Identification of novel selective P2Y6 receptor antagonists by high-throughput screening assay.

AIMS: The P2Y6 nucleotide receptor is widely involved in inflammatory responses, and is a promising molecular target for the treatment of inflammatory diseases. Although several P2Y6 receptor antagonists have been developed and evaluated thus far, none has successfully been developed into a therapeutic drug. In this study, we explored new promising compounds that inhibit the human P2Y6 receptor. MAIN METHODS: High-throughput screening (HTS) was used to study the effects of various compounds on human P2Y6 receptors expressed in 1321N1 human astrocytoma cells by monitoring intracellular Ca2+ concentration ([Ca2+]i) levels using an FDSS7000 real-time fluorescence detector. IL-8 concentration was measured by enzyme-linked immunosorbent assay. KEY FINDINGS: Among structurally diverse chemical libraries totalling 141,700 compounds, 43 compounds with an inhibitory activity against the P2Y6 receptor were identified. Further studies using a dose-response assay, receptor selectivity assay, and chemokine measurement assay revealed the selective P2Y6 receptor inhibitor TIM-38, which inhibited UDP-induced [Ca2+]i elevation in a dose-dependent manner. TIM-38 had an IC50 value of 4.3µM and inhibited P2Y6 without affecting the response induced by four other human P2Y or muscarinic receptors. In addition, TIM-38 inhibited UDP-induced interleukin-8 release in a dose-dependent manner without affecting releases caused by other stimulus such as interleukin-1ß or tumour necrosis factor-α. Analyses of TIM-38 derivatives revealed that the nitro moiety is vital to P2Y6 receptor inhibition. SIGNIFICANCE: TIM-38 acts as a novel structural antagonist of P2Y6 receptor and may be a good lead compound for developing a P2Y6 receptor-targeted anti-inflammatory drug.

An HTRF based high-throughput screening for discovering chemical compounds that inhibit the interaction between Trypanosoma brucei Pex5p and Pex14p.

The glycosome, a peroxisome-related organelle, is essential for the growth and survival of trypanosomatid protozoa. In glycosome biogenesis, Pex5p recognizes newly synthesized glycosomal matrix proteins via peroxisome-targeting signal type-1 (PTS-1) and transports them into glycosomes through an interaction with Pex14p, a component of the matrix protein import machinery on the glycosomal membrane. Knockdown of the PEX5 or PEX14 with RNAi has been shown to inhibit the growth of Trypanosoma brucei. Thus, compounds that inhibit the interaction of TbPex5p-TbPex14p are expected to become lead compounds in the development of anti-trypanosomal drugs. Here, we report a homogenous time-resolved fluorescence (HTRF) assay for the screening of compounds that inhibit the TbPex5p-TbPex14p interaction. The binding of GST-TbPex14p and TbPex5p-His with or without additional compounds was evaluated by measuring the energy transfer of the HTRF pair, using a terbium-labeled anti GST antibody as the donor and an FITC-labeled anti His antibody as the acceptor. The assay was performed in a 384-well plate platform and exhibits a Z'-factor of 0.85-0.91, while the coefficiency of variation is 1.1-7.7%, suggesting it can be readily adapted to a high-throughput format for the automated screening of chemical libraries. We screened 20,800 compounds and found 11 compounds that inhibited energy transfer. Among them, in a pull-down assay one compound exhibited selective inhibition of TbPex5p-TbPex14p without any HsPex5p-HsPex14p interaction.

Gliotoxin suppresses NF-κB activation by selectively inhibiting linear ubiquitin chain assembly complex (LUBAC).

A linear ubiquitin chain, which consists of ubiquitin molecules linked via their N- and C-termini, is formed by a linear ubiquitin chain assembly complex (LUBAC) composed of HOIP, HOIL-1L, and SHARPIN, and conjugation of a linear ubiquitin chain on the NF-κB essential modulator (NEMO) is deeply involved in NF-κB activation induced by various signals. Since abnormal activation of NF-κB is associated with inflammatory disease and malignancy, we searched for an inhibitor of LUBAC by high-throughput screening (HTS) with a Tb(3+)-fluorescein FRET system. As a result, we found that the fungal metabolite gliotoxin inhibits LUBAC selectively by binding to the RING-IBR-RING domain of HOIP, the catalytic center of LUBAC. Gliotoxin has been well-known as an inhibitor of NF-κB activation, though its action mechanism has remained elusive. Here, we show that gliotoxin inhibits signal-induced NF-κB activation by selectively inhibiting LUBAC-mediated linear ubiquitin chain formation.

A dual inhibitor against prolyl isomerase Pin1 and cyclophilin discovered by a novel real-time fluorescence detection method.

Pin1, a peptidyl prolyl cis/trans isomerase (PPIase), is a potential target molecule for cancer, infectious disease, and Alzheimer's disease. We established a high-throughput screening method for Pin1 inhibitors, which employs a real-time fluorescence detector. This screening method identified 66 compounds that inhibit Pin1 out of 9756 compounds from structurally diverse chemical libraries. Further evaluations of surface plasmon resonance methods and a cell proliferation assay were performed. We discovered a cell-active inhibitor, TME-001 (2-(3-chloro-4-fluoro-phenyl)-isothiazol-3-one). Surprisingly, kinetic analyses revealed that TME-001 is the first compound that exhibits dual inhibition of Pin1 (IC50=6.1 µM) and cyclophilin, another type of PPIase, (IC50=13.7 µM). This compound does not inhibit FKBP. This finding suggests the existence of similarities of structure and reaction mechanism between Pin1 and cyclophilin, and may lead to a more complete understanding of the active sites of PPIases.

Melanin-concentrating hormone 1-receptor antagonist suppresses body weight gain correlated with high receptor occupancy levels in diet-induced obesity mice.

Melanin-concentrating hormone (MCH), which is a neuropeptide expressed in the hypothalamus of the brain, is involved in regulating feeding behavior and energy homeostasis via the MCH(1) receptor in rodents. It is widely considered that MCH(1) receptor antagonists are worthy of development for medical treatment of obesity. Here we report on the development of an ex vivo receptor occupancy assay using a new radiolabeled MCH(1) receptor antagonist, [(35)S]-compound D. An MCH(1) receptor antagonist inhibited the binding of [(35)S]-compound D to brain slices in a dose-dependent manner. The result showed a good correlation between the receptor occupancy levels and plasma or brain levels of the MCH(1) receptor antagonist, suggesting that the ex vivo receptor binding assay using this radioligand is practical. Quantitative analysis in diet-induced obese mice showed that the efficacy of body weight reduction correlated with the receptor occupancy levels at 24h. Furthermore, more than 90% occupancy levels of MCH(1) receptor antagonists during 24h post-dosing are required for potent efficacy on body weight reduction. The present occupancy assay could be a useful pharmacodynamic marker to quantitatively estimate anti-obese efficacy, and would accelerate the development of MCH(1) receptor antagonists for treatment of obesity.

Synergistic interaction between neuropeptide Y1 and Y5 receptor pathways in regulation of energy homeostasis.

Neuropeptide Y plays a key role in the physiological control of energy homeostasis. Five neuropeptide Y receptor subtypes have been cloned, and multiple neuropeptide Y receptor subtypes are thought to mediate neuropeptide Y activity. However, interactions among neuropeptide Y receptor subtypes have not been elucidated to date. Herein, we examined the interaction between neuropeptide Y(1) and Y(5) receptors in feeding regulation by employing selective neuropeptide Y(1) and Y(5) receptor antagonists in C57BL/6 and neuropeptide Y(1) receptor knockout mice fed a high-fat diet. A single-dose of a neuropeptide Y(1) receptor antagonist (10-30 mg/kg) suppressed spontaneous food intake and reduced body weight in high-fat diet-fed C57BL/6 mice, while treatment with a neuropeptide Y(5) receptor antagonist did not significantly reduce food intake or body weight. Coadministration of a neuropeptide Y(1) receptor antagonist with a neuropeptide Y(5) receptor antagonist further suppressed food intake and reduced body weight. Next, we evaluated the chronic efficacy of a neuropeptide Y(5) receptor antagonist in high-fat diet-fed neuropeptide Y(1) receptor knockout mice in order to mimic chronic combination treatment with neuropeptide Y(1) and Y(5) receptor antagonists. The neuropeptide Y(5) receptor antagonist produced greater body weight reductions in high-fat diet-fed neuropeptide Y(1) receptor knockout mice than in wild-type C57BL/6 mice. These findings confirm an interaction between neuropeptide Y(1) and Y(5) receptors in the regulation of energy homeostasis, as blockade of both the neuropeptide Y(1) and Y(5) receptors produced a greater anti-obesity effect than blocking either receptor alone.

Identification and characterization of a selective radioligand for melanin-concentrating hormone 1-receptor (MCH1R).

We have developed and characterized [(35)S]4a as a potent and selective radioligand for melanin-concentrating hormone 1-receptor (MCH1R). Compound [(35)S]4a showed appreciable specific signals in brain slices prepared from wild-type mice but not from MCH1R deficient mice, confirming the specificity and utility of [(35)S]4a as a selective MCH1R radioligand for ex vivo receptor occupancy assays.

A neuropeptide Y Y5 antagonist selectively ameliorates body weight gain and associated parameters in diet-induced obese mice.

Neuropeptide Y (NPY) is thought to have a major role in the physiological control of energy homeostasis. Among five NPY receptors described, the NPY Y5 receptor (Y5R) is a prime candidate to mediate some of the effects of NPY on energy homeostasis, although its role in physiologically relevant rodent obesity models remains poorly defined. We examined the effect of a potent and highly selective Y5R antagonist in rodent obesity and dietary models. The Y5R antagonist selectively ameliorated diet-induced obesity (DIO) in rodents by suppressing body weight gain and adiposity while improving the DIO-associated hyperinsulinemia. The compound did not affect the body weight of lean mice fed a regular diet or genetically obese leptin receptor-deficient mice or rats, despite similarly high brain Y5R receptor occupancy. The Y5R antagonist acts in a mechanism-based manner, as the compound did not affect DIO of Y5R-deficient mice. These results indicate that Y5R is involved in the regulation and development of DIO and suggest utility for Y5R antagonists in the treatment of obesity.

CENP-B interacts with CENP-C domains containing Mif2 regions responsible for centromere localization.

Recently, human artificial chromosomes featuring functional centromeres have been generated efficiently from naked synthetic alphoid DNA containing CENP-B boxes as a de novo mechanism in a human cultured cell line, but not from the synthetic alphoid DNA only containing mutations within CENP-B boxes, indicating that CENP-B has some functions in assembling centromere/kinetochore components on alphoid DNA. To investigate whether any interactions exist between CENP-B and the other centromere proteins, we screened a cDNA library by yeast two-hybrid analysis. An interaction between CENP-B and CENP-C was detected, and the CENP-C domains required were determined to overlap with three Mif2 homologous regions, which were also revealed to be involved in the CENP-C assembly of centromeres by expression of truncated polypeptides in cultured cells. Overproduction of truncated CENP-B containing no CENP-C interaction domains caused abnormal duplication of CENP-C domains at G2 and cell cycle delay at metaphase. These results suggest that the interaction between CENP-B and CENP-C may be involved in the correct assembly of CENP-C on alphoid DNA. In other words, a possible molecular linkage may exist between one of the kinetochore components and human centromere DNA through CENP-B/CENP-B box interaction.

N-acyl 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline: the first orexin-2 receptor selective non-peptidic antagonist.

The identification of potent and selective orexin-2 receptor (OX(2)R) antagonists is described based on the modification of N-acyl 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline analogue 1, recently discovered during high throughput screening (HTS). Substitution of an acyl group in 1 with tert-Leucine (tert-Leu), and introduction of a 4-pyridylmethyl substituent onto the amino function of tert-Leu improved compound potency, selectivity, and water solubility. Thus, compound 29 is a promising tool to investigate the role of orexin-2 receptors.

Development of an orexin-2 receptor selective agonist, [Ala(11), D-Leu(15)]orexin-B.

Investigation of L-alanine and D-amino acid replacement of orexin-B revealed that three L-leucine residues at the positions of 11, 14, and 15 in orexin-B were important to show selectivity for the orexin-2 receptor (OX(2)) over the orexin-1 receptor (OX(1)). L-Alanine substitution at position 11 and D-leucine substitution at positions 14 and 15 maintained the potency of orexin-B to mobilize [Ca(2+)](i) in CHO cells expressing the OX(2), while their potency for the OX(1) was significantly reduced. In combined substitutions, we identified that [Ala(11), D-Leu(15)]orexin-B showed a 400-fold selectivity for the OX(2) (EC(50)=0.13nM) over OX(1) (EC(50)=52nM). [Ala(11), D-Leu(15)]orexin-B is a beneficial tool for addressing the functional roles of the OX(2).