Structure–function relationship studies of bovine parathyroid hormone [bPTH(1–34)] analogues containing α‐amino‐ iso ‐butyric acid (Aib) residues

The N‐terminal 1–34 fragments of the parathyroid hormone (PTH) and parathyroid hormone‐related protein (PTHrP) elicit the full spectrum of bone‐related biological activities of the intact native sequences. It has been suggested that the structural elements essential for bioactivity are two helical segments located at the N‐terminal and C‐terminal sequences, connected by hinges or flexible points around positions 12 and 19. In order to assess the relevance of the local conformation around Gly 12 upon biological function, we synthesized and characterized the following PTH(1–34) analogues containing Aib residues: ( I ) A‐V‐S‐E‐I‐Q‐F‐nL‐H‐N‐Aib‐G‐K‐H‐L‐S‐S‐nL‐E‐R‐V‐E‐Nal‐L‐R‐K‐K‐L‐Q‐D‐V‐H‐N‐Y‐NH 2 {[Nle 8,18 , Aib 11 , Nal 23 ,Tyr 34 ]bPTH(1–34)–NH 2 }; ( II ) A‐V‐S‐E‐I‐Q‐F‐nL‐H‐N‐L‐Aib‐K‐H‐L‐S‐S‐nL‐E‐R‐V‐E‐Nal‐L‐R‐K‐K‐L‐Q‐D‐V‐H‐N‐Y‐NH 2 {[Nle 8,18 , Aib 12 ,Nal 23 ,Tyr 34 ]bPTH(1–34)–NH 2 }; ( III ) A‐V‐S‐E‐I‐Q‐F‐nL‐H‐N‐L‐G‐Aib‐H‐L‐S‐S‐nL‐E‐R‐V‐E‐Nal‐L‐R‐K‐K‐L‐Q‐D‐V‐H‐N‐Y‐NH 2 {[Nle 8,18 , Aib 13 , Nal 23 ,Tyr 34 ]bPTH(1–34)–NH 2 }; ( IV ) A‐V‐S‐E‐I‐Q‐F‐nL‐H‐N‐Aib‐Aib‐K‐H‐L‐S‐S‐nL‐E‐R‐V‐E‐Nal‐L‐R‐K‐K‐L‐Q‐D‐V‐H‐N‐YNH 2 {[Nle 8,18 , Aib 11,12 , Nal 23 ,Tyr 34 ]bPTH(1–34)–NH 2 }; ( V ) A‐V‐S‐E‐I‐Q‐F‐nL‐H‐N‐L‐Aib‐Aib‐H‐L‐S‐S‐nL‐E‐R‐V‐E‐Nal‐L‐R‐K‐K‐L‐Q‐D‐V‐H‐N‐Y‐NH 2 {[Nle 8,18 , Aib 12,13 ,Nal 23 ,Tyr 34 ]bPTH(1–34)–NH 2 }. (nL= Nle; Nal= L‐(2‐naphthyl)‐alanine; Aib= α‐amino‐ iso butyric acid.) The introduction of Aib residues at position 11 in analogue I or at positions 11 and 12 in analogue IV resulted in a 5–20‐fold lower efficacy and a substantial loss of binding affinity compared to the parent compound [Nle 8,18 , Nal 23 ,Tyr 34 ]bPTH(1–34)–NH 2 . Both binding affinity and adenylyl cyclase stimulation activity are largely restored when the Aib residues are introduced at position 12 in analogue II , 13 in analogue III , and 12–13 in analogue V . The conformational properties of the analogues in aqueous solution containing dodecylphosphocholine micelles were studied by CD, two‐dimensional (2D) NMR and computer simulations. The results indicated the presence of two helical segments in all analogues, located at the N‐terminal and C‐terminal sequences. Insertion of Aib residues at positions 12 and 13, or of Aib dyads at positions 11–12 and 12–13, enhances the stability of the N‐terminal helix of all analogues. In all analogues the Aib residues are included in the helical segments. These results confirmed the importance of the helical structure in the N‐terminal activation domain, as well as of the presence of the Leu 11 hydrophobic side chain in the native sequence, for PTH‐like bioactivity. © 2003 Wiley Periodicals, Inc. Biopolymers: 437–457, 2003