Fm. Wisskirchen et al., Conformational restraints revealing bioactive beta-bend structures for h alpha CGRP(8-37) at the CGRP(2) receptor of the rat prostatic vas deferens, BR J PHARM, 126(5), 1999, pp. 1163-1170
1 The main aim of this study was to identify putative beta-bends and the ro
le of the N- and C-terminus in the CGRP receptor antagonist h alpha CGRP(8-
37), which was measured against h alpha CGRP inhibition of twitch responses
in the rat prostatic vas deferens.
2 With a bend-biasing residue (proline) at position 16 in h alpha CGRP(8-37
) (10(-5) M) an inactive compound was produced, while alanine at the same p
osition retained antagonist activity (apparent pK(B) 5.6+/-0.1 at 10(-5) M)
. Proline at position 19 within h alpha CGRP(8-37) (10(-5) M) was an antago
nist (apparent pK(B) 5.8+/-0.1).
3 Incorporation of a bend-forcing structure (beta-turn dipeptide or BTD) at
either positions 19,20 or 33,34 in h alpha CGRP(8-37) (10(-5) M) antagoniz
ed h alpha CGRP responses (apparent pK(B) 6.0+/-0.1 and 6.1+/-0.1, respecti
vely). Replacement by BTD at both positions 19,20 and 33,34 within h alpha
CGRP(8-37) competitively antagonized responses to h alpha CGRP (pA(2) 6.2;
Schild plot slope 1.0+/-0.1).
4 H alpha CGRP(8-37) analogues (10(-5) M), substituted at the N-terminus by
either glycine(8), or des-NH2 valines or prolines were all antagonists aga
inst h alpha CGRP (apparent pK(B) 6.1+/-0.1, 6.5+/-0.1 and 6.1+/-0.1, respe
ctively), while h alpha CGRP(8-37) (10(-5) M) substituted in three places b
y prolines and glutamic acid(10,14) was inactive.
5 Replacement of the C-terminus by alanine amide(37) in h alpha CGRP(8-37)
(10(-5) M) failed to antagonize h alpha CGRP responses.
6 Peptidase inhibitors did not alter either the agonist potency of h alpha
CGRP or the antagonist affinities of h alpha CGRP(8-37) BTD19,20 and (33,34
) and h alpha CGRP(8-37) Gly(8) (against h alpha CGRP responses).
7 In conclusion, two beta-bends at positions 18-21 and 32-35 are compatible
with high affinity by BTD and is the first approach of modelling the bioac
tive structure of ha CGRP(8-37). Further, the N-terminus of h alpha CGRP(8-
37) is not essential for antagonism, while the C-terminus interacts directl
y with CGRP receptor binding sites of the rat vas deferens.