Dennis R. Feller

Direct effects of ephedrine isomers on human β-adrenergic receptor subtypes

Ephedrine and its alkaloids are used for the treatment of asthma, nasal congestion, and obesity. Ephedrine, with two chiral centers, exists as four isomers that exhibit direct and indirect effects on both alpha- and beta-adrenergic receptors (AR). Our main goal was to study the direct effects of the ephedrine isomers on human beta1-, beta2-, and beta3-AR expressed in Chinese hamster ovary cells. Previous work indicated that the ephedrine isomers are inactive as agonists and that 1R,2S-ephedrine is more potent than the 1S,2R-isomer as an antagonist of catecholamine-induced lipolysis in rat adipose tissue (Lee et al., J Pharmacol Exp Ther 190: 249-259, 1974). Stimulation of adenylyl cyclase, associated with cyclic AMP accumulations, was measured by a luciferase reporter gene assay. On human beta1-AR, the rank order of potency (EC50 values, maximal response relative to isoproterenol = 100%) was 1R,2S-ephedrine (0.5 microM, 68%) > 1S,2R-ephedrine (72 microM, 66%) > 1S,2S-pseudoephedrine (309 microM, 53%) = 1R,2R-pseudoephedrine (1122 microM, 53%). On human beta2-AR, the rank order of potency was 1R,2S-ephedrine (0.36 microM, 78%) > 1R,2R-pseudoephedrine (7 microM, 50%) > or = 1S,2S-pseudoephedrine (10 microM, 47%) > 1S,2R-ephedrine (106 microM, 22%). Only 1R,2S-ephedrine showed significant agonist activity on human beta3-AR with an EC50 = 45 betaM and a maximal response of 31%. Our studies demonstrated that (a) stereoselective and rank order differences exist among the direct effects of ephedrine isomers; (b) 1R,2S-ephedrine is the most potent of the four ephedrine isomers on all three human beta-AR; and (c) 1R,2S- ephedrine was nearly equipotent as a beta1-/beta2-AR agonist and the only isomer possessing weak partial agonist activity on beta3-AR.

Evaluation of β-adrenergic receptor subtypes in the human prostate cancer cell line-LNCaP

Abstract The present study was undertaken to determine the effects of catecholamines, agonists, and antagonists of β-adrenergic receptors (AR) in the LNCaP cell line. Changes in cellular cyclic adenosine-3′,5′-monophosphate (cAMP) levels were quantified by the use of a 6 cAMP response element (CRE)-luciferase reporter gene assay. LNCaP cells were transiently transfected with this gene construct, incubated in 96-well microtiter plates for 24xa0hr, and then treated with β-AR agonists and/or antagonists for 4xa0hr. The rank order of potency for catecholamines and known β-AR agonists was terbutaline (3.31 nM )> isoproterenol (8.31 nM )≥ fenoterol (15 nM )= epinephrine (16.2 nM )> norepinephrine (77.5 nM )> BRL -37344 [( R ∗ , R ∗ )-(±)4-[2-[(2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxy acetic acid, sodium salt] (1000 nM )> dobutamine (1770 nM )> CGP 12177 (4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2 H -benzimidazole-2-one hydrochloride) (inactive). The non-selective β 1 -/-β 2 -AR antagonists; propranolol and CGP 12177, at 10 −7 xa0M, inhibited luciferase activity induced by these agonists by 80–96%. Propranolol blocked isoproterenol-induced luciferase responses in a competitive manner ( K B =1.4xa0nM). In addition, isoproterenol-activated luciferase expression was blocked more potently by ICI 118,551 [(±)-1-[2,3-(dihydro-7-methyl-1 H -inden-4-yl)oxy]-3-[(1-methylethy) amino]-2-butanol], a β 2 -AR antagonist than by ICI 89,406 [(±)- N -[2-[3-(2-cyanophenoxy-)]-2-hydroxypropylamino]ethyl- N -phenylurea], a β 1 -AR antagonist, giving K B values of 1.07 and 161xa0nM, respectively. These results suggest that the β 2 -AR is the major subtype mediating catecholamine-induced cAMP changes in LNCaP cells.

Yohimbine Dimers Exhibiting Selectivity for the Human α2c-Adrenoceptor Subtype

Yohimbine is a potent and selective α2- versus α1-adrenoceptor antagonist. To date, drugs with high specificity for the α2-adrenoceptor show marginal selectivity among the three α2-adrenoceptor subtypes. Initial studies showed that yohimbine was about 4- and 15-fold more selective for the human α2C-adrenoceptor in comparison with the α2A- and α2B-adrenoceptors, respectively. To improve on this α2-adrenoceptor subtype selectivity, a series of yohimbine dimers (varying fromn = 2 to 24 spacer atoms) were prepared and evaluated for receptor binding on human α2-adrenoceptor subtypes expressed in Chinese hamster ovary cells. Each dimeric analog showed higher affinities for α2A- and α2C-adrenoceptor versus the α2B-adrenoceptor; and yohimbine dimers with spacers ofn = 2, 3, 4, 18, and 24 exhibited selectivity for the α2C-adrenoceptor. The yohimbine dimersn = 3 and n = 24 showed the highest potency and selectivity (32- and 82-fold. respectively) for the α2C-adrenoceptor in receptor binding and in functional studies (42- and 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 α2C–adrenoceptor compared with the three α1-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 α2C-adrenoceptor.

Stereoselective effects of chiral clofibric acid analogs on rat peroxisome proliferator-activated receptor ? (rPPAR?) activation and peroxisomal fatty acid ?-oxidation

Enantiomers of a series of substituted analogs of 2-(4-chloronhenoxy)-acetic acid (CPAA) were synthesized and used to examine the influence of steric and structural parameters on peroxisome proliferation. The effects of these compounds were studied on the activation of the peroxisome proliferator-activated receptor α (PPARα) in CV-1 cells using an in vitro co-transfection assay. Selected sets of isomers were tested for their ability to increase peroxisomal fatty acyl-CoA oxidase (ACO) activity in H4IIEC3 (rat Reuber hepatoma) cells. Of the series of 2-substituted analogs studied, the isomers of the n-propyl and phenyl derivatives of CPAA showed a high degree of stereoselectivity [(S)-isomer ≫ (R)-isomer]. In general, the potency of the compound to activate the receptor increased with the size of the 2-alkyl substituent. Among the 4-chlorobenzyloxy- and 4-(4′-chlorophenyl)benzyloxy- analogs studied, 2-[4-(4′-chlorophenyl)-benzyloxy]-propanoic acid exhibited a high degree of stereoselectivity in both the biological systems studied [(R) ≫ (S)]. The congeners of 2-methyl substituted CPAA showed a reverse stereoselectivity [(R) > (S)] as compared to the other 2-substituted analogs [(S) > (R)]. Our results indicate that (1) both structural and steric characteristics of CPAA analogs play an important role in the activation of rPPARα and on stimulation of peroxisomal ACO activities, and (2) clofibric acid and analogs exert their peroxisome proliferative effects by interaction with a specific site on a protein. The enantiomers of the 2-n-propyl and the 2-phenyl CPAA analogs may be useful as mechanistic probes in elucidating the nature of this binding site. Chirality 9:37–47, 1997.

Yohimbine dimers exhibiting binding selectivities for human α2a- versus α2b-adrenergic receptors

Abstract A series of yohimbine dimers was prepared and evaluated at the human α2a- and α2b-adrenergic receptors (ARs) expressed in Chinese hamster ovary (CHO) cells. All dimers display higher binding selectivities for α2a versus α2b subtype than yohimbine, and four compounds ( 3d, 3e, 3g and 3i Download : Download high-res image (60KB) Download : Download full-size image Scheme 1 . ) represent the most potent and α2a- versus α2b-AR selective ligands identified so far.

AN EFFICIENT CYCLIC AMP ASSAY FOR THE FUNCTIONAL EVALUATION OF BETA -ADRENERGIC RECEPTOR LIGANDS

beta-Adrenoceptors are G-protein linked receptors that are positively coupled to adenylyl cyclase. We wanted to develop an alternative method to the detection of cAMP using radioimmunoassay for functional analysis of ligands for human beta-adrenoceptors (ARs). Chinese hamster ovary cells that stably expressed the beta 2-AR were transiently transfected with reporter plasmids containing the firefly luciferase gene under the transcriptional control of 6 or 12 cAMP response elements. The transiently transfected cells (electroporated at 150 volts, single pulse, 70 ms) were grown in 96-well microtiter plates (50,000 cells per well) for 20 hours, exposed to various ligands for 4 hours, and the luciferase activity was assayed. Stimulation of beta-ARs in these transfected cells resulted in a greater than 25-fold induction of luciferase activity. This activity was maximally increased in response to 20 microM forskolin, 1 mM 3-isobutyl-1-methylxanthine, and 10-30 nM (-)-isoproterenol. Exposure of cells to (-)-isoproterenol elicited a concentration dependent luciferase response and these effects of (-)-isoproterenol were blocked by propranolol (K(B) = 1.5 nM) and bupranolol (K(B) = 1.9 nM). The concentration of (-)-isoproterenol exhibiting half maximal response was 0.35 nM. This assay offers an excellent alternative to traditional methods of cAMP measurement and has applications to elucidate cAMP mediated signaling pathways in cells.

Tethered Yohimbine Analogs as Selective Human α2C-Adrenergic Receptor Ligands

Yohimbine is a potent and relatively nonselective α2-adrenergic receptor (AR) antagonist. In an earlier report, we demonstrated that dimeric yohimbine analogs containing methylene and methylene-diglycine tethers were highly selective human α2C-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 α2-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 α2-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 α2C- versus α2A- and α2B-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 α2C- versus α2A- and α2B-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 α2C-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 α2C-AR ligands. These compounds may be used as pharmacological probes and in the treatment of human disorders.

Bioisosteric phentolamine analogs as selective human α2- versus α1-adrenoceptor ligands☆

Phentolamine is known to act as a competitive, non-subtype-selective alpha-adrenoceptor antagonist. In an attempt to improve alpha(2)- versus alpha(1)-adrenoceptor selectivity and alpha(2)-adrenoceptor subtype-selectivity, two new chemical series of bioisosteric phentolamine analogs were prepared and evaluated. These compounds were evaluated for binding affinities on alpha(1)- (alpha(1A)-, alpha(1B)-, alpha(1D)-) and alpha(2)- (alpha(2A)-, alpha(2B)-, alpha(2C)-) adrenoceptor subtypes that had been stably expressed in human embryonic kidney and Chinese hamster ovary cell lines, respectively. Methylation of the phenolic hydroxy group and replacement of the 4-methyl group of phentolamine with varying lipophilic substituents yielded bioisosteric analogs selective for the alpha(2)- versus alpha(1)-adrenoceptors. Within the alpha(2)-adrenoceptors, these analogs bound with higher affinity at the alpha(2A)- and alpha(2C)-subtypes as compared to the alpha(2B)-subtype. In particular, the t-butyl analog was found to be the most selective, its binding at the alpha(2C)-adrenoceptor (Ki=3.6 nM) being 37- to 173-fold higher than that at the alpha(1)-adrenoceptors, and around 2- and 19-fold higher than at the alpha(2A)- and alpha(2B)-adrenoceptors, respectively. Data from luciferase reporter gene assays confirmed the functional antagonist activities of selected compounds from the bioisosteric series on human alpha(1A)- and alpha(2C)-adrenoceptors. Thus, the results with these bioisosteric analogs of phentolamine provide a lead to the rational design of potent and selective alpha(2)-adrenoceptor ligands that may be useful in improving the therapeutic profile of this drug class for human disorders.