Synthesis and structure–activity relationship of 2-phenyliminochromene derivatives as inhibitors for aldo–keto reductase (AKR) 1B10

Inhibitors of a human member (AKR1B10) of the aldo–keto reductase superfamily are regarded as promising therapeutics for the treatment of cancer. Recently, we have discovered (Z)-2-(4-methoxyphenylimino)-7-hydroxy-N-(pyridin-2-yl)-2H-chromene-3-carboxamide (1) as the potent competitive inhibitor using the virtual screening approach, and proposed its 4-methoxy group on the 2-phenylimino moiety as an essential structural prerequisite for the inhibition. In this study, 18 derivatives of 1 were synthesized and their inhibitory potency against AKR1B10 evaluated. Among them, 7-hydroxy-2-(4-methoxyphenylimino)-2H-chromene-3-carboxylic acid benzylamide (5n) was the most potent inhibitor showing a K_i value of 1.3 nM. The structure–activity relationship of the derivatives indicated that the 7-hydroxyl group on the chromene ring, but not the 4-methoxy group, was absolutely required for inhibitory activity, The molecular docking of 5n in AKR1B10 and site-directed mutagenesis of the enzyme residues suggested that the hydrogen-bond interactions between the 7-hydroxyl group of 5n and the catalytic residues (Tyr49 and His111) of the enzyme, together with a π-stacking interaction of the benzylamide moiety of 5n with Trp220, are important for the potent inhibition.     

Locally disordered conformer of the hamster prion protein: a crucial intermediate to PrPSc?

A crucial step for transformation of the normal cellular isoform of the prion protein (PrP(C)) to the infectious prion protein (PrP(Sc)) is thought to entail a previously uncharacterized intermediate conformer, PrP*, which interacts with a template PrP(Sc) molecule in the conversion process. By carrying out (15)N-(1)H two-dimensional NMR measurements under variable pressure on Syrian hamster prion protein rPrP(90-231), we found a metastable conformer of PrP(C) coexisting at a population of approximately 1% at pH 5.2 and 30 degrees C, in which helices B and C are preferentially disordered. While the identity is still unproven, this observed metastable conformer is most logically PrP* or a closely related precursor. The structural characteristics of this metastable conformer are consistent with available immunological and pathological information about the prion protein.     

Mutualistic association of Photorhabdus asymbiotica with Japanese heterorhabditid entomopathogenic nematodes

Gram-negative bacteria, Photorhabdus luminescens and P. temperata, form a mutualistic association with entomopathogenic heterorhabditid nematodes while P. asymbiotica is known as an opportunistic human pathogen that causes disseminated bacteremic spread on two continents, the United States and Australia. In the course of our phylogenetic study of Photorhabdus bacteria associated with Japanese Heterorhabditis nematodes, we found two Photorhabdus isolates (Photorhabdus sp. Cbkj163 and OnIr40) whose partial 16S rRNA gene sequence showed high similarities to clinical isolates of this pathogen from Heterorhabditis indica. The phylogenetic study, based upon the gyrase subunit B gene sequences of the two isolates, revealed clustering with these clinical isolates of P. asymbiotica from both the United States and Australia but not with other Photorhabdus bacteria associated with nematodes. The two bacterial isolates were also found to share microbiological and biochemical characteristics with clinical and entomopathogenic Photorhabdus strains. Moreover, not only the two novel Photorhabdus isolates but also an Australian clinical isolate of P. asymbiotica formed mutualistic association with H. indica isolates. These data suggest that the bacteria isolated from H. indica CbKj163 and OnIr40 are a novel subspecies of P. asymbiotica, and that some clinical isolates of P. asymbiotica could have originated from bacteria associated with entomopathogenic nematodes.     

Metastatic Recurrence in a Pancreatic Cancer Patient Derived Orthotopic Xenograft (PDOX) Nude Mouse Model Is Inhibited by Neoadjuvant Chemotherapy in Combination with Fluorescence-Guided Surgery with an Anti-CA 19-9-Conjugated Fluorophore

The aim of this study is to determine the efficacy of neoadjuvant chemotherapy (NAC) with gemcitabine (GEM) in combination with fluorescence-guided surgery (FGS) on a pancreatic cancer patient derived orthotopic xenograft (PDOX) model. A PDOX model was established from a CA19-9-positive, CEA-negative tumor from a patient who had undergone a pancreaticoduodenectomy for pancreatic adenocarcinoma. Mice were randomized to 4 groups: bright light surgery (BLS) only; BLS+NAC; FGS only; and FGS+NAC. An anti-CA19-9 or anti-CEA antibody conjugated to DyLight 650 was administered intravenously via the tail vein of mice with the pancreatic cancer PDOX 24 hours before surgery. The PDOX was brightly labeled with fluorophore-conjugated anti-CA19-9, but not with a fluorophore-conjugated anti-CEA antibody. FGS was performed using the fluorophore-conjugated anti-CA19-9 antibody. FGS had no benefit over BLS to prevent metastatic recurrence. NAC in combination with BLS did not convey an advantage over BLS to prevent metastatic recurrence. However, FGS+NAC significantly reduced the metastatic recurrence frequency to one of 8 mice, compared to FGS only after which metastasis recurred in 6 out of 8 mice, and BLS+NAC with metastatic recurrence in 7 out of 8 mice (p = 0.041). Thus NAC in combination with FGS can reduce or even eliminate metastatic recurrence of pancreatic cancer sensitive to NAC. The present study further emphasizes the power of the PDOX model which enables metastasis to occur and thereby identify the efficacy of NAC in combination with FGS on metastatic recurrence.     

Novel indole and benzothiophene ring derivatives showing differential modulatory activity against human epithelial sodium channel subunits,ENaC β and γ

The epithelial sodium channel (ENaC) plays a pivotal role in sodium homeostasis, and the development of drugs that modulate ENaC activity is of great potential therapeutic relevance. We screened 6100 chemicals for their ability to activate sodium permeability of ENaC. We used a two-step strategy: a high throughput cell-based assay and an electrophysiological assay. Five compounds were identified showing common structural features including an indole or benzothiophene ring. ENaC consists of three subunits: α, β, and γ. Changing the heteromeric combination of human and mouse ENaC αβγ subunits, we found that all five compounds activated the human β subunit but not the mouse subunit. However, four of them exhibited lower activity when the human γ subunit was substituted by the mouse γ subunit. Our findings provide a structural basis for designing human ENaC activity modulators.

Abbreviations: ENaC: Epithelial sodium channel; ΔRFU: delta relative fluorescence units; EC₅₀: Half-maximal effective concentration; E_max: maximum effect value.     

Heavy-ion beam irradiation is an effective technique for reducing major allergens in peanut seeds

Heavy-ion beam irradiation is an effective technique for mutation breeding to produce new cultivars. Heavy-ion beams have high linear energy transfer capable of breaking the double-stranded DNA molecules, thus inducing stable knockout mutants. Here, we report the first application of this technology to produce hypoallergenic peanut lacking major allergens, Ara h 2 and 3, from the Japanese Nakateyutaka variety. After irradiation with either N or C heavy-ion beams at a dose of 100?Gy, seventeen knockout mutants from 11,335 screened M2 seeds were obtained, eight of which lacked either one of the two isoforms of Ara h 2, and the other nine lacked one of the isoforms of Ara h 3. This result indicates that heavy-ion beam irradiation is a powerful means of producing knockout hypoallergenic peanuts.     

Synthesis and Evaluation of DMPO-Type Spin Traps

The spin traps substituted with some groups at the 4-position of dimethyl-1-pyrroline N-oxide(DMPO) were compared with DMPO itself regarding their abilities as spin traps and their physical properties. 4,5,5-Trimethyl-l-pyroHine N-oxide (4MDMPO) and 5,5-dimethyl- 4-phenyl-l-pyrolline N-oxide (4PDMPO) were synthesized by the Bonnett method, and 5,5-dimethyl-4-hydroxymethyl-l-pyrolline N-oxide (4HMDMPO) was made by a unique method from 2(5H)-furanone. The melting points of 4MDMPO, 4PDMPO and 4HMDMPO were higher than that of DMPO. The magnitude of hydrophilicity was in the order of 4HMDMPO, DMPO, 4MDMPO, and 4PDMPO based on the partition coefficient experiments in a 1-octanol-water system. Several radicals, O₂, HO-, -CH₃, -CH₂OH, -CH(CH₃)OH, (CH₃)₃ CO and H radicals, were trapped with these DMPO derivatives for comparison with the trapping by DMPO itself. Spin adducts of O J with the three DMPO derivatives showed ESR spectra similar to that of DMPO. In spite of the formation of diastereomers arising from spin trapping, the line-width enlargement was very small. The intensities and the decay rates of the spectra of 4MDMPO-O₂^-, 4PDMPO-O₂^- 4HMDMPO-O₂^- and DMPO-O₂^- were almost equal. In the trapping of the OH radical by 4MDMPO, 4PDMPO and 4HMDMPO, the eight-line ESR spectra observed were different from the well-known four-line spectrum of DMPO-OH.     

Group VIB Ca2+-independent phospholipase A2gamma promotes cellular membrane hydrolysis and prostaglandin production in a manner distinct from other intracellular phospholipases A2

Although group VIA Ca2+-independent phospholipase A2beta (iPLA2beta) has been implicated in various cellular events, the functions of other iPLA2 isozymes remain largely elusive. In this study, we examined the cellular functions of group VIB iPLA2gamma. Lentiviral transfection of iPLA2gamma into HEK293 cells resulted in marked increases in spontaneous, stimulus-coupled, and cell death-associated release of arachidonic acid (AA), which was converted to prostaglandin E2 with preferred cyclooxygenase (COX)-1 coupling. Conversely, treatment of HEK293 cells with iPLA2gamma small interfering RNA significantly reduced AA release, indicating the participation of endogenous iPLA2gamma. iPLA2gamma protein appeared in multiple sizes according to cell types, and a 63-kDa form was localized mainly in peroxisomes. Electrospray ionization mass spectrometry of cellular phospholipids revealed that iPLA2gamma and other intracellular PLA2 enzymes acted on different phospholipid subclasses. Transfection of iPLA2gamma into HCA-7 cells also led to increased AA release and prostaglandin E2 synthesis via both COX-1 and COX-2, with a concomitant increase in cell growth. Immunohistochemistry of human colorectal cancer tissues showed elevated expression of iPLA2gamma in adenocarcinoma cells. These results collectively suggest distinct roles for iPLA2beta and iPLA2gamma in cellular homeostasis and signaling, a functional link between peroxisomal AA release and eicosanoid generation, and a potential contribution of iPLA2gamma to tumorigenesis.     

High pressure NMR reveals a variety of fluctuating conformers in beta-lactoglobulin

High pressure 1H/15N two-dimensional NMR spectroscopy has been used to study conformational fluctuation in bovine beta-lactoglobulin at pH 2.0 and 36 degrees C. Pressure dependencies of 1H and 15N chemical shifts and cross-peak intensities were analyzed at more than 80 independent atom sites between 30 and 2000 bar. Unusually large and non-linear chemical shift pressure dependencies are found for residues centering in the hydrophobic core region, suggesting the existence of low-lying excited native states (N') of the protein. Measurement of 1H/15N cross-peak intensities at individual amide sites as a function of pressure suggests that unfolding events occur independently in two sides of the beta-barrel, i.e. the hydrophobic core side (betaF-H) (producing I2) and the non-core side (betaB-E) (producing I1). At 1 bar the stability is higher for the core region (DeltaG0 = 6.5(+/-2.0) kcal/mol) than for the non-core region (4.6(+/-1.3) kcal/mol), but at high pressure the stability is reversed due to a larger DeltaV value of unfolding for the core region (90.0(+/-35.2) ml/mol) than that for the non-core region (57.4(+/-14.4) ml/mol), possibly due to an uneven distribution of cavities. The DeltaG0 profile along the amino acid sequence obtained from the pressure experiment is found to coincide well with that estimated from hydrogen exchange experiments. Altogether, the high pressure NMR experiment has revealed a variety of fluctuating conformers of beta-lactoglobulin, notably N, N', I1, I2 and the totally unfolded conformer U. Fluctuation of N to I1 and I2 conformers with open barrel structures could be a common design of lipocalin family proteins which bind various hydrophobic compounds in its barrel structure.     

Identification of novel hypocholesterolemic peptides derived from bovine milk beta-lactoglobulin

This study was designed to clarify the mechanisms of hypocholesterolemic action of beta-lactoglobuline tryptic hydrolysate (LTH) and to identify the novel hypocholesterolemic peptide derived from LTH by screening using Caco-2 cells and animal studies. Serum and liver cholesterol levels were significantly lower in rats fed LTH than in those fed casein tryptic hydrolysate (CTH). The present study suggests that the inhibition of micellar solubility of cholesterol which causes the suppression of cholesterol absorption by a direct interaction between cholesterol mixed micelles, and LTH in the jejunal epithelia is part of the mechanism underlying the hypocholesterolemic action of LTH. Though no one could trace the hypocholesterolemic peptide to any protein origin, we identified, for the first time, a novel hypocholesterolemic peptide, Ile-Ile-Ala-Glu-Lys (IIAEK). Surprisingly, the present study provides the first direct evidence that a new hypocholesterolemic peptide derived from beta-lactoglobuline can powerfully influence serum cholesterol levels and exhibit a greater hypocholesterolemic activity in comparison with that of medicine, beta-sitosterol, in animal studies.