Inhibition of the Cardiac Sarcolemma Na+/Ca2+ Exchanger by Conformationally Constrained Small Cyclic Peptides

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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.