1521-0103/357/3/570–579$25.00 http://dx.doi.org/10.1124/jpet.115.228890THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 357:570–579, June 2016Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

Differential Potency of 2,6-Dimethylcyclohexanol Isomers forPositive Modulation of GABAA Receptor Currents

Luvana Chowdhury, Celine J. Croft, Shikha Goel, Naina Zaman, Angela C.-S. Tai,Erin M. Walch, Kelly Smith, Alexandra Page, Kevin M. Shea, C. Dennis Hall,D. Jishkariani, Girinath G. Pillai, and Adam C. HallNeuroscience Program, Departments of Biological Sciences (L.C., C.J.C., S.G., N.Z., A.C.-S.T., E.M.W., A.C.H.) and Chemistry(K.S., A.P., K.M.S.), Smith College, Northampton, Massachusetts; Department of Chemistry, University of Florida, Gainesville,Florida (C.D.H., D.J., G.G.P.); and Department of Chemistry, University of Tartu, Ravila, Estonia (G.G.P.)

Received September 9, 2015; accepted March 22, 2016

ABSTRACTGABAA receptors meet all of the pharmacological requirementsnecessary to be considered important targets for the action ofgeneral anesthetic agents in the mammalian brain. In thefollowing patch-clamp study, the relative modulatory effects of2,6-dimethylcyclohexanol diastereomers were investigated onhuman GABAA (a1b3g2s) receptor currents stably expressed inhuman embryonic kidney cells. Cis,cis-, trans,trans-, and cis,trans-isomers were isolated from commercially available 2,6-dimethylcyclohexanol and were tested for positive modulationof submaximal GABA responses. For example, the addition of30 mM cis,cis-isomer resulted in an approximately 2- to 3-foldenhancement of the EC20 GABA current. Coapplications of30 mM 2,6-dimethylcyclohexanol isomers produced a range ofpositive enhancements of control GABA responses with a rankorder for positive modulation: cis,cis . trans,trans $ mixture of

isomers.. cis,trans-isomer. Inmolecularmodeling studies, thethree cyclohexanol isomers bound with the highest bindingenergies to a pocket within transmembrane helices M1 and M2of the b3 subunit through hydrogen-bonding interactions with aglutamine at the 224 position and a tyrosine at the 220 position.The energies for binding to and hydrogen-bond lengths withinthis pocket corresponded with the relative potencies of theagents for positive modulation of GABAA receptor currents(cis,cis. trans,trans. cis,trans-2,6-dimethylcyclohexanol).In conclusion, the stereochemical configuration within thedimethylcyclohexanols is an important molecular feature inconferring positive modulation of GABAA receptor activity andfor binding to the receptor, a consideration that needs to betaken into account when designing novel anesthetics with en-hanced therapeutic indices.

IntroductionIntravenous sedatives and general anesthetics are some of

the most common therapeutic agents used during surgery.Several of these agents (e.g., propofol and etomidate) arepostulated to sedate patients and render them unconsciousthrough positive modulation of GABAA receptor currents inthe central nervous system (Franks and Lieb, 1994; KrasowskiandHarrison, 1999; Olsen and Li, 2011). GABAA receptors aremembrane-spanning chloride-selective ion channel complexesactivated through the binding of GABA (Barnard et al., 1998)and they are the predominant ionotropic receptor type forfast inhibitory neurotransmission in the mammalian centralnervous system. Their pentameric structure is composed ofdifferent subunits (a1–6, b1–4, g1–3, d, «, p, and u) with thepredominant GABAA receptor combination of a1b2g2 in mam-malian neurons (McKernan andWhiting, 1996). Investigations

of the action of anesthetics at GABAA receptors have revealedthat for select agents, the potentiation of GABA currentscorrelates with anesthetic potency in vivo (Krasowski et al.,2001; Watt et al., 2008; Hall et al., 2011).Given the interest in developing less toxic sedatives and

anesthetics, several studies have explored the structure-activity relationship for agents that enhance GABA-evokedcurrents and provide anesthesia (e.g., Krasowski et al., 2001;Pejo et al., 2014). Previously we demonstrated the potentialfor cyclohexanols to act as positive modulators of GABAA

receptor currents and as general anesthetics (Hall et al., 2004;Watt et al., 2008). The structure-activity relationship for arange of cyclohexanol analogs was further explored; amongthose tested, 2,6-dimethylcyclohexanol was determined to bethe most potent for both receptor modulation and as a generalanesthetic (Hall et al., 2011).Stereoselectivity for positive modulation of GABAA recep-

tor currents is not unprecedented, particularly in regardto enantiomers of general anesthetics (e.g., Hall et al., 1994;Tomlin et al., 1998). Likewise, cyclohexanol-based compounds(e.g., menthol) have also been shown to exhibit stereoselectivityof action for these receptors (Corvalán et al., 2009). In the

This work was supported by the Howard Hughes Medical Institute [Grant52007557 (to C.J.C. and S.G.)], a Smith College Tomlinson Award [(to L.C.)],the Blakeslee Foundation [(to A.C.H.)], and the European Social Fund [Project1.2.0401.09-0079, University of Tartu (to G.G.P.)].

dx.doi.org/10.1124/jpet.115.228890.

ABBREVIATIONS: GLIC, gloeobacter ligand-gated ion channel; HEK, human embryonic kidney; NMR, nuclear magnetic resonance; PDB, ProteinData Bank; TLC, thin-layer chromatography.

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following study, we usedWSS-1 cells to investigate modulationof wild-type GABAA receptors (a1b3g2s) by cis,cis-, trans,trans-,and cis,trans-diastereomers of 2,6-dimethylcyclohexanol(Fig. 1). In the most stable of the two possible chair confor-mations, cis,cis-dimethylcyclohexanol has both methyl groupsequatorial and the hydroxyl group axial. The cis,trans-isomerhas the hydroxyl group and onemethyl substituent equatorial,whereas the other methyl is axial. In the trans,trans-isomer, the hydroxyl group and both methyl groups are allequatorial, making it themost stable configuration and closestto planarity. Molecular modeling studies were carried out forthe cis,cis-, trans,trans-, and cis,trans-diastereomers of 2,6-dimethylcyclohexanol against the b3 subunit of the humanGABAA receptor. The results indicate that stereochemicalconfiguration within the dimethylcyclohexanols is an impor-tant molecular feature in conferring positive modulation ofGABAA receptor activity and for binding to the receptor.

Materials and MethodsCell Culture

WSS-1 cells (CRL-2029; American Type Culture Collection, Mana-ssas, VA) were used for all electrophysiology experiments. WSS-1 cellsare human embryonic kidney (HEK) cells that have been stablytransfected with cDNAs encoding for the rat a1 and g2s subunits ofthe GABAA receptor along with expression of an endogenous humanb3 subunit (Wong et al., 1992; Davies et al., 2000) and thus are aconvenient cell line for generating GABA-evoked currents consis-tently. WSS-1 cells were grown in standard media (90% Dulbecco’smodified Eagle’s medium and 10% fetal bovine serum, with 100 U/mlpenicillin and 100 mg/ml streptomycin) including 500 mg/ml geneticin(G-418) to select for cells expressing GABAA receptors (Wong et al.,1992). Cells were maintained in culture flasks in a humidifiedincubator with 5% CO2/95% air at 37°C and passaged on a weeklybasis. During passaging, cells were either plated on poly(L-lysine)(Trevigen, Gaithersburg, MD)–coated glass coverslips for electrophys-iological recording or were used to reseed another flask. Cells wereused for up to 30 passages after purchase from ATCC. All culturingreagents were purchased from Life Technologies (Carlsbad, CA)unless stated otherwise.

Electrophysiology

Electrophysiological recordings were performed using a standardwhole-cell patch-clamp technique at roomtemperature. Coverslipsweretransferred to a recording chamber that was continuously superfusedat 3 ml/min with extracellular recording medium containing 140 mMNaCl, 5 mM KCl, 1.2 mM MgCl2, 2.5 mM CaCl2, 11 mM glucose, and5 mM HEPES (pH 7.4 with NaOH). The electrode solution contained140 mM KCl, 2 mM MgCl2, 11 mM EGTA, 0.1 mM Mg21-ATP, and10 mM HEPES (pH 7.4 with KOH). Pipettes, fabricated using aFlaming/Brown micropipette puller (Sutter Instrument Company,Novato, CA), typically had resistances in the range of 2–4 MV. Pipettes

were maneuvered onto cells to form “gigaohm” seals using a microma-nipulator (MP-225; Sutter Instrument Company). Junction potentialswere zeroed in the chamber prior to all recordings, the liquid junctionpotential was negligible (approximately 2 mV), and cells were routinelyvoltage clamped at 250 mV.

Drugs were superfused onto cells using a motor-driven exchangedevice (Rapid Solution Changer, RSC-100; Bio-Logic Science Instru-ments, Claix, France) controlled via Clampex 10 acquisition software(Molecular Devices/Axon Instruments, Sunnyvale, CA). Flow ofextracellular solutions onto cells was driven by a multichannelinfusion pump (KD Scientific, Holliston, MA). Currents evoked byGABAwith and without the modulators in extracellular solution wereamplified via an Axopatch 200A (Molecular Devices/Axon Instru-ments), filtered at 1 kHz via a low-pass Bessel filter (FrequencyDevices, Ottawa, IL), and digitized using a Digidata 1440 (MolecularDevices/Axon Instruments). All currents were measured usingClampex 10 (Molecular Devices/Axon Instruments) and were furtheranalyzed using Origin software (OriginLab Corp., Northampton,MA). Data are expressed as the mean 6 S.E.M. calculated from atleast five individual cells for each data point reported (unless statedotherwise). Positive modulation of GABA-induced currents was de-fined as the percentage increase of the control GABA response(average of pre- and postdrug). Concentration-response data werefitted with the Hill equation (eq. 1) using Origin software (OriginLabCorp.):

I5 Imax: ½agonist�nH.�

½agonist�nH 1 ½EC50�nH�

(1)

where I is the agonist-evoked current at a given concentration, Imax

is the peak current at saturating [agonist], EC50 is the concentrationof agonist that elicits a half-maximal response, and nH is the Hillcoefficient.

Drugs and Reagents

All reagentswere purchased fromSigma-Aldrich (St. Louis,MO) unlessstated otherwise. During experiments, GABA and the cyclohexanolisomers were co-applied to assess the level of current modulation.Stock solutions (10–100 mM) of the 2,6-dimethylcyclohexanol isomersin dimethylsulfoxide were diluted daily to the required concentrationsin extracellular medium with dimethylsulfoxide concentrations infinal solutions never exceeding 0.1% (a concentration that had noeffect on GABA-activated currents).

Isolation of 2,6-Dimethylcyclohexanol Isomers

2,6-Dimethylcyclohexanol from Acros (Geel, Belgium) was a mixtureof 45% cis,cis-isomers, 32% trans,trans-isomers, and 23% cis,trans-isomers (Fig. 1). Thin-layer chromatography (TLC) was performed asfollows: eluant, 10% diethyl ether in petroleum ether; Rf 5 0.1, amixture of trans,trans- and cis,trans-diastereomers; Rf 5 0.2, cis,cis-diastereomer; both spots were visualized with vanillin.

2,6-Dimethylcyclohexanone was obtained from Sigma-Aldrich as amixture of 80% cis-isomers and 20% trans-isomers. TLCwas performedas follows: eluant, 5% diethyl ether in petroleum ether; Rf 5 0.23(trans), 0.42 (cis), visualized with KMnO4.

Fig. 1. Structures of 2,6-dimethylcyclohexanol diastereomers and enantiomers.

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2,6-Dimethylcyclohexanone (1.50 g, 11.9 mmol) was applied to acolumn of silica gel (150 g) and elutedwith 5% ethyl ether in petroleumether to yield cis-2,6-dimethylcyclohexanone (1.01 g, 8.00 mmol), amixture of both isomers consisting of 20% cis-2,6-dimethylcyclohex-anone and 80% trans-2,6-dimethylcyclohexanone (115 mg, 0.910mmol), and trans-2,6-dimethylcyclohexanone (72.1 mg, 0.570 mmol).

For cis-2,6-dimethylcyclohexanone, TLC was as follows: eluant, 5%diethyl ether in petroleum ether; and Rf 5 0.42, visualized withKMnO4.

1H nuclearmagnetic resonance (NMR) (300MHz, CDCl3) wasas follows: 1.0 (d, 6H), 1.2–1.4 (m, 2H), 1.7–1.9 (m, 2H), 2.0–2.2 (m, 2H),and 2.3–2.5 (m, 2 H)

For trans-2,6-dimethylcyclohexanone, TLC was as follows: eluant,5% diethyl ether in petroleum ether; and Rf 5 0.23, visualized withKMnO4.

1HNMR (300MHz, CDCl3) was as follows: 1.1 (d, 6H), 1.5–1.6(m, 2 H), 1.7–1.8 (s, 2 H), 1.9–2.0 (m, 2 H), and 2.5–2.6 (m, 2 H).

Reduction of cis-2,6-Dimethylcyclohexanone with LithiumAluminum Hydride.

An oven-dried 100-ml three-neck flask, equipped with magneticstirrer, rubber septum, and gas inlet was filled with N2. Tetrahydro-furan (anhydrous, 20 ml) was added followed by a 1 M solution oflithium aluminum hydride in tetrahydrofuran (13.1 ml, 13.1 mmol,1.10 Eq). cis-2,6-Dimethylcyclohexanone (1.46 g, 11.5 mmol) was thenadded dropwise via a syringe and the reaction was stirred at roomtemperature for 1 hour.

The reaction was quenched with 2 ml water added dropwise, thendiluted with 5ml 15%NaOH solution, followed by another 2ml water.Themixturewas filtered through silica gel under a vacuumand rinsedwith diethyl ether (approximately 50 ml). The filtrate was transferredto a 250-ml separatory funnel. The aqueous layer was extracted withethyl ether (3� 50 ml), and the combined ether layers were dried overanhydrous MgSO4.

1H NMR on the crude product (1.65 g, 100%)showed amixture of cis,cis-2,6-dimethylcyclohexanol (54%) and trans,trans-2,6-dimethylcyclohexanol (46%). Flash column chromatographyof the crude product using 240 g silica gel and 10:1 petroleum ether toethyl ether as eluant gave the cis,cis-isomer (646 mg, 42%), a com-bination of both isomers (41 mg, 3%), and the trans,trans-isomer(551 mg, 36%). The total yield was 1.24 g (9.65 mmol, 81%).

cis,cis-2,6-Dimethylcyclohexanol (Isomer 1). TLC was as fol-lows: Rf5 0.2, eluant 10% diethyl ether in petroleum ether, visualizedwith vanillin. 1H NMR (300 MHz, CDCl3) was as follows: 0.98 (d, 6 H),1.16 (d, 1 H), 1.28–1.38 (m, 5H), 1.45–1.59 (m, 2 H), 1.65–1.74 (m, 1H),and 3.51–3.56 (m, 1 H)

trans,trans-2,6-Dimethylcyclohexanol (Isomer 2). TLC wasas follows: Rf 5 0.1, eluant 10% diethyl ether in petroleum ether,visualized with vanillin. 1H NMR (300 MHz, CDCl3) was as follows:1.07 (d, 6 H), 1.20–1.29 (m, 2 H), 1.30–1.42 (m, 2 H), 1.47 (d, 1 H),1.56–1.65 (m, 2 H), 1.66–1.76 (m, 2 H), and 2.72 (t of d, 1 H).

Reduction of trans-2,6-Dimethylcyclohexanone with Lith-ium Aluminum Hydride.

(2)

It should be noted that 3 and 4 shown in eq. 2 are enantiomers andmay (or may not) have equal activity dependent on whether the site ischiral (not the same activity) or achiral (same activity).

An oven-dried 25-ml two-neck flask, equipped with magneticstirrer, rubber septum, and gas inlet, was filled with dry N2.Tetrahydrofuran (anhydrous, 1.5 ml) was added together with a 1 M

anhydrous solution of lithium aluminum hydride in tetrahydrofuran(1.10 ml, 1.10 mmol). A solution of trans-2,6-dimethyl cyclohexanone(128.5 mg, 1.01 mmol) in anhydrous tetrahydrofuran (1 ml) wastransferred by cannula from a pear-shaped flask and the mixture wasstirred at room temperature for 1 hour.

The reaction was quenched with a few drops of water and thentreated with 9% NaOH solution added dropwise. The mixture wasfiltered through silica gel under a vacuum, rinsed with diethyl ether(approximately 15 ml), and the filtrate was transferred to a 50-mlseparatory funnel. The aqueous layer was extracted with ethyl ether(3� 10ml). and the ether layerswere combined and dried overMgSO4.1H NMR on the crude product (42.9 mg, 33%) revealed a mixtureof cis,trans-2,6-dimethylcyclohexanol (98%) and trans,trans-2,6-dimethylcyclohexanol (2%).

cis,trans- and trans,cis-2,6-Dimethylcyclohexanol (Enantio-mers 3 and 4). TLCwas as follows: Rf5 0.1, eluant 10%diethyl etherin petroleum ether, visualized with vanillin. 1H NMR (300 MHz,CDCl3) was as follows: 0.97 (t, 6 H), 1.36–1.55 (m, 6 H), 1.63–1.80(m, 2 H), 1.90–2.04 (m, 1 H), and 3.28–3.35 (m, 1 H)

A sample of the cis,cis-isomer was also isolated directly from thecommercially available mixture via column chromatography of themixture (5 g) on silica gel (300 g) using hexane/ethyl acetate (20:1) toyield 1.1 g pure cis,cis-isomer (by 1H NMR) as a colorless liquid.Samples of the cis,cis-isomer from both isolation procedures producedsimilar modulation of GABA currents.

Molecular Modeling

Molecular docking studies were carried out to define the mode ofinteraction between the GABAA receptor propofol and each diastereo-mer of 2,6-dimethylcyclohexanol. Given previous literature highlight-ing the role of b subunits in the binding of propofol to GABAA receptors(Yip et al., 2013) and the availability of a crystal structure [ProteinData Bank (PDB) identifier 4COF; Miller and Aricescu, 2014], the b3

subunit of the humanGABAA receptor was the considered target. Thistarget was prepared for the docking process by protonating, minimiz-ing, and examining the missing side chain residues in the proteinusing Chimera Software (University of California, San Francisco)(Pettersen et al., 2004; Goddard et al., 2005). The prepared target wasuploaded to the ProBiS server (http://probis.nih.gov/; National Insti-tutes of Health, Bethesda, MD) (Carl et al., 2010) for the detectionof binding sites using protein binding site structure similarities. TheProBiS program aligns and superimposes protein binding sites, and itenables pairwise alignments and fast database searches for similarbinding sites.

We focused on propofol binding sites highlighted in previous studies(Nury et al., 2011; Yip et al., 2013; Chiara et al., 2014) and sites basedon the structural similarities of the following proteins (shown by PDBidentifiers): 2M6B (structure of transmembrane domains of humanglycine receptor a1 subunit; Mowrey et al., 2013), 4X5T (a1 glycinereceptor transmembrane structure fused to the extracellular domainof gloeobacter ligand-gated ion channel(GLIC); Moraga-Cid et al.,2015), and 3P50 (structure of propofol bound to GLIC; Nury et al.,2011). Based on these previous studies, we explored intrasubunitbinding sites within chain A of a single subunit of 4COF (Miller andAricescu, 2014). The best binding site with a Z score of 4.21 includedthe key amino acid residues Tyr220, Phe221, Gln224, His267, andThr271. A second binding site with key amino acid residues Tyr143,Thr225, Pro228, Ile264, and Leu268 gave a Z score of 4.01. The highscoring binding sites were then merged because there was consider-able spatial overlap between the key amino acids. Although it isrecognized that intersubunit sites have been proposed for propofolbinding to GABAA receptors (e.g., Bali and Akabas, 2004), sitesbetween subunits (e.g., chains A and B) were not considered becausesteric clashes were encountered when modeling multiple subunits atthese sites.

The protein was loaded to MGL-AutoDock Tools (Scripps ResearchInstitute, La Jolla, CA) to define the custom binding site grid box for

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the docking process. In brief, polar hydrogen atoms and Kollmancharges were assigned to the protein by converting to the PDBQTformat of AutoDock (Morris et al., 2009). The program AutoGrid wasused to generate grid maps for the custom binding site on the proteinand the grid was generated based on selected residues from bindingsite analysis. To generate gridmaps for different types of ligands (withpossible hydrogen bonding), the grid parameter file was modified toinclude O-H bond types from ligands. To generate the custom gridmaps, we defined grid points as x5 40, y5 40, and z5 50 and the gridcenter as x5 10.70, y5213.28, and z5 157.35 with a spacing of 0.38in the protein three-dimensional structure. The binding site for 2,6-dimethylcyclohexanols included Tyr143, Tyr220, Phe221, Gln224,Thr225, Pro228, Ile264, His267, Leu268, and Thr271 amino acidsfrom the M1 and M2 domains of chain A (Fig. 2).

Propofol, cis,cis-, trans,trans-, and cis,trans-diastereomers of 2,6-dimethylcyclohexanol were all considered as ligands. The geome-tries were drawn in MarvinSketch (Chemaxon, Cambridge, MA)and converted to three-dimensional structures using the MM2force field. The diastereomeric conformers were frozen for furtherquantum chemical optimization using the DFT/B3LYP level oftheory and the 6-31G basis set in HyperChem software (Hypercube,Inc., Gainesville, FL) (see Table 1 for quantum chemical proper-ties). The lowest energy conformer was uploaded to MGL Tools toassign Gasteiger partial charges and for the detection of torsionsto rotate the bonds during the docking procedure. For all ligands,random initial positions, fixed conformers, and torsions wereparameterized. The number of active torsions and the number oftorsional degrees of freedom were set to default values indicated inAutoDock. A Lamarckian genetic algorithm was used for minimi-zation using optimum parameters (from initial docking evalua-tions) to generate all possible energies to rank the conformers(Morris et al., 1998). For energy evaluations, we used the followingdocking parameters: 250,000 evaluations, 250 genetic algorithmiterations, and a population size of 150 to generate 250 dockedconformers. For reliable docking results, the root mean squaredeviation of the lowest energy conformer and the root mean squaredeviation to one another were analyzed to group families of similarconformations using clustering.

Resultscis,cis- and trans,trans-2,6-Dimethylcyclohexanols IsomersAre Positive Modulators of GABAA Receptor Currents

We investigated the modulation of submaximal GABAcurrents by the three isolated cis,cis-, cis,trans-, and trans,trans-2,6-dimethylcyclohexanol diastereomers along with themixture of the isomers. WSS-1 cells (HEK cells stably ex-pressing a1b3g2s GABAA receptors) were routinely exposed toapplications of 10 mM GABA that evoked approximate EC20

currents (effective concentration that evoked 20% of maximalcurrent). Coapplications of 30 mM 2,6-dimethylcyclohexanolsproduced potentiations of the GABA responses (Fig. 3). Forexample, the addition of 30 mM cis,cis-isomer resulted inapproximately 2- to 3-fold enhancement of the EC20 GABAcurrent (Fig. 3B). Pre-exposure to cyclohexanols did notaffect the extent of current modulation upon subsequentcoapplication and no direct activation of GABAA receptorcurrents was observed by the cyclohexanols even at the highestconcentrations (300mM) of the isomers tested (data not shown).Coapplications of 30 mM 2,6-dimethylcyclohexanol isomers

produced a range of positive enhancements of control GABAresponses with the rank order for positive modulation ofGABA EC20 currents: cis,cis . trans,trans $ mixture ofisomers . . cis,trans-isomer (Fig. 3). We confirmed therelative extent of the potentiations of the GABA receptoractivity in the presence of increasing concentrations of theisomers (1–300 mM, Fig. 4). For instance, on average with theaddition of 30 mM cis,cis-isomer, the positive modulation ofthe current (above control) was 165% 6 27% (n 5 6), whereasthe equivalent for the trans,trans-isomerwas92%624% (n5 5).Current modulation by the cis,trans-isomer was negligibleeven at the highest concentration tested (at 300 mM, 5%6 5%,n5 6). All of the cyclohexanol effects were fully reversible uponwashout (as previously reported; Hall et al., 2011).

Fig. 2. Binding site and hydrophobicity of the target GABAA b3-subunit protein chain A (PDB identifier 4COF) with cis,cis-2,6-dimethylcyclohexanol.Binding site is in the adjacent subunit of membrane region (M1 and M2) close to the Cys loop.

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The relative potencies for positive modulation of GABAcurrents were further supported by recording and plotting theleftward shifts in the GABA concentration-response curves inthe presence of 30mM2,6-dimethylcyclohexanol isomers (Fig. 5).For instance, the EC50 for the control GABA currents (approx-imately 21 mM) was shifted to approximately 10 mM by thetrans,trans-isomer and to approximately 7mMin the presence ofthe cis,cis-isomer. The cis,trans-isomer produced a negligibleshift in the concentration-response relationship (Fig. 5).

Modeling the Binding of the Isomers to the b3 Subunit of theGABAA Receptor

Molecular modeling focused on regions of the b3 subunit ofthe GABAA receptor (4COF; Miller and Aricescu, 2014) that,in previous studies, were implicated in propofol binding to

GABAA receptor b-subunits or GLIC channels (Nury et al.,2011; Yip et al., 2013; Chiara et al., 2014). The binding siteprediction server ProBiS (Carl et al., 2010) gave the bestbinding site score with key amino acid residues Tyr143,Tyr220, Phe221, Gln224, Thr225, Pro228, Ile264, His267,Thr271, and Leu268.This binding site is located between the transmembraneM1

and M2 helices of the b3 subunit (Fig. 2) and includes aphenylalanine at the 221 position (M1) and a histidine at the267 position (M2) that were previously photolabeled with anortho-propofol diazirine derivative (Yip et al., 2013). In thesame study, replacement of the phenylalanine at 221 by atryptophan residue was shown to attenuate propofol’s poten-tiation of GABA-evoked currents. Interestingly, the threecyclohexanol isomers were found to bind within this pocketthrough hydrogen-bonding interactions with a glutamine atthe 224 position and a tyrosine at the 220 position plushydrophobic interactions with the leucine at position 268,phenylalanine at position 221, and tyrosine at position 220(Fig. 6; Tables 1–3). By comparison, propofol’s hydrogenbonding was modeled only through the glutamine residue andhydrophobic interactions with the leucine at 268, tyrosineat 220, and threonine at 225 positions (Fig. 7). The ligandefficiency, van der Waals energy, and electrostatic and H-bondenergywere all considered in the calculation of binding energiesand intermolecular energies for this intrasubunit site. Withinthe site the binding energies for the interactions (Table 2) hada rank order of propofol. cis, cis. trans,trans. cis,trans-2,6-dimethylcyclohexanol corresponding with the rank order ofpotencies for positive modulation of GABAA receptor currentsrecorded electrophysiologically [comparewithFigs. 3 and 7 fromDavies et al. (2000) and Hall et al. (2011), respectively].

cis-,trans-2,6-Dimethylcyclohexanol as an Inhibitor ofPropofol’s Modulatory Action at GABAA Receptors

Finally, given the lack of modulation observed by cis,trans-2,6-dimethylcyclohexanol and the modeling of its binding tothe receptor at a site similar to that of propofol’s, we exploredthe possibility that this isomer might competitively inhibitpropofol’s modulatory action at the receptor. As expected,submaximal GABA (3 mM) responses were potently enhancedby propofol (10 mM; Fig. 8). However, this positive modulationwas only moderately attenuated by the coapplication of 100mM cis,trans-2,6-dimethylcyclohexanol (Fig. 8). In summary,positive modulation by propofol was attenuated by 14.1% 61.3% (n 5 4) and by 13.4% 6 2.5% (n 5 5) by 100 and 300 mMcis,trans-2,6-dimethylcyclohexanol, respectively.

DiscussionIn the search for novel anesthetic and sedative compounds,

this study investigated the potential of 2,6-dimethylcyclo-hexanol stereoisomers as positive modulators of GABAA

TABLE 1Quantum chemical properties in vacuum

Properties/Ligand E(RHF) Hatree Dipole Moment Debye E (Thermal) Heat Capacity Entropy Zero-Point Energy Correction Hatree

kcal/mol cal/mol·K cal/mol·K

cis,cis 2386.97113 1.815 161.11 35.239 91.083 0.247trans,trans 2386.97115 2.169 160.99 35.556 91.592 0.247cis,trans 2386.96832 1.891 161.18 35.374 90.988 0.247

Fig. 3. Current recordings illustrating modulation by 2,6-dimethylcyclo-hexanol isomers of GABAA receptor activity. WSS-1 cells were held at250mV and 10 mM GABA was applied for the duration (2 seconds) of theshaded boxes above each trace. Upper traces indicate current evoked inthe presence of GABA alone. Superimposed lower traces indicate currentevoked in presence of 10 mM GABA plus 30 mM of the 2,6-dimethylcyclo-hexanol isomer(s) specified. (A) With coapplication of the commerciallyavailable mixture of 2,6-dimethylcyclohexanol isomers. (B) With coappli-cation of cis,cis-2,6-dimethylcyclohexanol. (C) With coapplication of trans,trans-2,6-dimethylcyclohexanol. (D) With coapplication of cis,trans-2,6-dimethylcyclohexanol.

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receptor– mediated currents. In this study, the GABA EC50

in HEK cells expressing a1b3g2s receptors was determined tobe approximately 20 mMwith a Hill coefficient of 1.6, which isreasonably consistent with other reports for similar receptorcombinations using the same expression system (e.g., Uenoet al., 1996). The cyclohexanols in this study all have theiraliphatic groups in the 2,6-position relative to the hydroxylgroup equivalent to the commonly used intravenous anes-thetic, propofol (2,6-di-isopropylphenol). Following on fromstudies that demonstrated the efficacy of 2,6-dimethylcyclo-hexanol for positive modulation of GABAA receptor currentsand for inducing anesthesia (Hall et al., 2011), the majorelectrophysiological findings of our study are as follows.First, the mixture of 2,6-dimethylcyclohexanol isomers(3–300 mM) enhanced GABA currents evoked in HEK cellsexpressing a1b3g2s receptors. Second, the cis,cis- and trans,trans-isomers of 2,6-dimethylcyclohexanol positively modu-lated GABA currents, with the former beingmarginally morepotent. Finally, cis,trans-2,6-dimethylcyclohexanol had min-imal effects on GABA currents.The modulatory effects of cyclohexanols on GABA recep-

tors have been previously investigated (Hall et al., 2011); how-ever, in this instance, they were studied in oocytes expressinga1b2g2s receptors (the most prevalent combination found inthe mammalian brain). By comparison, the potentiation ofGABA currents by 30mM2,6-dimethylcyclohexanolwas greaterin the oocyte studies (approximately 4-fold enhancement; Hallet al., 2011) than in HEK cells expressing a1b3g2s receptorcomposition (approximately 1.5-fold enhancement, this study).Subunit composition, differences in expression systems andspeed of agonist/modulator application probably contribute tothe discrepancies in the extent of the modulations reported.Although the 2,6-dimethylcyclohexanol mixture of stereoiso-

mers enhanced GABA currents at concentrations of 3–300 mM,

the mixture is composed of isomers that may have differentmodulatory effects on the receptors. Stereoselective action hasbeen previously observed for GABA receptor modulation by an-esthetic agents. For instance, the S(1)-isoflurane isomer wasobserved to be more effective in potentiating GABA-inducedcurrents than the R(2)-isoflurane (Hall et al., 1994) withsteroselectivity also observed for (1)- and (2)-pentobarbital(Tomlin et al., 1998). It seemed plausible, therefore, thatindividual isomers of 2,6-di-methylcyclohexanol would exhibita range of potencies due to differing chemical configurations.2,6-Di-isopropylphenol (propofol), a potent positive modulatorof GABA receptor currents, consists of a hydroxyl group andtwo ortho isopropyl groups in the plane of the benzene ring.By contrast, all three 2,6-di-methylcyclohexanols are chairshaped and are not planar molecules. The trans,trans config-uration of 2,6-di-methylcyclohexanol is the closest to planaritywith the hydroxyl and twomethyl groups in equatorial positionsin the most stable conformer. Thus, we expected that the trans,trans-isomer would be themost potentmodulator since it mightfit most effectively into an equivalent propofol binding pocketwithin the GABAA receptor. Indeed, previous studies reportedthat cyclohexanols and propofol may share similar sites ofaction on GABAA receptors (Watt et al., 2008). The cis,cis con-figurationof 2,6-dimethylcyclohexanol was postulated to be thenextmost potent since themost stable conformationwould havethe hydroxyl group axial and the methyl groups equatorial.Finally, the cis,trans configuration of 2,6-dimethycyclohexanolwas expected to be the least potent since one methyl mustnecessarily be axial thereby reducing planarity. Contrary toexpectations, although the trans,trans-isomer was still aneffective modulator, the cis,cis-isomer was the most potentdiastereomer for positive modulation of GABA responses. Asanticipated, the cis,trans-isomer had minimal impact on themodulation of GABA receptor currents, suggesting that the

Fig. 5. GABA concentration-response curves are shifted to the left withcoapplication of cis,cis- and trans,trans-2,6-dimethylcyclohexanol isomers.Currents were normalized against the maximum current evoked by anyconcentration of agonist with or without coapplication of 2,6-dimethylcy-clohexanol. Data (n$ 5, mean6 S.E.M.) were fitted with the Hill equation(mentioned in the Materials and Methods) in the absence (filled square)and presence of cis,trans-2,6-dimethylcyclohexanol (open circles), cis,cis-2,6-dimethylcyclohexanol (open triangles), and trans,trans-2,6-dimethyl-cyclohexanol (closed inverted triangles).

Fig. 4. cis,cis-2,6-Dimethylcyclohexanol is the most potent of the isomersfor positive modulation of GABAA receptor currents. Relative modulationsof GABA (10 mM) responses were compared by coapplying increasingconcentrations (1–300 mM) of 2,6-dimethylcyclohexanol isomer(s). Withthe exception of cis,trans-2,6-dimethylcyclohexanol, the isomers wereeffective as positive modulators of GABA currents, with cis,cis-2,6-dimethylcyclohexanol demonstrating the most potent current enhance-ment. Data points represent mean 6 S.E.M. modulations for n $ 5 cells.

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23% of this isomer present in the commercially availablemixture acts as pharmacological ballast with regard to GABAA

receptor modulation.In previous studies, a range of 2,6-substituted cyclohexanols

with varying sizes of the aliphatic chains were assessed foranesthetic potency and for loss of righting reflex in a tadpole

assay (Hall et al., 2011). 2,6-Dimethylcyclohexanol was one ofthe most potent in this regard with an EC50 of approximately13 mM, with 2,6-di-isopropylcyclohexanol demonstratingequivalent potency (EC50 of approximately 14 mM). Todate, yields of isolated isomers from mixtures of both 2,6-dimethylcyclohexanol and 2,6-di-isopropylcyclohexanol

Fig. 6. Three-dimensional interpretations (A–C) and two-dimensional depictions (A’–C’) of molecular docking interactions between the ligands andGABAA receptor (b3-subunit). cis,cis-2,6-Dimethylcyclohexanol (A), trans,trans-2,6-dimethylcyclohexanol (B), and cis,trans-2,6-dimethylcyclohexanolwith hydrogen-bond interactions (red color), bond distance in Å (red color), and hydrophobic interaction regions in the blue-colored surfaces (C). H-Bonddistances were as follows: O, Gln224 = 1.898 Å (A and A’), 2.014 Å (B and B’), and 2.152 Å (C and C’), respectively; H, Tyr220 = 1.904 Å (A and A’), 1.891 Å(B and B’), and 2.054 Å (C and C’), respectively. Definitions for the various interacting elements represented in the two-dimensional depiction (A’–C’) areas follows. First, residues involved in hydrogen-bond, charge, or polar interactions are represented by purple circles. Second, residues involved in van derWaals interactions are represented by green circles. Third, the solvent accessible surface of an interacting residue is represented by a blue halo aroundthe residue with the diameter of the halo proportional to the solvent accessible surface. Finally, hydrogen-bond interactions with amino acid side chainsare represented by a blue arrows directed toward the electron donor. Circled residues in the two-dimensional diagrams are indicative of residues withinhydrophobic interaction regions and H-bonding interaction regions, whereas those outside are other relevant binding site residues. Note, in each case,only residues within 4-Å distance from the ligand are depicted.

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have been insufficient to enable similar analyses of the relativeanesthetic potencies of the cis,cis- versus trans,trans-isomers inin vivo tadpole assays.Although our electrophysiological studies were conducted

on a GABAA receptor consisting of a1, b3, and g2s subunits, theclosest approximating crystal structure for the molecularmodeling studies was for a homopentamer of human b3

subunits (4COF; Miller and Aricescu, 2014). The bindingpocket for the cyclohexanol isomers, revealed through ourmodeling studies of a GABAA receptor b3 subunit, wascomposed primarily of residues from M1 and M2 transmem-brane helices. This intrasubunit binding site was predicted byreference to previous propofol binding studies, by similarityin the templates used for the homology modeling and byidentifying key amino acid residues within two sites thatproduced the highest Z scores. The site included a phenylal-anine at the 221 position and a histidine at the 267 position(Fig. 7) that have already been implicated in propofol’spositive modulation of receptor currents and in propofol’sbinding (Yip et al., 2013). Both active isomers, cis,cis andtrans,trans, were also observed to bind to the receptor subunitvia the histidine 267 and through hydrophobic interactionswith the phenylalanine 221 (Fig. 6, A and B). It should benoted that Yip et al. (2013) observed interactions of an eq-uivalent binding site with the main chain of the neighboringsubunit. In our modeling studies, intersubunit sites of actionwere not explored because of steric clashes between subunits(e.g., chains A and B) at these sites that were severe even after

extensive protein preparation (i.e., minimization to refineprotein structure). Because this questions the availability ofthe proposed site in an assembled receptor, future studies willrequire a thorough protein minimization to refine the struc-ture and molecular dynamics simulations to address otherpotential intra- and intersubunit binding sites.Our modeling studies revealed several important points

regarding the binding of the 2,6-dimethylcyclohexanols atthe chosen site. First, the binding energies (Table 2) vary from24.42 through 24.38 to 24.21 kcal/mol for the cis,cis-, trans,trans-, and cis,trans-isomers, respectively. Such small differ-ences, although corresponding to the rank order of potency forreceptor modulation, are unlikely to explain changes in currentmodulation from approximately 300% (300 mM cis,cis) to 0%(300 mM cis,trans). Binding energy comprises a combination ofhydrophobic interactions, van der Waal’s forces, and hydrogenbonding. Figure 6 shows that the H-bond length to glutamine224 (Gln224) increases from 1.898 Å (cis,cis) through 2.014 Ǻ(trans,trans) to 2.152 Å (cis,trans). Interestingly, these H-bondlengths are inversely related to the extent of current enhance-ments derived electrophysiologically, suggesting that H bond-ing may be an important factor in determining the extent ofreceptor-positive modulation (Table 3).It is instructive to compare the modeling data for the

cyclohexanols with those for propofol, which shows a bindingenergy of 24.96 kcal/mol. If binding energy was the deter-mining factor, this only corresponds to an estimated modula-tion enhancement over cis,cis-2,6-dimethylcyclohexanol of

Fig. 7. Three-dimensional interpretations (left) and two-dimensional depictions (right) of molecular docking interactions between the propofol andGABAA receptor (b3 subunit). H-Bond distances were as follows: H, Gln224 = 1.874 Å. The green color discs represents van der Waals residueinteractions, and purple represents electrostatic interactions. Circled residues in the two-dimensional diagrams are indicative of residues withinhydrophobic interaction regions and H-bonding interaction regions, whereas those outside are other relevant binding site residues. Hydrogen-bondinteractions with the amino acid main chain is represented by a green arrow directed towards the electron donor Note, in each case, only residues within4-Å distance from the ligand are depicted.

TABLE 2Molecular docking results: docked ligand scoring parameters and interacting amino acid residues

Properties/Ligand BindingEnergy

Predicted InhibitionConstant

LigandEfficiency

IntermolecularEnergy

van der Waal’sDesolvation Energya H Bond Hydrophobic Interactions

kcal/mol mM kcal/mol kcal/mol

cis,cis 24.42 572.09 20.49 24.72 24.51 Tyr220, Gln224 Tyr220, Phe221, Leu268trans,trans 24.38 620.36 20.49 24.67 24.42 Tyr220, Gln224 Tyr220, Phe221, Leu268cis,trans 24.21 813.66 20.47 24.51 24.36 Tyr220, Gln224 Tyr220, Phe221, Leu268Propofol 24.96 231.96 20.38 25.85 25.71 Gln224 Tyr220, Thr225, Leu268, Gln224

avan der Waal’s desolvation energy = van der Waal’s + H bond + desolvation energy.

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approximately 3-fold, whereas the observed enhancementsusing propofol are considerably greater (e.g., compare Fig. 3Bwith Fig. 8). According to ourmodeling, propofol also forms aHbond with Gln224 with a bond length of 1.874 Ǻ only slightlyshorter than that with cis,cis-2,6-dimethylcyclohexanol. Thereis, however, a fundamental difference. TheH bond is describedbetween the hydrogen of the OH group and the basic amideoxygen of Gln224, possibly due to the acidity of propofol (pKa

of approximately 9). By contrast, the H bond between thecyclohexanols and Gln224 is between the NH2 group (inCONH2) of the glutamine and the oxygen atom of the OHgroup. Therefore, one may conclude that it is the nature andlengths of the hydrogen bonds that determines the degree ofreceptor-positivemodulation, althoughadditional contributionsfrom the hydrophobic and van der Waal’s interactions mustalso contribute to the binding energy.Although we focused on a site selected from predocking

studies with the highest Z scores, many other sites had thepotential to accommodate the isomers, albeit with lesserscores. Indeed, many other sites have been proposed forpropofol’s interactions and modulation of GABAA receptoractivity, including other sites in M1 (Chang et al., 2003), M2(Siegwart et al., 2002), and alsoM3 andM4helices (Krasowskiet al., 1998; Siegwart et al., 2002; Richardson et al., 2007). Forexample, in previous studies, a tyrosine in the 444 position ofb2 subunits proved important for modulation of the GABAcurrents by propofol (Richardson et al., 2007) and by menthol,a monoterpenoid with a neutral cyclohexanol chair structure(Watt et al., 2008).The drugs investigated in this study were all positive

modulators and, therefore, likely stabilize the “open” state ofthe ligand-gated ion channel through an allosteric mecha-nism. By contrast, the 4COF structure used for our modelingstudies is described by Miller and Aricescu (2014) as repre-sentative of a “desensitized” state. This state is evidently

distinct from that predicted to be stabilized by positivemodulators. Moreover, the 4COF structure represents amodified b3 subunit of the GABAA receptor with extensiveintracellular loop domains removed. Therefore, although4COF provides an approximation of the relevant structurefor binding studies, interpretation of the molecular modelingdata must be viewed with caution. Furthermore, the struc-ture of 4COF is that of a homopentameric receptor, whereasour electrophysiological recordings were derived from recep-tors with two additional subunits (a1 and g2s), which couldcontribute either directly to drug binding or indirectly tobinding site conformations. Given these caveats, othercandidate binding sites may be equally or more relevant toproducing the positive modulations of the receptor activityobserved electrophysiologically.Finally, we performed competition experiments to deter-

mine whether cis,trans-2,6-dimethylcyclohexanol could actas a competitive inhibitor of propofol’s action at the receptor(Fig. 8). In these recordings, we observed only modest inhibi-tion of the positive modulation induced by propofol. This re-sult suggests that although cis,trans-cyclohexanol producedno modulation of GABA responses, its ability to compete fora propofol binding site is limited, presenting a further caveatfor overinterpretation of the modeling data.In conclusion, the enhanced activity of cis,cis-2,6-dimethyl-

cyclohexanol presents an interesting lead in the developmentof novel anesthetics, given that cyclohexanols in general aretypically well tolerated (Thorup et al., 1983). Further refine-ment of such agents through isolation of individual isomersmay lead to novel anesthetics with improved therapeuticindices.

Acknowledgments

The authors thankHarrisonHunter and SalmaBargach for helpingto set up the patch-clamp electrophysiology. They also thank Dr.Andrew Jenkins (Department of Anesthesiology, Emory University,Atlanta, GA) for reading of and comments on the manuscript. G.G.Pillai thanks the University of Tartu Graduate School for FunctionalMaterials and Technologies.

Authorship Contributions

Participated in research design: Chowdhury, Croft, Goel, Zaman,Tai, Walch, Smith, Page, Shea, C.D. Hall, Jishkariani, Pillai, A.C.Hall.

Conducted experiments: Chowdhury, Croft, Goel, Zaman, Tai,Walch, Smith, Page, Shea, C.D. Hall, Jishkariani, Pillai, A.C. Hall.

Contributed new reagents or analytic tools: Smith, Page, Shea, C.D.Hall, Jishkariani.

Performed data analysis: Chowdhury, Croft, Goel, Zaman, Tai,Walch, Smith, Page, Shea, C.D. Hall, Jishkariani, Pillai, A.C. Hall.

Wrote or contributed to the writing of the manuscript: Chowdhury,Croft, Goel, Zaman, Tai, Walch, Smith, Page, Shea, C.D. Hall,Jishkariani, Pillai, A.C. Hall.

TABLE 3Molecular docking results: H-bond distance

Residues/Ligand Tyr220 Gln224

Å

cis,cis 1.904 1.898trans,trans 1.891 2.014cis,trans 2.054 2.152Propofol — 1.874

Fig. 8. Current recording illustrating minimal attenuation by cis,trans-2,6-dimethylcyclohexanol isomer of the positive modulation of GABAAreceptor activity by propofol. A WSS-1 cell was held at 250 mV and 3 mMGABAwas applied for the duration (2 seconds) of the open boxes above thetrace. During the period of the checkered box a current was evoked by thepresence of 3 mM GABA plus 10 mM propofol. During the filled box, acurrent was evoked by the presence of 3mMGABAplus 10mMpropofol plus100 mM cis,trans-2,6-dimethylcyclohexanol, resulting in only moderateinhibition of the propofol-induced positive modulation. Positive modulationby propofol was attenuated by 14.1% 6 1.3% (n = 4) and by 13.4% 6 2.5%(n = 5) by 100 and 300 mM cis,trans-2,6-dimethylcyclohexanol, respectively

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