PRIME 2006

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PRIME 2006

• Lisa Zhao
• Wednesday, August 23, 2006
• Final Presentation

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Introduction

• The Influenza A virus subtype H5N1 is currently
  the worlds largest pandemic threat due to its
  ability to cause illness in many animal species.
  Human cases are still relatively rare with only
  191 severe infections but with a high mortality
  rate. Commonly called the bird flu, the
  occurrence of this virus in humans is growing and
  thus the likelihood of a human adapted H5 virus
  to form is also increasing. There are currently
  16 avian and mammalian serotypes of HA known,
  only three (H1, H2, H3) have become adapted to
  the human population. (Stevens, Blixt et al. 2006)

Figure 1 Outbreaks of H5 Avian Influenza in Asia
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Introduction

• The Avian Influenza virus has an envelope with a
  host-derived lipid bilayer and covered with about
  500 projecting glycoprotein spikes with
  hemagglutinin and neuraminidase activities. These
  activities correspond to the two major surface
  viral glycoproteins the hemagglutinin (HA) and
  neuraminidase (NA), present as homotrimers and
  homotetramers, respectively.

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Introduction

• Hemagglutinin
• HA is a homotrimer, each monomer is created as a
  single polypeptide (HA0) that is cleaved by host
  proteases into two subunits (HA1 and HA2). HA
  binds to receptors containing glycans with
  terminal sialic acids, where their precise
  linkage determines species preference a switch
  in receptor specificity from sialic acids
  connected to galactose in a2,3 linkages (avian)
  and a2,6 linkages (human).
• Right
• Ribbon representation of the hemagglutinin HA
  trimer from the 1918 influenza virus.Receptor
  binding site (blue area) for virus attachment to
  the host lung epithelial cells via sialic acid
  containing host cell receptors.

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Methods Materials
LSTa and LSTc are sialo-pentasaccharides. They
are both natural sialosides from human milk that
have sequences common to complex oligosaccharides
on glycoproteins and glycolipids because of their
three terminal saccharides (Sia-Gal-GlcNAc). They
are used in this study when docking with the RBD
of HA because of their resemblance to the hosts
cell surface.
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Methods Materials

• AutoDock 3.05 is a automated docking software
  developed by the Scripps Institute that is
  designed to predict how ligands will bind to a
  macromolecule at its known receptor binding
  domain.
• Ligands were prepared by adding hydrogens,
  computing Gasteiger charges and uniting non-polar
  hydrogens. The macromolecule was prepared by
  first removing all water molecules, adding polar
  hydrogens only, and assigning Kollman charges. .
• AutoDockTools consists of AutoGrid, AutoTors, and
  AutoDock.
• AutoTors prepares the ligand by setting the
  number of rotatable bonds. For our study, the
  number of active torsions that move was set to
  the fewest number of atoms.
• AutoGrid then calculates the grid that describes
  the receptor binding domain of the macromolecule.
  All grid boxes were set at the default
  coordinates 4.58 x 4.58 x 4.58 angstroms.
• AutoDock performs the docking of the ligand to
  the pre-calculated grids on the macromolecule.
• AutoDock outputs estimated free energy of binding
  which includes the intermolecular energy and
  torsional free energy. It also outputs the
  docking energy which includes both the
  intramolecular and intermolecular energies.

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Methods Materials

• Began with Positive Control (PDB ID 1HGE) H3
  avian hemagglutinin bound to sialic acid analog.
  Separated the sialic acid analog (2 O Methyl 5 N
  Acetyl Alpha D Neuraminic Acid) from
  hemagglutinin and performed docking on receptor
  binding domain.
• Lowest Binding Energy -4.76 kcal/mol
• Lowest Docking Energy -6.67 kcal/mol
• Docked 2FK0 (Avian (H5N1) Hemagglutinin from
  Vietnam year 2004) with sialic acid in its chair
  conformation
• Lowest Binding Energy -4.75 kcal/mol
• Lowest Docking Energy -6.42 kcal/mol

Top Ribbon Representation of the Avian H3
hemagglutinin Bottom Visualization of the
Receptor binding domain and corresponding grid
box prior to docking.
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Results
MNA (best Conformation with 1HGE
Control) Lowest binding energy - 4.75 Arg 135,
Ser 228, Glu 190, His 183, Tyr 98, Asn 137, Ser
136 Hydrophobic interactions with Trp 153, Leu
194, Leu 226,
The Influenza a virus (a/viet nam/1203/2004(h5n1))
 receptorbinding domain (RBD) with the side
chains of key residues for receptor binding
labeled. The binding site comprises three
structural elements an a-helix (190-helix) and
two loops (130-loop and 220-loop).
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Materials Methods

• After analyzing the results from the positive
  control studies, further dockings were done on
  multiple subtypes of the avian influenza
  hemagglutinin.
• H1, H2, and H3 subtypes of the influenza A virus
  have adapted to cause pandemics in the human
  population and the likelihood of a human adapted
  H5 virus to form is increasing
• Docked with H1, H3, and H5 against LSTa and LSTc
  in order to discover whether or not there are
  binding affinity differences between these
  subtypes.

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Materials Methods

• Docking HA subtypes with LSTa and LSTc
• H1 1R8D Crystal Sructure of the 1918 Human H1
  Hemagglutinin Precursor (HA0)
• H3 2VIU A Reassortant Influenza Strain
  Containing Avian/aichi/68 (H3N2) Hemagglutinin
• H5 2FK0 Avian (H5N1) Hemagglutinin from Vietnam
  year 2004
• Set Grid Box centered on ligand in the original
  PDB, then deleted ligand leaving the RBD empty.
  Later when docking used LSTa and LSTc as ligand.
• This insures that the ligand being docked will go
  directly to the receptor binding domain and
  lessons the degree of human error.
• After docking H1, H3, and H5 with LSTa and LSTc.
• Examined with Ligplot and mapped the hydrophobic
  and hydrophilic surfaces.

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H1 1R8D Crystal Sructure of the 1918 Human H1
Hemagglutinin Precursor (HA0)
Results
LSTa (a 2,3 sialoside)
LSTc (a 2,6 sialoside)
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Results
Table of Interacting residues and hydrophobic
interactions for the Avian/aichi/68 (H3N2)
HemagglutininShown in Orange are the
dissimilarities between LSTa and LSTc binding.
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H3 2VIU A Reassortant Influenza Strain
Containing Avian/aichi/68 (H3N2) Hemagglutinin
Results
LSTa (a 2,3 sialoside)
LSTc (a 2,6 sialoside)
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Results
Table of Interacting residues and hydrophobic
interactions for the Avian/aichi/68 (H3N2)
HemagglutininShown in Orange are the
dissimilarities between LSTa and LSTc binding.
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H5 2FK0 Avian (H5N1) Hemagglutinin from Vietnam
year 2004
Results
LSTa (a 2,3 sialoside)
LSTc (a 2,6 sialoside)
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Results
Table of Interacting residues and hydrophobic
interactions for the Avian (H5N1) Hemagglutinin
from Vietnam year 2004 (PDB ID 2FK0) Shown in
Orange are the dissimilarities between LSTa and
LSTc binding.
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Results
Results from docking H3, and H5 hemagglutinins
with LSTa and LSTc are consistent with what has
been proposed by other experiments Above
Results from the study X-ray structures of H5
avian and H9 swine influenza virus hemagglutinins
bound to avian and human receptor analogs by Ya
Ha et al Examines binding of H5, H3, and H9 to
LSTa and LSTc.
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Comparison of H1, H3, and H5 Hemagglutinin
Receptor Binding Domain surfaces.
H1 Human HAPDB ID 1RD8
H3 Avian HAPDB ID 2VIU
H5 Avian HAPDB ID 2FK0
Illustrations (done on Chimera) showing the
molecular hydrophobic and hydrophilic surfaces of
the active cavity of three HA subtypes.
Hydrophobic (water hating) GreenHydrophilic
(water loving) BlueLSTa Receptor Analog
Magenta
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Comparison of HA Binding Energies

• H1 (human)
• lowest binding energy with LSTa -3.13 kcal/mol
• lowest binding energy with LSTc -2.89 kcal/mol
• H3 (avian)
• lowest binding energy with LSTa -2.78 kcal/mol
• lowest binding energy with LSTc -3.24 kcal/mol
• H5 (avian)
• lowest binding energy with LSTa -2.76 kcal/mol
• lowest binding energy with LSTc -3.52 kcal/mol

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Results
H1 Human
H5 Avian
Comparison of the lowest binding energies for
different sialic acid analogs docked with Avian
(H5N1) Hemagglutinin from Vietnam (2004) and the
1918 Human H1 Hemagglutinin In both cases, LSTa
and LSTc have significantly lower binding
energies than docking with sialic acid alone.
However, docking with the disaccharides O-SIALIC
ACID 2,3 D-GALACTOSE and O-SIALIC ACID 2,6
D-GALACTOSE gave slightly lower binding energies
than sialic acid alone.
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Methods Materials

• Virtual Screening
• Virtual Screening is a method using high
  performance computing to analyze large databases
  of chemical compounds for the purpose of finding
  lead drug candidates.
• In combination with molecular docking, the
  binding interactions and binding affinities can
  be outputted for every chemical compound from a
  data set. (Waszkowycz, Perkins et al. 2001)
• For this particular study, a collection of 1990
  compounds from the National Cancer Institute
  Diversity Set was used to screen against the H5
  Hemagglutinin (PDB ID 2FK0)
• The chemical data the individual compounds in
  the diversity set can be found on the National
  Cancer Institute webpage http//dtp.nci.nih.gov/d
  ocs/diversity/index.html
• Sorted top results by lowest binding energies and
  lowest docking energies
• Analyzed top results using ligplot to see which
  residues the ligand binds to on the hemagglutinin
  receptor binding domain.

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Results
Right Top Binding Result Diversity 1865
Lowest binding energy -11.12 kcal/molResidue
Interactions Glu 190, Tyr 98, His 183Top
Complex examined using Rasmol. Ligand (Red)
Hemagglutinin (Blue)
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Results

• Analysis of Top Binding Results from Virtual
  Screening
• Control - MNA (best Conformation with 1HGE)
• Lowest binding energy - 4.7 kcal/mol
• Residues Arg 135, Ser 228, Glu 190, His 183,
  Tyr 98, Asn 137, Ser 136
• Hydrophobic interactions with Trp 153, Leu 194,
  Leu 226,
• 1st . Diversity 1865 (best Conformation with
  2FK0)
• Lowest binding energy -11.12 kcal/mol
• Residues Glu 190, Tyr 98, His 183
• 3rd Diversity 1100 (best Conformation with 2FK0)
• Best binding energy -10.48 kcal/mol
• Residues Val 135, Gln 226, Tyr 98, Glu 190, His
  183,
• Hydrophobic interactions Lys 193, Ile 155, Lys
  156, Leu 194, Gly 228
• 4th Diversity 1986 (best Conformation with 2FK0)
• Best Binding energy - 10.47 kcal/mol
• Residues Gln 226, Glu 190, Tyr 98, Val 135, Ser
  137, His 183

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Discussion

• The results from Virtual Screening show that the
  best drug candidate for H5N1 should hydrogen bond
  to the following residues for maximum effect.
• Glu 190, Tyr 98, His 183, Val 135, and Gln 226
• Diversities 1865, 1100, and 1986 respectively,
  are the most promising candidates for further
  modification and structure-based drug design.
• Results from docking H1, H3, and H5
  hemagglutinins with LSTa and LSTc are consistent
  with what has been proposed by other experiments
• However there is not a significant difference in
  the binding affinities between the different
  subtypes.
• Further docking should be done on H2 and H9
  hemagglutinin and also more host species which
  may lead to a better conclusion.
• Surface analysis of H3, H1 and H5 show that H5
  seems to have more hydrophilic residues at the
  receptor binding domain whereas H3 and H1 have
  more hydrophobic residues.

Diversity 1865
Diversity 1100
Diversity 1986
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Advantages of the Database

• Allows the public to access all data and results
  from mine and Lilys research to compare and
  contrast with their own.
• With the proper software, users can download the
  .dlg file outputted from docking and view the
  binding conformations.
• Will be useful for next years PRIME students if
  they decide to pursue the same research topic.
• Results are organized such that each molecule is
  directly linked to its corresponding article.

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References

• http//www.agnr.umd.edu/avianflu/
• Scripps Research Institute Molecular Graphics
  Laboratory
• Stevens et al. Structure and Receptor
  Specificity of the Hemagglutinin from an H5N1
  Influenza Virus

Acknowledgements

• Computer Network Information Center, Chinese
  Academy of Sciences
• Dr. Wilfred Li, National Biomedical Computation
  Resource
• Dr. Bill Chang, National Science Foundation

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Thank you all for making this such a memorable
experience.
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