Structure−Function Studies of Analogues of Parathyroid Hormone (PTH)-1−34 Containing β-Amino Acid Residues in Positions 11−13<sup>†</sup><sup>,</sup><sup>‡</sup>

The 1−34 N-terminal fragments of human parathyroid hormone (PTH) and PTH-related protein (PTHrP) elicit the full spectrum of bone-relevant activities characteristic of the intact hormones. The structural elements believed to be required for receptor binding and biological activity are two helical segments, one N-terminal and one C-terminal, connected by hinges or flexible points located around positions 12 and 19. To test this hypothesis, we synthesized and characterized the following analogues of PTH-(1−34), each containing single or double substitutions with β-amino acid residues around the putative hinge located at position 12:  <b>I.</b> [Nle<sup>8,18</sup>,β-Ala<sup>11,12</sup>,Nal<sup>23</sup>,Tyr<sup>34</sup>]bPTH-(1−34)NH<sub>2</sub>; <b>II.</b> [Nle<sup>8,18</sup>,β-Ala<sup>12,13</sup>,Nal<sup>23</sup>,Tyr<sup>34</sup>]bPTH-(1−34)NH<sub>2</sub>; <b>III.</b> [Nle<sup>8,18</sup>,β-Ala<sup>11</sup>,Nal<sup>23</sup>,Tyr<sup>34</sup>]bPTH-(1−34)NH<sub>2</sub>; <b>IV. </b>[Nle<sup>8,18</sup>,β-hLeu<sup>11</sup>,Nal<sup>23</sup>,Tyr<sup>34</sup>]bPTH-(1−34)NH<sub>2</sub>; <b>V.</b> [Nle<sup>8,18</sup>,β-Ala<sup>12</sup>, Nal<sup>23</sup>,Tyr<sup>34</sup>]bPTH-(1−34)NH<sub>2</sub>; <b>VI. </b>[Nle<sup>8,18</sup>,β-Ala<sup>13</sup>, Nal<sup>23</sup>,Tyr<sup>34</sup>]bPTH-(1−34)NH<sub>2</sub> (β-hLeu = β-<i>homo</i>-leucine; β-Ala = β-alanine; Nal = l-2-naphthyl-alanine; Nle = norleucine). Analogues <b>I </b>and<b> III</b> exhibit very low binding affinity and are devoid of adenylyl cyclase activity. Analogue <b>II</b>, despite its very low binding capacity is an agonist. Biological activity and binding capacity are partially restored in analogue <b>IV</b>, and completely restored in analogues <b>V </b>and <b>VI</b>. The conformational properties of the analogues were investigated in aqueous solution containing dodecylphosphocholine (DPC) micelles as a membrane-mimetic environment using CD, 2D-NMR, and molecular dynamics calculations. All peptides fold partially into the α-helical conformation in the presence of DPC micelles, with a maximum helix content in the range of 30−35%. NMR analysis reveals the presence of two helical segments, one N-terminal and one C-terminal, as a common structural motif in all analogues. Incorporation of β-Ala dyads at positions 11,12 and 12,13 in analogues <b>I</b> and <b>II</b>, respectively, enhances the conformational disorder in this portion of the sequence but also destabilizes the N-terminal helix. This could be one of the possible reasons for the lack of biological activity in these analogues. The partial recovery of binding affinity and biological activity in analogue <b>IV</b>, compared to the structurally similar analogue <b>III</b>, is clearly the consequence of the reintroduction of Leu side-chain of the native sequence. In the fully active analogues <b>V</b> and <b>VI</b>, the helix stability at the N-terminus is further increased. Taken together, these results stress the functional importance of the conformational stability of the helical activation domain in PTH-(1−34). Contrary to expectation, insertion of a single β-amino acid residue in positions 11, 12, or 13 in analogues <b>III</b>−<b>VI</b> does not favor a disordered structure in this portion of the sequence.