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Chemoattractant receptor homologous molecule expressed on T helper type 2 cells (CRTH2) is a PGD2 receptor found on eosinophils, basophils, and Th2 type T cells which exhibits chemotaxis and functions in activation cascades. However, while a number of CRTH2 antagonists, including ramatroban, are known to exert activity in certain animal models, activity in a guinea pig model of EA-induced airway hyperresponsiveness has not been demonstrated. The newly developed CRTH2 antagonist ASP5642 has shown antagonistic activity against human and guinea pig CRTH2 in previous studies and has also been found effective in treating guinea pig models of airway inflammation and airway hyperresponsiveness. While previous studies have used animals such as rats and mice to evaluate CRTH2 antagonist effects, ours is the first attempt to evaluate CRTH2 function in a guinea pig asthma model, which may prove useful in evaluating the compound's effects in humans, given the comparable airway function between the two species taken together, these data from the present study strongly suggest the utility of ASP5642 in investigating the role of CRTH2 in inflammatory responses and as a drug treatment for human asthma., (Copyright © 2013 Elsevier B.V. All rights reserved.)

High throughput screening of our chemical library for CRTH2 antagonists provided a lead compound 1a. Initial optimization of the lead led to the discovery of a novel, potent and orally bioavailable CRTH2 antagonist 17., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Mechanisms by which GHRP stimulates ACTH release in corticotrope cells were investigated using mouse corticotrope AtT20 cells by focusing on the biological activity of BMP-4. GHRP-2 increased ACTH and cAMP secretion by AtT20 cells; however, its effects were less potent than the effects of CRH. BMP-4 suppressed basal ACTH production and POMC transcription, and the inhibition of endogenous BMP receptor signaling led to an increase in ACTH production. Of note, BMP-4 suppressed ACTH production and POMC-promoter activity induced by CRH more efficaciously than that induced by GHRP-2. BMP-4 had no significant effect on cAMP synthesis induced by CRH or GHRP-2. Stimulation with CRH, but not GHRP-2, activated ERK1/2, p38, SAPK/JNK and Akt phosphorylation, in which CRH-induced phosphorylation of ERK and p38 was suppressed by BMP-4. GHRP-2-induced ACTH secretion was not affected by inhibitors of ERK, p38 and Akt pathways, which effectively suppressed CRH-induced ACTH release. Blockage of the cAMP-PKA pathway reversed CRH- as well as GHRP-2-induced ACTH secretion. Furthermore, the inhibition of ERK and p38 significantly reduced cAMP synthesis induced by CRH but not by GHRP-2. Thus, CRH activates ACTH production through ERK and p38 pathways in addition to the cAMP-PKA pathway, which is also activated downstream of MAPK. On the other hand, GHRP-2-induced ACTH production was predominantly linked to the cAMP-PKA pathway. Moreover, CRH and GHRP-2 upregulated BMP receptor signaling, while BMP-4, CRH and GHRP-2 had no significant effect on the expression level of GHSR. In addition, GHRP-2 suppressed the expression of Smad7, which is an inhibitor of the BMP-Smad1/5/8 pathway. Collectively, the results revealed a functional interaction between GHRP-2 and BMP signaling, in which endogenous BMP may act as an autoregulatory system in controlling ACTH production., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Recent studies have shown that bone morphogenetic proteins (BMPs) are important regulators in the pituitary-gonadal endocrine axis. We here investigated the effects of BMPs on GNRH production controlled by estrogen using murine GT1-7 hypothalamic neuron cells. GT1-7 cells expressed estrogen receptor alpha (ERalpha; ESR1 as listed in MGI Database), ERbeta (ESR2 as listed in MGI Database), BMP receptors, SMADs, and a binding protein follistatin. Treatment with BMP2 and BMP4 had no effect on Gnrh mRNA expression; however, BMP6 and BMP7 significantly increased Gnrh mRNA expression as well as GnRH production by GT1-7 cells. Notably, the reduction of Gnrh expression caused by estradiol (E(2)) was restored by cotreatment with BMP2 and BMP4, whereas it was not affected by BMP6 or BMP7. E(2) activated extracellular signal-regulated kinase (ERK) 1/2 and stress-activated protein kinase/c-Jun NH(2)-terminal kinase (SAPK/JNK) signaling but did not activate p38-mitogen-activated protein kinase (MAPK) signaling in GT1-7 cells. Inhibition of ERK1/ERK2 reversed the inhibitory effect of estrogen on Gnrh expression, whereas SAPK/JNK inhibition did not affect the E(2) actions. Expression levels of Eralpha and Erbeta were reduced by BMP2 and BMP4, but were increased by BMP6 and BMP7. Treatment with an ER antagonist inhibited the E(2) effects on Gnrh suppression including reduction of E(2)-induced ERK phosphorylation, suggesting the involvement of genomic ER actions in Gnrh suppression. BMP2 and BMP4 also suppressed estrogen-induced phosphorylation of ERK1/ERK2 and SAPK/JNK signaling, suggesting that BMP2 and BMP4 downregulate estrogen effects by attenuating ER-MAPK signaling. Considering that BMP6 and BMP7 increased the expression of alpha1E-subunit of R-type calcium channel (Cacna1e), which is critical for GNRH secretion, it is possible that BMP6 and BMP7 directly stimulate GNRH release by GT1-7 cells. Collectively, a newly uncovered interaction of BMPs and ER may be involved in controlling hypothalamic GNRH production and secretion via an autocrine/paracrine mechanism.

A 35-year-old Japanese woman was referred for further examination of persistent hypertension with hypokalemia. Her serum aldosterone levels were high and her plasma renin activity markedly suppressed. Radiological examinations revealed the presence of a 3-cm diameter left adrenal tumor. (131)I-adosterol was specifically accumulated in the left adrenal tumor, whereas the accumulation in the right adrenal was completely suppressed. Low-dose dexamethasone failed to suppress cortisol secretion although the serum cortisol levels were within the normal range. Urinary excretion of 17-hydroxycorticosteroids but not 17-ketosteroids was increased. Levels of plasma adrenocorticotropin (ACTH) and serum dehydroepiandrosterone sulfate (DHEAS) were decreased. Upon diagnosis of left aldosteronoma with autonomous secretion of cortisol, left adrenalectomy was performed by laparoscopy. In the resected adenoma tissues, clear cells expressed P450c17 protein and the ratio of CYP17/CYP11B2 mRNA evaluated by quantitative real-time polymerase chain reaction (PCR) was apparently higher than that of typical aldosteronomas. Based on the corticotropin-releasing hormone (CRH) loading tests, the contra-lateral adrenal functions were restored 3 months after surgery. These results indicate that evaluation for autonomy of cortisol secretion and contra-lateral adrenal function is clinically important to avoid the risk of adrenal failure after surgery for primary aldosteronism.

Adenosine has anti-inflammatory activity. Adenosine deaminase (EC 3.5.4.4) metabolizes extracellular adenosine, resulting in an exacerbation of inflammation. Consequently, it was hypothesized that adenosine deaminase inhibitors produce anti-inflammatory activity by increasing extracellular adenosine concentration. This group recently developed a non-nucleoside adenosine deaminase inhibitor, FR234938, by using rational structure-based drug design. FR234938 inhibits recombinant human adenosine deaminase enzyme competitively. FR234938 inhibits interleukin (IL)-6-dependent immunoglobulin (Ig) M production by SKW6.4 cells, in the presence of adenosine. Inhibitory effect of FR234938/adenosine combination is blocked by an A2a adenosine receptor antagonist. FR234938 also inhibits anti-type II collagen delayed type hypersensitivity (DTH) in a dose-dependent manner, both in the presence and absence of recombinant human adenosine deaminase. Moreover, FR234938 inhibits tumor necrosis factor (TNF)-alpha and IL-10 production in a lipopolysaccharide (LPS)-induced cytokine production model in mice. These results indicate that FR234938 has potential anti-inflammatory activity. Non-nucleoside adenosine deaminase inhibitor FR234938 has good potential as a new type of anti-rheumatic and anti-inflammatory drug, by modulating host-defense concentrations of adenosine.

Structural snapshots corresponding to various states enable elucidation of the molecular recognition mechanism of enzymes. Adenosine deaminase has two distinct conformations, an open form and a closed form, although it has so far been unclear what factors influence adaptation of the alternative conformations. Herein, we have determined the first nonligated structure as an initial state, which was the open form, and have thereby rationally deduced the molecular recognition mechanism. Inspection of the active site in the nonligated and ligated states indicated that occupancy at one of the water-binding positions in the nonligated state was highly significant in determining alternate conformations. When this position is empty, subsequent movement of Phe65 toward the space induces the closed form. On the other hand, while occupied, the overall conformation remains in the open form. This structural understanding should greatly assist structure-oriented drug design and enable control of the enzymatic activity.

We disclose herein optimization efforts around the novel, highly potent non-nucleoside adenosine deaminase (ADA) inhibitor, 1-[(R)-1-hydroxy-4-(6-(3-(1-methylbenzimidazol-2-yl)propionylamino)indol-1-yl)-2-butyl]imidazole-4-carboxamide 1 (K(i)= 7.7 nM), which we recently reported. Structure-based drug design (SBDD) utilizing the crystal structure of the 1/ADA complex was performed in order to obtain structure-activity relationships (SAR) for this type of compound rationally and effectively. To utilize the newly formed hydrophobic space (F2), replacement of the benzimidazole ring of 1 with a n-propyl chain (4b) or a simple phenyl ring (4c) was tolerated in terms of binding activity, and the length of the methylene-spacer was shown to be optimal at two or three. Replacement of an amide with an ether as a linker was also well tolerated in terms of binding activity and moreover improved the oral absorption (6a and 6b). Finally, transformation of indol-1-yl to indol-3-yl resulted in discovery of a novel highly potent and orally bioavailable ADA inhibitor, 1-[(R)-4-(5-(3-(4-chlorophenyl)propoxy)-1-methylindol-3-yl)-1-hydroxy-2-butyl]imidazole-4-carboxamide 8c.

We disclose herein the rapid discovery of the first highly potent (Ki = 7.7 nM) non-nucleoside adenosine deaminase (ADA) inhibitor based on the rational hybridization of two structurally distinct leads. Two micromolar inhibitors were discovered by a parallel rational design and random screening program, and individual crystal structures of bovine ADA in complexation with these inhibitors revealed several unknown binding sites and distinct binding modes. Using this information as the starting point, highly effective lead hybridization was achieved in only two structure-based drug design iterations. The conceptual approach illustrated by this example promises to be broadly useful in the search for new chemical entities and can contribute greatly to improve the overall efficiency and speed of drug discovery.

We searched for non-nucleoside inhibitors of adenosine deaminase by rational structure-based de novo design and succeeded in the discovery of 1-(1-hydroxy-4-phenyl-2-butyl)imidazole-4-carboxamide (FR221647: K(i)=5.9 microM to human ADA) as a novel inhibitor with moderate activity and good pharmacokinetics compared with the known inhibitors pentostatin and EHNA.