The two proteases ?-secretase and ?-secretase generate the amyloid ? peptide and are drug targets for Alzheimer’s disease. Here we
has an acidic pH optimum and cleaves the amyloid precursor protein in the acidic endosomes. We identified two drugs, bepridil and
amiodarone, that are weak bases and are in clinical use as calcium antagonists. Independently of their calcium-blocking activity, both
compounds mildly raised the membrane-proximal, endosomal pH and inhibited ?-secretase cleavage at therapeutically achievable
ment could be a novel therapeutic strategy to inhibit ?-secretase. Surprisingly, bepridil and amiodarone also modulated ?-secretase
cleavage independently of endosomal alkalinization. Thus, both compounds act as dual modulators that simultaneously target ?- and
?-secretase through distinct molecular mechanisms. In addition to Alzheimer’s disease, compounds with dual properties may also be
Alzheimer’s disease (AD) is the most prevalent neurodegenera-
tive disorder. The amyloid hypothesis ascribes the pathogenesis
of the disease to a cascade of events, starting with the generation
and accumulation of the amyloid ? peptide (A?), a proteolytic
fragment of the amyloid precursor protein (APP) (Hardy and
processed by the two proteases, ?- and ?-secretase. A third pro-
tease, ?-secretase, cleaves APP within the A? domain and pre-
cludes A? generation. ?-Secretase is the membrane-bound
A? domain (Rossner et al., 2006; Cole and Vassar, 2008). This
cleavage generates the soluble APPs? and a C-terminal fragment
(C99), which undergoes a second cleavage at the C terminus of
bic transmembrane domain. ?-Secretase is a heterotetrameric
protein complex consisting of presenilin, nicastrin, PEN-2, and
APH-1 (Steiner et al., 2008). ?-Secretase mainly cleaves after
amino acid 40 of the A? sequence, resulting in the generation of
the A?40 peptide. To a lower extent, ?-secretase also generates
A?38 and A?42. Although a minor product, A?42 is considered
the key player in AD pathogenesis and is the main constituent of
the amyloid plaques found in AD brains (Hardy and Selkoe,
?-Secretase is an obvious drug target for AD, but most
?-secretase inhibitors do not reach sufficiently high concentra-
tions in the brain (Ghosh et al., 2008; Meredith et al., 2008). It is
therefore essential to identify alternative strategies for reducing
?-secretase cleavage. One possibility is the addition of a mem-
increase its local membrane concentration, resulting in a more
environment of ?-secretase cleavage instead of the ?-secretase
enzyme itself. ?-Secretase has an acidic pH optimum and mostly
where it can be blocked by the weak base ammonium chloride
(Haass et al., 1993; Schrader-Fischer and Paganetti, 1996). Inter-
estingly, several drugs in clinical use contain weakly basic amino
groups and thus have the potential to raise the endosomal pH.
Correspondence should be addressed to Stefan F. Lichtenthaler at the above address. E-mail: stefan.
8974 • TheJournalofNeuroscience,June30,2010 • 30(26):8974–8983
We screened such compounds for an inhibition of ?-secretase
cleavage and identified the drugs bepridil and amiodarone as
novel inhibitors of ?-secretase cleavage. Both compounds are
calcium channel blockers and calmodulin antagonists and are
used for the treatment of heart disease. Here we found them to
inhibit ?-secretase cleavage and A? generation independently of
their calcium channel- and calmodulin-blocking activities. The
inhibition of ?-secretase cleavage occurred at therapeutically
achievable and nontoxic concentrations by raising the endoso-
mal, membrane-proximal pH. Surprisingly, both compounds
additionally modulated the cleavage specificity of ?-secretase in
an inverse manner. Thus, bepridil and amiodarone define a new
class of drugs—the dual modulators—which may be used for
drug development targeting ?- and ?-secretase, either individu-
ally or simultaneously.
Antibodies and reagents. The following antibodies were used: 192wt and
192swe (APPs? wild-type and Swedish mutations, respectively, Elan
Pharmaceuticals), W02 (APPs?) and 22C11 [full-length APP (fl.APP),
provided by K. Beyreuther, University of Heidelberg, Heidelberg, Ger-
ogy), 3552 (A?, Eurogentec), 2D8 (A?1-16, provided by E. Kremmer,
Helmholtz Institute, Munich, Germany), ?-A?38 MSD-Tag (Meso Scale
Discovery), ?-A?40 BAP24-TAG and ?-A?42 BAP24-TAG (provided by
M. Brockhaus, Roche, Basel, Switzerland), ?-calmodulin (Millipore), and
?-?-actin (Sigma). The reagents used were as follows: bepridil hydro-
chloride, amiodarone hydrochloride, chloroquine, nimodipine, and ta-
moxifen (Sigma); AMI-AcOH (provided by H.-R. Ha, University
Hospital, Zurich, Switzerland); GL-189 (provided by K. Maskos and W.
phosphatidylcholine (PC) (Avanti Polar Lipids); fluorescein phos-
phatidylethanolamine (FPE) (Invitrogen); and siRNA pools targeting
Calmodulin genes calm1 and calm3 and nontargeting control pools
Cell culture, compound treatment, and immunoblots. Human embry-
onic kidney 293EBNA (HEK293) cells were cultured as described previ-
ously (Kuhn et al., 2007). Clonal H4 and U373 cells stably expressing
APP751 from the vector pRC/CMV hAPPwt were cultured in DMEM
cin, and 200 ?g/ml G418 (G418-medium) or supplemented with 15%
FCS, respectively. Clonal HEK293 cells stably expressing APP695 from
the vector pCEP4-APP695 (HEK293-APP) and polyclonal HEK293 cells
100 ?g/ml hygromycin. HEK293-APPswe cells were cultured in G418-
medium as described previously (Haass et al., 1995). Cells were plated
(for A? analysis by immunoblot). At confluence, cells were pretreated
with compound or vehicle for 45 min, then with fresh medium plus
(Schobel et al., 2008).
A? detection. For A? detection in the conditioned media, immuno-
precipitation with antibody 3552 or 4G8 was performed before SDS-
PAGE. Proteins were transferred to nitrocellulose membranes. Total A?
species were separated on 11% urea gels (Wiltfang et al., 1997). Signals
were quantified using a Fluorchem 8900 device with AlphaEaseFC soft-
ware. Sandwich immunoassay of individual A? species using the Meso
established fluorometric assay was used (Capell et al., 2002). The assay
was performed in a volume of 100 ?l containing 1 ?l of purified BACE1
ectodomain, 1 ?M substrate peptide (Cy3-SEVNLDAEFK(Cy5Q)-NH2,
inhibitor GL-189, or just the vehicle DMSO in 40 mM sodium acetate
2 h at room temperature (Fluoroskan Ascent, excitation 530 nm, emis-
sion 590 nm, Labsystems).
Lactate dehydrogenase release assay. HEK293-APP cells were plated
into poly-D-lysine-coated 96-well plates (BD Biosciences) at a density of
2 ? 104cells/well in medium containing heat-inactivated fetal calf se-
rum. On the next day, the medium was changed and the cells were incu-
wells/condition). Tamoxifen was used as a positive control. Forty-five
minutes before the end of the incubation, the cells from three wells of
incubation time, the amount of lactate dehydrogenase (LDH) in the
conditioned media of the cells was analyzed using the CytoTox kit (Pro-
mega) according to the manufacturer’s instructions. Toxicity was calcu-
lated as ØLDHsamples/ØLDHmaxfor each condition (where Ø is mean
fetal Tg2576 mouse brain at gestation day 16 as described previously
(Hartlage-Ru ¨bsamen et al., 2003). Briefly, brains from fetuses were pre-
pared and suspended in DMEM/Ham’s F12 by trituration with glass
pipettes. Neurons were separated by sequential passage of the cell sus-
pension through nylon membranes (150 and 20 ?m), centrifuged for 5
streptomycin, and neomycin). The cell number was determined and
neurons were seeded at a density of 1.5 million cells per milliliter of
culture medium in poly-L-lysine-coated 12-well plates. After 24 h of
cultivation, the seeding medium was replaced by cultivation medium
(25% astrocyte conditioned medium in DMEM/Ham’s F12, N2 supple-
ment, and 1? PSN). On day 3, in vitro neurons were used for pharma-
cological experiments. Cells were pretreated with compound or
vehicle for 45 min then with fresh medium plus vehicle or compound
for another 24 h.
Organotypic brain slice cultures. Organotypic brain slice cultures were
established from adult APP transgenic Tg2576 mice as described previ-
ously (Reimers et al., 2007). Briefly, mice were decapitated and brains
were quickly prepared and mounted in 2% agarose in distilled water.
Brain sections (350 ?m thick) were cut in the coronal plane using a
vibratome (Leica). Brain sections were collected and maintained in cul-
ture plate inserts (Millicell CM, 30 mm diameter; Millipore) in DMEM/
L-glutamine, and 0.3 mM HEPES. The medium was changed every other
day and pharmacological treatments were initiated after a cultivation
period of 4 d. Slices were incubated for 16 h with serum-free medium,
followed by pretreatment with vehicle or compound and then by incu-
bation with fresh medium plus vehicle or compound for another 16 h.
The conditioned medium and brain slices were collected and stored in
aliquots at ?20°C pending biochemical analyses.
Treatment of guinea pigs. Animal experiments were approved by Re-
gierungspra ¨sidium Leipzig (TVV 25/08). Female Duncin Hartley albino
guinea pigs with a weight of 350–400 g were used (Charles River Labo-
animals, baseline A?40 concentration was analyzed in each guinea pig.
Blood samples (400 ?l) were collected at 2:00 P.M. on the day before
bepridil injection by puncture of the vena saphena lateralis. Heparin
at 10:00 A.M., bepridil was injected intraperitoneally at 50 mg/kg body
100 g body weight. The positive control group received injections of
DAPT (60 mg/kg body weight). The negative control group received
vehicle injections. Four hours after injection, guinea pigs were killed by
described above and stored at ?80°C. In the time course experiment,
trations before and after treatments were quantified by ELISA using the
Biosource colorimetric assay kit according to the manufacturer’s proto-
col. For each animal, the percentage amount of A?40 present after treat-
Mitterreiteretal.•DualModulationofAPPProcessing J.Neurosci.,June30,2010 • 30(26):8974–8983 • 8975
A? were below the detection limit.
Measurement of the pH in the endolysosomal system of living cells.
HEK293-APP cells were incubated for 30 min with tetramethylrhodam-
were then washed three times with serum-free medium and reincubated
for 1 h in fresh medium containing the compounds. Cells were imaged
with a Leica SP2 AOBS inverted confocal microscope using a 63? oil
objective lens (numerical aperture ? 1.3), exciting with a 488 nm and a
lated from a calibration curve. The calibration curve was generated by
loading the cells as above with TMR-fluorescein-tagged dextran but
without compounds and incubating them for 15 min in a triple-
component calibration buffer containing HEPES, CHES, and citric acid
(0.1 ?M), and monensin (1 ?M). pH points between 4.5 and 7.0 with 0.5
increments were measured as described above (Di et al., 2006).
Measurement of the membrane-proximal pH using FPE. Twenty-five
milligrams of egg PC were dissolved in low-salt buffer (20 mM HEPES-
NaOH, pH 7.4) and hydrated for 1 h at room temperature (RT). Subse-
quently, small unilamellar vesicles (SUV) were prepared by sonication
for 20 min at RT under a stream of argon gas as described previously
(Kamp and Beyer, 2006). To incorporate the fluorescent probe FPE, a
film of FPE was prepared by dissolving 36 ?l of a stock solution of FPE
bottom flask followed by 1 h vacuum. The SUV suspension (1 ml, lipid
concentration 25 mM) was injected on top of the FPE film and gently
shaken overnight at RT. Remaining aggregates of unbound FPE were
removed by 5 min tabletop centrifugation. The supernatant contained
SUV loaded with FPE. Changes in the membrane-proximal pH were
of buffer (final lipid concentration, 250 ?M) and measuring the fluores-
cence of FPE (excitation at 490 nm, emission at 520 nm). Aliquots of the
hole of the fluorimeter (Jasco FP 6300).
lic Ca2?was measured in HEK293-APP cells using the fura 2/AM dye
(Invitrogen) as described previously (Hamid et al., 2007). Cells were
viewed with an upright microscope (BX50WI, Olympus) using a 20?
water-immersion objective. Excitation of the cells was performed at 340
nm for the Ca2?-bound form and 380 nm for the unbound form of fura
2 and the ratio of the emissions was obtained using a digital imaging
system (Till Photonics). Ca2?concentrations were calculated from
fluorescence-intensity ratios using the following equation (Grynkiewicz
et al., 1985):
[Ca2?] ? Kd? ?[(R ? Rmin)/(Rmax? R)],
is the fluorescence ratio of the 380 nm signal in the absence of Ca2?to
that in the presence of saturating Ca2?, R is the fluorescence ratio ob-
tained using a calcium calibration buffer kit (Invitrogen). All experi-
ments were performed at room temperature and drugs were applied by
Knockdown of calmodulin using siRNAs followed by compound treat-
ment. HEK293-APP cells were plated at a density of 1 ? 105cells/well
into poly-D-lysine-coated 24-well plates. After 4 h, cells were transfected
with a mixture of siRNA pools against calm1 and calm3 or control pools
using Lipofectamine2000 (Invitrogen) according to the manufacturer’s
instructions. Two days after transfection, cells were treated with bepridil
or vehicle as described above.
were performed as described previously (Winkler et al., 2009). The
Q-Sepharose eluate fraction of purified ?-secretase was used as enzyme
First, we verified that the known alkalizing drugs ammonium
APP cells (supplemental Fig. 1, available at www.jneurosci.org as
supplemental material). Next, we tested bepridil and amioda-
rone. Bepridil is in clinical use in France and Japan for the treat-
ment of angina pectoris. Amiodarone is widely used for the
treatment of arrhythmias. Both compounds are cationic am-
phiphilic drugs and have a hydrophobic core with two aromatic
rings (Fig. 1A), which allows them to partition into the mem-
brane. The hydrophobic core is linked through a short aliphatic
spacer to a protonatable tertiary amino group (Fig. 1A, arrows),
which acts as a weak base.
To investigate the effect of bepridil on ?-secretase cleavage in
detail, HEK293-APP cells were treated for 4 h with bepridil or
with DMSO as a control. Secreted APP and A? were detected in
the conditioned medium. Bepridil inhibited the generation of
APPs? and A? in a dose-dependent manner (Fig. 1B–D). In
contrast to the inhibition of ?-secretase cleavage, APPs? was
largely unaffected by bepridil treatment. The amount of full-
altered. Similar results for the inhibition of APPs? generation
were obtained for neuroglioma H4 and astroglioma U373 cells
stably expressing APP and for the endogenous APPs? derived
from HEK293 cells (supplemental Fig. 2, available at www.
jneurosci.org as supplemental material). The IC50for the inhibi-
tion of APPs? and A? generation was ?6 ?M (Fig. 1C,D), which
is in the concentration range of bepridil found in the plasma of
ity was observed below 50 ?M, as determined in a lactate dehy-
drogenase release assay (supplemental Fig. 3, available at www.
jneurosci.org as supplemental material). This is in line with the
fact that bepridil is used for chronic treatment. The inhibition of
pridil was not able to block the activity of the ?-secretase BACE1
in an established in vitro assay (Fig. 1E), which uses soluble
BACE1 and measures the cleavage of a fluorophor-tagged APP-
derived substrate peptide (Capell et al., 2002). In contrast, a
known active site inhibitor of BACE1, GL-189 (Capell et al.,
2002), fully blocked BACE1 activity in this assay (Fig. 1E). The
indirect inhibition of ?-secretase cleavage is in agreement with
the proposed alkalinization of the endosomal pH being the
mechanism of ?-secretase inhibition. Amiodarone inhibited
?-secretase cleavage in a similar manner as bepridil with an IC50
of ? 30 ?M (Fig. 1F,G). The therapeutically achievable plasma
concentration of amiodarone is 1.5–6 ?M (Freedman and Som-
berg, 1991). At this concentration, ?-secretase cleavage was in-
hibited by 10–20% (Fig. 1F,G). From the data above, we
odarone and bepridil are novel inhibitors of ?-secretase cleavage
of APP and of A? generation.
brain slice cultures, and in vivo in the plasma of guinea pigs
Next, we tested whether the inhibition of ?-secretase cleavage
neurons were prepared from Tg2576 mice, which are an estab-
lished mouse model of AD pathology and overexpress the Swedish
8976 • J.Neurosci.,June30,2010 • 30(26):8974–8983Mitterreiteretal.•DualModulationofAPPProcessing
and A? generation in the primary neurons in a dose-dependent
manner and with similar IC50values as in the cell lines (Fig.
organotypic brain slice cultures of Tg2576 mice. Also in this set-
ting, bepridil reduced A? generation in a dose-dependent man-
ner (Fig. 2C), demonstrating that the
inhibitory effect is not only seen in cul-
tured cells ex vivo, but also directly in the
brain tissue. Bepridil lowered A? produc-
tion to a similar extent as the well charac-
terized ?-secretase inhibitor DAPT (Fig.
2C) (Dovey et al., 2001). Compared with
cell lines and primary neurons, higher
concentrations of bepridil and DAPT
were required for inhibition in brain
slices. Presumably, both compounds do
at lower concentrations or are partly
bound by lipids and proteins in the slice.
At these concentrations, no toxicity was
were not significantly changed in the ly-
sates prepared from the slices (data not
To test whether bepridil also reduces
A? levels in vivo, guinea pigs were used.
Compared with mice, guinea pigs have a
larger body weight and blood volume,
which allows one to draw blood before
and after bepridil treatment and to de-
termine A? changes within one animal.
Guinea pigs were treated with bepridil (50
ing concentration of ?20 ?M in plasma
(Shiotani et al., 2005), or with the
nous A?40 in the plasma was measured by
ELISA. Plasma levels of endogenous A?38
and A?42 were below the detection limit.
group and three animals in the control
trol levels at 28 h after treatment (data not
Next, we examined a larger number of
guinea pigs (n ? 8) at the 4 h time point.
Twenty hours before treatment as well as
4 h after compound treatment, blood was
drawn from all animals and analyzed for
A?40. Bepridil reduced plasma levels of
A?40 by ?50% compared with control-
treated animals (Fig. 2D). A similar re-
duction of A?40 was observed for the
positive control DAPT (Fig. 2D).
onstrate that bepridil is able to block
?-secretase cleavage and lower A? gener-
ation in primary neurons, in organotypic
brain slice cultures, and in vivo in guinea
the pH in the acidic compartments of the endocytic pathway in
protonatable amino group is marked (arrow). The second amino group in bepridil cannot be protonated due to a low pKa.
APPs? were detected in the conditioned medium using antibodies 192wt and W02, respectively. A? was detected using the
experiments. E, The effect of bepridil was analyzed in an in vitro BACE1 activity assay using a fluorogenic APP-derived peptide
substrate. Bepridil did not inhibit BACE1 activity, whereas the BACE1 inhibitor GL-189 completely blocked the activity of the
Mitterreiteretal.•DualModulationofAPPProcessingJ.Neurosci.,June30,2010 • 30(26):8974–8983 • 8977
living cells, where ?-secretase cleavage
takes place (Rossner et al., 2006). To this
aim, HEK293-APP cells were loaded with
pH sensor, whereas tetramethylrhodam-
ine serves as a loading control and allows
dextran-fluorescein molecule is taken up
into cells by endocytosis and labels endo-
pridil led to a concentration-dependent
increase of the pH from 5.1 to 5.5 in the
endolysosomal pathway (Table 1). This
pH increase is in agreement with the ob-
served inhibition of BACE1, because
BACE1 has a narrow pH optimum at pH
4.5–4.8, and its activity can be inhibited
2008). Amiodarone had an effect on the
Table 1, available at www.jneurosci.org as
supplemental material). Together, these
odarone mildly alkalinize endosomal and
lysosomal compartments of living cells.
positively charged, basic amino group lo-
calizes closely to the membrane surface
where it can repel protons, leadingtoalo-
cal pH increase. This suggests that bepridil
marily in the membrane-proximal zone,
where the ?-secretase cleavage site in the APP sequence is lo-
cated, without affecting the bulk pH. Direct experimental proof
occurring in the bulk of the endolysosomal lumen versus those
that are restricted to the membrane-proximal area. To test for
possible pH changesinthemembrane-proximalarea,aninvitro
assay was used. A lipid-linked pH sensor was incorporated into
lipid vesicles prepared from egg phosphatidylcholine (for a
schematic drawing,seesupplementalFig. 4, available at www.j-
neurosci.org as supplemental material). The sensor consists of
the pH-sensitive fluorophor fluorescein coupled to phosphati-
dylethanolamine. The unprotonated form of fluorescein, which
is present under basic conditions, shows a higher fluorescence
than the protonated form, which is predominant under acidic
conditions. This system has been successfully used in the past for
the measurement of changes in surface potential and pH in the
membrane-proximal zone (Teissie et al., 1985; Simard et al.,
2008). Bepridil and amiodarone were used at concentrations of
10 and 20 ?M, which corresponds to the concentrations at which
and amiodarone increased the fluorescence of fluorescein in a
dose-dependent manner compared with control (Fig. 3A). The
increase in fluorescence was equivalent to an increase of the
membrane-proximal pH by 0.4 or 0.6 U for 20 ?M bepridil or
amiodarone, respectively, as determined by a pH titration curve
(supplemental Fig. 5, available at www.jneurosci.org as supple-
mental material). This increase is similar to the change observed
in the cellular system above (Table 1). Importantly, the com-
pounds did not affect the pH of the bulk solution, as measured
with a pH electrode. As a control for the assay, the lipid
1-tetradecylamine was used, which caused an increase in the
membrane-proximal pH, as expected for this amino group con-
ylic acid group instead of the amino group of amiodarone (Fig.
3C). AMI-AcOH lowered the membrane-proximal pH (Fig. 3B).
As expected from these data, AMI-AcOH did not block
?-secretase cleavage (Fig. 3D), which further supports that the
from Tg2576 mice were treated with vehicle control (con); with 20, 50, or 100 ?M bepridil (B20, B50, B100); or with 25 ?M
Bepridil inhibits A? generation in primary neurons, organotypic brain slice cultures, and in guinea pigs. A and B,
8978 • J.Neurosci.,June30,2010 • 30(26):8974–8983 Mitterreiteretal.•DualModulationofAPPProcessing
amino group and the alkalizing activity are required for blocking
Bepridil and amiodarone are known calcium channel blockers
and calmodulin (CaM) antagonists, but their mechanism of ac-
cleavage is indeed due to the compounds’ alkalizing activity, it
should be independent of calcium channel inhibition and CaM.
Indeed, the specific calcium channel blocker nimodipine, which
is unrelated to bepridil and amiodarone, did not affect APP pro-
cessing at concentrations where calcium channels in HEK293
cells are potently blocked (supplemental Fig. 6A, available at
www.jneurosci.org as supplemental material) (Berjukow et al.,
1996), which is in agreement with a previous publication (Fac-
lular calcium levels as determined in HEK293-APP cells loaded
with the calcium-indicator dye fura 2 (supplemental Fig. 6B, avail-
cells (supplemental Fig. 6C, available at www.jneurosci.org as sup-
plemental material). Together, these experiments exclude that
Generation of A? requires the action of both?-and?-secretase.
Because ?-secretase has multiple cleavage sites, A? species of dif-
A?40 and, to a lower extent, A?38 and
A?42. Some small molecule drugs, called
?-secretase modulators (GSM), shift the
?-secretase cleavage site. Straight GSMs
(sGSMs) lower A?42 and raise A?38
(Weggen et al., 2001), whereas inverse
GSMs (iGSM) do the opposite (Kukar et
al., 2005). To investigate whether bepri-
dil—in addition to its inhibitory effect on
?-secretase cleavage—modulates ?-secretase
cleavage, C99-expressing HEK293 cells were
used. C99 is the C-terminal APP fragment
lows monitoring ?-cleavage independently
of ?-cleavage. Total levels of A? as well as
levels of A?38, A?40, and A?42 were mea-
by approximately twofold and reduced
A?38 to ?50%, whereas levels of A?40
and total A? remained unchanged (Fig.
in vitro ?-secretase assay, where purified
?-secretase, reconstituted into lipid vesi-
cles together with C99 as a substrate, was
incubated with bepridil (Fig. 4C). Gener-
ation of the APP intracellular domain
(AICD), which is the second ?-secretase
cleavage product, was not affected in the
in vitro assay (supplemental Fig. 7, avail-
able at www.jneurosci.org as supplemen-
tal material), revealing that bepridil
does not inhibit ?-secretase activity, but
instead modulates the cleavage site of ?-secretase and the A?
species being generated. Together, these experiments demon-
strate that bepridil acts as an iGSM.
A? species in a sandwich immunoassay, HEK293-APPswe cells
cells. In contrast to C99-expressing cells, bepridil reduced total
A? levels (Fig. 4D) due to an inhibition of ?-secretase cleavage,
of the main A? species A?40 was inhibited to a similar extent as
total A?. However, A?38 was even more strongly inhibited,
whereas A?42 was only mildly affected by bepridil (Fig. 4D).
Thus, A?42 levels were highest, followed by A?40 and A?38,
cells (Fig. 4A,B). Similar results were observed for bepridil in
primary neurons obtained from Tg2576 mice (Fig. 4E). An even
stronger iGSM effect than for bepridil was observed for amioda-
rone. This compound strongly increased A?42, mildly reduced
(Fig. 4F). Compared with bepridil, the milder effect of amioda-
IC50value for ?-secretase inhibition. Together, the results dem-
onstrate that bepridil and amiodarone act as dual modulators.
They inhibit ?-secretase cleavage of APP and simultaneously
modulate ?-secretase cleavage.
To determine whether the GSM effect—similar to the inhibi-
tion of ?-secretase cleavage—requires the amino group, we
tested the effect of the amiodarone derivative AMI-AcOH on the
control 1-tda caused an increase in the fluorescence intensity of FPE, showing that they alkalinize the pH in the membrane-
proximal zone. In contrast, AMI-AcOH caused a decrease in the membrane-proximal pH. rel., Relative; a.u., arbitrary unit.
Bepridil and amiodarone raise the membrane-proximal pH. A and B, Measurements of the fluorescence of FPE
Mitterreiteretal.•DualModulationofAPPProcessingJ.Neurosci.,June30,2010 • 30(26):8974–8983 • 8979
group and is not able to raise the
membrane-proximal pH (Fig. 3B,C).
cleavage (Fig. 4G). This reveals that the
amino group is not required for GSM ac-
tivity, which is in agreement with the ob-
was also observed in the ?-secretase in
vitro assay (Fig. 4C). Surprisingly, AMI-
AcOH lowered A?42 and raised A?38
(Fig. 4G) and thus had the opposite GSM
effect (sGSM) compared with amioda-
ture of other known sGSMs and iGSMs
(Weggen et al., 2001; Kukar et al., 2005),
we observed that both classes have a hy-
additionally have a free carboxylic acid
group as it is found in AMI-AcOH (Fig.
3C). This suggests that the carboxylic acid
iGSM or a sGSM and thus defines a struc-
The proteases ?- and ?-secretase directly
generate the A? peptide and are consid-
ered main drug targets for Alzheimer’s
disease. However, clinically suitable in-
hibitors for both proteases have been dif-
ficult to find. Our study identifies and
mechanistically characterizes the two
clinically used compounds bepridil and
amiodarone, which we found to act as
inhibit ?-secretase cleavage and simulta-
neously modulate ?-secretase cleavage.
The mechanistic analysis shows that both
effects occur through distinct molecular
groups of the compounds. This provides a
?-Secretase inhibitors need to reach
the endosome where ?-secretase cleavage
of APP mostly takes place. For example, a
recent study showed that membrane-
anchoring of a BACE1 inhibitor allowed
its endocytosis, increased its local mem-
brane concentration in the endosome,
and inhibited BACE1 activity more effi-
ciently than the free inhibitor (Rajendran
et al., 2008). Here, we report that inhibi-
tion of ?-secretase cleavage can also be
pounds mildly raise the pH in acidic compartments of living cells
and increase the membrane-proximal pH in an in vitro membrane
assay. Interestingly, bepridil and amiodarone recently have been
shown to also block another pH-sensitive membrane-proximal en-
dosomal process—the insertion of anthrax toxin into endosomal
membranes—by raising the endosomal pH (Sanchez et al., 2007).
in guinea pigs occurred at concentrations of bepridil and amioda-
rone that are therapeutically achievable in the plasma of patients.
mice were incubated as in D with bepridil, which resulted in a strong reduction of A? 38, a milder reduction of A?40, and an
8980 • J.Neurosci.,June30,2010 • 30(26):8974–8983Mitterreiteretal.•DualModulationofAPPProcessing
Whether both compounds also block ?-secretase cleavage in the
is used for the treatment of the bacterial infection chronic Q fever,
Weak bases, such as bepridil and amiodarone, become con-
centrated in acidic organelles, where they are protonated and
retained. This principle may also help in the development of
active site-directed BACE1 inhibitors. One such compound was
found to have a higher potency in cells when it contained an
additional amino group (Yang et al., 2006). Although the reason
for the amino group requirement is not fully understood, it is
possible that the amino group targets the inhibitor to the endo-
some, resulting in higher local inhibitor concentrations at the
site of ?-secretase cleavage and a more efficient inhibition of
?-secretase. Potentially, such inhibitors also raise the endosomal
pH and thereby additionally inhibit ?-secretase cleavage.
Another outcome of our study is that amiodarone and bepri-
dil raised the membrane-proximal pH in the membrane assay,
endosomes in living cells. Direct experimental proof is difficult,
because the available technology to determine the pH in the en-
dolysosomal system in living cells, including the one used in this
study, does not allow to distinguish between pH changes occur-
ring in the bulk of the endosomal lumen versus those that are
restricted to the membrane-proximal area. However, the com-
pounds’ chemical properties fit well with an effect primarily on
the membrane-proximal pH. By inserting into the membrane,
amiodarone and bepridil repel protons from the membrane sur-
face and thereby raise the membrane-proximal pH. This puts
BACE1 in an environment where the pH is no longer favorable
for its activity. This localized area of action of bepridil and ami-
odarone may be helpful when the use of such compounds is
considered for therapeutic purposes, as other processes in the
place in the lumen of late endosomes, is not affected by amioda-
rone (Stadler et al., 2008).
Surprisingly, we found that amiodarone and bepridil—in ad-
dition to their inhibitory effect on ?-secretase cleavage—addi-
tionally modulate ?-secretase in an inverse manner (iGSM).
A?40, and only a minor reduction of A?42. The compounds’
modulatory effect on ?-secretase seems to be independent of
their alkalizing ability, because the effect also occurred in the
cell-free ?-secretase assay. Moreover, the basic amino group was
not required for the modulation of ?-secretase, since AMI-
specificity of ?-secretase. Additionally, other alkalizing com-
pounds, such as ammonium chloride, which inhibit ?-secretase
cleavage, do not affect the cleavage specificity of ?-secretase
(Vingtdeux et al., 2007). Furthermore, other compounds with
GSM activity, such as fenofibrate and flurbiprofen, do not con-
tain amino groups (Kukar et al., 2008). Thus, the dual modula-
The first GSMs were identified in 2001 (Weggen et al., 2001).
Proposed mechanisms of action include a direct binding to C99
2003) and a change in the dimerization strength of C99 (Munter
pounds. When comparing the structure of published GSMs, we
found that sGSMs contain a free carboxylic acid group, which is
absent in iGSMs. In line with this observation, we found that the
addition of a carboxyl group to amiodarone (AMI-AcOH) was
able to invert the specificity to ?-secretase. A similar result was
observed in a recent study, which showed that a hydrophobic
compound with iGSM activity could be converted to a sGSM by
the addition of a free carboxylic acid group (Narlawar et al.,
2007). Thus, the addition of a free acidic group may be a more
generally applicable strategy to convert an iGSM to an sGSM.
Mechanistically, it is not yet clear why the carboxyl group is
required for the sGSM effect. ?-Secretase seems to cleave first at
the C-terminal end of the transmembrane domain of C99 and
membrane domain, until the A? peptide is short enough to slip
charged free carboxylic acid of sGSMs binds and neutralizes
the positively charged lysine 28, which forms the luminal,
membrane-anchoring residue in the C99 sequence (Kukar et al.,
2008). This neutralization may allow C99 to insert more deeply
into the membrane. As a consequence, ?-secretase may proceed
up to more N-terminally located residues, resulting in more
A?38 and less A?42. Such a mechanism is in agreement with
the length of the C99 transmembrane domain has a profound
effect on the cleavage specificity of ?-secretase (Murphy et al.,
1999; Lichtenthaler et al., 2002). Based on our results, we con-
clude that the hydrophobic core of amiodarone and bepridil is
sufficient for modulation of ?-secretase and that the presence or
group—determines the sGSM or iGSM activity, respectively.
Together, our work serves as a proof-of-principle showing
that it is possible to identify dual modulators, which simulta-
neously target ?- and ?-secretase cleavage of APP. This indicates
that drugs developed against one of the two enzymes should al-
ways be inspected for potential additional effects on the other
rone and bepridil occurs at concentrations that are therapeuti-
cally reached in the plasma of patients, both compounds may be
four different classes of compounds. First, modification of the
hydrophobic core may yield drugs that lack GSM activity and
vides a new structural scaffold for sGSMs, which do not affect
?-secretase. The development of new sGSMs is needed because a
previously identified sGSM failed in a recent phase III clinical
ulators may be generated that simultaneously inhibit ?-secretase
cleavage and act as sGSMs on ?-secretase. An ideal dual modula-
tor should comprise of the following three structural elements:
(1) a hydrophobic core with aromatic rings for membrane-
targeting, (2) an acidic group to allow for sGSM activity, and (3)
itory effect on ?-secretase by raising the membrane-proximal
endosomal pH. Such compounds would allow the lowering of
total A? levels and the additional specific lowering of A?42,
which should result in an enhanced total reduction of A?42
levels. Fourth, given the accumulation of weak bases in acidic
organelles, we consider that the addition of an amino group
may be used as a more general strategy for endosomal and
Mitterreiteretal.•DualModulationofAPPProcessing J.Neurosci.,June30,2010 • 30(26):8974–8983 • 8981
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