Novel Carbohydrate-based Ligands with Immunostimulatory ... · novel carbohydrate-based ligands...
Transcript of Novel Carbohydrate-based Ligands with Immunostimulatory ... · novel carbohydrate-based ligands...
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NOVEL CARBOHYDRATE-BASED LIGANDS WITH
IMMUNOSTIMULATORY PROPERTIES
CECILIA H MARZABADI, VIKRAM BASAVA AND CONSTANTINE BITSAKTSIS
SETON HALL UNIVERSITY
SOUTH ORANGE, NJ USA
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INTRODUCTION
• THE ZWITTERIONIC POLYSACCHARIDE,
PSA1, ISOLATED FROM THE CAPSULE OF
BACTEROIDES FRAGILIS, HAS BEEN
SHOWN TO STIMULATE BOTH INNATE
AND ADAPTIVE IMMUNE RESPONSES IN
MAMMALS AND IS BELIEVED TO BE
IMPORTANT IN ESTABLISHING A
BALANCED IMMUNE SYSTEM WHICH IS
MEDIATED BY T-LYMPHOCYTES.
O
O
O
O
O
OO
CH3
CH3
NH3+
OH
O-
O
OH
NH
OH
NHO
O
OH
OHOH
OH H
OHAc
Ac
1
2
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INTRODUCTION
WORK IN OUR LABORATORY
INVOLVES THE SYNTHESIS OF SIMPLER
ZWITTERIONIC SACCHARIDE
ANALOGUES FOR EVALUATION AS
POTENTIAL STIMULANTS OF INNATE
AND/OR ADAPTIVE IMMUNITY.
RECENTLY, ONE SUCH ANALOG, 2, HAS
BEEN SYNTHESIZED AND HAS BEEN
SHOWN TO POSSESS
IMMUNOSTIMULATORY PROPERTIES.
O
O
O
O
OO
CH3
CH3
NH3
+
OH
O-
O
OH
NHAc
OH
2 3
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Synthesis of Subunit A:
(Proposed Scheme)
SYNTHETIC STUDIES
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ORTEP representation
of the anhydride3,6-Anhydride molecules showing intermolecular H-bonding
SYNTHETIC STUDIES
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SYNTHETIC STUDIES
Reactivity of 3,6-Anhydro-D-glucal:
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Reactivity of 3,6-Anhydro-D-glucal:
SYNTHETIC STUDIES
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SYNTHETIC STUDIES
Synthesis of Subunit A:
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SYNTHETIC STUDIES
Synthesis of Subunit B:
B
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SYNTHETIC STUDIES
Synthesis of Subunit C:
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SYNTHETIC STUDIES
Synthesis of Subunit C (cont):
C
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SYNTHETIC STUDIES
Coupling of Subunits A and B:
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SYNTHETIC STUDIES
Coupling of Subunit A+B with C:
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BIOLOGICAL EVALUATION
•PROTECTED ANALOG 2 WAS EVALUATED IN ELISA ASSAYS FOR
THE PRODUCTION OF THE PROINFLAMMATORY CYTOKINES TNF-
A AND IL-6 USING RAW 264.7 AND MOUSE PERITONEAL
MACROPHAGES (C57 BL/6, JACKSON)
Innate Immunity:
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BIOLOGICAL EVALUATION
TNF-A 24hrs
RAW
PEC
0
500
1000
1500015LPS***
*****[T
NF
-]
(pg
/ml)
Innate Immunity:
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BIOLOGICAL EVALUATION
IL-6
Innate Immunity:
24hrs
RAW
PEC
0
50
100
150
200
250015LPS
N.S.
N.S.
N.S.
N.S.
[IL
-6]
(pg
/ml)
48hrs
RAW
PEC
0
100
200
300
400
500015LPS**
****[I
L-6
] (p
g/m
l)
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BIOLOGICAL EVALUATION
•THE MOLECULE SYNTHESIZED WAS ALSO TESTED IN SPLEEN
CELLS FROM THE SAME MICE LOOKING FOR T AND B CELL
PROLIFERATION, LEVELS OF IFN-G IN THE SUPERNATANTS
(INDICATIVE OF A T CELL RESPONSE), AND POLYCLONAL IGM AND
IGG LEVELS IN THE SUPERNATANTS L (INDICATIVE OF A B CELL
RESPONSE) WERE MEASURED.
•NO T CELL OR B CELL RESPONSES WERE OBSERVED.
Adaptive Immunity:
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BIOLOGICAL EVALUATION
TO ELUCIDATE THE BACTERIAL TOLL LIKE RECEPTOR
RESPONSIBLE FOR CYTOKINE PRODUCTION, THE PERITONEAL
MACROPHAGES FROM TLR2 AND TLR4 KNOCKOUT MICE (FEMALE, 4-
6 WEEK OLD C57BL6) WERE DOSED WITH COMPOUND 2 AND, TNF-A
AND IL6 LEVELS WERE MEASURED OVERTIME IN THE
SUPERNATENTS.
Innate Immunity: TLR2 vs TLR4 Ligand
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BIOLOGICAL EVALUATION
TLR KO MICE: TNF- Production
2 hrs
WT
TLR-2
ko
TLR-4
ko
0
100
200
300
400015LPS
*****
[TN
F-
] (p
g/m
l)
24 hrs
WT
TLR-2
ko
TLR-4
ko
0
200
400
600015LPS
****
[TN
F-
] (p
g/m
l)
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BIOLOGICAL EVALUATION
TLR KO MICE: IL-6 Production
24hrs
WT
TLR-2
ko
TLR-4
ko
0
1000
2000
3000015LPS
****
[IL
-6]
(pg
/ml)
48hrs
WT
TLR-2
ko
TLR-4
ko
0
1000
2000
3000015LPS
****
[IL
-6]
(pg
/ml)
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THE EXTENT OF BINDING OF ANALOGUE 2 TO THE TLR-4
RECEPTORS WAS ILLUSTRATED BY TREATING THE PERITONEAL
EXUDATE CELLS (PEC) WITH DIFFERENT CONCENTRATIONS OF THE
COMPOUND. THE CELLS WERE PRE-INCUBATED WITH THE
COMPOUND, AFTER WHICH WERE WASHED. TO THE WASHED CELLS
WAS ADDED THE ANTIBODY FOLLOWED BY THE PHYTOERYTHRIN
DYE AND, THE NUMBER OF THE COLOURED CELLS WAS OBSERVED
BY FLOW CYTOMETRY.
TLR-4 EXPRESSION: Flow Cytometry
BIOLOGICAL EVALUATION
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BIOLOGICAL EVALUATION
TLR-4 EXPRESSION: Flow Cytometry
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TLR-4
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PECs
contr
ol
g/ml
1
g/ml
5
g/ml
10
0
20
40
60
80
N.S.***
% T
LR
-4 e
xp
res
sio
n
BIOLOGICAL EVALUATION
TLR-4 EXPRESSION: Flow Cytometry
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BIOLOGICAL EVALUATION
TLR-2 EXPRESSION: Flow Cytometry
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BIOLOGICAL EVALUATION
• A REDUCTION IN THE PRODUCTION OF BOTH TNF-A AND IL-6
WAS OBSERVED IN ONLY THE TLR4 KNOCKOUT MICE
SUGGESTING THAT SIGNALING OF 2 OCCURS THROUGH TLR4.
FURTHERMORE, THE MAGNITUDE OF TLR4 SIGNALING WITH 2
WAS GREATER THAN THE KNOWN TLR4 AGONIST, E. COLI LPS
(LIPOPOLYSACCHARIDE)
SUMMARY: TLR KO MICE
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CONCLUSIONS
• THE DESIRED ZWITTERIONIC POLYSACCHARIDE, AN ANALOGUE OF PSA1, WAS
SUCCESSFULLY SYNTHESIZED WITH BOTH AMINO AND CARBOXYLATE MOIETIES
• THE BIOLOGICAL EVALUATION CLEARLY DENOTES THAT THE ANALOGUE 2 SHOWS
IMMUNOSTIMULATORY PROPERTIES
• ANALOGUE 2 WAS FOUND TO STIMULATE THE INNATE IMMUNE SYSTEM, AND NOT THE
ADAPTIVE IMMUNE SYSTEM
• AN UNEXPECTED AND AN EFFICIENT SYNTHESIS OF 3, 6-ANHYDRO-D-GLUCAL PAVES A
ROUTE TO THE SYNTHESIS OF INTERESTING NEW CARBOHYDRATE MOIETIES
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