Molecular Pharmacology Fast Forward. Published on November 30, 2016 as DOI: 10.1124/mol.116.105874 This article has not been copyedited and formatted. The final version may differ from this version.
MOL #105874
Title Page
Homobivalent conjugation increases the allosteric effect of 9-aminoacridine at the α1 adrenergic receptors
Names and addresses of authors
Adrian P. Campbell, Laurence P.G. Wakelin, William A. Denny, Angela M. Finch Downloaded from Department of Pharmacology, School of Medical Sciences, UNSW Australia, Kensington,
NSW, 2052, Australia APC, LPGW, AMF, Auckland Cancer Society Research Centre, Faculty of
Medicine and Health Science, University of Auckland, 1142, New Zealand WAD molpharm.aspetjournals.org
at ASPET Journals on September 30, 2021
1 Molecular Pharmacology Fast Forward. Published on November 30, 2016 as DOI: 10.1124/mol.116.105874 This article has not been copyedited and formatted. The final version may differ from this version.
MOL #105874
Running Title Page
Running Title: Allosteric modulators of the α1 adrenergic receptors.
Corresponding author:
Dr Angela M Finch
Department of Pharmacology, School of Medical Sciences, UNSW Australia, Kensington,
NSW, 2052, Australia
Email: [email protected] Downloaded from
P: +61 (02) 9385 1325
molpharm.aspetjournals.org Number of text pages: 33
Number of tables: 4
Number of figures: 4
Number of references: 60 at ASPET Journals on September 30, 2021 Number of words in the Abstract: 199
Number of words in the Introduction: 622
Number of words in the Discussion: 1646
Abbreviations:
AR, adrenergic receptor; CNS, central nervous system; GPCR, G protein-coupled receptor; NE, norepinephrine; TM, transmembrane
2 Molecular Pharmacology Fast Forward. Published on November 30, 2016 as DOI: 10.1124/mol.116.105874 This article has not been copyedited and formatted. The final version may differ from this version.
MOL #105874
Abstract
The α1 adrenergic receptors are targets for a number of cardiovascular and CNS conditions, however 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- Downloaded from
3 aminoacridines increase the rate of [ H]prazosin dissociation from the α1A and α1B adrenergic receptors and non-competitively inhibit receptor activation by the endogenous agonist, molpharm.aspetjournals.org 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 GPCRs. Conjugation of two 9-aminoacridine pharmacophores, using linkers of at ASPET Journals on September 30, 2021 varying length, increases the potency and efficacy of the allosteric effects of this ligand, likely through optimisation 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.
3 Molecular Pharmacology Fast Forward. Published on November 30, 2016 as DOI: 10.1124/mol.116.105874 This article has not been copyedited and formatted. The final version may differ from this version.
MOL #105874
Introduction
Allosteric modulators offer a number of potential advantages over traditional, orthosterically acting drugs including maintenance of spatio-temporal patterns of physiological signalling (Christopoulos, 2002). These advantages are of particular value in the central nervous system (CNS) where perturbation of G protein-coupled receptor (GPCR) signalling, 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 Downloaded from growing interest as drugs to target validated, but otherwise intractable receptors, particularly in the CNS. molpharm.aspetjournals.org 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 at ASPET Journals on September 30, 2021 receptor family, where crystal structures are publically available for 4 of the 5 subtypes
(Kruse et al., 2012; Thal et al., 2016; Thorsen et al., 2014), one of which has an allosteric modulator bound (Kruse et al., 2013), and radiolabelled allosteric ligands are available
(Schober et al., 2014; Tränkle et al., 2003). 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 are 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
(Hillman et al., 2009; Pizzanelli et al., 2009; Zuscik et al., 2001), as well as for the treatment of heart failure (Du et al., 2006; Lin et al., 2001). However a common hurdle for the
4 Molecular Pharmacology Fast Forward. Published on November 30, 2016 as DOI: 10.1124/mol.116.105874 This article has not been copyedited and formatted. The final version may differ from this version.
MOL #105874 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 5-HT1A serotonin receptor
(GlaxoSmithKline, 2011; Shibata et al., 1995; Takei et al., 1999). Subtype selectivity is desirable as the α1A subtype which has the most therapeutic potential frequently exhibits
opposing actions to the α1B subtype (Hillman et al., 2009; Milano et al., 1994). Downloaded from
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 (Leppik et al., molpharm.aspetjournals.org 2000; Pfaffendorf et al., 2000; Sharpe et al., 2003; Waugh et al., 1999), and even less structural information regarding any allosteric site/s (Ragnarsson et al., 2015; Ragnarsson et al., 2013). Here we have employed an innovative approach to maximise the allosteric effect observable for a potential allosteric ligand. We have used a series of homobivalent at ASPET Journals on September 30, 2021 derivatives of 9-aminoacridine with increasing linker lengths (Figure 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., 1986; Adams et al.,
1985). 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)s depending on the length of the linker, and the potential existence of a second pocket, or cleft, on tissue specific receptors. We hypothesise that the observed differences in binding affinity are attributable to subtype selective binding of the bis-(9- aminoacridine)s, and that the postulated “cleft” constitutes an allosteric site on the α1 adrenergic receptors. Additionally, we predicted that an optimally sized bivalent ligand will maximise occupancy of an allosteric site, increasing its observable effects.
5 Molecular Pharmacology Fast Forward. Published on November 30, 2016 as DOI: 10.1124/mol.116.105874 This article has not been copyedited and formatted. The final version may differ from this version.
MOL #105874
Materials and Methods
Materials
COS-1 cells were purchased from ATCC (Virginia, USA). [3H]prazosin, [3H]myo- inositol, and Ultima Gold and Ultima Flo scintillation cocktails were purchased from Perkin
Elmer (Waltham, MA, USA). [3H]8-OH-DPAT was purchased from GE Healthcare
(Uppsala, Sweden). GF/C glass fibre filters were purchased from Whatman (Maidstone, UK). Downloaded from DMEM without inositol was purchased from MP Biomedicals (Santa Ana, CA, USA). FBS was purchased from Invitrogen (Carlsbad, CA, USA). (R)-epinephrine hydrochloride, (R)- norepinephrine hydrochloride, phentolamine hydrochloride, 5-methylurapidil, lithium molpharm.aspetjournals.org chloride, (S)-propranolol hydrochloride, tacrine hydrochloride, DEAE dextran, ammonium formate, chloroquine diphosphate, Tris and HEPES were purchased from Sigma Aldrich (St.
Louis, MO, USA). Bis(9-aminoacridine)s were synthesised as previously described
(Deshpande and Singh, 1972), and are referred to throughout by the designation Cn bis(9- at ASPET Journals on September 30, 2021 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, NSW, Australia). jetPEI was purchased from PolyPlus-transfection SA
(Illkirch, France). AG 1-X8 formate resin was from Bio-Rad (Hercules, CA, USA). Human
α1A adrenergic receptor, α1B adrenergic receptor and 5-HT1A receptor in pcDNA 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, USA).
Cell culture
COS-1 cells were maintained in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% (v/v) heat-treated FBS and 100 μg.mL-1 glutamine. Cultures were kept at 37°C and 5% CO2 and passaged as they approached confluence by standard tissue
6 Molecular Pharmacology Fast Forward. Published on November 30, 2016 as DOI: 10.1124/mol.116.105874 This article has not been copyedited and formatted. The final version may differ from this version.
MOL #105874 culture techniques.
Transfection and membrane preparation
Cells were seeded at a density of 4.5 x 106 cells per 175 cm2 flask and transfected 24 h later using 20 µg DNA per 175 cm2 flask as per the DEAE dextran method of Lopata et al.
(1984). 48-72 h after transfection, cells were harvested by scraping into cold PBS and pelleted by centrifugation at 500 x g for 5 min at 4°C. The pellet was resuspended in cold HE Downloaded from buffer (20 mM HEPES, 10 mM EDTA, pH 7.4) using 10 mL of buffer for each 175 cm2 plate scraped. Suspensions were homogenised on ice using an Ultra-Turax T125 homogeniser at
20,000 r.p.m. in three 10 s bursts. Lysate was centrifuged at 600 x g for 10 min at 4°C. molpharm.aspetjournals.org
Supernatant was then centrifuged at 40 000 x g for 1 h 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 homogenised on ice using an insulin syringe, aliquoted and stored at -80°C. Protein
concentration was measured using Bradford reagent. at ASPET Journals on September 30, 2021
Transfection and IP Accumulation
Resuspended COS-1 cells were diluted to a concentration of 1 x 105 cells.mL-1 DMEM and transfected with 1 μg construct and 2 μL jetPEI transfection reagent per 1 x 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 h following transfection to minimise uneven plating in the outmost wells (Lundholt et al., 2003), then transferred to a 37°C, 5% CO2 incubator. 16-24 h post-transfection, receptor activation was determined using total soluble inositol phosphate
(IP) production as previously described (Campbell et al., 2014) in the presence of 10 µM propranolol to inhibit any potential signalling from β adrenergic receptors.
Binding assays
All radioligand binding assays were performed at room temperature in duplicate. α1
7 Molecular Pharmacology Fast Forward. Published on November 30, 2016 as DOI: 10.1124/mol.116.105874 This article has not been copyedited and formatted. The final version may differ from this version.
MOL #105874 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. 5-HT1A receptor-expressing membranes were incubated in HC buffer (20 mM HEPES, 4 mM CaCl2, pH 7.4) using 10 μg protein per reaction. Reactions were terminated by the addition of 4°C PBS and vacuum filtration through Whatman GF/C glass
fibre filters. Downloaded from
Saturation binding
Saturation binding assays were performed in a final volume of 200 μL and allowed to molpharm.aspetjournals.org incubate for 1 h. α1 adrenergic receptor-expressing membranes were incubated in HEM buffer with 0.03 – 8 nM [3H]prazosin. Non-specific binding was determined using 100 μM phentolamine. 5-HT1A receptor-expressing membranes were incubated in HC buffer with 0.1
3 μ – 12 nM [ H]8-OH-DPAT. Non-specific binding was determined using 10 M serotonin. at ASPET Journals on September 30, 2021
Competition binding
Competition binding assays were performed in a final volume of 200 μL and allowed to incubate for 1 h in the presence of an approximate KD concentration of radioligand. α1 adrenergic receptor affinity was determined in HEM buffer in competition with 250 pM
3 [ H]prazosin. 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 min for the α1A adrenergic receptor, and TE buffer (50 mM Tris, 0.5 mM EDTA, pH 7.4) over 160 min for
3 the α1B adrenergic receptor. Receptors were pre-equilibrated with 250 pM [ H]prazosin for 1 h at room temperature in a volume of 400 μL. [3H]prazosin reassociation was then inhibited
8 Molecular Pharmacology Fast Forward. Published on November 30, 2016 as DOI: 10.1124/mol.116.105874 This article has not been copyedited and formatted. The final version may differ from this version.
MOL #105874 by the addition of 100 μL phentolamine for a final concentration of 100 μM in the absence or presence of test compounds.
Data analysis
All binding data was analysed in GraphPad Prism 6 by non-linear regression using standard one-site curves for saturation and dissociation kinetics assays. Competition binding assays were fit by a variable-slope competition binding model (Cheng and Prusoff, 1973; Downloaded from Hill, 1910). All values were compared by one-way ANOVA with Tukey post-test. IP accumulation data was analysed in GraphPad Prism 6 by non-linear regression using a variable slope, four-parameter fit. Inhibitor affinity was estimated by generating a double molpharm.aspetjournals.org 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). at ASPET Journals on September 30, 2021
9 Molecular Pharmacology Fast Forward. Published on November 30, 2016 as DOI: 10.1124/mol.116.105874 This article has not been copyedited and formatted. The final version may differ from this version.
MOL #105874
Results
Equilibrium binding of bis(9-aminoacridine)s
To address whether the tissue-specific differences in binding affinity originally observed for the bis(9-aminoacridine)s 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” Downloaded from 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
-1 at lower levels (Bmax: α1D 0.3 ± 0.1, 5-HT1A 0.2 ± 0.1 pmol.mg ). Radioligands bound with molpharm.aspetjournals.org
3 3 normal affinity ([ H]prazosin KD: α1A 1.1 ± 0.1, α1B 0.7 ± 0.1 and α1D 0.9 ± 0.2 nM; [ H]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 at ASPET Journals on September 30, 2021 previously reported values (Table 1) (Newman-Tancredi et al., 1998; Zhao et al., 1996). 9- aminoacridine, the parent monomer of the bis(9-aminoacridine)s, 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 greater than 400-fold selective over the 5-HT1A receptor (Ki: >100 µM) (Table 1, Figure 2).
To investigate whether the affinity of 9-aminoacridine is dependent upon it being fully aromatic, the affinity of its tetra-hydro derivative, tacrine, was also measured (Figure 1).
Tacrine displays affinities of 1.8, 19.4, and 6.4 μM at the α1A, α1B, and α1D adrenergic receptors, respectively (Table 1, Figure 2). Thus tacrine binding is consistently weaker than
9-aminoacridine at the α1A and α1B adrenergic receptors (P < 0.01), but it still maintains selectivity for α1A over α1B (P < 0.05).
In general, the bis(9-aminoacridine)s studied show low- to sub-micromolar affinity for
10 Molecular Pharmacology Fast Forward. Published on November 30, 2016 as DOI: 10.1124/mol.116.105874 This article has not been copyedited and formatted. The final version may differ from this version.
MOL #105874
all three α1 adrenergic receptor subtypes (Table 1, Figure 2). At the α1B and α1D adrenergic receptors the bis(9-aminoacridine)s 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 homologues (Table 1,
Figure 2). In contrast, at the α1A adrenergic receptor this profile is more pronounced, particularly for bis(9-aminoacridine)s with linkers between 2 and 7 carbons (Table 1, Figure
2). Whereas the bis(9-aminoacridine)s display only 5- and 7-fold differences between the Downloaded from 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 molpharm.aspetjournals.org 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), while 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)s with linkers longer than 6 at ASPET Journals on September 30, 2021 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)s at all of the tested receptors (Figure
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)s with a linker length of five carbons or fewer have significantly higher affinity for the three α1 adrenergic receptor subtypes than the 5-HT1A receptor. Affinities of the shorter bis(9-aminoacridine)s, 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)s at the α1 adrenergic receptors appear steeper than that generated by the competitive antagonist phentolamine (Table 2). The Hill coefficients of phentolamine
11 Molecular Pharmacology Fast Forward. Published on November 30, 2016 as DOI: 10.1124/mol.116.105874 This article has not been copyedited and formatted. The final version may differ from this version.
MOL #105874
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). The competition binding curves, in general, appear to increase in steepness as Downloaded from the linker increases in length, until it reaches 9 carbons, with significant correlation between
2 2 linker length and Hill slope (P < 0.05) seen at the α1A (r = 0.8) and α1B (r = 0.7) adrenergic molpharm.aspetjournals.org receptors. The magnitude of the Hill coefficient tends to a value of 2 for homologues 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, at ASPET Journals on September 30, 2021 2010). One possibility here, is that cooperativity arises from interaction with an allosteric site via bitopic binding of the bis(9-aminoacridine)s at the α1 adrenergic receptors. Accordingly, we sought experimental evidence for such a mechanism in the form of bis(9-aminoacridine)
3 modulation of the rate of dissociation of [ H]prazosin from the α1 adrenergic receptors.
3 Dissociation kinetics for [ H]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)s on prazosin dissociation rates from these two receptors were characterised.
In preliminary experiments to identify appropriate experimental conditions, we found that measurements in HEM buffer yielded a half-life of 14 min for the dissociation of
3 [ H]prazosin from the α1A adrenergic receptor: a value suitable for detecting both positive and
12 Molecular Pharmacology Fast Forward. Published on November 30, 2016 as DOI: 10.1124/mol.116.105874 This article has not been copyedited and formatted. The final version may differ from this version.
MOL #105874
negative allosteric modulation. However, dissociation from the α1B adrenergic receptor in this buffer was slow with less than 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 min. 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)s increase the dissociation rate of [3H]prazosin Downloaded from
The dissociation rate of [3H]prazosin was measured in the absence and presence of the bis(9-aminoacridine)s, 9-aminoacridine, and tacrine at 100 μM, this being the highest molpharm.aspetjournals.org 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 at ASPET Journals on September 30, 2021 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)s significantly increase
3 the dissociation rate of [ H]prazosin from the α1A adrenergic receptor (P < 0.05) in a linker-
2 length dependent manner (r =0.86, P<0.001) (Table 3). There is a similar finding for the α1B adrenergic receptor where all the bis(9-aminoacridine)s 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).
The C2-to-C9 bis(9-aminoacridine)s 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; Schroter et al., 2000).
13 Molecular Pharmacology Fast Forward. Published on November 30, 2016 as DOI: 10.1124/mol.116.105874 This article has not been copyedited and formatted. The final version may differ from this version.
MOL #105874
To investigate whether the potency of the bis(9-aminoacridine)s to modulate the dissociation rate of [3H]prazosin from the adrenergic receptors is correlated with their observed binding affinity, the α1A-selective C4 bis(9-aminoacridine), the non-selective C9 bis(9- aminoacridine), as well as the monomer 9-aminoacridine were chosen for a more thorough characterisation. 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 (Figure 3), and their Downloaded from allosteric effects appear at concentrations higher than their apparent affinities derived from competition binding data. 9-aminoacridine has an affinity of 247 nM at the α1A adrenergic molpharm.aspetjournals.org 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 at ASPET Journals on September 30, 2021 increase in dissociation at the α1B adrenergic receptor that appears to be close to saturating, producing an estimated EC50(diss) of 22 µM (Figure 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 (Figure 3). 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) (Figure 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 non-competitive antagonist of α1A adrenergic receptor
14 Molecular Pharmacology Fast Forward. Published on November 30, 2016 as DOI: 10.1124/mol.116.105874 This article has not been copyedited and formatted. The final version may differ from this version.
MOL #105874 activation
To test whether the bis(9-aminoacridine)s are also able to perturb activation and signalling of the receptor, activation of the α1A adrenergic receptor by norepinephrine was tested 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 Downloaded from the maximum observed response at concentrations of 30 μM and above (P<0.001) (Figure 4,
Table 4). 100 µM C9 also causes a small but significant increase in receptor signalling via the molpharm.aspetjournals.org
IP3 pathway in the absence of norepinephrine (Table 4). The IP accumulation data do not fit well to an operational model of allosterism (Leach et al., 2007), generating unrealistic or poor fit values e.g. 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 at ASPET Journals on September 30, 2021 applied to the IP accumulation data to estimate affinity of C9. 30 μM C9 produces an approximate 50% decrease in the maximum signal (Figure 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 non-competitive properties consistent with ligands capable of binding to the functionally distinct orthosteric and allosteric binding pockets.
15 Molecular Pharmacology Fast Forward. Published on November 30, 2016 as DOI: 10.1124/mol.116.105874 This article has not been copyedited and formatted. The final version may differ from this version.
MOL #105874
Discussion
The bis(9-aminoacridine)s were chosen for use in this study for their potential to engage a second site of the α1 adrenergic receptors. It was hypothesised that the tissue- specific differences in the binding affinity of the bis(9-aminoacridine)s 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 Downloaded from et al., 2009), however, 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). molpharm.aspetjournals.org 9-aminocridine and all bis(9-aminoacridine)s with linker lengths up to 5 carbons display the highest affinity at either the α1A or α1B subtypes, corresponding to the higher affinity displayed by these compounds for kidney tissue compared to cerebral cortex (Adams et al.,
1986), suggesting that their length is suitable to make critical interactions for selective at ASPET Journals on September 30, 2021 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)s in the rat cortex are consistently lower than kidney for all linker lengths less than 6 carbons. Affinities in the two tissues converge for linkers of 9-10 carbons, as do the affinities at the three human α1 adrenergic receptors showing 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
16 Molecular Pharmacology Fast Forward. Published on November 30, 2016 as DOI: 10.1124/mol.116.105874 This article has not been copyedited and formatted. The final version may differ from this version.
MOL #105874 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 Downloaded from the orthosteric and allosteric sites.
An interesting observation is that the potency and magnitude of the allosteric effect of molpharm.aspetjournals.org the bis(9-aminoacridine)s correlates better with linker length than apparent affinity, viz, 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 at ASPET Journals on September 30, 2021 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 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)s 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)s,
17 Molecular Pharmacology Fast Forward. Published on November 30, 2016 as DOI: 10.1124/mol.116.105874 This article has not been copyedited and formatted. The final version may differ from this version.
MOL #105874 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.
3 Using a [ H]prazosin concentration close to its KD i.e. 50% receptor occupancy, may leave dimer partners unoccupied and available for bitopic interaction with the bis(9- aminoacridine)s as would an asymmetrical binding profile. It has been demonstrated that the bitopic D2 dopamine receptor ligand, SB269652 binds to one receptor and negatively
modulates a dimer partner (Lane et al., 2014). D2 receptors have been demonstrated to form Downloaded from oligomers in native tissue (Zawarynski et al., 1998) while α1 adrenergic receptors have been demonstrated to form dimers in transfected cells (Vicentic et al., 2002) and, so this concept is molpharm.aspetjournals.org not unprecedented and we would suggest that the bis(9-aminoacridine)s 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 at ASPET Journals on September 30, 2021 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 (Jakubik et al., 2014; Pearce and Potter, 1988;
Tränkle et al., 2005). This observation would be in line with current understanding of ligand binding mechanisms. Recent structural biology efforts to crystallise GPCRs have yielded a structure of the M2 muscarinic receptor with a bound positive allosteric modulator,
LY2119620 (Kruse et al., 2013). This structure showed the allosteric modulator bound to an allosteric pocket comprised 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).
18 Molecular Pharmacology Fast Forward. Published on November 30, 2016 as DOI: 10.1124/mol.116.105874 This article has not been copyedited and formatted. The final version may differ from this version.
MOL #105874
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 to 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 Downloaded from equilibrium binding was 247 nM compared to the predicted allosteric affinity from the signalling assay of 19 µM, and the effective concentration required to increase [3H]prazosin molpharm.aspetjournals.org dissociation rate also appears to be in the mid 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 at ASPET Journals on September 30, 2021 possible that some effects of allosteric modulators may be attributable to interaction with the orthosteric site e.g. “allosteric agonists” may be allosteric ligands with additional, hitherto unappreciated weak orthosteric agonist activity. Such a multi-pocket mechanism of action has recently been described for some ligands of the FFA2 free fatty acid receptor (Grundmann et al.). 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 non-conserved sites of receptors, imparting increased binding selectivity (Christopoulos, 2002). However we have demonstrated that
19 Molecular Pharmacology Fast Forward. Published on November 30, 2016 as DOI: 10.1124/mol.116.105874 This article has not been copyedited and formatted. The final version may differ from this version.
MOL #105874 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 Downloaded from 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 molpharm.aspetjournals.org 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 binding of tacrine at ASPET Journals on September 30, 2021 induces a structural change in the orthosteric site that is less favourable 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 characterised, 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)s and the linker- length dependent changes in allosteric modulation will aide in identifying the location of this allosteric pocket and such an approach may facilitate a new approach to drug design and
20 Molecular Pharmacology Fast Forward. Published on November 30, 2016 as DOI: 10.1124/mol.116.105874 This article has not been copyedited and formatted. The final version may differ from this version.
MOL #105874
discovery for the α1 adrenergic receptors or GPCRs in general.
Downloaded from molpharm.aspetjournals.org at ASPET Journals on September 30, 2021
21 Molecular Pharmacology Fast Forward. Published on November 30, 2016 as DOI: 10.1124/mol.116.105874 This article has not been copyedited and formatted. The final version may differ from this version.
MOL #105874
Acknowledgements
The authors would like to thank Dr. Trevor Lewis for his valuable assistance in implementing custom models in GraphPad Prism.
Authorship contributions
Participated in research design: Campbell, Wakelin, Denny and Finch. Downloaded from
Conducted experiments: Campbell.
Performed data analysis: Campbell and Finch. molpharm.aspetjournals.org Wrote or contributed to the writing of the manuscript: Campbell, Wakelin, Denny and Finch.
at ASPET Journals on September 30, 2021
22 Molecular Pharmacology Fast Forward. Published on November 30, 2016 as DOI: 10.1124/mol.116.105874 This article has not been copyedited and formatted. The final version may differ from this version.
MOL #105874
References
Adams A, Jarrott B, Denny WA and Wakelin LPG (1986) Differences between central and peripheral rat α-adrenoceptors revealed using binuclear ligands. Eur J Pharmacol 127(1–2): 27-35.
Adams A, Jarrott B, Elmes BC, Denny WA and Wakelin LP (1985) Interaction of DNA- intercalating antitumor agents with adrenoceptors. Mol Pharmacol 27(4): 480-491.
Baker JG (2005) The selectivity of β-adrenoceptor antagonists at the human β1, β2 and β3 adrenoceptors. Br J Pharmacol 144(3): 317-322.
Campbell AP, MacDougall IJA, Griffith R and Finch AM (2014) An aspartate in the second Downloaded from extracellular loop of the α1B adrenoceptor regulates agonist binding. Eur J Pharmacol 733(0): 90-96.
Carruthers SG (1994) Adverse effects of α1-adrenergic blocking drugs. Drug Saf 11(1): 12- 20. molpharm.aspetjournals.org Cavalli A, Lattion A-L, Hummler E, Nenniger M, Pedrazzini T, Aubert J-F, Michel MC, Yang M, Lembo G, Vecchione C, Mostardini M, Schmidt A, Beermann F and Cotecchia S (1997) Decreased blood pressure response in mice deficient of the α1b-adrenergic receptor. Proc Natl Acad Sci U S A 94(21): 11589-11594.
Chen Z-j and Minneman KP (2005) Recent progress in α1-adrenergic receptor research. Acta Pharmacol Sin 26(11): 1281-1287. at ASPET Journals on September 30, 2021
Cheng Y-C and Prusoff WH (1973) Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol 22(23): 3099-3108.
Christopoulos A (2002) Allosteric binding sites on cell-surface receptors: novel targets for drug discovery. Nat Rev Drug Discov 1(3): 198-210.
Conn PJ, Lindsley CW, Meiler J and Niswender CM (2014) Opportunities and challenges in the discovery of allosteric modulators of GPCRs for treating CNS disorders. Nat Rev Drug Discov 13(9): 692-708.
Croy CH, Schober DA, Xiao H, Quets A, Christopoulos A and Felder CC (2014) Characterization of the novel positive allosteric modulator, LY2119620, at the muscarinic M(2) and M(4) receptors. Mol Pharmacol 86(1): 106-115.
Deshpande SM and Singh AK (1972) Synthesis of some N,N'-bis-(9-acridino)-α,θ- diaminoalkanes dihydrochloride as potential antibacterial, antitubercular and antileprotics. Chem Pharm Bull (Tokyo) 20(1): 206-208.
Dror RO, Pan AC, Arlow DH, Borhani DW, Maragakis P, Shan Y, Xu H and Shaw DE (2011) Pathway and mechanism of drug binding to G-protein-coupled receptors. Proc Natl Acad Sci U S A 108(32): 13118-13123.
Du X-J, Gao X-M, Kiriazis H, Moore X-L, Ming Z, Su Y, Finch AM, Hannan RA, Dart AM and Graham RM (2006) Transgenic α1A-adrenergic activation limits post-infarct ventricular
23 Molecular Pharmacology Fast Forward. Published on November 30, 2016 as DOI: 10.1124/mol.116.105874 This article has not been copyedited and formatted. The final version may differ from this version.
MOL #105874 remodeling and dysfunction and improves survival. Cardiovasc Res 71(4): 735-743.
Ehlert FJ (1988) Estimation of the affinities of allosteric ligands using radioligand binding and pharmacological null methods. Mol Pharmacol 33(2): 187-194.
Ellis J and Seidenberg M (2000) Interactions of alcuronium, TMB-8, and other allosteric ligands with muscarinic acetylcholine receptors: Studies with chimeric receptors. Mol Pharmacol 58(6): 1451-1460.
GlaxoSmithKline A (2011) Australian public assessment report for dutasteride/tamsulosin. Theraputic Goods Administration.
Grundmann M, Tikhonova IG, Hudson BD, Smith NJ, Mohr K, Ulven T, Milligan G, Kenakin T and Kostenis E (2016) A Molecular Mechanism for Sequential Activation of a G Downloaded from Protein-Coupled Receptor. Cell Chem Biol 23(3): 392-403.
Hill HV (1910) The possible effects of aggregation of the molecules of haemoglobin on its dissociation curves. J Physiol 40(suppl): iv-vii. molpharm.aspetjournals.org
Hillman KL, Lei S, Doze VA and Porter JE (2009) α1A adrenergic receptor activation increases inhibitory tone in CA1 hippocampus. Epilepsy Res 84(2–3): 97-109.
Jakubik J, Zimcik P, Randakova A, Fuksova K, El-Fakahany EE and Dolezal V (2014) Molecular mechanisms of methoctramine binding and selectivity at muscarinic acetylcholine receptors. Mol Pharmacol 86(2): 180-192.
Jensen B, Swigart P and Simpson P (2009) Ten commercial antibodies for α1-adrenergic at ASPET Journals on September 30, 2021 receptor subtypes are nonspecific. N-S Arch Pharmacol 379(4): 409-412.
Kenakin T (1997) Pharmacologic analysis of drug-receptor interaction. 3rd ed. Lippincott- Raven, Philadelphia, New York.
Kruse AC, Hu J, Pan AC, Arlow DH, Rosenbaum DM, Rosemond E, Green HF, Liu T, Chae PS, Dror RO, Shaw DE, Weis WI, Wess J and Kobilka BK (2012) Structure and dynamics of the M3 muscarinic acetylcholine receptor. Nature 482(7386): 552-556.
Kruse AC, Ring AM, Manglik A, Hu J, Hu K, Eitel K, Hubner H, Pardon E, Valant C, Sexton PM, Christopoulos A, Felder CC, Gmeiner P, Steyaert J, Weis WI, Garcia KC, Wess J and Kobilka BK (2013) Activation and allosteric modulation of a muscarinic acetylcholine receptor. Nature 504(7478): 101-106.
Lane JR, Donthamsetti P, Shonberg J, Draper-Joyce CJ, Dentry S, Michino M, Shi L, López L, Scammells PJ, Capuano B, Sexton PM, Javitch JA and Christopoulos A (2014) A new mechanism of allostery in a G protein–coupled receptor dimer. Nat Chem Biol 10(9): 745- 752.
Leach K, Sexton PM and Christopoulos A (2007) Allosteric GPCR modulators: taking advantage of permissive receptor pharmacology. Trends Pharmacol Sci 28(8): 382-389.
Leppik RA, Mynett A, Lazareno S and Birdsall NJ (2000) Allosteric interactions between the antagonist prazosin and amiloride analogs at the human α1A-adrenergic receptor. Mol Pharmacol 57(3): 436-445.
24 Molecular Pharmacology Fast Forward. Published on November 30, 2016 as DOI: 10.1124/mol.116.105874 This article has not been copyedited and formatted. The final version may differ from this version.
MOL #105874
Lin F, Owens WA, Chen S, Stevens ME, Kesteven S, Arthur JF, Woodcock EA, Feneley MP and Graham RM (2001) Targeted α1A-adrenergic receptor overexpression induces enhanced cardiac contractility but not hypertrophy. Circ Res 89(4): 343-350.
Lopata MA, Cleveland DW and Sollner-Webb B (1984) High level transient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment. Nucleic Acids Res 12(14): 5707-5717.
Lundholt BK, Scudder KM and Pagliaro L (2003) A simple technique for reducing edge effect in cell-based assays. J Biomol Screen 8(5): 566-570.
Milano CA, Dolber PC, Rockman HA, Bond RA, Venable ME, Allen LF and Lefkowitz RJ Downloaded from (1994) Myocardial expression of a constitutively active α1B-adrenergic receptor in transgenic mice induces cardiac hypertrophy. Proc Natl Acad Sci U S A 91(21): 10109-10113.
Newman-Tancredi A, Verrièle L, Chaput C and Millan MJ (1998) Labelling of recombinant 3 human and native rat serotonin 5-HT1A receptors by a novel, selective radioligand, [ H]-S 15535: Definition of its binding profile using agonists, antagonists and inverse agonists. N-S molpharm.aspetjournals.org Arch Pharmacol 357(3): 205-217.
Pearce BD and Potter LT (1988) Effects of tetrahydroaminoacridine on M1 and M2 muscarine receptors. Neurosci Lett 88(3): 281-285.
Perez DM and Doze VA (2011) Cardiac and neuroprotection regulated by α1-adrenergic receptor subtypes. J Recept Sig Transd 31(2): 98-110. at ASPET Journals on September 30, 2021 Pfaffendorf M, Batink HD, Trankle C, Mohr K and van Zwieten PA (2000) Probing the selectivity of allosteric modulators of muscarinic receptors at other G-protein-coupled receptors. J Auton Pharmacol 20(1): 55-62.
Pizzanelli C, Lazzeri G, Fulceri F, Giorgi FS, Pasquali L, Cifelli G, Murri L and Fornai F (2009) Lack of α1B adrenergic receptor protects against epileptic seizures. Epilepsia 50: 59- 64.
Potter LT, Ferrendelli CA, Hanchett HE, Hollifield MA and Lorenzi MV (1989) Tetrahydroaminoacridine and other allosteric antagonists of hippocampal M1 muscarine receptors. Mol Pharmacol 35(5): 652-660.
Prilla S, Schrobang J, Ellis J, Holtje HD and Mohr K (2006) Allosteric interactions with muscarinic acetylcholine receptors: complex role of the conserved tryptophan M2422Trp in a critical cluster of amino acids for baseline affinity, subtype selectivity, and cooperativity. Mol Pharmacol 70(1): 181-193.
Prinz H (2010) Hill coefficients, dose-response curves and allosteric mechanisms. J Chem Biol 3(1): 37-44.
Ragnarsson L, Andersson A, Thomas WG and Lewis RJ (2015) Extracellular surface residues of the α1B-adrenoceptor critical for G protein-coupled receptor function. Mol Pharmacol 87(1): 121-129.
Ragnarsson L, Wang C-IA, Andersson Å, Fajarningsih D, Monks T, Brust A, Rosengren KJ
25 Molecular Pharmacology Fast Forward. Published on November 30, 2016 as DOI: 10.1124/mol.116.105874 This article has not been copyedited and formatted. The final version may differ from this version.
MOL #105874 and Lewis RJ (2013) Conopeptide ρ-TIA defines a new allosteric site on the extracellular surface of the α1B-adrenoceptor. J Biol Chem 288(3): 1814-1827.
Rokosh DG, Bailey BA, Stewart AFR, Karns LR, Long CS and Simpson PC (1994) Distribution of α1C-adrenergic receptor mRNA in adult-rat tissues by RNase protection assay and comparison with α1B and α1D. Biochem Biophys Res Commun 200(3): 1177-1184.
Sato S, Hatanaka T, Yuyama H, Ukai M, Noguchi Y, Ohtake A, Taguchi K, Sasamata M and Miyata K (2012) Tamsulosin potently and selectively antagonizes human recombinant α1A/1D- adrenoceptors: Slow dissociation from the α1A-adrenoceptor may account for selectivity for α1A-adrenoceptor over α1B-adrenoceptor subtype. Biol Pharm Bull 35(1): 72-77.
Schober DA, Croy CH, Xiao H, Christopoulos A and Felder CC (2014) Development of a 3 Downloaded from radioligand, [ H]LY2119620, to probe the human M2 and M4 muscarinic receptor allosteric binding sites. Mol Pharmacol 86(1): 116-123.
Schroter A, Trankle C and Mohr K (2000) Modes of allosteric interactions with free and 3 [ H]N-methylscopolamine-occupied muscarinic M2 receptors as deduced from buffer- dependent potency shifts. N-S Arch Pharmacol 362(6): 512-519. molpharm.aspetjournals.org
Sharpe IA, Thomas L, Loughnan M, Motin L, Palant E, Croker DE, Alewood D, Chen S, Graham RM, Alewood PF, Adams DJ and Lewis RJ (2003) Allosteric α1-adrenoreceptor antagonism by the conopeptide ρ-TIA. J Biol Chem 278(36): 34451-34457.
Shibata K, Foglar R, Horie K, Obika K, Sakamoto A, Ogawa S and Tsujimoto G (1995) KMD-3213, a novel, potent, α1a-adrenoceptor-selective antagonist: characterization using recombinant human α1-adrenoceptors and native tissues. Mol Pharmacol 48(2): 250-258. at ASPET Journals on September 30, 2021
Steinfeld T, Hughes AD, Klein U, Smith JAM and Mammen M (2011) THRX-198321 is a bifunctional muscarinic receptor antagonist and β2-adrenoceptor agonist (MABA) that binds in a bimodal and multivalent manner. Mol Pharmacol 79(3): 389-399.
Takei R-i, Ikegaki I, Shibata K, Tsujimoto G and Asano T (1999) Naftopidil, a novel α1- adrenoceptor antagonist, displays selective inhibition of canine prostatic pressure and high affinity binding to cloned human α1-adrenoceptors. Jpn J Pharmacol 79(4): 447-454.
Thal DM, Sun B, Feng D, Nawaratne V, Leach K, Felder CC, Bures MG, Evans DA, Weis WI, Bachhawat P, Kobilka TS, Sexton PM, Kobilka BK and Christopoulos A (2016) Crystal structures of the M1 and M4 muscarinic acetylcholine receptors. Nature 531(7594): 335-340.
Thorsen Thor S, Matt R, Weis William I and Kobilka Brian K (2014) Modified T4 Lysozyme Fusion Proteins Facilitate G Protein-Coupled Receptor Crystallogenesis. Structure 22(11): 1657-1664.
Tränkle C, Dittmann A, Schulz U, Weyand O, Buller S, Jöhren K, Heller E, Birdsall NJM, Holzgrabe U, Ellis J, Höltje HD and Mohr K (2005) Atypical muscarinic allosteric modulation: Cooperativity between modulators and their atypical binding topology in muscarinic M2 and M2/M5 chimeric receptors. Mol Pharmacol 68(6): 1597-1610.
Tränkle C, Weyand O, Voigtländer U, Mynett A, Lazareno S, Birdsall NJM and Mohr K (2003) Interactions of orthosteric and allosteric ligands with [3H]dimethyl-W84 at the common allosteric site of muscarinic M2 receptors. Mol Pharmacol 64(1): 180-190.
26 Molecular Pharmacology Fast Forward. Published on November 30, 2016 as DOI: 10.1124/mol.116.105874 This article has not been copyedited and formatted. The final version may differ from this version.
MOL #105874
Valant C, Felder CC, Sexton PM and Christopoulos A (2012) Probe dependence in the allosteric modulation of a G protein-coupled receptor: implications for detection and validation of allosteric ligand effects. Mol Pharmacol 81(1): 41-52.
Vicentic A, Robeva A, Rogge G, Uberti M and Minneman KP (2002) Biochemistry and pharmacology of epitope-tagged α1-adrenergic receptor subtypes. J Pharmacol Exp Ther 302(1): 58-65.
Waugh DJ, Gaivin RJ, Damron DS, Murray PA and Perez DM (1999) Binding, partial agonism, and potentiation of α1-adrenergic receptor function by benzodiazepines: A potential site of allosteric modulation. J Pharmacol Exp Ther 291(3): 1164-1171.
Zawarynski P, Tallerico T, Seeman P, Lee SP, O'Dowd BF and George SR (1998) Dopamine Downloaded from D2 receptor dimers in human and rat brain. FEBS Lett 441(3): 383-386.
Zhao MM, Hwa J and Perez DM (1996) Identification of critical extracellular loop residues involved in α1 adrenergic receptor subtype-selective antagonist binding. Mol Pharmacol 50(5): 1118-1126. molpharm.aspetjournals.org Zuscik MJ, Chalothorn D, Hellard D, Deighan C, McGee A, Daly CJ, Waugh DJJ, Ross SA, Gaivin RJ, Morehead AJ, Thomas JD, Plow EF, McGrath JC, Piascik MT and Perez DM (2001) Hypotension, autonomic failure, and cardiac hypertrophy in transgenic mice overexpressing the α1B-adrenergic receptor. J Biol Chem 276(17): 13738-13743.
at ASPET Journals on September 30, 2021
27 Molecular Pharmacology Fast Forward. Published on November 30, 2016 as DOI: 10.1124/mol.116.105874 This article has not been copyedited and formatted. The final version may differ from this version.
MOL #105874
Figure legends
Figure 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.
Figure 2. Subtype selective binding of the bis(9-aminoacridine)s
Binding affinities of the bis(9-aminoacridine)s at all three α1 adrenergic receptors and 5-HT1A Downloaded from serotonin receptor. All binding curves were fit by a variable-slope competitive binding model. Affinities were compared by one-way ANOVA with Tukey post-test. a, b, d, s: P <
0.05 compared to α1A, α1B, α1D adrenergic receptors (AR), and 5-HT1A serotonin receptor, molpharm.aspetjournals.org respectively. Affinity is not shown where logKi is greater than -4.
3 Figure 3. Kinetics of dissociation of [ H]prazosin from α1 adrenergic receptors
[3H]prazosin dissociation from α adrenergic receptor membranes (A-H) and α adrenergic
1A 1B at ASPET Journals on September 30, 2021 receptor membranes (I-P) in the absence and presence of 9-aminoacridine (A, M), C4 bis(9- aminoacridine) (B, N), C9 bis(9-aminoacridine) (C, O), and tacrine (D, P). Membranes were preincubated with 250 pM [3H]prazosin for 1 h at room temperature in 400 µL HEM buffer.
[3H]prazosin reassociation was inhibited by the addition of 100 µL phentolamine with or without test compound at concentrations shown. A-D and I-L show time-dependent dissociation curves. E-H and M-P show observed dissociation rates (Kobs) normalized to the control dissociation rate (Koff) measured in the absence of modulator and plotted against the log concentration of modulator. Points and error bars are the mean ± SEM of 3 individual experiments performed in duplicate.
Figure 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
28 Molecular Pharmacology Fast Forward. Published on November 30, 2016 as DOI: 10.1124/mol.116.105874 This article has not been copyedited and formatted. The final version may differ from this version.
MOL #105874 overnight in the presence of [3H]myo-inositol. After 24 h of incubation, the medium was exchanged for serum-free DMEM in the presence or absence of C9 bis(9-aminoacridine) (C9) for 20 min followed by stimulation with norepinephrine for 30 min. Unstimulated points are shown in the left segment of the X-axis. All values were normalised between unstimulated response and maximum norepinephrine-induced response and fit by non-linear regression to a variable-slope dose-response curve using GraphPad Prism 6. Points and bars represent the
mean ± SEM of n=3-5 experiments performed in triplicate. Downloaded from
molpharm.aspetjournals.org
at ASPET Journals on September 30, 2021
29 MOL #105874
Tables Table 1. Equilibrium binding affinities Molecular PharmacologyFastForward.PublishedonNovember30,2016asDOI:10.1124/mol.116.105874
α1A adrenergic receptor α1B adrenergic receptor α1D adrenergic receptor 5-HT1A receptor This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion.
pKi n Ki (nM) pKi n Ki (nM) pKi n Ki (nM) pKi n Ki (nM) Phentolamine 8.4 ± 0.06*** 6 4 7.8 ± 0.2*** 6 14 7.7 ± 0.06*** 6 19 nd
Serotonin nd nd nd 9.0 ± 0.06 6 1
9-aminoacridine 6.6 ± 0.03 3 250 5.6 ± 0.05 3 2600 5.7 ± 0.1 3 1900 <4
C2 bis(9-aminoacridine) 5.7 ± 0.1*** 3 1900 6.6 ± 0.2*** 3 250 5.9 ± 0.09 3 1400 <4
C3 bis(9-aminoacridine) 6.2 ± 0.09 3 700 6.4 ± 0.2** 3 430 5.9 ± 0.06 3 1200 4.8 ± 0.2 3 18000
C4 bis(9-aminoacridine) 7.7 ± 0.2*** 3 21 6.7 ± 0.1*** 3 210 6.2 ± 0.03 3 650 5.5 ± 0.1 3 3000
C5 bis(9-aminoacridine) 7.5 ± 0.1*** 3 33 7.0 ± 0.2*** 3 94 6.4 ± 0.05* 3 360 5.8 ± 0.07 3 1800
C6 bis(9-aminoacridine) 7.2 ± 0.08 3 58 7.0 ± 0.1*** 3 110 6.7 ± 0.07*** 3 220 6.5 ± 0.05 3 340
C7 bis(9-aminoacridine) 6.9 ± 0.06 3 120 7.0 ± 0.2*** 3 110 6.7 ± 0.09*** 3 230 6.2 ± 0.09 3 620
C9 bis(9-aminoacridine) 6.6 ± 0.08 3 270 6.9 ± 0.2*** 3 130 6.5 ± 0.1* 3 330 5.7 ± 0.2 3 1800
C10 bis(9-aminoacridine) 6.5 ± 0.07 3 290 6.8 ± 0.1*** 3 150 6.4 ± 0.1* 3 370 6.0 ± 0.05 3 930
C12 bis(9-aminoacridine) 6.4 ± 0.2 3 400 6.9 ± 0.09*** 3 120 5.8 ± 0.06 3 1600 6.0 ± 0.09 3 950
Tacrine 5.7 ± 0.06*** 3 1900 4.7 ± 0.2*** 3 19000 5.2 ± 0.2 3 6400 nd pKi: Negative log of apparent affinity calculated according to the Cheng-Prusoff equation (Cheng and Prusoff, 1973). Ki: Calculated concentration of ligand required to occupy 50% of unoccupied receptors, derived from pKi. All data are presented as mean ± SEM for ‘n’ repeats, performed in duplicate. *, **, *** P < 0.05, 0.01, 0.001 compared to 9-aminoacridine, not determined for 5-HT1A receptor. nd: not determined.
30
Downloaded from from Downloaded molpharm.aspetjournals.org at ASPET Journals on September 30, 2021 30, September on Journals ASPET at MOL #105874
Table 2. Equilibrium binding assay slopes Molecular PharmacologyFastForward.PublishedonNovember30,2016asDOI:10.1124/mol.116.105874
α1A adrenergic receptor α1B adrenergic receptor α1D adrenergic receptor 5-HT1A receptor This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. Slope n Slope n Slope n Slope n
Phentolamine 1.03 ± 0.4 6 1.1 ± 0.06 5 1.0 ± 0.06 4 nd
Serotonin nd nd nd 0.8 ± 0.1 6
9-aminoacridine 1.0 ± 0.08 3 1.0 ± 0.07 3 1.2 ± 0.1 3 nd 4
C2 bis(9-aminoacridine) 1.2 ± 0.2 3 1.2 ± 0.060.089 3 1.4 ± 0.4 3 nd 3
C3 bis(9-aminoacridine) 1.1 ± 0.10 3 1.3 ± 0.04* 3 1.0 ± 0.08 3 0.7 ± 0.090.08 3
C4 bis(9-aminoacridine) 1.4 ± 0.3 3 1.3 ± 0.03* 3 1.3 ± 0.080.06 3 1.0 ± 0.07 3
C5 bis(9-aminoacridine) 1.6 ± 0.3 3 1.3 ± 0.10.09 3 1.3 ± 0.03* 3 1.0 ± 0.2 3
C6 bis(9-aminoacridine) 1.9 ± 0.6 3 2.0 ± 0.30.06 3 1.4 ± 0.06* 3 1.1 ± 0.2 3
C7 bis(9-aminoacridine) 1.5 ± 0.10.05 3 1.8 ± 0.20.06 3 1.5 ± 0.03** 3 0.8 ± 0.2 3
C9 bis(9-aminoacridine) 2.5 ± 0.40.06 3 2.3 ± 0.40.07 3 4.0 ± 1.6 3 1.0 ± 0.5 3
C10 bis(9-aminoacridine) 2.0 ± 0.06** 3 2.0 ± 0.1* 3 2.7 ± 0.8 3 1.4 ± 0.2 3
C12 bis(9-aminoacridine) 2.0 ± 1 3 1.0 ± 0.1 3 2.5 ± 0.40.07 3 1.7 ± 0.1* 3
Tacrine 1.1 ± 0.2 3 0.80 ± 0.08 3 0.9 ± 0.030.09 3 nd
Hill Slope coefficient of competitive binding data fit by a variable-slope competitive binding model as per section *, ** P < 0.05, 0.01 respectively compared to 1 by one sample t-test. P values between 0.05 and 0.1 are reported in superscript. All data are expressed as mean ± SEM for ‘n’ repeats
31
Downloaded from from Downloaded molpharm.aspetjournals.org at ASPET Journals on September 30, 2021 30, September on Journals ASPET at MOL #105874
3 Table 3. Modulation of dissociation rates of [ H]prazosin from the α1A and α1B adrenergic receptors Molecular PharmacologyFastForward.PublishedonNovember30,2016asDOI:10.1124/mol.116.105874 α adrenergic receptor α 1A 1B adrenergic receptor This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion.
Koff t1/2 Kobs/Koff log(Kobs/Koff) Koff t1/2 Kobs/Koff log(Kobs/Koff) Control 0.05 ± 0.01 13 1.0 0.00 0.02 ± 0.0 32 1.0 0.0
9-aminoacridine 0.17 ± 0.02 4.1 3.2 0.48** 0.14 ± 0.01 4.8 5.5 0.74***
C2 bis(9-aminoacridine) 0.08 ± 0.02 8.7 1.5 0.14 0.11 ± 0.01 6.1 4.4 0.63***
C3 bis(9-aminoacridine) 0.11 ± 0.03 6.2 2.1 0.28 0.13 ± 0.00 5.3 5.0 0.70***
C4 bis(9-aminoacridine) 0.18 ± 0.05 3.8 3.5 0.49** 0.23 ± 0.00 3.0 9.0 0.95***
C5 bis(9-aminoacridine) 0.17 ± 0.03 4.0 3.2 0.48** 0.36 ± 0.01 1.9 14 1.1***
C6 bis(9-aminoacridine) 0.40 ± 0.01 1.7 7.3 0.86*** 0.49 ± 0.07 4 1. 19 1.3***
C7 bis(9-aminoacridine) 0.37 ± 0.07 1.9 6.7 0.81*** 0.76 ± 0.04 0.9 29 1.5***
C9 bis(9-aminoacridine) 0.96 ± 0.09 0.7 18 1.2*** 1.7 ± 0.7 0.4 62 1.8***
C10 bis(9-aminoacridine) 1.2 ± 0.1 0.6 23 1.4*** 1.5 ± 0.7 0.5 63 1.7***
C12 bis(9-aminoacridine) 4.4 ± 1.4 0.2 83 1.9*** 0.72 ± 0.05 1.0 28 1.4***
Tacrine 0.10 ± 0.01 6.6 2.1 0.31* 0.07 ± 0.01 11 4.2 0.61***
3 -1 Koff: Dissociation rate of [ H]prazosin (min ).
Kobs/Koff: Ratio of the observed dissociation rates in the presence and absence of 100 µM modulator. 3 t1/2: Half-life of [ H]prazosin dissociation (minutes). *, **, *** P < 0.05, 0.01, 0.001 compared to control. Values are given as mean ± SEM n=3-6
32
Downloaded from from Downloaded molpharm.aspetjournals.org at ASPET Journals on September 30, 2021 30, September on Journals ASPET at MOL #105874
Table 4. C9 dependent changes in norepinephrine (NE) dependent and independent activation of the α1A adrenergic receptor Molecular PharmacologyFastForward.PublishedonNovember30,2016asDOI:10.1124/mol.116.105874 Control C9 10 μM C9 30 μM C9 100 μM This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion.
mean ± S.E.M n mean ± S.E.M n mean ± S.E.M n mean ± S.E.M n
NE pEC50 6.2 ± 0.1 5 5.6 ± 0.2* 4 5.4 ± 0.1** 4 5.0 ± 0.2*** 4
NE Slope 1.6 ± 0.2 5 1.4 ± 0.2 4 1.8 ± 0.3 4 1.8 ± 1 3
NE Emax (%) 100.8 ± 2 5 75 ± 10 4 46 ± 10*** 4 39 ± 5*** 4
Basal (%) 0.3 ± 0.3 5 5.4 ± 3 4 2.1 ± 1 4 17 ± 5** 5 pEC50: negative log of the concentration required to cause 50% maximal norepinephrine mediated activation. Slope: slope factor of norepinephrine mediated receptor activation
Emax: % maximum norepinephrine response measured in the absence of C9 bis(9-aminoacridine).
Basal: agonist independent receptor activity, expressed as % norepinephrine Emax. *, **, *** P < 0.05, 0.01, 0.001 compared to norepinephrine alone by one-way ANOVA with Tukey post-test.
33
Downloaded from from Downloaded molpharm.aspetjournals.org at ASPET Journals on September 30, 2021 30, September on Journals ASPET at Molecular Pharmacology Fast Forward. Published on November 30, 2016 as DOI: 10.1124/mol.116.105874 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from
Figure 1 molpharm.aspetjournals.org 9-aminoacridine Cn bis(9-aminoacridine) H+ H+ N N
NH2 NH at ASPET Journals on September 30, 2021
(CH2)n Tacrine NH H+ N
N H+ NH2 Molecular Pharmacology Fast Forward. Published on November 30, 2016 as DOI: 10.1124/mol.116.105874 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from
Figure 2 molpharm.aspetjournals.org
-4 α1A AR α AR a a,b,d 1B α1D AR -5 5-HT1A R a,b,d a,b,d
i a a b a,b,d b s b b -6 b b a,b,s b
ogK s a at ASPET Journals on September 30, 2021 l s s s a,d,s s b,d a,d -7 s s s s d,s s d,s -8 b,d,s
e 0 1 2 n C2 C3 C4 C5 C6 C7 C8 C9 ri C1 C1 C1 c 9-aa Ta Molecular Pharmacology Fast Forward. Published on November 30, 2016 as DOI: 10.1124/mol.116.105874 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from molpharm.aspetjournals.org at ASPET Journals on September 30, 2021 Molecular Pharmacology Fast Forward. Published on November 30, 2016 as DOI: 10.1124/mol.116.105874 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from molpharm.aspetjournals.org at ASPET Journals on September 30, 2021