Identifying Drugs That Bind Selectively to Intersubunit General Anesthetic Sites in the Α1β3γ2 GABAAR Transmembrane Domain

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1521-0111/95/6/615–628$35.00 https://doi.org/10.1124/mol.118.114975 MOLECULAR PHARMACOLOGY Mol Pharmacol 95:615–628, June 2019 Copyright ª 2019 by The American Society for Pharmacology and Experimental Therapeutics

Identifying Drugs that Bind Selectively to Intersubunit General Anesthetic Sites in the a1b3g2 GABAAR Transmembrane Domain

Selwyn S. Jayakar, Xiaojuan Zhou, David C. Chiara, Carlos Jarava-Barrera, Pavel Y. Savechenkov, Karol S. Bruzik, Mariola Tortosa, Keith W. Miller, and Jonathan B. Cohen Department of Neurobiology, Harvard Medical School, Boston, Massachusetts (S.S.J., D.C.C., J.B.C.); Department of

Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, Boston, Massachusetts (X.Z., K.W.M.); Downloaded from Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois (P.Y.S., K.S.B.); and the Departamento de Quimica Orgánica, Universidad Autónoma de Madrid, Madrid, Spain (C.J.-B., M.T.) Received October 19, 2018; accepted March 29, 2019

ABSTRACT molpharm.aspetjournals.org

GABAA receptors (GABAARs) are targets for important classes [4-(tert-butyl)-propofol and 4-(hydroxyl(phenyl)methyl)-propofol] of clinical agents (e.g., anxiolytics, anticonvulsants, and gen- bind with ∼10-fold higher affinity to b2 sites. Similar to R-mTFD- eral anesthetics) that act as positive allosteric modulators MPAB and propofol, these drugs bind in the presence of GABA (PAMs). Previously, using photoreactive analogs of etomidate with similar affinity to the a1–b2 and g1–b2 sites. However, we ([3H]azietomidate) and mephobarbital [[3H]1-methyl-5-allyl-5-(m- discovered four compounds that bind with different affinities to trifluoromethyl-diazirynylphenyl)barbituric acid ([3H]R-mTFD-MPAB)], the two b2 interface sites. Two of these bind with higher affinity we identified two homologous but pharmacologically distinct to one of the b2 sites than to the b1 sites. We deduce that classes of general anesthetic binding sites in the a1b3g2 4-benzoyl-propofol binds with .100-fold higher affinity to the 1 2 1 1 2 1 2 1 2 GABAAR transmembrane domain at b –a (b sites) and g –b site than to the a –b or b –a sites, whereas 1 2 1 2 2 a –b /g –b (b sites) subunit interfaces. We now use com- loreclezole, an anticonvulsant, binds with 5- and 100-fold higher at ASPET Journals on October 1, 2021 petition photolabeling with [3H]azietomidate and [3H]R-mTFD- affinity to the a1–b2 site than to the b1 and g1–b2 sites. These MPAB to identify para-substituted propofol analogs and other studies provide a first identification of PAMs that bind selectively drugs that bind selectively to intersubunit anesthetic sites. to a single intersubunit site in the GABAAR transmembrane Propofol and 4-chloro-propofol bind with 5-fold selectiv- domain, a property that may facilitate the development of 1 ity to b , while derivatives with bulkier lipophilic substitutions subtype selective GABAAR PAMs.

Introduction about anesthetic toxicities and undesirable side effects that makes important the development of safer anesthetics GABA receptors (GABA Rs), the receptors primarily re- A A (Forman, 2010; McKinstry-Wu et al., 2015). Other GABA R sponsible for fast inhibitory transmission in the brain, are A PAMs act as anticonvulsants, with use limited by side key targets for a structurally diverse class of drugs that act effects (Rogawski, 2006; Greenfield, 2013). Members of the as general anesthetics, including fluorinated ethers, pro- superfamily of pentameric ligand-gated ion channels, pofol, etomidate, barbiturates, and steroids, which all act as GABA Rs are assembled from five identical or homologous positive allosteric modulators (PAMs) (Franks, 2008; Zeller A subunits, and the 19 human GABA R subunit genes (in- et al., 2008). Despite their general efficacy, general anes- A cluding a1–6, b1–3, and g1–3) result in the expression of thetics are dangerous medicines that have therapeutic at least a dozen physiologic relevant subtypes that are indices ranging from 2 to 4, and there is increasing concern widely distributed throughout the central nervous system

(Chua and Chebib, 2017). With multiple GABAARsubtypes This work was supported, in whole or in part, by the National Institutes of as targets in vivo, anesthetics and anticonvulsants may Health National Institute of General Medical Sciences [Grant GM-58448] (J.B.C., K.W.M.). elicit desired actions through some subtypes and undesir- https://doi.org/10.1124/mol.118.114975. able actions through others (Bonin and Orser, 2008;

ABBREVIATIONS: 4-acetyl-propofol, 4-acetyl-2,6-diisopropylphenol; DAICc, corrected Akaike’s information criterion; 4-benzyl-propofol, 4-benzyl- 2,6-diisopropylphenol; 4-benzoyl-propofol, 4-benzoyl-2,6-diisopropylphenol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; 4-

Cl-propofol, 4-chloro-2,6-diisopropylphenol; ECD, extracellular domain; GABAAR, GABA type A receptor; 4-[hydroxyl(phenyl)methyl]-propofol, 4-(hydroxyl(phenyl)methyl)-2,6-diisopropylphenol; PAM, positive allosteric modulator; R-mTFD-MPAB, 1-methyl-5-allyl-5-(m-trifluoromethyl- diazirynylphenyl)barbituric acid; 4-(tert-butyl)-propofol, 4-(tert-butyl)-2,6-diisopropylphenol; TG-41, ethyl 2-(4-bromophenyl)-1-(2,4- dichlorophenyl)-1H-4-imidazolecarboxylate; TMD, transmembrane domain.

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Greenfield, 2013). Therefore, development of GABAAR subtype selective PAMs may lead to therapeutic agents with fewer undesirable side effects. Photolabeling studies with photoreactive anesthetics and mutational analyses, in conjunction with recently solved structures of GABAARs (Miller and Aricescu, 2014; Phulera et al., 2018; Zhu et al., 2018; Masiulis et al., 2019) and other pentameric ligand-gated ion channels, establish the presence of multiple binding sites for anesthetics and other PAMs within a single GABAAR (Sieghart, 2015; Chua and Chebib, 2017). In abg GABAARs, the most common subtype expressed at postsynaptic sites, GABA binding sites are located in the extracellular domain (ECD) at the two b1–a2 subunit interfaces, and benzodiazepines bind to a homologous site at the a1–g2 subunit interface (Sigel and Ernst, 2018) (Fig. 1). In the transmembrane domain (TMD), M2 helices from each Fig. 1. Locations in a a1b3g2 GABAAR of binding sites in the ECD for GABA and benzodiazepines and in the TMD for general anesthetics. subunit come together to form the ion channel. Amino acids Downloaded from Subunits are arranged b–a–b–a–g, counterclockwise, as viewed from the from the M1, M2, and M3 helices contribute to subunit extracellular space. GABA binds to sites at the interface between the b and interfaces, which contain binding sites for propofol, etomidate, a subunits, referred to as the b+–a2 subunit interface, and benzodiaze- + 2 and barbiturates in the extracellular third of the TMD (Chiara pines bind to a homologous site at the a –g interface. Indicated in the TMD are the four transmembrane helices (M1–M4) within each subunit et al., 2013; Forman and Miller, 2016) and distinct bind- and the high-affinity binding sites for azietomidate/etomidate at the b+–a2 ing sites for steroid anesthetics nearer the cytoplasmic sur- interfaces and for R-mTFD-MPAB at homologous sites at the a+–b2 and + 2 face (Hosie et al., 2006; Laverty et al., 2017; Miller et al., g –b subunit interfaces (Chiara et al., 2013; Forman and Miller, 2016). Propofol binds with similar affinity to the b+ and b2 sites (Chiara et al., molpharm.aspetjournals.org 2017). Photoaffinity labeling with analogs of etomidate 2013; Forman and Miller, 2016), and steroid anesthetics bind to a distinct 3 3 ([ H]azietomidate) and mephobarbital [[ H]1-methyl- site at the b+–a2 subunit interface (Hosie et al., 2006; Laverty et al., 2017; 5-allyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric Miller et al., 2017). acid ([3H]R-mTFD-MPAB)] identified two homologous, pharmacologically distinct classes of intersubunit general anes- 26 mM in ethanol), and [3H]azietomidate (19 Ci/mmol, 53 mMin thetic binding sites (Chiara et al., 2013) (Fig. 1). Azietomidate ethanol) were synthesized and tritiated previously (Savechenkov and etomidate bind with 100-fold selectivity to sites in the two et al., 2012; Jayakar et al., 2014). 4-(Tert-butyl)-2,6-diisopropylphenol 1 2 b –a subunit interfaces, while R-mTFD-MPAB binds with (4-(tert-butyl)-propofol) was from Chiron AS (Trondheim, Norway). 4-Acetyl- 1 2 1 2 50-fold selectivity to the sites within the a –b /g –b subunit 2,6-diisopropylphenol (4-acetyl-propofol), 4-chloro-2,6-diisopropylphenol interfaces. Competition photolabeling assays using these pho- (4-Cl-propofol), and 4-benozyl-propofol were synthesized as described at ASPET Journals on October 1, 2021 toprobes established that other barbiturates (pentobarbital and previously (Trapani et al., 1998; de Lassauniere et al., 2005), with the 1 thiopental) bind with less than 8-fold selectivity to the b2 sites, identity and purity of the compounds confirmed by HNMR.4-Benzyl- while propofol binds with ∼2-fold selectivity to the b1 sites. 2,6-diisopropylphenol (4-benzyl-propofol), racemic 4-(hydroxyl(phenyl)- methyl)-2,6-diisopropylphenol (4-[hydroxyl(phenyl)methyl]-propofol), Mutational analyses predict that the anticonvulsant PAMs 1 and the S-enantiomer were synthesized as described previously (Jarava- loreclezole and tracazolate also bind to the b sites (Wingrove Barrera et al., 2016). R-4-(hydroxyl(phenyl)methyl)-propofol was synthe- 1– 2 1– 2 et al., 1994; Thompson et al., 2002). The a b and g b sites sized following the same procedure (Jarava-Barrera et al., 2016), and are intrinsically nonequivalent due to differences in the amino the purity and structure were verified by NMR spectroscopy and by 1 2 acids contributed by aM3 and gM3 helices to the side of b measurement of specific rotation. (R)-Etomidate, propofol, GABA, interfaces, but in the presence of GABA, R-mTFD-MPAB binds stiripentol, topiramate, valnoctamide, felbamate, soybean asolectin, with similar high affinity to both sites (Jayakar et al., 2015). and the FLAG peptide (DYKDDDDK) were from Sigma-Aldrich. To further our understanding of the structural determi- Loreclezole and tracazolate were from Tocris. Bicuculline methochloride was from Abcam. Ethyl 2-(4-bromophenyl)-1-(2,4-dichlorophenyl)-1H- nants that contribute to selective binding of GABAAR PAMs to the intersubunit binding sites in the TMD, we used competi- 4-imidazolecarboxylate (TG-41) was a gift from Dr. Douglas Raines (Department of Anesthesia and Critical Care, Massachusetts General tion photolabeling assays with [3H]azietomidate and [3H]R- Hospital). The detergents n-dodecyl b-D-maltopyranoside and 3-[(3- mTFD-MPAB to determine the affinities of a panel of propofol cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) derivatives and structurally unrelated anticonvulsants for the 1 2 were from Anatrace-Affymetrix. b and b sites and to identify drugs that bind nonequiva- Expression and Purification of Human a1b3g2 GABA Rs. 2 A lently to the b intersubunit sites. Of the 11 agents studied, a1b3g2L GABAARs, with the a1 subunit containing a FLAG 1 four had the highest affinity for the b sites, whereas seven epitope at the N terminus of the mature subunit (MRK…SYG- had higher affinity for the b2 sites. Four agents had differ- DYKDDDDKQPS…), were expressed in a tetracycline-inducible, ent affinities for the a1–b2 and g1–b2 sites, with two of stably transfected HEK293S cell line and purified on an anti-FLAG these, 4-benzoyl-2,6-diisopropylphenol (4-benzoyl-propofol) and affinity resin as described previously (Chiara et al., 2013; Dostalova loreclezole, binding with the highest affinity to the g1–b2 et al., 2014). In brief, membranes were solubilized for 2.5 hours 1 2 in purification buffer (Chiara et al., 2013; Dostalova et al., 2014) and a –b sites, respectively. supplemented with 30 mM n-dodecyl b-D-maltopyranoside. Col- umn wash and elution buffers contained 5 mM CHAPS and 0.2 mM Materials and Methods asolectin, with 1.5 mM FLAG peptide used for elution of GABAAR. The concentration of GABAAR at the different stages of purifica- Materials. R-mTFD-MPAB (1-methyl-5-allyl-5-(m-trifluoromethyl- tion was assayed by determination of [3H]muscimol binding by diazirynylphenyl]barbituric acid), [3H]R-mTFD-MPAB (38 Ci/mmol, use of a filtration assay after precipitation with polyethylene glycol Binding Selectivity for a1b3g2 GABAAR General Anesthetic Sites 617

(Li et al., 2006; Chiara et al., 2013). The GABAARbindingsite R-mTFD-MPAB) in the presence of bicuculline compared with GABA concentration was measured with 250 nM [3H]muscimol, with varied from 20% to 80%. The source of this variability is unknown,

1 mM GABA added to determine nonspecific binding. For each although the ratio appeared to increase as GABAARs were purified purification, etomidate modulation of 2 nM [3H]muscimol binding from membranes isolated from later passage cells. For the results was determined as previously described (Chiara et al., 2013). For reported here, 16 GABAAR purifications were used, each character- 16 independent purifications, each starting with membrane frac- ized by specific incorporation ratios of ,45%. Each drug was tions containing ∼4nmolof[3H]muscimol binding sites, final characterized in at least three independent experiments utilizing at column eluates contained ∼1 nmol of binding sites. First and least two different receptor purifications. In four experiments using second elutions, each of 12 ml, contained site concentrations (mean three of these GABAAR purifications, photolabeling was also de- 6 S.D.)of546 8and316 7 nM, respectively. For elutions 1 and 2, termined in the absence of GABA or bicuculline (control). In these etomidate at 10 mM enhanced binding of 2 nM [3H]muscimol by experiments, the ratio of labeling in the presence of bicuculline 140% 6 20% and 134% 6 18%, respectively. Receptor aliquots compared with control was 72% 6 14%, consistent with bicuculline (1 ml) were flash frozen in liquid nitrogen and stored at 280°C acting as an inverse agonist, while labeling in the presence of GABA until use. was 184% 6 21% of control. 3 Photoaffinity Labeling of a1b3g2 GABAARs. To measure In a representative experiment, control [ H]azietomidate a subunit 6 1 6 1 photoincorporation at the subunit level, 30–60 mlofa1b3g2 GABAAR photolabeling was 1333 13 cpm ( GABA), 434 27 cpm ( bicucul- was photolabeled per condition with [3H]R-mTFD-MPAB (3 mCi) or line), and 205 6 23 cpm (nonspecific). For [3H]R-mTFD-MPAB, b [3H]azietomidate (2 mCi) at concentrations of 1–3 mM. After trans- subunit photolabeling was 4399 6 359 cpm (1GABA), 2244 6 310 cpm ferring appropriate volumes of [3H]azietomidate or [3H]R-mTFD- (1bicuculline), and 574 6 72 cpm (nonspecific). Depending upon the Downloaded from MPAB stock solutions to glass test tubes and evaporating solvent receptor purification and the concentration of radioligand, control under an argon stream, the radioligand was resuspended with [3H]azietomidate and [3H]R-mTFD-MPAB photolabeling in the pres- – GABAAR(∼0.7 ml) for 30 minutes on ice with occasional vortexing. ence of GABA varied from 1100 to 3100 cpm and 2000 10,000 cpm, Aliquots of 0.35 ml were then equilibrated for 15 minutes with GABA respectively, with labeling in the presence of bicuculline at 30%–40% (300 mM) or bicuculline methochloride (100 mM). During this time, of the GABA level. Nonspecific labeling was at 10%–20% of the GABA appropriate volumes of nonradioactive drugs were added by use of a level for [3H]azietomidate and at 10%–15% for [3H]R-mTFD-MPAB. molpharm.aspetjournals.org 1-ml syringe to 10-ml aliquots of GABAAR (without radioligand) in Quantitation of Concentration-Dependent Inhibition or 350-ml sample vials (catalog number CERT5000-69LV; Thermo Fisher Enhancement of Photolabeling. For GABAARs photolabeled with 3 Scientific). GABAAR equilibrated with radioligand was then added to [ H]azietomidate, parameters for the concentration dependence of samples containing nonradioactive drugs and further incubated for drug modulation were determined for 3H incorporation in the 56-kDa 30 minutes on ice to allow all binding and conformational transitions gel band, which reflects photolabeling of a1Met-236 in aM1 in the 1 2 to equilibrate before transfer to 96-well plastic plates (Corning catalog b –a interface sites. For [3H]R-mTFD-MPAB, parameters were number 2797) for photolabeling. Samples were irradiated using a determined for 3H incorporation in the 59- and 61-kDa gel bands, 365-nm lamp (Spectroline Model EN-16; Spectronics Corp, Westbury, which reflects photolabeling of bM1 b3Met-227 in the homologous but 1 2 1 1 NJ) for 30 minutes on ice at 0.5- to 1-cm distance from the lamp. Stock nonequivalent sites at the a –b /g –b interfaces (Chiara et al., solutions of 1 M propofol or 60 mM nonradioactive R-mTFD- 2013). For photolabeling in the presence of GABA, the concentration MPAB, loreclezole, tracazolate, ivermectin, and etomidate were pre- dependence of inhibition was fit using nonlinear least squares to at ASPET Journals on October 1, 2021 pared in ethanol. Stock solutions of the propofol analogs were single- or two-site models using eqs. 1 and 2, respectively: prepared at 60 mM in methanol. TG-41 (25 mM) was prepared in dimethylsulfoxide. Bicuculline methochloride (6 mM) and GABA 2 ð Þ 5 B0 Bns 1 (100 mM) were prepared in water. Ethanol or methanol at 0.5% (v/v) B x nH Bns (1) 1 1 x (or 0.2% dimethylsulfoxide for TG-41 experiments) was present in all IC50 samples during photolabeling. Those solvent concentrations altered ð 2 Þ ð 2 Þð 2 Þ , PH B0 Bns 1 PH B0 Bns photolabeling by 5%. All additions of nonradioactive drugs were to BðxÞ 5 1 1 Bns (2) GABA Rs in elution buffer containing 0.2 mM asolectin and 5 mM 1 1 x 1 1 x A IC50H IC50L CHAPS, a solution in which the drugs were soluble at the highest concentrations tested. where B(x) is the 3H in counts/min (cpm) incorporated into a subunit 3 Photolabeled samples were solubilized in SDS sample buffer and gel band at a total modulator concentration of x; and B0 is the H incubated for 30–60 minutes at room temperature before fraction- incorporation in the absence of inhibitor. Unless noted, the nonspecific ation by SDS-PAGE on Tris-glycine gels (6%) as previously described incorporation (Bns) was equal to the incorporation observed in the (Chiara et al., 2013). Gel bands were visualized with GelCode Blue presence of 300 mM etomidate for [3H]azietomidate or 60 mM R- 3 Safe Protein Stain (Thermo Fisher Scientific), with the gels treated mTFD-MPAB for [ H]R-mTFD-MPAB. IC50 is the total concentration with prestain solution [50% (v/v) methanol and 10% (v/v) glacial acetic of inhibitor reducing incorporated 3H by 50%, with H and L denoting acid] for 3 hours prior to staining to reduce background staining. The high- and low-affinity binding sites; PH is the fraction of specific three stained bands enriched in GABAAR a (56 kDa) and b (59 and photolabeling associated with the high-affinity binding site; and 3 61 kDa) subunits were excised separately, and H incorporation into nH is the Hill coefficient. To combinedatafrommultipleindepen- the excised gel bands was assayed by liquid scintillation counting as dent experiments, for each experiment 3H incorporation at each previously described (Chiara et al., 2013). inhibitor concentration was normalized to incorporation in the Allosteric Coupling and State Dependence of Drug Binding. absence of inhibitor (as percentage), and the full data set was fit to To quantify allosteric coupling between sites and the state dependence eq. 1 or eq. 2 using GraphPad Prism 7.0. The data plotted in the of drug binding, competition photolabeling experiments were carried figures are the mean (6S.D.) from N independent experiments. For out in parallel with GABAARs equilibrated with 300 mM GABA, which all fits to models, the best fit values (6S.E. in Results,95% under our assay conditions was expected to stabilize receptors in the asymmetric confidence interval in tables) of the variable parame- desensitized state, or with 100 mM bicuculline, an inverse agonist ters and the number of experiments are reported, with the plotted (Chang and Weiss, 1999; Thompson et al., 1999), which shifts curves calculated from those parameters. receptors toward the resting state. For photolabeling at anesthetic For drugs that inhibited photolabeling in the presence of bicucul- concentrations (∼1–3 mM), we found that in 21 independent GABAAR line, data were also fit to eq. 1, with nH set to 1 because of the small purifications, the ratio of specific incorporation of [3H]azietomidate or signal size compared with that in the presence of GABA. When [3H]R-mTFD-MPAB (i.e., inhibitable by 300 mM etomidate or 60 mM increasing drug concentrations first enhanced and then inhibited 618 Jayakar et al. photolabeling in the presence of bicuculline, data were fit to a model [3H]azietomidate and [3H]R-mTFD-MPAB photolabeling by a drug that assumes enhancement of photolabeling occurs as a result of the in the presence of GABA, an extra sum-of-squares F test (a 5 0.05) was tested drug binding with apparent affinity EC50 to a site different than used to compare separate fits for each radioligand to eq. 1 (nH 5 1) with the photolabeled site, where the drug binding inhibits photolabeling the fit for the pooled data (null hypothesis). This F test was also used to (eq. 3A): comparetheseparatefitstofitsofthe pooled data (null hypothesis) to determine the significance for differences of inhibition of [3H]azietomidate 2 3 BmaxÀ BbicÁ 1 2 or [ H]R-mTFD-MPAB photolabeling by a drug in the presence of GABA EC Bbic Bns 1 1 50 ð Þ 5 x 1 versus bicuculline. One-way ANOVA with Tukey’s multiple comparison B x nI Bns (3A) 1 x test was used to determine the significance of differences (a 5 0.05)oflog 1 IC 50 3 IC50 values for pairs of propofol analogs as inhibitors of [ H]azietomidate 3 3 1 with Bmax either fixed at the control H cpm incorporated in the or [ H]R-mTFD-MPAB photolabeling ( GABA). presence of GABA or, when noted, allowed to vary to determine 3 whether a better fit can be obtained. Bbic is the control H cpm Results incorporated in the presence of bicuculline, and Bns is the nonspecific incorporation. EC50 and IC50 are the total drug concentrations State Dependence of Photolabeling. We first compared producing half-maximal enhancement and inhibition of photolabeling, [3H]azietomidate and [3H]R-mTFD-MPAB photolabeling of respectively. Data from multiple independent experiments were a1b3g2 GABAARs in the presence of GABA, which stabilizes combined by normalizing (as percentage) to the control photolabeling

the desensitized state, with that in the presence of bicuculline, Downloaded from in the presence of GABA alone. Unless noted, the Hill coefficient for an inverse agonist (Chang and Weiss, 1999; Thompson et al., inhibition (nI) was set to 1, and Bns was fixed at the value seen in the presence of 300 mM etomidate or 60 mM R-mTFD-MPAB. 1999) that stabilizes the resting, closed-channel state 3 For four drugs (4-benzoyl-propofol, loreclezole, tracazolate, and TG-41) (Masiulis et al., 2019) (Fig. 2, A and D). For [ H]azietomidate, 3 that bind with different affinities to the two b2 sites in the presence of H incorporation was quantified in the a subunit gel GABA, enhancement and inhibition of [3H]R-mTFD-MPAB photolabel- band, which reflects photolabeling of a1Met-236 in aM1 in ing in the presence of bicuculline were fit to eq. 3A and to eq. 3B: the b1–a2 interface sites. For [3H]R-mTFD-MPAB, 3H in- molpharm.aspetjournals.org

2 corporation was quantified in the b subunit gel bands, which BmaxÀ BbicÁ EC ðB 2 B Þ 1 1 50 reflects photolabeling of b3Met-227 in bM1 in the homolo- ð Þ 5 bic ns 1 x 1 1 2 1 2 B x Bns (3B) gous but nonequivalent sites at the a –b /g –b interfaces 1 1 x 1 1 x IC50a IC50g (Chiara et al., 2013). For photolabeling at anesthetic concentrations (1–3 mM), specific photoincorporation of [3H]azietomidate This model takes into account the nonequivalent binding of 3 [3H]R-mTFD-MPAB to the b2 sites in the presence of bicuculline or [ H]R-mTFD-MPAB (i.e., etomidate or R-mTFD-MPAB inhib- (Jayakar et al., 2015). Under our photolabeling conditions at itable) in the presence of bicuculline was ∼30% of that seen in the 3 ∼1 mM[3H]R-mTFD-MPAB, it is the a1–b2 site that is occupied and presence of GABA. The lower level of [ H]azietomidate photo- photolabeled (Bbic-Bns), and the enhanced photolabeling in the pres- labeling in the presence of bicuculline resulted from its decreased ence of GABA or other modulator primarily reflects increased binding affinity for the b1–a2 sites. In addition, etomidate was less at ASPET Journals on October 1, 2021 1– 2 3 to the g b site (Bmax-Bbic). IC50a and IC50g are the apparent potent as an inhibitor of [ H]azietomidate photolabeling in affinities for the a1–b2 and g1–b2 sites, respectively, and EC is 50 the presence of bicuculline (IC50 5 5.5 6 1.4 mM) than in the the concentration producing half-maximal enhancement of photo- presence of GABA (IC 5 2.0 6 0.1 mM). For [3H]R-mTFD- labeling. For a drug binding with high affinity at the a1–b2 site that 50 1 2 MPAB in the presence of GABA, the b subunit photolabeling enhances photolabeling at the g –b site, we expect that EC50 = 1 2 1 2 1 2 resulted from labeling of the a –b and g –b sites at similar ∼IC50a. For a drug binding with the highest affinity to the g –b site, eq. 3B is not appropriate because the drug can only inhibit, not efficiency (Chiara et al., 2013; Jayakar et al., 2015). 3 enhance, photolabeling at that site. A drug binding with the highest R-mTFD-MPAB inhibited [ H]R-mTFD-MPAB photolabeling 1 2 affinity to the g –b site might produce a small enhancement of with IC50 5 0.66 6 0.05 mM and a Hill coefficient close to 1 (nH photolabeling at the a1–b2 site, which is not fully occupied under our 5 1.2 6 0.1), consistent with R-mTFD-MPAB binding with labeling conditions, in which case a fit to eq. 3A is appropriate, with similar affinities to the two b2 sites (Fig. 2D). In the presence 1 2 EC50 a measure of the apparent affinity for the g –b site and IC50 the 1 2 of bicuculline, R-mTFD-MPAB inhibited photolabeling with affinity for the a –b site. If the enhancement of photolabeling is at IC 5 3.1 6 1.0 mM. 1– 2 50 the a b site, the maximal enhancement from the fit (Bmax) should Allosteric Coupling between Intersubunit Anesthetic be ∼50%–60% of that seen in the presence of GABA, and values of Binding Sites. In the presence of bicuculline, etomidate at EC50 and IC50 should be close to the values of IC50H and IC50L 3 determined (eq. 2) by inhibition of [3H]R-mTFD-MPAB photolabeling concentrations from 1 to 100 mM increased [ H]R-mTFD-MPAB (1GABA). If a drug binds nonequivalently to the b2 sites with photolabeling to the level seen in the presence of GABA, with enhancement of photolabeling resulting from drug binding to the b1 inhibition then seen at higher concentrations (Fig. 2B). R-mTFD- 3 sites, fit to eq. 3B is appropriate. In this case, EC50 should be close MPAB at concentrations up to 3 mM enhanced [ H]azietomidate 1 1 2 to the IC50 for the b sites, and the affinity for the a –b site (IC50a) photolabeling, inhibiting it at higher concentrations (Fig. 2C). 1 2 may be less than or greater than the affinity for the g –b site (IC50g). The concentration-dependent enhancement of photolabeling by Data were fit to the models by nonlinear least squares using etomidate or R-mTFD-MPAB established that even in the GraphPad Prism 7.0 or SigmaPlot 11.0 (Systat Software, San Jose, presence of bicuculline, the binding of etomidate to the b1 sites 6 CA). For all fits, the best fit values of the variable parameters ( S.E. in is positively coupled energetically to the b2 sites, and the binding Results, 95% asymmetric confidence interval in tables) and the 2 of R-mTFD-MPAB to a b site is positively coupled energetically number of independent experiments are reported. The extra sum of 1 the squares principle (F test, a 5 0.05) was used to compare nested to the b sites. models with nonequivalent degrees of freedom. Otherwise, the A Photolabeling Assay to Determine Selectivity of difference in corrected Akaike’s information criterion values (DAICc) Drugs for Intersubunit Anesthetic Sites and to Detect 3 was used (GraphPad Prism 7.0) with the value of DAICc and Additional Sites. In the presence of GABA, [ H]R-mTFD- the predicted probability (%) reported. To compare inhibition of MPAB photoincorporation in the b subunit resulted from Binding Selectivity for a1b3g2 GABAAR General Anesthetic Sites 619 Downloaded from

+ 2 Fig. 2. Allosteric coupling between etomidate (b )andR-mTFD-MPAB (b ) intersubunit anesthetic binding sites. a1b3g2 GABAARs were photolabeled with [3H]azietomidate (A and C) or [3H]R-mTFD-MPAB (B and D) in the presence of etomidate (A and B) or nonradioactive R-mTFD-MPAB (C and D) and molpharm.aspetjournals.org bicuculline (100 mM, d) or GABA (300 mM, s). The receptor subunits were resolved by SDS-PAGE, and covalent incorporation of 3H was determined by 3 3 liquid scintillation counting of excised gel bands containing the GABAAR a ([ H]azietomidate) or b ([ H]R-mTFD-MPAB) subunits. Nonspecific photolabeling, indicated by the dashed lines, was determined in the presence of 300 mM etomidate or 60 mM R-mTFD-MPAB. For each independent experiment, data were normalized to the 3H cpm incorporated in the control condition (+GABA, no competitor), and the plotted data are the averages (6S.D.) from the independent experiments. As described under Materials and Methods, the pooled data from the independent experiments were fit to eq. 1 or, when enhancement of photolabeling was seen in the presence of bicuculline, to eq. 3A. Parameters for the fits and the number of independent experiments (N) are tabulated in Table 1. Based upon an F test comparison of fits of the data in the presence of GABA and bicuculline to eq. 1 3 to the same or separate IC50 values, separate fits were favored for etomidate inhibition of [ H]azietomidate [P = 0.0014, F(DFn,DFd) = 11.5(1,50)] and R-mTFD-MPAB inhibition of [3H]R-mTFD-MPAB photolabeling [P , 0.0001, F(DFn,DFd) = 246(1,68)]. at ASPET Journals on October 1, 2021 labeling of the a1–b2 and g1–b2 sites with similar efficiency increased [3H]R-mTFD-MPAB photolabeling, with inhibition (Chiara et al., 2013; Jayakar et al., 2015). The concentration occurring at higher concentrations (Fig. 3B). In contrast, dependence of inhibition of subunit photolabeling by drugs propofol did not enhance [3H]azietomidate photolabeling but that bind nonequivalently to the two sites will be character- inhibited it at concentrations above 10 mM (Fig. 3A). Since ized by Hill coefficients less than 1. For such drugs, fits of the propofol in the presence of GABA binds with 5-fold higher data to a two-site model will allow determination of the affinity to the b1 sites than to the b2 sites, propofol enhance- 1 2 1 2 3 affinities (IC50 values) for the a –b and g –b sites, and ment of [ H]R-mTFD-MPAB photolabeling is consistent with 3 1 2 the IC50 for inhibition of [ H]azietomidate photolabeling positive allosteric coupling between the b and b sites. The defines the affinity for the b1 sites. lack of enhancement of [3H]azietomidate photolabeling is The enhancement of [3H]R-mTFD-MPAB or [3H]- qualitatively consistent with propofol’s lower affinity for the azietomidate photolabeling by drugs in the presence of b2 sites. In addition, this lack of enhancement provides bicuculline provides an assay to identify PAMs that act with evidence that there are no other sites that both bind propofol a similar state dependence as GABA. Comparison of the with higher affinity than the b1 sites and are coupled 1 concentration dependence of enhancement to the IC50 for energetically to the b sites. For propofol and all other drugs inhibition of [3H]azietomidate or [3H]R-mTFD-MPAB photo- tested in the presence of bicuculline, quantitative fits of labeling in the presence of GABA provides a test of whether enhancement/inhibition are presented later. the enhancement results from the drug binding to either the Seeking para-Substituted Propofols that Bind Non- b1 or b2 intersubunit sites. For example, if a drug enhanced equivalently to a1–b2 and g1–b2 Sites. We characterized photolabeling at the b1 (etomidate) sites at concentrations a panel of 4-substituted (or para-substituted) propofol analogs, lower than those necessary to bind to the b2 (R-mTFD-MPAB) since many, including 4-Cl- and 4-benzoyl-propofol, act as potent sites, it would provide evidence that this PAM binds to sites GABAAR PAMs and general anesthetics (Trapani et al., other than the b1 and b2 intersubunit anesthetic sites. 1998; Krasowski et al., 2001a; Stewart et al., 2011). For five Propofol in the presence of GABA was more potent as an derivatives [4-Cl-propofol, 4-(tert-butyl)-propofol, 4-acetyl-propofol, 3 inhibitor of [ H]azietomidate (IC50 5 7.8 6 0.8 mM) than of 4-benzyl-propofol, and 4-[4-hydroxy(phenyl)methyl]-propofol], 3 3 [ H]R-mTFD-MPAB labeling (IC50 5 44 6 4 mM) (Fig. 3, A and the concentration dependence for inhibition of [ H]azietomidate B), consistent with previous results (Chiara et al., 2013). For and [3H]R-mTFD-MPAB labeling in the presence of GABA was both anesthetic photolabels, the concentration dependence of characterized by Hill coefficients close to 1 (Table 1). Similar to inhibition by propofol was fit by nH close to 1 (nH 5 1.1 6 0.1). propofol, 4-Cl-propofol bound with 4-fold higher affinity to the 1 2 In the presence of bicuculline, propofol from 3 to 30 mM b sites (IC50 5 5 6 1 mM) than to the b sites (Fig. 3, C and D). 620 Jayakar et al.

Fig. 3. Propofol (A and B) and 4-Cl-propofol (4-Cl-PPF) (C and D) bind preferentially to the b+ ([3H]azietomidate) sites and equivalently to the a+/g+–b2 ([3H]R-mTFD- MPAB) GABAAR anesthetic sites. GABAARs were photolabeled with [3H]azietomidate (A and C) or [3H] R-mTFD-MPAB (B and D) in the presence of bicuculline (d) or GABA (s). As described in Fig. 2, the 3H cpm incorporation in the GABAAR subunits was determined by liquid scintillation counting, and the data were then normalized for individual experiments that were combined, with the dashed lines indicating the nonspecific subunit photolabeling. The plotted data are the averages (6S.D.) from the independent experiments. The full data sets for each condition were fit to eq. 1, or to eq. 3A for [3H]R- mTFD-MPAB (+bicuculline). Parameters for the fits and the number of independent experiments are tabulated in Table 1. Based upon an F test comparison of fits of the data in the presence of GABA and bicucul-

line to eq. 1 to the same or separate IC50 values, Downloaded from separate fits were favored for inhibition of [3H]- azietomidate photolabeling by propofol [P = 0.003, F(DFn,DFd) = 9.2(1,69)] and 4-Cl-propofol [P = 0.016, F(DFn,DFd) = 6.2(1,62)]. molpharm.aspetjournals.org 4-Acetyl-propofol bound with little selectivity between the b1 and the b2 sites, enhanced [3H]azietomidate photolabeling with- 2 3 b sites (IC50 values ∼40 mM; Fig. 4, A and B). 4-Benzyl-propofol out enhancing [ H]R-mTFD-MPAB photolabeling. For drugs (Table 1), 4-(tert-butyl)-propofol (Fig. 4, C and D), and that only inhibited photolabeling in the presence of bicucul- 4-[4-hydroxy(phenyl)methyl]-propofol (Fig. 5, C and D) line, the differences in IC50 values in the presence of GABA each bound with ∼10-fold higher affinity to the b2 sites versus bicuculline were highly significant. Based upon F test (IC50 5 5.7 6 0.5, 17 6 3, and 10 6 1 mM, respectively). comparison of fits assuming common versus separate values of 3 For each of the drugs tested, the difference in IC50 values for IC50, separate fits were preferred for [ H]azietomidate inhibition [3H]azietomidate and [3H]R-mTFD-MPAB was statistically by propofol (P 5 0.003) and 4-Cl-propofol (P 5 0.016) and for [3H] significant. With the exception of 4-acetyl-propofol, there was R-mTFD-MPAB inhibition by 4-acetyl-propofol, 4-(tert-butyl)- no overlap of 95% confidence intervals (Table 1). Based upon F propofol (each P , 0.0001), and 4-[4-hydroxy(phenyl)methyl]- at ASPET Journals on October 1, 2021 test comparisons of fits assuming common versus separate propofol (P 5 0.001). values of IC50, separate fits were preferred. For 4-acetyl- 4-Benzoyl-propofol, which binds with the highest affinity propofol, P , 0.002 [F(DFn,DFd) 5 10.3(1,47)], and for the to one of the b2 sites, strongly enhanced [3H]azietomidate other drugs, P , 0.0001. photoincorporation, and at concentrations up to 10 mM, it also 4-Benzoyl-propofol was identified as a drug that distin- enhanced [3H]R-mTFD-MPAB photolabeling, with inhibition guished strongly between the two b2 sites. The concentration at higher concentrations as in the presence of GABA (Fig. 5, dependence of inhibition of [3H]R-mTFD-MPAB photolabel- A and B). Interpretation of the effects of 4-benzoyl-propofol on 3 ing was fit by a Hill coefficient (nH)of0.276 0.04 and [ H]R-mTFD-MPAB photolabeling in the presence of bicucul- IC50 5 21 6 6 mM (Fig. 5B). In a two-site model (eq. 2, line is complicated by the fact that the drug binds to one of Materials and Methods), this inhibition was characterized by the b2 sites with the highest affinity, to the b1 sites with 2 high-affinity (IC50H 5 0.5 6 0.3 mM; 45% 6 4% of specific intermediate affinity, and to the other b site with the lowest 2 incorporation) and low-affinity (IC50L 5 320 6 120 mM) affinity. The assignment of the b site associated with high- components. 4-Benzoyl-propofol was also unusual in that affinity binding will be considered later in conjunction with it was the only propofol analog studied that inhibited [3H]- quantitative analyses of the enhancement/inhibition profiles azietomidate photolabeling (IC50 5 99 6 8 mM) with a Hill for all drugs tested in the presence bicuculline. coefficient significantly greater than 1 (nH 5 2.0 6 0.3, P , Other Anesthetics and Sedative/Anticonvulsants 0.0001 [F(DFn,DFd) 5 25.6(1,38)]) (Fig. 5A). Can Distinguish between b2 Sites. We also identified For each propofol analog tested, the enhancement of [3H]- PAMs structurally unrelated to propofol that interact non- azietomidate or [3H]R-mTFD-MPAB photolabeling in the equivalently with the b2 sites: loreclezole, a triazole seda- presence of bicuculline was qualitatively consistent with tive/anticonvulsant (Wauquier et al., 1990); tracazolate, a the ligand selectivity for the b1 or b2 sites. 4-Cl-propofol, pyrazolopyridine anxiolytic and anticonvulsant with low which bound preferentially to the b1 sites, strongly en- sedative potency (Patel and Malick, 1982); and TG-41, an hanced [3H]R-mTFD-MPAB photolabeling without enhanc- imidazolecarboxylate that is a potent general anesthetic ing [3H]azietomidate photolabeling (Fig. 3, C and D). (Mascia et al., 2005). Loreclezole and tracazolate inhibited 4-Acetyl-propofol and 4-(tert-butyl)-propofol clearly enhanced [3H]azietomidate incorporation in the presence of GABA with 3 [ H]azietomidate photolabeling (Fig. 4, A and C) with little, IC50 values of 9.3 6 0.9 mM(nH 5 1.2 6 0.1) and 8.0 6 0.9 mM 3 if any, enhancement of [ H]R-mTFD-MPAB photolabeling (nH 5 1.0 6 0.1), respectively (Fig. 6, A and C). They each (Fig. 4, B and D). 4-[4-Hydroxy(phenyl)methyl]-propofol interacted nonequivalently with the b2 sites (Fig. 6, B and D). (Fig. 5, C and D), which binds with 10-fold higher affinity to The concentration dependence of loreclezole inhibition of TABLE 1 3 3 Inhibition and enhancement (+bicuculline) by para-substituted propofol analogs of a1b3g2 GABAAR photolabeling by [ H]azietomidate and [ H]R-mTFD-MPAB When drugs only inhibited photolabeling, data were fit to eq. 1 (Materials and Methods) to determine IC50 , the total drug concentration producing 50% inhibition of GABAAR photolabeling, and Hill coefficients (nH) and, when indicated, to eq. 2, a two-site model. For inhibition in the presence of bicuculline, nH was set to 1. When enhancement was seen in the presence of bicuculline, data were fit to eqs. 3A or 3B, as noted in the figure legends, to determine EC50 and IC50 values for 50% maximal enhancement and inhibition. Parameters [best fit 6 S.E. or 95% confidence interval (CI)] were determined from fits of data from N independent experiments from two or more GABAAR purifications. As bicuculline and GABA experiments were paired, their values of N are the same.

a 3 3 Ratio IC50(AziEt) /IC50(TFD-MPAB) [ H]R-mTFD-MPAB IC50 {95% CIs} (nH)[H]R-Azietomidate IC50 {95% CIs} (nH) Drug (NR-mTFD-MPAB , NAziET )

+GABA +GABA +Bicuculline EC50/IC50 +GABA +Bicuculline EC50 /IC50 mM mM mM mM Etomidate (5, 3) 0.01 6 0.002 430 {330, 580} (1.5 6 0.3) 3.0 6 0.7/440 6 50 2.0 {1.8, 2.3} (1.00 6 0.06) 5.5 {3.4, 9} R-mTFD-MPAB (5, 3) 45 6 12 0.7 {0.6, 0.8} (1.2 6 0.1) 3.1 {1.5, 5.0} 38 {29, 50} (1.0 6 0.1) 1.1 6 0.3/18 6 3 for Selectivity Binding Propofol (6, 6)b 0.2 6 0.04 44 {36, 53} (1.1 6 0.1) 2.5 6 0.8/57 6 11 7.8 {6, 10} (1.1 6 0.1) 29 {12, 97} 4-Cl-propofol (4, 4)b 0.25 6 0.08 20 {14, 28} (1.2 6 0.2) 1.5 6 0.4/19 6 3 4.9 {3.4, 7.0} (0.9 6 0.2) 20 {7, 90} 4-acetyl-propofol (4, 3)c 1.5 6 0.4 32 {25, 41} (0.9 6 0.1) 250 {100, 790} 48 {38, 61} (1.1 6 0.3) 17 6 2/41 6 4 4-(tert-butyl)-propofol (4, 3)c 8 6 3 17 {13, 23} (1.0 6 0.1) 120 {68, 220} 134 {100, 226} (1.2 6 0.2) 5.1 6 0.7/72 6 8 4-benzyl-propofol (2, 3) 5 6 1 5.7 {4.7, 6.9} (1.3 6 0.1) ND 30 {21, 53} (0.8 6 0.1) ND 4-benzoyl-propofol (6, 5)d 200 6 140e 21 {11, 38} (0.27 6 0.04) eq. 3A: 1.0 6 0.9/156 6 54 99 {82, 120} (2.0 6 0.3) 0.6 6 0.2/89 6 8 f eq. 2 IC50H = 0.5 6 0.3 Bmax =61%6 5% eq. 3B: Bmax =81%6 3% (45% 6 4%)/IC50L = 330 6 120 1.0 6 0.8/158 6 54 Bmax =87%6 5% 6 6 6 6 6 6 a

4-[hydroxyl(phenyl)methyl]-propofol 16 5 10 {8.5, 12} (1.1 0.1) 30 {15, 63} 157 33 (1.1 0.3) 8 2/41 8 1

(3,3) R-isomer (2) S-isomer (2)d 12 6 1; 7 6 1 ND; ND ND; ND ND; ND b 3 g

ND, not determined; GABA 2 a For the ratio of IC50 values (+GABA), uncertainties were determined by propagation of error from the individual parameter uncertainties. bData from Fig. 3. cData from Fig. 4.

d 3 A

Data from Fig. 5, with parameters for 4-benzoyl-propofol inhibition of [ H]R-mTFD-MPAB (+GABA) also fit to a two-site model, eq. 2. Sites Anesthetic General R e 3 The ratio calculated for the high-affinity component (IC50 H)of[H]R-mTFD-MPAB inhibition. f Parameters based upon fit of the data to eq. 3A with nI = 2, the Hill coefficient determined for inhibition in the presence of GABA.

621

Downloaded from from Downloaded molpharm.aspetjournals.org at ASPET Journals on October 1, 2021 1, October on Journals ASPET at 622 Jayakar et al.

Fig. 4. 4-Acetyl-propofol (A and B) binds nonselec- tively to b+ ([3H]azietomidate) and b2([3H]R-mTFD-MPAB) intersubunit sites, but 4-(tert-butyl)-propofol (4-tert-butyl- PPF) (C and D) binds preferentially to the b2 intersubunit anesthetic sites. GABAARs were photolabeled with [3H]azietomidate (A and C) or ([3H]R-mTFD- MPAB (B and D) in the presence of bicuculline (d)or GABA (s). As described in Fig. 2, the plotted data are the averages (6S.D.) from the independent experiments, with the dashed lines indicating the nonspecific subunit photolabeling. Data were fit to eq. 1 for [3H]R-mTFD-MPAB (both conditions) and to eqs. 1 and 3A for [3H]azietomidate in the presence of GABA and bicuculline, respectively. Parameters for the fits and the number of independent experiments are tabulated in Table 1. For fits of inhibition of [3H]R-mTFD-MPAB photolabeling in the presence of GABA vs. bicuculline by 4-acetyl-propofol and 4-tert-butyl-PPF, separate fits were favored Downloaded from [P , 0.0001, F(DFn,DFd) = 22(1,52) and 31(1,60), respectively].

3 molpharm.aspetjournals.org [ H]R-mTFD-MPAB photolabeling was characterized by a Hill inhibition characterized by IC50 5 18 6 7 mM (Fig. 6D). In the coefficient (nH) of 0.43 6 0.06 (IC50 5 116 6 35 mM) (Fig. 6B). presence of bicuculline, neither loreclezole nor tracazolate When fit to a two-site model, this inhibition was character- enhanced [3H]azietomidate labeling, while at concentrations 3 ized by a high-affinity component (IC50H 5 1.4 6 1.2 mM) from 1 to 30 mM, both enhanced [ H]R-mTFD-MPAB photo- accounting for 29% 6 7% of specific incorporation and a low- labeling, with inhibition at higher concentrations. 1 affinity component (IC50L 5 320 6 120 mM). Tracazolate at TG-41 bound with very high affinity to the b sites, 3 3 concentrations of 100 and 300 mM inhibited [ H]R-mTFD- inhibiting [ H]azietomidate photolabeling with IC50 values MPAB photolabeling maximally by ∼40%, with the observed of 16 6 3nM(1GABA) and 220 6 60 nM (1bicuculline) at ASPET Journals on October 1, 2021

Fig. 5. 4-Benzoyl-propofol (4-benzoyl-PPF) (A and B) binds nonequivalently to the b2 ([3H]R-mTFD-MPAB) intersubunit anesthetic sites, but 3 3 4-[hydroxyl(phenyl)methyl]-propofol (C and D) binds equivalently. GABAARs were photolabeled with [ H]azietomidate (A and C) or ([ H]R-mTFD-MPAB (B and D) in the presence of bicuculline (d) or GABA (s). The plotted data are the averages (6S.D.) from independent experiments, with the dashed lines indicating nonspecific subunit photolabeling. For experiments in the presence of GABA, the pooled data from the independent experiments were fit to eq. 1(Materials and Methods), and for 4-benzoyl-propofol ([3H]R-mTFD-MPAB) also to eq. 2, a two-site model (Table 1). Data for [3H]azietomidate 2 (+bicuculline) were fit to eq. 3A, with 4-benzoyl-propofol data fit to Bmax = 100% [dotted line, EC50 = 2.1 6 0.4 mM, IC50 =636 5 mM, nI =2,(R = 0.87, 2 F = 245)] or Bmax adjustable [solid line, Bmax =81%6 3%, EC50 = 0.6 6 0.2 mM, IC50 =896 8 mM, nI =2(R = 0.93)], a fit that was favored [P , 0.0001, F(DFn,DFd) = 30.6 (1,37)]. Since 4-benzoyl-propofol bound nonequivalently to the b2 sites in the presence of GABA, data for [3H]R-mTFD-MPAB photolabeling (+bicuculline) were fit to eqs. 3A and 3B, which fit the data similarly (DAICc = 0, (50%)). Equations 3A and 3B distinguish between preferential binding to the g+–b2 and a+–b2 sites, respectively (see Results and Discussion). Binding Selectivity for a1b3g2 GABAAR General Anesthetic Sites 623 Downloaded from

+ 3 2 3 Fig. 6. Loreclezole, tracazolate, and TG-41 bind with high affinity to the b ([ H]azietomidate) sites and to one b ([ H]R-mTFD-MPAB) site. GABAARs were photolabeled with [3H]azietomidate (A, C, and E) or [3H]R-mTFD-MPAB (B, D, and F) in the presence of bicuculline (d) or GABA (s). As described in Fig. 2, the plotted data are the averages (6S.D.) from independent experiments, with the dashed lines indicating nonspecific subunit photolabeling. The data for [3H]azietomidate were fit to eq. 1. For [3H]R-mTFD-MPAB (+GABA), loreclezole data were fit to eqs. 1 and 2 (Table 2), while tracazolate and 2 TG-41 data were fit to eq. 1 (nH = 1) with variable Bns. For tracazolate, Bns =56%6 5% and IC50 =186 7 mM(R = 0.68, F = 104); for TG-41, Bns =67%6 2% 2 3 + 2 and IC50 =0.16 0.04 mM(R = 0.75, F = 89). For [ H]R-mTFD-MPAB (+bicuculline), fit of the loreclezole data to eq. 3B with a –b as the high-affinity site molpharm.aspetjournals.org 2 + 2 (EC50 =IC50a =2.56 0.8 mM, IC50g =1606 30 mM, R = 0.70, F = 68) was favored over fit to eq. 3A with g –b as the high-affinity, enhancing site or to eq. 3B with g+–b2 as the high-affinity site and enhancement caused by binding to a site other than a b2 site (see Results). For tracazolate and TG-41, which each bound with highest affinity to the b+ sites and only to one of the b2 sites over the concentrations tested, data were fit to eq. 3B. For tracazolate, the fit + 2 2 with g –b as the high-affinity site [Bmax = 100%, EC50 =2.76 0.6 mM, IC50g =246 5 mM(R = 0.67, F = 96)] was strongly favored (DAICc = 12.7, (99.8%)) + 2 + 2 over the fit for a –b as the high-affinity site. For TG-41, the fit with a –b as the high-affinity site [Bmax = 100%, EC50 = 0.02 6 0.007 mM, IC50a =0.46 0.2 mM(R2 = 0.73, F = 79)] was slightly favored (DAICc = 2.5, (78%)) over the best fit for a high-affinity g+–b2 site.

(Fig. 6E). TG-41 in the presence of GABA, similar to GABAAR PAMs (Rho et al., 1997; Simeone et al., 2006; tracazolate, inhibited [3H]R-mTFD-MPAB maximally by Spampanato and Dudek, 2014). Stiripentol, which acted as only ∼35% at concentrations of 10–50 mM (Fig. 6F). The an a1b3g2 GABAAR PAM at concentrations of 30–100 mM 3 3 at ASPET Journals on October 1, 2021 observed inhibition was characterized by an IC50 of 100 6 (Fisher, 2009), inhibited [ H]azietomidate and [ H]R-mTFD- 40 nM. In the presence of bicuculline, TG-41 at concentrations MPAB photolabeling with IC50 values of 130 and 60 mM, above 30 nM increased [3H]R-mTFD-MPAB incorporation to respectively. the level seen at those concentrations in the presence GABA. Allosteric Coupling and Selectivity of Drugs for the We also screened additional anticonvulsants that act as Two b2 Intersubunit Sites, a1–b2 or g1–b2. For propofol GABAAR PAMs but do not bind to the GABA or benzodiaze- derivatives other than 4-benzoyl-propofol, which binds non- pine binding sites (Bonin and Orser, 2008; Greenfield, 2013) equivalently to the b2 sites, the concentration dependence (Fig. 7). Topiramate, felbamate, and valnoctamide did not of enhancement and inhibition of [3H]R-mTFD-MPAB or inhibit photolabeling at concentrations where they act as [3H]azietomidate photolabeling in the presence of bicuculline

Fig. 7. Inhibition of [3H]azietomidate and [3H]R-mTFD- MPAB photolabeling by sedatives/anticonvulsants. (A) GABAARs were photolabeled in the presence of GABA (300 mM) in the absence (control) or presence of 10 mM topiramate, 3 mM felbamate, 1 mM valnoctamide, or 300 mM stiripentol, and nonspecific photolabeling was determined in the presence of 300 etomidate or 60 mM R-mTFD-MPAB. Receptor subunits were resolved by SDS-PAGE, and covalent incorporation of 3H incorporation was determined by liquid scintillation counting of excised gel bands 3 containing the GABAAR a ([ H]azietomidate) or b ([3H]R-mTFD-MPAB) subunits. Specific subunit pho- 3 tolabeling ( H cpm, Btot – Bns), normalized to the control value, is plotted for two independent experiments (means 6 1/2 range). (B and C) Stiripentol inhibition of [3H]azietomidate or [3H]R-mTFD- MPAB photolabeling in the presence of bicuculline (100 mM, d) or GABA (300 mM, s). For each independent experiment, data were normalized to the 3H cpm incorporated in the control condition (+GABA, no competitor), and the plotted data are the averages (6S.D.) from the independent experiments. The pooled data were fit to eq. 1 or for [3H]azietomidate (+bicuculline) to eq. 3A (Table 2). 624 Jayakar et al. was fit to a model (Materials and Methods, eq. 3A) that assumes one site associated with enhancement (EC50) and a second site associated with inhibition (IC50). Unless noted in figure legends, better fits were obtained with Bmax equal to 100%, the photolabeling level in the presence of GABA, than with variable Bmax. For the drugs that bound preferentially to the [3H]azietomidate (b1) sites (propofol, 4-Cl-propofol) or to the [3H]R-mTFD-MPAB (b2) sites [4-(tert-butyl)-propofol and 4-[hydroxyl(phenyl)methyl]-propofol], the IC50 values for inhibition of the high-affinity site were 2- to 3-fold greater than the EC50 values for enhancement at the low-affinity site (Fig. 8A; Table 1). In the presence of GABA, 4-benzoyl-propofol distinguished strongly between the two b2 sites, inhibiting [3H]R-mTFD-MPAB photolabeling with IC50H 5 0.5 mMandIC50L 5 330 mM, whereas at the b1 sites, it inhibited [3H]azietomidate photolabeling 5 with IC50 99 mM (Fig. 5, A and B). In the presence of Downloaded from bicuculline, 4-benzoyl-propofol enhanced [3H]azietomidate photolabeling with EC50 5 0.6 6 0.2 mM (fit to eq. 3A), which was consistent with this action being mediated by binding to the high-affinity b2 site. In the presence of bicuculline, [3H]R-mTFD-MPAB photolabeling was enhanced by 4-benzoyl- propofol at concentrations sufficient to occupy the high- molpharm.aspetjournals.org affinity b2 site but 30-fold too low to occupy the b1 sites. To attempt to determine which of the two b2 sites is the high- affinity site for 4-benzoyl-propofol, we fit our data to eqs. 3A and 3B, reasoning the following as described in Materials and Methods: 1) if the high-affinity site is the a1–b2 site (eq. 3B), increasing 4-benzoyl-propofol concentrations would inhibit photolabeling at a1–b2, the site photolabeled in the presence of bicuculline (Jayakar et al., 2015), and enhance photolabeling 1 2 1 2 at the g –b site (a –b :IC50a 5 EC50 5 1.0 6 0.8 mM, Bmax 5 1 2 3 3 at ASPET Journals on October 1, 2021 87% 6 5%), with inhibition at high concentrations (g –b : Fig. 8. (A) Ratios of [ H]azietomidate IC50 (+GABA)/[ H]R-mTFD-MPAB 3 3 5 6 EC50 (+bicuculline) and of [ H]R-mTFD-MPAB IC50 (+GABA)/[ H]azieto- IC50g 158 54 mM); and 2) if the high-affinity site is the + 2 1– 2 midate EC50 (+bicuculline) for drugs selective for b and b subunit g b site (eq. 3A), 4-benzoyl-propofol cannot enhance interfaces. Ratios and error bars are calculated from the parameter fits 1– 2 photolabeling at g b , but it could produce small en- tabulated in Tables 1 and 2. (B) The relationship between a drug’sIC50 for 1 2 1 2 inhibiting [3H]azietomidate (d)or[3H]R-mTFD-MPAB (s) photolabeling hancement at the a –b site (g –b :EC50 5 1.0 6 0.8 mM, (+GABA) and the Connolly excluded volume of the propofol phenyl ring Bmax 5 60% 6 5%), with inhibition at high concentrations 1 2 substituent. Substituent volume is calculated (Discovery Studio; Biovia, (a –b :IC50 5 158 6 44 mM). Based upon Akaike’sinformation San Diego, CA) as the increase of the Connolly excluded volume over that of criterion, eqs. 3A and 3B fit the data equally (DAICc 5 0, (50%)), propofol. The lines are the linear least-squares fit of the data sets from 3 so our results do not allow an independent identification of the Table 1, with the plotted value of IC50H for [ H]R-mTFD-MPAB/4-benozyl- 2 propofol (4-benzoyl-PPF) excluded from the fit. One-way ANOVA with high-affinity b site (but see Discussion). Tukey’s multiple comparison test was used to determine the significance of Loreclezole, similar to 4-benzoyl-propofol, in the presence of differences (a = 0.05) of log IC50 values for pairs of propofol analogs as 2 5 inhibitors of [3H]azietomidate or [3H]R-mTFD-MPAB photolabeling GABA bound nonequivalently to the b sites (IC50H 1.4 mM, 3 5 1 (+GABA). For [ H]azietomidate, propofol/Cl- did not differ significantly IC50L 320 mM) but with the affinity for the b sites , 2 (P = 0.16), but each differed from the other tested drugs (P 0.0001). Other (IC50 5 9 6 1 mM) closer to that for the high-affinity b site significant differences were as follows: acetyl- vs. t-butyl- (P = 0.0003), (Fig. 7, A and B). In the presence of bicuculline, loreclezole benzoyl- (P = 0.006), or HO-phenyl- (P , 0.0001); t-butyl- vs. 3 benzyl- (P , 0.0001); benzyl- vs. benzoyl- or HO-phenyl- enhanced [ H]R-mTFD-MPAB photolabeling at concentra- 3 1 (P , 0.0001). For [ H]R-mTFD-MPAB (with omission of benzoyl-, which tions where it also bound to the b sites. In contrast to was fit to two-site model), propofol differed from each drug (P , 0.0001) 4-benzoyl-propofol, loreclezole binding to its high-affinity except acetyl-. Other significant differences were: Cl- vs. benzyl- b2 site did not enhance [3H]azietomidate photolabeling. (P , 0.0001) or HO-phenyl (P = 0.008); acetyl- vs. t-butyl- (P = 0.0066), 3 benzyl-, or HO-phenyl- (P , 0.0001); t-butyl- vs. benzyl- (P , 0.0001). The fit of the [ H]R-mTFD-MPAB data to eq. 3B with Bmax 5 1 2 1 2 100% (a –b ,IC50a 5 EC50 5 2.5 6 0.8 mM; g –b ,IC50g5 1 2 159 6 32 mM) was slightly favored (DAICC 5 2.6, (79%)) over results predict that a –b is the high-affinity site for the fit to eq. 3A with variable Bmax (Bmax 5 77% 6 7%, loreclezole, although the limited selectivity of loreclezole for 1 2 1 2 2 1 g –b :EC50 5 2.2 6 1.4 mM, a –b :IC50 5 172 6 62 mM) and a b site versus the b sites makes it likely that the 3 more strongly favored (DAICC 5 4.4, (90%)) over a fit to eq. 3B enhancement of [ H]R-mTFD-MPAB photolabeling is with g1–b2 being the high-affinity site and enhancement caused primarily by loreclezole binding to the b1 sites. 2 1 not resulting from binding to a b site (EC50 5 6 6 2 mM, IC50a 5 Consistent with this, loreclezole affinity for the b sites 620 6 480 mM, IC50g5 26 6 8 mM),afitthatresultedinavalue (IC50 5 9 mM) was ∼4-fold greater than the EC50 value of for the high-affinity b2 site not consistent with the value 2 mM for enhancement of [3H]R-mTFD-MPAB photolabel- determined in the presence of GABA (IC50H 5 1.4 mM). These ing from the preferred fit to eq. 3B, a ratio similar to that Binding Selectivity for a1b3g2 GABAAR General Anesthetic Sites 625

3 seen for propofol enhancement of [ H]R-mTFD-MPAB 50 photolabeling. 50 /IC 14

Tracazolate and TG-41 in the presence of GABA both bound 50 6

1 ) and, when 7 H 0.06 with the highest affinity to the b sites (Table 2), and 30 n M 3 6 6 6 m consistent with this, they both enhanced [ H]R-mTFD- ) H 14/84 n MPAB photolabeling in the presence of bicuculline (Fig. 6, D ( 6 and F). In the presence of GABA, high concentrations of either 50 drug inhibited [3H]R-mTFD-MPAB photolabeling maximally +Bicuculline EC by ∼40%. Control experiments established that this partial inhibition at high concentrations of either drug did not result from increased nonspecific photolabeling in the pres- -Azietomidate IC 0.3) 70 0.1) 44 0.1) 18 R purifications. As bicuculline and GABA R ence of 60 mM R-mTFD-MPAB. Thus, the incomplete in- A 6 6 6 H] hibition at high concentrations provided evidence that each 3 0.003 (1) 0.22

2 M drug bound nonequivalently to the b sites, and the data m 6 14 (1.8 +GABA 0.9 (1.2 0.9 (1.0 R photolabeling, and Hill coefficients (

allowed determination of an IC50 only for the high-affinity site. A 6 6 Because of this partial inhibition, data for tracazolate and 6 3

TG-41 inhibition of [ H]R-mTFD-MPAB in the presence of Downloaded from GABA were fit to eq. 1 with variable Bns rather than the value 5 in the presence of 60 mM R-mTFD-MPAB and with nH TFD-MPAB

1 because of the small amplitude of the signal. For tracazolate 0.2 0.016 30 9.3 R-m 50 6 6 6 5 8.0 and TG-41, the IC values were 18 7 and 0.1 0.04 mM, 6 50 H] /IC 3 6 respectively (Table 2). 50 Since tracazolate and TG-41 enhancement of [3H]R-mTFD- molpharm.aspetjournals.org M 0.007/0.4 39 134 0.8/160 0.6/24

MPAB photolabeling in the presence of bicuculline resulted m 6 6 6 from the drugs binding to the b1 sites, data were fit to eq. 3B 6 1 2 )[

– independent experiments, using two or more GABA to determine whether high-affinity binding to the a b or H n N 1– 2 ( g b site provided a better fit. For tracazolate, the fit with +Bicuculline EC 1 2 50 – 5 5 6 eq. 3B 2.5 g b as the high-affinity site (Bmax 100%, EC50 2.7 H]azietomidate and [ 3 0.6 mM, IC50g 5 24 6 5 mM) was strongly favored (DAICc 5 12.7, (99.8%)) over the best fit for the a1–b2 site as high affinity. The value of IC was close to the IC of 18 mM for 50g 50 2% eq. 3B 0.02 TFD-MPAB IC 3 120 m 5% eq. 3B 2.7 6

[ H]R-mTFD-MPAB inhibition in the presence of GABA, and - 6 at ASPET Journals on October 1, 2021 R 1.2 6 the value of EC was close to the IC of 8 mM for tracazolate , the total drug concentration producing 50% inhibition of GABA H] 50 50 0.06) 50 6 3 0.3) 73 3 1 [ 6

inhibition of [ H]azietomidate photolabeling ( GABA). In =67% =320 1 2 6 R photolabeling by [ = 56% ns contrast, for TG-41, fit to eq. 3B with a –b as the high- L A =1.4 M B 50 ns m 5 5 6 5 H affinity site (Bmax 100%, EC50 0.02 0.007 mM, IC50a B 7 (1.7 50 +GABA

6 5 35 (0.43

0.4 0.2 mM) was slightly favored (DAICc 2.5, (78%)) over 6 6 1 2 7%)/IC TFD-MPAB photolabeling. 2 GABA 7 (1)

– 5 6 0.04 (1) the best fit for the g b site as high affinity (B 85% ) to determine IC max g m 6 - 3 6 S.E.) were determined from fits of data from 6 116 R eq. 2 IC 3%, EC50 5 0.02 mM, IC50g 5 55 6 27 mM). Consistent with b 6 1 was set to 1. When enhancement was seen in the presence of bicuculline, data were fit to eq. 3B, as noted in the figure legends, to determine EC

1 2 H] a 3 – H

identification of a b as the high-affinity site, the value of (29% n 0.10 IC50a was close to the IC50 of 0.1 mM for TG-41 inhibition of [3H]R-mTFD-MPAB photolabeling in the presence of GABA, a whereas the best fit value of IC50g was not. Materials and Methods ) for inhibition of [ H TFD-MPAB)

Discussion 50 R-m 0.2 18 0.4 62 0.1 50(

In this report, we used competition photolabeling assays to c 6 6 6 6 identify structural features of propofol analogs and other /IC +GABA 0.2 GABAAR PAMs that determine selectivity between the ho- 50(AziEt)

mologous intersubunit anesthetic binding sites in the a1b3g2 are the same. N GABAAR transmembrane domain. Within a panel of TFD-MPAB photolabeling (+GABA), parameters are also included for the fit of the data to a two-site model, eq. 2. m - Ratio IC

para-substituted propofol analogs, drugs were identified R H] that bind with highest affinity to one (4-benzoyl-propofol) or 3 1 2

two [b : propofol, 4-Cl-propofol; b : 4-(tert-butyl)-propofol, )

4-[hydroxyl(phenyl)methyl]-propofol] sites per receptor, or aziet values (+GABA), uncertainties were determined by propagation of error from the individual parameter uncertainties.

1 2 N 50 ,

with similar affinity to 4 (b and b , 4-acetyl-propofol) sites. b We also identified PAMs structurally unrelated to propofol that bind nonequivalently to the b2 sites (loreclezole, tracazolate, and TG-41). Table 3 summarizes the relative affinities R-mTFD-MPAB

1 2 values for 50% maximal enhancement and inhibition. Parameters (best fit N

of the drugs for the b and b intersubunit sites. Similar to 50 For the ratio of IC For loreclezole inhibition of [ GABA, the drugs studied bind preferentially to a desensitized The ratio calculated for the high-affinity component (IC a b c Drug ( Loreclezole (4, 4) Tracazolate (6, 3) 0.4 Stiripentol (3, 3) 2 TG-41 (4, 3) indicated, to eq. 2, a two-site model. For inhibition in the presence of bicuculline, experiments were paired, their values of state of the receptor compared with the resting state stabilized TABLE 2 Sedative/anticonvulsant inhibition and enhancementWhen (+bicuculline) drugs of only inhibited photolabeling, data were fitand IC to eq. 1 ( 626 Jayakar et al. by bicuculline. However, electrophysiological studies are [35S]-tert-butylbicyclosphophorothionate binding to brain necessary to determine relative affinities of these drugs at membranes at concentrations [IC50 5 0.9 mM (Trapani et al., each site for open-channel compared with resting states. 1998)] consistent with binding to the high-affinity b2 site but Structural Determinants for Selective Binding to b1 100-fold lower than those necessary to occupy the b1 sites. Thus, or b2 Intersubunit Anesthetic Sites. This work provides 4-benzoyl-propofol may serve as a lead compound to identify a first identification of small structural modifications of other propofol derivatives that bind nonequivalently to the g1/ propofol that determine selectivity for the b1 versus b2 a1–b2 sites. intersubunit sites. Propofol and derivatives with para-sub- Amino acid differences on the plus side of the interface at stituents of small volume (H-, Cl-) bind with 5-fold selectivity homologous positions in aM3 (Ala, Tyr) and gM3 (Ser, Phe) to the b1 sites, while substituents with increasing volume contributing to the b2 binding sites (Chiara et al., 2013) are bind with lower affinity to the b1 sites and with higher affinity likely to contribute to the nonequivalent binding of 4-benzoyl- to the b2 sites (Table 1). The substituent volumes vary from propofol to the b2 sites. Our results establish that 4-benzoyl- 16 to 100 Å3, compared with propofol’s volume of 175 Å3, and propofol binding to its high-affinity b2 site is state-dependent over this range there is a reasonable linear correlation and leads to enhancement of [3H]azietomidate photolabeling 3 3 between substituent volume and log[IC50] for [ H]R-mTFD- (1bicuculline). Unfortunately, the [ H]R-mTFD-MPAB/4- MPAB (R2 5 0.75, P 5 0.03) or [3H]azietomidate (R2 5 0.65, benzoyl-propofol photolabeling data were fit equally well by 5 1– 2 P 0.05) inhibition (Fig. 8B). For para-substituted propofol equations assuming high-affinity binding to the g b Downloaded from analogs, including 4-Cl- and 4-benzoyl-propofol, as inhibitors (eq. 3A) or a1–b2 site (eq. 3B), which precludes identification of [35S]-tert-butylbicyclophosphorothionate binding to brain of the high-affinity b2 site. However, much published func- membranes, potency was enhanced by increased lipophilicity tional data suggest strongly that the g1–b2 site is the high- but reduced by substituent size (Trapani et al., 1998). affinity site that enhances [3H]azietomidate photolabeling. Quantitative structure-activity analyses identified the hydro- Drug interactions with the g1–b2 interface have been identi- phobic pockets accommodating the 2- and 6-iso-propyl groups fied as major determinants of abg GABA R conformational

A molpharm.aspetjournals.org as key determinants of propofol’s anesthetic and GABAAR transitions in other functional and photolabeling studies: 1) modulatory actions (Krasowski et al., 2002). While sec-butyl mutational analyses found the g1–b2 interface more important or tert-butyl replacements decreased affinity at the b1 and than the a1–b2 interface for propofol enhancement (Maldifassi b2 sites similarly (Chiara et al., 2013), studies with a broader et al., 2016); 2) mutational analyses established that ivermectin 1 2 range of substitutions can establish whether these positions binding to the g –b interface produced persistent GABAAR determine selectivity for b1 versus b2 sites. activation in the absence of GABA, while binding to the a1–b2 Nonequivalent Binding to b2 Intersubunit Sites. site enhanced GABA responses without persistent direct activa- This study’s novel finding is that some compounds bind tion (Estrada-Mondragon and Lynch, 2015); and 3) 1-methyl- selectively to the g1–b2 or a1–b2 site. The preference of 5-propyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid, 4-benzoyl-propofol for a single b2 site was unexpected in a convulsant differing in structure from R-mTFD-MPAB by at ASPET Journals on October 1, 2021 view of the nonselective binding of 4-benzyl-propofol chirality and the presence of a 5-propyl rather than 5-allyl (4-C6H5CH2-propofol) and 4-[hydroxyl(phenyl)methyl]- group, inhibited a1b3g2 while enhancing a1b3 GABAAR propofol. The most likely explanation for this difference responses (Desai et al., 2015; Jayakar et al., 2015), and is that, because of carbonyl-mediated electron resonance a1b3g2 GABAAR photolabeling established that 1-methyl- stabilization, 4-benzoyl-propofol’s structure is more rigid 5-propyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid than the other structures. 4-Benzoyl propofol inhibited binds with highest affinity to the g1–b2 site in the presence of

TABLE 3

Relative affinities of GABAAR PAMs for intersubunit anesthetic sites in the presence of GABA Data from Tables 1 and 2 are summarized by identifying the site with highest affinity (H), the sites binding with 2- to 10-fold lower affinity (I), or .15-fold lower affinity (L) than the high-affinity site. For the drugs that, in the presence of GABA, bind nonequivalently to the b2 sites, prediction of the high-affinity b2 site is based upon [3H]R-mTFD-MPAB photolabeling in the presence of bicuculline (see Results and Discussion).

b+ Interfaces b2 Interfaces Ligands + 2 + 2 + 2 b –a IC50 a –b IC50 g –b IC50

mM mM mM That distinguish the two b2 sites 4-benzoyl-propofol L 100 L 330 H 0.5 Loreclezole I 9 H 1 L 300 TG-41 H 0.02 I 0.1 L . 100 Tracazolate H 8 L . 300 I 18 That do not distinguish the two b2 sites Etomidate H 2.0 L 430 L 430 Propofol H 7.8 I 44 I 44 4-Cl-propofol H 4.9 I 20 I 20 4-acetyl-propofol H 48 H 32 H 32 4-(tert-butyl)-propofol I 130 H 17 H 17 4-benzyl-propofol I 30 H 6 H 6 Stiripentol I 130 H 60 H 60 4-OHPheMe-propofol L 160 H 10 H 10 R-mTFD-MPAB L 32 H 0.7 H 0.7 Binding Selectivity for a1b3g2 GABAAR General Anesthetic Sites 627 bicuculline, while R-mTFD-MPAB binds to that site preferen- photolabeled by a photoreactive propofol analog (Yip et al., tially in the presence of GABA and with little state dependence 2013). That propofol did not enhance [3H]azietomidate photo- to the a1–b2 site. labeling makes it unlikely that propofol binds to these sites in a Loreclezole, tracazolate, and TG-41 also bind with high state-dependent manner with higher affinity than the b1 sites. affinity to only one of the b2 sites, with neither tracazolate nor TG-41 inhibiting photolabeling at the other b2 site at the highest 2 concentrations tested. Loreclezole bound to its high-affinity b Conclusions site with ∼5-fold higher affinity than to the b1 sites but did not 3 1 Our study is the first to demonstrate that drugs can enhance [ H]azietomidate photolabeling ( bicuculline). Fits 2 of the [3H]R-mTFD-MPAB photolabeling data (1bicuculline) selectively target the b interfaces in the GABAAR TMD that for loreclezole slightly favored the a1–b2 site as its high-affinity are flanked by the a1org2 subunit. Together with previous b2 site. That loreclezole did not enhance [3H]azietomidate work (Chiara et al., 2013; Jayakar et al., 2015), this makes it photolabeling provides evidence that loreclezole, similar possible in principle to develop drugs selective for four of the to R-mTFD-MPAB, binds with little state dependence to the five TMD subunit interfaces in synaptic GABAARs. The 1– 2 potential for targeting those interfaces is great because there a b site. Tracazolate and TG-41 each bound with the 1 2 1 3 are six a and three b subunits, and they appear in both a –b highest affinity to the b sites and enhanced [ H]R-mTFD- 1 2 1 3 and b –a varieties. This principle has been recognized in the MPAB photolabeling ( bicuculline). Fits of the [ H]R-mTFD- Downloaded from 1 ECD, where agents are being developed that target sites MPAB photolabeling data ( bicuculline) predicted strongly 1 2 1 2 2 – that g –b is the high-affinity b site for tracazolate homologous to the benzodiazepine site in the a g interfaces 1 2 2 (Sieghart et al., 2012). However, the possibilities for allosteric (IC50g ∼25 mM), while a –b is the high-affinity b site for TG-41. For loreclezole, tracazolate, and TG-41, mutational action in the ECD are fewer because two of the interfaces analyses provided strong evidence that their enhancement of contain the orthosteric agonist sites. GABA R responses partially results from binding to the b1

A molpharm.aspetjournals.org sites. Thus, substitutions at b2/3Asn-265, position 159 in the Authorship Contributions bM2 helix, prevented enhancement by etomidate and propofol Participated in research design: Jayakar, Chiara, Cohen. (Belelli et al., 1997; Krasowski et al., 2001b; Bali and Akabas, Conducted experiments: Jayakar, Chiara. 2004) and reduced enhancement by loreclezole, tracazolate, Contributed new reagents or analytic tools: Zhou, Jarava-Barrera, and TG-41 (Wingrove et al., 1994; Thompson et al., 2002; Savechenkov, Bruzik, Tortosa, Miller. Asproni et al., 2005; Groves et al., 2006). b3Asn-265 contrib- Performed data analysis: Jayakar, Cohen. utes directly to the [3H]azietomidate/etomidate binding site Wrote or contributed to writing of the manuscript: Jayakar, Chiara, Miller, Cohen. (Chiara et al., 2012; Chiara et al., 2013; Stewart et al., 2014). While loreclezole, tracazolate, and TG-41 bind nonequiva-

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