Novel dengue virus NS2B/NS3 protease inhibitors

Article abstract of DOI:10.1128/AAC.03543-14

Dengue fever is a severe, widespread, and neglected disease with more than 2 million diagnosed infections per year. The dengue virus NS2B/NS3 protease (PR) represents a prime target for rational drug design. At the moment, there are no clinical PR inhibitors (PIs) available. We have identified diaryl (thio)ethers as candidates for a novel class of PIs. Here, we report the selective and noncompetitive inhibition of the serotype 2 and 3 dengue virus PR in vitro and in cells by benzothiazole derivatives exhibiting 50% inhibitory concentrations (IC₅₀s) in the low-micromolar range. Inhibition of replication of DENV serotypes 1 to 3 was specific, since all substances influenced neither hepatitis C virus (HCV) nor HIV-1 replication. Molecular docking suggests binding at a specific allosteric binding site. In addition to the in vitro assays, a cell-based PR assay was developed to test these substances in a replication-independent way. The new compounds inhibited the DENV PR with IC₅₀s in the low-micromolar or submicromolar range in cells. Furthermore, these novel PIs inhibit viral replication at submicromolar concentrations.

Full text of DOI:10.1128/AAC.03543-14

AAC Accepts, published online ahead of print on 8 December 2014
Antimicrob. Agents Chemother. doi:10.1128/AAC.03543-14
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Novel Dengue virus NS2B/NS3 protease inhibitors
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Hongmei Wu1#, Stefanie Bock2#, Mariya Snitko2, Thilo Berger1, Thomas Weidner1, Steven Holloway1,
Manuel Kanitz3, Wibke E. Diederich3, Holger Steuber4†, Christof Walter5, Daniela Hofmann2,
Benedikt Weißbrich2, Ralf Spannaus2, Eliana G. Acosta6, Ralf Bartenschlager6,7, Bernd Engels5, Tanja
Schirmeister1 and Jochen Bodem2*
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1Institut für Pharmazie und Biochemie, Johannes Gutenberg-Universität Mainz, Germany
2Institut für Virologie und Immunbiologie, Julius-Maximilians-Universität Würzburg, Germany
3Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Germany
4LOEWE-Zentrum für Synthetische Mikrobiologie, Philipps-Universität Marburg, Germany
5Institut für Physikalische und Theoretische Chemie, Julius-Maximilians-Universität Würzburg,
Germany
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6Department of Infectious Diseases, Molecular Virology, Heidelberg University
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7German Center for Infection Research, Heidelberg University
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†Present address: Bayer Pharma AG, Lead Discovery Berlin – Structural Biology, Germany
#H. W. and S. B. contributed equally to the paper
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*Corresponding author:
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Jochen Bodem
Mailing address: Universität Würzburg, Institut für Virologie und Immunbiologie,
Versbacher Str. 7,
97078 Würzburg, Germany
Tel: +49-931-201-49558
Fax: +49-931-201-49553
e-mail: Jochen.Bodem@vim.uni-wuerzburg.de
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ABSTRACT
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Dengue fever is a severe, widespread and neglected disease with more than 2 million diagnosed
infections per year. The Dengue virus NS2B/NS3 protease (PR) represents a prime target for rational
drug design. At the moment there are no clinical PR inhibitors (PIs) available. We have identified
diaryl (thio)ethers as candidates for a novel class of PIs. Here, we report the selective and non-
competitive inhibition of the serotypes 2 and 3 Dengue PR in vitro and in cells by benzothiazole
derivatives exhibiting IC50 values in the low micromolar range. Inhibition of DENV serotypes 1-3
replication was specific, since all substances neither influenced HCV, nor HIV-1 replication.
Molecular docking suggests binding at a specific allosteric binding site. In addition to the in vitro
assays a cell-based PR assay was developed to test these substances in a replication-independent way.
The new compounds inhibited the DENV PR with IC50 in the low micromolar or sub-micromolar
range in cells. Furthermore, these novel PIs inhibit viral replication at sub-micromolar concentrations.
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KEYWORDS. Dengue virus, protease, rational antiviral drug design, protease inhibitor, diaryl
(thio)ether
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INTRODUCTION
Dengue viruses (DENV) are enveloped positive strand RNA viruses and belong to the family
Flaviviridae. DENV is the most important arthropod-borne viral infection. Over one third of the world
population lives in DENV endemic areas and an estimated 390 million infections occur every year. In
addition, the number of countries having experienced DENV epidemics has risen from 9 in 1970 to
more than 100 nowadays (1, 2). Furthermore, diagnosed infections across America, South-east Asia
and Western Pacific nearly doubled from 1.2 million in 2008 to over 2.3 million in 2010 (2). Four
different DENV serotypes were identified so far. Recently, evidence for an additional subtype has
been presented (3). Serotypes 1-4 are now prevalent in Asia, Africa and America and the endemic
regions are still increasing (4-6) endangering even Europe and the USA due to vector spread. DENV
infections can be associated with Dengue fever, but up to 88% of the infections remain inapparent (7).
These non-persistent infected patients serve beside persistently infected mosquitoes as virus reservoir.
Severe DENV infections and especially reinfections may lead to the dengue hemorrhagic fever and the
dengue shock syndrome with lethality up to 5% (2, 8, 9). There is neither a vaccination nor a specific
treatment for DENV infections.
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The DENV genome contains a single open reading frame, which encodes the structural proteins
capsid, membrane precursor (prM), envelope and the non-structural (NS) proteins NS1, NS2, NS3,
NS4 and NS5 (10). Cellular proteases and the viral serine protease (PR) are responsible for cleaving
the viral precursor polyprotein into functional proteins. The DENV PR consists of the amino terminal
domain of the NS3 protein and requires NS2B, a 14kDa protein, as cofactor to form a stable complex.
This heterodimeric PR cleaves at the capsid-prM, NS2A/NS2B, NS2B/NS3, NS3/NS4A, NS4B/NS5,
internal NS2A, NS3 and NS4A cleavage sites (10, 11). PR inhibitors (PIs) were shown to be valuable
antiviral drugs especially in the treatment of HIV-1 and hepatitis C virus (HCV) (12-17). In case of
DENV, some already published substances showed inhibition of 50% in DENV replication in a mini
replicon system at submicromolar range (18) or inhibition to 10% of DENV replication with wild-type
virus at micromolar concentrations (19). Despite these promising developments, antiviral DENV
drugs are still not available.
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In this report, we describe the specific and selective inhibition of the DENV PR by diaryl
(thio)ethers. Evidence is provided for inhibition of the DENV-2 and -3 PR in vitro and for DENV-2
PR in cell culture. Furthermore, we show suppression of DENV-2 replication at submicromolar PI
concentrations.
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MATERIAL and METHODS
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DENV-2/DENV-3 PR expression and purification. Competent BL21Star E. coli (GE life
technologies) or BL21Gold(DE3) were transformed with either a DENV-2 PR encoding pET-28C (for
the DENV-2 enzyme used for fluorometric enzyme assays) (20), pET15b vector (for the DENV-2
enzyme used for microscale thermophoresis) (21) or a pGEX6P1 vector encoding the DENV-3 PR
(22). PR expression was induced by addition of 1 mM IPTG for 2 to 7 h at 37°C. Bacteria were lysed
by sonification in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0; 100 µM
PMSF and 3.08 µM aprotinin). The DENV-2 PR was purified either using Ni-NTA-beads (Qiagen)
and native wash buffer (lysis buffer with 20 mM imidazole); elution buffer (lysis buffer with 250 mM
imidazole) or loaded on HisTrap FF [DENV-2] or on GSTrap FF [DENV-3](GE Healthcare) columns
and eluted using the particular lysis buffers (Supplementary Material) containing either 300 mM
imidazole [DENV-2] or 10 mM reduced glutathione [DENV-3]. The PR was further purified by size
exclusion chromatography. Protein amounts were determined by Bradford assay and verified by
Coomassie stained PAGE. DENV PR purity was analyzed by silver stained PAGE.
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Chemistry. The synthesis of the compounds is described in the Supplementary information
(Supplementary Information and Supplementary Figures S1 and S2).
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Quantum mechanics computations. Single point calculations as well as full geometry
optimizations with several different structures and corresponding frequency calculations were
performed on the B3LYP-D3/cc-pVDZ (23-28) level of theory as implemented in Turbomole
(Turbomole GmbH, Germany). Additional constrained geometry optimizations of the docking
structures were performed with the B3LYP functional and the cc-pVDZ basis sets using the Gaussian
09 program package (Gaussian Inc, USA). To obtain the relative energies of all conformers we
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performed single point B3LYP-D3/cc-pVDZ calculation on the geometries obtained from the
constrained optimizations.
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Molecular Docking. For molecular docking of compounds 1–8, the recently solved crystal structure
of DENV-3 PR in complex with the aldehyde inhibitor Bz-nKKR-H (pdb accession code 3U1I) was
used (22). Possible docking modes between ligands and the PR were studied using the FlexX docking
approach of the LeadIT 2.2.6 suite (BioSolveIt, Germany). Energies of compound structures were
minimized using the MOE software (Molecular Operating Environment, 2012.10). All water and
ligand molecules were deleted from the PR structure. The binding site was defined on a proper protein
pocket, which was shown to be a specific allosteric binding site for other non-competitive inhibitors
near the catalytic site (29).
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Fluorometric DENV PR assays. For a preliminary screening of the substance library DENV-2 PR
assays using 100 µM of the fluorogenic substrate Boc-Gly-Arg-Arg-7-amino-4-methylcoumarin (20,
30) were performed. Assay conditions were taken from ref. (20). For the first screening approach
substrate hydrolysis was measured in absence (100 % enzyme activity) or presence of 50 µM
inhibitor. IC50-values for selected compounds were determined measuring the increase of fluorescence
at 10 different PI concentrations ranging from 0 - 500 µM, 0 - 100 µM or 0 - 10 µM depending on
their inhibitory effects. Fluorescence increase resulting from the product AMC by substrate hydrolysis
was measured for 10 min after starting the reaction using an Infinite 200 PRO (Tecan, Männedorf,
Switzerland) (DENV-2) or a Saphire² plate reader (Tecan) (DENV-3) at room temperature with 380
nm excitation and 460 nm emission wavelengths. For DENV-3 PR assays 50 mM Tris-HCl, 1 mM
Chaps at pH 9.0 and 40 µM substrate (PhAc-Lys-Arg-Arg-AMC) were used. The final assay volume
was 200 µl. The reactions were started by adding either DENV-2 or DENV-3 PR to a final
concentration of 50 nM. Each assay was performed as triplicates at room temperature and IC50-values
were calculated using GraFit (Erithacus Software Limited). IC50-values were obtained by fitting the
residual enzyme activities to the 4-parameter IC50 equation:
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(with y [dF/min] as the substrate hydrolysis rate, ymax as the maximum value of the dose-response
curve that is observed at very low inhibitor concentrations, ymin as the minimum value that is obtained
at high inhibitor concentrations, and s denotes the Hill coefficient (31)).
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HPLC-based enzyme assay. See Supplementary Material.
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Binding assay based on microscale thermophoresis. For compound 6 binding of the inhibitor to
the enzyme was quantified in a substrate-free assay using microscale thermophoresis (MST)
technology (32). For MST purified DENV-2/3 PR was labeled with the Monolith NTTM Protein
Labeling Kit Blue according to the supplied labeling protocol and 10 µl of a 95 nM stock solution of
DENV-2/3 PR were mixed with 10 µl of a serial dilution of compound 6 starting at 625 µM for
DENV-2 and 250 µM for DENV-3. Samples were diluted with buffers (for DENV-2 a 200 mM Tris-
HCl buffer pH 9.5 containing 30% glycerol; for DENV-3 a 50 mM Tris-HCl buffer pH 9.0 containing
1 mM CHAPS used) supplemented with DMSO at a final concentration of 5% (v/v) to ensure equal
DMSO concentrations. After incubation for 10 min samples were measured at an MST power of 40%
and a LED power of 20% with a laser-on time of 30 s and a laser-off time 5 s on a NanoTemper
Monolith NT.115 instrument using standard capillaries.
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DENV-2 replication, qRTPCR and cell culture based PR assays. The DENV was obtained from
J. Schneider-Schaulies (Virology, Würzburg). The PR encoding region was amplified and sequenced
in order to confirm the serotype. In order to obtain high titer DENV, the virus was amplified by co-
culture techniques as described before for HIV-1 (33). Vero cells were infected and incubated for 3
days. One fifth of the infected cells and one fifth of the cell culture supernatants were used to infect
Vero cell. This process was repeated for 10times and the resulting supernatant stored at -80 °C. To
analyze inhibition of viral replication, Vero cells were pre-incubated with the components at
decreasing concentrations below the cell toxicity. The cells were subsequently infected with DENV-2
(MOI 0.5). Cellular supernatants were collected after four days, centrifuged at 2000 rpm for 5 min to
remove detached cells and titrated on Vero cells. These cells were fixed with 4% paraformaldehyde
three days after infection. To visualize infected cells an immunofluorescence staining was performed
with monoclonal anti-DENV-2 E antibodies. Bound antibodies were visualized with a Cy3 conjugated
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donkey anti-mouse antibody (1:500) (Jackson ImmunoResearch, USA). Stained infected cells were
counted using a fluorescence microscope and viral titers were calculated. All experiments were
performed in three independent triplicates. The significances of differences in infectivity compared to
wild-type maturation efficiencies were determined by the paired two-sample t-test.
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To analyze the serotype specificity of the PIs DENV replication was monitored using a commercial
DENV qRTPCR kit (Genesig, Primerdesign LTD, Southampton, UK). Cells were infected with either
DENV-1 or DENV-2 (one genome copy per cell). The compounds were added subsequently and the
cells were incubated for 4 d. Cell culture supernatants were collected and centrifuged in order to
remove detached cells. Viral RNA was isolated using the QIAamp viral RNA mini kit, reverse
transcribed and amplified according to the manufactures’ instructions.
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A reporter system was constructed to analyze inhibition of the DENV-2 PR in cell culture. First, the
PR encoding sequence fused to a N-terminal FLAG Tag was introduced into a eukaryotic expression
vector, giving rise to the pDENV2-PR plasmid. Secondly, the DsRed2 open reading frame was
amplified with a sense primer encoding the DENV-2 NS2A/2B cleavage site. The amplicon was
introduced into the gfp encoding plasmid peGFP-C1 (Clontech) 3’ of the gfp gene. The resulting
peGFPCSDsRed reporter plasmid encodes a GFP-NS2A/2B-DsRed fusion protein. HEK 293T cells
were co-transfected with both the PR expression plasmid pDENV2-PR and the peGFPCSDsRed
reporter. PIs were added directly after transfection. Cells were harvested 2 d post-transfection and
lysed in RIPA buffer (150 mM NaCl, 1% NP40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0).
Three independent Western Blots of each of the triplicate samples were used to calculate relative
substrate and product amounts using AIDA software package (GE Healthcare). All experiments were
repeated at least three times. The significances of differences in processing compared to wild-type
maturation efficiencies were determined by the paired two-sample t-test. Expression of the viral PR
was monitored by anti-FLAG Tag Western blotting using monoclonal anti-FLAG M2 antibodies
(Sigma-Aldrich).
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HIV-1 replication assay. Viruses were produced by transfection of HEK 293T cells with proviral
plasmid pNL4-3 encoding infectious HIV-1 subtype B as described before (34). Viral titers were
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determined on TZM-bl indicator cells (CD4+, CCR5+, CXCR4+ HeLa cells) in single cycle infections
by serial dilutions of the cell culture supernatants as previously described (33, 34). Briefly, cell culture
supernatants were collected, centrifuged (1500 rpm, 5 min) to remove infected HEK 293T cells, and
titrated in serial dilutions on TZM-bl cells (96-well plate, 2x104 cells per well). TZM-bl cells carry a
stably integrated copy of a HIV LTR promoter driving a lacZ gene. Two days post-infection, TZM-bl
cells were fixed with ice-cold methanol/acetone for 5 min followed by a β-Galactosidase stain, using
X-gal as substrate. Stained cells were counted and the viral titers were calculated.
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HCV replication assay. Huh7.5 cells stably expressing the Firefly luciferase reporter gene
(Huh7.5-Fluc) were transfected by electroporation with in vitro transcribed RNA of the fully
functional Renilla luciferase-encoding reporter virus genome JcR2a (35). DMSO or different
concentrations of the compounds were added to the cells immediately after transfection. After 72 h of
incubation virus replication and cell viability were determined by measuring Renilla luciferase (Rluc)
and Firefly luciferase (Fluc) activity from cell lysates, respectively. Production of infectious particles
from transfected cells was determined by infection of naïve Huh7.5 cells with supernatants collected
from transfected cells and quantification of Rluc activity 72 h later. Experiments were performed three
times in triplicate (or sextuplicate in the case of DMSO treated cells). The HCV protease inhibitor
Boceprevir served as a positive control (36).
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Cell toxicity test. To exclude toxic side effects of the PIs the cell survival and metabolism was
measured by a cell proliferation assay (Promega, Germany). Vero and HEK 293T cells (2x104) were
incubated with decreasing amounts of compounds solubilized in DMSO or with DMSO alone as
control. The assays were performed in triplicates according to the manufacturer’s instructions. After
four days 20 µl of the MTS substrate were added, cells were further incubated for 90 min and the
OD490 was measured. Substance concentrations inhibiting the MTS substrate conversation were
excluded from further analyses.
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RESULTS AND DISCUSSION
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Inhibitor Design, Docking, and PR Inhibition. To identify new chemical scaffolds for inhibitors
of the DENV PR an in house library consisting of approximately 250 compounds was screened at 50
µM. A previously described DENV-2 PR assay was used to analyze the substance library using a
fluorogenic Boc-GRR-AMC substrate (20, 30). By this assay an active key substance (compound 1)
was discovered to inhibit the DENV-2 PR about 35% at 50 µM (Table 1). In a first approach to
improve affinity the thiophene moiety was replaced by alkyl chains or other (hetero)aromatic ring
systems, and the amide moiety was replaced by an ester or amine function. The diaryl thioethers were
prepared in a two-step synthesis (Table S1). HBTU served as coupling reagent for condensation of the
respective phenyl thiobenzoic acid with the amine parts of the inhibitors. Phenyl thiobenzoic acids
were prepared by nucleophilic aromatic substitution reaction from corresponding arylmercaptans and
arylhalides (37, 38).
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Thus, we obtained a small series of new compounds (Supporting Information). Replacement of the
amide linker by an amine or ester group led to loss of activity, and also compounds with alkyl chains
instead of the thiophene moiety turned out to be inactive. Among the new compounds with other
heteroaromatic moieties PI 2 with nitro-substituted benzothiazol fragment was identified as the most
potent (Table 1, Supporting Information and Supporting Information Figure S1). In a next step the
position of the thioether fragment was changed from ortho- to para- or meta-position. Furthermore, the
thioether moiety was replaced by a methylene group, and the nitro group at the phenyl ring was
replaced by hydrogen, primary amine, or a trifluoromethyl group. Among these new compounds the
meta-substituted dinitro derivative 4 and the trifluoromethyl derivative 3 exhibited an improved
inhibition (Supporting Information Figure S1). In order to explain these structure-activity relationships
and to propel optimization forward on a rational basis we determined the inhibition mechanisms of the
original lead compound 1, and the, to that point, most active inhibitor 3. Both compounds were found
to be non-competitive with respect to the substrate. This was shown by determination of IC50 values at
different substrate concentrations (20, 50, 100, 200 mM), and, the other way round, by determination
of Km and Vmax values at various inhibitor concentrations. The first assays provided no significant
differences of IC50 values, and plots from the second assays showed typical non-competitive graphs
with decreasing Vmax values and nearly constant Km values at increasing concentrations [I] of inhibitor
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3 ([I] = 0 µM: Vmax ΔF/min // Km µM = 52 // 75; [I] = 5 µM: 56 // 112; [I] = 25 µM: 32 // 108; [I] = 50
µM: 21 // 128; [I] = 75 µM: 14 // 95). Additionally, to exclude false positive results due to quenching
of the fluorescence of the hydrolysis product AMC by the nitro aromatics we determined inhibition of
the DENV-2 PR by compounds 2 and 3 using a semi-quantitative HPLC assay (Supporting
Information), which confirmed the inhibition of substrate hydrolysis observed with compound 2 and 3
(~70 - 75% inhibition in the HPLC assay vs. ~70% inhibition in fluorescence assay).
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The non-competitive inhibition raised the question of the binding region within the enzyme.
Previously a specific allosteric binding site for non-competitive and non-peptidic inhibitors has been
proposed. This deep binding pocket is located behind the active site (Ser135, Asp175, His51) and
mainly formed by the amino acids Trp89, Thr120, Gly121, Glu122, Ile123, Gly124, Gly164, Ile165,
Ala166, Gln167 on the one side and Lys73, Lys74, Asn152, Val78, Gly82, Met84 on the other side,
the last three of which are NS2B derived (Figure 1).
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This binding pocket was taken for docking studies with compound 3 using the FlexX docking
approach of the LeadIT 2.2.6 suite with the recently solved crystal structure of DENV-3 PR in
complex with the aldehyde inhibitor Bz-nKKR-H (pdb accession code 3U1I) (22). The structure of the
DENV-3 PR was taken since both PRs show a high degree of similarity and the used DENV-3 PR
structure is the only available structure of a DENV PR in complex with a low-molecular weight
inhibitor.
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Since diaryl thioethers are flexible compounds in respect to their thioether bond, quantum chemical
computations were performed in order to confirm that conformations obtained by docking studies are
plausible and this is indeed the case (Supporting Information). For compound 3 pose 3 is predicted to
be only about 1-5 kcal/mol higher than the global minima of the molecule, which is stabilized by
intramolecular π-π interactions. This energy difference can easily be compensated by inhibitor-enzyme
interactions. Pose 1 and 2 seem to be higher in energy since the amide unit is not or only partially in
conjugation with the other aromatic rings (see Supporting Information and Supporting Information
Figure S3).
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The docking studies proposed an interaction of the nitro group of compound 3 to the side chain of
Asn152 (Figure 2). In order to verify this binding mode by addressing additional possible binding
partners within the allosteric pocket we introduced a hydroxyl group adjacent to nitro group. This
should enable an additional hydrogen bond with the oxygen of the backbone carbonyl group in
Asn152. Additionally, instead of the nitro group a carboxylic acid function (compound 5) or a second
hydroxyl group (compound 8) were introduced to reduce possible quenching effects in the PR assays,
but to maintain the possible interaction to Asn152. While introduction of an acid and a hydroxyl
function at the benzothiazole moiety did not lead to improved affinity (compound 5), the dihydroxy
substituted inhibitor 8 was found to be highly active with an IC50 value of 3.6 µM. Also this PI was
shown to be a non-competitive inhibitor. It has to be mentioned that despite the catechol structure the
compound is stable and not sensitive to oxidation. The docking studies with this inhibitor propose
interactions of one hydroxyl group with the side chain of Asn152 and with the backbone of Lys74, and
an interaction of the other hydroxyl group with Lys73 (Figure 2).
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In agreement with the proposed binding mode the dimethoxy substituted compound 7 is not active
(10% inhibition at 50 µM). Also the derivatives with only one hydroxyl group are less active
corroborating the docking studies (see Supporting Information and Supporting Information Table S1).
Further docking studies proposed replacement of the trifluoromethyl substituted phenyl ring by a
hydroxyl substituted naphthyl moiety and replacement of the sulphur of the thioether bridge by oxygen
(Figure 3). This yielded compound 6, which was shown to be a non-competitive inhibitor with an IC50
value of ca. 4 µM. For that compound binding studies using microscale thermophoresis were
performed (Supporting Information Figure S4). Since these studies need labeling of the protein we
used the more stable mutant of the DENV-2 PR. With this substrate-independent binding assay we
found a dissociation constant of KD = 15.2 µM, which confirms the fluorometric enzyme assays
(Supporting Information Figure S4).
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For selected compounds, namely 1, 2, 3, 5, 6, 7 and 8, the inhibition of the PR of the serotype 3
virus was tested. With the exception of compound 8 DENV-3 PR is more sensitive to the inhibitors
with derivative 6 which is the most active one against DENV-2 PR being also the most potent DENV-
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3 PI (IC50 = 1 µM). This also underlines the results of the docking studies, which were done with the
structure of DENV-3 PR.
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Toxicity and antiviral Activities. Next, we analyzed inhibition of the viral replication by the PIs.
In order to exclude toxic side effects of the substances the cell survival and metabolism was measured
by a cell proliferation assay. Vero cells were incubated with decreasing amounts of the PIs solubilized
in DMSO or with DMSO alone as control (Table 1). Substance concentrations inhibiting the MTS
substrate conversation were excluded from further analyses. Substance 2 was not tested in cellular
assays, since it exhibited toxicity at concentrations below 1 µM (Table 1 and Supporting Information
Figure S5). Cell death due to cytotoxicity of the compounds was not observed at concentrations of
30µM. Higher concentration were not analyzed in cell culture.
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To analyze inhibition of viral replication, Vero cells were pre-incubated with the PIs at decreasing
concentrations below the cell toxicity (Table 1 and Supporting Information Figure S5) and
subsequently infected with DENV-2. Cellular supernatants were collected after four days and titrated
on Vero cells. Virus infected cells were visualized by immunofluorescence staining and viral titers
were calculated (Table 1). All PIs did significantly decrease viral replication. Furthermore, substances
3 and 4 inhibited viral replication more than 3 orders of magnitude at 1 µM and 3 µM, respectively
(Figure 4). This indicates that the PIs described here, except 2 and 8, suppressed viral replication quite
efficiently at low micromolar or even submicromolar concentrations. Especially the low EC50 values
for compounds 3, 4, 5, 6 and 8 showed that we have identified inhibitors of DENV-2 replication.
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In order to analyze, whether the compounds are able to inhibit PR of other DENV serotypes, effects
on replication was determined with DENV-1. Cells were infected with DENV-1 or DENV-2 in the
presence of the most effective compounds 3 and 4. Viral replication was determined by quantification
of viral RNA genomes in the cellular supernatants by qRTPCR. Both, compounds 3 and 4 decreased
DENV-2 genome copies to 27% and 3.1% of the DMSO control, and DENV-1 genome copies to 10%
and 3.5% indicating that DENV-1 exhibits a similar sensitivity to both substances (Figure 4). These
results together with the results of the in vitro PR analyses provide evidence that compounds 3 and 4
inhibit at least replication or PRs of DENV-1, DENV-2 and DENV-3.
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Interestingly the dimethoxy derivative 7 also exhibits antiviral activity against DENV-2 while it is
inactive against DENV-2 PR in the fluorometric enzyme assay (Table 1 and Figure 4). Vice versa, the
dihydroxy derivative 8 displays good inhibition of PR of serotypes 2 and 3, but is not active in cells at
concentrations below 3 µM. This observation leads to the hypotheses that compound 7 may act like a
prodrug being demethylated in cells yielding to the more potent dihydroxy derivative. This hypothesis
is supported by the fact that compound 7 inhibits DENV-1 replication as well (Figure 4) and the PR in
the cell-based assay (see below).
312
313
314
315
316
317
318
319
320
To further exclude that effects of our compounds on DENV-2 replication are due to cytotoxicity and
to prove the specificity of the observed inhibition of DENV-2 replication, we analyzed influences of
the PIs on HIV-1 replication. HIV-1 encodes an aspartate PR, which is neither in regard to its structure
nor to its substrate specificity similar to the DENV-PR. Thus, DENV PIs should not influence HIV-1
replication. HEK 293T cells were transfected with the proviral HIV-1 plasmid pNL4-3 and PIs were
added at concentrations above the EC50. Viral supernatants were collected 2 days post transfection and
titrated on TZM-bl indicator cells (Figure 5). TZM-bl indicator cells encoded a LacZ gene controlled
by the HIV-1 LTR promoter. Infected cells were identified by β-galactosidase stain. None of the PIs
influenced HIV-1 replication excluding cellular toxicity and unspecific inhibition of the HIV-1 PR.
321
322
323
324
325
326
327
328
329
To analyze the specificity of compounds, we determined antiviral activity against HCV, another
member of the family Flaviviridae. Human hepatoma cells expressing a firefly luciferase reporter that
was used to measure cytotoxicity (not shown) (Huh7.5-FLuc), were transfected with a Renilla
luciferase (RLuc)-encoding HCV reporter genome (JcR2a; Fig. 6A). After 72 h, virus replication was
assessed by quantification of RLuc activity in transfected cells (Fig. 6B). To determine virus titers,
naïve Huh7.5 cells were inoculated with culture supernatants collected 72 h after transfection and
RLuc expression was determined 3 days later (Figure 6C). None of the compounds influenced HCV
replication or production of infectious particles, showing that the PIs are specific for DENV and
inactive against the related HCV.
330
331
Inhibition of the DENV PR in cell culture. To measure inhibition of the DENV-2 PR in cells
quantitatively and without any influences of the viral replication, such as additional replication cycles
13

332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
etc. a PR reporter system was constructed. The coding regions of gfp and dsred2 were fused and a
NS2A/NS2B cleavage site was inserted by PCR (Figure 7). Cleavage of this GFP-NS2A/B cleavage
site-DsRed2 fusion protein by the DENV-2 PR would result in separated GFP and DsRed proteins.
HEK 293T cells were transfected with the reporter plasmid alone or in combination with a pDENV2-
PR expression plasmid. To monitor expression of the DENV PR a N-terminal M2 FLAG Tag was
added by PCR. Cleavage of the substrate was analyzed by quantitative Western blotting using anti-
GFP antibodies (Figure 7 and Supporting Information Figure S6). Determination of GAPH amounts
served as loading control. Expression of the substrate alone resulted in the expected protein of 55 kDa
protein. Co-expression of the DENV PR led to substrate cleavage of about 75%. Instead of the
expected single GFP derived product band, cleavage of the reporter fusion protein resulted in two
product bands (Figure 7). Therefore, we analyzed whether GFP was cleavage by the DENV-2 PR
(Supporting Information Figure S7) and could show that DENV-2 indeed cleaves the GFP protein at a
potential cleavage site KRDH. The lead substance 1 inhibited viral PR activity in cell culture already
with an IC50 at 15.6 µM. The optimized derivatives resulted in IC50 values in the low micromolar or
even submicromolar range (Table 1, Figure 7 and Supporting Information Figure S6). The high
inhibition of replication in comparison to the IC50 values for PR inhibition in cell culture could
indicate that an almost complete processing of viral proteins is required for viral infectivity. Similar
discrepancies between EC50 and IC50 values were observed by others, but attributed to the enzyme-
based DENV PR assay (18). Furthermore, our result might suggest that the PIs are enriched in cells.
Thus, concentrations less than the IC50 in cell culture led to the inhibition of viral replication.
352
353
354
355
356
357
358
359
Comparing the inhibition of the isolated proteases (column 1 and 2 in Table 1) with antiviral activity
(column 4) and PR inhibition in cell culture (column 5), no good correlation can be found between PR
inhibition (columns 1 and 2) and inhibition of viral replication (column 4) or PR in cell culture
(column 5). However, a very good correlation (Figure 8) can be found between antiviral activity
(column 4) and PR inhibition in cell culture (column 5) indicating that indeed the PR inhibition is the
main reason for the antiviral activity of the compounds, and furthermore substantiating the hypothesis
that the dimethoxy substituted compound 7 which is not active against the isolated proteases maybe a
prodrug for the dihydroxy compound 8. On the other hand, these results show that for rational design
14

360
361
362
of new PR inhibitors, both assays are necessary, the determination of inhibition of isolated proteases in
order to verify the docking hypotheses, and the determination of PR inhibition in cell culture which
can be used to predict the antiviral activity.
363
364
365
366
367
368
369
370
By analyzing influences of the PR inhibition in cell culture on DENV replication, a high sensitivity
of viral replication compared to the cell culture based cleavage assay was observed (Table 1 and
Figure 7). These results indicate that already partial inhibition of processing is detrimental for DENV,
which is similar to partial inhibition of HIV-1 maturation, which results in an almost complete
reduction of viral replication (39). Other viruses, such as foamy viruses, tolerate up to 40% of non-
processed proteins without any influences on viral replication (40). On the other hand, since the cell
culture based cleavage reporter system uses the NS2B/NS3 cleavage site, which was shown to be the
most efficient one, it can be assumed that the PIs will block less efficient cleavage sites at lower EC50.
371
372
373
In summary, we describe a novel class of DENV-2/3 PR inhibitors with an inhibition of the PR in
vitro and in vivo, which are shown to inhibit viral replication. Our substances may serve as promising
lead-compounds for further studies and optimization.
374
ACKNOWLEDGMENT
375
376
377
378
379
380
381
We would like to thank A. Kucharski (University of Würzburg, Institute of Pharmacy and Food
Chemistry), S. Mährlein and U. Nowe (University of Mainz, Institute of Pharmacy and Biochemistry)
for technical and the University of Mainz for financial support. C.W. was supported by the
Studienstiftung des Deutschen Volkes, T. S. and B. E. by the DFG (SFB630). The DENV-2 PR
bacterial expression plasmid was kindly provided by C. Klein and C. Steuer (University of
Heidelberg) and the anti-DENV-2 E antibodies and Dengue viruses by J. Schneider-Schaulies
(University of Würzburg).
382
ABBREVIATIONS
383
384
AMC, 7-Amino-4-methylcoumarin; DENV, Dengue virus; DMSO, Dimethylsulfoxid; HBTU,
N,N,N′,N′-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium
hexafluorophosphate;
HIV,
human
15

385
386
387
immunodeficiency virus; MOI, multiplicity of infection; MST, Microscale Thermophoresis; NS, non-
structural, PI, protease inhibitor; PR, protease
16

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19

509
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511
512
513
FIGURES
Figure 1. Allosteric site of DENV-3 PR with the docked compounds 3, 6 and 8. The NS2B unit is
shown in green, and the NS3 unit in cyan. Ligands are rendered as CPK-colored sticks with the
exception of carbon atoms (3: white, 6: yellow, 8: pink). Catalytic triad residues His51, Asp75, and
Ser135 in the active site, which is behind the allosteric site, are represented in balls and sticks.
514
515
516
517
518
Figure 2. Predicted binding modes of compounds 3 and 8. The PIs were docked into DENV-3 PR
by LeadIT-FlexX program. The image was generated with pymol. (A) Surface view of the allosteric
site with the docked compounds 3 and 8. Ligands are rendered as CPK-colored sticks with the
exception of carbon atoms (3: cyan, 8: pink). (B) Binding mode of compound 8 showing the H-bonds
between two the hydroxyl groups and the amino acids Lys73, Lys74 and Asn152.
519
520
521
Figure 3. Docking results for PI 6 within the DENV-3 PR. (A) Three-dimensional interaction
diagram of the compound 6 with the amino acids Lys73, Thr120, Asn152 and Gln167. (B) Schematic
view of all interactions (H-bonds and hydrophobic interactions) of compound 6.
522
523
524
525
526
527
528
529
530
531
Figure 4. Inhibition DENV-1 and DENV-2 replication. (A) Vero cells were pre-incubated with
decreasing amounts of the compounds. DMSO served as control. The cells were subsequently infected
with DENV-2. Cell culture supernatants were collected and viruses titrated on Vero cells. All
experiments were performed in triplicate assays. Error bars represent the standard deviation.
Significances of differences in viral titers compared to DMSO control were determined by the paired
two-sample t-test and indicated by two asterisks (p-value < 0.01) above the bars. (B) Subtype
specificity inhibition of replication by the PIs. Vero cells were pre-incubated with the compounds and
infected with DENV-1 or DENV-2 (one genome copy per cell). Cell culture supernatants were
collected and viral genome amounts were determined by qRTPCR. All experiments were performed in
independent triplicate assays. Error bars represent the standard deviation.
532
533
534
535
Figure 5. Compounds 3-7 do not influence HIV-1. HEK 293T cells were transfected with the
proviral HIV-1 plasmid pNL4-3 and subsequently incubated with the PIs. Viral titers were determined
on TZM-bl indicator cells, which encode a LacZ gene controlled by the HIV-1 LTR promoter. All
experiments were performed in triplicate assays. Error bars represent the standard deviation.
20

536
537
538
539
540
541
542
543
544
545
Figure 6. Components 1 and 3-7 do not influence HCV replication. (A) Scheme of the
experimental approach. Huh7.5-Fluc cells were transfected with in vitro transcribed HCV RNA of the
Renilla luciferase-encoding reporter virus JcR2a. (B) After 72 h virus replication and cell viability
were determined by measuring Renilla luciferase (Rluc) and Firefly luciferase (Fluc) activity from cell
lysates, respectively. (C) Production of infectious particles was determined by infection of naïve
Huh7.5 cells with supernatants collected from transfected cells and quantification of Rluc activity after
72 h. Experiments were performed three times in triplicate (or sextuplicate in the case of DMSO
treated cells). The HCV protease inhibitor Boceprevir was included as a positive control. Red line:
limit of detection. All data represent the mean of three independent experiments, which were
performed in triplicate assays. Error bars represent the standard deviation.
546
547
548
549
550
551
552
553
Figure 7. Compounds 1 and 3-7 inhibit DENV-2 PR in cells. (A) Scheme of the cleavage reporter
construct. (B) Representative quantitative Western blotting analyses of reporter cleavage by the
DENV-2 PR using a monoclonal anti-GFP antibody. Cells were transfected with the reporter vector
alone (lane 1), with the PR expression vector alone, or with the reporter vector and the PR (lanes 3 to
11). PIs were added after transfection at four non-toxic concentrations (indicated above the panel).
Substrate and products were visualized and the inhibition was calculated (ration of (fusion
protein)/((fusion protein)+(products)). Expression of the DENV PR was monitored by Western
blotting with monoclonal M2-antibodies. The positions of the molecular size markers are indicated.
554
555
556
557
Figure 8. Correlation of antiviral activity (expressed as log EC50, column 4 in Table 1) and PR
inhibition in cell culture (expressed as log IC50incell, column 5 in Table 1). For the diagram results for
compounds 1, 3, 4, 5, 6, and 7 were taken.
558
559
21

560
561
TABLES
Table 1. Structures and activities of the NS2B/NS3 protease inhibitors
562
1
2
3
4
5
IC50,
c
d
Compound
no.
IC50,a µM
DENV-2 e
IC50,a µM
DENV-3 e
Toxicity,b
[µM]
EC50
,
e
563
[µM]
Structure
[µM] e
564
1
98±4
31.8±4.5
30
3.5±0.3
15.6±3.4
565
2
3
34±5
22±1
5.4±2.9
21±4
<1
1
n.d.
n. d.
566
0.25±06.70
568
0.1±0.0
4
26±1
n.d.
3
0.3±0.1
0.7±0.1
569
2.35±07.07
571
5
6
66±3
12.3± 2.2
0.99±0.1
10
10
0.9±0.1
0.8±0.2
4.2±0.44
3.25±17.22
10%
inhibition
573
7
8
n.i.
30
3
2.5±0.1
>3
9.3±2.5
at 50 µM
3.6±0.11
574
9.1±1.02
>3
575
576
b
577
578
579
580
a, inhibition of isolated proteases determined by fluorometric enzymes assays with an AMC derived substrate;
,
concentration, where no influence on cellular metabolism was observed; c, antiviral activity, inhibition of viral
replication; d, biochemical inhibition of PR in cell culture; n.d., not done; n.i., no inhibition at 50 µM, e values are
indicated as mean of 3 independent experiments in triplicates ± standard deviation.
22

A
B
JcR2a
supernatants
Huh7.5-FLuc cells
Huh7.5 cells
3 days
6 days
Addition of
compounds
Cell lysates:
Rluc: virus replication
Fluc: cell viability
Cell lysates:
Rluc: virus production
100000000
10000000
1000000
100000
10000
**
**
1000
Virus production (n=3)
6
1
7
5
4
3
Boceprevir
C
100000000
10000000
1000000
100000
10000
*
**
**
1000
1
7
5
6
4
3
Boceprevir
Figure 6. Components 1 and 3-7 do not influence HCV replication. (A) Scheme of the experimental approach. Huh7.5-Fluc cells were
transfected with in vitro transcribed HCV RNA of the Renilla luciferase-encoding reporter virus JcR2a. (B) After 72 h virus replication and cell
viability were determined by measuring Renilla luciferase (Rluc) and Firefly luciferase (Fluc) activity from cell lysates, respectively. (C)
Production of infectious particles was determined by infection of naïve Huh7.5 cells with supernatants collected from transfected cells and
quantification of Rluc activity after 72 h. Experiments were performed three times in triplicate (or sextuplicate in the case of DMSO treated
cells). The HCV protease inhibitor Boceprevir was included as a positive control. Red line: limit of detection. All data represent the mean of
three independent experiments, which were performed in triplicate assays. Error bars represent the standard deviation.

1,2
1
0,8
0,6
0,4
0,2
0
-0,2
-0,4
-0,6
-0,8
-1 -0,8 -0,6 -0,4 -0,2
0
0,2 0,4 0,6
EC
log
50
Figure 8. Correlation of antiviral activity (expressed as log EC50, column 4 in Table 1) and PR inhibition in cell culture
(expressed as log IC50incell, column 5 in Table 1). For the diagram results for compounds 1, 3, 4, 5, 6, and 7 were taken.