Evaluation of superior BACE1 cleavage sequences containing unnatural amino acids

Article abstract of DOI:10.1016/j.bmc.2011.03.056

A recombinant form of BACE1 (β-site amyloid precursor protein cleaving enzyme-1) corresponding to positions 46-454 of the extracellular domain of the original membrane enzyme was prepared. The recombinant BACE1 (rBACE1) had the kinetic parameters K_m<

Full text of DOI:10.1016/j.bmc.2011.03.056

Bioorganic & Medicinal Chemistry 19 (2011) 2785–2789
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Evaluation of superior BACE1 cleavage sequences containing unnatural
amino acids
Taeko Kakizawa a, Akira Sanjoh b, Akane Kobayashi a, Yasunao Hattori a, Kenta Teruya a, Kenichi Akaji a,
⇑
a Department of Medicinal Chemistry, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kita-ku, Kyoto 603-8334, Japan
b R&D Center, Protein Wave Co. Minoo, Osaka 562-0035, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A recombinant form of BACE1 (b-site amyloid precursor protein cleaving enzyme-1) corresponding to
Received 5 March 2011
Revised 22 March 2011
Accepted 23 March 2011
Available online 29 March 2011
positions 46–454 of the extracellular domain of the original membrane enzyme was prepared. The
recombinant BACE1 (rBACE1) had the kinetic parameters Km = 5.5 l
M and kcat = 1719 sꢀ1. Using several
libraries of substrates containing unnatural amino acids, the specificity of rBACE1 was evaluated.
LC/MS of digests derived from the libraries clarified that a dodecapeptide containing unnatural amino
0
acids at P2 to P1 was a superior cleavage sequence.
Keywords:
Ó 2011 Elsevier Ltd. All rights reserved.
BACE1
Amyloid peptide
Substrate specificity
Unnatural amino acid
1. Introduction
2. Results and discussion
The increased production of amyloid peptide (Ab) followed by
oligomerization and accumulation in the brain is a major factor
underlying the pathogenesis of Alzheimer’s disease (AD).1 Ab
39–43 residues) derives from a larger membrane protein (amyloid
precursor protein, APP) which is cleaved by two proteases, b-secre-
To evaluate specificity with an assay system using conventional
HPLC, we first needed to obtain enough amount of highly active
rBACE1 corresponding to positions 46–454 of an extracellular
domain containing the pro sequence (Fig. 1). The corresponding
plasmid was introduced into Escherichia coli, and the 49-kDa pro-
BACE1 was obtained as a major product in inclusion bodies. The
tase (b-site APP cleaving enzyme, BACE1) and
cleaves APP at the N-terminus of Ab to yield a membrane-bound
C99 fragment, which is then cleaved by -secretase to produce
c
-secretase.2 BACE1
c
Ab. Since BACE1-mediated cleavage of APP is the first and rate-lim-
iting step in the processing of APP to yield Ab, BACE1 is considered
a major therapeutic target for treating AD.
BACE1 is a transmembrane aspartic protease and its catalytic
domain is located in the extra-cellular domain.3–6 Although
numerous X-ray structural analyses of the catalytic domain with
inhibitors have been conducted for the design of BACE1 inhibi-
tors,7,8 limited results on substrate specificity were obtained.9–11
The specificity was evaluated mainly by substituting parts of the
APP sequence with natural a-amino acids. More detailed informa-
tion on substrate specificity especially at the site of cleavage by
BACE1 would provide a useful starting point for the design of small
and tightly binding inhibitors. We report here superior BACE1
cleavage sequences containing unnatural amino acids based on
the combination of a conventional LC/MS assay procedure and a
recombinant BACE1 (rBACE1).
Figure 1. Amino acid sequence of BACE1:
clostripain; boxed sequences show the sites of catalytic Asp residues of the aspartic
protease BACE1; expressed sequence is 14–454.
; shows the site of cleavage by
⇑
Corresponding author. Tel./fax: +81 075 465 7659.
E-mail address: akaji@koto.kpu-m.ac.jp (K. Akaji).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.056

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T. Kakizawa et al. / Bioorg. Med. Chem. 19 (2011) 2785–2789
product in a solubilization buffer was refolded by dilution, and the
refolded pro-BACE1 was then treated with clostripain12 to yield
rBACE1. The desired 45-kDa product was further purified by anion
exchange chromatography to give a homogeneous rBACE1. The
purified protease was finally concentrated by ultrafiltration, and
an equal volume of glycerol was added. The rBACE1 solution was
stored at ꢀ20 °C without any loss of catalytic activity for at least
several years.
The catalytic activity of rBACE1 was confirmed by the cleavage
of an eicosa peptide amide (IKTEEISEVNLꢁDAEFRHDSG-NH2;
ꢁ shows the site of cleavage by BACE1) corresponding to the initial
cleavage site of Swedish (SW) mutant APP (Fig. 2). The peptide
amide (105 M) completely disappeared after digestion with
l
rBACE1 (100 nM) to give the expected fragments identified by
MALDI-TOF MS. From measurements of the initial digestion rate
and the plotting of [S]/v against [S], the kinetic parameters were
calculated to be Km = 5.5 l
M and kcat = 1719 sꢀ1. Compared to pub-
lished results,9–11 the catalytic activity of rBACE1 was considered
high enough to achieve peptide-library digestion and evaluation
with a conventional LC/MS procedure.
In the present evaluation of the specificity of rBACE1, we
0
focused on the P4 to P1 sites of its substrate and introduced
unnatural side-chain structures. Using a dodecapeptide amide, ISE-
VNLꢁDAEFRH-NH2, deduced from the SW mutant APP sequence as
0
a template, libraries of peptides containing diversity at P1 to P4
were designed (Fig. 3). Focusing mainly on the steric effects,
side-chain structures containing aliphatic or aromatic substitu-
tions were predominantly selected.
At position P1, the corresponding library sequence is
ISEVNX1ꢁDAEFRH-NH2, where X1 is replaced by the unnatural ami-
no acids listed in Figure 3B. For comparison, replacements with
previously reported favorable amino acids (Leu (in SW APP), Phe
and Tyr)9–11 were also included. The peptide library was prepared
by conventional Fmoc-based solid phase peptide synthesis (SPPS)13
using the split & mix procedure.14 The AcONa (pH 4.5, 40 mM) buf-
fered solution of the P1 library (300 lM) was then incubated with
rBACE1 (120 nM) at 37 °C. The mixture was analyzed with an ana-
lytical HPLC system connected online with an ESI-MS detector. A
typical HPLC profile of the reaction mixture is shown in Figure 4B.
The cleavage rates for substrates containing Thi as well as Leu (in
SW APP) and Phe at position P1 were much higher than for other
P1 components. Consistent with the reductions in peaks, the
corresponding fragments were clearly detected by LC/MS. For
quantitative evaluation, three substrates containing P1-Leu (SW),
Phe, or Thi were independently synthesized, purified, and then
mixed at an equal molar ratio. The mixture (300 lM) was treated
with rBACE1 (60 nM), and the decrease in each substrate was
monitored by HPLC. As summarized in Figure 4C, P1-Thi was most
rapidly cleaved by rBACE1 (ca. 70% cleavage after 3 h) compared to
the SW and P1-Phe substrates (ca. 50% after 3 h).
0
P4 to P1 libraries were then prepared and the cleavage by
rBACE1 was analyzed by LC/MS (Figs. S1–S4). Several substrates
cleaved more rapidly than the others were selected from each li-
brary and synthesized independently for quantitative evaluation.
These selected substrates were P2-Asn (in SW APP), -Asp, -Nva,
-Thi, -Met; P3-Val (in SW APP), -Nva, -Ile; P4-Glu, -Cit, -Phg; and
0
0
P1 -Asp, -Phg, -Cit, -Met, -Abu, -Nva. The residues at P3 to P1 were
clearly preferred by rBACE1 over the SW substrate: P3, Ile and Nva;
0
P2, Thi, Nva, and Met; P1 , Nva, Abu, and Met (Fig. 5). The P4-site Glu
Figure 2. Cleavage of the eicosa peptide amide (IKTEEISEVNLDAEFRHDSG-NH2⁄)⁄:
of the SW substrate was most preferred.
⁄
HPLC [System A, CH3CN (10–40%)/30 min] tR 17.80 min. The asterisks and
Finally, a dodecapeptide amide with the most preferred residues
indicate
a cleaved N-terminal fragment (IKTEEISEVNL, tR 17.36 min) and a
C-terminal fragment (DAEFRHDSG-NH2, tR 4.92 min), respectively.
0
at P4 to P1 , Ile-Ser-Glu-Ile-Thi-ThiꢁNva-Ala-Glu-Phe-Arg-His-NH2
Figure 3. (A) b-Site cleavage sequences of APP (WT; Wild-type, SW; Swedish mutant). (B) Unnatural amino acids used for the construction of libraries.

T. Kakizawa et al. / Bioorg. Med. Chem. 19 (2011) 2785–2789
2787
Glu-Phe-Arg-His-NH2 was the best substrate. The results should
facilitate the design and evaluation of substrate-based BACE1
inhibitors.
4. Experimental
Reagents were purchased commercially and used without fur-
ther purification. Fmoc amino acid derivatives were purchased
from Watanabe Chemical Inc. (Hiroshima, Japan). MALDI TOF-MS
spectra were obtained on an Autoflex-II (Bruker Daltonics), and
ESI-MS spectra, on a HCT plus spectrometer (Bruker Daltonics).
Analytical HPLC was performed using a C18 reversed phase column
in two different systems (system A: Cosmosil 5C18-AR-II column
(4.6 ꢂ 150 mm) with a binary solvent system: linear gradient of
CH3CN containing 0.1% trifluoroacetic acid (TFA) in 0.1% aqueous
TFA at a flow rate of 1.0 mL/min, detected at UV 220 nm; system
B: ZORBAX SB-C18 column (0.5 ꢂ 150 mm) with a binary solvent
system: linear gradient of CH3CN containing 0.1% TFA in 0.1% aque-
ous TFA at a flow rate of 20.0 lL/min, detected at UV 220 nm).
4.1. Construction of plasmid and expression
A cDNA fragment encoding residues 14–454 including the Pre-
(residues 14–21) and Pro(residues 22–45) regions6 of BACE was
synthesized (GenScript, USA) with a restriction enzyme sequence
at both N- and C-terminal sites. The cDNA was then subcloned into
the pET-11a expression vector (Novagen). The expression vector
containing the BACE construct was introduced into E. coli strain
BL21(DE3) (Novagen) by heat shock. The cells were grown at
37 °C for approximately 2.5 h in LB medium containing 100
mL of ampicillin to an optical density of 0.5. Protein expression
was induced by the addition of isopropyl-
lg/
D(ꢀ)-thiogalactopyrano-
side (IPTG) to a final concentration of 1.0 mM. The expression was
allowed to continue at 37 °C for 4 h. Cells were harvested by cen-
trifugation at 6500 rpm for 15 min at 4 °C, washed with Phos-
phate-buffered Saline (PBS) (140 mM NaCl, 2.7 mM KCl, 10 mM
Na2HPO4, and 1.8 mM KH2PO4, pH 7.3), centrifuged again under
the same conditions, and frozen at ꢀ80 °C.
Figure 4. (A) HPLC profiles of the P1 library before (A) and after (B) digestion (37 °C,
3 h) with rBACE1 (120 nM) [HPLC; Cosmosil 5C18 AR column (4.6 ꢂ 150 mm),
linear gradient of CH3CN containing 0.1% TFA (5–35% for 45 min) in 0.1% aqueous
4.2. Purification of the recombinant BACE1
⁄
⁄⁄
TFA at a flow rate of 1.0 mL minꢀ1, detected at 220 nm]. The asterisks and
Frozen cells (5 g) obtained from a 1-L culture were suspended
in 50 mL of sonication buffer (50 mM Tris–HCl, 100 mM NaCl,
1 mM EDTA, pH 7.5, and 50 lL of protease inhibitors (Sigma)),
indicate
a cleaved N-terminal fragment (ISEVNL and ISEVNThi, tR 25.75 min;
ISEVNF, tR 27.64 min) and a C-terminal fragment (DAEFRH-NH2, tR 13.00 min),
respectively. (C) Digestion of selected substrates of the P1 library by rBACE1.
and lysed by sonication (10 times). The lysate was collected and
centrifuged at 15,000 rpm for 1 h. An inclusion-body was
obtained as precipitant, dissolved in 125 mL of solubilization
buffer (100 mM Tris–HCl, 1 mM EDTA, 1 mM Glycine, 100 mM
b-mercaptoethanol, and 8 M Urea, pH 10.0), and centrifuged at
15,000 rpm for 1 h. The supernatant was concentrated and then
refolded at 4 °C by rapid dilution (20-fold) with refolding buffer
(20 mM Tris–HCl, 0.5 mM oxidized glutathione, and 1.25 mM re-
duced glutathione, pH 10.0). After 48 h, the solution was adjusted
to pH 9.0 and gently incubated at 4 °C for 24 h. The procedure
was repeated to adjust the pH to 8.0. The solution was concen-
trated and the product was loaded onto a Sephacryl S-300 size
exclusion column (GE Healthcare) pre-equilibrated in 20 mM
Tris–HCl, 0.4 M Urea, pH 8.0. Eluted fractions containing refolded
BACE were pooled, concentrated, and treated with clostripain
(Sigma).12 The product solution was loaded onto a Resource-Q
column (packed with SOURCE 15Q, GE Healthcare) pre-equili-
brated in 20 mM Tris–HCl, 0.4 M Urea, pH 8.0. rBACE1 was eluted
with a linear gradient from 0 to 500 mM NaCl in 20 mM Tris–HCl,
0.4 M Urea, pH 8.0 at a flow rate of 0.2 mL/min. The desired frac-
tions were pooled and concentrated to a volume of 3 mL. This
1, was synthesized and its cleavage by rBACE1 was evaluated. For
comparison, other sequences containing a preferred residue at P3
0
to P1 were prepared and evaluated (Fig. 6). As expected, all the sub-
strates (300 lM) were cleaved more quickly than the SW substrate.
The cleavage rate for 1 was highest, with ca. 90% of the substrate
cleaved within 3 h even using a decreased amount of rBACE
(10 nM), whereas only ca 10% of the SW substrate was cleaved un-
0
der the same conditions. The results also suggested the P1 substitu-
tion to have a significant effect on the recognition of the substrate
by rBACE1.
3. Conclusion
Using small libraries containing substitutions at specific sites,
several improved substrates for rBACE1 were obtained. Substitu-
0
tions by unnatural amino acids at P2 to P1 had remarkable effects
on how well the substrate was recognized. rBACE1 was useful for
on-line MS analyses of library digests using conventional HPLC.
Among the sequences examined, Ile-Ser-Glu-Ile-Thi-Thi-Nva-Ala-

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T. Kakizawa et al. / Bioorg. Med. Chem. 19 (2011) 2785–2789
0
Figure 5. Digestion of selected substrates of the P2-, P3-, P4-, and P1 -libraries by rBACE1.
Figure 6. Digestion of a superior substrate, Ile-Ser-Glu-Ile-Thi-Thi-Nva-Ala-Glu-Phe-Arg-His-NH2 (1) (300 lM), and related substrates by rBACE1 (10 nM).
solution was dialyzed for buffer exchange to 20 mM Tris–HCl,
150 mM NaCl, 2 mM DTT, pH 8.0.
The proteolytic activity of rBACE was detected by a cleavage as-
say with the eicosapeptide amide using analytical HPLC. The sub-
strate (105 lM) in 40 mM AcONa buffer pH 4.5 was incubated
4.3. Enzymatic activity
with rBACE1 (100 nM) at 37 °C and an aliquot was injected into
the analytical HPLC system [System A, CH3CN (10–50%)/30 min].
After 1 h of incubation, the substrate’s peak had decreased by
approximately 20%. After 6 h, the fragments derived from the
digestion [Fig. 2: detected by analytical HPLC, System A, CH3CN
(10–40%)/30 min] were identified by MALDI-TOF MS; IKTEEI-
SEVNL, tR 17.36 min, m/z 1274.361 for [M+H]+ (calcd 1274.685
for C55H96N13O21), DAEFRHDSG-NH2, tR 4.92 min, m/z 1032.241
for [M+H]+ (calcd 1032.450 for C42H62N15O16).
The substrate eicosapeptide amide (IKTEEISEVNLDAEFRHDSG-
NH2) was synthesized using Fmoc-based SPPS13 starting with Rink
amide resin (4-(20,40-dimethoxyphenyl-Fmoc aminomethyl)phen-
oxy resin)15 according to the procedure described in Section 4.4:
HPLC [System A, CH3CN (10–40%)/30 min] tR 17.80 min, MALDI
TOF-MS, m/z 2288.478 for [M+H]+ (calcd 2288.117 for
C97H155N28O36), Amino acid analysis after 6 M HCl (110 °C, 20 h)
hydrolysis; Asp 3.05, Thr 0.89, Ser 1.81, Glu 4.17, Gly 1.00, Ala
0.89, Val 1.03, Ile 1.82, Leu 1.03, Phe 1.05, Lys 0.90, His 1.01, Arg
1.00.
Kinetic parameters, Km and kcat, were determined by initial rate
measurements of the hydrolysis of the substrate at 37 °C. The reac-
tion was initiated by adding rBACE1 (33 nM) to various solutions

T. Kakizawa et al. / Bioorg. Med. Chem. 19 (2011) 2785–2789
2789
containing different final concentrations of the substrate
(0–105 M). After 15 min of incubation, aliquots were analyzed
A quantitative evaluation of selected sequences was performed
as above using 300 M of substrate and rBACE1 (60 nM for P1- and
l
l
by HPLC. The initial digestion rates were calculated from the de-
crease in the peak area of the substrate. Km was calculated from
the plot of [S]/v versus [S], where [S] is the concentration of the
P4-substrates, 30 nM for P2- and P3-substrates, and 10 nM for 1 and
0
P1 -substrate).
substrate and
three times and the results were averaged.
v is the initial reaction rate. Reactions were repeated
Acknowledgments
This work was supported by a Grant-in-aid for Young Scientists
(B, 22790119) from MEXT (The Ministry of Education, Culture,
Sports, Science and Technology) to T.K.
4.4. Peptide library
The peptide chain was elongated on Rink amide resin
(0.65 mmol/g resin) using a combination of Fmoc-deprotection
and coupling. The deprotection was performed through treatment
with piperidine/DMF (20% v/v) for 20 min at room temperature, fol-
lowed by washing with DMF (ꢂ10). Fmoc-amino acid (5 equiv) was
condensed in DMF in the presence of DIPCDI (5 equiv) and HOBt
(5 equiv) for 2 h at room temperature. The library was prepared by
the split and mix procedure by dividing the resin into equal portions
at a specific position and by mixing after the coupling. After elonga-
tion of the corresponding peptide chain, the protected peptide-resin
was treated with TFA/thioanisole (20:1, v/v) for 2 h at room temper-
ature. The mixture was filtered, and the solution was concentrated
in vacuo. Dry diethyl ether was added to the residue at 0 °C and
the mixture was centrifuged. The precipitate was suspended in
H2O, and lyophilized to give a crude product. Each component of
the library was identified by HPLC with ESI-MS. The data are de-
picted in Tables S1–S7 (see Supplementary data). Each library was
used for the rBACE1 assay without further purification.
Selected library components were similarly synthesized as
above, independently. The Fmoc-deprotection was carried out with
DBU/DMF (2% v/v, 5 min), and Fmoc-amino acid (2 equiv) was cou-
pled by a 1-h reaction using TBTU/HOBt16 (2 equiv each in DMF) as
coupling reagents. Each deprotected product was purified by pre-
parative reverse-phase HPLC, and the purified compound was
lyophilized to afford the target peptide as a white amorphous pow-
der: Ile-Ser-Glu-Ile-Thi-Thi-Nva-Ala-Glu-Phe-Arg-His-NH2 1; HPLC
[System A, CH3CN (5–35%)/45 min] tR 39.47 min, ESI-MS, m/z
1505.7024 for [M+H]+ (calcd 1505.7034 for C68H101N18O17S2). Ana-
lytical data for other selected library components were identical to
those in Tables S1–S7.
Supplementary data
Supplementary data (LC/MS data and HPLC profiles of Px
libraries and LC/MS data of the substrates) associated with this
article can be found, in the online version, at doi:10.1016/
j.bmc.2011.03.056.
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A mixture of the Px library (300
and P4, 60 nM for P2 and P3, and 30 nM for P4) in AcONa-buffered
solution (pH 4.5, 40 mM, 100 L) was incubated at 37 °C. At a spe-
cific time point (0, 1, 3 and 6 h), an aliquot (25 L) was collected
and the reaction was quenched by the addition of an equal volume
lM) and rBACE1 (120 nM for P1
l
l
of GuHCl solution (7 M, 25
ESI-MS.
lL). The mixture was analyzed by LC/
15. Rink, H. Tetrahedron Lett. 1987, 28, 3787.
16. Poulain, R. F.; Tartar, A. L.; Déprez, B. P. Tetrahedron Lett. 2001, 42, 1495.