Effect of ASP2205 fumarate, a novel 5-HT2C receptor agonist, on urethral closure function in rats.

The pharmacological profile of ASP2205 fumarate (ASP2205), a novel 5-HT2C receptor agonist, was evaluated in vitro and in vivo. ASP2205 showed potent and selective agonistic activity for the human 5-HT2C receptor, with an EC50 of 0.85 nM in the intracellular Ca2+ mobilization assay. Rat 5-HT2C receptor was also activated by ASP2205 with an EC50 of 2.5 nM. Intraduodenal administration (i.d.) of ASP2205 (0.1-1 mg/kg) significantly elevated the leak point pressure (LPP) in anesthetized rats in a dose-dependent manner. This ASP2205 (0.3 mg/kg i.d.)-induced LPP elevation was inhibited by SB242084 (0.3 mg/kg i.v.), a selective 5-HT2C receptor antagonist. Urethral closure responses induced by intravesical pressure loading in rats were enhanced by ASP2205 (0.3 mg/kg i.v.), which was abolished by pretreatment with SB242084 (0.3 mg/kg i.v.) and bilateral transection of the pudendal nerve. In contrast, ASP2205 (0.3 mg/kg i.v.) did not change the resting urethral pressure in rats. These results indicate that ASP2205 can enhance the pudendal nerve-mediated urethral closure reflex via the 5-HT2C receptor, resulting in the prevention of involuntary urine loss.

Design, synthesis, and biological evaluation of novel biphenyl-4-carboxamide derivatives as orally available TRPV1 antagonists.

A new series of transient receptor potential vanilloid type 1 (TRPV1) antagonists were designed and synthesized from N-(3-hydroxyphenyl)-2-(piperidin-1-ylmethyl)biphenyl-4-carboxamide hydrochloride (8). SAR studies identified (R)-N-(1-methyl-2-oxo-1,2,3,4-tetrahydro-7-quinolyl)-2-[(2-methylpyrrolidin-1-yl)methyl]biphenyl-4-carboxamide hydrochloride (ASP8370, 7), as a compound with high aqueous solubility, satisfactory stability in human liver microsomes, and reduced CYP3A4 inhibition. ASP8370 was selected as a clinical development candidate with significant ameliorative effects on neuropathic pain. SAR studies also revealed the structural mechanisms underlying the switching between TRPV1 antagonism and agonism.

Structural basis for recognition of cognate tRNA by tyrosyl-tRNA synthetase from three kingdoms.

The specific aminoacylation of tRNA by tyrosyl-tRNA synthetases (TyrRSs) relies on the identity determinants in the cognate tRNA(Tyr)s. We have determined the crystal structure of Saccharomyces cerevisiae TyrRS (SceTyrRS) complexed with a Tyr-AMP analog and the native tRNA(Tyr)(GPsiA). Structural information for TyrRS-tRNA(Tyr) complexes is now full-line for three kingdoms. Because the archaeal/eukaryotic TyrRSs-tRNA(Tyr)s pairs do not cross-react with their bacterial counterparts, the recognition modes of the identity determinants by the archaeal/eukaryotic TyrRSs were expected to be similar to each other but different from that by the bacterial TyrRSs. Interestingly, however, the tRNA(Tyr) recognition modes of SceTyrRS have both similarities and differences compared with those in the archaeal TyrRS: the recognition of the C1-G72 base pair by SceTyrRS is similar to that by the archaeal TyrRS, whereas the recognition of the A73 by SceTyrRS is different from that by the archaeal TyrRS but similar to that by the bacterial TyrRS. Thus, the lack of cross-reactivity between archaeal/eukaryotic and bacterial TyrRS-tRNA(Tyr) pairs most probably lies in the different sequence of the last base pair of the acceptor stem (C1-G72 vs G1-C72) of tRNA(Tyr). On the other hand, the recognition mode of Tyr-AMP is conserved among the TyrRSs from the three kingdoms.

Crystallization and preliminary X-ray crystallographic analysis of yeast tyrosyl-tRNA synthetase complexed with its cognate tRNA.

Yeast tyrosyl-tRNA synthetase (yTyrRS) has been crystallized by the vapor diffusion method in the presence of its cognate tRNA(Tyr). The crystals belong to a tetragonal space group P4(1)2(1)2 with cell dimensions of a = b = 63.85 Angstrom, and c = 330.3 Angstrom. The asymmetric unit contains one molecule each of yTyrRS and tRNA(Tyr) (one-half of a 2:2 complex). X-ray diffraction data have been collected up to 2.5 Angstrom resolution.

Phylogenetic Clades 6 and 8 of Enterohemorrhagic Escherichia coli O157:H7 With Particular stx Subtypes are More Frequently Found in Isolates From Hemolytic Uremic Syndrome Patients Than From Asymptomatic Carriers.

BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC) O157:H7 infection causes severe diseases such as bloody diarrhea and hemolytic uremic syndrome (HUS). Although EHEC O157:H7 strains have exhibited high genetic variability, their abilities to cause human diseases have not been fully examined. METHODS: Clade typing and stx subtyping of EHEC O157:H7 strains, which were isolated in Japan during 1999-2011 from 269 HUS patients and 387 asymptomatic carriers (ACs) and showed distinct pulsed-field gel electrophoresis patterns, were performed to determine relationships between specific lineages and clinical presentation. RESULTS: Clades 6 and 8 strains were more frequently found among the isolates from HUS cases than those from ACs (P = .00062 for clade 6, P < .0001 for clade 8). All clade 6 strains isolated from HUS patients harbored stx2a and/or stx2c, whereas all clade 8 strains harbored either stx2a or stx2a/stx2c. However, clade 7 strains were predominantly found among the AC isolates but less frequently found among the HUS isolates, suggesting a significant association between clade 7 and AC (P < .0001). Logistic regression analysis revealed that 0-9 year old age is a significant predictor of the association between clade 8 and HUS. We also found an intact norV gene, which encodes for a nitric oxide reductase that inhibits Shiga toxin activity under anaerobic condition, in all clades 1-3 isolates but not in clades 4-8 isolates. CONCLUSIONS: Early detection of EHEC O157:H7 strains that belonged to clades 6/8 and harbored specific stx subtypes may be important for defining the risk of disease progression in EHEC-infected 0- to 9-year-old children.

Genetic characterization of Escherichia coli O157: H7/- strains carrying the stx2 gene but not producing Shiga toxin 2.

Nine Escherichia coli O157: H7/- strains isolated primarily from non-clinical sources in Thailand and Japan carried the stx(2) gene but did not produce Stx2 toxin in a reversed passive latex agglutination (RPLA) assay. A strain (EDL933) bearing a stx(2) phage (933W) was compared to a strain (Thai-12) that was Stx2-negative but contained the stx(2) gene. To study the lack of Stx2 production, the Thai-12 stx(2) gene and its upstream nucleotide sequence were analyzed. The Thai-12 stx(2) coding region was intact and Stx2 was expressed from a cloned stx(2) gene using a plasmid vector and detected using RPLA. A lacZ fusion analysis found the Thai-12 stx(2) promoter non-functional. Because the stx(2) gene is downstream of the late promoter in the stx(2) phage genome, the antitermination activity of Q protein is essential for strong stx(2) transcription. Thai-12 had the q gene highly homologous to that of Phi21 phage but not to the 933W phage. High-level expression of exogenous q genes demonstrated Q antitermination activity was weak in Thai-12. Replication of stx(2) phage was not observed in Stx2-negative strains. The q-stx(2) gene sequence of Thai-12 was well conserved in all Stx2-negative strains. A PCR assay to detect the Thai-12 q-stx(2) sequence demonstrated that 30% of O157 strains from marketed Malaysian beef carried this sequence and they produced little or no Stx2. These results suggest that stx(2)-positive O157 strains that produce little or no Stx2 may be widely distributed in the Asian environment.

First synthesis and anticancer activity of phosmidosine and its related compounds.

This paper describes the first synthesis of phosmidosine and phosmidosine B, i.e., nucleotide antibiotics composed of 8-oxoadenosine and L-proline which are connected via a unique N-acyl phosphoramidate linkage. Phosmidosine has a yet-undetermined chiral center at the phosphorus atom of the N-acyl phosphoramidate linkage. Phosmidosine B is a demethylated phosmidosine derivative with no chirality on the phosphorus. Phosmidosine B was successfully synthesized by the reaction of an N-acetyl-8-oxoadenosine 5'-O-phosphoramidite derivative with an N-protected prolinamide in the presence of 5-(3,5-dinitrophenyl)-1H-tetrazole. The successful synthesis of phosmidosine was achieved by use of a tert-butoxycarbonyl (Boc) group, which was found to be selectively introduced into the 7-NH function of 8-oxoadenosine and to serve as a pseudo-protecting group due to its steric effect in such manner that the unmasked 6-amino group was not phosphitylated. Final coupling reaction of the 8-oxoadenosine 5'-phosphoramidite derivative with N-tritylprolinamide followed by full deprotection gave a mixture of phosmidosine and its diastereoisomer. The (13)C NMR spectra of the diastereomers suggest that the slow-eluted diastereomer 1b is the naturally occurring phosmidosine. The growth inhibitory activity of phosmidosine 1b, its diastereomer 1a, and phosmidosine B in various tumor cell lines was evaluated by the MTT assay. As the result, phosmidosine B showed high anticancer activities and both the diastereomers 1a and 1b of phosmidosine isolated were found to have similar but approximately 10 times higher anticancer activities than phosmidosine B. Moreover, it turned out that these phosmidosine derivatives showed characteristic inhibitory activities against cancer cells independent of their p53 phenotypes. These results suggest that phosmidosine and its related compounds would be promising as a new type of anticancer agents having a wide range of inhibitory activities against tumor cells.