17ß-Estradiol residues and estrogenic activities in the Hawkesbury River, Australia.

Two highly sensitive ELISAs for the specific detection of 17ß-estradiol (E2) residues were developed, showing the limits of detection (LOD, a concentration at 15% inhibition of color development) of 0.04 ±â€¯0.02 µg/L and 0.05 ±â€¯0.03 µg/L. The average recovery rate of the river water samples spiked with E2 at 1-50 ng/L range was 111.5% (68.6-252%) with the % relative standard deviation (RSD) of 0.5-86.3%. The ELISA demonstrated a good correlation with the GC-MS analyses of the spiked river water samples (r = 0.909). Applying the developed E2 ELISA assay to the monitoring of E2 residues in Hawkesbury River (New South Wales, Australia) found that all the tested creek samples contained E2 residues less than the biologically significant level of 10 ng/L. However, 25% of the water samples tested demonstrated the estrogen activity (determined by the yeast estrogen screening (YES) assay) above the levels that have been linked to the adverse effects in fish and other aquatic organisms (> 20 E2 Eq ng/L). It was apparent that the E2 residues together with the EE2 residues (reported in our previous study) contributed to most of the observed estrogenic activity in Hawkesbury River.

A survey of 17α-ethinylestradiol and mestranol residues in Hawkesbury River, Australia, using a highly specific enzyme-linked immunosorbent assay (ELISA) demonstrates the levels of potential biological significance.

This study reports on the potential status of 17α-ethinylestradiol (EE2) and mestranol (MeEE2) residues in aquatic environments in New South Wales (NSW), Australia, based on the analysis by a specific ELISA we developed. Polyclonal antibodies were raised against the EE2 hapten with a linker attached at the C3-position to direct the antibody binding towards the ring D of EE2/MeEE2. Using this approach, an ELISA highly specific to EE2 and MeEE2 was successfully developed, showing less than 3.1% cross-reactivity (% CR) with other major steroidal sex hormones and their derivatives. The assay performed with the limit of detection (LOD) of 0.04 ± 0.01µg/L for both EE2 and MeEE2, and the limit of quantitation (LOQ) of 0.05 ± 0.01ng/L when it was coupled with the SM2-Biobeads solid phase extraction. Prior to conducting the survey study, it was validated against the gas chromatography-mass spectrophotometry (GC-MS) method, which showed high correlation with R2 of 0.934. Fresh surface water samples collected at different sites along Hawkesbury River in New South Wales (NSW) were analyzed for the EE2/ MeEE2 residues using the developed ELISA. The EE2/MeEE2 levels were found to range between 4.1 and 8.3ng/L in Emigrant Creek, NSW, where the primary activity was macadamia plantation, and higher levels between 15 and 29ng/L in South Creek, NSW, Greater Western Sydney at sites upstream and downstream of the municipal sewage treatment plants.

Γ-aminobutyric acid(C) (GABAC) selective antagonists derived from the bioisosteric modification of 4-aminocyclopent-1-enecarboxylic acid: amides and hydroxamates.

Series of compounds were generated via the bioisosteric replacement of the carboxylate of 4-ACPCA (2) with hydroxamate or amide groups. All compounds from this study exhibited increased selectivity for GABAC, the most potent being 4-ACPHA (10a, IC50 = 13 µM) and 4-ACPAM (11a, IC50 = 10 µM). This provides evidence that a zwitterionic structure is not essential for GABAC antagonists, rather the emphasis lies in appropriate heteroatoms to participate in hydrogen bonding.

Structurally diverse GABA antagonists interact differently with open and closed conformational states of the ρ1 receptor.

Ligands acting on receptors are considered to induce a conformational change within the ligand-binding site by interacting with specific amino acids. In this study, tyrosine 102 (Y102) located in the GABA binding site of the ρ(1) subunit of the GABA(C) receptor was mutated to alanine (ρ(1Y102A)), serine (ρ(1Y102S)), and cysteine (ρ(1Y102C)) to assess the role of this amino acid in the action of 12 known and 2 novel antagonists. Of the mutated receptors, ρ(1Y102S) was constitutively active, providing an opportunity to assess the activity of antagonists on ρ(1) receptors with a proportion of receptors existing in the open conformational state compared to those existing predominantly in the closed conformational state. It was found that the majority of antagonists studied were able to inhibit the constitutive activity displayed by ρ(1Y102S), thus displaying inverse agonist activity. The exception was (±)-4-aminocyclopent-1-enecarboxamide ((±)-4-ACPAM) (8) not exhibiting any inverse agonist activity, but acting explicitly on the closed conformational state of ρ(1) receptors (ρ(1) wild-type, ρ(1Y102C) and ρ(1Y102A)). It was also found that the GABA antagonists were more potent at the closed compared to the open conformational states of ρ(1) receptors, suggesting that they may act by stabilizing closed conformational state and thus reducing activation by agonists. Furthermore, of the antagonists tested, Y102 was found to have the greatest influence on the antagonist activity of gabazine (SR-95531 (13)) and its analogue (SR-95813 (14)). This study contributes to our understanding of the mechanism of inverse agonism. This is important, as such agents are emerging as potential therapeutics.

GABA analogues derived from 4-aminocyclopent-1-enecarboxylic acid.

The incorporation of extra binding groups onto known ligands is a powerful tool for the development of more potent and selective agents at target sites such as the GABA receptors. In the present work we have attempted to build on the activity of the know potent GABA(A) agonist 4-ACP-3-CA and its cis and trans saturated analogues CACP and TACP. We have investigated reactions to add thiol substituents to the alpha,beta-unsaturated system of 4-ACP-3-CA. The reaction was successful with a limited number of thiols but gave products of mixed stereochemistry. The resultant thioether amino acids were screened for activity at human recombinant alpha(1)beta(2) gamma(2L) GABA(A) receptors. The most interesting derivative was the benzylthioether which acted as an antagonist with an IC(50) of 42 microM for the inhibition of a GABA EC(50) dose (50 microM). This study has shown that GABA analogues derived by thiol addition to 4-aminocyclopent-1-enecarboxylic acid display interesting antagonist activity at the alpha(1)beta(2)gamma(2L) GABA(A) receptor.

Guanidino acids act as rho1 GABA(C) receptor antagonists.

GABA(C) receptors play a role in myopia, memory-related disorders and circadian rhythms signifying a need to develop potent and selective agents for this class of receptors. Guanidino analogs related to glycine, beta-alanine and taurine were evaluated at human rho(1)GABA(C) receptors expressed in Xenopus oocytes using 2-electrode voltage clamp methods. Of the 12 analogs tested, 8 analogs were active as antagonists and the remaining were inactive. (S)-2-guanidinopropionic acid (IC(50) = 2.2 microM) and guanidinoacetic acid (IC(50) = 5.4 microM; K (B) = 7.75 microM [pK (B) = 5.11 +/- 0.06]) were the most potent being competitive antagonists at this receptor. In contrast, the beta-alanine and GABA guanidino analogs showed reduced activity, indicating the distance between the carboxyl carbon and terminal nitrogen of the guanidino group is critical for activity. Substituting the C2-position of guanidinoacetic acid with various alkyl groups reduced activity indicating that steric effects may impact on activity. The results of this study contribute to the structure-activity-relationship profile required in developing novel therapeutic agents.

3-hydroxypyridazine 1-oxides as carboxylate bioisosteres: a new series of subtype-selective AMPA receptor agonists.

Three positional isomers (compounds 1, 2, and 3) of 1-uracilylalanine (willardiine) based on a 3-hydroxypyridazine 1-oxide scaffold with an alanine side-chain at positions 4 (1), 5 (2) or 6 (3) were tested for binding to recombinant homomeric AMPA receptor (AMPA-R) subtypes GluR1-4, as well for excitatory activity on the rat cortical wedge preparation. 1 had approximately 30 times higher affinity than willardiine while showing a similar selectivity profile, i.e. 22-fold selectivity for GluR1/2 over GluR3/4. The GluR1-4 affinities of 3 were similar to 1, however, its 31-fold selectivity for GluR1/2 over GluR3/4 is the highest yet observed among azine-based glutamate analogues. The non-isosteric congener 2 showed weaker binding to AMPA-Rs. In the cortical wedge, 1 evokes similar responses to AMPA, while 3 and 2 are 10- and 100-fold weaker, respectively. Dose-response curves on Xenopus laevis oocytes expressing GluR1-4(flip) confirmed that 1 and 3 are potent GluR1/2 receptor agonists (EC(50)s from 0.26 to 1.7microM) but are 10- to 160-fold less potent at GluR3/4. The structures, potencies and selectivities of this new class of AMPA agonists are compared with those of willardiine, 5-fluorowillardiine and azawillardiine, referring to the binding mode observed in the crystal structure of willardiine bound to GluR2-S1S2. The results indicate that the 3-hydroxypyridazine 1-oxide moiety can function as an outstanding carboxylate mimic at AMPA-Rs, leading the way to further fine-tuning of subtype selectivity. This little-explored molecular motif may find wider application in medicinal chemistry.

Synthesis and in vivo evaluation of a novel peripheral benzodiazepine receptor PET radioligand.

The novel pyrazolopyrimidine ligand, N,N-diethyl-2-[2-(4-methoxyphenyl)-5,7-dimethyl-pyrazolo[1,5-a]pyrimidin-3-yl]-acetamide 1 (DPA-713), has been reported as a potent ligand for the peripheral benzodiazepine receptor (PBR) displaying an affinity of K(i)=4.7 nM. In this study, 1 was successfully synthesised and demethylated to form the phenolic derivative 6 as precursor for labelling with carbon-11 (t(1/2) = 20.4 min). [11C]1 was prepared by O-alkylation of 6 with [11C]methyl iodide. The radiochemical yield of [(11)C]1 was 9% (non-decay corrected) with a specific activity of 36 GBq/micromol at the end of synthesis. The average time of synthesis including formulation was 13.2 min with a radiochemical purity >98%. In vivo assessment of [11C]1 was performed in a healthy Papio hamadryas baboon using positron emission tomography (PET). Following iv administration of [11C]1, significant accumulation was observed in the baboon brain and peripheral organs. In the brain, the radioactivity peaked at 20 min and remained constant for the duration of the imaging experiment. Pre-treatment with the PBR-specific ligand, PK 11195 (5 mg/kg), effectively reduced the binding of [11C]1 at 60 min by 70% in the whole brain, whereas pre-treatment with the central benzodiazepine receptor ligand, flumazenil (1mg/kg), had no inhibitory effect on [11C]1 uptake. These results indicate that accumulation of [11C]1 in the baboon represents selective binding to the PBR. These exceptional in vivo binding properties suggest that [11C]1 may be useful for imaging the PBR in disease states. Furthermore, [11C]1 represents the first ligand of its pharmacological class to be labelled for PET studies and therefore has the potential to generate new information on the pathological role of the PBR in vivo.

Studies on pyridazine azide cyclisation reactions.

Reaction of sodium azide with 4-methyl-3,5,6-tribromopyridazine results in the formation of 3,5,6-triazide intermediate which could cyclise to give two possible bicyclic products while ab initio calculations show that the formation of a tricyclic compound is extremely energetically unfavourable. However, experimentally, only one major product is isolated. The structure of this unstable product has been conclusively established by X-ray crystallography as 3,5-diazido-4-methyl[1,5-b]tetrazolopyridazine confirming theoretical predictions.

A rapid aflatoxin B1 ELISA: development and validation with reduced matrix effects for peanuts, corn, pistachio, and Soybeans.

Among the competitive ELISAs for aflatoxins that have been described, few have been adequately validated for reduced matrix effects. Using an aflatoxin B(1) (AFB(1))-specific polyclonal antibody (produced from AFB(1)-oxime conjugated to bovine serum albumin (BSA)) and AFB(1)- and AFB(2)-enzyme conjugates, four direct competitive ELISAs based on 96-microwell plates (two standard assays and two rapid assays) were developed, paying special attention to producing a robust assay relatively free of interferences for a range of agricultural products. The antibody was AFB(1)-specific, detecting only AFB(1) in a mixture of four aflatoxins (AFB(1), AFB(2), AFG(1), and AFG(2)), but showed significant cross-reaction with AFG(1) (57-61%) when an individual compound was tested. Standard assays (long assays) exhibited higher sensitivities than rapid assays (short assays) with IC(50) values of 12 +/- 1.5 and 9 +/- 1.5 microg/kg in sample (with 1 in 5 dilution of sample extract) for AFB(1) and AFB(2)-enzyme conjugates, respectively. These assays have narrower detection ranges (7.1-55.5 microg/kg in sample) and required dilution of sample extracts to overcome solvent and matrix interferences, making these assays less ideal as analytical methods. Rapid assays exhibited IC(50) values of 21.6 +/- 2.7 and 12 microg/kg in sample for AFB(1)- and AFB(2)-enzyme conjugates, respectively. These assays have ideally broader detection ranges (4.2-99.9 microg/kg in sample) and showed no methanol effects up to 80% with significantly reduced matrix interferences as a result of the shorter incubation times and increasing the amounts of enzyme conjugate used. Therefore, the rapid assays were formatted to perform without a need for extract dilution. The rapid assays can be completed within 15 min, potentially suitable for receival bays where quick decision-making to segregate low and high contamination is critical. Further validation using the rapid assay with AFB(1)-enzyme conjugate indicated relatively good recoveries of AFB(1) spiked in corn, peanuts, pistachio, and soybeans, which were free from significant matrix effects. It can be concluded that this rapid assay would be suitable for monitoring aflatoxin AFB(1) at current legal maximum residue limits of 10 microg/kg in food such as corn, peanuts, pistachio, and soybeans.

Rapid enzyme immunoassays for the detection of carbaryl and methoprene in grains.

In order for grain handlers and traders to reliably estimate residues of grain protectants in the field, antibody-based rapid tests were developed for carbaryl (1-naphthyl methylcarbamate) and methoprene [isopropyl (E,E)-(RS)-11-methoxy-3,7,11-trimethyldodeca-2,4-dienoate]. To complement the rapid analysis, a simple and rapid extraction technique was developed. In these tests, a pesticide-containing methanol extract of the grain sample and an enzyme-labeled component are added to precoated strips. After a brief incubation, the strips are washed and a substrate/chromogen for the enzyme is added. The color developed is stopped by acidification and the results are read either by eye or in a portable field photometer. The overall test time is under 20 minutes. For carbaryl, the test had a limit of detection of 4.5 ppb (1.1 ppm in grain), while the methoprene test had a limit of detection of 4 ppb (1 ppm in grain) based on the lower datum point, which is 15% inhibition, in the standard curves. Both assays can be used as a screening test for carbaryl and methoprene in animal feed grains.