Conformational restraints revealing bioactive beta-bend structures for h alpha CGRP(8-37) at the CGRP(2) receptor of the rat prostatic vas deferens

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.