A series of nonsecosteroidal vitamin D receptor agonists for osteoporosis therapy

In an extension of our study on gamma hydroxy carboxylic acid analogs, we explored a series of nonsecosteroidal vitamin D receptor (VDR) agonists in which 1,3-diol of 1,25(OH)₂D₃ had been replaced by aryl acetic acid. These analogs showed very potent activity in vitro compared with 1,25(OH)₂D₃. An X-ray analysis of 8d showed that the inserted phenyl ring well mimicked the folded methylene linker of the gamma hydroxy carboxylic acid moiety but the carboxylic acid of 8d interacted with VDR in a different manner from gamma hydroxy carboxylic acids. Through our in vivo screening in an osteoporosis rat model using immature rats, we identified a potent active vitamin D₃ analog, compound 7e. In mature rats of the same model, compound 7e also showed good PK profiling and excellent ability to prevent bone mineral density loss without severe hypercalcemia. Our nonsecosteroidal VDR agonist 7e (CH5036249) could be a possible new drug candidate for treating osteoporosis in human.     

A novel,potent, and orally active VLA-4 antagonist with good aqueous solubility: trans-4-[1-[[2-(5-Fluoro-2-methylphenylamino)-7-fluoro-6-benzoxazolyl]acetyl]-(5S)-[methoxy(methyl)amino]methyl-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylic acid

We have carried out the optimization of substituents at the C-3 or the C-5 position on the pyrrolidine ring of VLA-4 antagonist 3 with 2-(phenylamino)-7-fluorobenzoxazolyl moiety for the purpose of improving in vivo efficacy while maintaining good aqueous solubility. As a result, we successfully increased in vitro activity in the presence of 3% human serum albumin and achieved an exquisite lipophilic and hydrophilic balance of compounds suitable for oral administrative regimen. The modification resulted in the identification of zwitterionic compound 7n with (5S)-[methoxy(methyl)amino]methylpyrrolidine, which significantly alleviated bronchial hyper-responsiveness to acetylcholine chloride at 12.5 mg/kg, p.o. in a murine asthma model and showed favorable aqueous solubility (JP1, 89 μg/mL; JP2, 462 μg/mL). Furthermore, this compound showed good oral bioavailability (F = 54%) in monkeys.     

Synthesis and properties of thymidines with six-membered amide bridge

Artificial thymidine monomers possessing amide or N-methylamide bridges were designed, synthesized, and introduced into oligonucleotides. UV-melting experiments showed that these oligonucleotides preferred single-stranded RNA (ssRNA) to single-stranded DNA (ssDNA) in duplex formation. Both amide- and N-methylamide-modified oligonucleotides led to a significant increase in the binding affinity to ssRNA by up to +4.7 and +3.7 °C of the T_m value per modification, respectively, compared with natural oligonucleotide. In addition, their oligonucleotides showed high stability against 3′-exonuclease.     

Purifications and Enzymatic Properties of β-Amylase and Pullulanase from Bacillus cereus var. mycoides

A β-amylase and a pullulanase produced by Bacillus cereus var. mycoides were purified by means of ammonium sulfate fractionation, adsorption on starch and celite and Sephadex G–100 column chromatography. The purified enzymes were homogeneous in disc electrophoresis.

The β-amylase released only maltose from amylose, amylopectin, starch and glycogen, and the released maltose was in β-form. The pullulanase released maltose, maltotriose and maltotetraose from β-limit dextrin and maltotriose from pullulan, but not amylose-like substance from amylopectin.

The optimum pHs of β-amylase and pullulanase were about 7 and 6~6.5, respectively. The optimum temperatures of the enzymes were about 50°C. The enzymes were inhibited by the sulfhydryl reagents such as mercuric chloride and p-chloromercuribenzoate, and the inhibitions with p-chloromercuribenzoate were restored by the addition of cysteine. The molecular weights of β-amylase and pullulanase were estimated to be 35,000±5,000 and 110,000±20,000, respectively.     

Quantification of Physical and Cyto-physiological Conditions for the Electrofusion of Saccharomyces cerevisiae

Various conditions for obtaining hybrids of the auxotrophic mutants SH1509 and SH1512 of Saccharomyces cerevisiae by electrofusion were investigated. An AC field of 400 V_p/cm and a DC field of 2 square pulses (7 kV/cm; 60/βsec each) at an interval of 0.5 sec were effective. Treatment with 0.2 (SH1509) or l.0 mg/ml (SH1512) Zymolyase for 1 or 1.5 hr was essential. As to the molarity of the osmotic stabilizer (sorbitol), the hybrid yield peaked at 0.6 m. The presence of CaCl₂ (up to 0.4 mm) or 0.1 mm CaCl₂ with 0.1 mm MgCl₂ enhanced the yield. The temperature of the spheroplast suspension during pulsations also affected the yield, the most suitable temperature being 28°C.     

Characterization of d-Alanyl-(d)-meso-2,6-diaminopimelic Acid Endopeptidase from Streptomyces globisporus 1829

d-Alanyl-(d)-meso-2,6-diaminopimelic acid endopeptidase was purified 47.4-fold with a yield of 40.5% from mutanolysin, which was partially purified from the cultural supernatant of Streptomyces globisporus 1829, by using ion exchange column chromatographies and a molecular sieve column. The purified enzyme was electrophoretically homogeneous. This enzyme had a molecular weight of 13,500 and an isoelectric point of pI 9.0. This enzyme was most active at pH 8.5 and stable between pHs 8.0 and 9.0. The hydrolyzing activity of this enzyme was enhanced by Co^{+ +} and Ca^{+ +} but inhibited appreciably by Zn^{+ +}, Cu^{+ +} and EDTA. The enzyme activity was not affected by β-lactam antibiotics and vancomycin. The Km values for bisdisaccharide heptapeptide and its derivative modified chemically by BOC-S were calculated to be 5.7 × 10^-4 and 4.0 × 10^-4 m, respectively.     

Preparation and Enzymatic Hydrolysis of Maltosyl-α-cyclodextrin

Maltosyl-α-cyclodextrin (6-α-maltosylcyclomaltohexaose, M-CD) was prepared from maltose and α-cyclodextrin by the reverse action of Bacillus pullulanase, and the action of α-amylases on this dextrin was examined. Among α-amylases tested, Thermoactinomyces vulgaris α-amylase (TVA) and Taka-amylase A (TAA) were found to attack the M-CD. Their action pattern on M-CD was studied. These α-amylases cleaved, first the cyclodextrin ring of M-CD, and the branched octasaccharides formed were immediately degraded to form glucose, branched tetraose, or pentaose, though the action pattern was different for TVA and TAA. In addition, TAA also split M-CD into glucose and glucosyl-α-cyclodextrin. Fission products at various stages of the reaction were separated and analyzed by paper chromatography and high performance liquid chromatography, their structures were analyzed, and the degradation pattern of M-CD was found.     

General Properties of Endo-N-acetylmuramidase of Bacillus subtilis YT-25

The enzymatic behaviour, amino acid composition and some physical properties of a new endo-N-acetylmuramidase (B-enzyme) of Bacillus subtilis YT–25 were determined and compared with hen’s egg white lysozyme. The molecular weight was estimated to be about 13000 by the sedimentation equilibrium method. The isoelectric point was pH 9.8. The amino acid composition indicates that the enzyme is rich in basic amino acids, especially lysin. Maximal activity on the lysis of cell walls of M. lysodeikticus occurred at pH 6.2. The enzyme was stable at pH 3.5 ~ 6.0. The specific activity for the lysis of cell walls of M. lysodeikticus was less than fourth part of that of hen’s egg white lysozyme. Digest of cell walls of M. lysodeikticus with B-enzyme consisted greater numbers of high molecular products than digest with egg white lysozyme. Substrate specificity of B-enzyme seemed to be different from that of egg white lysozyme.     

Formation of Glucose Isomerase by Streptomyces sp.

Sophoradin (I) [2′,4,4′-trihydroxy-3,3′,5-tris(3-methyl-2-butenyl)chalcone] which had been isolated from “Guang-Dou-Gen” (the root of Sophora subprostrata Chun et T. Chen) was synthesized through Claisen rearrangement. The reaction of p-hydroxybenzaldehyde and 3-chloro-3-methyl-1-butyne (III) gave 4-(1,1-dimethylpropargyloxy)benzaldehyde (VIII), which was catalytically hydrogenated over Lindlar catalyst to afford 4-(1,1-dimethylallyloxy)benzaldehyde (IX). IX was converted to 4-hydroxy-3-(3-methyl-2-butenyl)benzaldehyde (X) by Claisen rearrangement. The reaction of X and III gave 3-(3-methyl-2-butenyl)-4-(1,1-dimethylpropargyloxy)benzaldehyde (XI). Condensation of 2-hydroxy-4-(1,1-dimethylpropargyloxy)acetophenone (IV) and XI in alkaline solution gave a chalcone (XIII), which was catalytically hydrogenated over Lindlar catalyst to give 2′-hydroxy-4,4′-bis(1,-dimethylallyloxy)-3-(3-methyl-2-butenyl)chalcone (XIV). XIV was converted to I by Claisen rearrangement.     

SIZ1 deficiency causes reduced stomatal aperture and enhanced drought tolerance via controlling salicylic acid‐induced accumulation of reactive oxygen species in Arabidopsis

Transpiration and gas exchange occur through stomata. Thus, the control of stomatal aperture is important for the efficiency and regulation of water use, and for the response to drought. Here, we demonstrate that SIZ1‐mediated endogenous salicylic acid (SA) accumulation plays an important role in stomatal closure and drought tolerance. siz1 reduced stomatal apertures. The reduced stomatal apertures of siz1 were inhibited by the application of peroxidase inhibitors, salicylhydroxamic acid and azide, which inhibits SA‐dependent reactive oxygen species (ROS) production, but not by an NADPH oxidase inhibitor, diphenyl iodonium chloride, which inhibits ABA‐dependent ROS production. Furthermore, the introduction of nahG into siz1, which reduces SA accumulation, restored stomatal opening. Stomatal closure is generally induced by water deficit. The siz1 mutation caused drought tolerance, whereas nahG siz1 suppressed the tolerant phenotype. Drought stresses also induced expression of SA‐responsive genes, such as PR1 and PR2. Furthermore, other SA‐accumulating mutants, cpr5 and acd6, exhibited stomatal closure and drought tolerance, and nahG suppressed the phenotype of cpr5 and acd6, as did siz1 and nahG siz1. Together, these results suggest that SIZ1 negatively affects stomatal closure and drought tolerance through the accumulation of SA.