Differing activity profiles of the stereoisomers of 2,3,5,6TMP-TQS, a putative silent allosteric modulator of a7 nAChR

Article abstract of DOI:10.1124/mol.120.119958

Many synthetic compounds to which we attribute specific activities are produced as racemic mixtures of stereoisomers, and it may be that all the desired activity comes from a single enantiomer. We have previously shown this to be the case with the a7 nicotinic acetylcholine receptor positive allosteric modulator (PAM) 3a,4,5,9b-Tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[ c]quinoline-8-sulfonamide (TQS) and the a7 ago-PAM 4BP-TQS. Cis-trans-4-(2,3,5,6-tetramethylphenyl)-3a,4,5,9b-tetrahydro- 3H-cyclopenta[c]quinoline-8-sulfonamide (2,3,5,6TMPTQS), previously published as a "silent allosteric modulator" and an antagonist of a7 allosteric activation, shares the same scaffold with three chiral centers as the aforementioned compounds. We isolated the enantiomers of 2,3,5,6TMP-TQS and determined that the (2) isomer was a significantly better antagonist than the (+) isomer of the allosteric activation of both wild-type a7 and the nonorthosterically activatible C190A a7 mutant by the ago-PAM GAT107 (the active isomer of 4BP-TQS). In contrast, (+)2,3,5,6TMPTQS proved to be an a7 PAM. (2)2,3,5,6TMP-TQS was shown to antagonize the allosteric activation of a7 by the structurally unrelated ago-PAM B-973B as well as the allosteric activation of the TQS-sensitive a4b2L159M mutant. In silico docking of 2,3,5,6TMP-TQS in the putative allosteric activation binding site suggested a specific interaction of the (2) enantiomer with a7T106, and allosteric activation of a7T106 mutants was not inhibited by (2)2,3,5,6TMP-TQS, confirming the importance of this interaction and supporting the model of the allosteric binding site. Comparisons and contrasts between 2,3,5,6TMP-TQS isomers and active and inactive enantiomers of other TQS-related compounds identify the orientation of the cyclopentenyl ring to the plane of the core quinoline to be a crucial determinate of PAM activity.

Full text of DOI:10.1124/mol.120.119958

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Title Page
Differing activity profiles of the stereoisomers of 2,3,5,6TMP-TQS, a putative silent allosteric
modulator of a7 nAChR
Roger L. Papke*, Sumanta Garai, Clare Stokes, Nicole A. Horenstein, Arthur D. Zimmerman,
Khalil A. Abboud, and Ganesh A. Thakur*
Department of Pharmacology and Therapeutics, University of Florida, PO Box 100267,
Gainesville FL, 32610-0267 (RLP, CS, ADZ)
Department of Pharmaceutical Sciences, School of Pharmacy, Bouvé College of Health Sciences,
Northeastern University, Boston, MA 02115 (SG, GAT)
Department of Chemistry, University of Florida, Gainesville, FL 32611-7200 (NAH, KAA)
This research was supported by the National Institutes of Health Grants, R01-GM57481 and RO1-
EY024717
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Running title page
Running title: Stereochemistry of a7 nAChR PAMs
*To whom correspondence should be addressed:
Name: Roger L. Papke
Phone: 352-392-4712
Fax: 352-392-3558
E-mail: rlpapke@ufl.edu
Address: Department of Pharmacology and Therapeutics
University of Florida
P.O. Box 100267
Gainesville, FL 32610-0267
Name: Ganesh A. Thakur
Phone: 617-373-8163
E-mail: g.thakur@northeastern.edu
Department of Pharmaceutical Sciences
School of Pharmacy
Bouvé College of Health Sciences,
Northeastern University
Boston, MA 02115
Number of text pages:............................................................28
Number of tables:...................................................................1
Number of figures:.................................................................12
Number of references:............................................................36
Number of words in Abstract:................................................249
Number of words in Introduction: .........................................747
Number of words in Discussion:............................................1361
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Abbreviations: nAChR, nicotinic acetylcholine receptor; ACh, acetylcholine TQS 3a,4,5,9b-
Tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c]quinoline-8-sulfonamide; PAM, positive
allosteric modulator; AA site, allosteric activation binding site; 2,3,5,6TMP-TQS, Cis-trans-4-
(2,3,5,6-tetramethylphenyl)-3a,4,5,9b-te-trahydro-3H-cyclopenta[c]quinoline-8-sulfonamide;
2,4,MP-TQS, cis-cis-4-(2,4-dimethylphenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-
sulfonamide; GAT927, 4-(4,5,6,7-tetrahydrobenzo[b]thiophen-3-yl)-3a,4,5,9b-tetrahydro-3H-
cyclopenta[c]quinoline-8-sulfonamide; GAT107, (3aR,4S,9bS)-4-(4-bromophenyl)-3a,4,5,9b-
tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide; B-973B, (S)-3-(3,4-difluorophenyl)-N-(1-
(6-(4-(pyridin-2-yl)piperazin-1-yl)pyrazin-2-yl)ethyl)propanamide
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Abstract
Many synthetic compounds to which we attribute specific activities are produced as racemic
mixtures of stereoisomers, and it may be that all of the desired activity comes from a single
enantiomer. We have previously shown this to be the case with the a7 nicotinic acetylcholine
receptor positive allosteric modulator (PAM) TQS, and the a7 ago-PAM 4BP-TQS. 2,3,5,6TMP-
TQS, previously published as a "silent allosteric modulator" and an antagonist of a7 allosteric
activation, shares the same scaffold with three chiral centers as the aforementioned compounds.
We isolated the enantiomers of 2,3,5,6TMP-TQS and determined that the (-) isomer was a
significantly better antagonist than the (+) isomer of the allosteric activation of both wild-type a7
and the non-orthosterically activatible C190A a7 mutant by the ago-PAM GAT107 (the active
isomer of 4BP-TQS). In contrast (+)2,3,5,6TMP-TQS proved to be an a7 PAM. (-)2,3,5,6TMP-
TQS was shown to antagonize the allosteric activation of a7 by the structurally unrelated ago-
PAM B-973B as well as the allosteric activation of the TQS-sensitive a4b2L15'M mutant. In
silico docking of 2,3,5,6TMP-TQS in the putative allosteric activation binding site suggested a
specific interaction of the (-) enantiomer with a7T106, and allosteric activation of a7T106 mutants
was not inhibited by (-)2,3,5,6TMP-TQS, confirming the importance of this interaction and
supporting the model of the allosteric binding site. Comparisons and contrasts between
2,3,5,6TMP-TQS isomers and active and inactive enantiomers of other TQS-related compounds
identify the orientation of the cyclopentenyl ring to the plane of the core quinoline to be a crucial
determinate of PAM activity.
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Significance statement
Many synthetic ligands are in use as racemic preparations. We show that one enantiomer
of the TQS analog 2,3,5,6TMP-TQS, originally reported to lack activity when used as a racemic
preparation, is an a7 nAChR positive allosteric modulator (PAM). The other enantiomer is not a
PAM, but it is an effective allosteric antagonist. In silico studies and structural comparisons
identify essential elements of both the allosteric ligands and receptor binding sites important for
these allosteric activities.
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Introduction
Nicotinic acetylcholine receptors (nAChR) are pentameric assemblies of subunits that
function as ligand-gated ion channels (Papke, 2014). In the peripheral nervous system,
heteromeric nAChR mediate synaptic transmission at neuromuscular junctions and autonomic
ganglia. In the brain, heteromeric nAChR mediate both cognitive effects of ACh and addictive
effects of nicotine. A third class of nAChR are those that can form functional receptors as
homopentamers, and the best studied of these are composed of a7 subunits. Several features
distinguish homomeric a7 receptors from heteromeric nAChR. While all nAChR subtypes
desensitize in the continued presence of agonist, the desensitization of a7 nAChR to high
concentrations of agonist is especially rapid but also readily reversible. As a consequence of a7's
unique mode of desensitization, a7 receptors have an intrinsically low probability of channel
activation (Williams et al., 2012).
All nAChR are allosteric proteins, such that their functional properties are regulated by
sites other than the orthosteric sites where typical agonists bind (Bertrand and Gopalakrishnan,
2007; Changeux, 1981; Williams et al., 2011). Some of the most profound effects of allosteric
ligands have been described for a7 nAChR, and some positive allosteric modulators (PAMS)
reverse intrinsic limitations on channel activation (Williams et al., 2012). The binding site for a7
PAMs is within the receptor's transmembrane domains (Young et al., 2008) and requires the
presence of a methionine residue that is unique to a7 in the pore-forming second transmembrane
domain (Stokes et al., 2019).
One large class of PAMs is based on TQS, a sulfonamide-containing multi-ring molecule
(Gill et al., 2012; Gronlien et al., 2007). In addition to acting as a PAM, one analog, 4BP-TQS,
produces direct allosteric activation of a7 receptors as well as long-lived potentiation of orthosteric
agonist responses, identifying the compound as an "ago-PAM". GAT107 is the active isomer of
4BP-TQS (Papke et al., 2014), and both allosteric activation and positive modulation of a7 by
GAT107 require the specific sequence in the a7 transmembrane domains. While it was initially
hypothesized that this sequence alone was sufficient for allosteric activation (Gill et al., 2011), we
have provided evidence for a second allosteric activation binding site (AA site) in the receptor's
extracellular domain (Gulsevin et al., 2019; Horenstein et al., 2016; Papke et al., 2014).
2,3,5,6TMP-TQS
(Cis-trans-4-(2,3,5,6-tetramethylphenyl)-3a,4,5,9b-tetrahydro-3H-
cyclopenta[c]quinoline-8-sulfonamide) was previously identified as a “silent allosteric modulator”
of a7 nAChR (Gill-Thind et al., 2015), based on its apparent lack of positive or negative effect on
activation by 100 µM of ACh. Although apparently silent in regard to activation by 100 µM ACh,
2,3,5,6TMP-TQS blocked allosteric activation of a7 by the ago-PAM 2,4,MP-TQS. Subsequently
we used 2,3,5,6TMP-TQS (previously identified as 2,3,5,6MP-TQS (Gill-Thind et al., 2015)) as a
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tool to characterize the allosteric agonist activity of GAT107 (Papke et al., 2014; Thakur et al.,
2013).
The allosteric activation of a7 produced by an application of GAT107 persists only as long
as the drug is free in solution. Repeated applications of GAT107 produce additional episodes of
allosteric activation, suggesting that there is rapid binding and dissociation of GAT107 at the AA
site. In contrast to the rapid transient effects of GAT107 as an allosteric agonist, following a single
application of GAT107, subsequent applications of ACh alone are increased in amplitude and
duration. This "primed potentiation", can persist for at least{?} an hour (Papke et al., 2018). We
have observed that the co-application of GAT107 with 2,3,5,6TMP-TQS could specifically reduce
direct allosteric activation without apparent effects on primed potentiation, suggesting that
2,3,5,6TMP-TQS is a selective antagonist of the extracellular AA site with little or no effect at the
transmembrane site required for allosteric potentiation.
While allosteric activation by GAT107 does requires positive allosteric modulation at the
transmembrane PAM binding site, it does not require a functional binding site for orthosteric
agonists (Horenstein et al., 2016). We recently identified ligands previously classified as silent
agonists that produce PAM-dependent activation of a7 by binding to the same AA site as GAT107
(Gulsevin et al., 2019). Racemic 2,3,5,6TMP-TQS blocks this activity, but in the course of our
experiments, we also noted that 2,3,5,6TMP-TQS could additionally potentiate the responses to
low concentrations of ACh, contrary to initial reports. Since we have previously documented large
differences in the activity profiles of the stereoisomers of 4BP-TQS and TQS (Stokes et al., 2019),
we hypothesized that the two stereoisomers of 2,3,5,6TMP-TQS might discriminate between the
AA and PAM binding sites. Our results confirm that only (+)2,3,5,6TMP-TQS is a PAM and that
while (-)2,3,5,6TMP-TQS is not a PAM, it is a more potent allosteric antagonist than
(+)2,3,5,6TMP-TQS.
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Materials and Methods
Chemicals and reagents
Acetylcholine chloride (ACh) and buffer chemicals were purchased from Sigma-Aldrich
Chemical Company (St. Louis, MO). GAT107 and racemic 2,3,5,6TMP-TQS were synthesized in
Thakur laboratory by Dr. Sumanta Garai (Northeastern University, Boston, MA) following the
published procedures (Gill-Thind et al., 2015; Kulkarni and Thakur, 2013; Thakur et al., 2013).
PNU-120596 was synthesized in the Horenstein laboratory by Dr. Kinga Chojnacka following the
published procedure (Hurst et al., 2005).
The 2,3,5,6TMP-TQS isomers were synthesized and isolated as described in the
supplemental data.
Expression in Xenopus oocytes
The human a7 nAChR clone was obtained from Dr. J. Lindstrom (University of
Pennsylvania, Philadelphia, PA). The human resistance-to-cholinesterase 3 (RIC-3) clone was
obtained from Dr. M. Treinin (Hebrew University, Jerusalem, Israel) and co-injected with a7 to
improve the level and speed of a7 receptor expression without affecting the pharmacological
properties of the receptors (Halevi et al., 2003). Subsequent to linearization and purification of
the plasmid cDNAs, cRNAs were prepared using the mMessage mMachine in vitro RNA
transcription kit (Ambion, Austin, TX). The a7C190A mutant was made as previously described
with a C116S double mutation to prevent spurious disulfide bond formation with the free cysteine
(Papke et al., 2011). The b2L15'M mutant was prepared as previously described (Stokes et al.,
2019) and co-expressed with a b2-a4 concatamer (Zhou et al., 2003) to obtain receptors with a
single mutant b subunit in the accessory subunit position, outside the ACh binding sites.
Oocytes were surgically removed from mature female Xenopus laevis frogs (Nasco, Ft.
Atkinson, WI). Frogs were maintained in the Animal Care Service facility of the University of
Florida, and all procedures were approved by the University of Florida Institutional Animal Care
and Use Committee. In brief, the frog was first anesthetized for 15-20 min in 1.5 liters frog tank
water containing 1 g of 3-aminobenzoate methanesulfonate (MS-222) buffered with sodium
bicarbonate. The harvested oocytes were treated with 1.4 mg/ml Type 1 collagenase (Worthington
Biochemicals, Freehold NJ) for 2-4 h at room temperature in calcium-free Barth’s solution (88
mM NaCl, 1 mM KCl, 2.38 mM NaHCO3, 0.82 mM MgSO4, 15 mM HEPES, and 12 mg/l
tetracycline, pH 7.6) to remove the ovarian tissue and the follicular layers. Stage V oocytes were
subsequently isolated and injected with 50 nl of 5-20 ng nAChR subunit cRNA. Oocytes were
maintained in Barth’s solution with calcium (additional 0.32 mM Ca(NO3)2 and 0.41 mM CaCl2),
and recordings were carried out 1-14 days after injection.
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Two-electrode voltage clamp electrophysiology
Experiments were conducted using OpusXpress 6000A (Molecular Devices, Union City,
CA) (Papke and Stokes, 2010). Both the voltage and current electrodes were filled with 3 M KCl.
Oocytes were voltage-clamped at -60 mV at room temperature (24˚C). The oocytes were bath-
perfused with Ringer’s solution (115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2, 10 mM HEPES,
and 1 µM atropine, pH 7.2) at 2 ml/min for a7 and at 4 ml/min for a4b2. For wild-type a7 and
a4b2 mutant receptors, to evaluate the effects of experimental compounds compared to ACh-
evoked responses of various nAChR subtypes expressed in oocytes, control responses were
defined as the average of two initial applications of ACh made before test applications. The control
ACh concentrations were 60 µM for the wild type (WT) a7 receptor experiments and 10 µM ACh
for the a4b2 mutants. The average of independent 1 µM GAT107 responses were used as the
control for the a7C190A experiments.
Solutions were applied from 96-well plates via disposable tips. Drug applications were 12
s in duration followed by 181 s washout periods for a7 and 6 s in duration followed by 241 s
washout periods for a4b2. The responses were calculated as both peak current amplitudes and net
charge, as previously described (Papke and Papke, 2002). Data were collected at 50 Hz, filtered
at 20 Hz, and analyzed by Clampfit 9.2 or 10.0 (Molecular Devices) and Excel (Microsoft,
Redmond, WA). Data were expressed as means ± SEM from at least four oocytes for each
experiment and plotted with Kaleidagraph 4.5.2 (Abelbeck Software, Reading, PA). Multi-cell
averages were calculated for comparisons of complex responses. Averages of the normalized data
were calculated for each of the 10,322 points in each of the 206.44 s traces (acquired at 50 Hz), as
well as the standard errors for those averages.
Data and statistical analysis
The curve fits to concentration-response studies were generated using the Levenberg-
Marquardt algorithm to obtain the best Chi-Square fit to the Hill equation using the Kalidagraph
plotting program (V4.5.2). The errors reported in the supplemental data are the calculated standard
errors of the fit parameters based on the goodness of fit (Chi-Square and r values provided). Data
reported in Table 1a are the averaged values of the fit parameters for five cells under each condition
± standard deviation (SD).
For comparisons of results containing three or more groups, one-way and two-way
ANOVAs were used. For data where variances were different, as measured using the Brown-
Forsythe test, Brown-Forsythe ANOVAs were performed with Dunnett’s T3 multiple comparisons
test. For all other data requiring an ANOVA, ordinary one-way and two-way ANOVAs were
performed with Tukey’s posthoc multiple comparisons test. GraphPad Prism 8 software (San
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Diego, CA) was used to perform these statistical measures. GraphPad Prism 8 software was also
used to generate the difference between group means or "Gardner-Altman plot" (Figure 7B)(Ho et
al., 2019). Differences of mean and confidence interval values were generated from the Tukey’s
posthoc multiple comparisons test.
Comparisons of all other results were made using two-tailed t-tests between the pairs of
experimental measurements. A value of p <0.05 was used to constitute a minimum level of
significance. These statistics were calculated using an Excel template provided in Microsoft
Office.
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Results
Properties of racemic 2,3,5,6TMP-TQS
Shown in Figure 1 are the selective effects of racemic 2,3,5,6TMP-TQS on the direct
allosteric activation of a7 receptors by 10 µM GAT107 (p < 0.001). With this co-application
protocol, there was no effect on the long-lived potentiation of subsequent ACh responses.
Note that the previous work (Gill-Thind et al., 2015) that reported that 2,3,5,6TMP-TQS
had no effect on responses to "a sub-maximal concentration of ACh" relied on peak currents as
measurements of a7 responses to ACh applications. However, the a7 peak currents responses to
applications of ACh at concentrations ≥ 60 µM occur in advance of full solution delivery (Papke
and Thinschmidt, 1998), making them poor reporters of the concentration dependence of receptor
activation compared to measurements of the integrated net charge of agonist-evoked responses
(Papke and Papke, 2002). The concentration of 100 µM ACh used by Millar and co-workers is in
fact sufficient to produce maximal net-charge responses. Although 2,3,5,6TMP-TQS had no effect
when applied alone to cells expressing a7 nAChR (Gill et al., 2012), in the course of our work
with racemic 2,3,5,6TMP-TQS, we saw clear indications of PAM activity when the ACh
concentrations were lower (Figure 2).
Properties of 2,3,5,6TMP-TQS isomers on GAT107 activity with wild-type and mutant a7
When synthesized, 2,3,5,6TMP-TQS is a racemic mixture of isomers. In order to test the
hypothesis that PAM and allosteric antagonist activities of 2,3,5,6TMP-TQS might be associated
with specific enantiomers, we separated the (+) and (-) enantiomers and resolved their crystal
structures (Figure 3). Also shown in Figure 3 are the structures of the enantiomers of TQS and
4BP-TQS that we have previously shown to be the active α7 PAMs (Stokes et al., 2019; Thakur et
al., 2013). All of these compounds have three chiral centers, two that join the cyclopentenyl ring
to the quinoline, and a third that connects to the aromatic rings at the base. While the two that are
part of the cyclopentenyl ring are constrained to orient in concert, the lower group may be either
cis or trans relative to the cyclopentenyl ring of the molecule. As previously hypothesized (Gill et
al., 2012), due to the steric hindrances of two ortho methyl groups, through Povarov reaction we
only obtained trans isomers of 2,3,5,6TMP-TQS and not cis diastereomers. It was also originally
hypothesized (Gill et al., 2012) that the likely trans-configuration accounted for the apparent lack
of PAM activity for 2,3,5,6TMP-TQS.
We compared the effects of racemic 2,3,5,6TMP-TQS and the two isolated isomers on the
two phases of GAT107 activity using a protocol with a 30-second pre-application of 100 µM
2,3,5,6TMP-TQS prior to the co-application of 2,3,5,6TMP-TQS with 10 µM GAT107. The
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racemic 2,3,5,6TMP-TQS and each isomer inhibited the direct activation by GAT107 as
determined by a Brown-Forsythe ANOVA (F(3,27) = 15.17, p < 0.0001). The racemic
2,3,5,6TMP-TQS inhibited activation by GAT107 by 98%, while (+)2,3,5,6TMP-TQS and (-
)2,3,5,6TMP-TQS inhibited GAT107 by 93% and 99.8%, respectively. Therefore, the inhibition
by (+)2,3,5,6TMP-TQS was less than by the racemic (95% CI [-7.33, -0.38], p = 0.01, Figure 4A).
Note that with this pre-incubation protocol, there was also an inhibition of the potentiated
ACh response after the GAT107 (F(3, 27) = 9.11, p = 0.0002). Both (+)2,3,5,6TMP-TQS (95%
CI [34.65, 220.2], p = 0.004, Figure 4B) and (-)2,3,5,6TMP-TQS (95% CI [77.40, 262.9], p =
0.0002, Figure 4B) produced an inhibition of the potentiated ACh response after the GAT107. The
racemic 2,3,5,6TMP-TQS also produced a small inhibition of the potentiated ACh response after
the GAT107 (95% CI [-3.13, 188.9], p = 0.06, Figure 4B), and the inhibition with (-)2,3,5,6TMP-
TQS or (+)2,3,5,6TMP-TQS was equivalent to that produced by the racemic.
We tested the potency of (-)2,3,5,6TMP-TQS for inhibiting the direct activation of wild-
type a7 produced by 10 µM GAT107 with a co-application protocol (Figure 4C). There was
inhibition at all concentrations tested ≥ 3 µM using a Brown-Forsythe ANOVA (F(5, 11.55) =
22.79, p < 0.0001) with Dunnett’s T3 posthoc multiple comparisons test (3 µM: 95% CI [2.33,
29.70], p = 0.009; 10 µM: 95% CI [7.41, 36.00], p = 0.0009; 30 µM: 95% CI [11.27, 38.31], p =
0.0003; 100 µM: 95% CI [19.63, 47.58], p = 0.0002).
Key elements for the function of any nAChR are the disulfide-coupled vicinal cysteines
(C190 and C191 in the a7 sequence) at the tip of the alpha subunit C-loops. Disruption of this
link causes loss of function (Papke, 2014). We have previously shown that although a7C190A
mutants are insensitive to ACh, even when co-applied with PNU-120596, they are effectively
activated by GAT107 and that racemic 2,3,5,6TMP-TQS blocked that activation (Gulsevin et al.,
2019). In basic co-application experiments, we observed using a Brown-Forsythe ANOVA that
both isomers reduced GAT107-evoked currents of a7C190A (F(2, 9.09) = 11.25, p = 0.0035,
Figure 5), although the inhibition by (-)2,3,5,6TMP-TQS was greater than by (+)2,3,5,6TMP-TQS
(95% CI [-0.58, 11.64], p = 0.04).
We also tested the two 2,3,5,6TMP-TQS enantiomers for their ability to inhibit a7
allosteric activation by the structurally unrelated ago-PAM B-973B (Quadri et al., 2019). Like
GAT107, it is able to produce allosteric activation of a7 receptors and potentiate the responses to
ACh. We determined that (-)2,3,5,6TMP-TQS (95% CI [-0.45, 58.67], p = 0.05), but not
(+)2,3,5,6TMP-TQS (95% CI [-24.38, 36.68], p = 0.87), was also an effective antagonist of
allosteric activation by B-973B (F(2, 19) = 3.55, p = 0.049, Figure 6). Neither enantiomer had any
effect on the residual potentiation produced by this agent (F(2, 9) = 0.42, p = 0.67).
PAM activity of 2,3,5,6TMP-TQS isomers
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We conducted ACh concentration-response studies with or without co-application of the
2,3,5,6TMP-TQS isomers at 30 µM (Figure 7). Visual inspection of the curves suggests that
(+)2,3,5,6TMP-TQS functions as a PAM and (-)2,3,5,6TMP-TQS does not. We use several
statistical approaches to confirm this using both the averaged data (Figure 7) and an analysis of
the concentration-response data of individual cells (n=5 under each condition, see Supplemental
Data). Specifically, using two-way ANOVA, we found an interaction effect between ACh
concentration and co-application (F(12, 91) = 10.21, p < 0.0001, Figure 7A). Effects of
(+)2,3,5,6TMP-TQS were seen across almost the entire concentration range (10 µM - 1000 µM;
Figure 7B), including maximally effective concentrations ≥ 100 µM. When analyzing curve-fit
parameters by Brown-Forsythe ANOVA (EC50: (F(2, 12) = 6.97, p=0.05); Imax: (F(2, 12) = 11.69,
p=0.01)), the concentration-response function for net-charge responses (Papke and Papke, 2002)
was relatively unaffected by addition of (-)2,3,5,6TMP-TQS (EC50: p=0.17; Imax: p=0.53);
however, (+)2,3,5,6TMP-TQS increased ACh potency by 65% (p=0.0006) and efficacy by 91%
(p<0.0001) (Table 1a). We then ran an ANOVA for the logEC50 values with responses normalized
as a percentage of the average responses across five replicates (F(2, 12) = 26.10, p < 0.0001). We
saw that addition of (-)2,3,5,6TMP-TQS caused a similar decrease in potency (95% CI [-0.67, -
0.08], p=0.015), and an increase in potency with addition of (+)2,3,5,6TMP-TQS (95% CI, [0.18,
0.77], p=0.003). When calculating the inverse log from logEC50, values for EC50 using this
method were consistent, as ACh alone was 20.61 µM with the addition of (-)2,3,5,6TMP-TQS at
48.52 µM and (+)2,3,5,6TMP-TQS at 6.87 µM.
While all of these analyses confirm the PAM activity of (+)2,3,5,6TMP-TQS, it should be
noted that the magnitude of the (+)2,3,5,6TMP-TQS potentiation was at least an order of
magnitude less than previously reported for (-)TQS (Stokes et al., 2019) or GAT107 ((+)4BP-
TQS) (Papke et al., 2014).
2,3,5,6TMP-TQS isomer effects on mutant a4b2 receptors sensitive to a7 PAMs
We have recently described a point mutation (L15'M) in the second transmembrane domain
of neuronal b subunits that results in heteromeric neuronal nAChR that are sensitive to select a7
PAMs in the TQS family (Stokes et al., 2019). In a7, this residue is a methionine, and the opposite
mutation results in a loss of PAM sensitivity.
We have previously shown that the inclusion of a single mutant subunit is sufficient to
produce sensitivity to a7 PAMs and that the presence of additional mutant subunits reduces
receptor function (Stokes et al., 2019). In order to obtain a4b2 receptors with a single b2L15'M
mutant subunit, we co-expressed a b2-a4 concatamer (Zhou et al., 2003) with monomeric
b2L15'M. The co-application of (-)2,3,5,6TMP-TQS with ACh gave a small increase in peak
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current (p < 0.01) but had no effect on net charge (Figure 8). In contrast, co-application of
(+)2,3,5,6TMP-TQS with ACh produced large increases in both peak current and net-charge
responses (p< 0.001), as would be expected for a type II PAM (Gronlien et al., 2007). The co-
application of (+)2,3,5,6TMP-TQS increased peak current and net charge by an average 5.7 ± 0.3
and 4.6 ± 0.6 fold, respectively (n = 8, p < 0.001).
2,3,5,6TMP-TQS isomer effects on the allosteric activation of mutant a4b2 receptors
In our initial characterization of heteromeric neuronal nAChR sensitive to a7 PAMs
(Stokes et al., 2019), we identified GAT927 as an extremely potent and efficacious PAM for these
mutant heteromeric receptors. In addition to strongly potentiating ACh-evoked responses, we
noted that, when applied alone, high concentrations of GAT927 produced direct allosteric
activation, analogous to GAT107's effects on a7 (Figure 9A). We tested the hypothesis that (-
)2,3,5,6TMP-TQS, the isomer better for antagonizing a7 allosteric activation, would also more
effectively antagonize the allosteric activation of the a4b2 mutant receptor by GAT927. While (-
)2,3,5,6TMP-TQS did decrease GAT927 allosteric activation (p < 0.05, Figure 9B), activation by
GAT927 was totally eliminated by (+)2,3,5,6TMP-TQS (Figure 9C). Interestingly, the effects of
the 2,3,5,6TMP-TQS isomers were restricted to the period of direct allosteric activation by
GAT927, and the primed potentiation for a subsequent ACh-evoked response was unaffected
(Figure 9D). As noted above, neither of the isomers had an effect on the net charge or peak current
amplitude of these responses. However, while the responses shown in A and B had not decayed
fully to baseline after the GAT927 applications (baselines were 82 ± 27 and 107 ± 22% the
amplitude of the ACh controls, respectively), the trace in C (magenta in panel D) actually began
with a lower baseline current than the initial control (p < 0.001).
In silico model of (-)2,3,5,6TMP-TQS in the putative allosteric activation site of a7.
We (Gulsevin et al., 2019; Horenstein et al., 2016) and others (Spurny et al., 2015) have
proposed models for an allosteric ligand binding site in the extracellular vestibule of a7 receptors.
We compared the docking of GAT107 and the most active antagonist (-)2,3,5,6TMP-TQS into that
site (Figure 10) and found that (-)2,3,5,6TMP-TQS was notably different from GAT107 in that,
despite an overall similar binding mode, it presented its fused cyclopentenyl ring to T106 whereas
the other compound did not.
We hypothesized that this interaction would be lost with a T106A mutation (Figure 9D).
Using a co-application protocol, we confirmed that 30 µM (-)2,3,5,6TMP-TQS produced good
inhibition of GAT107 allosteric activation of wild-type a7 (p < 0.001) with no effect on the
potentiation of subsequent ACh responses (Figure 11 A). Using the same protocol with a7T106A,
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we saw no changes with 30 µM (-)2,3,5,6TMP-TQS on either GAT107 allosteric activation or
subsequent potentiation (Figure 11B).
Discussion
Stereoisomerism is a critical concern when designing drugs for enzymes and receptors
because stereoisomers can and often do display different pharmacology. Whereas molecules with
a single chiral center will be capable of existing as a pair of enantiomers, molecules with multiple
chiral centers N will have 2N maximum possible stereoisomers, as exemplified by the ring system
of 2,3,5,6TMP-TQS, which has 3 chiral centers and 8 theoretically possible stereoisomers
including enantiomers and diastereomers. However, as noted previously, constraints on the
cyclopentenyl ring and steric hindrances associated with two ortho methyl groups resulted in
exclusive formation and isolation of the trans diastereomer. Although most biological processes
that form organic molecules with chiral centers tend to produce single stereoisomers, such as D-
sugars and L-amino acids, test-tube reactions commonly produce racemic mixtures of isomers.
When these reaction products are then used experimentally, the biologic response may or may not
be deferentially sensitive to the component isomers.
In biological systems, the requirement for isomer specificity is a function of the receptor
and the flexibility of the ligand. Our right foot will readily accept either of a pair of socks but
shows a strong preference for only one of a pair of shoes. In the case of nAChR, while the ACh
binding site of muscle-type receptors shows a preference for the naturally occurring (-) isomer of
nicotine, both isomers are equally effective as low potency blockers of the ion channel (Rozental
et al., 1989). Likewise, both isomers of the neuronal nAChR non-competitive antagonist
mecamylamine have similar efficacy for blocking channel activity of a wide variety of heteromeric
neuronal nAChR (Papke et al., 2001; Papke et al., 2013).
The amino acid residues in neuronal b subunits that determine sensitivity to mecamylamine
face into the ion permeation pathway and are present in at least two subunits per heteromeric
pentamer (Webster et al., 1999). The dimensions of the open pore and the presence of multiple
binding sites may both be permissive factors for the lack of mecamylamine's stereo-selectivity. In
contrast, although the a7 PAM binding site is likely present in all subunits and requires a specific
residue in the pore-forming second transmembrane domain, the actual binding orientation of the
modulator is likely to be within a hydrophobic cavity within the transmembrane helices (Collins
and Millar, 2010; Newcombe et al., 2018; Young et al., 2008). In this tight pocket stereochemical
constraints appear to be very important. This was shown to be the case for three functionally
diverse analogs in the TQS family, TQS itself (Stokes et al., 2019), the ago-PAM 4BP-TQS (Papke
et al., 2014; Thakur et al., 2013), and the allosteric antagonist presented in this work, 2,3,5,6TMP-
TQS, as well as for the structurally unrelated ago-PAM B-973B (Garai et al., 2018; Quadri et al.,
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2019). Additionally, a recently published study of a family of sulfonamide-containing PAMs
showed that the stereochemical orientation of side groups around a critical central cyclopropyl ring
determine whether enantiomers function as Type I or Type II a7 PAMs (Harvey et al., 2019; Wang
et al., 2020).
In order to better understand the important structural elements of TQS-related PAMs, we
made a structural comparison of (-)TQS and (+)2,3,5,6TMP-TQS (Figure 12). The comparison
reveals considerable similarity in the overall three-dimensional shape of the fused ring system
consisting of the arylsulfonamide, piperidine, and cyclopentenyl rings. It is notable that, while
TQS is cis with regard to the cyclopentenyl and napthyl rings, (+)2,3,5,6TMP-TQS has a trans
relationship between the cyclopentenyl and tetramethylbenzene rings. Despite this, the
superposition revealed that the bulky napthyl and tetramethylphenyl rings occupied the region in
space in the overlay. This comparative analysis suggests that the basis for the PAM activity of
(+)2,3,5,6TMP-TQS arises from its overall similarity to the shape of TQS, despite the difference
in stereochemistry of the ring fusions. Further comparison with (+)4BP-TQS (GAT107) (Figure
3) suggests that possibly the most crucial feature for PAM activity is the orientation of
cyclopentenyl ring away from the plane of napthyl ring.
Structural models for the transmembrane domains of a7 have been proposed based on the
relatively low-resolution images of the muscle-type Torpedo receptor and suggestions made for
how PAMs may bind in these domains (Gill et al., 2011; Newcombe et al., 2018). However,
muscle-type receptors are not only insensitive to a7 PAMs, but the key L15'M mutation that
permits heteromeric neuronal nAChR to respond to TQS-type PAMs are ineffective in muscle
nAChR, so the predictive values of these models may be extremely limited. We are optimistic that
in the future, structures of PAM-sensitive mutant heteromeric receptors will become available,
which will allow further insights into the critical differences between the binding of active and
inactive isomers (Stokes et al., 2019), since both a7 and a4b2L15'M receptors discriminate the
same way between the isomers of both TQS and 2,3,5,6TMP-TQS.
While the PAM binding sites of both a7 and a4b2L15'M receptors may be similar, the
mechanism of allosteric activation of these receptors by GAT107 and GAT927, respectively,
seems to be different (Stokes et al., 2019). While we can suggest a specific allosteric agonist site
on a7 (Gulsevin et al., 2019), there is limited evidence for a similar site on a4b2L15'M receptors.
However, while GAT107 does not produce allosteric activation of these receptors (Stokes et al.,
2019), both isomers of 2,3,5,6TMP-TQS do antagonize allosteric activation by GAT927, albeit
with reverse stereo-selectivity than for allosteric antagonism of a7. The observation that neither
2,3,5,6TMP-TQS isomer reduced the potentiation of subsequent ACh response would suggest that
the antagonism of GAT927 allosteric activation is not due to binding at the transmembrane PAM
site.
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An important consideration is that previous work that relied on the use of racemic mixtures
of compounds from the TQS family (Gill et al., 2012; Gill-Thind et al., 2015; Horenstein et al.,
2016; Newcombe et al., 2018) should be viewed with circumspection. While racemic 4BP-TQS
is a mixture of active and inactive compounds that merely has the impact of lowering the effective
concentration, racemic TQS is in fact a mixture of a PAM and a molecule that antagonizes that
activity (Stokes et al., 2019). As we show in this study, racemic 2,3,5,6TMP-TQS carries a mixture
of two different activities. 2,3,5,6TMP-TQS was one of a total of seventeen TQS analogs with
varying activity that were reported in a single study (Gill et al., 2012), and all were characterized
as racemic mixtures, where one isomer may have obscured the properties of another. We have
also evaluated many additional TQS analogs as racemic mixtures including GAT154, GAT155,
GAT904, and GAT193 (Horenstein et al., 2016). Likewise, the ago-PAM of mutant heteromeric
receptors, GAT927, was used as a racemic mixture in these and previous experiments (Stokes et
al., 2019). It seems likely that the efforts required to isolate the isomers of various compounds
would be rewarded by the revelation of pharmacological diversity richer than we might imagine,
with the identification of relatively selective compounds that would not only inform further
structural studies but also have improved therapeutic potential.
The results of docking (Figure 10) suggested that the antagonist activity of (-)2,3,5,6TMP-
TQS at the allosteric activation site would, at least in part, arise from a unique interaction with
T106. The electrophysiological evaluation of mutation, T106A provided support for this
hypothesis via the observation that, relative to wild type, (-)2,3,5,6TMP-TQS lost the ability to
antagonize the allosteric activation by GAT107. The mechanism by which T106 is required for (-
)2,3,5,6TMP-TQS antagonism remains unclear. It is possible that the interaction with T106 is
required for (-)2,3,5,6TMP-TQS to bind with high affinity as an antagonist. Alternatively, T106
may cause (-)2,3,5,6TMP-TQS to adopt a conformation that is intrinsically inactive. Possibly both
of these things are factors. We have confirmed that (-)2,3,5,6TMP-TQS does not become an
allosteric agonist for a7T106A, since co-application with PNU-120596 does not evoke a response
(not shown).
An important conclusion that does emerge from the combination of docking and
mutagenesis studies is that they provide strong support for the location of the AA binding site. It
is instructive to learn that subtle changes in the interactions of a bound ligand can have such
divergent impact on receptor function, confirming that the AA site should be a continued target of
interest for development of new modulators of a7 function.
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Authorship Contributions
Participated in research design: RLP, CS
Conducted experiments: CS, SG, KAA
Contributed new reagents or analytic tools: SG, GAT
Performed data analysis: RLP, ADZ
Wrote or contributed to the writing of the manuscript: RLP, CS, KAA, ADZ, GAT
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Acknowledgements
We thank Lu Wenchi Corrie for conducting oocyte recordings
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Footnotes
This research was supported by the National Institutes of Health Grants, R01-GM57481 and RO1-
EY024717.
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Figure Legends
Figure 1. Co-application of 100 µM racemic 2,3,5,6TMP-TQS selectively inhibits the direct
allosteric activation of a7 nAChR by 10 µM of the ago-PAM GAT107 (p < 0.00001) with no
effect on subsequent potentiation of responses to ACh. Averaged raw data traces for cells (see
Methods) were normalized to the control responses to 60 µM ACh shown. The SEM of the
averaged normalized responses are represented by the tan colored areas.
Figure 2. Co-application of racemic 100 µM 2,3,5,6TMP-TQS potentiates a7 nAChR responses
to a low (10 µM) concentration of ACh. A) Averaged raw data traces for cells responding to 10
µM ACh alone or co-applied with 100 µM racemic 2,3,5,6TMP-TQS (n = 8 cells for each
condition). Data were normalized to the control responses to 60 µM ACh shown, and the
normalized peak current amplitudes were compared with a t-test, p < 0.001 (see Methods). The
SEM of the averaged normalized responses are represented by the tan-colored areas. B)
Superimposed responses to 10 µM ACh in panel A, normalized to 60 µM ACh controls. The data
in blue are those obtained with co-application of 100 µM 2,3,5,6TMP-TQS.
Figure 3. Structures of the 2,3,5,6TMP-TQS isomers. (-)2,3,5,6TMP-TQS has 3aS, 4S, 9bR
absolute stereochemistry. (+)2,3,5,6TMP-TQS has 3aR, 4R, 9bS absolute stereochemistry. Also
shown are the resolved structures of the active enantiomers of TQS (Stokes et al., 2019) and 4BP-
TQS (Thakur et al., 2013).
Figure 4. 2,3,5,6TMP-TQS racemic and isomer effects on GAT107 with a pre-application/co-
application protocol. Cells were treated with Ringer's solution or 100 µM 2,3,5,6TMP-TQS for
30 s prior to application of 10 µM GAT107 alone or co-applied with 100 µM 2,3,5,6TMP-TQS.
Data presented are averages (±SD) of the responses normalized to the initial ACh controls. A) The
inhibition by (+)2,3,5,6TMP-TQS was less than by the racemic (P = 0.01). The n values were 8, 7
,8, and 8. B) Inhibition of primed potentiation of subsequent ACh-evoked response, with pre-
application/co-applications. C) Effect of (-)2,3,5,6TMP-TQS concentration of inhibition of the
net charge of 10 µM GAT107-evoked a7 allosteric activation with a simple co-application
protocol. The n values were 7 for GAT107 alone and 8 for the co-applications. See supplemental
information for detailed results of the statistical analysis.
Figure 5. 2,3,5,6TMP-TQS isomer effects on allosteric activation of the non-orthosterically
activatible C190A a7 mutant by 10 µM GAT107 (GAT107 alone n = 7). Co-applications of 10
µM GAT107 with (-)2,3,5,6TMP-TQS isomer (n = 7) decreased peak current responses by 93%
(p = 0.015), and co-applications with (+)2,3,5,6TMP-TQS (n = 8) decreased peak current
24

Molecular Pharmacology Fast Forward. Published on July 20, 2020 as DOI: 10.1124/mol.120.119958
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MOL#119958
responses by 54% (p = 0.14). In addition, the inhibition by (-)2,3,5,6TMP-TQS was greater than
that by (+)2,3,5,6TMP-TQS (p = 0.04), and (+)2,3,5,6TMP-TQS did not reduce the net charge of
the 10 µM GAT107 responses. The data plotted are the individual values and the average
normalized (see Methods) net-charge responses ± SD. See supplemental information for detailed
results of the statistical analysis.
Figure 6. Effects of 2,3,5,6TMP-TQS enantiomers on the activation and potentiation of a7
nAChR by B-973B. Subsequent to the acquisition of two control responses to 60 µM ACh, cells
expressing human a7 were treated with 30 µM B-973B alone or co-applied with 100 µM of the
2,3,5,6TMP-TQS enantiomers. Direct allosteric activation was measured as the response to B-
973B ± 2,3,5,6TMP-TQS. Primed potentiation was measured by comparing the response to 60
µM ACh after the B-973B applications. The data shown are individual values with the average
net charge responses ± SD (n = 4-8). (-)2,3,5,6TMP-TQS reduced (p = 0.05) the B-973B allosteric
activation. See supplemental information for n values and detailed results of the statistical analysis.
Figure 7. Effect of 2,3,5,6TMP-TQS isomers on ACh responses of wild-type a7. A) Plotted are
a7 net-charge responses across a range of ACh concentrations, with or without the co-application
of 30 µM of the 2,3,5,6TMP-TQS isomers, normalized to the maximum response obtained with
the application of ACh alone. Data are the averages ± SD of 5 cells under each condition at each
concentration. The curves displayed are fits to the averaged data. See Table 1a for average curve
fit values (± SD) from the data from the individual cells and Supplemental Figure for the fit data
from the single cells. ANOVA of curve fit parameters from the individual replicates are discussed
in the text. Two-way ANOVA indicate that responses to ACh plus (+)2,3,5,6TMP-TQS were
greater than to ACh alone across most of the concentration range (P > 0.99, = 0.91, < 0.0001, <
0.0001, < 0.0001, < 0.0001, and < 0.0001 for 1, 3, 10, 30, 100, 300, and 1000 µM ACh,
respectively). Note that the parameters of the curve fits to the averaged data shown in the figure
compared to average fits of the replicates (±SD) are given in Table 1b. B) Differences between
group means are expressed as the differences between means of ACh alone and ACh plus (-
)2,3,5,6TMP-TQS or ACh plus (+)2,3,5,6TMP-TQS with 95% confidence intervals at each
concentration.
Figure 8. The sensitivity of a4b2 receptors containing a single b2L15'M subunit to co-applications
of 2,3,5,6TMP-TQS isomers. A) Averaged raw data traces (normalized to 10 µM control ACh
responses from the same cells (n = 8) to ACh alone or ACh co-applied with 30 µM of the
25

Molecular Pharmacology Fast Forward. Published on July 20, 2020 as DOI: 10.1124/mol.120.119958
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2,3,5,6TMP-TQS isomers. B) Superimposed traces from A. The data in blue are those obtained
with co-application of 30 µM 2,3,5,6TMP-TQS isomers.
Figure 9. The allosteric activation of a4(2)b2(2)b2L15'M receptors by GAT927 and sensitivity of
GAT927 responses to co-applications of 2,3,5,6TMP-TQS isomers. A) Application of the TQS
analog GAT927 at 30 µM to cells expressing a4b2 receptors with a mutant b2 subunit production
of allosteric activation and primed potentiation of subsequent ACh responses (n = 6). B) The co-
application of 100 µM (-)2,3,5,6TMP-TQS with 30 µM GAT927 (n = 7) reduced the allosteric
activation net charge (p < 0.005), with no effect on the primed potentiation of subsequent ACh
responses. C) The co-application of 100 µM (+)2,3,5,6TMP-TQS with 30 µM GAT927 (n = 7)
eliminated the allosteric activation net charge (p < 0.0001), also with no effect on the primed
potentiation of subsequent ACh responses. D) An overlay of the three post-GAT927 application
ACh responses.
Figure 10. Using methods previously described (Gulsevin et al., 2019), GAT107 (A) and (-
)2,3,5,6TMP-TQS (B) were docked into a homology model of the a7 extracellular domain. The
orientation of (-)2,3,5,6TMP-TQS in the putative allosteric activation site suggested a close
association with the T106 residue (shown), that was not present with GAT107. Panels C and D
show the same models with the threonine residue converted to alanine.
Figure 11. The effects of (-)2,3,5,6TMP-TQS on GAT107 responses of wild-type and T106A
mutant a7 receptors. A) Wild-type receptors (n = 6) show a selective inhibition (p ≤ 0.001) of
allosteric activation by 30 µM (-)2,3,5,6TMP-TQS. B) GAT107 responses of cells expressing
a7T106A were unaffected by co-application of 30 µM (-)2,3,5,6TMP-TQS. Averaged raw data
traces for cells (n = 4, see Methods) were normalized to the control responses to 60 µM ACh
shown. The SEM of the averaged normalized responses are represented by the tan colored areas.
Figure 12. Ball and stick figures and molecular superposition of the PAMs, (-)TQS and
(+)2,3,5,6TMP-TQS. A) (-)TQS, B) superposition, C) (+)2,3,5,6TMP-TQS.
26

Molecular Pharmacology Fast Forward. Published on July 20, 2020 as DOI: 10.1124/mol.120.119958
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Table 1a
Curve fit values of the data presented in Figure 7. These are the averages of the fits of five cells
under each condition ± SD. See Supplemental Data, Figure 1 for the multiple plots and
Supplemental Data, Table 1 for individual curve fits.
IMax
EC50
n
5
5
5
ACh alone
0.92 ± 0.10
1.16 ± 0.47
21 ± 3.8 µM
57 ± 37 µM
ACh + (-)2,3,5,6TMP-TQS
***
**
ACh + (+)2,3,5,6TMP-TQS 1.77 ± 0.10
7.2 ± 2.5 µM
*** p < 0.0001 compared to ACh alone
** p < 0.001 compared to ACh alone
Table 1b
Parameters of the fit shown in Figure 7 to the averaged data at each concentration, compared to
average values (±SD) of the fits of the replicates shown in Table 1a (see Supplemental Data). The
errors reported for the fit parameters to the averaged data are the calculated standard errors of the
fit parameters based on the goodness of fit (Chi-Square and r values, see Methods).
Fits of Averaged Data
IMax EC50
0.92 ± 0.005 20.65 ± 3.4 µM
Fits of Separate Replicates
IMax
EC50
N
5
5
ACh alone
0.92 ± 0.10
1.16 ± 0.47
1.77 ± 0.10
21 ± 3.8 µM
57 ± 37 µM
7.2 ± 2.5 µM
ACh + (-)2,3,5,6TMP-TQS
ACh + (+)2,3,5,6TMP-TQS
1.13 ± 0.06
1.73 ± 0.05
50.6 ± 7.9 µM
6.4 ± 0.64 µM
***
**
5
27

a t A S P E T J o u r n a l s o n J u l y m 2 7 o , l p 2 h 0 a 2 r 0 m . a s p e t j o u D r n o a w l s n . o l o r g a d e d f r o m

Molecular Pharmacology Fast Forward. Published on July 20, 2020 as DOI: 10.1124/mol.120.119958
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Molecular Pharmacology Fast Forward. Published on July 20, 2020 as DOI: 10.1124/mol.120.119958
This article has not been copyedited and formatted. The final version may differ from this version.