Constrained Corticotropin-Releasing Factor (CRF) Agonists and Antagonists with i−(i+3) G<UL>lu-Xaa-<SCP>d</SCP>Xbb-Ly</UL>s Bridges

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)[<SCP>d</SCP>Phe¹²,Glu²⁰,Lys²³,Nle^21,38]hCRF<INF>(12</INF><INF>-</INF><INF>41)</INF> and of astressin {cyclo(30−33)[<SCP>d</SCP>Phe¹²,Nle^21,38,Glu³⁰,Lys³³]hCRF<INF>(12</INF><INF>-</INF><INF>41)</INF>}, two potent CRF antagonists, and of cyclo(30−33)[Ac-Leu⁸,<SCP>d</SCP>Phe¹²,Nle²¹,Glu³⁰,Lys³³,Nle³⁸]hCRF<INF>(8</INF><INF>-</INF><INF>41)</INF>, the shortest sequence equipotent to CRF reported to date (Rivier et al. J. Med. Chem. <BO>1998</BO>, 41, 2614−2620 and references therein). To test the hypothesis that the G<UL>lu²⁰−Ly</UL>s²³ and G<UL>lu³⁰−Ly</UL>s³³ lactam rings were favoring an α-helical conformation rather than a turn, we introduced a <SCP>d</SCP>-amino acid at positions 22, 31, and 32 in the respective rings. Whereas the introduction of a <SCP>d</SCP>-residue at position 31 was only marginally deleterious to potency (ca. 2-fold decrease in potency), introduction of a <SCP>d</SCP>-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 [<SCP>d</SCP>His³²] (<BO>6</BO>) or acetylation of the N-terminus (<BO>7</BO>) 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<INF>13</INF>-NH<INF>2</INF> scaffold in order to quantify the structural influence of specific <SCP>l</SCP>- and <SCP>d</SCP>Ala⁶ and <SCP>l</SCP>- and <SCP>d</SCP>Ala⁷ substitutions in [Glu⁵,Lys⁸]Ac-Ala<INF>13</INF>-NH<INF>2</INF> in a standard α-helical configuration. Models of the general form [Glu⁵,<SCP>l</SCP>Ala⁶ or <SCP>d</SCP>Ala⁶,<SCP>l</SCP>Ala⁷ or <SCP>d</SCP>Ala⁷,Lys⁸]Ac-Ala<INF>13</INF>-NH<INF>2</INF> 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 Gln²⁶ in close enough proximity to Lys³⁶ to suggest introduction of a bridge between them. We synthesized dicyclo(26−36,30−33)[<SCP>d</SCP>Phe¹²,Nle²¹,Cys²⁶,Glu³⁰,Lys³³,Cys³⁶,Nle³⁸]Ac-hCRF<INF>(9</INF><INF>-</INF><INF>41)</INF> which showed significant α-helical content using circular dichroism (CD) and had low, but measurable potency {0.3% that of <BO>6</BO> or ca. 25% that of [<SCP>d</SCP>Phe¹²,Nle^21,38]hCRF<INF>(12</INF><INF>-</INF><INF>41)</INF>}. 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.