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CELLULAR AND MOLECULAR

Tethered Yohimbine Analogs as Selective Human _2C-Adrenergic Receptor Ligands

Department of Pharmacology (S.A.B., D.R.F.) and National Center for Natural Products Research (G.M., D.R.F.), School of Pharmacy, University of Mississippi, Oxford, Mississippi; and Department of Pharmaceutical Sciences (S.M.M., B.M.M., D.D.M.), University of Tennessee Health Science Center, Memphis, Tennessee

Received April 10, 2006; accepted July 20, 2006.

	Abstract

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Yohimbine is a potent and relatively nonselective ₂-adrenergic receptor (AR) antagonist. In an earlier report, we demonstrated that dimeric yohimbine analogs containing methylene and methylene-diglycine tethers were highly selective human _2C-AR ligands. Little work has been done to examine the role of the tether group or the absence of the second yohimbine pharmacophore on selectivity for human ₂-AR subtypes. The goal of our study was to determine the binding affinities and functional subtype selectivities of a series of tethered yohimbine ligands in the absence of the second pharmacophore. The profiles of pharmacological activity for the yohimbine analogs on the three human ₂-AR subtypes expressed in Chinese hamster ovary cells were examined using receptor binding and cAMP inhibition assays. All of the tethered yohimbine analogs exhibited higher binding affinities at the _2C- versus _2A- and _2B-AR subtypes. Notably, the benzyl carboxy alkyl amine and the carboxy alkyl amine analogs exhibited 43- and 1995-fold and 295- and 54-fold selectivities in binding to the _2C- versus _2A- and _2B-ARs, respectively. Data from luciferase reporter gene assays confirmed the functional antagonist activities and selectivity profiles of selected compounds from the tethered series. The data demonstrate that the second pharmacophore may not be essential to obtain _2C-AR subtype selectivity, previously observed with the dimers. Further changes in the nature of the tether will help in optimization of the structure-activity relationship to obtain potent and selective _2C-AR ligands. These compounds may be used as pharmacological probes and in the treatment of human disorders.

The increasing number of potential therapeutic uses has greatly stimulated interest in the design of ligands that interact selectively with the _2C-ARs. Lalchandani et al. (2002) have shown that dimers of the ₂-AR subtype nonselective antagonist ligand yohimbine exhibited selectivity for the _2C-AR. The n = 3 and n = 24 dimers exhibited the greatest _2C-AR selectivity in the series tested. Interestingly, none of the analogs surpassed the affinity of the parent compound, yohimbine. In addition, the exact mechanism underlying the _2C-AR selectivity observed for these dimeric compounds is unclear. An attempt to assess the role of the second pharmacophore and the spacer arm in the potency of dimeric ligands was recently made by Mustafa et al. (2005). To achieve this, a monomeric tethered ligand versus a dimeric ligand approach was used. Compounds having only one pharmacophore, i.e., only one yohimbine molecule, to which side chains of varying length and containing different patterns of hydrogen bond donors/acceptors, rigidity, hydrophobicity and/or charge had been appended, were evaluated for binding to the _2C-ARs. The data showed that even in the absence of the second pharmacophore, the tethered yohimbine analogs displayed high binding affinities at the _2C-AR.

In our study, our aims were to 1) determine the affinities of the tethered yohimbine ligands at all three ₂-ARs and examine subtype selectivities exhibited, if any, by the compounds and 2) elucidate the underlying physicochemical basis for the observed ₂-AR subtype selectivities. To start with, we have evaluated the standards for this study viz. the parent compound, yohimbine, and the n = 3 analog from the dimer series at the ₂-ARs. Furthermore, we have examined tethered analogs of yohimbine, which consist of various substitutions at the C-16 carbonyl position of yohimbine and yohimbinic acid (a nontethered analog of yohimbine possessing an acid functional group instead of a methyl ester at the C-16 position) at ₂-AR subtypes. The subtypes were stably expressed as homogeneous populations in Chinese hamster ovary (CHO) cells. Selected compounds were tested for binding affinities at ₁-AR subtypes, stably expressed as homogeneous populations in human embryonic kidney (HEK293) cells. Finally, functional activities of selected compounds were determined in CHO cells expressing the _2A- and _2C-ARs using a cAMP response element-luciferase (CRE-LUC) reporter gene assay.

	Materials and Methods

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Sources of Materials. All cell culture reagents were obtained from Invitrogen (Carlsbad, CA). CHO cells expressing homogeneous populations of human _2A-, _2B-, and _2C-ARs 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). HEK293 cells expressing homogeneous populations of human _1A-, _1B-, and _1D-ARs were obtained from Dr. Kenneth Minneman (Emory University School of Medicine, Atlanta, GA). The cAMP response element-luciferase gene construct (6 CRE-LUC) was provided by Dr. A. Himmler (Boehringer Ingelheim Research and Development, Vienna, Austria). Yohimbine and yohimbinic acid were obtained from ICN Biomedicals Inc. (Aurora, OH) and Aldrich Chemical Co. (Milwaukee, WI), respectively. Tethered yohimbine analogs and the n = 3 yohimbine dimer were provided by Dr. Duane D. Miller (Department of Pharmaceutical Sciences, University of Tennessee, Memphis, TN). The procedures for synthesis of the tethered yohimbine analogs and the n = 3 yohimbine dimer are as described by Mustafa et al. (2005), Zheng et al. (2000), and Zheng (1999). Solutions of the n = 3 yohimbine dimer were prepared as described previously (Lalchandani et al., 2002). Yohimbinic acid (2) and all the tethered yohimbine analogs, with the exception of the alkyl amine analog (8) and the carboxy alkyl amine analog (10), were dissolved in a mixture of water and dimethyl sulfoxide. Yohimbine (1), the alkyl amine analog (8), and the carboxy alkyl amine analog (10) were dissolved in water alone. Stock solutions (10^–2 M) were prepared and diluted in water to appropriate concentrations for the studies. [³H]Rauwolscine and [³H]prazosin were obtained from PerkinElmer Life and Analytical Sciences (Boston, MA), and all other chemicals were obtained from Sigma-Aldrich (St. Louis, MO).

Cell Culture. CHO cells stably expressing homogeneous populations of _2A-, _2B-, and _2C-ARs were grown in 150 cm² Corning flasks with Ham's F-12 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, penicillin (100 units/ml), streptomycin (100 µg/ml), and Geneticin (100 µg/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 trypsin (0.05% trypsin EDTA, 5 min).

HEK293 cells stably expressing homogeneous populations of _1A-, _1B-, and _1D-ARs were grown in 150 cm² Corning flasks with Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM glutamine, penicillin (100 units/ml), streptomycin (100 µg/ml), and Geneticin (100 µg/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 gentle scraping.

Radioligand Binding Assays. Radioligand binding studies were conducted in intact CHO cells expressing homogeneous populations of _2A-, _2B-, and _2C-ARs. Similar studies were performed in intact HEK293 cells expressing homogeneous populations of _1A-, _1B-, and _1D-ARs. In brief, CHO cells were harvested using Ham's F-12 media after trypsinization, whereas HEK293 cells were detached by simple scraping. Detached cells were washed and centrifuged with Tris-EDTA buffer, pH 7.4; containing 50 mM Tris, 20 mM disodium EDTA, and 154 mM NaCl, in which they were finally suspended. Competition binding assays were performed in duplicate by incubating 50,000 cells with [³H]rauwolscine (0.1 µCi, 0.7 nM) for human _2A-, _2B-, and _2C-ARs and [³H]prazosin (0.1 µCi, 0.7 nM) for human _1A-, _1B-, and _1D-ARs and varying concentrations of the analogs under investigation in a water bath at 37°C. The assays were conducted in a final volume of 2 ml. Nonspecific binding was determined in the presence of 10 µM phentolamine. Incubations were terminated at 60 min by rapid filtration over GF/C glass fiber filters (Whatman, Maidstone, UK) using a cell harvester (Brandel Inc., Gaithersburg, MD). The filter discs were washed three times with Tris-EDTA buffer, pH 7.4, at 4°C. The radioactivity was quantified by using a Packard Tri-Carb 2900 TR liquid scintillation analyzer (Packard Instrument Company, Meridian, CT), and data were analyzed using GraphPad Prism (GraphPad Software Inc., San Diego, CA). The displacement curves were plotted using a standard slope factor of 1.0; and the K_i values of the competing ligands were determined using the equation of Cheng and Prusoff (1973). The percentage of specific binding in the inhibition experiments was determined by dividing the difference between the total bound (disintegrations per minute) and nonspecific bound (disintegrations per minute) by the total bound (disintegrations per minute).

Scatchard analyses were carried out using varying concentrations, ranging from 0.03 to 3.6 nM, of selected radioligands to determine their affinities (K_d) and maximal binding characteristics (B_max). The saturation binding of [³H]rauwolscine to human _2A-, _2B-, and _2C-ARs and [³H]prazosin to human _1A-, _1B-, and _1D-ARs was conducted in a final volume of 1 ml. Nonspecific binding for the ₁- and ₂-ARs was determined in the presence of 10 µM phentolamine and 10 µM yohimbine, respectively. The total and nonspecific binding for each concentration was determined in triplicate. The specific binding, at each concentration of the radioligand, was established and plotted as bound ligand versus bound/free ligand and the corresponding K_d and B_max values calculated on each human -AR subtype. Data are expressed as the means ± S.E.M. of n = 6 to 9 experiments. The experimentally determined K_d (nanomolar concentrations) and B_max (picomoles per milligram of protein) values (means ± S.E.M., n = 6–9 experiments) of the radioligands on the AR subtypes were as follows: [³H]rauwolscine: _2A = 1.93 ± 0.12 nM and 8.20 ± 0.71 pmol/mg protein, _2B = 1.45 ± 0.08 nM and 1.64 ± 0.10 pmol/mg protein, and _2C = 0.32 ± 0.01 nM and 1.20 ± 0.13 pmol/mg protein in CHO cells; and [³H]prazosin: _1A = 0.22 ± 0.008 nM and 0.56 ± 0.01 pmol/mg protein, _1B = 0.24 ± 0.01 nM and 1.59 ± 0.10 pmol/mg protein, and _1D = 0.14 ± 0.01 nM and 0.46 ± 0.04 pmol/mg protein in HEK293 cells.

cAMP Response Element-Luciferase Reporter Gene Assay. To verify that the observed binding affinities of yohimbine and its selected analogs correlate with the functional responses in the ₂-ARs, functional responses of selected ligands were determined by using six copies of a cAMP response element-luciferase reporter gene construct (6 CRE-LUC, pADneo2-C6-BGL). The reporter gene assays were conducted in CHO cells expressing the human _2A- and _2C-AR subtypes. The cells were grown to confluence, upon which they were isolated and electroporated in the presence of the plasmid. The transfection procedure used was the same as that described previously in CHO cells (Vansal and Feller, 1999; Lalchandani et al., 2002). Cells were transiently transfected with the 6 CRE-LUC plasmid (5 µg/100 µl of cell suspension) using electroporation at 150 V, 70 ms, single pulse. Transfected cells were plated into a 96-well microplate at a density of approximately 50,000 cells per well and allowed to grow for 20 h. Fixed concentrations of the nonselective ₂-AR agonist medetomidine (Virtanen et al., 1988) (0.01 and 1 µM for the _2A- and _2C-ARs, respectively), which produced a submaximal inhibition of the forskolin response, were added directly to the medium 20 min before the addition of forskolin (3–5 µM) and then allowed to incubate for 4 h. Selected ligands were tested for antagonist activity and added 20 min before the addition of medetomidine. The media were then aspirated, the cells were lysed, and the luciferase activity was determined using the LucLite assay kit (Packard Biosciences, Meriden, CT). Changes in light production were measured on a Packard Topcount Luminescence Counter (Packard Biosciences) after addition of luciferin. Medetomidine inhibited the forskolin-induced cAMP changes by 50 to 70% in each of the two subtypes, and the antagonist effects of yohimbine and its selected analogs were determined by their ability to reverse the medetomidine action. The IC₅₀ values of yohimbine and its analogs for the concentration-dependent reversal of the medetomidine action against forskolin-induced cAMP responses at the human _2C-AR were calculated using GraphPad Prism and are expressed as the means ± S.E.M. of n = 4 experiments.

Data Accumulation and Statistical Analyses. For ligand binding studies in cell lines, varying concentrations of each of the compounds, ranging from 10^–12 to 10^–5 M, were added in duplicate within each experiment, and the individual molar IC₅₀ values were determined using GraphPad Prism. The displacement curves were plotted using a standard slope factor of 1.0, the K_i values of the competing ligands were determined using the equation of Cheng and Prusoff (1973), and the final data are presented as means ± S.E.M. of n = 4 or more experiments. In the functional studies, the IC₅₀ values of the antagonists for the concentration-dependent reversal of the action of the agonist, medetomidine, against forskolin-induced cAMP responses at the human _2C-AR were calculated using GraphPad Prism and are expressed as the means ± S.E.M. of n = 4 experiments. Differences between means of binding affinities and functional responses for individual ligands on the AR subtypes were analyzed by analysis of variance and Tukey's post hoc analysis test. When two means were compared, statistical analyses were done using Student's t test. Values were considered to be statistically significant when P < 0.05.

	Results

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Radioligand Binding Assay. Radioligand binding analyses of yohimbine (1), yohimbinic acid (2), the n = 3 yohimbine dimer (3), and tethered yohimbine analogs (4–10) were performed in CHO cells stably expressing homogeneous populations of human _2A-, _2B-, and _2C-ARs. Structures of all compounds are provided in Fig. 1. The binding affinities of the compounds are presented in Table 1.

View larger version (19K): [in this window] [in a new window]   Fig. 1. Chemical structures of yohimbine (Yoh) and analogs.

View this table: [in this window] [in a new window]   TABLE 1 Binding affinities of yohimbine, yohimbinic acid, the n = 3 yohimbine dimer, and tethered yohimbine analogs on human 2A-, 2B-, and 2C-AR subtypes that are stably expressed in CHO cells [3H]Rauwolscine was used as the radioligand in the equilibrium competition radioligand binding assays for the 2-ARs, and the nonspecific binding was measured in the presence of 10 µM phentolamine. pKi = –log Ki (Ki was calculated according to the Cheng-Prusoff equation). See Materials and Methods. The data are means ± S.E.M. of n = 4–6 experiments. Statistical analyses were carried out by one-way analysis of variance followed by Tukey's test (P < 0.05).

As described previously (Mustafa et al., 2005), the rank order of binding affinities exhibited by yohimbine (1) on the human ₂-AR subtypes was _{2C 2A} > _2B, with 2- and 7-fold higher binding affinities for the _2C- versus the _2A- and _2B-ARs. Interestingly, studies with yohimbinic acid (2) revealed that this compound exhibited a greatly decreased binding potency at the _2A-AR (345-fold) versus the _2B-AR (2-fold) and the _2C-AR (20-fold), compared with yohimbine. Furthermore, it was equipotent in binding to the _2B- and _2C-ARs, and its binding at the _2B-AR was 46-fold greater than its binding at the _2A-AR. Figure 2 shows the binding displacement curves for yohimbine (1) (A) and yohimbinic acid (2) (B) at the three ₂-AR receptor subtypes. The n = 3 dimeric analog (3) was 18- and 68-fold selective in binding to the _2C- versus _2A- and _2B-AR subtypes (Table 1). All of the tethered yohimbine analogs (4–10) exhibited significantly higher binding affinities at the _2C- versus _2A- and _2B-AR subtypes (Table 1). In particular, the benzyl carbamate alkyl amine (6), t-butyl carbamate alkyl amine (7), benzyl carboxy alkyl amine (9), and carboxy alkyl amine (10) analogs possessed binding affinities comparable with those of the parent molecule, yohimbine (1), at the _2C-AR (Table 1). The alkyl amine analog (8), however, was 32-fold less potent in binding to the _2C-AR compared with yohimbine (1). In addition, it was 129- and 316-fold less potent in binding to the _2A- and _2B-AR subtypes in comparison with yohimbine. The benzyl carbamate alkyl amine analog (6) and the t-butyl carbamate alkyl amine analog (7) were 11- and 59-fold and 3- and 33-fold selective in binding to the _2C- versus the _2A- and _2B-ARs, respectively (Table 1). The benzyl carboxy alkyl amine analog (9) and the carboxy alkyl amine analog (10) exhibited 43- and 1995-fold and 295- and 54-fold selectivities in binding to the _2C- versus _2A- and _2B-ARs, respectively. Figure 2, C and D, illustrates the binding displacement curves for the benzyl carboxy alkyl amine analog (9) and the carboxy alkyl amine analog (10) at the _2A-, _2B-, and _2C-AR subtypes, respectively. The alkyl amine analog (8) was 7- and 72-fold selective in binding to the _2C- versus the _2A- and _2B-ARs (Table 1).

View larger version (30K): [in this window] [in a new window]   Fig. 2. Binding displacement curves of (A) yohimbine, (B) yohimbinic acid, (C) yohimbine benzyl carboxy alkyl amine, and (D) yohimbine carboxy alkyl amine for human 2A- , 2B- , and 2C-ARs stably expressed in CHO cells. Plotted values are means ± S.E.M. (n = 4–6 experiments). Structures of compounds are shown in Fig. 1.

As has been observed with the yohimbine dimers (Lalchandani et al., 2002), all of the tethered yohimbine analogs, with the exception of the monoglycine ester (4) and the carboxy alkyl amine (10) analogs, displayed lower binding affinities at the _2B- versus the _2A- and _2C-ARs. The monoglycine ester analog (4) was equipotent in binding to the _2A- and _2B-ARs, whereas the carboxy alkyl amine analog (10) was 6-fold more potent in binding to the _2B- versus the _2A-AR (Table 1).

The binding affinities of yohimbine (1) and the two selected analogs, viz. the benzyl carbamate alkyl amine (6) and alkyl amine (8) analogs, were determined in HEK293 cells stably expressing homogeneous populations of human _1A-, _1B-, and _1D-ARs. Yohimbine and the selected analogs were found to bind with low affinities at all three ₁-AR subtypes (Table 2). The binding affinities of yohimbine (1), the benzyl carbamate alkyl amine (6), and the alkyl amine (8) analog for the _2C-AR subtype were at least 224-, 562-, and 100-fold greater than those on the ₁-AR subtypes, respectively (compare data in Tables 1 and 2). Taken collectively, the data confirm the binding selectivity of these ligands for the _2C-AR subtype.

View this table: [in this window] [in a new window]   TABLE 2 Binding affinities of yohimbine and selected tethered yohimbine analogs on human 1A-, 1B-, and 1D-AR subtypes that are stably expressed in HEK293 cells [3H]Prazosin was used as the radioligand in the equilibrium competition radioligand binding assays for the 1-ARs, and the nonspecific binding was measured in the presence of 10 µM phentolamine. pKi = –log Ki (Ki was calculated according to the Cheng-Prusoff equation). See Materials and Methods. The data are means ± S.E.M. of n = 4–6 experiments. Statistical analyses were carried out by one-way analysis of variance followed by Tukey's test (P < 0.05).

cAMP Response Element-Luciferase Reporter Gene Assay. The functional responses of yohimbine and selected tethered analogs in the human _2A- and _2C-ARs expressing CHO cells were determined using 6 CRE-LUC plasmid. The assays with both _2A- and _2C-ARs were conducted using the non-subtype-selective ₂-AR agonist, medetomidine, to block the cAMP changes induced by the adenylyl cyclase activator, forskolin. The concentration of forskolin (3–5 µM) for the assays was chosen such that it produced at least a 7- to 10-fold increase over basal levels. Basal values (solvent control) were subtracted from the forskolin values, and the resulting forskolin response was used as 100%. Likewise, basal values were subtracted from all other values obtained with ligands, and the data are expressed as a percentage luciferase response relative to that of forskolin alone.

Preliminary experiments with CHO cells stably expressing the _2A-ARs revealed a biphasic concentration-response curve with medetomidine (Fig. 3A). As shown, medetomidine showed inhibition of the forskolin-induced cAMP response at low concentrations (0.0001–0.01 µM), whereas higher concentrations (0.1–10 µM) reversed the inhibition of luciferase activity observed at the lower medetomidine concentrations. The maximal inhibition obtained for medetomidine was 56% at 0.01 µM. Medetomidine alone (in the absence of forskolin) increased cAMP levels by 17% when tested at a concentration of 10 µM (data not shown).

View larger version (14K): [in this window] [in a new window]   Fig. 3. Concentration-dependent effects of medetomidine on forskolin-induced cAMP elevations, as assessed by luciferase activity, on human (A) 2A- versus (B) 2C-ARs stably expressed in CHO cells. Plotted values are means ± S.E.M. (n = 4 experiments).

For functional studies with yohimbine and selected yohimbine analogs at the _2A- and _2C-ARs, the concentration of medetomidine was chosen such that it produced a submaximal (around 50–70%) inhibition of the forskolin response in these subtypes. From our data in the previous set of experiments (Fig. 3), the medetomidine concentration was fixed at 0.01 µM for the _2A-AR whereas it was fixed at 1 µM for the _2C-AR. The agonist ligand, medetomidine, was added directly to the medium 20 min before the addition of forskolin and then allowed to incubate for 4 h. Yohimbine analogs selected for functional testing at the ₂-ARs included the benzyl carbamate alkyl amine (6) and the alkyl amine (8) analogs. Concentrations of yohimbine (1), the benzyl carbamate alkyl amine (6), and the alkyl amine analog (8) were fixed at 0.1, 0.1, and 1 µM, respectively, for the _2A-AR assays whereas they were varied from 0.001 to 10 µM for the _2C-AR assays. These compounds were added 20 min before the addition of medetomidine.

On the _2A-AR, medetomidine (0.01 µM) inhibited forskolin-induced cAMP changes and the mean inhibition produced was 56 ± 2% for n = 5 experiments (Fig. 4). As noted in the graph, the mean percent inhibition produced after preincubation with yohimbine (1) (0.1 µM), the benzyl carbamate alkyl amine analog (6) (0.1 µM), and the alkyl amine analog (8) (1 µM) were 14.2, 54, and 53%, respectively. Thus, the changes in luciferase activity produced by medetomidine were blocked by yohimbine (1) (0.1 µM). Neither of the two analogs, however, blocked the medetomidine inhibition of forskolin-induced luciferase activity at the chosen concentrations. Controls of yohimbine and the two tethered yohimbine analogs were included for comparison (Fig. 4). In the presence of forskolin, none of the test antagonist ligands produced agonist activity at the concentrations tested (data not shown).

View larger version (12K): [in this window] [in a new window]   Fig. 4. Effects of yohimbine and selected tethered yohimbine analogs on medetomidine inhibition of forskolin-induced cAMP elevations, as assessed by luciferase activity, on human 2A-ARs stably expressed in CHO cells. Plotted values are means ± S.E.M. (n = 5 experiments). Structures of compounds are shown in Fig. 1. F, forskolin (5 µM); M, medetomidine (0.01 µM); Y, yohimbine (0.1 µM); BC, yohimbine benzyl carbamate alkyl amine analog (0.1 µM); AA, yohimbine alkyl amine analog (1 µM). *, P < 0.05 compared with F + M (1) using Student's t test.

A graphical representation of the concentration-dependent effects of yohimbine (1), the benzyl carbamate alkyl amine analog (6), and the alkyl amine analog (8), for the reversal of the forskolin-induced cAMP changes by medetomidine at the _2C-ARs is provided in Fig. 5, A, B, and C, respectively. As can be seen from these graphs, the mean percentage inhibition produced by medetomidine (1 µM) at the _2C-AR was 70 ± 2% for n = 4 experiments. Controls of yohimbine and the two tethered analogs were included for comparison (Fig. 5). In the presence of forskolin, none of the test antagonist ligands produced agonist activity at the highest concentration tested (data not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 5. Reversal of medetomidine inhibition of forskolin-induced cAMP elevations, as assessed by luciferase activity, by yohimbine (A), yohimbine benzyl carbamate alkyl amine analog (B), and yohimbine alkyl amine analog (C) on human 2C-ARs stably expressed in CHO cells. Plotted values are means ± S.E.M. (n = 4 experiments). Structures of compounds are shown in Fig. 1. F, forskolin (3 µM); M, medetomidine (1 µM); Y, yohimbine (0.001, 0.01, and 0.1 µM); BC, yohimbine benzyl carbamate alkyl amine analog (0.001, 0.01, and 0.1 µM); AA, yohimbine alkyl amine analog (0.1, 1, and 10 µM). *, P < 0.05 compared with F + M (1) using Student's t test.

View larger version (21K): [in this window] [in a new window]   Fig. 6. A comparison of the ability of yohimbine and selected tethered yohimbine analogs to reverse the medetomidine inhibition of forskolin-induced cAMP elevations, as assessed by luciferase activity, on human 2A- and 2C-ARs stably expressed in CHO cells. Plotted values are means ± S.E.M. (n = 4–5 experiments). Structures of compounds are shown in Fig. 1. F, forskolin (3–5 µM); M, medetomidine; Y, yohimbine (0.1 µM); BC, yohimbine benzyl carbamate alkyl amine analog (0.1 µM); AA, yohimbine alkyl amine analog (1 µM). The concentration of forskolin (3–5 µM) was chosen such that it produced at least a 7- to 10-fold increase over basal levels. The concentration of medetomidine was chosen such that it produced at least a 50% inhibition of the forskolin-induced cAMP response. The mean inhibition produced with the 2A-ARs was 56 ± 2% for n = 5 experiments, whereas at the 2C-ARs, the mean inhibition was 70 ± 2% for n = 4 experiments. *, response at the 2C-ARs is different from the response at 2A-ARs (P < 0.05 using Student's t test).

View this table: [in this window] [in a new window]   TABLE 3 IC50 values of yohimbine and selected tethered yohimbine analogs for reversing medetomidine effects on forskolin-induced cAMP elevations in CHO cells stably expressing the 2C-ARs IC50 and pIC50 values were calculated using GraphPad Prism and expressed as the means ± S.E.M. of n = 4 experiments. Values were determined as the effective concentration (IC50) and negative log (pIC50) of each compound that reversed the effect of medetomidine on the maximum cAMP response of forskolin.

	Discussion

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G protein-coupled receptors, traditionally considered to function as monomeric proteins, have now been proposed to exist as dimers or even higher-structure oligomers (Angers et al., 2002). The physiological significance of such dimerization and its implications in ligand pharmacology and, potentially, for drug design remain to be fully understood. However, there have been efforts to improve potency and/or subtype-selectivity by use of bivalent or dimeric ligands. Lalchandani et al. (2002) demonstrated that dimers of the potent and relatively nonselective ₂-AR antagonist yohimbine consisting of two pharmacophores linked through a spacer, exhibited a higher degree of ₂-AR subtype-selectivity than the parent molecule. The authors used the bivalent ligand approach, which is based on the concept that a bivalent ligand would first undergo univalent binding followed by binding of the second pharmacophore to a recognition site on a neighboring receptor. Thus, the bivalent ligand would exhibit a greater potency than that derived from the sum of its monovalent counterparts (Portoghese, 2001). In the yohimbine dimer study, the addition of methylene and methylene-diglycine spacer linkages produced analogs that were potent and selective _2C-AR ligands. It was proposed that one pharmacophore (or one yohimbine molecule) binds to the ligand receptor site, whereas the second pharmacophore may bind to either 1) an adjacent site of the ligand binding pocket, or transmembrane domain, or one of the extracellular loops on the same receptor or 2) a ligand-binding site or a recognition site on a neighboring receptor. With shorter spacer arms, however, an interaction of the second pharmacophore with an adjacent receptor molecule (dimer) seems less probable. Interestingly, none of the analogs in the yohimbine dimer series surpassed the affinity of the parent compound, yohimbine.

Recently, Mustafa et al. (2005) demonstrated that monomeric analogs of yohimbine, even in the absence of the second pharmacophore (or the second yohimbine molecule), with appendages or tethers of varying nature and composition retained high binding potencies, like that of the dimeric analogs, at the human _2C-AR. In fact, one of the tethered analogs, viz. the benzyl carboxy alkyl amine analog (9), displayed a greater binding affinity at the _2C-AR than the parent compound, yohimbine (Table 1). This result suggests that high binding affinities, previously observed with the dimeric ligands, may not be attributable to receptor dimerization and clustering, as proposed earlier (Lalchandani et al., 2002). The main focus of our study was to determine whether the second pharmacophore was essential for the _2C-AR selectivity of the yohimbine dimers; i.e., would truncating dimeric analogs to tethered analogs still retain the _2C-AR selectivity? For this purpose, we have extended the evaluation of the tethered yohimbine analogs (Mustafa et al., 2005) to all human ₂-AR subtypes. The importance of the charge of the tethers, in potency and/or subtype selectivity, was investigated using neutral, basic, and acidic functional groups.

In the yohimbine dimer series, the n = 3 and n = 24 dimers exhibited the greatest _2C-AR selectivity (Lalchandani et al., 2002). In functional assays, the n = 3 dimeric analog was found to be more potent as well as more _2C-AR selective than the n = 24 dimer. This finding, along with the idea that the n = 3 dimer would better fit the criteria for drug-like status (Lipinski et al., 1997) compared with the n = 24 dimer, led us to design the structure of the tethered analogs using the n = 3 yohimbine dimer as a standard. In our study, we have found the n = 3 dimer (3) to be 18- and 68-fold selective in binding to the _2C- versus the _2A- and _2B-ARs, respectively.

Our data for the parent compound, yohimbine (1) (Table 1; Fig. 2A), agree with those reported in the literature (Bylund et al., 1992; Lalchandani et al., 2002). We have also evaluated yohimbinic acid (2), a nontethered structural analog of yohimbine, for its binding to the ₂-ARs. Interestingly, there are no reports published in the literature on the interaction of yohimbinic acid with the ARs. Data from this study showed that a single structural change in the yohimbine molecule (changing the methyl ester at the C-16 carbonyl of yohimbine to an acid functionality), yielding yohimbinic acid, had a significant impact on its binding affinities at the ₂-AR subtypes. Yohimbinic acid exhibited greatly decreased binding potency at the _2A-AR (345-fold) versus the _2B-AR (2-fold) and the _2C-AR (20-fold), compared with yohimbine (Table 1; Fig. 2B). Furthermore, it was equipotent in binding to the _2B- and _2C-ARs, and its binding at the _2B-AR was 46-fold greater than its binding at the _2A-AR. It is noteworthy that yohimbinic acid is a selective _2C- versus _2A-AR ligand, with fold selectivity being 32-fold.

In the present study, as in the study reported previously (Lalchandani et al., 2000), the n = 3 dimer (3) did not surpass the binding affinity of yohimbine. In addition, although all of the tethered yohimbine analogs exhibited higher binding affinities at the _2C- versus _2A- and _2B-AR subtypes (Table 1), only one of the tethered analogs, the benzyl carboxy alkyl amine analog (9), exceeded the affinity of the parent compound, yohimbine (1) at the _2C-AR. The benzyl carbamate alkyl amine (6), the t-butyl carbamate alkyl amine (7) and the carboxy alkyl amine (10) analogs possessed binding affinities comparable with those of the parent molecule, yohimbine, at the _2C-AR. The alkyl amine analog (8) was 32-fold less potent in binding to the _2C-AR compared with yohimbine; however, it was 129- and 316-fold less potent in binding to the _2A- and _2B-AR subtypes in comparison with yohimbine (Table 1).

In regards to _2C-AR selectivities of the tethered analogs, the benzyl carbamate alkyl amine analog (6) and the alkyl amine analog (8) possessed affinities for the _2C-AR, which were 11- and 59-fold and 7- and 72-fold greater than their binding to the _2A- and _2B-ARs, respectively. The benzyl carboxy alkyl amine analog (9) and the carboxy alkyl amine analog (10) exhibited a 43- and 1995-fold and 295- and 54-fold selectivity in binding to the _2C- versus _2A- and _2B-ARs, respectively. Whether this enhanced _2C-AR selectivity observed with the benzyl carboxy alkyl amine analog and the carboxy alkyl amine analog represents additional interactions with basic residues in or around the ligand-binding pocket needs to be further investigated. Our binding results demonstrate that the tethered yohimbine analogs were more _2C-AR selective than the n = 3 dimer, suggesting that the second pharmacophore is not necessary to achieve the ₂-AR subtype selectivity with this chemical scaffold. We have also confirmed the functional antagonist activities of select compounds from this series at the _2A- and _2C-ARs using luciferase reporter gene assays (Figs. 3, 4, 5). Data from these assays also revealed that there is an _2C- versus _2A-AR selectivity for all of the tethered analogs tested (Fig. 6).

In conclusion, this study has yielded tethered analogs of yohimbine as selective and potent _2C-AR antagonists. This series of compounds may be interpreted as a novel series of yohimbine analogs with varying substitutions at the C-16 carbonyl position, yielding ligands with differing degrees of _2C-AR selectivity. The results also help to understand the underlying basis for the _2C-AR subtype-selectivity previously observed with the dimeric analogs and suggest the following: 1) The second pharmacophoric group, i.e., the second yohimbinic acid molecule, may not be essential for the _2C-AR subtype-selectivity previously seen with the dimeric analogs. We have shown that tethered yohimbine analogs, like dimers, exhibit _2C-AR selectivity. 2) Thus, an interaction of the second pharmacophoric group with either one of the extracellular loops on the same receptor or with a recognition site on a neighboring receptor, an adjacent site of the ligand-binding pocket on a single receptor, or an adjacent receptor molecule may not be the underlying mechanism(s) required for the observed synergy witnessed with the dimeric compounds, as proposed previously (Portoghese, 2001; Lalchandani et al., 2002). 3) Instead, we propose that differences in the physicochemical properties of the amino acid residues (e.g., steric or hydrogen bonding) constituting and surrounding the putative ligand-binding domain, are unique in each subtype, and this distinction can be exploited to develop receptor subtype selectivity. 4) Systematic alterations of the nature of the tether in a ligand on the yohimbine molecule scaffold may be crucial in maximizing unique ligand-receptor interactions in each ₂-AR subtype, and further optimization of the tether length and/or composition may possibly lead to the design of _2C-AR ligands that will be more potent than yohimbine. An additional advantage of using the monovalent ligand approach is that, compared with the dimeric compounds, the tethered analogs would have improved physicochemical and pharmacokinetic parameters (Lipinski et al., 1997).

	Acknowledgements

 
We thank the National Center for Natural Products Research and the United States Department of Agriculture at the University of Mississippi.

	Footnotes

 
This work was supported in part by U.S. Public Health Service Grant GM 29358 and U.S. Department of Agriculture ARS Agreement 58-6408-2-0009.

The work was part of the doctoral dissertation of Supriya A. Bavadekar at the University of Mississippi, University, MS.

The work has been previously presented, partially or entirely, at the following meetings: 1) Bavadekar SA, Ma G, Mustafa SM, Moore BM, Liggett SB, Miller DD, and Feller DR (2004) Tethered monomeric yohimbine analogs as selective human _2c-adrenergic receptor ligands, in Proceedings of the Southeastern Pharmacology Society Meeting; 2004 Nov 4–5; University, MI; 2) Mustafa SM, Bavadekar SA, Moore BM, Liggett SB, Feller DR, and Miller DD (2004) Synthesis and selectivity studies of yohimbine and its monomeric analogs on _2C-adrenergic receptors, in Proceedings of the 228th American Chemical Society National Meeting; 2004 Aug 22–26; Philadelphia; 3) Bavadekar SA, Suni MM, Moore BM, Liggett SB, Miller DD, and Feller DR (2004) Monomeric yohimbine analogs as selective human _2C-adrenergic receptor ligands, in Proceedings of Experimental Biology 2004; 2004 Apr 17–21; Washington DC.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.105981.

ABBREVIATIONS: AR, adrenoceptor; CHO, Chinese hamster ovary; HEK, human embryonic kidney; CRE-LUC, cAMP response element-luciferase.

Address correspondence to: Dr. Dennis R. Feller, 303 Faser Hall, Department of Pharmacology and National Center for Natural Products Research, School of Pharmacy, The University of Mississippi, University, MS 38677. E-mail: dfeller{at}olemiss.edu

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