Beruflich Dokumente
Kultur Dokumente
Departrnent of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai, 50200 Thaitand
Deparment of Chemistry, Faculty of Science, Chulalongkom University, Bangkok, 10330 Thailand
In order to understand the mechanisms of ligand binding and interaction between two commercial drugs (igands), zanamivir
and oseltamivir and H5N1 Influenza Virus Neurarninidase subtype N1, a three-dimensional model of Nl-ligand (GenBank
accession no. A,A,5654617) was initially generated by homology modeling using the 13 high-resolution X-ray structures of
neuraminidase N2 and N9 as the template. With the aid of the molecular mechanics and molecular dynamics methods, the
final implicit solvent refined model was obtained. It was, then, assessedby PROCIIECK, PROSA and VERIFY3D. With this
model, a flexible docking study was performed. The results show strong hydrogen bond interactions between the glycerol side
chains of zanamivir and Atg29 of the Nl. Common hydrogen bonds between the carboxyl groups and Arg279 were found for
both drugs. It was also found that the Glu30, Asp62, A1963, 1*9204, Trp310, Tyr313, Glu336, tre338, Trp348, Ala349 were
observed to facilitate the enzyme-ligand non-bonding interactions as they are located within the radius of 5 A from all atoms
of both drugs. Charge dishibution was evaluated using the semi-empirical AMl method. The results show that the total net
charges of the -NH side chain of zanamivir is less negative than that of oseltamivir. This is in contrast to what is observed for
the amide and alkyl (ether/glycerol) side chains. In comparison of the binding free energies between the X-ray N2-ligand and
N9-ligand complexes, N1-ligand binding is found to be less potent than N2 and N9 subtypes, while N2-ligand and N9-ligand
are roughly comparable. In addition, it is interesting to observe that the binding free energies for all three subtypes of the
zanamivir complexes are lower than those of oseltamivir.
Molcular Simulatim
ISSN 0892-7022 print/ISSN 1029-0435 onlire @ 2007 Taylor & Francis
http ://www.tandf .co.uVj omals
DOI: 10.1080/089n O2U 01255862
P Niwnanpipug et at.
ofinfected cells [4] so as to suppressthe viral population 2.2 Flexible docking study
and allow time for the host immune system to eliminate
Further flexible molecular dockings [13] with Genetic
the virus. Several successful drugs have been developed
Algorithm was performed using BioMedCache 2.0
based on the crystal structure of NA. However, some of the
Software [14] to find the most favorable binding
H5Nl strains bear high resistance for existing NA
interaction. Two drugs, oseltamivir and zanamivir, were
inhibitors. To understand the drug-resistance of H5N1
docked into the binding site of H5Nl-NA model yielded
virus at a deeper level, a structure-activity relationship for
from Section 2.I. T\e structure of zanamivir was obtained
some existing NA inhibitors is an emergent research topic
from X-ray crystallographic complex structure (PDB ID:
for the next possible pandemic influenza.
1A4G) [15], whereasoseltamivir was then generatedusing
As the experimental structural data for H5Nl
the initial conformation from the crystal structure of
neuraminidase is not available so far, we attempt to
zanamivir as a template. Both structures were optimized
build N1 neuraminidase from the human influenza H5N1
by the semi-empirical AMI method in the SPARIAN'04
viruses (Afl\ulandl2(SP33y2004(H5N1) isolated from
program [16]. The residues associating in the binding
Thailand [5]. In this study, a homology model of N1-NA
pocket was defined by conserved amino acid residues that
was built according to the crystal sfucture of N2-NA and
are found in all N1, N2 and N9 subtypes which are
N9-NA complexed with two commercial drugs, zanamivir
4'19106, Glu107, Asp139, Arg140, Try167, Arg2l3,
and oseltamivir. Subsequently, the structure-activity
Glu265, Glu266 and Arg281 according to their multiple
relationship was studied based on the modeled complex
sequencealignment in figure 1. These residues were also
structure of H5N1-NA with the two drugs. Finally, the
defined as the catalytic site of the NA by Wei et aI. from
drug-resistanceof H5Nl influenza was analyzedand some
difference fourier analysis of crystals soaked in sialic acid
insights were gained that might lead to new information to
solve the drug-resistance problem. [17]. Additionally, their neighbor residues in a radius of
3 A of these conserved amino acids were selected and
defined as the members of the binding pocket. The
potential mean force (PMF) scores of the drugs were
evaluated by a genetic algorithm with a population size of
50, crossoverrate of 0.80, elitism of 5, mutation rate of 0.2
2. Computational details and the maximum cycle generation is set tote 40,000. The
size of the grid box is 30 x 30 x 304. Finally, the
2.1 HSNI-NA model built complex stnrctures were analyzed and the interaction
energy between the ligand and protein was calculated.
The sequence of N1-NA (accession no. A45654617)
that was obtained from the National Center for
Biotechnology Information (NCBI, http://www.ncbi.
3. Results and discussion
nlm.nih.gov/) contains 457 arrino acids. Tolally 13
templates with high resolution values (>2.004,) of N2
(PDB code: linhA, lingA, linw, 1ivf, 1ivg, 1nn2 and 3.1 Modeling of 3-dimensional structure of HSNI-NA
2bat) and N9 (PDB code: INNB, INNA,7NN9, INNC, Multiple sequence alignment of N1-NA and its related
ILTFA and IMWE) neuraminidase were chosen from sequences were shown in figure 1. These alignments
the Research Collaboratory for Structural Bioinformatics reflect the structurally conseryed regions of the available
Protein Data Bank (http://www.rcsb.orglpdb) as crystal structures. The similarity of N1-NA with N2-NA
templates for homology modeling. Multiple sequence and N9-NA sequencesis about 46 and,497o,respeclively.
alignments were derived from Clustalx 1.83 [6]. The The overall sequence region that MODELLER used to
rough 3D model based on multiple templates was construct the initial model was set at residues 78-457.
constructed using the academic version of MODELLER The rough 3D model (model A) of 380 amino acids was
8.1 [7]. The model with the lowest objective function generated and the quality of the model was checked by
was chosen as the best proposed model (model A) and PROCHECK. Starting from model A, refinement was
used for further refinements to include all missing atoms performed using energy minimization and molecular
especially hydrogen atoms followed by energy mini- dynamic in implicit solvent. Then, models A1 and A,2
mization and to include the solvent effect by implicit were, respectively, yielded. The quality of the rough and
solvent molecular dynamics (MD) simulations using refined models were collected in table 1. There are more
the generalized born (GB) model at 300K for 500ps residues in core regions of the minimized model than
with a time step of 2.0fs using AMBER 8.0 force those of MD. Model A1 has the residues of 0.37o in
field paramete03 [8,9]. The overall quality of the disallowed regions, whereas,model ,A,2has no residuesin
refined model was evaluated by utilizing PROCHECK disallowed regions. The root mean square displacement
[10] for a evaluation of Ramachandran plot quality, (RMSD) of all atoms in A1 and 42 shows insignificant
PROSA [11] for interaction energy testing and structure difference. The Ramachandran plot of model A2
VERIFY3D [I2] for assessing the compatibility of was illustrated in figure 2. Based on^an analysis of 118
each amino acid residue. structuresof resolution of at least 2.0 A and R-factor lower
A computational H5Nl neurarninidnse model 489
gF'
f{,eyr- qg,q* *Ielix l:=p He.tF $ir*Fd * ffi,antdq** nt*r] tuee*i$ljr]" **sa*#: Ei*.id E i*r*4*i&*s
-
1ry
ru
m 9: 1 =:t-
Figure 1. Multiple sequence alignment of H5Nl-NA and its related sequences. The secondary structure was predicted by PROCTIECK.
than 20Vo, the results indicate reliability of model ,A'2in using the SPARIAN'04 program. Figure 4 shows the
which over 997o of the backbone dihedrals fall within the molecular structure and the atomic net charges around the
structurally favorable regions. The decomposition inter- central ring at the positions a, c, d and e for the zanamivir
action energies (DEfinteraction between residue i and and oseltamivir. The charge differences, subtract charges
the otherN - 1 residueswhereNdenotes total residuesof of atoms of zanamivir by those of oseltamivir, at the
enzyme-of models A1 and ,A,2 were further checked positionsa, c, d ande are 0.004, - 0.146,0.256 and 0. 168,
using PROSA and plotted in figure 3a. It appears that the respectively. This observation leads to the same
decomposition energies are almost negative indicating conclusion for both inhibitors, i.e. the net charges of the
stability of the enzyme. The overall DE of model ,{2 is -COO, -NH, amide and -R (ether/glycerol) side chains
lower than that of model A1. Two high DE regions were (summation of the atomic net charges of all atoms of each
found in the amino acid range 60-70 and 300-320 for functional group) of zanamivir are similar, less negative,
model A1 whereas only the first region was observed for more negative and more negative in comparison,
model ^A2.The above conclusions were confiflned by the respectively, to those of oseltamivir. The main difference
results evaluated by the VERIFY3D routine in the Amber in charge distribution was found on the position d andthe
progmm. Here, most of the residues in model A2 falls
within the criteria score ()0.1). Inappropriate region,
where criteria score )0.1, was observed at residues
> 360. However, these residues are not in the binding I
I ' ::,::
ss
3.2 Charge distribution on znnamiair and oseltamioir ;
jl6
0.$
0
0.8
1$
t H CI.?
Et
6l t n'u
ru -8,5 S e"+
s
t{ 0.4
€
8* H o.a
d
m
o.p
s,1
,
&-8
{I '! fifi 2.0* 3ns 4*0
Re*id*e Index R*eidueIndax
Figure 3. Changes of the Prosa Energy and 3D-lD average score as a function of residue indexes for models A1 (- - - - -) and
(- - - - -) (see text for more details).
amide group. Increasing in electron density at d and e In the investigated systems, hydrophobic interactions
positions as well as the corresponding connected side were found to change docking free energy between ligand
chains. causeseach inhibitor to bind to the amino acids at and enzyme significantly. The different nonpolar residues
the binding pocket differently. The details on the complex (from table 2 and figure 5) were observed, especially
structure with the complementally hydrophobic and around the -COO and -R (ether/glycerol group) of
hydrophilic interactions between the side chain and active inhibitors, indicating the hydrophobicity difference in
pocket were discussedin the next section. facilitating enzyme-ligand binding between the N2A.{9
and the N1 pockets. This can be clearly seen by the
binding free energy of the complexes shown in table 3 in
3.3 Comparison between N1-N4 N2-NA and N9-NA which those of NlJigand binding is found to be less
binding pocket potent than N2 and N9 subtypes, while N2Jigand and N9-
To understand the drug-resistance of H5N1 virus at a ligand are roughly comparable. This finding is ffue for
molecular level, zanamivir and oseltamivir were flexible- both inhibitors. In addition, it is interesting to observe that
docked into the binding pocket of the three different binding free energies for all three subtypes of the
neuraminidasesubtypes,our Nl, N2 (PDB ID: IIVF) [18] zanamivir complexes are lower than those of oseltamivir.
and N9 (PDB ID: INNB) models [19]. Amino acid In order to monitor the binding environment, the
residueslying within 5 A from ligands were evaluatedand positions of the seven conserved amino acid residues for
reported in table 2.The acidic, basic, polar and nonpolar the three subtypes were mapped to the side chain position
residues were shown in red, blue, green and black, of ligands. The results were shown in figure 5. It was
respectively. It was found that the seven conserved amino found that the zanamivir formed a similar binding pattern
acid residues around the active site in all case of N1. N2 to all three subtypes. In contrast, the binding of the -O-R
and N9 are Nl lArg29, Glu30, Asp62, A1963, Glu189, group of oseltamivir in the NI-NA complex was observed
Arg204 and Arg279l, N2 [Arg118, Glu119, Asp151, to be located at the position different from those of the N2-
Argl52, Glu211, Arg292 and Arg371l and N9 [Arg119, NA and N9-NA complexes. This indicates higher
GluI2O, Asp152, Arg153, Glu279, Arg294 and Arg372l. flexibility of the oseltamivir side chain that can be easier
The data shows similarity between binding pockets of N2 to adapt itself to the new environment, and hence, lower its
andN9. This is also shownby the flexible docking scorein resistance to enzyme mutation in comparison to those
table 3. events of zanamivir.
Thble 2' Numbers and qrpes of residues which were detected at the binding pockets
of the N I -NA, N2-NA and N9-NA complexed with zanamivir
and
oseltamivir.
7-anamioir Oseltamioir
NI subtype N2 subtypef N9 subtypel NI subtype M subtypef N9 subtypef
(1) Arg29 (a,e) (1) Arg118 (e) (1) Arg119 (e) (1) Arg29 (c) (1) Arg118 (a) (1) Arg119 (c)
(2) clu30 (a) (2) Glu119 (e) (2) Glu120 (c,e) (2) clu30 (c,d) (2) Glu119 (c) (2) clu120 (c)
tre150(e) Lys61(d)
(3) Asp62 (d) (3) Asplsl (d,e) (3) Aspls2 (c,e) (3) Asp62 (d) (3) Asplsl (c) (3) Asp152(c,d)
(a) A1963 (c,d) (4) Arg152 (a,c) (4) Arg153 (e) (4) A1963 (cd) (4) Argls2 (c) (4) Algls3 (d)
,419156(c) Arg157(e) 41967(d)
Trp178(c) A'19156(c) Arg157(c)
Trp180(e) Trp178(cd)
Ser179(c) Serl81(e) Trp180(d)
11e222(a) na22@) Serl8l (d)
te2A (a) Arg224 (d,e)
ArgzU (cd) 11e224.(e)
A19226(cd) rtu22s(d)
Thr225(cd) G\t229 (c) A19?2,6 (e)
Ght22:7(cd) (cd)
Gln22:7 G11229(d)
Ala%8 (d) Na24,6(e)
ltJa:246(a) Glu278 (d) A1a248(e)
Gra76 @) Glu276(d,e) Glu278(e)
(s) Gtu189 (c) (s) GIu277 (c) (s) clu279 (d)
(6) Arg20a (c) (6) Are2n@.,e) (s) Gru277(d) (s) ctu279 (d)
(6) Arg2e4 (d) (6) Arg2M (a,c) (6) Arg292(e)
Gly256 (c) Gly348(e) (6) Arg29a (e)
Asn296(d) Asn94(e)
Asn348(e) Asn296(e)
Gty349(e) Asn348(a,e)
Q) Are279 (a) (7) Arg371 (e) (7) Arg372 (e)
Trp310(e) Q) Arg279 (a) A Are37t(a) Q) Are372 (a)
Tyr406 (e) Tyr406 (e) Trp3l0 (e)
Ser311(a,e) Tyr406(a,c) Tyr406(a,c)
Tlr313 (a)
Gry3r2 (a)
Glu336(a)
Tyr313(a)
IIe338(a,e)
Glu336(a)
Trp348 (e)
tre338(a,e)
Ala349 (e)
Arg341(e)
Trp348(e)
Ala349(e)
Theacidic,basic,polarandnonpolarresidueswereshownilred,blu9.ereenandblack'respectively.rTheamino*,o.,,uo"ffi
residues(1)-(7) are the sevencinserved mino *ia
"oto-ootyTo*J;-.1il;.;, ;-"il"ti nk",h*ts indicatedthe ligmd binding position accordingto figure 4.
Arg29 o
Trp310 ^. I
Ser311 ,ccH3 Arg29
lle338 Arg63 ctu3o
Arg341 Glu189 Trp310
\
) A1963
Trp348 Arg204 1e'338 Ars2o4
Ala349 Gly256 Trp34B )l
Ala349
2.65
' :
Arsze I T Arg279
clu30 HN-
Ars279
Se r 3 1 l
--NH
I
\
Glu336
lle338
!
Gly312 -Arg279 Trp348
Tyr313
Glu336
lle338
Figure 6. Schematic illusfiations to show the binding interactions of the (3) zanamivil and (b) oseltamivir with H5N1-NA. The dotted lines represent
the hydrogen bond where the corresponding hydrogen bond distances (in A) were also given.
hydrogen bond was detected between Arg279-NH and structures of neuraminidase N2 and N9. The best
the O-carboxyl group of zanamivir. A stronger bond is refinement model was derived from implicit solvent MD
formed between Arg29-NH and O-carboxyl with a simulation. The overall quality of this model was
distance of 1.834. Therefore, the hydrogen bonds evaluated by PROCIIECK, PROSA and VERIFY3D.
between Arg29 and the hydroxyl group of the glycerol The reliability of the obtained model was shown by the
side chain might block the hydrophobic interaction Ramachandranplot in which 81.8, 17.6, 0.6 and 0.07o of
between the carbon and the hydrocarbon chain of the the residues are in the most favored region, additional
surrounding amino acids. More difficulty in adjusting allowed regions, generously allowed regions and dis-
the position of the glycerol group to the change in the allowed region, respectively. Meanwhile, the results from
hydrophobic environment in the NI-NA would be the PROSA and VERIFY3D are all well within their
expected. criteria. Evaluation scores from these tests confirm that
In addition, in contast to what is detected for zanamivir, this model is an adequate one for further investigation.
the -O-R group at the position e (figure 4) in oseltamivir Flexible docking studies of the two drugs show the
was found to replace the glycerol side chain. This side chain complementary hydrophobic and hydrophilic environ-
has a lower steric effect from the hydroxyl groups so that it ments between enzyme and drugs. In some cases,specific
can easierrotate the single bond between the oxygen and the mutations have been associated with drug resistance.
alkyl chain R in the partial hydrophobic pocket. As a result, However, the flexibility of the -O-R group in
the positions of conservedamino acids indexed 1-7 binding oseltamivir complex leads to the lower drug-resistance
to eacha-e side chains of oseltamivir are changedwhereas to enzyme mutation in the more hydrophobic pocket of N1
those of zanamivir are mostly fixed as a mutation occurs in comparison to those of N2 and N9. The hydrophobicity
from Nl to N2 or N9 as shown in figure 5. Only two of the inhibitors is not only an important factor for
hydrogen bonds, which arenot associatedwith the side chain determining the inhibitory activity but also for designing
interactions, between Arg279-NH and O-carboxyl are orally active drugs. Such a finding might provide the
formed. This data indicates that oseltamivir is able to adapt information for designing new drugs against these viruses.
itself to the change of hydrophobic environment in the Nl-
{
NA pocket. This is fully consistent with that observed by
Mase et al. l20l that the particular H5N1-NA did show
Acknowledgements
sensitivity to oseltamivir. The complementally hydrophobic
and hydrophilic interactions between the side chain at the
This work is supported by grants from the Thailand
position e and active pocket are determining the inhibiting
Research Funding (TRF) and National Center for Genetic
activitv.
Engineering and Biotechnology (BIOTEC), Postgraduate
Education and Research Program in Chemistry: Center of
Innovation in Chemistry (PERCH-CIC), Thailand and also
4. Conclusion supported by the computer simulation and modeling
research laboratory (CSML), Department of Chemistry,
Three dimensional structure models of neuraminidase Chiang Mai University. The authors also thank the
H5N1 isolated from Thailand were constructed by anonymous reviewers whose comments were very helpful
homology modeling with the 13 high-resolution X-ray to make the presentation of this study more accurate.
A compatational H5NI neuratninidase model