Yasuhisa Miyamoto

Identification of membrane-type receptor for bile acids (M-BAR)

Bile acids play an essential role in the solubilization and absorption of dietary fat and lipid-soluble vitamins. Bile acids also modulate the transcription of various genes for enzymes and transport proteins for their own and cholesterol homeostasis through binding to nuclear receptors. Here we report a novel category of bile acid receptor, a membrane-type G protein-coupled receptor (GPCR), BG37. Bile acids induced rapid and dose-dependent elevation of intracellular cAMP levels in BG37-expressing cells, but not in mock-transfected cells, independently of nuclear receptor expression. The rank order of potency of various bile acids for BG37-expressing cells was different from that for the nuclear receptor-mediated response. These observations demonstrate the presence of two independent signaling pathways for bile acids; membrane-type GPCR for rapid signaling and nuclear receptors for delayed signaling. Expression of BG37 was detected in various specific tissues, suggesting its physiological role, although it remains to be further characterized.

Therapeutic potential of histamine H3 receptor agonist for the treatment of obesity and diabetes mellitus

Histamine H3 receptors (H3Rs) are located on the presynaptic membranes and cell soma of histamine neurons, where they negatively regulate the synthesis and release of histamine. In addition, H3Rs are also located on nonhistaminergic neurons, acting as heteroreceptors to regulate the releases of other amines such as dopamine, serotonin, and norepinephrine. The present study investigated the effects of H3R ligands on appetite and body-weight regulation by using WT and H3R-deficient mice (H3RKO), because brain histamine plays a pivotal role in energy homeostasis. The results showed that thioperamide, an H3R inverse agonist, increases, whereas imetit, an H3R agonist, decreases appetite and body weight in diet-induced obese (DiO) WT mice. Moreover, in DiO WT mice, but not in DiO H3RKO mice, imetit reduced fat mass, plasma concentrations of leptin and insulin, and hepatic triglyceride content. The anorexigenic effects of imetit were associated with a reduction in histamine release, but a comparable reduction in histamine release with α-fluoromethylhistidine, an inhibitor of histamine synthesis, increased appetite. Moreover, the anorexigenic effects of imetit were independent of the melanocortin system, because imetit comparably reduced appetite in melanocortin 3 and 4 receptor-deficient mice. The results provide roles of H3Rs in energy homeostasis and suggest a therapeutic potential for H3R agonists in the treatment of obesity and diabetes mellitus.

AGOSTEROL A, A NOVEL POLYHYDROXYLATED STEROL ACETATE REVERSING MULTIDRUG RESISTANCE FROM A MARINE SPONGE OF SPONGIA SP.

Abstract Agosterol A (1) has been isolated from a marine sponge of Spongia sp. and the absolute stereo-structure elucidated. Agosterol A (1) is a novel polyhydroxylated sterol acetate, which completely reverses multidrug resistance in human carcinoma cells caused by overexpression of two kinds of membrane glycoprotein.

Identification of a stable chemerin analog with potent activity toward ChemR23.

Chemerin is a novel peptide that was identified as a natural ligand for ChemR23. As it has been reported to be involved in the regulation of immune responses and adipogenesis, chemerin may have a variety of physiological functions. Chemerin is synthesized as a precursor (prochemerin) and is proteolytically activated and inactivated in sequential steps, which control its physiological roles in a coordinated manner. Chemerin-9 (chemerin148-156) was previously identified as the smallest peptide with low nanomolar potency. However, like mature chemerin, chemerin-9 is rapidly degraded and inactivated in plasma, which has limited the use of chemerin-9 in in vivo experiments. In order to identify stable chemerin analogs that facilitate in vivo studies, we synthesized a series of chemerin-9 analogs and examined intrinsic activity and metabolic stability. We identified an agonistic and metabolically stable chemerin-9 analog (d-Tyr(147)-[d-Ser(151), d-Ala(154), Tic(155)]chemerin148-156) that shows enhanced plasma exposure with prolonged half-life in mice upon intraperitoneal administration. Improvement of metabolic stability resulted in a reduction in the plasma free fatty acid levels in fasted mice, which cannot be accomplished by unstable-mouse chemerin-9. This reduction in plasma free fatty acids reflects the anti-lipolysis activity of chemerin-9 and analogs in mouse primary adipocytes. The discovery of a metabolically stable chemerin analog will facilitate investigation of the pharmacological roles of chemerin in vivo. Moreover, this stable chemerin analog might provide new therapeutic approaches to inflammatory diseases such as asthma and metabolic disorders such as obesity and diabetes where ChemR23 activation may be of benefit.

Development of a High-Density Assay for Long-Chain Fatty Acyl-CoA Elongases

We established a convenient assay method for measuring elongation of very long chain fatty acids (ELOVLs) using a Unifilter-96 GF/C plate. The Unifilter GF/C plate preferentially interacts with hydrophobic end products of ELOVLs (i.e., long chain fatty acid), with minimal malonyl-CoA (C2 unit donor for fatty acid elongation) interaction. This new method results in the quick separation and detection of [14C] incorporated end products (e.g., [14C] palmitoyl-CoA) from reaction mixtures containing excessive amounts of [14C] malonyl-CoA. In the Unifilter-96 GF/C plate assay, recombinantly expressed human ELOVLs (i.e., ELOVL1,-2,-3,-5 and -6) displayed appreciable assay windows (>2-fold vs. mock-transfected control), enabling us to conduct comprehensive substrate profiling of ELOVLs. The substrate concentration profile of ELOVL6 in the Unifilter-96 GF/C plate assay is consistent with that obtained from the conventional liquid extraction method, thus, supporting the reliability of the Unifilter-96 GF/C plate assay. We then examined the substrate specificities of ELOVLs in a comprehensive fashion. As previously reported, ELOVL1, -3 and -6 preferably elongated the saturated fatty acyl-CoAs while ELOVL2 and ELOVL5 preferentially elongated the polyunsaturated fatty acyl-CoAs. This further confirms the Unifilter-96 GF/C plate assay reliability. Taken together, our newly developed assay provides a convenient and comprehensive assay platform for ELOVLs, allowing investigators to conduct high density screening and characterization of ELOVLs chemical tools.

Synthesis and evaluation of structurally constrained quinazolinone derivatives as potent and selective histamine H3 receptor inverse agonists.

A series of structurally constrained derivatives of the potent H 3 inverse agonist 1 was designed, synthesized, and evaluated as histamine H 3 receptor inverse agonists. As a result, the N-cyclobutylpiperidin-4-yloxy group as in 2f was identified as an optimal surrogate structure for the flexible 1-pyrrolidinopropoxy group of 1. Subsequent optimization of the quinazolinone core of 2f revealed that substitution at the 5-position of the quinazolinone ring influences potency. Representative derivatives 5a and 5s showed improved potency in a histamine release assay in rats and a receptor occupancy assay in mice.

Discovery and characterization of a novel potent, selective and orally active inhibitor for mammalian ELOVL6

The elongase of long chain fatty acids family 6 (ELOVL6) is a rate-limiting enzyme for the elongation of saturated and monounsaturated long chain fatty acids. ELOVL6 is abundantly expressed in lipogenic tissues such as liver, and its mRNA expression is up-regulated in obese model animals. ELOVL6 deficient mice are protected from high-fat-diet-induced insulin resistance, suggesting that ELOVL6 might be a new therapeutic target for diabetes. We previously identified an indoledione compound, Compound A, as the first inhibitor for mammalian ELOVL6. In this study, we discovered a novel compound, Compound B, and characterized its biochemical and pharmacological properties. Compound B has a more appropriate profile for use as a pharmacological tool compared to Compound A. Chronic treatment with Compound B in model animals, diet-induced obesity (DIO) and KKAy mice, showed significant reduction in hepatic fatty acid composition, suggesting that it effectively inhibits ELOVL6 activity in the liver. However, no improvement in insulin resistance by ELOVL6 inhibition was found in these model animals. Further studies need to address the impact of ELOVL6 inhibition on pharmacological abnormalities in several model animals. This is the first report on pharmacology data from chronic studies using a selective ELOVL6 inhibitor. Compound B appears to be a useful tool to further understand the physiological roles of ELOVL6 and to evaluate the therapeutic potential of ELOVL6 inhibitors.

Synthesis and evaluation of a novel indoledione class of long chain fatty acid elongase 6 (ELOVL6) inhibitors.

Novel indoledione derivatives were synthesized and evaluated as long chain fatty acid elongase 6 (ELOVL6) inhibitors. Systematic optimization of an indole class of lead 1 led to the identification of potent ELOVL6 selective inhibitors. Representative inhibitor 37 showed sustained plasma exposure and good liver penetrability in mice. After oral administration, 37 potently inhibited ELOVL6 activity in the liver in mice.

Synthesis and Biological Evaluation of a Novel 3-Sulfonyl-8-azabicyclo[3.2.1]octane Class of Long Chain Fatty Acid Elongase 6 (ELOVL6) Inhibitors

Long chain fatty acid elongase 6 (ELOVL6) catalyzes the elongation of long chain fatty acyl-CoAs and is a potential target for the treatment of metabolic disorders. The ultrahigh throughput screen of our corporate chemical collections resulted in the identification of a novel 3-sulfonyl-8-azabicyclo[3.2.1]octane class of ELOVL6 inhibitor 1a. Optimization of lead 1a led to the identification of the potent, selective, and orally available ELOVL6 inhibitor 1w.

Identification and Characterization of a Selective Radioligand for ELOVL6

ELOVL6, a member of the elongation of very long-chain fatty acids (ELOVL) family, has recently been identified as the rate-limiting enzyme for the elongation of palmitoyl-CoA. ELOVL6 deficient mice are protected from high-fat diet induced insulin resistance, suggesting that ELOVL6 might be a promising target for the treatment of metabolic disorders. Despite the increasing interest in Elovl6 as a therapeutic target, the lack of chemical tools for this enzyme has limited further elucidation of the biochemical and pharmacological properties of ELOVL6. We have identified Compound-A, a potent inhibitor for ELOVL6, by screening our company library and subsequently optimizing hit compounds. Compound-A potently inhibited human and mouse ELOVL6 and displayed >100-fold greater selectivity for ELOVL6 over other ELOVL family members. Consistent with its potent and selective inhibitory activity toward ELOVL6, [(3)H]Compound-A bound to ELOVL6 with high affinity while showing no specific binding to other ELOVL enzymes. The observation that [(3)H]Compound-A bound to ELOVL6 in a palmitoyl-CoA-dependent manner in the absence of malonyl-CoA and NADPH suggests that Compound-A might recognize an enzyme-substrate complex, e.g. an acyl-enzyme intermediate. Collectively, these observations demonstrate that Compound-A and its tritiated form are useful tools for biochemical and pharmacological characterization of ELOVL6.