INHIBITION OF THE CARDIAC SARCOLEMMA NA+ CA2+ EXCHANGER BY CONFORMATIONALLY CONSTRAINED SMALL CYCLIC-PEPTIDES/

Positively charged cyclic peptides (three to seven amino acids) have b een tested for their inhibitory effects on Na+/Ca2+ exchange in the ca rdiac sarcolemma vesicles. The lead structure of Phe-Arg-Cys-Arg-Cys-P he-CONH2 (FRCRCFa) has been systematically modified for identification of important pharmacophores. In cyclic peptides (intramolecular S-S b ond), the carboxyl terminal is locked with amide (CONH2), and positive charge is retained by one or two arginines, omithines, or lysines. Th irty-five different cyclic peptides show IC50 values in the range of 2 -800 mu M, suggesting that some specific structure-activity relationsh ips may determine the inhibitory effects. Shortening of the FRCRCFa le ngth to four amino acids decreases the inhibitory potency by 10-80-fol d. The substitution of Arg2 or Arg4 in FRCRCFa with lysine or ornithin e decreases the inhibitory potency by 5-12-fold, suggesting that both arginines are beneficial for inhibition. The substitution of Phe1 in F RCRCFa by 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid produces a potent inhibitor (IC50 = 2-4 mu M). The N-myristoylated FRCRCFa exhibi ts an inhibitory potency (IC50 = 8-10 mu M) similar to that of the par ent FRCRCFa peptide, thereby arousing a new possibility for the develo pment of a cell-permeable blocker of the Na+/Ca2+ exchanger. D-Arg4 or D-Cys5 substitutions in FRCRCFa do not alter the inhibitory effect, w hereas 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 CysS have a potential to increase the peptide stability t o proteolytic degradation. The insertion of proline outside of the rin g of FRCRCFa diminishes the inhibitory potency by 3-6-fold, whereas pr oline introduction into the ring decreases the inhibitory potency by 1 6-20-fold. The replacement of Cys3 and Cys5 in FRCRCFa with beta,beta- dimethylcystein has no significant effect on the inhibitory potency, s uggesting that the S-S bond is not exposed to the interface of the pep tide/receptor interaction. In conclusion, the current data support a p roposal that the conformationally constrained ArgCys-Arg-Cys structure is obligatory for inhibition of Na+/Ca2+ exchange, whereas hydrophobi c additions at the carboxyl and amino ends have limited effects in inc reasing the inhibitory potency.