Interaction of Flaviviruses with Reproduction Inhibitors Binding in β-OG Pocket: Insights from...

DOI: 10.1002/minf.201300185

Interaction of Flaviviruses with Reproduction InhibitorsBinding in b-OG Pocket: Insights from Molecular DynamicsSimulationsEvgenia V. Dueva,[a, b, c] Dmitry I. Osolodkin,[a, b, c] Liubov I. Kozlovskaya,[b] Vladimir A. Palyulin,*[a, c]

Vladimir M. Pentkovski,[c] and Nikolay S. Zefirov[a]

1 Introduction

Design of novel antiviral compounds is one of the most im-portant tasks of contemporary medicinal chemistry. Differ-ent strategies were suggested for solving this problem, andcertain successes were achieved.[1] One of prominent antivi-ral drug design strategies is the inhibition of viral fusion –the process of interaction between cellular and viral mem-branes resulting in the release of viral genetic material intothe host cell cytoplasm.[2] This strategy was successfully ap-plied to the design of entry inhibitors for HIV,[3] influenza vi-ruses,[2] alphaviruses,[2] and flaviviruses.[4] Nevertheless,mechanisms of the processes underlying fusion inhibitionare not sufficiently studied, thus hampering developmentof this research field.

Membrane fusion of enveloped viruses is a complexmulti-step process driven by conformational changes ofviral envelope proteins.[2,5] Three classes of fusion proteinsare known, exemplified by influenza virus, flaviviruses, andvesicular stomatitis virus, for which prefusion and postfu-sion conformations are studied by X-ray crystallography.[6]

Flavivirus fusion is of special interest due to the fast rate ofthis process,[7] making it most suitable for computationalstudies. The key step in the flavivirus fusion is the pH-de-pendent conformational rearrangement of envelope pro-teins E, which form dimers on the surface of the maturevirion. When virus particle interacts with the cellular recep-tors, an endosome is formed enclosing the virion. The en-dosomal pH level of about 6 launches the conformational

rearrangements of E proteins, leading to the formation ofspikes which attack the endosome membrane.

The so-called ‘histidine switch’ hypothesis was proposedto explain the driving force of such rearrangement, whichis supposed to be the protonation of histidine residues inthe endosome media.[8,9] The movement of E proteins leadsto the formation of a membrane stalk and then a mem-brane pore, which allows viral genetic material to be re-leased into the host cell.[2,5,7] Compounds, which bind enve-lope proteins in the sites important for the conformationalrearrangement, should prevent fusion, and thus can act aspotential antiviral drugs.

Abstract : Flaviviral diseases, including dengue fever, WestNile fever, yellow fever, tick-borne encephalitis, Omsk hae-morrhagic fever, and Powassan encephalitis, threatenhuman health all over the world. Lack of effective antiviralstargeting replication cycle of flaviviruses makes the searchof such compounds a challenging task. Recently we haveidentified a reproduction inhibitor effective against tick-borne encephalitis virus and Powassan virus (POWV) (ACSMed. Chem. Lett. , 2013, 4, 869–874). To enable using this in-hibitor as a template for 3D pharmacophore search, a bio-

logically active conformation of this molecule should havebeen established. Here we performed molecular dynamicssimulations of the complexes between the different enan-tiomers of the inhibitor and POWV envelope (E) proteins,putative targets of the inhibitor, in the different protona-tion states corresponding to the different stages of mem-brane fusion process. Several stable conformations of theinhibitor were identified, opening routes for further designof more advanced molecules.

Keywords: Powassan virus · Flavivirus · Antivirals · Envelope proteins · Molecular dynamics

[a] E. V. Dueva, D. I. Osolodkin, V. A. Palyulin, N. S. ZefirovDepartment of Chemistry, Lomonosov Moscow State UniversityMoscow 119991, Russiaphone: + 7-495-939-39-69, fax: + 7-495-939-02-90*e-mail : [email protected]

[b] E. V. Dueva, D. I. Osolodkin, L. I. KozlovskayaChumakov Institute of Poliomyelitis and Viral EncephalitidesMoscow 142782, Russia

[c] E. V. Dueva, D. I. Osolodkin, V. A. Palyulin, V. M. PentkovskiiScalare Laboratory, Moscow Institute of Physics and TechnologyDolgoprudny 141700, Russia

Supporting Information for this article is available on the WWWunder www.molinf.com.

� 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Mol. Inf. 2014, 33, 695 – 708 695

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Viruses belonging to the Flavivirus genus are the causa-tive agents of various infections, the most known amongthem being dengue fever, yellow fever, Japanese encephali-tis, West Nile fever and tick-borne encephalitis (TBE).[10]

Dengue virus (DENV), Japanese encephalitis virus (JEV),West Nile virus (WNV), and yellow fever virus (YFV) aretransmitted by mosquitoes, whereas TBE virus (TBEV)[11]

along with less known Powassan virus (POWV)[12–15] andOmsk hemorrhagic fever virus (OHFV)[16] are transmitted byticks. Tick-borne flaviviral diseases are endemic for Russia,Europe, and North America. The most studied among themis tick-borne encephalitis with about 9000 cases everyyear.[11] Vaccines are available for this disease, but postinfec-tion treatment is mostly symptomatic ; in many cases TBEresults in death or serious disability. Powassan encephalitisand Omsk hemorrhagic fever can be considered as veryrare diseases, however strong neurodegeneration that theycause and the unavailability of treatment makes them a seri-ous health concern for endemic regions (the Great Lakesregion and New England for the former, Omsk, Tyumen,and Novosibirsk regions for the latter).[12,13,15]

The common strategy of flavivirus fusion inhibitorsdesign was first applied by Yang et al. , who performed thedocking of the Comprehensive Medicinal Chemistry libraryinto a hydrophobic pocket of the X-ray structure of solubleectodomain sE of DENV (serotype 2) envelope protein andfound two tetracycline derivatives to inhibit DENV fusion.[16]

This pocket can accommodate a detergent (namely, n-octyl-b-d-glucoside (b-OG)) molecule.[17] It is located in theinterface between domains I and II of E protein, and bind-ing of a molecule in this pocket can disrupt the conforma-tional rearrangement that occurs during viral fusion. Fur-ther studies were performed in a similar manner, but dock-ing hits were tested not only against DENV, but also againstYFV,[18–22] WNV,[20,21] and JEV.[20] Competitive binding of thehits in the b-OG pocket was shown by saturation transferdifference NMR method.[18] Inhibitors that affect the latestages of fusion could also bind in this pocket in the trimer-ic state of E protein.[23]

The mechanism of fusion inhibition by b-OG pocketbinders was not simulated thoroughly. Stability of sE pro-tein dimers from TBEV[24,25] and DENV[26–28] was assessed inmolecular dynamics (MD) simulations at different pH levels(exemplified by different His protonation states). The pathfrom ‘high-pH’ to ‘low-pH’ conformation of sE protein mo-nomer and stability of these conformations in different en-vironments were studied with the help of metadynamicsmethodology.[29] It was shown that prefusion dimers arestable at neutral pH, whereas at low they are less stable,and postfusion trimers are favored at low pH but not atneutral pH.[26–29] Also, bending and twist motions of sEdimers were observed in neutral pH simulations.[24,25,27] Onthe other hand, such motions were not observed in theMonte-Carlo simulation of the full E dimer including trans-membrane parts and M proteins.[30] . It was shown thatcharacteristics of these motions (for example, bending

angle) correlate with virion properties such as thermostabil-ity; variants characterized by large-scale motions are moresusceptible to increased temperature and detergent treat-ment.[24,25]

Our recent efforts led to development of a series of re-production inhibitors for TBEV, POWV, and OHFV:[31] 2-(al-kylthio)-N,4-diaryl-3-cyano-6-methyl-1,4-dihydropyridine-5-carboxamides (1) and 8-aryl-6-oxo-3,4,7,8-tetrahydro-2H,6H-pyrido[2,1-b][1,3,5]thiadiazine-9-carbonitriles (2). Compound3 is the most potent inhibitor of both TBEV and POWV,showing IC50 of about 1.3 mM against POWV and 0.07 mMagainst TBEV in the plaque reduction assay. In this studywe analyze the binding mode of 3 in the b-OG pocket ofPOWV and the influence of its binding onto the conforma-tional stability of POWV sE protein dimer, using the implicitsolvent MD simulation protocol analogous to that used inour previous works.[24,25] Analysis of the inhibitor’s confor-mational space in the putative binding pockets would facil-itate the design of novel inhibitors based on shape similari-ty, bioisosteric replacement and scaffold hopping. Thestudy of POWV molecular dynamics is of special interest de-spite the rarity of this virus, because it is the flavivirus trans-mitted both by ticks and mosquitoes.[32]

2 Materials and Methods

2.1 Modeling of the Protein Structures

Homology modeling was performed for soluble ectodo-main (sE) dimers of POWV E protein in two states: ‘open’state resembling that of 1OKE X-ray structure with open b-OG pocket[17] and ‘closed’ state similar to the one observedin other X-ray structures of sE (particularly, 1OAN[17] fromDENV serotype 2 and 1SVB[33] from TBEV). Homology mod-eling procedure is the same as described in the literature[31]

and is based on the sequence alignment provided there.

Scheme 1.



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Amino acid sequences of the templates were extractedfrom the corresponding PDB files, and the target sequenceof POWV E protein was obtained from GenBank (accessioncode NC_003687.1[34]). Sequence alignment was performedin ClustalX 2.0.11[35] with BLOSUM62 substitution matrix.Homology modeling was performed in Modeller9v10[36]

based on this sequence alignment. The closed state modelof POWV E protein ectodomain was constructed based on1OAN and 1SVB templates; 1SVB template sequence identi-ty to the target is the highest among the available tem-plates, but one subunit of this template is a symmetriccopy of the other one, whereas 1OAN allows taking into ac-count possible asymmetry of the inter-subunit interactions.The open state POWV sE protein models were built basedon both the structure of DENV sE protein complex with b-OG (PDB ID 1OKE) and the closed state model of POWV sEwith kl loop region (residues 273–287) removed. Carbohy-drate moieties present in the templates were removed forsimplicity.

As soon as 1SVB template consists of a single subunit (itspartner in the dimer can be reconstructed using symmetryoperations), we call the corresponding subunit in ourmodels ‘subunit A’ and the reconstructed dimer subunit isreferred to as ‘subunit B’.

Fifty models of dimeric soluble domains were built forthe closed and open states of POWV sE protein, then simu-lated annealing procedure was applied to each model. Thequality of secondary structure packing was evaluated byProSa2003.[37] The choice of the best models was based onthe DOPE score implemented in Modeller and PROCHECKRamachandran plot quality.[38] The selected models weresubsequently optimized in SYBYL 8.0[39] by 200 steps of thePowell minimization in the Tripos force field.[40]

2.2 Ligand Docking

OpenEye software suite was used in the course of molecu-lar docking. The docking procedure consisted of the follow-ing steps: (1) tautomeric and protonation state cleanup inQUACPAC 1.5.0; (2) exhaustive conformer and enantiomergeneration in OMEGA 2.4.6;[41] (3) MMFF charges[43] assign-ment in QUACPAC 1.5.0[43] ; (4) rigid molecular docking ofthe pregenerated conformer ensembles into the rigid pro-tein site in FRED 2.2.5[44] with consensus of Pairwise LinearPotential (PLP),[45] Chemgauss3,[46] and Chemscore[47] scoringfunctions with equal weights used during pose selection,10 alternative poses were generated for each enantiomer;(5) scoring of alternative poses with Chemgauss3, Chem-score, OEChemscore, PLP, Screenscore,[48] Shapegauss,[46]

and consensus of these scoring functions; (6) visual analysisof the binding modes.

The docking was performed into the b-OG pocket of sub-unit A; then the selected conformer was docked rigidly intothe b-OG pocket of subunit B to obtain the final dockingsolutions for the use in further MD simulations.

2.3 Molecular Dynamics Simulations

Molecular dynamics simulations were performed usingAMBER 11 program suite[49] on iScalare supercomputer atMoscow Institute of Physics and Technology and SKIF MSU‘CHEBYSHEV’ supercomputer at the Research ComputingCenter, Lomonosov Moscow State University. Protocol ofthe simulation closely resembled the one used in our previ-ous works.[24,25] The AMBER ff99SB force field[50] was appliedfor the protein molecules, while GAFF[51] was used for thesmall molecules. Partial atomic charges were automaticallyassigned in LeAP module of AMBER suite to the proteinatoms, and AM1-BCC charges[52] were assigned to theligand molecules. Bonds involving hydrogen atoms wereconstrained using SHAKE algorithm.[53] Generalized Bornsolvent model was utilized to mimic solvent effects.[54] Dif-ferent pH levels were considered solely as different proto-nation states of His residues: in the normal pH model histi-dine residues were protonated on the Ne atom (AMBER HIEresidue), whereas in the low pH model all histidines wereprotonated on both N atoms of the imidazole moiety(AMBER HIP residue). For the sake of completeness, wehave also estimated His pKa values by PROPKA,[55] H + + ,[56]

and MCCE[57] (Supporting Information Table S1). Based onthese results all histidines were decided to be protonatedfor the simplicity of the model.

Energy minimization of the systems was performed priorto production dynamics run and consisted of 1000 itera-tions of steepest descent minimization and 1000 iterationsof conjugated gradient minimization. Production MD wasrun in sander with the following parameters: time step –2 fs, number of iterations – 15 millions (30 ns), system tem-perature was kept equal to 300 K by Langevin thermostatwith collision frequency of 1 ps�1. Visual analysis of trajecto-ries was performed with VMD;[58] statistical analysis wasmade in ptraj module of the AMBER suite.[59]

3 Results

3.1 Homology Modeling of Protein Structures

The POWV E protein ectodomain is more similar to sE pro-teins from tick-borne viruses compared to the proteinsfrom mosquito-borne viruses. According to the amino acidsequence alignment, there is 30 % identity of the DENV andPOWV sE protein sequences and 63 % identity between theTBEV and POWV sE sequences. POWV sE protein bears twoinsertions characteristic for tick-borne viruses compared tothe mosquito-borne viruses in the loops near the b-OGbinding region of E protein, namely, fg loop insertion of sixresidues (205–210) and the single residue 278 insertion inthe kl loop (Figure 1). Deletions appear in the loops E0F0, hi,and FG compared to DENV. A very specific feature of thePOWV sE protein is the presence of an additional residue337 in the domain III, which is typical for mosquito-trans-mitted viruses, while the main POWV vectors are ticks.



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Thus, the use of two templates (one from TBEV and onefrom DENV) for the closed form as well as for the openform models is clearly reasonable despite the lower se-quence homology of the DENV template.

As could be expected from the homology modeling,POWV sE protein models exhibited all characteristic struc-tural features of flavivirus sE proteins. Namely, they havethree well-defined domains with prevalent b-strand secon-dary structures[33] (Figure 1). The shape and size of themodels are also very similar to those of the template struc-tures. Mean all-atom RMSDs of 49 models from selectedstructures are 1.41 � and 0.47 � for closed and open formsrespectively (Supporting Information Table S2). Low RMSDshows small dispersion of candidates and reflects highstructural conservation. As far as the modeling was per-formed without symmetry restraints for twin subunits,heavy-atom RMSD for the subunits of the closed and openforms was 1.14 and 1.36 �, respectively.

Stereochemical quality of the models was assessed byPROCHECK and ProSa and compared with the templatecrystal structures (Table 1). In the closed form of POWV sEprotein no residues were found in the disallowed regionsof PROCHECK Ramachandran plot, whereas for the openstate three residues were observed in these regions(Ala249A, Ala249B, and Asp178A, where letter after the

number is the chain identifier), two of them (Ala249A,B)being inherited from the 1SVB template. However, theseresidues are located far from the b-OG binding pocket, andoverall stereochemical quality of the models was consid-ered as acceptable. It was also confirmed by the values ofProSa z-scores.

3.2 Docking of the Inhibitor

In order to obtain reasonable starting geometries of theligand-protein complex for the MD simulation, moleculardocking of both enantiomers of 3 was performed into theb-OG pocket of subunit A of the open state POWV sEmodel (Figure 2). The scores for the best-docked solutionsaccording to the consensus of functions are presented inTable 2. Three scoring functions (both Gaussian-based andScreenscore) showed strong preference for (S)-3 over (R)-3,whereas PLP showed negligible difference. On the otherhand, empirical Chemscore-based functions slightly pre-ferred (R)-isomer over (S)-isomer. Score contributions areprovided in the (Supporting Information Table S3).

The equivalent chemical groups of two enantiomerswere placed by the docking program in the opposite re-gions of the pocket in such a way that (R)-3 formed the hy-drogen bonds with Ser201 and Ser52 and its bicyclic core

Figure 1. POWV sE dimer model colored by domains: DI – red, DII – yellow, DIII – blue. Alignment of subunits A in the closed and openstates is shown on the lower subunit : kl loop in the open conformation is colored mauve and in the closed state cyan. Regions with inser-tions compared to templates are also marked (Lys337, kl loop, fg loop – orange for the open state and brown for the closed). Positions ofHis residues are shown at the top subunit of open sE. Fusion loops are colored green.

Table 1. Stereochemical quality of models and templates.

Protein Number ofresidues

Most favoredregion[a]

Additional allowedregion[a]

Generously allowedregion[a]

Disallowedregion[a]

z-score

1OAN 788 492 (73.2 %) 178 (26.5 %) 2 (0.3 %) 0 �11.391OKE 788 554 (82.4 %) 114 (17.0 %) 4 (0.6 %) 0 �11.021SVB 790 620 (91.2 %) 54 (7.9 %) 4 (0.6 %) 2 (0.3 %) �11.74POWV sE closed state 792 636 (92.4 %) 47 (6.8 %) 5 (0.7 %) 0 �11.41POWV sE open state 792 577 (83.9 %) 102 (14.8 %) 6 (0.9 %) 3 (0.4 %) �11.12

[a] Number of non-Gly residues in the mentioned regions and their percentage among all non-Gly residues.



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formed van der Waals contact with Val199, whereas (S)-3did not form any hydrogen bonds with protein residues(Figure 2).

The best-docked conformers of (S)-3 and (R)-3 were rigid-ly docked into the b-OG pocket of the subunit B to obtainthe starting structures for the MD simulations. The initialstates of the protein-inhibitor complexes at low pH wereobtained by changing HIE residues to HIP.

3.3 Molecular Dynamics Simulation

Thirty nanoseconds of molecular dynamics simulationswere performed for sE protein dimers in closed and open

states and for complexes with two molecules of inhibitor.Characteristics of the simulations are provided in Table 3.

3.3.1 Bending

The main visible component of protein movement in allsystems is bending (Figure 3a), which was also observed inearlier studies.[24,25] Superposition of all trajectory frames bymatching backbone atoms of domains I and III revealedthat bending can be attributed to the rotation of domain IIrelative to DI and DIII in the plane perpendicular to thedimer plane (Figure 3b). We characterized this movementanalogously to our previous papers[24,25] by bending angle

Figure 2. The best-scored poses of (R)-3 (dark green) and (S)-3 (light green). Ser275 belongs to kl loop and Ser201 is located in fg loop.

Table 2. Docking scores for the enantiomers of compound 3.

Shapegauss PLP Chemgauss3 Chemscore OEChemscore Screenscore

(S)-3 �482.7 �47.6 �50.7 �19.7 �34.2 �116.6(R)-3 �438.3 �47.4 �39.3 �21.1 �35.8 �91.8

Table 3. Parameters for systems utilized in MD simulations.

State of sE protein His residues state Abbreviation

Closed neutral apo_c_H0

Closed protonated apo_c_H+

Open neutral apo_o_H0

Open protonated apo_o_H+

Complexed with two molecules of (R)-3 neutral R_H0

Complexed with two molecules of (R)-3 protonated R_H+

Complexed with two molecules of (S)-3 neutral S_H0

Complexed with two molecules of (S)-3 protonated S_H+



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f measured between the centers of mass of DIII of sub-unit A, the whole dimer, and DIII of subunit B. In all systemsangle varies quasi-periodically (Figure 4).

Having started from almost a planar conformation (f=1768), sE dimers bend to different extents (Table 4). At neu-tral pH the bending amplitude of the free proteins is small-er compared to the systems with bound inhibitors (Fig-ure 4a), and the most significant angle change is observedfor simulation R_H0, where the extreme value of the bend-ing angle reaches 1278.

Although at neutral pH the closed form undergoes thesmallest bending, it loses the contact between the fusionloop of chain A and DIII of adjacent subunit after 22 ns ofsimulation in contrast to other systems (Figure 5 and Sup-porting Information Movie). The process of fusion loop sep-aration could be described as if subunit A adopted thebent conformation and the subunit B started to return to

the initial planar state by rotating back DII, that resulted inthe disruption of the contact between the fusion loop andsubunit B DIII. Such dissociation is not observed in the sim-ulation apo_c_H+ .

Simulations at acidic pH (with protonated His residues)lead to insignificant negative changes in values of meanand peak bending angles in apo_o_H+ and positivechanges in R_H+ , while a large increase in S_H+ and a de-crease in apo_c_H+ are observed. These values fall by 20degrees for the closed form and rise by about 10 degreesfor S_H+ (Figure 4b).

3.3.2 Inhibitor Behavior

Behavior of the inhibitor molecules in both b-OG pocketsof the dimer was observed separately along the MD trajec-tories (Figure 6).

Figure 3. Bending of sE dimer in the course of MD. a) Alignment of R_H0 starting frame and lowest f frame by chain A DI and DIII. b) Anal-ogous alignment of every 1000th R_H0 frame.

Table 4. Bending characteristics for different MD runs. Mean angle and standard deviation are calculated after first 5 ns.

apo_c_H0 apo_o_H0 R_H0 S_H0 apo_c_H+ apo_o_H+ R_H+ S_H+

Min f (8) 157 143 127 137 137 140 132 147Mean f (8) 170 162 144 153 150 157 149 161SD (8) 4.62 6.33 5.20 5.74 4.74 5.63 5.25 4.25

Figure 4. Bending angle vs. time plot: a) normal pH; b) acidic pH. Blue – closed form, orange – open form, dark green – complex with(R)-3, light green – complex with (S)-3.



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The orientation of (R)-3 in the subunit A pocket in R_H0

was not stable for the first 15 nanoseconds (Figure 6). Thebicyclic core of the molecule moved in the pocket, rotationof chlorophenyl and methoxy groups around C�O bondswas also observed. For the rest 15 ns the ligand adopteda stable orientation with tetrahydropyridine ring laying par-allel to the fg loop and forming van der Waals contact withVal199A (Figure 2); nitrile nitrogen atom was directed to-wards the Ser201A side chain, forming a weak hydrogen

bond with the hydroxyl group (Figure 7a). The key bindinginteraction, maintained along the trajectory, was formedbetween the ligand and Gln50A (Table 5). Its side chainpointed towards the ligand after 1 ns and formed the hy-drogen bond between amide NH and ligand’s carbonyloxygen atom (Figure 7a). A similar stable orientation of(R)-3 in the subunit B pocket was observed after 2.5 ns (Fig-ure 7b), though in this pocket the ligand does not forma hydrogen bond with Ser201.

Figure 5. Dissociation of chain B fusion loop (green) and chain A DIII. Domains are colored as in Figure 1.

Figure 6. RMSD vs. time plots for the ligands.



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Upon the protonation of histidine residues (trajectoryR_H+), the binding modes of the ligand were similar to theones observed in R_H0, except the lack of hydrogen bond-ing between the ligand nitrile moiety and Ser201. Thestable states were achieved much faster, after 1.5 and1.0 ns in the pockets A and B, respectively (Figures 6 and7c,d). In the pocket A RMSD of the ligand from the initialstructure varied greater than in the pocket B (Figure 6). Oc-cupancies of hydrogen bonds between the inhibitor andGln50 were slightly lower than in R_H0 (Table 5).

Orientation of (S)-3 became stable in the subunit Apocket after 5 ns in the simulation S_H0. The nitrile grouppointed to the kl loop and formed a weak hydrogen bondwith hydroxyl group of Ser275A after 7 ns (Figure 8a). An-other hydrogen bond was formed between the carbonylgroup of the ligand and Gln50A side chain amide NH. Inthe pocket B such interaction disappeared after 11 ns, the

inhibitor did not form other hydrogen bonds with the pro-tein and moved in the pocket vigorously. The orientation ofthe ligand in the pocket B was relatively stable only for thelast 4 ns (Figure 8b).

The trajectory S_H+ showed another way of behavior ofthe ligands. Between 2 and 16 ns the core of (S)-3 wasfixed in the subunit A pocket by hydrogen bonds with sidechains of Gln50A and Ser275A. After 18 ns the ligand rotat-

Figure 7. Stable orientations of (R)-3 : a) in the pocket A of R_H0 ; b) in the pocket B of R_H0; c) in the pocket A of R_H+ ; d) in the pocket Bof R_H+ . Hydrogen bonds are colored light green.

Table 5. Occupancy percent of hydrogen bonds between ligandcarbonyl oxygen atom and Gln50.

R_H0 R_H+ S_H0 S_H+

Subunit A 63 42 68 16 (1.8 to 15.6 ns)Subunit B 53 44 29 (0 to 11 ns) 73



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ed to direct the nitrile group inwards the pocket and todirect the carbonyl group outside the pocket without form-ing new hydrogen bonds. Chlorobenzoxy group was direct-ed into the hole between the subunits (Figure 8c). In thepocket B the ligand position was fixed by the hydrogenbond between Gln50B and ligand carbonyl group; nitrilemoiety was directed towards the Ser275B, but hydrogenbond was not identified (Figure 8d).

The conformations of core tetrahydropyridothiadiazinerings of 3 in the stable orientations were similar in all pock-ets (mean RMSD 0.123 � for (R)-3 and 0.258 � for (S)-3).

Thus, in the course of MD simulations both (R)- and (S)-enantiomers of 3 changed their orientation in the b-OGpocket and revealed different hydrogen bond patternscompared to the initial states. The orientation of (R)-iso-mers in both simulations was stabilized by the hydrogenbonds with Gln50 in both pockets of the sE dimer, while

for the (S)-isomer such fixation appeared only in onepocket.

3.3.3 Hydrogen Bonding

The overall number of hydrogen bonds in low pH trajecto-ries was higher as compared to that in neutral pH. Whilethe number of hydrogen bonds between subunits changesconsiderably for closed form and complex with (S)-3, itslightly increases for the complex with (R)-3 and for theopen form upon transition from neutral to low pH (Table 6).

Salt bridges between kl loop and domain II of the neigh-boring subunit were observed in apo_o_H0 simulation.They were formed by charged side chains of Glu277A andLys251B and by Glu277B and Lys251A (Figure 9), thesecond one being less occupied (Table 7). In the cases ofR_H0 and S_H0, only one salt bridge existed between kl

Figure 8. Stable orientations of (S)-3 : a) in the pocket A of S_H0 ; b) in the pocket B of S_H0 ; c) in the pocket A of S_H+ ; d) in the pocket Bof S_H+ . Hydrogen bonds are colored light green.



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loop and the adjacent subunit. In apo_o_H+ and S_H+

both bridges were highly occupied, but only one of themwas observed in R_H+ .

Interactions between Glu277 and His248 of differentchains were present simultaneously with the aforemen-tioned salt bridges for apo_o_H+ and S_H+ , while for R_H+

formation of a hydrogen bond between His248B and Glu7Awas observed.

In the forms with kl loop in closed state Glu277A andLys251B from different subunits were located quite far fromeach other and salt bridge was not formed.

In the closed form simulation apo_c_H0 both His248 didnot participate in hydrogen bonding with other residues,but such interactions appeared in apo_c_H+ : His248Aformed hydrogen bonds with the backbone carbonyls ofLys206B and Val276B during the first 15 ns, and for the restof the trajectory His248A interacted with Ser204B backboneand Asp207B. The hydrogen bond His248B�Glu7A, similarto R_H+ , was observed in the second subunit interface.

Other intersubunit interactions that appeared after histi-dine protonation were hydrogen bonds of His323 and His5

with Asp98B. These bonds tie together domains I and II ofsubunit A and the fusion loop of subunit B (Figure 10).

4 Discussion

POWV reproduction inhibitor 3 was identified during thedocking-based virtual screening of ChemEx databaseagainst b-OG pocket of open POWV sE protein model. Thiscompound showed the highest potency in the plaque for-mation inhibition assay against 280 PFU/mL POWV with IC50

of 1.3 mM.[31] This result was obtained for the racemic mix-ture of two enantiomers of this compound, (S)-3 and (R)-3.In this work we performed time of addition experiments,confirming the realization of inhibition through the interac-tion of 3 with virions prior to entry but not the cells (seeSupporting Information for the details).

Although we did not obtain the distinct evidence of theinteraction between compound 3 and the E protein as wellas of its binding in the hydrophobic pocket, we assumethis hypothesis to be reasonable as it allowed us to find

Table 6. Number of hydrogen bonds.

apo_c_H0 apo_o_H0 R_H0 S_H0 apo_c_H+ apo_o_H+ R_H+ S_H+

Overall number of hydrogen bonds[a] 160 161 163 162 175 170 168 170Trajectory average number of intersubunit hydrogen bonds[b] 4.5 4.7 5.6 5.6 7.9 5.8 6.1 9.1

[a] The standard deviation for the overall number of hydrogen bonds is equal to 11. [b] The standard deviation for the average number ofintersubunit hydrogen bonds is equal to 2.

Figure 9. a) The kl loop (green) in the closed state is included in b-sheet D0E0F0 (red) of domain I. b) The kl loop in the open state is includ-ed in b-sheet efg (yellow) of domain II, and its Glu277 forms a salt bridge with Lys251of the neighboring subunit.

Table 7. Occupancy of hydrogen bonds between the kl loop and adjacent subunit.

Occupancy of hydrogen bonds apo_o_H0 apo_o_H+ R_H0 R_H+ S_H0 S_H+

Glu277A–Lys251B 71 90 77 34 (10 to 20 ns) 0 89Glu277A–His248B 0 96 18 0 0 60Glu277B–Lys251A 43 (0 to 20 ns) 94 0 88 85 93Glu277B–His248A 0 63 0 0 0 92



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this structure via docking in the b-OG pocket. In a similarlydesigned study,[18] b-OG competitive YFV reproduction in-hibitor was identified by docking to DENV b-OG pocket,and the competition was shown in a saturation transfer dif-ference NMR experiment.

We have performed molecular dynamics simulationsaiming to reveal the mechanistic insights into the interac-tion pattern between the small molecule antiviral com-pound and the POWV envelope protein. Two protonationstates of the protein were studied, corresponding to theresting state (pH~7, His residues are neutral) and thefusion-ready state (pH~6, His residues are protonated). Dif-ferent states of b-OG pocket were studied, and both enan-tiomers of 3 were subjected to the simulation, becausethere are no experiment-grounded reasons to prefer oneenantiomer to another.

The previous MD studies of flavivirus sE protein dimersshowed that the main visible component of the proteinmovement is its bending.[24,27] It was shown that the bend-ing pattern correlates with such virion properties as stabili-ty and infectivity, despite the significant simplification ofthe model compared to a real-world system. Interactionwith the inhibitors consistently affected the bending pat-tern in all the current simulations, thus expanding the ap-plicability domain of the bending model.

4.1 Neutral pH Simulations

Molecular dynamics simulations of sE protein dimers in theclosed b-OG pocket state with uncharged histidine residueswere performed previously for DENV (8 ns)[26] and TBEV(10 ns).[24] Similarly to TBEV, bending of POWV sE protein

dimer with b-OG pocket closed by kl loop is observed. Aspecific feature of POWV sE protein dimer movement inthe course of MD is the loss of interaction between thefusion peptide of subunit A and domain III of subunit Bafter 22 ns. E protein dimers from DENV and TBEV did notshow the tendency for dissociation during 8 ns and 10 nssimulations respectively;[24,26] the 50 ns simulation of TBEVsE dimer also did not lead to the dissociation of the dimer(Osolodkin, unpublished results). Whereas the absence ofthe dimer dissociation in the short simulations could beclearly attributed to insufficient simulation time, it shouldbe taken into account that sE protein from West Nile virusexists as a monomer in solution in contrast to a monomer-dimer equilibrium at neutral pH of DENV sE protein.[60–62]

The X-ray structures (PDB ID 2HG0, 2I69) revealed that therelative orientation of DI and DIII of WNV sE is identical toother prefusion flavivirus dimers, but domain II is rotatedfor 13 to 208 in the plane of the dimer. In our case ofPOWV sE MD simulation, the rotation of DII takes place inthe plane perpendicular to the dimer plane.

Previous explicit solvent MD simulations of the DENV Eprotein dimer in the open b-OG pocket state for 60 ns and10 ns respectively based on the 1OKE structure at neutralpH did not reveal any significant structure changes.[27,28]

Bending of POWV sE in the open state is more pronouncedcompared to the closed state. Inhibitor binding in the b-OGpockets increases bending further, as shown by the de-crease of bending angle values. Despite the greater move-ment amplitude in apo_o_H0, R_H0, and S_H0 as comparedto apo_c_H0, the dissociation of the dimers is not observedin the open state simulations, being the result of dimer sta-bilization by open kl loops that partially fill in the holes be-tween subunits and form the intersubunit salt bridgesthrough Glu277 (from kl loop) and Lys251 (from the neigh-boring subunit ij hairpin). Even though, the occupancies ofthese salt bridges are not equivalent: they differ two-fold inapo_o_H0, and only one salt bridge per complex is presentin the cases of complexes with inhibitors dimer contactscan also be strengthened by van der Waals and hydropho-bic interactions between the open kl loop and the neigh-boring subunit. Fixation of the kl loop in the open state bythe salt bridge allows the inhibitor to reach the stable ori-entation quicker than in the non-stabilized pocket.

4.2 Low pH Simulations

According to the ‘histidine switch’ hypothesis, transitionfrom the dimeric resting state of the E protein to thefusion-ready trimeric state in low pH environment is con-trolled by the protonation of histidine residues and thesubsequent structure reorganization.[8,9] We have performedthe simplest assessment of this hypothesis via an MD simu-lation of POWV sE in the implicit solvent with the protonat-ed His residues. Such simplified system allows fast confor-mational sampling and direct comparison with other im-plicit solvent data. The separation of the dimer in the

Figure 10. Hydrogen bond cluster that supposedly stabilizes thedimer in apo_c_H+ .



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region of domain I was observed in the 10 ns explicit sol-vent open state DENV sE simulation with protonated histi-dines.[28] In an analogous study,[27] twisting and folding outmovement of the dimer were observed during a 60 ns sim-ulation. At neutral pH level only slight movements were ob-served in both studies.

His-protonated POWV sE dimers do bend in our simula-tions in the same way as the dimers with non-protonatedhistidine residues do. Difference in bending amplitude be-tween POWV sE dimers in apo- and ligand-bound forms be-comes less significant, and angle values come closer toeach other. The most considerable changes are observed inthe closed form and in the complex with (S)-3.

Intersubunit interactions of DENV sE protein dimers wereassessed by observing the hydrogen bonding pattern be-tween the subunits along the MD trajectories, and the de-crease of hydrogen bond number after the protonation ofHis and Glu residues was shown.[26] In our simulation, withonly His residues protonated, the average number of hy-drogen bonds in all the systems slightly increases, whilethe number of intersubunit hydrogen bonds increases onlyfor apo_c_H+ and S_H+ , which are also characterized bythe most significant, though opposite, changes in thebending pattern. Thus, there is no obvious correlation be-tween the bending and intersubunit interactions.

Salt bridges in POWV E protein apo_o_H+ betweenGlu277 and Lys251, which tie together kl loop and DII ofdimer partner, are stable and, in contrast to the neutral pHsystems, both of them exist with high occupancy duringthe whole trajectories. The same is correct for R_H+ and S_H+ . It is notable that there are no such options for stabiliz-ing kl loop in the open state of the DENV sE structure.

Among all studied systems, kl loop cannot perform as anadditional stabilising factor only in apo_c_H0 and apo_c_H+ simulations. Nevertheless, dissociation of the dimer inthe apo_c_H+ trajectory is not observed. Thus, stability ofthe protonated dimer may be induced by the additionalbonds formed by protonated His residues. There are actual-ly a number of hydrogen bonds formed by histidine resi-dues in apo_c_H+ that are absent in apo_c_H0. Protonationof these residues led to the formation of new hydrogenbonds that should strengthen the interaction between thefusion peptide and the neighboring subunit in the closedform. His323A (DIII) and His5A (DI) are involved in such in-teractions and form hydrogen bonds with Asp98B belong-ing to the fusion peptide, that tie together DI, DIII and thefusion peptide (Figure 10). The substitution H323A in TBEVE protein was shown to reduce fusion peptide exposureunder acidification and to influence the trimer stability.[9] Inour simulation His248 also makes a contribution to the in-crease of subunit coupling: this residue forms salt bridgeswith Glu7 (DI) of the other subunit in apo_c_H+ (His resi-dues that form hydrogen bonds in other systems but apo_c_H0 are listed in Table S2 Supporting Information). Thedouble mutant of TBEV E protein H248N–H287A was foundto be fusion negative, with impaired trimer formation and

trimer stability.[9] In view of these facts, His323 and His248should act as the dimer destabilizing agents at low pH, butin our study we observe the opposite behavior. Explicit sol-vent simulations of full-length E proteins with M proteinstaken into account will clarify this issue. It should be alsonoted that implicit solvent simulations are of limited relia-bility for the salt bridge analysis, because solvation of sol-vent-exposed charged residues makes salt bridge entropi-cally less favorable than its absence. Increase of salt bridgenumber upon His protonation, though slight, can be inter-preted in the course of this knowledge as the increase ofthe number of putative solvolysis sites.

5 Conclusions

Molecular dynamics simulations performed in the presentwork shed the light onto the character of interaction be-tween the Powassan virus reproduction inhibitors and en-velope proteins. The pattern of the protein thermal move-ment is changed when the inhibitor is present, and suchchanges can be coupled to the reproduction inhibition pro-cess. MD simulations with protonated histidine residues,despite being somewhat oversimplified models of the ini-tial prefusion states, correspond to the experimental dataobtained for the related viruses. The conformational spaceof both enantiomers of the inhibitor is sampled, and puta-tive biologically active conformations obtained this waycan be used for further 3D similarity-based searches fornovel antiviral compounds active against different flavivi-ruses.

Acknowledgements

The authors are grateful to Prof. Galina Karganova for thelong-time inspiration of this work. Free academic licencefor OpenEye software was kindly provided to the laboratoryof V. A. Palyulin by OpenEye Scientific Software, Inc. Free aca-demic licence for AMBER was kindly provided by Prof.David A. Case. This work was partially supported by RussianFoundation for Basic Research (Grants Number 14-03-00851and 14-03-31566). iScalare laboratory at MIPT was support-ed by the grant of the Government of the Russian Federation(Decree 220 of 09.04.2010). The authors express their grati-tude to the Research Computing Center at the LomonosovMoscow State University for providing the computing timefor the part of this work.

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