2.

Materials & methods

2.1. Homology model:
Homology modeling of the Neuraminidase of H1N1 was performed using Modeller software
[A. Sali, T.L. Blundell, Comparative protein modeling by satisfaction of spatial restraints, J.
Mol. Biol. 234 (1993) 779815]. The amino acid sequence of Neuraminidase was retrieved
from GenBank (accession number: ACP41952) in NCBI. The Neuraminidase was then
subjected to a BLAST search [S.F. Altschul, T.L. Madden, A.A. Schaffer, J.H. Zhang, Z.
Zhang, W. Miller, J.T. Lipman, Gapped BLAST and PSI-BLAST: a new generation of protein
database search programs, Nucleic Acids Res. 25 (1997) 33893402] in order to identify the
homologous proteins from the Brookhaven Protein Data Bank (PDB). An appropriate
template for SBD was identified based on the e-value and sequence identity. The template
and the target sequences were then aligned using ClustalW [Larkin MA, Blackshields G,
Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A,
Lopez R, Thompson JD, Gibson TJ and Higgins DG. Bioinformatics 2007 23(21): 29472948]. Subsequently, homology modeling was carried out for the Neuraminidase of H1N1
against the chosen template using Modeller. The outcomes of the modeled structures were
ranked on the basis of an internal scoring function, and those with the least internal scores
were identified and utilized for model validation.
2.2. Model validation:
In order to assess the reliability of the modeled structure of Neuraminidase, we calculated the
root mean square deviation (RMSD) by superimposing it on the template structure. The
backbone conformation of the modeled structure was calculated by analyzing the phi () and
psi () torsion angles using rampage [S.C. Lovell, I.W. Davis, W.B. Arendall III, P.I.W. de Bakker, J.M.
Word, M.G. Prisant, J.S. Richardson and D.C. Richardson (2002) Structure validation by Calpha geometry:
phi,psi and Cbeta deviation. Proteins: Structure, Function & Genetics. 50: 437-450],

as determined by

Ramachandran plot statistics. Finally, the statistics of non-bonded interactions was calculated
by ERRAT [Colovos C, Yeates TO. (1993). Verification of protein structures: patterns of nonbonded atomic
interactions. Protein Sci. 2, 1511-1519].

2.3. Molecular Docking & Virtual screening of Neuraminidase inhibitors:

Oseltamivir and zanamivir were constructed and minimized using the UCSF Chimera
modeling program [UCSF Chimera--a visualization system for exploratory research and
analysis. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin
TE. J Comput Chem. 2004 Oct;25(13):1605-12]. We aligned the structures of the model and
the template (PDB ID: 3B7E), and then used the orientation of zanamivir in the template to
identify the general binding pocket of the model.

The substrates were docked into the active site of the refined A(H1N1) neuraminidase
structure using AutoDock version 3.0.5 [G.M. Morris, D.S. Goodsell, R.S. Halliday, R. Huey,
W.E. Hart, R.K. Belew, A.J. Olson, Automated docking using a Lamarckian genetic algorithm
and an empirical binding free energy function, J. Comput. Chem. 19 (1998) 16391662.].
using the implemented empirical free energy function and the Lamarckian Genetic Algorithm
(LGA). The grid maps were calculated using AutoGrid. In all dockings, a grid map with
505050 points and a grid-point spacing of 0.375 was applied. Because the location of
the ligand in the complex was known, the maps were centered on the ligand's binding site.
One hundred solutions were generated for each docking with the default setting and clustered
with the RMSD cutoff set to 0.5 . The solution that has the lowest energy in the individual
cluster was chosen for further analysis.
The software DOCK6.2 software package [T.J.A. Ewing, I.D. Kuntz, Critical
evaluation of search algorithms for automated molecular docking and database screening, J.
Comput. Chem. 18 (1996) 11751189] was then applied in the virtual screening. The
molecular modeling program UCSF Chimera [UCSF Chimera--a visualization system for
exploratory research and analysis. Pettersen EF, Goddard TD, Huang CC, Couch GS,
Greenblatt DM, Meng EC, Ferrin TE. J Comput Chem. 2004 Oct;25(13):1605-12] was used
to prepare the receptor (Neuraminidase). The DOCK6 sphgen tool was used to create receptor
spheres with radii between 1.4 and 5.5 . Spheres within 10 of the center of the dimer
interface were selected for use in docking simulation, and a grid box was generated extending
out 5 from the spheres. Docking was performed by incorporating ligand flexibility, and
Amber scores were used for analysis. The lead-like compounds(523,366) were obtained from
ZINC database[Irwin and Shoichet, J. Chem. Inf. Model. 2005;45(1):177-82], a free database
of commercially-available compounds for virtual screening, provided by the Shoichet

Laboratory in the Department of Pharmaceutical Chemistry at the University of California,

San Francisco (UCSF).

2.4. Molecular Dynamics simulations:

The GROMACS 4.5.4 package [Hess, B.; Kutzner, C.; van der Spoel, D.; Lindahl, E.
GROMACS 4:Algorithms for highly efficient, load-balanced, and scalable molecular
simulation. J. Chem. Theory Comput. 2008, 4, 435447] was used to run Molecular
Dynamics simulations with the GROMOS96 43a1 force field [van Gunsteren, W.; Billeter, S.
R.; Eising, A. A.; Hunenberger, P. H.; Kruger, P.; Mark, A. E.; Scott, W. R. P.; Tironi., I. G.
Biomolecular Simulation: The GROMOS96 Manual and User Guide; Vdf Hochschulverlag
AG an der ETH Zurich: Zurich, Switzerland, 1996] and the SPC216 water model [Berendsen,
H. J. C.; Postma, J. P. M.; van Gunsteren, W. F.; Hermans, J. Intermolecular Forces; Reidel,
Dordrecht, The Netherlands, 1981]. The modeled Neuraminidase was energy minimized
using the Optimized Potentials for Liquid Simulations All Atom (OPLS) force field. This
preliminary energy minimization was done to discard the high-energy intramolecular
interactions. The overall geometry and atomic charges were optimized to avoid steric clashes.
Then the whole system was gradually heated from 0 to 300 K over 500 ps using the NVT
ensemble run with the Berendsen procedure[Berendsen, H. J. C.; Postma, J. P. M.; van
Gunsteren, W. F.; Dinola, A.; Haak, J. R. Molecular dynamics with coupling to an external
bath. J. Chem. Phys. 1984, 81, 36843690] and, subsequently in 500 ps NPT ensemble run at
pressure of 1atm. The Parrinello-Rahman pressure coupling [Parrinello, M.; Rahman, A.
Polymorphic transitions in single crystals: A new molecular dynamics method. J. Appl. Phys.
1981, 52, 71827190] has been used. Finally, an 10 MD simulation was carried out to
examine the changes and dynamic behaviour of the protein were analyzed by calculating the
RMSD and energy.
2.5. ADME Screening
The pharmacokinetic feature which gives idea about Absorption, Distribution,
Metabolism and Excretion/Toxicity (ADME/T) taken into account in this work. In fact, it
would be extremely advantageous if information about the ADME properties of the studied
molecules could be produced in the early stages of the drug discovery process. Once
obtained, this information is expected to help chemists to ameliorate the pharmacokinetic
profile of the compounds.

Results & Discussion

3.1. The Results of Homology modeling of Neuraminidase and its evaluation:
The neuraminidase sequence of A/H1N1/2009 was performed the NCBI BLAST.
Neuraminidase of A/Brevig Mission/1/1918 H1N1 strain was identified as homologous from
Protein Data Bank (PDB) (PDB ID: 3B7E). The automated sequence alignment (Fig. 1) and
analysis of the template and target was carried out using the ClustalW program [Larkin MA,
Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez
R, Thompson JD, Gibson TJ and Higgins DG. Bioinformatics 2007 23(21): 2947-2948.

]. Therefore, 3B7E

was identified as a promising template that had a sequence similarity of 88.8% to the
neuraminidase of A/H1N1/2009. The MODELLER package has been used to model the
structure of A (H1N1) neuraminidase.

3B7E_A|PDBID|CHAIN|SEQUENCE
A_H1N1__Neuraminidase

------VILTGNSSLCPISGWAIYSKDNGIRIGSKGDVFVIREPFISCSH 44
GQSVVSVKLAGNSSLCPVSGWAIYSKDNSVRIGSKGDVFVIREPFISCSP 50
* *:*******:**********.:*******************

3B7E_A|PDBID|CHAIN|SEQUENCE
A_H1N1__Neuraminidase

LECRTFFLTQGALLNDKHSNGTVKDRSPYRTLMSCPVGEAPSPYNSRFES 94
LECRTFFLTQGALLNDKHSNGTIKDRSPYRTLMSCPIGEVPSPYNSRFES 100
**********************:*************:**.**********

3B7E_A|PDBID|CHAIN|SEQUENCE
A_H1N1__Neuraminidase

VAWSASACHDGMGWLTIGISGPDNGAVAVLKYNGIITDTIKSWRNNILRT 144
VAWSASACHDGINWLTIGISGPDNGAVAVLKYNGIITDTIKSWRNNILRT 150
***********:.*************************************

3B7E_A|PDBID|CHAIN|SEQUENCE
A_H1N1__Neuraminidase

QESECACVNGSCFTIMTDGPSNGQASYKILKIEKGKVTKSIELNAPNYHY 194
QESECACVNGSCFTVMTDGPSNGQASYKIFRIEKGKIVKSVEMNAPNYHY 200
**************:**************::*****:.**:*:*******

3B7E_A|PDBID|CHAIN|SEQUENCE
A_H1N1__Neuraminidase

EECSCYPDTGKVMCVCRDNWHGSNRPWVSFDQNLDYQIGYICSGVFGDNP 244
EECSCYPDSSEITCVCRDNWHGSNRPWVSFNQNLEYQIGYICSGIFGDNP 250
********:.:: *****************:***:*********:*****

3B7E_A|PDBID|CHAIN|SEQUENCE
A_H1N1__Neuraminidase

RPNDGTGSCGPVSSNGANGIKGFSFRYDNGVWIGRTKSTSSRSGFEMIWD 294
RPNDKTGSCGPVSSNGANGVKGFSFKYGNGVWIGRTKSISSRNGFEMIWD 300
**** **************:*****:*.********** ***.*******

3B7E_A|PDBID|CHAIN|SEQUENCE
A_H1N1__Neuraminidase

PNGWTETDSSFSVRQDIVAITDWSGYSGSFVQHPELTGLDCMRPCFWVEL 344
PNGWTGTDNNFSIKQDIVGINEWSGYSGSFVQHPELTGLDCIRPCFWVEL 350
***** **..**::****.*.:*******************:********

3B7E_A|PDBID|CHAIN|SEQUENCE
A_H1N1__Neuraminidase

IRGQPKENTIWTSGSSISFCGVNSDTVGWSWPDGAELPFSI-- 385
IRGRPKENTIWTSGSSISFCGVNSDTVGWSWPDGAELPFTIDK 393
***:***********************************:*

Fig: 1 Structure-based sequence alignment of A (H1N1) neuraminidase and 3B7E.Active site residues are highlighted with black
triangles.

The modeled A (H1N1) neuraminidase was used for further optimization and validation. The
calculated root mean square deviation between the target and template structure was found to
be 1.2 (Fig. 2a). To assess the quality of the optimized models, Rampage [S.C. Lovell, I.W.
Davis, W.B. Arendall III, P.I.W. de Bakker, J.M. Word, M.G. Prisant, J.S. Richardson and D.C. Richardson
(2002) Structure validation by Calpha geometry: phi,psi and Cbeta deviation. Proteins: Structure, Function &
Genetics. 50: 437-450]

and ERRAT [Colovos C, Yeates TO. (1993). Verification of protein structures:

patterns of nonbonded atomic interactions. Protein Sci. 2, 1511-1519 ] analyses were also undertaken.
The Ramachandran plot(Fig:2b) of our model shows that 94.4% of residues were found in the
favored and 5.4% allowed regions and 0.3% were in the outlier region. The quality factor
(ERRAT) of our model was equal to 85.417, and thus we consider that the model is
acceptable for predicting the binding modes and interactions of oseltamivir and zanamivir
with A (H1N1) neuraminidase.

Fig: 2a Superimposition of template (green) and model protein (yellow).

modeled

2b Ramachandran plot for the

3.2. The Results of Molecular Dynamics simulations:

The modeled A (H1N1) neuraminidase was subjected to Molecular dynamics, in order to
explain protein structurefunction problems, such as folding, conformational flexibility and
structural stability. In the simulations, we monitored the backbone atoms and the C--helix of
the modeled protein. The RMSD values of the modeled structures backbone atoms were
plotted as a time-dependent function of the MD simulation. The results support our modeled
structure, as they show constant RMSD deviation throughout the whole simulation process.

The time dependence of the RMSD () of the backbone atoms of the modeled protein during
a 10 ns simulation is shown in Fig. 3 and the RMSD value variation with respect to the
simulation time. The RMSD value of the model has a low fluctuation during the entire time
process. The low RMSD and the simulation time indicate that, as expected, the 3D structural
model of A (H1N1) neuraminidase represents a stable folding conformation.

Fig. 3 RMSD of the backbone atoms of the A (H1N1) Neuraminidase over a time period of 10 ns

3.3. Binding modes of antiviral drugs with A (H1N1) Neuraminidase:

The two antiviral drugs, oseltamivir and zanamivir, drugs were docked into the active
site to explore the substrate binding modes. The initial binding modes were obtained by using
AutoDock. The obtained complex results are summarized in Table 1 and Fig. 4a and 4b. For
the zanamivir binding modes, five residues (ASN172, ASN219, ASN268, SER171, and
ARG292) form seven hydrogen bonds with the drug. For oseltamivir binding modes, four
residues (ARG76, SER171, GLU201, and ASN268) form six hydrogen bonds with the
drug, these residues were considered as binding site and the screening has been applied
against zinc database for identification of novel neuraminidase inhibitors.

Fig:4 Binding mode of a) Oseltamivir and b) Zanamivir with A (H1N1) Neuraminidase.

3.4. Results of Virtual screening of Neuraminidase inhibitors

Of the 523,366 compounds subject to the virtual screening with UCSF DOCK, we found that
the DOCK grid scores of ~30 compounds are better than those of the two drugs were shown
in Table 1. The chemical structures of these lead molecules are illustrated in Fig. 5, and the
binding modes of these 30 lead molecules and their interacting residues and docking
conformations of the 30 virtual hits around the binding site of A H1N1 Neuraminidase are
shown in Fig. 6, 7 and Table 1.

In addition, four compounds (ZINC03869914, ZINC03869917, ZINC12502585

and

ZINC53684003, Fig. 9) have best DOCK grid scores, which were nearly twice as low as the
two drugs and all the protein-ligand complexes for these four lead molecules possess multiple
hydrogen bonds when compared with two drugs. Consequently, these four compounds were
selected for subsequent ADMET studies.

Fig. 5. The chemical structures of the 30 candidates

Fig. 6. The docking poses of the 30 candidates

Fig. 7. Docking conformations of the 30 virtual hits (Color sticks) around the binding site (red box) of A H1N1
Neuraminidase (Blue Surface).

Table 1. Dock results for the 30 lead molecules and Oseltamivir and Zanamivir
Drug/Lead

DOCK grid

Amino acids involved in interactions

No.of HBs

Molecules
Oseltamivir
Zanamivir
ZINC03869914

scores (kcal/mol)
-31.097
-31.013
-53.074

ARG76, SER171, GLU201 and ASN268

ASN172, ASN219, ASN268, SER171and ARG292
ASN219, GLU201, ARG76, ARG149, ARG42, ARG292 and

6
7
8

ZINC03869917
ZINC12502585
ZINC53684003
ZINC00135466

-50.829
-53.833
-50.808
-84.382

GLU202
ASN219, GLU202, SER171 and ARG76
ARG76, ASN219, GLU201, ARG149, ARG292 and ARG42
SER171, ARG76, ARG217, GLY269, ARG292 and ARG42
N/A

7
7
8
N/A

ZINC02172775
ZINC03869234

-80.925
-52.423

N/A
ARG292, LYS74 and ARG42,

N/A
4

ZINC 03869916

-50.439

ARG76, ASP75, GLU202, ARG292, ARG42

ZINC03882070
ZINC04096400
ZINC05112750
ZINC05317111
ZINC12502589
ZINC12890057
ZINC13059497
ZINC13861587
ZINC16887666
ZINC20459160
ZINC23329815

-50.845
-52.128
-53.567
-91.123
-50.949
-51.868
-50.168
-52.736
-50.566
-55.286
-58.628

LYS74, ARG42 and ARG292

ASP75, ARG42 and ARG292
LYS74 and ARG354
N/A
GLU202, ARG292 and ARG42
ARG292, ARG42, LYS74 and ASP75
LYS74 and ARG354
ARG76, ARG149, ASP75, ARG42 and ARG292
LYS74, ARG42 and ARG292
ASP75, LYS74, ARG42, ARG354 and ARG292
ARG354

5
5
6
N/A
4
8
2
8
6
5
2

ZINC27558896
ZINC27558898
ZINC27558900
ZINC27645848
ZINC28866114
ZINC31392733
ZINC35288026
ZINC38541426
ZINC38665036
ZINC43198995
ZINC59514262

-56.343
-58.448
-56.515
-53.097
-51.910
-50.755
-53.430
-60.348
-51.376
-50.456
-50.835

ARG292, ARG42 and LYS74

LYS74 and ARG354
LYS74, ARG42 and ARG354
LYS74, ASP75 and ARG354
ARG42, ASP75 and LYS74
ARG149, ASP75 and LYS74
ARG42, ARG292 and LYS74
LYS74, ARG42 and ARG292
LYS74, ASP75, ARG292 and ARG42
GLY269, ARG292 and LYS74
ARG42, ARG292 and ARG354

3.5. Prediction of ADME properties

6
5
5
8
6
6
5
5
6
4
6