2. Background: a bit about… Important roles in: Glycoprotein biosynthesis, quality control & catabolism Multiple forms of α-mannosidases in mammalian cells, differing in specificity, function and cellular location Classified into 2 groups conserved through eukaryotic evolution Class I - Narrow specificity for α-1, 2- mannosidic linkages -Further specificity within family for isomeric species produced Class II - Broad specificity: hydrolyse α(1→ 2), α(1→3) and α(1→6) mannosidic linkages Manß1-4GlcNAcß1-4GlcNAcß1-Asn Man  1-6 Man  1-3 Man  1-6 Man  1-3 Man  1-2 Man  1-2 Man  1-2 Man  1-2

3. ERAD misfolded M8B / M8C persistently misfolded Monosaccharide Monosaccharide constituents ER Lysosome Cytosol CELL

4. G 1-3 M 8 N 1 G 1-3 M 5 N 1 G 1-3 M 4 N 1 ? lysosome Plasma membrane Chitobiase Proteasome M8N1M8N1 M5N1M5N1 Chitobiase Cytosolicα- mannosidase Cyt α-man ? Fast Slow Protein and Oligosaccharide Processing in the Cytosol of ERAD G 1-3 M 8 N 2 + M 8 N 2 + M4N1M4N1 Cyt α-man?

5. 148 266 149 265 N OH HO OH N OH HO OH Deoxymannojorimycin (DMJ)Kifunensin (Kif)Swainsonine (SW) 1, 4 deoxy-1,4-imino D- mannitol (DIM) Known α-mannosidase inhibitors Novel 7-membered imino-sugars: inhibitory effect on cellular α-mannosidases (Kukushkin & Butters; unpublished data) Weak general inhibitor of mammalian α-mannosidases Low conc.: Golgi mannosidase I and Man9-mannosidase Higher conc.: Will start to inhibit other α-mannosidases Potent and specific inhibitor of Golgi mannosidase I ER mannosidase I and Man9-mannosidase Inhibits lysosomal mannosidase and Golgi mannosidase

6. Project Aims The aims of my project were: To define α-mannosidase targets of novel inhibitors in cells and compare these to known α- mannosidase inhibitors This will be accomplished by: 1.Evaluation of cytosolic and lysosomal α-mannosidase inhibition by measuring free oligosaccharides (FOS) in HL60 and MDBK cell lines. 2.Increasing our knowledge of mannosidases within their cellular pathways in cell lines above. 3.Using glycoprotein precipitation, isolation and PNGase glycan release methods to observe effects of inhibitors on glycan species of total cellular glycoprotein. 4.Identification of lysosomal FOS species following cellular fractionation. Two cell lines will be used that have differing biosynthetic and catabolic pathways: HL60, a human promyelocytic leukaemia cell line and MDBK, a bovine kidney cell line. 5.Estimation of ER and Golgi α-mannosidase inhibition by using Fluorescence-activated cell sorting (FACS) to analysing glycoprotein derived oligosaccharides on the cell surfacee following treatment with α-mannosidase inhibitors.

7. Fluorescence (arbitrary units) M4N M5N G1M5 M9N M7N M4N M5N G1M5 M7N/N2 M8N/N2 M9N/N2 M7N/N2 M8N G1M9N? M8N/N2 M9N/N2 M7N/N2 Untreated HL60 DMJ treated (1mM) DIM treated (100μM) HPLC analysis of 2-AA fluorescently labelled free oligosaccharides (FOS) from HL60 cells treated with known α-mannosidase inhibitors

8. M5N M3N2 M4N2 M8N2 M5N M3N2 M5N2M9N/N2M6N2 M7N/N2 G1M5N M4N2 M4N M9N/N2 Untreated MDBK cells DIM treated (100μM) HPLC analysis of 2-AA fluorescently labelled free oligosaccharides (FOS) from MDBK cells treated with known α-mannosidase inhibitors 3000 6000 Fluorescence (arbitrary units)

9. Minutes HPLC analysis of 2-AA fluorescently labelled free oligosaccharides (FOS) from MDBK cells treated with Kif at increasing concentrations (1μM- 100μM) Untreated + 1μM Kif + 10μM Kif + 100μM Kif Fluorescence (arbitrary units)

10. SATIN-Fluorescence M9N M5N M4N M6NM7N/ N2 G1M5 2224262830323436384042 Minutes M5N M9N M7N/ N2 G1M9 G1M5 M6N G1M5 M8N M9N M4N M6N2 HPLC analysis of 2-AA fluorescently labelled free oligosaccharides (FOS) from HL60 cells treated with 148 and 149 Fluorescence (arbitrary units)

11. - M3N2 M4N2 M5N M7N2 18202224 26 28303234363840 42 M3N2 M4N2 M5N M6N2 M7N2 M8N2 6000 1800 1200 M9N/N2 G1M5 M9N/N2 M5N2 HPLC analysis of 2-AA fluorescently labelled free oligosaccharides (FOS) from MDBK cells treated with 148 and 149 Fluorescence (arbitrary units) Minutes

12. HPLC analysis of 2-AA fluorescently labelled free oligosaccharides (FOS) from HL60 cells treated with 265 and 266 2000 12141618202224262830323436384042 1600 4000 Minutes M5N M4N M3N M9N M6N Untreated + 100μM 265 + 100μM 266 Fluorescence (arbitrary units)

13. HPLC analysis of 2-AA fluorescently labelled free oligosaccharides (FOS) from MDBK cells treated with 265 and 266 SATIN - Fluorescence 6000 0 Minutes 12.0014.0016.0018.0020.0022.0024.0026.0028.0030.0032.0034.0036.0038.0040.0042.00 1300 M3N2 Fluorescence (arbitrary units)

14. Effect on total cellular glycoprotein upon inhibitor treatment Elution time (minutes) 600 1000 SATIN - Fluorescence 1200 1000 SATIN - Fluorescence 2500 222426283032343638404244 M9N2 fluorescence % increase in ConA binding after 24hrs % increase in ConA binding after 72hrs + 100μM Kif68.1%136% + 100μM 14813.9%34.2% Flow cytometry data representing % change in ConA binding in Kif and 148 treated HL60 cells over 24hrs and 72 hrs Untreated HL60 + DMJ (1mM) + 148 (100μM) + 265 (100μM) + 266 (100μM) + Kif (100μM)

15. Cellular fractionation of untreated HL60 and MDBK cells Fraction 9 42 Fluorescence(arbitrary units) 16 182022242628303234363840 Elution time (minutes) Fraction 8 121416182022242628303234363840 42

16. In Summary Known α-mannosidase inhibitors: -DMJ : low potency, low specificity α-mannosidase inhibitor. Does not inhibit cytosolic mannosidase at high concentration (1mM) but most likely inhibiting ER/Golgi and partially inhibiting lysosomal α-mannosidases. -Kif: Known strong inhibitor of ER Mannosidase I and Golgi mannosidases. No inhibition of ER Man II due to accumulation of M8C. -Very high potency, inhibitory effects observed from 1μM conc. No inhibition of cytosolic/lysosomal α- mannosidases. Selective for ER/Golgi mannosidases and the biosynthetic pathway for glycoprotein maturation. -DIM: Selective for lysosomal α-mannosidase and lysosomal α1,6 mannosidase. Partial inhibition of catabolism of high mannose structures, strong inhibition of breakdown of core N-linked glycan isomer M3aN2. Novel α-mannosidase inhibitors -148 & 149: Very similar inhibitory patterns in the cell. Inhibits cytosolic α-mannosidase, and lysosomal mannosidase in a similar way to DIM. Subtle inhibition of conversion of oligomannose to complex glycan maturation to the cell surface. -265: No inhibition of cytosolic α-mannosidase. Targets lysosomal α-mannosidase and lysosomal α1, 6 mannosidase with particularly potent inhibition for the catabolism of core M3aN2. -266: No inhibition of cytosolic α-mannosidase. Inhibits lysosomal α-mannosidase with particularly potent inhibition for the catabolism of core M3aN2. Partial inhibition of catabolism of high oligomannose glycans by lysosomal α- mannosidase. Low levels of inhibition for α1,6 mannosidase.

17. Future work Cellular fractionation with added inhibitor followed by FOS isolation and characterisation to confirm and clarify target compartments of novel inhibitors within the cell and to specify enzymes inhibited. Carry out Flow Cytometry experiment with 266 and 265 to analyse effect on glycoprotein maturation at the cell surface To confirm the cytosolic inhibition of 148 and 149. Proposed experiment: Addition of 148/149 following treatment of cells with glucosidase inhibitor such as NB-DMJ to create more tri- glucosylated species. If 148/149 inhibit in the cytosol, will generate more glucosylated high- mannose structures from FOS analysis. May want to develop a more selective or & more potent inhibitor of the cytosolic α-mannosidase: drug screening with a range of 148,149-like structures.

18. Acknowledgements Dr Terry Butters Dr David Neville Dr Dominic Alonzi Stephanie Boomkamp Professor Raymond Dwek