Marcus Schindler

CL/RAMP2 and CL/RAMP3 produce pharmacologically distinct adrenomedullin receptors: a comparison of effects of adrenomedullin22–52, CGRP8–37 and BIBN4096BS

Adrenomedullin (AM) has two known receptors formed by the calcitonin receptor‐like receptor (CL) and receptor activity‐modifying protein (RAMP) 2 or 3: We report the effects of the antagonist fragments of human AM and CGRP (AM22–52 and CGRP8–37) in inhibiting AM at human (h), rat (r) and mixed species CL/RAMP2 and CL/RAMP3 receptors transiently expressed in Cos 7 cells or endogenously expressed as rCL/rRAMP2 complexes by Rat 2 and L6 cells. AM22–52 (10 μM) antagonised AM at all CL/RAMP2 complexes (apparent pA2 values: 7.34±0.14 (hCL/hRAMP2), 7.28±0.06 (Rat 2), 7.00±0.05 (L6), 6.25±0.17 (rCL/hRAMP2)). CGRP8–37 (10 μM) resembled AM22–52 except on the rCL/hRAMP2 complex, where it did not antagonise AM (apparent pA2 values: 7.04±0.13 (hCL/hRAMP2), 6.72±0.06 (Rat2), 7.03±0.12 (L6)). On CL/RAMP3 receptors, 10 μM CGRP8–37 was an effective antagonist at all combinations (apparent pA2 values: 6.96±0.08 (hCL/hRAMP3), 6.18±0.18 (rCL/rRAMP3), 6.48±0.20 (rCL/hRAMP3)). However, 10 μM AM22–52 only antagonised AM at the hCL/hRAMP3 receptor (apparent pA2 6.73±0.14). BIBN4096BS (10 μM) did not antagonise AM at any of the receptors. Where investigated (all‐rat and rat/human combinations), the agonist potency order on the CL/RAMP3 receptor was AM∼βCGRP>αCGRP. rRAMP3 showed three apparent polymorphisms, none of which altered its coding sequence. This study shows that on CL/RAMP complexes, AM22–52 has significant selectivity for the CL/RAMP2 combination over the CL/RAMP3 combination. On the mixed species receptor, CGRP8–37 showed the opposite selectivity. Thus, depending on the species, it is possible to discriminate pharmacologically between CL/RAMP2 and CL/RAMP3 AM receptors.

A comparison of the actions of BIBN4096BS and CGRP8–37 on CGRP and adrenomedullin receptors expressed on SK-N-MC, L6, Col 29 and Rat 2 cells

The ability of the CGRP antagonist BIBN4096BS to antagonize CGRP and adrenomedullin has been investigated on cell lines endogenously expressing receptors of known composition. On human SK‐N‐MC cells (expressing human calcitonin receptor‐like receptor (CRLR) and receptor activity modifying protein 1 (RAMP1)), BIBN4096BS had a pA2 of 9.95 although the slope of the Schild plot (1.37±0.16) was significantly greater than 1. On rat L6 cells (expressing rat CRLR and RAMP1), BIBN4096BS had a pA2 of 9.25 and a Schild slope of 0.89±0.05, significantly less than 1. On human Colony (Col) 29 cells, CGRP8–37 had a significantly lower pA2 than on SK‐N‐MC cells (7.34±0.19 (n=7) compared to 8.35±0.18, (n=6)). BIBN4096BS had a pA2 of 9.98 and a Schild plot slope of 0.86±0.19 that was not significantly different from 1. At concentrations in excess of 3 nM, it was less potent on Col 29 cells than on SK‐N‐MC cells. On Rat 2 cells, expressing rat CRLR and RAMP2, BIBN4096BS was unable to antagonize adrenomedullin at concentrations up to 10 μM. CGRP8–37 had a pA2 of 6.72 against adrenomedullin. BIBN4096BS shows selectivity for the human CRLR/RAMP1 combination compared to the rat counterpart. It can discriminate between the CRLR/RAMP1 receptor expressed on SK‐N‐MC cells and the CGRP‐responsive receptor expressed by the Col 29 cells used in this study. Its slow kinetics may explain its apparent ‘non‐competive’ behaviour. At concentrations of up to 10 μM, it has no antagonist actions at the adrenomedullin, CRLR/RAMP2 receptor, unlike CGRP8–37.

Binding properties of the novel, non-peptide CGRP receptor antagonist radioligand, [3H]BIBN4096BS

BIBN4096BS [[R-(R,(R*,S*)]-N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl] pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl)-,1-Piperidinecarboxamide] is a selective calcitonin gene-related peptide (CGRP) receptor antagonist with a picomolar affinity to the CGRP receptor in human neuroblastoma SK-N-MC cells. Here, we describe the characterisation of the binding properties of the tritiated radioanalogue of BIBN4096BS in SK-N-MC cells as well as in marmoset tissue. [(3)H]BIBN4096BS showed reversible and saturable binding to SK-N-MC cells with a K(D) of 0.045 nM. In competition experiments, [3(H)]BIBN4096BS is concentration-dependently displaced from SK-N-MC cell membranes by BIBN4096BS as well as by the endogenous ligand CGRP and its analogues with the rank order of affinity BIBN4096BS>human alpha-CGRP=human beta-CGRP>[Cys(Et)(2,7)]human alpha-CGRP>adrenomedullin (high affinity site)=human alpha-CGRP-(8-37)=human beta-CGRP-(8-37)>calcitonin=amylin. In the marmoset cortex, saturable [(3)H]BIBN4096BS binding was observed with a K(D) of 0.077 nM. CGRP showed biphasic competition of [(3)H]BIBN4096BS binding, whilst BIBN4096BS monophasically displaced its radioanalogue with a K(i) of 0.099 nM. These data, using [(3)H]BIBN4096BS, confirm the high affinity of this novel antagonist for the primate CGRP receptor and demonstrate furthermore that this radioligand is a useful tool to study CGRP receptor pharmacology.

Novel 6,7,8,9-tetrahydro-5H-1,4,7,10a-tetraaza-cyclohepta[f]indene analogues as potent and selective 5-HT2C agonists for the treatment of metabolic disorders

The discovery of a novel series of 5-HT(2C) agonists based on a tricyclic pyrazolopyrimidine scaffold is described. Compounds with good levels of in vitro potency and moderate to good levels of selectivity with respect to the 5-HT(2A) and 5-HT(2B) receptors were identified. One of the analogues (7 g) was found to be efficacious in a sub-chronic weight loss model. A key limitation of the series of compounds was that they were found to be potent inhibitors of the hERG ion channel. Some compounds, bearing polar side chains were identified which showed a much reduced hERG liability however these compounds were sub-optimal in terms of their in vitro potency or selectivity.

Characterization of the NPGP receptor and identification of a novel short mRNA isoform in human hypothalamus

Recently, an orphan G protein coupled receptor (GPCR) termed NPGPR was described. A shorter variant of this receptor lacking exon 1 was shown to have subnanomolar affinity for neuropeptide FF (NPFF), a pain modulatory peptide, and therefore was named NPFF(2) receptor. Here, we characterize the full-length cloned NPGPR and identify a novel short form lacking exon 2 with a differential pattern of mRNA abundance in several tissues and organs. The NPGPR is most similar to the recently cloned neuropeptide FF (NPFF) receptor which lacks exon 1, but also shows high homology to the orexin and neuropeptide Y (NPY) receptor families, two neuropeptides involved in food intake regulation. Therefore, we used binding studies to examine the interaction of NPFF, orexin and NPY with the NPGPR. [125I] NPFF was displaced by NPFF with an IC(50) of 14.7 +/- 8.8 nM, whereas [125I] Orexin B was displaced by Orexin B with an IC(50) of 415 +/- 195 nM. We conclude that orexins interact with the NPGPR and that the affinity of NPFF for NPGPR is approximately 100-fold lower than for the NPFF2 receptor. We postulate that NPGPR is a splice variant of the family of NPFF receptors and displays a binding profile different from the other members of the NPFF receptor family due to the presence of exon 1. In order to evaluate whether NPGPR levels are affected by the feeding status, we examined the mRNA level using real-time PCR in two feeding models, i.e. before and after diet-induced body weight increase as well as after chronic food restriction in rats. However, hypothalamic NPGPR mRNA was unchanged in both models. Therefore, our evidence does not support the hypothesis that NPGPR is involved in feeding regulation.

Design, synthesis and evaluation of MCH receptor 1 antagonists—Part II: Optimization of pyridazines toward reduced phospholipidosis and hERG inhibition

Despite recent success there remains a high therapeutic need for the development of drugs targeting diseases associated with the metabolic syndrome. As part of our search for safe and effective MCH-R1 antagonists for the treatment of obesity, a series of 3,6-disubstituted pyridazines was evaluated. During optimization several issues of the initial lead structures had to be resolved, such as selectivity over related GPCRs, inhibition of the hERG channel as well as the potential to induce phospholipidosis. Utilizing property-based design, we could demonstrate that all parameters can significantly be improved by consequently increasing the polarity of the compounds. By this strategy, we succeeded in identifying potent and orally available MCH-R1 antagonists with good selectivity over M1 and 5-HT2A and an improved safety profile with respect to hERG inhibition and phospholipidosis.

Design, synthesis and evaluation of MCH receptor 1 antagonists—Part I: Optimization of HTS hits towards an in vivo efficacious tool compound BI 414

Despite recent approvals of anti-obesity drugs there is still a high therapeutic need for alternative options with higher efficacy in humans. As part of our MCH-R1 antagonist program for the treatment of obesity, a series of biphenylacetamide HTS hits was evaluated. Several issues of the initial lead structures had to be resolved, such as potency, selectivity over related GPCRs and P-gp efflux limiting brain exposure in this series. We could demonstrate that all parameters can be significantly improved by structural modifications resulting in BI 414 as a potent and orally available MCH-R1 antagonist tool compound with acceptable in vivo efficacy in an animal model of obesity.

Functional Characterisation of the CGRP Antagonist BIBN4096BS, in SK-N-MC Cells

The 37-amino acid vasodilatory peptide calcitonin gene-related peptide (CGRP) has been implicated in the pathophysiology of migraine. We have recently developed a nonpeptide CGRP antagonist, BIBN4096BS, with subnanomolar affinity for primate CGRP-1 receptors. BIBN4096BS proved to be a valuable tool to dissect CGRP receptor pharmacology[1]. Interestingly, in functional assays in some tissue, the pKB values for BIBN4096BS differ depending on the agonist used, αor β-CGRP[2]. We have therefore examined the potency of BIBN4096BS to block cyclic AMP accumulation caused by αor β-CGRP, in SK-N-MC human neuroblastoma cells. We have also used a novel radioligand, I-Tyr-β-CGRP to determine the binding affinity of BIBN4096BS to probe whether a different β-CGRP receptor binding site exists. I-Tyr-β-CGRP was custom synthesised by Biotrend. SK-N-MC were cultured as described[3]. Agonists (10 to 10 M) were given simultaneously with BIBN4096 BS (10 to 10 14 M), then incubated for 30 min at room temperature. Cyclic AMP was measured using Flashplates (NEN). Schild Plot analysis was carried out to determine pKB values (Graph Pad Prism). Radioligand binding on SK-N-MC cell membranes was carried out as described[3]. All experiments were carried out with n ≥ 3, in triplicate. BIBN4096BS blocked the cAMP stimulation evoked by α-CGRP with very high potency (pKB 13.4). BIBN4096BS was equally effective in blocking β-CGRP effects (pKB 13.7). The slopes for both Schild plots were not different from unity. In binding experiments, BIBN4096BS displaced I-Tyr-β-CGRP with an Ki value of 11.7, which is not significantly different to that previously determined using I-h -α-CGRP. BIBN4096BS is a highly potent full CGRP antagonist on SK-N-MC cells. The equipotency of BIBN4096BS versus α-or β-CGRP suggest that there are no differential binding sites present in this cell line.