Diaminodiacid bridge improves enzymatic and in vivo inhibitory activity of peptide CPI-1 against botulinum toxin serotype A

Article abstract of DOI:10.1016/j.cclet.2021.03.055

The replacement of the disulfide bridge of CPI-1, a peptide inhibitor of light chain of Botulinum toxin serotype A, with the thioether-containing and biscarba-containing diaminodiacid bridge leads to a significant decrease in the degradation by trypsin and increase in the detoxification activity in vivo, the addition of hydrophobic or positive amino acid at C-terminus of modified peptides further improves the inhibitory activity.

Full text of DOI:10.1016/j.cclet.2021.03.055

G Model
CCLET-6251; No. of Pages 4
Chinese Chemical Letters xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Communication
Diaminodiacid bridge improves enzymatic and in vivo inhibitory
activity of peptide CPI-1 against botulinum toxin serotype A
*
Jintao Shen, Jia Liu, Shuo Yu, Yunzhou Yu, Chao Huang, Xianghua Xiong, Junjie Yue ,
*
Qiuyun Dai
Beijing Institute of Biotechnology, Beijing 100071, China
A R T I C L E I N F O
A B S T R A C T
Article history:
The replacement of the disulfide bridge of CPI-1, a peptide inhibitor of light chain of Botulinum toxin
serotype A, with the thioether-containing and biscarba-containing diaminodiacid bridge leads to a
significant decrease in the degradation by trypsin and increase in the detoxification activity in vivo, the
addition of hydrophobic or positive amino acid at C-terminus of modified peptides further improves the
inhibitory activity.
Received 29 January 2021
Received in revised form 4 March 2021
Accepted 22 March 2021
Available online xxx
© 2021 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Published by Elsevier B.V. All rights reserved.
Keywords:
Botulinum neurotoxin serotype A
Light chain inhibitor
Peptide CPI-1
Diaminodiacid bridges
Inhibitory activity
BoNT/A is the most potent toxin known to mankind and an
important bio-threat agent. It is composed of a heavy chain (HC)
and a light chain (LC). The HC is responsible for receptor
recognition and toxin internalization, while the LC is a Zn2+
metalloprotease that selectively hydrolyzes SNAP-25 (synapto-
some-associated protein-25 kDa) [1–3]. As a result of SNAP-25
cleavage, the release of acetylcholine is inhibited, leading to
muscle paralysis and death.
The current treatment of BoNT/A poisoning is through antitoxin
administration, but the current antitoxins are ineffective once the
BoNT/A is internalized into neuromuscular cells [4], where the LC
persistently cleaves SNAP-25 and results in the prolonged
poisoning or even death of poison victims. Therefore, it is
important to develop non-antibody drugs for the treatment of
BoNT poisoning, especially drugs that are effective against the
BoNT/A LC [5]. To date, some LC inhibitors, such as hydroxamate
[6–8], quinolone derivatives [6,9,10], peptides [11–15] and other
small molecules [16,17] have been found to potently inhibit the LC
of BoNT/A in vitro, but no inhibitor has been reported to improve
survival rate in animal lethality assays, only few inhibitors showed
modest extension of time to death of mice [18,19]. CPI-1 [Dab(C)-
RWTKCL-NH2] is a cyclic peptide linked by a disulfide bridge, has
been found to be the most potent peptide inhibitor for the BoNT/A
LC, but it has no detoxification activity in vivo [15]. This loss of
activity is partly due to the reduction of the disulfide bridge by
reducing agents in vivo.
It has been reported that cyclic peptides possess higher activity
and stability compared to linear peptides [20]. The stability of
easily reduced disulfide bridge-containing peptides could be
improved by using disulfide bridges mimics, such as thioether
(S-C)- or biscarba (C-C) bridge, triazole bridge, etc. [21–24]. In order
to improve the inhibitory activity of CPI-1 in vivo against BoNT/A,
we used either thioether (S-C)- or biscarba (C-C)-bridged
diaminodiacid to replace the disulfide bridge of CPI-1 (Scheme 1).
Based on the structure–activity of CPI-1 (unpublished data),
hydrophobic and positively charged amino acids (e.g., Trp, Leu and
Arg) were further added at the C-terminus of CPI-1 disulfide bond
mimics (Table 1). We then determined the inhibitory activities of
CPI-1 derivatives against the LC of BoNT/A, the degradation of these
derivatives by trypsin and the detoxification activities of the
derivatives against BoNT/A in vivo.
The C-S diaminodiacids X1 and X2 [Scheme 1a] were
synthesized from protected
L-homoserine and L-cystine as
reported previously [22,25]. The C-C-bridged diaminodiacid
(X3) was prepared by using nickel-catalyzed reductive cross-
coupling reaction according to the literature [23]. The allyl/
allyloxycarbonyl (alloc) protecting group was employed
because it remains stable during the subsequent Fmoc solid-
phase peptide synthesis (SPPS) and can be easily removed by
[Pd(PPh3)4]/PhSiH3. The synthesis steps, mass and nuclear
magnetic resonance (NMR) spectra are shown in Figs. S1–S9
(Supporting information).
* Corresponding authors.
E-mail addresses: yue_junjie@126.com (J. Yue), daiqy@bmi.ac.cn (Q. Dai).
https://doi.org/10.1016/j.cclet.2021.03.055
1001-8417/© 2021 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.
Please cite this article as: J. Shen, J. Liu, S. Yu et al., Diaminodiacid bridge improves enzymatic and in vivo inhibitory activity of peptide CPI-1
against botulinum toxin serotype A, Chin. Chem. Lett., https://doi.org/10.1016/j.cclet.2021.03.055

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Fig. 1. HPLC analysis of the products of cyclization of typical linear CPI-1 disulfide
bridge mimics. (a) Linear peptide 5; (b) Cyclic products of linear peptide 5; (c)
Peptide 5 purified products. Samples were applied to a Calesil ODS-100 C18 column
(4.6 mm  250 mm) and eluted with a linear gradient of 0–1 min, 5%–10% B (B is
acetonitrile containing 0.1% TFA); 1–25 min, 10%–50% B; 25–28 min, 50%–95% B at a
flow rate of 1 mL/min, 214 nm of wavelength.
(Figs. S10–S17 in Supporting information). The mass spectra of CPI-
1 and its variants are consistent with the corresponding theoretical
molecular weight (Figs. S18–S25 in Supporting information). The
circular dichroism (CD) spectra of CPI-1 and its variants in
0.01 mol/L phosphate buffer show random coil structure around
190 and 260 nm (Fig. S26 in Supporting information).
Scheme 1. Synthesis route of CPI-1 disulfide bridge mimics. (a) Structures of
orthogonally protected diaminodiacids. (b) Solid-phase synthesis route of CPI-1
disulfide bridge mimic by using the protected diaminodiacids. The following
protecting groups for amino acid side chains were used: tert-butyl (for Thr),
2,2,4,6,7-pentamethyldihydrobenzofurane-5-sulfonyl (Pbf; for Arg) and tert-
butyloxycarbonyl (Boc; for Trp, Dab and Lys).
The inhibitory activities of peptides were evaluated with
BoNT/A light chain 1–424 (Balc424) [27], which was expressed
and purified according to the procedure described in supporting
information using SNAPtide as the enzymatic substrate [28]. Dose
response curves and the IC50 values of CPI-1 and its variants are
shown in Fig. 2a and Table 1, respectively. After the replacement of
its disulfide bridge by a C-S or C-C bridge, the IC50 values of
With purified diaminodiacid building blocks, we synthesized
CPI-1 disulfide bridge mimics (Table 1) using solid-phase method
as described previously [Scheme 1b] [22]. Fmoc-protected amino
acids were assembled on Rink resin by using the coupling reagent
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluor-
ophosphate (HBTU)/1-hydroxybenzotriazole (HOBt)/N,N-diiso-
propyl ethylamine (DIPEA). First, the alloc and allyl groups of
diaminodiacids on the protected linear peptide were removed with
[Pd(PPh3)4]/PhSiH3. The Fmoc group at the N-terminus was
subsequently removed with 20% piperidine in N,N-dimethylfor-
mamide. Cyclization was then conducted by lactamization using
peptides 1, 5 and 6 became 1.06, 1.32 and 2.72
decreased about 1–5 fold compared to CPI-1 (IC50 = 0.36
m
mol/L, which
mol/L).
m
On the other hand, the introduction of hydrophobic (Leu or Try) or
positive charge amino acid (Arg) at the C-terminus of peptide 1
(peptides 2–4) resulted in 1–2-fold increases in the inhibitory
activities. In addition, the inhibitory activities of CPI-1 and its
derivatives were also assessed with full length SNAP-25 as
enzymatic substrate. The peptides were pre-incubated with
Balc424 at 37 ꢀC for 1 h, and SNAP-25 was subsequently added
and incubated for another 1 h at the same temperature. The
reaction mixture was terminated by adding SDS-PAGE loading
buffer and the products were analyzed by electrophoresis on a 15%
polyacrylamide gel. We found that at the same concentrations,
CPI-1 derivatives 3 and 4 more efficiently inhibited the BoNT/A LC-
mediated cleavage of SNAP-25 compared to wild type CPI-1 and
other derivatives (Fig. 2b). It should be noted that the IC50 or Ki of
CPI-1 is higher than that (Ki =39.2 nmol/L) reported in the
literature [15], the reason is that the FRET substrate of CFP-
SNAP-25(141–206)-YFP is more sensitive than the SANPtide
[FITC–SNAP-25(189–202)-DABCYL] used in this study.
benzotriazole-1-yl-oxytripyrrolidinophosphonium
hexafluoro-
phosphate (PyBOP)/HOBt/N-methylmorpholine (NMM). Finally,
the peptide-resin was cleaved by trifluoroacetic acid, and the
crude peptide was further purified by semi-preparative high
performance liquid chromatography (HPLC). The linear CPI-1
(0.5 mg/mL) was allowed to fold into the cyclic peptide CPI-1 in the
NH4HCO3 buffer (0.1 mol/L, pH = 8.0–8.2) at room temperature,
and the cyclic peptides were then concentrated and purified by
reversed-phase (C18) HPLC [26]. HPLC analyses of the cyclization
products of linear CPI-1 derivative with the S-C bridge (peptide 5)
are shown in Fig. 1. Other CPI-1 derivatives were also individually
cyclized and the major products were purified. The SNAPtide [FITC-
T(D)RIDQANQRATK(DABCYL)Nle-NH2], which is an enzymatic
substrate of the BoNT/A LC used in a fluorescence resonance
energy transfer (FRET)-based assay of inhibitory activity, was
synthesized as described previously (Scheme S1 in Supporting
information) [27]. The purity of all final cyclic peptide
products were above 95%, as confirmed by HPLC with C18 column
To assess the stability of CPI-1 and its derivatives with disulfide
bridge mimics, trypsin degradation assays were conducted. The
Table 1
Amino acid sequence of CPI-1 and its derivatives and inhibitory activity.
Peptide
Amino acid sequence
Theoretical (experimental) MW (Da)#
IC50
(
mmol/L)
95% C.I. (
m
mol/L)
Ki (mmol/L)
CPI-1
Dab-(C)RWTKCL*
Dab-RWTKX1L*
Dab-RWTKX1LL*
Dab-RWTKX1LW*
Dab-RWTKX1LR*
Dab-RWTKX2L*
Dab-RWTKX3L*
1005.5 (1028.49)
987.54 (1010.53)
1100.63 (1101.37)
1173.64 (1174.58)
1143.68 (1144.53)
987.54 (1010.53)
969.62 (970.59)
0.36
1.06
0.63
0.82
0.37
1.32
2.72
0.33–0.39
0.90–1.25
0.50–0.80
0.62–1.08
0.31–0.43
1.06–1.63
1.58–4.71
0.24 Æ 0.01
0.75 Æ 0.07
0.45 Æ 0.06
0.59 Æ 0.10
0.25 Æ 0.02
0.93 Æ 0.12
2.14 Æ 0.66
1
2
3
4
5
6
X1 and X2 represent the C-S- and S-C-bridged diaminodiacid, respectively; X3 represents biscarba diaminodiacids. *, C-terminus is amidated; #, M + H or M + Na, isotopic; C.I. is
confidence interval; Ki is inhibition constant.
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Fig. 4. Survival curves of mice after the intraperitoneal injection of BoNT/A
(2 Â LD50) and CPI-1 or its derivatives. 2 Â LD50 BoNT/A was pre-incubated with
5 mg/kg different peptides for 30 min, and was then injected intraperitoneally into
mice. The death rates of mice were recorded every 12 h for 72 h. *P < 0.05, **P < 0.01
vs. CPI-1 group.
mice in CPI-1 or the control group died 24 h after challenge, while
the survival rate of peptide 1, 3, 4, 5 and 6 groups was 75%–100%.
After 72 h, 25%, 25%, 25%, 25% and 37.5% of the mice receiving
peptides 1, 3, 4, 5 and 6 were still alive, significantly higher than
that of CPI-1 and control groups.
To investigate why peptides 1, 5 and 6 have lower inhibitory
activity against the BoNT/A LC, and how the introduction of
hydrophobic or positive charge amino acid at the C-terminus of the
peptide enhances the inhibitory activity, we compared the
intermolecular interactions of Balc424 with CPI-1 and its
derivatives. The crystal structure of the enzyme-CPI-1 complex
shows that two salt bridge contacts exist [15], the Arg residue of
CPI-1 interacts with Asp370 of Balc424, and Lys201 of CPI-1
interacts with Asp159 of Balc424. However, the salt bridge
interaction between Arg of CPI-1 and Asp370 of Balc424 was
Fig. 2. Inhibitory activities of CPI-1 disulfide bridge mimics against LC of BoNT/A.
(a) Concentration-activity curves of Balc424-mediated (40 nmol/L) cleavage of
SNAPtide in a FRET assay (n = 3). (b) SDS-PAGE of cleavage products of SNAP-25
(10
(125
25 + LC + peptide.
m
m
mol/L) in the presence of Balc424 (1
mol/L). Left to right: marker, control (SNAP-25), SNAP-25 + LC and SNAP-
mmol/L) and peptide inhibitors
inhibitors were allowed to incubate with trypsin (100 ng/mL) in
the reaction buffer (50 mmol/L Tris-HCl, 20 mmol/L CaCl2, pH 8) at
37 ꢀC for 0, 1, 2 and 3 h, respectively. HPLC analysis was used to
determine the percent of degradation. As shown in Fig. 3, CPI-1
(0.25 mmol/L) was degraded by 73.4% after 8 h of incubation. The
derivatives with the C-S or S-C bridge (peptides 1, 2, 3, 5) and the
derivative with the C-C bridge (peptide 6) had much lower
degradation, at 37.4%, 30.4%, 37.5%, 58.8% and 16.06%, respectively.
Since arginine is a specific recognition site for trypsin, peptide 4
was degraded faster than other peptides. These results demon-
strate that the substitution of disulfide bridge with a S-C or C-S or
C-C bridge significantly improves the anti-trypsin degradation
properties of the peptides.
After observing the higher anti-protease degradation resistance
CPI-1 disulfide-bond mimics, we decided to further evaluate the
protective activities in vivo using Kunming mice. BoNT/A [2 Â
median lethal dose (LD50)/mice] was mixed with CPI-1 or its
derivatives (5 mg/kg), and then injected intraperitoneally into
mice. The survival rates and deaths were recorded every 12 h
during a time period of 72 h (Fig. 4). The results show that CPI-1
derivatives 1, 3, 4, 5 and 6 prolonged the survival time of mice. All
not observed in Balc424-peptide
6 complex (Fig. S28A in
Supporting information). CPI-1 disulfide bridge mimics have
smaller size compared to wild type CPI-1, mainly due to the
shorter carbon-carbon and carbon-sulfur bonds in mimics. This
decrease in size may disturb salt bridge interactions and decrease
the binding affinities of the peptides with the light chain of
BoNT/A. For peptide 4, after the introduction of Arg at the C-
terminal of peptide 1, this residue forms a salt bridge with Glu257
at Balc424 (Fig. S28B in Supporting information), which may
improve the binding affinity between the two molecules.
In conclusion, we have developed a series of CPI-1 derivatives
with thioether-containing and biscarba-containing diaminodiacid
bridges which are more resistant to trypsin degradation.
Fig. 3. HPLC analysis of the degradation products of CPI-1 and its derivatives in the presence of trypsin. (a) CPI-1; (b) Peptide 1; (c) Peptide 6; (d) Degradation rates–time
curves of CPI-1 and disulfide bridge mimics in the presence of trypsin. Analytical conditions were the same as described in Fig. 1.
3

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Importantly, these peptides can prolong the survival rate of mice
administered with 2 Â LD50 Â BoNT/A. The addition of hydrophic or
positive amino acid at C-terminus further improves the inhibitory
activity. This research provides an efficient strategy for developing
potent peptides inhibitors against BoNT/A.
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Declaration of competing interest
There are no conflicts to declare.
Acknowledgments
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Appendix A. Supplementary data
Supplementarymaterialrelatedto thisarticlecanbefound, inthe
online version, at doi:https://doi.org/10.1016/j.cclet.2021.03.055.
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