1
SUPPORTING INFORMATION
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S1 TEXT
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Potent allosteric dengue virus NS5 polymerase inhibitors: mechanism of action and
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resistance profiling
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Siew Pheng Lim1,*, Christian Noble1, Cheah Chen Seh1, Tingjin Sherryl Soh1,2, Abbas El
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Sahili2, Kar Yarn Grace Chan2, Julien Lescar2,3, Rishi Arora4, Timothy Benson4, Shahul
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Nilar1, Ujjini Manjunatha1, Kah Fei Wan1, Hongping Dong1, Xie Xuping1,#, Pei-Yong Shi1,#,
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Fumiaki Yokokawa1
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138670
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Singapore 637551
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3
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Maladies Infectieuses. Centre Hospitalier Universitaire Pitié-Salpêtrière, Faculté de Médecine
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Pierre et Marie Curie, Paris, France
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02139, USA
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#
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Structural Biology & Molecular Biophysics, University of Texas Medical Branch, Galveston,
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TX 77555, USA.
Novartis Institute for Tropical Diseases, #05-01 Chromos, 10 Biopolis Road, Singapore
School of Biological Sciences, Nanyang Technological University. 60 Nanyang Drive,
UPMC UMRS CR7 - CNRS ERL 8255-INSERM U1135 Centre d’Immunologie et des
Novartis Institute for Biomedical Research, 250 Massachusetts Avenue, Cambridge, MA
Present address: Department of Biochemistry & Molecular Biology, Sealy Center for
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*To whom correspondence may be addressed:
[email protected]; Tel: 65-
3
67222924.
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1
Supporting Methods.
2
Compound testing with DENV dnI FAPA assays. Final dnI assay reaction mixtures
3
contained 100 nM DENV 5´UTR-L-3´UTR IVT RNA, 20 µM ATP, 20 µM GTP, 20 µM
4
UTP, 5 µM ATTO-CTP, and 100 nM of DENV FL NS5 protein in 15 µl in assay buffer
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comprising 50 mM Tris/HCl, pH 7.5, 10 mM KCl, 1 mM MgCl2, 0.3 mM MnCl2 (DENV2
6
and 4) or 1 mM MnCl2 (DENV1 and 3), 0.001% Triton-X-100 and 10 µM cysteine [1]. To
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perform dose-response inhibition measurements, compounds from 0-20 or -100 µM
8
concentrations are two-fold serially diluted into 384-well black opaque plates (Corning
9
Costar), after which 100 nM DENV FL NS5 protein was added into respective wells. The
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plates were sealed, briefly shaken and centrifuged at 1500 rpm for 30 sec and incubated at RT
11
for 15 min. Thereafter, RNA and ATTO-CTP, ATP, GTP and UTP were added to the wells to
12
start the reactions. The plates were re-sealed, shaken and centrifuged as before, followed by
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incubation RT for 120 min. Reactions were stopped by addition of 10 µl of 2.5X STOP
14
buffer (200 mM NaCl, 25 mM MgCl2, 1.5M DEA, pH 10; Promega) with 25 nM calf
15
intestinal phosphatase (CIP), re- incubation at RT for 60 min and read on a Tecan Safire II
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microplate reader. For order-of-addition experiments, DENV4 FL NS5 was incubated for one
17
hour at RT with RNA, ATP, and GTP or RNA, ATP, GTP and ATTO-CTP, followed by
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exposure to serially diluted compounds for 20 min at RT. The missing components (ATTO-
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CTP and UTP or UTP alone) were added and the reactions continued for 120 min after which
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STOP buffer was added as before. All datapoints were performed in duplicate wells. Each
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compound was tested at least twice.
22 23
Compound testing in DENV1-4 infected cells by high content imaging. Approximately 7
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× 103 A549 cells per well were seeded into 384-well plates in Ham’s F-12K medium 3
1
containing 2 % FBS, 1mM L-glutamine and 1 % penicillin-streptomycin and incubated over-
2
night at 37 °C in 5 % CO2. At 24 h post-seeding, cells were infected with DENV1-4 at
3
multiplicity of infection [MOI] of 0.3-1 (DENV1, strain MY97-10245, MOI = 0.5; DENV2,
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strain MY97-10340, MOI = 0.3; DENV3 strain MY05-34640, MOI = 1; DENV4, strain
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MY01-22713; MOI = 0.5) and treated immediately with 10-point, 3-fold serially diluted
6
compounds (at final 0.5% DMSO concentration; 4). At 48 h post-infection, cells were fixed
7
with 4 % paraformaldehye and stained with 4G2 antibody conjugated with Dylight 488 (for
8
DENV envelope protein ) and DRAQ5 (for nuclear DNA). Quantifications of DENV
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envelope protein were determined using the Opera high content imaging system and analysis
10
software (Perkin Elmer, USA). Calculation of EC50 values were performed using Helios
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software package (Novartis, Basel).
12 13
Compound testing in DENV2 and HCV sub-genomic replicon assays. A549, BHK-21 and
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Huh7 DENV2 sub-genomic replicon cells [2] were seeded at a density of 3,000 cells per well
15
in a 384-well microplate. After incubation at 37 °C with 5 % CO2 overnight, the cells were
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treated with serially diluted compounds in a 10-point dose response starting from 20 or 50
17
µM concentrations. At 48 hr of post-incubation, renilla luciferase activities were measured
18
with the EndurRen live-cell substrate (Promega, USA) according to the manufacturer’s
19
protocol. Following luciferase activity measurements, the CellTiter-Glo reagent (Promega,
20
USA) was added to determine the cytotoxic effects of the compounds. For the HCV replicon
21
assay, Huh-7.5 cells harboring the HCV replicon [3] were seeded at a density of 20,000 cells
22
per well in a 96-well microplate. At 48 hr after compound treatment, the cells were assayed
23
for firefly luciferase activity by using a Bright-Glo luciferase assay (Promega, USA). NITD-
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008, a nucleoside inhibitor of DENV and HCV [4], was included as a control. Compounds
4
1
were tested up to 25 µM in HCV replicon cells due to limits in DMSO tolerability of these
2
cells, and up to 50 µM in other cell types, based on compound solubility.
3 4
Compound testing in cells transiently expressing DENV and WNV replicon. Renilla
5
luciferase sub-genomic replicon of DENV2 (strain New Guinea C) or WNV (New York
6
strain 3356) cloned into the pACYC plasmid, was in vitro transcribed using a T7 mMessage
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mMachine kit (Ambion, Austin, TX) after linearization with ClaI, as described previously
8
[5]. A549 cells were electroporated with 10 μg of replicon RNA using a GenePulser Xcell
9
system (Bio-Rad, Hercules, CA) with 3 pulses, at 450 V, 25 µF, at 5-10 sec intervals.
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Transfected cells were seeded into a 96-well plate at 1.5 × 104 cells per well, followed by
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treatment with the compound or the dimethyl sulfoxide (DMSO) control and incubation at
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37°C in 5 % CO2. At various time points post-transfection, cells were washed once with
13
phosphate-buffered saline (PBS) and lysed in 20 μl 1× lysis buffer (Promega, USA) and
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assayed for luciferase signals with a Clarity luminescence microplate reader (BioTek, USA)
15
using the renilla luciferase assay system (Promega, USA). CellTiter-Glo reagent was added to
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the lysates to determine the cytotoxicity effects of compounds. Duplicate wells were seeded
17
for each data point.
18 19
Compound testing in cells transiently expressing infectious DENV. Full length infectious
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virus of DENV2 (strain New Guinea C) cloned into the pACYC plasmid was in vitro
21
transcribed using a T7 mMessage mMachine kit (Ambion, Austin, TX) after linearization
22
with ClaI, as described previously [5]. BHK-21 cells were electroporated with 10 μg of
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replicon RNA using a GenePulser Xcell system (Bio-Rad, Hercules, CA) with 3 pulses, at
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850 V, 25 µF, at 5-10 sec intervals.
25
105 cells per well), followed by treatment with the compound or the dimethyl sulfoxide
Transfected cells were seeded into 96-well plates (1 ×
5
1
(DMSO) control and incubated at 37 °C in 5 % CO2. At various time points post-transfection
2
(p.t.), cells were washed once with phosphate-buffered saline (PBS) and lysed in 20 μl 1×
3
lysis buffer (Promega, USA) and assayed for luciferase signals with a Clarity luminescence
4
microplate reader (BioTek, USA). Cell viability was measured by using CellTiter-Glo kit
5
(Promega, USA) according to the manufacturer's protocols. Absorbance was measured at 450
6
nm by using a microplate reader (Tecan). Duplicate wells were seeded for each data point.
7 8
Plaque assay. Virus stock was produced by harvesting the supernatant of DENV2 full-length
9
RNA-transfected BHK-21 cells at 24, 48, 72, 96, and 120 hours post transfection. Virus titer
10
and morphology were determined by standard plaque assay. Briefly, a series of 10-fold
11
dilutions was prepared by first diluting 50 µl virus stock with 450 µl RPMI 1640 media
12
containing 2 % FBS to obtain 10-1 dilution and then further diluted until a final 10-6 dilution
13
was achieved. Confluent BHK-21 cells (1 × 105 cells per well, plated 2 days in advance)
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grown in 24-well plate was added with 200 µl of each dilution per well. Duplicates were
15
prepared for each time point and dilution factor. The infection was allowed to proceed at
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30°C for 1 hour, followed by removing the media and adding 500 µl 0.8% methyl-cellulose
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overlay (containing RPMI, 2 % FBS, 1 % P/S, 0.05 % NaHCO3, 25 mM Na Hepes, and
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0.5 % DMSO) into each well. The plates were incubated for 5 days at 37 °C in 5 % CO2
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before fixing in 3.7 % formaldehyde and staining with 1 % crystal violet. The viral titer was
20
calculated as plaque-forming unit (PFU) per ml.
21 22
Immunofluorescence assay (IFA). Cells harbouring DENV replicons or infectious virus
23
were seeded into an eight-well Lab-Tek chamber slide (Thermo Fisher Scientific) and
24
incubated at 37 °C in 5 % CO2. At various time-points, cells were washed twice in PBS, and
25
then fixed in cold methanol at -20 °C. Cells were blocked with PBS containing 1 % bovine 6
1
serum albumin, 1 % FBS and 0.05 % Tween-20, and washed thrice with PBS followed by
2
incubation with anti-rabbit DENV NS5 (GeneTex) or anti-mouse dsRNA (English &
3
Scientific Consulting Kft., Hungary), After 1 hr, cells were washed thrice with PBS, and
4
added with secondary antibodies, Alexa Fluor 488 goat anti-rabbit IgG or Alexa Fluor 568
5
donkey anti-mouse IgG (Invitrogen, USA). After washes with PBS, the cells were mounted in
6
mounting medium with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Inc.).
7
Fluorescence images were acquired with a Leica DM4000 B system.
8 9
Data visualization and analysis. Assay development data were generated in Microsoft
10
Excel. Data analysis and visualization was done with GraphPad Prism5 and Spotfire. Positive
11
(maximum RFU) and negative (minimum RFU) controls were averaged and subtracted from
12
sample data to calculate % inhibition by using equation 1 below:
13 14
% inhibition = (100 – ((sample signal – average Negative )/(average Positive – average
15
Negative )×100))
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The four parameter logistic equation was used for curve fitting to calculate IC50 according to
17
equation 2 below:
18 19
Y = Bottom + (Top -Bottom)/(1+((10^( logIC50 - X)^Hill slope))
20
where Bottom is the minimum Y value, Top is the maximum Y value, and Hill slope is the
21
slope of the linear portion of the semi-log curve. IC50 was extrapolated from logIC50
22
according to the GraphPad algorithm.
23
7
1
Co-crystallization of compounds with DENV3 FL NS5. Co-crystallization of either
2
compounds 27 or 29 with DENV3 FL NS5 was performed by hanging drop method mixing
3
1µl of mother liquor (16-18 % PEG 3350, 0.2 M Mg Acetate, 1 mM TCEP) to 1 µL of
4
protein mixture (2-3 mg/ml of protein with final concentration of 1-5 mM of each compound
5
diluted in 10 % DMSO).
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8
1
Supporting Figures.
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Fig. 1A. Representative IC50 curves for N-pocket inhibitors tested in DENV4 FL NS5 de
3
novo initiation FAPA assay [1]. Briefly, compounds (10-point, 3-fold serially diluted
4
compounds from 0- 20 or 100 µM) were incubated at RT for 20 min with enzyme alone, in
5
384-well plate opaque plates, after which reactions were started with addition of ssRNA and
6
nucleotide substrate components, and allowed to proceed for 2 hr. Reactions were stopped by
7
addition of 10 µl of 2.5X STOP buffer with 25 nM CIP, re- incubated at RT for 60 min and
8
read on a Tecan Safire II microplate reader (excitationmax and emissionmax wavelengths 422
9
nm and 566 nm). Data was fitted to the four parameter logistic equation and IC50 curves
10
plotted using Graphpad® Prism software. Table contains average IC50 values and hill slopes
11
obtained. All data points were measured in duplicates. 3 dGTP 15000
Best-fit values Bottom Top LogIC50 HillSlope IC50
12000 8000 4000
= 1980 22108 -0.2898 -0.9099 0.5131
A v e ra g e R F U
A v e ra g e R F U
16000
10000
5000
0
PKF024-680 = 2262 12785 2.896 -1.205 787.7
Bottom Top LogIC50 HillSlope IC50
12000 9000
-2
-1
0
1
2
3000
-1
0
1
2
3
4
Bottom Top LogIC50 HillSlope IC50
4000
LNG748 1917 9325 1.394 -1.196 24.75
0 -2
-1
0
lo g [c p d ],u M
(E) 15
1
2
3
-2
-1
lo g [c p d ],u M
0
1
2
3
0
1
lo g [c p d ],u M
(G) 26
(F) 25
NVP LXT442 NX1
24000
10000
24000
8000
20000
20000
A v e ra g e R F U
A v e ra g e R F U
8000
0
-2
lo g [c p d ]/u M
6000 Bottom Top LogIC50 HillSlope IC50
4000
2000
LOW911 1016 9248 0.1249 -0.6021 1.333
A v e ra g e R F U
-3
LNI017 = 2262 15349 1.490 -1.606 30.93
Bottom Top LogIC50 HillSlope IC50
6000
0 -4
A v e ra g e R F U
15000
20000
A v e ra g e R F U
12000
18000
24000
16000
Best-fit values Bottom Top LogIC50 HillSlope IC50
12000 8000 4000
0
= 1980 20930 -0.5515 -1.009 0.2809
16000
8000 4000
0
-3
-2
-1
0
1
2
3
= 1980 22628 -0.9410 -0.8121 0.1146
0 -4
-3
-2
lo g [c p d ],u M
-1
0
1
-4
-3
lo g [c p d ]/u M
(I) 27 18000
Best-fit values Bottom Top LogIC50 HillSlope IC50
12000
-2
-1
0
1
lo g [C p d ] /u M
(K) 29i
(J) 29
18000
20000
Best-fit values Bottom Top LogIC50 HillSlope IC50
6000
= 1980 16922 -0.7745 -1.058 0.1681
0
15000
Best-fit values Bottom Top LogIC50 HillSlope IC50
10000
5000
= 1980 18355 -0.7359 -0.9193 0.1837
-3
-2
-1
lo g [C p d ] /u M
0
1
16000 12000 Best-f it values Bottom Top LogIC50 HillSlope IC50
8000 4000
= 1980 19844 -1.535 -0.9626 0.02915
-4
-3
-2
-1
lo g [C p d ] /u M
0
1
-4
-3
-2
12000 Best-f it values Bottom Top LogIC50 HillSlope IC50
6000
= 1980 17707 -1.205 -0.8516 0.06241
0
0
0
-4
R F U 4 2 2 /5 6 6 n m
12000
R F U 4 2 2 /5 6 6 n m
A v e ra g e R F U
A v e ra g e R F U
20000
-1
lo g [C p d ] /u M
0
1
-4
-3
-2
-1
lo g [C p d ] /u M
12 13
9
1
Fig. 1B. Representative EC50 and CC50 curves for N-pocket inhibitors tested in HuH-7
2
DENV-2 replicon cell-based assay [2]. Cells were seeded over-night in white opaque 384-
3
well plates followed by incubation with increasing compound concentrations (10-point, 2-
4
fold serially diluted compounds from 0-50 µM) for 48 hr, after which cellular renilla
5
luciferase (EC50) or ATP (CC50) levels, measured as relative light units (RLU), were
6
determined using ViviRen and Cell Titer-Glo (Promega) according to manufacturer’s
7
protocol. All data points were measured in duplicates.
8 (B) 26 800000
A v e ra g e R L U
600000 80000 400000 40000 200000
CTG
800000
120000
A v e ra g e R L U
120000
600000 80000 400000 40000
V iv iR e n
V iv iR e n 0 -2
-1
0
1
2
0
0
0 -3
200000
CTG
-3
3
-2
-1
0
1
2
lo g 1 0 [ c p d ], µ M
lo g 1 0 [ c p d ], µ M
800000
140000
800000
105000
600000
105000
600000
70000
400000
70000
400000
CTG
35000
200000
A v e ra g e R L U
A v e ra g e R L U
(D) 27 140000
35000
V iv iR e n
0
0
0 -3
-2
200000
CTG
V iv iR e n
-1
0
1
0 -3
2
-2
lo g 1 0 [ c p d ], µ M
-1
0
1
2
lo g 1 0 [ c p d ], µ M
(F) 29i
400000 40000 200000
CTG
A v e ra g e R L U
A v e ra g e R L U
600000 80000
800000
120000
800000
120000
600000 80000 400000 40000
V iv iR e n
0
0 -3
9
-2
200000
CTG
V iv iR e n -1
0
lo g 1 0 [ c p d ], µ M
1
2
0
0 -3
-2
-1
0
1
2
lo g 1 0 [ c p d ], µ M
10 10
1
Fig. 2. Enzyme inhibition kinetics of N-pocket compounds against DENV polymerase.
2
DENV4 FL NS5 dnI FAPA assays [1] were performed in increasing concentrations of N-
3
pocket inhibitor, 15 or 29 or 3’dGTP (control) with 0-500 nM RNA or 0-50 µM GTP to
4
determine mechanism of inhibition with respect to either suibstrates. Representative
5
Michaelis-Menton plots were derived from non-linear regression curve fitting using
6
Graphpad Prism software.
7 8 500
R F U p e r m in
400
3 `d G T P (2 µ M ) 3 `d G T P (1 µ M )
300
3 `d G T P ( 0 .5 µ M ) 200
3 `d G T P (0 .2 5 µ M ) 3 `d G T P (0 .1 2 5 µ M )
100
R F U p e r m in
400
300
3 `d G T P (2 µ M ) 3 `d G T P (1 µ M ) 3 `d G T P ( 0 .5 µ M )
200
3 `d G T P (0 .2 5 µ M ) 3 `d G T P (0 .1 2 5 µ M )
100
3 `d G T P (0 .0 6 2 5 µ M )
3 `d G T P (0 .0 6 2 5 µ M )
0
0 0
100
200
300
400
500
0
5
R N A (n M )
10
15
20
25
G T P (µ M )
250
200
1 5 (2 0 µ M ) 150
1 5 (1 0 µ M ) 1 5 (5 µ M )
100
1 5 ( 2 .5 µ M ) 1 5 (1 .2 5 µ M )
50
R F U p e r m in
R F U p e r m in
200
150
1 5 (2 0 u M ) 1 5 (1 0 µ M )
100
1 5 (5 µ M ) 1 5 ( 2 .5 µ M ) 1 5 ( 1 .2 5 µ M )
50
1 5 (0 .6 2 5 µ M )
1 5 (0 .6 2 5 µ M ) 0
0 0
100
200
300
400
500
0
5
R N A (n M )
10
15
20
25
G T P (µ M )
2 9 -R N A 600
250
2 9 (1 2 5 n M ) 2 9 (6 2 .5 n M ) 2 9 ( 3 1 .2 5 n M )
200
2 9 (1 5 .6 2 5 n M )
R F U p e r m in
R F U p e r m in
200
2 9 (2 5 0 n M )
400
2 9 (2 5 0 n M ) 150
2 9 (1 2 5 n M ) 2 9 (6 2 .5 n M )
100
2 9 ( 3 1 .2 5 n M ) 2 9 (1 5 .6 2 5 n M )
50
2 9 (7 .8 1 2 5 n M ) 0 0
9
2 9 (7 .8 1 2 5 n M ) 0
100
200
300
R N A (n M )
400
500
0
5
10
15
20
25
G T P (µ M )
10 11 11
1
Fig. 3. Crystal structures of DENV3 FL NS5 were obtained by co-crystallization according
2
to conditions from [6] as described in Supplementary Materials. (A) Overall view of the
3
DENV3 FL NS5 structure displayed as ribbon with the methyltransferase domain in red, the
4
linker region in orange, palm, thumb and fingers subdomains colored in olive, green and blue
5
respectively. Both compounds 23 (magenta sticks) and 29 (yellow sticks) are overlaid in the
6
polymerase domain. Magnified views of compound 29 (B) and 27 (C) with Fo-Fc contoured
7
at 4 σ where each compound was omitted from the phase calculation. (D) Superimposition of
8
the RdRp domain from FL NS5 (green ribbon) and RdRp (pink ribbon) with bound
9
compounds 29 and 23 in sticks showing the absence of conformational changes. (E)
10
Superimposition of compound 29 bound to FL NS5 structure (magenta sticks) and bound to
11
RdRp structure (pink sticks) and compound 23 (panel F) bound to FL NS5 (grey sticks) and
12
bound to RdRp (orange sticks). The compound conformations are closely superimposable.
13
PDB codes for the FL NS5 structures with compounds 27 and 29 are 5JJS and 5JJR,
14
respectively.
15
12
1 2 3 4
13
1
Fig. 4. Phylogenetic tree representing relatedness of N-pocket amino acid residues from
2
different members of the Flavivirus family, derived from Clustal Omega program [8].
3 4 5 6
Fig. 5. Effects of N-pocket compounds on DENV polymerase thermo-stability. Melting
7
temperature (Tm) was assessed by thermo-denaturation in presence of the SYPRO Orange
8
dye as described in Materials and Methods. (A-F) Representative melting curves of DENV4
9
RdRp domain (aa 266-900; A-C) and FL NS5 (D-F) in presence of 50 µM N-pocket
10
inhibitors or 5 % DMSO control. (G) Cellular thermal shifts assays [9] were performed with
11
BHK-21 DENV2-NGC replicon cell lysates and 40 mM of 27 or 29. Briefly, lysates were
12
incubated with compounds or 5 % DMSO for 1 hr at 4 oC, followed by heating at 30-70 oC.
13
Samples were spun and the supernatants loaded onto 12 % SDS-PAGE gels, followed by gel
14
electrophoresis and western blotting with anti-DENV2 NS5 antibody (GeneTex, USA). Table
15
shows the changes in protein melting temperatures in presence of compounds compared to
16
controls treated with DMSO. 14
1 (A)
(B)
0
0
10
20
30
40
50
60
70
80
90
100
o
T e m p e r a tu r e ( C )
-1 0 0
- d R F U /d T
-1 5 0
0 10
-5 0
20
30
40
50
60
70
80
90
100
o
T e m p e r a tu r e ( C )
-1 0 0 -1 5 0
150 100
50
0
0
-5 0
10
20
30
40
50
60
70
80
90
100
o
-1 0 0
T e m p e r a tu r e ( C )
-1 5 0
- d R F U /d T
50
10
20
30
40
50
60
70
T e m p e r a tu r e ( C )
-1 5 0
-2 5 0
-3 0 0
-3 0 0
70
80
90
-1 5 0
DENV4 FL NS5 + 29
80
90
100
10 -5 0
20
30
40
50
60
70
80
T e m p (o C )
-1 0 0 -1 5 0 -2 0 0 -2 5 0
3 (G) DMSO control 30
32.5
35
37.5 40
42.5
45
30
32.5
35
37.5
42.5
45
47.5
50
27-40 µM 40
47.5
50
29-40 µM 19 32.5 30
35
37.5
100
DENV4 FL NS5 + DM SO
o
-2 5 0
60
0
-5 0
-2 0 0
50
o
50
D EN V4 FL N S5 + 2 9
-1 0 0
-2 0 0
40
T e m p e r a tu r e ( C )
100
DENV4 FL NS5 + DM SO
100
D EN V4 FL N S5 + 1 5
30
(F)
150
DENV4 FL NS5 + DM SO
20
-3 0 0
(E)
(D)
10
-2 5 0
-3 0 0
-3 0 0
-5 0 -1 0 0
-2 0 0
-2 5 0
-2 5 0
D EN V4 R dR p + 2 9
50
-2 0 0
-2 0 0
- d R F U /d T
D EN V4 R dR p + 2 7
- d R F U /d T
- d R F U /d T
50
D EN V4 R dR p + D M SO
100
DENV4 RdRp + 15
50
-5 0
150
D EN V4 R dR p + D M SO
100
100
2
(C)
150
D EN V4 R dR p + D M SO
- d R F U /d T
150
40
42.5
45
47.5
50
4 5 6 7 8 9 10
15
90
100
1
Fig. 6. Activity profiles of DENV polymerase bearing resistant phenotype amino acid
2
changes. Recombinant DENV2 (A, C) and DENV4 (B, D) FL NS5 proteins bearing single or
3
double amino acid changes in the N-pocket were tested in de novo initiation (A, B) and
4
elongation (C, D) FAPA assays and compared against activities of WT DENV2 or DENV4
5
FL NS5 proteins. Reactions were conducted over 2 hr at RT and from average relative
6
fluorescence units (RFU) obtained from one experiment. All data points were measured in
7
triplicate. (B)
(A) 30000
50000 DENV4 W T
d e n o v o in itia tio n
L511V E 802D 20000
L 5 1 1 V /E 8 0 2 D
10000
Q 803N 30000
L 5 1 2 V /Q 8 0 3 N
20000
10000
0 0 .0
L512V
40000
A v e ra g e R F U
A v e ra g e R F U
d e n o v o in itia tio n
DENV2 W T
0 0 .5
1 .0
1 .5
2 .0
0 .0
0 .5
T im e ( h r )
15000
8500
L511V E 802D
9000
L 5 1 1 V /E 8 0 2 D
6000
3000
0 0 .0
6800
A v e ra g e R F U
E lo n g a tio n A c tiv ity
DENV2 W T 12000
A v e ra g e R F U
1 .5
2 .0
1 .5
2 .0
(C)
(C) E lo n g a tio n A c tiv ity
1 .0
T im e ( h r )
DENV4 W T L512V Q 803N
5100
L 5 1 2 V /Q 8 0 3 N
3400
1700
0 0 .5
1 .0
T im e ( h r )
1 .5
2 .0
0 .0
0 .5
1 .0
T im e ( h r )
8 9 10 11 12 13 16
1
Fig. 7. Effects of compounds on DENV polymerase thermo-stability. Melting temperature
2
(Tm) was assessed by thermo-denaturation in presence of the SYPRO Orange dye as
3
described in Materials and Methods. (A-D) Representative melting curves of in vitro
4
expressed recombinant DENV2 FL NS5 WT or mutant proteins in presence of 50 µM
5
compound or 5 % DMSO control. Table shows the melting temperatures of DENV-2 and -4
6
FL NS5 WT and mutant proteins as well as the changes in their melting temperatures in
7
presence of compounds compared to controls treated with 5 % DMSO.
8
(A)
(B) 150
150
L511V + DM SO
W T + DMSO 100
W T + 29
50
0 20
40
60
-5 0
80
0
T e m p e ra tu re ,
d R F U /d T
d R F U /d T
50
100
W T + 27
100
C
-1 0 0
L511V + 27 L 511V + 29
0 20
40
60
-5 0
0
T e m p e ra tu re ,
80
100
80
100
C
-1 0 0
-1 5 0
-1 5 0
-2 0 0
-2 0 0 -2 5 0
(C)
(D)
150
150
E802D + DM SO 100
9
E 80 2D + 27
L 5 1 1 V /E 8 0 2 D + D M S O L 5 1 1 V /E 8 0 2 D + 2 7 L 5 1 1 V /E 8 0 2 D + 2 9
E 80 2D + 29
50
0 20
40
60
-5 0
T e m p e ra tu re ,
0
80
d R F U /d T
d R F U /d T
50
100
100
C
-1 0 0
0 20
40
60
-5 0
T e m p e ra tu re ,
0
C
-1 0 0
-1 5 0
-1 5 0
-2 0 0
-2 0 0
-2 5 0
-2 5 0
Protein melting temperatures ,Tm (oC) [Tm change compared to DMSO control] DENV2 FL NS5
DENV4 FL NS5
WT
L511V
E802D
L511V/E802D
WT
L512V
Q802N
L512V/Q802N
+ DMSO
35.5
35.5
36
35
37.5
36.5
37
36
+ 27
37 [1.5]
35.5 [0]
36 [0]
35 [0]
38.5 [1]
36.5 [0]
37.5 [0.5]
35 [-1]
+ 29
40 [4.5]
36.5 [1.0]
38 [2.0]
35.5 [0.5]
40.5 [3]
38 [1.5]
38.5 [1.5]
35.5 [-0.5]
10 17
1
Fig. 8. Analysis of viral and NS5 protein expressions from DENV WT and mutant
2
replicons and virus. Immuno-fluorescence stainings for DENV dsRNA and NS5 protein
3
were performed on BHK-21 cells at days 1-4 (D1-4) after electroporation of DENV-2 WT
4
and mutant (A) replicon or (B) full length viral IVT RNA. Cells were fixed and the stated
5
time-points and probed with mouse monoclonal anti-dsRNA (red; Scicons, USA) and rabbit
6
polyclonal anti-NS5 (green; GeneTex, USA) primary antibodies, and goat anti-mouse-IgG-
7
Alexa Fluor568 and goat anti-rabbit-IgG-FITC secondary antibodies (Invitrogen, USA).
8
Nuclear DNA was stained with DAPI (blue; Thermofisher, USA).
9
(A)
WT
L512V
E802D
L512V/E802D
D1
D2
D4 10 11 12 13 14 15 16 18
1
(B)
WT
L511V
E802D
L512V/E802D
D1
D2
D3
D4 2
19
1
Fig. 9. WNV (New York strain 3356) replicon cDNA was electroporated in BHK-21 cells,
2
after which cells were seeded into 96-well plates and treated with compounds, 26i, 27, 29 and
3
29i (10-point, 3-fold serially diluted compounds from 0- 50 µM), for 2 days. EC50 values
4
from replicon cells were determined by measuring cellular renilla luciferase levels. All data
5
points were measured in duplicates.
6
(A) 100000
26i 27
RFU
80000 60000 40000 20000 0 -3
-2
-1
0
1
2
Log (µM)
(B) 100000
29 29i
RFU
80000 60000 40000 20000 0 -3
7
-2
-1
0
1
2
Log (µM)
8 9 20
1
Table 1. Data collection and refinement statistics of DENV3 FL NS5 co-crystals.
2
PDB code
5JJR
5JJS
18 % PEG 3350, 0.2 M
18 % PEG 3350, 0.2 M Mg
Mg Acetate, 1 mM TCEP
Acetate, 1 mM TCEP
Wavelength (Å)
1.0
1.0
Resolution (Å)
50-1.99 (2.11-1.99)
50-1.65 (1.75-1.65)
Space group
P21212
P21212
Unit cell (Å)
94.6/150.4/69.1
94.7/150.2/68.5
Measured reflections
44525 (64401)
784305 (127110)
Unique reflections
67993 (10422)
225431 (36133)
Multiplicity
6.5 (6.1)
3.5 (3.5)
Completeness (%)
99.0 (95.2)
99.2 (98.3)
Mean I/signa I (I)
10.04 (3.01)
13.13 (1.65)
Rmerge (%)
19.0 (58.6)
6.01 (74.0)
CC (1/2) (%)
99.2 (83.5)
99.9 (64.3)
R-work (%)
17.9
17.1
R-free (%)
22.5
19.8
Macromolecule
6858
6920
Ligands
34 (L3) / 26 (SAH)
33 (L9) / 26 (SAH)
Solvent
473
628
Crystallization conditions
Number of non-hydrogen atoms
Protein residues
845
RMS (bonds) (Å)
0.010
0.010
21
RMS (angles) (°)
1.09
1.01
Ramachandran favoured (%)
99.3
99.3
Ramachandran outliers (%)
0.7
0.7
Macromolecules
23.2
26.9
Ligands
23.8
33.5
Solvent
28.2
35.9
Average B-factor (Å2)
1 2
Values in parenthesis are those for the last (highest resolution) shell
3
MR: Molecular replacement
4
CC1/2= percentage of correlation between intensities from random half‐dataset [7]
5 6 7 8 9 10 11 12 13
22
1
Table 2. Analysis of DENV2 (strain NGC) WT and mutant replicons and virus
2
replication.
3 4
(A)
5
kit (Promega, USA) from BHK-21 cells at days 1-4 after electroporation with DENV2 WT
6
and mutant replicons IVT RNA. Levels of secreted DENV2 (pfu/ml) were determined by
7
plaque assay from supernatants of BHK-21 cells at days 1-4 after electroporation with
8
DENV2 WT and mutant infectious viral IVT RNA.
Absolute relative renilla luciferase light units (RLU) measured using renilla luciferase
Renilla luciferase levels in
DENV2 levels (pfu/ml)
DENV2 replicon (RLU)
in culture supernatant
PostL512V/
L511V/ electroporation
WT
L511V
WT
E802D
L512V
E802D E802D
E802D (hr) 2276562.9
3061250.9
684487.8
2323574.8
± 14766.5
± 42578.2
± 9525.1
± 66485
1701190.5
2028336.0
1084181
1718929.8
±105595.8
± 136472
± 44392.6
± 25087.1
458693.7
618389.6
166496.1
467361.7
± 278.6
± 2494
± 577.4
± 12258.4
60752.2
141721.1
19744.6
67222.3
± 1488.5
± 5521.3
± 1073
± 9150
24
48
72
96
52500
1200
14000
65000
132500
55000
425000
775000
825000
200000
4500000
5250000
7000000
3250000
9750000
3750000
9 10 11
23
1
(B)
2
secreted virus titers, respectively, compared to WT replicon or virus levels (expressed as fold
3
increases over WT levels).
Mutant DENV2 replicons or virus replication measured by renilla luciferase levels or
Fold increase in DENV2 levels Renilla luciferase levels, fold increase
(pfu/ml) in culture
compared to WT replicon levels
supernatant compared to WT DENV2 levels
PostL511V/
L511V/ electroporation
L511V
L511V
E802D
E802D E802D
E802D (hr) 24
26.72
6.0
19.87
0.02
0.27
1.24
48
3.38
1.80
2.83
0.42
3.21
5.85
72
7.14
1.92
5.30
0.24
5.45
6.36
96
14.47
2.02
6.20
0.46
1.39
0.54
4 5 6 7 8 9 10 11 12 13 14 15 24
1
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4
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