Virtual screening and inhibition assay of human intestinal maltase and 3C-like protease of SARS using molecular docking on WISDOM production environment Thi-Thanh-Hanh NGUYEN 1, Sun LEE 1, Soonwook HWANG 2, Seungwoo RHO 2, Vincent BRETON 4, Doman KIM 1 1 Biotechnology and Bioengineering, Chonnam National Uiversity, Gwangju, South Korea 2 Korea Institute of Science and Technology Information, Daejeon, Korea, 3 HealthGrid LPC-Clermont-Ferrand, France, 4 LPC-Clermont-Ferrand, France TEL: , FAX: ,

Enabling Grids for E-sciencE WISDOM In silico Drug Discovery  WISDOM:  Goal: find new drugs for neglected and emerging diseases Neglected diseases lack R&D Emerging diseases require very rapid response time  Need for an optimized environment To achieve production in a limited time To optimize performances  Method: grid-enabled virtual docking Cheaper than in vitro tests Faster than in vitro tests Dr. Vincent Breton

Searching for new drugs  Drug development is a long (10-12 years) and expensive (~800 M US$) 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

Human intestinal maltase : N-terminal of Human maltase glucoamylase responsible for the hydrolysis of α (1-4)-linkages from maltooligosaccharide and belongs to glycosides hydrolase family 31 Inhibition of the enzyme activity → retardation of glucose absorption → decrease in postprandial blood glucose level Important target to discovery of new drug for treatment of type-2 diabetes. Sim L, Quezada-Calvillo R, Sterchi EE, Nichols BL, Rose DR. 2008, J Mol Biol. 375(3): Discoveries of novel inhibitor for human intestinal maltase

Data challenging on WISDOM production environment Total numbers of docking308,307 Total size of output results16.3 GBytes Estimated duration by 1 CPU22.4 years Duration of experiments3.2 days Maximum numbers of concurrent CPUs4700 CPUs Crunching Factor2556 Distribution Efficiency54.4 %

Processing in virtual screening Scoring based on docking score ( 308,307) Scoring based on docking score ( 308,307) 454,000 chemical compounds from Chembridge Interaction with key residues 2974 compounds selected 2574 compounds selected Key interactions binding models clustering Key interactions binding models clustering In vitro test In vitro test 42 compound selected Autodock 3 WISDOM Chimera and ligplot Wet Laboratory

Cloning and expression of human intestinal maltase in Pichia pastoris PCR M P 2.7Kb M 1 2 C 1 2 C Set 1 Set 2 Primer set 1 : α-factor - Internal Primer set 2 : α-factor – 3’AOX1  Conditions for HMA expression → Culture 500 ml in 2 L flask at 30 ℃ and 200 rpm → 0.5% methanol → ~4 days → enzyme reaction : 90 min at 37 ℃ (50 mM maltose) h Glc h ControlEnzyme activity

Primarily in vitro Inhibition assay Inhibition at 100 μM

Kinetic characterization of hit compounds → Competitive inhibitor → Ki = 19.8 ± 1.2 μM → Competitive inhibitor → Ki = 19.6 ± 0.9 μM → Competitive inhibitor → Ki ≒ 19.4 μM acarbose

Chemical structure, physiochemical properties and inhibition activity of the indentified hits with HMA Compound No Chemical structure Lowest energy M.W (g/mol) clogPK i (μM) IC 50 (µM) Type of inhibition ±1.2 58±4competitive ± ±3competitive Acarbose ±4competitive

Hydrogen bond interactions with key residues of two hit compounds in active site of protein (A) (B) (C) A)

Docking experiment of two hit compounds with human pancreatic α-amylase Human pancreatic α-amylase PDB ID: 1XCX Acarbose A C D NumberName of compounds Binding energy (kcal/mol) 1IAB Biotechnol. Lett Nov;33(11): Biotechnol. Lett Nov;33(11): Active site

 The possibility of the re-emergence of SARS is a serious threat, since efficient therapy and a vaccine are not currently available;  The 3C-like protease (3CL pro ) of severe acute respiratory syndrome associated coronavirus (SARS-CoV) is vital for SARS-CoV replication and is a promising drug target. Discovery of Novel inhibitor of 3CL protease of SARS

Processing in virtual screening Scoring based on docking score ( 308,307) Scoring based on docking score ( 308,307) 454,000 chemical compounds from Chembridge Interaction with key residues 1468 compounds selected 1065 compounds selected Key interactions binding models clustering Key interactions binding models clustering In vitro test In vitro test 53 compound selected Autodock 3.0 WISDOM Chimera and ligplot Wet Laboratory

Cloning and expression of 3CL-protease of SARS in E. coli BL21 (DE3) Transformation into E.coli DH5α RE digestion pET28a 3CL- 932bp 94 0 C 1min 53 0 C 30 s 72 0 C 94 0 C 5 min 1min 72 0 C 5 min 25 cycles Colony-PCR of E.coli BL21 (DE3) M B U W1 W2 W3 E1 E E3 E4 E5 E6 E7 E8 E9 M 3CL protease Ni-NTA purification 45 31

Primarily Inhibition study Km = ± 1. 4 μM (3CL protese from E.coli BL21(DE3) * Inhibitor at 100 μM

Compound No Free binding energy (kcal.mol -1 ) IC 50 (μM) ± ± ± ± ± ± ± 1.17 IC 50 of hit compounds against 3CL pro of SARS

Kinetic analysis of 3CL pro of SARS inhibition by compound 7 Fig. Lineweaver-Burk plot (A) and Dixon plot (B) of the inhibition of 3CL pro from E.coli BL21 (DE3) by compound 7. → Compound 7 inhibits 3CL pro as a competitive inhibitor → K i value for compound 7 is 9.93 ± 0.44 μM

Hydrogen bond interaction of compound 7 against 3CL pro

Hydrogen bond interaction of compound 6 against 3CL pro Bioorg. Med. Chem. Lett May 15;21(10): Bioorg. Med. Chem. Lett May 15;21(10): Inhibitors of SARS-coronavirus 3CL Protease for Severe Acute Respiratory Syndrome and Method for screening thereof. Korea Patent Pending, (Jan 11, 2011) Inhibitors of SARS-coronavirus 3CL Protease for Severe Acute Respiratory Syndrome and Method for screening thereof. Korea Patent Pending, (Jan 11, 2011)

Conclusion After datachallenge of 308,307 compounds, 42 compounds of HMA and 53 compounds of 3CL pro of SARS were select for in vitro assay; The 2 compounds and 7 compounds for HMA and SARS, respectively were identified IC 50 ; All of these compounds were showed the competitive inhibition. The inhibitors could be stabilized by the formation of H- bonds with catalytic residues and the establishment of hydrophobic contacts at the opposite regions of the active site.

Further study  Virtual screening of nature compounds, chembridge ligand library, Chemdiv ligand library, and Zinc with: -Influenza virus: N1 from H1N1. -Malaria: falcipain 2, 3. -Sars; -Diabetes type 2;

Acknowledgements Enzyme in vitro tests: Hwa-Ja Ryu, Hee-Kyoung Kang, Sun Lee (CNU, in vitro test), In silico data challenge and analyses (WISDOM): KISTI, Korea Soon-Wook HWANG, Seungwoo RHO, et al. CNRS-IN2P3-LPC, Clermont-Fd, France Vincent BRETON et al.

The Laboratory Functional Carbohydrate Enzymes and microbial Genomics.