1. Chonnam National University, Korea
Scientific results of WISDOM first data challenges on malaria and avian flu
Doman Kim
Chonnam National University, Korea
On the behalf of the WISDOM collaboration

2. Searching for new drugs
Drug development is a long (10-12 years) and expensive (~800 MDollars) process
In silico drug discovery opens new perspectives to speed it up and reduce its cost
From Dr. Vincent Breton

3. A first step towards in silico drug discovery: virtual screening
In silico virtual screening
Starting from millions of compounds, select a handful of compounds for in vitro testing
Very computationally intensive but potentially much cheaper and time effective than typical in vitro testing
From Dr. Vincent Breton

4. Wisdom I workflow for malaria inhibitor development
Molecular docking
Molecular dynamics
Re-ranking
MMPBSA-GBSA
Complex
visualization
In vitro
tests
Catalytic aspartic residues
4 H bonds
Amber
Ligand
2 Hydrogen
Bonds
AMBER
CHIMERA
WET LABORATORY
Millions
5000
180 30
FLEXX
AUTODOCK
From Ana & Vinod

5. Inhibition of Plasmepsin II
Areas with limited risk
No malaria
Areas where malaria transmission occurs

6. Plasmepsin II Hemoglobin (Hb) Heme Large fragments Hematin
Malaria, a dreadful disease is cause by protozoan parasite, plasmodium.
Plasmodium specise : Plasmodium falciparum, P. vivax, P. malariae, P. ovale
One of the crucial drug targets in malaria are plasmepsin.
Plasmepsins are involved in the hemoglobin degradation inside the food vacuole during the erythrocytic phase of the life cycle.
Ten different isoforms (PMI, II, III, IV, V, VI, VII, IX, X and HAP)
Plasmepsin II is responsible for initial attack on the hemoglobin α-chain between the residues Phe 33 and Leu 34, in the hinge region.
Hemoglobin (Hb)
Large fragments
Small peptides
Amino acids
Plasmepsins I, II, IV and HAP
Falcipain, plasmepsin
Falcilysin, aminopepdidases
Heme
Hematin
Hemozoin
oxidation
polymerization
(malarial pigment)
<Hemoglobin degradation in Plasmodium facipuram>

7. Expression and purification of recombinant plasmepsin II
- Plasmids name : rPMII (plasmepsin II),
- vector : pET-3d (4,640 bp, selection marker : ampicillin)
transformation of E. coli BL21(DE3)
kDa 66 44
- IPTG induction (1 L) : grown to an OD600 of 0.5 at 37oC
 addition of IPTG  18oC
37 kDa 31
Lane 1: cell supernatant after treatment of 8 M urea
Lane 2 : purified enzyme using Q-Sepharose column
Binding by 20 mM Tris buffer pH 8.0
Each fraction: 5 ml
Washing by same buffer
Each fractions: 30 ml
Elution by from 0 to 1 M NaCl in same buffer
Each fraction: 3 ml
OD280
NaCl concentration
Binding
Washing
Elution from 0 M to 1 M
Q-Sepharose chromatography
On UV

8. Malaria Aspartyl proteinase FRET assay
DABCYL-Glu-Arg-Nle-Phe-Leu-Ser-Phe-Pro-EDANS
cleavage site
Peptide
C NH
Dye O
Protease
COOH + H2N
<Carboxypeptidase reaction of “peptide-CO-NH-Dye” fluorogenic substrate>
Synthetic peptide designed to mimic the cleavage site present in hemoglobin
lane 1 : FRET substrate (10 μM)
lane 2 : active rPM2
lane 3 : FRET substrate + active rPM2
Before reaction After reaction

9. Pepstatin A vs. New potential plasmepsin inhibitors
General inhibitor of aspartic proteases
inhibition of hemoglobin degradation
by extracts of digestive vacuoles of P.
faciparum (Phe33 and Leu34 in the
hinge region of the α-chain)

10. rPM2 activity assay I FI
Rank　 NI
3418 PA
1059 1
2200 23 2
2074 22 3
2981 27 4
1439 16 5
2485 26 6
1297 11 7
1230 9 8
1402 13
3534 30 10
3430 28
1531 17 12
1808 21
2209 24 14
1025 　I FI
Rank　 15
1760 20 16
1214 8 17
1046 4 18
1173 7 19
1059 5
1026 3 21
1016 1 22
2322 25 23
1414 14 24
1642
1253 10 26
1341 12 27
1074 6 28
1426 29
1631 30
3499
Plasmepsin assay
- Plasmepsin activation : rPMII + assay buffer (pH 4.5) + 1 μl inhibitor (100 nM)
 37oC, 30 min induction
- FRET assay
1) Activated enzyme (75 ng) + 3 μM substrate peptide (50 μl final volume)
 30 min incubation at room temperature
2) measuring using a fluorescence microplate reader (excitation 405 nm, emission 510 nm)  detection of fluorescence spectra or UV spectra
I checked enzyme activity using 3 uM FRET substrate and hemoglobin and 100 nM inhibitors.
As shown in Figure, FRET substrate degradation by recombinant plasmepsin II was completely inhibited by 10 mM pepstatin A..
At the result of detection using Fluorescence spectra, best inhibitors are NO. 21, 14 and 20.
Next week, I will check enzyme activity using lower concentration of inhibitors.
I – Inhibitors; NI – w/o inhibitor
PA – w/ pepstatin A (reference)
* Red valued inhibitors – contain own fluorescence.
Similar or better inhibitions : 6/30 compounds [21,14, 20, 17, 19, (27)]

11. Inhibition test (hemoglobin degradation)
Recombinant plasmepsin II (75 ng) + assay buffer (pH 4.5) + 1 μl inhibitor
 37oC, 30 min incubation (pre-activation) (500 μM)
rPMII-inhibitor complex + Human hemoglobin (10 μg)
 37oC, 3 hr incubation
3. Digestion was terminated by addition of SDS-PAGE loading dye
4. SDS-PAGE analysis on a 15% polyacrylamide gel
H C P H
H C P
No detection of hemoglobin degradation fragments except inhibitors of 5, 9, 13, 21, 23, 24, 29
P – Pepstatin A, C – w/o Inhibitor, H – Hemoglobin only

12. Development of Neuraminidase Inhibitor by Grid-Enabled Virtual Screening
My subject is virtual screening of n…..
Especially In-vitro test
As you know well,
The shape of Avien virus is like this.
Two component is important for infected cell such as chicken …
One is Haemagglutinin spikers, which mediate with cell surface sialic acid receptor binidng to initiate virus infection
Nu removes sialic acid from virus and cellular glycoproteins to facilitate virus release and the spread of infection to new cells.
Then, we foucused on the nurmanidase, and virtual screening for Nu inhibition had already done by France co-workers.
WHO homepage

13. Modeling HTS against Inf-A NA on Grid
Neuramindase (NA) and replication of virion NA HA
An enzyme, cleaves host receptors
help release of new virions
From Prof. Ying-Ta Wu 13

14. GH Families by similarity of amino acid sequence
Classification of neuraminidase:
Coutinho, P.M. & Henrissat, B. (1999)
GH Families by similarity of amino acid sequence

15. Glycoside Hydrolase Family 33
…..mainly Pathogenic bacteria
Neuraminidase from Clostridium perfringens ATCC (Available 3D structure)
Streptococcus pneumoniae ATCC 6322, Salmonella typhimurium TA262
Vibrio cholera N16961….
Glycoside Hydrolase Family 34
…..mainly Influenza virus
Our target neurmanidase is largely 4 kinds and they belongs to GH family 33 such as strains Clostridium perfringens and Streptococcus pneumonia, and belongs to family 34 such H5N1.
Especially, 3D structure of Neuraminiase from Clostridium perfringens and H5N1 is available and good enzyme for inhibitor development.
Neu from Streptococcus pnemoniae focused on the function of pathogen and formation of different type capsular polysaccharide.
Neuraminidase from influenza A virus and B virus
about 6427 (Available 3D structure)

16. The deployment
Evaluate potential targets and model their 3D structures
Prepare the large-scale docking using Autodock.
Development of the grid environment for a large-scale deployment.
H5N1
From Prof. Ying-Ta Wu et al.

17. Preparation of neuraminidase 1:
GH family 34/H5N1
Ni-NTA column chromatography
E. coli Rosseta ( DE3 ) haboring-Neu1-23d
LB media (5 ml) containing
Ampicilline 50 ug/ml
Cultured until OD600＝0.5 at 37oC
Cool down in ice
Add IPTG
Cultured more …. 16oC
For purification, 4 L culture. sp
Neu1 : bp
Elution by from 25 to 500 mM IMD
Each fraction: 15 ml
SDS-PAGE (12%) of Neu1
kDa
209
124 80
Neuraminidase
50kDa
49.1
detect on the UV llumination
34.8
20.6
After 4 h SM SM T S 6 8 10 12 14 8

18. Preparation of neuraminidase 2:
GH family 33/ C. perfringens Neuraminidase
E. coli Rosseta ( DE3 ) haboring-CP42
LB media (5 ml) containing
Ampicilline 50 ug/ml
Cultured until OD600＝0.5 at 37oC
Cool down in ice
Add IPTG
Cultured more …. 18oC
For purification, 1 L culture.
Ni-NTA column chromatography
Elution by from 25 to 500 mM IMD
Each fraction: 15 ml
Expression vector
SDS-PAGE (10%) of CP42
kDa
209
124
Neuraminidase 80
In case of NamB, I tried to two kinds of construction plan like Neu22B/ Neu22B-M with or without signal peptide.
As like this figure, Amplified gene was cloned into pGemTeazy vector,
After digested with Nco1/Xhol, there are around 2 kb fragment.
As like this figure, 2 kb fragement constructed with pET23d as expression vector.
It will wait for expression confirmation.
In case of H5N1, Neuraminidase of H5N1, degenerated as NeuN1.
Digested Neul and pPICZaA as expression were ligated with EcoR1 and Not1.
As like this figure, 1.35kb for NeuN1 and 3.3kb fragment was shown.
Next slide show expression in E . Coli .
For expression in E. coli
I designed the three kinds of expression vector such as pET28a, pET23d, and pET32a depend on the N-, C-terminal histag or containing TrxA protein for high solubilization
Of expressed gene.
To insert in expression vector, NcoI contain the Neu1 gene,
I try to amplified using megar primer PCR.
As shown in Figure,
Ndel/Xhol product was amplified having 1.35 kb,
And FPCR product was obtained in approximatedly o.5 kB
After second PCR, mutated DNA was amplified in 1.35 kb
Now, I am doing cloning of amplified gene.
After preparation of recombinant enzyme, they will do inhibition’s test using amplex Red and 4MU-NANA as substrate.
That all
49.1
detect on the UV illumination/
Florescent at excitation at 332 nm emission 448 nm
42kDa
34.8
20.6 SM 8 10 12 14 S CE
CP42

19. Second screening (2 nmol)
Assay for neuraminidase activity 2: MU-NANA
4-Methylumbeliferyl-N-acetyl-a-D-neuramininic acid ammonium salt
[4MU-NANA]; Substrate
First screening
(200 nmol)
Recombinant Neuraminidase
Spectrofluorometric detector RF-551
362 nm excitation and 448 nm emission wavelengths
Second screening (2 nmol)
Red
Kinetic study
Inhibition
Blue

20. Screening of neuraminidase: First screening
Neu1 in GH 34
CP42 in GH 33
First Screening
116/308 compounds – 38%
First Screening
42/169 compounds – 25%

21. Screening of neuraminidase: Second screening
Neu1 in GH 34
CP42 in GH 33
Second screening
62/308 compounds-20.1% (Higher inhibition activity compare to Tamiflu)
Second screening
0/169 compounds-0%

22. Relative activity of Neu1
4MU-NANA
: 20 mM/RM
Neuraminidase
: 10 mU/reaction
Measure at excitation 362 nm and
emission at 448 nm
Rank
Compounds
Relative activity of Neu1 1
113 67 2 16 72 3 6 73 4
155 74 5 78 63
Tamiflu
100
On UV

23. Preparation of H5N1 mutant enzymes:
GH family 34/N1Mutation
Megar primer PCR
1. FPCR
2. Second
PCR
3. Cloning
7 mutants
E119G/A/D/V
H274Y/F
R292K
N294S
And I also mutant enzyme for inhibitor screening.
Recently, some mutant neurainidas show the resistantance for tamiflu
So, I also try to express mutant enzymes of N1 neruaminidase like 8 mutants.
Using this enzymes, I also use to search inhibitor for each mutants.
From Prof. Ying-Ta Wu et al.

24. Acknowledgements Enzyme in vitro tests:
Chonnam National University, Korea
Young-Min KIM (Nuraminidases), Hee-Kyoung KANG (Plasmepsin)
Nuraminidase, Plasmepsin In silico data challenge and analyses:
Academia Sinica, Taiwan
Hurng-Chun LEE, Simon C. LIN (Grid Computing Center)
Ying-Ta WU, Chon-Chen LEE (Genomic Research Center)
CNRS-IN2P3-LPC, Clermont-Fd, France
Vincent BRETON, Nicolas JACQ, Jean SALZEMANN
Vinod KASAM, Ana DA COSTA, Vincent BLOCH
Yannick LEGRE
HealthGrid
Nicolas SPALINGER
SCAI-Fraunhofer Institute, Germany
Martin HOFMANN
Modena University, Italy
Giulio RASTELLI