Adrenergic Receptor Ligands

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 2Cversus 2Aand 2B-AR subtypes. Notably, the benzyl carboxy alkyl amine and the carboxy alkyl amine analogs exhibited 43and 1995-fold and 295and 54-fold selectivities in binding to the 2Cversus 2Aand 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 structureactivity relationship to obtain potent and selective 2C-AR ligands. These compounds may be used as pharmacological probes and in the treatment of human disorders. Efforts made toward understanding the biological significance of each of the 2-adrenergic receptor (AR) subtypes ( 2A, 2B, and 2C) (Bylund et al., 1998) have resulted only in marginal success because of the lack of subtype-selective ligands. In recent years, this endeavor has been greatly assisted by genetic manipulation using mice with deletions, mutations, or overexpression of specific 2-AR subtypes. The role of the 2C-AR, in addition to the 2A-AR, in the feedback control of neurotransmitter release is a finding from one such study (Hein et al., 1999). Contribution of the 2C-ARs to 2-AR opioid synergy induced by certain agonists such as moxonidine is another finding (Fairbanks et al., 2002), suggesting that the 2C-AR may represent a better therapeutic target for analgesic therapy than the 2A-AR, since use of this subtype would also lead to fewer sedative effects. Peterhoff et al. (2003) used knockout mice to report that the 2Aand 2C-ARs mediate epinephrine-induced inhibition of insulin secretion in pancreatic islet cells. In the central nervous system, the 2C-ARs seem to have a distinct inhibitory role in various central nervous system-mediated behavioral and physiological responses including startle reactivity, aggressive behavior, and amphetamine-induced locomotor hyperactivity (Scheinin et al., 2001). Thus, increased 2C-AR activity may lead to or result from a constitutively stressful state, 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 elementluciferase. 0022-3565/06/3192-739–748$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 319, No. 2 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 105981/3144005 JPET 319:739–748, 2006 Printed in U.S.A. 739 at A PE T Jornals on N ovem er 3, 2017 jpet.asjournals.org D ow nladed from thereby causing depression, which suggests that 2C-AR subtype-selective drugs may be useful in a variety of neuropsychiatric disorders (Scheinin et al., 2001). Besides these findings derived from gene-targeted mice, a recent study (Chotani et al., 2000) has provided yet another potential therapeutic use for an 2C-AR antagonist. The study showed that at lower temperatures, the 2C-ARs are principally responsible for mediating the cold-induced augmented vasoconstrictor response. This subtype, however, did not contribute to 2-AR-dependent vasoconstriction at 37°C. A selective inhibition of the 2C-ARs in microvessels has, thus, been proposed to provide an effective treatment for cold-induced cutaneous arterial blood vessel constriction as observed in Raynaud’s phenomenon. 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 2CAR. 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 2CARs. 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 2Aand 2CARs using a cAMP response element-luciferase (CRE-LUC) reporter gene assay. Materials and Methods 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. [H]Rauwolscine and [H]prazosin were obtained from PerkinElmer Life and Analytical Sciences (Boston, MA), and all other chemicals were obtained from Sigma-Aldrich