The Biological Activity of a Novel Photoaffinity- Labelled Imino Sugar Jane Atkin St. Hilda’s College

OVERVIEW BackgroundMethods Results & Discussion AcknowledgementsQuestions

BACKGROUND (1) Glycoprotein formation: transfer of an oligosaccharide from a lipid precursor to a polypeptide Glycoprotein formation: transfer of an oligosaccharide from a lipid precursor to a polypeptide Processing of this glycan region – variable residues Processing of this glycan region – variable residues Endoplasmic reticulum and Golgi Endoplasmic reticulum and Golgi Glc Man Glc GlcNAc NH X-Asn-X-(Ser/Thr)---COO - Variable Core

BACKGROUND (2) Dwek et al, Nature Reviews Drug Discovery, 1, (2002)

BACKGROUND (3) Terminally misfolded proteins are broken down by ER- associated protein degradation (ERAD) Terminally misfolded proteins are broken down by ER- associated protein degradation (ERAD) Retrotranslocation, then deglycosylation Retrotranslocation, then deglycosylation Protein moiety is ubiquitinated and broken down by the proteasome Protein moiety is ubiquitinated and broken down by the proteasome Free oligosaccharides (FOS) broken down in the lysosome Free oligosaccharides (FOS) broken down in the lysosome Misfolded glycoprotein Proteasome PNGase ER lumen Cytosol Sec61 Figure modified from Dom Alonzi, Glycobiology Institute

BACKGROUND (4) NB-DNJ is an inhibitor of α -glucosidases I and II NB-DNJ is an inhibitor of α -glucosidases I and II Inhibition monitored by glucosylated FOS build-up Inhibition monitored by glucosylated FOS build-up Potential for anti-viral therapy: Potential for anti-viral therapy: HIV, Hepatitis B

BACKGROUND (5) DNJ-nitro-phenyl azide DNJ-nitro-phenyl azide (DNJ-NAP) made to investigate the behaviour of NB-DNJ Cross-links to neighbouring amide groups upon exposure to short-wave ultraviolet (UV) light Cross-links to neighbouring amide groups upon exposure to short-wave ultraviolet (UV) light fold better inhibitory activity than NB- DNJ on α -glucosidases in the cell fold better inhibitory activity than NB- DNJ on α -glucosidases in the cell

AIMS To determine the biological activity of this DNJ- NAP To determine the biological activity of this DNJ- NAP To observe the interactions this DNJ-NAP compound makes during its time in the cell by incubating it with cell samples for different time periods before exposure to UV light To observe the interactions this DNJ-NAP compound makes during its time in the cell by incubating it with cell samples for different time periods before exposure to UV light

METHODS Produced NB-DNJ Produced NB-DNJ derivative by reacting DNJ with the aldehyde linker Compared its inhibitory activity to that of an existing version of the compound and NB-DNJ, by incubating with HL60 cells and analysing the resulting glucosylated free oligosaccharides Compared its inhibitory activity to that of an existing version of the compound and NB-DNJ, by incubating with HL60 cells and analysing the resulting glucosylated free oligosaccharides Aldehyde Linker DNJ DNJ-NAP Reductive Amination

RESULTS Build-up of glucosylated FOS Build-up of glucosylated FOS My compound (NAP) is slightly more potent than previously synthesised compound (GS) My compound (NAP) is slightly more potent than previously synthesised compound (GS)

IN VITRO EXPERIMENTS (1) Radiolabelled the photo-activatable inhibitor with 3 H Radiolabelled the photo-activatable inhibitor with 3 H Bovine Serum Albumin (BSA) in vitro experiments: Bovine Serum Albumin (BSA) in vitro experiments: Denatured the protein Denatured the protein Added 3 H-DNJ-NAP to BSA and exposed to UV to see if it could react in the cell 1. Added 3 H-DNJ-NAP to BSA and exposed to UV to see if it could react in the cell Ran on SDS gel 2. Ran on SDS gel Measured radioactivity of gel slices using scintillation counter 3. Measured radioactivity of gel slices using scintillation counter No radioactivity associated with protein No radioactivity associated with protein Suggests BSA and DNJ-NAP do not interact – i.e. cross- linking is not random Suggests BSA and DNJ-NAP do not interact – i.e. cross- linking is not random

IN VITRO EXPERIMENTS (4) Therefore, repeated experiment with glucosidase enzymes – α, β Therefore, repeated experiment with glucosidase enzymes – α, β Also, crude rat liver extract to investigate how selective the binding is Also, crude rat liver extract to investigate how selective the binding is Analysed using a radioactivity detector plate Analysed using a radioactivity detector plate Could not see any radioactivity associated with protein bands in the gels Could not see any radioactivity associated with protein bands in the gels

IN VITRO EXPERIMENTS (5) Results e.g. β -glucosidase (impure) Results e.g. β -glucosidase (impure) Same for liver extract and α -glucosidase Same for liver extract and α -glucosidase β β-glucosidase + 3 H-DNJ- NAP + UV Free radioactivity

CELLULAR EXPERIMENTS (1) Incubated 3 H-DNJ-NAP with HL60 cells for different time periods before exposure to UV light Incubated 3 H-DNJ-NAP with HL60 cells for different time periods before exposure to UV light 0, 1, 2, 4, 10, 30, 60 minutes 0, 1, 2, 4, 10, 30, 60 minutes Control: 60 minute incubation but no UV exposure Control: 60 minute incubation but no UV exposure 2-day long-term experiment 2-day long-term experiment

CELLULAR EXPERIMENTS (2) Again, no radioactivity could be observed using the screen Again, no radioactivity could be observed using the screen Tried new technique Tried new technique Cut up dried gels into ~2mm slices and measured the radioactivity using scintillation counter Cut up dried gels into ~2mm slices and measured the radioactivity using scintillation counter Results promising… Results promising…

RESULTS All radioactivity was free and not associated with protein from 0-4 minutes All radioactivity was free and not associated with protein from 0-4 minutes

RESULTS (2) After 10 minutes, radioactivity was associated with a protein After 10 minutes, radioactivity was associated with a protein More binding after 30 minutes (~4x higher cpm) More binding after 30 minutes (~4x higher cpm)

RESULTS (3) After 60 minutes, there is no longer any radioactivity associated with protein. After 60 minutes, there is no longer any radioactivity associated with protein. Also seen after two days Also seen after two days

RESULTS (4) Used this technique to analyse β -glucosidase gel Used this technique to analyse β -glucosidase gel No radioactivity associated with protein No radioactivity associated with protein May be due to: May be due to: Lower affinity for DNJ-NAP Lower affinity for DNJ-NAP Not enough protein (and associated radioactivity) on the gel Not enough protein (and associated radioactivity) on the gel

CONCLUSIONS The photolabile DNJ-NAP will only cross-link to its substrate - specific The photolabile DNJ-NAP will only cross-link to its substrate - specific It seems to take minutes to reach its target and bind It seems to take minutes to reach its target and bind Binding is increased after 30 minutes Binding is increased after 30 minutes There is no longer binding after 60 minutes There is no longer binding after 60 minutes Most likely due to cell lysis Most likely due to cell lysis

FUTURE PERSPECTIVES Investigation into events between 30 and 60 minutes Investigation into events between 30 and 60 minutes More detailed analysis of the time-course of the binding reaction – repeats More detailed analysis of the time-course of the binding reaction – repeats Identify the species to which it is bound Identify the species to which it is bound

ACKNOWLEDGEMENTS Dr Terry Butters Dr Terry Butters Dr David Neville, Gabriele Reinkensmeier, Dom Alonzi, and Stephanie Boomkamp Dr David Neville, Gabriele Reinkensmeier, Dom Alonzi, and Stephanie Boomkamp Dr Mark Wormald Dr Mark Wormald Professor Raymond Dwek Professor Raymond Dwek

Thank you for listening Any Questions?