Activation and modulation of recombinant glycine and GABAAreceptors by 4-halogenated analogues of propofol

Article abstract of DOI:10.1111/bph.13566

Background and Purpose: Glycine receptors are important players in pain perception and movement disorders and therefore important therapeutic targets. Glycine receptors can be modulated by the intravenous anaesthetic propofol (2,6-diisopropylphenol). Howe

Full text of DOI:10.1111/bph.13566

British Journal of Pharmacology (2016) 173 3110–3120 3110
British Journal of
Pharmacology
BJP
RESEARCH PAPER
Activation and modulation of recombinant
glycine and GABAA receptors by
4-halogenated analogues of propofol
Correspondence Gustav Akk, Department of Anesthesiology, Washington University School of Medicine, Campus Box 8054, 660 South
Euclid Ave, St. Louis, MO 63110, USA. E-mail: akk@morpheus.wustl.edu
Received 6 May 2016; Revised 11 July 2016; Accepted 20 July 2016
Allison L Germann1*, Daniel J Shin1*, Brad D Manion1, Christopher J Edge2,3, Edward H Smith2,
Nicholas P Franks2, Alex S Evers1,4 and Gustav Akk1,4
1Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO, USA, 2Department of Life Sciences, Imperial College
London, South Kensington, UK, 3Department of Anaesthetics, Royal Berkshire NHS Foundation Trust, Reading, UK, and 4Taylor Family Institute for
Innovative Psychiatric Research, Washington University School of Medicine, St. Louis, MO, USA
*These authors contributed equally to this work.
BACKGROUND AND PURPOSE
Glycine receptors are important players in pain perception and movement disorders and therefore important therapeutic targets.
Glycine receptors can be modulated by the intravenous anaesthetic propofol (2,6-diisopropylphenol). However, the drug is more
potent, by at least one order of magnitude, on GABAA receptors. It has been proposed that halogenation of the propofol molecule
generates compounds with selective enhancement of glycinergic modulatory properties.
EXPERIMENTAL APPROACH
We synthesized 4-bromopropofol, 4-chloropropofol and 4-fluoropropofol. The direct activating and modulatory effects of these
drugs and propofol were compared on recombinant rat glycine and human GABAA receptors expressed in oocytes. Behavioural
effects of the compounds were compared in the tadpole loss-of-righting assay.
KEY RESULTS
Concentration–response curves for potentiation of homomeric α1, α2 and α3 glycine receptors were shifted to lower drug
concentrations, by 2–10-fold, for the halogenated compounds. Direct activation by all compounds was minimal with all subtypes
of the glycine receptor. The four compounds were essentially equally potent modulators of the α1β3γ2L GABAA receptor with EC50
between 4 and 7 μM. The EC50 for loss-of-righting in Xenopus tadpoles, a proxy for loss of consciousness and considered to be
mediated by actions on GABAA receptors, ranged from 0.35 to 0.87 μM.
CONCLUSIONS AND IMPLICATIONS
We confirm that halogenation of propofol more strongly affects modulation of homomeric glycine receptors than α1β3γ2L GABAA
receptors. However, the effective concentrations of all tested halogenated compounds remained lower for GABAA receptors. We
infer that 4-bromopropofol, 4-chloropropofol and 4-fluoropropofol are not selective homomeric glycine receptor modulators.
Abbreviations
4-BP, 4-bromopropofol; 4-CP, 4-chloropropofol; 4-FP, 4-fluoropropofol
DOI:10.1111/bph.13566
© 2016 The British Pharmacological Society

Actions of halogenated propofol analogues
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Tables of Links
TARGETS
LIGANDS
Ligand-gated ion channels
GABAA receptor
GABA
Glycine
Propofol
GABAA receptor α1 subunit
GABAA receptor β3 subunit
GABAA receptor γ2 subunit
Glycine receptor
Glycine receptor α1 subunit
Glycine receptor α2 subunit
Glycine receptor α3 subunit
These Tables list key protein targets and ligands in this article that are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the
common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Southan et al., 2016), and are permanently archived in the Concise Guide
to PHARMACOLOGY 2015/16 (Alexander et al., 2015).
et al., 2001). These actions likely underlie the loss-of-righting
reflex (LRR) and other anaesthetic endpoints following appli-
Introduction
Glycine receptors are members of the Cys-loop receptor
family (Lynch, 2004; Lynch, 2009). These Clꢀ-permeable
channels mediate inhibitory neurotransmission in the
retina, brainstem and spinal cord. Functional receptors are
formed as homopentamers of one of four α subunits or
heteropentamers consisting of α and β subunits. Subunit ex-
pression is region-specific and regulated developmentally.
For example, prenatal glycinergic transmission is largely me-
diated by α2 homomers, whose expression is reduced
sharply following birth (Malosio et al., 1991). Receptors con-
taining the α1 subunit are dominant in adult retina and spi-
nal cord. Interference with α1 expression, or introduction of
mutations disrupting receptor function, can result in the
hyperekplexia phenotype (Schaefer et al., 2013; Bode and
Lynch, 2014). Glycine receptors containing the α3 subunit
are expressed in the retina and in nociceptive sensory neu-
rons in the spinal cord dorsal horn where they may mediate
pain associated with chronic peripheral inflammation
(Haverkamp et al., 2003; Harvey et al., 2004). Thus, glycine
receptors can be important potential targets in a variety of
physiological processes.
Recent studies have reported that halogenated analogues
of propofol act as potent activators and modulators of the gly-
cine receptor. In mouse ventral horn neurons, submicromolar
concentrations of 4-bromopropofol (4-BP) elicit tonic current
that can be blocked by strychnine, but not bicuculline (Eckle
et al., 2014). 4-Chloropropofol (4-CP) reportedly potentiates
currents from heterologously expressed α1 receptor with the
concentration producing half-maximal effect below 1 nM
(de la Roche et al., 2012). These results led to the proposal that
these, and other novel halogenated analogues of propofol,
may be clinically useful in treating conditions mediated by
functional deficiencies of glycine receptors, such as
hyperekplexia, or employed as a starting point in the devel-
opment of non-sedative analgesics.
cation of propofol (Jurd et al., 2003). Previous work on halo-
genated analogues of propofol has demonstrated that
bromine substitution at the ortho position reduces anaes-
thetic and GABAergic activity of the analogues, while com-
pounds with halogen substituents para to the phenolic
hydroxyl group remain strong GABAergic modulators
(Krasowski et al., 2001; Trapani et al., 1998).
To provide a direct comparison of activation and modula-
tion of glycine and GABAA receptors by this class of
compounds and to test the potential for receptor selectivity,
we compared direct activation and modulation of
recombinant α1, α2 and α3 homomeric glycine
receptors, and α1β3γ2L GABAA receptors by propofol, 4-BP,
4-CP and 4-fluoropropofol (4-FP). We show that these com-
pounds are weak activators of the glycine receptor, with
maximal observed responses at approximately 20% of the re-
sponse to saturating glycine. All halogenated analogues were
more potent than propofol at potentiating responses to a low
concentration of glycine, with the EC50 for potentiation left-
shifted by up to an order of magnitude. 4-BP and 4-CP, but
not 4-FP, were also more potent than propofol at modulating
the α1β3γ2L GABAA receptor. The EC50 for the LRR, a proxy
for loss of consciousness in humans (Franks, 2008), were mea-
sured in Xenopus tadpoles and found to be 0.35–0.87 μM for
propofol and its analogues. We infer that while halogenation
of the propofol molecule does alter target selectivity, biasing
it towards modulation of glycine receptors, 4-halogenated
analogues of propofol do not attain sufficient selectivity for
use as selective glycinergic modulators.
Methods
Chemical syntheses
Propofol and its analogues can also activate and potenti-
ate the GABAA receptor (Hales and Lambert, 1991; Krasowski
4-Chloropropofol was synthesized according to the pub-
lished procedure (Wheatley and Holdrege, 1958).
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A L Germann et al.
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The synthesis of 4-BP was adapted from the published
procedure (Wheatley and Holdrege, 1958) as follows. 2,6-
Diisopropylphenol (1.85 mL, 1.78 g, 10 mmol) was added to
a stirred, orange solution of tetrabutylammonium tribromide
(4.82 g, 10 mmol) in chloroform (20 mL), at room tempera-
ture. After 30 min, the chloroform was evaporated from the
almost colourless solution, and the residue was treated with
water (30 mL) and diethyl ether (30 mL). The mixture was
shaken, and the two layers were separated. The organic layer
was washed with water (3×) and dried (MgSO4). Filtration
and concentration gave an orange oil which was
chromatographed on silica gel using hexane : ethyl acetate
(19:1) as eluent to give the product as a pale yellow oil (1.59
g, 62%).
(19:1) as eluent to give the product as a yellow oil (255 mg,
45%).
4-Fluoropropofol (Picard et al., 1996). The 4-fluoroanisole
(255 mg, 1.22 mmol) was dissolved in dichloromethane
(15 mL) and cooled in an ice bath. The stirred solution
was added to a solution of boron tribromide in dichloro-
methane (1 M, 5 mL, 5 mmole) and the resultant solution
stirred at 0°C for 45 min and then at room temperature for
3 h. The reaction mixture was cooled in an ice bath, then
water (15 mL) was added and the whole mixture was
poured into diethyl ether (50 mL). The layers were shaken
and separated, and the organic layer was washed with satu-
rated, aqueous sodium bicarbonate (1×) and water (1×) and
dried (MgSO4). The drying agent was filtered and the
filtrate was concentrated to a light brown oil (250 mg).
This was chromatographed on silica gel using hexane :
ethyl acetate (19:1) as eluent to give the product as an
off-white solid, m.p. 41–43°C, (178 mg, 74%).
4-Fluoropropofol was synthesized as follows.
4-Nitro-2,6-diisopropylanisole (Meek et al., 1968; Chastrette
et al., 1986): 2,6-Diisopropylanisole (760 mg, 4 mmol) was
dissolved in chloroform (5 mL) and treated with potassium
nitrate (456 mg, 4.56 mmol) and trifluoroacetic anhydride
(2 mL). The flask was loosely stoppered, and the mixture
was stirred at room temperature for 5 days. The mixture
was filtered; the solid washed with chloroform (1 × 5 mL)
and the combined filtrate and washings were concentrated
to a light brown oil (1.41 g). This was chromatographed on
silica gel using hexane : ethyl acetate (19:1) as eluent to give
the product as a yellow oil (910 mg, 96%).
Animals
All animal care and experimental protocols complied with
the Guide for the Care and Use of Laboratory Animals as
adopted and promulgated by the National Institutes of
Health and were approved by the Animal Studies Committee
of Washington University in St. Louis (Approval no. 20140
150 and no. 20150076). Frogs and tadpoles (from the
African clawed frog, Xenopus laevis) were housed and cared
for in a Washington University Animal Care Facility under
the supervision of the Washington University Division of
Comparative Medicine. Animal studies are reported in com-
pliance with the ARRIVE guidelines (Kilkenny et al., 2010;
McGrath and Lilley, 2015).
4-Amino-2,6-diisopropylanisole
(Ponomarev
and
Burmistrov, 1964): The 4-nitro derivative (910 mg, 3.84
mmol) was dissolved in ethanol (10 mL) and treated with
tin dichloride dihydrate (2.25 g, 10 mmol). The mixture was
stirred and heated at 90°C for 4 h. The reaction mixture was
cooled to room temperature, and then ice/water (50 mL)
was added followed by solid sodium hydroxide until the pH
of the mixture was >13. Dichloromethane (50 mL) was
added, and the mixture was vacuum filtered. The resultant
two phases were separated, and the aqueous layer was
extracted with dichloromethane (2 × 20 mL). The combined
organic layers were dried (Na2SO4), filtered and concentrated
to an orange–brown oil (900 mg). This was chromatographed
on silica gel using hexane : ethyl acetate (3:2) as eluent to give
the product as a red oil (564 mg, 71%).
Frogs were purchased from Xenopus 1 (Dexter, MI) and
oocytes were harvested from mature female frogs under
tricaine anaesthesia, twice per frog. Frogs were killed under
tricaine anaesthesia by rapid decapitation. Early prelimb-
bud stage tadpoles from Xenopus laevis were used in behav-
ioural assays. Tadpoles (purchased from Nasco, Fort
Atkinson, WI) were kept in continuously aerated tadpole
Ringer's solution in aquaria and used within 6 days of re-
ceipt. The aquaria are inspected and approved by the
Washington University Animal Care Committee. Behav-
ioural assays using Xenopus laevis tadpoles were conducted
in accordance with the Guide for the Care and Use of
Laboratory Animals as adopted and promulgated by the
4-Fluoro-2,6-diisopropylanisole
(Kriuchkova
and
Zavgorodnii, 1960): The 4-amino derivative (564 mg, 2.72
mmol) was dissolved in dry THF (3 mL), stirred, cooled in an
ice/acetone bath and treated with boron trifluoride etherate
(0.65 mL, 5.2 mmol) and then t-butyl nitrite (0.36 mL, 313
mg, 3.05 mmol). The dark brown solution was allowed to
warm to room temperature over 10 min then diluted
with perfluorodecalin (3 mL). The resultant two-phase
mixture was subjected to rotary evaporation to remove most
of the THF and t-BuOH (failure to do this or substituting a
hydrocarbon solvent for perfluorodecalin results in some
2,6-diisopropylanisole which is very difficult to remove from
its fluorinated analogue) and then stirred and heated to 115°C
over about 20 min. At 60–65°C, some gas evolution occurred.
Once 115°C had been attained, the flask was removed from
the oil bath and cooled to room temperature. Acetonitrile
(50 mL) was added. The lower, colourless, perfluorodecalin
layer was removed, and the acetonitrile evaporated to give a
very dark brown, viscous liquid (1.367 g). This was
chromatographed on silica gel using hexane : ethyl acetate
National Institutes of Health.
A total of 370 tadpoles
were used in the experiments described here. Tadpoles were
killed after each experiment by addition of lethal concen-
trations of tricaine.
Frog oocytes were used for expression of glycine and
GABAA receptors. Oocytes are a widely used and standard ex-
pression system for recombinant receptor channels. Their use
permits studies of a defined population of receptors to pro-
vide specific and reliable pharmacological information. The
use of tadpoles is justified because there are no cell lines or
computer modelling systems that replace the use of animals
for these behavioural studies.
Molecular biology and expression of receptors
The glycine and GABAA receptors were expressed in Xenopus
oocytes. The cDNAs in the pcDNA3 (glycine α3, GABAA α1,
3112 British Journal of Pharmacology (2016) 173 3110–3120

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β3, γ2L), pcDNA3.1 (glycine α2) or pGEMHE vector (glycine
α1) were linearized by digestion with Xba I, NheI, Hind III
or BglII (NEB Labs, Ipswich, MA). The cRNAs were produced
using mMessage mMachine (Ambion, Austin, TX). The oo-
cytes were injected with a total of 18–33 ng cRNA in a final
volume of 20–60 nl and incubated in ND96 (96 mM NaCl,
2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 2.5 mM Na pyruvate,
5 mM HEPES; pH 7.4) at 16°C. The ratio of GABAA receptor
cRNAs used for injection was 5:1:5 (α1:β3:γ2L). Electrophysio-
logical measurements were conducted within 1–3 days after
injection.
Data and statistical analysis
The data and statistical analysis comply with the recommen-
dations on experimental design and analysis in pharmacol-
ogy (Curtis et al., 2015).
Activation concentration–response curves were fitted
for data from each individual cell with the following equa-
tion:
ꢀ
ꢀ
ꢁꢁ
nH
Y ¼ Y max* ½drugꢁnH= ½drugꢁ þ EC50
;
(1)
nH
where EC50 is the concentration of drug producing a half-
maximal effect, nH characterizes the slope of relationship
and Ymax is the high-concentration asymptote. Fitting was
conducted using the NFIT software (The University of
Texas Medical Branch at Galveston, Galveston, TX).
Electrophysiology
Electrophysiological experiments were conducted using the
standard two-electrode voltage clamp approach. Voltage
and current electrodes were regular patch-clamp electrodes
that when filled with 3 M KCl had resistances of less than
1 MΩ. The oocytes were clamped at ꢀ60 mV. The chamber
(RC-1Z, Warner Instruments, Hamden, CT) was perfused
continuously at approximately 5 mL minꢀ1. Bath solution
(92.5 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, 10 mM HEPES;
pH 7.4) was perfused between all test applications. Solutions
were gravity-applied from 30 mL glass syringes with glass
luer slips via Teflon tubing to reduce adsorption and were
switched manually. A typical experiment consisted of re-
cording of a 10 s baseline, followed by a 20–60 s drug appli-
cation, and then a bath application (up to 10 min) until full
recovery. Duration of drug application depended on the na-
ture of drug and its concentration and was aimed at
reaching a saturated peak response. The current responses
were amplified with an Axoclamp 900 A (Molecular Devices,
Sunnyvale, CA) or OC-725C amplifier (Warner Instruments,
Hamden, CT), filtered at 40 Hz, digitized with a Digidata
Parameters of the fit are reported as mean
concentration–response data are presented as response rela-
tive to Ymax
SEM. The
.
Potentiation of concentration–response relationships
were determined by exposing cells to a low concentration of
agonist (EC4–14 of glycine or GABA), in the absence and pres-
ence of several concentrations of a modulator (4-BP, 4-CP, 4-
FP or propofol). Control applications to saturating glycine
or GABA were typically done after every 2–3 test applications,
to verify overall stability of responses. There were no appar-
ent changes in potentiation parameters for data obtained
within this range of EC values. The concentration–response
curves for potentiation are presented as response relative to
the peak response in the presence of saturating agonist. The
curves were fitted for data from each individual cell with the
following equation:
ꢀ
ꢀ
ꢁꢁ
nH
Y ¼ Y min þ ðY max ꢀ Y minÞ* ½drugꢁnH= ½drugꢁ þ EC50
;
(2)
nH
1320 or 1200 series digitizer (Molecular Devices) at
a
100 Hz sampling rate and stored using pClamp (Molecular
Devices). The traces were subsequently analysed with
Clampfit (Molecular Devices) to determine the maximal am-
plitude of current response.
The maximal final DMSO concentration in drug solutions
was 0.25%. Control experiments showed that 0.5% DMSO
was without effect on holding current (<0.1% of the response
to saturating transmitter). Co-application of 0.5% DMSO
with an EC50 concentration of a transmitter had no effect
(glycine α1, GABAA α1β3γ2L) or a small effect (<10%; glycine
α2, α3) on the peak response.
where EC50 is the concentration of drug producing a half-
maximal effect, nH characterizes the slope of relationship
and Ymin and Ymax are the low and high concentration as-
ymptotes respectively.
Statistical analysis was carried out with one-way ANOVA
and Bonferroni's correction (Stata/IC 12.1, StataCorp,
College Station, TX). P <0.05 was chosen to indicate signifi-
cant differences between group means.
Materials
Rat α1, α2 and α3 homomeric glycine receptors and human
α1β3γ2L GABAA receptors were expressed in Xenopus oo-
cytes. The glycine α1 clone was obtained from Dr J. Lynch
(The University of Queensland), α2 from Dr H. Akagi
(Gunma University) and α3 from Dr H. Betz (Max-Planck-
Institute for Brain Research). The GABAA receptor subunit
clones were provided by Drs G. White (Neurogen Corpora-
tion) and D. Weiss (University of Texas Health Science
Center, San Antonio). Inorganic salts used in buffers, gly-
cine and GABA were purchased from Sigma-Aldrich (St.
Louis, MO, USA). Stock solutions of glycine and GABA
were made in bath solution at 500 mM. The stock solu-
tions were stored in aliquots at ꢀ20°C and diluted as
needed on the day of experiment. Stock solutions of
propofol (200 mM) or its analogues (100–200 mM) were
Behavioural assays
Predetermined concentrations of 4-BP, 4-CP, 4-FP or propofol
were added to beakers containing 100 mL of oxygenated tad-
pole ringer's solution (5.8 mM NaCl, 67 μM KCl, 34 μM Ca
(NO3)2, 83 μM MgSO4, 419 μM Tris–HCl, 80 μM Tris-Base;
pH 7.5). Ten randomly chosen tadpoles were distributed into
each beaker and allowed to equilibrate in the tadpole ringer's
solution for 3 h. In the end of the equilibration period, the
LRR was measured by turning the tadpole over using a
hooked glass rod. LRR was defined as the inability of a tadpole
to right itself within 5 s on its back in three consecutive trials.
The operator was blinded to the concentration of compound
in the beaker.
British Journal of Pharmacology (2016) 173 3110–3120 3113

A L Germann et al.
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made in DMSO. Dilutions to working concentrations were
made on the day of experiment.
concentration tested) resulted in peak currents that were
12
2
3% (mean
SEM; n = 5 cells), 6
1% (n = 4) or
0.1% (n = 4) of the response to saturating glycine in cells
expressing α1, α2 or α3 homomeric glycine receptors respec-
tively. In oocytes expressing α1β3γ2L GABAA receptors,
100 μM 4-BP elicited a response that was 27 2% (n = 4) of
the response to saturating (1 mM) GABA.
Results
Direct activation of glycine and GABAA
receptors by propofol and its 4-halogenated
analogues
To attempt to gain insight into whether the failure of
these drugs to efficiently activate glycine receptors results
from impaired binding (low-affinity) or gating (low-efficacy),
we recorded current responses at lower drug concentrations.
Relative to peak responses to saturating glycine, the mean re-
sponses to 0.2 μM and 10 μM 4-BP were 0.03 0.02% (n = 5)
and 0.12 0.04% (n = 5) in α1, 0.01 0.004% (n = 4) and
0.23 0.12% (n = 4) in α2 and 0.22 0.15% (n = 5) and
0.32 0.18% (n = 6) in α3 glycine receptors. The responses
Oocytes expressing glycine or GABAA receptors responded
with inward current to applications of propofol or its haloge-
nated analogues. In general, the peak currents in response to
applications of 4-halogenated analogues were small,
compared with responses to saturating concentrations of
transmitter. Application of 100 μM 4-BP (the highest
Figure 1
Direct activation of glycine and GABAA receptors. (A) Sample currents from oocytes expressing α1, α2 or α3 glycine (GlyR) or α1β3γ2L GABAA re-
ceptors (GABAAR). The receptors were activated by 100 μM 4-BP, 4-CP or 4-FP (left trace in each pair) or a saturating concentration of glycine
(1 mM, 0.5 mM or 2 mM for α1, α2 and α3 respectively) or GABA (1 mM). In each pair, the traces for propofol analogue and glycine (or GABA)
are from the same cell. (B) Summary of direct activation of glycine and GABAA receptors by saturating concentrations of propofol and its ana-
logues. The responses (mean SEM) are normalized to responses to saturating concentrations of the transmitter from the same sets of cells.
3114 British Journal of Pharmacology (2016) 173 3110–3120

Actions of halogenated propofol analogues
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to 0.2 μM and 10 μM 4-BP in α1β3γ2L GABAA receptors were
0.02 0.01% (n = 4) and 6.2 0.6% (n = 8) of the peak re-
sponse to saturating GABA.
Application of 100 μM 4-CP elicited peak responses that
were 14 3% (n = 5), 21 6% (n = 5) or 6 1% (n = 5) of the
response to saturating glycine in oocytes expressing α1, α2
or α3 glycine receptors respectively. The α1β3γ2L GABAA re-
ceptors responded to application of 100 μM 4-CP with a peak
response that was 50 8% (n = 5) of the response to saturating
GABA.
The 4-fluorinated analogue of propofol was the weakest
activator. Exposure to 100 μM 4-FP resulted in peak responses
that were 1.6 0.2% (n = 4), 0.4 0.1% (n = 4) or 0.3 0.2%
(n = 5) of the response to saturating glycine in oocytes ex-
pressing α1, α2 or α3 glycine receptors respectively. In oocytes
expressing α1β3γ2L GABAA receptors, application of 100 μM
4-FP elicited a peak response that was 33 3% (n = 5) of the
response to 1 mM GABA. Sample current traces are shown in
Figure 1A.
We also tested the effect of the parent compound,
propofol. In agreement with previous data (Feng and
Macdonald, 2004), the α1β3γ2L GABAA receptors were effica-
ciously activated by 100 μM propofol, which produced a re-
sponse that was 90 3% (n = 7) of the response to saturating
GABA. In contrast, in glycine receptors, propofol was an inef-
fective agonist. At 100 μM, propofol elicited responses that
were 0.8 0.3% (n = 4), 1 0.3% (n = 4) or 0.2 0.01% (n = 4)
of the response to saturating glycine in α1, α2 or α3 receptors.
A summary of direct activation data showing the ratios of
responses to propofol and its analogues compared with satu-
rating glycine or GABA is given in Figure 1B.
Potentiation of glycine receptors by propofol and
its 4-halogenated analogues
We first determined the concentration–response relation-
ships for receptor activation by glycine. Cells expressing each
of the α subunits were exposed to 7–8 concentrations of gly-
cine. The raw current amplitudes were fitted with the Hill
equation for each cell individually. The EC50 of the glycine ac-
tivation curves were 79 14 μM (n = 5), 64 3 μM (n = 5) and
398 52 μM (n = 5) in oocytes expressing α1, α2 and α3 recep-
tors respectively. These values are similar to those already re-
ported (Downie et al., 1996; Mascia et al., 1996; Pistis et al.,
1997; Zhang et al., 2008).
We next investigated the effect of propofol or its
4-halogenated analogues on responses elicited by glycine.
Co-application of 4-BP, with a low concentration of glycine,
potentiated current responses (Figure 2). Homomeric α1 gly-
cine receptors were activated by 17.5–22 μM glycine
(<EC15) and additionally exposed to 1–500 μM 4-BP. The test
responses were normalized to control responses to a saturat-
ing concentration (0.5 mM) of glycine that was applied after
every 2–3 test responses and at the end of recordings from
each cell, to confirm overall stability of responses. The data
showed that 4-BP potentiated glycine-activated α1 receptors
with an EC50 of 42 μM (n = 6). In an analogous experimental
protocol, the EC50 for potentiation of α1 glycine receptors by
4-CP was 32 μM (n = 5), by 4-FP 78 μM (n = 5) and for poten-
tiation by propofol 138 μM (n = 6). Thus, the 4-halogenated
compounds were more potent than propofol at potentiating
α1 glycine receptors. The EC50, maximal current levels in
the presence of potentiator, and the Hill coefficients are given
in Table 1.
Figure 2
Potentiation of glycine and GABAA receptors. Sample currents from oocytes expressing α1, α2 or α3 glycine (GlyR) or α1β3γ2L GABAA receptors
(GABAAR). The receptors were activated by a low concentration (<EC15) of transmitter alone or in the presence of two concentrations of 4-BP
(A), 4-CP (B) or 4-FP (C). The concentrations of potentiators were selected to show minimal and near-maximal potentiating effects. Full
concentration–response relationships are shown in Figure 3 and fitting results summarized in Table 1.
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A L Germann et al.
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Table 1
Summary of concentration–response data for potentiation of glycine and GABAA receptors
Parameter
Gly α1
42 5 μM *
Gly α2
15 2 μM ≠
Gly α3
11 1 μM ≠
GABA α1β3γ2L
4-BP : EC50
*
*
*
*
4
1 μM ≠
*
nH
1.3 0.2
1.3 0.3
0.62 0.07
23 1 μM ≠
1.5 0.1
2.1 0.3
0.50 0.07
21 1 μM *
2.1 0.3
2.5 0.6
0.68 0.06 †
Imax
0.63 0.06 *
32 1 μM *
1.3 0.2
4-CP : EC50
5
1 μM ≠
*
nH
1.3 0.2
0.84 0.05 #
*
Imax
0.81 0.04
78 3 μM *
2.1 0.3
0.62 0.03
62 6 μM #†
0.56 0.05
42 7 μM #
1.9 0.2
4-FP : EC50
*
7
1 μM #†
nH
1.5 0.2
3.6 0.2
Imax
0.69 0.02
138 20 μM #†≠
1.2 0.2
0.59 0.05
95 9 μM #†≠
0.70 0.06
112 9 μM #†≠
0.70 0.02
Propofol : EC50
7
1 μM #†
nH
1.7 0.2
1.6 0.1
2.1 0.3
0.66 0.05 †
Imax
0.88 0.04 #
0.69 0.08
0.63 0.03
The Table summarizes the results from fitting the concentration-response curves for potentiation for glycine and GABAA receptors expressed in Xenopus
oocytes in the presence of 4-BP, 4-CP, 4-FP or propofol. EC50 is the concentration producing a half-maximal response, nH is the Hill coefficient and Imax
is the fitted high-concentration asymptote relative to the response to saturating concentration of transmitter. The data are presented as mean SEM for
five to seven cells. EC50 and Imax values for a given receptor were compared between 4-BP, 4-CP, 4-FP and propofol. The symbols next to the values
denote significant differences (P < 0.05) from the corresponding values for 4-BP (#), 4-CP (†), 4-FP (≠) or propofol (*); one-way ANOVA with Bonferroni
correction (Stata/IC 12.1, StataCorp, College Station, TX).
Figure 3
Concentration–response relationships for glycine and GABAA receptors. Oocytes expressing glycine α1, α2 or α3 subunit or GABAA α1β3γ2L sub-
units were activated by a low concentration of transmitter (<EC15) in the presence of 4-BP, 4-CP, 4-FP or propofol. The data points (mean SEM
from five to seven cells for each receptor-drug combination) show responses normalized to peak current in the presence of saturating transmitter
in the same cell. The curves show predicted concentration–response relationships based on data in Table 1.
3116 British Journal of Pharmacology (2016) 173 3110–3120

Actions of halogenated propofol analogues
BJP
The α2 subunit-containing glycine receptors were acti-
vated by 30–35 μM glycine (<EC15) in the presence of
1–200 μM 4-BP, 1–200 μM 4-CP, 5–500 μM 4-FP or
5–1000 μM propofol. The test responses were normalized to
control responses to a saturating concentration (0.5 mM) of
glycine. The half-maximal concentration for potentiation
by 4-BP was 15 μM (n = 5). 4-CP potentiated α2 receptors with
an EC50 of 23 μM (n = 5), and 4-FP potentiated the receptors
with an EC50 of 62 6 μM (n = 5). Potentiation in the presence
of propofol yielded an EC50 of 95 μM (n = 5). The data and the
results of statistical analyses are provided in Table 1.
The α3 receptors were activated by 85–100 μM glycine
(<EC15). Coapplication of 4-BP with glycine enhanced cur-
rent responses. The half-maximal concentration of 4-BP for
potentiation was 11 μM (n = 5). 4-CP and 4-FP produced
EC50s of 21 μM (n = 5) and 42 μM (n = 5) respectively. The
EC50 for potentiation by propofol was 112 μM (n = 5). Sample
traces are shown in Figure 2, and the data are summarized in
Figure 3 and Table 1.
0.1–50 μM 4-CP, 0.2–50 μM 4-FP or 0.2–100 μM propofol.
The current amplitudes were normalized to the response to
saturating (1 mM) GABA.
For propofol, recordings from five cells produced an EC50
of 7 μM. Potentiation data obtained in the presence of the ha-
logenated analogues of propofol yielded EC50s of 4 μM (n = 7),
5 μM (n = 5) and 7 μM (n = 5) for 4-BP, 4-CP and 4-FP respec-
tively. The concentration–response data are summarized in
Figure 3 and Table 1.
Propofol and its 4-halogenated analogues elicit
loss of the righting reflex (LRR) in Xenopus
tadpoles
GABAA receptor-mediated activity underlies the ability of
propofol to induce or maintain sedation (see Franks,
2008). To gain insight into the sedative actions of the halo-
genated analogues, we examined the tadpole LRR. This an-
aesthetic endpoint correlates very well with anaesthesia in
mammals, including humans (Franks and Lieb, 1993) and
is considered as surrogate for loss of consciousness models
(Franks, 2008).
Potentiation of α1β3γ2L GABAA receptors by
propofol and its 4-halogenated analogues
The tadpoles were incubated in beakers containing
0.1–10 μM of 4-BP, 0.06–3 μM 4-CP, 0.03–10 μM 4-FP or
0.03–10 μM propofol. The LRR was measured at the end of a
3 h exposure period. The number of tadpoles with LRR was
plotted as a fraction of total and the EC50 estimated using
the method described by Waud (1972).
We tested potentiation of α1β3γ2L GABAA receptors by
propofol and its halogenated analogues. The experiments
were conducted using the experimental protocol described
above for glycine receptors. A low concentration (1–3 μM)
of GABA was applied in the presence of 0.2–50 μM 4-BP,
Figure 4
Tadpole behavioural data. Exposure to 4-BP (A), 4-CP (B), 4-FP (C) or propofol (D) causes loss-of-righting in Xenopus tadpoles. The data are plotted
as quantal dose–response relationships with each tadpole represented as one circle. Ten tadpoles were used at each concentration. The ordinate
depicts the response as no effect (0) or LRR (1). The EC50 (and slopes) were 0.44 0.06 μM (3.32 0.93), 0.35 0.03 μM (6.01 1.69),
0.87 0.10 μM (4.36 1.30) and 0.52 0.08 μM (2.10 0.43) for 4-BP, 4-CP, 4-FP and propofol respectively.
British Journal of Pharmacology (2016) 173 3110–3120 3117

A L Germann et al.
BJP
All compounds were potent sedatives. The EC50 were
0.44 0.06 μM, 0.35 0.03 μM, 0.87 0.10 μM and
0.52 0.08 μM for 4-BP, 4-CP, 4-FP and propofol respectively.
The concentration–response data are shown in Figure 4.
11 μM in the midpoints of the concentration–response curves
(Table 1). The changes in EC50 were not accompanied by a
significant or systematic increase in relative current levels
from maximally potentiated receptors. We infer that haloge-
nation at the para position increases affinity of the com-
pound, rather than its efficacy, on glycine receptors.
Propofol and its 4-halogenated analogues were weak di-
rect activators of glycine receptors. Comparison of currents
obtained in the presence of 0.2, 10 and 100 μM 4-BP suggests
that the small responses are due to low affinity of the com-
pound rather than low efficacy.
Discussion and conclusions
The intravenous anaesthetic propofol directly activates and
potentiates currents from both glycine and GABAA receptors
(Pistis et al., 1997). The sensitivities of the two receptors to
the drug are, however, quite different, and it is considered
that propofol's actions at clinical doses are mediated by
GABAA, rather than glycine receptors (Rudolph and
Antkowiak, 2004). Previous work has indicated that para-
halogenation (at position 4) of the propofol molecule may
produce compounds with increased potency on glycine re-
ceptors. The 4-chloro substituted analogue was reported to
enhance currents elicited from heterologously expressed α1
homomeric glycine receptors at subnanomolar concentra-
tions (de la Roche et al., 2012). Nanomolar concentrations
of 4-BP, but not propofol, elicited strychnine-sensitive inward
currents in spinal slices and reduced the discharge rate of ven-
tral horn neurons (Eckle et al., 2014). In contrast, halogena-
tion of propofol may have little effect on its ability to
activate or modulate GABAA receptors. Compounds with
chloro-, bromo- or iodo- substituents at the para position of
propofol have been shown to directly activate and potentiate
the α1β1γ2 GABAA receptor with essentially the same
half-maximal concentrations and maximal effects as the
parent compound, propofol (Trapani et al., 1998). In tad-
poles, 4-iodopropofol and propofol induce LRR with similar
EC50 (Lingamaneni et al., 2001). Based on these findings, it
has been proposed that halogenated propofol analogues
may be considered selective glycinergic modulators.
Here, we have compared direct activation and modula-
tion of recombinant glycine and GABAA receptors by
propofol and its 4-halogenated analogues 4-BP, 4-CP and 4-
FP. We studied these actions on α1, α2 and α3 homomeric gly-
cine receptors. This choice was based on the previous study
that implicated spinal cord receptors producing tonic, rather
than synaptic, currents in the actions of 4-BP (Eckle et al.,
2014). Homomeric α subunit containing glycine receptors
are likely to be located presynaptically where they regulate
the release of transmitter or extrasynaptically where they me-
diate tonic glycinergic currents (Takahashi et al., 1992;
Turecek and Trussell, 2002; Grudzinska et al., 2005) because
synaptic localization requires a presence of the β subunit
(Meyer et al., 1995). The α4 subunit, that is expressed in the
chick brain and mouse retina, was not studied here. We also
note that inclusion of the β subunit is not expected to change
receptor sensitivity to propofol or its halogenated analogues
(Haeseleret al., 2005). We also used the α1β3γ2L GABAA recep-
tor. This subtype, along with one where β2 replaces β3, is the
major target for propofol to produce various anaesthetic end-
points (Jurd et al., 2003; Reynolds et al., 2003).
Halogenation of propofol weakly affected modulation of
GABAA receptors. In oocytes expressing α1β3γ2L GABAA re-
ceptors, the potentiation curves for 4-BP and 4-CP were shifted
to the left by less than two-fold compared with propofol,
whereas the concentration–response relationships for 4-FP
and propofol were indistinguishable. Halogenation of
propofol also had small effect on the EC50 for LRR in Xenopus
tadpoles. The EC50 for 4-BP and 4-CP were slightly lower while
that for 4-FP was higher than the EC50 for propofol. The LRR is
considered to be mediated by actions on the GABAA receptor
(Reith and Sillar, 1999; Belelli et al., 2003), and there is good
correlation between LRR in tadpoles and inhibition of bind-
ing of t-butylbicyclophosphorothionate or modulation of
GABAA receptor function (Krasowski et al., 2001; Akk et al.,
2007). Of note, the numerical values for EC50 of potentiation
by all tested analogues were lower for GABAA than glycine re-
ceptors. Thus, we infer that 4-BP, 4-CP or 4-FP cannot plausi-
bly be used as selective glycine receptor modulators.
This inference is not in agreement with conclusions made
in an earlier study that found significant effects on the dis-
charge rate of mouse ventral horn neurons and strychnine-
sensitive tonic currents in organotypic spinal cultures by
50–200 nM 4-BP (Eckle et al., 2014). In our experiments on re-
combinant homomeric glycine receptors, 200 nM 4-BP was
essentially inert in the absence of glycine (relative to saturat-
ing glycine mean responses ranged from 0.01–0.22%), while
potentiation of glycine-elicited currents was reliably ob-
served at micromolar concentrations. As Eckle et al. (2014)
did not provide a full concentration–response relationship
for their observed effects, precise comparison of our results
is difficult. The increase in tonic current in the presence of
200 nM 4-BP may have represented the low-concentration tail
of the potentiation curve. However, using the data in Table 1,
we calculate that potentiation by 200 nM 4-BP is minimal,
ranging from 0.2 to 4.1% for currents elicited by EC5 glycine.
Our data also contradict another study reporting
a
subnanomolar EC50 for potentiation of glycine α1 receptors by
4-CP (de la Roche et al., 2012). We found an EC50 of approxi-
mately 30 μM for potentiation of α1 receptors by 4-CP. The
cause for discrepancy is unclear to us. We note that other phe-
nol analogues with the 4-chloro substituent, 3-methyl-4-
chlorophenol and 3,5-dimethyl-4-chlorophenol also show po-
tentiation EC50 in the micromolar range (Haeseler et al., 2005).
In summary, our findings indicate that halogenation of
propofol at the para position generates compounds more
capable of potentiating glycine receptors than the parent
compound, propofol. The effect is manifested as a reduction
in the EC50 of the concentration–response curves for potenti-
ation. However, we emphasize that 4-halogenated analogues
of propofol remain more potent as potentiators of the
The key finding is that halogenation of propofol at the
para position leads to a 2–10-fold reduction in the EC50 for
potentiation of glycine receptors. The greatest effect was ob-
served with α3 receptors for which addition of the bromide
group to propofol resulted in a leftward shift from 112 to
3118 British Journal of Pharmacology (2016) 173 3110–3120

Actions of halogenated propofol analogues
BJP
Chastrette M, Zakarya D, Elmouaffek A (1986). Structure-odor
relations of the nitrobenzene musks. Eur J Med Chem 21: 505–510.
α1β3γ2L GABAA, than of homomeric glycine, receptors. It
may be argued that these compounds are less desirable as se-
lective clinical agents because halogenation at the para posi-
tion leads to reduction in selectivity for the GABAA
receptors that characterizes the parent compound propofol.
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Acknowledgements
We thank Amanda Taylor and Steve Mennerick for assistance
with harvesting Xenopus oocytes and Joe Henry Steinbach for
invaluable help with programming. This study was supported
by grants from the National Institutes of Health, USA
(GM108799 to A.S.E. and GM108580 to G.A.), by a grant
from the Medical Research Council, UK (G0901892 to N.P.
F.) and funds from the Taylor Family Institute for Innovative
Psychiatric Research (to A.S.E. and G.A.).
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inhalational general anaesthetics on native glycine receptors in rat
medullary neurones and recombinant glycine receptors in Xenopus
oocytes. Br J Pharmacol 118: 493–502.
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et al. (2014). 4-bromopropofol decreases action potential generation
in spinal neurons by inducing a glycine receptor-mediated tonic
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Author contributions
Franks NP (2008). General anaesthesia: from molecular targets to
neuronal pathways of sleep and arousal. Nat Rev Neurosci 9:
370–386.
N.P.F., A.S.E. and G.A. conceived the study and designed the
experiments; C.J.E. and E.H.S. synthesized the halogenated
analogues of propofol; A.G., D.S. and B.D.M. conducted the
experiments; A.G., D.S., B.D.M., C.J.E. and G.A. analysed
the data; and C.J.E., E.H.S., N.P.F., A.S.E. and G.A. wrote the
manuscript. All authors approved the final version of the
manuscript.
Franks NP, Lieb WR (1993). Selective actions of volatile general
anaesthetics at molecular and cellular levels. Br J Anaesth 71: 65–76.
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(2005). Structural features of phenol derivatives determining potency
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Conflict of interest
The authors declare no conflicts of interest.
Hales TG, Lambert JJ (1991). The actions of propofol on inhibitory
amino acid receptors of bovine adrenomedullary chromaffin cells
and rodent central neurones. Br J Pharmacol 104: 619–628.
Declaration of transparency and
scientific rigour
Harvey RJ, Depner UB, Wassle H, Ahmadi S, Heindl C, Reinold H et al.
(2004). GlyR α3: an essential target for spinal PGE2-mediated
inflammatory pain sensitization. Science 304: 884–887.
This Declaration acknowledges that this paper adheres to the
principles for transparent reporting and scientific rigour of
preclinical research recommended by funding agencies, pub-
lishers and other organisations engaged with supporting
research.
Haverkamp S, Muller U, Harvey K, Harvey RJ, Betz H, Wassle H (2003).
Diversity of glycine receptors in the mouse retina: localization of the
α3 subunit. J Comp Neurol 465: 524–539.
Jurd R, Arras M, Lambert S, Drexler B, Siegwart R, Crestani F et al.
(2003). General anesthetic actions in vivo strongly attenuated by a
point mutation in the GABAA receptor β3 subunit. FASEB J 17:
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