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.