Identification of a pyridoxine-derived small-molecule inhibitor targeting dengue virus RNA-dependent RNA polymerase

Article abstract of DOI:10.1128/AAC.02203-15

The viral RNA-dependent RNA polymerase (RdRp) activity of the dengue virus (DENV) NS5 protein is an attractive target for drug design. Here, we report the identification of a novel class of inhibitor (i.e., an active-site metal ion chelator) that acts aga

Full text of DOI:10.1128/AAC.02203-15

AAC Accepted Manuscript Posted Online 16 November 2015
Antimicrob. Agents Chemother. doi:10.1128/AAC.02203-15
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Identification of a pyridoxine-derived small-molecule inhibitor targeting dengue virus
RNA-dependent RNA polymerase
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Hong-Tao Xu, Susan P. Colby-Germinario, Said Hassounah , Peter K. Quashie, Yingshan
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Han, Maureen Oliveira, Brent R. Stranix, and Mark A. Wainberg
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McGill University AIDS Centre, Lady Davis Institute for Medical Research, Jewish General
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Hospital, Montreal, Quebec, Canada and Departments of Medicine and Microbiology and
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Immunology, McGill University, Montreal, Quebec, Canada. Champlain Exploration Pharma
Inc., Montreal, Quebec, Canada.
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Running title: Dengue virus RNA-dependent RNA polymerase inhibitor
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* To whom correspondence should be addressed: Mark A. Wainberg, Ph.D., McGill University
AIDS Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, 3755 Côte-
Ste-Catherine Rd, Montreal, Quebec, H3T 1E2, Canada Tel: (514) 340-8260; Fax: (514) 340-
7537; E-mail: mark.wainberg@mcgill.ca

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Word count:
Abstract: 250 words
Text:
5526 words
Tables: 6
Figures: 5
Acknowledgments
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This work was partly supported by research grants from the Canadian Institutes of Health
24 Research (CIHR). Mark A. Wainberg is a consultant to Champlain Biosciences Inc.
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ABSTRACT
The viral RNA-dependent RNA polymerase (RdRp) activity of the dengue virus (DENV)
27 NS5 protein is attractive target for drug design. Here, we report the identification of a
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novel class of inhibitor, i.e. an active-site metal-ion chelator, that acts against DENV
RdRp activity. DENV RdRp utilizes two-metal ion mechanism of catalysis; therefore, we
constructed a small library of compounds, through mechanism-based drug design, aimed
at chelating divalent metal ions in the catalytic site of DENV RdRp. We now describe a
pyridoxine-derived small-molecule inhibitor that targets DENV RdRp and show that 5-
benzenesulfonylmethyl-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylic
acid
hydroxyamide (termed DMB220) inhibited the RdRp activity of DENV sereoytypes 1-4
at low µM 50% inhibitory concentrations (IC50s of 5-6.7 µM) in an enzymatic assay. The
antiviral activity of DMB220 against DENV infection was also verified in cell-based
assay and showed a 50% effective concentration (EC50) <3 µM. Enzyme assays proved

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that DMB220 was competitive with nucleotide incorporation. DMB220 did not inhibit
the enzymatic activity of recombinant HIV-1 reverse transcriptase and showed only weak
inhibition of HIV-1 integrase strand-transfer activity, indicating high specificity for
41 DENV RdRp. S600T substitution in the DENV RdRp, that was previously shown to
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confer resistance to nucleoside analogue inhibitors (NI) conferred 3-fold hyper-
susceptibility to DMB220 and enzymatic analyses showed that this hyper-susceptibility
may arise from the decreased binding/incorporation efficiency of the natural NTP
substrate without significantly impacting on inhibitor binding. Thus, metal ion chelation
at the active site of DENV RdRp represents a viable anti-DENV strategy and DMB220
is a first of class DENV inhibitor.
Keywords
Dengue virus, antiviral, RNA-dependent RNA polymerase, non-nucleoside inhibitor,
metal-chelating inhibitor

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INTRODUCTION
Dengue virus (DENV) belongs to the family Flaviviridae, a group of enveloped positive-
sense single-stranded RNA viruses that includes the genera Hepacivirus (prototype,
58 hepatitis C virus), Flavivirus (prototype, yellow fever virus), and Pestivirus (prototype,
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bovine virus diarrhoea) (1). Distinct from the hepaciviruses and pestiviruses which are
not arthropod-borne, the flaviviruses are transmitted by mosquitos and ticks. Dengue, the
most prevalent arthropod-borne viral disease of humans, is caused by four serotypes
(DENV 1-4) and has had major impact on global public health (2-4).
Infection with any of the DENV serotypes may result in a wide spectrum of clinical
symptoms ranging from a mild flu-like syndrome (known as dengue fever [DF]) to the
most severe forms of the disease, which are characterized by coagulopathy, increased
vascular fragility, and permeability (dengue hemorrhagic fever [DHF]). The latter may
progress to hypovolemic shock (dengue shock syndrome [DSS])(3, 5). Among the four
serotypes, DENV-2 is the most prevalent on a global scale, followed by DENV-3,
70 DENV-1 and DENV-4(6). Dengue is endemic in over 100 tropical and sub-tropical
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countries and the global incidence of dengue has grown dramatically in recent
decades(7).
Half of the world's population is now at risk of dengue infection and ~400 million people
experience DENV infections annually (8), with 500,000 cases of DHF/DSS and 22,000
deaths (http://www.who.int/csr/disease/dengue/impact/en/, accessed July 6, 2015).There
is currently no specific antiviral treatment or preventive vaccine for dengue. Despite

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decades of efforts, developing a preventive dengue vaccine remains challenging because
a vaccine must provide long-lasting protection against all four DENV serotypes
80 (tetravalent dengue vaccine) and not be prone to potential side effects of non-
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neutralizing, serotype-cross-reactive immune responses (9-11).
Secondary heterotypic DENV infection is associated with an increased risk of severe
disease as a result of an immune-pathological component in dengue pathogenesis, which
is referred to as antibody-dependent enhancement. Although progress in vaccine
development has been made, there is an obvious need to also develop anti-DENV
antivirals (for reviews see (12-21)).
The DENV NS5 protein is an important target of DENV drug discovery efforts (15).NS5
is about 900 amino acids long and comprises a methyl transferase domain at its N
terminus and an RNA-dependent RNA polymerase (RdRp) domain at the C terminus.
NS5 is the most conserved of DENV viral proteins and the crystal structures of NS5 and
its polymerase domain have been solved (22-24). The DENV RdRp possesses a half-
closed right hand architecture, which is conserved among different classes of DNA and
95 RNA polymerases (24). Within the subdomains termed finger, palm and thumb, 6
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conserved motifs, termed A-F, play key roles in RNA, NTP, and metal-ion binding and
catalysis (24). The amino acids in the catalytic site of DENV RdRp are located within
motifs A (aspartate at position 533, D533) and a catalytic triad GDD at position 662–664
in motif C. These aspartate residues are involved in the coordination of two divalent
Mg++ cofactors that are essential to the catalytic process, i.e. the “two-metal-ion
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mechanism” (25).The DENV RdRp, like those of other members of the Flaviviridae
family, carries out viral RNA synthesis through a de novo initiation mechanism(26).
Moreover, RdRp activity is essential for viral replication. Since there is no mammalian
host cellular enzyme equivalent, no issues of toxicity should be anticipated if specific
inhibitors of DENV RdRp activity can be developed (15, 19).
Investigational DENV RdRp inhibitors can be grouped into two classes, nucleoside
analogue inhibitors (NIs) and non-nucleoside analogue inhibitors (NNIs), (for reviews
see (15, 16, 19, 21)). NIs require intracellular phosphorylation to a 5’-triphosphate form
by cellular kinases; they then bind at the enzyme active site and compete with natural
substrates for incorporation and this is followed by chain termination (27, 28). NNIs do
not require intracellular activation and provide an alternative mechanism by binding to
allosteric sites remote from the active site of the RdRp and interfere with the chemical
step of RNA synthesis (15, 16, 19). No NIs or NNIs have been approved for clinical
treatment of DENV infections.
Metal chelating agents represent an important class of viral enzyme inhibitors (29-33).
Chelation of the divalent Mg++ cofactors of HIV integrase has proven to be a successful
strategy in the design of HIV integrase inhibitors and resulted in approval of the chelating
inhibitors raltegravir (RAL), elvitegravir (EVG) and dolutagravir (DTG) (for review, see
(31)). Metal chelation was also effective at inhibiting HCV polymerase (34, 35) and the
influenza PA endonuclease activity (33). A similar concept for the development of

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compounds bearing chelating motifs that are able to bind the bivalent metal ions in the
catalytic site of DENV RdRp has not previously been tested.
125 In this study, we report on
a
novel inhibitor of DENV RdRp, i.e. 5-
benzenesulfonylmethyl-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylic acid
hydroxyamide (termed DMB220). The pyridoxine-derived small-molecule inhibitor
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128 DMB220 was identified through the screening of a small library of small molecule
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compounds, constructed through mechanism-based drug design aimed at chelating
divalent metal ions at the catalytic sites of such viral enzymes as HIV integrase and viral
RNA-dependent RNA polymerase (36). DMB220 has a broad spectrum of inhibitory
activity against the RdRp enzymes all of four DENV serotypes. The inhibitory activity of
DMB220 can be reversed by enhancing concentrations of the natural ribonucleotide
substrate. Furthermore, we show that the DENV RdRp variant S600T, which was shown
to be resistant to nucleoside analogue inhibitors (27), is hyper-susceptible to DMB220.
Thus, metal ion chelation is a potential approach in anti-DENV antiviral development
137 and DMB220 may be a first of class potent pan-serotypic RdRp inhibitor for DENV
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replication.
Materials and Methods
Reagents and nucleic acids
The (-)3’UTR RNA, which contains the last 380 nucleotides (nt) of the negative strand
of DENV2 genome plus 3 extra guanines upstream of its 5’ terminus, was synthesized
using an Ambion T7-MEGAscript kit (Invitrogen, Burlington, ON, Canada) as described

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146 (27). T7 transcription was performed on PCR product amplified from plasmid pBAC-
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DENV-FL DNA which contains the full length cDNA of the DENV NGC strain (37)
(kindly provided by Drs. Jose A. Usme-Ciro and Juan C. Gallego-Gomez, Universidad de
149 Antioquia, Colombia). RNA was purified by 8% polyacrylamide-urea gel electrophoresis.
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3’dGTP was obtained from TriLink BioTechnologies (San Diego, CA). Mycophenolic
acid (MPA) was obtained from Sigma-Aldrich (Markham, ON, Canada). The plasmid
152 pET21a[His-Strep-DENV2_NS5 DNA] (38) was kindly provided by Dr. Matthias Götte,
153 University of Alberta, Canada.
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Preparation of DMB-220
DMB220 was identified through screening a small library of small molecule compounds
157 (36) constructed through mechanism-based drug design aimed at chelating the divalent
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metal ions within the catalytic site of viral enzymes such as HIV integrase and viral
RNA-dependent RNA polymerase. For chemical preparation of DMB220 (Fig. 1.), 550
mg isopropylidene pyridoxine dissolved in 30 mL DCM was reacted with 2.5 equivalents
of methane sulfonyl chloride in the presence of 5 equivalents triethyl amine. Extraction
with 5% citric acid, drying over Na2SO4, and evaporation yielded 500mg of desired
mesylate. This was immediately reacted with 400 mg of benzene sulfinic acid in 2 mL
164 DMF. The product was isolated by precipitation in water and filtration to give a 85%
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yield. The crude was dissolved in CHCl330mL and 400 mg mCPBA was added. After 1
h stirring, the reaction was extracted using K2CO3 and the organic phase dried over
CaCO3 and the evaporated. The residue was dissolved in 3 mL DCM and 3mL
168 trifluoroactetic anhydride (TFAA) was added. Stirring at reflux 45oC for 20 h affording

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the rearranged product, isolated through quantitative evaporation of solvent. The residue
was then added to a solution of MnO2 2g in CHCl3 (30 ml) and stirred at reflux 1h.
Filtration and evaporation yielded the aldehyde (250 mg). This was placed in 10 mL
MeOH with 1.2 eq I2 and 3 eq KOTMS. Stirring at room temperature for 1h yielded the
173 ester via quantitative conversion. The product was purified on silica gel. Then 100 mg of
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the ester was reacted with excess (hydroxylamine 50% aq) in pyridine to give the
hydroxamate. Dilution in EtOAc and extraction via 5% citric acid gave the desired
intermediate. The final product was obtained by adding 50 mg of the above acetonide to
neat 70% formic acid. After 15 min the reaction was complete, the formic acid was
evaporated off and the residue triturated with water to yield DMB220as a white powder
179 (41% yield). Stock solution at 10 mM was prepared by dissolving DMB220 in 100%
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DMSO. Storage was in small aliquots at -80oC.
Purification of His6-tagged recombinant DENVNS5 polymerase domains
183 The expression vectors used in this study are all T7 promoter-driven. Plasmid
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pET21a[His-Strep-DENV2_NS5 DNA] containing the NS5 polymerase domain of
DENV2 NGC strain (38) was transformed into Novagen Rosetta 2 (DE3) competent cells
(EMD Millipore, Etobicoke, ON, Canada)and used for purification of the NS5 RNA-
dependent RNA polymerase (RdRp) domain with a N-terminal His6 tag of the DENV2
NGC strain, based on previously described protocols with minor modifications (38, 39).
For construction of an S600T mutant DENV2 NS5 polymerase, we performed site-
directed mutagenesis using the Stratagene Quick Change II XL site-directed mutagenesis

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kit on pET21a[His-Strep-DENV2_NS5 DNA]. DNA sequencing of the complete RdRp
domain was performed to verify the absence of spurious substitutions .
For purification of the RdRp domains of the other three serotypes DENV1
(US/Hawaii/1944), 3 (MY00-22366) and 4 (MY01-22713), cDNAs encoding the RdRp
domains of DENV1, 3 and 4 (23) were cloned into a pNIC-Bsa4 vector bearing a TEV
197 cleavage site downstream of the N-terminal His6 tag (kindly provided by Dr. Julien
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Lescar, Nanyang Technological University, Singapore); the plasmid DNAs pNIC-
Bsa4[DENV1_NS5], pNIC-Bsa4[DENV3_NS5] and pNIC-Bsa4[DENV4_NS5] were
transformed into Rosetta 2 (DE3) competent cells (EMD Millipore-Novagen, Etobicoke,
ON, Canada).The bacterial cells were cultured in LB medium supplemented with
carbenicillin (100 µg/ml) for the pET21a construct or kanamycin (50 µg/ml)for the
203 pNIC-Bsa4 construct at 37oC until the optical density at 600 nM reached 0.6-0.8. After
204 induction of expression with 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) in
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the presence of 50 µM MgCL2 and 50 µM ZnCL2, the cells were grown for 18 hours at
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15 C with a shaking speed of 250 rpm. Cells were harvested by centrifugation, re-
suspended in lysis buffer containing 20 mM Tris-HCL pH7.5, 5% glycerol, 10 mM
imidazole, 300 mM NaCL, 0.1% Igepal-CA630, 10 U/ml Benzonase, and 1 x complete
EDTA-free protease inhibitors cocktail (Roche, Mississauga, ON, Canada). Cells were
lysed by sonication, and the extract was cleared by centrifugation at 18,500 x g for 30
min at 4 oC. The clarified supernatant was loaded onto a Ni-nitrilotriacetic acid (Ni-NTA)
column (Novagen) equilibrated with lysis buffer. The Ni-NTA column was washed and
eluted with lysis buffer containing a gradient of imidazole from 20 mM to 500 mM.

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Fractions containing polymerase were combined and dialyzed overnight against dialysis
215 buffer (10 mM Tris-HCL, pH 7.5, 300 mM NaCL, 20% glycerol, 5 mM MgCL2). After
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dialysis, proteins were diluted with an equal volume of dialysis buffer without NaCl and
bound to Heparin Sepharose 6 Fast Flow (GE Healthcare). The bound proteins were then
eluted with a gradient of NaCl (0–0.6M) in dialysis buffer. Protein concentrations were
measured by a Bradford protein assay kit (Bio-Rad Laboratories, Saint-Laurent, QC,
Canada). After final dialysis, the purified DENV NS5 polymerase domains were stored
at −80°C .
In vitro filter-binding DENV RdRp assay
The filter-binding DENV RdRp assay was adapted from a published protocol(27) with
modifications. Briefly, the reaction samples consisted of 40 nM enzyme, 10 nM (-)
3’UTR RNA, mixed together in RdRp assay buffer containing 40 mM Tris-HCL, pH 7.0,
10 mM NaCL, 2 mM MgCl2, 0.001% Triton X-100, and 10 µM cysteine. Reactions were
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228 initiated at 30oC by adding 200µM each of ATP, GTP, UTP and 0.9 µM H-CTP in a
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final volume of 20 µL. The reactions were allowed to proceed up to 180 min and were
230 terminated by adding 0.2 mL of 10% cold trichloracetic acid (TCA) and 20 mM sodium
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pyrophosphate containing 5µg herring sperm DNA carrier and incubated for at least 30
min on ice. The precipitated products were filtered onto a 96-well MutiScreen HTS FC
filter plate (Millipore, Etobicoke, ON). The filter plate was pre-wet with 150 µL assay
buffer prior to use and sequentially washed with 200 µL of 10% TCA and 150 µL of 95%
ethanol. The radioactivity of incorporated products was analyzed by a 1450 MicroBeta

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TriLux Microplate Scintillation and Luminescence Counter (Perkin Elmer, Waltham,
MA).
239 Steady-state kinetic analysis
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Measurements of steady-state kinetic parameters were performed as described (27). To
determine the Michaelis-Menten constants (Km) and Vmax for the NTP substrate in the
filter-binding DENV RdRp assay, NTPs were serially diluted to provide a range of
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243 substrate concentrations. The NTPs (containing H -CTP as tracer) concentrations tested
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ranged from 0.02 µM to 50 µM at 3-fold serial dilutions, and the RNA concentration was
245 fixed at 200 nM. All data were analyzed using GraphPad Prism5.0 software (GraphPad
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Software, San Diego, CA) according to the instructions of the software provider.
Inhibitory effects of DMB220 on DENV RdRp activity
Measurement of the inhibitory effect of compounds on DENV NS5RdRp activity was
accomplished by evaluating the amount of radiolabeled CTP incorporated by the enzyme
into newly synthesized RNA using the filter-binding RdRp assay and a DENV
(-)3’UTR RNA template essentially as described (27). The final assay reaction mixtures
contained multiple concentrations of serially diluted test compounds,40 nM enzyme, and
254 10 nM (-) 3’UTR RNA in assay buffer containing 40 mM Tris-HCL, pH 7.0, 10 mM
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NaCL, 1 mM MgCl2 and 0.5 mM MnCL2, 0.001% Triton X-100, and 10 µM cysteine.
Reactions were started at 30oC by adding NTP substrate containing 3H-CTP as tracer in a
final volume of 20 µL. The reactions were terminated and processed as described above
for the RdRp assay. The IC50 values were determined by nonlinear regression using

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GraphPad Prism5.0 software (GraphPad Software, San Diego, CA) according to
instructions of the software provider.
Evaluation of inhibitory efficiencies of compounds against dengue virus replication
in cell culture
The viruses DENV1 (Hawaii, TVP 17788), DENV2 NGC strain, DENV3 (H87, TVP
15322), and DENV4 (TVP 13886) (kindly provided by Dr. Robert B. Tesh, University of
Texas Medical Branch) and BHK-21 cells (Baby hamster kidney cells; ATCC CCL10)
were used in the cell-based antiviral assays. The viral stock was prepared by inoculation
268 of C6/36 cells (Aedes albopictus clone C6/36cells; ATCC CRL166) as described (40).
269 Infected cells were maintained in RPMI-1640 (Invitrogen, Burlington, ON, Canada)
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medium containing 2% fetal bovine serum (FBS) and 0.5% penicillin/streptomycin stock
at 28oC in a 5% CO2 incubator for 7 days until the cells showed cytopathic effects (CPE).
The supernatant was cleared by centrifugation at 2,000 x g for 5 min to remove cell
debris and adjusted to 20 % FBS. Aliquots of virus were stored at -80oC. The 50% tissue
culture infective dose (TCID50) was determined by the Promega Viral ToxGlo Assay
(Fisher Scientific, Ottawa, ON, Canada) using BHK-21 cells as instructed by the
manufacturer. Cell-based assays for evaluation of antiviral activity against DENV were
essentially performed as described (41, 42) with modifications using the Viral ToxGlo
Assay (Fisher Scientific, Ottawa, ON) as instructed by the manufacturer. Briefly, BHK-
21 cells were plated into 96 well plates at 2,500 cells/well in DMEM containing 10%
FBS and 1% penicillin/streptomycin. Cells were incubated at 37°C overnight and
infected with 100 TCID50 of DENV for 90 min. The media were then replaced by DMEM

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containing 2.5 % FBS with variable concentrations of test compounds. Cell cultures were
maintained at 37°C in a 5% CO2 incubator for 3 days. The Viral Tox reagent was
prepared fresh and 100µl reagent were added to each well. Plates were incubated for at
least 10 minutes and then quantitated by a 1450 MicroBeta TriLux Microplate
Scintillation and Luminescence Counter (Perkin Elmer, Waltham, MA). For an off-target
cytotoxicity assay, test compounds were processed in the same way as described above
except without DENV infection of the cells. The luminescence readings were plotted
289 against the log transformation of the concentration of the compound. Data were analyzed
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by nonlinear regression using GraphPad Prism5.0software (GraphPad Software, San
Diego, CA).
DMB-220 inhibition of HIV-1 RT activity
294 Subtype B HIV-1 wild-type RT and that containing the M184V substitution were
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generated as described previously(43, 44).A test of the inhibitory effect of DMB-220 on
HIV-1 RT activity was performed as described (45).The reaction mixture (50 µl)
contained 50 mM Tris-HCL (pH 7.8), 5 mM MgC12, 2 mM ATP, 60 mM KCL, 5 mM
DTT, 1 µM [3H]-dTTP, 5 µg/ml of template/primer poly (rA)/(oligo(dT)12-18(Midland
Certified Reagent Company, Midland, TX), recombinant RTs of the same activity, and
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variable amounts of DMB-220. After incubation for30 min at 37 C. The reactions were
terminated and processed as described above for the RdRp assay.
DMB-220 inhibition of HIV-1 integrase strand transfer activity

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304 Recombinant subtype B HIV-1 integrase was expressed in the BL21 (DE3) bacterial
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strain and purified as published (46, 47). The susceptibility of HIV recombinant integrase
to DMB220 and integrase strand transfer inhibitors (INSTIs) was assessed by strand
transfer assay (46, 47) in the presence of DMB220 or INSTIs including DTG, EVG and
RAL. Compounds were diluted in compound assay buffer (50 mM MOPS, pH 6.8, 50
μg/mL BSA, 0.15% CHAPS, 50 mM NaCl, and 30 mM MnCl2) plus 10% DMSO to
concentrations between 0-1000 nM for INSTIs and 0-100 μM for DMB220. DNA-BIND
96-well plates (Corning) were coated with pre-processed LTR DNA blocked and washed
before the addition of purified recombinant proteins. Purified HIV integrase protein in
assay buffer supplemented with 5 mM DTT was added to each well and incubated for 30
min at room temperature. 25 μL of diluted INSTIs or DMB220 were added to each well
315 followed by 25 μL of appropriately diluted biotinylated target DNA duplex. This was
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followed by a 1 h incubation at 37°C. Integrated target DNA was detected after washes
through the use of europium-labeled streptavidin molecules as described (46, 47). The
50% inhibitory concentrations (IC50s) of test compounds were determined by nonlinear
regression analysis using GraphPad Prism5.0 software (GraphPad Software, San Diego,
CA).
Molecular modeling
The Dengue polymerase structure PDB ID:2J7U was prepared as an Autodock receptor
using autodock tools. Docking of the compound was performed using methods similar to
those of previous publications (27), (48). The possible location of a putative second
active site Mg++ ion was superimposed into the 2J7U structure of the HIV RT structure

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1RTD by partial active site overlays. The minimal region for active site overlays was
obtained by multiple partial sequence alignments between the dengue polymerase and
329 HIV RT polymerase active sites using T-COFFEE (49). Prior to compound docking, a
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short single RNA strand (5'UUUAGUU) was retrieved from the PDB structure 1WMQ
(50) and docked into the prepared 2J7U structure. All twenty docked poses showed that
the RNA inserted into the RNA binding groove.
Subsequent compound docking was performed on the top two conformations. The 3-
dimensional structure of the inhibitory compound (5-Benzenesulfonylmethyl-3-hydroxy-
4-hydroxymethyl-pyridine-2-carboxylic acid hydroxyamide) was modeled and optimized
for pH7.0 using the program ChemBioDrawUltra 12.0 (www.cambridgesoft.com). The
compound was then docked into the active site residue of dengue polymerase, into a grid
box centered on the D533 and D663 residues, with grid box dimensions (x,y,z) of 30Å,
30Å, 30Å. All ligands and receptors were prepared using Autodock Tools V1.5.6 and
docked using Autodock Vina. Image processing was performed using PyMol.
Results
Purification and characterization of recombinant NS5 polymerase from DENV1, 2,
344 3, and 4
345 Recombinant NS5 polymerase domains from DENV1, 2, 3 and 4 were purified to >95%
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homogeneity as demonstrated by SDS-PAGE (Fig.2A). For characterization of enzyme
activity, the rate of RNA synthesis was analyzed by following the incorporation of
348 radiolabelled nucleotide into the newly synthesized RNA, which was separated from free
349 radiolabeled nucleotide by TCA precipitation and filtration. Time course experiments

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were initiated by mixing together 40 nM purified NS5 polymerase from each of the four
DENV serotypes with 10 nM RNA in RdRp assay buffer; this permitted a comparison of
352 the rates of nucleotide polymerization by NS5 polymerase for all four serotypes(Fig. 2B).
353 We observed that the NS5 polymerase of DENV4 was the most active among the four
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enzymes with polymerization rates following the order:
DENV4>DENV2>DENV3>DENV1(Fig. 2B). This result is somewhat different from
those of previous reports (27), which showed that the ranking order was
DENV2>DENV3>DENV4>DENV1, a discrepancy that may arise from differences in
the NS5 polymerase sequences used in the two studies. Steady-state kinetic analyses
showed that the Km values for NTPs of NS5 polymerase of all four serotypes ranged
from 3.3 - 4.0 µM (Table 1). The catalytic efficiency (Kcat/Km) of the polymerase of the
four serotypes followed the same ranking order as observed in the time course RdRp
assay (Fig. 2B).
Inhibitory effects of DMB220 on DENV RdRp activity.
We used an in vitro filter-binding DENV RdRp assay to determine the inhibitory effects
of DMB220 on DENV NS5 polymerase activity. We showed that DMB220 was potent
against NS5 polymerase activity of all four serotypes (Fig. 3.) with IC50s ranging
between 5.0-6.7 µM (Table 2).We also observed a 3-fold decrease in IC50 for the S600T
mutated RdRp, suggesting that this variant was hyper-susceptible to DMB220. The
S600T mutant was chosen for this study based on the fact that this residue is highly
conserved in the RdRp B motif and was previously shown to be resistant to nucleoside
analogue inhibitors(27). As an internal control for the filter-binding RdRp assay, we

18
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375
376
measured the inhibitory effect of a nucleoside chain terminator 3’dGTP on DENV1-4
NS5 polymerase. We showed that 3’dGTP inhibited RdRp activity with similar potency
with IC50s obtained ranging between 0.4-0.6 µM, in agreement with previous reports on
the inhibitory potency of 3’dGTP on DENV NS5 polymerase activity (51). The S600T
377 mutant enzyme did not show resistance to 3’dGTP (Table 2).
378
379 Antiviral activity of DMB220 against DENV in a cell-based assay
380
381
382
383
To validate the anti-DENV activity of DMB220 in cell culture, we used the Promega
Viral ToxGlo Assay and BHK-21 cells. DENV-infected cells were treated with
increasing concentrations of DMB220 and protection from cytopathic effect was
measured. We used mycophenolic acid (MPA) as a positive control of inhibition of viral
384 replication. The data show that DMB220 was able to inhibit all four serotypes of DENV
385
386
387
with EC50s ranging between 2.2-2.8 µM (Table 3),whereas the anti-DENV potency of
MPA ranged between 0.4 to 0.6 µM across the four serotypes, in agreement with
previous reports (52, 53).The CC50 of DMB220 was 18 µM as assayed in BHK-21 cells
388 using the Viral ToxGlo Assay (Table 3). Thus, the selectivity index (SI) values of
389
390
391
392
393
394
DMB220 were 6.4-8.2 as determined by the ratio of CC50 to EC50, which were lower
than those of the reference compound MPA (SI: 83-125). Accordingly, DMB220 is
potentially more toxic than MPA.
Effect of NTP concentration on inhibitory potency of DMB220 against DENV
polymerase activity

19
395
396
397
398
DMB220 was designed to be able to chelate divalent metal ions at the active site of the
DENV RdRp, therefore, its binding to the RdRp active site might be competitive with
that of natural NTP substrates. To assess this in reactions that were initiated by adding
o
3’(-)UTP RNA template at 30 C over 120 min, we varied NTP concentrations. We
399 found that the IC50 of DMP220 increased as higher concentrations of the nucleotide
400
401
402
403
404
405
406
substrate were used, which suggests a competitive mechanism of action for DMB220
(Table 4). We also tested the inhibitory potency of DMB220 at various concentrations of
the RNA substrate. In this circumstance, we did not observe variations in IC50s (data
not shown).
Mechanism of mutated S600T RdRp hyper-susceptibility to DMB220
The S600T substitution resulted in 3-fold hyper-susceptibility to DMB220. This hyper-
407 susceptibility may arise from facilitation of inhibitor binding and/or from diminished
408 binding/incorporation of the natural NTP substrate. To determine the underlining
409 mechanism of DMB220 hyper-susceptibility associated with S600T substitution, we
410 compared the enzyme kinetic parameters of the DENV2 NS5 WT and S600T enzymes
411
412
413
414
415
416
417
(Table 5). We measured the steady-state kinetic constant Km for the natural nucleoside
substrate NTP and the inhibition constant Ki for DMB220 using (-)3’UTR RNA as
template. Determinations of Ki were performed by the Dixon Plot method (Fig. 4.).
Ratios of Ki/Km were calculated to determine the ability of each enzyme to selectively
bind DMB220 relative to the natural nucleotide substrate. The steady state Km value of
the S600T enzyme was shown to be 3-fold that of the WT, suggesting that the S600T
mutant enzyme binds/incorporates the natural nucleotide substrate with decreased

20
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425
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427
428
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430
431
432
433
434
435
436
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440
efficiency compared to that of WT RT. However, the Ki value of the S600T enzyme for
DMB220 was similar to that of the WT counterpart. The S600T enzyme showed
decreases in Ki/Km of 0.3-fold compared with WT enzyme for DMB220, suggesting
increased binding of this inhibitor. These results further support the notion that DMB220
competes with NTP at the active site of the RdRp. Our work also showed that the S600T
mutant enzyme shows a 7-1-fold drop in catalytic efficiency (Kcat/Km) compared to WT,
in agreement with previous studies (27). These findings indicate that the S600T
substitution in DENV RdRp enhances susceptibility to DMB220 not through increased
binding but rather by decreasing binding and/or incorporation of the natural nucleotide
substrate.
Molecular docking
The compound docked into the modified active site of the dengue polymerase structure
2J7U with a relatively high score and in interactions with the active site residues D533,
D663 and D664 (Fig.5.). The compound is coordinated to both magnesium ions though
interactions with its sulfoxide oxygens as well as through hydroxyl groups on the
pyrimidine ring. The sulfoxide oxygens potentially have electrostatic interactions with
the RNA template but minimal hydrophobic contacts. There are several other interactions
with active site residues such as the main chain hydroxyl of A531 and the side chain of
S710, with a possible induced dipole interaction with E733 on the phenyl portion. The
docking thus supports the mechanism of action of this compound as a competitive
inhibitor of Dengue NS5b polymerase. While the active site of Dengue polymerase is
very much like that of HCV polymerase, the alignment of the active site of the recent

21
441
442
443
444
445
446
447
448
449
450
451
452
453
454
HCV ternary complex (54) with dengue structures 2J7U and 4V0R (59) reveals a
different Mg coordination orientation from dengue; thus, this compound may have weak
activity against HCV polymerase with altered interactions. Like integrase strand transfer
inhibitors, this compound coordinates towards both Mg++ ions. Unlike integrase
inhibitors with minimal hydrophobic contacts as observed in our simulations, this
compound may not have broad class specificity or may bind to other polymerases
differently. The lack of significant hydrophobic contacts may thus affect its potency. The
addition of halogen groups on the phenyl ring may conceivably augment potency.
Inhibitory effects of DMB220 on HIV-1 integrase and reverse transcriptase
We also determined whether DMB220 can inhibit HIV-1integrase and reverse
transcriptase, which also require metal ion cofactors at the active sites for catalysis. We
observed that DMB 220 did not inhibit HIV-1 RT activity at concentrations as high as
100 µM. In the integrase strand transfer assay, DMB220 showed a weak inhibitory effect
455 with an IC50 of 14 µM, in comparison with IC50s of the approved HIV integrase
456
457
458
459
460
461
inhibitors raltegravir, elvitegravir and dolutagravir, which ranged between 2.8 nM to 8.8
nM (Table 6). Thus, DMB220 possesses apparent specificity for the DENV NS5
polymerase.
Discussion
462 We have shown that a novel compound, DMB220, effectively inhibited the RdRp activity
463 of NS5 polymerase of all four serotypes of DENV. Use of a cell-based assay has

22
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465
466
467
468
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470
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473
474
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477
478
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481
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486
validated this result, showing that DMB 220 possesses activity with EC50s in the low
micomolar range. DMB220 did not inhibit the reverse transcriptase activity of HIV-1 and
exhibited only minimal inhibitory activity against HIV-1 integrase. These findings
suggest that DMB220 is a selective anti DENV pan-serotype RdRp inhibitor.
DMB 220 is a pyridoxine-derived small-molecule inhibitorand represents a novel class of
anti-RdRp agent. DMB220 was designed to chelate metal ion cofactors at the active site
of RdRp. Our enzymatic and molecular modeling data prove that DMB220 competes
with the natural NTP substrate for binding to the RdRp active site. Thus, this active-site
non-nucleoside analogue inhibitor is different from classic NNIs that do not bind at the
active site of the RdRp. Metal chelating inhibitors that target the active site of viral RdRp
have been reported for hepatitis C virus. Derivatives of 4,5-dihydroxypyrimidine
carboxylic acid and α,γ-diketo acid (DKA) (34, 35) have been developed to target HCV
RdRp by chelating divalent metal ions at the active site of the polymerase. These anti-
HCV inhibitors are competitive with nucleotides (55-57). Future work will assess
whether DMB 220 possesses inhibitory effects against other polymerases from other
viruses including that of HCV.
We have also demonstrated that the S600T substitution in RdRp can result in is hyper
susceptibility to DMB220 by decreasing binding and/or incorporation of the natural
nucleotide substrate rather than through increased binding of the inhibitor. This further
supports the notion that DMB220 competes with natural nucleotide substrates for active-
site binding. The serine residue at position 600 of DENV RdRp is located in the B motif

23
487
488
489
490
491
492
493
494
495
496
497
of the palm subdomain and is highly conserved among all viral RdRp enzymes. S600T
was previously shown to be able to confer resistance to a nucleoside inhibitor of DENV
RdRp, termed beta-d-2'-ethynyl-7-deaza-adenosine triphosphate (2'E-7D-ATP)(27). The
counterpart S282T substitution in HCV RdRp NS5B was also shown to confer resistance
to nucleoside analogue inhibitors (58-60). Our findings strongly suggest that DMB220 or
its derivatives may also have the potential to mechanistically complement nucleoside
analogue inhibitors in regard to inhibition of DENV replication. Hyper-susceptibility is
defined as an increase in susceptibility to a particular drug, i.e., lower concentrations of
drug are needed to inhibit the replication of a mutated virus compared with wild-type.
Currently, HIV represents the most extensively investigated agent in regard to antiviral
drug-resistance and hyper-susceptibility and it is generally accepted that hyper-
498 susceptibility can occur when the fold-change (FC) of EC50/IC50 is <0.4. In the case of
499
500
501
502
503
504
505
506
507
508
509
DENV antiviral research, no FDA-approved drug is yet available. Thus, it seems
reasonable to state that the 3-fold change (FC=0.33) represents hyper-susceptibility.
The prototype molecule DMB-220 exhibits cytotoxicity in the low micro molar range
which is not unusual for antiviral compounds. However its low selectivity index, and the
ratio of cytotoxicity to antiviral activity suggests that further development of this class of
molecules is warranted. A more in depth study on the structure-activity relationship is
being pursued and should result in better identification of the crucial chemical moieties.
Our aim is to improve the SI to above 1000 before undertaking preclinical studies.

24
510
511
512
513
514
515
516
517
518
519
In summary, we have shown that the pyridoxine-derived active site inhibitor DMB-220
can inhibit DENV RdRp activity and viral replication in tissue culture. The compound
apparently acts as a competitive inhibitor of natural nucleotide substrates for binding to
the active site of the enzyme. Our study demonstrates that chelation of active site metals
within the viral polymerase may be a valid strategy for the development of novel and
efficacious anti-DENV compounds.This is also the first report of an active-site non-
nucleoside inhibitor of DENV RdRp against which an NI-resistant substitution can confer
hyper-susceptibility.
520
FIGURE LEGENDS
521
522
523
Fig.1. Structure and reaction scheme of the chemical synthesis of compound DMB220.
Reagents and conditions: a) MS-Cl, DCM TEA R.T; b) ArSO2-Na, DMF; c) MCPBA
70%, DCM; d) TFAA CHCl3; e) MnO2 CHCl3; f) TMSOK, I2, MeOH; g) 70% HCOOH
524 aq; h) H2NOH aq, Py.
525 Fig.2. Purification and activity of recombinant NS5 polymerase domains of the four
526 serotypes of DENV. (A) Coomassie brilliant blue staining of purified NS5 polymerase
527
528
529
530
domains after 10% SDS-PAGE. MW, molecular size standards, in kilo daltons. The
positions of purified recombinant NS5 polymerase domains are indicated on the right. (B)
Activity of recombinant DENV NS5 polymerase domains as assessed using a(-) 3’UTR
RNA template by filter-binding RdRp assay as described in Materials and Methods.

25
531 Values are means of two independent experiments. Error bars represent standard
532
deviations.
533
534
535
536
Fig. 3. Dose-response curves showing inhibition by DMB220 of the activity of DENV1-4
polymerase and the S600T mutant. Inhibitory effect was determined by in vitro filter
binding RdRp assay using purified enzymes and DENV (-) 3’UTR RNA template in
assay buffer containing 40 mM Tris-HCL, pH 7.0, 10 mM NaCL, 1 mM MgCL2, 0.5
3
537 mM MnCL2, 0.001% Triton X-100, 10 µM cysteine and 4 µM NTPs including H -CTP
538 as tracer. The data shown are from one representative experiment. The IC50 values were
539
540
determined from the results of three independent experiments and are summarized in
Table 2.
541
542
543
544
545
546
547
548
549
Fig. 4. The inhibition constants (Ki) of DMB220 for WT (A) and S600T (B) enzymes
were determined by graphical methods and use of a Dixon plot. The plots demonstrate
the inhibitory effect of DMB220 in the presence of varying NTPs added at concentrations
of 1.3 µM, 4 µM and 8 µM, respectively. The Ki values of DMB220 for the WT and
S600T enzymes are 3.2± 0.7 µM and 2.9 ± 0.6 µM respectively. The graphical
representations resulted from one representative experiment performed in duplicate and
data are presented as means ± SD. The reported Ki was calculated from the results of
three independent experiments and is expressed as mean ± SD in Table 5.
550
551
Fig. 5. Docking of the compound into the active site of the Dengue polymerase structure
(PDB ID:2J7U) (24) (Blue cartoon structure). The compound docks in close proximity to

26
552
553
554
and interacting with active site residues D663 and S661. The active site is shown as
surface coloured by standard CPK coloration. Blue represents surface-exposed nitrogen
groups and red represents surface-exposed oxygen groups. The possible location of a
555 putative second active site Mg++ ion was superimposed into the 2J7U structure of the
556
557
558
559
560
561
HIV RT structure 1RTD by multiple sequential partial sequence and structure overlay.
Prior to compound docking, a short single RNA strand (5'UUUAGUU) was retrieved
from the PDB structure 1WMQ and docked into the prepared 2J7U structure using
Autodock Tools and Autodock Vina. Structure visualization was by PyMol.
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