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Research ArticleArticle

A Series of α7 Nicotinic Acetylcholine Receptor Allosteric Modulators with Close Chemical Similarity but Diverse Pharmacological Properties

JasKiran K. Gill, Persis Dhankher, Tom D. Sheppard, Emanuele Sher and Neil S. Millar
Molecular Pharmacology May 2012, 81 (5) 710-718; DOI: https://doi.org/10.1124/mol.111.076026

Abstract

Acetylcholine activates nicotinic acetylcholine receptors (nAChRs) by binding to an extracellular site located at the interface of two adjacent subunits. In contrast, recent studies have provided evidence that positive allosteric modulators (PAMs) such as TQS (4-(naphthalen-2-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide) and allosteric agonists such as 4BP-TQS (4-(4-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide) interact at an intrasubunit transmembrane site. Here, we describe the synthesis and pharmacological characterization of a series of chemically related allosteric modulators of the α7 nAChR. Minimal changes in the chemical structure of these compounds have been found to exert profound effects on their pharmacological properties. For example, compounds containing a bromine atom at either the ortho or meta position on the phenyl ring, such as 2BP-TQS (4-(2-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide) and 3BP-TQS (4-(3-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide), rather than at the para position (4BP-TQS), display no allosteric agonist activity but retain PAM activity on α7 nAChRs, demonstrating the importance of the location of the halogen atom on pharmacological properties. Replacement of the bromine atom in 4BP-TQS with either a chlorine [4CP-TQS (4-(4-chloroophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide)] or an iodine atom [4IP-TQS (4-(4-iodoophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide)] results in compounds that have pharmacological properties characteristic of allosteric agonists but display differences in activation rates, in inactivation rates, and in levels of desensitization. In contrast, replacement of the bromine atom in 4BP-TQS with a fluorine atom [4FP-TQS (4-(4-fluorophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide)] generated a compound that lacks allosteric agonist activity but acts a potentiator of responses to acetylcholine. In addition, 4FP-TQS was found to act as an antagonist of responses evoked by allosteric agonists such as 4BP-TQS. These findings provide evidence of the pharmacological diversity of compounds interacting with the allosteric transmembrane site on α7 nAChRs.

Introduction

Nicotinic acetylcholine receptors (nAChRs) are transmembrane receptors for the neurotransmitter acetylcholine. They are members of a structurally related family of “Cys-loop” ligand-gated ion channels that also includes receptors for neurotransmitters such as 5-hydroxytryptamine (serotonin), GABA, and glycine (Lester et al., 2004). In common with other members of the Cys-loop family of receptors, nAChRs are pentameric complexes in which the conventional “orthosteric” agonist binding site is located at an extracellular location at the interface of two subunits (Arias, 2000; Unwin, 2005).

Nicotinic receptors comprise a diverse family of receptors. In vertebrates, 17 subunits have been identified that assemble into a large number of receptor subtypes with distinct subunit composition (Millar and Gotti, 2009). In addition to the well characterized nAChRs expressed at the mammalian neuromuscular junction, a diverse family of neuronal nAChRs are expressed in the mammalian central and peripheral nervous system (Millar and Gotti, 2009). Muscle nAChRs and most neuronal nAChRs are heteromeric complexes of more than one type of subunit. In addition, some neuronal nAChR subunits, such as α7, are able to form functional homomeric nAChRs (Couturier et al., 1990).

A considerable amount of effort has been devoted to the characterization of compounds (including agonists and competitive antagonists) that interact with the conventional orthosteric binding site of nAChRs (Jensen et al., 2005; Arneric et al., 2007; Haydar and Dunlop, 2010). In addition, studies have begun to explore the pharmacological diversity of compounds acting at allosteric binding sites (Bertrand and Gopalakrishnan, 2007; Haydar and Dunlop, 2010; Williams et al., 2011). For example, recent studies have revealed that allosteric modulators of α7 nAChRs have cognitive enhancing effects that may potentially be useful in the treatment of neurological and psychiatric disorders such as Alzheimer's disease and schizophrenia (Ng et al., 2007; Timmermann et al., 2007; Haydar and Dunlop, 2010).

The term positive allosteric modulator (PAM) has been used to describe compounds that act at a site that is distinct from the orthosteric agonist binding site (Bertrand and Gopalakrishnan, 2007). Typically, such compounds lack agonist activity themselves but potentiate responses evoked by conventional agonists such as acetylcholine. Two types of α7-selective PAMs (type I and type II) have been identified (Bertrand and Gopalakrishnan, 2007). Both types potentiate peak agonist-induced responses but they have different effects on the rate of agonist-induced receptor desensitization. Type I PAMs have little or no effect on the rapid rate of desensitization that is characteristic of α7 nAChRs, whereas type II PAMs cause a dramatic slowing of receptor desensitization. In a recent study, evidence has emerged to demonstrate that compounds acting at an allosteric site can activate α7 nAChRs in the absence of conventional agonists. Such compounds have been described as nAChR allosteric agonists (Gill et al., 2011) to distinguish them from agonists that act at the classic extracellular orthosteric site. In many respects, such compounds are analogous to the allosteric agonists (sometimes referred as ago-allosteric compounds) that have been described for G-protein coupled receptors (Langmead and Christopoulos, 2006; Schwartz and Holst, 2006). It is probable that nAChRs contain a variety of distinct allosteric binding sites (Taly et al., 2009), but studies have provided evidence that one such site is located in an intrasubunit cavity located between the four α-helical transmembrane domains of a single α7 subunit (Young et al., 2008; Gill et al., 2011). In addition, there is evidence that this is a site at which allosteric agonists, as well as both type I and type II potentiators, can act (Young et al., 2008; Collins et al., 2011; Gill et al., 2011).

Here, we have examined the pharmacological properties of a series of compounds (Fig. 1), all with close chemical similarity to two previously described α7-selective allosteric modulators: TQS, a PAM displaying no agonist activity (Grønlien et al., 2007; Gill et al., 2011) and 4BP-TQS a potent nondesensitizing allosteric agonist (Gill et al., 2011). We have found that small changes to the structure of these compounds (for example, changing the position or nature of a single halogen atom) can result in dramatic changes in pharmacological properties. These pharmacologic effects include the loss of allosteric agonist activity (while retaining PAM activity) and more subtle changes in allosteric agonist activation rates, inactivation rate, and desensitization rates.

Fig. 1.
Fig. 1.

Chemical structure of α7 nAChR allosteric modulators examined in the present study.

Materials and Methods

Chemical Synthesis.

TQS compounds were prepared by an InCl3-catalyzed reaction of sulfanilamide, cyclopentadiene, and the corresponding substituted benzaldehyde according to methods described previously (Becker et al., 2004). In all cases, the cis-cis diastereoisomer was obtained as shown in Fig. 1. Details concerning the synthesis of these compounds is provided in Supplemental Fig. 1.

Subunit cDNAs and Plasmid Expression Vectors.

All experiments were performed with human α7 nAChRs. Human wild-type and mutant (M253L) nAChR α7 subunit cDNA constructs in plasmid pSP64GL have been described previously (Broadbent et al., 2006; Gill et al., 2011).

Xenopus laevis Oocyte Electrophysiology.

X. laevis oocytes were isolated and defolliculated as described previously (Young et al., 2007) by treatment with collagenase (2 mg/ml; Worthington Biochemicals, Freehold, NJ) in calcium-free Barth's solution containing 88 mM NaCl, 2.4 mM NaHCO3, 1 mM KCl, 0.82 mM MgSO4, and 15 mM HEPES, pH 7.6. To express human α7 nAChRs, in vitro-transcribed cRNA was injected into the oocyte cytoplasm. In vitro transcription of cRNA was performed using a mMESSAGE mMACHINE SP6 transcription kit (Ambion, Huntington, UK). Oocytes were injected with 6 to 12 ng of cRNA per oocyte in a volume of 32.2 nl using a Drummond variable volume microinjector. After injection, oocytes were incubated at 18°C in a calcium-containing Barth's solution (composition, as above, but with 0.77 mM CaCl2) supplemented with antibiotics (100 units/ml penicillin, 100 μg/ml streptomycin, 4 μg/ml kanamycin, and 50 μg/ml tetracycline). Experiments were performed on oocytes after 3 to 5 days of incubation. Oocytes were placed in a recording chamber and continuously perfused with a saline solution (115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2, and 10 mM HEPES, pH 7.3) with a flow rate of approximately 15 ml/min. Two electrode voltage-clamp recordings were performed (with the oocyte membrane potential held at −60 mV), as described previously (Young et al., 2007) using a OC-725C amplifier (Warner Instruments, Hamden, CT), PowerLab 8SP and Chart 5 software (AD Instruments, Oxford, UK). Agonists and PAMs were applied to oocytes using a BPS-8 solenoid valve solution exchange system (ALA Scientific Inc., Westbury, NY). For multiple comparisons, statistical significance was determined by ANOVA with Tukey's post hoc test. Student's unpaired t tests were used for pairwise comparisons.

All TQS compounds were dissolved in dimethyl sulfoxide (to generate a 100 mM stock solution) before dilution into saline recording solution. Because of their limited solubility in aqueous solution, the maximum concentration that could be tested was 100 μM for all compounds. A problem we have encountered is that all of these compounds have a tendency to stick to plastic tubing and to plastic apparatus used for oocyte perfusion. This was found to be a particular problem with silicone tubing. We have found that the problem could be reduced somewhat by using polyurethane tubing (e.g., color-coded, 1/16 i.d. × 8 channels; World Precision Instruments, Inc., Sarasota, FL) rather than silicone tubing, but it was still necessary to rinse all perfusion tubing (and also the Perspex recording chamber) with ethanol after each use. When solenoid pinch valves were used, a small section of silicone tubing was used (e.g., versilic tubing, 1.5 mm i.d.; 3 mm o.d.; Scientific Laboratory Supplies, Nottingham, UK). However, because of problems in washing compounds off the silicone tubing, all silicone tubing was replaced after each change of TQS compound in the perfusion system.

Results

Agonist Activation of α7 nAChRs.

A series of compounds with close chemical similarity to TQS (a PAM of α7 nAChRs) and 4BP-TQS (an allosteric agonist of α7 nAChRs) have been synthesized (Fig. 1). The pharmacological properties of these compounds have been examined by two-electrode voltage-clamp recordings using X. laevis oocytes expressing recombinant α7 nAChRs.

Initial studies were focused on a series of compounds containing different halogen atoms at the 4-position of the phenyl ring (Fig. 1). As has been shown previously for 4BP-TQS (Gill et al., 2011), compounds containing either a chlorine or an iodine atom at this position (4CP-TQS and 4IP-TQS, respectively) were found to have potent agonist activity on α7 nAChRs (Fig. 2A). In contrast, replacement of the bromine atom with a fluorine atom at the 4-position (4FP-TQS) resulted in the complete loss of agonist activity (Fig. 2A). The EC50 values determined for 4BP-TQS, 4CP-TQS, and 4IP-TQS were not significantly different from one another (Table 1). However, as has been reported previously for 4BP-TQS (Gill et al., 2011), agonist concentrations of these compounds causing half-maximal activation are significantly lower than that of acetylcholine (Table 1).

Fig. 2.
Fig. 2.

Pharmacological properties of agonists on α7 nAChRs expressed in X. laevis oocytes. A, dose-response data are presented for a range of concentrations of acetylcholine (●), 4BP-TQS (○), 4CP-TQS (▵), 4FP-TQS (×), 4IP-TQS (□), and 4MP-TQS (♢). Data are means ± S.E.M. of at least three independent experiments. B, representative recordings obtained in the continued presence of a maximum concentration of agonist, illustrating activation and desensitization rates. C, representative recordings of agonists (at a maximum concentration) that were applied only until the peak response is reached, thereby illustrating activation rates and rates of recovery after agonist wash-off. Responses have been normalized to their peak response.

View this table:
TABLE 1

Pharmacological and kinetic properties of allosteric agonists

Data are means of 7 to 23 independent experiments ± S.E.M. EC50 values of all compounds were significantly different from that for acetylcholine (ANOVA; P < 0.001). For all compounds examined, Hill coefficients were significantly different from that for acetylcholine in pairwise comparisons (t test; P < 0.05). Imax (maximal current) was normalized to a maximal (3 mM) acetylcholine response. Desensitization rate indicates time for response to decline from the peak to half of the peak in the continuous presence of agonist; all compounds were significantly different from acetylcholine by ANOVA (P < 0.05) or by pairwise comparisons to acetylcholine (t test; P < 0.01). Activation rate indicates time from the start of agonist application to peak response; all compounds except 4CP-TQS were significantly different from acetylcholine by ANOVA (P < 0.05), and in pairwise comparisons, all compounds were significantly different from acetylcholine (t test; P < 0.001). Inactivation rate indicates time for recovery to half of the peak response after removal of agonist (this parameter could not be determined for acetylcholine because of the rapid rate of desensitization).

Previous studies have reported that, whereas activation of α7 nAChRs by acetylcholine results in rapidly desensitizing responses (Couturier et al., 1990), activation by the allosteric agonist 4BP-TQS results in largely nondesensitizing responses (Gill et al., 2011). Here we have examined longer applications of 4BP-TQS and have detected very slow desensitization that occurs over a period of many minutes (Fig. 2B). By examining similarly long applications of 4CP-TQS and 4IP-TQS we have observed a progressive slowing of the rate of receptor desensitization as the size of the halogen atom increases (Fig. 2B; Table 1). In addition, the rate at which receptors return to their resting state after agonist activation differs between the three halogen-containing agonists (Fig. 2C; Table 1). A further difference in activation by acetylcholine and by 4CP-TQS, 4BP-TQS, or 4IP-TQS is the slower activation rate that is observed with the latter compounds (Fig. 2, B and C; Table 1).

Differences in the activation rates prompted us to examine the consequence of coapplication of acetylcholine with allosteric agonists (Fig. 3). It is noteworthy that coapplication of these compounds with acetylcholine resulted in receptor activation that can be resolved into two components (Fig. 3). An initial desensitizing response is seen, which is typical of activation by acetylcholine. This is followed by a secondary response that displays slower activation and slower desensitization and is typical of activation by 4CP-TQS, 4BP-TQS, and 4IP-TQS.

Fig. 3.
Fig. 3.

Agonist activation of α7 nAChRs. Application of acetylcholine (3 mM) results in receptor activation, followed by rapid desensitization (left). Coapplication of acetylcholine (3 mM) with 4IP-TQS (100 μM) results in a two-component response: an initial desensitizing acetylcholine response followed by a secondary response with a slower activation, which is a typical of an allosteric agonist such as 4IP-TQS (right). Agonist applications are indicated by horizontal lines. Data shown for 4IP-TQS are typical of data obtained with all allosteric agonists examined.

To examine further the relative influence of the size or the chemical properties of groups attached at the 4-position of the phenyl ring, several additional compounds were synthesized and characterized. Insertion of a methyl or trifluoromethyl group at the 4-position of the phenyl ring (4MP-TQS and 4TF-TQS) generated compounds that retained agonist activity (Figs. 2 and 4), whereas inclusion of a hydrogen atom or hydroxyl group at this position (P-TQS and 4HP-TQS) resulted in complete loss of agonist activity (Fig. 4). These findings support the conclusion that a relatively large group attached at the 4-position of the phenyl ring is required for agonist activity. In addition, a further compound (2N-TQS) was synthesized in which the naphthyl group was attached in an orientation different from that in TQS (Fig. 1). Whereas no agonist activity is observed with TQS (Gill et al., 2011), 2N-TQS displayed clear agonist activity (Fig. 4). Taken together, these findings provide strong evidence that a relatively large group is required at the 4-position of the phenyl ring to confer agonist activity. We also examined the importance of the position at which halogen atoms were attached on the phenyl ring by synthesis and characterization of compounds in which bromine was attached at either the ortho or meta position (2BP-TQS and 3BP-TQS, respectively) (Fig. 1) or in which iodine was attached at the meta position (3IP-TQS) (Fig. 1). In contrast to the potent agonist activity observed with 4BP-TQS, no agonist activation was detected with either 2BP-TQS, 3BP-TQS, or 3IP-TQS (Fig. 4), indicating that the position, as well as the size, of groups attached to the phenyl ring is critical in determining agonist activity. In contrast to the potent agonist activity observed with 4BP-TQS, no agonist activation was detected with a compound containing a bromine atom at both the meta and para positions on the phenyl ring (3,4BP-TQS). Taken together, these results clearly show that minor changes to the structure of these compounds have substantial influence on pharmacological properties.

Fig. 4.
Fig. 4.

Agonist activity of TQS derivatives on α7 nAChRs. A, agonist responses observed with a maximum concentration of each compound (100 μM) are normalized to responses with a maximum concentration of acetylcholine (3 mM). Data are means ± S.E.M. of 7 to 23 independent experiments (Table 1). B, representative recordings obtained in the continued presence of a maximum concentration of 2N-TQS and 4TF-TQS, illustrating activation and desensitization rates. See Fig. 2 for representative responses for other agonists. C, representative recordings of 2N-TQS and 4TF-TQS (at a maximum concentration) that were applied only until the peak response is reached, thereby illustrating activation rates and rates of recovery after agonist wash-off. Responses have been normalized to their peak response.

We have reported previously (Gill et al., 2011) that at high concentrations of 4BP-TQS (greater than ∼30 μM) an increase in agonist response is observed after agonist application ceases. It seems likely that this is a consequence of receptor/channel-blocking activity and is a feature that is also observed with high concentrations of orthosteric agonists such as acetylcholine, for which it has been described as a “hump current” (Liu et al., 2008). Among the agonists examined in the present study, we have seen a wide variation in the extent of this phenomenon. As is illustrated in Fig. 4, a maximal concentration of 4TF-TQS (100 μM) produced a very large hump current (252 ± 78% of the agonist response), whereas a maximal concentration of 2N-TQS (100 μM) produced a relatively small hump current (2.6 ± 1.9% of the agonist response). It is possible that these differences reflect different abilities of these compounds to block the receptor, perhaps by interacting at a site other than the allosteric agonist site.

Positive Allosteric Modulation of α7 nAChRs.

It is clear that alterations at the 4-position of the benzene ring have a dramatic effect on agonist effects of this series of compounds. It is also of interest that several of the compounds tested had no agonist activity, despite close chemical similarity. For each of the compounds that lacked agonist activity (2BP-TQS, 3BP-TQS, 3IP-TQS, 3,4BP-TQS, 4FP-TQS, 4HP-TQS, and P-TQS), we examined whether they were able to act as PAMs. In all cases, these compounds potentiated acetylcholine-evoked responses (Fig. 5), as has been reported previously for TQS (Grønlien et al., 2007; Gill et al., 2011).

Fig. 5.
Fig. 5.

Positive allosteric modulation of α7 nAChRs. A, bar graphs illustrate levels of potentiation caused by a maximum concentration of each compound (100 μM). Potentiation of agonist-evoked responses was determined with a submaximal (EC50) concentration of acetylcholine (100 μM). B, representative recordings are shown, illustrating the differences in activation and desensitization rates for various PAMs. The PAM (100 μM) was preapplied for 5 s and then coapplied with a submaximal (EC50) concentration of acetylcholine (100 μM). Traces are normalized to the same peak response. Data are means ± S.E.M. of 3 to 13 independent experiments (Table 2).

Previous studies have demonstrated that TQS acts as a classic “type II” PAM, causing potentiation of acetylcholine-evoked responses and a dramatic loss of receptor desensitization (Grønlien et al., 2007; Gill et al., 2011). Of interest, just as we had observed differences in rates of desensitization of α7 nAChRs after activation by 4BP-TQS, 4CP-TQS, and 4IP-TQS, marked differences were apparent in the rate of desensitization after potentiation of acetylcholine-evoked responses with different PAMs (Fig. 5B; Table 2).

View this table:
TABLE 2

Pharmacological and kinetic properties of positive allosteric modulators

Data are means of 3 to 13 independent experiments ± S.E.M. Fold potentiation refers to submaximal (EC50) concentration of acetylcholine (100 μM). Desensitization rate indicates time for response to decline from peak to half of the peak in the continuous presence of agonist; all compounds were significantly different from TQS (ANOVA; P < 0.001). Activation rate indicates time from the start of agonist application to peak response; all compounds were significantly different from TQS (ANOVA; P < 0.001).

Influence of the Transmembrane M253L Mutation.

It is well established that acetylcholine activates nAChRs by binding to an extracellular site. In contrast, recent studies have proposed that α7-selective PAMs such as TQS and allosteric agonists such as 4BP-TQS act via a transmembrane binding site (Young et al., 2008; Collins et al., 2011; Gill et al., 2011). One of the lines of evidence supporting this proposal is that potentiation by TQS and agonist activation by 4BP-TQS are completely abolished on α7 receptors containing the transmembrane mutation M253L (Gill et al., 2011). In contrast, M253L has been shown to have no significant effect on activation by the conventional orthosteric agonist acetylcholine (Young et al., 2008; Gill et al., 2011). The effect of M253L on agonist activation by 4CP-TQS, 4IP-TQS, and 4MP-TQS was examined, and it was found to cause complete loss of agonist activation. In addition, the effect of M253L on allosteric potentiation by 2BP-TQS, 3BP-TQS, 3IP-TQS, 3,4BP-TQS, 4FP-TQS, 4HP-TQS, and P-TQS was examined, and it was found to cause complete loss of PAM activity of all of these compounds. These findings support the conclusion the all of the TQS-related compounds examined in this study act by a similar mechanism of action. The simplest explanation is that they all act via a shared allosteric binding site, as has been proposed previously for TQS and 4BP-TQS (Gill et al., 2011).

Antagonism by 4FP-TQS of 4BP-TQS Evoked Responses.

As has been described above, the replacement of a single bromine atom with a fluorine atom converts the allosteric agonist 4BP-TQS into a PAM that lacks agonist activity (4FP-TQS). On the basis of previous studies with α7-selective allosteric modulators (Young et al., 2008; Gill et al., 2011), it seems reasonable to hypothesize that 4BP-TQS and 4FP-TQS might bind competitively at a common allosteric site. If this assumption is correct, we would predict that 4FP-TQS would act as an antagonist if coapplied with 4BP-TQS. We have tested this hypothesis by applying an EC50 concentration of 4BP-TQS (10 μM) and then coapplying 4BP-TQS with a range of concentrations of 4FP-TQS (Fig. 6). As predicted, the coapplication of 4FP-TQS resulted in a dose-dependent inhibition of responses evoked by 4BP-TQS, with an IC50 concentration of 4.4 ± 1.3 μM (Fig. 6, A and B). In contrast, coapplication of 4FP-TQS with an EC50 concentration of acetylcholine results in a dose-dependent potentiation of responses evoked by acetylcholine, with an EC50 concentration of 23 ± 8.1 μM (Fig. 6C). These findings suggest that 4BP-TQS and 4FP-TQS bind to a common site, which is distinct from that of acetylcholine.

Fig. 6.
Fig. 6.

Antagonism by 4FP-TQS of 4BP-TQS-evoked responses on α7 nAChRs. A, representative trace showing activation of wild-type α7 nAChRs by 4BP-TQS (10 μM) followed by the coapplication of 4FP-TQS (100 μM). Applications of allosteric modulators are indicated by horizontal lines. B, dose-response data illustrating the ability of 4FP-TQS to inhibit responses evoked by a submaximal (EC50) concentration of 4BP-TQS (10 μM). C, dose-response data are presented for a range of concentrations of 4FP-TQS on responses evoked by a submaximal (EC50) concentration of acetylcholine with either wild-type α7 nAChRs (●) or α7 nAChRs containing the M253L mutation (○). Data are means ± S.E.M. of at least three independent experiments, each from different oocytes.

Discussion

There is extensive evidence demonstrating that conventional nAChR agonists such as acetylcholine bind to an extracellular orthosteric site located at the interface between two subunits (Arias, 2000; Karlin, 2002; Sine, 2002). Typically, heteromeric nAChRs contain two or three potential orthosteric agonist binding sites. In the case of the well characterized nAChR subtype from the Torpedo spp. electric organ, there are two acetylcholine binding sites, located at the α/δ and the α/γ subunit interfaces (Blount and Merlie, 1989; Sine and Claudio, 1991). In contrast, in homomeric nAChRs, such as α7, there are five potential binding sites for agonists such as acetylcholine or for other ligands that interact with the orthosteric binding site ligands, such as competitive antagonists (Palma et al., 1996).

More recently, activation of nAChRs by allosteric agonists has been described and has been proposed to occur by the interaction with a transmembrane binding site located within an intrasubunit cavity (Gill et al., 2011). This allosteric site has also been proposed as being the binding site for a series of α7 PAMs (Young et al., 2008; Collins et al., 2011). In addition, photoaffinity labeling studies, conducted with nAChRs purified from the Torpedo spp. electric organ, have provided further evidence that a variety of ligands (including volatile anesthetics) interact with a transmembrane modulatory site (Ziebell et al., 2004; Garcia et al., 2007). Indeed, there is increasing evidence that transmembrane sites are important modulatory sites in a range of Cys-loop neurotransmitter receptors, including those gated by GABA and glycine (Ye et al., 1998; Hosie et al., 2006).

In previous studies (Gill et al., 2011), one of the most obvious differences between agonist activation of α7 by acetylcholine and by the allosteric agonist 4BP-TQS was the marked difference in rates of agonist-induced receptor desensitization. Here we have examined a series of compounds, all chemically related to 4BP-TQS. Significant differences were observed in the rates of receptor desensitization when the bromine atom of 4BP-TQS was replaced by either a chlorine or iodine atom (Fig. 2B); however, in all cases, levels of desensitization caused by these compounds were much slower than the very rapid desensitization that is characteristic of α7 nAChRs when activated by acetylcholine (Couturier et al., 1990). Rates of desensitization, rates of activation, and also rates of recovery after removal of agonist were found to increase as the size of the halogen atom decreased (Fig. 2C). However, introduction of the smallest halogen atom (fluorine) at this position (4FP-TQS) resulted in a complete loss of agonist activity (Fig. 4), as did the introduction of a hydrogen atom (P-TQS) or a hydroxyl group (4HP-TQS). However, in all cases, PAM activity was retained (Fig. 5). It seems, therefore, that relatively minor changes to the structure of these compounds can have a profound influence on their pharmacological properties. It is possible that these differences can be explained entirely by steric effects. For example, the slower activation rate observed with 4IP-TQS than with either 4BP-TQS or 4CP-TQS may be a consequence of reduced accessibility to its binding site.

Although it is possible that the differences in size between fluorine and the other three halogens could explain the differences in agonist activity observed, differences in chemical properties may also be significant. For example, halogens differ in their electrostatic surface potential. Indeed, such differences have been suggested to be responsible for the ability of organic compounds containing chlorine, bromine, or iodine (but not those containing fluorine) to form halogen bonds (Auffinger et al., 2004; Politzer et al., 2007). However, the fact that agonist activity was seen when the halogen atom was replaced by a methyl group (4MP-TQS) or a trifluoromethyl group (4TF-TQS) but not when it was replaced with a hydrogen atom (P-TQS) or a hydroxyl group (4HP-TQS) argues that the size of the group attached to the 4-position of the phenyl ring may be more important than its chemical or electrostatic properties in determining allosteric agonist activity.

Of particular note was the much faster activation rate observed with acetylcholine than with any of the allosteric agonists examined. This difference in activation rates prompted us to examine the consequence of coapplying acetylcholine with allosteric agonists (Fig. 3). Of interest, coapplication of acetylcholine with an allosteric agonist resulted in receptor activation that could be resolved into two components (Fig. 3). An initial desensitizing response was seen, which is typical of activation by acetylcholine, and was followed by a secondary response displaying slower activation and slower desensitization. This secondary response is typical of activation by allosteric agonists (Gill et al., 2011). These two components of the agonist response are, presumably, a consequence of the ability of acetylcholine to access its extracellular orthosteric binding site more rapidly than allosteric agonists are able to access their binding sites. This observation is further evidence for a difference in the mechanism of action of these two classes of agonist. In previous studies (Gill et al., 2011), we have shown that the response to a submaximal concentration of 4BP-TQS is greatly potentiated by the subsequent coapplication of acetylcholine, indicating that 4BP-TQS may be more potent as a positive allosteric modulator than as an allosteric agonist. Similar experiments conducted with other allosteric agonists described in the present study suggest that this is a common phenomenon for this series of compounds.

In addition to the size of the halogen atom being important in determining pharmacological properties, the position of the halogen is also critical. This was illustrated by the finding that changing the location of the bromine from the para position to either the ortho or meta position resulted in loss of agonist activity. Likewise, no agonist activity was observed for 3,4BP-TQS, indicating that the allosteric agonist properties of 4BP-TQS can also be lost by the addition of a second halogen atom to the phenyl ring. Taken together, these findings illustrate that the arrangement of groups attached to the phenyl ring is critical in conferring allosteric agonist properties on these compounds.

As was described under Materials and Methods, studies conducted with the TQS series of compounds pose some technical problems, due in particular to their low solubility and tendency to stick to plastic tubing and apparatus, which has complicated construction of dose-responses curves. However, in all cases examined (Table 1), activation by allosteric agonists resulted in significantly steeper dose-response curves (and significantly greater Hill coefficients) than observed with acetylcholine. This was particularly apparent for 4CP-TQS, which produced particularly steep and highly reproducible dose-response curves. It is possible that the Hill coefficient estimated for the other allosteric agonists (Table 1) may be an underestimate because of the problems associated with their slow association rates and difficulty in defining the maximum response with the maximum concentrations that could be used.

Several of the compounds examined in this study lacked allosteric agonist activity but caused dramatic potentiation of responses evoked by acetylcholine. Such effects are typical of a range of compounds that have been described as nAChR PAMs. Relatively small changes in chemical structure result in clear pharmacological differences. For example, as has been reported previously (Grønlien et al., 2007; Gill et al., 2011), minimal desensitization is observed with TQS (Fig. 5B). In contrast, progressively faster rates of desensitization were observed with the other PAMs examined (Fig. 5B). The terms “type I” and “type II” have been used extensively to describe PAMs acting on α7 nAChRs that either have no effect on receptor desensitization (type I) or cause a loss of desensitization (type II). However, it seems that this system of classification may be an oversimplification (Dunlop et al., 2009; Malysz et al., 2009; Williams et al., 2011), and data obtained in this study support this conclusion. Indeed, it has been suggested that α7 nAChR PAMs may have a continuous spectrum of effects ranging from those with minimal effects on desensitization to those that cause complete loss (Dinklo et al., 2011).

We have also demonstrated that α7 nAChR PAMs such as 4FP-TQS can act as antagonists of responses evoked by allosteric agonists such as 4BP-TQS (Fig. 6). The simplest explanation for this observation is that these two chemically similar allosteric modulators interact at a common site. The binding of a PAM such as 4FP-TQS to its allosteric site can have two opposing effects: allosteric potentiation of responses to orthosteric agonists such as acetylcholine and antagonism of responses to allosteric agonists such as 4BP-TQS. The former is presumably a consequence of 4FP-TQS and acetylcholine binding to different sites, and the latter is a consequence of 4FP-TQS and 4BP-TQS binding competitively to a common site. Of interest, in cases in which this was examined, the range of EC50 values determined for the allosteric agonists (Table 1) was not significantly different from that of the related compounds with only PAM activity (Table 2). This finding would argue that, in addition to evidence that allosteric agonists and PAMs may be acting at a common site, they seem to do so with broadly similar apparent affinities.

Work described here and elsewhere supports the conclusion that allosteric agonists and PAMs of α7 nAChRs can bind to a common transmembrane site (Young et al., 2008; Collins et al., 2011; Gill et al., 2011). This is supported by evidence that the effects of both allosteric agonists and PAMs can be blocked completely by a single point mutation (M253L) located in the transmembrane region: a mutation that has no significant effect on agonist activation by acetylcholine (Young et al., 2008). Both allosteric agonists and PAMs are presumably able to reduce the energy barrier for transitions between open and closed states of the receptor. The difference between the two classes of allosteric modulator is that those compounds that lack agonist activity are able to stabilize the open conformation of the receptor efficiently only in the presence an orthosteric agonist such as acetylcholine.

Authorship Contributions

Participated in research design: Gill, Dhankher, Sheppard, Sher, and Millar.

Conducted experiments: Gill and Dhankher.

Contributed new reagents: Dhankher and Sheppard.

Performed data analysis: Gill, Dhankher, Sheppard, Sher, and Millar.

Wrote or contributed to the writing of the manuscript: Gill, Dhankher, Sheppard, Sher, and Millar.

Footnotes

  • ↵Embedded Image The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.

  • This work was supported by the Biotechnology and Biological Sciences Research Council [Grant BB/F017146/1]; an Engineering and Physical Sciences Research Council Advanced Research Fellowship [Grant EP/E052789/1] (to T.S.); a Biotechnology and Biological Sciences Research Council (BBSRC) Collaborative Award in Science and Engineering Ph.D. studentship cofunded by BBSRC and Eli Lilly (to J.K.G.); and a Ph.D. studentship from the University College London Ph.D. program in drug discovery (P.D.).

  • Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

    http://dx.doi.org/10.1124/mol.111.076026.

  • ABBREVIATIONS:

    nAChR
    nicotinic acetylcholine receptor
    2BP-TQS
    4-(2-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide
    2N-TQS
    4-(naphthalen-2-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide
    3,4BP-TQS
    4-(3,4-dibromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide
    3BP-TQS
    4-(3-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide
    3IP-TQS
    4-(3-iodophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide
    4BP-TQS
    4-(4-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide
    4CP-TQS
    4-(4-chloroophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide
    4FP-TQS
    4-(4-fluorophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide
    4HP-TQS
    4-(4-hydroxyphenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide
    4IP-TQS
    4-(4-iodoophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide
    4MP-TQS
    4-p-tolyl-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide
    4TF-TQS
    4-(4-(trifluoromethyl)phenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide
    ANOVA
    analysis of variance
    cRNA
    complementary RNA
    PAM
    positive allosteric modulator
    P-TQS
    4-phenyl-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide
    TQS
    4-(napthalen-1-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide.

  • Received January 16, 2012.
  • Accepted February 10, 2012.

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Allosteric Modulation of the α7 Nicotinic Receptor

JasKiran K. Gill, Persis Dhankher, Tom D. Sheppard, Emanuele Sher and Neil S. Millar
Molecular Pharmacology May 1, 2012, 81 (5) 710-718; DOI: https://doi.org/10.1124/mol.111.076026
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