title: Inhibition of the cardiac sarcolemma Na+/Ca2+ exchanger by conformationally constrained small cyclic peptides.
abstract: Positively charged cyclic peptides (three to seven amino acids) have been tested for their inhibitory effects on Na+/Ca2+ exchange in the cardiac sarcolemma vesicles. The lead structure of Phe-Arg-Cys-Arg-Cys-Phe-CONH2 (FRCRCFa) has been systematically modified for identification of important pharmacophores. In cyclic peptides (intramolecular S-S bond, the carboxyl terminal is locked with amide (CONH2), and positive charge is retained by one or two arginines, ornithines, or lysines. Thirty-five different cyclic peptides show IC50 values in the range of 2-800 microM, suggesting that some specific structure-activity relationships may determine the inhibitory effects. Shortening of the FRCRCFa length to four amino acids decreases the inhibitory potency by 10-80-fold. The substitution of Arg2 or Arg4 in FRCRCFa with lysine or ornithine decreases the inhibitory potency by 5-12-fold, suggesting that both arginines are beneficial for inhibition. The substitution of Phe1 in FRCRCFa by 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid produces a potent inhibitor (IC50 = 2-4 microM). The N-myristoylated FRCRCFa exhibits an inhibitory potency (IC50 = 8-10 microM) similar to that of the parent FRCRCFa peptide, thereby arousing a new possibility for the development of a cell-permeable blocker of the Na+/Ca2+ exchanger, D-Arg4 or D-Cys5 substitutions in FRCRCFa do not alter the inhibitory effect, whereas the L-to-D substitutions of other amino acids in FRCRCFa reduce the inhibitory potency by 4-5-fold. Thus, the L-to-D substitutions of Arg4 and/or Cys5 have a potential to increase the peptide stability to proteolytic degradation. The insertion of proline outside of the ring of FRCRCFa diminishes the inhibitory potency by 3-6-fold, whereas proline introduction into the ring decreases the inhibitory potency by 16-20-fold. The replacement of Cys3 and Cys5 in FRCRCFa with beta, beta-dimethylcystein has no significant effect on the inhibitory potency, suggesting that the S-S bond is not exposed to the interface of the peptide/receptor interaction. In conclusion, the current data support a proposal that the conformationally constrained Arg-Cys-Arg-Cys structure is obligatory for inhibition of Na+/Ca2+ exchange, whereas hydrophobic additions at the carboxyl and amino ends have limited effects in increasing the inhibitory potency.
authors.affiliation: Department of Physiology and Pharmacology, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv, Israel. dhanan@post.tau.ac.il
authors.forename: D
authors.initials: D
authors.lastname: Khananshvili
authors.forename: B
authors.initials: B
authors.lastname: Mester
authors.forename: M
authors.initials: M
authors.lastname: Saltoun
authors.forename: X
authors.initials: X
authors.lastname: Wang
authors.forename: G
authors.initials: G
authors.lastname: Shaulov
authors.forename: D
authors.initials: D
authors.lastname: Baazov
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors*; D002417:Cattle; D009206:Myocardium / Q000201:enzymology*; D010456:Peptides, Cyclic / Q000737:chemistry / Q000494:pharmacology*; D011487:Protein Conformation; D012508:Sarcolemma / Q000201:enzymology*; D012964:Sodium / Q000378:metabolism*; D019831:Sodium-Calcium Exchanger; D013329:Structure-Activity Relationship
chemical_list: D002352:Carrier Proteins; D010456:Peptides, Cyclic; D019831:Sodium-Calcium Exchanger; D012964:Sodium; D002118:Calcium