From the Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115
Received for publication, January 16, 2003, and in revised form, March 13, 2003
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ABSTRACT |
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Cerebral accumulation of the amyloid- A body of work (11) suggests that The recent discovery of structurally diverse inhibitors suggests
that these new compounds may also be important molecular probes for the
protease complex. Several non-transition state analog inhibitors are
very potent; however, unlike transition state analogs, their site(s) of
interaction within the Compound Synthesis--
Photoprobe III-63 and analog III-31-C
were prepared using methods described previously (8). JLK2 and JLK6
were synthesized according to published procedures (15). DAPT (16) and
Compound E (17) were synthesized as described previously.
BocLys(Cbz)Ile-Leu-epoxide was synthesized using a
procedure similar to the one reported before (18), only employing
Boc-Lys(Cbz)-OH (Bachem) instead of Boc-Lys(Dnp)-OH. All
D-peptide Boc-D-Val-Gly- Cell-based and Cell-free A Preparation of Cell Lysates and Photoaffinity Labeling--
HeLa
cells were lysed in buffer containing 50 mM MES, pH 6.0, 150 mM NaCl, 5 mM MgCl2, 5 mM CaCl2, 1% CHAPSO, and protease inhibitors.
The cell homogenate was centrifuged at 20,000 × g, and
the supernatant was spun at 100,000 × g for 1 h.
The final supernatant was diluted with PIPES buffer (50 mM
PIPES, pH 7.0, 150 mM NaCl, 5 mM
MgCl2, 5 mM CaCl2) to a final
0.25% CHAPSO solution. The photolabeling was performed essentially as
described previously (4), only the labeled proteins were released from
streptavidin beads with 8 M guanidinium hydrochloride, pH
2.3. The solvent was then exchanged to phosphate-buffered saline
by using Microcone centrifugation tubes. The labeled proteins were
detected by SDS-PAGE/immunoblotting using antibody AB14 to the PS1 N
terminus and antibody 4627 to the PS1 C terminus and with anti-biotin
antibody to the biotinylated species. The concentration of the
competitor was 25 times the cell-free IC50 for active
compounds or at a maximum of 200 µM for compounds
inactive in the cell-free assay.
To fully characterize the inhibitors used in this study and to
provide consistency in the data analysis, we evaluated the inhibitory
potency of each compound in our cell-based and cell-free assays. The
ability of inhibitors to decrease A-Secretase is a protease complex of four
integral membrane proteins, with presenilin (PS) as the apparent
catalytic component, and this enzyme processes the transmembrane
domains of a variety of substrates, including the amyloid
-protein
precursor and the Notch receptor. Here we explore the mechanisms
of structurally diverse
-secretase inhibitors by examining their
ability to displace an active site-directed photoprobe from PS
heterodimers. Most
-secretase inhibitors, including a potent
inhibitor of the PS-like signal peptide peptidase, blocked the
photoprobe from binding to PS1, indicating that these compounds either
bind directly to the active site or alter it through an allosteric
interaction. Conversely, some reported inhibitors failed to displace
this interaction, demonstrating that these compounds do not interfere
with the protease by affecting its active site. Differential effects of
the inhibitors with respect to photoprobe displacement and in
cell-based and cell-free assays suggest that these compounds are
important mechanistic tools for deciphering the workings of this
intramembrane-cleaving protease complex and its similarity to other
polytopic aspartyl proteases.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
protein
(A
)1 is considered a
central event in the pathogenesis of Alzheimer's disease (AD). A
is
produced via
- and
-secretases, proteases that have become
important therapeutic targets for AD (1).
-Secretase plays a crucial
role in determining the proportion of two forms of A
,
A
40 and A
42. The 42-residue
A
42 is more prone to fibril formation and is
disproportionately present in the plaques characteristic of the AD
brain. Accumulating evidence (2-4) strongly suggests that
-secretase is an intramembrane-cleaving aspartyl protease with
presenilin (PS) as the catalytic component. Three other multipass membrane proteins, nicastrin, Aph-1, and Pen-2, are genetically linked
to
-secretase activity (5-7), and biochemical isolation has
provided evidence that these proteins are indeed necessary members of
the protease complex (8-10). Despite the remarkable progress in
uncovering the identity of
-secretase, its mechanism of action
remains unclear.
-secretase cleaves amide bonds
within the transmembrane regions of its substrates, a poorly understood
process of hydrolysis within a hydrophobic environment. Elucidating the
molecular interaction between an inhibitor and its enzyme target can
help identify the enzyme and provide insight into the catalytic
mechanism. The study of peptidomimetic inhibitors of
-secretase that
contain classic aspartyl protease transition state-mimicking moieties
led to the suggestion that
-secretase is an aspartyl protease and
that two conserved aspartates in presenilins are catalytic residues
(12). PS is processed into N-terminal (NTF) and C-terminal (CTF)
fragments. These fragments are metabolically stable, remain associated,
and their formation is tightly regulated, suggesting that together they
are the bioactive form of PS (12). The direct binding of transition
state analog
-secretase inhibitors to these fragments strongly
suggests that the active site is at the NTF/CTF heterodimeric interface
(3, 4), consistent with the fact that each subunit contributes one of
the two critical aspartates (2).
-secretase complex is unclear. To probe the
mechanism of
-secretase inhibitor action, we studied the ability of
structurally diverse compounds to displace a transition state-based
photoactive molecule from its target, the
-secretase active site at
the PS1 NTF/CTF interface. Differential effects of these compounds
suggest that they inhibit
-secretase by distinct mechanisms and are
thus important new probes to elucidate the workings of this complex
protease. Moreover, the ability of a transition state analog inhibitor
of signal peptide peptidase (SPP), a multipass membrane aspartyl
protease with presenilin-like motifs (13, 14), to displace the
-secretase photoprobe suggests that the active site topographies of
these two proteases are similar.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-aminoisobutyric acid
(Aib)-D-Val-D-Val-D-Ile-Aib-D-Thr(O-benzyl)-D-Val-Aib-OMe (D-294) was prepared by solution-phase peptide synthesis
using D-amino acids and the achiral Aib. SPP inhibitor
(Z-LL)2-ketone was purchased from
Calbiochem. All compounds were dissolved in Me2SO to
make stock solutions.
Production--
Inhibition of A
production in cells and measurement by sandwich ELISA were performed as
described previously (19). The cell-free assay was performed as
reported previously (8). IC50 values were estimated by
plotting the ELISA data on SigmaPlot and fitting it to a sigmoidal function.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
production in cell culture was
tested in Chinese hamster ovary cells stably transfected with human
APP. The IC50 values are reported below for each compound,
and all values are summarized in Table I. In cell-free assays CHAPSO-solubilized HeLa cell membrane preparations were used to evaluate the ability of inhibitors to reduce
-secretase proteolysis of recombinant APP-based substrate (Fig.
1).
Chemical structures, inhibition properties, and the displacement
ability of the -secretase inhibitors used in the study
production were
determined using Chinese hamster ovary cells stably expressing human
APP751. Cell-free IC50 values were measured using
solubilized
-secretase prepared from HeLa cells and a recombinant
APP-based (C100FLAG) substrate (see Fig. 1). Displacement indicates the
ability of the inhibitor to prevent the transition state-based
photolabel from binding to PS1 heterodimer (see Fig. 3).
View larger version (22K):
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Fig. 1.
Inhibitory properties of
-secretase inhibitors in a cell-free assay.
Recombinant substrate C100FLAG was incubated with varying
concentrations of Compound E (open circle), III-31-C
(filled square), helical 294 (filled triangle),
epoxide (open triangle), and
(Z-LL)2-ketone (dotted
circle) in CHAPSO-solubilized HeLa cell membranes. After 4 h
the samples were assayed for A
by sandwich ELISA.
Photoactivatable Transition State Analog Inhibitor III-63
Covalently Labels PS1--
We have developed and optimized the
labeling of PS1 with a derivative of a highly potent aspartyl protease
transition state analog inhibitor of -secretase, called III-31-C, a
(hydroxyethyl)urea peptidomimetic (8). III-31-C inhibits A
production with an IC50 of 10 nM in the
cell-free
-secretase assay and 200 nM in APP-transfected
cells. We modified this compound with a photoactivatable benzophenone
and with biotin at the C terminus of the molecule (20). The derivatized
molecule, called III-63, exhibited an in vitro
IC50 value similar to the parent compound, indicating that
the modifications did not affect the ability of the compound to bind
the
-secretase active site.
Compound III-63 was used for photolabeling of PS1 under in
vitro conditions that preserve -secretase activity. We used
lysates as well as microsomes isolated from HeLa cells by lysing and/or solubilizing with the detergent CHAPSO (21). The labeled species were
precipitated with streptavidin beads and analyzed by Western blot,
detecting with anti-PS1 antibodies. The observed biotinylated proteins,
a major band at about 21 kDa and a minor band at about 31 kDa, were
identified as PS1 CTF and PS1 NTF, respectively (Fig. 2, lane 1). These results
strongly suggest that the III-63 photolabel directly binds to the
interface of PS1 heterodimers. This observation is essentially the same
as seen by Li et al. (4), who used a nearly identical
photoprobe and labeled the CTF of PS1 exclusively. Apparently, our
closely related compound can also label small amounts of NTF. When the
unbiotinylated parent compound III-31-C (250 nM) was used
as a competitor, no labeled PS1 NTF and PS1 CTF species were detected
(Fig. 2, lane 2). No labeled proteins were detected when
irradiation was not applied (data not shown). These observations
demonstrated specific labeling of PS1 heterodimers by III-63. This
direct photoprobe binding provided a means of assessing the mechanism
of structurally diverse
-secretase inhibitors by testing the ability
of these inhibitors to displace the photoprobe from its molecular
target. All subsequent competition experiments were performed in cell
lysates, because this method requires smaller amounts of initial
cellular material, but yields similar results. We used competitor
concentrations equal to 25 times their IC50 value in
vitro. The latter was chosen to compare the degree of displacement
with the positive control of displacement by III-31-C at such a
concentration (250 nM, 25 times above the 10 nM
IC50 value).
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Effects of DAPT and Compound E--
Several reported inhibitors
were identified from small molecule library screening and subsequent
optimization, but the means by which they inhibit the protease are
unclear. For example, the dipeptide DAPT (Table I) is a non-transition
state analog, but nevertheless a very potent inhibitor, that
substantially decreased A levels in the brains of APP transgenic
mice (16). In our hands, this compound inhibited A
production with
an IC50 of 10 nM in the cell-free
-secretase
assay and with an IC50 of 20 nM in
APP-transfected cells. After addition of DAPT as a competitor to
photoprobe III-63, somewhat less biotinylated species were observed at
a competitor concentration of 250 nM (25 times the cell-free IC50), and much less labeled species were
detected when 2 µM DAPT (200 times the IC50)
was used (Fig. 3A). This
observation that DAPT prevents labeling with a transition state analog
affinity reagent suggests that DAPT either directly binds to the active site between PS1 heterodimers or alters it through an allosteric interaction (20). Interestingly, DAPT does not displace the photoprobe
as well as III-31-C does at the same relative concentration (25 times
the IC50), despite being essentially equivalent inhibitors in the solubilized
-secretase assay. This intriguing observation suggests that transition state analog III-31-C and non-transition state
analog DAPT act at partially, but not completely, overlapping sites.
Similar results were obtained with Compound E, a non-transition state
analog inhibitor containing a benzodiazepine moiety. In our assays,
this compound inhibited A
production with an IC50 of 3 nM in microsomes and with an IC50 of 0.3 nM in APP-transfected cells. After addition of Compound E
as a competitor to photoprobe, the level of labeled species was reduced
somewhat (Fig. 3A) at a competitor concentration of 75 nM (25 times the IC50), and almost no labeled
species were detected when 600 nM (200 times the
IC50) Compound E was used.
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Isocoumarin-based Compounds--
Isocoumarin "JLK" compounds
(Table I) reported by Petit et al. (15) were also examined
in the displacement assay. These compounds were reported to inhibit
A production in cells without affecting proteolysis of the Notch
receptor. The Notch signaling pathway is involved in cell fate
decisions, and presenilins are linked to proteolysis of the
transmembrane region of the Notch receptor as a key step in the
signaling mechanism (22). As we reported previously (23), we confirmed
that JLK compounds lower A
production in cell culture with an
IC50 of 80 µM; however, the isocoumarins
failed to block
-secretase activity in our cell-free assays.
Moreover, we observed no change in the degree of PS1 heterodimers labeling by the photoprobe (Fig. 3B) when the most active
analogs, JLK2 and JLK6, were used as the competitors at concentrations as high as 0.5 mM. This observation indicates that JLKs
fail to block the interaction of III-63 with its molecular target,
suggesting that these compounds are not likely interacting with or
otherwise affecting the active site of
-secretase. Taken together,
our evidence implies that the isocoumarins probably work upstream of
-secretase.
Aib-containing Peptides--
We also examined APP-based peptides
designed to assume a helical conformation and mimic the APP
transmembrane domain upon initial interaction with -secretase. Thus,
these compounds were designed to inhibit the protease by a different
mechanism than transition-state analogs do. Specifically, these
peptides are based on the sequence of the APP transmembrane domain,
modified with the helix promoting residue Aib. One of the most potent
helical peptides, D-294 (Table I), displays
IC50 values of 3 µM in cells and 100 nM in the in vitro assay (Fig. 1). When this
peptide was run as a competitor to the photoprobe at concentrations as
high as 6 µM (60 times IC50), it
did not affect the photoprobe binding to PS1 heterodimers (Fig.
3B). When D-294 was used at 100 µM
(1000 times IC50), little or no change in the degree of
labeling was observed (data not shown). Therefore, despite the fact
that this helical peptide directly inhibits
-secretase activity, it
does not interact with or otherwise affect the active site. These
observations are consistent with the helical peptide inhibitor
competing with the substrate for a separate initial docking site
(8).
Epoxide Peptidomimetic--
Another class of -secretase
inhibitor with a distinct structure are epoxide-containing molecules. A
number of small epoxide molecules have been identified as irreversible
inhibitors of aspartyl proteases that act by alkylating the catalytic
aspartates (24). One such molecule (18) was reported by Golde et
al. to be an inhibitor of
-secretase, an unconventional
aspartyl protease. We synthesized a similar epoxide (Table I) and
analyzed its inhibitory properties toward
-secretase. This epoxide
inhibited
-secretase activity in the cell-based assay
(IC50 of 20 µM) and in the cell-free assay
(IC50 of 20 µM) (Fig. 1). When tested in the
displacement assay under standard conditions, it did not prevent the
photoprobe from binding to the active site (Fig. 3C). However, when the
epoxide was pre-incubated with lysate for 2 h before the addition
of photoprobe and irradiation, much less photolabeling was observed.
Such time-dependent displacement was not observed with
compound III-31-C. This result suggests that the epoxide is a
mechanism-based inhibitor, inactivating
-secretase by irreversible
binding to the active site.
SPP Inhibitor--
Finally, we tested
(Z-LL)2-ketone (Table I), an
aspartyl protease transition state analog inhibitor of SPP, which is a
recently discovered presenilin-like aspartyl protease (13). We found this compound to be only moderately active in the -secretase cell-free assay, with an IC50 value of 30 µM
(Fig. 1). The (Z-LL)2-ketone similarly inhibited the formation of the other (FLAG-tagged)
proteolytic product generated in this assay (data not shown). When
tested in the displacement assay, 200 µM
(Z-LL)2-ketone (only seven times IC50) completely prevented the photoprobe from binding
(Fig. 3D), suggesting that it successfully competes for the
active site. Thus, this SPP inhibitor directly affects
-secretase
activity by binding to or allosterically altering the catalytic site.
This finding indicates that the topographies of the SPP and presenilin active sites share similarities.
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DISCUSSION |
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The use of a photoactivatable probe directed to the active site of
-secretase provides a convenient means to probe the inhibitory mechanism of action of structurally diverse molecules. Such studies are
particularly important in this case, because
-secretase is a
multicomponent membrane protease that has eluded purification and is
unlikely to be crystallized in the near future. Certain small organic
inhibitors (e.g. transition state analogs) of this enzyme
have served as important mechanistic probes, and the more recently
identified agents will likely do so as well. By analyzing the ability
of structurally different
-secretase inhibitors to displace the
active site-directed photoprobe from its target, we show here that not
all inhibitors act by means of binding or altering the protease active
site. Indeed, different
-secretase inhibitors fell into different
mechanistic classes, summarized in Table I. Most
-secretase
inhibitors interfered with the ability of the photoprobe to bind PS1,
indicating that these compounds either bind directly to the active site
or alter the active site through an allosteric interaction.
Importantly, some non-transition state analogs, namely DAPT and
Compound E, are very potent in vitro and successfully
compete for the active site in the displacement assay. However, these
non-transition state analogs do not displace the photoprobe as well as
the transition state analog III-31-C does at a comparable
concentration. Such behavior suggests that these non-transition state
analogs bind at a site overlapping with that of transition state mimics.
Some reported -secretase inhibitors, the isocoumarins and the
Aib-containing helical peptides, failed to displace the photoprobe from
the active site, demonstrating that these compounds do not block A
production by affecting the active site of the protease. However, these
two types of compounds apparently do not share the same mechanism.
Unlike the Aib-containing helical peptide D-294, the
isocoumarins are not active in the solubilized enzyme assay, indicating
that these inhibitors probably do not target
-secretase directly. In
contrast, helical peptide D-294 does inhibit A
production in the cell-free assay but does not compete with the
photoaffinity probe, suggesting that D-294 acts directly on
-secretase, but not at the active site. Overall, these findings show
that there are apparently several distinct mechanisms for inhibiting
-secretase.
These studies do not distinguish between compounds that bind to the
active site and those that allosterically alter it. To gain insight
into interaction specifics, each inhibitor class should be modified
with a photoactive or chemically reactive moiety to identify its direct
target(s). Such studies should also provide additional characterization
of the initial substrate binding site of -secretase. Recently,
Greenberg and co-workers (25) reported that a number of
-secretase
inhibitors of diverse structures show non-competitive inhibitory
kinetics. This finding suggested the existence of another
substrate binding site (i.e. for initial docking) besides
the active site, which is consistent with other evidence from our
laboratory (8). It is possible that the initial binding site may be a
target of the helical peptide inhibitors (e.g.
D-294), which do not affect the active site. Inhibitors that target the initial substrate docking site may be selective for
different
-secretase substrates, if different substrates do not
share the same initial binding site. These future mechanistic studies
may uncover the possibility of finding
-secretase inhibitors selective for APP over other substrates such as Notch.
We also found that an SPP inhibitor displays pharmacological crossover,
blocking -secretase activity as well. Sequence homology between PS
and an entire family of so-called "PS homologs," which includes
SPP, is found primarily at the transmembrane motifs YD and LGLGD that
contain the critical and conserved aspartates (14). (Z-LL)2-ketone is a transition state
analog that directly interacts with SPP (13). By showing that this
compound can inhibit
-secretase and compete for the active site on
PS, we provide evidence that
-secretase and SPP have similar active
sites and likely share the same proteolytic mechanism. Other than the
short, conserved aspartate-containing motifs, SPP and PS share very
little homology, suggesting that the two proteins arrived at their
aspartyl protease mechanisms via independent evolutionary paths.
Finally, analyzing an epoxide inhibitor in our displacement assay
revealed that this molecule affects the active site of -secretase in
a time-dependent manner. The epoxide may inactivate
-secretase by irreversible binding to the active site aspartates due
to its chemical properties. Further work will focus on studying this interaction in detail, as epoxides might be good chemical probes for
the active site of
-secretase or other unknown intramembrane aspartate proteases awaiting a discovery.
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ACKNOWLEDGEMENTS |
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We thank S. Gandy for AB14 antibody; W. T. Kimberly, W. P. Esler, and M. LaVoie for helpful discussions; and I. Buldyrev and T. S. Diehl for expert technical assistance.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants NS41355 and AG17574 and Alzheimer's Association Grant IIRG-02-4047 (to M. S. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. E-mail:
mwolfe@rics.bwh.harvard.edu.
Published, JBC Papers in Press, March 18, 2003, DOI 10.1074/jbc.C300019200
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ABBREVIATIONS |
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The abbreviations used are:
A, amyloid-
protein;
AD, Alzheimer's disease;
PS, presenilin;
NTF, N-terminal fragment;
CTF, C-terminal fragment;
SPP, signal peptide
peptidase;
DAPT, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine
t-butyl ester;
Boc, t-butoxycarbonyl;
Aib,
-aminoisobutyric acid;
ELISA, enzyme-linked immunosorbent assay;
MES, 4-morpholineethanesulfonic acid;
CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic
acid;
PIPES, 1,4-piperazinediethanesulfonic acid;
APP, amyloid
-protein precursor;
Cbz, carboxybenzoyl;
Dnp, 2,4-dinitrophenyl.
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