Rational design, conformational studies and bioactivity of highly potent conformationally constrained calcitonin analogues

Calcitonin is known for its hypocalcaemic effect and the inhibition of bone resorption, and is used therapeutically for the treatment of osteoporosis and Paget's disease. Our studies on the conformational features of human ca lcitonin (hCt) bioactivity have led to the conformationally constrained hCt analogue cyclo17,21-[Asp17,Lys21]hCt (1), which had a 5-10 times higher in vivo hypocalcaemic potency than hCt [Kapurniotu, A. & Taylor, J.W. (1995) J. Med. Chem. 38, 836-847]. We hypothesized that a stabilized, possibly typ e I beta turn/beta sheet conformation between residues 17 and 21 could play a crucial role in hCt bioactivity. Here, we designed, synthesized and stud ied the conformation and bioactivity of 19-member to 17-member ring-size an alogues of 1 with the structure cyclo17,21-[Asp17,XX21]hCt with XX = Orn (2 ), Dab (3) and Dap (4), of the control peptide [Asp17,Orn21]hCt (5), and of the 19-member cyclo17,21-[Glu17,Dab21]hCt (6). Analyses of the far-UV CD s pectra indicated increased type I beta turn and antiparallel beta sheet con tent in the bicyclic analogues compared with hCt. In the in vivo hypocalcae mic assay, cyclo17,21-[Asp17,Orn21]hCt (2) was found to have a 400-fold hig her potency than hCt and was fourfold more potent than salmon calcitonin (s Ct), which has been the most potent known Ct. Analogue 3 had a 30-fold high er potency than hCt, whereas the highly constrained analogue 3 was as poten t as hCt. Bioactivity was not enhanced for the nonbridged compound [Asp17,O rn21]hCt (5), whereas cyclo17,21-[Glu17,Dab21]hCt (6) showed the same bioac tivity as 1. This study identifies 2 as exhibiting the highest in vivo pote ncy among currently known Cts, while it differs in only one amino acid resi due from hCt, strongly suggesting that the introduced constraint may have s erved in 'freezing' hCt in a bioactive conformation. Our findings provide e vidence for the first time that a beta turn/beta sheet conformation in regi on 17-21 of hCt and the topological features of the side chain of Asn17 are strongly associated with in vivo bioactivity, and offer a novel lead struc ture for a hCt-based drug for the treatment of osteoporosis and other bone- disorder-related diseases.