Inhibitors of tick-borne flavivirus reproduction from structure-based virtual screening

Article abstract of DOI:10.1021/ml400226s

Flaviviruses form a large family of enveloped viruses affecting millions of people over the world. To date, no specific therapy was suggested for the infected people, making the treatment exclusively symptomatic. Several attempts were performed earlier for the design of fusion inhibitors for mosquito-borne flaviviruses, whereas for the tick-borne flaviviruses such design had not been performed. We have constructed homology models of envelope glycoproteins of tick-transmitted flaviviruses with the detergent binding pocket in the open state. Molecular docking of substituted 1,4-dihydropyridines and pyrido[2,1-b][1,3,5]thiadiazines was made against these models, and 89 hits were selected for the in vitro experimental evaluation. Seventeen compounds showed significant inhibition against tick-borne encephalitis virus, Powassan virus, or Omsk hemorrhagic fever virus in the 50% plaque reduction test in PEK cells. These compounds identified through rational design are the first ones possessing reproduction inhibition activity against tick-borne flaviviruses.

Full text of DOI:10.1021/ml400226s

Letter
pubs.acs.org/acsmedchemlett
Inhibitors of Tick-Borne Flavivirus Reproduction from Structure-
Based Virtual Screening
Dmitry I. Osolodkin,†,‡,§ Liubov I. Kozlovskaya,‡ Evgenia V. Dueva,†,‡,§ Victor V. Dotsenko,∥
Yulia V. Rogova,‡ Konstantin A. Frolov,∥ Sergey G. Krivokolysko,∥ Ekaterina G. Romanova,†
Alexey S. Morozov,† Galina G. Karganova,*,‡,⊥ Vladimir A. Palyulin,*,†,§ Vladimir M. Pentkovski,§,#
and Nikolay S. Zefirov†,§
†Department of Chemistry, Lomonosov Moscow State University, Moscow 119991, Russia
‡FSBI Chumakov Institute of Poliomyelitis and Viral Encephalitides RAMS, Moscow 142782, Russia
§iSCALARE Laboratory, Moscow Institute of Physics and Technology, Dolgoprudny 141700, Russia
∥ChemEx Laboratory, Vladimir Dal’ East Ukrainian National University, Lugansk 91034, Ukraine
⊥Department of Biology, Lomonosov Moscow State University, Moscow 119991, Russia
S
* Supporting Information
ABSTRACT: Flaviviruses form a large family of enveloped viruses affecting millions of people
over the world. To date, no specific therapy was suggested for the infected people, making the
treatment exclusively symptomatic. Several attempts were performed earlier for the design of
fusion inhibitors for mosquito-borne flaviviruses, whereas for the tick-borne flaviviruses such
design had not been performed. We have constructed homology models of envelope
glycoproteins of tick-transmitted flaviviruses with the detergent binding pocket in the open
state. Molecular docking of substituted 1,4-dihydropyridines and pyrido[2,1-b][1,3,5]-
thiadiazines was made against these models, and 89 hits were selected for the in vitro
experimental evaluation. Seventeen compounds showed significant inhibition against tick-borne
encephalitis virus, Powassan virus, or Omsk hemorrhagic fever virus in the 50% plaque
reduction test in PEK cells. These compounds identified through rational design are the first ones possessing reproduction
inhibition activity against tick-borne flaviviruses.
KEYWORDS: Antiviral compounds, flavivirus, 1,4-dihydropyridines, pyrido[2,1-b][1,3,5]thiadiazines
important flaviviruses, specific vaccines development would
never be done due to a small number of cases. Thus, other
more specific and effective ways of treatment of tick-borne
flaviviral infections are urgently needed.
he Flavivirus genus includes a range of viral pathogens
1−4
Tcausing severe diseases in humans.
These viruses are
transmitted by arthropod vectors to mammalian hosts.
Apparently the most studied among flaviviruses are the
mosquito-borne viruses such as dengue virus (DENV), West
Nile virus (WNV), yellow fever virus (YFV), and Japanese
encephalitis virus (JEV). Tick-borne flaviviral diseases such as
tick-borne encephalitis, being a serious health concern in Russia
and Europe,4,5 and Omsk hemorrhagic fever4,6 and Powassan
encephalitis,4,7,8 showing low incidence and local importance in
Russia, US, and Canada, are associated with severe symptoms.
Nevertheless, their causing agents are less studied. The
situation is additionally complicated by unavailability of specific
drugs affecting flaviviruses. Despite that vaccines are available
against tick-borne encephalitis virus (TBEV), YFV, and JEV,9
they can be used successfully only as prophylaxis measure
before the infection. Other treatment options include
symptomatic treatment schemes and immunoglobulin therapy
of tick-borne encephalitis cases,10 several immunomodulating
agents (e.g., jodantipyrin11 and luromarin12) are on a preclinical
stage, but their applicability in the course of flaviviral infection
is limited. Moreover, in the case of Omsk hemorrhagic fever
virus (OHFV), Powassan virus (POWV), and other locally
Inhibition of the virus entry and replication is a widely
accepted strategy in the design of antivirals.13−15 The entry of
flavivirus particle into the host cell via receptor-mediated
endocytosis requires the fusion of viral and cellular membranes.
This process is ruled by pH decrease in the endosome and
mediated mostly by envelope glycoprotein E. During the fusion
E protein dimers initially arranged in a herringbone pattern on
the virion surface undergo a large-scale conformational
rearrangement leading to the formation of protein spikes,
which attack the host cell membrane.16 A detergent n-octyl-β-D-
glucoside (β-OG) binding pocket has been identified in one of
DENV E protein ectodomain structures (PDB ID 1OKE17),
whereas in the other structures this pocket is closed by the kl
loop.18 It was assumed that this pocket can be exploited as a
putative site for DENV fusion inhibitors. As far as flaviviral E
Received: June 13, 2013
Accepted: July 25, 2013
© XXXX American Chemical Society
A
dx.doi.org/10.1021/ml400226s | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Figure 1. Superposition of E protein structures and models for DENV (subunit 1 colored by domains (red, domain I; yellow, domain II; blue,
domain III), subunit 2 colored magenta), TBEV (white), POWV (green), and OHFV (orange). Loops kl and fg are colored in subunit 1 and shown
as ribbons in subunit 2. β-OG molecules are shown spacefilled.
Scheme 1.a
a
Reagents and conditions: (a) N-methylmorpholine, EtOH, 20 °C, 70−93%. (b) 10% aq. KOH, R1C(O)CH2Cl, EtOH, 20−40 °C, 56−87%. (c) N-
Methylmorpholine, EtOH, 20 °C, 1 h. (d) EtOH, reflux, 4 h, 68−78%. (e) One equiv of RNH2, 37% aq. HCHO, EtOH, reflux, 51−70%. BH+ = N-
methylmorpholinium.
proteins are rather similar,1 this hypothesis has been extended
to the other genus members. Blind docking studies against this
pocket led to a successful identification of DENV, WNV, and
YFV replication inhibitors.19−24 In all these cases, docking was
performed into 1OKE structure, whereas activity was tested
with different assays. Binding of the inhibitors in the β-OG
pocket was shown by NMR.20 According to a Monte Carlo
simulation, the open state of β-OG pocket may be preferred
over the closed one in the dynamic native conditions.25
Alternative binding sites were also searched for on the surface
of the DENV E ectodomain dimer, and several inhibitors were
identified.26 A teicoplanin analogue LCTA-949 has been shown
to prevent cell entry of various flaviviruses, being the most
effective against TBEV, but no data is available on the mode of
action of this molecule.27
constructed homology models of E proteins for the selected
viruses with β-OG pocket in the open state. Subsequently, a
virtual screening campaign of a compound library was
performed, and hit compounds were selected according to
the scores and visual analysis of the binding modes. In vitro
assays of the hits revealed several compounds that had shown
inhibition of viral reproduction. These data confirmed the
validity of the chosen methodology and could be used for
further design and development of novel antiflaviviral drugs.
Homology models of E proteins were constructed for the
representative strains of TBEV (Absettarov), POWV (Powas-
san24), and OHFV (Nikitina) (Figure 1). The homology
models were built in Modeller 9.1028 based on the TBEV E
protein dimer template structure (PDB ID 1SVB18) for the
whole sequence except the kl loop, which was modeled based
on 1OKE template.17 All models built possessed good
stereochemical quality.
We tried to identify small molecule compounds able to
inhibit the reproduction of tick-borne flaviviruses (namely,
TBEV, POWV, and OHFV) via interaction with the envelope
protein E in the β-OG pocket. To achieve this goal, we
Given the absence of known compounds targeting the
TBEV, POWV, and OHFV E proteins, we chose a library of
B
dx.doi.org/10.1021/ml400226s | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Table 1. Structures and Antiviral Activity of the Compounds That Passed Preliminary Assays
IC50, μM
CC50, μM
(acute)
CC50, μM
(chronic)
compd
Ar
R
R1
TBEV
POWV
OHFV
1a
1b
1c
1d
1e
1f
2-furyl
2-furyl
2-furyl
2-furyl
2-furyl
2-furyl
2-furyl
4-EtOC6H4
4-H2NSO2C6H4
4-ClC6H4
4-EtC6H4
2-MeC6H4
Ph
4-nBuC6H4NH
4-EtC6H4NH
4-EtC6H4NH
4-EtC6H4NH
2-naphthyl-NH
3,4-Me2C6H3
64
14
2.5 0.5
>10
>10
>10
>10
>10
>10
>10
>10
>10
a
>250
153
27
10 7.8
>10
26
>10
57
33
>10
>10
>250
>250
>250
>250
19
>10
5.3 0.1
3.2 0.8
7.1 0.1
>10
1g
2,6-Me2C6H3
2-benzothiazolyl-
NH
7
>10
1h
2-furyl
2-furyl
2-benzothiazolyl
2-benzothiazolyl-
NH
>250
52
>10
>10
2.5 0.9
1i
2-benzothiazolyl
2-MeOC6H4
Ph
4-iPrC6H4NH
4-BrC6H4NH
Ph
>250
248
29
34
17
97
41
38
20
89
31
39
116
236
35
53
>10
>10
2.5 0.1
3.7 0.4
>10
1j
5-Me-2-furyl
2-thienyl
>10
>10
1k
1l
>250
>250
>250
>250
111
>10
>10
2-thienyl
2-MeOC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
2-MeC6H4
Ph
4-MeC6H4NH
3-MeC6H4NH
4-PhOC6H4NH
2-naphthyl-NH
4-MeOC6H4NH
4-EtC6H4NH
>10
>10
5.5 0.9
>10
1m
1n
1o
1p
1q
2a
2b
2c
2d
2e
Ph
2.0 0.4
2.8 0.6
>10
>10
Ph
>10
>10
Ph
>10
>10
2-FC6H4
>250
114
>10
>10
7.2 0.5
1.8 0.4
>10
4-HO-3-MeOC6H3
4-(4-ClC6H4CH2O)C6H4
3-BnOC6H4
3-BnOC6H4
4-BnOC6H4
4-BnO-3-MeOC6H3
>10
>10
2,4-(MeO)2C6H3
2-EtOC6H4
4-EtC6H4
109
0.07 0.02
2.6 0.4
>10
1.3 0.1
2.2 0.3
>10
>250
>250
>250
>250
>10
>11.9
>10
4-nBuC6H4
4-MeOC6H4
1.9 0.4
0.09 0.01
>10
>10
>10
a
The 250 μM concentration is the highest concentration studied.
elongated heterocyclic compounds synthesized in ChemEx
laboratory for further virtual screening (VS) studies. The
molecular docking of the library containing 5886 compounds
was performed with FRED 2.2.5.29 The predicted binding
modes for POWV E protein were scored with six scoring
functions implemented in FRED. Top 500 hit lists were
analyzed for each scoring function, and the compounds
included into at least three hit lists were selected for further
analysis along with top compounds selected by each scoring
function; the total number of compounds was 201. These
compounds were then docked into TBEV and OHFV E protein
models. Predicted binding modes of the compounds were
analyzed visually, and compounds with unacceptable binding
modes (e.g., forming hydrogen bond donor−donor or
acceptor−acceptor interactions) were excluded along with
compounds belonging to scarcely represented classes. The final
hit list consisted of 89 compounds belonging to two related
classes, 1 and 2.
Prior to in vitro studies, cytotoxicity of the compounds was
assessed in porcine embryo kidney (PEK) cell line (Table 1);
acute (24 h) and chronic (7 days) toxicities were evaluated. For
the majority of compounds, CC50 values were much over 10
μM. It was considered as generally acceptable for the first
compounds in the class.
Preliminary assessment of antiviral activity of hit compounds
in 10 μM concentration was studied in 50% plaque reduction
test (PRT50) in PEK cell line against selected strains of TBEV,
POWV, or OHFV. Twenty-two compounds showing over 50%
antiviral activity in the preliminary tests were promoted for the
IC50 determination stage performed as the same assay with
sequential dilutions of the studied compound starting from 10
μM. Tested compounds showed dose-dependent inhibition of
viral reproduction. PRT50 IC50 values are given in Table 1.
Detailed computational and experimental procedures and
compound characterization data are provided in the Supporting
Information.
The Hantzsch-type 1,4-dihydropyridines 1a−q were synthe-
sized in two steps by analogy with known procedures.30,31 First,
the reaction of cyanothioacetamide 3 with aromatic aldehydes
and acetoacetanilides 4 in the presence of 1.5-fold excess of N-
methylmorpholine gave 1,4-dihydropyridine-2-thiolates 5. The
next step included regioselective S-alkylation by treatment with
alkyl chlorides to give target compounds 1a−q (Scheme 1.a).
Pyridothiadiazines 2a−e were obtained in three steps starting
from cyanothioacetamide 3 and Meldrum’s acid 6 by analogy
with the known procedure.32 The stable adducts 7 were
cyclized in ethanol under reflux to furnish 4,5-dihydropyridone-
2-thiolates 8.33,34 Double Mannich-type aminomethylation of
these intermediates was achieved by treatment with 1 equiv of a
primary amine and an excess of formaldehyde under short-term
heating to form compounds 2 as sole products.
Compounds from 1,4-dihydropyridine series 1 have shown
the inhibitory activity against OHFV or TBEV (Table 1). These
inhibitors were ranked high in the hit lists for the
corresponding viruses, showing high selectivity despite 93%
sequence identity between E protein ectodomains. Compound
1q, the most active against OHFV, was the only one in the
series 1 containing hydroxy and methoxy groups in the Ar
substituent. According to the docking results, this moiety
occupied a cavity between Tyr49 and the kl loop (Figure 2).
The presence of many aromatic and/or hydroxyl bearing
residues in this region (Ser47, Tyr49, Thr279, and Tyr281)
suggested that binding of such moiety is favored there. The
presence of hydrogen bond acceptors in the variable moieties
was the only requirement that seems to be important for the
C
dx.doi.org/10.1021/ml400226s | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
preference for a specific enantiomer over another one: (R)-
isomers were usually ranked higher for series 2.
Structure−activity relationships for the compounds seem to
be rather obscure, being more complicated by the absence of a
consistent binding mode of the compounds: even similar
compounds are predicted to interact with the proteins in
different orientations (Figure 2). The compounds, protecting
the cells against OHFV, do not protect the cells against TBEV
and POWV, and vice versa. It should be attributed to E protein
peculiarities resulting in different cellular tropism and therefore
clinical outcome. These peculiarities also reveal themselves in
the docking results: usually, compounds active against certain
viruses form more hydrogen bonds with the corresponding E
proteins (Figure 2) and are ranked higher against them.
Because of the significant similarity of OHFV and TBEV and
the compounds themselves, this situation is counterintuitive
and may be attributed to a rather small number of compounds
assessed in this study and evaluation of activity only for
racemates.
Whether the mode of interaction of the identified inhibitors
with the flavivirus envelope proteins is realized as predicted, i.e.,
via the binding in the β-OG pocket, cannot be unambiguously
established in this experiment. Even a nonspecific mode of
binding cannot be unanticipated. Rigorous study of the
reproduction inhibition mechanism and the binding mode
will be reported elsewhere.
Our virtual screening campaign against homology models of
the envelope proteins from tick-borne flaviviruses resulted in
the identification of the small molecule compounds preventing
TBEV, POWV, and OHFV reproduction in the host cells.
These compounds belong to the series of 1,4-dihydropyridines,
showing activity against TBEV or OHFV, and pyridothiadia-
zines, showing submicromolar activity against TBEV and
micromolar against POWV. Further optimization of these
compounds guided by in vitro and in vivo studies will be
performed with the aim to obtain drugs for flaviviral fevers and
encephalitises treatment.
ASSOCIATED CONTENT
* Supporting Information
■
S
Detailed experimental procedures for the homology modeling,
virtual screening, synthesis and purification of compounds, and
antiviral assays. Preliminary experimental data for inactive
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
Figure 2. Putative binding modes of 2a in the β-OG pockets according
to the docking results. Hydrogen bonds are shown as green lines.
AUTHOR INFORMATION
Corresponding Author
■
*(V.A.P.) Tel: +74959393969. Fax: +74959390290. E-mail:
vap@qsar.chem.msu.ru. (G.G.K.) Tel: +74958419327. Fax:
+74958419330. E-mail: karganova@bk.ru.
compound potency; the only active compound in the series 1
without such groups was 1m.
Pyridothiadiazines 2 showed inhibitory activity against TBEV
and POWV (Table 1); they were generally more potent despite
their lower abundance in the hit lists compared to 1,4-
dihydropyridines. Compounds 2a and 2b showed significant
activity against both TBEV and POWV despite the limited
similarity between these viruses. The only inactive compound
in this series, 2c, was ranked high only against the OHFV E
protein. Subtle differences in the 2e structure compared to 2a
led to the elimination of anti-POWV activity in 2e but did not
affect submicromolar anti-TBEV activity. Such ambiguities
could be attributed to the fact that racemic mixtures were
studied because molecular docking simulations showed some
Author Contributions
The study was initiated and designed by D.I.O., L.I.K., G.G.K.,
and V.A.P. Computational study was performed by D.I.O.,
E.V.D., and E.G.R. Compounds were synthesized and
characterized by V.V.D., K.A.F., S.G.K., and A.S.M. Biological
assessment was performed by L.I.K. and Y.V.R. The study was
supervised by V.A.P., G.G.K., V.M.P., and N.S.Z. All authors
discussed and approved the publication of the manuscript.
Funding
iSCALARE laboratory is supported by the Grant of the
Government of Russian Federation (decree 220 of April 09,
D
dx.doi.org/10.1021/ml400226s | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
(14) Stevens, A. J.; Gahan, M. E.; Mahalingam, S.; Keller, P. A. The
Medicinal Chemistry of Dengue Fever. J. Med. Chem. 2009, 52, 7911−
7926.
(15) Teissier, E.; Penin, F.; Pecheur, E.-I. Targeting Cell Entry of
́
Enveloped Viruses as an Antiviral Strategy. Molecules 2011, 16, 221−
250.
2010). The present work was partially supported by RFBR
grants 11-03-01174a and 12-04-31317a and by the Program of
Development of Lomonosov Moscow State University.
Notes
The authors declare no competing financial interest.
#Vladimir M. Pentkovski deceased on December 24, 2012. We
dedicate this paper to his memory.
(16) Smit, J. M.; Moesker, B.; Rodenhuis-Zybert, I.; Wilschut, J.
Flavivirus Cell Entry and Membrane Fusion. Viruses 2011, 3, 160−171.
(17) Modis, Y.; Ogata, S.; Clements, D.; Harrison, S. C. A Ligand-
Binding Pocket in the Dengue Virus Envelope Glycoprotein. Proc.
Natl. Acad. Sci. U.S.A. 2003, 100, 6986−6991.
(18) Rey, F. A.; Heinz, F. X.; Mandl, C.; Kunz, C.; Harrison, S. C.
The Envelope Glycoprotein from Tick-Borne Encephalitis Virus at 2 Å
Resolution. Nature 1995, 375, 291−298.
(19) Yang, J.-M.; Chen, Y.-F.; Tu, Y.-Y.; Yen, K.-R.; Yang, Y.-L.
Combinatorial Computational Approaches to Identify Tetracycline
Derivatives as Flavivirus Inhibitors. PLoS One 2007, 2, e428.
(20) Zhou, Z.; Khaliq, M.; Suk, J.-E.; Patkar, C.; Li, L.; Kuhn, R. J.;
Post, C. B. Antiviral Compounds Discovered by Virtual Screening of
Small-Molecule Libraries against Dengue Virus E Protein. ACS Chem.
Biol. 2008, 3, 765−775.
(21) Li, Z.; Khaliq, M.; Zhou, Z.; Post, C. B.; Kuhn, R. J.; Cushman,
M. Design, Synthesis and Biological Evaluation of Antiviral Agents
Targeting Flavivirus Envelope Proteins. J. Med. Chem. 2008, 51, 4660−
4671.
(22) Kampmann, T.; Yennamalli, R.; Campbell, P.; Stoermer, M. J.;
Fairlie, D. P.; Kobe, B.; Young, P. R. In Silico Screening of Small
Molecule Libraries Using the Dengue Virus Envelope E Protein Has
Identified Compounds with Antiviral Activity against Multiple
Flaviviruses. Antiviral Res.. 2009, 84, 234−241.
(23) Poh, M. K.; Yip, A.; Zhang, S.; Priestle, J. P.; Ma, N. L.; Smit, J.
M.; Wilschut, J.; Shi, P.-Y.; Wenk, M. R.; Schul, W. A Small Molecule
Fusion Inhibitor of Dengue Virus. Antiviral Res. 2009, 84, 260−266.
(24) Mayhoub, A. S.; Khaliq, M.; Botting, C.; Li, Z.; Kuhn, R. J.;
Cushman, M. An Investigation of Phenylthiazole Antiflaviviral Agents.
Bioorg. Med. Chem. 2011, 19, 3845−3854.
(25) Osolodkin, D. I.; Kozlovskaya, L. I.; Palyulin, V. A.; Pentkovski,
V. M.; Karganova, G. G.; Zefirov, N. S. A Molecular Model and Monte
Carlo Simulation of Flavivirus Envelope Building Block. Biochem.
Biophys. Res. Commun. 2012, 425, 207−211.
(26) Yennamalli, R.; Subbarao, N.; Kampmann, T.; McGeary, R. P.;
Young, P. R.; Kobe, B. Identification of Novel Target Sites and an
Inhibitor of the Dengue Virus E Protein. J. Comput.-Aided Mol. Des.
2009, 23, 333−341.
ACKNOWLEDGMENTS
■
The authors are grateful to Dr. Mstislav I. Lavrov and Dr.
Alexey D. Averin (Department of Chemistry, Lomonosov
Moscow State University) for assistance with compound
characterisation and to Sergey A. Pisarev for manuscript
reading and discussion. Free academic licence for OpenEye
software is provided by OpenEye Scientific Software, Inc.
ABBREVIATIONS
■
β-OG, n-octyl-β-D-glucoside; DENV, dengue virus; JEV,
Japanese encephalitis virus; OHFV, Omsk hemorrhagic fever
virus; PEK, porcine embryo kidney; POWV, Powassan virus;
TBEV, tick-borne encephalitis virus; VS, virtual screening;
WNV, West Nile virus; YFV, Yellow fever virus
REFERENCES
■
(1) Lindebach, B. D.; Thiel, H.-J.; Rice, C. M. Flaviviridae: The
Viruses and Their Replication. In Fields Virology, 5th ed.; Knippe, D.
M., Howley, P. M., Eds.; Lippincott Williams and Wilkins and Wolters
Kluwer Business Publishers: Philadelphia, PA, 2007; pp 1101−1152.
(2) Turtle, L.; Griffiths, M. J.; Solomon, T. Encephalitis Caused by
Flaviviruses. QJM 2012, 105, 219−223.
(3) Sips, G. J.; Wilschut, J.; Smit, J. M. Neuroinvasive Flavivirus
Infections. Rev. Med. Virol. 2011, 22, 69−87.
(4) Gritsun, T. S.; Nuttall, P. A.; Gould, E. A. Tick-Borne
Flaviviruses. Adv. Virus Res. 2003, 61, 317−371.
(5) Mansfield, K. L.; Johnson, N.; Phipps, L. P.; Stephenson, J. R.;
Fooks, A. R.; Solomon, T. Tick-Borne Encephalitis Virus: A Review of
an Emerging Zoonosis. J. Gen. Virol. 2009, 90, 1781−1794.
(6) Ruzek, D.; Yakimenko, V. V.; Karan, L. S.; Tkachev, S. E. Omsk
̊
̌
Haemorrhagic Fever. Lancet. 2010, 376, 2104−2113.
(7) Gholam, B. I. A.; Puksa, S.; Provias, J. P. Powassan Encephalitis:
A Case Report with Neuropathology and Literature Review. Can. Med.
Assoc. J. 1999, 161, 1419−1422.
(8) Raval, M.; Singhal, M.; Guerrero, D.; Alonto, A. Powassan Virus
Infection: Case Series and Literature Review from a Single Institution.
BMC Res. Notes 2012, 5, 594.
(27) De Burghgraeve, T.; Kaptein, S. J. F.; Ayala-Nunez, N. V.;
Mondotte, J. A.; Pastorino, B.; Printsevskaya, S. S.; de Lamballerie, X.;
Jacobs, M.; Preobrazhenskaya, M.; Gamarnik, A. V.; Smit, J. M.; Neyts,
J. An Analogue of Antibiotic Teicoplanin Prevents Flavivirus Entry In
Vitro. PLoS One 2012, 7, e37244.
̌
(28) Sali, A.; Blundell, T. L. Comparative Protein Modelling by
(9) Heinz, F. X.; Stiasny, K. Flaviviruses and Flavivirus Vaccines.
Satisfaction of Spatial Restraints. J. Mol. Biol. 1993, 234, 779−815.
(29) McGann, M. FRED Pose Prediction and Virtual Screening
Accuracy. J. Chem. Inf. Model. 2011, 51, 578−596.
(30) Dyachenko, V. D.; Krivokolysko, S. G.; Nesterov, V. N.;
Litvinov, V. P. Synthesis and Properties of N-Methylmorpholinium 4-
Aryl-6-methyl-5-phenylcarbamoyl-3-cyano-1,4-dihydropyridine-2-thio-
lates. Molecular and Crystalline Structure of 2-Allylthio-6-methyl-5-
phenylcarbamoyl-4-(2-chlorophenyl)-3-cyano-1,4-dihydropyridine.
Chem. Heterocycl. Compd. 1996, 32, 1066−1074.
(31) Dyachenko, V. D.; Krivokolysko, S. G.; Litvinov, V. P. Synthesis
of N-Methylmorpholinium 6-Methyl-4-(2-thienyl)-5-phenylcarbamo-
yl-3-cyano-1,4-dihydropyridine-2-thiolate and Its Reaction with
Various Functionally Substituted Methyl Halides. Chem. Heterocycl.
Compd. 1997, 33, 577−582.
Vaccine 2012, 30, 4301−4306.
(10) Pen’evskaia, N. A.; Rudakov, N. V. Efficiency of Use of
Immunoglobulin Preparations for the Postexposure Prevention of
Tick-Borne Encephalitis in Russia (A Review of Semi-Centennial
Experience). Med. Parazitol. 2010, 1, 53−59.
(11) Khudoley, V. N.; Saratikov, A. S.; Lepekhin, A. V.; Yavorskaya,
V. E.; Evstropov, A. N.; Portnyagina, E. V.; Pomogaeva, A. D.;
Beloborodova, E. I.; Vnushinkaia, M. A.; Schmidt, E. V.; Krilova, N. V.;
Khunafina, D. K.; Mezenzeva, M. V.; Ershov, F. I.; Raevski, K. K.;
Vlasova, E. V.; Abdulova, G. A.; Kropotkina, E. A. Antiviral Activity of
Jodantipyrin: An Anti-Inflammatory Oral Therapeutic with Interferon-
Inducing Properties. Anti-Inflammatory Anti-Allergy Agents Med. Chem.
2008, 7, 106−115.
(12) Krylova, N. V.; Leonova, G. N.; Maistrovskaia, O. S.; Popov, A.
M.; Artiukov, A. A.; Kozlovskaia, E. P. In Vitro Activity of Luromarin
against Tick-Borne Encephalitis Virus. Antibiot. Khimioter. 2010, 55,
17−19.
(13) De Clercq, E. Strategies in the Design of Antiviral Drugs. Nat.
Rev. Drug Discovery 2002, 1, 13−25.
(32) Dotsenko, V. V.; Krivokolysko, S. G.; Chernega, A. N.; Litvinov,
V. P. Synthesis and Structure of Pyrido[2,1-b][1,3,5]thiadiazine
Derivatives. Dokl. Chem. 2003, 389 (No. 4−6), 92−96.
(33) Nesterov, V. N.; Krivokolysko, S. G.; Dyachenko, V. D.;
Dotsenko, V. V.; Litvinov, V. P. Synthesis, Properties, and Structures
E
dx.doi.org/10.1021/ml400226s | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
of Ammonium 4-Aryl-5-cyano-2-oxo-1,2,3,4-tetrahydropyridine-6-thi-
olates. Russ. Chem. Bull. 1997, 46, 990−996.
(34) Krivokolysko, S. G.; Dyachenko, V. D.; Litvinov, V. P. A New
Method for the Synthesis of N-Methylmorpholinium 4-Aryl-5-cyano-
2-oxo-1,2,3,4-tetrahydropyridine-6-thiolates and Their Properties.
Russ. Chem. Bull. 1997, 46, 1758−1762.
F
dx.doi.org/10.1021/ml400226s | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX