1. Structure-activity relationships of some alkylated iminosugars Nikolay Kukushkin, St.-Petersburg State University

2. Iminosugar Ceramide-specific glucosyltransferase α -glucosidases I & II Glucose NB-DNJ

3. Ceramide-specific glucosyltransferase Biosynthesis Catabolism GSL pool Treatment of glycosphingolipid (GSL) storage diseases

4. Ceramide-specific glucosyltransferase Key enzyme in GSL biosynthesis Key enzyme in GSL biosynthesis

5. Unfolded proteinMisfolded protein α-glucosidases I & II Unfolded protein Glucose Mannose N-acetylglucosamine α-glucosidase Calnexin/calreticulincycle ERAD Potential antiviral strategies Potential antiviral strategies

6. N -pentyl idonojirimycin (NP-IDJ) NB-DNJNP-IDJ Glucose geometry Idose geometry

7. Lys-Lys-Ala-Ala(KKAA) ER retention signal ER retention signal

8. KKAA CytosolER KKAA ?

9. N HOOH HO OH N OH OH HO HO N HOOH HO N HOOH HO 148149265266 N-alkylated 7-membered iminosugars Mannosidase inhibitors?

10. Materials and methods HL60 (human myeloid leukaemia) CGT inhibition Glycosphingolipid analysis Glycosphingolipid analysis RPMI medium RPMI medium + foetal calf serum, + penicillin/streptomycin + L-glutamine 96 h incubation 96 h incubation Initial cell concentration: Initial cell concentration: 2×10 4 cells/ml

11. Fluorescent labelling with anthralinic acid (2-AA) Fluorescent labelling with anthralinic acid (2-AA) HPLC analysis HPLC analysis Ceramide Glycan Hirudo medicinalis (leech) Ceramide glycanase Extracted with chloroform-methanol Extracted with chloroform-methanol Purified using reverse-phase C18 columns Purified using reverse-phase C18 columns Analysis Materials and methods CGT inhibition

12. Materials and methods Glucosidase inhibition Free oligosaccharide analysis Free oligosaccharide analysis Misfolded protein Glucose Mannose N-acetylglucosamine PNGase ENGase Mannosidase M5N Lysosome G3M5N Free oligosaccharide (FOS)

13. NB-DNJ 500 µM Untreated HL60 M5N G1M5N M4N G3M5N Materials and methods Glucosidase inhibition Default pathway for ERAD

14. Untreated control NP-IDJ 500 µM NB-DNJ 500 µM Results: NP-IDJ NP-IDJ vs. NB-DNJ: HL60 cells FOS profile M5N M4N G3M5N

15. NP-IDJ 500 µM NP-IDJ 100 µM NP-IDJ 50 µM NP-IDJ 5 µM Results: NP-IDJ NP-IDJ: HL60 cells FOS profile, 24h incubation M5N M4N G3M5N

16. Results: NP-IDJ NP-IDJ vs. NB-DNJ: HL60 cells FOS profile

17. Untreated control NP-IDJ 500 µM NB-DNJ 500 µM Results: NP-IDJ NP-IDJ vs. NB-DNJ: HL60 cells GSL profile Lac-Cer GM3 pG Sialyl-pG

18. Results: NP-IDJ NP-IDJ vs. NB-DNJ: HL60 cells GSL profile

19. Untreated control KKAA-DNJ 100 µM NB-DNJ 100 µM Results: KKAA-DNJ KKAA-DNJ: HL60 cells FOS profile, 24h incubation G3M5N M5N M4N G1M5N

20. KKAA-DNJ 100 µM KKAA-DNJ 10 µM KKAA-DNJ 1 µM Results: KKAA-DNJ KKAA-DNJ: HL60 cells FOS profile, 24h incubation M4N M5N G1M5N

21. Untreated control KKAA-DNJ 100 µM NB-DNJ 100 µM Results: KKAA-DNJ KKAA-DNJ vs. NB-DNJ: HL60 cells FOS profile, 72h incubation M4N M5N G1M5N G3M5N

22. Results: KKAA-DNJ KKAA-DNJ vs. NB-DNJ: HL60 cells FOS profile, 24h incubation

23. Results: KKAA-DNJ KKAA-DNJ vs. NB-DNJ: HL60 cells FOS profile, 72h incubation

24. ? Cytosol ER KKAA ? Ceramide-specific glucosyltransferase

25. Untreated control KKAA-DNJ 200 µM NB-DNJ 200 µM Results: KKAA-DNJ KKAA-DNJ vs. NB-DNJ: HL60 cells GSL profile Lac-Cer GM3 Sialyl-pG pG

26. Results: KKAA-DNJ KKAA-DNJ vs. NB-DNJ: HL60 cells GSL profile

27. Results: KKAA-DNJ KKAA-DNJ: in vitro α-glucosidase inhibition IC 50 : 83 µM NB-DNJ IC 50 : 53±16 µM

28. Results: 7-membered iminosugars 148 vs control: HL60 cells FOS profile Untreated control 148 100 µM M9N M8N M7N M6N M5N M4N

29. M9N Kifunensine: mannosidase inhibitor Results: 7-membered iminosugars Untreated control Kifunensine 100 µM

30. Results: 7-membered iminosugars 149 vs control: HL60 cells FOS profile M9N Untreated control 149 100 µM

31. Results: 7-membered iminosugars 266 vs control: HL60 cells FOS profile Untreated control 266 100 µM

32. 265 vs control: HL60 cells FOS profile Results: 7-membered iminosugars M5N M4N M3N M2N M6N M7N M8N M9N Untreated control 265 100 µM

33. OH N OHHO OH OHConclusions NP-IDJ No effect on FOS production No inhibition of α-glucosidases No inhibition of α-glucosidases Significant decrease of GSL levels Effective inhibition of CGT Effective inhibition of CGT Possible therapeutic alternative to NB-DNJ Possible therapeutic alternative to NB-DNJ

34. Conclusions KKAA-DNJ N OH HO HO OH O KKAA No effect on FOS production Low rates of uptake Low rates of glucosidase inhibition Normal in vitro glucosidase inhibition Low effect on GSL biosynthesis Low rates of CGT inhibition Permeabilization with transfectin?

35. Strong increase of M2N-M6N concentrations Strong increase of M6N-M9N concentrations Decrease of M4N-M5N concentrations Low or no increase of M7N-M9N concentrations Conclusions N-alkylated 7-membered iminosugars N HOOH HO OH N OH OH HO HO N HOOH HO N HOOH HO 148149265266 No effect

36. Acknowledgements Dr. Terry Butters Dr. Terry Butters Group Group Oxford university Oxford university St.-Petersburg State University St.-Petersburg State University

37. St. Mary Le Bow