Efficient inhibition of human papillomavirus 16 L1 pentamer formation by a carboxylatopillarene and a p-sulfonatocalixarene

Article abstract of DOI:10.1039/c3cc49789e

Pillarenes and calixarenes showed obvious inhibition of HPV16 L1 pentamer formation via their selective binding to Arg and Lys residues at the monomer interface, which was reversible after the release of cyclic arenes. Pillarenes are more effective than calixarenes in terms of the inhibition efficiency, attributing to the different kinetics and binding affinity. The Royal Society of Chemistry 2014.

Full text of DOI:10.1039/c3cc49789e

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Efficient inhibition of human papillomavirus 16 L1
pentamer formation by a carboxylatopillarene and
a p-sulfonatocalixarene†
Cite this: Chem. Commun., 2014,
50, 3201
Received 26th December 2013,
Accepted 29th January 2014
Dong-Dong Zheng,a Ding-Yi Fu,a Yuqing Wu,*a Yu-Long Sun,a Li-Li Tan,a
Ting Zhou,a Shi-Qi Ma,b Xiao Zhab and Ying-Wei Yang*a
DOI: 10.1039/c3cc49789e
www.rsc.org/chemcomm
Pillarenes and calixarenes showed obvious inhibition of HPV16 L1 pentamer replace current treatments. Although inhibitors have been widely used
formation via their selective binding to Arg and Lys residues at the for controlling the assembly process of other health-related viruses,9
monomer interface, which was reversible after the release of cyclic arenes. few/no small-molecular inhibitors have been identified for HPV
Pillarenes are more effective than calixarenes in terms of the inhibition assembly mainly due to the insufficient target sites and unavailability
efficiency, attributing to the different kinetics and binding affinity.
of agents. Small molecules that recognize protein surfaces specifically
are important tools for modifying protein interactions. Molecules that
Human papillomavirus (HPV) is the causative agent of cervical cancer target the cationic side chains of lysine and arginine have great
and other anogenital and oropharyngeal cancers.1 The major capsid potential as generic surface binders.10 Pillarenes,11 as a new class of
protein L1 and minor capsid protein L2 assemble into virus particles synthetic macrocycles, have exhibited intriguing and peculiar host–
essentially and mediate the primary attachment of viral particles to guest properties and many potential applications in chemistry,
cells2 and the extracellular matrix,3 emphasizing their significance in biology and materials science.12 Pillarene-based molecular recognition
viral infections. Current HPV vaccines target the major capsid protein in organic solvents has been extensively investigated, but few exam-
L1 of the most common cancer-causing types and protect humans ples have been shown in aqueous solutions,12d–g particularly the
against HPV infection and the development of neoplasias4 success- complexation with biologically important entities such as proteins
fully, but they are type-specific, expensive and require cold chain has rarely been reported yet.12d
transportation.5 Therefore, there is an urgent need for the develop-
ment of cost-effective and/or more broad-spectrum alternatives.
A negatively charged pillarene derivative, i.e., a carboxylato-
pillar[5]arene sodium salt (CP5A), possessing a three-dimensional,
Creating disturbances in virus assembly is a particularly attractive rigid and p-rich cavity, as well as two anionic portals, has shown high
method for antiviral intervention because viral structures are formed water solubility and good selective binding towards basic amino acids
by multiple, relatively weak noncovalent interactions,6 and essentially (AAs), such as L-lysine (Lys), L-arginine (Arg) and L-histidine (His).12d
the viral infectivity is critically dependent on capsid formation and its These results inspired us to employ CP5A to bind to the arginine/
stability.7 The cleavage of the glutathione-S-transferase (GST) fusion lysine-rich HPV major capsid protein, L1, and further inhibit the
L1, i.e., GST-L1, by PreScissiont Protease (PPase) liberates the capsid formation of L1 pentamers and VLPs. The controlled assembly of L1
proteins for self-assembly into pentamers and further virus-like pentamers by CP5As has been examined by static light scattering
particles (VLPs).6a,8 Structural determination of L1 pentamers6a indi- (SLS) and fast protein liquid chromatography (FPLC). In the presence
cated that the L1 monomer represents individual subdomains within of CP5A, the L1 pentamer formation was efficiently suppressed. The
pentamers. Image reconstruction of in vitro assembled VLPs shows comparison of CP5A with a water-soluble sulfonatocalix[4]arene
that VLPs are organized by interaction between helices 2 and 4 of L1 (SC4A) by monitoring the L1 pentamer formation was also performed,
and linked to each other via the monomer interface of L1. Therefore, and big differences in the intrinsic binding mechanisms and kinetics
novel small-molecule inhibitors of HPV, targeting specific sites of were also explored. So far, CP5A and SC4A are the first family of small
proteins to stop further virus assembly, are needed to complement or molecular inhibitors directed against pentamer formation of HPV L1.
The HPV L1, with the GST fusion, was coded, expressed, and
a State Key Laboratory of Supramolecular Structure and Materials,
College of Chemistry, Jilin University, 2699 Qianjin Street, Changchun, 130012,
China. E-mail: yqwu@jlu.edu.cn, ywyang@jlu.edu.cn
b Sichuan Tumor Hospital & Institute, Chengdu, 610041, China
† Electronic supplementary information (ESI) available: Experimental proce-
dures, SLS, NMR, IC50, TEM, DLS, FPLC data and structural analysis of the target
protein interaction. See DOI: 10.1039/c3cc49789e
purified using the previously reported protocols.6a After removing the
GST-tag by PPase cleavage, the liberated L1 can assemble into penta-
mers with structural features that resemble ‘‘donuts’’.6a,8 The pentamer
formation leads to an increase in sample turbidity, as monitored by
fluorescence scattering intensity,8 which was further used to probe the
potential inhibitory effects of CP5A on L1 pentamer formation.
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Fig. 2 Examination of the pentamer formation of L1 by size-exclusion
chromatography. The GST-L1 (0.1 mg mLÀ1) was treated with different
molar ratios of CP5A : GST-L1 (0 : 1, 10 : 1, 20 : 1, 40 : 1 and 80 : 1). The graph
on the right shows the decrease in the yield of L1-p to L1-m ratios upon
addition of CP5A.
peaks that appeared to the right of the monomer peak correspond to
the excess amount of CP5A used in the systems. The yield of L1-p to
L1-m ratio was in positive correlation with that of the CP5A concen-
tration in the samples (Fig. 2B). More precisely, the rate of pentamer
Fig. 1 Monitoring of the pentamer formation from GST-L1 monomers
(0.1 mg mLÀ1) by time-dependent static light scattering (SLS) after addition of
the PPase for GST cleavage. The protein was incubated with CP5A in buffer L
(50 mM Tris-HCl, pH = 8.0, 0.2 M NaCl, 1 mM DTT and 1 mM EDTA) at
different concentrations for 30 min before PPase addition and measurement. formation was inhibited by CP5A in a dose-dependent manner,
The curves in different colours represent the molar ratios of CP5A to GST-L1
supplying a half-inhibition concentration (IC50) of CP5A at 0.62 mM
for 3 mg mLÀ1 GST-L1 (Fig. S7, ESI†). Once CP5A was released by
at 0:1, 10:1, 20:1, 40:1 and 80:1, from top to bottom, respectively.
dialysis, the cleaved L1 could further form L1 pentamers and VLPs, as
revealed by dynamic light scattering (DLS, Fig. S9, ESI†) and transmis-
In the absence of CP5A, the scattering intensity increases gradually
sion electron microscopy (TEM, Fig. 3). Similar inhibitory effects of
with time after addition of PPase for GST cleavage (Fig. 1), suggesting
SC4A on the L1-p formation and its reversibility were also observed for
that the particle size increases with L1 pentamer formation. The
increase in rate was restrained in the presence of CP5A and the
the SC4A–L1 system (Fig. S10, ESI†).
By using the same molar ratios of SC4A/GST-L1 as compared
tendency was enhanced with the addition of more CP5As, which was
with CP5A/GST-L1, we observed that SC4A has a similar (Fig. S2,
demonstrated more clearly by the corresponding curve fitting
ESI†) but less pronounced efficacy in assembly inhibition (Fig. 4).
plots (Fig. S1, ESI†). The pronounced inhibitory effects of CP5A
That is, the inhibition rate of L1-p formation was enhanced in a
on L1-pentamer formation suggest that tight binding exists
dose-dependent manner by both CP5A and SC4A, while CP5A has a
between CP5A and L1, where the exposed residues of basic AAs
more distinct effect on it. The kinetics of L1-p formation under CP5A
might be the main binding sites.
and SC4A are compared using in situ SLS plots (Fig. 4). At the same
Based on the standard methods,13 1H NMR spectra of a peptide of
concentration, both CP5A and SC4A have similar effects on L1-p
HPV L1 helix 5 (DLDQFPLGRKFLLQ), a representative one of L1, and
formation initially; however, with time especially several hours after
the peptide with CP5A/SC4A were obtained, and the proton peaks
addition of PPase, they exhibited obvious differences. The enhanced
were assigned (Tables S1–S3, ESI†). The NH regions of the 1H NMR
spectra of 1 mM peptide with CP5A/SC4A at different molar ratios are
shown in Fig. S3 and S4 (ESI†). The differences between the chemical
shifts of the amide NH protons of the peptide backbone in the
absence and presence of CP5A are pronounced (Fig. S5, ESI†). In
other words, the chemical shifts of NH protons in the backbone and
increase rate for SC4A indicates the formation of more L1-p,
suggesting the less efficiency of SC4A to bind L1 when competing
with the intermolecular binding between L1 monomers. In contrast,
CP5A showed a more efficient inhibition with the increase of the
L1particle size, suggesting a higher binding affinity of CP5A and L1.
Thus, the binding of CP5A–L1 should be stronger than that of
side chains of AAs, especially Arg and Lys, are markedly affected by
CP5A/SC4A (Fig. S6, ESI†). Obviously, the results demonstrate that
CP5A/SC4A can bind strongly to the basic residues of AAs in L1.
After treatment with different molar ratios of CP5A to GST-L1 and
then digestion with PPase to remove the GST-tag, the sample was
injected into a gel filtration column to obtain the FPLC elution profile
(Fig. 2). Without CP5A treatment, the results showed an intense peak
corresponding to B284 kDa in the FPLC elution profile (peak1 in
Fig. 2A), which was in a good agreement with the mass of the HPV L1
pentamer (L1-p). Also eluted was a peak corresponding to B58 kDa
Fig. 3 Comparison of the electronic micrograms (EM) of HPV L1 in
(A) monomer (L1-m, after incubated with CP5A at the molar ratios of
(peak2 in Fig. 2A) that could be attributed to the L1 monomer (L1-m);
both show a decreased ratio of the integrated peak area, Sp to Sm. Pre-
1 : 80 and GST cleavage); (B) pentamer (L1-p, after removal of CP5A by
incubation of GST-L1 with CP5A resulted in a decrease of the L1-p
peak and a simultaneous increase of the L1-m peak. The intense
dialysis in buffer L) and (C) VLPs (after further assembly with L1-p in assembly
buffer G). The concentration of initial GST-L1 was 0.1 mg mLÀ1
.
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monomer interface. The results show that CP5A is more efficient
than SC4A in inhibiting the L1 pentamer formation, with different
kinetics and binding affinity to L1. The GST-released L1 could
form L1 pentamers and VLPs well after CP5A was removed by
dialysis. These findings lay the groundwork for the development
of assembly inhibitors as a new class of prophylactic and/or
therapeutic agents for the treatment of HPV infections.
This work was supported by the National Natural Science
Foundation of China (91027027, 20934002, and 21272093) and the
Innovation Program of the State Key Laboratory of Supramolecular
Structure and Materials.
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In conclusion, CP5A and SC4A showed obvious inhibition of
HPV L1 pentamer formation via binding to Arg and Lys at the
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 3201--3203 | 3203