The Biological The Biological Activity of a Novel Activity of a Novel

Photoaffinity-Photoaffinity-Labelled Imino Labelled Imino

SugarSugarJane AtkinJane Atkin

St. Hilda’s St. Hilda’s CollegeCollege

OVERVIEWOVERVIEW

BackgroundBackground

Methods Methods

Results & DiscussionResults & Discussion

AcknowledgementsAcknowledgements

QuestionsQuestions

BACKGROUND (1)BACKGROUND (1)

Glycoprotein Glycoprotein formationformation: transfer of : transfer of an oligosaccharide from an oligosaccharide from a lipid precursor to a a lipid precursor to a polypeptidepolypeptide

Processing of this glycan Processing of this glycan region – variable residuesregion – variable residues

Endoplasmic reticulum Endoplasmic reticulum and Golgiand Golgi

Glc

Glc

Man

Man

Man

Man

Man

Man

Man

Man

Man

Glc

GlcNAc

GlcNAc

NH3+---X-Asn-X-(Ser/Thr)---

COO-

VariableCore

BACKGROUND (2)BACKGROUND (2)

Dw

ek e

t al,

Natu

re R

evi

ew

s D

rug

Dis

cove

ry,

Dw

ek e

t al,

Natu

re R

evi

ew

s D

rug

Dis

cove

ry,

1,

65

-75

(2

00

2)

1,

65

-75

(2

00

2)

BACKGROUND (3)BACKGROUND (3) Terminally misfolded proteins are broken down by Terminally misfolded proteins are broken down by

ER-associated protein degradation (ERAD)ER-associated protein degradation (ERAD)

RetrotranslocatioRetrotranslocation, then n, then deglycosylation deglycosylation Protein moiety is Protein moiety is ubiquitinated and ubiquitinated and broken down by broken down by the proteasomethe proteasome Free Free oligosaccharides oligosaccharides (FOS) broken (FOS) broken down in the down in the lysosomelysosome

Misfolded glycoprotein

Proteasome

PNGase

ER lumen

Cytosol

Sec61

Fig

ure

mod

ified

fro

m D

om

Alo

nzi

, G

lyco

bio

log

y In

stit

ute

BACKGROUND (4)BACKGROUND (4)

NB-DNJ is an inhibitor of NB-DNJ is an inhibitor of αα--glucosidases I and IIglucosidases I and II

Inhibition monitored by glucosylated Inhibition monitored by glucosylated FOS build-upFOS build-up

Potential for anti-viral therapy:Potential for anti-viral therapy:

HIV, Hepatitis BHIV, Hepatitis B

BACKGROUND (5)BACKGROUND (5) DNJ-nitro-phenyl azide DNJ-nitro-phenyl azide

(DNJ-NAP) made to (DNJ-NAP) made to investigate the behaviour investigate the behaviour of NB-DNJof NB-DNJ

Cross-links to neighbouring amide groups Cross-links to neighbouring amide groups upon exposure to short-wave ultraviolet upon exposure to short-wave ultraviolet (UV) light (UV) light

20-100-fold better inhibitory activity20-100-fold better inhibitory activity than NB-DNJ on than NB-DNJ on αα-glucosidases in the -glucosidases in the cellcell

AIMSAIMS

To determine the biological activity To determine the biological activity of this DNJ-NAPof this DNJ-NAP

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

METHODSMETHODS

Produced NB-DNJ Produced NB-DNJ derivative by reacting derivative by reacting DNJ with the aldehydeDNJ with the aldehydelinker linker

Compared its inhibitory activity to that of an Compared its inhibitory activity to that of an existing version of the compound and NB-existing version of the compound and NB-DNJ, by incubating with HL60 cells and DNJ, by incubating with HL60 cells and analysing the resulting glucosylated free analysing the resulting glucosylated free oligosaccharidesoligosaccharides

Aldehyde Linker

DNJ

DNJ-NAP

Reductive Amination

RESULTSRESULTS

Build-up of glucosylated Build-up of glucosylated FOSFOS

My compound (NAP) is My compound (NAP) is slightly more potent than slightly more potent than previously synthesised previously synthesised compound (GS)compound (GS)

IN VITROIN VITRO EXPERIMENTS EXPERIMENTS (1)(1)

Radiolabelled the photo-activatable inhibitor with Radiolabelled the photo-activatable inhibitor with 33HH Bovine Serum Albumin (BSA) Bovine Serum Albumin (BSA) in vitroin vitro experiments: experiments:

• Denatured the proteinDenatured the protein

1. Added Added 33H-DNJ-NAP to H-DNJ-NAP to BSA and exposed to UV BSA and exposed to UV to see if it could react in to see if it could react in the cellthe cell

2. Ran on SDS gelRan on SDS gel

3. Measured Measured radioactivity of gel radioactivity of gel slices using scintillation slices using scintillation countercounter

• No No radioactivity radioactivity associated with associated with proteinprotein • Suggests BSA Suggests BSA and DNJ-NAP do and DNJ-NAP do not interact – i.e. not interact – i.e. cross-linking is cross-linking is not randomnot random

IN VITROIN VITRO EXPERIMENTS EXPERIMENTS (4)(4)

Therefore, repeated experiment with Therefore, repeated experiment with glucosidase enzymes – glucosidase enzymes – αα, , ββ

Also, crude rat liver extract to investigate Also, crude rat liver extract to investigate how selective the binding ishow selective the binding is

Analysed using a radioactivity detector Analysed using a radioactivity detector plateplate

Could not see any radioactivity Could not see any radioactivity associated with protein bands in the gelsassociated with protein bands in the gels

IN VITROIN VITRO EXPERIMENTS EXPERIMENTS (5)(5)

Results e.g. Results e.g. ββ-glucosidase (impure) -glucosidase (impure) Same for liver extract and Same for liver extract and αα-glucosidase-glucosidase

ββ-glucosidase + 3H-DNJ-NAP + UV Free

radioactivity

CELLULAR EXPERIMENTS CELLULAR EXPERIMENTS (1)(1)

Incubated Incubated 33H-DNJ-NAP with HL60 H-DNJ-NAP with HL60 cells for different time periods cells for different time periods before exposure to UV lightbefore 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 Control: 60 minute incubation but no UV exposureUV exposure

2-day long-term experiment2-day long-term experiment

CELLULAR EXPERIMENTS CELLULAR EXPERIMENTS (2)(2)

Again, no radioactivity could be Again, no radioactivity could be observed using the screenobserved using the screen

Tried new techniqueTried new technique Cut up dried gels into ~2mm slices Cut up dried gels into ~2mm slices

and measured the radioactivity using and measured the radioactivity using scintillation counterscintillation counter

Results promising…Results promising…

RESULTSRESULTS All radioactivity was free and not associated All radioactivity was free and not associated

with protein from 0-4 minutes with protein from 0-4 minutes Radioactivity of Gel Slices From 4 Minute Incubation

0

50

100

150

200

250

300

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38

Slice Number

Ave

rage

cpm

RESULTS (2)RESULTS (2) After 10 minutes, radioactivity was associated After 10 minutes, radioactivity was associated

with a protein with a protein More binding after 30 minutes (~4x higher More binding after 30 minutes (~4x higher

cpm)cpm)Radioactivity of Gel Slices From 10 Minute Incubation

0

50

100

150

200

250

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

Slice Number

Avera

ge c

pm

Radioactivity of Gel Slices From 30 Minute Incubation

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46

Slice Number

Avera

ge c

pm

RESULTS (3)RESULTS (3) After 60 minutes, there is no longer any After 60 minutes, there is no longer any

radioactivity associated with protein. radioactivity associated with protein. Also seen after two daysAlso seen after two days

Radioactivity of Gel Slices from 60 Minute Incubation

0

10

20

30

40

50

60

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

Slice Number

Ave

rage

cpm

RESULTS (4)RESULTS (4)

Used this technique to analyse Used this technique to analyse ββ--glucosidase gelglucosidase gel

No radioactivity associated with proteinNo 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 Not enough protein (and associated

radioactivity) on the gelradioactivity) on the gel

CONCLUSIONSCONCLUSIONS The photolabile DNJ-NAP will only cross-The photolabile DNJ-NAP will only cross-

link to its substrate - specificlink to its substrate - specific

It seems to take 4 - 10 minutes to reach It seems to take 4 - 10 minutes to reach

its target and bindits target and bind

Binding is increased after 30 minutesBinding is increased after 30 minutes

There is no longer binding after 60 There is no longer binding after 60

minutesminutes

Most likely due to cell lysisMost likely due to cell lysis

FUTURE PERSPECTIVESFUTURE PERSPECTIVES

Investigation into events between 30 Investigation into events between 30

and 60 minutesand 60 minutes

More detailed analysis of the time-More detailed analysis of the time-

course of the binding reaction – repeatscourse of the binding reaction – repeats

Identify the species to which it is boundIdentify the species to which it is bound

ACKNOWLEDGEMENTSACKNOWLEDGEMENTS

Dr Terry Butters Dr Terry Butters

Dr David Neville, Gabriele Dr David Neville, Gabriele

Reinkensmeier, Dom Alonzi, and Reinkensmeier, Dom Alonzi, and

Stephanie BoomkampStephanie Boomkamp

Dr Mark Wormald Dr Mark Wormald

Professor Raymond DwekProfessor Raymond Dwek

Thank you for listeningThank you for listening

Any Questions?Any Questions?