Detection and quantification of microcystins from cyanobacteria strains isolated from reservoirs and ponds in Morocco

In Morocco, the occurrence of toxic cyanobacteria blooms is confirmed in some water bodies used for recreational and/or as drinking water reservoirs. According to WHO recommendations, the establishment of a monitoring program for microcystins is a necessity. This paper presents toxicological studies of 19 toxic cyanobacteria strains of Microcystis, Synechocystis, Pseudanabaena, and Oscillatoria. These strains were isolated from various water bodies including natural lakes, reservoirs, and ponds located in central regions of Morocco. The isolation, culture, and biomass production of these strains was made on Z8 or BG13 media under laboratory controlled conditions. The hepatotoxicity of cyanobacterial lyophilized material was confirmed by mouse bioassays. The amount of microcystins produced by each strain was determined by the enzyme-linked immunosorbent assay (ELISA). The detection and identification of microcystin variants was performed by high performance liquid chromatography (HPLC) with photodiode array detection. Almost all strains showed medium to high toxicity, the estimated LD50 i.p. mice bioassay ranged between 28 to 350 mg/kg body weight. The concentrations of microcystins varied between 2.16 to 944 micrograms/g and 26.8 to 1884 micrograms/g dry weight determined by ELISA and HPLC, respectively. The screening of bloom-forming and microcystin producer cyanobacteria strains in these fresh water bodies leads us to propose the need for the establishment of a survey of cyanobacteria and a cyanotoxin-monitoring program.     

A colorimetric protein phosphatase inhibition assay for the determination of cyanobacterial peptide hepatotoxins based on the dephosphorylation of phosvitin by recombinant protein phosphatase 1

A colorimetric protein phosphatase inhibition assay based on the dephosphorylation of phosvitin by recombinant protein phosphatase 1 was developed for analysis of waters for cyanobacterial hepatotoxins. The phosphate released in the assay was determined using a malachite green reagent. Good agreement with toxin concentrations determined by HPLC was obtained. The assay was capable of determining these toxins at concentrations around 1 μg/L with high precision and without sample concentration. This is of considerable benefit as the World Health Organisation specifies a provisional guideline of 1 μg/L for microcystin‐LR. There was evidence, however, that the sample matrix might affect quantification, leading to false positive results. Thus the assay should be viewed as a screening procedure, and confirmatory analyses by an alternative procedure should be carried out for positive results. Further work is required to resolve the question of matrix interferences if phosphatase inhibition assays are used directly for measuring toxin levels in water, especially if this information is used to check compliance with water quality guidelines. © 2001 John Wiley & Sons, Inc. Environ Toxicol 16: 242–252, 2001     

Initial studies on the occurrence of cyanobacteria and microcystins in Irish lakes

We report the results of a synoptic survey at 14 sites across the north of Ireland undertaken to determine the occurrence of cyanobacteria and their constituent microcystin cyanotoxins. Seven microcystin toxins were tested for, and five of which were found, with MC‐LR, MC‐RR, and MC‐YR being the most prevalent. Gomphosphaeria spp and Microcystis aeruginosa were the most dominant cyanobacterial species encountered. Together with Aphanizomenon flos‐aquae, these were the cyanobacteria associated with the highest microcystin concentrations. The occurrence of several microcystin toxins indicates that there may potentially be more than one cyanobacteria species producing microcystins at many sites. Total microcystin concentrations varied over three orders of magnitude dividing the sites into two groups of high (>1000 ngMC/μgChla, six sites) or low toxicity (<200 ngMC/μgChla, eight sites). © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2010.     

Comparative study on in vitro inhibition of grass carp (Ctenopharyngodon idellus) and mouse protein phosphatases by microcystins

Microcystins isolated from toxic cyanobacteria are potent inhibitors of protein phosphatases 1 and 2A (PP1 and PP2A). The inhibitory effects of three structural variants of microcystins (microcystin‐LR, ‐YR, and ‐RR) on protein phosphatases isolated and purified from the liver and kidney of grass carp (Ctenopharyngodon idellus) were investigated using the ³²P radiometric assay. The relationships between percentage inhibition of protein phosphatase activity and microcystin levels followed a typical dose‐dependent sigmoid curve. These results were compared to those obtained from mouse PP2A. The degree and pattern of inhibition of both fish and mouse protein phosphatases by microcystins were similar. Protein phosphatases in crude fish tissue homogenates showed similar inhibition patterns as purified fish PP2A toward microcystins. © 2000 John Wiley & Sons, Inc. Environ Toxicol 15: 71–75, 2000     

Sampling and analysis of microcystins: Implications for the development of standardized methods

Microcystins (MC), a group of cyanotoxins, have been found in lakes and rivers worldwide. One goal of MC research is to develop models which predict MC concentrations, but these efforts have been hampered by a lack of standardized methods necessary for comparing data across studies. Here, we investigate the effect of chemical analysis (HPLC-PDA and ELISA), sample collection (whole water, plankton tow and surface scum), and choice of normalizing parameter (volume, dry weight, and chlorophyll a) on reported MC concentrations. Samples were collected over three years from a temperate mesotrophic, shallow lake with episodic blooms of cyanobacteria. We found that microcystins were up to four times higher in lake samples when analyzed by ELISA relative to HPLC-PDA and that MC concentration measured by HPLC explained less than half of the variation in MC concentrations measured by ELISA. Also, samples collected by plankton tow gave consistently higher concentrations than whole water samples. An additional HPLC analysis of two chlorophyte cultures revealed the presence of compounds with a similar UV absorbance spectrum to MC-LR, suggesting that identifying MC based solely on UV absorbance is not valid. Our results document the discrepancy in MC concentrations that can arise by using different methods throughout all stages of sampling, analysis, and reporting of MC concentrations.     

Identification of microcystins in waters used for daily life by people who live on Tai Lake during a serious cyanobacteria dominated bloom with risk analysis to human health

Tai Lake is the third largest freshwater lake in China with annual cyanobacteria blooms. Microcystins produced by these blooms have serious health risks for populations surrounding the lake, especially for people living on Tai Lake, because they usually drink raw lake water after a simple alum treatment. This study presents data on the detection and identification of microcystins in waters used for daily life by people living on Tai Lake, during the cyanobacterial blooming in July 2007. The health risks from drinking these microcystin-polluted waters were also calculated. The main microcystins detected by high-performance liquid chromatography-electrospray ionization mass spectrometry in the water samples collected from two parts of Tai Lake (Wuli Lake and Meiliang Bay) were MC-LR (4.33-12.27 microg/L), MC-RR (8.36-16.91 microg/L) and MC-YR (1.41-5.57 microg/L). Risk assessment showed that the drinking water simply treated by alum was not safe. The lowest calculated hazards ratios in all water samples was 6.4, which indicated that the risk of microcystins exposure from drinking water was over six times higher than the tolerable daily intake (TDI) recommended by The World Health Organization (WHO). Further studies should be conducted to elucidate the relationships between the epidemiology of people living on Tai Lake and microcystins exposure from drinking water.     

Insight into the Molecular Mechanism for the Discrepant Inhibition of Microcystins (MCLR,LA, LF,LW, LY) on Protein Phosphatase 2A

Microcystins (MCs) exhibit diversified inhibition effects on protein phosphatases (PPs) due to their structural differences. To fully evaluate the potential mechanism for the discrepant inhibition effects, the five most frequent MCs with varying residues at position Z⁴ were selected as the tested toxins. Their inhibition sequence on PP2A was detected as follows: MCLR > MCLW > MCLA > MCLF > MCLY. Combined with homology modeling and molecular docking technology, the major interaction parameters between the MCs and PP2A were obtained. The correlation analysis for the major interaction parameters and inhibition effects showed that the hydrophobicity of Z⁴ had an important influence on the interaction of the MCs to PP2A. The introduction of hydrophobic Z⁴ directly weakened hydrogen bonds Z⁴→Pro₂₁₃ and Z⁴←Arg₂₁₄, indirectly weakened hydrogen bonds Adda⁵←Asn₁₁₇, Glu⁶←Arg₈₉, and MeAsp³←Arg₈₉, but indirectly enhanced ionic bonds Glu⁶←Arg₈₉, Glu⁶-Mn₁²⁺, and Glu⁶-Mn₂²⁺. In this way, the combination of the MCs with PP2A was blocked, and thus, the interactions between PP2A and the Mn²⁺ ions (in the catalytic center) were further affected; metal bonds Asp₈₅-Mn₁²⁺ and Asp₈₅-Mn₂²⁺ were weakened, while metal bond His₂₄₁-Mn₁²⁺ was enhanced. As a result, the interactions in the catalytic center were inhibited to varying degrees, resulting in the reduced toxicity of MCs.     

High grazer toxicity of [D-Asp(3),(E)-Dhb(7)]microcystin-RR of Planktothrix rubescens as compared to different microcystins.

Planktothrix rubescens, the dominant cyanobacterium in Lake Zürich, is generally considered to be toxic to zooplankton. The major toxin was determined by NMR spectroscopy and chemical analysis to be [D-Asp³,(E)-Dhb⁷]microcystin-RR. The compound was isolated in high purity, and its 24-h acute grazer toxicity was compared with microcystin-LR, microcystin-RR, microcystin-YR, and nodularin using a Thamnocephalus platyurus bioassay. Based on LC₅₀ values [D-Asp³,(E)-Dhb⁷]microcystin-RR was the most toxic microcystin tested. Nodularin was slightly more toxic under the conditions of the assay. The large number of individuals available for the grazer bioassay allowed the determination of dose-response curves of the different microcystins. These curves showed marked differences in their steepness. Microcystin-RR, which had nearly the same LC₅₀ as microcystin-LR and microcystin-YR, exhibited a very flat dose-response curve. This flat curve indicates that, for some individuals, lower concentrations of this microcystin are much more toxic than are the other two microcystins. Mortality of 100% requires much higher concentrations of microcystin-RR, indicating the resistance of some animals to the toxin. The purified [D-Asp³,(E)-Dhb⁷]microcystin-RR exhibited a higher molar absorption coefficient determined by quantitative amino acid analysis than the coefficients generally used for other microcystins. This observation has consequences for the risk assessment for microcystins and makes a structural determination of microcystins an absolute requirement. The presence of the dehydrobutyrine residue may be the reason for the higher specific toxicity of [D-Asp³,(E)-Dhb⁷]microcystin-RR when compared to the N-methyldehydroalanine-containing microcystins.     

A protein phosphatase 2A (PP2A) inhibition assay using a recombinant enzyme for rapid detection of microcystins

Worldwide blooms of toxic cyanobacteria (blue-green algae) commonly occur in freshwater, often in drinking water sources, necessitating routine monitoring of water quality. Microcystin-LR and related cyanobacterial toxins strongly inhibit protein phosphatase 2A (PP2A) and are therefore assayable by measuring the extent of PP2A inhibition. In this study, we evaluated the suitability of the catalytic subunit of recombinant PP2A (rPP2Ac) expressed with a baculovirus system for use in a microplate microcystin assay. Five microcystin analogs, microcystin-LR, -RR, -YR, -LF, and -LW, and nodularin strongly inhibited rPP2Ac activity with IC(50) values of 0.048, 0.072, 0.147, 0.096, 0.114, and 0.54 nM, respectively. Microcystin-LR in a water sample could be assayed from 0.005 to 5 ng/ml. The assay could detect the toxin at a far lower level than required by the World Health Organization for regulation of microcystin-LR or its equivalent (1 microg/L). Pretreatment or concentration of water samples with low toxin concentrations was not necessary. The microplate assay using rPP2Ac was more sensitive than an enzyme-linked immunosorbent assay (ELISA) method and a cytotoxicity assay. The genetically engineered rPP2Ac was more stable than a commercially available dimeric enzyme, producing accurate and reproducible results. Our results confirm that the rPP2Ac we prepared is an excellent tool for detecting and quantifying microcystins in water.     

Accumulation of microcystins in water and fish tissues: An estimation of risks associated with microcystins in most of the Greek Lakes

Toxin‐producing cyanobacteria in lakes and reservoirs form a threat to humans as well as various forms of aquatic life. This study is an investigation into the occurrence and distribution of Microcystins (MCYST) in 13 Greek Lakes. The distribution of MCYST in water and surface scum and toxin bioaccumulations in the omnivorous fish species Carassius gibelio were surveyed in all lakes. Considerable amounts of MCYST were found in water and scum of all lakes, irrespective of the trophic state, the type of the lake, and the reported dominant cyanobacterial species. Toxin accumulation in six tissues (liver, brain, intestine, kidney, ovary, and muscle) of C. gibelio was also analyzed. Even though the target organ for MCYST is the liver, in our study, MCYST were found also in the rest of C. gibelio tissues in the following order: liver > intestine > kidney > brain > ovaries > muscle. Risk assessments were carried out, taking into account the WHO guidelines and the tolerable daily intake (TDI) for MCYST. Our findings suggest that the amounts of MCYST found in water of Lakes Kastoria, Koronia, Pamvotis, Doirani, Mikri Prespa, Petron, and Zazari, pose adverse health risks. Also, it is likely to be unsafe to consume C. gibelio in Lakes Koronia, Kastoria, Pamvotis, and Mikri Prespa due to the high concentrations of accumulated MCYST. © 2009 Wiley Periodicals, Inc. Environ Toxicol 25: 418–427, 2010.     

Effects of a saxitoxin-producer strain of Cylindrospermopsis raciborskii (cyanobacteria) on the swimming movements of cladocerans

This study evaluated the effects of a saxitoxin-producer strain (T3) of the cyanobacteria species Cylindrospermopsis raciborskii on the swimming movements of three cladoceran species (Daphnia gessneri, D. pulex, and Moina micrura). Acute toxicity bioassays were designed to access the effects of T3 strain, of a nonsaxitoxin producer strain (NPLP-1) of the same species and of a raw water sample from Funil reservoir (Rio de Janeiro, Brazil), that contained this and other cyanobacteria. In the acute bioassays, animals were exposed to C. raciborskii filaments or Funil water for 24-48 h and then transferred to food suspensions without cyanobacterial filaments for a further 48 h. During the exposure time to T3 strain filaments there was a decrease in the number of swimming individuals, with animals showing progressive immobilization. The same effect was observed with Funil water sample. Animals stayed alive on the bottom of the test tube and recovered swimming movements when transferred to food suspensions without toxic cells. This effect was not observed with the strain NPLP-1. The cladoceran D. pulex showed to be extremely sensitive to T3 strain and to Funil water containing C. raciborskii filaments, showing complete paralysis after 24-h exposure to T3 cell densities of 10(3) and 10(4) cells mL(-1), and after 24-h exposure to only 10% of raw water. However, D. gessneri was not sensitive to both T3 and to Funil water, whereas M. micrura was intermediate in sensitivity. This is the first report on the effects of cyanobacterial saxitoxins on movements of freshwater cladocerans, showing also difference in sensitivity among closely related Daphnia species.     

A new morphospecies of Microcystis sp. forming bloom in the Cheffia dam (Algeria): seasonal variation of microcystin concentrations in raw water and their removal in a full-scale treatment plant

Toxic cyanobacterial blooms are an increasing problem in Algeria. The production of cyanotoxins (microcystins) and their presence in drinking water represent growing hazards to human health. In this study, seasonal variations in the concentrations of total microcystins and physicochemical parameters (pH, temperature, dissolved oxygen, nitrate, orthophosphate, and chlorophyll-a) were analyzed in the Cheffia dam (Algeria), mainly used to supply drinking water. The removal of cyanobacterial cells and microcystins was also evaluated in full-scale plant associated with the Cheffia reservoir. The levels of microcystins (MCYSTs) in both raw and drinking water were evaluated using the protein phosphatase type 2A (PP2A) inhibition test as MCYST-LR equivalents. Identification of microcystin variants was achieved by LC/MS/MS. During the period of study (March-December 2004), microscopic observation showed the dominance in the autumn months (September-November) of a new morphospecies of Microcystis sp. The MCYST-LR equivalent concentrations in raw water varied between 50.8 and 28,886 ng L(-1). The highest level of toxins was observed in October 2004 and was significantly correlated with the chlorophyll-a. Three variants of microcystins assigned as microcystin-YR (MCYST-YR), microcystin-LR (MCYST-LR), and 6Z-Adda stereoisomer of MCYST-LR were observed in the crude extract of the Microcystis sp. bloom sample. During the bloom period, total elimination of Microcystis sp. and toxins were achieved through a classical treatment plant comprised of coagulation and flocculation, powdered activated carbon at 15 mg L(-1), slow sand filtration and chlorination before storage.     

First observation of cylindrospermopsin in Anabaena lapponica isolated from the boreal environment (Finland)

The cyanobacterial cytotoxin cylindrospermopsin has been mostly associated with cyanobacteria present in tropical and subtropical regions. Cylindrospermopsin has recently been found in cyanobacterial samples in central and southern Europe but the possible presence of the toxin in northern Europe has been unknown. Fifty-eight field and laboratory culture samples of Finnish cyanobacteria were analyzed by high-performance liquid chromatography combined with UV diode-array detection, multiple reactant monitoring in a triple-quadrupole mass spectrometer (MS), and accurate mass measurements using a time-of-flight MS instrument. Cylindrospermopsin was confirmed by all three techniques in a culture sample of Anabaena lapponica at a concentration of 242 microg cylindrospermopsin per g freeze-dried cyanobacterial material.     

Toxic threshold of dietary microcystin (-LR) for quart medaka

This study was designed to estimate the toxic threshold of male and female fish to microcystins based on different biomarkers. Japanese medaka (Oryzias latipes) were fed dietary Microcystin-LR (0, 0.46, 0.85, 2.01 and 3.93 μg MC-LR/g dry diet for 8 weeks at 25 °C. The results revealed that dietary MC-LR inhibited growth at the end of 8 weeks. The survival of embryos and the RNA/DNA ratio of whole fish decreased significantly (P < 0.05) in fish fed 3.93 μg MC-LR/g dry diet. Heat shock protein (Hsp60) expression was induced in the liver of female and male fish fed diets containing ≥0.85 and 0.46 μg MC-LR/g diet, respectively. The activity of liver caspase 3/7 was significantly higher in female fish fed 3.93 μg MC-LR/g diet and in males fed 2.01 MC-LR μg/g dry diet than fish fed the control diet. The threshold for inhibition of liver protein phosphatase expression was lower in female (2.01 μg/g diet) than that in male fish (3.93 μg/g diet). Histopathological examination showed significant single-cell necrosis in female and male medaka fed diets containing 0.85 and 3.93 μg MC-LR/g diet, respectively. Based on different biomarkers, this study demonstrated that dietary MC-LR is toxic to Medaka and the effects are gender dependent.     

An advanced technique for immuno-labelling of microcystins in cryosectioned cells of Microcystis aeruginosa PCC 7806 (cyanobacteria): implementations of an experiment with varying light scenarios and culture densities.

The intracellular localisation of cyanobacterial toxins might well indicate production sites and possible shifts to destination points, thus giving information on possible functions of these toxins within algal cells or at the ecological level beyond. By preparing cells of Microcystis aeruginosa PCC 7806 by cryofixation/cryosectioning and using purified high quality antibodies for immunogold-localisation, excellent ultrastructural integrity and labelling of microcystins was shown. Compared to conventional techniques, including organic solvents, possible dislocation/extraction was significantly minimised, hence, the labelling density was enhanced and the labelling pattern changed. The microcystins were mainly localised within the inner nucleoplasmic area and accumulations of epitopes could be detected around/within intracellular inclusions, such as polyphosphate bodies and carboxysomes. Photosynthetic active radiation (PAR) had a significant effect on microcystin biosynthesis, and the medium light intensity of 25 microE m(-2) s(-1) induced the highest intracellular microcystin contents (up to 160 epitopes per cell and 26 epitopes per microm2). The restriction of the full light spectrum to blue (400-500 nm) or red (>610 nm) wavelengths did not result in any significant effect on microcystin production. However, the subcultures harvested at higher optical densities (>0.5) revealed significantly higher microcystin labelling compared to the less dense cell cultures (OD < 0.5). Altogether, the possibility was discussed whether microcystin might function as an inhibitor of RUBISCO under conditions of C-limitations. The effects of light intensity and cell suspension density on intracellular microcystin shown by immuno-detection matched the pattern of microcystin concentrations determined in parallel by HPLC and ELISA.     

A diclofenac suppository–nabumetone combination therapy for arthritic pain relief and a monitoring method for the diclofenac binding capacity of HSA site II in rheumatoid arthritis

Diclofenac suppository, a non‐steroidal anti‐inflammatory drug (NSAID), is used widely in rheumatoid arthritis (RA) patients with severe arthritic pain. As the binding percentage of diclofenac to serum proteins is high, its free (unbound) concentration after rectal administration is low. To increase temporarily the free concentration of diclofenac and to enhance its analgesic effect by inhibiting the protein binding of diclofenac, the analgesic effect of diclofenac was examined before and after the start of an inhibitor administration to RA patients with insufficient control of arthritic pain, and the protein binding capacity of diclofenac was evaluated. Binding experiments were performed by ultrafiltration, and arthritic pain was recorded by the face scale. Free fractions of diazepam and diclofenac were augmented by increasing 6‐methoxy‐2‐naphthylacetic acid (6‐MNA; the active metabolite of the NSAID nabumetone) concentrations. The free fraction of diazepam increased after the start of nabumetone administration to RA patients, and arthritic pain relief was observed. These results suggest that 6‐MNA has an inhibitory effect on the protein binding of diclofenac and the free fraction of diazepam can be used to evaluate the binding capacity of diclofenac. It is considered that diclofenac suppository–nabumetone combination therapy and the method for protein binding monitoring by diazepam can positively benefit RA patients with insufficient control of arthritic pain. Copyright © 2013 John Wiley & Sons, Ltd.     

Neonatal serotonin (5-HT) depletion does not disrupt prepulse inhibition of the startle response in rats

The neurodevelopmental hypothesis of many brain disorders is based on the notion that environmental factors have significant effects on brain maturation. Because serotonin (5-HT) dysfunction in development may be involved in disease etiology, the present investigation assessed the effects of neonatal 5-HT depletion on prepulse inhibition of the startle response (PPI) in rats. Three-day-old Sprague-Dawley rats were pretreated with desipramine (20 mg/kg), followed by an intraventricular injection of the selective 5-HT neurotoxin 5,7-dihydroxytryptamine (5,7-DHT, 70 μg dissolved in 2 μl of 0.1% saline solution in ascorbic acid) on each side. Three months later, the rats' PPI was tested. Despite a severe and permanent decrease (80-100%) in hippocampal, prefrontal and striatal 5-HT levels, treatment with 5,7-DHT caused no disruption of PPI. In contrast to this lack of effect, the 5,7-DHT treatment increased basal startle activity, as measured in response to a 120 dB stimulus. Thus, our results clearly indicate that neonatal 5-HT depletion does not interrupt prepulse inhibition in rats. Studies involving lesions of brain structures or chemical systems run the risk of inducing compensatory changes in brain function, resulting in an amelioration of any deficit. The development of such compensatory mechanisms seems likely in the current study, due to the severe and long-lasting effect of neonatal 5,7-DHT-induced reduction on 5-HT levels.     

113Cd Nuclear Magnetic Resonance (NMR) Study of the Inhibitory Effect of Methylvinylether/Maleic Acid (PVM/MA) Copolymer on the Alkaline Phosphatase of Escherichia coli

The inhibitory effect of PVM/MA copolymer on the alkaline phosphatase (AP) of E. coli was investigated. Kinetic studies indicated that enzyme inhibition was characterized by a reduction in both the Vmax and the Km. Addition of 1 mM zinc or magnesium ions to the reaction prevented inhibition of the enzyme by the copolymer. The inhibitory effect of the copolymer on alkaline phosphatase was also investigated using 113Cd NMR after exchange of the active center metal ions with 113Cd. The resulting Cd(II)6AP exhibited characteristic 113Cd resonances reflecting the environment of the A, B, and C metal binding sites of the enzyme's active center. Addition of copolymer resulted in a 113Cd NMR spectrum which indicated removal of 113Cd from the C site and formation of two distinct forms of the enzyme. Possible explanations for the 113Cd NMR results are discussed.     

The role of biotransformation on the pharmacology of the monoamineoxidase inhibitor 5-oxo-N-(d-trans-2-phenylcyclopropyl)-1-2-pyrrolidone-carboxamide (EX-4883)

5-oxo-N-(d-trans-2-phenylcyclopropyl)-1-2-pyrrolidone-carboxamide (EX 4883) is a potent inhibitor of monoamine oxidase in vivo. Upon being activated by a soluble fraction preparation of rat liver homogenate, it is also an inhibitor of monoamine oxidase in vitro. The biotransformation products are hypothesized to be tranylcypromine and pyrrolidone carboxylic acid.Tranylcypromine sulfate and EX-4883 were added to several test preparations in order to compare any pharmacological responses. Comparing the responses on a molar basis, it was impossible to distinguish between the stimulatory effects of the two drugs in the following pharmacological test systems: (1) rat blood pressure, (2) cat blood pressure, (3) cat nictitating membrane, and (4) rat Langendorff heart.The first dramatic pharmacological evidence of non-identical responses to equimolar concentrations of tranylcypromine and EX-4883 came in experiments with isolated rat atria. Tranylcypromine was more potent in eliciting positive inotropic responses from the atria than was EX-4883, but the dose-response curves were parallel. Prior bioactivation of EX-4883 by a soluble fraction component of rat liver homogenate caused a shift of the control curve toward the tranylcypromine control curve.The results support the conclusion that EX-4883 must be biotransformed before it is a pharmacologically active compound, and, that once it is activated, EX-4883 has a pharmacology analogous to that of tranylcypromine.     

Establishment of robust functional assays for the characterization of neuropeptide Y (NPY) receptors: identification of 3-(5-benzoyl-thiazol-2-ylamino)-benzonitrile as selective NPY type 5 receptor antagonist

The human Neuropeptide Y (NPY) receptors 1 (hY1), 2 (hY2), 4 (hY4), and the mouse type 5 (mY5) receptor were expressed in human embryonic kidney 293 (HEK293) cells. The receptors bound a radioiodinated NPY ligand with high affinity and various NPY analogs competed for binding in a receptor selective-manner. Similarly, cAMP-inhibition and GTPgammaS binding assays were established. The four NPY receptors were further tested in the fluorimetric imaging plate reader (FLIPR) format, a cellular high-throughput assay, in the absence and presence of chimeric G proteins, Gqo5, Gqi5 and Gqi9. The receptors stimulated transient calcium release only in the presence of chimeric G proteins. While hY1, hY2 and hY4 receptors coupled to Gqo5, Gqi5 and Gqi9, the mY5 receptor stimulated transient calcium release only when co-expressed with Gqi9. Using an in silico screening approach we identified a small molecule 3-(5-benzoyl-thiazol-2-ylamino)-benzonitrile (compound 1), which bound to the mY5 receptor with high affinity (Ki=32.1+/-1.8 nM), competitively antagonized NPY-mediated GTPgammaS binding and calcium stimulation with high potency, and had no affinity for other NPY receptors. These data show that NPY receptors can be functionally coupled to the FLIPR readout, allowing for high throughput compound testing and identification of novel molecules.