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-C̅y̅s̅-̅A̅r̅g̅-̅C̅y̅s̅-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 IC₅₀values in the range of 2–800 μm, 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 μm). TheN-myristoylated FRCRCFa exhibits an inhibitory potency (IC₅₀ = 8–10 μm) 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 ord-Cys5 substitutions in FRCRCFa do not alter the inhibitory effect, whereas the l-to-dsubstitutions of other amino acids in FRCRCFa reduce the inhibitory potency by 4–5-fold. Thus, thel-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 β,β-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-C̅y̅s̅-̅A̅r̅g̅-̅C̅y̅s̅ 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.