Synthesis and relative potencies of new constrained CRF antagonists

Abstract

Two series of CRF antagonists with N alpha- and C alpha-methylated alanine and leucines were evaluated for their biological activities in vitro and in vivo in several systems. The poly-N-methylated analogue of alpha-helical-CRF9-41, [N alpha MeLeu10,15,27,37,N alpha MeAla22,32,41]-alpha-Hel-CRF9-41, was found to be considerably less potent than the parent non-N-methylated analogue. This result was expected on the basis that alpha-helicity was thought to be required for biological activity and the prediction that backbone substitutions on the nitrogen have a tendency to break alpha-helices (a hypothesis that was confirmed by circular dichroism). Next, a series of constrained analogues of the potent CRF antagonist, [DPhe12,Nle21,38]h/rCRF12-41, was synthesized that contained C alpha-methylleucine and/or C alpha-methylalanine (Aib) residues at selected positions. Because C alpha-methylation is recognized to increase alpha-helicity, and because there is now strong NMR data suggesting that residues 6-36 assume a well-defined alpha-helix, it was expected that these analogues would be more potent. Although usual solid-phase peptide synthesis procedures were followed, success in coupling the C alpha-methyl amino acids was obtained only with a 1:1 mixture of BOP/HOBt. In vitro potencies of the synthesized compounds were measured in a collagenase-dispersed anterior pituitary cell culture bioassay. Monosubstituted analogues were shown to be twice to one fourth as potent as the parent compound; while the pluri-substituted peptides were slightly less potent. This decrease in potency might be correlated to an unexpected lower helical content of the pluri-substituted compounds (as determined by CD spectroscopy), as it was suggested that the bioactive conformation of the CRF was predominantly alpha-helical. Interestingly, one analogue, [DPhe12,Nle21,38,C alpha-MeLeu37]h/rCRF12-41, was found to be more potent and longer acting than the parent compound in two in vivo assays measuring ACTH release after intravenous administration to adrenalectomized rats and reversal of stress-induced delay in gastric emptying in the rat after intracisternal administration. The molecular basis for this increased duration of action and potency is being investigated.

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 …”

Corticotropin-Releasing Hormone Antagonists, Astressin B and Antalarmin: Differing Profiles of Activity in Rhesus Monkeys

“…A variety of CRH antagonists have been synthesized, beginning with the early modifications of CRH itself that resulted in a-helical CRH (Rivier et al, 1984) and D-Phe CRH (Hernandez et al, 1993;Menzaghi et al, 1994), and extending to the more recent analogs, astressin (Gulyas et al, 1995) and the longer-lasting astressin B . Nonpeptidic compounds, the so-called 'small molecule' CRH-R1 selective antagonists, such as CP-154,526 (Schulz et al, 1996), its methyl analog, antalarmin (Webster et al, 1996; see also review by McCarthy et al, 1999), and SSR125543A (Griebel et al, 2002) have been investigated preclinically using animal models of depression and anxiety.…”

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.…”