Isolation and Characterization of a Hyperbranched Proteoglycan from Ganoderma Lucidum for Anti-Diabetes

1. Isolation and characterization of a hyperbranched proteoglycan from Ganoderma Lucidum for anti-diabetes 1 Ping Zhou Fudan University, Shanghai, China 25/7/2016 Natural Products Conferences in Melbourne, Australia, July 25-27, 2016

2. Background Natural Products Conferences in Melbourne, Australia, July 25-27, 2016

3. 3 Insulin signaling pathway and PTP1B PTP1B • Protein tyrosine phosphatase 1B (PTP1B) dephosphorylate the insulin receptor, and negatively regulates the insulin signal transduction. • Overactivation of PTP1B inhibits the insulin receptor signaling cascade. Therefore, PTP1B is an insulin-sensitive drug target for anti-diabetes. • Developing PTP1B inhibitor is a promising strategy for diabetes treatment.

4. Current development of PTP1B inhibitors • Synthesized small molecules: • Hydrophilic fluorine methylene phosphoric tyrosine analogues • Carboxylic phosphate tyrosine analogues • Heterocyclic phosphoric tyrosine analogues • Characteristic: • Significantly inhibitory effect on PTP1B activity in vitro • Good selectivity for PTP1B • Shortcoming: • Bad cell penetration • Not safe in vivo • New strategy: • Inhibitor better cell penetrative and significantly inhibitory on PTP1B in vivo and in clinic • Natural product highly safe in clinic Andrew P. Combs Recent Advances in the Discovery of Competitive Protein Tyrosine Phosphatase 1B Inhibitors for the Treatment of Diabetes, Obesity, and Cancer J.Med.Chem.2010.53.2333-2344 4

5. Our Results Natural Products Conferences in Melbourne, Australia, July 25-27, 2016

6. 6 PTP1B inhibitor extracted from G. lucidum • FYGL is a water soluble macromolecule PTP1B inhibition Extraction FYGL—— Fudan-Yueyang G. lucidum Ganoderma lucidum, used for diabetes treatment for two thousand years • BothUV and refraction index in GPC analysis demonstrated the FYGLcontent of 90%, and molecular weight of 106with both protein and sugar present. Incubated plant

7. 7 7 PTP1B inhibition of FYGL in vitro • PTP1B inhibition and mechanism of FYGL • PTP1B inhibition IC50 = 5.12 g/mL • Lineweaver--Burk plots indicated FYGL is a competitive inhibitor of PTP1B. Bao-Song Teng, Ping Zhou,* et al.J. Agric. Food Chem. 2011, 59(12), 6492-6500.

8. FYGL effect on insulin pathway • FYGL treatment was associated with an increase in phosphorylation of insulin receptor IRS1, and its down stream signaling, AKT and ERK kinases. • The data suggest that FYGL have effects on increasing the insulin signaling upon insulin stimulation in adipocytes. Western blot analysis for phosphorylation of IRS1, AKT and ERK in 3T3 cells.

9. 9 9 Pharmacology trials of FYGL in vivo • Plasma glucose level of STZ-induced type II diabetes rats Bao-Song Teng, Ping Zhou,* et al.Eur Rev Med Pharmacol Sci. 2012; 16: 166-175; J. Agric. Food Chem.2011, 59, 6492-6500

10. 10 10 Pharmacology trials of FYGL in vivo • Plasma glucose level of db/db genetic type II diabetes mice Chendong Wang, Ping Zhou,* et al. Brit J Nutr, 2012, 108, 2014-2025.

11. 9 Pharmacology trials of FYGL in vivo • Glycosylated hemoglobin (HbAlc) level of db/db mice n = 8, *p < 0.05 vs. control，**p < 0.01 vs. control • HbAlc is considered a “golden index” indicating the plasma glucose level. After 8 weeks，HbA1c level was significantly decreased dose-dependently for the mice treated by FYGL and metformin. Deng Pan, Ping Zhou,* et al. PLoS One, 2013, 8(7), e68332. 11

12. 10 Pharmacology trials of FYGL in vivo • Oral glucose tolerance of db/db genetic type II diabetes mice n = 8, *p < 0.05 vs. control，**p < 0.01 vs. control • BothFYGL and metformin can recovery the glucose level of postprandial 2h, improving the oral glucose tolerance of diabetes mice. Deng Pan, Ping Zhou,* et al. PLoS One, 2013, 8(7), e68332. 12

13. 11 Pharmacology trials of FYGL in vivo Radioimmuno-assay,*p < 0.05vs. control， **p < 0.01 vs. control • Insulin conc., HOMA- and HOMA-IR in STZ-induced diabetes rats （b）HOMA- （a）insulin conc. (homeostasis model assessment of -cell function) • After the rats were treated with FYGL, metformin and rosiglitazone for 30 days, insulin concentration and HOMA- were increased dose-dependently. • while HOMA-IR were significantly decreased • Indicating that FYGL, metformin and rosiglitazone could repair the -cells. （c）HOMA-IR 13 (homeostasis model assessment of insulin resistance)

14. 12 Pharmacology trials of FYGL in vivo Radioimmuno-assay,*p < 0.05vs. control， **p < 0.01 vs. control • Insulin concentration, HOMA- and HOMA-IR in db/db mice （b）HOMA- （a）insulin conc. • After the mice were treated with FYGL for 4weeks, HOMA-IR were decreased dose-dependently. • Whilenot like metformin group, in FYGL group, insulin concentration was decreased dose-dependently, whileHOMA-  was not restored. • Indicating that FYGLis an insulin sensitive agent, but, metformin is not. （c）HOMA-IR 14

15. 13 Pharmacology trials of FYGL in vivo • Effects on the serum lipid profile in STZ-induced diabetes rats *p < 0.05 vs. control • The levels of FFA, TG, TC, LDL-C and HDL-C were significantly improved dose-dependently in STZ-induced diabetic rats treated with FYGL, better than that treated by metformin and rosiglitazone.

16. 16 16 Mechanism of PTP1B target in vivo • PTP1B expression and activity in skeletal muscle in STZ-induced diabetic rats • Compared with control group, PTP1B expression and activity were inhibited for FYGL group, while not for metformin and rosiglitazone groups, indicating that the target of FYGL is PTP1B in vivo. Bao-Song Teng, Ping Zhou*et al, Eur. Rev. Med. Pharmacol. Sci, 2012, 16(2), 166-175.

17. 17 17 Mechanism of PTP1B target in vivo • PTP1B expression and activity in skeletal muscle in db/db mice • Compared with control group, PTP1B expression and activity were inhibited dose-dependently for FYGL group, also indicating that the target of FYGL is PTP1B in vivo. Chendong Wang, Ping Zhou,* et al. Brit J Nutr, 2012, 108, 2014-2025.

18. 16 FYGLsafety in vivo • Acute toxicity test of FYGL • LD50 = 6 g/Kg • highly safe （generally, LD50 > 2 g/Kgis considered safe） 18

19. 19 Characterization of FYGL Components and Structures of FYGL

20. 20 20 Analytical methods • Anion exchange resin DEAE-52 cellulose — Isolation of polysaccharide • Gel permeation chromatographic (GPC) — components and molecular weight • Lowry and phenol-sulfuric acid colorimetry —protein and polysaccharide contents • IC, GC-MS——amino acid and monosaccharide residues • -elimination reaction——covalent linkages between saccharide and protein • Methylation, periodate oxidation and Smith degradation—glycosidic linkage • 1D & 2D NMR—— residues and their sequence

21. 21 FYGLcomponents • Isolation of FYGL FYGL-2 FYGL-3 FYGL-1 Three components were isolated by DEAE-52 cellulose with 0, 0.1, 0.3M NaClgradient eluent，monitored by both Lowry and phenol-sulfuric acid colorimetry

22. 22 Bioactivity of FYGL-1, 2, 3on PTP1B in vitro • The higher protein content in FYGL, the better inhibition on PTP1B.

23. 23 23 Amino acids of FYGL-1a，2，3 a: FYGL-1is a polysaccharide dominant. • Most of amino acids are present in FYGL.

24. 24 24 Monosaccharide residues of FYGL-1,2,3 (A) (B) (C) (A) FYGL-1；(B) FYGL-2; (C) FYGL-3 monosaccharide residues Deng Pan, Ping Zhou,* et al. Food Chem. 2012, 135, 1097–1103 .

25. 25 Configuration of monosaccharide in FYGL-1, 2, 3 Methylated sugars in FYGL-3 Methylated sugars in FYGL-1 Methylated sugars in FYGL-2

26. Structures of FYGL-1,2,3 Example of FYGL-3 Structure

27. Covalent linkage between protein and saccharide 27 -elimination reaction probed by UV Amino acid contents before and after -elimination reaction • The figure indicates that protein is bound with saccharide by O-glycosidic linkage. • The table show that after -elimination reaction, both Thr and Ser contents were decreased, while Ala increased, indicating that proteins bind covalently with saccharides by Thr and Ser residues.

28. Structure determination by 1D & 2D NMR 28 • There are many peaks in anomeric region in 1Hand 13C NMR, suggesting FYGL-3 be a heteropolysaccharide with α and  linkages.

29. Structure determination by 1D & 2D NMR 29 1H-1H COSY 1H-13C HSQC 1H-1H TOCSY 1H-1H NOESY

30. 30 Structure determination by 1D & 2D NMR 1H-13C HMBC Connection between sugar residues deduced from HMBC

31. 31 A hyperbranchedproteoglycan of FYGL-3structure • Hyperbranched polysaccharide in backbone, proteins in side chain. Deng Pan, Ping Zhou,* et al. Carbohydrate Polymer, 2015, 117, 106–114.

32. Structure of FYGL-1 FYGL-1is a branched polysaccharide 32 Deng Pan, Ping Zhou*, et al. Food Chemistry2012, 135, 1097–1103.

33. Structure of FYGL-2 FYGL-2is a branched proteoglycan 33 Deng Pan, Ping Zhou,* et al. Biopolymers, 2014, 101(6), 613-623.

34. 32 Conclusion • FYGL, screened from Ganoderma lucidum, is an efficient PTP1B inhibitor in vivo • FYGL can decrease the plasma glucose level through inhibiting the PTP1B expression and activity, consequently, regulating the tyrosine phosphorylation level of the IR β-subunit. • FYGL can decrease indexes of FFA, TG, TC and LDL-C, and increase HDL-C. • FYGL contain branched heteropolysaccharide and hyperbranched proteoglycan, which may play special roles for its bioactivities of PTP1B inhibition and antihyperglycemic potency. • FYGL is a biomacromolecule which is more water soluble and safe than most of small molecule PTP1B inhibitors. 34

35. Acknowledgment 35 Collaboration： • Shanghai University of Traditional Chinese Medicine, China • East China Normal University, China • University of Melbourne, Australia Funding: • NSFC • Science and Technology Innovation of Shanghai • Senior Visiting Scholar Foundation of Key Laboratory of Fudan University

36. 36 36 The End Thanks for your attention!