Frontal analysis of cell-membrane chromatography for determination of drug-α1D adrenergic receptor affinity

The aim of the present study was to determine drug-α_1D adrenergic receptor (AR) affinity by frontal analysis of cell-membrane chromatography (CMC). The cell-membrane stationary phase (CMSP) was prepared by immobilizing rat aorta cell membranes on porous silica, and the resulting CMSP was used to determine drug binding affinity to α_1D-AR by frontal analysis. The CMSP of rat aorta was stable and reproducible. Relative binding affinities (dissociation constant, K_d) were determined by frontal chromatography for prazosin (166.13 ± 18.36 nmol), BMY7378 (537.40 ± 30.84 nmol), phentolamine (646.92 ± 23.17 nmol), 5-methylurapidil (725.66 ± 25.48 nmol), oxymetazoline (910.56 ± 40.62 nmol) and methoxamine (1299.27 ± 51.73 nmol). These results were consistent with the affinity rank order and showed a good correlation with the affinity of the same compounds for the cloned α_1D-AR subtype obtained from radioligand-binding assay. The study demonstrates that frontal analysis of CMC may be used for direct determination of drug–receptor binding interactions, and that CMC is an alternative reliable method to quantitatively study ligand–receptor interactions.     

Inhibition of serotonin outflow by nociceptin/orphaninFQ in dorsal raphe nucleus slices from normal and stressed rats: Role of corticotropin releasing factor

In the dorsal raphe nucleus (DRN) many inputs converge and interact to modulate serotonergic neuronal activity and the behavioral responses to stress. The effects exerted by two stress-related neuropeptides, corticotropin releasing factor (CRF) and nociceptin/orphaninFQ (N/OFQ), on the outflow of [³H]5-hydroxytryptamine were investigated in superfused rat dorsal raphe nucleus slices.Electrical stimulation (100 mA, 1 ms for 2 min) evoked a frequency-dependent peak of [³H]5-hydroxytryptamine outflow, which was sodium and calcium-dependent. Corticotropin releasing factor (1–100 nM), concentration-dependently inhibited the stimulation (3 Hz)-evoked [³H]5-hydroxytryptamine outflow; the inhibition by 30 nM corticotropin releasing factor (to 68 ± 5.7%) was prevented both by the non selective CRF receptor antagonist alpha-helicalCRF(9-41) (α-HEL) (300 nM) and by the CRF₁ receptor antagonist antalarmin (ANT) (100 nM). The CRF₂ agonist urocortin II (10 nM) did not modify [³H]5-hydroxytryptamine outflow, ruling out the involvement of CRF₂ receptors. Bicuculline (BIC), a GABA_A antagonist (10 μM), prevented the inhibitory effect of corticotropin releasing factor (30 nM), supporting the hypothesis that the inhibition was mediated by increased γ-aminobutyric acid (GABA) release. Nociceptin/orphaninFQ (1 nM–1 μM) exerted an antalarmin- and bicuculline-insensitive inhibition on [³H]5-hydroxytryptamine outflow, with the maximum at 100 nM (to 63 ± 4.2%), antagonized by the NOP receptor antagonist UFP-101 (1 μM). Dorsal raphe nucleus slices prepared from rats exposed to 15 min of forced swim stress displayed a reduced [³H]5-hydroxytryptamine outflow, in part reversed by antalarmin and further inhibited by nociceptin/orphaninFQ. These findings indicate that (i) both corticotropin releasing factor and nociceptin/orphaninFQ exert an inhibitory control on dorsal raphe nucleus serotonergic neurons; (ii) the inhibition by corticotropin releasing factor involves γ-aminobutyric acid neurons; (iii) nociceptin/orphaninFQ inhibits dorsal raphe nucleus serotonin system in a corticotropin releasing factor- and γ-aminobutyric acid-independent manner; (iv) nociceptin/orphaninFQ modulation is still operant in slices prepared from stressed rats. The nociceptin/orphaninFQ-NOP receptor system could represent a new target for drugs effective in stress-related disorders.     

Binding of diarrheic shellfish poisoning toxins to okadaic acid binding proteins purified from the sponge Halichondria okadai

Okadaic acid (OA) and dinophysistoxin-1 (DTX1) cause diarrheic shellfish poisoning. This article examines the biochemical interactions of the two toxins with novel okadaic acid binding proteins (OABPs) 2.1 and 2.3, originally isolated from the marine sponge Halichondria okadai. First, recombinant OABPs 2.1 and 2.3 were expressed in Escherichia coli BL21 (DE3) cells. Binding assays using [24-³H]OA and the recombinant OABP 2.1 or 2.3 demonstrated the dissociation constant K_d of 1.30 ± 0.56 nM and 1.54 ± 0.35 nM, respectively. Binding of [24-³H]okadaic acid to recombinant OABP2.1 was almost equally replaced with OA and DTX1. OA-induced cytotoxicity in mouse leukemia P388 cells was inhibited in the presence of the recombinant OABPs 2.1 and 2.3 with an EC₅₀ of 92 ± 8.4 nM and 87 ± 13 nM, respectively. These results suggest that the blockage of OA-induced cytotoxicity by OABPs 2.1 and 2.3 may be involved in regulating symbiotic relationships present in the sponge H. okadai.     

Kinematics of cross-country sit skiing during a Paralympic race

The study had three purposes: to verify a hypothesized speed decrease during the 15 km cross-country sit skiing (CCSS) race; documenting this possible fatigue effect (speed decrease), to evaluate changes among the four laps in kinematics parameters (cycle speed, cycle duration, cycle length, duty cycle (percentage ratio between pushing and total cycle duration), pole inclination, trunk inclination and shoulder–hand distance); to compare the kinematics parameters in cross-country sit skiers of different level. Video recordings were carried out during the 2006 Turin Winter Paralympic Games with two conventional digital video-cameras positioned on a flat and an uphill (8.3°) track, respectively. Better performing skiers (G1) had significantly higher speeds than worse performers (G2) both in the flat (6.54 ± 0.13 vs. 5.89 ± 0.50 m s^?1 and 5.55 ± 0.14 vs. 4.62 ± 0.22 m s^?1 in the first and last lap, respectively) and in the uphill track (3.67 ± 0.45 vs. 3.05 ± 0.59 m s^?1 and 3.20 ± 0.36 vs. 2.26 ± 0.36 m s^?1 in the first and last lap, respectively). The G1 athletes were able to maintain the high-speed better than the G2 over the entire race. Significant differences in cycle length and duty cycle between groups would be justified by the higher physical fitness of G1 skiers.     

Effects of intraspecific competition on growth and photosynthesis of Atriplex prostrata

We investigated the effect of intraspecific competition on growth parameters and photosynthesis of the salt marsh species Atriplex prostrata Boucher in order to distinguish the effects of density-dependent growth inhibition from salt stress. High plant density caused a reduction of 30% in height, 82% in stem dry mass, 80% in leaf dry mass, and 95% in root dry mass. High density also induced a pronounced 72% reduction in leaf area, 29% decrease in length of mature internodes and 50% decline in net photosynthetic rate. The alteration of net photosynthesis paralleled growth inhibition, decreasing from 7.6 ± 0.9 μmol CO₂ m⁻² s⁻¹ at low density to 3.5 ± 0.4 μmol CO₂ m⁻² s⁻¹ at high density, indicating growth inhibition caused by intraspecific competition is mainly due to a decline in net photosynthesis rate. Plants grown at high density also exhibited a reduction in stomatal conductance from 0.7 ± 0.1 mol H₂O m⁻² s⁻¹ at low density to 0.3 ± 0.1 mol H₂O m⁻² s⁻¹ at high density and a reduction in transpiration rate from 6.0 ± 0.3 mmol H₂O m⁻² s⁻¹ at low density to 4.3 ± 0.3 mmol H₂O m⁻² s⁻¹ at high density. Biomass production was inhibited by an increase in plant density, which reduced the rate of photosynthesis, stomatal conductance and leaf area of plants.     

Design,synthesis and biological evaluation of novel non-covalent piperidine-containing peptidyl proteasome inhibitors

A series of novel non-covalent piperidine-containing dipeptidyl derivatives were designed, synthesized and evaluated as proteasome inhibitors. All target compounds were tested for their proteasome chymotrypsin-like inhibitory activities, and selected derivatives were evaluated for the anti-proliferation activities against two multiple myeloma (MM) cell lines RPMI 8226 and MM-1S. Among all of these compounds, eight exhibited significant proteasome inhibitory activities with IC₅₀ less than 20 nM, and four are more potent than the positive control Carfilzomib. Compound 28 displayed the most potent proteasome inhibitory activity (IC₅₀: 1.4 ± 0.1 nM) and cytotoxicities with IC₅₀ values at 13.9 ± 1.8 nM and 9.5 ± 0.5 nM against RPMI 8226 and MM-1S, respectively. Additionally, the ex vivo blood cell proteasome inhibitory activities of compounds 24 and 27–29 demonstrated that the enzymatic metabolism in the whole blood could be well tolerated. All these experiments confirmed that the piperidine-containing non-covalent proteasome inhibitors are potential leads for exploring new anti-cancer drugs.     

Synthesis and Characterization of Time-resolved Fluorescence Probes for Evaluation of Competitive Binding to Melanocortin Receptors

Probes for use in time-resolved fluorescence competitive binding assays at melanocortin receptors based on the parental ligands MSH(4), MSH(7), and NDP-α-MSH were prepared by solid phase synthesis methods, purified, and characterized. The saturation binding of these probes was studied using HEK-293 cells engineered to overexpress the human melanocortin 4 receptor (hMC4R) as well as the human cholecystokinin 2 receptor (hCCK2R). The ratios of non-specific binding to total binding approached unity at high concentrations for each probe. At low probe concentrations, receptor-mediated binding and uptake was discernable, and so probe concentrations were kept as low as possible in determining K_d values. The Eu-DTPA-PEGO-MSH(4) probe exhibited low specific binding relative to non-specific binding, even at low nanomolar concentrations, and was deemed unsuitable for use in competition binding assays. The Eu-DTPA-PEGO probes based on MSH(7) and NDP-α-MSH exhibited K_d values of 27 ± 3.9 nM and 4.2 ± 0.48 nM, respectively, for binding with hMC4R. These probes were employed in competitive binding assays to characterize the interactions of hMC4R with monovalent and divalent MSH(4), MSH(7), and NDP-α-MSH constructs derived from squalene. Results from assays with both probes reflected only statistical enhancements, suggesting improper ligand spacing on the squalene scaffold for the divalent constructs. The K_i values from competitive binding assays that employed the MSH(7)-based probe were generally lower than the K_i values obtained when the probe based on NDP-α-MSH was employed, which is consistent with the greater potency of the latter probe. The probe based on MSH(7) was also competed with monovalent, divalent, and trivalent MSH(4) constructs that previously demonstrated multivalent binding in competitive binding assays against a variant of the probe based on NDP-α-MSH. Results from these assays confirm multivalent binding, but suggest a more modest increase in avidity for these MSH(4) constructs than was previously reported.     

An aromatic amino acid within intracellular loop 2 of the prostaglandin EP2 receptor is a prerequisite for selective association and activation of Gαs

We previously demonstrated that the aromatic moiety of Tyr¹⁴³ within the intracellular loop 2 (ICL2) region of the prostaglandin EP2 receptor plays a crucial role in Gs coupling. Here we investigated whether the ICL2 of the EP2 receptor directly binds to Gαs and whether an aromatic moiety affects this interaction. In Chinese hamster ovary cells, mutations of Tyr¹⁴³ reduced the ability of the EP2 receptor to interact with G proteins as demonstrated by GTPγS sensitivity, as well as the ability of agonist-induced cAMP formation, with the rank order of Phe > Tyr (wild-type) = Trp > Leu > Ala (= 0). We found that the wild-type ICL2 peptide (i2Y) and its mutant with Phe at Tyr¹⁴³ (i2F) inhibited receptor-G protein complex formation of wild-type EP2 in membranes, whereas the Ala-substituted mutant (i2A) did not. Specific interactions between these peptides and the Gαs protein were detected by surface plasmon resonance, but Gαs showed different association rates, with a rank order of i2F > i2Y ≫ i2A, with similar dissociation rates. Moreover, i2F and i2Y, but not i2A activated membrane adenylyl cyclase. These results indicate that the ICL2 region of the EP2 receptor is its potential interaction site with Gαs, and that the aromatic side chain moiety at position 143 is a determinant for the accessibility of the ICL2 to the Gαs protein.     

New potently bioactive alkaloids from Crinum erubescens

Antimalarial bioassay-guided fractionation of the swamp lily Crinum erubescens led to the isolation of four compounds with potent antiplasmodial activity. Compounds 1 and 2 were determined from their spectroscopic data to be the known pesticidal compound cripowellin A and the known pesticidal and antiproliferative compound cripowellin B. 1D and 2D-NMR techniques were used to determine the identities of 3 and 4 as the new compounds cripowellin C and D. A fifth compound was identified as the known alkaloid hippadine, which was inactive against Plasmodium falciparum. The antiplasmodial IC₅₀ values of compounds 1–4 were determined to be 30 ± 2, 180 ± 20, 26 ± 2, and 260 ± 20 nM, respectively, and their antiproliferative IC₅₀ values against the A2780 human ovarian cancer cell line were 11.1 ± 0.4, 16.4 ± 0.1, 25 ± 2, and 28 ± 1 nM.     

DNA binding and photocleavage properties and apoptosis-inducing activities of a ruthenium porphyrin complex [(Py-3′)TPP-Ru(phen)2Cl]Cl and its heterometallic derivatives

The interactions of a ruthenium porphyrin complex [(Py-3′)TPP-Ru(phen)₂Cl]Cl (phen = 1,10-phenanthroline, (Py-3′)TPP = 5-(3′-pyridyl-10,15,20-triphenylporphyrin) (1) and its heterometallic derivatives, [Ni(Py-3′)TPP-Ru(phen)₂Cl][PF₆] (2) and [Cu(Py-3′)TPP-Ru(phen)₂Cl][PF₆] (3), with calf thymus DNA have been investigated by spectroscopic and viscosity measurements in this study. The results showed that these synthetic complexes can bind to double strand helix DNA in groove binding mode, and the intrinsic binding constants of complexes 1, 2 and 3, as calculated according to the decay of the Soret absorption, are (1.35 ± 0.5) ×10⁵ M^?1 (s = 4.2), (1.29 ± 0.5) × 10⁵ M^?1 (s = 5.6) and (1.22 ± 0.5) × 10⁵ M^?1 (s = 6.2) (s is the binding-site size), respectively, which are consistent with those obtained from ethidium bromide-quenching experiments. Further investigations on the photocleavage properties of these complexes on plasmid pBR 322 DNA showed that complexes 1, 2 and 3 could cleave single chain DNA and convert DNA molecules from supercoiled form to the nicked form. As determined by MTT assay, the complexes were also identified as potent antiproliferative agents against A375 human melanoma cells, MCF-7 human breast adrenocarcinoma cells, Colo201 human colon adenocarcinoma cells and HepG2 human liver cancer cells. Complex 1 inhibits the growth of A375 cells through induction of apoptotic cell death and G0/G1 cell cycle arrest. Further investigation on intracellular mechanisms indicated that Complex 1 induced depletion of mitochondrial membrane potential (ΔΨ_m) in A375 cells through regulating the expression of pro-survival and pro-apoptotic Bcl-2 family members. Our results suggest that ruthenium porphyrin complexes could be candidates for further evaluation as chemopreventive and chemotherapeutic agents for human cancers.     

Free fatty acid receptor 1 (GPR40) agonists containing spirocyclic periphery inspired by LY2881835

The free fatty acid receptor 1 (FFA1), a G protein-coupled receptor (GPCR) naturally activated by long-chain fatty acids is a novel target for the treatment of metabolic diseases. The basic amine spirocyclic periphery of Eli Lilly’s drug candidate LY2881835 for treatment of type 2 diabetes mellitus (which reached phase I clinical trials) inspired a series of novel FFA1 agonists. These were designed to incorporate the 3-[4-(benzyloxy)phenyl]propanoic acid pharmacophore core decorated with a range of spirocyclic motifs. The latter were prepared via the Prins cyclization and subsequent modification of the 4-hydroxytetrahydropyran moiety in the Prins product. Here, we synthesize 19 compounds and test for FFA1 activity. Within this pilot set, a nanomolar potency (EC₅₀ = 55 nM) was reached. Four lead compounds (EC₅₀ range 55–410 nM) were characterized for aqueous solubility, metabolic stability, plasma protein binding and Caco-2 permeability. While some instability in the presence of mouse liver microsomes was noted, mouse pharmacokinetic profile of the compound having the best overall ADME properties was evaluated to reveal acceptable bioavailability (F = 10.3%) and plasma levels achieved on oral administration.     

Heterobivalent dual-target probe for targeting GRP and Y1 receptors on tumor cells

Receptor targeting ligands for imaging and/or therapy of cancer are limited by heterogeneity of receptor expression by tumor cells, both inter-patient and intra-patient. It is often more important for imaging agents to identify local and distant spread of disease than it is to identify a specific receptor presence. Two natural hormone peptide receptors, GRPR and Y1, are specifically interesting because expression of GRPR, Y1 or both is up-regulated in most breast cancers. We describe here the design and development of a new heterobivalent peptide ligand, truncated bombesin (t-BBN)/BVD15-DO3A, for dual-targeting of GRPR and Y1, and validation of its dual binding capability. Such a probe should be useful in imaging cells, tissues and tumors that are GRPR and/or Y1 positive and should target radioisotopes, for example, ⁶⁸Ga and/or ¹⁷⁷Lu, to more tumors cells than single GRPR or Y1 targeted probes. A GRP targeting ligand, J-G-Abz4-QWAVGHLM-NH₂ (J-G-Abz4-t-BBN), and an Y1 targeting ligand, INP-K[ε-J-(α-DO3A-ε-DGa)-K]-YRLRY-NH₂([ε-J-(α-DO3A-ε-DGa)-K]-BVD-15), were synthesized and coupled to produce the heterobivalent ligand, t-BBN/BVD15-DO3A. Competitive displacement binding assays using t-BBN/BVD15-DO3A against ¹²⁵I-Tyr⁴-BBN yielded an IC₅₀ value of 18 ± 0.7 nM for GRPR in T-47D cells, a human breast cancer cell line. A similar assay using t-BBN/BVD15-DO3A against porcine ¹²⁵I-NPY showed IC₅₀ values of 80 ± 11 nM for Y1 receptor in MCF7 cells, another human breast cancer cell line. In conclusion, it is possible to construct a single DO3A chelate containing probe that can target both GRPR and Y1 on human tumor cells.     

Synthesis of alpha amylase inhibitors based on privileged indole scaffold

Twenty five derivatives of indole carbohydrazide (1–25) had been synthesized. These compounds were characterized using ¹H NMR and EI-MS, and further evaluated for their α-amylase inhibitory potential. The analogs (1–25) showed varying degree of α-amylase inhibitory potential.ranging between 9.28 and 599.0 µM when compared with standard acarbose having IC₅₀ value 8.78 ± 0.16 µM. Six analogs, 25 (IC₅₀ = 9.28 ± 0.153 µM), 22 (IC₅₀ = 9.79 ± 0.43 µM), 4 (IC₅₀ = 11.08 ± 0.357 µM), 1 (IC₅₀ = 12.65 ± 0.169 µM), 8 (IC₅₀ = 21.37 ± 0.07 µM) and 14 (IC₅₀ = 43.21 ± 0.14 µM) showed potent α-amylase inhibition as compared to the standard acarbose (IC₅₀ = 8.78 ± 0.16 µM). All other analogs displayed good to moderate inhibitory potential. Structure-activity relationship was established through the interaction of the active compounds with enzyme active site with the help of docking studies.     

Angiotensin III stimulates high stretch-induced ANP secretion via angiotensin type 2 receptor

Angiotensin III (Ang III) is metabolized from Ang II by aminopeptidase (AP) A and in turn, Ang III is metabolized to Ang IV by APN. Ang III is known to have a similar effect to Ang II on aldosterone secretion, but the effect of Ang III on atrial natriuretic peptide (ANP) secretion from cardiac atria is not known. The aim of the present study is to define the effect of Ang III on ANP secretion and its receptor subtype using isolated perfused beating atria. The volume load was achieved by elevating the height of outflow catheter connected with isolated atria from 5 cmH₂O to 7.5 cmH₂O. Atrial stretch by volume load increased atrial contractility and ANP secretion. Ang III stimulated stretch-induced ANP secretion in a dose-dependent manner without change in atrial contractility. The stimulated effect of Ang III (1 μM) on stretch-induced ANP secretion was blocked by the pretreatment of Ang II type 2 (AT₂) receptor antagonist but not by AT₁ or Mas receptor antagonist. Pretreatment with inhibitor of phosphoinositide 3-kinase (PI3K), Akt, nitric oxide synthase, soluble guanylyl cyclase, or protein kinase G (PKG) attenuated Ang III-stimulated ANP secretion. When Ang III (40 nM) or Ang II (4 nM) was infused for 10 min into anesthetized rats, mean arterial pressure was increased about 10%. However, Ang III increased plasma ANP level by 35.81 ± 10.19% but Ang II decreased plasma ANP level by 30.41 ± 7.27%. Therefore, we suggest that Ang III, opposite to Ang II, stimulated stretch-induced ANP secretion through AT₂ receptor/PI3K/Akt/nitric oxide/PKG pathway.     

Isatin based Schiff bases as inhibitors of α-glucosidase: Synthesis,characterization, in vitro evaluation and molecular docking studies

Isatin base Schiff bases (1–20) were synthesized, characterized by ¹H NMR and EI/MS and evaluated for α-glucosidase inhibitory potential. Out of these twenty (20) compounds only six analogs showed potent α-glucosidase inhibitory potential with IC₅₀ value ranging in between 2.2 ± 0.25 and 83.5 ± 1.0 μM when compared with the standard acarbose (IC₅₀ = 840 ± 1.73 μM). Among the series compound 2 having IC₅₀ value (18.3 ± 0.56 μM), 9 (83.5 ± 1.0 μM), 11 (3.3 ± 0.25 μM), 12 (2.2 ± 0.25 μM), 14 (11.8 ± 0.15 μM), and 20 (3.0 ± 0.15 μM) showed excellent inhibitory potential many fold better than the standard acarbose. The binding interactions of these active analogs were confirmed through molecular docking.     

Dihydropyrano [2,3-c] pyrazole: Novel in vitro inhibitors of yeast α-glucosidase

Inhibition of α-glucosidase enzyme activity is a reliable approach towards controlling post-prandial hyperglycemia associated risk factors. During the current study, a series of dihydropyrano[2,3-c] pyrazoles (1–35) were synthesized and evaluated for their α-glucosidase inhibitory activity. Compounds 1, 4, 22, 30, and 33 were found to be the potent inhibitors of the yeast α-glucosidase enzyme. Mechanistic studies on most potent compounds reveled that 1, 4, and 30 were non-competitive inhibitors (K_i = 9.75 ± 0.07, 46 ± 0.0001, and 69.16 ± 0.01 μM, respectively), compound 22 is a competitive inhibitor (K_i = 190 ± 0.016 μM), while 33 was an uncompetitive inhibitor (K_i = 45 ± 0.0014 μM) of the enzyme. Finally, the cytotoxicity of potent compounds (i.e. compounds 1, 4, 22, 30, and 33) was also evaluated against mouse fibroblast 3T3 cell line assay, and no toxicity was observed. This study identifies non-cytotoxic novel inhibitors of α-glucosidase enzyme for further investigation as anti-diabetic agents.     

Redesigning the active site of a carboxyl esterase from the archaeon Archaeoglobus fulgidus to improve sensitivity to organophosphorus compounds

Organophosphorus compounds (OPs) are widely used as pesticides because of their ability to inhibit the activity of acetylcholinesterase (AChE) in the nervous system. Thus, AChE is generally used as a biosensor for pesticide detection. Due to the instability of AChE a more stable enzyme would be desirable for robust applications. We investigated the sensitivity of a thermostable carboxylesterase from the archaeon Archaeoglobus fulgidus (AFEST) to seven selected OPs. The IC₅₀ of dichlorvos against AFEST (50.8 ± 2.6 nM) was 10-fold lower than that of the commercially obtained AChE, indicating that AFEST had higher sensitivity. Its sensitivity for the other OPs was lower than AChE. To enhance the sensitivity of AFEST to OPs, site-directed mutations were introduced in the cap domain of AFEST. The sensitivity of mutant N44S/S48V was enhanced toward all seven OPs compared to the wild-type and was higher than AChE for four OPs, including paraoxon (3.3 ± 0.01 nM), dichlorvos (28.0 ± 0.6 nM), profenofos (43.0 ± 1.0 nM), and diazinon (3.0 ± 0.2 nM). The half-lives of AFEST and the mutant N44S/S48V at 37 °C were over 15 d. The interactions between the enzymes and select OPs were investigated by molecular docking. The results demonstrated that AFEST and the mutant N44S/S48V have the potential to be biosensor for OP detection.     

Identification of dihydrostilbenes in Pholidota chinensis as a new scaffold for GABAA receptor modulators

A dichloromethane extract of stems and roots of Pholidota chinensis (Orchidaceae) enhanced GABA-induced chloride currents (IGABA) by 132.75 ± 36.69% when tested at 100 μg/mL in a two-microelectrode voltage clamp assay, on Xenopus laevis oocytes expressing recombinant α₁β₂γ_2S GABA_A receptors. By means of an HPLC-based activity profiling approach, the three structurally related stilbenoids coelonin (1), batatasin III (2), and pholidotol D (3) were identified in the active fractions of the extract. Dihydrostilbene 2 enhanced I_GABA by 1512.19 ± 176.47% at 300 μM, with an EC₅₀ of 52.51 ± 16.96 μM, while compounds 1 and 3 showed much lower activity. The relevance of conformational flexibility for receptor modulation by stilbenoids was confirmed with a series of 13 commercially available stilbenes and their corresponding semisynthetic dihydro derivatives. Dihydrostilbenes showed higher activity in the oocyte assay than their corresponding stilbenes. The dihydro derivatives of tetramethoxy-piceatannol (12) and pterostilbene (20) were the most active among these derivatives, but they showed lower efficiencies than compound 2. Batatasin III (2) showed high efficiency but no significant subunit specificity when tested on the receptor subtypes α₁β₂γ_2s, α₂β₂γ_2s, α₃β₂γ_2s, α₄β₂γ_2s, α₅β₂γ_2s, α₁β₁γ_2s, and α₁β₃γ_2s. Dihydrostilbenes represent a new scaffold for GABA_A receptor modulators.     

The binding of okadaic acid analogs to recombinant OABP2.1 originally isolated from the marine sponge Halichondria okadai

The binding between [24-³H]okadaic acid (OA) and a recombinant OA binding protein OABP2.1 was examined using various OA analog, including methyl okadaate, norokadanone, 7-deoxy OA, and 14,15-dihydro OA, 7-O-palmitoyl DTX1, to investigate the structure activity relationship. Among them, 7-O-palmitoyl DTX1, which is one of the diarrhetic shellfish poisoning (DSP) toxins identified in shellfish, displayed an IC₅₀ for [24-³H]OA binding at 51 ± 6.3 nM (Mean ± SD). In addition, a synthetic compound, N-pyrenylmethyl okadamide, exhibited its IC₅₀ at 10 ± 2.9 nM (Mean ± SD). These results suggested that the recombinant OABP2.1 and the N-pyrenylmethyl okadamide might be core substances in a novel assay for the DSP toxins.