Short cyclic peptides derived from the C-terminal sequence of α1-antitrypsin exhibit significant anti-HIV-1 activity

Article abstract of DOI:10.1016/j.bmcl.2012.02.037

Serpin A1 (α1-AT), the largest subgroup of serpins, presents in human plasma at high concentration and plays important regulatory roles in physiological and pathological processes. Accumulated evidence suggests that α1-AT may play a role in controlling HI

Full text of DOI:10.1016/j.bmcl.2012.02.037

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2393–2395
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Short cyclic peptides derived from the C-terminal sequence
of a1-antitrypsin exhibit significant anti-HIV-1 activity
Qiyan Jia a, Xifeng Jiang a, Fei Yu b,c, Jiayin Qiu c, Xiaoyu Kang a, Lifeng Cai a, Lin Li c, Weiguo Shi a,
b,d,
a,
Shuwen Liu c, , Shibo Jiang
, Keliang Liu
⇑
⇑
⇑
a Beijing Institute of Pharmacology & Toxicology, Pharmaceutical Chemistry, 27 Taiping Road, Beijing 100850, China
b Lindsley F. Kimball Research Institute, New York Blood Center, NY 10065, USA
c School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China
d MOE/MOH Key Laboratory of Medical Molecular Virology, Institutes of Medical Microbiology, Shanghai Medical College, Fudan University, Shanghai 200032, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Serpin A1 (
plays important regulatory roles in physiological and pathological processes. Accumulated evidence sug-
gests that 1-AT may play a role in controlling HIV-1 infection. In this study, we designed and synthe-
sized a set of short linear peptides derived from the C-terminal sequence of 1-AT. Since none of them
showed significant anti-HIV-1activity, we proceeded to synthesize four short cyclic peptides having 7
amino acids, and we found that three of them exhibited significant anti-HIV-1 activity. One of these cyclic
a1-AT), the largest subgroup of serpins, presents in human plasma at high concentration and
Received 11 November 2011
Revised 30 January 2012
Accepted 14 February 2012
Available online 22 February 2012
a
a
Keywords:
peptides, designated CPM, inhibited HIV-1 entry and infection at low
cyclic peptides could serve as leads for the development of novel anti-HIV-1 therapeutics.
Ó 2012 Elsevier Ltd. All rights reserved.
lM level, indicating that these short
a1-Antitrypsin
HIV-1
Peptides
Entry inhibitors
Acquired immune deficiency syndrome (AIDS) is a lethal dis-
ease caused by the human immunodeficiency virus (HIV). So far,
25 anti-HIV entities have been licensed by the US Food and Drug
Administration (FDA) (http://www.fda.gov/oashi/aids/virals.html).
However, drug resistance and adverse reactions during long-term
medication make it essential to continue developing novel anti-
HIV drugs with new mechanisms of action.
all effectively inhibit HIV-1 infection. Although these results sug-
gest the potential for further development as anti-HIV-1 therapeu-
tics, such peptide drugs could not now be administered orally and
would lack oral availability.
The carboxyl terminal fragment of al-AT (residues 370–374),
FVFLM (Fig. 1), is the binding site of the serpin–enzyme complex
(SEC) to its receptor.10 Binding of FVFLM to the receptor could lead
to proteinase inactivation, serving as ‘hydrophobic core’ for cellular
uptake and nucleolar localization. A synthetic analogue of this pen-
tapeptide, peptide 105C (FVYLI), was able to block the binding and
internalization of al-AT-1261-trypsin complexes by HepG2 cells.9
We thus hypothesized that the peptides containing the se-
quences of FVFLM and FVYLI might have anti-HIV-1 activities.
Based on this supposition, we synthesized two short linear pep-
tides, FM5 (FVFLM) and PI6 (PFVYLI), as previously described.14
Because these two peptides exhibited low solubility, we then
Serpins (SERine Proteinase INhibitors), which comprise 330ꢀ
400 amino acids and represent a superfamily of approximately
500 proteins, fold into a conserved structure consisting of three b-
sheets and at least seven
antitrypsin ( 1-AT), is the largest subgroup of serpins. It presents
a a1-
-helices.1 Serpin A1, also called
a
in human plasma at high concentration and plays important regu-
latory roles in physiological and pathological processes.2–5 Accu-
mulated evidence suggests that
a1-AT plays roles in the
inhibition of the HIV-1 fusion with the target cells6,7 and the sup-
pression of HIV-1 infection.8,9 Besides the whole protein, the pep-
tides derived from
activity. For example, peptide A1-C26 that derives from the C-ter-
a1-AT also exhibited potent anti-HIV-1
360
370
390
1AT Partial
LEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK
Amino Acid Sequence
minal 26-residue sequence
a
1-AT,10,11 its analogue C105Y (CSIP-
VIRIP
A1-C36
A1-C26
PEVKFNKPFVYLI),12 and VIRIP (VIRus-Inhibitory Peptide)13 could
⇑
Corresponding authors. Tel.: +86 10 6816 9363; fax: +86 10 8827 0677 (K.L.);
tel.: +86 20 6164 8538; fax: 86 20 6164 8655 (S.L.); tel.: +1 212 570 3058; fax: +1
212 570 3099 (S.J.).
Figure 1. Bioactive peptides derived from the C-terminus of serpin A1. VIRIP
corresponds to 1-AT residues 353–372. Peptide A1-C36 corresponds to residues
a
E-mail addresses: liusw@smu.edu.cn (S. Liu), sjiang@nybloodcenter.org (S. Jiang),
keliangliu55@126.com (K. Liu).
359–394. Peptide A1-C26 corresponds to residues 369–394. The FVFLM sequence
(underlined) corresponds to the binding site of SEC to its receptor.8
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.02.037

2394
Q. Jia et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2393–2395
Table 2
designed several analogous peptides by extending toward the N-
termini of FM5, such as PM6 (PFVFLM), KM7 (KPFVFLM), and
NM8 (NKPFVFLM), or by introducing polar amino acids in peptide
sequences (Glu or Arg), including PE6 (PFVFLE), EM6 (EFVFLM),
PEM6 (PEVFLM), and PR6 (PFVFLR) (Table 1). Their anti-HIV-1
activities were tested by a virus inhibition assay as previously de-
scribed.15 However, none of these peptides exhibited inhibitory
activity on HIV-1IIIB replication, even at concentrations as high as
Inhibition of the cyclic peptides on HIV-1IIIB replication
Peptide
Sequence
MW
IC50
(
l
M)
CC50(lM)
CPM
CPE
CPR
Cyclo-(CPFVFLM)-SH
Cyclo-(CPFVFLE)-SH
Cyclo-(CPFVFLR)-SH
Cyclo-(CPEVFLM)-SH
837.8
835.6
862.9
819.7
8.96 2.23
55.84 3.31
74.07 22.84
>100
>200
>200
>200
>200
CPEM
100 lM (Table 1), suggesting that these short linear peptides might
not maintain their active conformation.
Table 3
Inhibition of the bioactive peptides on cell–cell fusion
However, some reports16–18 have demonstrated that cyclization
is a promising strategy to enhance bioavailability of peptides. Spe-
cifically, with constrained conformations, the cyclic peptides pos-
sessed improved in vivo stability and better selectivity.19–21
Therefore, we selected four linear peptides, including PM6, PE6,
PR6, and PEM6, for synthesis of the cyclopeptides CPM, CPE, CPR,
and CPEM, respectively (Table 2), using the thioester method22
with some modifications. The inhibitory activities of the cyclic pep-
tides were then determined using an HIV-1IIIB replication assay, as
described above. Surprisingly, three of the four cyclic peptides
exhibited anti-HIV-1 activity. CPM, the most active peptide,
Peptide
Sequence
IC50 (lM)
PM6
PE6
PFVFLM
PFVFLE
>200
>200
>200
>200
67.20 4.05
115.58 6.28
73.98 7.44
>200
PR6
PFVFLR
PEM6
CPM
CPE
CPR
CPEM
PEVFLM
Cyclo-(CPFVFLM)-SH
Cyclo-(CPFVFLE)-SH
Cyclo-(CPFVFLR)-SH
Cyclo-(CPEVFLM)-SH
showed an IC50 (50% inhibitory concentration) of 8.96
HIV-1IIIB replication (Table 2). All the four peptides exhibited low
cytotoxicity with
CC50 (50% inhibitory concentration)15 of
>200 M to MT-2 cells (Table 2).
lM against
100
80
60
40
20
0
a
l
To determine the mechanism of action, we tested the activity of
cyclic peptides on HIV-1 envelope protein (Env)-mediated cell–cell
fusion, as previously described23 and the IC50 was calculated using
the CalcuSyn software.24 CPM showed an IC50 of 67.2
lM in the
cell–cell fusion assay, which is reasonable, based on its anti-HIV-
1 activity, as described above, and results of the pseudovirus repli-
cation assay, which is explained below (Table 3). Noticeably, CPM
was about 5- to 6-fold more potent than CPE and CPR in inhibiting
HIV-1IIIB replication, while former was almost equally potent as
latter, suggesting that CPM may target not only the HIV-1 fusion
step, but also other step of HIV-1 replication.
-20
To further determine whether a cyclic peptide could block HIV-
1 entry, we constructed a pseudovirus expressing the envelop pro-
tein gp160 of the HIV-1 R5 virus SF162 and tested the inhibitory
activity of peptide CPM on the single cycle infection of the pseudo-
typed HIV-1 as previously described.25 The results showed that the
cyclic peptide CPM significantly inhibited HIV-1 pseudovirus infec-
1
10
Concentration of peptide (µM)
100
Figure 2. The inhibitory activity of CPM on HIV-1 pseudovirus infection assay. The
peptide was tested in triplicate and the data presented as mean SD.
tion with an IC50 of 5.95 lM (Fig. 2), suggesting that these cyclic
peptides inhibited HIV-1 infection by blocking virus entry, an out-
cyclic peptide inhibited HIV-1 infection by blocking virus fusion
with and entry into the target cells, suggesting that that CPM
and other short cyclic peptides could serve as leads for the devel-
opment of novel anti-HIV therapeutics.
come which is consistent with the mechanisms of action of the
long linear peptides derived from
further modify the peptide sequences by introducing unnatural
amino acids, such as -amino acids, so that the short cyclic antivi-
a
1-AT, such as VIRIP.13 We will
D
Acknowledgments
ral peptide can be orally administered.
In conclusion, a set of linear and cyclic peptides derived from
This work was supported by grants from Key Tech. of National
S&T Major Project of Original New Drug Research
(2012ZX09301003–001) to KL and National Natural Science Foun-
dation of China (U0832001 to SL and 81173098 to SJ).
the C-terminal sequence of
A cyclic peptide CPM showed low
a
1-AT were designed and synthesized.
M inhibitory activity against
l
HIV-1 infection. The mechanism study showed that this short
Supplementary data
Table 1
Inhibition of the designed peptides on HIV-1IIIB replication
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2012.02.037.
Peptides
Sequences
MW
696.9
IC50 (lM)
FM5
PM6
KM7
NM8
PI6
FVFLM
>100
>100
>100
>100
>100
>100
>100
>100
>100
PFVFLM
KPFVFLM
NKPFVFLM
PFVYLI
794.0
922.2
1,036.3
792.0
791.9
826.0
776.0
819.0
References and notes
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EM6
PEM6
PR6
EFVFLM
PEVFLM
PFVFLR

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Their N- and C-termini were acetylated and amidated, respectively. All the
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chromatography to >95% homogeneity. The molecular weight of the peptides
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15. Jiang S.; Lu H.; Liu S.; Zhao Q.; He Y.; Debnath A. K. Antimicrob. Agents
Chemother. 2004, 48, 4349. For measuring the inhibitory activity of the
peptides on infection of HIV-1 IIIB infection, 1 x 104 MT-2 cells were infected
with 100 TCID50 HIV-1IIIB in the presence or absence of the peptides at graded
concentrations. On the fourth day post-infection, the culture supernatants
were collected for detection of HIV-1 gag antigen p24 antigen using an
enzyme-linked immunosorbent assay (ELISA). For determining the cytotoxicity
19. Perlmutter, D. H.; Joslin, G.; Nelson, P.; Schasteen, C.; Adams, S. P.; Fallon, R. J. J.
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22. Zhang L.; Tam J. P. J. Am. Chem. Soc. 1997, 119, 2363. The thioester peptides
were synthesized as described by Zhang et al with the following modifications:
(1) The Trityl(Trt) was used as protecting group to protect the thiol group of
HSCH2CH2COOH and then attached it to onto the resin by N, N’-
dicyclohexylcarbodiimide (DCC)/1-hydroxybenzotriazole (HOBt). (2) The Trt
protecting group was removed using 10%TIS/TFA and then the C-terminal
amino acid was coupled to the thiol group to get thioester by 2-(1H-
benzotriazol-1)-yl-1,1,3,3-tetramethyluronium hexafluoro-phosphate(HBTU)/
diisopropylethylamine(DIEA). The lyophilized linear thiolester peptide was
dissolved in 50% acetonitrile/H2O containing 0.1 M Na2HPO4 at the final
concentration of 2 mg/mL, followed by addition of 3 equiv TCEP and
adjustment of the pH to 7.0. The reaction was monitored by HPLC. The cyclic
peptides were then purified by reverse phase HPLC to >95% homogeneity. The
molecular weight of the peptides was confirmed by MALDI-TOF-MS (Autoflex
III, Bruker Daltonics).
23. Jiang S.; Lin K.; Strick N.; Neurath A. R. Nature 1993, 365, 113. For testing the
inhibitory activity of the peptides on HIV-1-mediated cell-to-cell fusion, the
chronically HIV-1IIIB-infected H9 (H9/HIV-1IIIB) cells were labeled with
Calcein-Am (Molecular Probes, Inc., Eugene, OR). After washes, the
fluorescence-labeled H9/HIV-1IIIB cells were incubated with MT-2 cells at
1:5 ratio at 37°C for 2 h in the absence or presence of the peptides at a graded
concentration. The percentage of fused cells was counted under a fluorescence
microscope (Zeiss, Germany) and the percentage of inhibition of cell fusion by
the peptides was calculated as previously described.14
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11129. For determining the inhibitory activity of the peptides on infection by a
pseudovirus expressing the envelop protein gp160 of the HIV-1 R5 virus SF162,
U87-CCR5 cells (1 Â 104/well) were seeded in 96-well plates and grown
of the peptides, 100
equal volumes of MT-2 cells (5 Â 105/ml) in a well of 96-well plate. After
incubation at 37 °C for 4 days, XTT (1 mg/ml; 50 l/well; Poly-Sciences, Inc.,
ll of a peptide at a graded concentration was added to
l
overnight. A peptide at indicated concentration was incubated with
Warrington, PA) was added. Four hours later, the soluble intracellular
formazan was quantitated colorimetrically at 450 nm with a reference at
650 nm. The 50% cytotoxicity concentrations (CC50) was calculated by using
the software Calcusyn.
pseudotyped particles For construction and expression of a pseudovirus
expressing the envelop protein gp160 of the HIV-1 R5 virus SF162, (1 ng
p24/well) for 30 min at 37 °C. Subsequently, the virus-compound mixture was
transferred to the cells and incubated for an additional 48 h. Cells were washed
with PBS and lysed with the lysing reagent included in the luciferase kit
(Promega, Madison, WI). Aliquots of cell lysates were transferred to
luminometer plates, followed by the addition of luciferase substrate. The
luciferase activity was measured in a microplate luminometer (Genios Pro,
Tecan).
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