Fluorometric Imaging Plate Reader Research Articles

Generation and phenotype analysis of CysLTR1 L118F mutant mice.

Cysteinyl leukotrienes (CysLTs) are a group of eicosanoids that regulate the pathogenesis of various human diseases, mainly by signaling through the cysteinyl leukotriene receptor 1 (CysLTR1). The aim of this study was to generate and examine the phenotype of CysLTR1 L118F mutant mice. CysLTR1 L118F mutant mice were generated by the simultaneous microinjection of single guide RNA, Cas9 messenger RNA, and donor plasmid into fertilized mouse eggs. The morphological and behavioral characteristics of the resultant CysLTR1 L118F mutant mice were analyzed using an animal phenotype analysis platform, which included the assessment of body length, tail length, grip strength, and locomotor activity. Immunoprecipitation coupled with mass spectrometry was performed to identify CysLTR1-interacting proteins, and the intracellular calcium levels were determined using fluorometric imaging plate reader assays. The body length and tail length of CysLTR1 L118F mutant mice were significantly increased compared with wild-type mice. In addition, the grip strength and locomotor activity were remarkably elevated in L118F mutant mice compared with wild-type mice. Only three proteins were found to interact with both wild-type and CysLTR1 L118F proteins, whereas 4 and 13 additional proteins interacted exclusively with wild-type and mutant CysLTR1, respectively. Lastly, the responsiveness of cardiac muscle cells to CysLTs were significantly impaired by the L118F substitution in CysLTR1 proteins. The CysLTR1 L118F point mutation induced significant changes in the mouse morphology and behavior, which might be mediated by alterations of its protein interaction profile.

A FLIPR Assay for Discovery of GABAA Receptor Modulators of Natural Origin.

A fluorometric imaging plate reader (FLIPR) assay utilizing Chinese hamster ovary (CHO) cells stably transfected with GABAA receptors of α 1 β 2 γ 2 subunit composition was evaluated and validated for rapid screening of plant extract libraries and efficient localization of active compounds in extracts. Validation was performed with pure compounds and extracts known to contain allosteric GABAA receptor modulators. Plants extracts that had been previously reported as active in an assay using Xenopus laevis oocytes transiently expressing GABAA receptors of α 1 β 2 γ 2 subunit composition were also active in the FLIPR assay. A protocol for HPLC-based activity profiling was developed, whereby separations of 0.4 - 1.2 mg of extracts on an analytical HPLC column were found to be sufficient for the sensitivity of the bioassay. The protocol successfully localized the activity of known GABAergic natural products, such as magnolol in Magnolia officinalis, valerenic acid in Valeriana officinalis, and piperine in Piper nigrum extract. EC50 values of compounds (magnolol: 4.81 ± 1.0 µM, valerenic acid: 12.56 ± 1.2 µM, and piperine: 5.76 ± 0.7 µM) were found to be comparable or lower than those reported using Xenopus oocyte assays.

Synthesis of 21‐Alkylidenes and 21‐Alkylol Analogues of Uscharin and Their Effects on Intracellular Calcium in Cardiac Cells

AbstractFive 21‐alkylidenes (2‐6) and a 21‐alkylol (7) derivatives of uscharin (1) were synthesized by aldol condensation reaction and aldol reaction of uscharin with aromatic aldehydes, respectively. We investigated the ability of uscharin and its derivatives to interfere with the intracellular calcium level by flow cytometry and contractile characteristics at the single cell level by time‐lapse microscopy in H9c2 cells. Changes of spontaneous calcium oscillations in beating HL‐1 cells were also studied by a high‐throughput Fluorometric Imaging Plate Reader (FLIPR) assay. We reported for the first time that uscharin could produce increased contraction strength in cardiac muscle cells while it showed a significant cytotoxicity to atrial cells. Unlike uscharin, 6 induced a marked increase of myocardial contractility, whereas 6 neither caused irregular frequency of oscillations nor cardiac cytotoxic effects under tested conditions. Structural modification on the butenolide moiety of uscharin showed a reduction of cardiac cytotoxicity and exhibited potential for use as an inotropic agent for the treatment of heart failure.

Functional analysis of a biflavone, as a novel inhibitor of TRPV4‐dependent macrophages foam cell formation

Cardiovascular disease is the number one cause of death in United States, and atherosclerosis, a chronic inflammatory disease, is the most dominant underlying pathology. Macrophages, an essential cell type in atherogenesis, are known to directly respond to changes to their surrounding mechanical environment. As TRPV4 (transient receptor potential channel of the vanilloid subfamily) channels can be sensitized by changes in matrix stiffness, we hypothesize that, TRPV4 may upregulate inflammatory/atherogenic macrophage functions. We performed a high‐throughput screening for antagonists of TRPV4 from a library of 2000 compounds using a fluorometric imaging plate reader (FLIPR)‐based Ca2+ influx assay; we identified ginkgetin, a biflavone, as a novel antagonist to TRPV4. Primary and secondary screening was performed with normal human dermal fibroblasts and mouse bone marrow derived macrophages (BMDMs) to assess the ability of ginkgetin and other candidates to inhibit TRPV4‐elicited Ca2+ influx after TRPV4 activation with its selective agonist GSK1016790. In our hand's, ginkgetin demonstrated an IC50 of 0.5 μM for inhibition of TRPV4‐elicited Ca2+ influx. We show that antagonism of TRPV4 channel by ginkgetin blocks oxidized LDL (oxLDL)‐induced macrophage foam cell formation in a concentration dependent manner. Mechanistically, we show that ginkgetin attenuated oxLDL uptake but not its cell surface binding in BMDMs. Altogether, our results show that ginkgetin is a novel inhibitor of TRPV4‐mediated macrophage foam cell formation by modulating uptake of oxLDL.Support or Funding InformationAHA (13SDG17310007), Startup grant from University of Maryland, NIH (1R01EB024556‐01), and NSF (CMMI‐1662776) grants to Shaik O RahamanThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

New-generation azaindole-adamantyl-derived synthetic cannabinoids

PurposeThis work reports the synthesis and pharmacological and analytical data for a new series of recently identified azaindole-adamantyl-derived synthetic cannabinoids (SCs).MethodsEach SC was synthesised using an efficient and divergent synthesis, and assessed by electron ionisation mass spectrometry (EIMS). The cannabimimetic activity of each compound was conducted using a fluorometric imaging plate reader (FLIPR) assay.ResultsThe described EIMS method and retention time by gas chromatography were able to effectively differentiate each of the analogues regardless of the bicyclic core. For the first time in these SC structures, the bicyclic ring system was shown to have an impact on the cannabimimetic activities in the fluorometric assay of membrane potential. Analogues ranged from moderately potent at both CB1 and CB2 (e.g., AP4AIC EC50 = 160 nM and EC50 = 64 nM, respectively) to not active at either cannabinoid receptor (AP4AICA, AP5AICA, and APIC).ConclusionsFurther investigation into receptor selectivity surrounding these bicyclic cores could prove useful for future therapeutic applications.

Identification of Alkaloids from Corydalis yanhusuo W. T. Wang as Dopamine D₁ Receptor Antagonists by Using CRE-Luciferase Reporter Gene Assay.

Corydalis yanhusuo W. T. Wang (C. yanhusuo) has been traditionally used for drug addiction and pain relief in China. In our previous study, we showed that the extract of C. yanhusuo blocks dopamine receptors, demonstrating that its pharmacological activities are mostly due to the antagonistic effects of some of its components at dopamine receptors. As part of our ongoing project on C. yanhusuo, the aim of the present study is to establish a high-throughput and low-cost screening assay system and test the abilities of the isolated alkaloids from C. yanhusuo to inhibit dopamine-induced dopamine D1 receptor activity. By using our established cyclic adenosine monophosphate (cAMP)-response element (CRE)-luciferase reporter gene assay system, we identified eight alkaloids from C. yanhusuo with D1 receptor antagonistic activities. We next validated the activities of these compounds using fluorometric imaging plate reader (FLIPR) assay by measuring the intracellular Ca2+ change. Six out of eight compounds, including tetrahydropalmatine, corydaline, 13-methyldehydrocorydalmine, dehydrocorybubine, dehydrocorydaline, and columbamine, can be confirmed for their inhibitory activities. The dopamine-receptor-antagonistic effects of four compounds, including 13-methyldehydrocorydalmine, dehydrocorydaline, columbamine, and corydaline, are reported for the first time. The present study provides an important pharmacological basis to support the traditional use of C. yanhusuo in China.

Abstract 241: In Vitro Disease Modeling and Discovery of Small Molecules for the Treatment of Duchenne Muscular Dystrophy Utilizing Patient iPSC-derived Cardiomyocytes

Duchenne Muscular Dystrophy (DMD) is a fatal disease of skeletal and cardiac muscle characterized by progressive muscle weakness and atrophy. Current average life expectancy is in the mid-20s and mortality is often linked to end stage cardiomyopathy. DMD is a monogenic disease caused by loss-of-function mutations in the dystrophin gene. While dystrophin is critical for muscle function by connecting the cytoskeleton to the sarcolemma and extracellular matrix, its absence can be largely compensated for by up-regulation of utrophin (UTRN), the fetal orthologue of dystrophin. Because of the importance of UTRN expression in the heart in both DMD patients and preclinical models, we utilized DMD patient iPSC-derived cardiomyocytes as a relevant cellular model to perform in vitro pharmacology as well as functional assessment. Human iPSC-derived cardiomyocytes recapitulate key properties of bona fide cardiomyocytes, such as expression of cardiac genes, spontaneous contractility, and response to chronotropic drugs (eg. isoproterenol and hERG channel blockers). We have created an isogenic pair of healthy control and DMD iPSC lines with exon 51 deletion using CRISPR/Cas9 technology. Functional measurements of cardiomyocyte electrophysiology were performed by multielectrode array (MEA) and fluorometric imaging plate reader (FLIPR) platforms. High-throughput assays were developed to measure UTRN expression in cardiomyocyte culture in 384-well plates. Importantly, we have identified small molecules that increased UTRN expression in both patient and isogenic DMD iPSC-derived cardiomyocytes. Our study represents a valuable strategy for small molecule drug discovery for the treatment of DMD cardiomyopathy based on iPSC technology.

Reduction in ultraviolet B light-induced erythema by oxymetazoline and brimonidine is mediated by different α-adrenoceptors.

When applied topically, oxymetazoline and brimonidine reduce the persistent facial erythema of rosacea; this effect is mediated by cutaneous vasoconstriction induced by postsynaptic activation of α-adrenoceptors. We investigated the α-adrenergic pharmacology of oxymetazoline and brimonidine. Functional activity on α-adrenoceptors was evaluated in vitro in HEK293 cells stably expressing single receptor subtypes using a fluorometric imaging plate reader Ca2+ influx assay. Oxymetazoline was an α1 -adrenoceptor agonist with partial α2 -adrenoceptor activity, whereas brimonidine was a highly selective full α2 -adrenoceptor agonist. In vivo pharmacology was investigated in a mouse model of ultraviolet B light (UVB)-induced skin erythema. To selectively inhibit α-adrenoceptor subtypes, mice were injected with prazosin (an α1 -selective antagonist) or rauwolscine (an α2 -selective antagonist) following UVB exposure. Oxymetazoline cream 1.0%, brimonidine gel 0.33% or vehicle control was applied topically, and erythema was measured using a chromameter. Oxymetazoline and brimonidine reduced UVB-induced erythema compared with vehicle control (P<.01). The effect of oxymetazoline was impaired in prazosin-pretreated but not rauwolscine-pretreated mice. Conversely, the effect of brimonidine was impaired in rauwolscine-pretreated but not prazosin-pretreated mice. These data suggest that while oxymetazoline and brimonidine produce cutaneous vasoconstriction, they do so through different α-adrenergic mechanisms, with oxymetazoline primarily acting via α1 -adrenoceptors and brimonidine acting via α2 -adrenoceptors.

Calcium imaging with genetically encoded sensor Case12: Facile analysis of α7/α9 nAChR mutants.

Elucidation of the structural basis of pharmacological differences for highly homologous α7 and α9 nicotinic acetylcholine receptors (nAChRs) may shed light on their involvement in different physiological functions and diseases. Combination of site-directed mutagenesis and electrophysiology is a powerful tool to pinpoint the key amino-acid residues in the receptor ligand-binding site, but for α7 and α9 nAChRs it is complicated by their poor expression and fast desensitization. Here, we probed the ligand-binding properties of α7/α9 nAChR mutants by a proposed simple and fast calcium imaging method. The method is based on transient co-expression of α7/α9 nAChR mutants in neuroblastoma cells together with Ric-3 or NACHO chaperones and Case12 fluorescent calcium ion sensor followed by analysis of their pharmacology using a fluorescence microscope or a fluorometric imaging plate reader (FLIPR) with a GFP filter set. The results obtained were confirmed by electrophysiology and by calcium imaging with the conventional calcium indicator Fluo-4. The affinities for acetylcholine and epibatidine were determined for human and rat α7 nAChRs, and for their mutants with homologous residues of α9 nAChR incorporated at positions 117–119, 184, 185, 187, and 189, which are anticipated to be involved in ligand binding. The strongest decrease in the affinity was observed for mutations at positions 187 and 119. The L119D mutation of α7 nAChR, showing a larger effect for epibatidine than for acetylcholine, may implicate this position in pharmacological differences between α7 and α9 nAChRs.

Rapid Throughput Analysis of GABAA Receptor Subtype Modulators and Blockers Using DiSBAC1(3) Membrane Potential Red Dye.

Fluorometric imaging plate reader membrane potential dye (FMP-Red-Dye) is a proprietary tool for basic discovery and high-throughput drug screening for G-protein-coupled receptors and ion channels. We optimized and validated this potentiometric probe to assay functional modulators of heterologous expressed GABAA receptor (GABAAR) isoforms (synaptic α1β3γ2, extrasynaptic α4β3δ, and β3 homopentomers). High-resolution mass spectrometry identified FMP-Red-Dye as 5,5'-(1-propen-1-yl-3-ylidene)bis[1,3-dimethyl-2-thio-barbituric acid]. GABAAR-expressing cells equilibrated with FMP-Red-Dye exhibited depolarized equilibrium membrane potentials compared with GABAAR-null cells. The channel blockers picrotoxin, fipronil, and tetramethylenedisulfotetramine, and the competitive antagonist bicuculline reduced fluorescence near the levels in GABAAR-null cells indicating that FMR-Red-Dye, a barbiturate derivative, activates GABAAR-mediated outward Cl- current in the absence of GABA. GABA caused concentration-dependent increases in fluorescence with rank order of potencies among GABAAR isoforms consistent with results from voltage-clamp experiments (EC50 values for α4β3δ, α1β3γ2, and β3 homopentamers were 6 ± 1, 40 ± 11, and >18 mM, respectively), whereas GABAAR-null cells were unresponsive. Neuroactive steroids (NAS) increased fluorescence of GABAAR expressing cells in the absence of GABA and demonstrated positive allosteric modulation in the presence of GABA, whereas benzodiazepines only exhibited positive allosteric modulator (PAM) activity. Of 20 NAS tested, allopregnanolone, (3α,5α,20E)-3-hydroxy-13,24-cyclo-18-norcholan-20-ene-21-carbonitrile, eltanolone, 5β-pregnan-3α,21-diol-20-one, and ganaxolone showed the highest potency. The FMP-Red-Dye-based assay described here provides a sensitive and quantitative method of assessing the activity of GABAAR agonists, antagonists, and PAMs on diverse GABAAR isoforms. The assay has a wide range of applications, including screening for antiseizure agents and identifying channel blockers of interest to insecticide discovery or biosecurity.

High-throughput screen detects calcium signaling dysfunction in typical sporadic autism spectrum disorder

Autism spectrum disorder (ASD) is a heterogeneous group of neurodevelopmental disorders without any defined uniting pathophysiology. Ca2+ signaling is emerging as a potential node in the genetic architecture of the disorder. We previously reported decreased inositol trisphosphate (IP3)-mediated Ca2+ release from the endoplasmic reticulum in several rare monogenic syndromes highly comorbid with autism – fragile X and tuberous sclerosis types 1 and 2 syndromes. We now extend those findings to a cohort of subjects with sporadic ASD without any known mutations. We developed and applied a high throughput Fluorometric Imaging Plate Reader (FLIPR) assay to monitor agonist-evoked Ca2+ signals in human primary skin fibroblasts. Our results indicate that IP3 -mediated Ca2+ release from the endoplasmic reticulum in response to activation of purinergic receptors is significantly depressed in subjects with sporadic as well as rare syndromic forms of ASD. We propose that deficits in IP3-mediated Ca2+ signaling represent a convergent hub function shared across the spectrum of autistic disorders – whether caused by rare highly penetrant mutations or sporadic forms – and holds promise as a biomarker for diagnosis and novel drug discovery.

Molecular Mechanisms of Toxicity and Cell Damage by Chemicals in a Human Pancreatic Beta Cell Line, 1.1B4.

Mechanisms of toxicity and cell damage were investigated in novel clonal human pancreatic beta cell line, 1.1B4, after exposure to streptozotocin, alloxan, ninhydrin, and hydrogen peroxide. Viability, DNA damage, insulin secretion/content, [Ca]i, and glucokinase/hexokinase, mRNA expression were measured by MTT assay, comet assay, radioimmunoassay, fluorometric imaging plate reader, enzyme-coupled photometry, and real-time polymerase chain reaction, respectively. Chemicals significantly reduced 1.1B4 cell viability in a time/concentration-dependent manner. Chronic 18-hour exposure decreased cellular insulin, glucokinase, and hexokinase activities. Chemicals decreased transcription of INS, GCK, PCSK1, PCSK2, and GJA1 (involved in secretory function). Insulin release and [Ca]i responses to nutrients and membrane-depolarizing agents were impaired. Streptozotocin and alloxan up-regulated transcription of genes, SOD1 and SOD2 (antioxidant enzymes). Ninhydrin and hydrogen peroxide up-regulated SOD2 transcription, whereas alloxan and hydrogen peroxide increased CAT transcription. Chemicals induced DNA damage, apoptosis, and increased caspase 3/7 activity. Streptozotocin and alloxan decreased transcription of BCL2 while increasing transcription of BAX. Chemicals did not affect transcription of HSPA4 and HSPA5 and nitrite production. 1.1B4 cells represent a useful model of human beta cells. Chemicals impaired 1.1B4 cell secretory function and activated antioxidant defense and apoptotic pathways without activating endoplasmic reticulum stress response/nitrosative stress.

Characterization of a Novel M1 Muscarinic Acetylcholine Receptor Positive Allosteric Modulator Radioligand, [3H]PT-1284.

Selective activation of the M1 muscarinic acetylcholine receptor (mAChR) via a positive allosteric modulator (PAM) is a new approach for the treatment of the cognitive impairments associated with schizophrenia and Alzheimer's disease. Herein, we describe the characterization of an M1 PAM radioligand, 8-((1S,2S)-2-hydroxycyclohexyl)-5-((6-(methyl-t3)pyridin-3-yl)methyl)-8,9-dihydro-7H-pyrrolo[3,4-hour]quinolin-7-one ([(3)H]PT-1284), as a tool for characterizing the M1 allosteric binding site, as well as profiling novel M1 PAMs. 8-((1S,2S)-2-Hydroxycyclohexyl)-5-((6-methylpyridin-3-yl)methyl)-8,9-dihydro-7H-pyrrolo[3,4-hour]quinolin-7-one (PT-1284 ( 1: )) was shown to potentiate acetylcholine (ACh) in an M1 fluorometric imaging plate reader (FLIPR) functional assay (EC50, 36 nM) and carbachol in a hippocampal slice electrophysiology assay (EC50, 165 nM). PT-1284 ( 1: ) also reduced the concentration of ACh required to inhibit [(3)H]N-methylscopolamine ([(3)H]NMS) binding to M1, left-shifting the ACh Ki approximately 19-fold at 10 μM. Saturation analysis of a human M1 mAChR stable cell line showed that [(3)H]PT-1284 bound to M1 mAChR in the presence of 1 mM ACh with Kd, 4.23 nM, and saturable binding capacity (Bmax), 6.38 pmol/mg protein. M1 selective PAMs were shown to inhibit [(3)H]PT-1284 binding in a concentration-responsive manner, whereas M1 allosteric and orthosteric agonists showed weak affinity (>30 μM). A strong positive correlation (R(2) = 0.86) was found to exist between affinity values generated for nineteen M1 PAMs in the [(3)H]PT-1284 binding assay and the EC50 values of these ligands in a FLIPR functional potentiation assay. These data indicate that there is a strong positive correlation between M1 PAM binding affinity and functional activity, and that [(3)H]PT-1284 can serve as a tool for pharmacological investigation of M1 mAChR PAMs.

Abstract B57: Assessment of gene expression of calcium pumps, channels, and channel regulators in human breast cancer-associated fibroblasts

Abstract Fibroblasts represent one of the predominant stromal cell populations present in the breast tumor microenvironment. Cancer-associated fibroblasts found growing in close proximity to the tumor site are thought to influence tumor behavior. While a number of protein and mRNA markers showing differential expression between cancer-associated and non-cancer associated fibroblasts have been proposed, current understanding of the phenotypic differences between these two cell populations remains incomplete. Calcium signaling plays an important role in each of the cancer hallmarks and is altered in a variety of disease states including cardiovascular disease. The aim of this study was to investigate the potential remodeling of the calcium signal and calcium channel and pump expression as a consequence of the transition from a normal to cancer-associated phenotype in human breast fibroblasts. To investigate expressional changes, RNA was isolated from primary cultures of matched normal and cancer-associated fibroblasts established from human breast cancer tissue specimens. Real time RT-PCR was used to assess mRNA levels of a total of 36 proteins involved in the movement of Ca2+ into and out of the cytoplasmic space, and included different classes of plasma membrane localized Ca2+ channels, as well as Ca2+ pumps of intracellular organelles. To further study potential differences in Ca2+ signaling between normal and cancer-associated fibroblast phenotypes, the immortalized human breast fibroblast line HMF3S was treated with transforming growth factor beta 1 (TGFβ1) (0, 0.01, 0.1, 1 and 10 ng/mL, 48 h), a growth factor implicated in the tumor microenvironment and myofibroblast differentiation. Successful TGFβ1-mediated activation of fibroblasts to a cancer-associated phenotype was confirmed by induction of smooth muscle alpha actin (SMαA) protein via immunoblotting. Real time RT-PCR was used to assess mRNA levels of classes of Ca2+ channels and pumps showing altered expression in patient matched normal and cancer-associated fibroblast pairs. In addition to assessing expression changes, potential functional differences in intracellular Ca2+ signaling profiles of HMF3S cells treated with TGFβ1 were assessed using a fluorometric imaging plate reader (FLIPRTETRA) and cells loaded with the Ca2+ sensitive indicator Fluo-4. Using both clinical samples, and an in vitro model of cancer-associated fibroblasts, our findings reveal a differential remodeling of plasma membrane calcium channels and organellar pumps of different classes in cells representative of cancer-associated relative to non-cancer associated fibroblasts. In addition to expression changes, there also appears to be a functional remodeling of the calcium signal in HMF3S fibroblasts induced to transition from a normal to cancer-associated phenotype by TGFβ1. This is the first study to assess changes in calcium pumps, channels and channel regulators, as well as remodeling of the calcium signal, in breast cancer-associated fibroblasts. This work may suggest that the breast cancer-associated fibroblast phenotype is associated with altered Ca2+ signaling. Citation Format: Teneale A. Stewart, Patsy S. Soon, Sarah J. Roberts-Thomson, Gregory R. Monteith. Assessment of gene expression of calcium pumps, channels, and channel regulators in human breast cancer-associated fibroblasts. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B57.

Structure–activity relationships of synthetic cannabinoid designer drug RCS-4 and its regioisomers and C4 homologues

RCS-4 [(4-methoxyphenyl)-1-yl-(1-pentyl-1H-indol-3-yl)methanone] represents the first of several N-alkyl-3-(methoxybenzoyl)indoles identified by forensic scientists as synthetic cannabinoid (SC) designer drugs. Despite the detection of RCS-4 and several analogues (RCS-2, RCS-3, RCS-2-C4, RCS-3-C4, and RCS-4-C4) in products intended for human consumption, relatively little is known about this class of cannabinoids. The synthesis of all regioisomers of RCS-4 and their C4 homologues is described. This study also systematically explored the structure–activity relationships of this class of SCs at human CB1 and CB2 receptors using an in vitro fluorometric imaging plate reader membrane potential assay. All compounds demonstrated agonist activity at CB1 (EC50 = 54–574 nM) and CB2 (EC50 = 4.5–46 nM) receptors, with the C4 homologues showing a preference for CB2 receptors over CB1 receptors (31–42 times). Since most of the analogues (RCS-2, RCS-3, RCS-2-C4, RCS-3-C4 and RCS-4-C4) are not subject to regulation in much of the world, despite their activities towards CB1 and CB2 receptors, there is a possibility that these analogues will emerge on the black market.

Gonadotropin-releasing hormone receptor-targeted paclitaxel-degarelix conjugate: synthesis and in vitro evaluation.

To increase the selectivity of chemotherapeutic agents, receptor-mediated tumor-targeting approaches have been developed. Here, degarelix [Ac-D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(Cbm)-Leu-ILys-Pro-D-Ala-NH2], a gonadotropin-releasing hormone antagonist, was employed as a targeting moiety for paclitaxel (PTX). Five PTX-degarelix conjugates were synthesized, in which PTX was attached via disulfide bond to the different position in the degarelix sequence. All of the PTX-degarelix conjugates exhibited a half-life greater than 10 h determined in human serum. A fluorometric imaging plate reader assay showed that the conjugates LK-MY-9 and LK-MY-10 had an antagonism efficacy similar to that of degarelix. The in vitro cytostatic effects of the conjugates were determined by a (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay, and the 50% inhibitory concentration value of the conjugates on 3T3 mouse embryonic fibroblast cells were one order of magnitude higher than the 50% inhibitory concentration values of the conjugates on MCF-7 human breast cancer cells and HT-29 human colon cancer cells. Receptor saturation tests further demonstrated that pre-incubation of the cells with degarelix reduced the efficacy of LK-MY-10 in a concentration-dependent manner. In conclusion, degarelix is a valid and stable moiety that has great potential for targeting chemotherapy drugs.

Discovery of N-methyltetrahydroprotoberberines with κ-opioid receptor agonists-opioid receptor agonist activities from corydalis yanhusuo W. T. Wang by using two-dimensional liquid chromatography

Ethnopharmacological relevanceThe need for an efficacious analgesic without unwanted side effects is urgent. κ-opioid receptor agonists are known to exhibit potent analgesic effects and elicited fewer side effects than other opioid agonists. Thus in this study we chose the κ-opioid receptor as the target to identify the active components from traditional Chinese medicines (TCMs). Materials and methodsThe κ-opioid receptor was expressed in human embryonic kidney-293 T cells (HEK293T). Fluorometric Imaging Plate Reader (FLIPR) assay was used for the determination of Ca2+ response when κ-opioid receptor was activated. A novel 2D separation system employing C18HCE as the first dimension and a strong cation exchange column (SCX) as the second dimension was conducted for the purification of the active principles. ResultsWith the aid of HPLC-based activity profiling, activities could be linked to two peaks from Corydalis yanhusuo W. T. Wang (C. yanhusuo) extract. Two N-methyltetrahydroprotoberberines with κ-opioid receptor agonist activities were isolated for the first time from C. yanhusuo by using 2D-LC. ConclusionsOur study suggests that N-methyltetrahydroprotoberberines may serve as a new scaffold for κ-opioid receptor ligands. The strategy that we adopted can be applied to other naturally-occurring active alkaloids acting at different receptors.

Natural compounds with P2X7 receptor-modulating properties.

The adenosine 5'-triphosphate (ATP)-gated P2X7 receptor is a membrane-bound, non-selective cation channel, expressed in a variety of cell types. The P2X7 senses high extracellular ATP concentrations and seems to be implicated in a wide range of cellular functions as well as pathophysiological processes, including immune responses and inflammation, release of gliotransmitters and cytokines, cancer cell growth or development of neurodegenerative diseases. In the present study, we identified natural compounds and analogues that can block or sensitize the ATP (1 mM)-induced Ca(2+) response using a HEK293 cell line stably expressing human P2X7 and fluorometric imaging plate reader technology. For instance, teniposide potently blocked the human P2X7 at sub-miromolar concentrations, but not human P2X4 or rat P2X2. A marked block of ATP-induced Ca(2+) entry and Yo-Pro-1 uptake was also observed in human A375 melanoma cells and mouse microglial cells, both expressing P2X7. On the other hand, agelasine (AGL) and garcinolic acid (GA) facilitated the P2X7 response to ATP in all three cell populations. GA also enhanced the YO-PRO-1 uptake, whereas AGL did not affect the ATP-stimulated intracellular accumulation of this dye. According to the pathophysiological role of P2X7 in various diseases, selective modulators may have potential for further development, e.g. as neuroprotective or antineoplastic drugs.

The design and synthesis of novel, phosphonate-containing transient receptor potential melastatin 8 (TRPM8) antagonists

A series of benzothiophene-based phosphonates was synthesized and many analogs within the series were shown to be potent antagonists of the TRPM8 channel. The compounds were obtained as a racemic mixture in 5 synthetic steps, and were tested for TRPM8 antagonist activity in a recombinant, canine TRPM8-expressing cell line using a fluorometric imaging plate reader (FLIPR) assay. Structure–activity relationships were developed initially by modification of the core structure and subsequently by variation of the aromatic substituents and the phosphonate ester. Compound 9l was administered intraperitoneally to rats and demonstrated engagement of the TRPM8 target in both prevention and reversal-modes in an icilin-induced ‘wet-dog’ shake model.

Discovery of Novel P2Y14 Agonist and Antagonist Using Conventional and Nonconventional Methods

P2Y14 is a member of the pyrimidinergic GPCR family. UDP-Glc has been previously shown to activate human P2Y14, whereas UDP was unable to activate the receptor. In this study, the authors used conventional and nonconventional methods to further characterize P2Y14 and its ligands. Conventional calcium mobilization and nonconventional cellular impedance functional assays revealed that UMP and UDP selectively activated HEK cells coexpressing P2Y14 and Gα(qi5). In the impedance assays, the presence of exogenous Gα(qi5) resulted in agonist-induced Gq signaling, whereas in the absence of exogenous Gα(qi5), the signal was indicative of Gi. The authors established the first P2Y14 membrane filtration binding assay using a novel optimized expression vector and [(3)H]UDP as radioligand. UDP-Glc, UMP, and UDP dose dependently inhibited [(3)H]UDP binding in the binding assay, and saturation analysis revealed that UDP bound P2Y14 with a K(D) = 10 nM and a B(max) = 110 pmol/mg. The authors screened a phosphonate library and identified compound A, which inhibited UDP-Glc-mediated calcium signaling in the fluorometric imaging plate reader assay (IC(50) = 2.3 µM) and competed for [(3)H]UDP binding in the novel binding assay with a K(i) = 1280 nM.