Conformational restraints revealing bioactive beta-bend structures for halpha CGRP8-37 at the CGRP2 receptor of the rat prostatic vas deferens

Abstract

1. The main aim of this study was to identify putative beta-bends and the role of the N- and C-terminus in the CGRP receptor antagonist halpha CGRP8-37, which was measured against halpha CGRP inhibition of twitch responses in the rat prostatic vas deferens. 2. With a bend-biasing residue (proline) at position 16 in halpha CGRP8-37 (10(-5) M) an inactive compound was produced, while alanine at the same position retained antagonist activity (apparent pKB 5.6+/-0.1 at 10(-5) M). Proline at position 19 within halpha CGRP8-37 (10(-5) M) was an antagonist (apparent pKB 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 halpha CGRP8-37 (10(-5) M) antagonized halpha CGRP responses (apparent pKB 6.0+/-0.1 and 6.1+/-0.1, respectively). Replacement by BTD at both positions 19,20 and 33,34 within halpha CGRP8-37 competitively antagonized responses to halpha CGRP (pA2 6.2; Schild plot slope 1.0+/-0.1). 4. Halpha CGRP8-37 analogues (10(-5) M), substituted at the N-terminus by either glycine8, or des-NH2 valine8 or proline8 were all antagonists against halpha CGRP (apparent pKB 6.1+/-0.1, 6.5+/-0.1 and 6.1+/-0.1, respectively), while halpha CGRP8-37 (10(-5) M) substituted in three places by proline8 and glutamic acid10,14 was inactive. 5. Replacement of the C-terminus by alanine amide37 in halpha CGRP8-37 (10(-5) M) failed to antagonize halpha CGRP responses. 6. Peptidase inhibitors did not alter either the agonist potency of halpha CGRP or the antagonist affinities of halpha CGRP8-37 BTD19,20 and 33,34 and halpha CGRP8-37 Gly8 (against halpha 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 bioactive structure of halpha CGRP8-37. Further, the N-terminus of halpha CGRP8-37 is not essential for antagonism, while the C-terminus interacts directly with CGRP receptor binding sites of the rat vas deferens.

Figure 1

Chemical structure of the bend-biasing amino acid proline and the BTD (beta-turn dipeptide) peptidomimetic. Bold lines illustrate bend-biasing (proline) and bend-forcing (BTD) regions. The BTD mimic replaces the i+1 and i+2 amino acid residues of a four residue β-turn with its backbone conformation based on a 1-thioindolizine structure. Dotted lines represent hydrogen bonding between C=O of residue i and NH of residue i+3.

Figure 2

Effect of replacement by either proline¹⁶, alanine¹⁶ or proline¹⁹ in hα CGRP_8–37 on hα CGRP responses in the rat prostatic vas deferens. Concentration response curves to hα CGRP on twitch responses, and in the presence of 10⁻⁵M of (a) hα CGRP_8–37 Pro¹⁶ (b) hα CGRP_8–37 Ala¹⁶ and (c) hα CGRP_8–37 Pro¹⁹. Results are expressed as percentage inhibition of twitch responses. Points and error bars represent the mean±s.e.mean of four or five separate experiments.

Figure 3

Effect of replacement by BTD at either positions 19,20 or 33,34 in hα CGRP_8–37 on hα CGRP responses in the rat prostatic vas deferens. Concentration response curves to hα CGRP on twitch responses, and in the presence of 10⁻⁵M of (a) hα CGRP_8–37 BTD^19,20 and (b) hα CGRP_8–37 BTD^33,34. Results are expressed as percentage inhibition of twitch responses. Points and error bars represent the mean±s.e.mean of four or five separate experiments.

Figure 4

Effect of replacement by BTD at positions 19,20 and 33,34 in hα CGRP_8–37 on hα CGRP responses in rat prostatic vas deferens. Graph (left) showing a concentration response curve to hα CGRP on twitch responses, and in the presence of hα CGRP_8–37 BTD^{19,20 and 33,34} at 3×10⁻⁶M and 10⁻⁵M. The Schild plot (right) for hα CGRP_8–37 BTD^{19,20 and 33,34} against hα CGRP. Results on the graph are expressed as percentage inhibition of twitch responses. Points and error bars represent the mean±s.e.mean of four separate experiments. On the Schild plot, points represent individual data of eight separate experiments.

Figure 5

Effect of replacement by either glycine⁸, des-NH₂ valine⁸, proline⁸ or proline⁸ and glutamic acid^10,14 in hα CGRP_8–37 on hα CGRP responses in the rat prostatic vas deferens. Concentration response curves to hα CGRP on twitch responses, and in the presence of 10⁻⁵M of (a) hα CGRP_8–37 Gly⁸, (b) hα CGRP_8–37 des-NH₂ Val⁸, (c) hα CGRP_8–37 Pro⁸ and (d) hα CGRP_8–37 Pro⁸ Glu^10,14. Results are expressed as percentage inhibition of twitch responses. Points and error bars represent the mean±s.e.mean of four or five separate experiments.

Figure 6

Effect of peptidase inhibitors (amastatin, bestatin, captopril, phosphoramidon, thiorphan; 10⁻⁶M) on antagonism by hα CGRP_8–37 BTD^{19,20 and 33,34} and hα CGRP_8–37 Gly⁸ against hα CGRP responses in the rat prostatic vas deferens. Concentration response curves to hα CGRP on twitch responses, and in the presence of 10⁻⁵M of (a) hα CGRP_8–37 BTD^{19,20 and 33,34} and (b) hα CGRP_8–37 Gly⁸, before and after treatment with peptidase inhibitors. Results are expressed as percentage inhibition of twitch responses. Points and error bars represent the mean±s.e.mean of four separate experiments.