New anti-HIV aptamers based on tetra-end-linked DNA G-quadruplexes: Effect of the base sequence on anti-HIV activity

Article abstract of DOI:10.1039/c2cc34399a

This communication reports on the synthesis and biophysical, biological and SAR studies of a small library of new anti-HIV aptamers based on the tetra-end-linked G-quadruplex structure. The new aptamers showed EC₅₀ values against HIV-1 in the r

Full text of DOI:10.1039/c2cc34399a

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COMMUNICATION
New anti-HIV aptamers based on tetra-end-linked DNA G-quadruplexes:
effect of the base sequence on anti-HIV activityw
Valentina D’Atri,a Giorgia Oliviero,a Jussara Amato,a Nicola Borbone,*a Stefano D’Errico,a
Luciano Mayol,a Vincenzo Piccialli,b Shozeb Haider,c Bart Hoorelbeke,d Jan Balzarinid and
Gennaro Picciallia
Received 19th June 2012, Accepted 9th August 2012
DOI: 10.1039/c2cc34399a
This communication reports on the synthesis and biophysical,
biological and SAR studies of a small library of new anti-HIV
aptamers based on the tetra-end-linked G-quadruplex structure.
The new aptamers showed EC50 values against HIV-1 in the
range of 0.04–0.15 lM as well as affinities for the HIV-1 gp120
envelope in the same order of magnitude.
large pools of nucleic acid sequences for their ability to bind
selectively and with high affinity to a biomedically relevant
target. For their properties, aptamers can be considered as the
nucleic acid analogues of antibodies. Like the antibodies,
several aptamers proved to be valuable diagnostic tools5 and
promising therapeutics.6,7
In 1994 H. Hotoda (SA-1042)8 and J. R. Wyatt (ISIS 5320)9
independently reported the first anti-HIV aptamers targeted
against the V3 loop. In both cases the ODN sequences were
chemically modified to improve their resistance against nucleases
by capping the 50-ends with DMT groups (SA-1042) or by using
the phosphorothioate backbone (ISIS 5320). Subsequently, other
analogues of SA-1042 were obtained and tested,10,11 and the 6-mer
TGGGAG ODN (known as ‘‘Hotoda’s sequence’’) bearing
3,4-dibenzyloxybenzyl groups at the 50-ends and 2-hydroxy-
ethylphosphates at the 30-ends (R-95288) showed the highest
anti-HIV activity.11 CD investigations on ISIS 5320, R95288
and their analogues suggested their structuration into parallel
tetramolecular quadruplexes. Following biophysical studies
established that the presence of aromatic12,13 moieties at 50-
and/or 30-ends of TGGGAG dramatically enhances the rate of
formation of quadruplex complexes. Moreover, the overall
stability of quadruplex complexes was found to correlate with
the reported anti-HIV activity.12
The first stage of the HIV infection requires the entry of
human immunodeficiency virus (HIV) into host cells. This
stage involves the sequential interaction of the virion surface
glycoprotein gp120 with the CD4 glycoprotein and a chemokine
receptor, either CCR5 or CXCR4, on the host cell surface.1 The
CD4 glycoprotein is expressed on the surface of T-lymphocytes,
monocytes, dendritic cells and brain microglia, and its expression
makes these cells a target for HIV in vivo. Furthermore, an
interesting function of CD4 binding is to induce conformational
changes in gp120 that allow binding to the co-receptor, which is
essential for viral entry.2 The third variable region of gp120
(from now on designated V3 loop) is a pivotal component of
the co-receptor binding site, typically consisting of a 35 amino
acid-loop (range 31 to 39), closed by two cysteines that form a
disulfide bridge. The crystal structure of gp120 complexed with
the CD4 receptor and a neutralizing antibody (PDB ID 2B4C)
revealed that the V3 loop is extended away from the gp120
protein and it is involved in co-receptor binding and selection,
acting as a ‘‘molecular hook’’ that organizes associations
within the viral spike.3 The importance of gp120 and the role
of the V3 loop in HIV-1 entry and pathogenesis have led to the
recent pursuit of drugs targeted against it. One of the most
studied alternatives is the use of aptamer technology.4 Aptamers
are single stranded DNA or RNA molecules selected among
In 2010, with the aim to overcome the unfavourable entropic
factor and to stabilize the A-tetrad in Hotoda’s analogues, we
synthesized a series of new monomolecular anti-HIV aptamers
by using the Tetra-End-Linker (TEL) strategy proposed by
some of us in 2004.14,15 Several TEL-(TGGGAG)4 aptamers
were prepared and analyzed in order to probe the influence of
lipophilic groups and TEL size and position on the structural
and anti-HIV properties of the resulting TEL-quadruplexes.16
The results showed that (i) the presence of the TEL at either
50- or 30-ends was required for the anti-HIV activity, and (ii)
lipophilic tert-butyldiphenylsilyl (TBDPS) groups at the 50-ends
strongly enhanced both the stability of TEL-quadruplexes and
their anti-HIV activity. The EC50 of the best aptamer (I, Fig. 1),
bearing TBDPS groups at the 50-ends and the longer TEL at the
30-ends was 0.082 ꢀ 0.04 mM.
a
`
Dipartimento di Chimica delle Sostanze Naturali, Universita degli
Studi di Napoli Federico II, Via D. Montesano 49, I-80131 Napoli,
Italy. E-mail: Nicola.borbone@unina.it
b
`
Dipartimento di Chimica, Universita degli Studi di Napoli Federico
II, Via Cynthia 4, I-80126 Napoli, Italy
c Centre for Cancer Research and Cell Biology, Queen’s University of
Belfast, 97 Lisburn Road, BT9 7BL, Belfast, UK
d Rega Institute for Medical Research, KU Leuven,
Minderbroedersstraat 10, B-3000 Leuven, Belgium
With the aim to further improve the anti-HIV activity of
TEL-ODN aptamers, we used I as a molecular synthon for the
synthesis of a series of new TEL-ODN aptamers embodying
w Electronic supplementary information (ESI) available: Synthesis
and characterization of TEL-ODNs, as well as CD, SPR and docking
studies and biological evaluation assays. See DOI: 10.1039/c2cc34399a
c
9516 Chem. Commun., 2012, 48, 9516–9518
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The antiviral activity of II–IV against HIV-1 and HIV-2 was
determined as previously reported for I16 and is shown in Table 1.
The binding properties of active aptamers II–IV to HIV-1(IIIB)
gp120 were also determined through Surface Plasmon Resonance
(SPR) technology (Table 1; Fig. S6 in ESIw).
All new TEL-(TGGGXG)4 aptamers reported in this
communication (II–IV) retained potent anti-HIV-1 activity
(nanomolar EC50 values). They displayed comparable on
(ka) and off (kd) rates for HIV-1 gp120 binding resulting in
very similar gp120 binding affinities (KD) to I, thus confirming
that the residues of the gp120 V3 loop interact mainly with the
grooves and the sugar–phosphate backbone of the aptamers.
However, considering that the EC50’s of II and IV were
significantly lower than those of I and III (0.039–0.041 versus
0.11–0.15 mM), and that V proved to be markedly less active
notwithstanding its structuration in a stable TEL-quadruplex
structure, we hypothesized that some specific interactions between
the X5G6 bases of I–IV and the V3 loop must occur. It is well-
described that binding of agents (i.e. monoclonal antibodies) to the
viral envelope does not necessarily result in efficient virus neutra-
lization. Their effect on viral infectivity depends on the molecular
epitope that is recognized on gp120.20 Thus, it seems that I and III
bind nearly as efficiently to gp120 as II and IV but neutralize
B3-fold less efficiently HIV-1 possibly due to subtile differences
in epitope recognition. Alternatively, it cannot be excluded that
the different TEL-quadruplexes have slightly different cellular
uptake efficiencies or intracellular stability.
Fig. 1 Schematic representation of investigated TEL-ODNs.
the TGGGXG sequence (II–IV, Fig. 1). TEL-ODNs V and VI
(a TEL-(TGGGAG)4 analogue unable to form a TEL-quadruplex)
were also synthesized and studied in order to investigate the role
played by the G-tetrad at the 30-terminus of TEL-quadruplexes
I–IV and to further corroborate the requirement of quadruplex
formation for the inhibition of HIV cytopathicity. In this
communication we report the results of biophysical, biological
and SAR studies performed on I–VI.
To obtain insight into the nature of the atomistic interactions
between the TEL-aptamers I–IV and the V3 loop, we carried
out molecular modelling studies by docking I–IV to the V3 loop
of gp120 (PDB ID 2B4C). Our results revealed that (i) the V3
loop interacts with all aptamers with a similar binding mode
involving the 30-end tetrad and the TEL (Fig. S7 in ESIw),
(ii) the type of nucleobases at the aptamer-V3 loop interface
determines the chemical groups available for the interaction,
and (iii) the number and type of interactions between the
aptamer and the protein are responsible for the subtle differences
in the binding energies (Table S1 in ESIw).
All products were synthesized and characterized following
the previously described approach.14,16 In order to verify that
the presence of one (II–IV) or two (V) different bases did not
affect the parallel quadruplex arrangement observed for the
parent TEL-ODN I, CD measurements were performed
(Fig. S2 in ESIw). The overall CD profiles, recorded in
100 mM K+ buffer, were in agreement with the formation
of G-quadruplex structures, showing two positive maxima at
220 and 260 nm and a negative minimum close to 240 nm, which
are characteristic of head-to-tail arrangement of guanines, as
typically found in parallel G-quadruplexes.17–19 Unexpectedly,
CD spectra of VI were similar to those of a random coil, thus
evidencing that the insertion of a propylphosphate moiety
between the OH at 30-ends and TBDPS groups is detrimental
for quadruplex stability. CD melting analyses on II–IV
confirmed the expected great thermal stability of the new
TEL-quadruplexes (Fig. S3 in ESIw). In fact, as for I, the
apparent melting temperature of II–IV could not be determined
since no derivatizable melting curve was obtained.
As reported by Honig and co-workers,21 charged residues
are important in protein–DNA interactions. In this case, an
important role is played by the side chain of residue R181 in the
V3 loop that binds into the groove created by the phosphodiester
Table 1 Anti-HIV and SPR studies on I–V
a
EC50 (mM)
SPR vs. HIV-1(IIIB) gp120b
KD (nM) ka (1/Ms) kd (1/s)
ODN HIV-1
HIV-2
The formation of thermally stable TEL-quadruplexes for
1
II–IV was also confirmed by H NMR evidence (Fig. S4 and
I
0.11 ꢀ 0.05
1.4 ꢀ 0.9 256 ꢀ 63 (1.72 ꢀ 0.28) ꢁ 104
(4.31 ꢀ 0.37) ꢁ 10ꢂ3
II
0.041 ꢀ 0.007 Z 2
0.15 ꢀ 0.0015 1.4 ꢀ 0.87 248 ꢀ 28 (2.55 ꢀ 0.35) ꢁ 104
183 ꢀ 40 (2.40 ꢀ 0.20) ꢁ 104
(4.35 ꢀ 0.61) ꢁ 10ꢂ3
S5 in ESIw). The G-quadruplex diagnostic exchange-protected
imino proton signals were observed in all recorded spectra up
to 90 1C (100 mM K+ buffer; H2O/D2O 9 : 1). As expected,
the intensity of imino proton signals reflected the stability of
the X-tetrad within the G-quadruplex structure. Stronger
imino signals were observed for II (five stacked G-tetrads),
whereas weaker imino signals were observed for I, III and IV
due to the presence of the less stable A-, T- or C-tetrad,
respectively, that accounts for the faster exchange of imino
protons with the solvent.
III
IV
(6.29 ꢀ 0.14) ꢁ 10ꢂ3
0.039 ꢀ 0.005 0.73 ꢀ 0.16 196 ꢀ 69 (2.15 ꢀ 0.40) ꢁ 104
(4.07 ꢀ 0.71) ꢁ 10ꢂ3
V
Z 2
2
—
—
a
EC50 represents the 50% effective concentration required to inhibit
b
virus-induced cytopathicity in CEM cell cultures by 50%. HIV-1(IIIB)
gp120 was obtained from recombinantly-expressed gp120 in CHO cell
cultures.
c
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Table 2 Interaction energies of top docks (Kcal molꢂ1) and number
of H bonds between I–V and the V3 loop of gp120
interaction of the V3 loop with both the backbone and the
TEL of the aptamers is required. Furthermore, the direct
involvement of nucleobases in the interaction with the V3
loop gives additional stability to the complexes and results in a
better biological activity. Overall, the here reported results
expand our knowledge about anti-HIV G-quadruplexes and
provide the rational basis for the design of novel anti-HIV
aptamers with improved biological activity.
Aptamer
Interactions energy
Number of H bonds
I
ꢂ24.11
ꢂ28.45
ꢂ13.74
ꢂ28.09
ꢂ13.74
8
15
9
11
8
II
III
IV
V
The European Commission through the COST Action
MP0802, the Italian MURST (PRIN 2009), KU Leuven
(PF 10/18 and GOA 10/14) and the FWO are gratefully
acknowledged for the financial support. Dr Luisa Cuorvo is
also acknowledged for technical assistance.
backbone atoms (see Table S1 in ESIw). In particular, in II and
IV, R181 makes multiple interactions with both phosphates
and purine bases (G5 and G6 in II, G6 in IV). Furthermore,
the side chains of R190, T195 and E197 and the nitrogen
backbone atom of T194 of the V3 loop interact with the
oxygen atoms of the TEL, giving additional stability to the
complexes. In the II–V3 loop complex, the side chain of Y193
and the nitrogen backbone atom of Y193, T195 and I198
established additional interactions with the TEL, thereby
resulting in lower interaction energy of this complex with
respect to the other ones (Table 2). It should also be noted
that when the side chain of Y193 is involved in the interaction
(I, II and IV), the resulting complexes are found to have a
better biological activity. Thus, the G5 nucleobase in II
presents additional points for hydrogen bonding with the V3
loop. This is not seen in other bases. Therefore we infer that
the differences in activity can arise from the thermal stability
of the structures. Furthermore, this is also rationalised by the
SPR experiments.
Notes and references
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In order to better understand the structural features critical
for the biological activity, we also carried out a molecular
modelling study between the V3 loop and V, a quadruplex
structure lacking marked anti-HIV activity. Our results revealed
that the V3 loop interacts with V by using a different binding
mode (Fig. S7 in ESIw). Differently from what was observed in
the above-described complexes, V interacts with the V3 loop
primarily via the TEL atoms and no atom of nucleobases is
involved. Furthermore, except for R181, different residues of
the V3 loop, such as K182, S183, I184, and I186, are involved in
the interactions (Table S1 in ESIw). A plausible reason for the
different binding mode of V is due to the presence of the
T-tetrad at the 30-terminus. As shown in Fig. S7 in ESIw, when
there is a G-tetrad at the 30-position, the oxygen atoms of
guanines are involved in the interaction with the V3 loop
through the side chains of R181 (and Y193 in the case of IV).
In the V–V3 loop complex the methyl groups of the thymines
are positioned in the groove formed by the phosphodiester
backbone atoms, not allowing the formation of H bonds with
R181 and Y193 of the V3 loop. All together, the structural
evidence suggests that the T-tetrad at the 30-position markedly
affects the biological activity. In accordance with this finding
and with the experimental data, the aptamer II showed the best
docking score and the highest number of hydrogen bonds with
the protein (Table 2).
The anti-HIV activity against HIV-1 and HIV-2 and the
binding properties with HIV gp120 of a small library of
new TEL-aptamers (II–IV) have been reported. Results from
TEL-ODNs V and VI confirmed that the formation of a
quadruplex species by the aptamer is required, but not sufficient
to exert the anti-HIV activity. The docking data suggest that the
21 R. Rohs, S. M. West, A. Sosinsky, P. Liu, R. S. Mann and
B. Honig, Nature, 2009, 461, 1248–1253.
c
9518 Chem. Commun., 2012, 48, 9516–9518
This journal is The Royal Society of Chemistry 2012