The discovery of biaryl carboxamides as novel small molecule agonists of the motilin receptor

Optimisation of urea (5), identified from high throughput screening and subsequent array chemistry, has resulted in the identification of pyridine carboxamide (33) which is a potent motilin receptor agonist possessing favourable physicochemical and ADME profiles. Compound (33) has demonstrated prokinetic-like activity both in vitro and in vivo in the rabbit and therefore represents a promising novel small molecule motilin receptor agonist for further evaluation as a gastroprokinetic agent.     

9-Dihydroerythromycins as non-antibiotic motilin receptor agonists

A series of 9-dihydroerythromycin A and B analogues with modification of the desosamine nitrogen have been synthesized and screened for motilin agonist activity, antibiotic activity, tachyphylaxis and hERG channel current inhibition. Small alkyl groups resulted in the potency while compounds with a primary or secondary amine resulted in the low motilin agonist potency. Several compounds were identified as non-antibiotic motilin receptor agonists with minimal tachyphylaxis and low hERG interaction.     

Design and synthesis of novel tetra-peptide motilin agonists

A series of novel tetra-peptide motilin agonists, having the general structure H-Phe-Val-X-Ile-NH(2), were designed, on the basis of structure-activity relationship studies of motilin. Peptides, in which X is a side chain substituted tryptophan residue, have agonistic activity. H-Phe-Val-Trp(2'-CH(2)CH(2)OH)-Ile-NH(2)(7), H-Phe-Val-Trp(2'-SCH(3))-Ile-NH(2)(8), and H-Phe-Val-Trp(2'-SCH(2)CH(2)CH(3))-Ile-NH(2)(9), showed an EC(50) for contractile activity in the rabbit smooth muscle of 14.1+/-3.2, 12.9+/-4.1, and 4.6+/-1.6 microM, respectively. Interaction of the tryptophan aliphatic side chain with motilin receptor appears to influence the signal transduction via motilin receptor.     

Mutational analysis of predicted intracellular loop domains of human motilin receptor

Motilin is an important endogenous regulator of gastrointestinal motor function, mediated by the class I G protein-coupled motilin receptor. Motilin and erythromycin, two chemically distinct full agonists of the motilin receptor, are known to bind to distinct regions of this receptor, based on previous systematic mutagenesis of extracellular regions that dissociated the effects on these two agents. In the present work, we examined the predicted intracellular loop regions of this receptor for effects on motilin- and erythromycin-stimulated activity. We prepared motilin receptor constructs that included sequential deletions throughout the predicted first, second, and third intracellular loops, as well as replacing the residues in key regions with alanine, phenylalanine, or histidine. Each construct was transiently expressed in COS cells and characterized for motilin- and erythromycin-stimulated intracellular calcium responses and for motilin binding. Deletions of receptor residues 63-66, 135-137, and 296-301 each resulted in substantial loss of intracellular calcium responses to stimulation by both motilin and erythromycin. Constructs with mutations of residues Tyr66, Arg136, and Val299 were responsible for the negative impact on biological activity stimulated by both agonists. These data suggest that action by different chemical classes of agonists that are known to interact with distinct regions of the motilin receptor likely yield a common activation state of the cytosolic face of this receptor that is responsible for interaction with its G protein. The identification of functionally important residues in the predicted cytosolic face provides strong candidates for playing roles in receptor-G protein interaction.     

The motilin pharmacophore in CHO cells expressing the human motilin receptor

We performed a structure-activity study with the human motilin receptor, which was recently cloned from thyroid tissue. N-terminal fragments, Ala-analogs of motilin, and motilides were tested in a cell line that expresses the cloned human motilin receptor and apoaequorin. Full potency to induce calcium fluxes was obtained with N-terminal fragments of 14 amino acids. Motilin fragments 1-14 in which residues 1 (Phe), 4 (Ile), and 7 (Tyr) were replaced by Ala showed the largest reduction in potency. Only motilides with an enol configuration had markedly higher potencies compared to erythromycin A. The potencies to induce Ca(2+) fluxes correlated strongly with rabbit binding and contractility data, suggesting that the cloned receptor is indeed the motilin receptor, responsible for contractile effects. Conservation of the motilin pharmacophore in evolution indicates an important physiological role of motilin.     

Synthesis and SAR of 1,3-disubstituted cyclohexylmethyl urea and amide derivatives as non-peptidic motilin receptor antagonists

A series of 1,3-disubstituted cyclohexylmethyl urea and amide derivatives were synthesized as motilin receptor antagonists. Starting from known motilin antagonists, 1a and 1b, the cyclopentene scaffold was replaced and the four recognition elements optimized to arrive at a potent novel series.     

Identification of peptide ligand-binding domains within the human motilin receptor using photoaffinity labeling.

The cDNA encoding the human motilin receptor was recently cloned and found to represent a G protein-coupled receptor that is structurally related to the growth hormone secretagogue receptors. Together, these represent a new Class I receptor family. Our aim in the present work is to gain insight into the molecular basis of binding of motilin to its receptor using photoaffinity labeling. To achieve this, we developed a Chinese hamster ovary cell line that overexpressed functional motilin receptor (CHO-MtlR; 175,000 sites per cell, with K(i) = 2.3 +/- 0.4 nm motilin and EC(50) = 0.3 +/- 0.1 nm motilin) and a radioiodinatable peptide analogue of human motilin that incorporated a photolabile p-benzoyl-l-phenylalanine (Bpa) residue into its pharmacophoric domain. This probe, [Bpa(1),Ile(13)]motilin, was a full agonist at the motilin receptor that increased intracellular calcium in a concentration-dependent manner (EC(50) = 1.5 +/- 0.4 nm). This photolabile ligand bound specifically and with high affinity to the motilin receptor (K(i) = 12.4 +/- 1.0 nm), and covalently labeled that molecule within its M(r) = 45,000 deglycosylated core. Cyanogen bromide cleavage demonstrated its covalent attachment to fragments of the receptor having apparent M(r) = 6,000 and M(r) = 31,000. These were demonstrated to represent fragments that included both the first and the large second extracellular loop domains, with the latter representing a unique structural feature of this receptor. The spatial approximation of the pharmacophoric domain of motilin with these receptor domains support their functional importance as well.     

The discovery and optimisation of benzazepine sulfonamide and sulfones as potent agonists of the motilin receptor

Optimisation of a series of benzazepine sulfonamide hit compounds identified from high throughput screening led to the discovery of a new series of tractable, potent motilin receptor agonists.     

The erythromycin derivative EM-523 is a potent motilin agonist in man and in rabbit

Erythromycin may stimulate gastrointestinal motor activity via its effect upon motilin receptors. We have studied the ability of the derivative EM-523 [de(N-methyl)-N-ethyl-8,9-anhydroerythromycin A 6,9-hemiacetal] to induce contractions in duodenal smooth muscle strips and to displace labeled motilin bound to antral smooth muscle, in man and in rabbit. In both species EM-523 approached the potency of motilin for inducing contractions. Thus pED50 values were 7.84 +/- 0.11 and 8.69 +/- 0.12 for motilin in, respectively, man and rabbit, against 6.08 +/- 0.13 and 8.19 +/- 0.10 for EM-523. In rabbit the efficacy of both compounds decreased in parallel aborally, the responses to EM-523 could not be blocked by atropine (10(-7) M) or TTX (10(-7) M), and both compounds were unable to further enhance the maximum effect to the other compound. In binding studies the order of potency was the same as in the contraction studies. The pIC50 values were: motilin (8.84 +/- 0.31, 9.17 +/- 0.20) greater than EM-523 (7.89 +/- 0.1, 8.40 +/- 0.10). A Schild plot revealed that EM-523 was a competitive inhibitor of motilin receptor binding in man and in rabbit. We conclude that EM-523 is a potent motilin agonist.     

Anxiolytic Actions of Motilin in the Basolateral Amygdala

Motilin is a 22-amino-acid gastrointestinal polypeptide that was first isolated from the porcine intestine. We identified that motilin receptor is highly expressed in GABAergic interneurons in the basolateral nucleus (BLA) of the amygdala, the structure of which is closely involved in assigning stress disorder and anxiety. However, little is known about the role of motilin in BLA neuronal circuits and the molecular mechanisms of stress-related anxiety. Whole-cell recordings from amygdala slices showed that motilin depolarized the interneurons and facilitated GABAergic transmission in the BLA, which is mimicked by the motilin receptor agonist, erythromycin. BLA local injection of erythromycin or motilin can reduce the anxiety-like behavior in mice after acute stress. Therefore, motilin is essential in regulating interneuron excitability and GABAergic transmission in BLA. Moreover, the anxiolytic actions of motilin can partly be explained by modulating the BLA neuronal circuits. The present data demonstrate the importance of motilin in anxiety and the development of motilin receptor non-peptide agonist as a clear target for the potential treatment of anxiety disorders.     

A new strategy for metabolic stabilization of motilin using the C-terminal part of ghrelin

Ghrelin consists of 28 amino acid residues with an octanoyl modification at the third serine residue. Recently we have found that the C-terminal part of ghrelin protects the ester bond of 3-octanoyled serine from plasma esterases and plays the essential role to prolong the plasma half-life and to show its biological activity in vivo. In the present study, we researched whether the C-terminal part of ghrelin has a potential to prolong the plasma half-life of motilin, by comparing the pharmacokinetics of various chimeric peptides of ghrelin and motilin. Motilin is another gastro-intestinal peptide hormone related with ghrelin structurally, binding to the same family of G protein-coupled receptors. Chimeric peptides were designed to be composed of motilin(1-12) fragment, the active core binding to the motilin receptor, GPR38, and C-terminal part of ghrelin. The modification of motilin(1-12) fragment by C-terminal part of ghrelin hardly influenced its agonist activity to GPR38 and almost all these chimeric peptides showed more than two times longer plasma half-lives than motilin in rats. From the relationship between structures of chimeric peptides and their corresponding plasma half-lives, the mid-region of ghrelin rich in basic amino acids ((15)RKESKK(20)) was considered to be the most important in prolonging the plasma half-life of motilin. The deletion of these fragments or replacement of 17th glutamic acid with a neutral amino acid resulted in short plasma half-lives. In conclusion, our data suggested that the C-terminal part of ghrelin has a potential to improve the biokinetics of motilin probably by a metabolic stabilizing effect.     

Stable Expression of a Synthetic Gene for the Human Motilin Receptor: Use in an Aequorin-Based Receptor Activation Assay

A synthetic gene for the human motilin receptor containing 33 unique restriction sites was designed and stably coexpressed in HEK293 cells with the bioluminescent Ca(2+) indicator protein aequorin. The dose-dependent response of the receptor to motilin was demonstrated using transient transfections, and a stable cell line was selected. [(125)I]Motilin binding was used to estimate receptor expression level for the stable cell line, and titration of a membrane preparation indicated a K(d) value of 0.8 nM. The same cell line was used to evaluate a panel of erythromycin-derived agonists and provided EC(50) values for receptor activation that agree closely with data obtained in contractility assays. The peptide antagonist ANQ11125 (Phe3Leu13 motilin 1-14) inhibited motilin induced response with a K(i) value of 10 nM. The system is well-suited for the screening of compound libraries and receptor mutagenesis studies.     

Discovery of 4-(dimethylamino)quinazolines as potent and selective antagonists for the melanin-concentrating hormone receptor 1

A series of 4-(dimethylamino)quinazoline based antagonists of the melanin-concentrating hormone receptor 1 (MCH-R1) is described. This series was derived from a lead compound, AR129330, identified by HTS of a GPCR-directed library using a functional assay with a constitutively activated (CART) form of the receptor. The preliminary optimization resulted in the identification of compounds 20, 21, and 23.     

Effects of intrathecal opioids combined with low-dose naloxone on motilin and its receptor in a rat model of postoperative pain

Aims

To investigate the effects of intrathecal morphine and fentanyl combined with low-dose naloxone on the expression of motilin and its receptor in a rat model of postoperative pain.

Main methods

An intrathecal catheter was implanted, and saline, opioids (morphine and fentanyl) and naloxone were intrathecally administered 7 days later. An incisional pain model was established to induce pain behaviors in rats by unilateral plantar incision. Thermal hyperalgesia and mechanical allodynia were measured by using a radiant heat and electronic Von Frey filament, respectively. The expression of motilin in the hippocampus, stomach, duodenum, and plasma was determined by ELISA; and the expression of motilin receptor in the hippocampus was detected by Western blot assay.

Key findings

Motilin and its receptor were detected in the hippocampus. Acute incisional pain increased the motilin expression in the hippocampus and duodenum, while decreasing its expression in the gastric body and plasma. Postoperative analgesia with morphine + fentanyl upregulated the expression of motilin in the hippocampus; however, motilin was downregulated in peripheral sites. Naloxone at 1 ng/kg restored motilin to baseline levels. Acute pain, morphine + fentanyl, and naloxone all induced the expression of motilin receptor in the hippocampus.

Significance

Acute pain, postoperative analgesia with opioids, and naloxone significantly impacted the expression of hippocampal and peripheral motilin. Variation trends in all sites were not identical. Intrathecal injection of low-dose naloxone upregulated paw withdrawal thermal latency and enhanced the analgesic effects of opioids. The findings presented here provide a new basis for central and peripheral regulations in GI motility, clinical postoperative analgesia, and management of analgesic complications.     

Molecular identification and characterization of the dog motilin receptor

Motilin, a 22-amino acid peptide hormone secreted by endocrine cells of the intestinal mucosa, plays an important role in the regulation of gastrointestinal motility. The actions of motilin agonists have been extensively investigated in dogs due to physiological similarities between the dog and human alimentary tracts. The amino acid sequence of the dog motilin receptor, however, was previously unknown. We have cloned a cDNA from dog stomach corresponding to the motilin receptor. The deduced protein shared 71% and 72% sequence identity with the human and rabbit motilin receptors, respectively. Expression of the dog motilin receptor in CHO cells promoted the typical cellular responses to the agonists, motilin and erythromycin. The rank order of potency determined for these agonists was similar to that found for the human motilin receptor, with motilin being more potent than erythromycin. Immunohistochemistry of the dog stomach revealed that the motilin receptor was localized in neuronal cell bodies and fibers. This is the first study detailing the cloning, expression, and functional characterization of the dog motilin receptor. Determination of the full sequence and functional properties of the dog motilin receptor will provide useful information enabling us to interpret previous and future studies of motilin agonists in dogs.     

Motilin Stimulates Gastric Acid Secretion in Coordination with Ghrelin in Suncus murinus

Motilin and ghrelin constitute a peptide family, and these hormones are important for the regulation of gastrointestinal motility. In this study, we examined the effect of motilin and ghrelin on gastric acid secretion in anesthetized suncus (house musk shrew, Suncus murinus), a ghrelin- and motilin-producing mammal. We first established a gastric lumen-perfusion system in the suncus and confirmed that intravenous (i.v.) administration of histamine (1 mg/kg body weight) stimulated acid secretion. Motilin (0.1, 1.0, and 10 μg/kg BW) stimulated the acid output in a dose-dependent manner in suncus, whereas ghrelin (0.1, 1.0, and 10 μg/kg BW) alone did not induce acid output. Furthermore, in comparison with the vehicle administration, the co-administration of low-dose (1 μg/kg BW) motilin and ghrelin significantly stimulated gastric acid secretion, whereas either motilin (1 μg/kg BW) or ghrelin (1 μg/kg BW) alone did not significantly induce gastric acid secretion. This indicates an additive role of ghrelin in motilin-induced gastric acid secretion. We then investigated the pathways of motilin/motilin and ghrelin-stimulated acid secretion using receptor antagonists. Treatment with YM 022 (a CCK-B receptor antagonist) and atropine (a muscarinic acetylcholine receptor antagonist) had no effect on motilin or motilin-ghrelin co-administration-induced acid output. In contrast, famotidine (a histamine H₂ receptor antagonist) completely inhibited motilin-stimulated acid secretion and co-administration of motilin and ghrelin induced gastric acid output. This is the first report demonstrating that motilin stimulates gastric secretion in mammals. Our results also suggest that motilin and co-administration of motilin and ghrelin stimulate gastric acid secretion via the histamine-mediated pathway in suncus.     

Hypotensive mechanism of [Leu13]motilin in dogs in vivo and in vitro.

The effects of [Leu13]motilin were examined in vivo after its intravenous administration into anesthetized dogs and in vitro with isolated preparations of canine mesenteric artery. [Leu13]Motilin (0.1-10 nmol x kg(-1), i.v.) induced both strong and clustered phasic contractions in the gastric antrum and duodenum. At doses of over 1 nmol x kg(-1), [Leu13]motilin also produced transient decreases in arterial blood pressure, left ventricular pressure, maximum rate of rise of left ventricular pressure, and total peripheral resistance, and an increase in aortic blood flow and heart rate. A selective motilin antagonist, GM-109 (Phe-cyclo[Lys-Tyr(3-tBu)-betaAla] trifluoroacetate), completely abolished the gastric antrum and duodenal motor responses induced by [Leu13]motilin. In contrast, hypotension induced by [Leu13]motilin (1 nmol x kg(-1)) was unchanged in the presence of GM-109. In isolated mesenteric artery preparations precontracted with U-46619 (10(-7) M), [Leu13]motilin (10(-8)-10(-5) M) induced an endothelium-dependent relaxation, and this was inhibited by a pretreatment with N(omega)-nitro-L-arginine, a competitive inhibitor of NO synthase (10(-4) M). A high dose (10(-4) M) of GM-109 slightly decreased [Leu13]motilin-induced relaxation, and shifted the concentration-response curve of [Leu13]motilin to the right. However, the pA2 value (4.09) of GM-109 for [Leu13]motilin in the present study was conspicuously lower than that previously demonstrated in the rabbit duodenum (7.37). These results suggest that [Leu13]motilin induces hypotension via the endothelial NO-dependent relaxation mechanism and not through the receptor type that causes upper gastrointestinal contractions.     

Motilin receptors in the human antrum

Motilin is an intestinal peptide that stimulates contraction of gut smooth muscle. The motilin receptor has not been cloned yet, but motilin-receptor agonists appear to be potent prokinetic agents for the treatment of dysmotility disorders. The aim of this study was to determine neural or muscular localization of motilin receptors in human upper gastrointestinal tract and to investigate their pharmacological characteristics. The binding of (125)I-labeled motilin to tissue membranes prepared from human stomach and duodenum was studied; rabbit tissues were used for comparison. Solutions enriched in neural synaptosomes or in smooth muscle plasma membranes were obtained. Various motilin analogs were used to displace the motilin radioligand from the various tissue membranes. The highest concentration of human motilin receptors was found in the antrum, predominantly in the neural preparation. Human motilin receptors were sensitive to the NH(2)-terminal portion of the motilin molecule, but comparison with rabbit showed that both species had specific affinities for various motilin analogs [i.e., Mot-(1-9), Mot-(1-12), Mot-(1-12) (CH(2)NH)(10-11), and erythromycin]. Motilin receptors obtained from synaptosomes or muscular plasma membranes of human antrum expressed different affinity for two motilin-receptor agonists, Mot-(1-12) and Mot-(1-12) (CH(2)NH)(10-11), suggesting that they correspond to specific receptor subtypes. We conclude that human motilin receptors are located predominantly in nerves of the antral wall, are functionally (and probably structurally) different from those found in other species such as the rabbit, and express specific functional (and probably structural) characteristics dependent on their localization on antral nerves or muscles, suggesting the existence of specific receptor subtypes, potentially of significant physiological or pharmacological relevance.     

A relationship between motilin and growth hormone secretagogue receptors

The motilin receptor (MR) belongs to a family of Class I G protein-coupled receptors that also includes growth hormone secretagogue receptor (GHSR). Their potentially unique structure and the molecular basis of their binding and activation are not yet clear. We previously reported that the perimembranous residues in the predicted extracellular loops and amino-terminal tail of the MR were important for responses to the natural peptide ligand, motilin, and the transmembrane domains of the MR were important for a non-peptidyl ligand, erythromycin. We also reported that the perimembranous residues in the second extracellular loop of the GHSR were critical for natural ligand ghrelin binding and activity. The MR is 52% identical to GHSR, with 86% sequence identity in the transmembrane domains. In the current work, to gain insight into a relationship between MR and GHSR, we studied functional responses to motilin, erythromycin and ghrelin of expression cells of chimeric constructs of MR and GHSR and co-expression cells of both MR and GHSR. We also generated human MR transgenic mice, and clarified a relationship between motilin and ghrelin. MR(1-62)/GHSR(68-366) construct responded only to ghrelin, MR(1-102)/GHSR(108-366) responded to ghrelin and erythromycin, and MR(1-129)/GHSR(135-366) and MR(1-178)/GHSR(184-366) responded to erythromycin, while GHSR(1-183)/MR(179-412) responded to neither motilin, erythromycin nor ghrelin. MR and GHSR co-expression cells have no additional responses to these ligands. Motilin or erythromycin administration to human MR transgenic mice resulted in a decrease of serum acyl-ghrelin levels, while MR and GHSR mRNA expression in the gastrointestinal tracts were not changed. These data suggested that in species expressing both motilin-MR and ghrelin-GHSR, there is a compensatory relationship in vivo.