Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 848-854
doi:10.1111/j.1745-7270.2008.00470.x
Design of peptide inhibitors
for furin based on the C-terminal fragment of histone H1.2
Suming Wang1,3#, Jinbo Han2,3#, Yanfang Wang2#, Wuyuan Lu4, and Chengwu Chi2,3*
1
School of Life Sciences,
University of Science and Technology of China, Anhui 230027, China
2
Institute of Protein
Research, College of Life Sciences and Technology, Tongji University, Shanghai
200092, China
3 Institute of Biochemistry and Cell Biology,
Shanghai Institute of Biological Sciences, Chinese Academy of Sciences,
Shanghai 200031, China
4 Institute of Human Virology, University of
Maryland Biotechnology Institute, Baltimore, Maryland 21201, USA
Received: June 2, 2008 Accepted: August
10, 2008
This work was supported by grants from
the National Basic Research Program of China (No. 2004CB719904) and the
National Natural Science Foundation of China (No. U0632001)#
These
authors contributed equally to this work
*Corresponding
author: Tel, 86-21-54921165; Fax, 86-21-54921011; E-mail, [email protected]
The mammalian
proprotein convertase furin has been found to play an important role in diverse
physiological and pathological events, such as the activation of viral glycoproteins
and bacterial exotoxins. Small, non-toxic and highly active, furin inhibitors
are considered to be attractive drug candidates for diseases caused by virus
and bacteria. In this study, a series of peptide inhibitors were designed and
synthesized based on the C-terminal fragment of histone H1.2, which has an
inhibitory effect on furin. Replacing the reactive site of inhibitors with the
consensus substrate recognition sequence of furin has been found to increase
inhibitory activity greatly. The most potent inhibitor , I4, with 14 amino acid residues has a Ki value of 17 nM for furin. Although most of the synthesized
peptides were temporary inhibitors, the inhibitor I5, with nine
amino acids, retained its full potency, even after a 3 h incubation period with
furin at 37 ?C. These inhibitors may potentially lead to the development of
anti-viral and anti-bacterial drug compounds.
Keywords furin; inhibitor; histone H1.2; peptide synthesis
In the secretory pathway, proproteins are limited and cleaved by a
family of proteolytic enzymes called proprotein convertases (PCs). PCs are
calcium-dependent serine proteases whose catalytic domain shares some sequence
similarities with that of the bacterial subtilisin [1]. This cleavage is an
important process widely used to regulate the activation of peptides and
proteins that play significant roles in various biological events that are
implicated in both homeostasis and various diseases [2]. Furin, a mammalian
PC, was the first to be identified, and it has been extensively studied. Furin
has been shown to have effects on different substrates, such as blood-clotting factors,
growth factors, hormone receptors, matrix metalloproteinases and ion channels
[3–5].
Bacterial exotoxins, such as diphtheria toxin, anthrax toxin, and viral
envelope glycoproteins of HIV and the SARS virus, are also processed by furin
[2,6–9]. Furthermore, many studies have indicated that increased furin
activity is closely related to the malignancy of various tumors [10]. Thus,
furin is an attractive target for therapeutic drugs.Many furin inhibitors have been studied, including small molecular
PC inhibitors and protein-based inhibitors [11]. Each small molecular PC
inhibitor is categorized as a peptide inhibitor, peptidomimetic inhibitor or
non-peptide inhibitor [12–14], while protein-based inhibitors include polypeptides derived
from the prodomain of PC [15–17], bioengineered proteins [18–21], and some endogenous proteins
[22–26].
Among them, a1-antitrypsin Portland and polyarginine have been used to prevent the
activation of bacterial toxin, the processing of envelope glycoprotein in viral
replication and the metastasis of cancer [10,27,28]. By comparison, small peptide inhibitors are more attractive furin
inhibitors, since they are more potent but have low toxicity. Many peptide
inhibitors have been investigated; for example, some of them were designed
based on the sequence of PC prodomain or PC partner proteins and the lysine
active domain of the mung bean trypsin inhibitor (MBTI) [29,30]. Other peptide
inhibitors include the consensus substrate recognition sequence of furin, the
C-terminals of which are modified by an active group (–CMK, –CHO, =NOH or –CH=NNHCONH2) [17,31]. Meanwhile, the stability of small peptide inhibitors has
been improved by cyclic peptide inhibitors, such as chymotrypsin inhibitor 2
from the barley serine proteinase inhibitor-2 [32], sunflower trypsin
inhibitor-1 [33], and the Lys fragment of mung bean trypsin inhibitor [34]. In our previous study, three highly active inhibitors against furin
were purified from porcine liver and identified as C-terminal truncated
fragments with different sizes of histone H1.2. The inhibitory activities of
these fragments were greater than that of the full-length histone H1.2, and it
has been suggested that inhibitory activity against furin relies upon the
C-terminal domain [35]. In the same study, a synthesized 36 amino acid peptide
of the C-terminal fragment retained inhibitory activity against furin;
however, this 36 amino acid peptide with a Ki value
of 5.1?10–7 M is too long for wide application and lacks the ability to inhibit
the activity of furin efficiently. In this study, we used this small amino acid
peptide as a template to design a shorter but more potent and stable furin
inhibitor. Seven peptide inhibitors derived from the 36 amino acid peptide were
synthesized, and their potency and stability against furin were characterized.
Of them, we found a nonapeptide with high stability and a Ki value of 2.7?10–8 M, which may serve as a leading
compound for the development of therapeutic drugs for furin-mediated diseases,
such as HIV.
Materials and methods
Materials
The fluorogenic substrate
pyrArg-Thr-Lys-Arg-7-amino-4-methylcoumarin (MCA) was purchased from Bachem
Bioscience (San Diego, USA). All Fmoc amino acids and Fmoc resins were obtained
from Applied Biosystems (Foster City, USA).
Peptide synthesis
All the linear peptides were synthesized using the standard Fmoc
chemistry. The protected peptide was independently grown on a Wang-resin, using
the HBTU (O-Benzotriazole-N,N,N‘,N‘-tetramethyl-uronium-hexafluoro-phosphate)/HOBT
(N-Hydroxybenzotriazole) amino acid activation method. Solid phase peptide
synthesis was performed on a 433A peptide synthesizer (Applied Biosystems). The
protected amino acids were Fmoc-Ser (tBu), Fmoc-Lys (tBoc), Fmoc-Thr (tBu),
Fmoc-Arg (Pbf) and Fmoc-Asp (otBu). The resin was incubated in TFA containing
5% p-cresol and a few drops of triethylsilane and thioanisole for 1.5 h at room
temperature for cleavage. The crude peptides were precipitated by cool
anhydrous diethyl ether and purified by reverse phase HPLC.All the cyclic peptides were synthesized through their corresponding
linear peptide thioester precursors by intramolecular native chemical ligation
[36]. The linear peptide thioester precursors were prepared using the Boc
solid phase method with in situ neutralization [37]. Typically,
S-trityl-b-mercaptopropionic acid was preactivated with HBTU/DIEA (N,N-diisopropylethylamine)
and introduced to Leu-Pam resin. After deprotection with neat TFA, the first
amino acid from the C-terminal was coupled to the resin with a free thiol group
using HBTU/DIEA as coupling reagent. After the chain elongation was finished,
all the protection groups were removed and peptides were cleaved from resin by
HF (Hydrogen Fluoride)/p-cresol (90:10) at 0 ?C. The peptides were precipitated
and washed with cold diethyl ether and purified by reverse phase HPLC. The
cyclization of the linear peptides was performed on 0.25 M phosphate buffer
containing 6 M guanidine hydrochloride, pH 7.4, overnight. The reaction was
monitored by RP-HPLC and the cyclic product was purified by HPLC and
identified by electrospray ionization-mass spectrometry.
Peptide purification
Peptide purification
The synthetic peptides were desalted on a Sephadex G15 column
(Amersham Biosciences, Piscataway, USA), washed with buffer A (0.1% TFA in
water), lyophilized, dissolved in buffer A and then purified on a Zorbax C18
column (9.4250 mm) (Agilent, Palo Alto, USA) by HPLC. The peptides were
equilibrated with buffer A at a flow rate of 2 ml/min and eluted in a gradient
of 0% buffer B (0.1% TFA in acetonitrile) for 5 min and 0%–30% buffer B for
25 min. The molecular masses of all synthetic peptides were determined with an
ABI API2000 Q-trap mass spectroscope (Applied Biosystems).
Ki measurement and stability assay
The fluorogenic MCA substrate (pyrArg-Thr-Lys-Arg-MCA) was used for
the furin activity assay. To determine the inhibitory activity, different
amounts of the inhibitors were first incubated with a fixed amount of enzyme
(1.7 mM) at 37 ?C for 3 min in a final volume of 1 ml HEPES buffer (100 mM
HEPES, pH7.5, 1 mM CaCl2, 0.5% Triton X-100, and 1 mM b-mercaptoethanol),
and the residual enzyme activity was then measured with an F-2500 fluorescence
spectrophotometer (Hitachi, Tokyo, Japan). For stability assay, the inhibitors
were incubated with furin for different periods (0, 30, 60, 90, 120, 150 and
180 min), and then the inhibitory activity was measured. Enzymes incubated
without inhibitors were measured as control. The excitation and emission
wavelengths were 370 nm (slit width, 10 nm) and 460 nm (slit width, 10 nm),
respectively. The Ki values of inhibitors against furin were
determined by Dixon’s plot (1/V against I) using two different concentrations
of substrate (1.0 mM, 1.5 mM). The substrate concentration for stability assay was 1.0 mM. Data from
three measurements were averaged and graphically analyzed with an equation to
obtain the equilibrium inhibition constant Ki.
HPLC assay of stability of
peptide inhibitors
HPLC was used to study the stability of peptide inhibitors; 20 mg peptide
inhibitors with or without incubation with furin (1.7 mM) in HEPES buffer at 37 ?C
for 3 h were placed into 300 ml buffer A and centrifugated. The supernate was then loaded to a
PepMap C18 column (4.6250 mm) (Applied Biosystems). I5 was
equilibrated with buffer A at a flow rate of 0.8 ml/min and eluted in a
gradient of 0% buffer B for 5 min and 0%–50% buffer B for 25 min. I4 was equilibrated with 10% buffer B at a flow rate of 0.8 ml/min and
eluted in a gradient of 10% buffer B for 5 min and 10%–50% buffer B for 20 min.
The molecular masses of all peaks were determined with an ABI API2000 Q-trap
mass spectroscope.
Results
Optimization of inhibitor
As reported in our previous work, a peptide, bearing a Ki value of 5.1?10–7 M, with 36 amino acid residues (PAAATVTKKVAKSPKKAKAAKPKKAAKSAAKAVKPK)
derived from the C-terminal fragment of histone H1.2 possesses a potent
inhibitory activity against furin [35]. Based on this 36 amino acid peptide
template (termed I1), a series of shorter peptides was designed
and synthesized (Table 1) (Fig, 1). The first step in
optimization was to shorten the 36 amino acid peptide from both the N- and
C-terminals. The resulting peptide termed I2, with
14 amino acid residues exhibited a 10-fold lower inhibitory activity than I1. To improve the potency of I2, the
second step introduced the consensus substrate recognition sequence of furin
into the reactive site. Furin recognizes a specific RXRAKR? site. The peptide I3 was then designed by replacing
the P2 residue with Lys and P1, P4 and P6 residues with Arg. These replacements led to
a decrease in the Ki value of I3 by
approximately 5.8?10–8 M, suggesting that the consensus substrate sequence of furin is
essential for the inhibitor. When two alanine residues at the P1‘ and P2‘ positions of I3 were replaced with Asp (P1‘) and Leu (P2‘), respectively, to achieve I4, the Ki value for furin further decreased three-fold, indicating that a
negatively charged residue at the P1‘ site is favorable.
Though the 14 amino acid peptides I3 and I4 have appropriate inhibitory activities, their relatively large
sizes restrain their application. The third step was to remove the N-terminal
Thr-Lys-Lys-Val and C-terminal Ala residues flanking the reactive site of the
inhibitor I3 to obtain I5. The truncation at both
termini had no apparent impact on the inhibitory activity of I5, resulting in a nonapeptide inhibitor with a Ki value of 2.7?10–8 M. To protect the peptides from
possible in vivo degradation by exopeptidase, three cyclic peptide
inhibitors with 10, 12 and 14 amino acid residues were also synthesized in the
thioester formation, between the N-terminal cysteine and the C-terminal Leu.
Unexpectedly, the inhibitory potencies of the peptides I6, I7 and I8
decreased by 160, 35 and 5 folds, respectively, compared to I4 (Table 2).
Stability analysis
Stability assays were carried out to measure the stability of the
inhibitors over several hours. To measure the stability of these inhibitors,
the IC50 concentrations of the inhibitors were used based on their Ki values. The substrate concentration for the stability assay was
1.0 mM. Enzymes incubated without inhibitors were used as a control to
confirm that furin activity would not change during incubation in buffer at 37
?C. The initial inhibitory activity of each inhibitor was marked as 100%; their
inhibitory activities at indicated time points were then compared with initial
activity and normalized as percentage values. Inhibitory activities were
measured three times.When the synthesized peptides (I1–I8) were
incubated with furin for an indicated time, their inhibitory activities
gradually decreased in a time-dependent manner, with the exception of I5. The most stable inhibitor, I5,
retained 100% potency against furin, even after a 3 h incubation period. In
contrast, the inhibitor I6 was the least stable with a
50% activity loss during the same time period. Among inhibitors I2, I3 and I4, the
activity of the one with the highest inhibitory activity (Ki-I4
however, I5 is more stable than I4. Compared with I4, the cyclic peptides I6 and I8 lost their potencies much more quickly, suggesting that the
cyclization of peptide is not helpful in the optimization of a furin inhibitor
(Fig. 2).To confirm the stability analysis results further, HPLC was also
used to measure stability. Since I4 and I5 are the most active peptide inhibitors, they were selected to be
incubated with furin for 0 h or 3 h, and then separated by HPLC. As shown in Fig.
3(A), after 3 h incubation with furin at 37 ?C (right panel), the HPLC
profile of I5 was the same as that of I5 incubated with furin for
0 h (left panel). The figure inserted on the right shows the molecular weight
of the peak marked I5, as measured by mass spectrum. Consistent
with our stability assay, the HPLC profile of I4 showed
that it decreased and a new peptide was generated, with a retention time of 9.5
min on HPLC, after incubating with furin at 37 ?C for 3 h [fig. 3(B)]. The molecular weight of
this newly generated peptide was 1,438.6 kDa [Fig. 3(C)], which is a
good match with the calculated molecular weight of peptide cleaved from I4 between P1 and P1‘. This indicates that the instability of these inhibitors was
caused by the cleavage of furin at the C-terminal of P1.
Discussion
Since furin has been found to be related to bacterial and viral
infections, the development of atherosclerosis [38], Alzheimer’s disease [39],
and the metastasis of cancer [10], it has become an important therapeutic
target for those types of diseases. Furin inhibitors are capable of
neutralizing bacterial exotoxins and preventing viral infections. Until now,
proteinase inhibitor 8 and histone H1.2 have been reported as naturally
synthesized possible inhibitors of furin in mammals [23,35]. The C-terminal
fragment of histone H1.2 was found to be more potent than full-length histone
H1.2, with a Ki value of 310
nM against furin. This C-terminal fragment of histone H1.2 has many advantages
over other furin inhibitors; for example, its small size and lack of a
disulfide bond means it is easily synthesized and purified. However,
modifications are needed to promote its potency and stability before it can be
applied therapeutically. In this study, we successfully optimized this
C-terminal fragment to be a more potent and stable furin inhibitor, and thus
made it an attractive and potential candidate for use as a therapeutic drug.The crystal structure of the catalytic domain of mouse furin
indicates that the active site of furin forms an extended substrate-binding
groove that is lined with many negatively charged residues [40]. Studies of
furin inhibitors have shown that peptides comprised of positively charged
residues are better furin inhibitors [13,41]. There are three pockets in the
substrate binding sites of furin [4,42]: S1, S2 and S4. In general, the S1 pocket
of furin needs Arg in the P1 site of the
substrate/inhibitor, the S2 pocket interacts with Lys in
the P2 site, and the S4 pocket favorably interacts
with Arg in the P4 site. As furin does not have an S3 pocket, the P3 site of the
substrate/inhibitor is optional; thus, a favorable substrate of furin would
have the conserved RAKR sequence. Furin also has another secondary pocket in
the substrate binding sites, the S6 pocket. It can interact
with Arg in the P6 site of the substrate/inhibitor. Our previous study showed
that only one site cleaved by furin exists in the C-terminal of histone H1.2
(K175–K178) [35]. Based on this cleavage site, I1 with 36
amino acids was designed and found to be a potent furin inhibitor, with a Ki value of 5.1?10–7 M. To shorten the original I1 peptide,
the 14 amino acid peptide inhibitor I2 was then designed by removing the N- and C-terminal residues
flanking the reactive site of I1; the inhibitory activity of I2 was thus reduced almost 10-fold. In order to increase inhibitory
potency, we further designed I3 and I4 based
on the optimal cleavage site (RXRAKR DL). The mutations at the reactive site
markedly increased the inhibitory activities of I3 and I4, indicating that the consensus substrate sequence of furin is
preferable to achieve high inhibitory activity. At the same time, substitution
with Asp and Leu at the P1‘ and P2‘ positions
may also increase inhibitory activity three-fold (Table 2). By docking
the nonapeptide (RERRRKKRG) with furin [43], the S1, S2, S4 and S6 pockets
are at one side. The cyclic peptides (I6–I8) in our
study form rigid structures, and not all the amino acids in the reactive site
are able to bind to the S1, S2, S4 and S6 pockets of furin. The cyclic peptides achieved
greater structural flexibility when the length of the circle increased, and
accordingly, the inhibitory activity increases with the elongation of peptide
from I6 to I8.Like other peptide inhibitors of furin, most of the synthesized
peptides (I1–I8) were temporary inhibitors, as their inhibitory activities
gradually decreased in a time-dependent manner. Notably, the lower the Ki value, the more quickly activity decayed (Fig. 2). One
exception was nonapeptide I5, with a Ki value of 2.7?10–8 M, in which no apparent change in inhibitory activity was found,
even after a 3 h incubation period with furin at 37 ?C. Three cyclic peptides
were also designed to improve inhibitor stability. Unexpectedly, cyclization
increased neither the potency nor the stability of the inhibitor.In summary, based on the C-terminal fragment of Histone H1.2, a
series of furin inhibitors were designed. Among them, I4
exhibited the highest inhibitory activity, and I5 was the
most stable. These inhibitors may serve as ideal lead compounds for the
development of therapeutic drugs used in the fight against furin-mediated
diseases, such as HIV.
Acknowledgement
We would like to thank Dr. Iris. Lindberg (Louisiana State University,
New Orleans, USA) for the purified recombinant mouse furin.
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