BACE1- and BACE2-expressing Human Cells
CHARACTERIZATION OF
-AMYLOID PRECURSOR PROTEIN-DERIVED CATABOLITES, DESIGN OF A NOVEL FLUORIMETRIC ASSAY, AND IDENTIFICATION OF NEW IN VITRO INHIBITORS*
David Andrau
,
Cécile Dumanchin-Njock
,
Erwan Ayral ¶ ||,
Jean Vizzavona ||,
Mike Farzan **,
Michel Boisbrun ¶,
Pierre Fulcrand ¶,
Jean-François Hernandez ¶,
Jean Martinez ¶,
Solveig Lefranc-Jullien

and
Frédéric Checler

From the
Institut de Pharmacologie
Moléculaire et Cellulaire of Centre National de la Recherche
Scientifique, UMR6097, 06560 Valbonne, France, the
¶Laboratoire des Amino Acides, Peptides et
Protéines, CNRS UMR5810, 34093 Montpellier, France, and
**Harvard Medical School, Boston, Massachusetts
02115
Received for publication, March 14, 2003
, and in revised form, April 14, 2003.
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ABSTRACT
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We have set up stably transfected HEK293 cells overexpressing the
-secretases BACE1 and BACE2 either alone or in combination with
wild-type
-amyloid precursor protein (
APP). The characterization
of the
APP-derived catabolites indicates that cells expressing BACEs
produce less genuine A
1 40/42 but higher amounts of secreted
sAPP
and N-terminal-truncated A
species. This was accompanied by a
concomitant modulation of the C-terminal counterpart products C89 and C79 for
BACE1 and BACE2, respectively. These cells were used to set up a novel BACE
assay based on two quenched fluorimetric substrates mimicking the wild-type
(JMV2235) and Swedish-mutated (JMV2236)
APP sequences targeted by BACE
activities. We show that BACEs activities are enhanced by the Swedish mutation
and maximal at pH 4.5. The specificity of this double assay for genuine
-secretase activity was demonstrated by means of cathepsin D, a
"false positive" BACE candidate. Thus, cathepsin D was unable to
cleave preferentially the JMV2236-mutated substrate. The selectivity of the
assay was also emphasized by the lack of JMV cleavage triggered by other
"secretases" candidates such as ADAM10 (A
disintegrin and metalloprotease 10), tumor
necrosis
-converting enzyme, and presenilins 1 and 2. Finally, the
assay was used to screen for putative in vitro BACE inhibitors. We
identified a series of statine-derived sequences that dose-dependently
inhibited BACE1 and BACE2 activities with IC50 in the micromolar
range, some of which displaying selectivity for either BACE1 or BACE2.
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INTRODUCTION
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Alzheimer's disease is characterized by the cortical and sub-cortical
accumulation of proteinaceous deposits called senile plaques
(1). The main constituent of
these aggregates is referred to as amyloid
peptide
(A
).1 A
is
generated from the
amyloid precursor protein (
APP) by the
subsequent attacks by
- and
-secretases, which liberate the N-
and C-terminal moieties, respectively
(2). It is now clear that under
this generic terminology, "A
" gathers a series of distinct
"A
-related species," some of them truncated at their
-secretase-derived N terminus
(3). Although the nature of the
-secretase is still discussed
(4,
5), the identity of the
-secretase candidate is more consensual.
BACE1 (
-site APP-cleaving enzyme) is an aspartyl protease recently
identified by several groups
(69),
which displays all the properties expected from a genuine
-secretase
candidate (for review, see Refs.
10 and
11). Thus, BACE1 is an acidic
protease mainly localized in the Golgi apparatus and in endo-somal
compartments and exhibits a lumenal active site fitting well with the lumenal
cleavage of
APP occurring in these acidic compartments where A
had
been detected. The overexpression of BACE1 leads to increased recovery of
A
-related fragments, mainly 1 40 and 11 40 that are also
observed when recombinant BACE is incubated with
APP
(7,
9). The opposite phenotype is
observed when BACE is down-regulated by an antisense approach
(7,
9). BACE2 is a parent protease
(12) that seems to contribute
poorly to neuronal A
production. However, BACE2 could be important in
Down syndrome pathology because the enzyme is encoded by chromosome 21
(13,
14) and elevated BACE2
expression is observed in trisomic brains
(15).
BACE1 not only behaves as a
-secretase, in vitro, but likely
corresponds to the main contributor of the
-secretase pathway, in
vivo. Thus, it was evidenced that the invalidation of the BACE
gene led to abolishment of A
production in knockout mice
(16) and neurons
(17). That this deletion
appeared totally innocuous
(18) makes BACE likely the
most interesting target of an A
-directed AD therapy. Here we fully
characterize the
APP-derived catabolites generated by BACE1 and
BACE2-expressing cells. Furthermore, we describe a novel fluorimetric assay
and new in vitro inhibitors of potential interest to further
characterize BACE1 and BACE2 properties.
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EXPERIMENTAL PROCEDURES
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Chemical ReagentsFmoc-amino acids, HBTU, and the resin were
purchased from Senn Chemicals (Gentilly, France). The reagents and solvents
for solid-phase peptide synthesis were obtained from Acros (Noisy-le-Grand,
France) or from SDS (Peypin, France). All other chemicals were of the purest
grade available.
Synthesis of Peptides and InhibitorsThe intramolecularly
quenched fluorogenic peptidic substrates Abz-Val-Lys-Met-Asp-Ala-Glu-EDDnp
(JMV2235) and Abz-Val-Asn-Leu-Asp-Ala-Glu-EDDnp (JMV2236) contain the
ortho-aminobenzoyl (Abz)/dinitrophenyl groups as the donor/acceptor
pair (19). They were
synthesized essentially by solid phase methods
(20), starting from the
C-terminal residue Glu-EDDnp. The latter was first assembled in solution by
coupling Fmoc-Glu(
-tBu)-OH to EDDnp using HBTU as the coupling agent,
in the presence of diisopropylethylamine. The resulting
Fmoc-Glu(
-tBu)-EDDnp was treated by trifluoroacetic acid to free the
carboxylate group that was subsequently attached to the linker of a
Wang resin by the symmetric anhydride method. After manual assembling of the
substrates using the solid phase Fmoc strategy and HBTU as the coupling agent,
cleavage of the peptides from the resin and simultaneous deprotection of side
chains were carried out by treatment with a solution containing
trifluoroacetic acid, thioanisole, water, phenol (8.5/0.5/0.5/0.5) for 2 h at
room temperature. The final deprotected peptides were purified by
reverse-phase chromatography on a C18 column (Deltapack Waters, 40 x 100
mm) by means of a linear gradient of 20 35% acetonitrile in 0.1%
aqueous trifluoroacetic acid over 30 min (flow rate 50 ml/min), and their
purity and identity were assessed by reverse-phase HPLC and electrospray mass
spectrometry (Abz-VKMDAE-EDDnp: experimental mass 1018.7 ± 0.2,
calculated mass 1018.4; Abz-VNLDAE-EDDnp: experimental mass 986.6 ±
0.2, calculated mass 986.4).
All peptidic inhibitors were amidated at their C-terminal end and blocked
at their N terminus, either by an acetyl or by a Boc group (excepted the
Qui-containing compounds), to protect them from exopeptidasic attacks. The
peptide backbone of the compounds was stepwise assembled by classical methods,
using Boc as the
-amino protecting group and benzotriazol-1-yloxy
tris(dimethylamino)phosphonium hexafluorophosphate as the coupling reagent,
either in homogeneous phase or on solid phase with methylbenzhydrylamine
resin, necessitating a final HF cleavage procedure. Published protocols were
followed for the formation of the peptide bond isostere moieties, reduced
amide bond (Leu-
(CHsNH)-Asp in
JMV963) (21,
22), norstatine
(Norsta-containing compounds)
(23,
24), and statine and analogs
(Sta-, AHPPA-, ACHPA-containing compounds)
(25,
26). All synthetic inhibitors
were purified on C18 reverse-phase HPLC, and their purity and identity were
assessed by reverse-phase HPLC and electrospray mass spectrometry.
HEK293 Culture and Stable TransfectionHEK293 and stably
transfected HEK293 cells overexpressing wt
APP751 (WT) and
sw-
APP751 (SW) (27) were
stably transfected with DAC30 (according to the manufacturer's instruction
Eurogentec) containing 2 µg of pcDNA3 vector encoding either 1D4-tagged
BACE1 (WTB1) or BACE2 (WTB2) and zeocin resistance. 1D4 is a 10-amino acid
C-terminal tag derived from bovine rhodopsin
(28). Medium was replaced 48 h
after transfection with selective medium containing neomycin and/or zeonin (1
g/liter). Transfectants were screened by 10% SDS-PAGE gels analysis and
Western blotting (see below). Positive clones overexpress 75- and 50-kDa
immunoreactive proteins, respectively, corresponding to BACE1 and BACE2
(28). Tumor necrosis
-converting enzyme (TACE), A disintegrin
and metalloprotease 10 (ADAM10), and presenilin 1 and
presenilin 2-expressing cells were previously described
(2931).
Western Blot AnalysesHEK293 cells were scraped and lysed in
RIPA 1x buffer (10 mM Tris, pH 7.5, containing 150
mM NaCl, 5 mM EDTA, 0.1% deoxycholate, and 1% Nonidet
P-40). Equal quantity of proteins were separated on a 10% SDS-PAGE gel for the
detection of BACE1 and BACE2 proteins and transferred to Hybond-C (Amersham
Biosciences) membranes. After transfer, membranes were blocked with 5% nonfat
milk and incubated overnight with the primary antibody anti-1D4 (1:1000)
(mouse monoclonal antibody donated by Scott Waniger at the National Cell
Culture Center, National Cell Culture Center, Minneapolis, MN). Immunological
complexes were revealed with an anti-mouse peroxidase (Amersham Biosciences)
antibody followed by enhanced chemiluminescence (Amersham Biosciences). All
protein concentrations were determined by the Bradford
(32) procedure as
described.
Measurements of Total A
Stably transfected
WT, WTB1, WTB2, and SW HEK293 cells (see above) were incubated for7hinthe
presence of phosphoramidon (1 µM) (Sigma). Media were collected,
diluted in 1/10 RIPA 10x buffer (10 mM Tris, pH 7.5,
containing 150 mM NaCl, 5 mM EDTA, 0.1% SDS, and 1%
Nonidet P-40), and incubated overnight with a 200-fold dilution of FCA18
(total A
) as described
(33). After further incubation
for 3 h with protein A-Sepharose (Zymed Laboratories Inc.) and centrifugation,
pelleted proteins were submitted to 16.5% Tris/Tricine gels and then Western
blotted on Hybond C membranes (Amersham Biosciences) for 45 min.
Nitrocellulose sheets were heated in boiling phosphate-buffered saline for 5
min and capped with 5% skim milk in phosphate-buffered saline containing 0.05%
Tween 20 for 1 h. Membranes were then incubated overnight with WO2 antibody
(ABETA GmbH, Heidelberg, Germany) specific for the 5 8 N-terminal
region of A
(34)) and
detected by enhanced chemiluminescence.
Measurements of sAPP
, sAPP
, and
C-terminal ProductsWT, WTB1, and WTB2 cells were allowed to
secrete for 7 h as above and then 15 (sAPP
/
'2) or 50 µl
(sAPP
1/2) of secretate were loaded on 8% SDS-PAGE and Western blotted
with 10D5C or WT192 (kind gift from Dr. D. Schenk, Elan Pharmaceuticals),
respectively. Immunological complexes were revealed with the adequate IgG
coupled to peroxidase. C-terminal products were analyzed from the same cell
lysates and were separated on large Tris/Tricine 16.5% gels, Western blotted,
and probed with BR188 as described
(35).
Fluorimetric AssayDifferent cell types were lysed, in 10
mM Tris, pH 7, and then various amounts of total homogenate
proteins were preincubated for 10 min with a commercial
-secretase
inhibitor (KTEEISEVN-(Sta)-VAEF-OH, Enzyme System Products,
Livermore, CA) or with distinct JMV inhibitors (10 µM) in
96-well plates. The JMV2235, JMV2236, or a
-secretase commercial
substrate (Mca-SEVNLDAEFRK-(dinitrophenyl)-RR-NH2, R
& D Systems) were then added (10 µM) and incubated for
various times at 37 °C. At the end of the incubation, fluorescence was
recorded at 320 and 420 nm as excitation and emission wavelengths,
respectively. When the effect of pH was monitored, lysed cells were pelleted,
then resuspended in 25 mM Na+-acetate/MES/Tris/TES
adjusted at pH values ranging from 2 to 10 and then incubations were performed
as above. Controls include membrane proteins or substrate alone and background
fluorescence was subtracted to recorded BACE activities. This fluorescence was
very low and did not change with the time of incubation at 37 °C.
Cathepsin D activity (Sigma, 0.5 µg) was assayed as above with JMV2235,
JMV2336, or with the commercial substrate.
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RESULTS
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Characterization of Secreted and Intracellular
APP-derived Products Generated by BACE1 and BACE2-expressing Human
CellsWe have set up stably transfected HEK293 human cells
overexpressing 1D4-tagged BACE1 and BACE2 with expected molecular weights
(Fig. 1A), either
alone (called B1 and B2) or in combination with wild-type
APP (referred
to as WTB1 and WTB2). We have used WT192 monoclonal antibodies that recognize
the two last amino acids of the secreted products derived from the canonical
-secretase cleavages taking place at the N terminus of the Asp-1 residue
of A
(see
1 and
2 sites in
Fig. 1, E and
F). sAPP
1 and sAPP
2 generated by BACE1 and
BACE2 were identical (Fig. 1, E
and F) and theoretically both were recognized by WT192.
As expected, secreted WT192-immunoreactive products were recovered in higher
amounts in both BACE1- and BACE2-expressing cells than in WT cells
(Fig. 1B). Additional
'1- and
'2-derived cleavages targeted by BACE1 and
BACE2, respectively, occur inside the A
sequence and theoretically
liberate sAPP
'1- and sAPP
'2-secreted fragments (see
Fig. 1, E and
F). sAPP
'2, as well as sAPP
, the
physiological
-secretase-derived APP fragment, retain a 10D5C epitope
that was disrupted by
'1 cleavage (see
Fig. 1, E and
F). As expected, 10D5C-positive fragments secreted by WT
and WTB2 cells (corresponding to sAPP
/
'2) were recovered in
much higher amounts than with WTB1 cells
(Fig. 1C).
The C-terminal counterparts of the above products were probed in cell
lysates using BR188, a polyclonal antibody that interacts with the C terminus
of all fragments (Fig. 1,
EG). As expected,
1- and
2-derived
cleavages (Fig. 1, E and
F) increase C99 in WTB1 and WTB2
(Fig. 1D), in
agreement with higher production of their N-terminal counterpart sAPP
1/2
(Fig. 1B). The nature
of C99 was confirmed by means of FCA18
(36), a polyclonal antibody
that specifically interacts with the free Asp-1 residue of A
and C99
(not shown). A fragment of lower molecular weight was specifically recovered
with WTB1 cells (Fig.
1D). Although not definitively identified, this product
likely corresponds to C89, the
'1-derived specific product
generated by WTB1 cells (Fig. 1, D
and E). Finally, a low molecular weight product present
in BACE2 but not BACE1 cells could derive from the
'2 cleavage
(Fig. 1F). This
product tentatively ascribed to C79 co-migrates with the C83 major product
generated by
-secretase in WT cells
(Fig. 1, D and
G). C89- and C79-like products were FCA18-negative (not
shown), confirming that the fragments lack the intact N terminus present in
C99.
Characterization of Genuine A
in BACE1- and
BACE2-expressing HEK293 CellsThe use of FCA18 was proved useful to
monitor secreted genuine A
. Thus, this antibody only recognizes free
Asp-1 residue of A
or C99 (Fig.
2) because acetylation or removal of this aspartyl residue
abolishes recognition by FCA18
(36). Therefore, FCA18 does
not label N-terminal truncated A
-related species. As expected, the
overexpression of wild-type
APP increases A
1 40/42 (compare
mock and WT in Fig. 2).
Interestingly, as previously described
(27,
3739)
the introduction of the Swedish mutation triggers increased A
secretion
(compare WT and SW). Both WTB1 and WTB2 cells secrete less A
than WT
cells (Fig. 2), indicating that
the
'1 and
'2 cleavages are likely more efficient than
the
1 and
2 breakdowns elicited by BACE1 and BACE2 (see
Fig. 2), respectively. It is
noteworthy that while "complete" A
is still detectable in
BACE1-expressing cells, it was not recovered in WTB2. The fact that sAPP
was augmented in BACE2 (Fig.
1B) suggests that the
2 cleavage before Asp-1 of
A
sequence indeed occurs. However, the relatively faint augmentation of
the C-terminal counterpart C99 and the accumulation of C79
(Fig. 1D) suggests
that BACE2-elicited
'2 cleavage was particularly efficient and
could take place using C99 as a substrate, thereby explaining the low recovery
of "full-length A
" observed with these cells
(Fig. 2).
Design of a Novel BACE Fluorimetric AssaySeveral studies
have indicated that the Swedish mutation responsible for a familial form of
Alzheimer's disease leads to increased production of A
via an
exacerbation of the
-secretase-derived cleavage
(3739).
We have set up a new assay based on the cleavage of quenched fluorimetric
substrates mimicking the wild-type (JMV2235) and Swedish-mutated (JMV2236)
sequences (Fig. 3) targeted by
-secretase (s) with the assumption that a good assay should be reflected
by a favored hydrolysis of mutated JMV2236.
At acidic pH, B1 and B2 cell extracts (see
Fig. 1) hydrolyze both JMV2235
and JMV2236 in a time- and dose-dependent manner
(Fig. 4). It should be noted
that kinetic analyses indicate that activities recovered with JMV2236
plateaued at the same fluorescence value
(Fig. 4). This could be because
of either depletion of available substrate or, alternatively, to an inhibitory
effect by one of the products of the reaction. The latter hypothesis was ruled
out by the fact that the rates of hydrolyzes of JMV2235 by B1 and B2 cells
were not affected by 100 µM Bz-VNL, Bz-VKL,
Bz-VNL-NH2, and Bz-VKL-NH2. These N-terminal degradation
products lack the N-terminal fluorescent moiety and were either free or
amidated at the C terminus (to prevent it from putative carboxypeptidase
attack (not shown). In agreement with a substrate extinction, standard
fluorescence observed with synthetic Abz-VNL (10 µM) corresponds
to the value observed at plateau when 10 µM substrate is used,
indicating that this fluorescence indeed corresponds to 100% hydrolysis of the
substrate (not shown).

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FIG. 4. Kinetic analyses of JMV2235 and JMV2236 hydrolysis by BACE1- and
BACE2-expressing cells. BACE1- (upper panels) and BACE2-
(lower panels) expressing cells (30 µg of proteins) were assayed
for their JMV2235- (left panels) or JMV2236- (right panels)
hydrolyzing activities for the times indicated at pH 4.5 as described under
"Experimental Procedures." Insets, activities were
measured for 30 min at pH 4.5 with the indicated varying amounts of proteins.
Fluorescence was recorded at 320 and 420 nm as excitation and emission
wavelengths, respectively, as described under "Experimental
Procedures."
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We used a
-secretase commercial inhibitor to further validate our
assay. Interestingly, mock-transfected cells exhibit faint JMV2235- and
JMV2236-hydrolyzing activities that remained insensitive to the inhibitor
(Fig. 5, A and
B), indicating that endogenous
-secretase activity
was low in HEK293 cells. The JMV2235- and JMV2236-hydrolyzing activities of B1
cells returned to the mock-transfected cell value in the presence of the
inhibitor (Fig. 5, A and
B), indicating that most of the fluorescence recorded was
indeed because of BACE1 in B1 cells. This conclusion also stands for
BACE2-expressing cells (Fig.
5C) although the extent of inhibition was slightly lower
(69 versus 97% of inhibition for B2 and B1 cells, respectively, see
Fig. 6B).
Interestingly, in B1 cells, hydrolysis of JMV2236-mutated substrate is twice
as efficient as that observed for the non-mutated analog
(Fig. 5C).

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FIG. 6. Inhibitor-sensitive JMV2236 hydrolysis by BACE1- and BACE2-expressing
cells is pH-dependent. BACE1- (HEKB1) or BACE2- (HEKB2)
expressing cell proteins (30 µg) were incubated with 10 µM
JMV2236 (A) for 30 min at pH 4.5 or 8, in the absence () or
presence (+) of a commercial -secretase inhibitor (CI, 10
µM), then the activity was fluorimetrically recorded as
described under "Experimental Procedures." Bars are the
mean ± S.E. of eight determinations. Specific activities in B
correspond to initial velocity measurements of the JMV2236-hydrolyzing
activity obtained with or without inhibitor. The % of inhibition was
calculated after subtraction of the CI-insensitive activity obtained in
mock-transfected cells (see "Results"). A fluorescence of 700
corresponds to 1 nmol of Abz-VNL-OH liberated.
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Another clue for stating that our novel assay was
-secretase-specific
was the strong pH requirement observed. We have carried out the assay at pH
ranging from 2 to 10 (in 25 mM Na+-acetate/MES/Tris/TES
to avoid any intrinsic influence other than the pH). The activity was sharply
maximal at pH 44.5 for JMV2236-
(Fig. 6A) and JMV2235-
(not shown) hydrolyzing activities in both B1 and B2 cells. It was interesting
to note that at pH 8, very high JMV2236-cleaving activities were displayed by
B1 and B2 calls but that, unlike at pH 4.5, remains totally insensitive to the
-secretase commercial inhibitor (Fig.
6, A and B). This further indicates that our
assay allows selective dosage of
-secretase only at pH relevant for BACE
biological activity.
Comparison of JMV-based Assay with a Commercial
-Secretase Assay by Use of Cathepsin DBACE1 and BACE2
hydrolyze another commercial fluorimetric substrate (CS) with identical pH
(Fig. 7A) and
inhibitor-sensitive (Fig. 7, A and
C) manners. At acidic pH, CS appeared even better cleaved
by B1 and B2 cells than JMV2235 and JMV2236 substrates
(Fig. 7B). We took
advantage of the description of cathepsin D as an in vitro
-secretase-like activity
(40,
41) to further compare the
usefulness and accuracy of these two assays. Interestingly, although purified
cathepsin D potently hydrolyzed CS and JMV2235, this enzyme was unable to
hydrolyze the mutated substrate JMV2236
(Fig. 8, A and
B). This shows that on the basis of the hydrolysis of CS
or JMV2235 only, cathepsin D would have behaved as a good
-secretase
candidate. The dual screening with JMV2235 and -2236 makes it fall in the
category of the
-secretase "false positive" candidates.
These data indicate the drastic improvement brought by our new assay for the
monitoring of putative unknown
-secretase-like candidates and also
confirm the fact that cathepsin D is not
-secretase. The selectivity of
the assay was further emphasized by the inability of ADAM10, TACE, and
presenilins 1 and 2, the
- and
-secretases candidates to cleave
JMV2236 (Fig. 9).

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FIG. 8. Hydrolysis of JMV2235, JMV2236, and the -secretase commercial
substrate by purified cathepsin D. A, purified cathepsin D
activity (0.5 µg) was assayed for the indicated time periods at pH 4.5 with
10 µM JMV2235, JMV2336, or commercial substrate (CS).
In B, bars are the mean ± S.E. of six determinations and
compare the fluorescence generated from the indicated substrates by cathepsin
D and BACE1. A fluorescence of 700 corresponds to 1 nmol/30 min of Abz-VNL-OH
liberated.
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FIG. 9. Hydrolysis of JMV2236 by several "secretase" candidates
reveals selectivity for -secretase. Mock-transfected HEK293 cells or
cells expressing BACE1 (B1), presenilin 1 (PS1), presenilin
2 (PS2), ADAM10 or TACE were obtained as described under
"Experimental Procedures." Thirty µg of proteins were assayed
for 30 min at pH 4.5 (B1 cells) or neutral pH (other cells) with 10
µM JMV2236 and then the activity was fluorimetrically recorded
as described under "Experimental Procedures." Bars are
the mean ± S.E. of eight determinations.
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Novel in Vitro Inhibitors of BACE1 and BACE2The above data
suggested the use of JMV2236 as a good probe to screen for putative in
vitro
-secretase inhibitors. We have studied the putative
inhibitory effect of 26 peptidic sequences modified at the
-secretase
site and harboring various N- and C-terminal lengths
(Table I). Most of the
molecules are displaying a statine-derived group, a non-cleavable residue that
mimics the tetrahedral intermediate of catalysis by aspartyl proteases. The
statine group can be replaced by an AHPPA
((3S,4S)-4-amino-3-hydroxy-5-phenyl-pentanoic acid) moiety
without influencing the inhibitory activity toward both BACE1 and BACE2
(compare JMV1197 and JMV1200 in Table
I and Fig. 10).
JMV1195 and JMV1197 block BACE1 (Fig.
10A) and BACE2 (Fig.
10B) with IC50 values in the micromolar range
(Fig. 10C and
Table II), suggesting that
shortening the N-terminal part of the inhibitor does not significantly alter
its potency. The C-terminal length appears more important because shortening
it by only one amino acid leads to an inactive inhibitor on BACE2 and less
potent agent against BACE1 (compare JMV1196 and JMV1197;
Fig. 10, A and
B). It is noteworthy that this screening led to the
identification of molecules that appear to discriminate between BACE1 and
BACE2. This appears to be the case for JMV1321 which is more potent on BACE2
than on BACE1 whereas the contrary is true for JMV1196
(Fig. 10, A and
B).

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FIG. 10. Effect of new inhibitors toward BACE1 and BACE2. BACE1- (A)
or BACE2- (B) expressing cell proteins (30 µg) were incubated with
JMV2236 (10 µM) for 30 min at pH 4.5 in the presence of the
indicated JMV inhibitors (10 µM) and then the activity was
fluorimetrically recorded as described under "Experimental
Procedures." Bars are the mean ± S.E. of four
determinations. In C, a complete dose-response curve of the indicated
JMV inhibitors obtained with BACE2-expressing cells is shown.
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 |
DISCUSSION
|
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Although the etiology of Alzheimer's disease is not formally known, it is
difficult to consider the overproduction of the amyloid
peptide
(A
) as an innocuous and inert event in the progression of the disease.
Causative or not, A
is the most obvious biochemical common denominator
between sporadic and familial forms of this disease. Thus, the mutations
triggering the genetic forms of Alzheimer all lead to an acceleration of the
disease progression that, if not directly because of a modulation of A
production, appears at least linked to it (for reviews, see Ref.
4245).
This so called "amyloid cascade" hypothesis
(46) implies that the
secretases that generate A
could be considered as major targets of
therapeutic strategies aimed at slowing down the onset and progression of the
disease.
A
is generated from a transmembrane precursor, the
-amyloid
precursor protein, by subsequent attacks by
- and
-secretases,
which liberate the N- and C-terminal moieties of A
, respectively (for
reviews, see Refs. 2 and
4749)).
The nature of the
-secretase is still discussed
(4,
5,
50,
51) and awaits definitive
identification whereas the
-secretase function is more consensually
ascribed to BACE1 (
-site APP cleaving enzyme also called memapsin 2 or
Asp2,
(69)).
Both
- and
-secretase inhibition could be theoretically seen as a
means to prevent A
production but several apparently uncircumventable
problems lead to the conclusion that
-secretase is not an adequate
target. Thus, inhibitors of the
-secretase activity not only prevent
A
production but also alter the processing of various proteins involved
in vital functions at adulthood
(5256).
Furthermore, preventing
APP processing at the
-secretase site
increases the recovery of the highly toxic C99 product
(3) that accumulates in AD
brains (57).
At first sight,
-secretase appears as a much better target. First, it
is noteworthy that BACE expression and activity are elevated in sporadic
Alzheimer's disease brains, particularly in the cortical and hippocampal areas
affected in the disease
(5860).
Second, the abrogation of the BACE1 gene totally abolishes the
formation of A
-related species and C99 product in knockout neurons
(17) and BACE1-deficient mice
brain (16). Of most interest,
mice devoid of BACE1 develop normally and have an unaltered phenotype
(16,
18). The latter indicates that
even if BACE1 specificity for
APP is not exclusive, the other targeted
substrates do not share essential functions or that another enzyme can
complement for BACE-mediated proteolysis. This contrasts with
presenilins-dependent
-secretase-like cleavages, which when abolished,
trigger lethality at the embryonic stage
(6163)
and severe alterations at adulthood, particularly in thymocyte development
(52,
64).
We have set up stable transfectants overexpressing BACE1. When expressed
together with
APP, we observed that the production of sAPP
and
C99, the two
-secretase-derived complementary products were increased,
but to a much lesser extent for the latter. This is likely because of the
subsequent cleavage of C99 inside the A
domain, leading to an
N-terminal-truncated fragment as previously described
(7,
65,
66). This agreed well with our
observation that genuine A
, i.e. A
starting at the
canonical Asp-1 residue, was drastically reduced after overexpression of
BACE1. These features also stand and were even accentuated when studying cells
overexpressing BACE2, the BACE1 parent protein. In this case, full-length
A
appeared barely detectable in agreement with studies indicating that
BACE2 mainly cleaved in the middle of the A
sequence, after the
19th and 20th residues
(28,
67,
68), thereby leading to
N-terminal truncated products that escaped immunological detection in our
conditions. In line with these data, N-terminal truncated C89 and C79
accumulated in BACE1- and BACE2-expressing cells, respectively.
We have set up a new
-secretase assay based on the hydrolysis of two
quenched fluorimetric substrates, one of which harboring the Swedish mutation
is thought to enhance
-secretase cleavage (see Introduction). The assay
allows to monitor a time- and dose-dependent fluorescence specifically
increased by BACE1 and BACE2 overexpression. More important, we establish that
this activity was maximal at acidic pH and enhanced by the Swedish mutation,
in agreement with the reported properties of
-secretase activity (for
reviews, see Refs. 10,
11, and
69). It should be noted that
the effect of the mutation on BACE activity appears weaker with these
fluorigenic substrates than with
APP itself. This is likely because of
the fact that BACEs act better as protease rather than peptidases. This
semantic discrimination implies that these proteases hydrolyze preferentially
proteins rather than small peptides because the former likely fit better with
a relatively larger recognition/catalytic pocket
(70).
It is interesting to note that the signature of a genuine
-secretase
activity is indeed revealed by the mutation-induced differential fluorescence
monitored by this dual assay. Thus, cathepsin D, a protease with in
vitro-like
-secretase activity
(40,
41), indeed cleaves
efficiently a commercial substrate mimicking the
-secretase-targeted
sequence. However, our assay demonstrated that this protease did not behave as
a good
-secretase candidate because it did not hydrolyze JMV2236, the
fluorimetric substrate bearing the Swedish mutation. This dual assay therefore
proved useful to monitor genuine
-secretase activities. In this context,
it is interesting to note that recently, splice variants of BACE1 have been
identified in human brain and pancreas
(71,
72). Our assay should allow
the monitoring of other putative BACE-like activities and help reveal yet
unknown functions of these activities.
The most potential interest of our assay would be to design highly potent,
bioavailable and metabolically stable inhibitors of BACE1. This is a real
challenge because until now, the inhibitors designed are mostly peptide-based
(for reviews see Refs. 10,
11, and 73), and therefore
poorly enter the blood-brain barrier and are susceptible to proteolysis. Our
rapid, reproducible and sensitive assays should allow to screen for numerous
inhibitor candidates. Our data allow to establish that the length of the
C-terminal tails adjacent of a stabilizing statine group is a more drastic
structural requirement to maintain full efficiency than length of the
N-terminal moiety. Our data also showed the potential of designing fully
specific BACE1 or BACE2 inhibitors. Thus, we found inhibitors of BACE1 or
BACE2 that appeared inactive on the parent protease. Although these compounds
clearly did not fully discriminate between the two enzymes, they constitute
the starting point of a rational design leading to the selection of fully
selective blocking agents. This is not only a biochemical challenge but indeed
an important issue. Thus, BACE2 cleaves mainly inside the A
sequence, at
a nonamyloidogenic site and therefore likely contributes to the depletion of
A
or BACE1-generated A
-related species. In this case that likely
reflects most of sporadic AD, inhibition of BACE1 but not BACE2 is an
important issue. On the other hand, BACE2 is encoded by chromosome 21 and
therefore possibly contributes to the neuropathological AD-like stigmata that
take place in trisomic brains. Thus it has been shown that brains affected
with Down's syndrome display elevated BACE2 expression
(15). BACE2 activity also
appears increased by the Flemish AD mutation
(28). In these cases, a
BACE2-specific inhibitor would be likely very useful.
 |
FOOTNOTES
|
---|
* This work was supported in part by the CNRS and INSERM. The costs of
publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
Supported by the Fondation Alzheimer. Present address: INSERM EMI 9906,
Rouen, France. 
|| Present address: Centre Medical Universitaire, Département de Chimie
Médicinale, Genève, Switzerland. 

Supported by Aventis Pharma. 

To whom correspondence should be addressed: Institut de Pharmacologie
Moléculaire et Cellulaire, UMR6097 du CNRS, 660 Route des Lucioles,
Sophia Antipolis, 06560 Valbonne, France. Tel.: 33-493957760; Fax:
33-493957708; E-mail:
checler{at}ipmc.cnrs.fr.
1 The abbreviations used are: A
, amyloid
peptide;
APP,
-amyloid precursor protein; Abz, p-aminobenzoic acid; tBu,
tertio-butyl; EDDnp, N-(2,4-dinitrophenyl)ethylenediamine;
Fmoc, N-(9-fluorenyl)methoxycarbonyl; HBTU,
O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate; HPLC, high pressure liquid chromatography; Boc,
tert-butyloxycarbonyl; Sta, statine; Norsta,
norstatine; AHPPA,
(3S,4S)-4-amino-3-hydroxy-5-phenyl-pentanoic acid;
ACHPA,
(3S,4S)-4-amino-5-cyclohexyl-3-hydroxy-pentanoic acid;
Qui, quinolein-2-carboxylic acid; TACE, tumor necrosis
-converting enzyme; CS, commercial substrate; Tricine,
N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; TES,
2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; MES,
4-morpholine-ethanesulfonic acid; Bz, benzoyl. 
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to Dr. D. Shenk (Elan Pharmaceuticals) for providing WT192
and 10D5 antibodies. Dr. M. Goedert (MRC, Cambridge, UK) kindly provided
BR188. Anti-1D4 antibodies were provided by the National Cell Culture Center,
Minneapolis, MN. We thank Dr. Luiz Juliano (Sao Paulo, Brazil) for help with
the synthesis of EDDnp. Drs. C. Alves da Costa, C. Sunyach, and B. Vincent are
thanked for critical reading of the manuscript.
 |
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