Characterization of the Allosteric Interactions between Antagonists and Amiloride Analogues at the Human a2A-Adrenergic Receptor

The purpose of this study was to determine whether there is a well-defined allosteric site on the human a2A-adrenergic receptor. To explore this question, we examined the effects of amiloride analogues on the dissociation of [H]yohimbine, [H]rauwolscine, and [H]RX821002. The dissociation data fitted well to an equation derived from the ternary complex allosteric model with amiloride analogue concentration and time as two independent variables. The estimated maximal increase in the [H]yohimbine dissociation rate caused by the 5-N-alkyl amilorides varied from 2-fold for the parent amiloride to 140and 160-fold for 5-(N,N-hexamethylene)-amiloride and 5-(N-ethylN-isopropyl)-amiloride, respectively. The calculated log affinities at the yohimbine-occupied receptor ranged from 1.75 for 5-(N-ethyl-N-isopropyl)-amiloride to 2.5 for 5-(N,N-hexamethylene)-amiloride. The increase in affinity found at the yohimbine-occupied receptor was not correlated with increase in size of the 5-N-alkyl side chain, in contrast to the situation found at the unoccupied receptor. The effect of competition between two amilorides on yohimbine dissociation also was explored. The data obtained were well fitted by the equation derived from the relevant model, with the off-rate increases caused by 5-(N,N-hexamethylene)-amiloride being either decreased or increased by the competing amiloride analogue in line with predictions, and the parameters derived from the fits were in good agreement with those obtained in the above dissociation assays. Thus, the data are compatible with the amilorides competing at the one allosteric site on the a2A-adrenergic receptor and rules out the possibility that the amilorides are acting in a nonspecific fashion. Within many subtypes of G protein-coupled receptors, molecular modeling studies predict that there is a high degree of amino acid identity around the primary neurotransmitter or hormone binding site (Trumpp-Kallmeyer et al., 1992). Thus, the development of drugs with subtype selectivity can be a daunting task, and within the muscarinic receptor field, for example, it has proved to be very difficult to design antagonists selective for one subtype over the other subtypes and so far impossible to discover subtype-selective agonists (Caulfield and Birdsall, 1998; Tucek and Proska, 1995). Within the adrenergic field, a limited number of compounds are known with 10–100-fold higher affinity for certain subtypes (Bylund et al., 1994). However, also in this field, there remains a need for the development of further and more subtype-selective drugs (Ruffolo et al., 1995). It thus would be desirable to have an alternative target for the development of such drugs, and such an alternative could be an allosteric site. The potential usefulness of such a site for drug design is emphasized by the success of the benzodiazepines, which act at an allosteric site on the g-aminobutyric acidA receptors, enhancing the response to g-aminobutyric acid (Barker et al., 1986). A schematic representation of allosterism is shown in Fig. 1. In this model, the binding of an allosteric agent X to the allosteric site modulates the binding of a ligand L at the primary binding site. Depending on the magnitude and direction of the changes in the equilibrium and kinetic binding constants, the affinity of L at the primary site will either increase (a . 1, positive cooperativity), decrease (a , 1, negative cooperativity), or remain unchanged (a 5 1, neutral cooperativity). This opens the possibility for the modulation of the action of the natural agonist of a receptor in a subtypespecific manner if, for example, the agonist activity is enhanced at one subtype (positive cooperativity) but inhibited at other subtypes (negative cooperativity) (Lazareno and Birdsall, 1995). This work was supported by a ROPA Research Grant (R.A.L., A.M.). ABBREVIATIONS: DMA, 5-(N,N-dimethyl)-amiloride; CHO, Chinese hamster ovary; BZA, benzamil; EPA, 5-(N-ethyl-N-isopropyl)-amiloride; HMA, 5-(N,N-hexamethylene)-amiloride; MBA, 5-(N-methyl-N-isobutyl)-amiloride; DMF, N,N-dimethylformamide; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. 0026-895X/98/050916-10$3.00/0 Copyright © by The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 53:916–925 (1998). 916 at A PE T Jornals on M ay 3, 2017 m oharm .aspeurnals.org D ow nladed from Allosteric interactions have been best characterized with the muscarinic family (Stockton et al., 1983; Lee and ElFakahany, 1991a; Leppik et al., 1994; Lazareno and Birdsall, 1995; Tucek and Proska, 1995), and it is of interest to note that many of the somewhat subtype-selective compounds discovered to date have also been reported to act at the allosteric site. These include the m2 selective compounds gallamine (Stockton et al., 1983), himbacine (Lee and ElFakahany, 1990; Matsui et al., 1995), and methoctramine (Lee and El-Fakahany, 1991b; Matsui et al., 1995), thus highlighting the potential value of the allosteric site in generating subtype selectivity. In regard to the enhancement of agonist activity, it has recently been found that the alkaloid brucine allosterically enhances the action of acetylcholine at only the m1 muscarinic receptor subtype, whereas N-chloromethyl-brucine allosterically enhances acetylcholine action at only the m3 muscarinic receptor subtype (Birdsall et al.,