Co-reporter:Thanigaimalai Pillaiyar, Meryem Köse, Katharina Sylvester, Heike Weighardt, Dominik Thimm, Gleice Borges, Irmgard Förster, Ivar von Kügelgen, and Christa E. Müller
Journal of Medicinal Chemistry May 11, 2017 Volume 60(Issue 9) pp:3636-3636
Publication Date(Web):April 13, 2017
DOI:10.1021/acs.jmedchem.6b01593
The Gi protein-coupled receptor GPR84, which is activated by (hydroxy)fatty acids, is highly expressed on immune cells. Recently, 3,3′-diindolylmethane was identified as a heterocyclic, nonlipid-like GPR84 agonist. We synthesized a broad range of diindolylmethane derivatives by condensation of indoles with formaldehyde in water under microwave irradiation. The products were evaluated at the human GPR84 in cAMP and β-arrestin assays. Structure–activity relationships (SARs) were steep. 3,3′-Diindolylmethanes bearing small lipophilic residues at the 5- and/or 7-position of the indole rings displayed the highest activity in cAMP assays, the most potent agonists being di(5-fluoro-1H-indole-3-yl)methane (38, PSB-15160, EC50 80.0 nM) and di(5,7-difluoro-1H-indole-3-yl)methane (57, PSB-16671, EC50 41.3 nM). In β-arrestin assays, SARs were different, indicating biased agonism. The new compounds were selective versus related fatty acid receptors and the arylhydrocarbon receptor. Selected compounds were further investigated and found to display an ago-allosteric mechanism of action and increased stability in comparison to the lead structure.
Co-reporter:Muhammad Rafehi, Enas M. Malik, Alexander Neumann, Aliaa Abdelrahman, Theodor Hanck, Vigneshwaran Namasivayam, Christa E. Müller, and Younis Baqi
Journal of Medicinal Chemistry April 13, 2017 Volume 60(Issue 7) pp:3020-3020
Publication Date(Web):March 17, 2017
DOI:10.1021/acs.jmedchem.7b00030
P2Y4 is a Gq protein-coupled receptor activated by uridine-5′-triphosphate (UTP), which is widely expressed in the body, e.g., in intestine, heart, and brain. No selective P2Y4 receptor antagonist has been described so far. Therefore, we developed and optimized P2Y4 receptor antagonists based on an anthraquinone scaffold. Potency was assessed by a fluorescence-based assay measuring inhibition of UTP-induced intracellular calcium release in 1321N1 astrocytoma cells stably transfected with the human P2Y4 receptor. The most potent compound of the present series, sodium 1-amino-4-[4-(2,4-dimethylphenylthio)phenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (PSB-16133, 61) exhibited an IC50 value of 233 nM, selectivity versus other P2Y receptor subtypes, and is thought to act as an allosteric antagonist. A receptor homology model was built and docking studies were performed to analyze ligand–receptor interactions. Compound 64 (PSB-1699, sodium 1-amino-4-[4-(3-pyridin-3-ylmethylthio)phenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate) represents the most selective P2Y4 receptor antagonist known to date. Compounds 61 and 64 are therefore anticipated to become useful tools for studying this scarcely investigated receptor.
Co-reporter:Azeem Danish;Sang-Yong Lee;Christa E. Müller
Analyst (1876-Present) 2017 vol. 142(Issue 19) pp:3648-3655
Publication Date(Web):2017/09/25
DOI:10.1039/C7AN00981J
A fast and robust procedure for the quantification of GFP-tagged membrane proteins in cell homogenates was developed employing capillary gel electrophoresis coupled to laser-induced fluorescence detection (CGE-LIF). The new method was found to be highly sensitive and applicable to structurally diverse membrane proteins including synaptic vesicle protein 2A (SV2A), adenosine A2A receptor (A2AAR), and connexin 43 (Cx43). Quantification of SV2A and A2AAR using radioligand binding assays confirmed the results obtained with CGE-LIF. The CGE-LIF method showed significantly higher sensitivity as compared to fluorimetric measurement in a microplate. Importantly, CGE-LIF involves separation of the target proteins and their degradation products prior to quantification and thereby ensures specificity. We anticipate broad applicability of the method for any fluorophore-tagged protein.
Co-reporter:Muhammad Rafehi;Joachim C. Burbiel;Isaac Y. Attah
Purinergic Signalling 2017 Volume 13( Issue 1) pp:89-103
Publication Date(Web):2017 March
DOI:10.1007/s11302-016-9542-3
The Gq protein-coupled, ATP- and UTP-activated P2Y2 receptor is a potential drug target for a range of different disorders, including tumor metastasis, inflammation, atherosclerosis, kidney disorders, and osteoporosis, but pharmacological studies are impeded by the limited availability of suitable antagonists. One of the most potent and selective antagonists is the thiouracil derivative AR-C118925. However, this compound was until recently not commercially available and little is known about its properties. We therefore developed an improved procedure for the synthesis of AR-C118925 and two derivatives to allow up-scaling and assessed their potency in calcium mobilization assays on the human and rat P2Y2 receptors recombinantly expressed in 1321N1 astrocytoma cells. The compound was further evaluated for inhibition of P2Y2 receptor-induced β-arrestin translocation. AR-C118925 behaved as a competitive antagonist with pA2 values of 37.2 nM (calcium assay) and 51.3 nM (β-arrestin assay). Selectivity was assessed vs. related receptors including P2X, P2Y, and adenosine receptor subtypes, as well as ectonucleotidases. AR-C118925 showed at least 50-fold selectivity against the other investigated targets, except for the P2X1 and P2X3 receptors which were blocked by AR-C118925 at concentrations of about 1 μM. AR-C118925 is soluble in buffer at pH 7.4 (124 μM) and was found to be metabolically highly stable in human and mouse liver microsomes. In Caco2 cell experiments, the compound displayed moderate permeability indicating that it may show limited peroral bioavailability. AR-C118925 appears to be a useful pharmacological tool for in vitro and in vivo studies.
Co-reporter:Aliaa Abdelrahman, Vigneshwaran Namasivayam, Sonja Hinz, Anke C. Schiedel, ... Christa E. Müller
Biochemical Pharmacology 2017 Volume 125(Volume 125) pp:
Publication Date(Web):1 February 2017
DOI:10.1016/j.bcp.2016.11.016
Antagonists for ATP-activated P2X4 ion channel receptors are currently in the focus as novel drug targets, in particular for the treatment of neuropathic and inflammatory pain. We stably expressed the human, rat and mouse P2X4 receptors in 1321N1 astrocytoma cells, which is devoid of functional nucleotide receptors, by retroviral transfection, and established monoclonal cell lines. Calcium flux assay conditions were optimized for high-throughput screening resulting in a Z′-factor of >0.8. The application of ready-to-use frozen cells did not negatively affect the results of the calcium assays, which is of great advantage for the screening of compound libraries. Species differences were observed, the rat P2X4 receptor being particularly insensitive to many ATP derivatives. Membrane preparations of the cell lines showed high levels of specific [35S]ATPγS binding with low nonspecific binding (<5% of total binding), while non-transfected cells were devoid of specific binding sites for the radioligand. Conditions were employed which allow binding studies to be performed at room temperature. While a variety of nucleotide-derived agonists and the antagonist TNP-ATP displaced [35S]ATPγS from its binding site at human P2X4 receptors, the non-nucleotidic antagonists paroxetine and 5-BDBD did not compete with radioligand binding and were therefore characterized as allosteric antagonists. Homology modeling was applied to find an explanation for the observed species differences.Download high-res image (197KB)Download full-size image
Co-reporter:Enas M. Malik, Mahmoud Rashed, Lukas Wingen, Younis Baqi, Christa E. Müller
Dyes and Pigments 2016 Volume 131() pp:33-40
Publication Date(Web):August 2016
DOI:10.1016/j.dyepig.2016.03.023
•Synthesis of novel 1-amino-4-(ar)alkylaminoanthraquinones having a Br, CH2OH, CN, or CH3 in the 2-position of the scaffold.•The target compounds were obtained by Ullmann condensation of bromaminic acid analogues with a variety of amines.•Optimization of the conditions for the different reactions has been conducted.•Comparison of the different bromaminic acid analogues in terms of chemical reactivity and reaction conditions is presented.•The anthraquinone derivatives may be useful as new dyes or represent bioactive compounds for experimental pharmacology.Novel 1-amino-4-(ar)alkylaminoanthraquinone derivatives bearing different substituents (Br, CH2OH, CN, or CH3) at the 2-position of the anthraquinone scaffold were synthesized by copper-catalyzed Ullmann condensation. The 2-substituted 1-amino-4-bromoanthraquinone derivatives (bromaminic acid analogues) were reacted with a variety of alkyl-, aryl-, and aralkylamines. Different reaction conditions were applied depending on the physical state of the amine and the nature of the substituent at the 2-position of the anthraquinone scaffold. A solvent-free synthetic procedure was developed for reactions with liquid amines. Condensation with solid amines required the addition of a solvent, and reaction conditions had to be optimized for each of the anthraquinone derivatives. Our results emphasize that copper-catalyzed reactions are highly variable depending on the molecules and anions present in the medium, since different reaction mechanisms apply. The synthesized anthraquinone derivatives may be useful as new dyes, or may show biological activity and could be employed as tool compounds in experimental pharmacology.
Co-reporter:Andreas Brunschweiger, Pierre Koch, Miriam Schlenk, Muhammad Rafehi, Hamid Radjainia, Petra Küppers, Sonja Hinz, Felipe Pineda, Michael Wiese, Jörg Hockemeyer, Jag Heer, Frédéric Denonne, Christa E. Müller
Bioorganic & Medicinal Chemistry 2016 Volume 24(Issue 21) pp:5462-5480
Publication Date(Web):1 November 2016
DOI:10.1016/j.bmc.2016.09.003
•A series of 61 diversely substituted pyrazinopurinediones was synthesized.•Aromatic residues were connected to the core heterocyclic structure by different linkers.•Their adenosine receptor affinities and monoamine oxidase inhibitory potencies were investigated.•Several compounds showed higher water-solubility than the isomeric pyrimidopurinediones.•Multi-target drugs were designed and optimized for neurodegenerative diseases.Multitarget approaches, i.e., addressing two or more targets simultaneously with a therapeutic agent, are hypothesized to offer additive therapeutic benefit for the treatment of neurodegenerative diseases. Validated targets for the treatment of Parkinson’s disease are, among others, the A2A adenosine receptor (AR) and the enzyme monoamine oxidase B (MAO-B). Additional blockade of brain A1 ARs may also be beneficial. We recently described 8-benzyl-substituted tetrahydropyrazino[2,1-f]purinediones as a new lead structure for the development of such multi-target drugs. We have now designed a new series of tetrahydropyrazino[2,1-f]purinediones to extensively explore their structure–activity-relationships. Several compounds blocked human and rat A1 and A2AARs at similar concentrations representing dual A1/A2A antagonists with high selectivity versus the other AR subtypes. Among the best dual A1/A2AAR antagonists were 8-(3-(4-chlorophenyl)propyl)-1,3-dimethyl-6,7,8,9-tetrahydropyrazino[2,1-f]purine-2,4(1H,3H)-dione (41, Ki human A1: 65.5 nM, A2A: 230 nM; Ki rat A1: 352 nM, A2A: 316 nM) and 1,3-dimethyl-8-((2-(thiophen-2-yl)thiazol-4-yl)methyl)-6,7,8,9-tetrahydropyrazino[2,1-f]purine-2,4(1H,3H)-dione (57, Ki human A1: 642 nM, A2A: 203 nM; Ki rat A1: 166 nM, A2A: 121 nM). Compound 57 was found to be well water-soluble (0.7 mg/mL) at a physiological pH value of 7.4. One of the new compounds showed triple-target inhibition: (R)-1,3-dimethyl-8-(2,1,3,4-tetrahydronaphthalen-1-yl)-6,7,8,9-tetrahydropyrazino[2,1-f]purine-2,4(1H,3H)-dione (49) was about equipotent at A1 and A2AARs and at MAO-B (Ki human A1: 393 nM, human A2A: 595 nM, IC50 human MAO-B: 210 nM) thus allowing future in vivo explorations of the intended multi-target approach.
Co-reporter:Amelie Fiene, Younis Baqi, Enas M. Malik, Patrice Newton, Wenjin Li, Sang-Yong Lee, Elizabeth L. Hartland, Christa E. Müller
Bioorganic & Medicinal Chemistry 2016 Volume 24(Issue 18) pp:4363-4371
Publication Date(Web):15 September 2016
DOI:10.1016/j.bmc.2016.07.027
Legionella pneumophila is an aerobic, Gram-negative bacterium of the genus Legionella, which constitutes the major causative agent of Legionnaires’ disease. Recently a nucleoside triphosphate diphosphohydrolase (NTPDase) from L. pneumophila was identified and termed Lp1NTPDase; it was found to be a structural and functional homolog of mammalian NTPDases catalyzing the hydrolysis of ATP to ADP and ADP to AMP. Its activity is believed to contribute to the virulence of Legionella pneumophila. Therefore Lp1NTPDase inhibitors are considered as novel antibacterial drugs. However, only weakly potent compounds are available so far. In the present study, a capillary electrophoresis (CE)-based enzyme assay for monitoring the Lp1NTPDase activity was established. The enzymatic reaction was performed in a test tube followed by separation of substrate and products by CE and subsequent quantification by UV analysis. After kinetic characterization of the enzyme, a series of 1-amino-4-ar(alk)ylamino-2-sulfoanthraquinone derivatives structurally related to the anthraquinone dye Reactive Blue 2, a non-selective ecto-NTPDase inhibitor, was investigated for inhibitory activity on Lp1NTPDase using the CE-based enzyme assay. Derivatives bearing a large lipophilic substituent (e.g., fused aromatic rings) in the 4-position of the 1-amino-2-sulfoanthraquinone showed the highest inhibitory activity. Compounds with IC50 values in the low micromolar range were identified. The most potent inhibitor was 1-amino-4-[phenanthrene-9-yl-amino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (28, PSB-16131), with an IC50-value of 4.24 μM. It represents the most potent Lp1NTPDase inhibitor described to date. These findings may serve as a starting point for further optimization. Lp1NTPDase inhibition provides a novel approach for the (immuno)therapy of Legionella infections.
Co-reporter:Cornelius Hess;Clara T. Schoeder;Thanigaimalai Pillaiyar;Burkhard Madea;Christa E. Müller
Forensic Toxicology 2016 Volume 34( Issue 2) pp:329-343
Publication Date(Web):2016/07/01
DOI:10.1007/s11419-016-0320-2
In recent years, many synthetic cannabinoid (CB) receptor agonists have appeared on the market as constituents of herbal incense mixtures known as “spice”. Contrary to the declared use, they are perorally consumed as a replacement for marijuana to get “high”. In many cases, detailed information on the physicochemical and pharmacological properties of the synthetic compounds found in spice preparations is lacking. We have now evaluated a large series of heterocyclic compounds, 1,3-disubstituted indole and 2-azaindole derivatives known or assumed to be CB1 receptor agonists, many of which have previously been identified in forensic samples. The mainly observed structural variations to circumvent restriction by law were bioisosteric exchanges of functional groups in known CB1 agonists. We analyzed the structure-activity relationships of compounds at human CB1 and CB2 receptors based on affinities obtained in radioligand binding studies, and determined their efficacy in cAMP accumulation assays. Moreover, we investigated the activities of the compounds at the orphan G protein-coupled receptors GPR18 and GPR55 both of which are known to interact with cannabinoids. Most of the investigated compounds behaved as potent full agonists of CB1 and CB2 receptors with affinities in the low nanomolar to subnanomolar concentration range. Some compounds were moderately potent GPR55 antagonists, while none interacted with GPR18. Most derivatives were predicted to cross the blood–brain barrier as determined by bioinformatics tools. These data are useful for assessing synthetic cannabinoids and will be helpful for predicting pharmacological properties of novel compounds that appear on the illicit drug market.
Co-reporter:Elisabetta De Filippo;Vigneshwaran Namasivayam;Lukas Zappe
Purinergic Signalling 2016 Volume 12( Issue 2) pp:313-329
Publication Date(Web):2016 June
DOI:10.1007/s11302-016-9506-7
The G protein-coupled A2A adenosine receptor represents an important drug target. Crystal structures and modeling studies indicated that three disulfide bonds are formed between ECL1 and ECL2 (I, Cys712.69-Cys15945.43; II, Cys743.22-Cys14645.30, and III, Cys773.25-Cys16645.50). However, the A2BAR subtype appears to require only disulfide bond III for proper function. In this study, each of the three disulfide bonds in the A2AAR was disrupted by mutation of one of the cysteine residues to serine. The mutant receptors were stably expressed in Chinese hamster ovary cells and analyzed in cyclic adenosine monophosphate (cAMP) accumulation and radioligand binding studies using structurally diverse agonists: adenosine, NECA, CGS21680, and PSB-15826. Results were rationalized by molecular modeling. The observed effects were dependent on the investigated agonist. Loss of disulfide bond I led to a widening of the orthosteric binding pocket resulting in a strong reduction in the potency of adenosine, but not of NECA or 2-substituted nucleosides. Disruption of disulfide bond II led to a significant reduction in the agonists’ efficacy indicating its importance for receptor activation. Disulfide bond III disruption reduced potency and affinity of the small adenosine agonists and NECA, but not of the larger 2-substituted agonists. While all the three disulfide bonds were essential for high potency or efficacy of adenosine, structural modification of the nucleoside could rescue affinity or efficacy at the mutant receptors. At present, it cannot be excluded that formation of the extracellular disulfide bonds in the A2AAR is dynamic. This might add another level of G protein-coupled receptor (GPCR) modulation, in particular for the cysteine-rich A2A and A2BARs.
Co-reporter:Sang-Yong Lee, Arianna Perotti, Steven De Jonghe, Piet Herdewijn, Theodor Hanck, Christa E. Müller
Bioorganic & Medicinal Chemistry 2016 24(14) pp: 3157-3165
Publication Date(Web):15 July 2016
DOI:10.1016/j.bmc.2016.05.046
Ecto-nucleotide pyrophosphatase/phosphodiesterase1 (NPP1) is the most important member of the NPP family, which consists of seven closely related proteins (NPP1–NPP7). This glycoprotein is a membrane-associated or secreted enzyme, which catalyzes the hydrolysis of a wide range of phosphodiester bonds, e.g., in nucleoside triphosphates, dinucleotides and nucleotide sugars. NPP1 plays a crucial role in various physiological functions including bone mineralization, soft-tissue calcification, and insulin receptor signaling. Recently, an upregulated expression of NPP1 has been observed in astrocytic brain cancers. Therefore, NPP1 has been proposed as a novel drug target for the treatment of glioblastoma. Despite their therapeutic potential, only few NPP1 inhibitors have been reported to date, which are in most cases non- or only moderately selective. The best investigated NPP1 inhibitors so far are nucleotide derivatives and analogs, however they are not orally bioavailable due to their high polarity. We identified thiazolo[3,2-a]benzimidazol-3(2H)-one derivatives as a new class of NPP1 inhibitors with drug-like properties. Among the 25 derivatives investigated in the present study, 2-[(5-iodo-2-furanyl)methylene]thiazolo[3,2-a]benzimidazol-3(2H)-one (17) was found to be the most potent NPP1 inhibitor with a Ki value of 467 nM versus ATP as a substrate and an un-competitive mechanism of inhibition. Compound 17 did not inhibit other human ecto-nucleotidases, including NTPDase1 (CD39), NTPDases2-3, NPP2, NPP3, tissue-nonspecific alkaline phosphatase (TNAP), and ecto-5′-nucleotidase (eN, CD73), and is thus highly selective for NPP1.
Co-reporter:Sanjay Bhattarai; Marianne Freundlieb; Jan Pippel; Anne Meyer; Aliaa Abdelrahman; Amelie Fiene; Sang-Yong Lee; Herbert Zimmermann; Gennady G. Yegutkin; Norbert Sträter; Ali El-Tayeb;Christa E. Müller
Journal of Medicinal Chemistry 2015 Volume 58(Issue 15) pp:6248-6263
Publication Date(Web):July 6, 2015
DOI:10.1021/acs.jmedchem.5b00802
ecto-5′-Nucleotidase (eN, CD73) catalyzes the hydrolysis of extracellular AMP to adenosine. eN inhibitors have potential for use as cancer therapeutics. The eN inhibitor α,β-methylene-ADP (AOPCP, adenosine-5′-O-[(phosphonomethyl)phosphonic acid]) was used as a lead structure, and derivatives modified in various positions were prepared. Products were tested at rat recombinant eN. 6-(Ar)alkylamino substitution led to the largest improvement in potency. N6-Monosubstitution was superior to symmetrical N6,N6-disubstitution. The most potent inhibitors were N6-(4-chlorobenzyl)- (10l, PSB-12441, Ki 7.23 nM), N6-phenylethyl- (10h, PSB-12425, Ki 8.04 nM), and N6-benzyl-adenosine-5′-O-[(phosphonomethyl)phosphonic acid] (10g, PSB-12379, Ki 9.03 nM). Replacement of the 6-NH group in 10g by O (10q, PSB-12431) or S (10r, PSB-12553) yielded equally potent inhibitors (10q, 9.20 nM; 10r, 9.50 nM). Selected compounds investigated at the human enzyme did not show species differences; they displayed high selectivity versus other ecto-nucleotidases and ADP-activated P2Y receptors. Moreover, high metabolic stability was observed. These compounds represent the most potent eN inhibitors described to date.
Co-reporter:Wenjin Li, Isabell Zech, Volkmar Gieselmann, Christa E. Müller
Journal of Chromatography A 2015 Volume 1407() pp:222-227
Publication Date(Web):14 August 2015
DOI:10.1016/j.chroma.2015.06.053
•The first validated CE method for monitoring CST activity was developed.•A LOD for PAP of 66.6 nM was achieved by dynamic pH junction stacking.•The new assay is suitable for enzyme characterization and inhibitor screening.•The method will help to develop drugs for MLD, a rare and severe genetic disease.Metachromatic leukodystrophy (MLD) is a rare and severe genetic disease. Inhibition of cerebroside sulfotransferase (CST) has been proposed as a promising new therapeutic strategy for the treatment of MLD. CST catalyzes the transfer of a sulfate group from the coenzyme 3′-phosphoadenosine-5′-phosphosulfate (PAPS) to cerebroside yielding cerebroside sulfate and adenosine-3′,5′-diphosphate (PAP). So far only a few weak CST inhibitors have been described. The goal of the present study was to establish a suitable assay for identifying and characterizing novel CST inhibitors. To this end, we developed and optimized a capillary electrophoresis (CE) based assay for monitoring the catalytic activity of CST by measuring the formation of PAP. A sample matrix consisting of 5 mM phosphate buffer with about 0.0001% polybrene at pH 7.4 and a background electrolyte (BGE) containing 75 mM phosphate buffer with 0.002% polybrene at pH 5.6 were utilized to achieve a stacking effect for PAP by dynamic pH junction. This led to a limit of detection for the enzymatic product PAP of 66.6 nM. The CE method was sensitive, robust, and suitable for CST inhibitor screening, Ki value determination, and enzyme kinetic studies. Selected reference compounds were tested in order to validate the assay, including the substrates cerebroside and psychosine, and the inhibitor Congo Red. The newly developed CE method will be useful for the identification and development of novel CST inhibitors which are urgently needed for the treatment of MLD.
Co-reporter:Amelie Fiene, Younis Baqi, Joanna Lecka, Jean Sévigny and Christa E. Müller
Analyst 2015 vol. 140(Issue 1) pp:140-148
Publication Date(Web):21 Oct 2014
DOI:10.1039/C4AN01694G
The following members of the ecto-nucleoside triphosphate diphosphohydrolase family, NTPDase1 (CD39), NTPDase-2, -3, and -8, play an important role in purinergic signal transduction by regulating extracellular nucleotide levels. Potent and selective NTPDase inhibitors are required as pharmacological tools and have potential as novel drugs, e.g. for anti-cancer and anti-bacterial therapy. We have developed fast and sensitive NTPDase fluorescence polarization (FP) immunoassays using the natural substrates (ATP or ADP). During the NTPDase1-catalyzed reaction, the substrate is dephosphorylated to ADP which is further dephosphorylated yielding AMP as the final product (by NTPDase1). NTPDase3 and -8 yield AMP and ADP, while NTPDase2 results mainly in the formation of ADP. Direct quantification of the respective product, AMP or ADP, is achieved by displacement of an appropriate fluorescent tracer nucleotide from a specific antibody leading to a change in fluorescence polarization. The assays are highly sensitive and can be performed with low substrate concentrations (20 μM ATP or 10 μM ADP) below the KM values of NTPDases, which simplifies the identification of novel competitive inhibitors. Optimized antibody and enzyme concentrations allow the reproducible detection of 2 μM ADP and 1 μM AMP (at 10% substrate conversion). Validation of the assays yielded excellent Z′-factors greater than 0.70 for all investigated NTPDase subtypes indicating high robustness of the analytical method. Furthermore, we tested a standard inhibitor and performed a first exemplary screening campaign with a library consisting of >400 compounds (Z′-factor: 0.87, hit rate 0.5%). Thereby we demonstrated the suitability of the FP assay for IC50 value determination and high-throughput screening in a 384-well format. The new FP assays were shown to be superior to current standard assays.
Co-reporter:Mohamad Wessam Alnouri;Stephan Jepards;Alessandro Casari
Purinergic Signalling 2015 Volume 11( Issue 3) pp:389-407
Publication Date(Web):2015 September
DOI:10.1007/s11302-015-9460-9
Adenosine receptors (ARs) have emerged as new drug targets. The majority of data on affinity/potency and selectivity of AR ligands described in the literature has been obtained for the human species. However, preclinical studies are mostly performed in mouse or rat, and standard AR agonists and antagonists are frequently used for studies in rodents without knowing their selectivity in the investigated species. In the present study, we selected a set of frequently used standard AR ligands, 8 agonists and 16 antagonists, and investigated them in radioligand binding studies at all four AR subtypes, A1, A2A, A2B, and A3, of three species, human, rat, and mouse. Recommended, selective agonists include CCPA (for A1AR of rat and mouse), CGS-21680 (for A2A AR of rat), and Cl-IB-MECA (for A3AR of all three species). The functionally selective partial A2B agonist BAY60-6583 was found to additionally bind to A1 and A3AR and act as an antagonist at both receptor subtypes. The antagonists PSB-36 (A1), preladenant (A2A), and PSB-603 (A2B) displayed high selectivity in all three investigated species. MRS-1523 acts as a selective A3AR antagonist in human and rat, but is only moderately selective in mouse. The comprehensive data presented herein provide a solid basis for selecting suitable AR ligands for biological studies.
Co-reporter:Lei Chang ; Sang-Yong Lee ; Piotr Leonczak ; Jef Rozenski ; Steven De Jonghe ; Theodor Hanck ; Christa E. Müller ;Piet Herdewijn
Journal of Medicinal Chemistry 2014 Volume 57(Issue 23) pp:10080-10100
Publication Date(Web):November 5, 2014
DOI:10.1021/jm501434y
Nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) belongs to the family of ecto-nucleotidases, which control extracellular nucleotide, nucleoside, and (di)phosphate levels. To study the (patho)physiological roles of NPP1 potent and selective inhibitors with drug-like properties are required. Therefore, a compound library was screened for NPP1 inhibitors using a colorimetric assay with p-nitrophenyl 5′-thymidine monophosphate (p-Nph-5′-TMP) as an artificial substrate. This led to the discovery of 2-(3H-imidazo[4,5-b]pyridin-2-ylthio)-N-(3,4-dimethoxyphenyl)acetamide (5a) as a hit compound with a Ki value of 217 nM. Subsequent structure–activity relationship studies led to the development of purine and imidazo[4,5-b]pyridine analogues with high inhibitory potency (Ki values of 5.00 nM and 29.6 nM, respectively) when assayed with p-Nph-5′-TMP as a substrate. Surprisingly, the compounds were significantly less potent when tested versus ATP as a substrate, with Ki values in the low micromolar range. A prototypic inhibitor was investigated for its mechanism of inhibition and found to be competitive versus both substrates.
Co-reporter:Nikolay T. Tzvetkov ; Sonja Hinz ; Petra Küppers ; Marcus Gastreich ;Christa E. Müller
Journal of Medicinal Chemistry 2014 Volume 57(Issue 15) pp:6679-6703
Publication Date(Web):June 23, 2014
DOI:10.1021/jm500729a
Indazole- and indole-carboxamides were discovered as highly potent, selective, competitive, and reversible inhibitors of monoamine oxidase B (MAO-B). The compounds are easily accessible by standard synthetic procedures with high overall yields. The most potent derivatives were N-(3,4-dichlorophenyl)-1-methyl-1H-indazole-5-carboxamide (38a, PSB-1491, IC50 human MAO-B 0.386 nM, >25000-fold selective versus MAO-A) and N-(3,4-dichlorophenyl)-1H-indole-5-carboxamide (53, PSB-1410, IC50 human MAO-B 0.227 nM, >5700-fold selective versus MAO-A). Replacement of the carboxamide linker with a methanimine spacer leading to (E)-N-(3,4-dichlorophenyl)-1-(1H-indazol-5-yl)methanimine (58) represents a further novel class of highly potent and selective MAO-B inhibitors (IC50 human MAO-B 0.612 nM, >16000-fold selective versus MAO-A). In N-(3,4-difluorophenyl-1H-indazole-5-carboxamide (30, PSB-1434, IC50 human MAO-B 1.59 nM, selectivity versus MAO-A >6000-fold), high potency and selectivity are optimally combined with superior physicochemical properties. Computational docking studies provided insights into the inhibitors’ interaction with the enzyme binding site and a rationale for their high potency despite their small molecular size.
Co-reporter:Henrik Harms ; Viktor Rempel ; Stefan Kehraus ; Marcel Kaiser ; Peter Hufendiek ; Christa E. Müller ;Gabriele M. König
Journal of Natural Products 2014 Volume 77(Issue 3) pp:673-677
Publication Date(Web):January 28, 2014
DOI:10.1021/np400850g
A marine-derived strain of Dichotomomyces cejpii produces the new compounds emindole SB beta-mannoside (1) and 27-O-methylasporyzin C (2), as well as the known indoloditerpenes JBIR-03 (3) and emindole SB (4). Indole derivative 1 was found to be a CB2 antagonist, while 2 was identified as the first selective GPR18 antagonist with an indole structure. Compound 4 was found to be a nonselective CB1/CB2 antagonist. The new natural indole derivatives may serve as lead structures for the development of GPR18- and CB receptor-blocking drugs.
Co-reporter:Maoqun Tian, Aliaa Abdelrahman, Stephanie Weinhausen, Sonja Hinz, Stefanie Weyer, Stefan Dosa, Ali El-Tayeb, Christa E. Müller
Bioorganic & Medicinal Chemistry 2014 Volume 22(Issue 3) pp:1077-1088
Publication Date(Web):1 February 2014
DOI:10.1016/j.bmc.2013.12.035
Antagonists for the P2 receptor subtype P2X4, an ATP-activated cation channel receptor, have potential as novel drugs for the treatment of neuropathic pain and other inflammatory diseases. In the present study, a series of 47 carbamazepine derivatives including 32 novel compounds were designed, synthesized, and evaluated as P2X4 receptor antagonists. Their potency to inhibit ATP-induced calcium influx in 1321N1 astrocytoma cells stably transfected with the human P2X4 receptor was determined. Additionally, species selectivity (human, rat, mouse) and receptor subtype selectivity (P2X4 vs P2X1, 2, 3, 7) were investigated for selected derivatives. The most potent compound of the present series, which exhibited an allosteric mechanism of P2X4 inhibition, was N,N-diisopropyl-5H-dibenz[b,f]azepine-5-carboxamide (34, IC50 of 3.44 μM). The present study extends the so far very limited knowledge on structure–activity relationships of P2X4 receptor antagonists.
Co-reporter:Meryem Köse, Kirsten Ritter, Katharina Thiemke, Michel Gillard, Evi Kostenis, and Christa E. Müller
ACS Medicinal Chemistry Letters 2014 Volume 5(Issue 4) pp:326-330
Publication Date(Web):January 16, 2014
DOI:10.1021/ml400399f
The recently described synthetic GPR17 agonist 2-carboxy-4,6-dichloro-1H-indole-3-propionic acid (1) was prepared in tritium-labeled form by catalytic hydrogenation of the corresponding propenoic acid derivative 8 with tritium gas. The radioligand [3H]PSB-12150 (9) was obtained with a specific activity of 17 Ci/mmol (629 GBq/mmol). It showed specific and saturable binding to a single binding site in membrane preparations from Chinese hamster ovary cells recombinantly expressing the human GPR17. A competition assay procedure was established, which allows the determination of ligand binding affinities.Keywords: MDL29,951; montelukast; multiple sclerosis; nucleotide; orphan GPCR; pranlukast; radioligand;
Co-reporter:Matthias D. Mertens, Sonja Hinz, Christa E. Müller, Michael Gütschow
Bioorganic & Medicinal Chemistry 2014 Volume 22(Issue 6) pp:1916-1928
Publication Date(Web):15 March 2014
DOI:10.1016/j.bmc.2014.01.046
In this study, alkynyl–coumarinyl ethers were developed as inhibitors of human monoamine oxidase B (MAO-B). A series of 31 new, ether-connected coumarin derivatives was synthesized via hydroxycoumarins, whose phenolic group at position 6, 7 or 8 was converted by means of the Mitsunobu reaction. The majority of the final products were produced from primary alcohols with a terminal alkyne group. The inhibitors were optimized with respect to the structure of the alkynyloxy chain and its position at the fused benzene ring as well as the residue at position 3 of the pyran-2H-one part. A hex-5-ynyloxy chain at position 7 was found to be particular advantageous. Among the 7-hex-5-ynyloxy-coumarins, the 3-methoxycarbonyl derivative 36 was characterized as a dual-acting inhibitor with IC50 values of less than 10 nM towards MAO-A and MAO-B, and the 3-(4-methoxy)phenyl derivative 44 was shown to combine strong anti-MAO-B potency (IC50 = 3.0 nM) and selectivity for MAO-B over MAO-A (selectivity >3400-fold).
Co-reporter:Younis Baqi, Samer Alshaibani, Kirsten Ritter, Aliaa Abdelrahman, Andreas Spinrath, Evi Kostenis and Christa E. Müller
MedChemComm 2014 vol. 5(Issue 1) pp:86-92
Publication Date(Web):22 Nov 2013
DOI:10.1039/C3MD00309D
The orphan G protein-coupled receptor GPR17 was shown to be involved in myelin repair and has been proposed as a novel drug target for the treatment of brain and spinal cord injury and for multiple sclerosis. Recently, 3-(2-carboxy-4,6-dichloro-indol-3-yl)propionic acid (MDL29,951, 1a) was discovered and characterized as a potent synthetic GPR17 agonist. In the present study we substantially optimized the preparation of 1a, which is carried out via Japp–Klingemann condensation of 3,5-dichlorophenyldiazonium chloride and deprotonated 2-(ethoxycarbonyl)cyclopentanone yielding phenylhydrazone derivative 5a followed by Fischer indole (diaza-Cope) rearrangement. A robust synthesis of 1a (75% yield) was developed to allow upscaling of the procedure. The developed method was applied to the synthesis of a series of 10 derivatives, eight of which represent new compounds. Biological evaluation in calcium mobilization assays using 1321N1-astrocytoma cells recombinantly expressing the human GPR17 provided first insights into their structure–activity relationships. 3-(2-Carboxy-4,6-dibromo-indol-3-yl)propionic acid (1b) showed similar potency to 1a and represents the most potent synthetic GPR17 agonist described to date with an EC50 value of 202 nM.
Co-reporter:Marianne Freundlieb, Herbert Zimmermann, Christa E. Müller
Analytical Biochemistry 2014 Volume 446() pp:53-58
Publication Date(Web):1 February 2014
DOI:10.1016/j.ab.2013.10.012
Abstract
Ecto-5′-nucleotidase (eN) is a membrane-bound enzyme that hydrolyzes extracellular nucleoside-5′-monophosphates yielding the respective nucleoside and phosphate. Increased levels of eN expression have been observed in many cancer cells. By increasing extracellular adenosine concentrations, they contribute to their proliferative, angiogenic, metastatic, and immunosuppressive effects. Therefore, eN is of considerable interest as a novel drug target for the treatment of cancer as well as of inflammatory diseases. In this study, we developed, optimized, and applied a highly sensitive radiometric assay using [3H]adenosine-5′-monophosphate (AMP) as a substrate. The reaction product [3H]adenosine was separated from [3H]AMP by precipitation of the latter with lanthanum chloride and subsequent filtration through glass fiber filters. Conditions were optimized to reproducibly collect the [3H]adenosine-containing filtrate used for quantitative determination. Validation of the assay yielded a mean Z′ factor of 0.73, which demonstrates its suitability for high-throughput screening. The new assay shows a limit of detection that is at least 30-fold lower than those of common colorimetric methods (e.g., optimized malachite green assay and capillary electrophoresis-based assay procedures), and it is also superior to a recently developed luciferase-based assay.
Co-reporter:V. Rempel, K. Atzler, A. Behrenswerth, T. Karcz, C. Schoeder, S. Hinz, M. Kaleta, D. Thimm, K. Kiec-Kononowicz and C. E. Müller
MedChemComm 2014 vol. 5(Issue 5) pp:632-649
Publication Date(Web):11 Feb 2014
DOI:10.1039/C3MD00394A
GPR18 and GPR55 are orphan G protein-coupled receptors (GPCRs) that interact with certain cannabinoid (CB) receptor ligands. In the present study bicyclic imidazole-4-one derivatives were discovered as new scaffolds for the development of antagonists for GPR18 and GPR55. Interaction with CB1 and CB2 receptors was also studied to assess selectivity. The presented extensive structure–activity relationship study of 49 derivatives investigated at all four GPCRs revealed structural requirements for the development of potent and selective GPR18 and GPR55 antagonists. (Z)-(2,3-Difluorobenzylidene)-6,7-dihydro-2H-imidazo[2,1b][1,3]thiazin-3(5H)-one (18) was identified as a selective GPR55 antagonist (IC50 3.15 μM). The most potent GPR18 antagonist was (Z)-2-(3-(4-chlorobenzyloxy)benzylidene)-6,7-dihydro-2H-imidazo[2,1b][1,3]-thiazin-3(5H)-one (32, IC50 0.279 μM, >36-fold selective vs. CB1 and GPR55, 14-fold selective vs. CB2) representing the first selective GPR18 antagonist. The new compounds may serve as lead structures and as tools to explore the (patho-)physiological roles of these orphan GPCRs and their potential as drug targets.
Co-reporter:Dr. Andreas Brunschweiger;Dr. Pierre Koch;Dr. Miriam Schlenk;Dr. Felipe Pineda;Dr. Petra Küppers;Dr. Sonja Hinz;Dr. Meryem Köse;Dr. Stefan Ullrich;Dr. Jörg Hockemeyer;Dr. Michael Wiese;Dr. Jag Heer;Dr. Christa E. Müller
ChemMedChem 2014 Volume 9( Issue 8) pp:1704-1724
Publication Date(Web):
DOI:10.1002/cmdc.201402082
Abstract
8-Benzyl-substituted tetrahydropyrazino[2,1-f]purinediones were designed as tricyclic xanthine derivatives containing a basic nitrogen atom in the tetrahydropyrazine ring to improve water solubility. A library of 69 derivatives was prepared and evaluated in radioligand binding studies at adenosine receptor (AR) subtypes and for their ability to inhibit monoamine oxidases (MAO). Potent dual-target-directed A1/A2A adenosine receptor antagonists were identified. Several compounds showed triple-target inhibition; one of the best compounds was 8-(2,4-dichloro-5-fluorobenzyl)-1,3-dimethyl-6,7,8,9-tetrahydropyrazino[2,1-f]purine-2,4(1H,3H)-dione (72) (human AR: Ki A1 217 nM, A2A 233 nM; IC50 MAO-B: 508 nM). Dichlorinated compound 36 [8-(3,4-dichlorobenzyl)-1,3-dimethyl-6,7,8,9-tetrahydropyrazino[2,1-f]purine-2,4(1H,3H)-dione] was found to be the best triple-target drug in rat (Ki A1 351 nM, A2A 322 nm; IC50 MAO-B: 260 nM), and may serve as a useful tool for preclinical proof-of-principle studies. Compounds that act at multiple targets relevant for symptomatic as well as disease-modifying treatment of neurodegenerative diseases are expected to show advantages over single-target therapeutics.
Co-reporter:Christa E. Müller;Anke C. Schiedel;Herbert Zimmermann
Purinergic Signalling 2014 Volume 10( Issue 4) pp:651-656
Publication Date(Web):2014 December
DOI:10.1007/s11302-014-9433-4
Co-reporter:Alexander Fuchs, Roland Baur, Clara Schoeder, Erwin Sigel, Christa E. Müller
Bioorganic & Medicinal Chemistry 2014 22(24) pp: 6908-6917
Publication Date(Web):
DOI:10.1016/j.bmc.2014.10.027
Co-reporter:Anne Stößel ; Miriam Schlenk ; Sonja Hinz ; Petra Küppers ; Jag Heer ; Michael Gütschow ;Christa E. Müller
Journal of Medicinal Chemistry 2013 Volume 56(Issue 11) pp:4580-4596
Publication Date(Web):April 30, 2013
DOI:10.1021/jm400336x
Blockade of A2A adenosine receptors (A2AARs) and inhibition of monoamine oxidase B (MAO-B) in the brain are considered attractive strategies for the treatment of neurodegenerative diseases such as Parkinson’s disease (PD). In the present study, benzothiazinones, e.g., 2-(3-chlorophenoxy)-N-(4-oxo-4H-3,1-benzothiazin-2-yl)acetamide (13), were identified as a novel class of potent MAO-B inhibitors (IC50 human MAO-B: 1.63 nM). Benzothiazinones with large substituents in the 2-position, e.g., methoxycinnamoylamino, phenylbutyrylamino, or chlorobenzylpiperazinylbenzamido residues (14, 17, 27, and 28), showed high affinity and selectivity for A2AARs (Ki human A2AAR: 39.5–69.5 nM). By optimizing benzothiazinones for both targets, the first potent, dual-acting A2AAR/MAO-B inhibitors with a nonxanthine structure were developed. The best derivative was N-(4-oxo-4H-3,1-benzothiazin-2-yl)-4-phenylbutanamide (17, Ki human A2A, 39.5 nM; IC50 human MAO-B, 34.9 nM; selective versus other AR subtypes and MAO-A), which inhibited A2AAR-induced cAMP accumulation and showed competitive, reversible MAO-B inhibition. The new compounds may be useful tools for validating the A2AAR/MAO-B dual target approach in PD.
Co-reporter:Mario Funke ; Dominik Thimm ; Anke C. Schiedel ;Christa E. Müller
Journal of Medicinal Chemistry 2013 Volume 56(Issue 12) pp:5182-5197
Publication Date(Web):May 28, 2013
DOI:10.1021/jm400587g
8-Amido-chromen-4-one-2-carboxylic acid derivatives were identified as novel agonists at the G protein-coupled orphan receptor GPR35. They were characterized by a β-arrestin recruitment assay and optimized to obtain agonists with nanomolar potency for the human GPR35. The compounds were found to exhibit high selectivity versus the related GPR55. The most potent agonists were 6-bromo-8-(4-methoxybenzamido)-4-oxo-4H-chromene-2-carboxylic acid (85, EC50 12.1 nM) and 6-bromo-8-(2-chloro-4-methoxybenzamido)-4-oxo-4H-chromene-2-carboxylic acid (90, EC50 11.1 nM), both of which were >1700-fold selective versus GPR55. Most compounds were considerably less potent at rat and mouse than at human GPR35. 6-Bromo-8-(2-methoxybenzamido)-4-oxo-4H-chromene-2-carboxylic acid (87) was the only derivative that activated GPR35 of all three species at similar, low micromolar concentration. Compounds 85 and 90 are the most potent agonists at the human GPR35 known to date and might thus serve as powerful pharmacological tools to further elucidate the receptor’s (patho)physiological role and its potential as a future drug target.
Co-reporter:Dominik Thimm ; Mario Funke ; Anne Meyer ;Christa E. Müller
Journal of Medicinal Chemistry 2013 Volume 56(Issue 17) pp:7084-7099
Publication Date(Web):July 26, 2013
DOI:10.1021/jm4009373
The potent and selective GPR35 agonist 6-bromo-8-(4-methoxybenzamido)-4-oxo-4H-chromene-2-carboxylic acid (12) was obtained in tritium-labeled form, designated [3H]PSB-13253, with a specific activity of 36 Ci (1.33 TBq)/mmol. Radiolabeling was achieved by methylation of ethyl 6-bromo-8-(4-((tert-butyldimethylsilyl)oxy)benzamido)-4-oxo-4H-chromene-2-carboxylate (19) with [3H]methyl tosylate followed by ester hydrolysis. The radioligand was characterized by kinetic, saturation, and competition assays at membrane preparations of Chinese hamster ovary cells recombinantly expressing the human GPR35. [3H]12 labeled the receptor with high affinity (KD = 5.27 nM). Binding was saturable (Bmax = 12.6 pmol/mg of protein) and reversible. Affinities of selected standard ligands and a library of amidochromen-4-one-2-carboxylates were determined. Binding data mostly correlated with potencies determined in β-arrestin assays. On the basis of the test results, several new fluorine-substituted 6-bromo-8-benzamidochromen-4-one-2-carboxylic acids were obtained, which represent the most potent GPR35 agonists known to date. 6-Bromo-8-(2,6-difluoro-4-methoxybenzamido)-4-oxo-4H-chromene-2-carboxylic acid (83; Ki = 0.589 nM, EC50 = 5.54 nM) showed the highest affinity with a Ki value in the subnanomolar range.
Co-reporter:Pierre Koch, Rhalid Akkari, Andreas Brunschweiger, Thomas Borrmann, Miriam Schlenk, Petra Küppers, Meryem Köse, Hamid Radjainia, Jörg Hockemeyer, Anna Drabczyńska, Katarzyna Kieć-Kononowicz, Christa E. Müller
Bioorganic & Medicinal Chemistry 2013 Volume 21(Issue 23) pp:7435-7452
Publication Date(Web):1 December 2013
DOI:10.1016/j.bmc.2013.09.044
Adenosine receptors and monoamine oxidases are drug targets for neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease. In the present study we prepared a library of 55 mostly novel tetrahydropyrimido[2,1-f]purinediones with various substituents in the 1- and 3-position (1,3-dimethyl, 1,3-diethyl, 1,3-dipropyl, 1-methyl-3-propargyl) and broad variation in the 9-position. A synthetic strategy to obtain 3-propargyl-substituted tetrahydropyrimido[2,1-f]purinedione derivatives was developed. The new compounds were evaluated for their interaction with all four adenosine receptor subtypes and for their ability to inhibit monoamine oxidases (MAO). Introduction of mono- or di-chloro-substituted phenyl, benzyl or phenethyl residues at N9 of the 1,3-dimethyl series led to the discovery of a novel class of potent MAO-B inhibitors, the most potent compound being 9-(3,4-dichlorobenzyl)-1,3-dimethyl-6,7,8,9-tetrahydropyrimido[1,2-f]purine-2,4(1H,3H)-dione (21g, IC50 human MAO-B: 0.0629 μM), which displayed high selectivity versus the other investigated targets. Potent dually active A1/A2A adenosine receptor antagonists were identified, for example, 9-benzyl-1-methyl-3-propargyl-6,7,8,9-tetrahydropyrimido[1,2-f]purine-2,4(1H,3H)dione (19f, Ki, human receptors, A1: 0.249 μM, A2A: 0.253 μM). Several compounds showed triple-target inhibition, the best compound being 9-(2-methoxybenzyl)-1-methyl-3-(prop-2-ynyl)-6,7,8,9-tetrahydro pyrimido [1,2-f]purine-2,4(1H,3H)-dione (19g, Ki A1: 0.605 μM, Ki A2A: 0.417 μM, IC50 MAO-B: 1.80 μM). Compounds inhibiting several different targets involved in neurodegeneration may exhibit additive or even synergistic effects in vivo.
Co-reporter:Viktor Rempel, Alexander Fuchs, Sonja Hinz, Tadeusz Karcz, Matthias Lehr, Uwe Koetter, and Christa E. Müller
ACS Medicinal Chemistry Letters 2013 Volume 4(Issue 1) pp:41-45
Publication Date(Web):November 14, 2012
DOI:10.1021/ml300235q
The bark of Magnolia officinalis is used in Asian traditional medicine for the treatment of anxiety, sleeping disorders, and allergic diseases. We found that the extract and its main bioactive constituents, magnolol and honokiol, can activate cannabinoid (CB) receptors. In cAMP accumulation studies, magnolol behaved as a partial agonist (EC50 = 3.28 μM) with selectivity for the CB2 subtype, while honokiol was less potent showing full agonistic activity at CB1 and antagonistic properties at CB2. We subsequently synthesized the major metabolites of magnolol and found that tetrahydromagnolol (7) was 19-fold more potent than magnolol (EC50 CB2 = 0.170 μM) exhibiting high selectivity versus CB1. Additionally, 7 behaved as an antagonist at GPR55, a CB-related orphan receptor (KB = 13.3 μM, β-arrestin translocation assay). Magnolol and its metabolites may contribute to the biological activities of Magnolia extract via the observed mechanisms of action. Furthermore, the biphenylic compound magnolol provides a simple novel lead structure for the development of agonists for CB receptors and antagonists for the related GPR55.Keywords: bioactivation; biphenyls; CB2 receptor agonists; Chinese traditional medicine; honokiol; magnolia extract; Magnolia officinalis; magnolol; magnolol metabolites;
Co-reporter:Anke C. Schiedel;Svenja K. Lacher;Carsten Linnemann
Purinergic Signalling 2013 Volume 9( Issue 3) pp:351-365
Publication Date(Web):2013 September
DOI:10.1007/s11302-013-9354-7
The effects of standard adenosine receptor (AR) agonists and antagonists on the proliferation of human T lymphocytes, unstimulated and phytohemagglutinin-stimulated human peripheral blood lymphocytes (PBL), and Jurkat T cells were investigated. Real-time PCR measurements confirmed the presence of all four AR subtypes on the investigated cells, although at different expression levels. A2A ARs were predominantly expressed in PBL and further upregulated upon stimulation, while malignant Jurkat T cells showed high expression levels of A1, A2A, and A2B ARs. Cell proliferation was measured by [3H]-thymidine incorporation assays. Several ligands, including the subtype-selective agonists CPA (A1), BAY60-6583 (A2B), and IB-MECA (A3), and the antagonists PSB-36 (A1), MSX-2 (A2A), and PSB-10 (A3) significantly inhibited cell proliferation at micromolar concentrations, which were about three orders of magnitude higher than their AR affinities. In contrast, further investigated AR ligands, including the agonists NECA (nonselective) and CGS21680 (A2A), and the antagonists preladenant (SCH-420814, A2A), PSB-1115 (A2B), and PSB-603 (A2B) showed no or only minor effects on lymphocyte proliferation. The anti-proliferative effects of the AR agonists could not be blocked by the corresponding antagonists. The non-selective AR antagonist caffeine stimulated phytohemagglutinin-activated PBL with an EC50 value of 104 μM. This is the first study to compare a complete set of commonly used AR ligands for all subtypes on lymphocyte proliferation. Our results strongly suggest that these compounds induce an inhibition of lymphocyte proliferation and cell death through AR-independent mechanisms.
Co-reporter:Melanie Knospe;Christa E. Müller;Patrizia Rosa
Purinergic Signalling 2013 Volume 9( Issue 3) pp:367-381
Publication Date(Web):2013 September
DOI:10.1007/s11302-013-9355-6
The rat adenine receptor (rAdeR) was the first member of a family of G protein-coupled receptors (GPCRs) activated by adenine and designated as P0-purine receptors. The present study aimed at gaining insights into structural aspects of ligand binding and function of the rAdeR. We exchanged amino acid residues predicted to be involved in ligand binding (Phe1103.24, Asn1153.29, Asn1734.60, Phe17945.39, Asn1945.40, Phe1955.41, Leu2015.47, His2526.54, and Tyr2687.32) for alanine and expressed them in Spodoptera frugiperda (Sf9) insect cells. Membrane preparations subjected to [3H]adenine binding studies revealed only minor effects indicating that none of the exchanged amino acids is part of the ligand binding pocket, at least in the inactive state of the receptor. Furthermore, we coexpressed the rAdeR and its mutants with mammalian Gi proteins in Sf9 insect cells to probe receptor activation. Two amino acid residues, Asn1945.40 and Leu2015.47, were found to be crucial for activation since their alanine mutants did not respond to adenine. Moreover we showed that—in contrast to most other rhodopsin-like GPCRs—the rAdeR does not contain essential disulfide bonds since preincubation with dithiothreitol neither altered adenine binding in Sf9 cell membranes, nor adenine-induced inhibition of adenylate cyclase in 1321N1 astrocytoma cells transfected with the rAdeR. To detect rAdeRs by Western blot analysis, we developed a specific antibody. Finally, we were able to show that the extended N-terminal sequence of the rAdeR constitutes a putative signal peptide of unknown function that is cleaved off in the mature receptor. Our results provide important insights into this new, poorly investigated family of purinergic receptors.
Co-reporter:Peter Ripphausen ; Marianne Freundlieb ; Andreas Brunschweiger ; Herbert Zimmermann ; Christa E. Müller ;Jürgen Bajorath
Journal of Medicinal Chemistry 2012 Volume 55(Issue 14) pp:6576-6581
Publication Date(Web):June 25, 2012
DOI:10.1021/jm300658n
We aimed to identify inhibitors of ecto-5′-nucleotidase (ecto-5′-NT, CD73), a membrane-bound metallophosphoesterase that is implicated in the control of purinergic receptor signaling and a number of associated therapeutically relevant effects. Currently, only very few compounds, including ADP, its more stable analogue α,β-methylene-ADP, ATP, and anthraquinone derivatives are known to inhibit this enzyme. In the search for inhibitors with more drug-like properties, we applied a model structure-based virtual screening approach augmented by chemical similarity searching. On the basis of this analysis, 51 candidate compounds were finally selected for experimental evaluation. A total of 13 of these molecules were confirmed to have competitive inhibitory activity. The most potent inhibitor, 6-chloro-2-oxo-N-(4-sulfamoylphenyl)-2H-chromene-3-carboxylic acid amide (17), showed an IC50 value of 1.90 μM. In contrast to the nucleotide- and anthraquinone-derived antagonists, the newly identified competitive inhibitors are uncharged at physiological pH values, possess a drug-like structure, and are structurally distinct from known active compounds.
Co-reporter:Michael Gütschow ; Miriam Schlenk ; Jürgen Gäb ; Minka Paskaleva ; Mohamad Wessam Alnouri ; Silvia Scolari ; Jamshed Iqbal ;Christa E. Müller
Journal of Medicinal Chemistry 2012 Volume 55(Issue 7) pp:3331-3341
Publication Date(Web):March 12, 2012
DOI:10.1021/jm300029s
2-(Acyl)amino-4H-3,1-benzothiazin-4-ones and related thienothiazinones were identified as structurally novel antagonists at adenosine receptors (ARs). 6-Methyl-2-benzoylamino-4H-3,1-benzothiazin-4-one (10d) was found to be a balanced AR antagonist with affinity for all human (h) subtypes (Ki hA1 65.6 nM; hA2A 120 nM; hA2B 360 nM; hA3 30.4 nM), while in rat (r), 10d was a highly potent A1-selective antagonist (rA1 7.7 nM; rA2A 546 nM; rA2B 679 nM, rA3 >10000 nM). 2-(4-Methylbenzoylamino)-4H-3,1-benzothiazin-4-one (10g) was found to be a potent antagonist at human A2A (68.8 nM) and A3 ARs (23.0 nM) with high selectivity versus the other human AR subtypes. In contrast to A1 and A3 ARs, A2A and A2B ARs tolerated bulky 2-acyl substituents. tert-Butyl (4-oxo-4H-3,1-benzothiazin-2-ylcarbamoyl)benzylcarbamate (15g, Ki hA2B 186 nM; hA2A 603 nM) and 4-(4-benzylpiperazine-1-carbonyl)-N-(4-oxo-4H-3,1-benzothiazin-2-yl)benzamide (15k, hA2A 69.5 nM; hA2B 178 nM) were highly selective versus the other AR subtypes. 2-Acylamino-3,1-benzothiazin-4-ones represent novel scaffolds suitable for the development of potent and selective AR antagonists for each of the four receptor subtypes.
Co-reporter:Viktor Rempel ; Nicole Volz ; Sonja Hinz ; Tadeusz Karcz ; Irene Meliciani +; Martin Nieger ; Wolfgang Wenzel +; Stefan Bräse ;Christa E. Müller
Journal of Medicinal Chemistry 2012 Volume 55(Issue 18) pp:7967-7977
Publication Date(Web):August 23, 2012
DOI:10.1021/jm3008213
A series of 7-alkyl-3-benzylcoumarins was designed, synthesized, and tested at cannabinoid CB1 and CB2 receptors in radioligand binding and cAMP accumulation studies. 7-Alkyl-3-benzylcoumarins were found to constitute a versatile scaffold for obtaining potent CB receptor ligands with high potency at either CB1 or CB2 and a broad spectrum of efficacies. Fine-tuning of compound properties was achieved by small modifications of the substitution pattern. The most potent compounds of the present series include 5-methoxy-3-(2-methylbenzyl)-7-pentyl-2H-chromen-2-one (19a, PSB-SB-1201), a selective CB1antagonist (Ki CB1 0.022 μM), 5-methoxy-3-(2-methoxybenzyl)-7-pentyl-2H-chromen-2-one (21a, PSB-SB-1202), a dual CB1/CB2agonist (CB1Ki 0.032 μM, EC50 0.056 μM; CB2Ki 0.049 μM, EC50 0.014 μM), 5-hydroxy-3-(2-hydroxybenzyl)-7-(2-methyloct-2-yl)-2H-chromen-2-one (25b, PSB-SB-1203), a dual CB1/CB2 ligand that blocks CB1 but activates CB2 receptors (CB1Ki 0.244 μM; CB2Ki 0.210 μM, EC50 0.054 μM), and 7-(1-butylcyclopentyl)-5-hydroxy-3-(2-hydroxybenzyl)-2H-chromen-2-one (27b, PSB-SB-1204), a selective CB2 receptor agonist (CB1Ki 1.59 μM; CB2Ki 0.068 μM, EC50 0.048 μM).
Co-reporter:Victor Hernandez-Olmos ; Aliaa Abdelrahman ; Ali El-Tayeb ; Diana Freudendahl ; Stephanie Weinhausen ;Christa E. Müller
Journal of Medicinal Chemistry 2012 Volume 55(Issue 22) pp:9576-9588
Publication Date(Web):October 17, 2012
DOI:10.1021/jm300845v
P2X4 receptor antagonists have potential as drugs for the treatment of neuropathic pain and neurodegenerative diseases. In the present study the discovery of phenoxazine derivatives as potent P2X4 antagonists is described. N-Substituted phenoxazine and related acridone and benzoxazine derivatives were synthesized and optimized with regard to their potency to inhibit ATP-induced calcium influx in 1321N1 astrocytoma cells stably transfected with the human P2X4 receptor. In addition, species selectivity (rat, mouse, human) and receptor subtype selectivity (versus P2X1,2,3,7) were investigated. The most potent P2X4 antagonist of the present series was N-(benzyloxycarbonyl)phenoxazine (26, PSB-12054) with an IC50 of 0.189 μM and good selectivity versus the other human P2X receptor subtypes. N-(p-Methylphenylsulfonyl)phenoxazine (21, PSB-12062) was identified as a selective P2X4 antagonist that was equally potent in all three species (IC50: 0.928–1.76 μM). The compounds showed an allosteric mechanism of action. The present study represents the first structure–activity relationship analysis of P2X4 antagonists.
Co-reporter:Nikolay T. Tzvetkov, Christa E. Müller
Tetrahedron Letters 2012 Volume 53(Issue 42) pp:5597-5601
Publication Date(Web):17 October 2012
DOI:10.1016/j.tetlet.2012.07.140
An efficient approach for the formation of 5-amino- and 7-amino-6-azaoxindole derivatives was developed. 2-Amino-4-chloro-3-nitropyridine (8), and its 5-nitro-substituted regioisomer (9), respectively, were obtained by reaction with ethyl malonate. The resulting 2-amino-3/5-nitropyridine derivatives substituted in the 4-position with malonic acid diethyl ester (10, 11) were subjected to reductive cyclization yielding 3-ethoxycarbonyl-6-azaoxindole derivatives 4a and 5a. Protection of the amino function was not required. Intermediates 10 and 11 could also be converted to the corresponding 4-acetic acid ethyl esters 12 and 13 by dealkoxycarbonylation with LiCl, and subsequently cyclized under reductive conditions yielding 3-unsubstituted 5-/7-aminooxazindoles.
Co-reporter:Simone Hildenbr;Dr. Susanne Schoch;Dr. Marec vonLehe;Dr. Rainer Surges;Dr. Christa E. Müller
ChemMedChem 2012 Volume 7( Issue 8) pp:1369-1374
Publication Date(Web):
DOI:10.1002/cmdc.201200183
Co-reporter:Renata Ciccarelli;Christa E. Müller;Herbert Zimmermann
Purinergic Signalling 2012 Volume 8( Issue 1) pp:105-106
Publication Date(Web):2012 March
DOI:10.1007/s11302-011-9256-5
Co-reporter:Simone Hildenbr;Younis Baqi ;Christa E. Müller
Journal of Labelled Compounds and Radiopharmaceuticals 2012 Volume 55( Issue 1) pp:48-51
Publication Date(Web):
DOI:10.1002/jlcr.1942
A method for the preparation of [³H]levetiracetam with a high specific activity of 98 Ci/mmol (3.6 TBq/mmol) is described. The radioligand proved to be highly useful for the labeling of specific levetiracetam binding sites in rat brain membrane preparations. Copyright © 2011 John Wiley & Sons, Ltd.
Co-reporter:Sang-Yong Lee, Sébastien A. Lévesque, Jean Sévigny, Christa E. Müller
Journal of Chromatography B 2012 Volume 911() pp:162-169
Publication Date(Web):12 December 2012
DOI:10.1016/j.jchromb.2012.10.044
A highly sensitive capillary electrophoresis method has been developed to monitor the activity of nucleotide pyrophosphatases/phosphodiesterases (NPPs) and screen for NPP inhibitors. In this method, p-nitrophenyl 5′-thymidine monophosphate (p-Nph-5′-TMP) was used as an artificial substrate, and separation of reaction products was performed on a dynamically coated capillary. We found that the optimal capillary electrophoresis (CE) conditions were as follows: fused-silica capillary (20 cm effective length × 75.5 μm (id)), electrokinetic injection for 60 s, 70 mM phosphate buffer containing polybrene 0.002%, pH 9.2, constant current of −80 μA, constant capillary temperature of 15 °C and detection at 400 nm. To allow precise quantification, 2-methyl-4,6-dinitrophenol (dinitrocresol) was applied as an internal standard. The limit of detection (LOD) and the limit of quantification (LOQ) were 137 and 415 nM, respectively. This new method was shown to be over 8-fold more sensitive than the conventional spectrophotometric assays and 16-fold more than the previously reported CE procedure, and the results (Km values for NPP1 and NPP3, Ki values for standard inhibitors) obtained were in accordance with previous literature data. Therefore, this new method is an improvement of actual techniques and could be used as a quick and standard analytical technique for the identification and characterization of NPP inhibitors.Highlights► A sensitive nucleotide pyrophosphatase (NPP) assay was developed. ► p-Nitrophenyl 5′-thymidine monophosphate was used as an artificial substrate. ► A CE-VIS method coupled with dynamically polybrene-coated capillaries was used. ► A combination of phosphate–HEPES buffer notably increased the method sensitivity. ► The obtained LOD and LOQ for p-nitrophenolate are in the nanomolar range.
Co-reporter:Younis Baqi ; Ralf Hausmann ; Christiane Rosefort ; Jürgen Rettinger ; Günther Schmalzing ;Christa E. Müller
Journal of Medicinal Chemistry 2011 Volume 54(Issue 3) pp:817-830
Publication Date(Web):January 5, 2011
DOI:10.1021/jm1012193
Evaluation and optimization of anthraquinone derivatives related to Reactive Blue 2 at P2X2 receptors yielded the first potent and selective P2X2 receptor antagonists. The compounds were tested for inhibition of ATP (10 μM) mediated currents in Xenopus oocytes expressing the rat P2X2 receptor. The most potent antagonists were sodium 1-amino-4-[3-(4,6-dichloro[1,3,5]triazine-2-ylamino)phenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (63, PSB-10211, IC50 86 nM) and disodium 1-amino-4-[3-(4,6-dichloro[1,3,5]triazine-2-ylamino)-4-sulfophenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (57, PSB-1011, IC50 79 nM). Compound 57 exhibited a competitive mechanism of action (pA2 7.49). It was >100-fold selective versus P2X4, P2X7, and several investigated P2Y receptor subtypes (P2Y2,4,6,12); selectivity versus P2X1 and P2X3 receptors was moderate (>5-fold). Compound 57 was >13-fold more potent at the homomeric P2X2 than at the heteromeric P2X2/3 receptor. Several anthraquinone derivatives were found to act as positive modulators of ATP effects at P2X2 receptors, for example, sodium 1-amino-4-(3-phenoxyphenylamino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (51, PSB-10129, EC50 489 nM), which led to about a 3-fold increase in the ATP-elicited current.
Co-reporter:Ali El-Tayeb ; Aidong Qi ; Robert A. Nicholas ;Christa E. Müller
Journal of Medicinal Chemistry 2011 Volume 54(Issue 8) pp:2878-2890
Publication Date(Web):March 21, 2011
DOI:10.1021/jm1016297
A large series of derivatives and analogues of the uracil nucleotides UMP, UDP, and UTP with modifications in various positions of the uracil moiety and/or the phosphate groups were synthesized and evaluated at human P2Y2, P2Y4, and P2Y6 receptors. 2-(Ar)alkylthio substitution of UMP and UDP was best tolerated by the P2Y2 receptor. 2-Phenethylthio-UMP (13e) showed an EC50 value of 1.3 μM at P2Y2 and >70-fold selectivity versus P2Y4 and P2Y6 receptors. Substitution of the 2-keto group in UMP by NH (13g, iso-CMP) resulted in the first potent and selective P2Y4 agonist (EC50 4.98 μM, >20-fold selective vs P2Y2 and P2Y6). In contrast, replacement of the 2-keto function in UDP by NH yielded a potent P2Y2 agonist (12g, iso-CDP, EC50 = 0.604 μM, >100-fold selective). In an attempt to obtain metabolically stable UTP analogues, β,γ-dichloro- and β,γ-difluoro-methylene-UTP derivatives were synthesized. The triphosphate modifications were much better tolerated by P2Y2, and in some cases also by P2Y6, than by P2Y4 receptors. 4-Thio-β,γ-difluoromethylene-UTP (14g) was a potent P2Y2 agonist with an EC50 value of 0.134 μM and >50-fold selectivity. N3-Phenacyl-β,γ-dichloromethylene-UTP (14b) proved to be a potent P2Y6 receptor agonist (EC50 0.142 μM) with high selectivity versus P2Y4 (50-fold) and moderate selectivity versus P2Y2 receptors (6-fold).
Co-reporter:Jamshed Iqbal, Christa E. Müller
Journal of Chromatography A 2011 Volume 1218(Issue 29) pp:4764-4771
Publication Date(Web):22 July 2011
DOI:10.1016/j.chroma.2011.04.085
A simple, efficient, and highly sensitive in-line CE method was developed for the characterization and for inhibition studies of the nucleoside-metabolizing enzymes purine nucleoside phosphorylase (PNP) and adenosine deaminase (ADA) present in membrane preparations of human 1539 melanoma cells. After filling the running buffer (50 mM borate buffer, 100 mM SDS, pH 9.10) into a fused-silica capillary (50 cm effective length × 75 μm), a large sample volume was loaded by hydrodynamic injection (5 psi, 36 s), followed by the removal of the large plug of sample matrix from the capillary using polarity switching (−20 kV). The current was monitored and the polarity was reversed when 95% of the current had been recovered. The separation of the neutral analytes (nucleosides and nucleobases) was performed by applying a voltage of 15 kV. An about 10-fold improvement of sensitivity for the five investigated analytes (adenosine, inosine, adenine, hypoxanthine, xanthine) was achieved by large-volume stacking with polarity switching when compared with CE without stacking. For inosine and adenine detection limits as low as 60 nM were achieved. To the best of our knowledge, this represents the highest sensitivity for nucleoside and nucleobase analysis using CE with UV detection reported so far. The Michaelis–Menten constants (Km) for PNP and ADA and the inhibition constants (Ki) for standard inhibitors determined with the new method were consistent with literature data.
Co-reporter:Mahmoud Fahmi Elsebai, Viktor Rempel, Gregor Schnakenburg, Stefan Kehraus, Christa E. Müller, and Gabriele M. König
ACS Medicinal Chemistry Letters 2011 Volume 2(Issue 11) pp:866
Publication Date(Web):September 17, 2011
DOI:10.1021/ml200183z
The fungus Auxarthron reticulatum derived from the marine sponge Ircinia variabilis produced the diketopiperazine alkaloid amauromine (1) and the quinolinone methyl-penicinoline (2). Compound 2 is identical to the reported methyl-marinamide, whose structure is herewith revised. In radioligand binding studies at human cannabinoid CB1 and CB2 receptors recombinantly expressed in Chinese hamster ovary (CHO) cells, amauromine (1) was found to exhibit high affinity and selectivity for the CB1 receptor (Ki = 178 nM). The compound was shown to be a neutral CB1 antagonist with a Kb value of 66.6 nM determined in cAMP assays. Compound 2 exhibited only weak or no effects at CB receptors. To the best of our knowledge, compound 1 is the first fungal natural product that shows affinity for cannabinoid CB1 receptors. Because of its high antagonistic potency and selectivity, it may have potential for use as a drug and/or serve as a lead structure for drug development.Keywords: amauromine; Auxarthron reticulatum; cannabinoid receptors; CB1; CB2; natural products
Co-reporter:Ali El-Tayeb, Sebastian Michael, Aliaa Abdelrahman, Andrea Behrenswerth, Sabrina Gollos, Karen Nieber, and Christa E. Müller
ACS Medicinal Chemistry Letters 2011 Volume 2(Issue 12) pp:890
Publication Date(Web):October 10, 2011
DOI:10.1021/ml200189u
Adenosine A2A receptor agonists for the local treatment of inflammatory bowel disease (IBS) were designed and synthesized. Polar groups were introduced to prevent peroral absorption and subsequent systemic, e.g., hypotensive, side effects. 4-(2-{6-Amino-9-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-9H-purin-2-ylthio}ethyl)benzenesulfonic acid (7, PSB-0777) was selected for further evaluation in rat ileum/jejunum preparations in ex vivo experiments. Compound 7 significantly improved impaired acetylcholine-induced contractions induced by 2,4,6-trinitrobenzenesulfonic acid and showed synergism with an A2B-selective antagonist. Thus, nonabsorbable, locally active A2A agonists, as a monotherapy or in combination with an A2B antagonist, may be an efficient novel treatment for IBS, preventing the severe systemic side effects of known A2A agonists.Keywords: A2A receptor agonist; A2B receptor antagonist; anti-inflammatory drug; inflammatory bowel disease; nonabsorbable A2A receptor agonist
Co-reporter:Christa E. Müller
BIOspektrum 2011 Volume 17( Issue 6) pp:
Publication Date(Web):2011 October
DOI:10.1007/s12268-011-0103-6
Membranständige Purinrezeptoren sind aufgrund der zahlreichen Subtypen und ihrer weiten Verbreitung im Organismus für eine große Bandbreite an medizinischen Indikationen von Interesse.Membrane-localized purine receptors represent a new class of drug targets. Due to the large number of different purine receptor subtypes and their wide distribution in the body they are of interest for a wide range of medical indications.
Co-reporter:Younis Baqi ; Sang-Yong Lee ; Jamshed Iqbal ; Peter Ripphausen ; Anne Lehr ; Anja B. Scheiff ; Herbert Zimmermann ; Jürgen Bajorath ;Christa E. Müller
Journal of Medicinal Chemistry 2010 Volume 53(Issue 5) pp:2076-2086
Publication Date(Web):February 10, 2010
DOI:10.1021/jm901851t
ecto-5′-Nucleotidase (eN, CD73) plays a major role in controlling extracellular adenosine levels. eN inhibitors have potential as novel drugs, for example, for the treatment of cancer. In the present study, we synthesized and investigated a series of 55 anthraquinone derivatives as potential inhibitors of eN, 11 of which are novel compounds and another 11 of which had previously been described but have now been synthesized by an improved method. We identified several potent inhibitors of rat eN. The most potent compounds were 1-amino-4-[4-fluoro-2-carboxyphenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (45, PSB-0952, Ki = 260 nM) and 1-amino-4-[2-anthracenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (52, PSB-0963, 150 nM), with 52 being the most potent eN inhibitor described to date. Selected compounds were further characterized and found to exhibit a competitive mechanism of inhibition. Investigations of ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) and the P2Y receptor subtypes P2Y2, P2Y4, P2Y6, and P2Y12 showed that compound 45 exhibited the highest degree of selectivity (>150-fold).
Co-reporter:Jamshed Iqbal, Aileen F. Knowles, Christa E. Müller
Journal of Chromatography A 2010 Volume 1217(Issue 4) pp:600-604
Publication Date(Web):22 January 2010
DOI:10.1016/j.chroma.2009.11.100
A simple and fast method of immobilization of cell membrane suspension containing human ecto-nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) on a polyacrylamide-coated capillary was developed. The enzyme microbioreactor was prepared by hydrodynamic injection of a small plug of the polycationic electrolyte hexadimethrine bromide (HDB) followed by a suspension of an enzyme-containing membrane preparation. In order to shorten the enzyme assay time and to increase the throughput of the assay, the capillary was coated from the outlet end and all injections were performed from the outlet end of the capillary. For the monitoring of the enzymatic reaction, the substrate ATP dissolved in reaction buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, and 10 mM Hepes, pH 7.4, internal standard: 10 μM UMP) in the absence or presence of inhibitor was injected electrokinetically and incubated in the microbioreactor for 1 min with 1 kV of applied voltage. Then, the electrophoretic separation of the reaction products was initiated by applying a constant current of 60 μA. A 50 mM phosphate buffer (pH 6.5) was used for the separations and the products were detected by UV absorbance at 260 nm. The new method was compared with an at-capillary-inlet method without immobilization of the enzyme. The results (Km values, Ki values for inhibitor) obtained with both methods were similar and comparable with literature data. The developed outlet immobilized enzyme microreactor using a coated capillary is very fast, simple and most economic allowing multiple use of the enzyme.
Co-reporter:Anja B. Scheiff, Swapnil G. Yerande, Ali El-Tayeb, Wenjin Li, Gajanan S. Inamdar, Kamala K. Vasu, Vasudevan Sudarsanam, Christa E. Müller
Bioorganic & Medicinal Chemistry 2010 Volume 18(Issue 6) pp:2195-2203
Publication Date(Web):15 March 2010
DOI:10.1016/j.bmc.2010.01.072
A series of 2-amino-5-benzoyl-4-phenylthiazole derivatives was investigated in radioligand binding studies at adenosine receptor (AdoR) subtypes with the goal to obtain potent and A1-selective antagonists. Acylation of the 2-amino group was found to be crucial for high A1 affinity. The best compound of the present series was 2-benzoylamino-5-p-methylbenzoyl-4-phenylthiazole (16m) showing a Ki value of 4.83 nM at rat and 57.4 nM at human A1 receptors combined with high selectivity versus the other AdoR subtypes. The compound behaved as an antagonist in GTP shift assays at A1 receptors. Compound 16m may serve as a new lead structure for the development of second-generation non-xanthine-derived A1 antagonists which have potential as novel drugs.
Co-reporter:Christa E. Müller
Purinergic Signalling 2010 Volume 6( Issue 2) pp:145-148
Publication Date(Web):2010 June
DOI:10.1007/s11302-010-9182-y
Co-reporter:Thomas Borrmann ; Sonja Hinz ; Daniela C. G. Bertarelli ; Wenjin Li ; Nicole C. Florin ; Anja B. Scheiff ;Christa E. Müller
Journal of Medicinal Chemistry 2009 Volume 52(Issue 13) pp:3994-4006
Publication Date(Web):May 27, 2009
DOI:10.1021/jm900413e
A new series of 1-alkyl-8-(piperazine-1-sulfonyl)phenylxanthines was designed, synthesized, and characterized in radioligand binding and functional assays at A2B adenosine receptors. A2B antagonists with subnanomolar affinity and high selectivity were discovered. The most potent compounds were 1-ethyl-8-(4-(4-(4-trifluoromethylbenzyl)piperazine-1-sulfonyl)phenyl)xanthine (24, PSB-09120, Ki (human A2B) = 0.157 nM) and 8-(4-(4-(4-chlorobenzyl)piperazine-1-sulfonyl)phenyl)-1-propylxanthine (17, PSB-0788, Ki (human A2B) = 0.393 nM). Moreover, 8-(4-(4-(4-chlorophenyl)piperazine-1-sulfonyl)phenyl)-1-propylxanthine (35, PSB-603) was developed as an A2B-specific antagonist exhibiting a Ki value of 0.553 nM at the human A2B receptor and virtually no affinity for the human and rat A1 and A2A and the human A3 receptors up to a concentration of 10 μM. A tritiated form of the compound was prepared as a new radioligand and characterized in kinetic, saturation, and competition studies. It was shown to be a useful pharmacological tool for the selective labeling of human as well as rodent A2B receptors (KD human A2B 0.403 nM, mouse A2B 0.351 nM).
Co-reporter:Younis Baqi ; Kerstin Atzler ; Meryem Köse ; Markus Glänzel ;Christa E. Müller
Journal of Medicinal Chemistry 2009 Volume 52(Issue 12) pp:3784-3793
Publication Date(Web):May 22, 2009
DOI:10.1021/jm9003297
Anthraquinone derivatives related to the moderately potent, nonselective P2Y12 receptor antagonist reactive blue 2 (6) have been synthesized and optimized with respect to P2Y12 receptor affinity. A radioligand binding assay utilizing human blood platelet membranes and the P2Y12 receptor-selective antagonist radioligand [3H]2-propylthioadenosine-5′-adenylic acid (1,1-dichloro-1-phosphonomethyl-1-phosphonyl) anhydride ([3H]PSB-0413) was applied for compound testing. 1-Amino-2-sulfoanthraquinone derivatives bearing a (p-phenylamino)anilino substitution in the 4-position and an additional acidic function in the meta-position of the aniline ring showed high P2Y12 receptor affinity. These new anthraquinone derivatives became accessible by a recently developed copper(0)-catalyzed Ullmann coupling reaction of 1-amino-4-bromoanthraquinone derivatives with anilines in phosphate buffer under microwave irradiation. The most potent compounds exhibited Ki values of 24.9 nM (1-amino-4-[4-phenylamino-3-sulfophenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate, PSB-0739, 39), and 21.0 nM (1-amino-4-[4-phenylamino-3-carboxyphenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate, PSB-0702, 41), respectively. 1-Amino-2-sulfo-4-anilinoanthraquinone derivatives appeared to be noncytotoxic, as shown for selected derivatives at two human cell lines (melanoma and astrocytoma). Compounds 39 and 41 represent new lead structures for the development of antithrombotic drugs.
Co-reporter:Petra Hillmann ; Geun-Yung Ko ; Andreas Spinrath ; Alexandra Raulf ; Ivar von Kügelgen ; Samuel C. Wolff ; Robert A. Nicholas ; Evi Kostenis ; Hans-Dieter Höltje ;Christa E. Müller
Journal of Medicinal Chemistry 2009 Volume 52(Issue 9) pp:2762-2775
Publication Date(Web):April 1, 2009
DOI:10.1021/jm801442p
The P2Y2 receptor, which is activated by UTP, ATP, and dinucleotides, was studied as a prototypical nucleotide-activated GPCR. A combination of receptor mutagenesis, determination of its effects on potency and efficacy of agonists and antagonists, homology modeling, and chemical experiments was applied. R272 (extracellular loop EL3) was found to play a gatekeeper role, presumably responsible for recognition and orientation of the nucleotides. R272 is also directly involved in binding of dinucleotides, which behaved as partial agonists. Y118A (3.37) mutation led to dramatically reduced efficacy of agonists; it is part of the entry channel as well as the triphosphate binding site. While the Y114A (3.33) mutation did not have any effect on agonist activities, the antagonist Reactive Blue 2 (6) was completely inactive at that mutant. The disulfide bridge Cys25−Cys278 was found to be important for agonist potency but neither for agonist efficacy nor for antagonist potency.
Co-reporter:Thomas Borrmann ; Aliaa Abdelrahman ; Rosaria Volpini ; Catia Lambertucci ; Edgars Alksnis ; Simone Gorzalka ; Melanie Knospe ; Anke C. Schiedel ; Gloria Cristalli ;Christa E. Müller
Journal of Medicinal Chemistry 2009 Volume 52(Issue 19) pp:5974-5989
Publication Date(Web):September 4, 2009
DOI:10.1021/jm9006356
Adenine derivatives bearing substituents in the 2-, N6-, 7-, 8-, and/or 9-position and a series of deazapurines were synthesized and investigated in [3H]adenine binding studies at the adenine receptor in rat brain cortical membrane preparations (rAde1R). Steep structure−activity relationships were observed. Substitution in the 8-position (amino, dimethylamino, piperidinyl, piperazinyl) or in the 9-position (2-morpholinoethyl) with basic residues or introduction of polar substituents at the 6-amino function (hydroxy, amino, acetyl) represented the best modifications. Functional evaluation of selected adenine derivatives in adenylate cyclase assays at 1321N1 astrocytoma cells stably expressing the rAde1R showed that all compounds investigated were agonists or partial agonists. A subset of compounds was additionally investigated in binding studies at human embryonic kidney (HEK293) cells, which also express a high-affinity adenine binding site. Structure−affinity relationships at the human cell line were similar to those at the rAde1R, but not identical. In particular, N6-acetyladenine (25, Ki rat: 2.85 μM; Ki human: 0.515 μM) and 8-aminoadenine (33, Ki rat: 6.51 μM; Ki human: 0.0341 μM) were much more potent at the human as compared to the rat binding site. The new AdeR ligands may serve as lead structures and contribute to the elucidation of the functions of the adenine receptor family.
Co-reporter:Ali El-Tayeb ; Jamshed Iqbal ; Andrea Behrenswerth ; Michael Romio ; Marion Schneider ; Herbert Zimmermann ; Jürgen Schrader ;Christa E. Müller
Journal of Medicinal Chemistry 2009 Volume 52(Issue 23) pp:7669-7677
Publication Date(Web):July 6, 2009
DOI:10.1021/jm900538v
Prodrugs of adenosine A2A receptor agonists were developed that are activated by ecto-5′-nucleotidase (ecto-5′-NT, CD73). Because ecto-5′-NT is upregulated in inflamed tissue, the A2A agonists are expected to be released from their prodrug form at the sites of inflammation. 2-(Ar)alkyl-substituted AMP derivatives were synthesized and investigated. Certain 2-substituted AMP derivatives, including 2-hexylthio-AMP, 2-cyclopentylthio-AMP, 2-cyclohexylmethylthio-AMP, and 2-cyclohexylethylthio-AMP were accepted as substrates by ecto-5′-NT and readily converted to the corresponding 2-substituted adenosine derivatives. The 2-cyclohexylethylthio substitution was a good compromise between the requirements of the ecto-5′-NT and the adenosine A2A receptor. The corresponding AMP derivative (12g) was a similarly good substrate as AMP itself, while the resulting adenosine derivative (11g) was a relatively potent A2A agonist (radioligand binding to rat brain striatal membranes: Ki = 372 nM; inhibition of anti-CD3/anti-CD28-induced IFN-γ release in mouse CD4+ cells: EC50 = 50 nM). Compound 11g was released from 12g by incubation with CD4+ cells isolated from wild-type mice but only to a much smaller extent by cells from ecto-5′-NT knockout mice. Compound 12g will be a new lead structure for the development of more potent and selective ecto-5′-NT-activated prodrugs of selective anti-inflammatory A2A receptor agonists.
Co-reporter:Roland Sauer, Ali El-Tayeb, Marko Kaulich, Christa E. Müller
Bioorganic & Medicinal Chemistry 2009 Volume 17(Issue 14) pp:5071-5079
Publication Date(Web):15 July 2009
DOI:10.1016/j.bmc.2009.05.062
A series of new uracil nucleotide analogs (monophosphates, triphosphates, and phosphonates) was synthesized, in which the ribose moiety was replaced by acyclic chains, including branched or linear alkyl or dialkylether linkers. 1-ω-Bromoalkyluracil derivatives (2) were converted to the corresponding alcohols by treatment with sodium hydroxide and subsequently phosphorylated using phosphorus oxychloride followed by hydrolysis to yield the monophosphates, or by coupling with diphosphate to form the triphosphates. Reaction of 2 with triethyl phosphite followed by deprotection with trimethylsilyl bromide led to the ω-phosphonylalkyluracil derivatives. These products could be further phosphorylated by converting them into their imidazolides and subsequent treatment with diphosphate yielding the corresponding UTP analogs. Nucleoside analogs with an oxygen atom in the 2′-position, which are more similar to the natural ribosides, were synthesized from silylated uracil and trimethylsilyl iodide-treated 1,3-dioxolane, or 1,3-dioxane, respectively, and subsequently phosphorylated by standard procedures. The nucleotide analogs were investigated in a functional assay at NG108-15 cells, a neuroblastoma × glioma hybrid cell line which expresses the UTP- and ATP-activated nucleotide receptor subtype P2Y2. The acyclic nucleotide analogs were generally weaker ligands than UTP, and—in contrast to UTP—they were antagonistic. The most potent compound was diphosphoric 5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)pentylphosphonic anhydride (5c) with an IC50 value of 92 μM showing that the replacement of the α-phosphate by phosphonate, which leads to enhanced stability, was well tolerated.
Co-reporter:Andrea Behrenswerth, Nicole Volz, Jakob Toräng, Sonja Hinz, Stefan Bräse, Christa E. Müller
Bioorganic & Medicinal Chemistry 2009 Volume 17(Issue 7) pp:2842-2851
Publication Date(Web):1 April 2009
DOI:10.1016/j.bmc.2009.02.027
In the present study we synthesized 36 coumarin and 2H-chromene derivatives applying a recently developed umpoled domino reaction using substituted salicylaldehyde and α,β-unsaturated aldehyde derivatives as starting compounds. In radioligand binding studies 5-substituted 3-benzylcoumarin derivatives showed affinity to cannabinoid CB1 and CB2 receptors and were identified as new lead structures. In further GTPγS binding studies selected compounds were shown to be antagonists or inverse agonists.3-Benzylcoumarin derivatives synthesized from substituted salicylaldehydes and α,β-unsaturated aldehydes by an umpoled domino reaction were identified as new lead structures for the development of cannabinoid receptor antagonists/inverse agonists.
Co-reporter:ChristaE. Müller
Chemistry & Biodiversity 2009 Volume 6( Issue 11) pp:2071-2083
Publication Date(Web):
DOI:10.1002/cbdv.200900114
Abstract
Low water solubility and low bioavailability are frequent problems in drug development, particularly in the area of central nervous system (CNS) drugs. This short review describes selected prodrug approaches which have been developed to enhance the bioavailability of drugs, especially that of poorly soluble drugs. Some of the most successful drugs on the market are prodrugs. With a better understanding of active-transport processes at cell membranes in the gut as well as at the blood–brain barrier, the importance of prodrug approaches will further increase in the future. Prodrug approaches will already be considered in the early phase of drug discovery.
Co-reporter:Younis Baqi;Stefanie Weyler;Jamshed Iqbal;Herbert Zimmermann
Purinergic Signalling 2009 Volume 5( Issue 1) pp:
Publication Date(Web):2009 March
DOI:10.1007/s11302-008-9103-5
Reactive blue 2 (RB-2) had been characterized as a relatively potent ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) inhibitor with some selectivity for NTPDase3. In search for the pharmacophore and to analyze structure-activity relationships we synthesized a series of truncated derivatives and analogs of RB-2, including 1-amino-2-sulfo-4-ar(alk)ylaminoanthraquinones, 1-amino-2-methyl-4-arylaminoanthraquinones, 1-amino-4-bromoanthraquinone 2-sulfonic acid esters and sulfonamides, and bis-(1-amino-4-bromoanthraquinone) sulfonamides, and investigated them in preparations of rat NTPDase1, 2, and 3 using a capillary electrophoresis assay. Several 1-amino-2-sulfo-4-ar(alk)ylaminoanthraquinone derivatives inhibited E-NTPDases in a concentration-dependent manner. The 2-sulfonate group was found to be required for inhibitory activity, since 2-methyl-substituted derivatives were inactive. 1-Amino-2-sulfo-4-p-chloroanilinoanthraquinone (18) was identified as a nonselective competitive blocker of NTPDases1, 2, and 3 (Ki 16–18 μM), while 1-amino-2-sulfo-4-(2-naphthylamino)anthraquinone (21) was a potent inhibitor with preference for NTPDase1 (Ki 0.328 μM) and NTPDase3 (Ki 2.22 μM). Its isomer, 1-amino-2-sulfo-4-(1-naphthylamino)anthraquinone (20), was a potent and selective inhibitor of rat NTPDase3 (Ki 1.5 μM).
Co-reporter:Stefanie Weyler, Younis Baqi, Petra Hillmann, Marko Kaulich, Andrea M. Hunder, Ingrid A. Müller, Christa E. Müller
Bioorganic & Medicinal Chemistry Letters 2008 Volume 18(Issue 1) pp:223-227
Publication Date(Web):1 January 2008
DOI:10.1016/j.bmcl.2007.10.082
A library of anilinoanthraquinone derivatives was synthesized by parallel Ullmann coupling reaction of bromaminic acid with aniline derivatives in solution using a compact parallel synthesizer. The products were purified by HPLC and evaluated as antagonists at mouse and human P2Y2 receptors. 4-Phenylamino-substituted 1-amino-2-sulfoanthraquinones, for example, 1-amino-4-(2-methoxyphenyl)-2-sulfoanthraquinone (PSB-716), were potent P2Y2 antagonists with IC50 values in the low micromolar range.A library of anilinoanthraquinone derivatives was synthesized by a parallel Ullmann coupling reaction in solution and evaluated as P2Y2 receptor antagonists. PSB-716 was found to be a potent P2Y2 antagonist with an IC50 of 9 μM.
Co-reporter:Jamshed Iqbal, David Jirovsky, Sang-Yong Lee, Herbert Zimmermann, Christa E. Müller
Analytical Biochemistry 2008 Volume 373(Issue 1) pp:129-140
Publication Date(Web):1 February 2008
DOI:10.1016/j.ab.2007.09.028
Powerful capillary electrophoresis (CE) methods were developed for monitoring the reaction of ecto-5′-nucleotidase (ecto-5′-NT, CD73), a (patho)biochemically important enzyme that hydrolyzes nucleoside-5′-monophosphates to the corresponding nucleosides. The enzymatic reaction was performed either before injection into the capillary (method A) or directly within the capillary (method B). In method A, separation of substrates and products was achieved within 8 min using an eCAP fused-silica capillary (20 cm effective length, 75 μM i.d., UV detection at 260 nm), 40 mM sodium borate buffer (pH 9.1), normal polarity, and a constant voltage of 15 kV. In method B, the sandwich technique was applied; substrate dissolved in reaction buffer (10 mM Hepes [pH 7.4], 2 mM MgCl2, and 1 mM CaCl2) was hydrodynamically injected into a fused-silica capillary (30 cm, 75 μM i.d.), followed by enzyme (recombinant rat ecto-5′-NT) and subsequent injection of substrate solution. The reaction was initiated by the application of 1 kV voltage for 1 min. The voltage was turned off for 1 min and again turned on at a constant voltage of 15 kV to elute products (nucleosides) within 4 min using borate buffer (40 mM, pH 9.1). Thus, assays could be performed within 6 min, including enzymatic reaction, separation, and quantification of the formed nucleoside. The CE methods were used for measuring enzyme kinetics and for assaying inhibitors and substrates. In addition, the online assay was successfully applied to melanoma cell membrane preparations natively expressing the human ecto-5′-NT.
Co-reporter:Jamshed Iqbal, Leonardo Scapozza, Gerd Folkers, Christa E. Müller
Journal of Chromatography B 2007 Volume 846(1–2) pp:281-290
Publication Date(Web):1 February 2007
DOI:10.1016/j.jchromb.2006.09.018
A fast, convenient capillary electrophoresis (CE) method was developed for monitoring the enzymatic reaction of herpes simplex virus type 1 thymidine kinase (HSV-1 TK). The reaction was performed in a test tube followed by quantitative analysis of the products. The optimized CE conditions were as follows: polyacrylamide-coated capillary (20 cm effective length × 50 μm), electrokinetic injection for 30 s, 50 mM phosphate buffer at pH 6.5, constant current of −60 μA, UV detection at 210 nm, UMP or cAMP were used as internal standards. Phosphorylated products eluted within less than 7 min. The limits of detection were 0.36 μM for dTMP and 0.86 μM for GMP. The method was used to study enzyme kinetics, and to investigate alternative substrates and inhibitors.
Co-reporter:Anke C. Schiedel;Heiko Meyer;Bernt B. A. Alsdorf
Purinergic Signalling 2007 Volume 3( Issue 4) pp:347-358
Publication Date(Web):2007 September
DOI:10.1007/s11302-007-9060-4
[3H]Adenine has previously been used to label the newly discovered G protein-coupled murine adenine receptors. Recent reports have questioned the suitability of [3H]adenine for adenine receptor binding studies because of curious results, e.g. high specific binding even in the absence of mammalian protein. In this study, we showed that specific [3H]adenine binding to various mammalian membrane preparations increased linearly with protein concentration. Furthermore, we found that Tris-buffer solutions typically used for radioligand binding studies (50 mM, pH 7.4) that have not been freshly prepared but stored at 4°C for some time may contain bacterial contaminations that exhibit high affinity binding for [3H]adenine. Specific binding is abolished by heating the contaminated buffer or filtering it through 0.2-μm filters. Three different, aerobic, gram-negative bacteria were isolated from a contaminated buffer solution and identified as Achromobacter xylosoxidans, A. denitrificans, and Acinetobacter lwoffii. A. xylosoxidans, a common bacterium that can cause nosocomial infections, showed a particularly high affinity for [3H]adenine in the low nanomolar range. Structure–activity relationships revealed that hypoxanthine also bound with high affinity to A. xylosoxidans, whereas other nucleobases (uracil, xanthine) and nucleosides (adenosine, uridine) did not. The nature of the labeled site in bacteria is not known, but preliminary results indicate that it may be a high-affinity purine transporter. We conclude that [3H]adenine is a well-suitable radioligand for adenine receptor binding studies but that bacterial contamination of the employed buffer solutions must be avoided.
Co-reporter:Christa E. Müller, Jamshed Iqbal, Younis Baqi, Herbert Zimmermann, Anita Röllich, Holger Stephan
Bioorganic & Medicinal Chemistry Letters 2006 Volume 16(Issue 23) pp:5943-5947
Publication Date(Web):1 December 2006
DOI:10.1016/j.bmcl.2006.09.003
Polyoxotungstates were identified as potent inhibitors of NTPDases1, 2, and 3. The most potent compound was K6H2[TiW11CoO40], exhibiting Ki values of 0.140 μM (NTPDase1), 0.910 μM (NTPDase2), and 0.563 μM (NTPDase3). One of the compounds, (NH4)18[NaSb9W21O86], was selective for NTPDases2 and 3 versus NTPDase1. NTPDase inhibition might contribute to the described biological effects of polyoxometalates, including their anti-cancer activity.Polyoxotungstates were identified as novel potent inhibitors of ecto-NTPDases. [TiW11CoO40]8− (7) exhibited a Ki value of 140 nM at rat E-NTPDase1.
Co-reporter:Stefanie Weyler Dr.;Friederike Fülle Dr.;Martina Diekmann Dr.;Britta Schumacher Dr.;Sonja Hinz;Karl-Norbert Klotz Dr.;Christa E. Müller Dr.
ChemMedChem 2006 Volume 1(Issue 8) pp:
Publication Date(Web):10 JUL 2006
DOI:10.1002/cmdc.200600066
The structure–activity relationships of xanthine derivatives related to the adenosine A1 receptor antagonists 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and 1,3-dipropyl-8-(3-noradamantyl)xanthine (KW3902) were investigated by focusing on variations of the 3-substituent. Aromatic residues were well tolerated by the A1 receptor in that position. A moderate effect of stereochemistry was found for the 3-(1-phenylethyl)-substituted analogue of DPCPX (S>R) at A1 and A3 receptors, whereas the opposite stereoselectivity was observed at the A2 receptor subtypes. A 3-hydroxypropyl substituent was found to be optimal for high A1 affinity and selectivity. The most potent compound of the present series was 1-butyl-3-(3-hydroxypropyl)-8-(3-noradamantyl)xanthine (10 c), which exhibits a Ki value of 0.124 nM at rat, and 0.7 nM at human adenosine A1 receptors, combined with high selectivity (≫200-fold) versus the other receptor subtypes. The similarly potent 8-cyclopentyl-3-(3-hydroxypropyl)-1-propylxanthine was converted into a water-soluble phosphate prodrug, which may become a useful pharmacological tool for in vivo studies. 8-Alkyl-2-(3-noradamantyl)pyrimido[1,2,3-cd]purine-8,10-diones, which can be envisaged as xanthine analogues with a fixed 3-propyl substituent, were identified as a new class of potent, selective adenosine A1 receptor antagonists. For example, compound 14 (8-butyl-substituted) exhibits a Ki value of 13.8 nM at human A1 receptors. A selection of the most potent compounds was investigated in [35S]GTPγS binding assays and showed inverse agonistic activity. Their efficacy was generally lower than that of the full inverse agonist DPCPX, and depended on subtle structural changes. Some of the new compounds belong to the most potent and selective A1 antagonists described to date.
Co-reporter:Vita Ozola, Mark Thorand, Martina Diekmann, Ramatullah Qurishi, Britta Schumacher, Kenneth A. Jacobson, Christa E. Müller
Bioorganic & Medicinal Chemistry 2003 Volume 11(Issue 3) pp:347-356
Publication Date(Web):6 February 2003
DOI:10.1016/S0968-0896(02)00456-X
Structure–activity relationships of 2-phenyl-imidazo[2,1-i]purin-5-ones as ligands for human A3 adenosine receptors (ARs) were investigated. An ethyl group in the 8-position of the imidazoline ring of 4-methyl-2-phenyl-imidazopurinone leading to chiral compounds was found to increase affinity for human A3 ARs by several thousand-fold. Propyl substitution instead of methyl at N4 decreased A3 affinity but increased A1 affinity leading to potent A1-selective AR antagonists. The most potent A1 antagonist of the present series was (S)-8-ethyl-2-phenyl-4-propyl-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]purin-5-one (S-3) exhibiting a Ki value of 7.4 nM at rat A1 ARs and greater than 100-fold selectivity versus rat A2A and human A3 ARs. At human A1 ARs 2-phenylimidazo[2,1-i]purin-5-ones were generally less potent and therefore less A1-selective (S-3: Ki=98 nM). 2-, 3-, or 4-Mono-chlorination of the 2-phenyl ring reduced A3 affinity but led to an increase in affinity for A1 ARs, whereas di- (3,4-dichloro) or polychlorination (2,3,5-trichloro) increased A3 affinity. The most potent and selective A3 antagonist of the present series was the trichlorophenyl derivative (R)-8-ethyl-4-methyl-2-(2,3,5-trichlorophenyl)-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]purin-5-one (R-8) exhibiting a subnanomolar Ki value at human A3 ARs and greater than 800-fold selectivity versus the other AR subtypes. Methylation of 4-alkyl-2-phenyl-substituted imidazo[2,1-i]purin-5-ones led exclusively to the N9-methyl derivatives, which exhibited largely reduced AR affinities as compared to the unmethylated compounds. [35S]GTPγS binding studies of the most potent 2-phenyl-imidazo[2,1-i]purin-5-ones at membranes of Chinese hamster ovary cells expressing the human A3 AR revealed that the compounds were inverse agonists at A3 receptors under standard test conditions. Due to their high A3 affinity, selectivity, and relatively high water-solubility, 2-phenyl-imidazo[2,1-i]purin-5-ones may become useful research tools.Graphic
Co-reporter:Christa E. Müller, Anke C. Schiedel, Younis Baqi
Pharmacology & Therapeutics (September 2012) Volume 135(Issue 3) pp:292-315
Publication Date(Web):1 September 2012
DOI:10.1016/j.pharmthera.2012.06.002
Rhodopsin-like (class A) G protein-coupled receptors (GPCRs) are one of the most important classes of drug targets. The discovery that these GPCRs can be allosterically modulated by small drug molecules has opened up new opportunities in drug development. It will allow the drugability of “difficult targets”, such as GPCRs activated by large (glyco)proteins, or by very polar or highly lipophilic physiological agonists. Receptor subtype selectivity should be more easily achievable with allosteric than with orthosteric ligands. Allosteric modulation will allow a broad spectrum of pharmacological effects largely expanding that of orthosteric ligands. Furthermore, allosteric modulators may show an improved safety profile as compared to orthosteric ligands. Only recently, the explicit search for allosteric modulators has been started for only a few rhodopsin-like GPCRs. The first negative allosteric modulators (allosteric antagonists) of chemokine receptors, maraviroc (CCR5 receptor), used in HIV therapy, and plerixafor (CXCR4 receptor) for stem cell mobilization, have been approved as drugs. The development of allosteric modulators for rhodopsin-like GPCRs as novel drugs is still at an early stage; it appears highly promising.
Co-reporter:Wenjin Li, Hannah Ulm, Marvin Rausch, Xue Li, Katie O’Riordan, Jean C. Lee, Tanja Schneider, Christa E. Müller
International Journal of Medical Microbiology (November 2014) Volume 304(Issue 8) pp:958-969
Publication Date(Web):1 November 2014
DOI:10.1016/j.ijmm.2014.06.002
Polysaccharide capsules significantly contribute to virulence of invasive pathogens, and inhibition of capsule biosynthesis may offer a valuable strategy for novel anti-infective treatment. We purified and characterized the enzymes CapD and CapE of the Staphylococcus aureus serotype 5 biosynthesis cluster, which catalyze the first steps in the synthesis of the soluble capsule precursors UDP-d-FucNAc and UDP-l-FucNAc, respectively. CapD is an integral membrane protein and was obtained for the first time in a purified, active form. A capillary electrophoresis (CE)-based method applying micellar electrokinetic chromatography (MEKC) coupled with UV detection at 260 nm was developed for functional characterization of the enzymes using a fused-silica capillary, electrokinetic injection, and dynamic coating with polybrene at pH 12.4. The limits of detection for the CapD and CapE products UDP-2-acetamido-2,6-dideoxy-α-d-xylo-hex-4-ulose and UDP-2-acetamido-2,6-dideoxy-β-l-arabino-hex-4-ulose, respectively, were below 1 μM. Using this new, robust and sensitive method we performed kinetic studies for CapD and CapE and screened a compound library in search for enzyme inhibitors. Several active compounds were identified and characterized, including suramin (IC50 at CapE 1.82 μM) and ampicillin (IC50 at CapD 40.1 μM). Furthermore, the cell wall precursors UDP-d-MurNAc-pentapeptide and lipid II appear to function as inhibitors of CapD enzymatic activity, suggesting an integrated mechanism of regulation for cell envelope biosynthesis pathways in S. aureus. Corroborating the in vitro findings, staphylococcal cells grown in the presence of subinhibitory concentrations of ampicillin displayed drastically reduced CP production. Our studies contribute to a profound understanding of the capsule biosynthesis in pathogenic bacteria. This approach may lead to the identification of novel anti-virulence and antibiotic drugs.
Co-reporter:Christa E. Müller, Kenneth A. Jacobson
Biochimica et Biophysica Acta (BBA) - Biomembranes (May 2011) Volume 1808(Issue 5) pp:1290-1308
Publication Date(Web):May 2011
DOI:10.1016/j.bbamem.2010.12.017
Co-reporter:Vigneshwaran Namasivayam, Sang-Yong Lee, Christa E. Müller
Biochimica et Biophysica Acta (BBA) - General Subjects (March 2017) Volume 1861(Issue 3) pp:603-614
Publication Date(Web):March 2017
DOI:10.1016/j.bbagen.2016.12.019
Co-reporter:Viktor Rempel ; Nicole Volz ; Franziska Gläser ; Martin Nieger ; Stefan Bräse ;Christa E. Müller
Journal of Medicinal Chemistry () pp:
Publication Date(Web):May 16, 2013
DOI:10.1021/jm4005175
The orphan G-protein-coupled receptor GPR55, which is activated by 1-lysophosphatidylinositol and interacts with cannabinoid (CB) receptor ligands, has been proposed as a new potential drug target for the treatment of diabetes, Parkinson’s disease, neuropathic pain, and cancer. We applied β-arrestin assays to identify 3-substituted coumarins as a novel class of antagonists and performed an extensive structure–activity relationship study for GPR55. Selectivity versus the related receptors CB1, CB2, and GPR18 was assessed. Among the 7-unsubstituted coumarins selective, competitive GPR55 antagonists were identified, such as 3-(2-hydroxybenzyl)-5-isopropyl-8-methyl-2H-chromen-2-one (12, PSB-SB-489, IC50 = 1.77 μM, pA2 = 0.547 μM). Derivatives with long alkyl chains in position 7 were potent, possibly allosteric GPR55 antagonists which showed ancillary CB receptor affinity. 7-(1,1-Dimethyloctyl)-5-hydroxy-3-(2-hydroxybenzyl)-2H-chromen-2-one (69, PSB-SB-487, IC50 = 0.113 μM, KB = 0.561 μM) and 7-(1,1-dimethylheptyl)-5-hydroxy-3-(2-hydroxybenzyl)-2H-chromen-2-one (67, PSB-SB-1203, IC50 = 0.261 μM) were the most potent GPR55 antagonists of the present series.
![8-{4-[(4-Benzyl-1-piperazinyl)sulfonyl]phenyl}-1-propyl-3,7-dihyd ro-1H-purine-2,6-dione](http://img.cochemist.com/ccimg/905400/905311-24-2.png)
![8-{4-[(4-Benzyl-1-piperazinyl)sulfonyl]phenyl}-1-propyl-3,7-dihyd ro-1H-purine-2,6-dione](http://img.cochemist.com/ccimg/905400/905311-24-2_b.png)
![7-[(3-chlorobenzyl)oxy]-4-[(methylamino)methyl]-2h-chromen-2-one](http://img.cochemist.com/ccimg/911300/911290-20-5.png)
![7-[(3-chlorobenzyl)oxy]-4-[(methylamino)methyl]-2h-chromen-2-one](http://img.cochemist.com/ccimg/911300/911290-20-5_b.png)
![3-[(1S,9AR)-OCTAHYDRO-2H-QUINOLIZIN-1-YL]PROPANOIC ACID](http://img.cochemist.com/ccimg/771600/771573-02-5.png)
![3-[(1S,9AR)-OCTAHYDRO-2H-QUINOLIZIN-1-YL]PROPANOIC ACID](http://img.cochemist.com/ccimg/771600/771573-02-5_b.png)
![[3-(1，3-Thiazol-2-yl)phenyl]methylamine](http://img.cochemist.com/ccimg/672400/672324-88-8.png)
![[3-(1，3-Thiazol-2-yl)phenyl]methylamine](http://img.cochemist.com/ccimg/672400/672324-88-8_b.png)
![2-(FURAN-2-YL)-7-(2-(4-(4-(2-METHOXYETHOXY)PHENYL)PIPERAZIN-1-YL)-ETHYL)-7H-PYRAZOLO[4,3-E][1,2,4]TRIAZOLO[1,5-C]PYRIMIDIN-5-AMINE](http://img.cochemist.com/ccimg/377800/377727-87-2.png)
![2-(FURAN-2-YL)-7-(2-(4-(4-(2-METHOXYETHOXY)PHENYL)PIPERAZIN-1-YL)-ETHYL)-7H-PYRAZOLO[4,3-E][1,2,4]TRIAZOLO[1,5-C]PYRIMIDIN-5-AMINE](http://img.cochemist.com/ccimg/377800/377727-87-2_b.png)
![Acetamide,N-(4-cyanophenyl)-2-[4-(2,3,6,9-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]-](http://img.cochemist.com/ccimg/264700/264622-58-4.png)
![Acetamide,N-(4-cyanophenyl)-2-[4-(2,3,6,9-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]-](http://img.cochemist.com/ccimg/264700/264622-58-4_b.png)
![Glycine,N-[(5Z,8Z,11Z,14Z)-1-oxo-5,8,11,14-eicosatetraen-1-yl]-](http://img.cochemist.com/ccimg/179200/179113-91-8.png)
![Glycine,N-[(5Z,8Z,11Z,14Z)-1-oxo-5,8,11,14-eicosatetraen-1-yl]-](http://img.cochemist.com/ccimg/179200/179113-91-8_b.png)
![b-D-Ribofuranuronamide,1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-](http://img.cochemist.com/ccimg/153000/152918-18-8.png)
![b-D-Ribofuranuronamide,1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-](http://img.cochemist.com/ccimg/153000/152918-18-8_b.png)
![Boron,[2-[1-(3,5-dimethyl-2H-pyrrol-2-ylidene-kN)ethyl]-3,5-dimethyl-1H-pyrrolato-kN]difluoro-, (T-4)-](/data/chemimg/110400/121207-31-6.png)
![Boron,[2-[1-(3,5-dimethyl-2H-pyrrol-2-ylidene-kN)ethyl]-3,5-dimethyl-1H-pyrrolato-kN]difluoro-, (T-4)-](/data/chemimg/110400/121207-31-6_b.png)
![Benz[6,7]indeno[1,2-b]indol-3-ol,1,2,3,4,4a,5,6,6a,7,12,12b,12c-dodecahydro-4,12b,12c-trimethyl-4-(4-methyl-3-penten-1-yl)-,(3S,4S,4aR,6aS,12bS,12cS)-](http://img.cochemist.com/ccimg/113000/112900-04-6.png)
![Benz[6,7]indeno[1,2-b]indol-3-ol,1,2,3,4,4a,5,6,6a,7,12,12b,12c-dodecahydro-4,12b,12c-trimethyl-4-(4-methyl-3-penten-1-yl)-,(3S,4S,4aR,6aS,12bS,12cS)-](http://img.cochemist.com/ccimg/113000/112900-04-6_b.png)
![2-Anthracenecarbonitrile, 1-amino-4-[(3-chlorophenyl)amino]-9,10-dihydro-9,10-dioxo-](http://img.cochemist.com/ccimg/112100/112007-87-1.png)
![2-Anthracenecarbonitrile, 1-amino-4-[(3-chlorophenyl)amino]-9,10-dihydro-9,10-dioxo-](http://img.cochemist.com/ccimg/112100/112007-87-1_b.png)
![[1,2,4]Triazolo[1,5-c]quinazolin-5-amine,9-chloro-2-(2-furanyl)-](http://img.cochemist.com/ccimg/104700/104615-18-1.png)
![[1,2,4]Triazolo[1,5-c]quinazolin-5-amine,9-chloro-2-(2-furanyl)-](http://img.cochemist.com/ccimg/104700/104615-18-1_b.png)
![Propanoic acid, 2-[(3,5-dichlorophenyl)hydrazono]-, ethyl ester](http://img.cochemist.com/ccimg/103900/103855-01-2.png)
![Propanoic acid, 2-[(3,5-dichlorophenyl)hydrazono]-, ethyl ester](http://img.cochemist.com/ccimg/103900/103855-01-2_b.png)
![Pyrazino[1'',2'':1,5;4'',5'':1',5']dipyrrolo[2,3-b:2',3'-b']diindole-7,15(5H,7aH)-dione,8a,16a-bis(1,1-dimethyl-2-propen-1-yl)-5a,8,8a,13,13a,15a,16,16a-octahydro-,(5aS,7aS,8aR,13aS,15aS,16aR)-](http://img.cochemist.com/ccimg/88400/88360-87-6.png)
![Pyrazino[1'',2'':1,5;4'',5'':1',5']dipyrrolo[2,3-b:2',3'-b']diindole-7,15(5H,7aH)-dione,8a,16a-bis(1,1-dimethyl-2-propen-1-yl)-5a,8,8a,13,13a,15a,16,16a-octahydro-,(5aS,7aS,8aR,13aS,15aS,16aR)-](http://img.cochemist.com/ccimg/88400/88360-87-6_b.png)
![Benzoic acid,2-[(4-amino-3-bromo-9,10-dihydro-9,10-dioxo-1-anthracenyl)amino]-](http://img.cochemist.com/ccimg/84000/83929-40-2.png)
![Benzoic acid,2-[(4-amino-3-bromo-9,10-dihydro-9,10-dioxo-1-anthracenyl)amino]-](http://img.cochemist.com/ccimg/84000/83929-40-2_b.png)
![N,N-dimethyl-9-[2,3-O-(1-methylethylidene)-beta-D-glycero-pentofuranosyl]-9H-purin-6-amine](http://img.cochemist.com/ccimg/19100/19083-21-7.png)
![N,N-dimethyl-9-[2,3-O-(1-methylethylidene)-beta-D-glycero-pentofuranosyl]-9H-purin-6-amine](http://img.cochemist.com/ccimg/19100/19083-21-7_b.png)
![2-[6-amino-8-(methylamino)purin-9-yl]-5-(hydroxymethyl)oxolane-3,4-diol](http://img.cochemist.com/ccimg/13400/13389-13-4.png)
![2-[6-amino-8-(methylamino)purin-9-yl]-5-(hydroxymethyl)oxolane-3,4-diol](http://img.cochemist.com/ccimg/13400/13389-13-4_b.png)
![((3aR,4R,6R,6aR)-2,2-dimethyl-6-(6-(methylamino)-9H-purin-9-yl)tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methanol](http://img.cochemist.com/ccimg/4600/4566-77-2.png)
![((3aR,4R,6R,6aR)-2,2-dimethyl-6-(6-(methylamino)-9H-purin-9-yl)tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methanol](http://img.cochemist.com/ccimg/4600/4566-77-2_b.png)
![2-Anthracenesulfonicacid, 1-amino-4-[[4-[[4-chloro-6-[[3(or4)-sulfophenyl]amino]-1,3,5-triazin-2-yl]amino]-3-sulfophenyl]amino]-9,10-dihydro-9,10-dioxo-](http://img.cochemist.com/ccimg/12300/12236-82-7.png)
![2-Anthracenesulfonicacid, 1-amino-4-[[4-[[4-chloro-6-[[3(or4)-sulfophenyl]amino]-1,3,5-triazin-2-yl]amino]-3-sulfophenyl]amino]-9,10-dihydro-9,10-dioxo-](http://img.cochemist.com/ccimg/12300/12236-82-7_b.png)
