title: 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.
authors.affiliation: Department of Chemistry and Chemical Biology, Rutgers University, 610 Taylor Road, Piscataway, New Jersey 08854, USA. taylor@rutchem.rutgers.edu
authors.forename: John W
authors.initials: JW
authors.lastname: Taylor
authors.forename: Qian K
authors.initials: QK
authors.lastname: Jin
authors.forename: Massimo
authors.initials: M
authors.lastname: Sbacchi
authors.forename: Lu
authors.initials: L
authors.lastname: Wang
authors.forename: Piero
authors.initials: P
authors.lastname: Belfiore
authors.forename: Martine
authors.initials: M
authors.lastname: Garnier
authors.forename: Athanasios
authors.initials: A
authors.lastname: Kazantzis
authors.forename: Aphrodite
authors.initials: A
authors.lastname: Kapurniotu
authors.forename: Paola F
authors.initials: PF
authors.lastname: Zaratin
authors.forename: Mark A
authors.initials: MA
authors.lastname: Scheideler
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism; D002116:Calcitonin / Q000031:analogs & derivatives* / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D002460:Cell Line; D002942:Circular Dichroism; D000242:Cyclic AMP / Q000378:metabolism; D015195:Drug Design; D017930:Genes, Reporter; D006801:Humans; D066298:In Vitro Techniques; D007769:Lactams / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D051381:Rats; D017207:Rats, Sprague-Dawley; D018003:Receptors, Calcitonin / Q000819:agonists / Q000378:metabolism; D012474:Salmon; D014162:Transfection
chemical_list: D007769:Lactams; D018003:Receptors, Calcitonin; D002116:Calcitonin; D000242:Cyclic AMP; D008156:Luciferases