specific hibitor series. Although it remains a formal possibility that the in-
resonances upon the addition of inhibitor P02, suggesting hibitors bind elsewhere on E and are allosterically affected by the
displacement of bOG by the inhibitor, but whether P02 directly mutations, this seems unlikely since an allosteric mechanism
or allosterically affects binding of bOG to E has remained un- would be expected to affect all compounds equivalently. The
clear. We used photocrosslinking experiments to show that distinct differences in how mutations in the pocket affect each
JBJ-16-111 interacts with residues of E contained within tryptic of the inhibitors are instead more consistent with a model in
peptide 247–284 and that the likely site of crosslinking is within which the 2,4-diamino pyrimidine, 4,6-disubstituted pyrimidine,
residues 274–283. These residues include the l
o
region of the and cyanohydrazone inhibitors form specific and distinct inter-
protein immediately adjacent to the bOG molecule in the PDB: actions with the bOG pocket.
Cell Chemical Biology 25, 1–11, August 16, 2018
7

Please cite this article in press as: de Wispelaere et al., Inhibition of Flaviviruses by Targeting a Conserved Pocket on the Viral Envelope Protein, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.05.011
Mechanistic Implications of the Resistance Profile for
DENV2 E-M196V
In particular, although residues that interact with the glucose
ring near the top of the pocket are divergent, those deep within
While the absence of reported mutations conferring antiviral the pocket and proximal to the viral membrane exhibit conserva-
resistance can be a hindrance in identifying inhibitor target tion within DENV strains as well as within the ZIKV, WNV,
site(s), it also suggests that the natural barrier to resistance and JEV.
may be high for these compounds. Although we attempted to
Despite this apparent conservation of primary structure, co-
select for resistance against multiple pyrimidines (8 individual crystallization of bOG with other flavivirus E proteins has not
compounds, R10 passages), we obtained a resistant quasispe- been reported despite deliberate efforts in some cases to obtain
cies only in the experiment that resulted in isolation of DENV2 co-crystals with bOG (Kanai et al., 2006; Modis et al., 2005; Ny-
E-M196V. This suggests that conservation of residues in the bakken et al., 2006; Zhang et al., 2004). The absence of structural
pocket is required to maintain E’s function in viral entry. We veri- or pharmacological data indicating that the entry of other flavivi-
fied that the E-M196V mutation confers resistance to 7-148-6 ruses can be inhibited by this strategy has indirectly suggested
and the related 2,4-diamino pyrimidine 2-12-2, and reduces that the pocket on other flavivirus E proteins is inaccessible
the affinity of the interaction between both compounds and re- and/or that the analogous sites on other flavivirus E proteins
combinant, soluble DENV2 E (sE). The DENV2 E-M196V muta- have insufficient molecular complementarity to bind to bOG or
tion was also previously identified as one conferring resistance other candidate ligands. Our demonstration that cyanohydra-
to multiple neutralizing antibodies targeting DENV2 (Sukupolvi- zone 3-110-22, as well as compounds from both of the pyrimi-
Petty et al., 2010), suggesting that the E-M196V substitution dine series, inhibit ZIKV, WNV, and JEV, and that 3-110-22 inter-
perturbs E’s structure and/or conformational dynamics rather acts with recombinant Zika sE in biochemical assays, indicates
than by altering specific epitopes. This could suggest that that the ligand-binding pocket between domains I and II is a gen-
the reductions in antiviral sensitivity and affinity caused by the eral feature of flavivirus E proteins and presents a defined molec-
E-M196V mutation are due to direct or allosteric effects of the ular target for antiviral intervention.
mutation on accessibility of the bOG pocket through gross ef-
Interestingly, the three inhibitor series we examined exhibit
fects on E structure, dimerization, and conformational dynamics. distinct viral specificity profiles, with the cyanohydrazone series
Arguing against this, however, are the absence of changes in in particular exhibiting broad-spectrum activity against multiple
hydrogen-deuterium exchange kinetics we observe for sE- flaviviruses, whereas both pyrimidine series exhibit greater
M196V versus wild-type protein (Data S1) and the greater effect selectivity for DENV. That the three inhibitor series also exhibit
of this substitution on binding of the 4,6-disubstituted pyrimi- different sensitivities to mutations introduced to the bOG pocket
dines and cyanohydrazone 3-110-22 (R10-fold loss of affinity) reinforces the idea that specific interactions drive inhibitor bind-
versus the 2,4-diamino pyrimidines (%5-fold loss of affinity). ing and motivate current efforts to understand the structure-ac-
Since methionine and valine have different van der Waals vol- tivity relationships driving broad versus narrow spectrum activity
umes, an alternate hypothesis is that the methionine to valine against flaviviruses.
substitution alters hydrophobic packing around the small-
molecule inhibitor. Although efforts to acquire high-resolution Antivirals that Inhibit Flavivirus Fusion by Targeting E
structural data for all three inhibitor series bound to sE have A major advantage of the viral envelope protein as a molecular
been unsuccessful to date, we note that there have been no target is that drugs can engage E extracellularly under conditions
co-crystal structures of DENV E with other reported inhibitors of physiological temperature and pH, and non-membrane per-
(
Clark et al., 2016; Kampmann et al., 2009; Poh et al., 2009; meant compounds can be devised to obviate the need for selec-
Schmidt et al., 2012; Wang et al., 2009; Zhou et al., 2008). A likely tivity over the vast array of intracellular host targets. Conserva-
confounding factor has been the incompatibility of the conditions tion of residues in the bOG pocket open the possibility of
under which the protein crystallizes and the hydrophobicity of discovery and development of antivirals efficacious against mul-
the small molecules. Our findings are currently driving renewed tiple flavivirus pathogens. Our demonstration that 3-110-22’s in-
efforts in this area using inhibitors modified with solubilizing hibition of multiple viruses is correlated with its binding to both
groups along with additional efforts to define resistance profiles DENV and ZIKV sE proteins (Clark et al., 2016 and Figure 5E)
for all three of our inhibitor series.
in a competitive luminescence proximity assay suggests that
this may serve as a tool for target-based screening and medici-
nal chemistry optimization of inhibitors against these and other
flavivirus E proteins. Collectively, these efforts provide pharma-
The Ligand-Binding Pocket Targeted by DENV E
Inhibitors Is Conserved across Flaviviruses
The high apparent barrier to antiviral resistance likely reflects the cological validation of the ligand-binding site between domains
functional importance of the bOG-binding region of E in viral en- I and II as an anti-flaviviral target and provide a foundation for
try and/or in viral assembly. Consistent with this inference, we developing inhibitors to test whether inhibition of E’s fusogenic
have found that many of the residues proximal to bOG in the function is an efficacious antiviral strategy in preclinical murine
crystal structure could not tolerate substitutions, as we were un- models of flavivirus infection.
able to recover infectious viral particles when we targeted the
bOG-binding pocket by alanine scanning mutagenesis (J.B.K. SIGNIFICANCE
and M.J.C., unpublished data). Perhaps even more compelling
is the observation of significant conservation of the DENV2 res- Dengue virus (DENV), Zika virus (ZIKV), and other flavivi-
idues proximal to bOG when aligned with the analogous se- ruses are human pathogens that cause significant disease.
quences from other members of the Flavivirus genus (Figure 5A). Due to their transmission by widespread mosquito species,
8
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Please cite this article in press as: de Wispelaere et al., Inhibition of Flaviviruses by Targeting a Conserved Pocket on the Viral Envelope Protein, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.05.011
flaviviruses can spread rapidly when introduced into new
geographical locations where prior immunity does not exist.
As illustrated by the explosive spread of Zika virus in South
America and the Caribbean, rapid emergence of these vi-
ruses can have a devastating impact on public health. There
are no approved drugs to counteract any of the flaviviruses.
Dengvaxia, the only marketed vaccine against dengue, is
effective in boosting natural immunity for those with prior
dengue exposure, but actually sensitizes non-immune
children to more severe disease and hospitalization once
they are infected. Countermeasures to prevent or to treat
dengue, Zika, and other flavivirus infections thus represent
a large, unmet medical need, one made even more critical
by the lack of success of conventional vaccine and antivirals
approaches to date. Viral envelope proteins have conserved
structures and functions but lack ‘‘druggable’’ active sites
that can be rationally targeted with small molecules. In addi-
tion, the surface of viral envelope proteins can be highly var-
iable due to immune selection. In this study, we show that
small molecules can bind extracellularly to the envelope pro-
tein, E, on the surface of virions and prevent infection by
blocking E-mediated membrane fusion during viral entry.
Our study demonstrates that these compounds bind in a
pocket located between domains I and II that appears to
be conserved across dengue, West Nile, Zika, and Japanese
encephalitis viruses. The low frequency of resistance muta-
tions in serial passaging and site-directed mutagenesis ex-
periments supports the idea that the pocket is functionally
important and thus conserved. These findings provide phar-
macological evidence for the pocket as a target for devel-
oping broad-spectrum antivirals against multiple, mos-
quito-borne flavivirus pathogens.
d
B Determination of Dissociation Constant (K ) Values by
Bio-Layer Interferometry (BLI)
B Protein Crosslinking and Capillary Electrophoresis/-
Mass Spectrometry Analysis
B AlphaScreen Competition Assay
B Hydrogen-Deuterium Exchange (HDX) Analysis
d QUANTIFICATION AND STATISTICAL ANALYSIS
B Viral Infectivity Assays for IC90 Determination
D
B Determination of Dissociation Constant (K ) Values by
Bio-Layer Interferometry (BLI)
B Hydrogen-Deuterium Exchange (HDX) Analysis
B Statistical Analysis
SUPPLEMENTAL INFORMATION
Supplemental Information includes five figures, two tables, and one data
file and can be found with this article online at https://doi.org/10.1016/j.
chembiol.2018.05.011.
ACKNOWLEDGMENTS
Stephen C. Harrison, Aaron Schmidt, Nicholas Kwiatkowski, and members of
the Yang, Harrison, and Gray labs are thanked for scientific discussions. Ste-
phen C. Harrison, Ian Walker, and Daryl Klein are also thanked for technical
assistance in expression of recombinant sE proteins. We thank the following
colleagues for sharing reagents: Gregory Gromowski for JEV SA14-14-2; Did-
ier Musso and Nathalie Pardigon for ZIKV PF13-251013-18; John Aaskov for
the hybridoma cell line producing Mab 6F3.1 against DENV core; Theodore
Pierson for plasmids to produce DENV and WNV single-cycle reporter viruses;
Lee Gehrke for DENV2 New Guinea C virus; Eva Harris for BHK-21 cells; Ste-
phen C. Harrison for the synthetic DENV2 sE gene and cell lines. Kelly Arnett
and the Center for Macromolecular Interactions and Jennifer Smith and the
ICCB-Longwood Screening Facility provided access to instruments and tech-
nical support. We thank Matthew Henn, Sinead Chapman, and the Viral Geno-
mics Sequencing group at The Broad Institute of Harvard and MIT for
sequencing of dengue virus quasispecies. The Nancy Lurie Marks Medicinal
Chemistry Laboratory at the Dana-Farber Cancer Institute provided access
to compounds. We thank Congressman Michael Capuano and his staff for
assistance importing ZIKV PF13-251013-18 virus. We thank Wendy Jackelow
for assistance with graphic art and figure preparation. This work was sup-
ported by NIH R01AI095499 (P.L.Y.), NIH U19AI109740, NIH U54AI057159,
NIH CA188881 (J.A.M.), NIH GM101135 (J.R.E.), and a research collaboration
with the Waters Corp (J.R.E.). J.D.P. was supported by NIH T32AI007245.
J.B.K. was supported by a Diversity Supplement to NIH R01AI095499
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE
d CONTACT FOR REAGENT AND RESOURCE SHARING
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
d METHOD DETAILS
(
P.L.Y.) and the Harvard Medical School Dean’s Postdoctoral Fellowship.
B Viruses
AUTHOR CONTRIBUTIONS
B Antibodies
B Compound Synthesis and Characterization
B Virus Infections
Conceptualization, P.L.Y.; Validation, M.d.W., W.L., P.-C.L., J.J., S.B.F.,
M.J.C., X.Z., J.B.K., J.D.P., and T.E.W.; Formal Analysis, M.d.W., W.L.,
S.P., P.-C.L., and T.E.W.; Investigation, M.d.W., W.L., S.P., P.-C.L., J.J.,
S.B.F., M.J.C., J.B.K., J.D.P., and T.E.W.; Resources, J.A.M., N.S.G., and
P.L.Y.; Data Curation, M.d.W., W.L., and J.W.; Writing – Original Draft,
P.L.Y.; Writing – Review and Editing, All Authors; Visualization, S.B.F.,
T.E.W., and P.L.Y.; Supervision, J.R.E., J.A.M., N.S.G., and P.L.Y.; Funding
Acquisition, J.R.E., J.A.M., N.S.G., and P.L.Y.
B Viral Plaque-Formation Assay
B Viral Infectivity Assays for IC90 Determination
B Selection of DENV2 Resistant to 7-148-6
B Single-Cycle Reporter Viral Particles (RVP) Production
B DENV2 RVP Titration Assay
B Single-Cycle Reporter Virus (RVP) Infectivity Assays
B Virion Attachment Assay
DECLARATION OF INTERESTS
B Capsid Protection Assay to Monitor Fusion of Virions
with Liposomes
P.L.Y. and N.S.G. are co-inventors on patent application US20160031826A1.
B Expression and Purification of 6His-tagged DENV2 and
ZIKV Soluble Envelope Protein (sE) Expression and Pu-
rification
Received: February 28, 2018
Revised: April 23, 2018
Accepted: May 16, 2018
Published: June 21, 2018
TM
B Biotinylation of AviTag sE Proteins
Cell Chemical Biology 25, 1–11, August 16, 2018
9

Please cite this article in press as: de Wispelaere et al., Inhibition of Flaviviruses by Targeting a Conserved Pocket on the Viral Envelope Protein, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.05.011
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10 Cell Chemical Biology 25, 1–11, August 16, 2018

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Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.05.011
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and Kobe, B. (2009). Identification of novel target sites and an inhibitor of
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Cell Chemical Biology 25, 1–11, August 16, 2018 11

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STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE
Antibodies
SOURCE
IDENTIFIER
Mouse monoclonal antibody against DENV
envelope protein
Supernatants of hybridoma
D1-4G2-4-15; ATCC
Cat#HB-112; RRID:CVCL_J890
N/A
Mouse monoclonal antibody against
DENV2 core protein
Supernatants of hybridoma 6F3.1;
John Aaskov, Queensland University of
Technology (Bulich and Aaskov, 1992)
Bacterial and Virus Strains
Dengue virus serotype 2 strain New Guinea C
Lee Gehrke, Massachusetts Institute
of Technology
N/A
N/A
N/A
Zika virus strain PF-251013-18
Didier Musso, Institut Louis Malarde´
(Aubry et al., 2016)
Japanese encephalitis virus strain SA14-14-2
Gregory Gromowski, Walter Reed
Army Institute of Research
(Gromowski et al., 2014)
DH10Bac
Thermo Fisher Scientific
Cat#10361012
Chemicals, Peptides, and Recombinant Proteins
2
-12-2
Clark et al., 2016
Clark et al., 2016
Clark et al., 2016
Clark et al., 2016
Clark et al., 2016
Schmidt et al., 2012
This paper
N/A
7-148-6
N/A
GNF-2
N/A
GNF-2-biotin
N/A
1-100-1
N/A
3-110-22
N/A
JBJ-16-111
-palmitoyl-2-oleoyl-sn-glycero-3-
phosphocholine (POPC)
-palmitoyl-2-oleoyl-sn-glycero-3-
N/A
1
Avanti Polar Lipids
Cat#850457C
1
Avanti Polar Lipids
Cat# 850757C
phosphoethanolamine (POPE)
Cholesterol
Avanti Polar Lipids
Broadpharm
Cat#700000P
Biotin-PEG2-NHS ester
Critical Commercial Assays
Renilla luciferase assay system
BirA-500 kit
Cat# BP-22608
Promega
Cat#E2810
Avidity
Cat#BirA500
Cat#18-5101
Cat#18-5057
Cat#6760619C
Ni-NTA biosensors
PALL Fort e´ Bio
PALL Fort e´ Bio
PerkinElmer
Super Streptavidin biosensors
AlphaScreen histidine (nickel chelate)
detection kit
Experimental Models: Cell Lines
Vero
ATCC
ATCC
Cat#CCL-81; RRID:CVCL_0059
Cat#CRL-3216; RRID:CVCL_0063
N/A
HEK293T
BHK-21
Eva Harris, University of California
Berkeley
C6/36
ATCC
Cat#CRL-1660; RRID:CVCL_Z230
Cat#B825-01; RRID:CVCL_0549
Cat#B85502; RRID:CVCL_C190
Sf9
Invitrogen / Thermo Fisher Scientific
Thermo Fisher Scientific
High Fiveꢀ
Oligonucleotides
Primers, see Table S2
IDT DNA, this paper
N/A
(Continued on next page)
e1 Cell Chemical Biology 25, 1–11.e1–e8, August 16, 2018

Please cite this article in press as: de Wispelaere et al., Inhibition of Flaviviruses by Targeting a Conserved Pocket on the Viral Envelope Protein, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.05.011
Continued
REAGENT or RESOURCE
Recombinant DNA
SOURCE
IDENTIFIER
pCDNA6.2-D2.CprME
Theodore Pierson, N.I.H.
N/A
N/A
N/A
(
Ansarah-Sobrinho et al., 2008)
Theodore Pierson, N.I.H.
Ansarah-Sobrinho et al., 2008)
Theodore Pierson, N.I.H.
Pierson et al., 2006)
pWNI-CprME-IH2
pWIIrep-REN-IB
(
(
pFastBac-DENV2-sE-His6Tag
pFastBac-DENV2-DI-DII-His6Tag
pFastBac-DENV2-sE-AviTag
pFastBac-ZIKV-sE-AviTag
Software and Algorithms
Prism
This paper
This paper
This paper
This paper
N/A
N/A
N/A
N/A
GraphPad Software
PALL Fort e´ Bio
RRID:SCR_002798 https://www.graphpad.com/
scientific-software/prism/
Octet Data Analysis Software
N/A https://www.fortebio.com/
octet-software.html
mzStudio
Ficarro et al., 2017
Matrix Science
N/A
Mascot 2.6
RRID:SCR_014322
http://www.matrixscience.com
Multiplierz
Alexander et al., 2017
Waters
RRID:SCR_012058
ProteinLynx Global Server (PLGS) 3.0
N/A
http://www.waters.com/waters/
en_US/ProteinLynx-Global-SERVER-
%
28PLGS%29/nav.htm?cid=
513821&locale=en_US
DynamX HDX Data Analysis Software 3.0
Waters
N/A
http://www.waters.com/waters/
library.htm?cid=511436&lid=
134832928&locale=en_US
CONTACT FOR REAGENT AND RESOURCE SHARING
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Priscilla L.
Yang (priscilla_yang@hms.harvard.edu).
EXPERIMENTAL MODEL AND SUBJECT DETAILS
African green monkey kidney-derived Vero cells (ATCC, RRID:CVCL_0059) and human kidney-derived HEK293T cells (ATCC,
ꢀ
RRID:CVCL_0063) were cultured in Dulbecco modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) at 37 C with
5
in Eagle minimum essential medium (MEM-a) supplemented with 5% FBS at 37 C with 5% CO
% CO
2
. Baby hamster kidney-derived BHK-21 cells (laboratory of Eva Harris, University of California, Berkeley) were maintained
ꢀ
2
. C6/36 cells, a continuous mosquito
cell line derived from Aedes albopictus (Diptera: Culicidae) embryonic tissue (ATCC, RRID:CVCL_Z230), were maintained in Leibovitz
ꢀ
medium (L-15) containing 10% FBS at 28 C. Spodoptera frugiperda cells (Sf9) (Cat # B825-01 Invitrogen/Thermo Fisher Scientific)
ꢀ
were cultured in Sf-900ꢀ II SFM media (Thermo Fisher Scientific) at 27 C. Trichoplusia ni cells (High Fiveꢀ, Thermo Fisher Scientific)
ꢀ
were maintained at 27 C in EX-CELLꢁ 405 Serum-Free Medium (Sigma). The sex of the cell lines is not known.
METHOD DETAILS
Viruses
All work with infectious virus was performed in a biosafety level 2 (BSL2) laboratory using additional safety practices as approved by
the Harvard Committee on Microbiological Safety.
Dengue virus serotype 2 strain New Guinea C (DENV2 NGC) was a kind gift from Lee Gehrke (Massachusetts Institute of Technol-
ogy). Virus stocks were propagated in C6/36 cells.
Cell Chemical Biology 25, 1–11.e1–e8, August 16, 2018 e2

Please cite this article in press as: de Wispelaere et al., Inhibition of Flaviviruses by Targeting a Conserved Pocket on the Viral Envelope Protein, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.05.011
The Zika virus (ZIKV) strain PF-251013-18 was obtained from Didier Musso (Institut Louis Malard e´ , Tahiti Island, French Polynesia),
and was kindly provided by Nathalie Pardigon (Institut Pasteur, France). The ZIKV strain PF-251013-18 (Aubry et al., 2016) was iso-
lated from a viremic patient in French Polynesia in 2013, and was propagated three times on Vero cells. The viral stocks used in the
current study were produced in C6/36 cells, and were not propagated on cells more than six times.
The Japanese encephalitis virus (JEV) vaccine strain SA14-14-2 was kindly provided by Gregory Gromowski (Walter Reed Army
Institute of Research). The viral stocks used in the current study were produced in C6/36 cells, and were not propagated more
than twice.
Antibodies
Monoclonal antibody 4G2 against the DENV E protein was produced from culture supernatants of hybridoma D1-4G2-4-15 (ATCC
HB-112, RRID:CVCL_J890). Mouse hybridoma producing monoclonal antibody 6F3.1 against DENV2 core protein were generously
provided by John Aaskov (Queensland University of Technology) (Bulich and Aaskov, 1992).
Compound Synthesis and Characterization
Unless otherwise noted, reagents and solvents were obtained from commercial suppliers and were used without further purification.
1
H NMR spectra were recorded on 600 MHz (Varian AS600), and chemical shifts are reported in parts per million (ppm, d) downfield
from tetramethylsilane (TMS). Coupling constants (J) are reported in Hz. Spin multiplicities are described as s (singlet), brs (broad
singlet), t (triplet), q (quartet), and m (multiplet). Mass spectra were obtained on a Waters Micromass ZQ instrument. Preparative
HPLC was performed on a Waters Symmetry C18 column (19 x 50 mm, 5mM) using a gradient of 5-95% acetonitrile in water contain-
ing 0.05% trifluoacetic acid (TFA) over 8 min (10 min run time) at a flow rate of 30 mL/min. Purities of assayed compounds were in all
cases greater than 95%, as determined by reverse-phase HPLC analysis.
Synthesis of JBJ-16-111: To a solution of 3-((6-((4-(trifluoromethoxy)phenyl)amino)pyrimidin-4-yl)oxy)benzoic acid (60 mg,
0
3
.153 mmol), HATU (116 mg, 0.306 mmol) and DIEA (0.1 ml, 0.612 mmol) in N,N-dimethylformamide (1 ml) was added (3-methyl-
H-diazirin-3-yl)methanamine (20 mg, 0.230 mmol). After stirring for 30 min, the reaction mixture was diluted with EtOAc and washed
with brine and water three times. The organic layer was dried over Na
2 4
SO , filtered and concentrated under reduced pressure. The
residue was purified by preparative HPLC to obtain N-((3-methyl-3H-diazirin-3-yl)methyl)-3-((6-((4-(trifluoromethoxy)phenyl)amino)
1
pyrimidin-4-yl)oxy)benzamide (JBJ-16-111, 39 mg, 56 %). H NMR 500 MHz (DMSO-d
6
) d 9.80 (s, 1H), 8.59 (t, J = 6.0 Hz, 1H),
8.39 (s, 1H), 7.77 - 7.70 (m, 3H), 7.62 (t, J = 1.8 Hz, 1H), 7.57 (t, J = 9.0 Hz, 1H), 7.40 (dd, J = 7.8, 2.0 Hz, 1H), 7.32 (d, J = 8.5 Hz,
2H), 6.17 (s, 1H), 3.29 (d, J = 6.1 Hz, 2H), 1.06 (s, 3H).
Other small molecule inhibitors were synthesized using previously reported methods (Clark et al., 2016; Schmidt et al., 2012; Adri a´ n
et al., 2006).
1
Synthesis of 2-12-2 (Clark et al., 2016): H NMR (600 MHz, CDCl3) d 8.41 (dd, J = 9.0, 1.2 Hz, 1H), 8.04 (d, J = 5.4 Hz, 1H), 7.35
m, 2H), 7.15-7.20 (m, 4H), 6.94 (dt, J = 9.0, 1.8 Hz, 1H), 6.54 (br s, 1H), 6.14 (d, J = 6.0 Hz, 1H).
(
(
(
1
Synthesis of 7-148-6 (Clark et al., 2016): H NMR (600 MHz, DMSO) d 8.17 (br s, 1H), 8.05 (d, J = 6.6 Hz, 1H), 7.85 (m, 4H), 7.55
m, 4H),7.50-7.41 (m, 4H), 6.50 (d, J = 6.6 Hz, 1H).
1
Synthesis of 1-100-1 (Clark et al., 2016): H NMR (600 MHz, CDCl3) d 9.97 (br s, 1H), 8.34 (s, 1H), 7.43 (m, 2H), 7.36 (m, 2H), 7.28
m, 3H), 7.11 (m, 2H), 6.24 (s, 1H).
Synthesis of GNF-2 (Adri a´ n et al., 2006): GNF-2, [3-(6)-(4-Trifluoromethoxy-phenylamino)-pyrimidin-4-yl]-benzamide]: A mixture
of (6-Chloro-pyrimidin-4-yl)-(4-trifluoromethoxy-phenyl)-amine (200 mg, 0.69 mmol), 3-carbamoylphenylboronic acid (115 mg,
0.69 mmol), palladium tetrakis (-triphenylphosphine) (40 mg, 0.034 mmol) and sodium carbonate (292 mg, 2.76 mmol) in acetoni-
ꢀ
trile/water (1:1, v/v, 10 mL) was heated to 90 C under an argon atmosphere. After 8h, the solution was cooled to room temperature
and the solvent was removed in vacuo. The resulting crude product was purified by flash chromatography using ethyl acetate/hex-
1
anes (1:1, v/v) to yield 206 mg of GNF-2 (yield: 80 %). H NMR (400 MHz, DMSO-d6): d8.79 (s, 1H), 8.53 (s, 1H), 8.23 (d, J = 8.5Hz, 1H),
7
.96 (d, J = 5.1Hz, 1H), 7.85 (d, J = 6.9Hz, 2H), 7.75 (t, J = 7.9Hz, 1H), 7.48 (s, 2H), 7.36 (d, J =8.2Hz, 2H), 7.33 (s, 1H). MS: m/z 375.1
+
M+H) . GNF-2 is also available from commercial vendors (Sigma-Aldrich, catalog number G9420; Cayman Chemical catalog num-
(
ber 16253).
Synthesis of GNF-2-biotin (Clark et al., 2016): A reaction mixture of compound cGNF-2 3-(6-(4-(trifluoromethoxy)phenylamino)
pyrimidin-4-yl)benzoic acid (61 mg, 0.18 mmol), 17-azido-3,6,9,12,15-pentaoxaheptadecan-1-amine (56 mg, 0.15 mmol), O-(7-Aza-
0
0
benzotriazol-1- yl)-N,N,N ,N -tetramethyluronium hexafluorophosphate (HATU) (86 mg, 0.23 mmol), and DIEA (78 mL, 0.45 mmol) in
DMF (3 mL) was stirred at room temperature overnight. Once the reaction was completed, the reaction mixture was diluted with ethyl
2 4
acetate (20 mL) and washed with brine (20 mL). The organic phase was dried over Na SO and evaporated. The residue was purified
by flash chromatography to afford the desired product 13 N-(17- azido-3,6,9,12,15-pentaoxaheptadecyl)-3-(6-(4-(trifluoromethoxy)
phenylamino)pyrimidin-4-yl)benzamide (57 mg, 58% yield). To a solution of N-(17-azido-3,6,9,12,15-pentaoxaheptadecyl)-3-(6-(4-
(
trifluoromethoxy)phenylamino)pyrimidin-4-yl)benzamide (17 mg, 0.026 mmol) in 5 mL of methanol was added 5 mg Pd/C. The re-
sulting suspension was degassed three times and then was stirred with a balloon filled with H for 1 hour. The crude reaction mixture
2
was filtered through a short pad of Celite, which was washed with 10 mL of methanol. The combined filtrates were concentrated and
used for the next step without further purification. To a solution of above amine in DMF (2 mL) were added NHS-Biotin (Thermo
Scientific), 2,5-dioxopyrrolidin-1-yl 5-(2-oxo-hexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate, 8.8 mg, 0.026 mmol) and
DIEA (4.5 mL, 0.026 mmol). After stirring at room temperature for 2 h, the reaction mixture was concentrated and purified by
e3 Cell Chemical Biology 25, 1–11.e1–e8, August 16, 2018

Please cite this article in press as: de Wispelaere et al., Inhibition of Flaviviruses by Targeting a Conserved Pocket on the Viral Envelope Protein, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.05.011
1
prep-HPLC to afford GNF-2-biotin (6.8 mg, 31% yield). H NMR (600 MHz, DMSO-d6) d 9.97 (s, 1H), 8.75 (s, 1H), 8.70 (t, J = 6.0 Hz,
1H), 8.51 (s, 1H), 8.17 (d, J = 8.4 Hz, 1H), 7.98 (d, J = 7.8 Hz, 1H), 7.83 – 7.85 (m, 2H), 7.80 (t, J = 7.8 Hz, 1H), 7.62 (t, J = 7.8 Hz, 1H),
7.33 – 7.35 (m, 2H), 6.39 (s, 1H), 6.33 (s, 1H), 5.74 (s, 1H), 4.26 – 4.30 (m, 1H), 4.09 – 4.13 (m, 1H), 3.49 – 3.57 (m, 20H), 3.36 (t, J =
6.0 Hz, 2H), 3.14 – 3.17 (m, 2H), 3.05 – 3.08 (m, 1H), 2.79 (dd, J = 12.0, 5.4 Hz, 1H), 2.53 – 2.57 (m, 1H), 2.04 (t, J = 7.2 Hz, 2H),
1.56 -1.60 (m, 1H), 1.41 – 1.49 (m, 3H), 1.24 – 1.30 (m, 2H).
Synthesis of 3-110-22 (Schmidt et al., 2012): To a solution of 3-(trifluoromethyl)aniline (0.45 mL, 3.6 mmol) in 6-N hydrochloric acid
o
10 mL) was added dropwise a solution of sodium nitrite (248 mg, 3.6 mmol) in water (5 mL) at 5 C. After the addition, the mixture was
(
stirred at ambient temperature for 15 min before the resulting solution was divided into 20 2-dram vials. To each vial was added a
mixture of 2-aroylacetonitrile (0.15 mmol) and sodium acetate (74 mg, 0.90 mmol) in dioxane (1 mL). The vials were then vortexed
at ambient temperature for 4 hrs, when the reaction mixtures were diluted by water (1 mL) and ethyl acetate (1 mL). The upper organic
layer was taken by a pipette and transferred through a filter cartridge containing magnesium sulfate into a culture tube. The filtrates
1
were concentrated in GeneVac and the residues were purified on silica SPE. H NMR (600 MHz, CDCl
3
) d 8.30 (s, 1H), 7.80 (d, 1H,
J=8.4), 7.75 (s, 1H), 7.67-7.63 (m, 2H), 7.60-7.53 (m, 3H), 7.39-7.36 (m, 1H).
Virus Infections
All DENV2 and JEV infectivity assays were performed on BHK-21 cells, while ZIKV infectivity assays were performed on Vero cells.
Confluent monolayers of cells were infected by incubation of viral inoculum diluted in Earle’s balanced salt solution (EBSS) at MOI of 1
ꢀ
(
unless otherwise indicated) for 1 hour at 37 C. The inoculum was then removed and the cells were washed twice with 1X PBS. The
appropriate medium containing 2% FBS was then overlaid on cells. Unless stated otherwise, the viral supernatants were harvested at
ꢀ
2
4 hours post-infection and stored at -80 C.
Viral Plaque-Formation Assay
DENV2 and JEV titers were quantified by plaque-formation assay (PFA) on BHK-21 cells. ZIKV titers were quantified by PFA on Vero
ꢀ
cells. Cells were seeded in 24-well plates and incubated overnight at 37 C until formation of confluent monolayers. Aliquots from viral
stocks or infections were thawed at room temperature. Ten-fold dilutions in EBSS were prepared in duplicate, and 100 mL of each
ꢀ
dilution was added to the cells. The plates were incubated for 1 hour at 37 C and rocked every 15 minutes. Non-adsorbed virus was
removed, after which 1 mL of DMEM (Vero cells) or MEMa (BHK-21 cells) supplemented with 1.05% carboxymethyl cellulose (CMC),
ꢀ
4
4 mM sodium bicarbonate, and 2% FBS was added to each well, followed by incubation at 37 C for 2.5 days (ZIKV), 3 days (JEV) or
.5 days (DENV2). The CMC overlay was aspirated, the cells were washed with 1X PBS and stained with crystal violet, and viral pla-
ques were counted.
4
Viral Infectivity Assays for IC90 Determination
Virus inocula were diluted in EBSS to achieve an MOI of 1, and were pre-incubated with the given small molecule at varying concen-
ꢀ
ꢀ
trations for 45 min at 37 C. The mixture was then added to cells for 1 hour at 37 C to allow infection, after which the inoculum was
removed and the cells were washed with 1X PBS to remove unbound virus and compound. Cells were overlaid with medium lacking
ꢀ
inhibitor and incubated at 37 C for 24 hours, corresponding to a single cycle of infection. Culture supernatants were harvested at this
time, and the yield of infectious particles produced was quantified by PFA.
Selection of DENV2 Resistant to 7-148-6
DENV2 (MOI 0.1) was incubated with 20 mM 7-148-6 for 45 min at 37 C before addition to Vero cells in a T25 flask. After a 1 hour
ꢀ
infection, 5 mL medium was added (2% FBS in DMEM) containing 20 mM 7-148-6 diluted from DMSO stock; the vehicle control
was passaged in an equivalent percentage of DMSO. Infections proceeded for 4 days, at which point supernatant was harvested
and spun briefly to remove cell debris. 500 mL of supernatant was used to infect a fresh T25 of Vero cells for 1 hour, at which point
4
.5 mL of medium containing compound (or vehicle) was added. Viral titer of each passage was determined by PFA. Cell superna-
tants containing the resistant quasi-species or the corresponding DMSO-passaged control quasispecies were collected at pas-
sage 7, viral RNA was extracted (QIAamp Viral RNA kit, Qiagen), and cDNA was synthesized. Consensus sequencing of the
cDNA sample (The Broad Institute Viral Genomics Sequencing Core) led to the identification of an A to T mutation resulting in sub-
stitution of methionine to valine (M196V) in the E protein. The mutant virus was triply plaque purified, and viral stocks of the clonal virus
that contains the E-M196V mutation were propagated as described above. The presence of the mutation was monitored by extrac-
tion of viral RNA from the supernatants, followed by reverse transcription-PCR and sequencing.
Single-Cycle Reporter Viral Particles (RVP) Production
To produce reporter viral particles (RVP), HEK293T cells were plated in a 10-cm dish and then co-transfected using Lipofectamine
2
000 (Life Technologies), according to the manufacturer’s instructions, with two plasmids. The first plasmid encodes the structural
proteins of either DENV2 (pCDNA6.2-D2.CprME (Ansarah-Sobrinho et al., 2008)), or WNV (pWNI-CprME-IH2 (Pierson et al., 2006)).
The second plasmid encodes a WNV subgenomic replicon (pWIIrep-REN-IB (Pierson et al., 2006)) that expresses the virus non-struc-
tural proteins and a Renilla luciferase reporter. To produce the mutant pCDNA6.2-D2.CprME that carries the E-M196V mutation, site-
directed mutagenesis of the plasmid was performed using the Quikchange II kit according to manufacturer’s instructions (Agilent),
and using the primers listed in Table S2.
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Please cite this article in press as: de Wispelaere et al., Inhibition of Flaviviruses by Targeting a Conserved Pocket on the Viral Envelope Protein, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.05.011
To produce DENV2 RVP, the plasmids pCDNA6.2-D2.CprME and pWIIrep-REN-IB were transfected to cells at a 3:1 ratio. To pro-
duce WNV RVP, the plasmids pWNI-CprME-IH2 and pWIIrep-REN-IB were transfected to cells at a 1:2 ratio.
For all of the RVP preparations, fresh medium was added at 24 hours post-transfection. The supernatants were collected at
ꢀ
ꢀ
48 hours post-transfection, clarified by centrifugation at 1,000 x g for 5 minutes at 4 C, and aliquots were stored at -80 C. The
genome copy number per mL was determined for each RVP stock by RT-qPCR on the supernatants using primers specific for
the Renilla luciferase gene, listed in Table S2.
DENV2 RVP Titration Assay
BHK-21 cells were plated in 48-well plates the day before infection and incubated until ꢁ70% confluency. On the day of infection,
8
0 mL of a dilution series of RVP was added to each well (in triplicate) and incubated for 1hour. Cells were then washed with 1X PBS
and MEMa media supplemented with 2% FBS was added to the cells. At 24 hours post-infection, the cells were washed with 1X PBS
and the samples were processed according to the manufacturer’s instructions of the Renilla luciferase assay system (Promega). The
Renilla luciferase signal was read using a Perkin Elmer EnVision plate reader. Signal from non-infected sample was considered back-
ground. For DENV2 RVPs, the wells were scored as positive or negative for signal to determine the tissue culture infectious dose
(
TCID50).
Single-Cycle Reporter Virus (RVP) Infectivity Assays
BHK-21 cells were seeded in 48-well tissue culture plates in MEMa supplemented with 2% FBS. The RVPs were diluted with EBSS,
ꢀ
and treated with compounds for 45 minutes at 37 C. For the DENV2 RVPs, the cells were infected at MOI of 0.007 based on TCID50
values. For the WNV RVPs, each well was infected with 4000 genome copies. The RVP-compound mixture was then added to the
ꢀ
cells, and the plates were incubated for 1 hour at 37 C. Unadsorbed RVPs were washed with 1X PBS and removed, after which
ꢀ
MEMa supplemented with 2% FBS was added to the cells, followed by incubation at 37 C. At 24 hours post-infection, the samples
were processed according to the manufacturer’s instructions of the Renilla luciferase assay system (Promega). The Renilla luciferase
signal was read using a Perkin Elmer EnVision plate reader.
Virion Attachment Assay
DENV2 was incubated with either 5 mM of 2-12-2, 10 mM of 7-148-6, 20 mg/ml of heparin, or DMSO for 45 minutes at 37 C, then added
ꢀ
ꢀ
to BHK-21 cells (MOI 1) at 4 C for 1 hour with constant rocking. Cells were washed three times with cold 1X PBS to remove unbound
virus and compound. For quantitation of viral genomes bound, total RNA was extracted using TRIzol (Thermo Fisher Scientific) ac-
cording to the manufacturer’s instructions. Total RNA was used as a template for cDNA synthesis using iScript (Bio-Rad), according
ꢀ
to the manufacturer’s instructions. The resulting cDNA was digested by RNaseH (NEB) for 1 hour at 37 C. Quantitative PCR (qPCR)
assays utilized the iQSYBR green Supermix (Bio-Rad) and were performed on a MyiQ iCycler machine (Bio-Rad) as follow: 5 minutes
ꢀ
ꢀ
ꢀ
ꢀ
at 95 C and 40 cycles of 15 seconds at 95 C and 30 seconds at 60 C, followed by the melt curve that started at 50 C and increased
ꢀ
0
.5 C every 10 seconds for 90 cycles. The primers used targeted the DENV2 C gene (Table S2).
Capsid Protection Assay to Monitor Fusion of Virions with Liposomes
Liposomes were made with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) (Avanti Polar Lipids), 1-palmitoyl-2-oleoyl-
sn-glycero-3-phosphoethanolamine (POPE) (Avanti Polar Lipids), and cholesterol (Avanti Polar Lipids) at 1:1:1 molar ratio in TAN
buffer (20 mM triethanolamine, 100 mM NaCl, pH 8.0). Lipids were dried down and resuspended thoroughly by vortexing and son-
ication. Liposomes were prepared by extrusion through a 0.2 micron filter after five freeze/thaw cycles. For trypsin-containing lipo-
somes, 10 mg of trypsin was added to 1 mL of lipids (3 mg each) after the third freeze/thaw cycle, prior to extrusion. Liposomes were
separated from unincorporated trypsin by size-exclusion chromatography using a 10/300 Increase Superdex 200 column (GE
Healthcare) on an Akta fast performance liquid chromatography (FPLC) system. Trypsin-containing liposomes were used within
2
4 hours.
Purified DENV2 virus (300 ng) was incubated with various concentrations of compounds (5-40 mM) for 45 min at 37 C in TAN buffer
pH 8.0) prior to addition of liposomes (7.5 mL of peak fraction from column) for 10 minutes. After incubation with liposomes, 7.5 mL 1M
ꢀ
(
sodium acetate (pH 5.0) was added to drop the pH to 5.5 for 10 minutes. Samples were back-neutralized with 15 mL 1M triethanol-
ꢀ
amine (unbuffered) and incubated for 45 minutes at 37 C to allow trypsin digestion. SDS sample buffer was added, and samples were
boiled for 20 minutes before separation by SDS-PAGE on a 12% gel. Proteins were transferred to a nitrocellulose membrane using a
semi-dry transfer apparatus. 4G2 and 6F3.1 antibodies were used for detection of DENV envelope and capsid protein, respectively.
Expression and Purification of 6His-tagged DENV2 and ZIKV Soluble Envelope Protein (sE) Expression and
Purification
The coding sequence for the soluble portion of the DENV2 NGC envelope protein (sE), fused to a N-terminal signal peptide was codon
optimized for expression in insect cells was synthesized and generously donated by Daryl Klein and Stephen C. Harrison. The coding
sequences of full length or domain I and II of DENV2 envelope protein were cloned into a pFastBacTM/CT-TOPOꢁ vector (Thermo
Fisher Scientific) to enable expression of sE or DI-DII with a TEV-His6 tag at its C terminus. The sequence coding for the soluble
portion of the mutant DENV2 NGC envelope protein carrying the E-M196V mutation (sE-M196V) and fused to an N-terminal signal
peptide and to a C-terminal TEV-His6 tag was codon optimized for expression in insect cells and cloned into a pFastBac1 vector
e5 Cell Chemical Biology 25, 1–11.e1–e8, August 16, 2018

Please cite this article in press as: de Wispelaere et al., Inhibition of Flaviviruses by Targeting a Conserved Pocket on the Viral Envelope Protein, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.05.011
by Life Technologies (now Thermo Fisher Scientific). We then modified the pFastBac-DENV2-sE-His6Tag plasmid to introduce a
TM
TM
C-terminal AviTag , as described previously (Clark et al., 2016) to allow production of AviTag DENV2 sE protein. Site-directed
mutagenesis was performed on this plasmid using primers listed in Table S2 to introduce the following mutations: Q52A, T171A,
F193L, M196V, Q200A, Q200E, Q271A, M272S, and F279S.
The sequence coding for the soluble portion of the ZIKV H/PF/2013 envelope protein (E) (GenBank accession number KJ776791),
fused to a N-terminal signal peptide and to a C-terminal TEV His6 tag, was codon-optimized for expression in insect cells and cloned
into a pFastBac1 vector by GenScript.
The plasmids were used to transform DH10Bac cells (Thermo Fisher Scientific, 10361012). The transformed cells were grown in
ꢀ
presence of 50 mg/mL kanamycin, 7 mg/mL gentamicin, 10 mg/mL tetracycline, 100 mg/mL Bluo-gal, and 40 mg/mL IPTG at 37 C for 2
to 3 days. The white colonies, containing the insert, were selected and grown in Luria broth supplemented with 50 mg/mL kanamycin,
ꢀ
7
mg/mL gentamicin, 10 mg/mL tetracycline overnight at 37 C in the dark. The bacmids were extracted after lysis of cells with 50 mM
Tris-Cl, pH 8.0, 10mM EDTA, 100mg/mL RNase A, followed by precipitation with 0.8 mL isopropanol. The pellets containing the bac-
mid DNA were washed with 70% ethanol, dried and dissolved in 40 mL TE buffer pH 8.
6
To produce baculoviruses expressing DENV2 or ZIKV proteins, 2-5 mg of the appropriate bacmid were used to transfect in 2x10
ꢀ
Sf9 cells in 5 mL Sf-900ꢀ II SFM medium using Cellfectin II Reagent (Life Technologies). The cells were incubated at 27 C for 3 days,
and the supernatants that contained the first production of baculovirus (P1) were collected by centrifugation at 150 x g for 5 min at
ꢀ
7
4 C. The baculoviruses were then amplified by inoculating 1 to 2 mL of P1 in 2x10 Sf9 cells in 30 mL of Sf-900ꢀ II SFM medium. The
supernatants containing the second passage of baculovirus (P2) were collected after 3 days and clarified by centrifugation at 150 x g
8
for 10 min. The third passage of baculovirus (P3) was produced by inoculating 10-15 mL of P2 in 2x10 Sf9 cells in 400 mL of Sf-900ꢀ
II SFM medium and incubated under rotation at 130 rpm, at 27 C, for 3 days. The culture supernatants were centrifuged at 1400 x g
ꢀ
ꢀ
for 10 min, and stored at 4 C protected from light.
To express DENV2 sE or DI-DII proteins, High Five cells were cultured in 3-6 L of EX-CELLꢁ 405 Serum-Free Medium until they
reached a density of 1 to 1.5 million cells/mL, and infected with 20 mL of P3 baculoviruses per liter of medium. At 72 hours post-infec-
ꢀ
tion, the supernatants were collected by centrifugation at 3,000 x g for 20 min at 4 C. Proteins were purified by passing over 25 mL
TALON metal-affinity resin slurry (Clontech). The beads were washed once with 1X PBS pH 7.4, 10 mM imidazole and the proteins
were eluted with 200 mM imidazole pH 8. The protein was further purified by size-exclusion chromatography using a Superdex 200
grade 10/300 GL column (GE Healthcare) with buffer containing 20 mM triethanolamine hydrochloride pH 8.0 and 100 mM NaCl.
Fractions containing DENV2 sE or DI-DII were pooled and concentrated to approximately 1.2 to 2.5 mg/mL (20 to 54 mM), then
ꢀ
flash-frozen in liquid nitrogen and stored at -80 C.
Soluble ZIKV sE protein was expressed in the same manner as DENV2 sE protein, except only 10 mL of P3 baculovirus was used
ꢀ
per liter of culture. The culture was harvested at 72 hours post-infection by centrifugation at 4000 x g for 30 minutes at 4 C. The su-
pernatants were then filtered through a 0.22 micron filter (Corning) before loading onto 15 mL Ni-NTA agarose (Qiagen) that had been
pre-equilibrated in 1x PBS buffer pH 7.4. The unbound fraction was passed over the Ni-NTA resin a second time followed by 2
washes with 5 column volumes of 1X PBS pH 7.4, 10 mM imidazole. Bound protein was then eluted from the column with 2–-3 column
volumes of 200 mM imidazole pH 8.0. The buffer was exchanged to a buffer containing 20 mM triethanolamine hydrochloride pH 8.0
and 100 mM NaCl and concentrated down to 10 mL using a 30 kDa amicon filter (Millipore). Size-exclusion chromatography was used
as a polishing step on a Superdex 200 grade 16/600 pg column (GE Healthcare) pre-equilibrated with 20 mM triethanolamine hydro-
chloride pH 8.0 and 100 mM NaCl. Fractions containing ZIKV sE protein were pooled and concentrated to 1.6-2.3 mg/mL (35-50 mM),
ꢀ
flash frozen in liquid nitrogen, and stored at -80 C.
TM
Biotinylation of AviTag sE Proteins
TM
The purified AviTag DENV2 sE proteins were biotinylated using the BirA-500 kit (Avidity). Briefly, the protein was concentrated to
around 400 mM using a 30 kDa Amicon filter (Millipore) and was then resuspended in 50 mM Biocine pH 8.5. The proteins were incu-
ꢀ
bated for 60 minutes at 30 C in the presence of 2.5 mg BirA per 10 nmol of purified protein, according to the manufacturer’s instruc-
tions. The biotinylated protein was then purified from the reaction using a Superdex 200 grade 10/300 GL column (GE Healthcare)
pre-equilibrated with 20 mM triethanolamine hydrochloride (pH 8.0) and 100 mM NaCl.
Purified ZIKV sE protein was biotinylated as follows. 4 nmol of ZIKV sE protein was mixed with 20 nmol of biotin-PEG2-NHS (9-bio-
tinlamino-4,7-dioxanonanoic acid N-hydroxysuccinimidyl ester, Broadpharm) in 1X PBS to which 20 mM NaHCO
ꢀ
3
, pH 8.3 was added
to adjust the solution to slightly basic pH. The solution was incubated at 4 C overnight. Excess biotin was removed by dialysis in 1X
PBS buffer (Thermo Scientific Slide-A-Lyzer Dialysis Kit, 10000 MWCO, 0.1-0.5 mL).
d
Determination of Dissociation Constant (K ) Values by Bio-Layer Interferometry (BLI)
BLI mixtures (80 mL) were prepared in wells of a 384-well black tilted-bottom plate (Fort e´ Bio) and the measures were monitored by
Octet RED384 system (Fort e´ Bio).
Ni-NTA Biosensor Reactions for Characterization of Binding of Small Molecules 2-12-2 and 7-148-6 to DENV2
sE-Wildtype and sE- M196V Proteins Carrying a 6His Tag
1
.6 mg of DENV2 sE protein was loaded on Ni-NTA biosensors (Fort e´ Bio) for 600 seconds and then quenched with 5 mM imidazole for
1
80 seconds. The Ni-NTA biosensors were then equilibrated in reaction buffer [1X Kinetic buffer (Fort e´ Bio), 20 mM triethanolamine
Cell Chemical Biology 25, 1–11.e1–e8, August 16, 2018 e6

Please cite this article in press as: de Wispelaere et al., Inhibition of Flaviviruses by Targeting a Conserved Pocket on the Viral Envelope Protein, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.05.011
hydrochloride pH 8, 100 mM NaCl, 2% DMSO] for 600 seconds prior to baseline collection. Small molecule concentrations ranged
from 50 nM to 30 mM with association and dissociation times both set at 120 seconds.
Super Streptavidin (SSA) Biosensor Reaction for Determination of Binding of Small Molecule Inhibitors to Biotinylated
DENV2 and ZIKV sE Proteins
1
.6 mg of the biotinylated protein was loaded on an SSA biosensor tip (Fort e´ Bio) for 600 seconds and then quenched with 0.8 mg
biocytin for 120 seconds. The SSA biosensors were then equilibrated in reaction buffer [1X Kinetic buffer (Fort e´ Bio), 1X PBS, 2%
DMSO] for 180 seconds prior to baseline collection. For DENV2 sE proteins, association with small molecules was monitored for
120 seconds with inhibitor concentrations that ranged from 50 nM to 20 mM; dissociation was performed in reaction buffer and moni-
tored for 120 seconds. For ZIKV sE protein, association with 3-110-22 was monitored for 150 seconds at inhibitor concentrations
between 0.1 to 15 mM; dissociation was monitored in reaction buffer for 10 seconds.
Protein Crosslinking and Capillary Electrophoresis/Mass Spectrometry Analysis
DENV2 DI-DII protein was treated with a five-fold molar excess of JBJ-16-111, incubated for 30 minutes at 37 C, and irradiated with
ꢀ
long-wave UV light for 10 minutes using a Spectrolinker UV Crosslinker (model XL1000, Spectronics Corp., Westbury, NY). After irra-
diation, protein was denatured with Rapigest (0.1% final concentration; Waters, Milford, MA), reduced with 10 mM dithiothreitol for
ꢀ
3
3
4
0 minutes at 56 C, alkylated with 22.5 mM iodoacetamide for 30 minutes protected from light, and digested with trypsin overnight at
ꢀ
ꢀ
7 C. Rapigest was cleaved by adding 10% TFA and incubating at 37 C for 30 minutes. The digest was centrifuged for 10 minutes at
C and the supernatant subjected to desalting by C18. Eluted peptides were dried by vacuum centrifugation, reconstituted in 50%
acetonitrile, 1% formic acid with 100 mM ammonium acetate and analyzed by CE-MS using a ZipChip CE system and autosampler
908 Devices, Boston, MA) interfaced to an Orbitrap Lumos mass spectrometer (Thermo Fisher Scientific). Peptide solutions were
(
loaded for 30 seconds and separation performed at 500 V/cm for 10 minutes using an HR chip (22 cm separation channel) with
a background electrolyte composed of 1% formic acid in 50% acetonitrile. Pressure assist was utilized and started at 1 minute.
The mass spectrometer was operated in data-dependent mode, where the five most abundant ions in each MS scan (orbitrap detec-
tion, 120K resolution) were subjected to MS/MS (HCD, 30% collision energy, 2 Da isolation width, 2E5 target, 100 ms max fill time,
FTMS detection at 15K resolution). Dynamic exclusion was enabled with a repeat count of 1 and an exclusion time of 6 seconds. To
allow more accurate filtering of data to a 1% FDR, multiplierz (Alexander et al., 2017) generated .mgf files were appended to an .mgf
file derived from an analysis of K562 total protein tryptic peptides, and the merged .mgf was searched using Mascot against a for-
ward-reverse human protein database (NCBI refseq) appended with the sequence of the DI-DII protein. Search parameters specified
a precursor ion mass tolerance of 10 ppm, a product ion mass tolerance of 25 mmu, fixed carbamidomethylation of cysteine, variable
oxidation of methionine, and variable JBJ-16-111 modification of lysine, serine, threonine, or tyrosine. Search results were filtered to
1
% FDR using a multiplierz script. Spectral analysis was performed using mzStudio (Ficarro et al., 2017).
AlphaScreen Competition Assay
pmol of ZIKV sE protein was mixed with AlphaScreen nickel chelate acceptor beads (15 ng/mL, PerkinElmer) in HEPES buffer
25 mM HEPES, 200 mM NaCl, pH 7.4), in the presence of 10.5 pmol of GNF-2-biotin (Choi et al., 2009; Clark et al., 2016) per
7
(
well. The solution was dispensed at 30 mL/well into Perkin Elmer standard OptiPlates 384. Small molecule 3-110-22 was dispensed
into the plate at concentrations ranging from 0.5 to 10 mM. The plate was centrifuged at 233 x g for 1 min and incubated at room
temperature for 2.5 hours. AlphaScreen streptavidin donor beads (Perkin Elmer) were diluted at 15 ng/mL in HEPES buffer and
5 mL was distributed to the plate. The plate was centrifuged at 233 x g for 1 minute and incubated at room temperature for 2.5 hours.
The fluorescence signal (excitation 680 nm, emission 520-620 nm) was measured using a Perkin Elmer EnVision plate reader.
Hydrogen-Deuterium Exchange (HDX) Analysis
Stock solutions of DENV2 sE (wild-type 69.5 mM; M272S 68.6 mM; M196V 47.2 mM; Q52A 30 mM) were prepared in equilibration buffer
(
10 mM potassium phosphate, 100 mM NaCl, pH 7.0, H
0 mL (15-fold) labeling buffer (10 mM potassium phosphate, 100 mM NaCl, pD 7.0, 99.8% D
2
O). To initiate labeling, 2 mL of each protein were independently diluted with
O) and incubated for predetermined
3
2
times (10 sec, 1 min, 10 min, 1 hour, and 4 hours). At the end of each labeling time, the entire deuterium labeling reaction was added
ꢀ
to 30 mL of quench buffer (200 mM sodium phosphate, 4M guanidine hydrochloride, 0.72 M TCEP, pH 2.3, 0 C) to quench the reac-
ꢀ
tion. All steps from quenching forward were performed at 0 C.
Quenched samples were immediately digested online with an immobilized pepsin column (prepared in house according to (Wang
et al., 2002) and directed into a Waters nanoAcquity UPLC with HDX technology (Wales et al., 2008). Peptides were trapped on a
Waters UPLC BEH C18 1.7 mm VanGuard BEH column and desalted with 0.1% formic acid in water for 3 minutes at 100 mL/min.
Peptides were eluted over 10 minutes using a 5-35% gradient of water:acetonitrile with 0.1% formic acid flowing at a rate of
9
0 mL/min using a Waters HSS T3 1.8 mm C18, 1.0 mm x 50 mm analytical column. Deuterium incorporation was measured using
E
a Waters Synapt G2Si equipped with a standard ESI source in HDMS mode. Mass spectra were acquired over a m/z range of
0-2000, and mass accuracy was confirmed by calibration with 500 fmol/mL of human glu-fibrinopeptide. Peptic peptides were iden-
tified using ProteinLynx Global Server (PLGS) 3.0 (Waters) and deuterium incorporation measured using DynamX 3.0 (Waters).
5
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Please cite this article in press as: de Wispelaere et al., Inhibition of Flaviviruses by Targeting a Conserved Pocket on the Viral Envelope Protein, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.05.011
QUANTIFICATION AND STATISTICAL ANALYSIS
Viral Infectivity Assays for IC90 Determination
The yield of infectious particles produced was quantified by PFA and plotted relative to DMSO-treated samples. The concentrations
that caused 90% JEV, ZIKV or DENV2 inhibition (IC90) were calculated using the nonlinear fit variable slope model (Prism, GraphPad
Software). Differences in IC90 values for E-M196V mutant versus wildtype viruses were evaluated for significance using the Student’s
T-test.
Determination of Dissociation Constant (K
Equilibrium dissociation constants (K ) values were determined by plotting the local fit maximum response (nm) as a function of small
D
) Values by Bio-Layer Interferometry (BLI)
D
molecule concentrations (mM) using Octet Data Analaysis Software (Fort e´ Bio) and Prism (GraphPad Software). Titration curves were
fit to the following steady-state analysis equation:
D
Response = (Rmax*Conc)/K + Conc
D
where Rmax is the local fit response maximum; Conc is the concentration of small molecule; and K is the equilibrium dissociation
constant.
Maximum compound concentrations employed in these experiments were set based on compound aggregation, as assessed by
deterioration of signal-to-noise at higher concentrations in the BLI assay and empirical observation of compound aggregation by dy-
namic light scattering (data not shown). In several cases, we were unable to saturate inhibitor-binding with a given mutant. Where
D
clear concentration-dependent interaction was observed, we have estimated a lower bound for the K based on the fit of available
data and the highest concentrations that yielded well-behaved data in the biolayer interferometry experiments. In other cases, we
were unable to reliably detect signal over background, presumably due to low affinity of the E-inhibitor interaction and or due to ag-
D
gregation of the compound at concentrations relevant for K measurement. These are reported as ‘‘N.D.’’ in Table S1.
Hydrogen-Deuterium Exchange (HDX) Analysis
As all experiments were comparisons between states and measurements were taken close in time using nearly identical experimental
conditions, the deuterium levels were not corrected for back exchange and are reported as relative (Wales and Engen, 2006). All ex-
periments were performed in duplicate. The average error for each data point in the instrumental system so described did not
exceed ± 0.15 Da and is consistent with previously reported values (Houde et al., 2011). Average differences exceeding 0.5 Da
were considered significant.
Statistical Analysis
Statistical tests and the associated error bars are identified in the corresponding figure legends. ‘‘Independent’’ experiments
describe the number of biological replicates, which were repeat experiments performed on different days. Statistical analysis was
performed using Prism (GraphPad Software).
Cell Chemical Biology 25, 1–11.e1–e8, August 16, 2018 e8