BACE1 inhibitor peptides: Can an infinitely small k cat value turn the substrate of an enzyme into its inhibitor?

Article abstract of DOI:10.1021/ml2002373

Recently, we reported substrate-based pentapeptidic β-secretase (BACE1) inhibitors with a hydroxymethylcarbonyl isostere as a substrate transition-state mimic. These inhibitors showed potent BACE1 inhibitory activity in enzyme and cell assays, with KMI-429 showing in vivo inhibition of Aβ production. We also designed and synthesized nonpeptidic and small-sized BACE1 inhibitors using "in-silico conformational structure-based designa€'. By studying the structure-activity relationship of these inhibitors, we found that the σ-φ interaction of an inhibitor with the BACE1-Arg235 side chain played a key role in the inhibition of BACE1. We speculated that a peptide capable of binding to the BACE1-Arg235 side chain via the σ-φ interaction might exhibit BACE1 inhibitory activity. Hence, we designed and synthesized a series of peptides that were modified at the P ₂ position and found that some of these peptides exhibited a potent BACE1 inhibitory activity despite their structural similarity to the BACE1 substrate.

Full text of DOI:10.1021/ml2002373

Letter
pubs.acs.org/acsmedchemlett
BACE1 Inhibitor Peptides: Can an Infinitely Small kcat Value Turn the
Substrate of an Enzyme into Its Inhibitor?
Yoshio Hamada,*,†,‡ Shoichi Ishiura,§ and Yoshiaki Kiso*,†,‡,∥
†Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Minatojima, Chuo-ku, Kobe 650-8586, Japan
‡Center for Frontier Research in Medicinal Science, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8412, Japan
§Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Meguro-ku, Tokyo 153-8902, Japan
∥Laboratory of Peptide Science, Nagahama Institute of Bio-Science and Technology, Tamura-cho, Nagahama 526-0829, Japan
ABSTRACT: Recently, we reported substrate-based pentapeptidic β-secretase (BACE1)
inhibitors with a hydroxymethylcarbonyl isostere as a substrate transition-state mimic. These
inhibitors showed potent BACE1 inhibitory activity in enzyme and cell assays, with KMI-429
showing in vivo inhibition of Aβ production. We also designed and synthesized nonpeptidic
and small-sized BACE1 inhibitors using “in-silico conformational structure-based design”. By
studying the structure−activity relationship of these inhibitors, we found that the σ−π
interaction of an inhibitor with the BACE1-Arg235 side chain played a key role in the
inhibition of BACE1. We speculated that a peptide capable of binding to the BACE1-Arg235
side chain via the σ−π interaction might exhibit BACE1 inhibitory activity. Hence, we
designed and synthesized a series of peptides that were modified at the P2 position and found
that some of these peptides exhibited a potent BACE1 inhibitory activity despite their
structural similarity to the BACE1 substrate.
KEYWORDS: Alzheimer’s disease, β-secretase, BACE1, BACE1 inhibitor, substrate, inhibitor peptide
β-Secretase, also known as β-site amyloid precursor protein
cleaving enzyme 1 (BACE1), is a promising molecular target
for developing anti-Alzheimer’s disease drugs,1−7 mainly
because it triggers amyloid β (Aβ) peptide formation by
cleaving the amyloid precursor protein (APP) at the N-
terminus of the Aβ domain.8−13 Recently, we reported potent
pentapeptidic BACE1 inhibitors5,14−19 (KMI-420, KMI-429,
KMI-684, and KMI-574) possessing phenylnorstatine [Pns:
(2R,3S)-3-amino-2-hydroxy-4-phenylbutyric acid] to be sub-
strate transition-state mimics as shown in Figure 1. Among
them, KMI-42915,16 exhibited strong inhibition of BACE1
activity in cultured cells, and on direct administration into the
hippocampi of APP transgenic and wild-type mice, it caused
significant reduction in Aβ production.16 We also designed and
reported nonpeptidic and small-sized BACE1 inhibitors, KMI-
1027 (IC50 = 50 nM)20 and KMI-1303 (IC50 = 9 nM),21
possessing a heterocyclic scaffold at the P2 position as shown in
Figure 2. KMI-1027 was designed from KMI-429 as a lead
compound using the in-silico conformational structure-based
design, which focused on a conformer of docked inhibitor in
BACE1. On establishing that a slightly attractive force such as
stacking or σ−π interaction between the guanidino-plane of
BACE1-Arg235 and the P2 region of the inhibitor plays a key
role in the mechanism of BACE1 inhibition,20 we designed a
potent BACE1 inhibitor, KMI-1303, which possesses an
electron-rich halogen atom that seemed to interact with the
electron-poor guanidino-π orbital of BACE1-Arg235 by
Coulomb’s force.20
Figure 1. Peptidic BACE1 inhibitor with a substrate transition-state
analogue.
by us, have been designed from the Swedish mutant APP
sequence (EVNL*D), which is cleaved faster than the wild-type
APP sequence (EVKM*D) by BACE1.6 The Km values of the
Swedish mutant APP type substrate (9 μM) and wild-type APP
type substrate (7 μM) are similar, suggesting that the affinities
Received: October 10, 2011
Accepted: December 26, 2011
Published: December 26, 2011
Until now, most of the peptidic BACE inhibitors possessing a
substrate transition-state analogue, including the ones reported
© 2011 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
Figure 2. Nonpeptidic BACE1 inhibitors.
for both types of substrates against BACE1 are comparable.
However, the Swedish mutant type substrate that has very high
kcat value (0.02 s−1) elicited higher catalytic efficiency by
BACE1 than did the wild-type APP type substrate (0.002
s−1).22 We speculated that the hydrogen-bonding interaction
between BACE1-Arg235 and the P2-Asn side chain of the
Swedish mutant substrates activates the turnover required for
enzymatic catalysis, thereby improving the kcat value. However,
strong attractive forces, such as a hydrogen bonding, seem to be
unfavorable for enzyme inhibition. In fact, our KMI compounds
possessing a hydrogen-bond acceptor or donor, such as the Asn
residue, at the P2 position showed the lowest BACE1 inhibitory
activity.5,21 As described above, we found the interaction
between BACE1-Arg235 and its inhibitors to be significant; the
guanidino-plane of BACE1-Arg235 in most publicly available
X-ray crystal structures of BACE1−inhibitor complexes showed
similar figures flopping over the P2 region of the inhibitors.
Moreover, the nearest distances between the guanidino-plane
and the P2 regions showed similar values of about 3 Å. We
hypothesized that the guanidino-plane of Arg235 pushed down
on the P2 region of the inhibitors, causing them to be affixed in
the active site of BACE1. In addition, a slightly attractive force,
such as stacking or σ−π interaction, might pack down the
inhibitors effectively in the active site. Hence, we speculated
that such an interaction at the S2 site reduces the turnover of an
enzyme, and a substrate possessing a vastly reduced kcat value
might turn into an inhibitor (Figure 3). Herein, we designed a
series of peptides that were modified at the P2 position.
Figure 3. Interaction of substrates with the BACE1-Arg235. (A)
Swedish mutant type substrate. (B) BACE1 inhibitor peptide with a P2
Leu residue. The P2 Leu side chain seemed to interact with the
guanidino-plane of BACE1-Arg235 by a weak σ−π interaction.
a
Scheme 1. Synthesis of Inhibitor 15
Peptides 1−22 were synthesized by 9-fluorenylmethoxycar-
bonyl (Fmoc)-based solid-phase peptide synthesis methods;
the synthesis of peptide 15 is outlined in Scheme 1. In brief, the
Fmoc-protected amino acid was attached to 2-chlorotrityl
chloride resin using diisopropylethylamine (DIPEA) in 1,2-
dichloroethane (DCE). The Fmoc group was removed with
20% piperidine in DMF, and peptide bonds were formed using
diisopropylcarbodiimide (DIPCDI) as a coupling reagent in the
presence of 1-hydroxybenzotriazole (HOBt). After the peptide
chain was elongated, cleavage from resin was achieved using
trifluoroacetic acid (TFA) in the presence of m-cresol,
thioanisole, and water. All peptides were purified by preparative
RP-HPLC. BACE1 inhibitory activity was determined by an
enzyme assay using a recombinant human BACE1 and a
fluorescence resonance energy transfer (FRET) substrate as
previously reported.5,14−21 The enzymatic reaction was
performed by incubating BACE1 and the FRET substrate (7-
methoxycoumarin-4-yl)acetyl-Ser-Glu-Val-Asn-Leu*Asp-Ala-
a
Reagents and conditions: (a) 2-Chlorotrityl chloride resin, DIPEA,
DCE; (b) 20% piperidine/DMF; (c) protected amino acid, DIPCDI,
HOBt/DMF; (d) 5-fluoroorotic acid, DIPCDI, HOBt/DMF; (e)
TFA, m-cresol, thioanisole, H2O.
Glu-Phe-Arg-Lys(2,4-dinitrophenyl)-Arg-Arg-NH2) together
with 2.0 or 0.2 μM KMI compounds, in incubation buffer,
and the N-terminal cleavage fragment of the substrate was
analyzed by RP-HPLC with fluorescence detection.
Because our KMI compounds with a P2 Leu residue and P1
substrate transition-state analogue exhibited potent BACE1
inhibitory activity and the side chain of P2 Leu seemed to
interact with the guanidino-plane of BACE1-Arg235 by a weak
σ−π interaction, we first synthesized some peptides possessing
a Leu residue at the P2 position as shown in Table1. The
octapeptides 1−6 exhibited weak inhibitory activity. Because
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Table 1. Peptides That Showed BACE1 Inhibitory Activities
Table 2. Peptides with a 5-Fluoroorotyl Moiety at the P4 Position
peptides 2, 4, and 6 containing a D-amino acid residue showed
slightly higher inhibitory activity, pentapeptides 7 and 8
possessing a D-amino acid were synthesized, and they replicated
the inhibitory activity of the octapeptides. Peptide 9, which
lacked the P1′ residue, also showed slightly weak inhibitory
activity; on the other hand, it is noteworthy that peptide 11,
with the P2−Asn residue corresponding to the Swedish mutant
APP sequence, showed no inhibitory activity. Peptide 10,
however, with the P2-Lys residue corresponding to the wild-
type APP sequence, showed slightly higher inhibitory activity
than did 9 and 11. This supports the fact that the Km value of
the wild-type APP type substrate is slightly lower than that of
the Swedish mutant APP type substrate, and thereby, the wild-
type APP sequence, rather than the Swedish mutant APP
sequence, possesses higher affinity against BACE1.
Previously, we reported that replacing the carboxylic acid of
the P4-Glu residue in peptidic KMI-compounds with its
bioisostere, such as a tetrazole ring or a 5-fluoroorotyl group,
improved their BACE1 inhibitory activities.17,18 Hence, we
designed a series of BACE1 inhibitors containing a 5-
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ACS Medicinal Chemistry Letters
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fluoroorotyl group in the P4-amino acid residue as shown in
Table 2. Peptides 12−15 and 17−20 showed potent BACE1
inhibitory activity. In particular, peptides 13, 15, and 17−20,
possessing a D-amino acid residue at the P4 position, exhibited
potent inhibitory activity comparable to that of KMI-44618
(99% inhibition at 2 μM, IC50 = 26 nM) with a substrate
transition-state analogue. In contrast, peptide 16, with the P2−
Asn residue corresponding to the Swedish mutant APP
sequence, showed no inhibitory activity, as predicted. Although
we reported that KMI compounds possessing a cyclo-
hexylalanine (Cha) residue at the P2 position showed improved
BACE1 inhibitory activity,18 peptides 19 and 20 with a Cha
residue showed inhibitory activity similar to that of the
corresponding peptides 13 and 15. Because peptide 21 lacking
the P1′ residue also showed potent BACE1 inhibitory activity,
peptide 21 that is released from peptides 12−15, 17, and 18 by
cleavage at their P1−P1′ amide bonds might inhibit BACE1.
Hence, we performed the incubation experiment using the
same concentrations of BACE1 and BACE1 inhibitors used in
our BACE1 inhibitory assay. However, peptides 12−15 were
not cleaved by incubation for 1 or 24 h (1 h corresponds to the
BACE1 inhibition assay incubation time), and therefore,
peptide 21 was not detected by HPLC analysis of the incubated
buffer solution.
How can a common peptide without a substrate transition-
state analogue inhibit enzyme activity? One possible mecha-
nism of inhibition is that the transition-state of these peptides
might stably bind to BACE1 because of their vastly reduced kcat
value, which is a result of the σ−π interaction between the P2
region of peptides and BACE1-Arg235. Although this
hypothesis seems reasonably explained, it is difficult to prove.
However, examples of inhibitors with the trifluoromethylketone
moiety as a transition-state analogue that can bind to an
enzyme, such as cysteine or serine protease, and form
complexes with the tetrahedral structure of trifluoromethylke-
tone’s carbonyl carbon at the C-terminus of inhibitors, have
already been reported.23 Another possibility is that inhibitor
peptides might bind to an active site of BACE1 without change
in the peptide bond. To investigate this, we performed the
docking simulation study under the MMFF94x force field using
MOE software (Chemical Computing Group Inc., Canada).
Octapeptide 2 bound in BACE1 (PDB ID: 1FKN) was shown
in Figure 4A. The reason that octapeptides 2, 4, and 6, with a D-
amino acid, showed higher inhibitory activity than peptides 1,
3, and 5, with a corresponding L-amino acid, seems to be the
small space corresponding to a water molecule between the
peptides and the two catalytic side chains of BACE1. This space
might be important for the cleavage mechanism of an aspartic
protease. The hydrophilic P1′-side chains of octapeptides 2, 4,
and 6 might occupy this space, allowing them to bind stably to
BACE1. On the other hand, pentapeptides 7, 8, 13, 15, and
17−20 with a D-amino acid also showed higher inhibitory
activity than did pentapeptides with a corresponding L-amino
acid. We considered that the same phenomenon occurred in
octapeptides and pentapeptides but for different reasons. It is
possible that the α-carboxylic group and the side chain at the
P1′ position of these pentapeptides counterchange their
positions, permitting effective binding to the active site of
BACE1 as shown in Figure 4B. This is supported by the fact
that pentapeptide 18, with a bulky Val residue at the same
position, also showed potent inhibitory activity and is unable to
fit into the small space. Additionally, the α-carboxylic group of
Figure 4. Octapeptide 2 (A) and pentapeptide 13 (B) bound in
BACE1 (PDB ID: 1FKN). Inhibitor peptides and Arg235 were
depicted by space-filling modeling. The cartoon and stick models
indicate BACE1 enzyme. The P1′ regions of the inhibitors are behind
the terms “Thr72” situated in the right upper parts of flap domains in
both models A and B.
the P1′ residue might interact with the hydrophilic S1′-pocket of
BACE1.
In conclusion, we designed a series of peptides possessing a
P2-Leu residue that seemed to reduce the turnover in the
BACE1 inhibitory mechanism, and we found some peptides
with weak inhibitory activity. We also synthesized a series of
peptides with a 5-fluoroorotyl moiety in the P4-amino acid
residue. Peptides KMI-1706, KMI-1709, KMI-1793, and KMI-
1794 showed potent BACE1 inhibitory activity, comparable to
that of KMI-446 with a substrate transition-state analogue.
KMI-1858, possessing the same P2-Asn residue as the Swedish
mutant APP that seemed to have higher affinity for the active
site of BACE1, showed no inhibitory activity, suggesting that
the interaction of inhibitors and substrates with BACE1-Arg235
plays a critical role in the inhibition and catalytic action of
BACE1, respectively. These findings, describing the interaction
of BACE1-Arg235 with the P2 position of inhibitors/substrates,
may be important for developing the next generation of BACE1
inhibitors. To the best of our knowledge, this is the first study
to report that the concepts for designing substrates and
inhibitors are fundamentally different. Thus, the finding that a
common peptide without a substrate transition-state analogue
can inhibit BACE1 activity can spur future research toward
developing “a gene-based therapy using a DNA that codes for
peptide sequence with BACE1 inhibitory activity”.
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Letter
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AUTHOR INFORMATION
■
Corresponding Author
*Y.H.: telephone, +81-78-974-4907; e-mail, pynden@gmail.
com. Y.K.: telephone, +81-749-64-8113; fax, +81-749-64-8140;
e-mail, y_kiso@nagahama-i-bio.ac.jp.
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Funding
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production and secretion. Proc. Natl. Acad. Sci. U.S.A. 1999, 96,
11049−11053.
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This study was supported in part by Grants-in-Aid for Scientific
Research (A) and (C) from MEXT (Ministry of Education,
Culture, Sports, Science and Technology), Japan (KAKENHI
No. 21249007 and No. 23590137, respectively).
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We thank T. Hamada and Dr. J.-T. Nguyen for performing the
in vitro enzyme assays and his help in preparing the manuscript,
respectively.
(16) Asai, M.; Hattori, C.; Iwata, N.; Saido, T. C.; Sasagawa, N.;
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