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Research ArticleArticle

Homobivalent Conjugation Increases the Allosteric Effect of 9-aminoacridine at the α1-Adrenergic Receptors

Adrian P. Campbell, Laurence P. G. Wakelin, William A. Denny and Angela M. Finch
Molecular Pharmacology February 2017, 91 (2) 135-144; DOI: https://doi.org/10.1124/mol.116.105874

Abstract

The α1-adrenergic receptors are targets for a number of cardiovascular and central nervous system conditions, but the current drugs for these receptors lack specificity to be of optimal clinical value. Allosteric modulators offer an alternative mechanism of action to traditional α1-adrenergic ligands, yet there is little information describing this drug class at the α1-adrenergic receptors. We have identified a series of 9-aminoacridine compounds that demonstrate allosteric modulation of the α1A- and α1B-adrenergic receptors. The 9-aminoacridines increase the rate of [3H]prazosin dissociation from the α1A- and α1B-adrenergic receptors and noncompetitively inhibit receptor activation by the endogenous agonist norepinephrine. The structurally similar compound, tacrine, which is a known allosteric modulator of the muscarinic receptors, is also shown to be a modulator of the α1-adrenergic receptors, which suggests a general lack of selectivity for allosteric binding sites across aminergic G protein-coupled receptor. Conjugation of two 9-aminoacridine pharmacophores, using linkers of varying length, increases the potency and efficacy of the allosteric effects of this ligand, likely through optimization of bitopic engagement of the allosteric and orthosteric binding sites of the receptor. Such a bivalent approach may provide a mechanism for fine tuning the efficacy of allosteric compounds in future drug design efforts.

Introduction

Allosteric modulators offer a number of potential advantages over traditional, orthosterically acting drugs, including maintenance of spatiotemporal patterns of physiological signaling (Christopoulos, 2002). These advantages are of particular value in the central nervous system (CNS) where perturbation of G protein-coupled receptor (GPCR) signaling, or direct and sustained stimulation/inhibition of receptors frequently leads to limiting side effects (Conn et al., 2014). Therefore, allosteric modulators of GPCRs are of growing interest as drugs to target validated but otherwise intractable receptors, particularly in the CNS.

One of the main hurdles to the use of allosteric modulators remains their identification from drug screens. This, coupled with the general lack of structural information about allosteric pockets on GPCRs, means allosteric modulators remain relatively underrepresented as drugs for GPCRs. One of the few exceptions to this is the muscarinic acetylcholine receptor family, where crystal structures are publically available for four of the five subtypes (Kruse et al., 2012; Thorsen et al., 2014; Thal et al., 2016), one of which has an allosteric modulator bound (Kruse et al., 2013), and radiolabeled allosteric ligands are available (Tränkle et al., 2003; Schober et al., 2014). These serve as excellent models for fundamental properties of allosteric modulators, but many other GPCR families still lack defined allosteric sites or many examples of allosteric ligands.

One family of GPCRs with potential CNS as well as peripheral applications is the α1-adrenergic receptors. The α1-adrenergic receptors are three of the nine GPCRs that bind and respond to the neurohormones epinephrine and norepinephrine. The α1A-adrenergic receptor subtype has been validated as a potential target to treat seizure disorders arising in the CNS (Zuscik et al., 2001; Hillman et al., 2009; Pizzanelli et al., 2009) as well as for the treatment of heart failure (Lin et al., 2001; Du et al., 2006). However a common hurdle for the successful targeting of this receptor family is subtype selectivity (Chen and Minneman, 2005). Even clinically relevant drugs such as prazosin, silodosin, and naftopidil possess less than 100-fold selectivity for their target receptor over other α1-adrenergic receptor subtypes, as well as other biogenic amine receptors such as the serotonin 1A (5-hydroxytryptamine or 5-HT1A) receptor (Shibata et al., 1995; Takei et al., 1999; https://www.tga.gov.au/sites/default/files/auspar-duodart.pdf). Subtype selectivity is desirable as the α1A subtype, which has the most therapeutic potential, frequently exhibits opposing actions to the α1B subtype (Milano et al., 1994; Hillman et al., 2009).

Allosteric modulators have the potential to overcome many of these barriers, yet there are few examples of allosteric modulators for the α1-adrenergic receptor family (Waugh et al., 1999; Leppik et al., 2000; Pfaffendorf et al., 2000; Sharpe et al., 2003), and even less structural information regarding any allosteric sites (Ragnarsson et al., 2013, 2015). Here we have employed an innovative approach to maximize the allosteric effect observable for a potential allosteric ligand. We have used a series of homobivalent derivatives of 9-aminoacridine with increasing linker lengths (Fig. 1), which were previously identified as having unexpectedly high affinity for the α1-adrenergic receptors with some tissue-specific differences in binding affinity (Adams et al., 1985, 1986). The three subtypes of the α1-adrenergic receptors had not been described at that time, but the observed differences in affinity were attributed to different binding modes of the bis(9-aminoacridine) compounds depending on the length of the linker, and the potential existence of a second pocket, or cleft, on tissue-specific receptors. We hypothesize that the observed differences in binding affinity are attributable to subtype selective binding of the bis-(9-aminoacridine) compounds, and that the postulated “cleft” constitutes an allosteric site on the α1-adrenergic receptors. Additionally, we predicted that an optimally sized bivalent ligand will maximize occupancy of an allosteric site, increasing its observable effects.

Fig. 1.
Fig. 1.

Structures of 9-aminoacridine and derivatives. Structure of 9-aminoacridine, tacrine (tetrahydro-9-aminoacridine), and the general structure for bis(9-aminoacridine) in their protonated state.

Materials and Methods

Materials.

COS-1 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA). [3H]prazosin, [3H]myo-inositol, and Ultima Gold and Ultima Flo scintillation cocktails were purchased from Perkin Elmer (Waltham, MA). [3H]8-OH-DPAT was purchased from GE Healthcare (Uppsala, Sweden). Glass fiber filters (GF/C grade) were purchased from Whatman (Maidstone, United Kingdom). Dulbecco’s modified Eagle’s medium (DMEM) without inositol was purchased from MP Biomedicals (Santa Ana, CA). Fetal bovine serum was purchased from Invitrogen (Carlsbad, CA). (R)-epinephrine hydrochloride, (R)-norepinephrine hydrochloride, phentolamine hydrochloride, 5-methylurapidil, lithium chloride, (S)-propranolol hydrochloride, tacrine hydrochloride, diethylaminoethyl (DEAE) dextran, ammonium formate, chloroquine diphosphate, Tris, and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) were purchased from Sigma-Aldrich (St. Louis, MO). Bis(9-aminoacridine) compounds were synthesized as previously described by Deshpande and Singh (1972), and are referred to throughout by the designation Cn bis(9-aminoacridine), where n is the number of methylenes in the linker chain. EDTA, EGTA, MgCl2, NaCl, KCl, Na2HPO4, KH2PO4, and glycerol were purchased from Ajax Finechem (Taren Point, Australia). The transfection reagent jetPEI was purchased from PolyPlus-Transfection SA (Illkirch, France). AG 1-X8 formate resin was obtained from Bio-Rad Laboratories (Hercules, CA). Human α1A-adrenergic receptor, α1B-adrenergic receptor, and 5-HT1A receptor in pcDNA 3.1+ were purchased from Missouri S&T cDNA Resource Centre (www.cdna.org). Human α1D-adrenergic receptor in pMV6-XL5 vector was obtained from Origene (Rockville, MO).

Cell Culture.

COS-1 cells were maintained in DMEM supplemented with 10% (v/v) heat-treated fetal bovine serum and 100 μg.ml−1 glutamine. Cultures were kept at 37°C and 5% CO2 and passaged as they approached confluence by standard tissue culture techniques.

Transfection and Membrane Preparation.

COS-1 cells were seeded at a density of 4.5 × 106 cells per 175 cm2 flask and transfected 24 hours later using 20 µg DNA per 175 cm2 flask as per the DEAE dextran method of Lopata et al. (1984). At 48–72 hours after transfection, the cells were harvested by scraping into cold phosphate-buffered saline and pelleted by centrifugation at 500g for 5 minutes at 4°C. The pellet was resuspended in cold HE buffer (20 mM HEPES, 10 mM EDTA, pH 7.4) using 10 ml of buffer for each 175 cm2 plate scraped. Suspensions were homogenized on ice using an Ultra-Turax T125 homogenizer at 20,000 r.p.m. in three 10-second bursts. Lysate was centrifuged at 600g for 10 minutes at 4°C. The supernatant was then centrifuged at 40,000g for 1 hour at 4°C. The final pellet was resuspended in cold 20 mM HEPES (pH 7.4) + 10% glycerol (v/v) using 200 μl per 175 cm2 flask then homogenized on ice using an insulin syringe, aliquoted, and stored at −80°C. The protein concentration was measured using Bradford reagent.

Transfection and Inositol Phosphate Accumulation.

Resuspended COS-1 cells were diluted to a concentration of 1 × 105 cells.ml−1 DMEM and transfected with 1 μg construct and 2 μl jetPEI transfection reagent per 1 × 105 cells as per the manufacturer’s instructions. Cells were plated into 96-well plates (220 μl per well) and left at room temperature for 1 hour after transfection to minimize uneven plating in the outmost wells (Lundholt et al., 2003), then transferred to a 37°C, 5% CO2 incubator. At 16–24 hours after transfection, receptor activation was determined using total soluble inositol phosphate (IP) production as described elsewhere (Campbell et al., 2014) in the presence of 10 µM propranolol to inhibit any potential signaling from β-adrenergic receptors.

Binding Assays.

All radioligand binding assays were performed at room temperature in duplicate. The α1-adrenergic receptor-expressing membranes were incubated in HEM buffer (20 mM HEPES, 1.4 mM EGTA, 12.5 mM MgCl2, pH 7.4), unless otherwise stated, using 1–2 μg protein per reaction for α1A- and α1B-adrenergic receptors and 10–15 μg protein per reaction for the α1D-adrenergic receptor. The 5-HT1A receptor-expressing membranes were incubated in HC buffer (20 mM HEPES, 4 mM CaCl2, pH 7.4) using 10 μg of protein per reaction. Reactions were terminated by the addition of 4°C phosphate-buffered saline and vacuum filtration through Whatman GF/C glass fiber filters.

Saturation Binding.

Saturation binding assays were performed in a final volume of 200 μl and allowed to incubate for 1 hour. α1-adrenergic receptor-expressing membranes were incubated in HEM buffer with 0.03–8 nM [3H]prazosin. Nonspecific binding was determined using 100 μM phentolamine. The 5-HT1A receptor-expressing membranes were incubated in HC buffer with 0.1–12 nM [3H]8-OH-DPAT. Nonspecific binding was determined using 10 μM serotonin.

Competition Binding.

Competition binding assays were performed in a final volume of 200 μl and allowed to incubate for 1 hour in the presence of an approximate KD concentration of radioligand. α1-adrenergic receptor affinity was determined in HEM buffer in competition with 250 pM [3H]prazosin. The ligand binding affinity for 5-HT1A receptors was determined in HC buffer in competition with 1 nM [3H]8-OH-DPAT.

Dissociation Binding Kinetics.

Dissociation kinetics assays were performed in HEM buffer over 80 minutes for the α1A-adrenergic receptor, and in TE buffer (50 mM Tris, 0.5 mM EDTA, pH 7.4) over 160 minutes for the α1B-adrenergic receptor. The receptors were pre-equilibrated with 250 pM [3H]prazosin for 1 hour at room temperature in a volume of 400 μl. Reassociation of [3H]prazosin was then inhibited by the addition of 100 μl of phentolamine for a final concentration of 100 μM in the absence or presence of the test compounds.

Data Analysis.

All binding data were analyzed in GraphPad Prism 6 (GraphPad Software, San Diego, CA) by nonlinear regression using standard one-site curves for saturation and dissociation kinetics assays. Competition binding assays were fit by a variable-slope competition binding model (Hill, 1910; Cheng and Prusoff, 1973). All values were compared by one-way analysis of variance with Tukey post-test. The IP accumulation data were analyzed in GraphPad Prism 6 by nonlinear regression using a variable slope, four-parameter fit. The inhibitor affinity was estimated by generating a double reciprocal plot of equi-effective concentrations of agonist in the absence and presence of inhibitor at a concentration that produces an approximate 50% decrease in the maximum signal (Ehlert, 1988; Kenakin, 1997).

Results

Equilibrium Binding of bis(9-aminoacridine) Compounds.

To address whether the tissue-specific differences in binding affinity originally observed for the bis(9-aminoacridine) compounds is attributable to subtype selectivity, their apparent affinities were determined via coincubation with radioligands for membrane-expressed human α1-adrenergic receptors as well as the 5-HT1A receptor to assess for “off-target” binding.

The α1A- and α1B-adrenergic receptors expressed at similar levels (Bmax: α1A 2.3 ± 0.2, α1B 2.4 ± 0.2 pmol.mg−1) while the α1D-adrenergic receptors and 5-HT1A receptor expressed at lower levels (Bmax: α1D 0.3 ± 0.1, 5-HT1A 0.2 ± 0.1 pmol.mg−1). Radioligands bound with normal affinity ([3H]prazosin KD: α1A 1.1 ± 0.1, α1B 0.7 ± 0.1, and α1D 0.9 ± 0.2 nM; [3H]8-OH-DPAT KD 2.2 ± 0.1 nM), and all assays were titrated so that specific binding of radioligands constituted less than 10% of free ligand.

Known competitive ligands of the α1-adrenergic receptors and 5-HT1A receptor were found to display affinities comparable to previously reported values (Table 1) (Zhao et al., 1996; Newman-Tancredi et al., 1998). We found that 9-aminoacridine, the parent monomer of the bis(9-aminoacridine) compounds, has an affinity of 247 nM for the α1A-adrenergic receptor, is 10-fold selective over the α1B-adrenergic receptor (Ki: 2.6 µM, P < 0.01), 8-fold selective over the α1D-adrenergic receptor (Ki: 1.9 µM, P < 0.001), and >400-fold selective over the 5-HT1A receptor (Ki: > 100 µM) (Fig. 2; Table 1).

View this table:
TABLE 1

Equilibrium binding affinities

All data are presented as mean ± S.E.M. for n repeats, performed in duplicate.

Fig. 2.
Fig. 2.

Subtype selective binding of the bis(9-aminoacridine) compounds. Binding affinities of the bis(9-aminoacridine) compounds at all three α1-adrenergic receptors and 5-HT1A serotonin receptor. All binding curves were fit by a variable-slope competitive binding model. Affinities were compared by one-way analysis of variance with Tukey post-test. a, b, d, s: P < 0.05 compared with α1A-, α1B-, α1D-adrenergic receptors (AR), and 5-HT1A serotonin receptor, respectively. Affinity is not shown where logKi is greater than −4.

To investigate whether the affinity of 9-aminoacridine depends on it being fully aromatic, the affinity of its tetra-hydro derivative, tacrine, was also measured (Fig. 1). Tacrine displays affinities of 1.8, 19.4, and 6.4 μM at the α1A-, α1B-, and α1D-adrenergic receptors, respectively (Fig. 2; Table 1). Thus, tacrine binding is consistently weaker than 9-aminoacridine at the α1A and α1B-adrenergic receptors (P < 0.01) but still maintains selectivity for α1A over α1B (P < 0.05).

In general, the bis(9-aminoacridine) compounds studied show low- to sub-micromolar affinity for all three α1-adrenergic receptor subtypes (Fig. 2; Table 1). At the α1B- and α1D-adrenergic receptors the bis(9-aminoacridine) compounds have similar or greater affinity than 9-aminoacridine for all linker lengths tested, their affinity-chain length relationship producing a shallow, concave profile with maximum affinity occurring around the C5, C6, and C7 homologs (Fig. 2; Table 1). In contrast, at the α1A-adrenergic receptor this profile is more pronounced, particularly for bis(9-aminoacridine) compounds with linkers between two and seven carbons (Fig. 2; Table 1). Whereas the bis(9-aminoacridine) compounds display only 5- and 7-fold differences between the highest and lowest affinity compounds at the α1B- and α1D-adrenergic receptors, at the α1A-adrenergic receptor there is a 91-fold difference between the highest and lowest affinity compounds, C4 and C2 bis(9-aminoacridine), respectively.

At the α1A-adrenergic receptor C2 has an affinity of 1.9 μM, which is significantly lower than that of 9-aminoacridine (P < 0.01); the affinity of C4, at 21 nM, is significantly higher than that of 9-aminoacridine as well as that of C2, C3, and all other bis(9-aminoacridine) compounds with linkers longer than six carbons (P < 0.05). The affinity of C4 bis(9-aminoacridine) at the α1A-adrenergic receptor is the highest observed for any of the bis(9-aminoacridine) compounds at all the tested receptors (Fig. 2), the ligand being 10-fold selective over the α1B subtype (P < 0.001), 30-fold selective over the α1D-adrenergic receptor (P < 0.001), and 145-fold selective over the 5-HT1A receptor (P < 0.001).

All bis(9-aminoacridine) compounds with a linker length of five carbons or fewer have a significantly higher affinity for the three α1-adrenergic receptor subtypes compared with the 5-HT1A receptor. Affinities of the shorter bis(9-aminoacridine) compounds C2 and C3 as well as the monovalent 9-aminoacridines for the 5-HT1A receptor are some of the lowest observed in the test set, resulting in substantial selectivity for the α1-adrenergic receptor subtypes.

Close observation reveals that the slopes of the competitive binding curves for some of the bis(9-aminoacridine) compounds at the α1-adrenergic receptors appear steeper than that generated by the competitive antagonist phentolamine (Table 2). The Hill coefficients of phentolamine binding do not differ significantly from unity for any α1-adrenergic receptor, nor for serotonin binding at the 5-HT1A receptor (Table 2). There is a similar finding for the monomers 9-aminoacridine and tacrine (Table 2). In contrast, the Hill slope for C10 bis(9-aminoacridine) is significantly greater than 1 at the α1A- and α1B-adrenergic receptors (P < 0.01, P < 0.05, respectively). Likewise, C3 and C4 have Hill slopes significantly greater than 1 at the α1B-adrenergic receptor (P < 0.05), as do C7 and C9 at both the α1A- and α1B-adrenergic receptors (P = 0.05–0.07).

View this table:
TABLE 2

Equilibrium binding assay slopes

Hill Slope coefficient of competitive binding data fit by a variable-slope competitive binding model. All data are expressed as mean ± S.E.M. for n repeats. P values between 0.05 and 0.1 are reported in superscript.

The competition binding curves, in general, appear to increase in steepness as the linker increases in length, until it reaches nine carbons, with significant correlation between linker length and Hill slope (P < 0.05) seen at the α1A- (r2 = 0.8) and α1B- (r2 = 0.7) adrenergic receptors. The magnitude of the Hill coefficient tends to a value of 2 for homologs from C6 to C12 for the α1-adrenergic receptors, a feature not noted for binding to the 5-HT1A receptor (Table 2). Hill coefficients greater than 1 imply the existence of multiple ligand binding sites on the receptor, and may implicate the involvement of cooperative modes of binding (Prinz, 2010).

One possibility here is that cooperativity arises from interaction with an allosteric site via bitopic binding of the bis(9-aminoacridine) compounds at the α1-adrenergic receptors. Accordingly, we sought experimental evidence for such a mechanism in the form of bis(9-aminoacridine) modulation of the rate of dissociation of [3H]prazosin from the α1-adrenergic receptors.

Dissociation Kinetics for [3H]Prazosin Binding to the α1-Adrenergic Receptors.

Given that the α1A subtype is the most validated therapeutic α1-adrenergic receptor target (Perez and Doze, 2011) and that the α1B subtype is a common source of off-target activity (Carruthers, 1994; Cavalli et al., 1997), the modulatory effects of the bis(9-aminoacridine) compounds on prazosin dissociation rates from these two receptors were characterized. In preliminary experiments to identify appropriate experimental conditions, we found that measurements in HEM buffer yielded a half-life of 14 minutes for the dissociation of [3H]prazosin from the α1A-adrenergic receptor: a value suitable for detecting both positive and negative allosteric modulation. However, dissociation from the α1B-adrenergic receptor in this buffer was slow with <50% dissociation observed after 2 hours (data not shown). This difficulty was overcome by the use of TE buffer for this receptor, as described by Sato et al. (2012), in which the half-life was 33 minutes. Accordingly, HEM buffer was used for kinetic experiments with the α1A-adrenergic receptor, and TE buffer for experiments with the α1B-adrenergic receptor.

Bis(9-aminoacridine) Compounds Increase the Dissociation Rate of [3H]Prazosin.

The dissociation rate of [3H]prazosin was measured in the absence and presence of the bis(9-aminoacridine) compounds 9-aminoacridine and tacrine at 100 μM, this being the highest concentration possible, limited by bis(9-aminoacridine) solubility. At 100 µM, 9-aminoacridine causes a 3.2-fold increase in dissociation rate at the α1A-adrenergic receptor and a 5.5-fold increase in dissociation rate at the α1B-adrenergic receptor (Table 3). Tacrine shows a similar, albeit less efficacious, profile resulting in a 2.1-fold increase in dissociation rate from the α1A-adrenergic receptor and a 4.2-fold increase from the α1B-adrenergic receptor (Table 3). With the exception of C2 and C3, the bis(9-aminoacridine) compounds significantly increase the dissociation rate of [3H]prazosin from the α1A-adrenergic receptor (P < 0.05) in a linker-length dependent manner (r2 = 0.86, P < 0.001) (Table 3). There is a similar finding for the α1B-adrenergic receptor where all the bis(9-aminoacridine) compounds increase the dissociation rate of [3H]prazosin (P < 0.001) in a manner dependent on chain length (r2 = 0.74, P < 0.001) (Table 3).

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TABLE 3

Modulation of dissociation rates of [3H]prazosin from the α1A- and α1B-adrenergic receptors

Values are given as mean ± S.E.M., n = 3–6.

The C2-to-C9 bis(9-aminoacridine) compounds all produce a greater proportional increase in dissociation rate at the α1B-adrenergic receptor (P < 0.01), while C12 gives the greatest observed effect at the α1A-adrenergic receptor (Table 3). However, the use of different buffers in the dissociation kinetics assays for each of the subtypes makes it difficult to compare potency between the receptor subtypes as affinity and efficacy of allosteric ligands are known to change in different buffers (Ellis and Seidenberg, 2000; Schröter et al., 2000).

To investigate whether the potency of the bis(9-aminoacridine) compounds to modulate the dissociation rate of [3H]prazosin from the adrenergic receptors is correlated with their observed binding affinity, we chose the α1A-selective C4 bis(9-aminoacridine), the nonselective C9 bis(9-aminoacridine), and the monomer 9-aminoacridine for a more thorough characterization. Tacrine was again included to investigate whether planarity of the molecule is a necessary feature for modulation.

The increase in [3H]prazosin dissociation rate caused by the monovalent ligands does not seem to be saturating at 100 µM (Fig. 3), and their allosteric effects appear at concentrations higher than their apparent affinities derived from competition binding data. For example, 9-aminoacridine has an affinity of 247 nM at the α1A-adrenergic receptor, yet allosteric properties only emerge at a minimum concentration of 30 µM. C4 bis(9-aminoacridine) causes a maximum 3.5-fold increase in dissociation rate at the α1A-adrenergic receptor but does not cause an observable increase in rate at 10 μM, well above its apparent affinity of 21 nM for this receptor. In contrast, it causes a maximum 9.0-fold increase in dissociation at the α1B-adrenergic receptor that appears to be close to saturating, producing an estimated EC50(diss) of 22 µM (Fig. 3), a concentration ∼100-fold higher than its apparent affinity. C9 bis(9-aminoacridine) causes a 17.6-fold increase in dissociation rate from the α1A-adrenergic receptor at 100 μM (P < 0.001) and a 62.3-fold increase in dissociation rate from the α1B-adrenergic receptor (P < 0.001). This produces EC50(diss) estimates of 32 and 6 µM for the α1A- and α1B-adrenergic receptors, respectively (Fig. 3).

Fig. 3.
Fig. 3.

Kinetics of dissociation of [3H]prazosin from α1-adrenergic receptors. [3H]prazosin dissociation from α1A-adrenergic receptor membranes (A–H) and α1B-adrenergic receptor membranes (I–P) in the absence and presence of 9-aminoacridine (A, I), C4 bis(9-aminoacridine) (B, J), C9 bis(9-aminoacridine) (C, K), and tacrine (D, L). Membranes were preincubated with 250 pM [3H]prazosin for 1 hour at room temperature in 400 µl of HEM buffer. Reassociation of [3H]prazosin was then inhibited by the addition of 100 µl of phentolamine with or without the test compound at the concentrations shown. Observed dissociation rates (Kobs) were normalized to the control dissociation rate (Koff) measured in the absence of modulator and were plotted against the log concentration of modulator (E-H, M-P). Points and error bars are the mean ± S.E.M. of three individual experiments performed in duplicate.

Despite C4 bis(9-aminoacridine) having higher affinity than C9 at the α1A-adrenergic receptor, this was not translated into increased modulation potency. C9 bis(9-aminoacridine) consistently causes greater increases in [3H]prazosin dissociation, at lower concentrations, than C4 bis(9-aminoacridine) (Fig. 3), implying that these two properties are dictated by ligand binding to distinct sites on the receptor, which have differing affinity for the acridine dimers.

C9 bis(9-aminoacridine) Is a Noncompetitive Antagonist of α1A-Adrenergic Receptor Activation.

To test whether the bis(9-aminoacridine) compounds are also able to perturb activation and signaling of the receptor, we tested the activation of the α1A-adrenergic receptor by norepinephrine in the absence and presence of C9 bis(9-aminoacridine). C9 was chosen as it is one of the most potent and efficacious modulators of [3H]prazosin dissociation identified in this study. We found it causes a significant reduction in the potency of norepinephrine at the α1A-adrenergic receptor at concentrations of 10 μM and above (P < 0.05), as well as a decrease in the maximum observed response at concentrations of 30 μM and above (P < 0.001) (Fig. 4; Table 4). We found that 100 µM C9 also causes a small but significant increase in receptor signaling via the inositol trisphosphate pathway in the absence of norepinephrine (Table 4).

Fig. 4.
Fig. 4.

C9 bis(9-aminoacridine) modulation of norepinephrine-induced total soluble inositol phosphate accumulation. COS-1 cells were transiently transfected with the α1A-adrenergic receptor then incubated overnight in the presence of [3H]myo-inositol. After 24 hours of incubation, the medium was exchanged for serum-free DMEM in the presence or absence of C9 bis(9-aminoacridine) (C9) for 20 minutes followed by stimulation with norepinephrine for 30 minutes. Unstimulated points are shown in the left segment of the x-axis. All values were normalized between unstimulated response and maximum norepinephrine-induced response and fit by nonlinear regression to a variable-slope dose-response curve using GraphPad Prism 6. Points and bars represent the mean ± S.E.M. of n = 3–5 experiments performed in triplicate.

View this table:
TABLE 4

C9-dependent changes in norepinephrine-dependent and -independent activation of the α1A-adrenergic receptor

The IP accumulation data do not fit well to an operational model of allosterism (Leach et al., 2007), generating unrealistic or poor fit values: for instance, R2 < 0.5, β < 0, or α > 1012. This is possibly due to a bitopic, rather than purely allosteric binding mode of C9 bis(9-aminoacridine). Instead, the method of Ehlert (1988) was applied to the IP accumulation data to estimate the affinity of C9. We found that 30 μM C9 produces an approximately 50% decrease in the maximum signal (Fig. 4), making it the most ideal concentration for use with this method (Kenakin, 1997).

Generating a double reciprocal plot of equi-effective concentrations of agonist in the absence and presence of C9 bis(9-aminoacridine) yields an affinity estimate of 19.0 ± 4.9 μM, which is 71-fold higher than the apparent affinity observed for C9 at the α1A-adrenergic receptor in equilibrium binding experiments. Substituting this value for KB in the operational model does not further improve the fit. Taking all the available data together, the monovalent and homobivalent 9-aminoacridines appear to display a combination of competitive and noncompetitive properties consistent with ligands capable of binding to the functionally distinct orthosteric and allosteric binding pockets.

Discussion

The bis(9-aminoacridine) compounds were chosen for use in this study for their potential to engage a second site of the α1-adrenergic receptors. It was hypothesized that the tissue-specific differences in the binding affinity of the bis(9-aminoacridine) compounds observed between rat brain and kidney (Adams et al., 1986) could be explained by subtype-selective binding of the ligands that reflects the tissue-specific distribution of the subtypes. Protein levels of α1-adrenergic receptors, like GPCRs in general, are difficult to quantify by Western blot (Jensen et al., 2009), but mRNA transcript levels in the rat suggest that the predominant α1-adrenergic receptors found in the kidney are the α1A and α1B subtypes (Rokosh et al., 1994). All the bis(9-aminoacridine) compounds with linker lengths up to five carbons and 9-aminoacridine display the highest affinity at either the α1A or α1B subtypes, corresponding to the higher affinity displayed by these compounds for kidney tissue compared with cerebral cortex (Adams et al., 1986), which suggests that their length is suitable to make critical interactions for selective binding.

In the rat brain, mRNA suggests that the predominant subtype is the α1D-adrenergic receptor (Rokosh et al., 1994). Similar to the binding profile seen for the α1D-adrenergic receptor, binding affinities of the bis(9-aminoacridine) compounds in the rat cortex are consistently lower than in the kidney for all linker lengths less than six carbons. Affinities in the two tissues converge for linkers of 9–10 carbons, as do the affinities at the three human α1-adrenergic receptors, which shows general consensus with the original observations made in mixed receptor tissue preparations.

In addition to competitive binding, the acridines also demonstrate allosteric effects at the α1-adrenergic receptors, supporting the prediction that the 9-aminoacridines are able to interact with a second site. These allosteric effects occur at free ligand concentrations well above the apparent affinity of the 9-aminoacridines, indicating a relatively low affinity interaction with the allosteric site. The lack of any observable binding cooperativity for the monovalent ligands is also consistent with a low-affinity, allosteric interaction; the orthosteric site would be saturated before any significant allosteric effect can be observed.

Cooperative binding may only emerge for the bivalent ligands as a result of one acridine moiety being tethered to the higher-affinity orthosteric site, creating a high local concentration of ligand around the allosteric pocket and increasing the likelihood of an allosteric binding event. The Hill slopes most different from 1 at the α1-adrenergic receptors occur around the 9-carbon linker, suggesting that this length is optimal for promoting concomitant engagement of both the orthosteric and allosteric sites.

An interesting observation is that the potency and magnitude of the allosteric effect of the bis(9-aminoacridine) compounds correlates better with linker length than apparent affinity—namely, C4 bis(9-aminoacridine) is not the most potent modulator despite displaying the highest affinity. This implies that the allosteric interactions are associated with a binding site that is distinct from the site that is imparting high-affinity binding interactions. For the α1B subtype, the allosteric effect appears to peak at the 9-carbon length, further reinforcing this as the optimal linker length for bitopic binding at the α1-adrenergic receptors. This supports our prediction that an optimally sized bivalent ligand will promote engagement of an allosteric site to exaggerate the allosteric effects of a ligand with otherwise poor efficacy or potency. Interestingly, 9 carbons was also found to be the optimum length to promote bitopic engagement of the reciprocally bivalent compound THRX-198321 (biphenyl-2-yl-carbamic acid 1-{9-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydro-quinolin-5-yl)-ethylamino]-nonyl}-piperidin-4-yl ester), a dual β2-adrenergic receptor agonist and muscarinic receptor antagonist (Steinfeld et al., 2011), perhaps suggesting a conserved distance between orthosteric and allosteric pockets in the biogenic amine receptor family.

One caveat to our model of bitopic binding for the bis(9-aminoacridine) compounds is that it may require the existence of dimers, or higher order oligomers, of the α1-adrenergic receptors. A bitopic model best satisfies the observed pharmacology of the bis(9-aminoacridine) compounds; however, in the experimental kinetics paradigm, the orthosteric site of the receptors would already be occupied by the probe ligand [3H]prazosin, precluding any potential for tethering. However, using a [3H]prazosin concentration close to its KD, resulting in 50% receptor occupancy, may leave dimer partners unoccupied and available for bitopic interaction with the bis(9-aminoacridine) compounds, as would an asymmetrical binding profile. It has been demonstrated that the bitopic D2 dopamine receptor ligand, SB269652 (N-((trans)-4-(2-(7-cyano-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)cyclohexyl)-1H-indole-2-carboxamide) binds to one receptor and negatively modulates a dimer partner (Lane et al., 2014). D2 receptors have been demonstrated to form oligomers in native tissue (Zawarynski et al., 1998), and α1-adrenergic receptors have been demonstrated to form dimers in transfected cells (Vicentic et al., 2002). Thus, this concept is not unprecedented, and we would suggest that the bis(9-aminoacridine) compounds are acting in a similar fashion.

We have demonstrated that a single pharmacophore can have actions at two distinct receptor binding sites. One of the most significant implications of this observed pharmacology for the 9-aminoacridines is that the orthosteric and allosteric sites of the α1-adrenergic receptors, and potentially many other GPCRs, share some degree of similarity. In addition, monovalent tacrine, tacrine dimers, and methoctramine also show similar effects at muscarinic receptors, with mixed orthosteric-allosteric actions and the homobivalent ligands are also suggested to bind in a bitopic manner (Pearce and Potter, 1988; Tränkle et al., 2005; Jakubík et al., 2014). This observation would be in line with current understanding of ligand binding mechanisms. Recent structural biology efforts to crystallize GPCRs have yielded a structure of the M2 muscarinic receptor with a bound positive allosteric modulator, LY2119620 (3-amino-5-chloro-N-cyclopropyl-4-methyl-6-[2-(4-methylpiperazin-1-yl)-2-oxoethoxy]thieno[2,3-b]pyridine-2-carboxamide) (Kruse et al., 2013). This structure showed the allosteric modulator bound to an allosteric pocket composed of residues from transmembrane (TM) helices TMV, TMVI, TMVII, and the second extracellular loop, which have previously been shown to constitute the primary allosteric site for the muscarinic receptors (Prilla et al., 2006; Valant et al., 2012). This site on the M2 muscarinic receptor also corresponds to the extracellular vestibule of the β2-adrenergic receptor, which is a low-affinity pocket identified in molecular dynamics simulations where orthosteric ligands are postulated to bind transiently before progressing to their final pose in the orthosteric pocket (Dror et al., 2011). The molecular dynamics analysis of the β2-adrenergic receptor predicted the affinity of alprenolol to be 5 µM at the binding vestibule compared with 0.9 nM affinity observed in competitive binding assays (Baker, 2005; Dror et al., 2011). In a similar pattern, the affinity of 9-aminoacridine determined from equilibrium binding was 247 nM compared with the predicted allosteric affinity from the signaling assay of 19 µM, and the effective concentration required to increase [3H]prazosin dissociation rate also appears to be in the middle to high micromolar range.

“Pure” orthosteric and allosteric ligands may then simply be ligands with significant selectivity for one site over the other, and the differences in 9-aminoacridine affinity between the two sites (∼100-fold) is not substantial enough to give a single binding profile. It is possible that some effects of allosteric modulators may be attributable to interaction with the orthosteric site—for instance, “allosteric agonists” may be allosteric ligands with additional, hitherto unappreciated weak orthosteric agonist activity. Such a multipocket mechanism of action has recently been described for some ligands of the FFA2 free fatty acid receptor (Grundmann et al., 2016). The two identified pockets are considerably more superficial on the receptor surface, are in closer proximity, and have more similar affinities than is expected for the aminergic receptors, but nonetheless demonstrate that a single ligand can have distinct actions at topographically distinct sites on a single receptor.

The observed effects of tacrine as well as the other 9-aminoacridines at the α1-adrenergic receptors present an interesting conundrum. One of the purported advantages of allosteric ligands is the ability to bind to nonconserved sites of receptors, imparting increased binding selectivity (Christopoulos, 2002). However we have demonstrated that tacrine, an allosteric ligand of the muscarinic receptors (Pearce and Potter, 1988; Potter et al., 1989; Tränkle et al., 2005), is also an allosteric ligand of the α1-adrenergic receptors, in addition to its orthosteric activity at the two receptor families. There may therefore be conservation of allosteric sites in related receptor families, which would also explain why there appears to be a conserved distance between the orthosteric and allosteric sites in α1- and β2-adrenergic receptors as well as muscarinic receptors.

One notable difference between the α1-adrenergic and muscarinic receptors, however, is that tacrine acts as a positive modulator of ligand binding at the muscarinic receptors (Croy et al., 2014), while it is a negative modulator of ligand binding at the α1-adrenergic receptors. The structural basis of positive modulation of the M2 muscarinic receptor appears to be a “capping” effect where LY2119620 binds to the M2 receptor in a location that directly hinders the egress of the ligand from the orthosteric binding pocket (Kruse et al., 2013). Tacrine’s actions as a negative modulator of the α1-adrenergic receptors would imply a different mechanism whereby the binding of tacrine induces a structural change in the orthosteric site that is less favorable for ligand binding. We believe the 9-aminoacridine compounds are more likely to mimic the binding mode of the D2 dopamine receptor negative allosteric modulator SB269652, where the allosteric binding pocket is located closer to TMII than TMVII (Lane et al., 2014).

While the allosteric site of the α1-adrenergic receptors remains to be fully characterized, it is clear from the observed pharmacology that the 9-aminoacridines are interacting with more than one site on the α1-adrenergic receptors. Furthermore, by increasing or decreasing the distance between orthosteric and allosteric moieties using an appropriate linker, the magnitude of an allosteric effect can be controlled, imposing a level of control over the size of the allosteric component. The bivalent nature of the bis(9-aminoacridine) compounds and the linker-length-dependent changes in allosteric modulation will aid in identifying the location of this allosteric pocket, and such an approach may facilitate a new approach to drug design and discovery for the α1-adrenergic receptors or GPCRs in general.

Acknowledgments

The authors thank Dr. Trevor Lewis for his valuable assistance in implementing custom models in GraphPad Prism.

Authorship Contributions

Participated in research design: Campbell, Wakelin, Denny, Finch.

Conducted experiments: Campbell.

Performed data analysis: Campbell, Finch.

Wrote or contributed to the writing of the manuscript: Campbell, Wakelin, Denny, Finch.

Footnotes

Abbreviations

CNS
central nervous system
DEAE
diethylaminoethyl
DMEM
Dulbecco's modified Eagle’s medium
GPCR
G protein-coupled receptor
HEPES
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
5-HT
serotonin, 5-hydroxytryptamine
IP
inositol phosphate
LY2119620, 3-amino-5-chloro-N-cyclopropyl-4-methyl-6-[2-(4-methylpiperazin-1-yl)-2-oxoethoxy]thieno[2
3-b]pyridine-2-carboxamide
SB269652
N-((trans)-4-(2-(7-cyano-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)cyclohexyl)-1H-indole-2-carboxamide
THRX-198321
biphenyl-2-yl-carbamic acid 1-{9-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydro-quinolin-5-yl)-ethylamino]-nonyl}-piperidin-4-yl ester
TM
transmembrane

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Allosteric Modulators of the α1 Adrenergic Receptors

Adrian P. Campbell, Laurence P. G. Wakelin, William A. Denny and Angela M. Finch
Molecular Pharmacology February 1, 2017, 91 (2) 135-144; DOI: https://doi.org/10.1124/mol.116.105874
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