Structural and conformational requirements for human calcitonin activity: design, synthesis, and study of lactam-bridged analogues.

The conformational and pharmacological effects of the introduction of conformational constraints in the form of i-(i + 4) lactam-bridges in the potential amphiphilic alpha-helical region (8-21) of human calcitonin (hCT) were studied. The following three cyclic hCT analogues were synthesized: cyclo17,21-[Lys17,Asp21]hCT (1), cyclo17,21- [Asp17,Lys21]hCT (2) and cyclo10,14-[Lys10,-Asp14]hCT (3). For their syntheses, solid-phase methodology was used in combination with either direct side chain to side chain cyclization on the solid support or a segment-condensation strategy. Circular dichroism studies in aqueous buffer, pH 7.0, indicated that the conformational effects were different for each lactam bridge introduced. Significant induction of alpha-helical structure was observed only for peptide 3. In contrast, peptide 1 and hCT had similar CD spectra, indicative of mixed disordered and beta-sheet conformations, and peptide 2 had a weaker spectrum consistent with the formation of a more ordered but nonhelical structure. In rat brain receptor binding assays, peptide 2 showed a nearly 80-fold higher potency than hCT or peptides 1 and 3. All three analogues stimulated adenylyl cyclase in the rat kidney membrane at 5-fold lower concentrations than hCT and with similar maximal effects. In vivo hypocalcemic assays, performed in mice by analysis of serum calcium levels 1 h after sc injection, indicated that peptide 2 had similar maximal effects to hCT and was 10-20 times more potent than hCT at doses giving half-maximal effects. In contrast, peptides 1 and 3 were not significantly more potent than hCT. Our findings indicate compatibility of all three lactam bridges and, most probably, also the amphiphilic alpha-helix, with the pharmacological activities of hCT. However, the properties of peptide 2 also suggest that another conformation, possibly a type I beta-turn involving residues 17-20, may play an important role. A multistep mechanism of receptor recognition by hCT that might account for these results is discussed.