Transcript Therapeutic properties of NB-DNJ as a drug for Guillain Barré

Inhibition of ganglioside
biosynthesis by imino sugars
reduces binding of Guillain-Barré
Syndrome autoantibodies
Part II project by Katherine WarreCornish
Guillain Barré syndrome:
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Auto-immune disease
Presence of anti-ganglioside antibodies
Causes immune system to attack neurons
Symptoms: limb-weakness and pain, impaired reflexes,
coordination and respiration
85% of patients make a full recovery within 6 to 12 months
Mortality rate of approximately 5% and 7-15% of patients left
with persistent minor symptoms
Incidence:1-3 cases in 100 000 people per year
Current therapies:
– plasma exchange (total exchange of about 5 plasma volumes)
– high dose intravenous IgG
Causes of GBS
• Antecedent bacterial infection (1-2 weeks
before GBS onset)
– Most commonly associated with Campylobacter jejuni
– Haemophilus influenzae? Cytomegalovirus? EpsteinBarr?
• Molecular mimicry
– Lipo-oligosaccharide (LOS) is the principal cell surface
structure that is recognised by the host.
– Forms of LOS have been isolated with identical terminal
sugar residues to various human gangliosides
Human gangliosides and C.jejuni lipo-oligo
saccharides
Bacterial
Human
GM1-like LOS
Core-lipid A
Ceramide
GM1
GD1a-like LOS
Core-lipid A
Ceramide
GD1a
GD1c-like LOS
Core-lipid A
Ceramide
GQ1b
Galactose
Glucose
N-Acetylneuraminic acid
N-Acetylgalactosamine
Immune system attacks neurons
with high levels of ganglioside
• Due to cross reactivity with LOS, antibodies
against self ganglioside are present in patients.
• IgG antibodies to GM1 and GD1a strongly
associated with GBS
• IgG antibodies to GQ1b associated with MillerFischer syndrome (MFS) (caused similarly to
GBS, different neurological symptoms)
• Neurological features of the disease and sites of
neurological damage are determined by the
antibody specificities.
Is it possible to treat an autoimmune disease by metabolically
decreasing levels of autoepitope?
N-alkylated imino-sugars:
inhibitors of ceramide specific glucosyltransferase
Glycolipid Synthesis
NB-DNJ and NBDGJ are glucosyl
transferase
inhibitors
NB-DNJ: IC50 of
20.4uM
NB-DGJ: IC50 of
30uM
NB-DNJ/ NBDGJ Inhibition
Butters et al, 2000, Chemical Reviews
V100, 12, pp4683-4696
Results
ELISA – determination of the degree of patient vs
control sera antibody binding to gangliosides
p=0.06
p=0.27
p=.16
p=.09
p=0.4
GD1a
GQ1b
GD1b
2.5
2
1.5
1
0.5
0
GM1
GM2
• 12 patient sera (black dots) and 4 control sera (white dots) tested
against 5 GSLs
• Most significant differences between patient and control Ab binding
observed with GM1 and GQ1b
ELISAs carried out using GM1
and GQ1b with increasing
dilution factors of selected patient
sera
Patient sera binding curves to GM1
GM1
3
A(450nm)
2.5
control2
control3
patient8
patient10
patient13
patient15
2
1.5
1
0.5
0
0.00001
0.0001
0.001
sera dilution
0.01
Patient sera binding curve to GQ1b
A(450nm)
GQ1b
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0.00001
control3
control4
patient8
patient9
patient10
p11av
0.0001
0.001
sera dilution
0.01
Does inhibition of ganglioside
biosynthesis reduce antibody binding?
• Thin Layer Chromatography: demonstrate
decrease in levels of antibody binding to GM1 in
extract from drug treated cells
• Glycolipids were extracted from RAW cells (high
GM1 content) treated with a range of
concentrations of drugs: NB-DNJ and NB-DGJ
• Components were separated by TLC. Extracts
were run parallel to GM1 standard.
Immuno-overlays: detection of patient
antibody binding to TLC plate
• Analogous to a western blot
• TLC plates were incubated
a 1:1000 dilution of patient
sera
• Plates were then incubated
a 1:1000 dilution of
secondary anti-human
antibody, labelled with
horse-radish peroxidase
• Bands were detected by
ECL
Horse-radish
peroxidase
Goat antihuman
antibody
Patient sera
IgG
GM1
TLC - results
Orcinol staining (proportional to carbohydrate concentration)
confirms high levels of GM1 in RAW cells
GM1
Conc(uM)Standard
Drug:
0
10
20
50 100 1000
NB-DNJ
10
50 100 1000
NB-DGJ
TLC - results
Patient sera
GM1
Conc(uM)Standard
Drug:
0
10
20
50 100 1000
NB-DNJ
Control sera
10
50 100 1000
NB-DGJ
GM1
0
Standard
10
20
50 100 1000 10
NB-DNJ
50 100 1000
NB-DGJ
Metabolic inhibition reduces patient
serum IgG binding.
• Conclusions from TLC
• Decrease in patient antibody binding as
concentrations of NB-DNJ or NB-DGJ are
increased.
• Effect appears to be more pronounced with NBDGJ than NB-DNJ
• Physiologically, IgG binding is enhanced by
glycolipid clustering. Relatively low concentrations
of drug could disrupt this clustering (TLCglycolipids are artificially clustered together)
• Lower drug concentrations may be required than
results suggest.
Current work:
Can this approach be extended to
other cell lines…?
• Glycosphingolipids were extracted from
PC12 cells treated with NB-DNJ and NBDGJ
• Extracted GSLs fluorescently labelled
• Separated by HPLC
• Spectra obtained
• Levels of gangliosides quantified by
measuring peak areas
HPLC results NB-DNJ
Decrease in ganglioside levels with NB-DNJ
600000
500000
peak areas
400000
0M NB-DNJ
300000
50uM NB-DNJ
1mM NB-DNJ
200000
100000
0
1
GQ1b
2
GD1b
3
GD1c
4
GM1a
5
Gb3
HPLC results NB-DGJ
Decrease in ganglioside levels with NB-DGJ
1200000
1000000
peak areas
800000
0M NB-DGJ
600000
50uM NB-DGJ
1mM NB-DGJ
400000
200000
0
1
GQ1b
2
GD1b
3
GD1c
4
GM1a
5
Gb3
General Conclusions
• Use of a metabolic inhibitor of GSL synthesis
could potentially be used as a treatment for
Guillain-Barré syndrome
– This depends on:
• Levels of dosage required for decreased antibody binding –
can these be tolerated physiologically
• Turnover rate of gangliosides in the affected neurons?
– NBDGJ appears better than NBDNJ
• Further research:
– additional, eg neuronal cell lines
– Animal models – (GBS mice)
Acknowledgements
• Dr Chris Scanlan
• Dr Terry Butters