RESEARCH ARTICLE
Potent Allosteric Dengue Virus NS5
Polymerase Inhibitors: Mechanism of Action
and Resistance Profiling
Siew Pheng Lim
1
*, Christian Guy Noble
1
, Cheah Chen Seh
1
, Tingjin Sherryl Soh
1,2
,
Abbas El Sahili
2
, Grace Kar Yarn Chan
2
, Julien Lescar
2,3
, Rishi Arora
4
, Timothy Benson
4
,
Shahul Nilar
1
, Ujjini Manjunatha
1
, Kah Fei Wan
1
, Hongping Dong
1
, Xuping Xie
1¤
, Pei-
Yong Shi
1¤
, Fumiaki Yokokawa
1
1 Novartis Institute for Tropical Diseases, Singapore, 2 School of Biological Sciences, Nanyang
Technological University, Singapore, 3 UPMC UMRS CR7—CNRS ERL 8255-INSERM U1135 Centre
d’Immunologie et des Maladies Infectieuses, Centre Hospitalier Universitaire Pitié-Salpêtrière, Faculté de
Médecine Pierre et Marie Curie, Paris, France, 4 Novartis Institute for Biomedical Research, Cambridge,
Massachusetts, United States of America
¤ Current address: Department of Biochemistry & Molecular Biology, Sealy Center for Structural Biology &
Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555, USA.
*
siew_pheng.lim@novartis.com
Abstract
Flaviviruses comprise major emerging pathogens such as dengue virus (DENV) or Zika
virus (ZIKV). The flavivirus RNA genome is replicated by the RNA-dependent-RNA poly-
merase (RdRp) domain of non-structural protein 5 (NS5). This essential enzymatic activity
renders the RdRp attractive for antiviral therapy. NS5 synthesizes viral RNA via a “de novo”
initiation mechanism. Crystal structures of the flavivirus RdRp revealed a “closed” confor-
mation reminiscent of a pre-initiation state, with a well ordered priming loop that extrudes
from the thumb subdo main into the dsRNA exit tunnel, close to the “GDD” active site. To-
date, no allosteric pockets have been identified for the RdRp, and compound screening
campaigns did not yield suitable drug candidates. Using fragment-based screening via X-
ray crystallography, we found a fragment that bound to a pocket of the apo-DENV RdRp
close to its active site (termed “N pocket”). Structure-guided improvements yielded DENV
pan-serotype inhibitors of the RdRp de novo initiation activity with nano-molar potency that
also impeded elongation activity at micro-molar concentrations. Inhibitors exhibited mixed
inhibition kinetics with respect to competition with the RNA or GTP substrate. The best com-
pounds have EC
50
values of 1–2 μM against all four DENV serotypes in cell culture assays.
Genome-sequencing of compound-resistant DENV replicons, identified amino acid
changes that mapped to the N pocket. Since inhibitors bind at the thumb/palm interface of
the RdRp, this class of compounds is proposed to hinder RdRp conformational changes
during its transition from initiation to elongation. This is the first report of a class of pan-sero-
type and cell-active DENV RdRp inhibitors. Given the evolutionary conservation of residues
lining the N pocket, these molecules offer insights to treat other serious conditions caused
by flaviviruses.
PLOS Pathogens | DOI:10.1371/journal.ppat.1005737 August 8, 2016 1/25
a11111
OPEN ACCESS
Citation: Lim SP, Noble CG, Seh CC, Soh TS, El
Sahili A, Chan GKY, et al. (2016) Potent Allosteric
Dengue Virus NS5 Polymerase Inhibitors:
Mechanism of Action and Resistance Profiling. PLoS
Pathog 12(8): e1005737. doi:10.1371/journal.
ppat.1005737
Editor: Daved H Fremont, Washington University,
UNITED STATES
Received: February 16, 2016
Accepted: June 9, 2016
Published: August 8, 2016
Copyright: © 2016 Lim et al. This is an open access
article distributed under the terms of the Creative
Commons Attribution License
, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: Refined coordinates for
DENV3 RdRp co-crystals with compounds 27 and 29
as well as those for DENV2-NGC RdRp co-crystal
with compound 27, have been deposited in the
Protein Databank (PDB) under the PDB codes, 5I3P,
5I3Q and 5K5M respectively. PDB codes for the FL
NS5 structures with compounds 27 and 29 are 5JJS
and 5JJR, respectively.
Funding: Funding sources that supported this work
were obtained from Novartis Institute for Tropical
Diseases, Novartis Institute for Biomedical Research,
and Singapore Economic Development Board. The
Author Summary
Dengue virus (DENV) is the world’s most prevalent mosquito-borne viral disease and
nearly 40% of the world’s population is at risk of infection. Currently, no specific drugs are
available to treat dengue or other flaviviral diseases. DENV NS5 is a large protein of 900
amino acids composed of two domains with key enzymatic activities for viral RNA replica-
tion in the host cell and constitutes a prime target for the design of anti-viral inhibitors.
We performed a fra gment-based screening by X-ray crystallography targeting the DENV
NS5 polymerase and identified an allosteric binding pocket at the base of the thumb sub-
domain close to the enzyme active site. Potent inhibitors active in both DENV polymerase
biochemical and cell-based assays were developed through structure-guided design. Resis-
tant virus replicons grown in the presence of the inhibitor, harbored amino acid changes
that mapped to the compound binding site. The proposed mode of action for this class of
inhibitors is by impeding RdRp protein conformational changes during the transition
from initiation to elongation phase of enzyme activity.
Introduction
Several flaviviruses, such as DENV, Japanese Encephalitis virus (JEV), West Nile virus (WNV),
Yellow Fever virus (YFV) or Tick-borne encephalitis virus (TBEV) are major human patho-
gens, whilst Zika (ZIKV) is an emerging flavivirus of global signifi cance causing severe neuro-
logical conditions in infected adults and newborn babies, most likely by mother-to-child
transmission [
1]. The mosquito-borne DENV causes widesp read epidemics in over 100 coun-
tries, with *390 million infections each year [
2]. Infection by any of the four DENV serotypes
can lead to several outcomes, ranging from asymptomatic infection, dengue fever, to dengue
hemorrhagic fever and dengue shock syndrome. After several decades of efforts, the first vac-
cine was recently licensed for use, but confers only partial cross protection for the four DENV
serotypes [
3, 4]. No antivirals have been approved to treat dengue or other flaviviral diseases
[
5].
Flavivirus RNA replication occurs in host cells on endoplasmic reticulum-derived mem-
branes within a multi-protein replication complex (RC) consisting of viral NS proteins and
host cofactors [
6–8]. Comprising 900 amino acid residues, NS5 is the largest and most con-
served protein component of the flavivirus RC. Its N-terminal domain (residues 1–265 in
DENV3) is an S-adenosyl-L-methionine (SAM)-dependent methyltransferase (MTase) that
methylates the viral RNA genome cap [
9–15]. A guanylyltransferase activity was also proposed
for the N-terminal domain of NS5 [
16, 17]. Its C-terminal RdRp domain (residues 267–900)
synthesizes the viral genomic RNA [
18–22]. A potentially flexible linker region that connects
the two catalytic domains of NS5 regulates RdRp activities and virus replication by modulating
MTase-RdRp interactions [23–25]. In addition to its enzymatic functions, NS5 inhibits host
interferon-mediated signaling by promoting degradation of STAT2 [
26]. In DENV, NS5 local-
izes to the nucleus of infected cells in a serotype-dependent manner that modulates host pro-
cesses [
27].
Following DENV infection, the RdRp synthesizes viral RNA in the absence of a primer
strand, via a de novo initiation mechanism, in which the (+) strand viral RNA template is tran-
scribed into a complementary RNA strand of (-) polarity [
18, 19]. This duplex in turn serves as
a template for synthesis of additional RNA strands of (+) polarity that either act as mRNA for
protein translation or are packaged into virions. DENV RdRp possesses a right hand-like archi-
tecture conserved across different polymerase families [
21, 22, 25], with three subdomains
Profiling Dengue Virus NS5 Polymerase Inhibitors from Rational Design
PLOS Pathogens | DOI:10.1371/journal.ppat.1005737 August 8, 2016 2/25
funders had no role in study design, data collection
and analysis, decision to publish, or preparation of
the manuscript.
Competing Interests: I have read the journal's policy
and the authors of this manuscript have the following
competing interests: SPL, CGN, CCS, SN, UM, KFW,
HD, XX, PYS, FY are employees of Novartis Institute
for Tropical Diseases. RA, TB are employees of
Novartis Institute of Biomedical Research. JL is an
employee of Nanyang Technological University of
Singapore and a consultant of Novartis Institute for
Tropical Diseases. TSS is a PhD student funded by
the Novartis Institute for Tropical Diseases and
Singapore Economic Development Board. SPL and
JL are editorial board members for ANti-viral
Research Journal. PYS has served as Editor (ACS
Infectious Diseases, Journal of General Virology, and
Nature Vaccine) and Editorial Board member (Journal
of Virology, Virology, and Antiviral Research).
termed ‘‘fingers”, ‘‘palm” and ‘‘thumb”. Within these subdomains, seven conserved amino-acid
sequence motifs play key roles for binding RNA, NTPs and metal-io ns and for catalysis [
28,
29]. Structures of the apo-DENV RdRp were found to adopt a “closed” pre-initiation state con-
formation, with a well-ordered priming loop projecting into a narrow RNA binding tunnel.
Disordered peptide segments were observed in motifs F, G and at the C-terminal end [
21, 22,
25].
The importance of NS5 for viral replication makes it an ideal target for developing inhibitors
to treat diseases caused by flaviviruses [
30–32]. Although several high-throughput screening
campaigns have been performed, only a few DENV RdRp non-nucleoside inhibitors have been
described [
33–36]. From these latter efforts, we previously identified two compounds that bind
to the RNA tunnel but did not succeed in improving their lead-like properties [
34, 35]. Here,
using fragment-based screening via X-ray crys tallography targeting the apo-DENV RdRp, we
identified a fragment that bound to a pocket located in the thumb subdomain, close to the
enzyme active site, which we term as the “N pocket” [
37, 38]. Using a structure-guided
approach that combines biochemical, biophysical and cell-based assays, we designed potent
inhibitors that bound to this allosteric site, and inhibited DENV1-4 viral replication across var-
ious cell-based assays. Resistant DENV replicons with amino acid changes in the “N” pocket
were raised with two compounds, confirming that the NS5 polymerase was the specific target
for this class of inhibitors in DENV infected cells. To our knowledge, this is the first report of a
Flavivirus RdRp allosteric pocket and the successful use of structure-guided approach for
designing potent inhibitors targeting NS5. This work has major implications for the design of
much-needed flavivirus anti-viral inhibitors.
Results
Structure-guided design of a novel chemical scaffold that binds to the
DENV RdRp N pocket
Following fragment-based screening using X-ray crystallography, we identified 3 , a bi-phenyl
acetic acid fragment, that bound to a pocket in the DENV3 RdRp thumb subdomain (IC
50
734 μM;
Fig 1 and Table 1; 37). Iterative rounds of structure-guided design led to compounds
that inhibited both DENV polymerase activity and viral replication in cells (
Fig 1 and Table 1;
Fig 1A and 1B in
S1 Text). Firstly, switching the distal unsubstituted phenyl ring in 3, with a
thiophene ring (3i) improved compound potency by >12-fold in DENV1-4 polymerase de
novo initiation ( dn I) enzym e assays [
38, 39]. Substitution of the methoxyl group on the outer
phenyl ring with a second acid moiety increased potency in DENV- 1 and -3 (compare 3i and
11). Replacement of the chloro-substituent on the thiophene ring in 11, with a propargyl alco-
hol, markedly increased compound inhibitory property. Compound 15, which bears this moi-
ety, was >16-fold more active across DENV1-4 enzymes. Whilst subsequent derivatives,
exemplified by compound 15, displayed low nano-molar potencies across DENV1-4 dnI poly-
merase assays, they failed to inhibit DENV replication in cells. This is probably due to unfavor-
able physicochemical properties that limited their cell permeability (likely due to the presence
of bis-carboxylic acid groups in 15).
Successive design strategies produced compounds with acyl-sulfonamide derivatives
(replacing the charged acid groups with the acyl-sulfonamide bio-isosteres increases lipophili-
city) with EC
50
2 μM, in a HuH-7 DENV2 replicon cell-based assay (Fig 1 and Table 1; Fig
1B in
S1 Text). The most active compounds in this series, 29 and 29i, bear the 8-quinolinol
moiety, and demonstrated IC
50
values ranging from 0.013 to 0.074 μM across DENV1-4 poly-
merase, with EC
50
value of ~2 μM in the DENV2 replicon cell-based assay (Table 1).
Profiling Dengue Virus NS5 Polymerase Inhibitors from Rational Design
PLOS Pathogens | DOI:10.1371/journal.ppat.1005737 August 8, 2016 3/25
Fig 1. Structures of N-pocket inhibitors. N-pocket inhibitors listed in order of increasing potency in DENV
enzyme and replicon cell-based assays. Compound 3 is the original hit identified from the fragment-based
screening [
37]. Compounds 3i to 29i were synthesized through rational design [38] following compound
testing in DENV4 FL NS5 dnI assay, SPR analyses and X-ray co-crystallography.
doi:10.1371/journal.ppat.1005737.g001
Table 1. Inhibitory properties of DENV polymerase N-pocket compounds.
DENV FL NS5 de novo initiation FAPA, IC
50
(μM) HuH-7 DENV Replicon (μM)
DENV4 DENV1 DENV2 DENV3 DENV2
Compound IC
50
IC
50
IC
50
IC
50
EC
50
CC
50
3'dGTP 0.79 ± 0.20 0.52 0.62 0.27 >100 >100
3 733.5 ± 10.6* 768.8 768.9 796 >100 >100
3i 30.7 ± 9.4 46.62 37.25 66.76 >100 >100
11 26.0 ± 3.7* 7.1 39.74 10.7 >100 >100
15 1.66 ± 0.35* 0.30 2.20 0.46 >100 >100
25 0.165 ± 0.108* 0.033 0.106 0.040 11.4 >100
26 0.251 ± 0.129* 0.075 0.145 0.057 21 >100
26i 0.109 ± 0.061 0.024 0.086 0.054 12.1 >50
27 0.172 ± 0.097* 0.068* 0.106* 0.048* 3.9 ± 0.62 >100
29 0.023 ± 0.001* 0.013 0.038 0.016 1.9 ± 0.2 >50
29i 0.074 ± 0.031 0.027 0.057 0.026 2.1 ± 0.6 >50
N-pocket inhibitors listed in order of increasing potency in DENV enzyme and replicon cell-based assays. IC
50
values from DENV de novo initiation FAPA
assay were obtained from dose response testing of compounds (10-point, 3-fold serially diluted compounds from 0–20 μM) and are averaged from 3
independent experiments with DENV4 FL NS5 or from one experiment each with DENV-1, -2 and -3 FL NS5 [
22]. Briefly, compounds were incubated for 20
min with enzyme alone, after which reactions were started with the ssRNA and nucleotide substrate components, and allowed to proceed for 2 hr [
39]. Hill
slopes for IC
50
curves (Fig 1A in S1 Text) ranged from -0.7 to -1.6. Determination of compound inhibition (EC
50
) and cytotoxicity (CC
50
) in stable HuH-7
DENV-2 replicon cells [
40] was performed 1–2 times. Cells were incubated for 48 hr in increasing compound concentration (10-point, 2-fold serially diluted
compounds from 0–100 μM) after which cellular renilla luciferase (EC
50
) or ATP (CC
50
) levels measured as relative light units (RLU) were determined. All
data points were measured in duplicates.
*DENV4 FL NS5 IC
50
value for compound 3 was reported in [37, 38]. DENV4 FL NS5 IC
50
value for compounds 11, 15, 25, 26 and 29, as well as DENV1-4
FL NS5 IC
50
values for compound 27 were reported in [38].
doi:10.1371/journal.ppat.1005737.t001
Profiling Dengue Virus NS5 Polymerase Inhibitors from Rational Design
PLOS Pathogens | DOI:10.1371/journal.ppat.1005737 August 8, 2016 4/25
Kinetic studies of DENV RdRp inhibition
To better understand the inhibition mode of this class of compounds, order-of-reagent addi-
tion experiments were performed using the DENV dnI FAPA assay (
Table 2). The standard
assay format involved compounds exposed to enzyme alone followed by reaction initiations
with ssRNA template and NTPs [39]. In the first experiments, compounds 15, 27 and 29 were
exposed to pre-formed enzyme-ssRNA complexes, followed by reaction initiation with NTPs.
IC
50
values generated for 15 and 27 were similar to the standard assay format, suggesting that
these compounds do not discriminate between the apo-enzyme and the polymerase bound to
ssRNA. Compound 29, showed about 3-fold reduction in potency. Next, compounds were
exposed to elongated enzyme-dsRNA complexes, in which the active site was occupied by the
ssRNA template and newly synthesized short RNA products AGAA or AGAACC. Resulting
compound inhibitory potencies dropped by 8–15 fold. The change was most pron ounced in
compound 29 (10 – 15 fold decline). These findings imply that during transition from initiation
to the elongation complex, to accommodate the growing dsRNA product, the N-pocket
Table 2. Inhibitory and binding properties of DENV polymerase N-pocket compounds.
Experiments with DENV4 FL NS5 Compounds 3’dGTP 15 27 29
de novo IC
50
(μM); order of addition
experiments [fold change]
Enzyme + compound 0.79 ± 0.20 1.66 ± 0.35* 0.172 ± 0.097* 0.023 ± 0.001*
[Enzyme + RNA] + compound 0.44 ± 0.02 1.93 ± 0.77
[1.1X]
0.20 ± 0.07
[0.84X]
0.073 ± 0.02
[3.2X]
[Enzyme + RNA+ATP+GTP]
+ compound
0.74 ± 0.26 12.9 ± 4.0
[7.8X]
2.2 ± 1.91 [9.3X] 0.338 ± 0.12
[14.7X]
[Enzyme + RNA+ ATP+GTP
+ATTO-CTP] + compound
0.71 ± 0.20 13.3 ± 2.6 [8X] 1.89 ± 1.56 [8X] 0.239 [10.4X]
Elongation IC
50
(μM) Enzyme + compound 0.43 ± 0.29 16.2 ± 4.7
[9.8X]
5.46 ± 2.14 [23X] 0.427 ± 0.013
[18.6X]
SPR K
d, μ
M n/a 1.44 0.0745* 0.00725*
K
off, μ
M n/a n/a 2.38 0.642
K
on, μ
M n/a n/a 32000000 88500000
%R
max
n/a 66.8 20.9
Change in protein melting temperature, Tm
[+ DMSO ! +50 μM compound], °C
RdRp domain n/a 15 [50!65] 8 [50!58] 14 [50!64]
FL NS5 n/a 9.5 [42.5!52] 4 [42.5!46.5] 9 [42.5!51.5]
FL NS5 from BHK-21 DENV2-NGC
replicon cell lysate
n/a nd 5 [42.5!47.5] 7.5 [42.5!50]
Order-of-addition experiments were performed with DENV4 FL NS5 de novo initiation FAPA assay with 10-point, 3-fold serially diluted compounds from
0–20 μM, to determine effects on inhibitory properties of compound 15 , 27 and 29, as described in Materials and Methods. Compounds were incubated for
20 min with enzyme alone, enzyme-ssRNA complex, enzyme-dsRNA complex (comprising ssRNA template and newly synthesized AG or AGC RNA
products), after which reactions were started with the corresponding missing ssRNA and/or nucleotide components, and allowed to proceed for 2 hr. IC
50
values were averaged from 3 independent experiments with compound 15 and 27, and determined from at least one experiment for 29.IC
50
values
obtained from elongation assays were averaged from >3 independent experiments for all three compounds. Hill slopes for IC
50
curves ranged from -0.7 to
-1.6. All data points were measured in duplicates. Binding affinities (K
d
) of compounds were determined by surface plasmon resonance using a Biacore
T200 instrument as described in Materials and Methods and analyzed using Biacore T200 evalution with affinity-kinetics analysis. Effects of compounds on
protein thermo-stability (melting temperature, T
m
) was assessed using the thermo-denaturation assay as described in Materials and Methods, with in vitro
expressed recombinant DENV4 FL NS5, RdRp domain (aa 268–900), or cell lysates from BHK-21 DENV2 (strain NGC) replicon cells following the cellular
thermal shift assay described previously [
41]. Proteins or cell lysates were incubated with 50 μM compound or control 5% DMSO alone, followed by thermo-
denaturation. Experiments were performed in duplicates.
“n/a” and “nd” respectively denote “not applicable” and “experiment not done”.
*DENV4 FL NS5 IC
50
value for compounds 15, 27 and 29 and K
d
values for compounds 27 and 29 previously reported in [38].
doi:10.1371/journal.ppat.1005737.t002
Profiling Dengue Virus NS5 Polymerase Inhibitors from Rational Design
PLOS Pathogens | DOI:10.1371/journal.ppat.1005737 August 8, 2016 5/25