Zebrafish as an Animal Model for Ocular Toxicity Testing: A Review of Ocular Anatomy and Functional Assays.

Xenobiotics make their way into organisms from diverse sources including diet, medication, and pollution. Our understanding of ocular toxicities from xenobiotics in humans, livestock, and wildlife is growing thanks to laboratory animal models. Anatomy and physiology are conserved among vertebrate eyes, and studies with common mammalian preclinical species (rodent, dog) can predict human ocular toxicity. However, since the eye is susceptible to toxicities that may not involve a histological correlate, and these species rely heavily on smell and hearing to navigate their world, discovering visual deficits can be challenging with traditional animal models. Alternative models capable of identifying functional impacts on vision and requiring minimal amounts of chemical are valuable assets to toxicology. Human and zebrafish eyes are anatomically and functionally similar, and it has been reported that several common human ocular toxicants cause comparable toxicity in zebrafish. Vision develops rapidly in zebrafish; the tiny larvae rely on visual cues as early as 4 days, and behavioral responses to those cues can be monitored in high-throughput fashion. This article describes the comparative anatomy of the zebrafish eye, the notable differences from the mammalian eye, and presents practical applications of this underutilized model for assessment of ocular toxicity.

Use of Zebrafish in Drug Discovery Toxicology.

Unpredicted human safety events in clinical trials for new drugs are costly in terms of human health and money. The drug discovery industry attempts to minimize those events with diligent preclinical safety testing. Current standard practices are good at preventing toxic compounds from being tested in the clinic; however, false negative preclinical toxicity results are still a reality. Continual improvement must be pursued in the preclinical realm. Higher-quality therapies can be brought forward with more information about potential toxicities and associated mechanisms. The zebrafish model is a bridge between in vitro assays and mammalian in vivo studies. This model is powerful in its breadth of application and tractability for research. In the past two decades, our understanding of disease biology and drug toxicity has grown significantly owing to thousands of studies on this tiny vertebrate. This Review summarizes challenges and strengths of the model, discusses the 3Rs value that it can deliver, highlights translatable and untranslatable biology, and brings together reports from recent studies with zebrafish focusing on new drug discovery toxicology.

Design of Aminobenzothiazole Inhibitors of Rho Kinases 1 and 2 by Using Protein Kinase†A as a Structure Surrogate.

We describe the design, synthesis, and structure-activity relationships (SARs) of a series of 2-aminobenzothiazole inhibitors of Rho kinases (ROCKs) 1 and 2, which were optimized to low nanomolar potencies by use of protein kinase A (PKA) as a structure surrogate to guide compound design. A subset of these molecules also showed robust activity in a cell-based myosin phosphatase assay and in a mechanical hyperalgesia in vivo pain model.

Pharmacological modulation of brain activity in a preclinical model of osteoarthritis.

The earliest stages of osteoarthritis are characterized by peripheral pathology; however, during disease progression chronic pain emerges-a major symptom of osteoarthritis linked to neuroplasticity. Recent clinical imaging studies involving chronic pain patients, including osteoarthritis patients, have demonstrated that functional properties of the brain are altered, and these functional changes are correlated with subjective behavioral pain measures. Currently, preclinical osteoarthritis studies have not assessed if functional properties of supraspinal pain circuitry are altered, and if these functional properties can be modulated by pharmacological therapy either by direct or indirect action on brain systems. In the current study, functional connectivity was first assessed in order to characterize the functional neuroplasticity occurring in the rodent medial meniscus tear (MMT) model of osteoarthritis-a surgical model of osteoarthritis possessing peripheral joint trauma and a hypersensitive pain state. In addition to knee joint trauma at week 3 post-MMT surgery, we observed that supraspinal networks have increased functional connectivity relative to sham animals. Importantly, we observed that early and sustained treatment with a novel, peripherally acting broad-spectrum matrix metalloproteinase (MMP) inhibitor (MMPi) significantly attenuates knee joint trauma (cartilage degradation) as well as supraspinal functional connectivity increases in MMT animals. At week 5 post-MMT surgery, the acute pharmacodynamic effects of celecoxib (selective cyclooxygenase-2 inhibitor) on brain function were evaluated using pharmacological magnetic resonance imaging (phMRI) and functional connectivity analysis. Celecoxib was chosen as a comparator, given its clinical efficacy for alleviating pain in osteoarthritis patients and its peripheral and central pharmacological action. Relative to the vehicle condition, acute celecoxib treatment in MMT animals yielded decreased phMRI infusion responses and decreased functional connectivity, the latter observation being similar to what was detected following chronic MMPi treatment. These findings demonstrate that an assessment of brain function may provide an objective means by which to further evaluate the pathology of an osteoarthritis state as well as measure the pharmacodynamic effects of therapies with peripheral or peripheral and central pharmacological action.

Optimization of Zebrafish Larvae Sectioning for Mass Spectrometry Imaging.

The utility of zebrafish is becoming more frequent due to lower costs and high similarities to humans. Zebrafish larvae are attractive subjects for drug screening and drug metabolism research. However, obtaining good quality zebrafish larvae sections for batch samples at designated planes, angles, and locations for comparison purposes is a challenging task. We report here the optimization of fresh frozen zebrafish larvae sectioning for mass spectrometry imaging. We utilized the gelatin solutions that were created at two different temperatures (50 and 85 °C) as embedding media. Gelatin-50 (gelatin created under 50 °C, solid gel under room temperature) was used to make a larvae-shaped mold and gelatin-85 (gelatin created under 85 °C, liquid under room temperature) was used to embed the larvae. H&E staining of sections shows well-preserved morphology and minimal histological interference. More importantly, the position of the larvae was well controlled resulting in more consistent sectioning of the larvae.

Molecular determinants of species-specific activation or blockade of TRPA1 channels.

TRPA1 is an excitatory, nonselective cation channel implicated in somatosensory function, pain, and neurogenic inflammation. Through covalent modification of cysteine and lysine residues, TRPA1 can be activated by electrophilic compounds, including active ingredients of pungent natural products (e.g., allyl isothiocyanate), environmental irritants (e.g., acrolein), and endogenous ligands (4-hydroxynonenal). However, how covalent modification leads to channel opening is not understood. Here, we report that electrophilic, thioaminal-containing compounds [e.g., CMP1 (4-methyl-N-[2,2,2-trichloro-1-(4-nitro-phenylsulfanyl)-ethyl]-benzamide)] covalently modify cysteine residues but produce striking species-specific effects [i.e., activation of rat TRPA1 (rTRPA1) and blockade of human TRPA1 (hTRPA1) activation by reactive and nonreactive agonists]. Through characterizing rTRPA1 and hTRPA1 chimeric channels and point mutations, we identified several residues in the upper portion of the S6 transmembrane domains as critical determinants of the opposite channel gating: Ala-946 and Met-949 of rTRPA1 determine channel activation, whereas equivalent residues of hTRPA1 (Ser-943 and Ile-946) determine channel block. Furthermore, side-chain replacements at these critical residues profoundly affect channel function. Therefore, our findings reveal a molecular basis of species-specific channel gating and provide novel insights into how TRPA1 respond to stimuli.

A multi-institutional study benchmarking the zebrafish developmental assay for prediction of embryotoxic plasma concentrations from rat embryo-fetal development studies.

Predicting embryotoxicity of pharmaceutical compounds or industrial chemicals is crucial for public safety. Conventional studies which monitor embryo-fetal development in rats and rabbits are costly and time consuming. Alternative assays which are simpler and less costly are being pursued. The purpose of this research was to assess the capacity for the zebrafish development assay to predict mammalian plasma levels that are embryotoxic. Previously published data on rat plasma levels associated with embryotoxicity were used to guide concentration ranges for each of 25 chemicals dissolved in the media bathing developing zebrafish embryos. Embryotoxic media concentrations were compared to embryotoxic rat plasma concentrations. Assays were conducted in parallel at multiple sites as a consortium effort through the Health and Environmental Sciences Institute (HESI). Considering results from all sites, the zebrafish embryo development assay predicted (within 1-log) the rat maternal exposure levels associated with embryotoxicity 75% of the time.

Positive allosteric modulation of the alpha7 nicotinic acetylcholine receptor: ligand interactions with distinct binding sites and evidence for a prominent role of the M2-M3 segment.

The alpha7 nicotinic acetylcholine receptor (nAChR), a homopentameric, rapidly activating and desensitizing ligand-gated ion channel with relatively high degree of calcium permeability, is expressed in the mammalian central nervous system, including regions associated with cognitive processing. Selective agonists targeting the alpha7 nAChR have shown efficacy in animal models of cognitive dysfunction. Use of positive allosteric modulators selective for the alpha7 receptor is another strategy that is envisaged in the design of active compounds aiming at improving attention and cognitive dysfunction. The recent discovery of novel positive allosteric modulators such as 1-(5-chloro-2-hydroxyphenyl)-3-(2-chloro-5-trifluoromethylphenyl)urea (NS-1738) and 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)urea (PNU-120596) that are selective for the alpha7 nAChRs but display significant phenotypic differences in their profile of allosteric modulation, suggests that these molecules may act at different sites on the receptor. Taking advantage of the possibility to obtain functional receptors by the fusion of proteins domains from the alpha7 and the 5-HT(3) receptor, we examined the structural determinants required for positive allosteric modulation. This strategy revealed that the extracellular N-terminal domain of alpha7 plays a critical role in allosteric modulation by NS-1738. In addition, alpha7-5HT(3) chimeras harboring the M2-M3 segment showed that spontaneous activity in response to NS-1738, which confirmed the critical contribution of this small extracellular segment in the receptor gating. In contrast to NS-1738, positive allosteric modulation by PNU-120596 could not be restored in the alpha7-5HT(3) chimeras but was selectively observed in the reverse 5HT(3)-alpha7 chimera. All together, these data illustrate the existence of distinct allosteric binding sites with specificity of different profiles of allosteric modulators and open new possibilities to investigate the alpha7 receptor function.

High-throughput Measurement of Gut Transit Time Using Larval Zebrafish.

Zebrafish are used as alternative model organisms for drug safety testing. The gastrointestinal (GI) tract of zebrafish has genetic, neuronal, and pharmacological similarities to that of the mammals. GI intolerance during clinical testing of drug candidates is common and may pose a serious threat to human health. Testing for GI toxicity in preclinical mammalian models can be expensive in terms of time, test compound, and labor. The high-throughput method presented here may be used to predict GI safety issues. Compared to mammalian models, this method allows for more expedient assessment of test compound effects on GI transit while using low quantities of compound. In this method, larval zebrafish (7 days post fertilization) are fed food containing a fluorescent label. After feeding, each larval fish is placed into a well of a 96-conical-bottom-well plate and dosed with test compound (dissolved in water) or the vehicle. As gut transit occurs, fecal matter accumulates on the bottom of the wells, and the rate at which this happens is monitored by measuring fluorescence from the bottom of the well repeatedly over time using a plate spectrophotometer. The fluorescence from larvae in a given treatment group are averaged and these values are graphed along with standard error, for each measurement time, yielding a curve representing average transit of food over time. Effects on gut transit time are identified by comparing the area under the curve for each treatment group to that of the vehicle-treated group. This method detected changes in zebrafish GI transit time induced by drugs with known clinical GI effects; it can be employed to interrogate dozens of treatments for GI effects per day. As such, safer compounds can be quickly prioritized for mammalian testing, which expedites discovery and proffers 3Rs advancement.

From the Cover: Inhibitors of Nicotinamide Phosphoribosyltransferase Cause Retinal Damage in Larval Zebrafish.

Nicotinamide phosphoribosyltransferase (NAMPT) has been investigated as a target for oncology because it catalyzes a rate-limiting step in cellular energy metabolism to produce nicotinamide adenine dinucleotide. Small molecule inhibitors of NAMPT have been promising drug candidates but preclinical development has been hindered due to associated retinal toxicity. Here we demonstrate that larval zebrafish can predict retinal toxicity associated with this mechanism revealing an attractive alternative method for identifying such toxicities. Zebrafish permit higher throughput testing while using far lower quantities of test article compared with mammalian systems. NAMPT inhibitor-associated toxicity manifested in zebrafish as a loss of response to visual cues compared with auditory cues. Zebrafish retinal damage associated with NAMPT inhibitor treatment was confirmed through histopathology. Ranking 6 NAMPT inhibitors according to their impact on zebrafish vision revealed a positive correlation with their in vitro potencies on human tumor cells. This correlation indicates translatable pharmacodynamics between zebrafish and human NAMPT and is consistent with on-target activity as the cause of retinal toxicity associated with NAMPT inhibition. Together, these data illustrate the utility of zebrafish for identifying compounds that may cause ocular toxicity in mammals, and, likewise, for accelerating development of compounds with improved safety margins.

Utility of large-scale transiently transfected cells for cell-based high-throughput screens to identify transient receptor potential channel A1 (TRPA1) antagonists.

Despite increasing use of cell-based assays in high-throughput screening (HTS) and lead optimization, one challenge is the adequate supply of high-quality cells expressing the target of interest. To this end, cell lines stably expressing targets are often established, maintained, and scaled up by cell culture. These steps require large investments of time and resources. Moreover, significant variability invariably occurs in cell yield, viability, expression levels, and target activities. In particular, stable expression of targets such as transient receptor potential A1 (TRPA1) causes toxicity, cell line degeneration, and loss of functional activity. Therefore, in an effort to identify TRPA1 antagonists, the authors used large-scale transiently transfected (LSTT) cells, enabling rapid establishment of assays suitable for HTS. LSTT cells, which could- be stored frozen for a long period of time (e.g., at least 42 weeks), retained TRPA1 protein expression and could be easily revived to produce robust and consistent signals in calcium influx and electrophysiological assays. Using cells from a single transfection, a chemical library of 700,000 compounds was screened, and TRPA1 antagonists were identified. The use of LSTT circumvented issues associated with stable TRPA1 expression, increased flexibility and consistency, and greatly reduced labor and cost. This approach will also be applicable to other pharmaceutical targets.

Measuring drug absorption improves interpretation of behavioral responses in a larval zebrafish locomotor assay for predicting seizure liability.

INTRODUCTION: Unanticipated effects on the central nervous system are a concern during new drug development. A larval zebrafish locomotor assay can reveal seizure liability of experimental molecules before testing in mammals. Relative absorption of compounds by larvae is lacking in prior reports of such assays; having those data may be valuable for interpreting seizure liability assay performance. METHODS: Twenty-eight reference drugs were tested at multiple dose levels in fish water and analyzed by a blinded investigator. Responses of larval zebrafish were quantified during a 30min dosing period. Predictive metrics were calculated by comparing fish activity to mammalian seizure liability for each drug. Drug level analysis was performed to calculate concentrations in dose solutions and larvae. Fifteen drug candidates with neuronal targets, some having preclinical convulsion findings in mammals, were tested similarly. RESULTS: The assay has good predictive value of established mammalian responses for reference drugs. Analysis of drug absorption by larval fish revealed a positive correlation between hyperactive behavior and pro-convulsive drug absorption. False negative results were associated with significantly lower compound absorption compared to true negative, or true positive results. The predictive value for preclinical toxicology findings was inferior to that suggested by reference drugs. DISCUSSION: Disproportionately low exposures in larvae giving false negative results demonstrate that drug exposure analysis can help interpret results. Due to the rigorous testing commonly performed in preclinical toxicology, predicting convulsions in those studies may be more difficult than predicting effects from marketed drugs.

Detection of multiple H3 receptor affinity states utilizing [3H]A-349821, a novel, selective, non-imidazole histamine H3 receptor inverse agonist radioligand.

1. A-349821 is a selective histamine H3 receptor antagonist/inverse agonist. Herein, binding of the novel non-imidazole H3 receptor radioligand [3H]A-349821 to membranes expressing native or recombinant H3 receptors from rat or human sources was characterized and compared with the binding of the agonist [3H]N--methylhistamine ([3H]NMH). 2. [3H]A-349821 bound with high affinity and specificity to an apparent single class of saturable sites and recognized human H3 receptors with 10-fold higher affinity compared to rat H3 receptors. [3H]A-349821 detected larger populations of receptors compared to [3H]NMH. 3. Displacement of [3H]A-349821 binding by H3 receptor antagonists/inverse agonists was monophasic, suggesting recognition of a single binding site, while that of H3 receptor agonists was biphasic, suggesting recognition of both high- and low-affinity H3 receptor sites. 4. pKi values of high-affinity binding sites for H3 receptor competitors utilizing [3H]A-349821 were highly correlated with pKi values obtained with [3H]NalphaMH, consistent with labelling of H3 receptors by [3H]A-349821. 5. Unlike assays utilizing [3H]NMH, addition of GDP had no effect on saturation parameters measured with [3H]A-349821, while displacement of [3H]A-349821 binding by the H3 receptor agonist histamine was sensitive to GDP. 6. In conclusion, [3H]A-349821 labels interconvertible high- and low-affinity states of the H3 receptor, and displays improved selectivity over imidazole-containing H3 receptor antagonist radioligands. [3H]A-349821 competition studies showed significant differences in the proportions and potencies of high- and low-affinity sites across species, providing new information about the fundamental pharmacological nature of H3 receptors.

A high-throughput method for predicting drug effects on gut transit time using larval zebrafish.

INTRODUCTION: Zebrafish are an attractive vertebrate model due to their small size, transparency through organogenesis, and high fecundity. The zebrafish gastrointestinal (GI) tract is similar to the mammalian GI tract in gene expression, nervous system control, and response to chemical challenges. GI intolerance is a common preclinical finding and can be a serious clinical safety concern. Mammalian GI liability tests are conducted at the expense of time, test article, and labor. We developed a high-throughput method to predict mammalian GI safety issues using larval zebrafish. METHODS: Fluorescent food is fed to larval zebrafish (7 days post fertilization). After feeding, larvae are placed individually into wells of a 96-well plate and dosed with test compounds. Fluorescence is measured from the bottom of the wells repeatedly over the course of 24h and thus fecal accumulation is tracked over time. The area under the curve is compared between treated and vehicle-treated groups. RESULTS: Drugs with established clinical GI effects significantly impacted zebrafish GI transit time as measured by this method; tegaserod and metoclopramide accelerated transit time, while atropine and amitriptyline slowed transit time. This method is sensitive enough to reflect dose-level associated effects as demonstrated using atropine. Using a suite of 24 compounds with known (positive or negative) mammalian GI effects, we characterized this method as having a high positive predictive value. DISCUSSION: Here we present an efficient assay for predicting mammalian GI transit liabilities using larval zebrafish. With this assay, an investigator can evaluate dozens of compounds in a single day using very little amount of each test article. As such, safe drug candidates can be prioritized for mammalian testing, which expedites the discovery process and provides 3 Rs impact.

CYP3A5 genotype has a dose-dependent effect on ABT-773 plasma levels.

BACKGROUND: The metabolizing enzyme cytochrome P450 (CYP) 3A5 is polymorphically expressed as a result of genetic variants that do not encode functional protein. Because of overlapping substrate specificity with CYP3A4 and the multidrug efflux pump P-glycoprotein, the importance of CYP3A5 genetic polymorphism for pharmacokinetics is controversial. OBJECTIVE: Our objective was to determine whether genetic polymorphisms in CYP3A5 or MDR-1 (which encodes P-glycoprotein) influence the drug levels of ABT-773, a ketolide antibiotic that is a substrate for both CYP3A and P-glycoprotein. METHODS: Healthy volunteers given 3 different oral dose levels of ABT-773 were genotyped at 2 common CYP3A5 and 7 common MDR-1 polymorphisms. Individuals were categorized as CYP3A5-positive if they carried at least 1 functional CYP3A5*1 allele and as CYP3A5-negative if they did not. Area under the plasma concentration-time curves (AUCs) from 0 to 6 hours (AUC(t)) and maximum postdose plasma concentration (C(max)) after a single dose and on day 5 of a twice-daily regimen were calculated and correlated with genotypes. RESULTS: ABT-773 AUC(t) and C(max) were, on average, higher in CYP3A5-negative subjects given 450 mg ABT-773 (n = 9) than in CYP3A5-positive subjects with identical doses (n = 8). The relationship for AUC(t) was statistically significant both after a single dose (geometric mean and 95% confidence interval [CI], 5.0 microg.h/mL [3.9-6.4 microg.h/mL] versus 2.8 microg.h/mL [1.8-4.3 microg.h/mL]; P =.03) and on the fifth day of twice-daily dosing (12.4 microg.h/mL [8.7-17.6 microg.h/mL] versus 7.4 microg.h/mL [5.5-9.8 microg.h/mL], P =.04). The relationship for C(max) was statistically significant after a single dose (1220 microg/mL [867-1167 microg/mL] versus 727 microg/mL [506-1044 microg/mL], P =.04) and showed a trend in the same direction on the fifth day of twice-daily dosing (2566 microg/mL [1813-3631 microg/mL] versus 1621 microg/mL [1122-2343 microg/mL], P =.07). In contrast, AUC(t) and C(max) were not significantly different between CYP3A5-positive and CYP3A5-negative individuals given 150 mg or 300 mg ABT-773. ABT-773 plasma levels did not trend with MDR-1 genotypes. CONCLUSIONS: These results suggest that CYP3A5 genotype may be an important determinant of in vivo drug disposition and that this effect may be dose-dependent.

Use of a fluorescent imaging plate reader--based calcium assay to assess pharmacological differences between the human and rat vanilloid receptor.

The cloned vanilloid receptor 1 (VR1) is a ligand-gated calcium channel that is believed to be the capsaicin-activated vanilloid receptor found in native tissues, based on similarities regarding molecular mass, tissue distribution, and electrophysiological properties. Using a Fluorescent Imaging Plate Reader (FLIPR), along with Fluo-3 to signal intracellular calcium levels ([Ca(++)](i)), rat VR1 (rVR1) and a human orthologue (hVR1) were pharmacologically characterized with various VR1 ligands. HEK-293 cells, stably expressing rVR1 or hVR1, exhibited dose-dependent increases in [Ca(++)](i) when challenged with capsaicin (EC(50)s congruent with 10 nM). Responses to capsaicin were blocked by the VR1 antagonist capsazepine and were dependent on VR1 expression. Potencies for 10 structurally diverse VR1 agonists revealed rVR1 potencies highly correlated to that of hVR1 (R(2) = 0.973). However, a subset of agonists (tinyatoxin, gingerol, and zingerone) was approximately 10-fold more potent for rVR1 compared to hVR1. Schild analysis for blockade of capsaicin-induced responses by capsazepine was consistent with competitive antagonism, whereas ruthenium red displayed noncompetitive antagonism. Compared to rVR1, hVR1 was more sensitive to blockade by both antagonists. For both rVR1 and hVR1, time-response waveforms elicited by resiniferatoxin increased more gradually compared to other agonists. Tinyatoxin also displayed slow responses with hVR1 but showed rapid responses with rVR1. Thus, FLIPR technology can be used to readily reveal differences between rVR1 and hVR1 pharmacology with respect to potencies, efficacies, and kinetics for several VR1 ligands.

Tissue specific expression of alternative splice forms of human cyclic nucleotide gated channel subunit CNGA3.

PURPOSE: The cyclic nucleotide gated cation channel subunit, CNGA3, (also known as the cone alpha CNG subunit), plays a vital role in signal transduction of human cone cells. Owing to its well established role in vision, the human CNGA3 isoform studied thus far was cloned from retinal tissue. However, non-human homologs of CNGA3 have been cloned from a variety of other tissues including kidney, heart, pineal gland, adrenal gland and testes. Among these, alternative splice forms of CNGA3 have been identified. The objective of this study was to explore alternative splice forms of human CNGA3 and determine their distribution among various human tissues. METHODS: RT-PCR was used to amplify full length open reading frames of CNGA3 from human testes RNA and to detect and distinguish among splice forms in 23 tissues. DNA sequencing was used to characterize full length splice forms and to confirm the identity of RT-PCR products from a number of tissues. RESULTS: Two new full length alternatively spliced forms of hCNGA3 (referred to as Variant 2 and 3) were isolated. These splice variants are different from the cone hCNGA3 (Variant 1) in that they lack exon 5. In addition, they differ from each other in that Variant 3 contains an extra exon that originates from the intron preceding exon 4. We demonstrate that CNGA3 transcripts are detectable in all 23 human tissues examined. In all tissues, except retina, Variant 2 specific PCR products had the brightest band intensity. In retina, the band from Variant 1 (containing exon 5) was most intense. In fact, among all tissues examined, Variant 1 can only be detected strongly in the retina. CONCLUSIONS: The extensive distribution of CNGA3 and the tissue specific expression of alternative splice forms indicate widespread and diverse roles for CNGA3. The unique expression of Variant 1 in the retina implies a significance to the amino acids encoded by exon 5 that may be necessary for the function of CNGA3 in human cone cells.

Cloning and pharmacological characterization of the monkey histamine H3 receptor.

Species differences have been described previously for histamine H(3) receptor pharmacology. Rat selective histamine H(3) receptor ligands such as ciproxifan and A-304121 (2-amino-1-[4-[3-(4-cyclopropanecarbonyl-phenoxy)-propyl]-piperazin-1-yl]-propan-1-one) show over 100-fold selectivity for the rat receptor compared to the human receptor. To date, however, the pharmacology of the cloned monkey histamine H(3) receptor has not been examined. In this study, we cloned the monkey histamine H(3) receptor gene (H(3)R) and evaluated the receptor pharmacology in binding and functional assays. The monkey histamine H(3) receptor is highly homologous to the human receptor with 438 identities in their 445 amino acid sequences, but less homologous to the rat receptor. However, unlike the human or rat, we found no evidence for additional splicing for the monkey H(3)R. Pharmacological analysis indicated that the monkey receptor exhibited similar pharmacological profiles to those of the human receptor, providing critical information for characterizing histamine H(3) receptor ligands in monkey behavioral models.

Species comparison and pharmacological characterization of rat and human CB2 cannabinoid receptors.

Pharmacological effects of cannabinoid ligands are thought to be mediated through cannabinoid CB1 and CB2 receptor subtypes. Sequence analysis revealed that rat and human cannabinoid CB2 receptors are divergent and share 81% amino acid homology. Pharmacological analysis of the possible species differences between rat and human cannabinoid CB2 receptors was performed using radioligand binding and functional assays. Pronounced species selectivity at the rat cannabinoid CB2 receptor (50- to 140-fold) was observed with AM-1710 (3-(1,1-Dimethyl-heptyl)-1-hydroxy-9-methoxy-benzo[c]chromen-6-one) and AM-1714 (3-(1,1-Dimethyl-heptyl)-1-9-dihydroxy-benzo[c]chromen-6-one). In contrast, JWH-015 ((2-Methyl-1-propyl-1H-indol-3-yl)-napthalen-1-yl-methanone) was 3- to 10-fold selective at the human cannabinoid CB2 receptor. Endocannabinoid ligands were more human receptor selective. Cannabinoid CB2 receptor antagonist, AM-630 ((6-Iodo-2-methyl-1-(2-morpholin-4-yl-ethyl)-1H-indol-3-yl)-(4-methoxy-phenyl)-methanone) was more potent at the rat receptor in radioligand binding and functional assays than that of the human receptor. The findings of the pharmacological differences between the human and rat cannabinoid CB2 receptors in this study provide critical information for characterizing cannabinoid ligands in in vivo rodent models for drug discovery purpose.

Importance of M2-M3 loop in governing properties of genistein at the α7 nicotinic acetylcholine receptor inferred from α7/5-HT3A chimera.

Genistein and 5-hydroxyindole (5-HI) potentiate the α7 nicotinic acetylcholine receptor current by primarily increasing peak amplitude, a property of type I α7 positive allosteric modulation. In this study, the effects of these two compounds were investigated at two different α7/5-HT(3) chimeras (chimera 1, comprising of extracellular α7 N-terminus fused to the remainder of 5-HT(3A), and chimera 2 containing an additional α7 encoded M2-M3 loop), and wild-type α7 and 5-HT(3A) receptors. Agonist-evoked responses, examined by expression of the chimeras in Xenopus laevis oocytes or HEK-293 cells, revealed that currents decayed slower and compounds {rank order: N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-282987)~2-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5-phenyl-1,3,4-oxadiazole (NS6784)>acetylcholine>choline} were more potent in chimera 2 than chimera 1 or α7 receptors. In chimera 2, genistein and 5-HI potentiated agonist-evoked responses (EC(50): 4-5 µM for genistein and 300-500 µM for 5-HI) and at higher concentrations evoked current directly consistent with ago-allosteric modulation. At chimera 1 and 5-HT(3A) receptors, neither compound directly evoked any current and 5-HI, only at chimera 1, was able to potentiate agonist-evoked responses. Genistein and 5-HI did not inhibit the binding of the α7 agonist [(3)H](1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1] heptane ([(3)H]A-585539) to rat brain or chimera 2. In summary, this study supports the role of the M2-M3 loop being critical for the positive allosteric effect of genistein, but not 5-HI, and in agonist-evoked response fine-tuning. The identification of distinct α7 receptor modulatory sites offers unique opportunities for developing CNS therapeutics and understanding its pharmacology.