Class B1 GPCR activation by an intracellular agonist.

G protein-coupled receptors (GPCRs) generally accommodate specific ligands in the orthosteric-binding pockets. Ligand binding triggers a receptor allosteric conformational change that leads to the activation of intracellular transducers, G proteins and ß-arrestins. Because these signals often induce adverse effects, the selective activation mechanism for each transducer must be elucidated. Thus, many orthosteric-biased agonists have been developed, and intracellular-biased agonists have recently attracted broad interest. These agonists bind within the receptor intracellular cavity and preferentially tune the specific signalling pathway over other signalling pathways, without allosteric rearrangement of the receptor from the extracellular side1-3. However, only antagonist-bound structures are currently available1,4-6, and there is no evidence to support that biased agonist binding occurs within the intracellular cavity. This limits the comprehension of intracellular-biased agonism and potential drug development. Here we report the cryogenic electron microscopy structure of a complex of Gs and the human parathyroid hormone type 1 receptor (PTH1R) bound to a PTH1R agonist, PCO371. PCO371 binds within an intracellular pocket of PTH1R and directly interacts with Gs. The PCO371-binding mode rearranges the intracellular region towards the active conformation without extracellularly induced allosteric signal propagation. PCO371 stabilizes the significantly outward-bent conformation of transmembrane helix 6, which facilitates binding to G proteins rather than ß-arrestins. Furthermore, PCO371 binds within the highly conserved intracellular pocket, activating 7 out of the 15 class B1 GPCRs. Our study identifies a new and conserved intracellular agonist-binding pocket and provides evidence of a biased signalling mechanism that targets the receptor-transducer interface.

[Intervention and assistance by doctor for smoking cessation].

Patients with nicotine dependence require suitable intervention and assistance for smoking cessation. We should try to provide every patient with the appropriate way to quit smoking. I'm making the patient classification (motivation-confidence typing) with a questionnaire. When the patient has poor motivation, the motivational interviewing and intervention are required. If the patient has poor self-confidence, I will encourage him and admire his efforts. So varenicline significantly reduce smoking satisfaction, that I allow patients to smoke intentionally during two weeks after starting smoking cessation program. Smoking under the influence of varenicline may be useful to quit without regret.

Novel myostatin-specific antibody enhances muscle strength in muscle disease models.

Myostatin, a member of the transforming growth factor-ß superfamily, is an attractive target for muscle disease therapy because of its role as a negative regulator of muscle growth and strength. Here, we describe a novel antibody therapeutic approach that maximizes the potential of myostatin-targeted therapy. We generated an antibody, GYM329, that specifically binds the latent form of myostatin and inhibits its activation. Additionally, via "sweeping antibody technology", GYM329 reduces or "sweeps" myostatin in the muscle and plasma. Compared with conventional anti-myostatin agents, GYM329 and its surrogate antibody exhibit superior muscle strength-improvement effects in three different mouse disease models. We also demonstrate that the superior efficacy of GYM329 is due to its myostatin specificity and sweeping capability. Furthermore, we show that a GYM329 surrogate increases muscle mass in normal cynomolgus monkeys without any obvious toxicity. Our findings indicate the potential of GYM329 to improve muscle strength in patients with muscular disorders.

Smoking cessation increases levels of osteocalcin and uncarboxylated osteocalcin in human sera.

Smoking is thought to be a risk factor for osteoporosis development; however, the consequences of stopping smoking for bone homeostasis remain unknown. Here we conducted two separate human studies and show that bone mineral density was significantly lower in smokers than in non-smokers. The first was an observational study of pre- and post-menopausal healthy female smokers and non-smokers; the second included 139 current smokers determined to stop smoking. In the second study, levels of bone formation markers such as osteocalcin and uncarboxylated osteocalcin significantly increased after successful smoking cessation, as verified by significantly reduced levels of serum cotinine, a nicotine metabolite. Moreover, nicotine administration to mice reduced bone mineral density and significantly increased the number of osteoclasts in bone. Reduced bone mass phenotypes seen in nicotine-treated mice were significantly increased following nicotine withdrawal, an outcome accompanied by significantly reduced serum levels of tartrate-resistant acid phosphatase, a bone resorption marker. Taken together, our findings suggest that bone homeostasis is perturbed but can be rescued by smoking cessation.

Oral administration of MA-2029, a novel selective and competitive motilin receptor antagonist, inhibits motilin-induced intestinal contractions and visceral pain in rabbits.

The pharmacological properties of MA-2029, a novel motilin receptor antagonist, were investigated. In vitro, MA-2029 (1 to 30 nM) competitively inhibited motilin-induced contractions in isolated rabbit duodenal longitudinal muscle strips, with a pA2 value of 9.17+/-0.01 (n=5). However, contractile responses to acetylcholine and substance P were unaffected even at 1 microM of MA-2029. MA-2029 concentration-dependently inhibited the binding of [125 I]motilin to motilin receptors in a homogenate of rabbit colon smooth muscle tissue and membranes of HEK 293 cells expressing human motilin receptors. The pKi of MA-2029 was 8.58+/-0.04 in the rabbit colon homogenate (n=4) and 8.39 in the HEK 293 cells (mean of duplicate experiments). In vivo, orally-administered MA-2029 (3 to 30 mg/kg) dose-dependently inhibited colonic contractions induced by motilin (3 microg/kg, i.v.) in conscious rabbits. Inhibition was caused by all doses at 30 min after administration and by 10 mg/kg or more at 4 h after administration. The plasma concentration of MA-2029 correlated with its inhibitory effect. Furthermore, the oral administration of MA-2029 (0.3 to 3 mg/kg) also inhibited abdominal muscle contractions (an index of the visceral pain) induced by intravenous infusion of motilin (3 microg/kg/h) during colorectal distension in conscious rabbits. These results indicate that MA-2029 is an orally active, selective and competitive motilin receptor antagonist. It is suggested that this compound may be useful for gastrointestinal disorders associated with disturbed gastrointestinal motility such as irritable bowel syndrome.

Mitemcinal (GM-611), an orally active motilin receptor agonist, improves delayed gastric emptying in a canine model of diabetic gastroparesis.

1. The aim of the present study was to evaluate the effects of mitemcinal (GM-611), an orally active motilin receptor agonist, on delayed gastric emptying in a canine model of diabetic gastroparesis and to compare these effects with those of cisapride. 2. Moderate hyperglycaemia was induced by a single intravenous injection of a mixture of streptozotocin (30 mg/kg) and alloxan (50 mg/kg). Dogs that maintained moderate hyperglycaemia (fasting plasma glucose 200-300 mg/dL) without insulin treatment were selected and gastric emptying in these dogs was determined by the paracetamol method. 3. One year after the onset of diabetes, there was no difference in the gastric emptying of normal and diabetic dogs. However, after 5 years, the diabetic dogs showed delayed gastric emptying. The motor nerve conduction velocity of the tibial nerve was significantly lower in diabetic dogs compared with normal dogs at both time points. 4. Histopathological examination at the end of the study showed that there were fewer nerve fibres in both dorsal vagal and tibial nerves of diabetic dogs compared with normal dogs. The onset of delayed gastric emptying is thought to have occurred gradually, in parallel with abnormal autonomic nerve function induced by the long period of moderate hyperglycaemia. 5. Oral administration of mitemcinal (0.125, 0.25 or 0.5 mg/kg) dose-dependently accelerated delayed gastric emptying, significant at 0.5 mg/kg, in diabetic dogs, whereas cisapride (1, 3 or 10 mg/kg) had no significant effect. These results add to the existing evidence that mitemcinal is likely to be useful for treating diabetic gastroparesis.

Inhibition of MEK1 Signaling Pathway in the Liver Ameliorates Insulin Resistance.

Although mitogen-activated protein kinase kinase (MEK) is a key signaling molecule and a negative regulator of insulin action, it is still uncertain whether MEK can be a therapeutic target for amelioration of insulin resistance (IR) in type 2 diabetes (T2D) in vivo. To clarify whether MEK inhibition improves T2D, we examined the effect of continuous MEK inhibition with two structurally different MEK inhibitors, RO5126766 and RO4987655, in mouse models of T2D. RO5126766 and RO4987655 were administered via dietary admixture. Both compounds decreased blood glucose and improved glucose tolerance in doses sufficient to sustain inhibition of extracellular signal-regulated kinase (ERK)1/2 phosphorylation downstream of MEK in insulin-responsive tissues in db/db mice. A hyperinsulinemic-euglycemic clamp test showed increased glucose infusion rate (GIR) in db/db mice treated with these compounds, and about 60% of the increase was attributed to the inhibition of endogenous glucose production, suggesting that the liver is responsible for the improvement of IR. By means of adenovirus-mediated Mek1 shRNA expression, we confirmed that blood glucose levels are reduced by suppression of MEK1 expression in the liver of db/db mice. Taken together, these results suggested that the MEK signaling pathway could be a novel therapeutic target for novel antidiabetic agents.

An orally active motilin receptor antagonist, MA-2029, inhibits motilin-induced gastrointestinal motility, increase in fundic tone, and diarrhea in conscious dogs without affecting gastric emptying.

The pharmacological properties of MA-2029, a selective and competitive motilin receptor antagonist, were investigated in conscious dogs after oral administration. Gastrointestinal contractile activity was recorded by chronically implanted force transducers. The proximal gastric volume was measured with a barostat under constant pressure. Gastric emptying was examined using the paracetamol absorption test. MA-2029 (0.3-10 mg/kg, p.o.) administered in the interdigestive state inhibited gastrointestinal contractions induced by motilin (3 microg/kg, i.v.) in a dose-dependent manner. MA-2029 (0.3-3 mg/kg, p.o.) also inhibited the occurrence of spontaneous phase III contractions, even though MA-2029 had no effect on basal gastrointestinal motility or basal gastric emptying even at 10 and 30 mg/kg p.o. The inhibitory effect of MA-2029 on motilin-induced gastrointestinal motility corresponded to its plasma concentration. Motilin (0.3 microg/kg/h, i.v. infusion) reduced the proximal gastric volume by about 50% of control during isobaric distension. This effect was also inhibited by MA-2029 (1-10 mg/kg, p.o.) in a dose-dependent manner. In the digestive state, injection of motilin (3 microg/kg, i.v.) induced diarrhea in 9 of 11 dogs. MA-2029 (1-30 mg/kg, p.o.) reduced the incidence of diarrhea induced by motilin in a dose-dependent manner. The results indicate that MA-2029 inhibits hypermotility induced by motilin in conscious dogs without having an effect on the basal gastrointestinal tone or gastric emptying rate. MA-2029 may be useful in treating gastrointestinal disorders in which the pathogenesis involves the elevation of circulating motilin.

Effects of mitemcinal (GM-611), an orally active erythromycin-derived prokinetic agent, on delayed gastric emptying and postprandial glucose in a new minipig model of diabetes.

AIMS: This study was conducted to evaluate the suitability of a new minipig model for investigating aspects of diabetes such as delayed gastric emptying and glucose metabolism abnormalities, and to test the effects of mitemcinal (GM-611), an orally active erythromycin-derived motilin receptor agonist, on gastric emptying and postprandial glucose in normal and diabetic minipigs. METHODS AND RESULTS: Intravenous injection of 300 mg/kg streptozotocin (STZ) to 5-week-old minipigs induced moderate hyperglycemia (about 200 mg/dl) for >80 weeks without insulin treatment. Decreased insulin production (P<.05), increased area under the glucose curve (P<.05), and slower glucose disappearance (P<.05) were demonstrated, and there was no severe inhibition of body weight gain, liver failure, or renal failure. Gastric emptying was significantly delayed in diabetic minipigs (P<.05) at 80 weeks, but not at 40 weeks, post-STZ. Oral administration of mitemcinal (5 mg/kg) at 80 weeks accelerated gastric emptying and induced a similar postprandial glucose profile in normal and diabetic minipigs with delayed gastric emptying. CONCLUSIONS: The new diabetic minipig model showed suitability for investigating diabetes, gastric emptying, and plasma glucose excursions. Since delayed gastric emptying and irregular plasma glucose excursions are characteristic of diabetic gastroparesis, the accelerating and regulating effects of mitemcinal on this model add to the existing evidence that mitemcinal is likely to be useful for treating diabetic gastroparesis.