Carbohydrate-Based Ligands with Immunostimulatory Properties
Carbohydrate-based ligands, specifically the zwitterionic polysaccharide PSA1 isolated from Bacteroides fragilis, have been researched for their role in stimulating immune responses. The synthesis of simpler analogues like 2 has shown promising immunostimulatory properties. Synthetic studies have focused on subunit synthesis and reactivity evaluations, paving the way for potential immune stimulants. Research presented by Cecilia H. Marzabadi, Vikram Basava, and Constantine Bitsaktsis from Seton Hall University aims to understand and harness the immune-modulating capabilities of these 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
INTRODUCTION + H3 N H3 C O THE ZWITTERIONIC POLYSACCHARIDE, PSA1, ISOLATED FROM THE CAPSULE OF BACTEROIDES BACTEROIDES FRAGILIS 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. H O NH Ac O OH O O NH OH Ac OH O O H O H O OH O OO H O OH O- CH3 1 2
INTRODUCTION + H3 N H3 C O WORK IN OUR LABORATORY INVOLVES THE SYNTHESIS OF SIMPLER ZWITTERIONIC SACCHARIDE ANALOGUES FOR EVALUATION AS POTENTIAL STIMULANTS OF INNATE AND/OR ADAPTIVE IMMUNITY. H O Ac NH O OH OH O RECENTLY, ONE SUCH ANALOG, 2, 2, HAS BEEN SYNTHESIZED AND HAS BEEN SHOWN TO POSSESS IMMUNOSTIMULATORY PROPERTIES. O O O O O- CH3 2 3
SYNTHETIC STUDIES Synthesis of Subunit A: (Proposed Scheme) 4
SYNTHETIC STUDIES ORTEP representation of the anhydride 3,6-Anhydride molecules showing intermolecular H-bonding 5
SYNTHETIC STUDIES Reactivity of 3,6-Anhydro-D-glucal: 6
SYNTHETIC STUDIES Reactivity of 3,6-Anhydro-D-glucal: 7
SYNTHETIC STUDIES Synthesis of Subunit A: 9
SYNTHETIC STUDIES Synthesis of Subunit B: B 10
SYNTHETIC STUDIES Synthesis of Subunit C: 11
SYNTHETIC STUDIES Synthesis of Subunit C (cont): C 12
SYNTHETIC STUDIES Coupling of Subunits A and B: 13
SYNTHETIC STUDIES Coupling of Subunit A+B with C: 14
BIOLOGICAL EVALUATION Innate Immunity: PROTECTED ANALOG 2 WA THE PRODUCTION OF THE PROINFLAMMATORY CYTOKINES TNF- AND IL-6 USING RAW 264.7 AND MOUSE PERITONEAL MACROPHAGES (C57 BL/6, JACKSON) 2 WAS EVALUATED IN ELISA ASSAYS FOR 15
BIOLOGICAL EVALUATION Innate Immunity: TNF- 24hrs [TNF- ] (pg/ml) 1500 0 1 5 LPS *** ** 1000 ** * 500 0 PEC RAW 16
BIOLOGICAL EVALUATION Innate Immunity: 48hrs IL-6 24hrs 500 [IL-6] (pg/ml) 250 0 1 5 LPS 0 1 5 LPS [IL-6] (pg/ml) 400 200 ** * ** * N.S. 300 150 N.S. 200 100 N.S. N.S. 50 100 0 0 PEC RAW PEC RAW 17
BIOLOGICAL EVALUATION Adaptive Immunity: 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. 18
BIOLOGICAL EVALUATION Innate Immunity: TLR2 vs TLR4 Ligand 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 2 AND, TNF-A AND IL6 LEVELS WERE MEASURED OVERTIME IN THE SUPERNATENTS. 19
BIOLOGICAL EVALUATION TLR KO MICE: TNF- Production 2 hrs *** ** 24 hrs ** ** [TNF- ] (pg/ml) [TNF- ] (pg/ml) 400 600 0 1 5 LPS 0 1 5 LPS 300 400 200 200 100 0 0 WT WT TLR-2ko TLR-4ko TLR-2ko TLR-4ko 20
BIOLOGICAL EVALUATION TLR KO MICE: IL-6 Production 24hrs ** ** 48hrs ** ** 3000 3000 0 1 5 LPS 0 1 5 LPS [IL-6] (pg/ml) [IL-6] (pg/ml) 2000 2000 1000 1000 0 0 WT WT TLR-2ko TLR-4ko TLR-2ko TLR-4ko 21
BIOLOGICAL EVALUATION TLR-4 EXPRESSION: Flow Cytometry THE EXTENT OF BINDING OF ANALOGUE 2 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. 22
BIOLOGICAL EVALUATION TLR-4 EXPRESSION: Flow Cytometry TLR-4 23
BIOLOGICAL EVALUATION TLR-4 EXPRESSION: Flow Cytometry PECs N.S.*** % TLR-4 expression 80 60 40 20 0 g/ml 1 g/ml 5 g/ml 10 control 24
BIOLOGICAL EVALUATION TLR-2 EXPRESSION: Flow Cytometry 25
BIOLOGICAL EVALUATION SUMMARY: TLR KO MICE 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 2 OCCURS THROUGH TLR4. FURTHERMORE, THE MAGNITUDE OF TLR4 SIGNALING WITH 2 2 WAS GREATER THAN THE KNOWN TLR4 AGONIST, E. COLI LPS (LIPOPOLYSACCHARIDE) 26
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 2 SHOWS IMMUNOSTIMULATORY PROPERTIES ANALOGUE 2 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 27
