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Vol. 297, Issue 1, 338-351, April 2001


General Anesthetic Potencies of a Series of Propofol Analogs Correlate with Potency for Potentiation of gamma -Aminobutyric Acid (GABA) Current at the GABAA Receptor but Not with Lipid Solubility

Matthew D. Krasowski , Andrew Jenkins, Pamela Flood, Amiinah Y. Kung, Anton J. Hopfinger and Neil L. Harrison

Department of Anesthesiology, Weill Medical College of Cornell University, New York City, New York (A.J., N.L.H.); Department of Anesthesia and Critical Care (M.D.K., A.Y.K.) and the Committee on Neurobiology (M.D.K.), University of Chicago, Chicago, Illinois; Department of Anesthesiology, Columbia University, New York City, New York (P.F.); and Laboratory of Molecular Modeling and Design, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois (A.J.H.)

    Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

A series of 27 analogs of the general anesthetic propofol (2,6-diisopropylphenol) were examined for general anesthetic activity in Xenopus laevis tadpoles and for the ability to produce enhancement of submaximal GABA responses and/or direct activation at recombinant GABAA receptors. Fourteen of the propofol analogs produced loss of righting reflex in the tadpoles, whereas 13 were inactive as anesthetics. The same pattern of activity was noted with the actions of the compounds at the GABAA alpha 1beta 2gamma 2s receptor. The potencies of the analogs as general anesthetics in tadpoles correlated better with potentiation of GABA responses than direct activation at the GABAA alpha 1beta 2gamma 2s receptor. The calculated octanol/water partition coefficients for the analogs did not explain the lack of activity exhibited by the 13 nonanesthetic analogs, although this physicochemical parameter did correlate modestly with in vivo anesthetic potency. The actions of one nonanesthetic analog, 2,6-di-tert-butylphenol, were examined in detail. 2,6-Di-tert-butylphenol was inactive at GABAA receptors, did not function as an anesthetic in the tadpoles, and did not antagonize any of the actions of propofol at GABAA receptors or in tadpoles. A key influence on the potency of propofol analogs appears to be the size and shape of the alkyl groups at positions 2 and 6 of the aromatic ring relative to the substituent at position 1. These data suggest steric constraints for the binding site for propofol on the GABAA receptor.

    Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Since its discovery in 1980, propofol (2,6-diisopropylphenol) has proven to be a clinically useful general anesthetic. The advantages of propofol include a rapid onset and offset of action and relatively low toxicity, which has led to the use of propofol in many surgical and critical care settings (Langley and Heel, 1988). There have been a number of efforts to understand the molecular mechanism of action of this clinically useful drug (Trapani et al., 2000).

One hypothesis, supported by substantial experimental evidence, cites the ability of propofol to positively modulate the function of gamma -aminobutyric acid type A (GABAA) receptors, a property common to many other general anesthetics (Franks and Lieb, 1994; Krasowski and Harrison, 1999; Trapani et al., 2000). Propofol has been shown in electrophysiological assays to allosterically enhance ("potentiate") the actions of GABA at the GABAA receptor (Hales and Lambert, 1991) and also to prolong inhibitory postsynaptic currents mediated by GABAA receptors (Orser et al., 1994). Propofol can also open the GABAA receptor ion channel in the absence of GABA (termed "direct activation") although this usually occurs at higher concentrations of propofol than necessary to potentiate submaximal receptor responses to GABA (Hales and Lambert, 1991; Hara et al., 1993; Jones et al., 1995).

The general anesthetic properties of propofol were initially discovered during a screen of 97 alkylphenols in mice and rabbits, following up on the initial observation that 2,6-diethylphenol possessed potent anesthetic effects (James and Glen, 1980). There have been few studies using structure-activity relationship (SAR) analysis for propofol (Trapani et al., 1998; Sanna et al., 1999; Lingamaneni et al., 2001), even though the relatively simple molecular structure of propofol would seem to favor SAR analysis.

The studies in this manuscript report concentration-response relationships for three actions of 27 propofol analogs (Fig. 1): loss of righting reflex in Xenopus laevis tadpoles, potentiation of submaximal GABA responses at the GABAA alpha 1beta 2gamma 2s receptor, and direct activation of the GABAA alpha 1beta 2gamma 2s receptor. X. laevis tadpoles were chosen for the determination of in vivo anesthetic potency because propofol and other phenols have very complicated pharmacokinetics in mammals, including extensive binding to plasma proteins (Langley and Heel, 1988; Trapani et al., 2000). The loss of righting reflex assay in tadpoles, which has a long history with respect to the study of general anesthetics, was therefore chosen to generate a self-consistent set of potencies for the immobilizing properties of the propofol analogs (Downes and Courogen, 1996). The GABAA alpha 1beta 2gamma 2s receptor was selected for the electrophysiological studies because this represents the most common subunit combination in the mammalian central nervous system (McKernan and Whiting, 1996).


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  		Fig. 1.   Chemical structures of the 27 propofol analogs analyzed in this study. The top structure illustrates propofol, depicting the numbering of the carbon atoms on the aromatic ring.

Additional studies characterized the pharmacological properties of GABAA receptors in dissociated spinal neurons from X. laevis tadpoles. Spinal neurons were chosen because spinal cord circuitry is involved in mediating the immobilization produced by general anesthetics (for review, see Collins et al., 1995), although it is not yet clear if the neuronal circuitry underlying nocifensive movements in mammals and righting reflex in tadpoles is the same. The results of these experiments further justified the use of tadpoles as a model system for studying general anesthesia and the role of GABAA receptors in the effects of propofol and its analogs.

We also compared the potencies of the propofol analogs with their calculated octanol/water partition coefficients, a measure of lipid solubility. The relationship between general anesthetic potency and the octanol/water partition coefficient, the "Meyer-Overton correlation", has often been invoked to suggest that general anesthetics act at lipid, as opposed to protein, targets. Traditional theories of general anesthesia, however, have proved increasingly untenable in the face of experimental evidence (Franks and Lieb, 1994; Krasowski and Harrison, 1999), including the discovery of compounds known as "nonimmobilizers", which possess high lipid solubility and yet lack anesthetic activity, despite being structurally related to ether and alkane general anesthetics (Koblin et al., 1994). The propofol analogs selected for investigation here included compounds such as 2,6-di-tert-butylphenol known to lack anesthetic activity in mice (James and Glen, 1980). The experiments thus tested what molecular effect best accounts for both the anesthetic activity and inactivity of a series of propofol analogs.

    Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Determination of Anesthetic Potencies for Loss of Righting Reflex in X. laevis Tadpoles. General anesthetic potencies were determined as previously described (Krasowski and Harrison, 2000) for X. laevis tadpoles (Xenopus 1; Ann Arbor, MI) in the prelimb-bud stage of development, corresponding to stages 43 to 50 of the standard nomenclature for X. laevis development (Nieuwkoop and Faber, 1956). Tadpoles were maintained in dechlorinated tap water in an aerated aquarium at room temperature.

The assay for loss of righting reflex in tadpoles has historically been a very popular assay for determining the in vivo potency of general anesthetics (Downes and Courogen, 1996). One major advantage of determining anesthetic potencies in tadpoles is that, in theory, the passage of drugs across the gills or skin of amphibians depends on the same physicochemical parameters as does equilibration across the mammalian blood-brain barrier (Downes and Courogen, 1996). At steady state, there should be an equilibrium between the drug in the bath and in the plasma of the tadpole. Drug metabolism in tadpoles appears to be far less efficient than in mature frogs, even relative to body weight. The role of such metabolism in tadpoles is therefore negligible in comparison with uptake and elimination across the skin and gills (Brodie and Maickel, 1962).

The anesthetic endpoint referred to as loss of righting reflex, a measure of immobility, is defined as a lack of purposeful and sustained swimming response after a gentle inversion with a smooth glass rod (Downes and Courogen, 1996). During randomized blind experiments, approximately 10 tadpoles were placed in each of a number of beakers containing 300 ml of tap water, with or without the addition of anesthetic compounds. Except for a tap water control, all beakers contained 0.2% (v/v) dimethyl sulfoxide (Sigma, St. Louis, MO) to control for the highest dimethyl sulfoxide concentration that would be present in any experiment. No anesthetic actions or lethality due to dimethyl sulfoxide were observed at 0.2% (v/v) or lower.

The number of anesthetized tadpoles was recorded every 10 min for up to 120 min, with equilibrium usually reached within 20 to 60 min, after which the tadpoles were returned to fresh tap water, and recovery was monitored. To lessen the risk of lethality, tadpoles were removed from beakers in which the drug completely ablated all tadpole movement, including twitching, before 120 min. Because anesthesia is defined as a reversible phenomenon, instances in which a tadpole failed to recover from a particular drug concentration were scored as a lethal event and not as anesthesia. Drugs that produced no reversible loss of righting reflex at any concentration, even though they might produce lethality at high concentrations, were defined as "inactive" with respect to anesthetic activity. To ensure that anesthetic potency did not vary depending on developmental stage, propofol was tested at several different developmental stages, with very little variation in results (M. D. Krasowski and A. Jenkins, unpublished observations).

Tadpole concentration-response data were fitted to a quantal analysis ("Waud") equation of the form p = (100 × In) [In + (EC50)n]-1 where p is the percentage of the population anesthetized, I is the anesthetic concentration, n is the slope factor, and EC50 is the concentration for a half-maximal anesthetic effect (Waud, 1972). Quantal analysis used software written by one of us (A.J.).

Estimates of the LC50 (concentration producing lethality in 50% of the tadpole population) were calculated using the Waud equation for those drugs where sufficient data existed for such a determination. Because these experiments were not designed to assess lethality in detail, in some cases there was only sufficient data to estimate a range of concentrations for the LC50.

Dissociation and Culturing of Tadpole Spinal Neurons. The method used to dissociate X. laevis tadpole spinal neurons was adapted from a procedure previously described (Dale, 1991). The following saline solutions were used: HEPES saline, 115 mM NaCl, 3 mM KCl, 1 mM Ca(NO3)2, 1 mM CaCl2, 1 mM MgCl2, 2.4 mM NaHCO3, 10 mM glucose, 10 mM HEPES, adjusted to pH 7.6 with NaOH; PIPES saline, 115 mM NaCl, 3 mM KCl, 0.1 mM CaCl2, 0.1 mM MgCl2, 25 mM glucose, 10 mM piperazine-N,N'-bis[2-ethanesulfonic acid] (PIPES), adjusted to pH 7.0 with HCl; and dissociation saline, 115 mM NaCl, 3 mM KCl, 2 mM EDTA, 25 mM glucose, 10 mM PIPES, adjusted to pH 7.0 with HCl. All saline solutions were saturated with 100% O2 before use.

Stage 45 to 50 X. laevis tadpoles (Nieuwkoop and Faber, 1956) were anesthetized with 0.5 g/l 3-aminobenzoic acid ethyl ester (Sigma) dissolved in dechlorinated tap water. For each tadpole, the head was removed and the spinal cord, often with ganglia clearly visible, was carefully dissected out using fine forceps and placed into HEPES saline. Spinal cords from up to five animals were combined to increase the yield of neurons. The spinal cords were transferred to a dish containing 1.25 mg/ml type XI trypsin (Sigma) in HEPES saline and incubated for 3 min at room temperature, then to a dish containing dissociation saline for 1 min, and finally to a dish containing PIPES saline for 5 to 6 min. The cords were manually agitated in both the dissociation and PIPES saline. Any obvious connective tissue or pigment cells were removed with fine tungsten pins, and the cords were then transferred to a dish of HEPES saline, aspirated in 2 to 3 ml of HEPES saline, and triturated with a fire-polished Pasteur pipette to dissociate the tissue.

The dissociated cords were plated onto coverslips coated with poly(D-lysine). The neurons were allowed to settle for at least 4 h before electrophysiological recordings and were used for up to 16 h after plating. The neurons used for electrophysiological analysis had somata approximately 6 to 10 µm in diameter and often possessed a single prominent neurite less than 5 µm in length. Electrophysiological recordings from dissociated neurons were performed as described below for recordings of HEK 293 cells.

Cell Culture and Transfection of Receptor cDNAs. The GABAA alpha 1 (Schofield et al., 1989) and gamma 2s (Pritchett et al., 1989) receptor subunits cDNAs are of human origin. The GABAA beta 2 receptor subunit cDNA is from the rat (Ymer et al., 1989). The human GABAA alpha 1 and gamma 2s receptor subunit cDNAs were generously provided by the late Dr. Dolan Pritchett (University of Pennsylvania, Philadelphia, PA). The rat GABAA beta 2 subunit cDNA was provided by Dr. Dennis Grayson (University of Illinois at Chicago, Chicago, IL).

GABAA receptor cDNAs were expressed via the vector pCIS2, which contains one copy of the strong promoter from cytomegalovirus and a polyadenylation sequence from simian virus 40. HEK 293 cells (American Type Culture Collection, Rockville, MD) were maintained in culture and passaged weekly by trypsin treatment for a maximum of 20 times before being discarded and replaced with early passage cells. HEK 293 cells were maintained in Eagle's minimal essential medium (Sigma) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), L-glutamine (0.292 µg/ml; Life Technologies, Grand Island, NY), penicillin G sulfate (100 units/ml; Life Technologies), and streptomycin sulfate (100 µg/ml; Life Technologies).

For electrophysiological experiments, cells were plated on glass coverslips coated with poly(D-lysine) (Sigma). Each coverslip of cells was individually transfected by the calcium phosphate precipitation technique as previously described (Krasowski et al., 1998). Each transfection used 1 to 5 µg of each cDNA; the cDNA was in contact with the cells for 24 h under an atmosphere containing 3% CO2 before being removed and replaced with fresh culture medium in an atmosphere of 5% CO2.

Electrophysiology and Design of Pharmacology Experiments. Electrophysiological recordings were performed at room temperature using the whole-cell patch-clamp technique as previously described (Krasowski et al., 1998). The coverslips were transferred 48 to 96 h after removal of the cDNA to a large chamber that was continuously perfused (2-3 ml/min) with extracellular medium containing 145 mM NaCl, 3 mM KCl, 1.5 mM CaCl2, 1 mM MgCl2, 5.5 mM D-glucose, and 10 HEPES, pH 7.4, osmolarity 320 to 330 mosmol. The electrode solution contained 145 mM N-methyl-D-glucamine hydrochloride, 5 mM K2ATP, 5 mM HEPES/KOH, 2 mM MgCl2, 0.1 mM CaCl2, and 1.1 mM EGTA, pH 7.2, osmolarity 315 mosmol. Pipette-to-bath resistance was 4 to 6 MOmega . Cells were voltage-clamped at -60 mV. Since the intracellular and extracellular solutions contained symmetrical chloride concentrations, the chloride equilibrium potential was approximately 0 mV.

All drugs were applied to the cell by local perfusion (Krasowski et al., 1998) using a motor-driven solution exchange device (Bio Logic rapid solution changer RSC-100; Molecular Kinetics, Pullman, WA). Laminar flow was maintained by applying all solutions at identical flow rates via a multichannel infusion pump (Stoelting, Wood Dale, IL). The solution changer was driven by protocols in the acquisition program pCLAMP5 (Axon Instruments, Foster City, CA). Responses were digitized (TL-1-125 interface; Axon Instruments) using pCLAMP5 and stored for off-line analysis.

Potentiation of GABA responses by propofol and other drugs was always assessed by coapplication of the drug to be tested with an EC20 GABA concentration (i.e., the concentration of GABA that produces 20% of the maximal response to GABA). Propofol and its analogs were always preapplied before coapplication with GABA, to ensure that the drugs were in equilibrium with the receptor. Average pre- and postanesthetic agonist control responses had to differ by less than 15% from each other for the data to be included for analysis (15% from the larger of the two average current responses). At the end of each potentiation experiment, a maximal GABA response was elicited by an appropriately high concentration of GABA, to verify that the average control GABA response fell between EC10 and EC30. Propofol "direct activation" currents were expressed as a fraction of the maximal current response to GABA.

To verify that responses to GABA and propofol in HEK 293 cells were mediated solely by the transfected GABAA receptor subunits, we undertook several control experiments. Untransfected HEK 293 cells did not respond to application of either GABA (1-5000 µM; n = 23) or propofol (1-200 µM; n = 8). Sham-transfected HEK 293 cells, which were treated with the calcium phosphate precipitation method minus the addition of receptor subunit cDNAs, also did not respond to GABA (1-5000 µM; n = 12) or propofol (1-200 µM; n = 6). In HEK 293 cells transfected with GABAA alpha 1, beta 2, and gamma 2s receptor subunit cDNAs, currents elicited by GABA (50 µM) were blocked by the GABAA receptor antagonist picrotoxin (50 µM) to 12.2 ± 6.6% of control (n = 4). Similarly, currents elicited by propofol (100 µM) were blocked by picrotoxin (50 µM) to 15.4 ± 8.0% of control (n = 4). In addition, the reversal potential for propofol-elicited currents (2.9 ± 2.1 mV; n = 5) was similar to that for GABA-activated currents (2.2 ± 2.8; n = 5).

These data demonstrate that the current responses elicited by propofol and GABA in HEK 293 cells expressing GABAA alpha 1beta 2gamma 2s receptors were in fact exclusively mediated by GABAA receptors. These data are similar to previous investigations of propofol direct activation of recombinant GABAA receptors expressed in HEK 293 cells (Jones et al., 1995; Davies et al., 1997; Krasowski et al., 1998). Thus, even though propofol at high concentrations has been shown to affect ion channels other than GABAA receptors, such as neuronal sodium channels (Rehberg and Duch, 1999) and ionotropic glutamate receptors (Orser et al., 1995), this is not a confounding issue in our experiments on HEK 293 cells expressing recombinant GABAA receptor subunits.

Data Analysis. Drug-induced potentiation of a GABA-induced current was defined as the percentage increase of the control GABA response (defined as the average of the predrug and postdrug GABA-induced currents). Concentration response data were fitted (KaleidaGraph, Reading, PA) with the equation: I/Imax = 100 × [drug]nH/([drug]nH + (EC50)nH), where I/Imax is the percentage of the maximum obtainable response, EC50 is the concentration producing a half-maximal response, and nH is the Hill coefficient. Pooled data are presented throughout as mean ± S.E. Statistical significance was determined by one-way analysis of variance with Dunnett's post hoc test, unless otherwise specified.

Drugs. Stock solutions of GABA (Sigma) and the propofol analogs were diluted into extracellular solution daily before use. With the exception of phenol, the propofol analogs were all prepared as stock solutions in dimethyl sulfoxide as carrier before being dissolved in the extracellular medium. The maximum final concentration of dimethyl sulfoxide was 0.2% (v/v), which was determined during control experiments to have no significant effect on GABA-induced currents in the recombinant or neuronal GABAA receptors analyzed in this study.

The sources of the propofol analogs and other drugs were as follows: 2,6-di-sec-butylphenol (Acros Organics, Pittsburgh, PA); 2-cyclopentylphenol, 2,6-dibromophenol, 1,3-diisopropylbenzene, 3,5-diisopropylcatechol, 2,6-dimethoxyphenol, 2,6-dimethylphenol, 2,6-dimethylthiophenol, 2,4-di-tert-butylphenol, 3,5-di-tert-butylphenol, 2-tert-butyl-6-methylphenol, 2-hydroxy-3-isopropylbenzoic acid, 2-isopropylphenol, 2-isopropylthiophenol, and phenol (Aldrich Chemical Co., Milwaukee, WI); 2,6-diethylphenyl isocyanate, 2,6-diethylphenyl isothiocyanate, 2,6-diisopropylphenyl isocyanate, 2,6-diisopropylphenyl isothiocyanate, and 2,6-diethylphenyl bromide (Lancaster Synthesis, Windham, NH); picrotoxin (Research Biochemicals International, Natick, MA); midazolam hydrochloride (Versed intravenous/intramuscular preparation; Roche Pharmaceuticals, Manati, Puerto Rico); and 2,4-di-sec-butylphenol (Sigma-Aldrich Rare Chemicals Library, Milwaukee, IL). Each analog was of the highest purity grade commercially available.

Propofol, 2,6-diethylphenol, 2,6-di-tert-butylphenol, 2,6-dicyclopentylphenol, and 2,6-dicyclohexylphenol were generously provided by Drs. J. B. Glen and Roger James of Zeneca Pharmaceuticals (Macclesfield, Cheshire, UK). 4-Iodo-2,6-diisopropylphenol (4-iodopropofol) was kindly provided by Drs. Hugh Hemmings and Ratnakumari Lingamaneni of Weill Medical College of Cornell University (New York, NY; see Lingamaneni et al., 2001 for details on synthesis of 4-iodopropofol). Loreclezole was a gift from Janssen Pharmaceutica (Beerse, Belgium).

Calculated Physicochemical Properties of the Propofol Analogs. Log P (where P = octanol/water partition coefficient) and the molecular volume for each molecule were calculated using the QSAR Properties program version 1.6, used in conjunction with HyperChem 5.0 software (Hypercube Inc., Gainesville, FL).

    Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Potencies for the Propofol Analogs in Producing Loss of Righting Reflex in Tadpoles. Potencies for producing loss of righting reflex in X. laevis tadpoles were determined for propofol and related analogs (Table 1). The concentration-response curve for the loss of righting reflex elicited by propofol and three analogs is shown in Fig. 2A. A previous study has determined the EC50 for propofol-induced loss of righting reflex in X. laevis tadpoles to be 1.9 ± 0.1 µM (Tonner et al., 1997), identical to the EC50 value described here. The EC50 for loss of righting reflex in tadpoles differs by severalfold from the general anesthetic potency of propofol in mammals, although the potency in mammals is complicated by extensive binding to serum proteins (Franks and Lieb, 1994).


                              
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  		TABLE 1
Physicochemical properties of the propofol analogs and potencies of the analogs for loss of righting reflex in X. laevis tadpoles and for potentiation of GABA responses and direct activation at GABAA alpha 1beta 2gamma 2s receptors expressed in HEK 293 cells

Molecular volume and log P are calculated parameters (under Materials and Methods). The EC50 and maximal effect values are presented as mean ± S.E. n = 5-12 for all data points in the potentiation of GABA responses and direct activation experiments. The LC50 values in tadpoles are given as mean ± S.E., when possible, or otherwise as an approximate range of concentrations.


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  		Fig. 2.   A, concentration-response curves for loss of righting reflex in X. laevis tadpoles for propofol, 2,6-diethylphenol, 2,6-dimethylphenol, and 2,6-di-tert-butylphenol. 2,6-Di-tert-butylphenol did not produce loss of righting reflex at any concentration. All curves are for 30 min of exposure. Curves for propofol, 2,6-diethylphenol, and 2,6-dimethylphenol are fit by quantal dose-response relationships as described under Materials and Methods, with the curve fit parameters listed in Table 1. B, time course for loss of righting reflex in tadpoles for propofol, 4-iodopropofol, and 2,4-di-sec-butylphenol. The ordinate depicts the estimate of the EC50 for loss of righting reflex at a particular time point of drug exposure.

The propofol analog 2,6-di-tert-butylphenol failed to produce loss of righting reflex at any concentration tested (Fig. 2A). 2,6-Di-tert-butylphenol also failed to antagonize the loss of righting reflex produced by propofol. Propofol had an EC50 for loss of righting reflex of 1.9 ± 0.2 µM with a slope factor of 3.4 ± 0.8. In the presence of 200 µM 2,6-di-tert-butylphenol, propofol had an EC50 of 1.9 ± 0.2 µM with a slope factor of 2.7 ± 0.6.

The rate of obtundation produced by the propofol analogs varied between compounds. This is expressed in Fig. 2B in terms of the rate of change of the EC50 for loss of righting reflex over time. For instance, the anesthetic action of propofol reaches equilibrium within 20 min, whereas the anesthetic actions of 4-iodo-2,6-diisopropylphenol (4-iodopropofol) and 2,4-di-sec-butylphenol plateau by 60 and 70 min, respectively, underscoring the importance of allowing time for the action of a particular drug to equilibrate.

Table 1 also lists the estimated LC50 values and the derived "therapeutic indices" (LC50/EC50 for loss of righting reflex) for each analog. The experiments were not specifically designed to determine LC50, and some lethality measurements are necessarily biased by the fact that tadpoles were not exposed to all concentrations of a particular compound for equal lengths of time (under Materials and Methods). Nonetheless, the reported LC50 values give an approximation of the toxicity of the agents, and of the proximity of the lethal concentration range to the anesthetic concentration range. Most agents that produced loss of righting reflex had therapeutic indices less than 10. Propofol, 2,6-di-sec-butylphenol, and phenol had the highest therapeutic indices in tadpoles (Table 1).

Pharmacology of GABAA Receptors from Dissociated Tadpole Spinal Neurons. The pharmacology of tadpole GABAA receptors was assessed in acutely dissociated spinal neurons. Dissociation of tadpole spinal neurons by the method of Dale (1991) yielded healthy neurons that were suitable for patch-clamp analysis from 4 h after dissociation. Application of GABA elicited current responses in approximately 70% of such neurons. Desensitization during GABA application was especially noticeable at high GABA concentrations (>500 µM), and recovery from desensitization occurred over several minutes following application of 1 mM GABA. GABA concentration-response data for the spinal neurons is depicted in Fig. 3. The maximal response to GABA, for those neurons that responded to GABA, was 488 ± 149 pA (n = 16). Figure 3B also displays the GABA concentration-response curve for the GABAA alpha 1beta 2gamma 2s receptor expressed in HEK 293 cells.


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  		Fig. 3.   GABA concentration-response relationships for GABAA receptors of dissociated X. laevis tadpole spinal neurons. A, representative traces from an individual tadpole spinal neuron in response to application of 5, 20, 50, 100, 1000, and 2000 µM GABA. Note that the responses to 1000 and 2000 µM GABA overlap. B, concentration-response curve for GABA at dissociated tadpole spinal neurons with the GABA concentration curve for the GABAA alpha 1beta 2gamma 2s receptor expressed in HEK 293 cells shown for comparison. The EC50 for GABA in the dissociated spinal neurons was 28.3 ± 2.1 µM with a Hill slope of 1.7 ± 0.2 (n = 6). The EC50 for the GABAA alpha 1beta 2gamma 2s receptor expressed in HEK 293 cells was 29.5 ± 2.4 µM with a Hill slope of 1.4 ± 0.1 (n = 7).

The next set of experiments tested the pharmacology of the GABAA receptors in the tadpole spinal neurons. Submaximal (EC20) GABA responses in tadpole neurons were enhanced by both 5 µM loreclezole and 0.5 µM midazolam (Fig. 4, A and C). Sensitivity to submicromolar concentrations of the benzodiazepine midazolam suggests the presence of both a GABAA gamma 2-like subunit (Pritchett et al., 1989) and an alpha -subunit similar to mammalian alpha 1-, alpha 2-, alpha 3-, or alpha 5-subunits (Sigel and Buhr, 1997). In addition, sensitivity to low micromolar concentrations of the anticonvulsant loreclezole implies the presence of a beta -subunit isoform similar to mammalian GABAA beta 2- or beta 3-subunit isoforms (Wingrove et al., 1994). Therefore, tadpole spinal neurons appear to express GABAA receptors containing subunits similar to mammalian beta 2/beta 3- and gamma 2-subunit isoforms.


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  		Fig. 4.   Submaximal (EC20) GABA current responses in tadpole spinal neurons are enhanced by loreclezole (5 µM), the benzodiazepine midazolam (0.5 µM) (A), and propofol (2 µM), but not by 2,6-di-tert-butylphenol (100 µM) (B). In addition, 2,6-di-tert-butylphenol failed to produce any direct activation during preapplication. Traces shown in A and B are individual recordings from tadpole spinal neurons. C, summary of the effects of loreclezole, midazolam, propofol, and 2,6-di-tert-butylphenol (2,6-DTBP) on submaximal GABA responses at tadpole spinal neurons (n = 5 for all points). The ordinate depicts percentage of potentiation of an EC20 test concentration of GABA by coapplication with drug.

There are no previous published reports of the sensitivity of tadpole or frog neuronal GABAA receptors to the modulatory actions of propofol. Submaximal GABA currents in the tadpole spinal neurons were enhanced by coapplication of 2 µM propofol but not by 100 µM of the propofol analog 2,6-di-tert-butylphenol (Fig. 4, B and C). Propofol also directly activated the tadpole GABAA receptors, with 50 µM propofol (in the absence of GABA) eliciting a response 44 ± 6% of the magnitude of the maximal GABA current (n = 5). In contrast, 500 µM 2,6-di-tert-butylphenol failed to produce any direct activation of the GABAA receptors in the tadpole neurons (n = 6).

Potentiation of GABA Responses and Direct Activation by the Propofol Analogs at GABAA alpha 1beta 2gamma 2s Receptors. Concentration-response curves for potentiation of GABA responses and direct activation by all 27 propofol analogs were determined at GABAA alpha 1beta 2gamma 2s receptors (Table 1; see Fig. 3B for the GABA concentration-response curve for the GABAA alpha 1beta 2gamma 2s receptor). Log P (octanol/water partition coefficients) and molecular volume values for the molecules are also included in Table 1. The compounds in Table 1 are sorted by molecular volume.

Figure 5, A and B, shows representative records of potentiation of responses to 12 µM GABA by propofol and 2,6-di-sec-butylphenol at GABAA alpha 1beta 2gamma 2s receptors. Both of these compounds also directly activated the GABAA alpha 1beta 2gamma 2s receptor, which is noticeable during anesthetic preapplication of 1 and 5 µM propofol and 5 and 20 µM 2,6-di-sec-butylphenol. The effects of all compounds that potentiated GABA responses or produced direct activation were reversible on washout with extracellular medium.


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  		Fig. 5.   Propofol and 2,6-di-sec-butylphenol potentiate GABA responses and directly activate GABAA alpha 1beta 2gamma 2s receptors, whereas 2,6-di-tert-butylphenol is inactive. A, potentiation of EC20 GABA responses by propofol at GABAA alpha 1beta 2gamma 2s receptors. Propofol (1 and 5 µM) elicits direct activation during preapplication. B, potentiation of EC20 GABA responses by 2,6-di-sec-butylphenol at GABAA alpha 1beta 2gamma 2s receptors. 2,6-Di-sec-butylphenol (5 and 20 µM) elicits direct activation during preapplication. C, in contrast, 2,6-di-tert-butylphenol at 1, 100, and 500 µM does not potentiate responses to 12 µM GABA. The 500 µM application of 2,6-di-tert-butylphenol illustrates the complete lack of direct receptor activation produced by this compound. Traces shown in A through C are individual recordings from HEK 293 cells transfected with cDNAs encoding the GABAA alpha 1, beta 2, and gamma 2s subunits.

Thirteen analogs did not potentiate submaximal GABA currents at any concentration tested. These 13 analogs were the exact same compounds that did not produce loss of righting reflex in the tadpoles (Table 1). Fourteen analogs failed to directly activate the GABAA alpha 1beta 2gamma 2s receptor (Table 1). The only difference between the overall pattern of activity for potentiation of GABA responses and direct activation was that phenol potentiated GABA-evoked currents but did not elicit direct activation at any concentration tested. Phenol also had the lowest potency for potentiation of GABA responses of any of the 14 analogs that potentiated GABA-evoked currents (Table 1). Figure 5C demonstrates that 2,6-di-tert-butylphenol failed to potentiate GABA responses or directly activate the GABAA alpha 1beta 2gamma 2s receptor at concentrations up to 500 µM, a striking contrast with the potent effects of propofol and 2,6-di-sec-butylphenol.

Figure 6 depicts concentration-response curves for potentiation of GABA responses and direct activation by propofol and four analogs. These four analogs vary in the alkyl substituents at positions 2 and 6 of the aromatic ring. In this series of alkylphenols, the apparent affinity for potentiation of GABA responses and direct activation decreased as the size of the alkyl substituent at positions 2 and 6 decreased. In addition, the maximal percentage of potentiation or direct activation for some compounds were less than that for propofol (i.e., a lower "relative efficacy"; see Table 1 and Fig. 6, particularly 2-isopropylphenol).


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  		Fig. 6.   Concentration-response relationships for potentiation of GABA responses (A) and direct activation (B) at GABAA alpha 1beta 2gamma 2s receptors by propofol, 2,6-diethylphenol, 2,6-dimethylphenol, 2-isopropylphenol, and 2,6-di-tert-butylphenol (n = 5-12 for all points). 2,6-Di-tert-butylphenol failed to potentiate GABA responses or directly activate GABAA alpha 1beta 2gamma 2s receptors at all concentrations tested. See Table 1 for summary data on the curve fits.

Lack of Negative Allosteric or Null Modulation by the Propofol Analogs. We next looked for evidence that any of the propofol analogs functioned as negative allosteric or null modulators (i.e., a "propofol antagonist" that blocks the effects of propofol but has no intrinsic action at the GABAA receptor). The model for this is the impressive range of ligands that has been shown to compete for a common benzodiazepine "binding site" at GABAA receptors. Positive, negative, and null benzodiazepine modulators have all been described (Sigel and Buhr, 1997).

No propofol analog tested reversibly inhibited GABA responses, suggesting that none was a negative allosteric modulator. The possibility was then tested that 2,6-di-tert-butylphenol might have a null modulatory action analogous to the actions of flumazenil at the benzodiazepine binding site (Sigel and Buhr, 1997). As shown above, 2,6-di-tert-butylphenol was inactive as an anesthetic in tadpoles and did not potentiate GABA responses or produce direct activation at the GABAA alpha 1beta 2gamma 2s receptor (Figs. 5C and 6), despite differing from propofol only by the addition of two methyl groups. This compound also did not antagonize the loss of righting reflex induced by propofol in the X. laevis tadpoles (see above; Fig. 7A).


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  		Fig. 7.   2,6-Di-tert-butylphenol does not antagonize the loss of righting reflex by propofol in X. laevis tadpoles, nor does 2,6-di-tert-butylphenol antagonize the potentiation of GABA responses or direct activation by propofol at GABAA alpha 1beta 2gamma 2s receptors. A, concentration-response curves for loss of righting reflex in tadpoles for propofol alone and for propofol in the presence of 200 µM 2,6-di-tert-butylphenol. Both curves are for 20 min of exposure. Curves in A are fit by quantal dose-response relationships as described under Materials and Methods. Concentration-response curves for potentiation of GABA responses (B) and direct activation (C) by propofol at GABAA alpha 1beta 2gamma 2s receptors in the presence and absence of 500 µM 2,6-di-tert-butylphenol. See Results for summary data on the curve fits in A-C.

Concentration-response curves for potentiation of GABA responses and direct activation by propofol were determined in the presence and absence of 500 µM 2,6-di-tert-butylphenol. Pre- and coapplication of 500 µM 2,6-di-tert-butylphenol had no effect on the concentration-response curves for either potentiation of GABA responses or direct activation by propofol (Fig. 7, B and C). Potentiation of GABA responses by propofol at GABAA alpha 1beta 2gamma 2s receptors had an EC50 of 1.9 ± 0.4 µM with a Hill slope of 1.6 ± 0.4 and an Emax of 242 ± 18% (n = 8; Fig. 7B). In the presence of 500 µM 2,6-di-tert-butylphenol, potentiation of GABA responses by propofol had an EC50 of 2.2 ± 0.5 µM with a Hill slope of 1.3 ± 0.3 and an Emax of 273 ± 23% of the maximal GABA current (n = 7; Fig. 7B).

Direct activation of GABAA alpha 1beta 2gamma 2s receptors by propofol had an EC50 of 10.6 ± 1.3 µM with a Hill slope of 1.3 ± 0.2 and an Emax of 67 ± 3% of the maximal GABA current (n = 4-8; Fig. 7C). In the presence of 500 µM 2,6-di-tert-butylphenol, direct activation by propofol had an EC50 of 8.7 ± 2.7 µM with a Hill slope of 1.5 ± 0.2 and an Emax of 64 ± 1% (n = 6; Fig. 7C). Coapplication of 500 µM 2,6-di-tert-butylphenol had no significant effect on the EC50, Hill slope, or Emax for potentiation of GABA responses or direct activation by propofol at GABAA alpha 1beta 2gamma 2s receptors. Consequently, 2,6-di-tert-butylphenol has no detectable null modulatory action at the propofol "site" of action, in contrast to the action of flumazenil at the benzodiazepine binding site (Sigel and Buhr, 1997).

Correlation Analysis of the Potencies of the Propofol Analogs. We next evaluated the correlations between the potencies of the propofol analogs in producing loss of righting reflex in tadpoles and for producing potentiation of GABA responses and direct activation of GABAA alpha 1beta 2gamma 2s receptors. These potencies were also compared with two physicochemical parameters, molecular volume and log P. The aim was to assess the impact of molecular size and lipophilicity of the propofol analogs on biological activity.

Figure 8 shows how the various experimentally determined potencies of the propofol analogs varied with respect to the molecular volume and log P values of the analogs. In each panel of Fig. 8, the analogs have been divided into "active" (squares) and "inactive" (circles), with the inactive analogs arbitrarily given a -log(EC50) value of 1. Active here simply refers to compounds for which a biological effect could be detected and quantified in terms of an EC50 value. It is at once clear that molecular volume and log P do not account for the lack of activity exhibited by some of the analogs; for instance, the log P values of both active and inactive analogs span more than 4 orders of magnitude (Fig. 8, A, C, and E).


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  		Fig. 8.   Correlation of propofol analog log P and molecular volume values with the potency for producing loss of righting reflex in tadpoles (A and B), potentiation of GABA responses at GABAA alpha 1beta 2gamma 2s receptors (C and D), and direct activation of GABAA alpha 1beta 2gamma 2s receptors (E and F). Potencies are expressed as -log(EC50), with EC50 in molar units. The inactive analogs (open circle ) have been arbitrarily given a -log(EC50) value of 1. The lines drawn through the data points on the graphs indicated linear regression of the relationship between the potencies of the active compounds () and the independent variable (log P or molecular volume). The correlations of the active compounds and the independent variables were subjected to statistical testing (under Materials and Methods), the results of which are indicated on the graphs. Statistical significance is indicated by *p < 0.05, **p < 0.01, or ***p < 0.001.

In considering the entire group of active compounds, log P and molecular volume each correlate significantly, albeit somewhat weakly, with the potency for loss of righting reflex in tadpoles (r2 = 0.48 and 0.50, p < 0.01, respectively; Fig. 8, A and B). In contrast, both log P and molecular volume correlate poorly with the potencies for potentiation of GABA responses or direct activation, with r2 values ranging from 0.02 to 0.25 (Fig. 8, C-F).

We also used multiple regression analysis to determine whether two-term correlations of molecular volume and log P with biological activity provided any improvement over single correlations with either log P or molecular volume alone. The two-term correlations of log P and molecular volume with potencies for tadpole loss of righting reflex (r2 = 0.51, p < 0.05), potentiation of GABA responses (r2 = 0.25, p = 0.21), and direct activation (r2 = 0.06, p = 0.74) were all not improved, or only marginally improved, over the correlations to a single independent variable (compare with Fig. 8). This is perhaps not surprising given that molecular volume and log P cross-correlate significantly with one another for the 14 active compounds (r2 = 0.85, p < 0.001).

The appearance of the data points in Fig. 8, C and D, suggested a parabolic dependence of potency for potentiation of GABA current on log P or molecular volume. Indeed, a parabolic function yielded a better fit, compared with linear regression, of the potency data for potentiation of GABA responses versus log P (r2 = 0.51, p < 0.05) or molecular volume (r2 = 0.47, p < 0.05; compare with Fig. 8). The traditional interpretation of the parabolic dependence upon log P is that at low values of log P, the ligand is too aqueous to partition into the target environment; conversely, if log P is too high, the ligand is insoluble and there are no ligands available to reach the target (Hansch et al., 1967). The parabolic dependence with molecule volume in Fig. 8D probably reflects the strong cross-correlation between log P and molecule volume. There was no improvement of fit, compared with linear regression, when parabolic functions were applied to the potency data for tadpole loss of righting reflex versus log P (r2 = 0.49, p < 0.05) or molecular volume (r2 = 0.51, p < 0.05), or for direct activation potency versus log P (r2 = 0.07, p = 0.71) or molecular volume (r2 = 0.03, p = 0.88; compare with Fig. 8, A, B, E, and F).

As mentioned above, there was a one-to-one correspondence between the group of compounds that potentiated GABA responses and those that produced loss of righting reflex in tadpoles. Conversely, all 13 analogs that did not potentiate GABA responses were also inactive in producing loss of righting reflex (Table 1). A similar situation was observed for direct receptor activation, with the exception that phenol failed to produce direct activation.

Figure 9 shows how the potencies of the active propofol analogs for producing loss of righting reflex, potentiation of GABA responses, and direct activation co-vary. The potencies for loss of righting reflex in tadpoles correlated significantly with both potentiation of GABA responses and with direct receptor activation (Fig. 9, A and B). The dashed lines drawn in Fig. 9 illustrate lines of unity. Of the three correlation plots, potentiation of GABA responses versus potency for tadpole loss of righting reflex (Fig. 9A) is the closest to a one-to-one correspondence.


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  		Fig. 9.   Cross-correlation of the potencies of the propofol analogs for producing loss of righting reflex in tadpoles, potentiation of GABA responses at GABAA alpha 1beta 2gamma 2s receptors, and direct activation of GABAA alpha 1beta 2gamma 2s receptors. Potencies are expressed as -log(EC50), with EC50 in molar units. Only compounds for which EC50 values could be determined are included in these graphs (i.e., the active compounds). In each graph, the dashed line indicates the line of unity, whereas the solid line is from linear regression analysis. The correlations were subjected to statistical testing, the results of which are indicated on the graphs. Statistical significance is indicated by *p < 0.05, **p < 0.01, or ***p < 0.001.

    Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

This is the first study to make rigorous comparisons over a chemically diverse series of propofol analogs between their anesthetic potency in vivo and their electrophysiological actions at GABAA receptors. The SAR data for the propofol analogs reported here are clearly compatible with the hypothesis that GABAA receptors play a significant role in the anesthetic actions of propofol and related analogs. The results also are consistent with previous in vivo studies in mice (James and Glen, 1980).

The best correlation was between the potencies for potentiation of GABA responses and loss of righting reflex in tadpoles, particularly because the 13 analogs that did not potentiate submaximal GABA currents also failed to produce loss of righting reflex in the tadpoles. Despite the overall correlation between the potencies for potentiation of GABA responses and loss of righting reflex in tadpoles (Fig. 9A), there are certainly compounds that deviate significantly from perfect linear correspondence. There are several possible explanations for this. The experimental design used in this study cannot, obviously, model all of the complexities of the actions of general anesthetics at intact neuronal circuits (Mody et al., 1994; Antkowiak, 1999). In addition, the distinction between potentiation of GABA responses and direct activation by propofol, which can be easily made in this in vitro system, is, in fact, somewhat artificial. In a living animal, both effects are expected to occur with some qualitative differences between the two effects. For instance, direct receptor activation would be expected to occur continuously at all GABAA receptors, even those located extrasynaptically, whereas potentiating actions would only occur at synapses when GABA is present.

In addition, this study cannot address whether molecular targets other than GABAA receptors contribute to the anesthetic effects of propofol and its analogs in tadpoles. So far, convincing effects of propofol at clinically relevant concentrations have only been demonstrated at GABAA and strychnine-sensitive glycine receptors (for review, see Krasowski and Harrison, 1999; although see discussion about "clinically relevant concentrations" by Eckenhoff and Johansson, 1999). It is currently not known whether other molecular targets, such as the two-pore domain potassium channels recently shown to be sensitive to volatile anesthetics (Patel et al., 1999; Sirois et al., 2000), will also be shown to be sensitive to propofol.

The electrophysiological studies of native GABAA receptors in dissociated tadpole spinal neurons are in agreement with previous reports demonstrating that the pharmacology of GABAA receptors in frog neurons (Akaike et al., 1985) closely parallels that of mammalian GABAA receptors. This manuscript is the first published report of the effects of propofol on GABAA receptors expressed in neurons from tadpoles or frogs. The potentiation of GABA-evoked responses and direct activation by propofol, but not by 2,6-di-tert-butylphenol, in the tadpole spinal neurons is also consistent with the hypothesis that alteration of GABAA receptor function is responsible, at least in part, for the anesthetic actions (or lack thereof) of propofol and related analogs in tadpoles.

One of the most striking observations from the SAR is that the presence of bulky or nonplanar alkyl substituents at positions 2 and 6 of the aromatic ring abolished activity of the 2,6-dialkylphenol analogs (Fig. 10). This is illustrated by the complete inactivity of 2,6-di-tert-butylphenol, 2,6-dicyclopentylphenol, and 2,6-dicyclohexylphenol. An intriguing finding is that 2,6-di-sec-butylphenol (an isomer of 2,6-di-tert-butylphenol) has high potency at GABAA receptors and for loss of righting reflex in tadpoles. Similar findings were originally noted by James and Glen (1980), who demonstrated that 2,6-di-tert-butylphenol, 2,6-dicyclopentylphenol, and 2,6-dicyclohexylphenol were all ineffective as intravenous anesthetics in mice, whereas 2,6-di-sec-butylphenol was highly potent. To explain the inactivity of 2,6-di-tert-butylphenol, James and Glen (1980) suggested that the tert-butyl moieties crowd the phenol hydroxyl and interfere with a critical interaction between the phenol hydroxyl and the target receptor. Cyclopentyl and cyclohexyl groups would similarly "sterically hinder" the phenol hydroxyl more than a sec-butyl or isopropyl group.


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  		Fig. 10.   Summary of the pattern of activity for the propofol analogs. Active here refers to analogs for which EC50 values could be determined for loss of righting in tadpoles, potentiation of GABA responses at GABAA alpha 1beta 2gamma 2s receptors, and direct activation of GABAA alpha 1beta 2gamma 2s receptors. Note that direct activation of GABAA alpha 1beta 2gamma 2s receptors was not detected for phenol, although phenol produced loss of righting reflex in tadpoles and potentiated GABA responses at GABAA alpha 1beta 2gamma 2s receptors. Phenol was classified as an active compound.

Although bulky alkyl groups at positions 2 and 6 of the aromatic ring interfere with the activity of the propofol analogs, the potencies of the propofol analogs clearly increase with the size of alkyl groups up to a point, as illustrated by the following series: phenol, 2,6-dimethylphenol, 2-isopropylphenol, 2,6-diethylphenol, propofol, and 2,6-di-sec-butylphenol. In fact, the potencies of the compounds within this small group increase almost linearly with molecular volume and log P, a finding also noted by James and Glen (1980). James and Glen (1980) considered only simple alkylphenols in their structure-activity studies, which probably accounts for their observation that log P correlated very strongly with in vivo anesthetic potency for the alkylphenols.

Log P and molecular volume did not correlate well with the biological potencies of the active compounds as an entire group (i.e., beyond the simple 2,6-dialkylphenols). For example, 2,6-di-sec-butylphenol and 2,4-di-sec-butylphenol have nearly identical log P values and molecular volumes yet have biological potencies that differ by more than one order of magnitude. Log P and molecular volume also do not explain the lack of activity exhibited by the remaining compounds such as 2,6-di-tert-butylphenol. These observations appear difficult to reconcile with a "nonspecific" or lipid-based mechanism of action for the propofol analogs, which would predict that compounds such as 2,6-di-tert-butylphenol would have high anesthetic potency. Compounds such as 2,6-di-tert-butylphenol and 2,6-dicyclopentylphenol may be analogous to the volatile "nonimmobilizers" (Koblin et al., 1994), in that these compounds have high lipid solubility, yet do not produce loss of righting reflex in tadpoles.

The SAR studies suggest a critical interplay between substituents at positions 1, 2, and 6 of the aromatic ring (Fig. 10). Removal of the phenolic hydroxyl group of propofol abolished activity (1,3-diisopropylbenzene), whereas bromide, isocyanate, and isothiocyanate could all substitute for the hydroxyl group, although this usually resulted in a drop in potency at GABAA receptors or for producing tadpole loss of righting reflex (Fig. 10; Table 1). An interesting observation was that 2,6-diisopropylphenyl isocyanate and 2,6-diisopropylphenyl isothiocyanate were completely inactive both at the GABAA alpha 1beta 2gamma 2s receptor and in tadpoles. The inactivity of 2,6-diisopropylphenyl isocyanate (molecular volume = 681 Å3) and 2,6-diisopropylphenyl isothiocyanate (713 Å3) is especially striking compared with the activities of propofol (641 Å3), 2,6-diethylphenyl isocyanate (602 Å3), and 2,6-diethylphenyl isothiocyanate (638 Å3). A key influence on the interactions of propofol with the target site, thus, appears to be the size and shape of the alkyl groups at positions 2 and 6 of the aromatic ring relative to the substituent at position 1.

Three other studies have examined the activity of propofol analogs at GABAA receptors (Trapani et al., 1998; Sanna et al., 1999; Lingamaneni et al., 2001; for review, see Trapani et al., 2000). Most of the analogs analyzed by Trapani et al. (1998) involved groups added to position 4 of the propofol aromatic ring (i.e., the para-position). Two compounds analyzed in this study, 4-iodopropofol and 2-hydroxy-3-isopropylbenzoic acid, were also studied by Trapani et al. (1998). Most substitutions at the para-position are remarkably well tolerated, even groups as large as -CO(Phenyl). One possible explanation for this is that the substituents at the para-position do not form critical interactions with the target receptor and, thus, diverse chemical groups may be added to the para-position without abolishing activity.

We have previously reported that mutation of a methionine residue at position 286 of the GABAA beta 1-subunit to tryptophan abolishes potentiation of GABA responses by propofol at GABAA receptors (Krasowski et al., 1998). This methionine residue, thought to be near the interface of transmembrane domain 3 with the extracellular fluid (Williams and Akabas, 1999), is also necessary for the actions of volatile ether anesthetic and alcohols at GABAA receptors (Mihic et al., 1997; Krasowski and Harrison, 2000). A potential synthesis of the available data is that propofol and related analogs interact with a binding site formed by the extracellular region of the transmembrane domains of the beta -subunit. Substituents at the para-position may "hang off" into the extracellular space, whereas positions 1, 2, and 6 of the propofol molecule form the major interactions with a conformationally restricted binding pocket formed in part by methionine 286 of the beta -subunit. This hypothesis will be tested in subsequent studies.

    Acknowledgments

We thank Steve Lopez, Audrey Lin, and Natalia Nikolaeva for invaluable technical support. We also thank Drs. J. B. Glen and Roger James of Zeneca Pharmaceuticals for supplying five of the propofol analogs, Drs. Hugh Hemmings and Ratnakumari Lingamaneni of Weill Medical College of Cornell University for providing 4-iodopropofol, and Janssen Pharmaceutica for the gift of loreclezole.

    Footnotes

Accepted for publication January 9, 2001.

Received for publication August 18, 2000.

This study was funded by the C. V. Starr Foundation (New York City, NY) and the Rice Foundation (Chicago, IL) to N.L.H., by National Institutes of Health Grants GM56850 and GM62195 to N.L.H. and K08-GM00695 to P.F., by National Institute of Mental Health training fellowship MH11504 to M.D.K., and by the Procter and Gamble Company and The Chem21 Group, Inc., to A.J.H.

Send reprint requests to: Neil L. Harrison, Ph.D., Director, Laboratory of Molecular Neuropharmacology, Department of Anesthesiology, A-1050, Weill Medical College of Cornell University, 525 East 68th St., New York, NY 10021. E-mail: neh2001{at}med.cornell.edu

    Abbreviations

GABAA, gamma -aminobutyric acid type A; SAR, structure-activity relationship; PIPES, piperazine-N,N'-bis[2-ethanesulfonic acid]; cDNA, complimentary DNA; HEK, human embryonic kidney; log P, log10 of the octanol/water partition coefficient.

    References
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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D. B. Williams and M. H. Akabas
Structural Evidence that Propofol Stabilizes Different GABAA Receptor States at Potentiating and Activating Concentrations
J. Neurosci., September 1, 2002; 22(17): 7417 - 7424.
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