10.1021/jm980350k.s001 Steven C. Koerber Steven C. Koerber Jozsef Gulyas Jozsef Gulyas Sabine L. Lahrichi Sabine L. Lahrichi Anne Corrigan Anne Corrigan A. Grey Craig A. Grey Craig Catherine Rivier Catherine Rivier Wylie Vale Wylie Vale Jean Rivier Jean Rivier Constrained Corticotropin-Releasing Factor (CRF) Agonists and Antagonists with <i>i</i>−(<i>i</i>+3) Glu-Xaa-dXbb-Lys Bridges<sup>†</sup> American Chemical Society 1998 Nle 21 NH 2 scaffold Glu 5 antagonist CRF 33 lactam rings residue hCRF dAla 7 substitutions NH 2 helical introduction dAla 6 Lys 33 Glu 30 Lys 8 Nle 38 dPhe 12 cyclo potency 1998-11-12 00:00:00 Journal contribution https://acs.figshare.com/articles/journal_contribution/Constrained_Corticotropin-Releasing_Factor_CRF_Agonists_and_Antagonists_with_i_i_i_i_i_i_3_Glu-Xaa-dXbb-Lys_Bridges_sup_sup_/3683919 We hypothesized that covalent constraints such as side-chain to side-chain lactam rings would stabilize an α-helical conformation shown to be important for the recognition and binding of the human corticotropin-releasing factor (hCRF) C-terminal 33 residues to CRF receptors. These studies led to the discovery of cyclo(20−23)[dPhe<sup>12</sup>,Glu<sup>20</sup>,Lys<sup>23</sup>,Nle<sup>21,38</sup>]hCRF<sub>(12</sub><sub>-</sub><sub>41)</sub> and of astressin {cyclo(30−33)[dPhe<sup>12</sup>,Nle<sup>21,38</sup>,Glu<sup>30</sup>,Lys<sup>33</sup>]hCRF<sub>(12</sub><sub>-</sub><sub>41)</sub>}, two potent CRF antagonists, and of cyclo(30−33)[Ac-Leu<sup>8</sup>,dPhe<sup>12</sup>,Nle<sup>21</sup>,Glu<sup>30</sup>,Lys<sup>33</sup>,Nle<sup>38</sup>]hCRF<sub>(8</sub><sub>-</sub><sub>41)</sub>, the shortest sequence equipotent to CRF reported to date (Rivier et al. <i>J.</i> <i>Med.</i> <i>Chem.</i> <b>1998</b>, <i>41</i>, 2614−2620 and references therein). To test the hypothesis that the G<u>lu<sup>20</sup>−Ly</u>s<sup>23</sup> and G<u>lu<sup>30</sup>−Ly</u>s<sup>33</sup> lactam rings were favoring an α-helical conformation rather than a turn, we introduced a d-amino acid at positions 22, 31, and 32 in the respective rings. Whereas the introduction of a d-residue at position 31 was only marginally deleterious to potency (ca. 2-fold decrease in potency), introduction of a d-residue at position 22 and/or 32 was favorable (up to 2-fold increase in potency) in most of the cyclic hCRF, α-helical CRF, urotensin, and urocortin agonists and antagonists that were tested and was also favorable in linear agonists but not in linear antagonists; this suggested a unique and stabilizing role for the lactam ring. Introduction of a [dHis<sup>32</sup>] (<b>6</b>) or acetylation of the N-terminus (<b>7</b>) of astressin had a minor deleterious or a favorable influence, respectively, on duration of action. In the absence of structural data on these analogues, we conducted molecular modeling on an Ac-Ala<sub>13</sub>-NH<sub>2</sub> scaffold in order to quantify the structural influence of specific l- and dAla<sup>6</sup> and l- and dAla<sup>7</sup> substitutions in [Glu<sup>5</sup>,Lys<sup>8</sup>]Ac-Ala<sub>13</sub>-NH<sub>2</sub> in a standard α-helical configuration. Models of the general form [Glu<sup>5</sup>,lAla<sup>6</sup> or dAla<sup>6</sup>,lAla<sup>7</sup> or dAla<sup>7</sup>,Lys<sup>8</sup>]Ac-Ala<sub>13</sub>-NH<sub>2</sub> were subjected to high-temperature molecular dynamics followed by annealing dynamics and minimization in a conformational search. A gentle restraint was applied to the 0−4, 1−5, and 8−12 O−H hydrogen bond donor−acceptor pairs to maintain α-helical features at the N- and C-termini. From these studies we derived a model in which the helical N- and C-termini of hCRF form a helix−turn−helix motif around a turn centered at residue 31. Such a turn brings Gln<sup>26</sup> in close enough proximity to Lys<sup>36</sup> to suggest introduction of a bridge between them. We synthesized dicyclo(26−36,30−33)[dPhe<sup>12</sup>,Nle<sup>21</sup>,Cys<sup>26</sup>,Glu<sup>30</sup>,Lys<sup>33</sup>,Cys<sup>36</sup>,Nle<sup>38</sup>]Ac-hCRF<sub>(9</sub><sub>-</sub><sub>41)</sub> which showed significant α-helical content using circular dichroism (CD) and had low, but measurable potency {0.3% that of <b>6</b> or ca. 25% that of [dPhe<sup>12</sup>,Nle<sup>21,38</sup>]hCRF<sub>(12</sub><sub>-</sub><sub>41)</sub>}. Since the 26−36 disulfide bridge is incompatible with a continuous α-helix, the postulate of a turn starting at residue 31 will need to be further documented.