WISDOM In silico Drug Discovery

1. 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 NGUYEN1, Sun LEE1, Soonwook HWANG2, Seungwoo RHO2, Vincent BRETON4, Doman KIM1 1Biotechnology and Bioengineering, Chonnam National Uiversity, Gwangju, South Korea2Korea Institute of Science and Technology Information, Daejeon, Korea, 3HealthGrid LPC-Clermont-Ferrand, France, 4 LPC-Clermont-Ferrand, France TEL: +82-62-530-1844, FAX: +82-62-530-1949 , E-mail: dmkim@chonnam.ac.kr

2. Enabling Grids for E-sciencE WISDOM In silico Drug Discovery • WISDOM: http://wisdom.healthgrid.org/ • 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

3. 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

4. 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

5. Discoveries of novel inhibitor for human intestinal maltase • 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):782-92

6. Data challenging on WISDOM production environment

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

8. Cloning and expression of human intestinal maltase in Pichiapastoris Set 1 Set 2 M 1 2 C 1 2 C Control 0 24 40 48 96h Glc 0 24 40 48 96h PCR M P 2.7Kb Enzyme activity • 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) Primer set 1 : α-factor - Internal Primer set 2 : α-factor – 3’AOX1

9. Primarily in vitro Inhibition assay Inhibition at 100 μM

10. Kinetic characterization of hit compounds 18 17 acarbose → Competitive inhibitor → Ki ≒ 19.4 μM → Competitive inhibitor → Ki = 19.8 ± 1.2 μM → Competitive inhibitor → Ki = 19.6 ± 0.9 μM

11. Chemical structure, physiochemical properties and inhibition activity of the indentified hits with HMA

12. Hydrogen bond interactions with key residues of two hit compounds in active site of protein (B) A) (A) (C) www.themegallery.com

13. Docking experiment of two hit compounds with human pancreatic α-amylase A Active site Human pancreatic α-amylase PDB ID: 1XCX 187899 258532 Acarbose C D • Biotechnol. Lett. 2011 Nov;33(11):2185-9

14. Discovery of Novel inhibitor of 3CL protease of SARS • 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 (3CLpro) of severe acute respiratory syndrome associated coronavirus (SARS-CoV) is vital for SARS-CoV replication and is a promising drug target.

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

16. Cloning and expression of 3CL-protease of SARS in E. coli BL21 (DE3) Colony-PCR of E.coli BL21 (DE3) Transformation into E.coli DH5α pET28a M B U W1 W2 W3 E1 E E3 E4 E5 E6 E7 E8 E9 M 3CL-932bp RE digestion 940C 940 C 1min 5 min 720 C 720 C 1min 5 min 530C 30 s 25 cycles 45 3CL protease 31 Ni-NTA purification

17. Primarily Inhibition study * Inhibitor at 100 μM Km = 10.17 ± 1. 4 μM (3CL protese from E.coli BL21(DE3)

18. IC50 of hit compounds against 3CLpro of SARS

19. Kinetic analysis of 3CLpro of SARS inhibition by compound 7 Fig. Lineweaver-Burk plot (A) and Dixon plot (B) of the inhibition of 3CLpro from E.coli BL21 (DE3) by compound 7. → Compound 7 inhibits 3CLproas a competitive inhibitor → Kivalue for compound 7 is 9.93 ± 0.44 μM

20. Hydrogen bond interaction of compound 7 against 3CLpro

21. Hydrogen bond interaction of compound 6 against 3CLpro • Bioorg. Med. Chem. Lett. 2011 May 15;21(10):3088-91 • Inhibitors of SARS-coronavirus 3CL Protease for Severe Acute Respiratory Syndrome and Method for screening thereof. Korea Patent Pending, 10-2011-0003078 (Jan 11, 2011)

22. Conclusion After datachallenge of 308,307 compounds, 42 compounds of HMA and 53 compounds of 3CLpro of SARS were select for in vitro assay; The 2 compounds and 7 compounds for HMA and SARS, respectively were identified IC50; 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.

23. 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;

24. Acknowledgements Enzyme in vitro tests: Hwa-JaRyu, 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.

25. The Laboratory Functional Carbohydrate Enzymes and microbial Genomics.