D(TGGGAG) with 5′-nucleobase-attached large hydrophobic groups as potent inhibitors for HIV-1 envelop proteins mediated cell-cell fusion

Article abstract of DOI:10.1016/j.bmcl.2011.08.007

The Hotoda's sequence substituted with TBDPS via 5′-end nucleobase existed as parallel quadruplex structure and exhibited inhibitory activities in an HIV-1 envelop proteins mediated cell-cell fusion assay. This result demonstrated that the 5′-aromatic gro

Full text of DOI:10.1016/j.bmcl.2011.08.007

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5762–5764
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Bioorganic & Medicinal Chemistry Letters
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d(TGGGAG) with 50-nucleobase-attached large hydrophobic groups as
potent inhibitors for HIV-1 envelop proteins mediated cell–cell fusion
⇑
⇑
Wei Chen, Liang Xu, Lifeng Cai, Baohua Zheng, Kun Wang, Junlin He , Keliang Liu
Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Beijing 100850, China
a r t i c l e i n f o
a b s t r a c t
The Hotoda’s sequence substituted with TBDPS via 50-end nucleobase existed as parallel quadruplex
structure and exhibited inhibitory activities in an HIV-1 envelop proteins mediated cell–cell fusion assay.
This result demonstrated that the 50-aromatic groups of the Hotoda’s sequence are allowed to have a
large spatial freedom and remain to be optimized for its role in the binding to HIV-1 envelop proteins.
Ó 2011 Elsevier Ltd. All rights reserved.
Article history:
Received 12 May 2011
Revised 18 July 2011
Accepted 2 August 2011
Available online 8 August 2011
Keywords:
Hotoda’s sequence
TBDPS
Anti-fusion activity
Nucleoside analogues
G-quadruplexes are recognized as biologically important nu-
cleic acid structures, their existence in vivo have been shown to
be related to gene expression control and cell cycling.1–8 Ex vivo
mimicking of the specific recognition and binding of G-quadru-
plexes with target proteins have also been developed by in vitro
selection. For example, oligonucleotide-based aptamers have been
utilized as therapeutic strategies, in which complex G-quadruplex
motif is thought to be crucial for the activity.9,10 Several short
G-quadruplexes have been evaluated as potential anti-HIV drug
candidates, by binding with proteins related to HIV-1 infec-
tion.11–22 T30177 (or AR177, a 17-mer G-rich sequence) was
reported to be active in inhibiting HIV replication cycles.11–15
PS-ISIS5320 (TTGGGGTT) with phosphorothioate backbone modifi-
cation17 and Hotoda’s 6-mer sequence, d(TGGGAG)4, were assessed
to be active in inhibiting HIV-1 entry by interacting with the V3
loop of the gp120 envelop protein of HIV-1.18–24 Their intramolec-
ular or intermolecular quadruplex structural motifs were supposed
to be essential for the activities. In the shortest Hotoda’s sequence,
the 50-dangling large hydrophobic group, including DMT (4,40-
dimethoxytriphenyl), TBDPS (tert-butyldiphenylsilyl), DBB (3,4-
dibenzyloxybenzyl) is another essential moiety for the anti-HIV
activity. They were linked to the sequence either with 50-O-ether
linkage or via phosphodiester linkage.19,24 On the contrary, their
attachment at 30-end of the quadruplex conducted much less posi-
tive influence. Modification with LNA (locked nucleic acids) or
TINA (twisted intercalating nucleic acids) was demonstrated to
be beneficial for its activity.25 In this study, the hydrophobic group
TBDPS was introduced to the 50-end nucleobase, as shown in
20-deoxythymidine analogues 1–3 (Fig. 1),26 and two TBDPS groups
were combined in compounds 4 and 5, all of them were used to
replace 50-dT residue in the Hotoda’s sequence for the purpose of
facilitating the design of more efficient anti-HIV structures.
The corresponding phosphoramidites of compounds 1–5 (Ref.
26 and supplementary data) were prepared and used for the syn-
thesis of ODNs 9–13 (Table 1). Unmodified ODN 7 and DMT-ODN
8 were used as the control. Deprotection of the sequences was con-
ducted with aq. ammonia at 55 °C for 2.5 h, and purified by re-
verse-phase HPLC (A: 0.1 M TEAA/5% acetonitrile, B: acetonitrile,
B in A from 10% to 80% in 20 min, at 1 ml/min), and characterized
with MALDI-TOF MS or ESI MS (Table 1).
To clarify the quadruplex formation of these modified struc-
tures in PBS, their CD spectra were measured in 10 mM PBS buffer
(pH 7.2) with the concentration of 2 OD/ml at 260 nm at 10 °C. The
widely accepted CD spectrum of a parallel tetramer has a positive
ellipticity maximum around 260 nm and a negative minimum
around 240 nm.27,28 And it is extremely sensitive to small changes
in quadruplex structure and environmental factors, such as ionic
conditions and the formation of multiple quadruplex forms. The
CD spectrum (Fig. 2, A) indicated that sequences 7 and 8 formed
quadruplexes under present conditions.19 With TBDPS on the
50-end thymine base of sequence 7 by different lengths of alkyl
linkers, quadruplexes 9–11 was also formed (Fig. 2B). Furthermore,
their CD spectrum demonstrated the significant influence of the
linkage of the hydrophobic groups on quadruplex conformation,
the greatest shift of the positive lobe was observed for quadruplex
10 with two-carbon linkage, and the least shift for quadruplex 11
⇑
Corresponding authors. Tel.: +86 010 6816 9363; fax: +86 010 6821 1656 (K.L.),
tel.: +86 010 6823 7761; fax: +86 010 6821 1656 (J.H.).
E-mail addresses: hejunlin@yahoo.com (J. He), keliangliu55@126.com (K. Liu).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.08.007

W. Chen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5762–5764
5763
O
N
O
N
O
N
O
N
RO
RO
NH
NH
RO
NH
NH
O
O
O
O
R'O
R'O
R'O
DMTO
O
O
O
O
OH
1: R = TBDPS, R' = H
4: R = TBDPS, R' = TBDPS
OH
2: R = TBDPS, R' = H
5: R = TBDPS, R' = TBDPS
OH
3: R = TBDPS, R' = H
OH
6
Figure 1. 20-Deoxythymidine analogues for chemical modification of Hotoda’s sequence.
Table 1
were listed in Table 2. The quadruplex 7 without attaching any
hydrophobic group was void of activity, while its 50-DMT deriva-
tive 8 was found to be effective. The quadruplex 8 was supposed
to interfere with the cell–cell membrane fusion by interacting with
V3 loop of gp120 through its negatively charged backbone and the
50-end large hydrophobic group, and the hydrophobic group TBDPS
acts similarly with DMT in the context.19 Fabio and his coworkers
reported that Hotoda’s sequences with 50-phosphodiester-conju-
gated aromatic groups bound to HIV-1 gp120 and gp41 specifi-
cally.24 In the present research, when the TBDPS was linked to
the 50-end nucleobase in the quadruplexes 9–11, potent anti-fusion
activities were also observed, as shown in Table 2. Especially, the
anti-fusion activity of 9 and 11 was better than that of 8.
Based on such modification method and the anti-fusion activi-
ties of 9–11, further addition of other large hydrophobic groups
could be realized at the 50-end of the quadruplexes for more candi-
dates of anti-fusion activity, as exemplified by quadruplexes 12
and 13. They also exhibited inhibitory activity. In addition, no obvi-
ous cytotoxicities were observed with all of these modified quad-
MALDI-TOF characterization of ODNs
Sequence (50–30)
MS (calc)
MS (found)
d(TGGGAG) (7)
d(6GGGAG) (8)
d(1GGGAG) (9)
d(2GGGAG) (10)
d(3GGGAG) (11)
d(4GGGAG) (12)
d(5GGGAG) (13)
1872.3
2173.5
2125.5
2139.5
2153.5
2365.1
2379.1
1871.6
2172.6
2126.5
2140.6
2155.2
2366.1
2380.9
with the one-carbon linkage. With two hydrophobic groups at the
50-end of Hotoda’s sequence, the quadruplexes 12 and 13 also
exhibited similar CD spectrum characteristic of parallel quadruplex
(Fig. 2C). The influence of the number of hydrophobic groups on
the CD spectrum was not significant in the context, as observed
between 9 and 12, and as well as between 10 and 13.
An HIV-1 envelop proteins mediated cell–cell fusion assay was
used for determining the anti-fusion activities of the quadruplexes
as reported, using HL2/3 cell (effecter cell, expressing HIV-1 gp120
and gp41 in the cell surface) and TZM-bl cell (target cell expressing
CD4 and coreceptors), and the cell–cell fusion was quantitated by a
luciferease assay.29 The inhibitory activities of the quadruplexes
ruplexes under their highest test concentration (30 lM).
The anti-fusion effect of Hotoda’s quadruplexes with different
positioned TBDPS, either at 50-hydroxyl group or 50-end nucleo-
base, indicated that 50-end hydrophobic groups might be permitted
Figure 2. CD spectra of modified Hotoda’s sequences 7–15 obtained with a 2 OD of strand concentration in 1.5 ml of 10 mM PBS at 10 °C.

5764
W. Chen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5762–5764
Table 2
A. Supplementary data
The in vitro anti-cell fusion activity of modified Hotoda’s sequences
Sequence (50–30)
IC50
(
l
M)
Sequence (50–30)
IC50
(
l
M)
Supplementary data associated with this article can be found,
in the online version, at doi:10.1016/j.bmcl.2011.08.007.
d(TGGGAG)4 (7)
d(7GGGAG)4 (8)
d(1GGGAG)4 (9)
d(2GGGAG)4 (10)
d(3GGGAG)4 (11)
ꢀ30
d(4GGGAG)4 (12)
d(5GGGAG)4 (13)
4.97 1.13
1.42 0.22
1.05 0.38
0.33 0.028
1.44 0.21
0.62 0.12
References and notes
d(TGG1GAG) (14)
d(G2GGGAG)4 (15)
ꢀ30
2.02 0.73
1. Huppert, J. L.; Balasubramanian, S. Nucleic Acids Res. 2005, 33, 2908.
2. Eddy, J.; Maizels, N. Nucleic Acids Res. 2006, 34, 3887.
3. Patel, D. J.; Phan, A. T.; Kuryavyi, V. Nucleic Acids Res. 2007, 35, 7429.
4. Han, H.; Hurley, L. H. TiPS 2000, 21, 136.
5. Gomez, D.; Guédin, A.; Mergny, J. L.; Salles, B.; Riou, J.-F.; Teulade-Fichou, M. P.;
Calsou, P. Nucleic Acids Res. 2010, 38, 7187.
6. Huppert, J. L.; Balasubramanian, S. Nucleic Acids Res. 2007, 35, 406.
7. Cogoi, S.; Paramasivam, M.; Filichev, V.; Géci, I.; Pedersen, E. B.; Xodo, L. E. J.
Med. Chem. 2009, 52, 564.
8. Sun, D.; Guo, K.; Shin, Y. J. Nucleic Acids Res. 2011, 39, 1256.
9. Navani, N. K.; Li, Y. Curr. Opin. Chem. Biol. 2006, 10, 272.
10. White, R. R.; Sullenger, B. A.; Rusconi, C. P. J. Clin. Invest. 2000, 106, 929.
11. Jing, N.; Hogan, M. E. J. Biol. Chem. 1998, 273, 34992.
12. Phan, A. T.; Kuryavyi, V.; Ma, J. B.; Faure, A.; Andréola, M. L.; Patel, D. J. Proc.
Natl. Acad. Sci. U.S.A. 2005, 102, 634.
13. Jing, N.; Rando, R. F.; Pommier, Y.; Hogan, M. E. Biochemistry 1997, 36, 12498.
14. Mazumder, A.; Neamati, N.; Ojwang, J. O.; Sunder, S.; Rando, R. F.; Pommier, Y.
Biochemistry 1996, 35, 13762.
15. Esté, J. A.; Cabrera, C.; Schols, D.; Cherepanov, P.; Gutierrez, A.; Witvrouw, M.;
Pannecouque, C.; Debyser, Z.; Rando, R. F.; Clotet, B.; Desmyter, J.; De Clercq, E.
Mol. Pharmacol. 1998, 53, 340.
16. Urata, H.; Kumashiro, T.; Kawahata, T.; Otake, T.; Akagi, M. Biochem. Biophys.
Res. Commun. 2004, 313, 55.
to have a relatively large flexibility in spacial occupation, which is
consistent with the similar effect of DBB when linked at both 50-su-
gar moiety19,20 and 50-end phosphate.24 Therefore, the spacial
occupation of the 50-hydrophobic groups could be optimized, such
as by the linkage between TBDPS and the nucleobase. As observed
in the present study, the quadruplexes with one-carbon-linked
TBDPS was the most potent of all the structures listed in Table 2.
The 50-end nucleobase was further testified with 7-deaza-20-
deoxyguanosine (G1),30 in which 7-N atom was replaced with a
carbon atom and no stable quartet could be formed as evidenced
by sequence 14 (Table 2 and Fig. 2D). When it was located at the
50-end in its 50-DMT form (G2), as shown in structure 15 (Table
2), a stable quadruplex formed (Fig. 2D) and a moderate inhibitory
effect was observed. The effect of G2 seems to be similar with that
of DMT-containing dT (6) in sequence 8. This result indicated that
50-dT in the Hotoda’s sequence is not necessary for its anti-fusion
activity, other nucleobases could play the same role.
17. Wyatt, J. R.; Vicker, T. A.; Roberson, J. L.; Buckheit, J. R. W.; Klimkait, T.;
DeBaets, E.; Davis, P. W.; Rayner, B.; Imbach, J. L.; Ecker, D. J. Proc. Natl. Acad. Sci.
U.S.A. 1994, 91, 1356.
In conclusion, a group of quadruplexes with 50-nucleobase-
modifications were synthesized, and their ability as anti-HIV-1
fusion inhibitors was examined with HIV-1 envelop proteins med-
iated cell–cell fusion assay. The 50-nucleobase-located hydrophobic
groups play a positive role in binding with HIV-1 envelop proteins,
offering a new choice for the position of hydrophobic groups in the
context of the Hotoda’s sequence. Even the 50-thymine residue
could be replaced by other nucleobase. The CD spectra demon-
strated the distinct influence of the position of the 50-end hydro-
phobic groups on the conformation of quadruplexes, although it
is not clear how the conformational change was related to the
anti-fusion activity of the quadruplexes. The anti-HIV-1 activity
of the Hotoda’s sequence could be improved by modulating the
hydrophobic groups.
18. Hotoda, H.; Koizumi, M.; Koga, R.; Momota, K.; Ohmine, T.; Furukawa, H.;
Nishigaki, T.; Kinoshita, T.; Kaneko, M. Nucleosides Nucleotides 1996, 15, 531.
19. Hotoda, H.; Koizumi, M.; Koga, R.; Kaneko, M.; Momota, K.; Ohmine, T.;
Furukawa, H.; Agatsuma, T.; Nishigaki, T.; Sone, J. J. Med. Chem. 1998, 41, 3655.
20. Koizumi, M.; Koga, R.; Hotoda, H.; Momota, K.; Ohmine, T.; Furukawa, H.;
Agatsuma, T.; Nishigaki, T.; Abe, K.; Kosaka, T.; Tsutsumi, S.; Sone, J.; Kaneko,
M.; Kimura, S.; Shimada, K. Bioorg. Med. Chem. Lett. 1997, 5, 2235.
21. Jakša, S.; Kralj, B.; Pannecouque, C.; Balzarini, J.; De Clercq, E.; Kobe, J.
Nucleosides Nucleotides Nucleic Acids 2004, 23, 77.
22. D’Onofrio, J.; Petraccone, L.; Martino, L.; Di Fabio, G.; Iadonisi, A.; Balzarini, J.;
Giancola, C.; Montesarchio, D. Bioconjugate Chem. 2008, 19, 607.
23. D’Onofrio, J.; Petraccone, L.; Erra, E.; Martino, L.; Di Fabio, G.; De Napoli, L.;
Giancola, C.; Montesarchio, D. Bioconjugate Chem. 2007, 18, 1194.
24. Di Fabio, G.; D’Onofrio, J.; Chiapparelli, M.; Hoorelbeke, B.; Montesarchio, D.;
Balzarini, J.; De Napoli, L. Chem. Commun. 2011, 2363.
25. Pedersen, E. B.; Nielsen, J. T.; Nielsen, C.; Filichev, V. V. Nucleic Acids Res. 2011,
39, 2470.
26. Zhang, D.; Xu, L.; Wei, X.; Li, Y.; He, J.; Liu, K. Nucleosides Nucleotides Nucleic
Acids 2009, 28, 924.
Acknowledgments
27. Masiero, S.; Trotta, R.; Pieraccini, S.; Tito, S. D.; Perone, R.; Randazzo, A.; Spada,
G. P. Org. Biomol. Chem. 2010, 8, 2683.
28. Balagurumoorthy, P.; Brahmachari, S. K.; Mohanty, D.; Bansal, M.;
Sasisekharan, V. Nucleic Acids Res. 1992, 20, 4061.
29. Wexler-Cohen, Y.; Shai, Y. FASEB J. 2007, 21, 1.
This work was supported by grants from the National Key
Technologies R&D Program for New Drugs 2009ZX09301-002 and
National Natural Science Foundation of China 21072229.
30. Winkeler, H. D.; Seela, F. J. Org. Chem. 1983, 48, 3119.