Post on 04-Jan-2016
Structure-activity Structure-activity relationships of some relationships of some alkylated iminosugarsalkylated iminosugars
Nikolay Kukushkin, St.-Petersburg State UniversityNikolay Kukushkin, St.-Petersburg State University
N
OHHO
OH
OH
IminosugarIminosugar
NH
OHHO
OH
OHO
OHHO
OH
OHHO
Ceramide-specificglucosyltransferase
α-glucosidases I & II
GlucoseGlucose
NB-DNJNB-DNJ
Ceramide-specificglucosyltransferase
BiosynthesisBiosynthesis Catabolism
GSL poolGSL pool
Treatment of glycosphingolipid Treatment of glycosphingolipid (GSL) storage diseases(GSL) storage diseases
Ceramide-specificglucosyltransferase Key enzyme in GSL Key enzyme in GSL
biosynthesisbiosynthesis
Unfolded proteinMisfolded protein
α-glucosidases I & II
Unfolded protein
GlucoseGlucose
MannoseMannose
N-acetylglucosamineN-acetylglucosamine
α-glucosidase
Calnexin/calreticulinCalnexin/calreticulincyclecycle
ERAD
ERAD
Potential antiviral Potential antiviral strategiesstrategies
N
OHHO
OH
OHN
OHHO
OH
OH
NN-pentyl idonojirimycin-pentyl idonojirimycin(NP-IDJ)(NP-IDJ)
NB-DNJNB-DNJNP-IDJNP-IDJGlucose geometryGlucose geometryIdose geometryIdose geometry
N
OH
HO
HO
OH
O
NH
HN
NH
HN
OHO
CH3O
O
O
NH2
NH2
CH3
Lys-Lys-Ala-AlaLys-Lys-Ala-Ala(KKAA)(KKAA)
N
OH
HO
HO
OH
O
KKAA
ER retention ER retention signalsignal
KKAA
CytosolCytosol ERER
N
OH
HO
HO
OH
O
KKAAKKAA
?
N
HO OH
HOOH
N
OH
OHHO
HO
N
HO OH
HO
N
HO OH
HO
148148 149149 265265 262666
N-alkylated 7-membered iminosugarsN-alkylated 7-membered iminosugars
Mannosidase inhibitors?Mannosidase inhibitors?
Materials and methodsMaterials and methods
HL60HL60(human myeloid leukaemia)(human myeloid leukaemia)
CGT inhibitionCGT inhibition Glycosphingolipid analysisGlycosphingolipid analysis
•RPMI mediumRPMI medium+ foetal calf serum, + foetal calf serum, + penicillin/streptomycin+ penicillin/streptomycin+ L-glutamine+ L-glutamine
•96 h incubation96 h incubation
•Initial cell concentration: Initial cell concentration: 2×102×104 4 cells/mlcells/ml
•Fluorescent labelling with Fluorescent labelling with anthralinic acid (2-AA)anthralinic acid (2-AA)•HPLC analysisHPLC analysis
Ceramide
GlycanGlycan
Hirudo medicinalisHirudo medicinalis (leech) (leech)
Ceramideglycanase
•Extracted with chloroform-methanolExtracted with chloroform-methanol•Purified using reverse-phase C18 Purified using reverse-phase C18 columnscolumns
AnalysisAnalysis
Materials and methodsMaterials and methodsCGT inhibitionCGT inhibition
Materials and methodsMaterials and methodsGlucosidase inhibitionGlucosidase inhibition Free oligosaccharide Free oligosaccharide
analysisanalysis
Misfolded protein
GlucoseGlucose
MannoseMannose
N-acetylglucosamineN-acetylglucosaminePN
Gas
e
ENGase
Mannosidase
M5NM5NLysosomeLysosome
G3M5NG3M5N
Free Free oligosaccharidoligosaccharid
e (FOS)e (FOS)
mV
0.00
200.00
400.00
600.00
800.00
1000.00
1200.00
1400.00
1600.00
1800.00
2000.00
2200.00
2400.00
2600.00
Minutes20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00
mV
200.00
400.00
600.00
800.00
1000.00
1200.00
1400.00
1600.00
1800.00
2000.00
Minutes20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00
NB-DNJ 500 NB-DNJ 500 µMµMUntreated HL60Untreated HL60 M5M5NN
G1M5G1M5NN
M4M4NN
G3M5G3M5NN
Materials and methodsMaterials and methodsGlucosidase inhibitionGlucosidase inhibition
Default pathway for ERADDefault pathway for ERAD
mV
0.00
200.00
400.00
600.00
800.00
1000.00
1200.00
1400.00
1600.00
1800.00
2000.00
2200.00
2400.00
2600.00
Minutes20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00
Untreated control
NP-IDJ 500 µM
NB-DNJ 500 µM
Results: NP-IDJResults: NP-IDJNP-IDJ vs. NB-DNJ: HL60 cells FOS profileNP-IDJ vs. NB-DNJ: HL60 cells FOS profile
M5M5NN
M4M4NN
G3M5G3M5NN
mV
200.00
400.00
600.00
800.00
1000.00
1200.00
1400.00
1600.00
1800.00
2000.00
2200.00
2400.00
Minutes20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00
NP-IDJ 500 µM
NP-IDJ 100 µM
NP-IDJ 50 µM
NP-IDJ 5 µM
Results: NP-IDJResults: NP-IDJNP-IDJ: HL60 cells FOS profile, 24h incubationNP-IDJ: HL60 cells FOS profile, 24h incubation
M5M5NN
M4M4NN
G3M5NG3M5N
Peak areas
0
10
20
30
40
50
60
70
Compound, μM
% to
tota
l FO
S pe
ak a
reas
M4N
M5N
G1M4N
Total FOS peak area, 24h
0
50
100
150
200
250
300
350
Compound, μM%
to
co
ntr
ol
Results: NP-IDJResults: NP-IDJNP-IDJ vs. NB-DNJ: HL60 cells FOS profileNP-IDJ vs. NB-DNJ: HL60 cells FOS profile
mV
0.00
500.00
1000.00
1500.00
2000.00
2500.00
3000.00
3500.00
4000.00
Minutes8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00
Untreated control
NP-IDJ 500 µM
NB-DNJ 500 µM
Results: NP-IDJResults: NP-IDJNP-IDJ vs. NB-DNJ: HL60 cells GSL profileNP-IDJ vs. NB-DNJ: HL60 cells GSL profile
Lac-Lac-CerCer
GM3GM3
pGpGSialyl-Sialyl-
pGpG
Results: NP-IDJResults: NP-IDJNP-IDJ vs. NB-DNJ: HL60 cells GSL profileNP-IDJ vs. NB-DNJ: HL60 cells GSL profile
NP-IDJ
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
100.00
GM3 pG Syalil-pG%
to
co
ntr
ol
NP-IDJ 500 µM
NP-IDJ 100 µM
NP-IDJ 50 µM
NP-IDJ 5 µM
NB-DNJ
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
100.00
GM3 pG Syalil-pG
% t
o c
on
tro
l
NB-DNJ 500 µM
NB-DNJ 100 µM
NB-DNJ 50 µM
NB-DNJ 5 µM
mV
200.00
400.00
600.00
800.00
1000.00
1200.00
1400.00
1600.00
1800.00
2000.00
2200.00
Minutes20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00
Untreated control
KKAA-DNJ 100 µM
NB-DNJ 100 µM
Results: KKAA-DNJResults: KKAA-DNJKKAA-DNJ: HL60 cells FOS profile, 24h incubationKKAA-DNJ: HL60 cells FOS profile, 24h incubation
G3M5G3M5NN
M5M5NN
M4M4NN
G1M5G1M5NN
mV
200.00
400.00
600.00
800.00
1000.00
1200.00
1400.00
1600.00
1800.00
Minutes20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00
KKAA-DNJ 100 µM
KKAA-DNJ 10 µM
KKAA-DNJ 1 µM
Results: KKAA-DNJResults: KKAA-DNJKKAA-DNJ: HL60 cells FOS profile, 24h incubationKKAA-DNJ: HL60 cells FOS profile, 24h incubation
M4M4NN
M5M5NN
G1M5G1M5NN
mV
0.00
200.00
400.00
600.00
800.00
1000.00
1200.00
1400.00
1600.00
1800.00
2000.00
2200.00
2400.00
2600.00
Minutes
20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00
Untreated control
KKAA-DNJ 100 µM
NB-DNJ 100 µM
Results: KKAA-DNJResults: KKAA-DNJKKAA-DNJ vs. NB-DNJ: HL60 cells FOS profile, 72h incubationKKAA-DNJ vs. NB-DNJ: HL60 cells FOS profile, 72h incubation
M4M4NN
M5M5NN
G1M5G1M5NN
G3M5G3M5NN
Peak areas, 24h
0
10
20
30
40
50
60
Compound, μM
%
to t
ota
l F
OS
pea
k ar
eas
M4N
M5N
G1M5N
Total FOS, 24h
0
20
40
60
80
100
120
140
160
Compound, μM%
to
con
trol
Results: KKAA-DNJResults: KKAA-DNJKKAA-DNJ vs. NB-DNJ: HL60 cells FOS profile, 24h incubationKKAA-DNJ vs. NB-DNJ: HL60 cells FOS profile, 24h incubation
Peak areas, 72h
0
10
20
30
40
50
60
Compound, μM
% t
o to
tal F
OS
pea
k ar
eas
M4N
M5N
G1M5N
Total FOS, 72h
0
20
40
60
80
100
120
140
160
180
200
Compound, μM%
to
con
trol
Results: KKAA-DNJResults: KKAA-DNJKKAA-DNJ vs. NB-DNJ: HL60 cells FOS profile, 72h incubationKKAA-DNJ vs. NB-DNJ: HL60 cells FOS profile, 72h incubation
?
CytosolCytosol ERER
N
OH
HO
HO
OH
O
KKAAKKAA
N
OH
HO
HO
OH
O
KKAAKKAA
?
Ceramide-specificglucosyltransferase
mV
0.00
500.00
1000.00
1500.00
2000.00
2500.00
3000.00
3500.00
Minutes8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00
Untreated control
KKAA-DNJ 200 µM
NB-DNJ 200 µM
Results: KKAA-DNJResults: KKAA-DNJKKAA-DNJ vs. NB-DNJ: HL60 cells GSL profileKKAA-DNJ vs. NB-DNJ: HL60 cells GSL profile
Lac-Lac-CerCer
GM3GM3
Sialyl-Sialyl-pGpG
pGpG
NB-DNJ
0.0
20.0
40.0
60.0
80.0
100.0
120.0
GM3 pG sialyl-pG
% t
o c
on
tro
l
NB-DNJ, 200 µM
NB-DNJ, 100 µM
NB-DNJ, 50 µM
NB-DNJ, 5 µM
KKAA-DNJ
0.0
20.0
40.0
60.0
80.0
100.0
120.0
GM3 pG sialyl-pG%
to
co
ntr
ol
KKAA-DNJ, 200 µM
KKAA-DNJ, 100 µM
KKAA-DNJ, 50 µM
KKAA-DNJ, 5 µM
Results: KKAA-DNJResults: KKAA-DNJKKAA-DNJ vs. NB-DNJ: HL60 cells GSL profileKKAA-DNJ vs. NB-DNJ: HL60 cells GSL profile
Results: KKAA-DNJResults: KKAA-DNJKKAA-DNJ: KKAA-DNJ: in vitroin vitro αα-glucosidase inhibition-glucosidase inhibition
In vitro inhibition of α-glucosidase by KKAA-DNJ
0
10
20
30
40
50
60
70
80
90
100
1 10 100
KKAA-DNJ, µM
% i
nh
ibit
ion
ICIC5050: 83 µM: 83 µMNB-DNJ ICNB-DNJ IC5050: 53±16 µM: 53±16 µM
mV
200.00
400.00
600.00
800.00
1000.00
1200.00
1400.00
1600.00
Minutes20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00
Results: Results: 7-membered iminosugars7-membered iminosugars 148 vs control: HL60 cells FOS profile148 vs control: HL60 cells FOS profile
Untreated control
148 100 µM
M9NM9NM8NM8N
M7NM7NM6NM6NM5NM5N
M4NM4N
mV
100.00
200.00
300.00
400.00
500.00
600.00
700.00
800.00
900.00
1000.00
1100.00
Minutes20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00
M9M9NN
Kifunensine: mannosidase inhibitorKifunensine: mannosidase inhibitor
Results: Results: 7-membered iminosugars7-membered iminosugars
Untreated control
Kifunensine 100 µM
mV
200.00
400.00
600.00
800.00
1000.00
1200.00
1400.00
1600.00
Minutes20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00
Results: Results: 7-membered iminosugars7-membered iminosugars 149 vs control: HL60 cells FOS profile149 vs control: HL60 cells FOS profile
M9M9NN
Untreated control
149 100 µM
mV
200.00
400.00
600.00
800.00
1000.00
1200.00
1400.00
1600.00
Minutes20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00
Results: Results: 7-membered iminosugars7-membered iminosugars 266 vs control: HL60 cells FOS profile266 vs control: HL60 cells FOS profile
Untreated control
266 100 µM
mV
0.00
200.00
400.00
600.00
800.00
1000.00
1200.00
1400.00
1600.00
1800.00
2000.00
2200.00
2400.00
2600.00
2800.00
3000.00
Minutes12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00
265 vs control: HL60 cells FOS profile265 vs control: HL60 cells FOS profile
Results: Results: 7-membered iminosugars7-membered iminosugars
M5NM5NM4NM4N
M3NM3N
M2NM2N
M6NM6N M7NM7N
M8NM8NM9NM9N
Untreated control
265 100 µM
OHN
OHHO
OH
OH
ConclusionsConclusions
NP-IDJNP-IDJ
No effect on FOS productionNo effect on FOS production
No inhibition of No inhibition of αα--glucosidasesglucosidases
Significant Significant decrease of GSL decrease of GSL levelslevels Effective inhibition of Effective inhibition of
CGTCGT
Possible therapeutic alternative to Possible therapeutic alternative to NB-DNJNB-DNJ
ConclusionsConclusions
KKAA-DNJKKAA-DNJ
N
OH
HO
HO
OH
O
KKAA
No effect on FOS productionNo effect on FOS production
Low rates Low rates of uptakeof uptake
Low rates of Low rates of glucosidase glucosidase inhibitioninhibition
Normal Normal in vitroin vitro glucosidase inhibition glucosidase inhibitionLow effect on GSL biosynthesisLow effect on GSL biosynthesis
Low rates Low rates of CGT of CGT
inhibitioninhibition
Permeabilization with transfectin?Permeabilization with transfectin?
Strong Strong increase of increase of M2N-M6NM2N-M6N
concentratioconcentrationsns
Strong Strong increase of increase of M6N-M9NM6N-M9N
concentratioconcentrationsns
Decrease of Decrease of M4N-M5N M4N-M5N
concentratioconcentrationsns
Low or no Low or no increase of increase of M7N-M7N-
M9NM9N concentrationsconcentrations
ConclusionsConclusionsN-alkylated 7-membered iminosugarsN-alkylated 7-membered iminosugars
N
HO OH
HOOH
N
OH
OHHO
HO
N
HO OH
HO
N
HO OH
HO
148148 149149 265265 262666
No effectNo effect
AcknowledgementsAcknowledgements
• Dr. Terry ButtersDr. Terry Butters
• GroupGroup
• Oxford universityOxford university
• St.-Petersburg State St.-Petersburg State UniversityUniversity
St. Mary Le BowSt. Mary Le Bow