Design, synthesis, and anti-tumor activities of novel Brevinin-1BYa peptidomimetics

Article abstract of DOI:10.1016/j.bmcl.2021.127831

Brevinin-1BYa is an amphibian skin-derived peptide that exhibits promising anti-microbial activity against gram-positive and -negative bacteria. However, the anti-tumor activity of Brevinin-1BYa remains unclear, and, more importantly, its therapeutic application is limited owing to its poor protease and reduction stability. In this study, a series of novel Brevinin-1BYa derivatives, including O-linked N-acetyl-glucosamine glyclopeptides and disulfide bond mimetics, were designed and synthesized. Additionally, their anti-tumor activity against human prostate cancer cell line C4-2B, human NSCLC cell line A549 (adenocarcinoma), and human hepatoma cells line HuH-7 was investigated. Among these, the thioether bridge substituted peptidomimetic Brevinin-1BYa-3 displayed improved reduction stability, more stable secondary structure, greater protease stability, and increased anti-tumor activity compared with the original peptide, rendering it a promising leading compound for drug development, particularly for applications against malignant tumors.

Full text of DOI:10.1016/j.bmcl.2021.127831

Bioorg. Med. Chem. Lett. 37 (2021) 127831
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and anti-tumor activities of novel
Brevinin-1BYa peptidomimetics
,
,
Shili Xionga d, Nan Wangb d, Chao Liub, Huaxing Shenb, Zengqiang Quc, Lijun Zhuc,
Xiaosong Baia, Hong-gang Hub, Wei Congb *, Liang Zhaoa
,
,*
a Luodian Clinical Drug Research Center, Institute for Translational Medicine Research, Shanghai University, Shanghai, China
b Institute of Translational Medicine, Shanghai University, Shanghai, China
c Department of Invasive Technology, Eastern Hepatobiliary Surgery Hospital, Shanghai, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Brevinin-1BYa is an amphibian skin-derived peptide that exhibits promising anti-microbial activity against gram-
positive and -negative bacteria. However, the anti-tumor activity of Brevinin-1BYa remains unclear, and, more
importantly, its therapeutic application is limited owing to its poor protease and reduction stability. In this study,
a series of novel Brevinin-1BYa derivatives, including O-linked N-acetyl-glucosamine glyclopeptides and disul-
fide bond mimetics, were designed and synthesized. Additionally, their anti-tumor activity against human
prostate cancer cell line C4-2B, human NSCLC cell line A549 (adenocarcinoma), and human hepatoma cells line
HuH-7 was investigated. Among these, the thioether bridge substituted peptidomimetic Brevinin-1BYa-3 dis-
played improved reduction stability, more stable secondary structure, greater protease stability, and increased
anti-tumor activity compared with the original peptide, rendering it a promising leading compound for drug
development, particularly for applications against malignant tumors.
Brevinin-1BYa
Glyclopeptides
Disulfide bond mimetics
Anti-tumor activity
Amphibian skin has been revealed to be a valuable source for
discovering novel drug candidates with various biological activities,
including anti-bacterial, anti-viral, anti-fungal, and anti-tumor activ-
ities, among others.1–4 Several compounds with unusual architecture
and remarkable biological activity, especially a number of peptides
including Aurein2.2,5 Magainin II,6 Ascaphin8,7 Dermaseptin-S1,8
Temporin A,9 and Brevinin-1BYa,10 have become the focus of recent
research.11
focused on its anti-tumor activity. Considering the good anti-tumor ac-
tivity of Brevinin-2R1, another AMP from the Brevinin family,13 the
anti-tumor activity of Brevinin-1BYa is explored herein. Meanwhile, it
was also noticed that Brevinin-1BYa contains a cyclic disulfide-bridged
loop at the C-terminal end of the peptide that was unstable in vivo
because of a reducing circumstance, disulfide isomerases, and enzymatic
cleavage and the degradation then leads to structural distortion and
activity loss.14,15 To overcome these difficulties, synthetic disulfide
surrogates including thioether, amide, and triazole bridges with
improved reduction stability and conformational rigidity have been
introduced into the peptide backbone to obtain Brevinin-1BYa-1, Bre-
vinin-1BYa-2, and Brevinin-1BYa-3 (Fig. 1).16–19 In addition, it has been
reported that glycosylation could improve the chemical properties of
peptides, such as hydrophilicity, oral bioavailability, and conformation
stability,20 and thus two glycopeptides, Brevinin-1BYa-4 and Brevinin-
1BYa-5, were designed and synthesized by acetylglucosamine glyco-
sylation at serine and threonine residues, respectively (Fig. 1).
Brevinin-1BYa is an anti-microbial peptide (AMP) that was originally
isolated from the skin secretions of the North American foothill yellow-
legged frog (Rana boylii).12 It has been reported that Brevinin-1BYa
performed with high potential against gram-positive and gram-
negative bacteria, including methicillin-resistant Staphylococcus
aureus (MRSA), as well as against a range of opportunistic yeast path-
ogens such as Candida albicans, Candida parapsilosis, Candida krusei, and
Candida tropicalis.10 Brevinin-1BYa has been regarded as a promising
candidate for anti-bacterial drug development, but few studies have
* Corresponding authors.
E-mail addresses: viviencong@shu.edu.cn (W. Cong), zhaoliangphar@163.com (L. Zhao).
d
Shili Xiong and Nan Wang contribute equally to this work.
https://doi.org/10.1016/j.bmcl.2021.127831
Received 2 November 2020; Received in revised form 6 January 2021; Accepted 22 January 2021
Available online 5 February 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

S. Xiong et al.
Bioorganic & Medicinal Chemistry Letters 37 (2021) 127831
Fig. 1. Structures of Brevinin-1BYa and derivatives.
Scheme 1. Synthesis route of Brevinin-1BYa and Brevinin-1BYa-1, -2, -4 -5. Conditions and reagents: a) TFA/TIPS/phenol/water (88:5:5:2, v/v/v/v), 4 h, RT; b)
Guanidine hydrochloride, sodium dihydrogen phosphate, DMSO, PBS buffer, 24 h, RT; c) 2,6-lutidine, DIPEA, sodium ascorbate, CuBr acetonitrile/DMF, 8 h, rt; d)
PyAOP, HOAt, NMM, NMP, 12 h, RT. Resin-bound peptides were protected on side chains at asterisk sites. The following protecting groups for amino acid side chains
were used: tert-butyl (tBu; for Thr and Ser) and tert-butyloxycarbonyl (Boc; for Lys).
2

S. Xiong et al.
Bioorganic & Medicinal Chemistry Letters 37 (2021) 127831
Scheme 2. Synthesis route of diaminodiacid (A) and Brevinin-1BYa-3 (B). Conditions and reagents: a) i) Na2CO3, FmocOSu, water/1,4-dioxane, 12 h, RT; ii) tert-
butyl trichloroacetimidate, DCM, 12 h, rt; b) CBr4, PPh3, DCM, 12 h, RT; c) i) Na2CO3, allyl chloroformate, water/acetonitrile, 12 h, RT; ii) Allyl bromide, NaHCO3,
DMF, 12 h, RT; d) i) TFA, TIPs, DCM, 1 h, RT; ii) TBAB, NaHCO3, water/EA, 12 h, RT; e) TFA/DCM (1:1, v/v), 2 h, RT; f) i) Pd(PPh3)4, PhSiH3, DMF/DCM, 3 h, RT; ii)
PyAOP, HOAt, NMM, NMP, 12 h, RT; g) TFA/TIPS/phenol/water (88:5:5:2, v/v/v/v), 4 h, RT. Resin-bound peptides were protected on side chains at asterisk sites.
The following protecting groups for amino acid side chains were used: tert-butyl (tBu; for Thr and Ser) and tert-butyloxycarbonyl (Boc; for Lys).
The prototype peptide Brevinin-1BYa and its derivative were synthe-
sized through 9-fluorenylmethoxycarbonyl solid-phase peptide synthesis
(Fmoc-SPPS, Scheme 1). First, Brevinin-1BYa was realized via a solid-
phase-coupling/liquid-phase-cyclization strategy. The on-resin peptide 1
was synthesized through SPPS using 5-chloro-1-[bis(dimethylamino)
methylene]-1H-benzotriazolium 3-oxide hexafluorophosphate (HCTU)
and N,N-diisopropylethylamine (DIPEA) as coupling reagents, and it was
cleaved from the solid support to obtain the free peptide 2. The following
oxidative folding of 2 with 10% DMSO in buffer provided the disulfide
peptide Brevinin-1BYa in an overall yield of 53.2%. Next, by incorporating
Fmoc-β-azido alanine and Fmoc-L-propargly glycine into peptide backbone
through standard SPPS, the protected on-resin peptide 3 was assembled.
On-resin intermediate 4 was obtained through a copper(I)-catalyzed azide-
alkyne cycloaddition (CuAAC) reaction subjected to the treatment of
lutidine, DIPEA, sodium ascorbate, and CuBr. After cleavage from the solid
support and global sidechain deprotection with B reagent, Brevinin-1BYa-
1 was obtained in 56.4% yield. The synthetic route of Brevinin-1BYa-2 was
similar to that of Brevinin-1BYa-1. Fmoc-Lys(Alloc)-OH and Fmoc-Asp
(OAllyl)-OH were coupled into the peptide to provide on-resin interme-
diate 5. De-protection of Alloc/Allyl protective groups with Pd(PPh3)4/
PhSiH3 in DMF/DCM was conducted and, following macrocyclization
promoted by (7-Azabenzotriazol-1-yloxy)tripyrrolidinophosphonium
noticed that not all types of disulfide surrogates could be obtained
through a post-chain-assembly cyclization strategy in which the disul-
fide bond or its substitutes were formed after the peptide backbone had
already been completed, especially the thioether bridge-substituted
Brevinin-1BYa-3. The residual containing halogen sidechain hardly
tolerated the base condition required by Fmoc SPPS because of its
nucleophilicity. Hence, our interest was focused on the diaminodiacid
strategy based on pre-prepared diaminodiacids for synthesis of peptide
mimics with thioether as disulfide surrogates. Since every step was
achieved via peptide coupling reactions, the difficulty in generating non-
peptidic structures on peptides could be avoided in this strategy. Briefly,
installation of the tert-butyl (tBu) protective group followed by capping
of the Fmoc group on L-Homoser-OH (11) provided Fmoc-Homoser-
OtBu (12). Compound 12 was subjected to carbon tetrabromide and
triphenylphosphine, which provided bromine-substituted homoserine
Fmoc-Homoser(Br)-OtBu (13). L-Cys(Trt)-OH was further protected with
allyloxycarbonyl (Alloc) and allyl groups to obtain Alloc-Cys(Trt)-OAllyl
(15). After removal of trityl protection, Alloc-Cys(Trt)-OAllyl (15) was
reacted with compound 13 to obtain protected diaminodiacid 16 in a
high chemical yield. Finally, the diaminodiacid building block 17 was
prepared from diaminodiacid 16 by removing the tert-butyl protection
group using trifluoroacetic acid (Scheme 2A). Next, to obtain on-resin
peptide 18, the key intermediate 17 was successfully attached to resin
through standard Fmoc SPPS using 2-(7-azabenzotriazol-1-yl)-N,N,N′,
N′-tetramethyluronium hexafluorophosphate (HATU) and 3H-[1,2,3]-
triazolo[4,5-b]pyridin-3-ol (HOAt) as the coupling reagent. After allyl
hexafluorophosphate
(PyAOP)/3H-[1,2,3]-triazolo[4,5-b]pyridin-3-ol
(HOAt)/N-methylmorpholine (NMM)/N-methyl-2-pyrrolidone (NMP),
precursor 6 was obtained. Final de-protection and cleavage provided
amide-bridged derivative Brevinin-1BYa-2 with a yield of 52.5%.
During synthesis of the designed derivatives of Brevinin-1BYa, it was
and
Alloc
group
de-protection
via
[Pd(PPh3)4]/PhSiH3,
3

S. Xiong et al.
Bioorganic & Medicinal Chemistry Letters 37 (2021) 127831
Fig. 2. A) Reduction stability of Brevinin-1BYa vs. its derivatives under DTT treatment. B) Protease stability of Brevinin-1BYa vs. its derivatives under α-chymo-
trypsin treatment. C) CD spectra of Brevinin-1BYa vs. its derivatives in TFE aqueous solution. D) Hemolytic activity of Brevinin-1BYa vs. its derivatives with fresh
rabbit blood. Data points are displayed as the mean ± standard error of mean (SEM) of duplicate independent experiments. Percentage of residual peptide was
monitored via HPLC.
Table 1
Degradation half-life, helicity, HC50 and anti-tumor activity of Brevinin-1BYa and derivatives.
Peptide
DTT-t1/2 (h)
α-chymo-trypsin-t1/2 (h)
Helicity (%)
HC50
(μM)
IC50 (μM)
C4-2B
A549
Huh-7
Brevinin-1BYa
Brevinin-1BYa-1
Brevinin-1BYa-2
Brevinin-1BYa-3
Brevinin-1BYa-4
Brevinin-1BYa-5
3.35
>12
>12
>12
3.32
3.31
1.62
2.93
1.81
2.51
4.33
4.32
22.57
94.41
70.43
>100
70.43
>100
>100
13.96
12.63
16.77
9.233
20.51
18.72
9.240
21.68
17.10
11.15
20.93
32.65
14.86
11.33
16.59
10.83
23.37
39.60
–
26.34
83.65
–
–
macrocyclization was successfully accomplished with PyAOP, NMM,
and HOAt as lactamization reagents to achieve cyclic peptide 19.
Elongation with subsequent amino acids gave on-resin peptide 20.
Finally, one-step global de-protection and cleavage with the TFA cock-
tail delivered Brevinin-1BYa-3 in a yield of 55.6% (Scheme 2B).
The synthesis of glycopeptide Brevinin-1BYa-4 and Brevinin-1BYa-5
was based on the key glycoamino-acid building block Fmoc-Ser(GlcNAc
(Ac3))-OH, which was readily incorporated into the peptide backbone
through standard Fmoc SPPS to obtain on-resin peptides 7 and 8. The
following on-resin de-protection of the acetyl protection group utilizing
hydrazine hydrate provided the on-resin intermediates 9 and 10. After
acidic cleavage, global de-protection, and oxidative folding, Brevinin-
1BYa-4 and Brevinin-1BYa-5 were obtained in yields of 54.1% and
54.3%, respectively (Scheme 1).
stability was observed between Brevinin-1BYa and its disulfide bond
mimetics, indicating that the disulfide mimics could significantly
improve the reduction stability of peptides rather than the protease
stability. However, it was also noticed that glycopeptides Brevinin-
1BYa-4 and Brevinin-1BYa-5 performed 1.67- and 1.66-fold longer
enzyme degradation half-lives, respectively, compared with the proto-
type peptide (Fig. 2B and Table 1), indicating the significant roles of
glycosylation moieties for peptide protease stability as previously re-
ported.23 Next, the secondary structure of Brevinin-1BYa derivatives
was measured by circular dichroism (CD, Fig. 2C and Table 1). CD
analysis indicated that prototype Brevinin-1BYa performs an
α-helix
structure with a helicity of 22.57% which was consistent with previous
report.12 Brevinin-1BYa-1, -4 and -5 lost their
α-helix structure and
showed random conformations. Brevinin-1BYa-2, whose amide bond
substitution was similar in size to disulfide bonds, maintained the sec-
ondary structure of the prototype drug. More importantly, it is worth
noting that a huge improvement of helicity was observed for Brevinin-
1BYa-3 (83.65%, 2.70-fold improvements compared to Brevinin-1BYa)
whose thioether substitution was only one atom difference with disul-
fide bond, indicating that large steric disulfide bond replacements or
inappropriate glycosylation sites were dramatically detrimental to their
secondary structure maintaining.
To investigate the influence of disulfide substitutions on the redox
stability of Brevinin-1BYa, a reduction stability experiment was carried
out using dithiothreitol (DTT) as a reducing agent.21 It was suggested
that natural disulfide bridge-containing peptides, including Brevinin-
1BYa, Brevinin-1BYa-4, and Brevinin-1BYa-5, were completely
reduced in 48 h in aqueous DTT solution, while peptidomimetics
Brevinin-1BYa-1, Brevinin-1BYa-2, and Brevinin-1BYa-3 remained
intact after 48 h of treatment (Fig. 2A and Table 1). Furthermore, the
protease stability experiment was investigated using
α
-chymotrypsin,
With Brevinin-1BYa and its analogs in hand, their anti-tumor activ-
ities were measured using the CCK-8 test with the human prostate cancer
cell line C4-2B, the human NSCLC cell line A549 (adenocarcinoma), and
the human hepatoma cell line HuH-7. The results are summarized in
which is a protease that preferentially cleaves large, hydrophobic amino
acids such as tryptophan, phenylalanine, and leucine.22 It was found
that after 24 h of protease exposure, only slight improvement of protease
4

S. Xiong et al.
Bioorganic & Medicinal Chemistry Letters 37 (2021) 127831
Table 1. Among the disulfide mimics, Brevinin-1BYa-1 and Brevinin-
1BYa-2 showed a slight loss of anti-tumor activities compared to pro-
totype peptide, except for Brevinin-1BYa-3. The thioether bridge con-
taining Brevinin-1BYa-3 showed the highest anti-tumor activities
peptidomimetics containing different bridges, such as diselenium and
olefin bond, are currently under way.25–27 Meanwhile, Brevinin-1BYa
glycosylated derivatives focusing on the different spatial locations and
types of carbohydrate moiety will also be investigated and the results
disclosed in due course.
against the C4-2B and HuH-7 cell lines (IC50 = 9.233 and 10.83 μM,
respectively), with values that are 1.50- and 1.38-fold greater, respec-
tively, than those of Brevinin-1BYa. These results suggested that a
spatially appropriate linker is indispensable for the anti-tumor activities
of Brevinin-1BYa.24 Meanwhile, excessive biological activity decreases
were observed with the glycopeptides Brevinin-1BYa-4 and Brevinin-
1BYa-5, suggesting that an inappropriate spatial location of the carbo-
hydrate block might impart significant structural and conformational
changes upon the peptide and perturb the interactions between the
peptide and target protein. However, the carbohydrate block could
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
indeed improve enzyme stability.23 Further hemolytic activity (HC50
,
Fig. 2D and Table 1) was evaluated by fresh rabbit blood hemoglobin
releasing from erythrocyte suspensions. Data indicated that the HC50 of
We thank LetPub (www.letpub.com) for its linguistic assistance
during the preparation of this manuscript.
initial Brevinin-1BYa was 94.41
μM and that of the derivative peptides
ranged from 65.19 M to >100 μM. Among them, Brevinin-1BYa-4 and
μ
Appendix A. Supplementary data
-5 displayed the lowest hemolytic activity. These results demonstrated
that glycosylation moiety could dramatically ameliorate the hemolytic
activity which was a significant characteristic of AMPs. All in all, both of
the peptide derivatives showed no obvious hemolytic activity at the
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2021.127831.
IC50
.
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investigated, and a novel series of O-linked N-acetyl-glucosamine gly-
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were synthesized through either a post-chain assembly cyclization
strategy or diaminodiacid-based strategy to improve the structural ri-
gidity and redox stability of the prototype peptide. In vitro data sug-
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Brevinin-1BYa-2, and Brevinin-1BYa-3 was significantly improved,
while all of the peptidomimetics exhibited slightly improved protease
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the CD data, these results indicated that inappropriate non-peptidic
bridges may possibly change the bridge length, which distorted the
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5