Yohimbine Dimers Exhibiting Selectivity for the Human 2c-Adrenoceptor Subtype

Yohimbine is a potent and selective 2versus 1-adrenoceptor antagonist. To date, drugs with high specificity for the 2-adrenoceptor show marginal selectivity among the three 2-adrenoceptor subtypes. Initial studies showed that yohimbine was about 4and 15-fold more selective for the human 2C-adrenoceptor in comparison with the 2Aand 2B-adrenoceptors, respectively. To improve on this 2-adrenoceptor subtype selectivity, a series of yohimbine dimers (varying from n 2 to 24 spacer atoms) were prepared and evaluated for receptor binding on human 2-adrenoceptor subtypes expressed in Chinese hamster ovary cells. Each dimeric analog showed higher affinities for 2Aand 2C-adrenoceptor versus the 2B-adrenoceptor; and yohimbine dimers with spacers of n 2, 3, 4, 18, and 24 exhibited selectivity for the 2C-adrenoceptor. The yohimbine dimers n 3 and n 24 showed the highest potency and selectivity (32and 82-fold. respectively) for the 2C-adrenoceptor in receptor binding and in functional studies (42and 29-fold, respectively) measuring cAMP changes using a cell-based luciferase reporter gene assay. The dimers (n 3 and n 24) had high selectivity ( 1000-fold) for the 2C–adrenoceptor compared with the three 1-adrenoceptor subtypes. These findings demonstrate that the addition of spacer linkages to bivalent yohimbine molecules provides a successful approach to the development of ligands that are potent and highly selective for the 2C-adrenoceptor. The lack of 2-adrenoceptor (AR) subtype-selective antagonists has precluded clarification of the role of each subtype in various physiological and behavioral studies (MacDonald et al., 1997). Homologous recombination strategies that lead either to gene knockouts or a substitution of a mutant receptor at the wild-type locus in the mouse genome have been developed. Using genetically engineered mouse strains it has been suggested that the 2C-AR subtype has a distinct inhibitory role in the processing of sensory information and in the control of motor and emotion-related activities in the central nervous system (Scheinin et al., 2001). It is therefore possible that 2C-AR-selective drugs may have therapeutic value in the treatment of various neuropsychiatric disorders. In addition to the central nervous system effects associated with 2C-AR, a remarkable role of 2C-AR in vascular dysfunction has recently been discovered. The cold-induced cutaneous arterial blood vessel constriction observed in Raynaud’s disease may be linked to increased reactivity of smooth muscle 2C-AR. 2C-ARs are not functionally responsive at 37°C, i.e., silent in normal regulation of the vascular function, but are activated by catecholamines at 28°C (Chotani et al., 2000). Thus, selective blockade of these receptors by using 2C-ARselective antagonists may provide a new therapeutic intervention for the treatment of Raynaud’s disease. Findings from cardiovascular studies using 2A-, 2B-, and 2C-AR knockout mice indicated that although the peripheral 2Aand 2B-AR modulate blood pressure, the 2C-AR does not play a measurable role in the control of systemic blood pressure (MacDonald et al., 1997). This finding is consistent with the notion that an 2C-AR-selective drug may offer beneficial clinical uses with a minimal potential for systemic blood pressure effects (Rizzo et al., 2001). 2C-AR subtype-selective antagonists will help to further elucidate the physiological roles of this receptor and may provide leads for a variety of therapeutic disorders. The present article illustrates the identification of potent and selective human 2C-AR antagonists using the bivalent This work was supported in part by the National Institute of Health Grant R01GM 29358. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.102.039057. ABBREVIATIONS: AR, adrenoceptor; CHO, Chinese hamster ovary; HEK, human embryonic kidney; CRE, cyclic AMP response element; GCPR, G protein-coupled receptor. 0022-3565/02/3033-979–984$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 303, No. 3 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 39057/1024603 JPET 303:979–984, 2002 Printed in U.S.A. 979 at A PE T Jornals on M ay 3, 2017 jpet.asjournals.org D ow nladed from ligand approach. The term bivalent ligand (or pharmacophore dimer) is defined as a molecule that contains two pharmacophores linked through a spacer. This approach to receptor ligand design would be predicted to generate ligands that exhibit a potency that is greater than that derived from the sum of its two monovalent counterparts. The design relies on the concept that a bivalent ligand should first undergo univalent binding, followed by the binding of the second pharmacophore to a recognition site on a neighboring receptor (Portoghese, 2001). Here, we selected the bivalent pharmacophore of yohimbine because this compound is a selective and potent 2-AR antagonist (Bylund, 1985). The yohimbine dimers used were evaluated on human 2A-, 2B-, and 2C-AR subtypes expressed stably in Chinese hamster ovary (CHO) cells using a radioligand binding assay. Selective yohimbine dimers were also evaluated for binding affinities on human 1A-, 1B-, and 1D-AR subtypes expressed stably in human embryonic kidney (HEK) cells. Finally, cell systems containing cDNA clones of the 2-ARs, were used to assay agonist and antagonist agents using a CRE reporter gene functional assay. Our approach was to evaluate changes in luciferase activities, as an index of cAMP accumulations, for highly selective yohimbine dimers on human 2-AR subtypes expressed in CHO cells. Materials and Methods Sources of Materials. All cell culture reagents were obtained from Invitrogen (Carlsbad, CA). Human 2A-, 2B-, and 2C-AR subtypes expressed in CHO cells were obtained from Drs. Marc Caron and Robert Lefkowitz (Duke University Medical Center, Durham, NC) and Dr. Stephen Liggett (College of Medicine, University of Cincinnati, Cincinnati, OH). Human 1A-, 1B-, and 1D-AR subtypes expressed in HEK cells were obtained from Dr. Kenneth Minneman (Department of Pharmacology, Emory University School of Medicine, Atlanta, GA). MK-912 was obtained from Merck (Rahway, NJ). Yohimbine dimers (Fig. 1) were provided by Dr. Duane D. Miller (Department of Pharmaceutical Sciences, University of Tennessee, Memphis, TN). The yohimbine dimers (n 2, 3, 4, 5, 6, and 9) were prepared as diamides by coupling the commercially available yohimbinic acid with aliphatic , -diamines under standard peptide coupling condition (Zheng et al., 2000). The procedures for the synthesis of the glycinamide derivatives (n 18 and n 24) are as described by Zheng (1999). All the yohimbine dimers were hygroscopic and light-sensitive, and dimers, with the exception of n 18 and n 24, were provided as dihydrochloride salts and were soluble in water. The dimers n 18 and n 24, prepared as glycinamide derivatives and supplied as the free base, were dissolved in a 1:5 mixture of dimethyl sulfoxide/water. Stock solutions of 10 mM were prepared and diluted in water to appropriate concentrations for these studies. Samples and drug solutions were protected from light. [H]Rauwolscine and [H]prazosin were obtained from PerkinElmer Life Sciences (Boston, MA). All other chemicals were obtained from SigmaAldrich (St. Louis, MO). Cell Culture. CHO cells stably expressing human 2A-, 2B-, and 2C-AR subtypes were grown in 150-cm 2 Corning flasks with Ham’s F-12 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, penicillin (100 units/ml), and streptomycin 100 g/ml). The flasks were incubated at 37°C (5% CO2). Media were changed every 48 h until the cells were confluent. Upon confluence, the cells were detached by addition of 0.05% trypsin EDTA for 3 to 5 min. HEK cells stably expressing human 1A-, 1B-, and 1D-AR subtypes were grown in 150-cm Corning flasks with Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin. The flasks were incubated at 37°C (5% CO2). Media were changed every 48 h until the cells were confluent. Upon confluence, the cells were detached by gentle scraping. Radioligand Binding Assays. Radioligand binding studies were performed in CHO cells expressing human 2A-, 2B-, and 2C-AR subtypes and HEK cells expressing human 1A-, 1B-, and 1D-AR subtypes. The detached cells were washed and centrifuged with Tris-EDTA buffer, pH 7.4, in which they were finally suspended. The competition binding assays were performed in duplicate by incubating 50,000 cells with [H]rauwolscine and [H]prazosin for the 2and 1-ARs, respectively, and varying concentrations of the compounds under investigation for 1 h in a water bath at 37°C. Nonspecific binding was defined by addition of 10 M yohimbine and 10 M phentolamine for the 2and 1-ARs, respectively. Cell suspensions were filtered using GF/B glass fiber filters (Whatman, Maidstone, UK) using a cell harvester (model 12-R; Brandel Inc., Gaithersburg, MD). The filter discs were washed three times with 5 ml of TrisEDTA buffer, pH 7.4, at 4°C. The radioactivity was determined using a TriCarb 2900TR liquid scintillation counter (Packard Instrument Company, Inc., Downers Grove, IL). The displacement curves were generated using GraphPad Prism software (GraphPad Software, San Diego, CA). The displacement curves were plotted using a standard slope factor of 1.0; and the Ki values of the competing ligands were determined using the equation of Cheng and Prusoff (1973). The percentage of specific binding was determined by dividing the difference between total bound (dpm) and nonspecific bound (dpm) by the total bound (dpm). Scatchard analyses for radioligands were determined using varyFig. 1. Structures of yohimbine dimers of varying spacer atom lengths (n 2, 3, 4, 5, 6, 7, 9, 18, and 24). 980 Lalchandani et al. at A PE T Jornals on M ay 3, 2017 jpet.asjournals.org D ow nladed from ing concentrations of [H]rauwolscine and [H]prazosin, alone or in the presence of high concentrations of yohimbine or phentolamine. The specific binding was established at each concentration and plotted as bound ligand versus bound/free ligand and the corresponding Bmax and Kd values calculated on each human adrenoceptor subtype. Data are expressed as the mean S.E.M. of at least n 3 experiments. Each concentration was done in triplicate. The experimentally determined Kd (nanomolar) and Bmax (picomoles per milligram) values (mean S.E.M.) of the radioligands on the AR subtypes were as follows: [H]rauwolscine: 2A 1.14 0.15 and 0.21 0.03; 2B 0.63 0.07 and 0.19 0.07; 2C 0.28 0.05 and 0.072 0.003 in CHO cells; and [H]prazosin: 1A 0.20 0.02 and 0.22 0.02; 1B 0.11 0.01 and 0.38 0.01; 1D 0.17 0.07 and 0.11 0.04, respectively, in HEK cells. The Kd values of these radioligands on these AR subtypes agree very favorably with published reports (Bylund et al., 1998). Cyclic AMP Response Element-Luciferase Reporter Gene Assay. The functional responses of the yohimbine analogs in these 2-AR subtypes were determined with the use of six copies of a cAMP response element-luciferase reporter gene construct (6 CRE-LUC, pADneo2-C6-BGL) provided by Dr. A. Himmler (Boehringer Ingelheim Research and Demonstration, Vienna, Austria) by the same transfection procedures described previously in CHO cells (Vansal and Feller, 1999). The antagonist effects of yohimbine and the two highly selective bivalent analogs (n 3 and n 24) were examined for their concentration-dependent reversal of medetomidine on forskolin-induced cAMP levels, as assessed by luciferase activity changes in CHO cells expressing the human 2Aand 2C-AR subtypes. The compounds were tested under the following conditions: the 6 CRE-LUC plasmid (5 g/100 l of cell suspension) was transiently transfected into CHO cells expressing the 2Aand 2C-AR subtypes using electroporation, at 150 V, 70 ms, single pulse. The transfected cells were plated at a density of 50,000 cells/200 l/well in a 96-well microtiter plate and allowed to grow for 20 h. The compounds under investigation for agonist activity were added directly to the medium and allowed to incubate for 4 h. When tested for antagonist activity, the compounds were added at varying concentrations 20 min before addition of a fixed concentration of medetomidine (10 nM). Subsequently, the media were aspirated, the cells lysed, and the luciferase activity determined using a TopCount (Packard BioScience, Meriden, CT) luminometer after adding luciferin. Data were analyzed using GraphPad Prism software and expressed as a mean S.E.M. The response produced by 5 M forskolin in each experiment was used as 100%. Data Accumulation and Statistical Analyses. For ligand binding studies in cell lines, varying concentrations of each drug/ligand (ranging from 10 –10 11 M) were added in duplicate within each experiment, and the individual molar IC50 values were determined using GraphPad Prism. The Ki values of each ligand were determined according to the equation described by Cheng and Prusoff (1973), and final data are presented as pKi S.E.M. of n 4 experiments. The concentration-dependent reversal of medetomidine actions on luciferase activity changes in CHO cells by yohimbine and dimers (n 3 and n 24) were determined as molar EC50 and negative log molar effective concentration-50 (pEC50) values S.E.M. of n 3 to 4 experiments. Differences of means of binding affinities and functional responses for individual ligands on the AR subtypes were done by analysis of variance and Tukey’s post hoc analysis test.