Modifications to the N-Terminus but Not the C-Terminus of Calcitonin Gene-Related Peptide(8-37) Produce Antagonists with Increased Affinity

Abstract

Seventeen novel analogues of human calcitonin gene-related peptide(8-37) (hCGRP(8-37)) were synthesized by solid-phase methods and purified to apparent homogeneity by semipreparative cation exchange and/or reversed-phase high-performance liquid chromatography. The C-terminal Phe was replaced by Gly, cyclohexylalanine (Cha), Tyr, all four isomers of beta-methylphenylalanine (beta-MePhe), and l- and d-tetrahydroisoquinoline carboxylic acid (Tic), resulting in analogues 3-11. For the synthesis of the beta-MePhe-containing analogues 6-9, crystallization was used to separate a mixture of all four isomers of beta-MePhe into the erythro pair of enantiomers (2S,3S, 2R,3R) and the threo pair of enantiomers (2S,3R, 2R,3S), which were then converted to Fmoc derivatives and used in two separate syntheses. Two diastereomeric peptides were obtained from each synthesis and were separated by RP-HPLC to yield enantiomerically pure 6-9. Substitution of Tyr for Phe caused no change in binding affinity at CGRP receptors. All other substitutions for Phe resulted in substantial reductions in binding affinity. Indeed, no binding was observed for analogues 7, 9, and 11, all of which contained a d-amino acid residue in the C-terminal position, and the binding affinities of the remaining analogues were >10-fold lower than that of h-alpha-CGRP(8-37). These data suggest that a conformationally flexible phenyl ring in the C-terminal position of h-alpha-CGRP(8-37) is preferred for high-affinity binding to CGRP receptors. Acetylation, benzoylation, and benzylation of the N-termini of h-alpha-CGRP(8-37) and h-beta-CGRP(8-37) produced analogues 12-14 and 16-18, respectively. A byproduct was isolated by RP-HPLC from the resin-cleaved crude product of each benzylated analogue, which was characterized as the dibenzylated derivative of h-alpha-CGRP(8-37) and h-beta-CGRP(8-37) (analogues 15 and 19, respectively). Amino acid analysis and (1)H NMR showed that the second benzyl group was located on the C4 carbon of the imidazole ring of His(10). Radioligand binding experiments showed that derivatizing the N-termini substantially increased binding affinities at CGRP receptors. The benzoylated and dibenzylated derivatives had the highest affinities, which were approximately 50-fold greater than those of h-alpha-CGRP(8-37). Functional experiments confirmed that the N-terminally derivatized analogues of h-alpha-CGRP(8-37) are antagonists that are more potent than h-alpha-CGRP(8-37). In conclusion, these studies underscore the importance of Phe(37) of h-alpha-CGRP(8-37) for binding to CGRP receptors and have identified the N-terminus and His(10) as two positions that can be used for the design of antagonists with increased affinity for CGRP receptors.

Pharmacological Characterization of Novel α-Calcitonin Gene-Related Peptide (CGRP) Receptor Peptide Antagonists That Are Selective for Human CGRP Receptors

“…Thus, unlike blood vessels from some other species TABLE 2 Antagonist equilibrium dissociation constants (K B and pA 2 ) and Schild slopes for CGRP (8 -37) , bzl-CGRP (8 -37) , and bzl-bn-CGRP (8 -37) jpet.aspetjournals.org Downloaded from (Wisskirchen et al, 1999), we found that the mouse has typical CGRP-1 receptor-mediated vascular relaxation as has been reported in isolated blood vessels from humans (Hasbak et al, 2003). In addition to the prototypical CGRP receptor antagonist CGRP (8 -37) , we also quantitatively characterized a CGRP receptor antagonist, bzl-CGRP (8 -37) , previously identified by our laboratory (Smith et al, 2003), and a novel antagonist bzl-bn-CGRP (8 -37) . These three antagonists all acted in a competitive manner in blocking functional responses in mouse aorta and in human SK-N-MC cells, and the rank order of affinity at CGRP receptors in both of these assay systems was bzl-bn-CGRP (8 -37) Ն bzl-CGRP (8 -37) Ͼ CGRP (8 -37) .…”

“…Peptides were synthesized by Merrifield's solid-phase methods using in situ neutralization (Schnolzer et al, 1992) using Boc amino acids and para-methylbenzhydrylamine resin. Details of peptide synthesis are provided in previous publications (Smith et al, 2003;Taylor et al, 2005). Peptides were purified to Ͼ98% by semipreparative reversed-phase high-performance liquid chromatography (RP-HPLC) on a Vydac 218TP510 C 18 column (1 ϫ 25 cm) from the Separations Group (Hesperia, CA).…”

“…CGRP (8 -37) and bzl-CGRP (8 -37) were synthesized and purified as described previously (Smith et al, 2003;Taylor et al, 2005). Synthesis of bzl-bn-CGRP (8 -37) was carried out manually in a 30-ml glass reaction vessel on a 0.5-mmol scale.…”

“…Synthesis of bzl-bn-CGRP (8 -37) was carried out manually in a 30-ml glass reaction vessel on a 0.5-mmol scale. A portion of the fully protected peptide-resin (100 mg, 0.02 mmol) was benzylated and then benzoylated as described previously (Smith et al, 2003). The peptide was cleaved from the resin, and the crude product was loaded onto a C 18 RP-HPLC column.…”

“…Therefore, we characterized the standard CGRP receptor antagonist CGRP (8 -37) , our previously reported high-affinity competitive antagonist [N-␣-benzoyl]human ␣-CGRP (8 -37) ] (Smith et al, 2003) and the new antagonist, bzl-bn-CGRP (8 -37) at mouse CGRP receptors, using the mouse thoracic aorta, and at human CGRP receptors, using the human SK-N-MC cell line.…”