Minimal-Size, Constrained Corticotropin-Releasing Factor Agonists withi−(i+3) Glu−Lys and Lys−Glu Bridges

Abstract

In three earlier publications (Miranda et al. J. Med. Chem. 1994, 37, 1450-1459; 1997, 40, 3651-3658; Gulyas et al. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 10575-10579) we have hypothesized that covalent constraints such as side-chain-to-side-chain lactam rings would stabilize an alpha-helical conformation shown to be important for the recognition and binding of the CRF C-terminus 30 residues, to CRF receptors. These studies led to the discovery of useful CRF antagonists such as alpha-helical CRF (alpha-hel-CRF) and Astressin both in vitro and in vivo. To test the hypothesis that such lactam rings may also be modulating activation of the receptor when introduced at the N-terminus of CRF, we studied the influence of the successive introduction from residues 4 to 14 of a cyclo(i, i+3)[Lysi-Glu(i+3)] and a cyclo(i,i+3)[Glui-Lys(i+3)] bridge on the in vitro potency of the agonist [Ac-Pro4,dPhe12,Nle21,38]hCRF(4-41) and related compounds. We have also introduced the favored cyclo(Glu30-Lys33) substitution found to be remarkable in several families of antagonists (such as Astressin) and in a number of CRF agonists and investigated the role of residues 4-8 on receptor activation using successive deletions. Earlier studies had shown that in both oCRF and alpha-helical CRF, deletion of residues 1-6, 1-7, and 1-8 led to gradual loss of intrinsic activity (IA) (from 50% IA to <10% IA) resulting in alpha-hel-CRF being a potent competitive antagonist. We show that acetylation of the N-terminus of these fragments generally increases potency by a factor of 2-3 with no influence on IA. While cyclo(30-33)[Ac-Leu8,dPhe12,Nle21, Glu30,Lys33,Nle38]hCRF(8-41) (30) is the shortest reported analogue of CRF to be equipotent to CRF (70% IA), the corresponding linear analogue (31) is 120 times less potent (59% IA). Addition of one amino acid at the N-terminus ¿cyclo(30-33)[Ac-Ser7,dPhe12,Nle21, Glu30,Lys33,Nle38]hCRF(7-41) (28)¿ results in a 5-fold increase in agonist potency and full intrinsic activity (113%). The most favored modifications were also introduced in other members of the CRF family including sauvagine (Sau), urotensin (Utn), urocortin (Ucn), and alpha-hel-CRF. Parallel and consistent results were obtained suggesting that the lactam cyclization at residues 29-32 and 30-33 (for the members of the CRF family with 40 and 41 amino acid residues, respectively) will induce (in the shortened agonists) a structural constraint (alpha-helix) that stabilizes a bioactive conformation similar to that shown in the Astressin family of CRF antagonists and that residue 8 (leucine or isoleucine) bears the sole responsibility for activation of the receptor since deletion of that residue leads to potent antagonists (Gulyas et al. Proc. Natl. Acad.Sci. U.S.A. 1995, 92, 10575-10579).

Biomimetic Screening of Class-B G Protein-Coupled Receptors

“…Our findings in the UCN 4-15 template correlate perfectly with an early single point D-substitution study on the full length CRH endogenous hormone . It is noteworthy that the D-Phe substitution was systematically used for the synthesis and SAR studies of CRHR 1 peptide agonists and antagonists (Cervini et al 1999;Gulyas et al 1995;Hernandez et al 1993;Koerber et al 1998;Miranda et al 1994;Miranda et al 1997;Rivier et al 1998a;Rivier et al 1998b …”

A Role for a Helical Connector between Two Receptor Binding Sites of a Long-chain Peptide Hormone

“…17 for review), previously published structure-activity relationship studies of single-point substituted (18 -20) and terminally truncated CRF analogues (7,21) showed that the N-terminal sequence (9 -19) represents a receptor binding site, since substitutions in this region resulted in a significant decrease in receptor binding. The most Nterminal amino acid residues are thought to be responsible for receptor activation (7), since truncation of these residues produced antagonists.…”

Biomimetic screening of class B G protein-coupled receptors

“…The high-affinity carrier peptides 1-3 share common and specific structural features: all three possess an intramolecular lactam bridge. This conformational constrain is well characterized and has been shown to stabilize the peptide's helical conformation thus enhancing its affinity for the CRHR 1 -ECD1 (Gulyas et al 1995;Hernandez et al 1993;Koerber et al 1998;Miranda et al 1994;Rivier et al 1998b). Similarly, recent structure-activity-relationship studies have identified the cyclohexyl-alanine (X) substitution to be greatly potency enhancing (Yamada et al 2004).…”

“…Our findings in the UCN 4-15 template correlate perfectly with an early single point D-substitution study on the full length CRH endogenous hormone . It is noteworthy that the D-Phe substitution was systematically used for the synthesis and SAR studies of CRHR 1 peptide agonists and antagonists (Cervini et al 1999;Gulyas et al 1995;Hernandez et al 1993;Koerber et al 1998;Miranda et al 1994;Miranda et al 1997;Rivier et al 1998a;Rivier et al 1998b). To further probe the role of the Phe 11 residue for the potency of ligands, we decided to synthesize a library of UCN 4-15 conjugates bearing Phe 11 substitutions.…”