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Table 1. Inhibition of FP-2 (%) at a given concentration by compounds 1-13Peptides1 2 3 4 5 6 7 8 9 10 11 12 1350 µM 64 56 64 60 72 60 73 70 58 56 57 50 4025 µM 61 53 63 52 69 60 71 70 55 53 57 41 2810 µM 60 50 59 47 68 49 68 65 52 49 45 15 155 µM 57 44 49 32 63 50 60 57 50 39 47 5 10Results and DiscussionThe crystal structure of FP-2 in complex with chicken egg white cystatin (CEWC) showsthree regions of CEWC interacting with the active site of FP-2: the N-terminal sequence( 6 RLLGAPV 12 - Figure 1a) and a β-hairpin loop ( 52 RQLVSGI 58 - Figure 1b) are in closecontact with the active site of FP-2, while a second β-hairpin loop ( 103 PW 104 - Figure 1c) issituated at a greater distance [3]. Since the distance between the N-terminal sequence andthe first β-hairpin loop is about 4-5 Å, we reasoned that the cystatin protein scaffold couldbe replaced by an appropriate linker joining the interacting domains.Cystatin/FP-2 interaction mimics were designed linking the neighboring amino acidicpairs of the N-terminal sequence ( 6 RLLGAPV 12 ) and the β-hairpin loop ( 52 RQLVSGI 58 )with cyclic and acyclic amino acids selected based on docking studies. Pro 11 -Gln 53 andVal 12 -Arg 52 were therefore joined with γ-aminobutyric acid (GABA) and cis-4-aminocyclohexanecarboxylicacid (ACHC) (1, 2, 5 and 6).Compounds 3, 4, 7, 8 are analogous of compounds 1, 2, 5 and 6 in which the dipeptidePro-Trp has been added to the carboxyl terminus in order to evaluate the importance of thesecond loop and two β-alanines have been introduced to fill up the distance between theloops. In a third series of compounds (9-11) the conformational freedom of GABA wasreduced by replacing the amino acids bound to GABA with two cysteines and a disulphidebridge was then build. Finally the N-terminal ( 6 RLLGAPV 12 ) and the loop ( 52 RQLVSGI 58 )sequences were synthesized as control peptides (12, 13).Inhibition of FP-2 has been determined and peptides 5-8 containing the cis-ACHClinker were the most potent inhibitors, while most of the peptides containing the GABAlinker and the disulphide bridge (1-4, 9-11) were less potent, with the exception of peptide1 that showed 57% inhibition at 5 µM. Compounds 1, 3, 5, 7 having two additional aminoacids were always more active. Insertion of the Pro-Trp dipeptide (3, 4, 7, 8) did not havesignificant effect on FP-2 inhibition. Control peptides (12-13) showed only weak inhibitoryactivities (Table 1).Treatment of parasites in in vitro culture at late ring stage with cystatin mimicsresulted in phenotype similar to that seen in parasite treated with other cysteine proteaseinhibitors such as E-64/leupeptin [4,5]: food vacuole at early trophozoite stage was swollenand in some cases it also showed clumps of malarial pigment (Figure 2).AcknowledgmentsThis publication was generated in the context of the AntiMal project, funded under the 6 th FrameworkProgramme of the European Community (contract no.IP-018834). LR was funded by “Compagnia SanPaolo” in the context of the Italian Malaria Network.References1. Rosenthal, P.J. Int. J. Parasitol. 34, 1489-1499 (2004).2. Stubbs, M.T., et al. EMBO J. 9, 1939-1947 (1990).3. Wang, S.X., et al. Proc. Natl. Acad. Sci. U.S.A. 103, 11503-11508 (2006).4. Sijwali, P.S., et al. Proc. Natl. Acad. Sci. U.S.A. 101, 4384-4389 (2004).5. Rosenthal, P. J. Exp. Parasitol. 80, 272-281 (1995).239