MOTILIN (HUMAN, PORCINE)

The human MLN gene is mapped to 6p21.31. It is a single copy gene composed of five exons, spanning approximately 9 kb. The 22-aa MLN peptide gene has a structure similar to that of ghrelin, and is encoded by exon 3 and part of exon 2 . The aa sequences of preproMLN from the pig, rabbit, human, sheep, and monkey have been deduced from the cDNA sequence. The C-terminal MAP is largely encoded by parts of exons 3 and 4, with the last two aa of the MLN precursor and the 30 untranslated region encoded by exon 5. MLN is identified throughout the gastrointestinal (GI) tract of numerous species, and is found predominantly not only in the endocrine M cells of the duodenal mucosa but also in the myenteric plexus, where it is colocalized with neurons immunoreactive for neuronal nitric oxide synthase. MLN-producing cells decrease distally in the small intestine. MLN is also present in the thyroid and the brain, where the highest concentration is found in the hypothalamus.

MLN is released in circulation at approximately 100- min intervals in the interdigestive state, and the ingestion of food during this period prevents the secretion of MLN. The cholinergic pathway is an important regulator of the release of MLN. Muscarinic3 (M3) receptors have been found responsible for MLN release from canine MLN cells in the perifusion system. Exogenous MLN treatment stimulates endogenous MLN release through the muscarinic receptors on MLN-producing cells via preganglionic pathways involving 5-hydroxytryptamine 3 receptors.

Feighner and colleagues first identified the orphan GPCR, GPR38, as the human MLN receptor (MTLR, MLNR), for which two alternatively spliced forms exist. An mRNA, GPR38-A (splice variant 1a), encodes a 412-aa protein with seven predicted α-helical transmembrane domains, the hallmark feature of GPCRs, and is an active form of the receptor, whereas GPR38-B (variant 1b) mRNA encodes a 386-aa protein with five predicted transmembrane domains. The signal transduction pathway of MLNR is unknown. However, the molecular and cellular mechanisms involved in MLN-induced MLNR desensitization have been observed. After MLN stimulation, the MLNR becomes phosphorylated, probably via GPCR kinases. This leads to the recruitment of β-arrestin-2, which targets the receptor to clathrin-coated pits. Upon internalization, the β-arrestin dissociates from the receptor, and the MLN receptor complex is subsequently sorted to the recycling endosomes that transport the MLNR back to the plasma membrane.

Erythromycin is extensively used as an effective agent to accelerate the gastric emptying of food in patients with diabetic gastroparesis through MLNR. Several pharmaceutical companies have generated MLN-like macrolides (motilides) that are erythromycin derivatives devoid of antibiotic activity but with strong affinity to MLNR. However, the first drugs, EM-523 and its successor EM-574, failed because of their chemical instability and low bioavailability. Another compound, ABT-229, was also unsuccessful for treating functional dyspepsia and diabetic gastroparesis, possibly because of its strong desensitizing properties. To overcome these limitations, the second-generation compounds were developed. Based on the N-terminal region of MLN and its biological activity, a novel synthetic human MLN analog, atilmotin, which lacks the C-terminal end, was developed to accelerate gastric emptying in healthy subjects. However, its effect was poor and only detected during the first 30min. An acid-resistant nonpeptidyl MLN agonist, mitemcinal, was developed that is orally active and could be beneficial for the treatment of delayed gastric emptying and transit. However, symptom relief occurred only in a subset of patients. Two MLNR agonists, RQ-00201894 and camicinal (GSK962040), have been developed. RQ-00201894, a novel nonmacrolide MLN agonist, demonstrates agonistic activity similar to that of MLN in human MLNR expressed in CHO cells, and facilitates cholinergically mediated human antral muscle contractions evoked by electrical field stimulation. However, its structure remains unknown. Camicinal, in contrast, is derived from a benzylpiperazine molecular structure that selectively activates the recombinant human MLNR, induces contractions in human and rabbit isolated stomach preparations, and increases the fecal output in conscious rabbits.

The macrocyclic peptidomimetic TZP-201 is a potent semisynthetic nonpeptidyl MLN antagonist that has been 326 30B. Motilin I-3. Gastrointestinal hormones used for the treatment of various forms of moderate to severe diarrhea associated with irritable bowel syndrome, cancer, and infectious diseases. Preclinical data show that TZP-201 is efficacious in a dog model of chemotherapy-induced diarrhea. Similarly, an orally active MLNR antagonist, MA-2029, inhibits MLN induced GI motility without affecting the basal GI tone or gastric emptying rate.

MLNR is mainly found in the GI tract, but the exact localization is species-dependent. In humans, through binding experiments with iodinated porcine [Leu13] MLN, the MTLR density was found to be the highest in the gastroduodenal region, and decreased distally in the small intestine toward the colon. MLNR immunoreactivity is present in muscle cells and the myenteric plexus, but not in mucosal or submucosal cells, in humans. In rabbits, the highest MLNR density is found in the colon. MLNR has been found outside the GI tract in the hypothalamus, nodose ganglion, thyroid, and bone marrow. Mlnr gene expression has been found in the lung and heart in Suncus murinus, suggesting that MLN could have an unknown function in the respiratory and cardiovascular systems.The main biological functions are to increase lower esophageal sphincter (LES) pressure and to induce the interdigestive motor complex (IMC) for removing debris and cleaning the GI tract. Recent studies in humans show that MLN-induced gastric contractions stimulate hunger.

Plasma MLN concentrations increase significantly in patients with diabetic gastroparesis who maintain a normal migrating motor complex (MMC), even without antral phase-III activity. Similarly, a higher plasma MLN concentration is reported in hypergastrinemic chronic atrophic gastritis and chronic renal failure, whereas decreased MLN release has been observed in patients with functional bowel disorders such as chronic idiopathic constipation or idiopathic megacolon. Abnormal fluctuations of MLN also occur with severe pancreatic insufficiency. Fasting and postprandial levels of MLN are significantly raised in patients with infectious diarrhea. Hypermotilinemia is often associated with Crohn’s disease, ulcerative colitis, and tropical malabsorption.

Motilin was isolated from the duodenojejunal mucosa and found to control the motor activity of the digestive tract. It is a potential therapeutic drug target for improving digestive dysmotility. Motilin was isolated from a side fraction produced during the purification of secretin in 1971 and was found to stimulate contractility in the fundus of the stomach. Complete porcine and human motilin were purified and sequenced in 1973 and 1983 respectively.

Motilin is a 22-residue polypeptide isolated from the duodenum.Its secretion is stimulated by the presence of acid inthe duodenum. Motilin inhibits gastric motor activity anddelays gastric emptying.

To date, almost all macrolides have lacked effectiveness as MLN agonists. However, pharmacies have been trying to develop new types of macrolides, such as PF-04548043 (formerly known as KOS-2187), for the treatment of GI motility disorders such as gastroparesis and gastroesophageal reflux disease. Moreover, the new MLNR agonist RQ-00201894 is a promising drug for the treatment of gastroparesis, postoperative ileus, and functional dyspepsia.

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Motilin is highly conserved across species, and is synthesized as a part of a larger inactive prohormone. Structure-activity studies with analogs and fragments of porcine motilin have shown an N-terminal region, which is considered a physiological and biological active site, and a C-terminal α-helical domain. Human motilin is synthesized as a preprohormone composed of 133 aa residues, each consisting of a 25-aa signal peptide followed by a 22-aa (mature motilin) and a C-terminal motilin-associated peptide (MAP). In the N-terminal region, identical aa sequences exist in human and porcine motilin, but differ from canine motilin at positions 7, 8, 12, 13, and 14 . Chicken MLN also differs from human and porcine sequences by six residues at positions 4, 7–10, and 12, and the binding affinity and pharmacological potency against the chicken MLNR differ from those for mammalian MLN.