Side-Chain Lactam-Bridge Conformational Constraints Differentiate the Activities of Salmon and Human Calcitonins and Reveal a New Design Concept for Potent Calcitonin Analogues

Abstract

We have recently reported the potent hypocalcemic effects of side-chain lactam-bridged analogues of human calcitonin (hCT) (Kapurniotu, A.; et al. Eur. J. Biochem. 1999, 265, 606-618). To extend these studies, we have now synthesized a new series of (Asp(17), Lys(21)) and (Asp(17), Orn(21)) side-chain bridged salmon calcitonin (sCT) and hCT analogues. The affinities of these analogues for the human calcitonin receptor, hCTR(I1)(-), and for rat-brain membrane receptors were assayed in competitive binding assays, and agonist potencies at the hCTR(I1)(-) receptors were assessed, using a cAMP-responsive gene-reporter assay. The bridged sCT analogues had activities similar to sCT itself. In contrast, an (Asp(17), Orn(21)) side-chain bridged hCT analogue, cyclo(17-21)-[Nle(8), Phe(12), Asp(17), Orn,(21) Tyr(22))-hCT, was 80 and 450 times more active than hCT in the hCTR(I1)(-) and rat-brain receptor binding assays, respectively, and was 90 times more potent than hCT and 16 times more potent than sCT in initiating receptor signaling. An uncyclized, isosteric analogue of this peptide was also more potent than hCT, demonstrating that the cyclization constraint and these single-residue substitutions enhance the activities of hCT in an additive fashion. This study demonstrates that the potency-enhancing effects of lactam-bridge constraints at hCT residues 17-21 are not transferable to sCT. We also show that, in comparison to the hCT analogues, sCT and its analogues are less potent agonists than expected from their hCTR(I1)(-) affinities. This suggests that it may be possible to preserve the efficient signal transduction of hCT while introducing additional receptor affinity-enhancing elements from sCT into our potent lactam-bridged hCT analogue, thereby creating new super-potent, hCT-based agonists.

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

“…Our findings with regard to the importance of nature and chirality of residues 18 and 19 for hCt receptor binding and bioactivity were confirmed by SAR studies of linear substitution analogs of hCt carrying β-turn-inducing substitutions for these residues [109]. These latter studies also suggested that nature and chirality of residues 18 and 19 plays an important role not only for receptor binding, but also for adenylate cyclase activation.…”

“…Together, the SAR studies were in support of both the type I β-turn/β-sheet "hypothesis" and of a proposed model of ligand-CtR interaction according to which all three domains, i.e. the Nterminal region 1-7, the region 8 to 22, and the C-terminal region 22-32 may interact with distinct domains of the CtR [20,43,46,47,107,109]. (Fig.…”

“…designed, synthesized, and studied with regard to conformation and bioactivity [108]. In addition to the above SAR studies, J. Taylor, M. Scheideler and coworkers have recently designed and studied a series of linear hCt analogs carrying β-turn-inducing substituents in positions 18 and 19 and a novel substitution analog of cyclo 1 7 , 2 1 -[Asp 17 ,Orn 21 ]hCt (B2) [109]. In addition, a series of cyclic sCt analogs carrying an Asp 17 to Lys 21 or an Asp 17 to Orn 21 bridge were also studied [109].…”

“…Similarly to the results obtained with hCtR I1-, the effects of the substituents alone (in D 8 ) and the lactam bridge constraint (D7) on binding affinity to rat brain membranes were also additive. The signal transducing potencies of the analogs at hCtR I1-were assessed in SH-SY5Y cells with a cAMP-responsive gene-reporter assay [109]. Thereby, D8 showed a 2-fold (ED 50 = 0.27 nM) and D7 a 90-fold (ED 50 = 0.007 nM) higher potency than hCt (ED 50 = 0.60 nM).…”

Calcitonin Increases Tumorigenicity of Prostate Cancer Cells: Evidence for the Role of Protein Kinase A and Urokinase-Type Plasminogen Receptor

“…There are also reports of differential alterations in the secretion of both CT species in secretions of PC as well as other human cancers (13,27,32). However, synthetic sCT is widely used clinically in the treatment of bone-related disorders and as an agonist for hCTR in human tissues and cell lines (15,(33)(34)(35)(36)(37). sCT stimulates the same signaling mechanisms as hCT and produces similar biological effects in human tissues and cell lines.…”