Conformationally constrained human calcitonin (hCt) analogues reveal a critical role of sequence 17–21 for the oligomerization state and bioactivity of hCt

Calcitonin (Ct) is a 32‐residue peptide hormone that is mainly known for its hypocalcemic effect and the inhibition of bone resorption. Our previous studies have led to potent, side‐chain lactam‐bridged human Ct (hCt) analogues [Kapurniotu, A. Kayed, R., Taylor, J.W. & Voelter W. (1999) Eur. J. Biochem. 265 , 606–618; Kapurniotu, A. & Taylor, J.W. (1995) J. Med. Chem. 38 , 836–847]. We have hypothesized that a possibly type I β turn/β sheet conformation in the region 17–21 may play an important role in hCt bioactivity. To investigate this hypothesis, analogues of the potent hCt agonist cyclo17,21‐[Asp17,Lys21]hCt ( 1 ) bearing type I (and II′) or II β turn‐promoting substituents at positions 18 and 19 were designed, synthesized and their solution conformations, human Ct receptor binding affinities and in vivo hypocalcemic potencies were assessed. The novel analogues include cyclo17,21‐[Asp17, d ‐Phe19, Lys21]hCt ( 2 ), cyclo17,21‐[Asp17,Aib18,Lys21]hCt ( 3 ), cyclo17,21‐[Asp17, d ‐Lys18,Lys21]hCt ( 4 ), corresponding partial sequence peptides containing the lactam‐bridged region 16–22, and nonbridged control peptides. Only 1 showed a higher Ct receptor binding affinity than hCt, whereas analogues 2–4 had similar receptor affinities to hCt. In the in vivo hypocalcemic assay, 3 and 4 were as potent as 1 , whereas 2 completely lost the high potency of 1 , suggesting that type I (and II′) β turn‐promoting substituents are fully compatible with in vivo bioactivity. CD spectroscopy showed that analogues 1 – 4 were markedly β sheet‐stabilized compared to hCt and indicated the presence of distinct β turn conformeric populations in each of the analogues. Unexpectedly, the d ‐amino acid‐ or Aib‐containing cyclic analogues 2 – 4 but not 1 or hCt self‐associated into SDS denaturation‐stable dimers. Our results demonstrate a crucial role of the conformational and topological features of the residues in sequence 17–21 and in particular of residues 18 and 19 for human Ct receptor binding and in vivo bioactivity and also for the self association state of hCt. These results may assist to delineate the structure‐function relationships of hCt and to design novel hCt agonists for the treatment of osteoporosis and other bone‐disorder‐related diseases.