A rapid competitive binding nonseparation electrochemical enzyme immunoassay (NEEIA) test strip for microcystin-LR (MCLR) determination

A competitive binding nonseparation electrochemical enzyme immunoassay (NEEIA) is described for the determination of microcystin-LR (MCLR) using a double-sided microporous gold electrode in cartridge-type cells. A gold film sputtered on one side of porous nylon membrane constitutes a working electrode, while another gold film formed on the opposite side serves as a pseudo reference electrode. After immobilizing MCLR antibody on working electrode by physical adsorption, the double-sided electrode was placed simply in a diffusion U-type or within a dry strip-type cell with a conjugate pad pre-loaded with a glucose oxidase labeled MCLR (GOx-MCLR) on working electrode side. Assays were performed in two steps: an MCLR-containing sample mixed with a known amount of GOx-MCLR conjugate either in buffer solution or in pre-loaded dry pad was incubated for an appropriate period (about 10 min) to induce competitive reaction with an immobilized anti-MCLR antibody on working electrode, and a fixed concentration of glucose solution (substrate) was then added to the backside of the working electrode. Due to the competitive nature of the assay, enzymatically generated product, hydrogen peroxide (H2O2), was detected at the working gold electrode (at +800 mV versus Au) by oxidation, and the magnitude of amperometric current was inversely proportional to the concentration of MCLR in the sample. The response time after substrate addition was about 30s. Mean recovery of MCLR added to tap water was 93.5%, with a coefficient of variation (CV) of 6.6%. The proposed competitive NEEIA system is in general comparable to existing heterogeneous enzyme immunoassays with a similar detection limit (100 pg/mL MCLR), and suitable for developing a disposable type biosensor for on-site monitoring of environment.     

Parallel synthesis and SAR study of novel oxa-steroids as potent and selective progesterone receptor antagonists

Efficient parallel synthesis of novel 7-oxa-steroids 4 has been achieved from the key intermediate 3 via a one-pot four-step sequence. oxa-Steroids 4 with various ortho-, meta-, and para-monosubstituents on the phenyl ring, as well as disubstituted phenyl and heterocycles, were evaluated for progesterone receptor (PR) and glucocorticoid receptor (GR) antagonist activities. SAR study demonstrated that the para-fluorinated substituents on the phenyl ring not only increased the potency for PR in a T47D cell functional assay, but also improved the selectivity over GR in an A549 cell functional assay. The para-fluorophenyl oxa-steroid 4l and the para-trifluoromethylphenyl oxa-steroid 4p were found to be remarkably more potent and more selective PR antagonists than mifepristone, with subnanomolar potency and about 140-fold selectivity over GR. Molecular modeling of the oxa-steroid bound to PR provided meaningful insight for the SAR study. oxa-Steroids 4a and 4b were found to be more efficacious than mifepristone in vivo in a rat uterine complement C3 assay via the oral route, although they were less than or equally potent to mifepristone in the T47D assay.     

HYPERSENSITIVE TO RED AND BLUE 1 and its C-terminal regulatory function control FLOWERING LOCUS T expression

The red and far-red light-absorbing phytochromes and UV-A/blue light-absorbing cryptochromes regulate seedling de-etiolation and flowering responses. The signaling steps that mediate the photoreceptor regulation on key flowering genes remain largely unknown. We report that a previously identified photomorphogenic mutant, hypersensitive to red and blue 1 (hrb1), flowered late and showed attenuated expression of FLOWERING LOCUS T (FT) over both long days and short days. Transgenic plants that overexpress the full-length HRB1, or its C-terminal half, flowered early and accumulated more FT messages under short-day conditions. The transgenic plants also displayed hyposensitive de-etiolation phenotypes, and the expression of these phenotypes requires the action of PIF4. The double mutant of hrb1/cry2 showed a flowering phenotype and an FT expression pattern similar to hrb1 under long-day conditions, suggesting that HRB1 may function downstream of cry2 under long-day conditions. In contrast, hrb1/phyB-9 showed a flowering phenotype and an FT expression pattern similar to phyB-9 over both long days and short days, indicating a modulatory role of HRB1 in the flowering pathway mediated by phyB. Overexpression of HRB1 did not affect the expression of the central clock oscillators, TOC1 and CCA1. HRB1 therefore represents a signaling step that regulates FT expression downstream of red and blue light perception.     

Docking-based 3D-QSAR study for selectivity of DPP4, DPP8, and DPP9 inhibitors

In order to obtain information regarding the design of selective DPP4 inhibitors, a 3D-QSAR study was conducted using DPP4, DPP8, and DPP9 inhibitors including newly synthesized six- and seven-membered cyclic hydrazine derivatives (KR64300, KR64301), which were evaluated in vitro for their inhibition of DPP4, DPP8, and DPP9. In this study, a highly predictive CoMFA model based on the fast-docking for DPP4, DPP8, and DPP9 inhibitors was obtained. This reliable model showed leave-one-out cross-validation q(2) and conventional r(2) values of 0.68 and 0.96 for the DPP4 inhibitors, 0.58 and 0.98 for the DPP8 inhibitors, and 0.68 and 0.97 for the DPP9 inhibitors, respectively. The validation of the CoMFA model was confirmed by the compounds in the test set, including the synthesized six- and seven-membered cyclic hydrazines. According to this study, to obtain selective DPP4 inhibitors compared to their isozymes, the interaction of the inhibitors with the S3 site and S1' site in DPP4 must be considered. The proposed newly synthesized compounds, KR64300 and KR64301, interact well with the sites mentioned above, showing excellent selectivity.     

Discovery of orally bioavailable and novel urea agonists of the high affinity niacin receptor GPR109A

A urea class of high affinity niacin receptor agonists was discovered. Compound 1a displayed good PK, better in vivo efficacy in reducing FFA in mouse than niacin, and no vasodilation in a mouse model. Compound 1q demonstrated equal affinity to GPR109A as niacin.     

Platelet-activating factor (PAF) receptor binding antagonists from Alpinia officinarum

The bioassay-guided purification of ether extracts of Alpinia officinarum led to the isolation of two new compounds 6-hydroxy-1,7-diphenyl-4-en-3-heptanone (1) and 6-(2-hydroxy-phenyl)-4-methoxy-2-pyrone (4) as well as three known compounds 1,7-diphenyl-4-en-3-heptanone (2), 1,7-diphenyl-5-methoxy-3-heptanone (3), and apigenin (5). Their structures were established on the basis of spectral methods. All three diarylheptanoids 1, 2, and 3 exhibited potent PAF receptor binding inhibitory activities with an IC₅₀ of 1.3, 5.0, and 1.6 μM, respectively. These studies have identified diarylheptanoids as a novel class of potent PAF antagonists.     

Modification of cap group in delta-lactam-based histone deacetylase (HDAC) inhibitors

Novel delta-lactam-based HDAC inhibitors which have various substituted benzyl, bi-aromatic cap groups were prepared using ring closure metathesis reaction, and evaluated their HDAC inhibitory activities and anti-proliferative effects. Among prepared analogues, 11m and 11o have very strong HDAC enzymatic inhibition and showed the most potent growth inhibitory activity to five human tumor cell lines including PC-3, ACHN, NUGC-3, HCT-15, and MBA-MB-231 tumor cell lines. Compounds 11m and 11o also showed good tumor growth inhibition of MDA-MB-231 cells in in vivo xenograft model. Structure-activity relationship study using docking model explained the significance of hydrophobic aromatic cap groups for their in vitro activities.     

Synthesis and identification of novel oxa-steroids as progesterone receptor antagonists

A novel series of oxa-steroids 6 derived from (8S, 13S, 14R)-7-oxa-estra-4,9-diene-3,17-dione 1 have been synthesized and identified as potent and selective progesterone receptor antagonists. These novel oxa-steroids showed similar potency to mifepristone. Preliminary SAR study resulted in the most potent 17-phenylethynyl oxa-steroid 6i wih an IC(50) of 1.4nM. In contrast to the equipotent mifepristone toward the progesterone receptor (PR) and glucocorticoid receptor (GR), compound 6i had over 200-fold selectivity for PR over GR.     

Alpha-substituted N-(4-tert-butylbenzyl)-N'-[4-(methylsulfonylamino)benzyl]thiourea analogues as potent and stereospecific TRPV1 antagonists

A series of alpha-substituted N-(4-tert-butylbenzyl)-N'-[4-(methylsulfonylamino)benzyl]thiourea analogues have been investigated as TRPV1 receptor antagonists. alpha-Methyl substituted analogues showed potent and stereospecific antagonism to the action of capsaicin on rat TRPV1 heterologously expressed in Chinese hamster ovary cells. In particular, compounds 14 and 18, which possess the R-configuration, exhibited excellent potencies (respectively, K(i)=41 and 39.2 nM and K(i(ant))=4.5 and 37 nM).