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