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

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

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

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

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

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>

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

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 1 2 3 1 2 3 lane 1 : FRET substrate (10 μM) lane 2 : active rPM2 lane 3 : FRET substrate + active rPM2 Before reaction After reaction

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)

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)]

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 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 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

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

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

GH Families by similarity of amino acid sequence Classification of neuraminidase: http://www.cazy.org/ http://www.cazy.org/ Coutinho, P.M. & Henrissat, B. (1999) GH Families by similarity of amino acid sequence

Glycoside Hydrolase Family 33 …..mainly Pathogenic bacteria Neuraminidase from Clostridium perfringens ATCC 13124 (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)

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.

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 : 1350 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

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

Second screening (2 nmol) Assay for neuraminidase activity 2: 4-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

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%

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%

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

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.

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