TEMPLATE DESIGN 2007

www.PosterPresentations.com
Computer-aided Discovery of Novel Influenza Endonuclease Inhibitors
to Combat Flu Pandemic


Introduction
Aim 1. To identify new influenza endonuclease inhibitors
by combining computer-aided virtual screening and
biological assays.
Aim 2. To obtain a structural basis for further optimization
of the lead influenza endonuclease inhibitors.
Abstract
Materials and Methods
Relevant Applications to Biotechnology
Conclusions
Acknowledgements
1. The endonuclease inhibitors are new potentially lifesaving
anti-influenza medicine with a huge global market.
2. My discovery is patented by UCSD and the intellectual
property will likely attract biotech/pharmaceutical
companies to further develop the endonuclease inhibitors
into new anti-influenza drugs.
3. Similar strategies can be applied to identify drugs targeting
other influenza proteins, such as PB1, PB2 and NP.
4. Finding new, less toxic drugs may lead to cocktail therapy
similar to the successful HIV treatment, which may control
influenza more effectively and avoid viral resistance.
Eric Chen, Canyon Crest Academy, San Diego, CA
I initiated a project targeting the influenza PA
endonuclease to develop new anti-flu drugs. I believe by
tapping into the immense power of computers, we can
expedite the discovery of new anti-flu drugs. I used an
interdisciplinary approach combining computational
research and biological studies to discover new
endonuclease inhibitors.
I built pharmacophore models to perform virtual
screening of compound libraries (>450,000 compounds). I
also set up a fluorescence-based assay to validate virtual
screening hits as endonuclease inhibitors. In parallel, I
performed molecular dynamics (MD) simulations and
solvent mapping of the endonuclease to construct a
comprehensive database of binding pockets and druggable
hot spots. Molecular docking of the new inhibitors to the PA
endonuclease active sites revealed interacting residues. I
also examined structure and activity relationship using 21
analogs of the inhibitors.
Through this approach, I identified a number of new,
potent and structurally diverse endonuclease inhibitors with
great potential to be developed into new anti-influenza
drugs. The structural study also provided valuable
information for designing even more potent inhibitors.
A patent has been filed on my discovery by the
University of California, San Diego (UCSD).

Computer-aided drug discovery
MD simulation: MD simulation was performed in explicit solvent on the
TACC Ranger supercomputer. From 100 nanosecond MD runs of H5N1
PAN, thousands of frames of possible conformations were obtained at
atomic levels. RMSD clustering was performed using the GROMOS
analysis software
Computational solvent mapping: FTMap, a program searching the
protein surface for areas that can bind probe molecules was used with
MD conformations or crystal structures of PAN.
Pharmacophore model and virtual screening: Pharmacophore
models were generated using the Openeye ROCS program based on
nine known inhibitors and the best scored model is used to screen
compound libraries.
Molecular docking: Docking of compounds into the active sites of PAN
was carried out using the Glide module in Schrodinger software. The X-
ray structures and the MD conformations of PAN were used in the
analysis with the presence of two Mn
2+
ions and water molecules.
Results and Interpretation
Dr. Rommie Amaro, Assistant Professor
Department of Chemistry and Biochemistry
Dr. Gen-Sheng Feng, Professor
Department of Pathology
University of California, San Diego
The solvent mapping algorithm moves a
number of small, solvent-like organic probes
around the protein surface to find energetically
the most favorable binding positions, which
are druggable hot spots.
1. Combining virtual screening with biological assays
demonstrated a quick and efficient way to identify new
endonuclease inhibitors from large compound libraries,
saving time and resources.
2. In this study, I identified a number of new potent
influenza endonuclease inhibitors with diverse structures
representing four unique scaffolds. Some of them are
the most potent endonuclease inhibitors discovered so
far and are confirmed to have anti-viral activity. Thus,
these inhibitors can serve as leads to be further
optimized and developed into new anti-influenza drugs.
3. Structural studies and molecular docking of the new
inhibitors to the endonuclease active sites, together with
structure-activity relationship studies, laid the ground
work for new drug design and optimization.
Molecular docking experiments of new inhibitors provide key information for drug optimization.
Pharmacophore models were generated using ROCS program based on nine known
inhibitors. 3D overlay of the model with the best overall validation score is displayed
and the three compounds are colored in green, pink and orange. The principal feature
of the model is a set of consensus oxygen atoms, which are capable of chelating the
two metal ions at the active-site. The top scored model was used for virtual screening. Objective
The World Health Organization warns that flu
pandemics remain a constant threat to public health. If the
highly lethal avian influenza H5N1 virus acquires the ability
to transmit efficiently from human to human, it could cause
a disaster like the 1918 Spanish flu, which killed over 40
million people. As current anti-flu drugs are losing their
effectiveness due to emerging resistant strains, new anti-
influenza drugs are urgently needed to prepare for the
impending flu pandemics.
The influenza endonuclease is an ideal target to
develop new anti-flu medicine, because it is both well-
conserved and essential for viral propagation. The inhibitors
of this enzyme should block viral transcription and stop
propagation of any influenza virus including new pandemic
strains. Specific inhibitors could have minimal toxicity and
reduce the chance of developing resistance.
Six compounds were found to
inhibit PA endonuclease with
IC50 below 50M.
MD simulation can
illuminate the way
proteins move in
solution, and provide
more conformations for
drug development than
a single snapshot from
crystal structure.
Structural study by molecular dynamic (MD) simulation
Results and Interpretation
Based on molecular docking analysis, 21
available analogs of the new inhibitors
were selected for endonuclease activity
assay. Consistent with the predictions
from docking studies, modifications at the
two arms of D03 (chlorine and methoxy
group) diminished the inhibitory activity.
Meanwhile, four additional new inhibitors
with IC50 below 2 M were identified.
Two compounds C03 and C09 with minimal cytotoxicity were confirmed to
have good antiviral activity in reducing cytopathic effect in MDCK cells
caused by influenza virus 48 hours after infection. Experiments with
viruses were performed by a supervising scientist. More compounds are
being tested for their anti-viral activities.
Pharmacophore model and virtual screening
Surface representation of the N terminal endonuclease domain of the PA subunit (PAN ,1-209 residues) from
H5N1 for the X-ray structure and the three most populous cluster centroids (MD-C1, MD-C2 and MD-C3)
from MD simulation analysis. The two red spheres in each image represent Mn2+ ions.
Solvent mapping of PAN endonuclease on the
surfaces around the binding sites of the two Mn2+
ions (indicated as two pink balls). The results are
shown on the ribbon structure. The solvent probe
carbons are colored in green and the adjacent
amino acids of PAN are displayed. These results
will be useful for a fragment-based drug design
strategy called fragment growth. When
combined with the new scaffolds of inhibitor
candidates identified in this study, it can guide
lead optimization and rational design of new
inhibitors with better affinity.
Solvent mapping of druggable hot spots
Molecular docking of new inhibitors to PA
N

The Glide program was used to determine the binding modes of the new compounds with the X-ray
tertiary structures and the MD ensembles of the PA endonuclease. An optimal docking model of
compound D03 is depicted here. D03 has a chelating group with three oxygen atoms binding to the
two Mn2+ ions at the active pocket of PAN as predicted by the pharmacophore model. Different from all
known inhibitors, D03 has two arms extending into two pockets of the enzyme and forming
interactions (charge, van der Waals, hydrophobic) with residues conserved in the PA of influenza A
and B. Therefore, these new inhibitors are predicted to be effective to all influenza A and B strains
including new emerging potentially pandemic influenza viruses.
PA endonuclease assays
FRET (fluorescence resonance energy transfer) assay:
A single-stranded DNA oligonucleotide dual-labeled with 5-FAM fluorescence dye
and 3-TAMRA quencher dye was used as a reporter. Cleavage of the substrate
would separate the fluorescence dye from the quencher dye resulting in an increase
of the fluorescence signal. The assay is adapted to a 96- or 384-well format,
allowing measurement of hundreds of samples at a time. The assay was validated
by using the wildtype PA endonuclease and its inactive mutant control (PA-E119A).
FRET-based endonuclease assay of
compound D03. Traces shown are the
time course of fluorescence signal in the
FRET assay with different
concentrations of compound D03.
The IC50 of FRET assay and cytotoxic
CC50 in MDCK cells were determined for
each compounds. All experiments were
repeated three times.
M13mp18 single-stranded
circular phage DNA was
used as the substrate for
analyzing PA endonuclease
activity. The inhibitory effect
of six new inhibitors on PA
was confirmed by this gel-
assay. A gel result is shown
for the assays of compounds
C03 and D03.
Structural and Activity Relationship (SAR) study
Biological assays
PAN protein production: His tagged H1N1 PAN (1-220) was expressed in E. coli strain BL21
and purified by Ni-agarose affinity column to near homogeneity, confirmed by protein gel
electrophoresis.
Endonuclease FRET assay: A 17 base dual-labeled DNA oligonucleotide (FAM-TCT CTA
GCA GTG GCG CC-TAM) was used as the substrate. 1uM PAN, various concentrations of
inhibitors and 0.5uM Oligo substrate was added in the reaction. Fluorescence signal was read
at 485 nm excitation and 535 nm emission wavelength in the BioTek plate reader Flx-800 for
30min at 1min intervals at 37
0
C. The rate Vm was calculated by software with the instrument.
IC50 (50%inhibitory concentration) was determined by nonlinear curve fitting using
GraphPad Prism software.
Endonuclease gel-assay: M13mp18 single-stranded circular phage DNA was used as the
substrate and incubated with PAN and various concentrations of inhibitors at 37
0
C for 1 hr and
analyzed by agarose gel (1.0%) electrophoresis.
Compound toxicity assay: 1.5x10
4
Madin-Darby canine kidney (MDCK) cells were plated to
each well of a 96-well plate. One day later, compounds at various concentrations were added
and incubated for 48 hours, followed by staining with methylene blue dye. The plate was read
at 630nm on a plate reader and CC50 (50%cytotoxic concentration) was calculated.
Anti-influenza activity
filter undesirable compounds
Compound libraries
>450,000
>100,000
compounds
Pharmacophore model
237 compounds
or hits
Based on Lipinski Rule of Five
PAN
Inhibitor C03 D03
0 0.1 1 10 100 0.1 1 10 100 uM
DNA
only
Confirmation by single-strand DNA cleavage gel-assay
I would like to thank my mentors for their support.
Compound FRET IC50
M
Cytotoxic
CC50 M
C03 5.93+1.06 >50
D03 0.74+0.08 >100
H08 2.02+0.17 12.59+3.32
C09 13.69+3.63 >100
D09 10.78+2.68 20.76+5.93
C06 45.69+5.82 >100
X-Ray MD-C1 MD-C2 MD-C3
Cap Snatching by PA endonuclease
Cellular capped
Pre-mRNA
PA
PB1
PB2
vRNA
3
5
PA
PB1
PB2
vRNA
Viral transcript
Influenza viral RNA polymerase is composed of PA, PB1 and
PB2 subunits. PA has endonuclease activity, which cleaves
capped host pre-mRNA to start viral RNA transcription.
Leads optimization
Toxicity test
Preclinical
Clinical Trial
New PA inhibitors
FDA approved drugs
Efforts from both
universities and
pharmaceutical companies
Analog
Compound
FRET IC50
(M)
D03A 0.62+0.13
D03B >100
D03C >100
C09A 4.67+1.77
C09B 1.93+0.18
C09C 0.66+0.16
D09B 1.48+0.20