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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010TAMRASelective Targeting of Extracellular Cyclophilins by NovelCyclosporin A DerivativesMiroslav Malešević 1 , Jan Kühling 1 , Viktoria Kahlert 1 ,Frank Erdmann 1 , Molly A. Balsley 2 , Michael I. Bukrinsky 2 ,Stephanie L. Constant 2 , and Gunter Fischer 11 Max Planck Research Unit for Enzymology of Protein Folding, Halle (Saale), 06120,Germany; 2 Department of Microbiology, Immunology, and Tropical Medicine,The George Washington University, Washington, DC, 20037, U.S.A.IntroductionCyclosporin A (CsA), a cyclic undecapeptide extracted from the fungus Tolypocladiuminflatum, is an indiscriminately tight binding inhibitor of Cyclophilins (Cyps). Cyclophilinstogether with parvulins and FK-506 binding protein belongs to the family of peptidyl-prolylcis/trans isomerases (PPIases EC 5.2.1.8). These ubiquitous chaperone enzymes, thatinfluence de novo protein folding, and refolding of denatured proteins, are found invirtually all organisms. There are 20 different human Cyp isoforms; the most abundantmember of them is cyclophilin A (CypA, Cyp18). Cyp A and Cyp B are also extracellularenzymes that cause chemotactic responses of eosinophils, neutrophils, and T-cells(mediated by the PPIase active site of cyclophilins) probably through the CD-147 receptor.The mechanism and stimuli needed for Cyp release from cell is unknown. However,elevated levels of Cyp have been detected in different inflammatory diseases, like asthma,severe sepsis, rheumatoid arthritis, or psoriasis.Although human Cyps are involved in a multitude of cellular functions like cellgrowth, proliferation, and motility, Cyp-inhibitors, like e.g. CsA, are considered as possibleantiviral or antiparasitic therapeutics. Also, Cyp inhibition is a promising strategy fortreating several pathological processes like cancer or inflammatory diseases. However,major obstacles for a broader use of Cyp inhibitors are lack of selectivity andimmunosuppression.Results and DiscussionA novel CsA derivative (Figure 1) for selective targeting of extracellular Cyps wasdesigned and synthesized [1]. A trifunctional template, obtained from trimesic acid [2], wasfunctionalized with the fluorescent marker 5(6)-carboxytetramethylrhodamine and a sideONHOOOHNNHOOOHNO[D-Ser8]-CsAO(D-Glu) 6-Gly-OHFig. 1. The novel CsA derivativeMM-218.chain-extended [D-Ser8]-CsA analog. Theremaining third position was occupied with a highlynegatively charged (D-Glu) 6 -Gly-OH moiety, whichwas used to improve the derivative solubilities atphysiological conditions and to diminish its cellpermeabilities. As a cell-permeable reference, (Cs9-Rhd), the same [D-Ser8]-CsA analog and5(6)-carboxytetramethylrhodamine connected withthe cadaverin linker [3], was synthesized.Both confocal laser scanning microscopy(Figure 2) and fluorescence-activated cell sortingexperiments confirmed the cell impermeability ofthe MM-218 compound. Contrary, the Cs9-Rhd wasaccumulated mostly in the cell cytoplasm. However,fluorescent clusters were observed on the cell membrane upon incubation with bothcompounds. In a protease-coupled PPIase assay [4] MM-218 (Ki=1.8± 0.6 nM CypA, 1.3 ±0.5 nM CypB) and Cs9-Rhd (Ki=4.3±0.5 nM CypA, 12.0± 2.8 nM CypB) were even betterreversible Cyp inhibitors than CsA itself (Ki=8.4±2.5 nM CypA, 6.9±2.1 nM CypB).However, in a RII phosphopeptide dephosphorylation assay [3], the Calcineurin (CaN) wasbest inhibited by a CsA/CypA binary complex (MM-218/CypA Ki=26.2 ±1.2 µM,Cs9-Rhd/CypA Ki=0.8±0.1, CsA/CypA Ki=0.09±0.005 µM). Probably because of its cellimpermeability the MM-218 compound could not inhibit the proliferation of theconcanavalin A stimulated splenic lymphocytes and appeared to be non-immunosuppresive24