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

Abstract

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 calcitonin (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 type I b turn/b sheet conformation between residues 17 and 21 could play a crucial role in hCt bioactivity. Here, we designed, synthesized and studied the conformation and bioactivity of 19-member to 17-member ring-size analogues 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 spectra indicated increased type I b turn and antiparallel b sheet content in the bicyclic analogues compared with hCt. In the in vivo hypocalcaemic assay, cyclo17,21-[Asp17,Orn21]hCt (2) was found to have a 400-fold higher potency than hCt and was fourfold more potent than salmon calcitonin (sCt), which has been the most potent known Ct. Analogue 3 had a 30-fold higher potency than hCt, whereas the highly constrained analogue 4 was as potent as hCt. Bioactivity was not enhanced for the nonbridged compound [Asp17, Orn21]hCt (5), whereas cyclo17,21-[Glu17,Dab21]hCt (6) showed the same bioactivity as 1. This study identifies 2 as exhibiting the highest in vivo potency among currently known Cts, while it differs in only one amino acid residue from hCt, strongly suggesting that the introduced constraint may have served in`freezing' hCt in a bioactive conformation. Our findings provide evidence for the first time that a b turn/b sheet conformation in region 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 structure for a hCt-based drug for the treatment of osteoporosis and other bonedisorder-related diseases.

The biological potency of a series of analogues of human calcitonin correlates with their interactions with phospholipids

“…An alternative approach is to produce a conformationally constained analog of hCT. 4 However, producing an analog that is too conformationally rigid can result in the loss of biological potency. 5,6 It is recognized that compensatory features, such as conformational flexibility, can result in a calcitonin analog that has minimal interaction with phospholipids but retains a high biological potency.…”

Contribution of Conformationally Constrained Calcitonin (Ct) Analogs to the Understanding of the Structural and Conformational Requirements of Calcitonin Bioactivity and to the Design of Potent Agonists

“…(3). Far-UV CD spectra of hCt and analogs A2, B2-B5 in aqueous buffer, pH 7.4 (A), and in 50% TFE in aqueous buffer, pH 7.4 (B) [107]. orientation of the lactam-bridged residues on conformation and bioactivity, analogs A1 and A2 were designed.…”

“…To begin to systematically investigate the hypothesis that a β-turn/β-sheet conformation between residues 16 or 17 and 21 may play a crucial role for hCt bioactivity and to obtain novel hCt agonists, we followed two ways: first, we aimed at studying the effect of further constraining the conformation induced by the 20-member ring in cyclo 17,21 -[Asp 17 ,Lys 21 ]hCt (A2) on conformation and bioactivity of hCt and we designed, synthesized, and studied a series of 19-to 17-membered ring-size analogs of cyclo 17,21 -[Asp 17 ,Lys 21 ]hCt (A2) [107]. By the second strategy, we aimed at investigating in more detail the effect on conformation and bioactivity of type I (and II') and type II β-turn-promoting amino acid residues in positions i+2 and i+3 of the hypothetical β-turn structure in A2.…”

“…(4). Primary structures of hCt and analogs A2, and B2-B6 [107]. Numbers above the hCt sequence indicate the positions of the substituted residues.…”

Expression of Human Calcitonin by Microencapsulated Recombinant Myoblasts

“…For therapeutic purposes, these patients require frequent administration of calcitonin over a prolonged period of time. In addition, salmon peptides are only 50% homologous to the human hormone; they are antigenic, which limits their use because of resistance or desensitization to the treatment (Kapurniotu et al 1999). To address these above problems, somatic gene therapy for delivering a constant, therapeutic level of human calcitonin is an attractive alternative treatment of postmenopausal osteoporosis.…”