Azo-propofols: Photochromic potentiators of GABAA receptors

Full text of DOI:10.1002/anie.201205475

Angewandte
Chemie
DOI: 10.1002/anie.201205475
Photopharmacology
Azo-Propofols: Photochromic Potentiators of GABAA Receptors**
Marco Stein, Simon J. Middendorp, Valentina Carta, Ervin Pejo, Douglas E. Raines,
Stuart A. Forman, Erwin Sigel,* and Dirk Trauner*
GABAA receptors are pentameric ligand-gated ion channels
that are activated by the major inhibitory neurotransmitter in
the mammalian brain, g-aminobutyric acid (GABA).[1] Bind-
ing of GABA results in the opening of a chloride ion selective
pore, thus hyperpolarizing the postsynaptic neuron and
decreasing the likelihood of action-potential firing. As such,
GABAA receptors are a prominent target for anesthetic,
hypnotic, and anticonvulsant drugs (Scheme 1).[2,3]
While agonists, antagonists, and blockers of GABAA
receptors, such as muscimol, gabazine, or picrotoxinin,
respectively, have proven to be valuable research tools, their
impact on human medicine has been limited. Drugs that
target these receptors are dominated by allosteric modulators
that potentiate, that is, increase, chloride currents elicited by
the neurotransmitter. Well-established potentiators include
benzodiazepines (e.g. clonazepam), barbiturates (e.g. pheno-
barbital), the imidazopyridine zolpidem, and the simple
phenol propofol.[2] These drugs bind to distinct allosteric
sites on GABAA receptors, thereby increasing the mean open
time or the opening frequency of the channel. However, the
analysis of their exact binding sites at a molecular level has
been complicated by a lack of detailed structural data.
After its discovery in 1980, propofol has become the most
widely used intravenous general anesthetic.[4] Although its
mode of action has not been fully elucidated, it is commonly
Scheme 1. Agonists (GABA, muscimol), antagonists (gabazine), block-
ers (picrotoxinin), or potentiators (clonazepam, phenobarbital, zolpi-
dem, propofol) of GABAA receptors.
accepted that the anesthesia induced by this unusually
lipophilic drug mostly results from potentiation of GABA-
[*] M. Stein,[+] D. Trauner
Department of Chemistry, Ludwig-Maximilians-Universitꢀt
Mꢁnchen and Center of Integrated Protein Science
81377 Munich (Germany)
E-mail: dirk.trauner@lmu.de
induced currents, as well as a direct activation of the chloride
ion channel at high concentrations. Propofol has a rapid onset
and offset of action and shows only minimal accumulation
S. J. Middendorp,[+] V. Carta, E. Sigel
upon prolonged use. The intravenous administration of
Institute for Biochemistry and Molecular Medicine
propofol is also associated with reduced postoperative
University of Bern
Bꢁhlstr. 28, CH-3012 Bern (Switzerland)
nausea and vomiting.[5]
While GABAA receptors respond to a variety of ligands,
they are normally not sensitive toward light. It would be
fascinating to confer light sensitivity to these ion channels,
since light is unsurpassed in terms of the temporal and spatial
precision it provides. This light sensitivity could be indirectly
achieved by using ligands that act on the receptors but can be
optically switched between an active and an inactive form.
Photochromic ligands of GABAA receptors could be agonists,
antagonists, or allosteric modulators. In principle, these
ligands could be covalently attached as photoswitched
tethered ligands (PTLs) or act as soluble photochromic
ligands (PCLs).[6] Indeed, both approaches have been used to
convert neuronal[7] and neuromuscular[8] nicotinic acetylcho-
line receptors, another type of pentameric ligand-gated ion
E-mail: erwin.sigel@ibmm.unibe.ch
E. Pejo, D. E. Raines, S. A. Forman
Department of Anesthesia, Critical Care and Pain Medicine
Massachusetts General Hospital
32 Fruit Street, Boston, MA 02114 (USA)
[+] These authors contributed equally to this work.
[**] We thank Dr. Peter Mayer (LMU Munich) for the determination of
the X-ray structure. M. S. is grateful to the Fonds der Chemischen
Industrie for a Ph.D. fellowship. This work was supported by the
Swiss National Science Foundation grants 31003A-132806/1 (E.S.)
and the European Science Foundation (ERC grant No 268795 to
D.T.) and the National Institutes of Health (GM089745 to S.A.F and
GM087316 to D.E.R.). GABA=g-aminobutyric acid.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201205475.
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channels, as well as ionotropic glutamate receptors[9] into
artificial photoreceptors. Tethered and soluble photochromic
blockers of K+, Na+, and Ca2+ ion channels have been
described as well and have been used to control heartbeat,[10]
pain sensation[11] and visual responses[12] in different animals
with light.
We now report photochromic potentiators of GABA
currents that change the strength of GABA-induced currents
in a light-dependent fashion. Our program was prompted by
a recent report on a photoaffinity probe based on propofol, p-
4-aziC5-propofol that underscored that a relatively large
substituent in the para-position of the phenol would be
tolerated and that the propofol pharmacophor would be
compatible with photochemistry (Scheme 2a).[13] Accord-
ingly, we designed a series of azobenzene derivatives of
propofol; in these derivatives an aryldiazene unit is directly
coupled to the pharmacophor. These molecules, termed azo-
propofols 1–16 (AP1–16) are shown in Scheme 2a. Their
varying substituent in the 4’-position of the azobenzene core
determines their pharmacodynamic as well as spectral
properties.
Compounds AP1–16 were synthesized using classical
diazo-coupling chemistry, as shown in the representative
synthesis of AP1 and AP2 (Scheme 2b, also see the Support-
ing Information).[14] The X-ray structure of AP2 is displayed
in Scheme 2c.[15] Owing to crystal packing effects, the trans-
azobenzene is not fully planar in this solid-state structure (see
the Supporting Information for a more detailed discussion of
these effects).
Although several of the azo-propofols shown in Scheme 2
function as photochromic potentiators, AP2 emerged early on
as our most promising candidate owing to its favorable
pharmacological and photochemical features. By virtue of its
amino substituent, the photoswitch has a red-shifted absorp-
tion spectrum, which means that a maximum cis content is
achieved at irradiation with 404 nm light. However, owing to
the broad absorption spectrum, slightly longer or shorter
wavelengths could be used effectively (see the Supporting
Information). In addition to this, the substitution of the
azobenzene core with electron-donating substituents greatly
decreases the thermal stability of the cis isomer. Therefore,
AP2 quickly reverts to its trans form once the light is switched
off. Since the absorption spectra of the cis and trans isomers
are very similar (see the Supporting Information), this process
cannot be accelerated by irradiation with a different wave-
length. Other APs studied have less favorable photophysical
properties, show decreased potency (e.g. AP3, AP9, AP10),
no activity at all (e.g. AP4),[14c] or unfavorable solubility and
distribution (e.g. AP6).
The effect of AP2 on ClÀ currents was investigated with
electrophysiology using a1b2g2 GABAA receptors expressed
in Xenopus oocytes (Figure 1).[16] This receptor subtype
represents the most prevalent form in the human brain.[17]
First, the heterologously expressed GABAA receptors were
exposed to GABA at a concentration eliciting 0.3% of the
maximal current amplitude in combination with increasing
concentrations of propofol or AP2 in the dark to compare the
relative effect of the compounds. From the resulting dose–
response curves, we extracted an EC50 value of (17.1 Æ 2.9) mm
for propofol and of (6.1 Æ 0.4) mm for AP2 (mean Æ standard
error of the mean (SEM), n = 4; Figure 1a). Thus, AP2 in its
dark-adapted trans form has a significantly higher affinity
than propofol itself, albeit its efficacy is reduced by about
twofold when compared with its parent compound.
Having established that AP2, in its dark-adapted form,
has an effect on GABAA receptors, we investigated the light
dependency of the current potentiation. UV/Vis light from an
Ultrafire 1 Watt UV LED pocket lamp (YonC Trading,
Zꢀrich; emission wavelength 390–450 nm) had no effect on
the GABA response or the combined GABA/propofol
response (data not shown). Figure 1b illustrates the effect
of UV/Vis light on currents elicited by the combined
application of GABA and AP2. Stimulation of GABA
currents by AP2 (1.5 mm) was (159 Æ 25)% (mean Æ SEM,
n = 6). Exposure to light decreased the residual stimulation to
(18 Æ 3)% (mean Æ SEM, n = 6). Similar observations were
made using a UV high power LED pocket lamp, 5 Watt
(Uveco GmbH, Bruckmꢀhl, Germany), emission wavelength
355-380 nm equipped with a CHROMA bandpass filter D365/
10 ꢁ , to limit light emission to 360-370 nm. The possibility to
Scheme 2. a) p-4-aziC5-propofol, a photoreactive derivative of propofol,
and AP1–16, photoswitchable derivatives of propofol. b) Synthesis of
AP1 and AP2, which is shown in its trans and cis configuration. c) X-ray
structure of trans-AP2; C green, O red, H white, N blue.
2
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use these different light sources reflects the broad absorption
spectrum of AP2. Owing to redistribution of the hydrophobic
compound AP2 into egg yolk, the rate of photoswitching
could not be determined in Xenopus oocytes. For this
purpose, we expressed a1b2g2 GABAA receptors in HEK
cells and performed experiments using the whole-cell patch-
clamp technique. GABA was co-applied with AP2. Subse-
quently, the perfusion was stopped to prevent arrival of new
trans-AP2 during the measurement, and the cells were
exposed to the light. The current amplitude decreased rapidly
and increased again upon turning off the light source. Current
traces were fitted with a mono-exponential function. The time
constant t amounted to (1.1 Æ 0.4) s (mean Æ SD, n = 7) for
the trans-to-cis transition and (2.0 Æ 0.7) s (mean Æ SD, n = 6)
for the cis-to-trans transition.
Next, we investigated anesthetic activity and photorever-
sibility of both propofol and AP2 in a small animal model,
albino Xenopus laevis tadpoles. Groups of animals were
placed in aqueous solutions containing either propofol or
AP2 and tested every five minutes for loss of righting reflexes
(LORR), a standard assay for anesthesia.[13] Steady-state
LORR results were observed at 10 min for propofol and
25 min for AP2. After 30 min in drug solution, each animal
was exposed for five to ten seconds to 360–370 nm bandpass
filtered UV light (details in the Supporting Information)
while retesting for LORR. Propofol alone produced LORR
with an EC50 of 1.3 mm (Figure 2a). Illumination induced
vigorous swimming activity in unanesthetized tadpoles (thus
suggesting that illumination represents a noxious stimulus)
and a small rightward shift in the corresponding plot of
LORR versus propofol concentration was observed (EC50 ca.
2.0 mm; Figure 2a). AP2 alone produced LORR with an EC50
value similar to that of propofol (1.1 mm; Figure 2b). How-
ever, illumination produced a large rightward shift in the AP2
EC50 value to 4.6 mm (Figure 2b). All animals recovered from
anesthesia when returned to water alone. In an independent
set of experiments (see video in the Supporting Information),
propofol (3 mm) produced LORR in all tadpoles with or
without light, whereas in AP2 (3 mm), all animals showed
LORR without light and all spontaneously righted themselves
and swam during illumination with UV light.
Figure 1. a) a1b2g2 GABAA receptors were expressed in Xenopus
oocytes. Currents were activated with a concentration of GABA eliciting
0.3% of the maximal current amplitude (EC0.3) with increasing
amounts of either propofol or AP2. Mean ÆSEM of four experiments
is shown. b) GABA (1 mm) was applied repetitively until a stable
current response was observed. Co-application of AP2 (1.5 mm) with
GABA resulted in current potentiation. During co-application, the
oocyte was exposed to a light source emanating 390–450 nm light. As
a consequence, current stimulation rapidly decreased until it reached
a steady level. When the light-source was turned off, the amplitude
increased again. This procedure was repeated. This experiment was
repeated independently six times using different oocytes. c) a1b2g2
GABAA receptors were expressed in HEK cells. GABA (0.5 mm) was co-
applied with AP2 (5 mm). Subsequently, the perfusion was stopped and
the cells were exposed to the light source. The inward current
amplitude decreased rapidly and increased again upon turning off the
light source (trace left). Current decrease (trace top right) and increase
(trace bottom right) were each fitted with a mono-exponential func-
tion.
Figure 2. Light-dependent anesthesia in tadpoles. Loss of righting
reflexes is plotted against aqueous anesthetic concentration, overlayed
with logistic fits. Each point represents data from ten animals. a) 360–
370 nm light, an apparently noxious stimulus in Xenopus tadpoles,
produces a small rightward shift in propofol-dependent loss of righting
reflexes (LORR) from (1.1Æ0.1) mm (circles) to (2.0Æ0.1) mm (stars).
b) 360–370 nm light shifts the AP2-dependent LORR curve to the right
from (1.1Æ0.1) mm (squares) to 4.6Æ0.2 mm (crosses). This larger
shift is due to photoisomerization of AP2.
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The photoreversibility of both AP2-induced GABAA
receptor modulation and its anesthetic action in animals
supports the hypothesis that anesthesia caused by AP2 and
propofol is largely mediated by GABAA receptors. However,
evidence also implicates other targets, including HCN1
channels (hyperpolarization-activated cation channels),[18] in
propofolꢂs anesthetic actions. The examination of the effects
of AP2 on these other targets and the investigation of the
photoreversibility of the modulation of these targets might
help to further elucidate their roles in the pharmacology of
general anesthesia.
In summary, we have developed photoswitchable versions
of propofol that allow the indirect optical control of GABAA
receptors. Functionally, our compounds differ from previ-
ously introduced PCLs, because they act as photochromic
potentiators rather than photochromic agonists, antagonists,
or channel blockers. Application of our lead compound, AP2,
in the dark potentiates GABA-induced ClÀ currents, which
can be reversed upon irradiation with violet light. The ability
of azo-propofols to control neural systems has been demon-
strated, since AP2 functions as a light-dependent anesthetic
in translucent tadpoles. Future work will address the useful-
ness of azo-propofols in other systems, such as brain slices and
retinas lacking innate photoreceptors, wherein photochromic
potentiators could restore visual responses through their
action on neurons expressing GABAA receptors.
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Received: July 11, 2012
Published online: && &&, &&&&
Keywords: azo compounds · GABA receptors · ion channels ·
[15] AP2: C18H23N3O, Mr = 297.395 gmolÀ1, red block, 0.14 ꢁ 0.25 ꢁ
0.30 mm, monoclinic, P21, a = 10.5454(3), b = 9.5435(3), c =
.
photochromism · photopharmacology
17.0651(4) ꢃ,
a = 90,
b = 90.0955(16),
g = 908,
V=
1704.28(8) ꢃ3, Z = 4, 1 = 1.159 gcmÀ3, m(MoKa) = 0.073 mmÀ1
,
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4
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Communications
Photopharmacology
M. Stein, S. J. Middendorp, V. Carta,
E. Pejo, D. E. Raines, S. A. Forman,
E. Sigel,* D. Trauner*
&&&& — &&&&
Azo-Propofols: Photochromic
Potentiators of GABAA Receptors
Shine and rise! GABAA receptors are
ligand-gated chloride ion channels that
respond to g-aminobutyric acid (GABA),
which is the major inhibitory neurotrans-
mitter of the mammalian central nervous
system. Azobenzene derivatives of pro-
pofol, such as compound 1 (see scheme),
increase GABA-induced currents in the
dark form and loose this property upon
light exposure and thus function as
photochromic potentiators. Compound
1 can be employed as a light-dependent
general anesthetic in translucent tad-
poles.
Angew. Chem. Int. Ed. 2012, 51, 1 – 5
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