Determinants of 1-Piperidinecarboxamide, N-[2-[[5-Amino-l-[[4-(4-pyridinyl)-l-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl) (BIBN4096BS) Affinity for Calcitonin Gene-Related Peptide and Amylin Receptors—The Role of Receptor Activity Modifying Protein 1

Abstract

1-Piperidinecarboxamide, N-[2- [[5-amino-l-[[4-(4-pyridinyl) -lpiperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl) (BIBN4096BS), a calcitonin gene-related peptide (CGRP) receptor antagonist, can alleviate the symptoms of migraine and is highly selective for CGRP over adrenomedullin (AM) receptors. These receptors are heterodimers of the calcitonin receptor-like receptor (CL) and receptor activity modifying proteins (RAMPs), with the pharmacological properties determined by the RAMP subunit. BIBN4096BS-sensitive CGRP 1 receptors are CL/RAMP1, whereas BIBN4096BS-insensitive AM receptors are CL/RAMP2 or CL/RAMP3 (AM 1 and AM 2 , respectively), implicating RAMP1 in conferring BIB-N4096BS sensitivity. Because calcitonin receptors [CT (a) ] also interact with RAMP1 [AMY 1(a) receptors], BIBN4096BS could also have affinity for these receptors. To test this, receptors were transfected into COS-7 cells and agonist-stimulated cAMP levels measured in the presence and absence of antagonists. We found that AMY 1(a) receptors were ϳ150-fold less sensitive to BIBN4096BS antagonism than CGRP 1 receptors. In contrast, AMY 3(a) [CT (a) /RAMP3] or AM 2 receptors were not sensitive to BIBN4096BS antagonism. We investigated Trp74 in RAMP1, a residue implicated in the species selectivity of BIBN4096BS. BIBN4096BS affinity was reduced at AMY 1(a) and CGRP 1 receptors when this residue was mutated to lysine or alanine. The equivalent residue in RAMP3, Glu74, when mutated to tryptophan (E74W), induced BIBN4096BS sensitivity at AM 2 and AMY 3(a) receptors. It is interesting that a selective reduction in AM potency was observed at E74W AM 2 receptors, implicating this residue in AM interactions with this receptor. These data support the importance of Trp74 in RAMP1 in the interaction of BIBN4096BS with CGRP 1 and AMY 1(a) receptors and identified Glu74 in RAMP3 as the first amino acid in RAMP important for agonist interactions with calcitonin-family receptors.Calcitonin (CT) gene-related peptide (CGRP), a 37-amino acid neuropeptide, has potent effects in the vasculature and has been implicated in migraine (Brain and Grant, 2004). CGRP-like immunoreactivity is elevated in a migraine attack, and triptans normalize these levels (Edvinsson, 2001). Furthermore, infusion of CGRP into subjects prone to migraine can trigger an attack (Lassen et al., 2002). However, the most compelling evidence for the in- ABBREVIATIONS:CT, calcitonin; AM, adrenomedullin; AMY, amylin receptor phenotype; Amy, amylin; BIBN4096BS, 1-piperidinecarboxamide, N-[2[[5-amino-l-[[4-(4-pyridinyl)-l-piperazinyl]

Role of Calcitonin Gene-Related Peptide in Light-Aversive Behavior: Implications for Migraine

“…For example, hRAMP1 can interact with the calcitonin receptor to generate the AMY 1(a) amylin receptor, which has a relatively high affinity for CGRP (Hay et al, 2006a). Nevertheless, olcegepant has ~150-fold lower affinity for AMY 1(a) than the CGRP receptor (Hay et al, 2006b). However, given that the drug concentrations at the relevant sites are not known, it is possible that the nestin/hRAMP1 phenotype may reflect a combination of CGRP and AMY 1(a) receptor actions in the nervous system.…”

Receptor activity‐modifying protein‐dependent effects of mutations in the calcitonin receptor‐like receptor: implications for adrenomedullin and calcitonin gene‐related peptide pharmacology

“…The RAMP residue W84/F111 is similarly involved in all of the binding pockets and is conserved within each RAMP across a wide range of species (Hay et al ., 2006a). Interestingly, the W74/E101/E74 position is only hydrophobic in RAMP1 where its mutation causes an increase in AM and AM2 binding but has no effect on CGRP binding (Figure 8) (Qi et al ., 2008; Moore et al ., 2013).…”

“…Its equivalent in RAMP2 (F111) has been implicated in AM binding, as has E101 (Kusano et al ., 2012). Mutagenesis studies of RAMP1 and RAMP3 indicate that W74 and E74 (the analogous residue in each protein to E101 of RAMP2) are involved in AM selectivity (Hay et al ., 2006a; Qi et al ., 2008). …”

Induction of multiple photophobic behaviors in a transgenic mouse sensitized to CGRP

“…In addition, RAMP1 interacts with the calcitonin receptor to generate the amylin receptor, AMY 1(a) . AMY 1(a) has a relatively high affinity for CGRP (Hay et al, 2006a), but olcegepant has approximately 150-fold higher affinity for the CGRP receptor than for the AMY 1(a) receptor (Hay et al, 2006b). A potential caveat is that the drug concentrations at the relevant sites are not known; thus, we cannot exclude a combination of CGRP and AMY 1(a) receptor actions contributing to the light-aversive phenotype.…”