NOVEL CARBOHYDRATE-BASED LIGANDS WITH IMMUNOSTIMULATORY PROPERTIES

Transcription

1 NVEL CARBHYDRATE-BASED LIGANDS WITH IMMUNSTIMULATRY PRPERTIES CECILIA H MARZABADI, VIKRAM BASAVA AND CNSTANTINE BITSAKTSIS SETN HALL UNIVERSITY SUTH RANGE, NJ USA

2 INTRDUCTIN THE ZWITTERINIC PLYSACCHARIDE, PSA1, ISLATED FRM THE CAPSULE F BACTERIDES FRAGILIS, HAS BEEN SHWN T STIMULATE BTH INNATE AND ADAPTIVE IMMUNE RESPNSES IN MAMMALS AND IS BELIEVED T BE + H 3 N H 3 C H H H NH Ac H H Ac NH H H IMPRTANT IN ESTABLISHING A BALANCED IMMUNE SYSTEM WHICH IS MEDIATED BY T-LYMPHCYTES. H H 1 - CH 3 2

3 INTRDUCTIN WRK IN UR LABRATRY INVLVES THE SYNTHESIS F SIMPLER ZWITTERINIC SACCHARIDE ANALGUES FR EVALUATIN AS PTENTIAL STIMULANTS F INNATE AND/R ADAPTIVE IMMUNITY. RECENTLY, NE SUCH ANALG, 2, HAS BEEN SYNTHESIZED AND HAS BEEN SHWN T PSSESS IMMUNSTIMULATRY PRPERTIES. + H 3 N H 3 C H AcNH - CH 3 H H 2 3

4 SYNTHETIC STUDIES Synthesis of Subunit A: (Proposed Scheme) 4

5 SYNTHETIC STUDIES RTEP representation of the anhydride 3,6-Anhydride molecules showing intermolecular H-bonding 5

6 Reactivity of 3,6-Anhydro-D-glucal: SYNTHETIC STUDIES 6

7 Reactivity of 3,6-Anhydro-D-glucal: SYNTHETIC STUDIES 7

8 8

9 SYNTHETIC STUDIES Synthesis of Subunit A: 9

10 SYNTHETIC STUDIES Synthesis of Subunit B: B 1

11 Synthesis of Subunit C: SYNTHETIC STUDIES 11

12 SYNTHETIC STUDIES Synthesis of Subunit C (cont): C 12

13 Coupling of Subunits A and B: SYNTHETIC STUDIES 13

14 Coupling of Subunit A+B with C: SYNTHETIC STUDIES 14

15 Innate Immunity: BILGICAL EVALUATIN PRTECTED ANALG 2 WAS EVALUATED IN ELISA ASSAYS FR THE PRDUCTIN F THE PRINFLAMMATRY CYTKINES TNF- A AND IL-6 USING RAW AND MUSE PERITNEAL MACRPHAGES (C57 BL/6, JACKSN) 15

16 Innate Immunity: TNF-A BILGICAL EVALUATIN [TNF- ] (pg/ml) *** ** RAW 24hrs ** * PEC 1 5 LPS 16

17 Innate Immunity: BILGICAL EVALUATIN IL-6 [IL-6] (pg/ml) hrs N.S. N.S. N.S. N.S. RAW PEC 1 5 LPS [IL-6] (pg/ml) RAW 48hrs ** ** * * PEC 1 5 LPS 17

18 Adaptive Immunity: BILGICAL EVALUATIN THE MLECULE SYNTHESIZED WAS ALS TESTED IN SPLEEN CELLS FRM THE SAME MICE LKING FR T AND B CELL PRLIFERATIN, LEVELS F IFN-G IN THE SUPERNATANTS (INDICATIVE F A T CELL RESPNSE), AND PLYCLNAL IGM AND IGG LEVELS IN THE SUPERNATANTS L (INDICATIVE F A B CELL RESPNSE) WERE MEASURED. N T CELL R B CELL RESPNSES WERE BSERVED. 18

19 BILGICAL EVALUATIN Innate Immunity: TLR2 vs TLR4 Ligand T ELUCIDATE THE BACTERIAL TLL LIKE RECEPTR RESPNSIBLE FR CYTKINE PRDUCTIN, THE PERITNEAL MACRPHAGES FRM TLR2 AND TLR4 KNCKUT MICE (FEMALE, 4-6 WEEK LD C57BL6) WERE DSED WITH CMPUND 2 AND, TNF-A AND IL6 LEVELS WERE MEASURED VERTIME IN THE SUPERNATENTS. 19

20 BILGICAL EVALUATIN TLR K MICE: TNF- Production [TNF- ] (pg/ml) WT 2 hrs *** ** TLR-2ko TLR-4ko 1 5 LPS [TNF- ] (pg/ml) WT 24 hrs ** ** TLR-2ko TLR-4ko 1 5 LPS 2

21 BILGICAL EVALUATIN TLR K MICE: IL-6 Production [IL-6] (pg/ml) WT 24hrs ** ** TLR-2ko TLR-4ko 1 5 LPS [IL-6] (pg/ml) WT 48hrs ** ** TLR-2ko TLR-4ko 1 5 LPS 21

22 BILGICAL EVALUATIN TLR-4 EXPRESSIN: Flow Cytometry THE EXTENT F BINDING F ANALGUE 2 T THE TLR-4 RECEPTRS WAS ILLUSTRATED BY TREATING THE PERITNEAL EXUDATE CELLS (PEC) WITH DIFFERENT CNCENTRATINS F THE CMPUND. THE CELLS WERE PRE-INCUBATED WITH THE CMPUND, AFTER WHICH WERE WASHED. T THE WASHED CELLS WAS ADDED THE ANTIBDY FLLWED BY THE PHYTERYTHRIN DYE AND, THE NUMBER F THE CLURED CELLS WAS BSERVED BY FLW CYTMETRY. 22

23 BILGICAL EVALUATIN TLR-4 EXPRESSIN: Flow Cytometry TLR-4 23

24 BILGICAL EVALUATIN TLR-4 EXPRESSIN: Flow Cytometry % TLR-4 expression control PECs N.S.* ** 1 g/ml 5 g/ml 1 g/ml 24

25 BILGICAL EVALUATIN TLR-2 EXPRESSIN: Flow Cytometry 25

26 SUMMARY: TLR K MICE BILGICAL EVALUATIN A REDUCTIN IN THE PRDUCTIN F BTH TNF-A AND IL-6 WAS BSERVED IN NLY THE TLR4 KNCKUT MICE SUGGESTING THAT SIGNALING F 2 CCURS THRUGH TLR4. FURTHERMRE, THE MAGNITUDE F TLR4 SIGNALING WITH 2 WAS GREATER THAN THE KNWN TLR4 AGNIST, E. CLI LPS (LIPPLYSACCHARIDE) 26

27 CNCLUSINS THE DESIRED ZWITTERINIC PLYSACCHARIDE, AN ANALGUE F PSA1, WAS SUCCESSFULLY SYNTHESIZED WITH BTH AMIN AND CARBXYLATE MIETIES THE BILGICAL EVALUATIN CLEARLY DENTES THAT THE ANALGUE 2 SHWS IMMUNSTIMULATRY PRPERTIES ANALGUE 2 WAS FUND T STIMULATE THE INNATE IMMUNE SYSTEM, AND NT THE ADAPTIVE IMMUNE SYSTEM AN UNEXPECTED AND AN EFFICIENT SYNTHESIS F 3, 6-ANHYDR-D-GLUCAL PAVES A RUTE T THE SYNTHESIS F INTERESTING NEW CARBHYDRATE MIETIES 27