전남대학 교 carboenz 기능성 탄수화물 효소 및 미생물 유전체 연구실 scientific...

전남대학전남대학교교

http://altair.chonnam.ac.kr/http://altair.chonnam.ac.kr/~carboenz~carboenz

기능성 탄수화물 효소 및 미생물 유전체 연구실기능성 탄수화물 효소 및 미생물 유전체 연구실

Scientific results of WISDOM first

data challenges on malaria and

avian flu

Doman KimChonnam National University, Korea

On the behalf of the WISDOM collaboration




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

TargetIdentification and validation- 2/5 years- 30% success rate

Leadidentification- 0.5 year- 65% success rate

Leadoptimization- 2/4 years- 55% success rate

Target discovery Lead discovery

Gene expression analysis,Target function prediction,Target structure prediction

De novo design,Virtual screening

Virtual screening,QSAR

TargetIdentification and validation- 2/5 years- 30% success rate

Leadidentification- 0.5 year- 65% success rate

Leadoptimization- 2/4 years- 55% success rate

Target discovery Lead discovery

Gene expression analysis,Target function prediction,Target structure prediction

De novo design,Virtual screening

Virtual screening,QSAR

From Dr. Vincent Breton




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

Com

putationaldemand

Starting compound database

Starting target structure model

Filter, preparation

Docking, scoring, filter

Predicted binding models

Post-analysis

Define binding site

Visual evaluation

Visual evaluation

Visual evaluation

Compounds for assay

Protein surface

Ligand

Water

Protein surface

Ligand

Water

From Dr. Vincent Breton




Wisdom I workflow for malaria inhibitor development

FLEXXAUTODOCK

Molecular docking

Molecular dynamics

Re-ranking MMPBSA-GBSA

Complexvisualization

In vitro tests

Catalytic aspartic residuesCatalytic aspartic residues4 H bonds

AmberLigand

Ligand2 Hydrogen Bonds

Ligand

Catalytic aspartic residues

AMBER

CHIMERA

WET LABORATORY

Millions

5000

180

30

From Ana & Vinod




Inhibition of Plasmepsin II

Areas with limited risk No malaria

Areas where malaria transmission occurs




Plasmepsin IIPlasmepsin II

-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>




Expression and purification of recombinant plasmepsin II

M 1 2

66

44

31

kDa

37 kDa

Lane 1: cell supernatant after treatment of 8 M ureaLane 2 : purified enzyme using Q-Sepharose column

- IPTG induction (1 L) : grown to an OD600 of 0.5 at 37oC addition of IPTG 18oC

- Plasmids name : rPMII (plasmepsin II), - vector : pET-3d (4,640 bp, selection marker : ampicillin)- transformation of E. coli BL21(DE3)

Binding by 20 mM Tris buffer pH 8.0Each 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

0

0.5

1

1.5

2

2.5

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 160

Fraction No.

0

0.2

0.4

0.6

0.8

1

1.2

OD

28

0

NaC

l co

nce

ntr

ati

on

BindingWashing Elution from 0 M to 1 M

Q-Sepharose chromatography

On UV




Malaria Aspartyl proteinase FRET assay

DABCYL-Glu-Arg-Nle-Phe-Leu-Ser-Phe-Pro-EDANS

cleavage site

- Synthetic peptide designed to mimic the cleavage site present in hemoglobin

Peptide C NH Dye

OProtease

Peptide COOH + H2N Dye

<Carboxypeptidase reaction of “peptide-CO-NH-Dye” fluorogenic substrate>

1 2 3 1 2 3

Before reaction After reaction

lane 1 : FRET substrate (10 μM)lane 2 : active rPM2lane 3 : FRET substrate + active rPM2




Pepstatin A- 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)

Pepstatin A vs. New potential plasmepsin Pepstatin A vs. New potential plasmepsin inhibitorsinhibitors




rPM2 activity assay

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 FIRank

　

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

9 3534 30

10 3430 28

11 1531 17

12 1808 21

13 2209 24

14 1025 2

I FI Rank 　

15 1760 20

16 1214 8

17 1046 4

18 1173 7

19 1059 5

20 1026 3

21 1016 1

22 2322 25

23 1414 14

24 1642 19

25 1253 10

26 1341 12

27 1074 6

28 1426 15

29 1631 18

30 3499 29

I – Inhibitors; NI – w/o inhibitorPA – w/ pepstatin A (reference)* Red valued inhibitors – contain own fluorescence.

Similar or better inhibitions : 6/30 compounds [21,14, 20, 17, 19, (27)]




Inhibition test (hemoglobin degradation)

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

H C P 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 H H C P 24 25 26 27 28 29 30

1. Recombinant plasmepsin II (75 ng) + assay buffer (pH 4.5) + 1 μl inhibitor

37oC, 30 min incubation (pre-activation) (500 μM)

2. rPMII-inhibitor complex + Human hemoglobin (10 μg) 37oC, 3 hr incubation3. Digestion was terminated by addition of SDS-PAGE loading dye4. SDS-PAGE analysis on a 15% polyacrylamide gel



기능성 탄수화물 효소 및 미생물 유전체 연구실기능성 탄수화물 효소 및 미생물 유전체 연구실WHO homepage

Development of Neuraminidase Inhibitor by Grid-Enabled Virtual Screening




HA

Neuramindase (NA) and replication of virion

An enzyme, cleaves host receptorshelp release of new virions

NA

Modeling HTS against Inf-A NA on Grid

From Prof. Ying-Ta Wu




GH Families by similarity of amino acid sequence

http://www.cazy.org/ Coutinho, P.M. &

Henrissat, B. (1999)

Classification of neuraminidase: http://www.cazy.org/




Neuraminidase from Clostridium perfringens ATCC 13124 (Available 3D structure)

Streptococcus pneumoniae ATCC 6322, Salmonella typhimurium TA262

Vibrio cholera N16961….

Neuraminidase from influenza A virus and B virus

about 6427 (Available 3D structure)

…..mainly Pathogenic bacteria

…..mainly Influenza virus

Glycoside Hydrolase Family 33

Glycoside Hydrolase Family 34




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.

The deployment

H5N1

EGEE Grid Resources

Web Interface

DIANEMaster Process

Resource Broker

Grid Job Submission

Docking task pullingDocking complex returning

Virtual Cluster (DIANE workers)

Interactive scoringVisualization

From Prof. Ying-Ta Wu et al.




Elution by from 25 to 500 mM IMDEach fraction: 15 ml

Ni-NTA column chromatography

E. coli Rosseta ( DE3 ) haboring-Neu1-23dLB media (5 ml) containing Ampicilline 50 ug/ml

Cultured until OD600 ＝ 0.5 at 37oC Cool down in iceAdd IPTGCultured more …. 16oCFor purification, 4 L culture.

detect on the UV llumination

Neuraminidase

GH family 34/H5N1Preparation of neuraminidase 1:

sp

Neu1 : 1350 bp

SDS-PAGE (12%) of Neu1

SM SM ST

20.6

34.8

49.1

80124 209

kDa

0

0.2

0.4

0.6

0.8

0 5 10 15 20 25 30 35 40

Fraction nos

ab

s28

0

6 8 1210

50kDa

14 8

After 4 h




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 iceAdd IPTGCultured more …. 18oCFor purification, 1 L culture.

GH family 33/ C. perfringens NeuraminidasePreparation of neuraminidase 2:

0

0.2

0.4

0.6

0.8

1

0 10 20 30 40

Fraction nos.

ab

s28

0

Elution by from 25 to 500 mM IMDEach fraction: 15 ml

Ni-NTA column chromatography

SDS-PAGE (10%) of CP42

SM 8 141210 S CE

42kDa

20.6

34.8

49.1

80124 209

kDa

CP42

detect on the UV illumination/Florescent at excitation at 332 nm emission 448 nm

Neuraminidase

Expression vector




Spectrofluorometric detector RF-551362 nm excitation and 448 nm emission wavelengths

4-Methylumbeliferyl-N-acetyl--D-neuramininic acid ammonium salt [4MU-NANA]; Substrate

Recombinant Neuraminidase

Assay for neuraminidase activity 2: 4-MU-NANA

Red

BlueInhibition

First screening

(200 nmol)

Second screening (2 nmol)

Kinetic study




CP42 in GH 33Neu1 in GH 34

Screening of neuraminidase: First screening

First Screening116/308 compounds – 38%

First Screening42/169 compounds – 25%




Neu1 in GH 34 CP42 in GH 33

Screening of neuraminidase: Second screening

Second screening 62/308 compounds-20.1% (Higher inhibition activity compare to Tamiflu)

Second screening 0/169 compounds-0%




Measure at excitation 362 nm andemission at 448 nm

4MU-NANA: 20 M/RM

Neuraminidase: 10 mU/reaction

Rank Compounds Relative activity of Neu1

1 113 67

2 16 72

3 6 73

4 155 74

5 78 78

63 Tamiflu 100

On UV




GH family 34/N1MutationPreparation of H5N1 mutant enzymes:

1. FPCR

2. Second PCR

Megar primer PCR

3. Cloning

E119G/A/D/VH274Y/FR292KN294S

7 mutants

From Prof. Ying-Ta Wu et al.




Enzyme in vitro tests: Chonnam National University, KoreaYoung-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, FranceVincent BRETON, Nicolas JACQ, Jean SALZEMANNVinod KASAM, Ana DA COSTA, Vincent BLOCHYannick LEGRE

HealthGrid Nicolas SPALINGER

SCAI-Fraunhofer Institute, GermanyMartin HOFMANN

Modena University, Italy Giulio RASTELLI

Acknowledgements

전남대학 교 carboenz 기능성 탄수화물 효소 및 미생물 유전체 연구실 scientific...

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