Multivalent binding oligomers inhibit HIV Tat-TAR interaction critical for viral replication

Article abstract of DOI:10.1016/j.bmcl.2009.10.078

We describe the development of a new type of scaffold to target RNA structures. Multivalent binding oligomers (MBOs) are molecules in which multiple sidechains extend from a polyamine backbone such that favorable RNA binding occurs. We have used this stra

Full text of DOI:10.1016/j.bmcl.2009.10.078

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6893–6897
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Multivalent binding oligomers inhibit HIV Tat–TAR interaction critical
for viral replication
Deyun Wang a, Jaclyn Iera a, Heather Baker a, Priscilla Hogan b, Roger Ptak b, Lu Yang c, Tracy Hartman c,
Robert W. Buckheit Jr. c, Alexandre Desjardins d, Ao Yang d, Pascale Legault d, Venkat Yedavalli e,
Kuan-Teh Jeang e, Daniel H. Appella a,
*
a Laboratory of Bioorganic Chemistry, NIDDK, NIH, DHHS, Bethesda, MD 20892, United States
b Southern Research Institute, Frederick, MD 21701, United States
c ImQuest Biosciences Inc., Frederick, MD 21704, United States
d Département de Biochimie, Université de Montréal, Qc, Canada H3C 3J7
e Molecular Virology Section, NIAID, NIH, DHHS, Bethesda, MD 20892, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
We describe the development of a new type of scaffold to target RNA structures. Multivalent binding olig-
omers (MBOs) are molecules in which multiple sidechains extend from a polyamine backbone such that
favorable RNA binding occurs. We have used this strategy to develop MBO-based inhibitors to prevent the
association of a protein–RNA complex, Tat–TAR, that is essential for HIV replication. In vitro binding
assays combined with model cell-based assays demonstrate that the optimal MBOs inhibit Tat–TAR bind-
ing at low micromolar concentrations. Antiviral studies are also consistent with the in vitro and cell-
based assays. MBOs provide a framework for the development of future RNA-targeting molecules.
Published by Elsevier Ltd.
Received 10 September 2009
Accepted 19 October 2009
Available online 23 October 2009
Keywords:
HIV
RNA
TAR
Multivalency
While chemists have excelled at the design and synthesis of
organic molecules that inhibit protein functions by binding to active
sites, there exists a lack of basic knowledge about how one should
design a molecule to target a folded RNA.1–4 The fact that proteins
have folded, three-dimensional structures with unique binding
pockets allows chemists to develop small organic molecules that
bind with high affinity and specificity to a target protein. Because
RNA can also have folded, three-dimensional structures, it should
be possible for chemists to design new molecules that bind a target
RNA with high affinity and specificity. A wealth of structural infor-
mation on RNA has demonstrated that this biopolymer can adopt a
multitude of folded structures.5,6 In the cell, RNA often has folded
structures to create protein or small molecule binding sites or to per-
form catalytic functions.6 In many cases, the folded RNA structures
approach the complexity of folded protein structures. Despite the
amount of structural information, RNA continues to be underuti-
lized as a target for drug development because there is a lack of syn-
thetic RNA-binding molecules with well-defined molecular
recognition properties associated with biological activity.7
of cationic charge or aromatic density in each of these molecular
types, excellent binding affinity to a target RNA can be achieved;
however, affinity is usually attained at the expense of specificity
for the target. Other approaches to identify RNA-binding molecules
have explored high-throughput screening of chemical libraries
(either in vitro or in silico).8–12 While a few interesting leads from
such studies have been identified, most results contribute more to
the techniques of screening rather than identifying new molecular
frameworks for development into RNA-binding drugs. The emerg-
ing picture from these pioneering studies indicates that new types
of RNA-specific chemical scaffolds must be developed.7
We are studying the RNA-binding properties of a novel class of
molecules termed Multivalent Binding Oligomers (MBOs). These
molecules are derived from amino acids and their synthesis is sim-
ilar to that of polypeptides. MBOs are oligomeric, like polypeptides,
but an amine linkage holds the adjacent amino acids together
instead of an amide bond. MBOs were designed to bind to RNA tar-
gets while avoiding the disadvantages of other classes of RNA-
binding molecules. Specifically, MBOs contain sidechains with
non-ionic functional groups to direct the specificity of binding
through hydrogen bonding or aromatic–aromatic interactions,
while the amines in the backbone contribute ionic interactions to
facilitate binding to the anionic RNA backbone. At the same time,
the presence of the sidechains is hypothesized to reduce the
amount of non-specific binding to non-target RNAs when interac-
tions between the sidechains and RNA are unfavorable.
The most common types of molecules that have been designed
for RNA binding include aminoglycosides, polypeptides, and poly-
cyclic aromatic molecules.1 By incorporating a significant amount
* Corresponding author.
E-mail address: appellad@niddk.nih.gov (D.H. Appella).
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2009.10.078

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D. Wang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6893–6897
We report in this Letter the development and characterization
In vitro inhibition assays and optimization. Binding between TAR
and Tat is an important part of HIV replication. Before studying
MBOs in cell-based antiviral assays, the ability of MBOs to prevent
Tat–TAR formation in vitro was optimized. Using an established
competition assay,32 a series of MBO derivatives were investigated
for their ability to inhibit association between TAR and a fluores-
cently-labeled peptide derived from Tat. Each inhibition curve
was fit to a single-site binding model to provide an EC50 value. This
system was used as the principal method to evaluate the inhibitory
potential of each MBO.
As shown in Table 1, the length of the MBO has a significant
effect on inhibitory activity. For instance, the EC50 improves by
two orders of magnitude as the length increases from a YYY trimer
to a YYYYYYYY octamer. Next, the importance of side chains for
inhibition of Tat binding was investigated using a series of MBO
hexamers. Systematic replacement of a tyrosine side chain with
alanine shows only modest decreases in activity, and there is no
change in activity when this modification is introduced at positions
2 and 4 within the sequence. A derivative that consists of one tyro-
sine and five alanine side chains displays considerably weaker
activity compared to hexamers composed mostly of tyrosines. To
test whether MBOs can selectively inhibit Tat–TAR over another
protein–RNA complex, an established competition assay that mon-
itors Rev–RRE binding was used.33–35 Using the fluorescence-based
competition assay, the MBOs YYYYYY and YYYAYY displayed no
inhibition of Rev binding to RRE up to an MBO concentration of
of MBOs to target TAR, an HIV RNA that forms a stem-loop with
a three nucleotide bulge (Fig. 1A).13,14 The HIV protein Tat must
bind to TAR for viral transcription to proceed efficiently, and mol-
ecules that inhibit the association of Tat with TAR block replication
of the virus.15 TAR is present in all HIV-1 transcripts, is highly con-
served, and must fold into a stem-bulge-loop structure to be recog-
nized by Tat.16 Therefore, molecules that can bind with high
affinity and selectivity to the bulge of TAR could evolve into new
treatments for HIV infection.17 Previously developed TAR-binding
molecules have demonstrated that this is a good target to inhibit
HIV,18–21 but most of the currently available TAR binders are not
suitable for further drug development due to non-specific bind-
ing,22,23 toxicity,24,25 poor biological activity (sometimes due to
poor cellular uptake),26 or a combination of these problems. The
activity of a number of other TAR binders is still being investigated,
and in some cases only limited characterization of biological activ-
ity has been performed.27–30 The results presented below demon-
strate that proper selection of side chains and oligomer length
lead to MBOs that inhibit Tat–TAR association in vitro, are cell-per-
meable, maintain activity in a cell-based model system, and exert
anti-HIV activity in infected white blood cells across a range of dif-
ferent clinically-derived strains of HIV-1. Structural data are also
presented that confirm specific interactions between one of the
most active MBOs and the bulge of TAR.
Trimer and cell-permeability. Our starting point to develop MBO
inhibitors of Tat–TAR association was the molecule YYY (Fig. 1B),
which is derived from tyrosines and binds to the bulge of TAR with
20 lM (see Supplementary data for details). Therefore, these two
MBOs are at least 20 times more selective for inhibition of Tat–
moderate affinity (Kd ꢀ5
lM). Previously, we had developed the
TAR over Rev–RRE.
chemistry to make such molecules and provided initial character-
ization of their binding to TAR.31 Before developing more potent
derivatives, YYY was investigated to ascertain if this type of mole-
cule could be taken up by cells. Therefore, a fluorescein-labeled
version of this molecule was synthesized (YYY-Fl), and cell-uptake
studies were performed using HeLa cells. Fluorescence imaging
showed reasonable cellular penetration, with concentration of
the molecule in the nucleus and nucleolus (Fig. 1C). Based on these
encouraging initial results, we felt that other MBO derivatives
would similarly be able to permeate cells.
Other amino-acid-derived side chains were incorporated into
hexameric MBOs to determine their effects on inhibition of Tat–
TAR binding. Lysine and tryptophan have commonly been used
in peptides to improve RNA binding by increasing the amount of
cationic charge or pi-stacking between the peptide and the RNA.
Introducing one lysine sidechain at position 4 (YYYKYY) improved
the activity three times compared to the analog made entirely from
tyrosine, but incorporation of an additional lysine or a tryptophan
sidechain did not further improve the activity. Since position 4 was
amenable to sidechain variation in the MBO hexamer, the same
A
B
C
Dapi
DIC
YYY-Fl
overlay
G G
OH
U
C
G
A
O
C
G
H
N
NH2
H2N
N
N
H
G
A
G
C
U
C
H
OH
O
OH
U
YYY
C
bulge
HO
U
A
U
OH
G
A
C
C
C
U
G
G
OH
O
Me
O
H
N
H
N
H2N
N
N
H
O
H
O
G
G
C
C
16
46
OH
OH
5' 3'
TAR
YYY-Fl
Figure 1. (A) Sequence and secondary structure of TAR. (B) Initial lead MBO (YYY) with Kd ꢀ5
l
M for binding to the bulge of TAR and the fluorescein-labeled derivative (YYY-
Fl). (C) Three separate views of cellular uptake of YYY-Fl in HeLa cells (top three rows show 2
lM of YYY-Fl incubated for 30 min at 37 °C followed by washing, fixing,
permeabilization and staining; bottom row shows results with fluorescein alone).

D. Wang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6893–6897
6895
Table 1
EC50 values for MBO inhibition of Tat-peptide binding to TAR and for MBO-promoted aggregation of TAR in vitro (for fluorescence competition 100 nM TAR(49–57) and 100 nM
Tat-peptide were used; aggregation observed by native gel electrophoresis using 32P-labeled TAR (residues 17–45))
Length variation
Alanine scan
MBO sequence EC50
Sidechain variation
TAR aggregation
MBO sequence
EC50
(lM) in vitro
(lM) in vitro
MBO sequence
EC50
(l
M) in vitro
MBO sequence
EC50 (lM) in vitro
YYY
YYYY
YYYYY
YYYYYY
YYYYYYY
YYYYYYYY
83.8 3.7
8.9 1.0
3.0 0.7
1.7 0.4
0.5 0.2
0.2 0.1
YAYYYY
YYAYYY
YYYAYY
YYYYAY
YYYYYA
YAAAAA
1.9 0.3
3.2 0.4
1.6 0.3
3.9 0.5
3.3 0.5
YYYKYY
YYYKWY
YKYKYY
YKYKWY
YYYAYYY
YYYAYYYY
0.5 0.1
0.3 0.1
0.4 0.1
0.2 0.1
0.5 0.1
0.20 0.02
YYYY
YYYYY
YYYYYY
450 20
220 10
40 10
14
2
Single letter amino acid codes are used to represent the sidechain, but the backbone for each entry is similar to the structures shown in Figure 1.
position was changed to an alanine in the corresponding heptamer
and octamer (YYYAYYY and YYYAYYYY). Similarly, no detectable
change in inhibition of Tat–TAR association was observed.
and line-broadening effects (not shown), further supporting that
MBOs specifically target the Tat-binding area of TAR. Given the
modest chemical-shift changes observed upon addition of YYY
and YYYAYY, it is likely that binding of these MBOs does not signif-
icantly alter the structure of free TAR, in contrast with the binding
of argininamide and small Tat-derived peptides that cause a major
conformational change in the TAR bulge.14,36,37
The MBOs of the current study all contain a polyamine back-
bone that may be protonated under the conditions of the binding
assays and could cause non-specific RNA aggregation similar to
other types of highly charged molecules. Using native gel electro-
phoresis with 32P-labeled TAR (residues 17–45), the concentration
at which representative MBOs induced TAR aggregation was deter-
mined (see Supplementary data). The studies showed that the
aggregation of TAR is dependent on the length of the MBO; lower
EC50 values were observed for longer MBOs (Table 1). Since the
EC50 values for MBO-induced TAR aggregation are significantly
higher than the IC50 values reported in Table 1, it is likely that
the fluorescence-based competition assays correctly report specific
inhibition of the Tat–TAR complex by MBOs.
Additional methods were employed to further characterize the
association of the most active MBOs with TAR. Heteronuclear NMR
spectroscopy has been used to examine the structure of HIV-1 TAR
in both ligand-bound and free states.14,36–38 Therefore, following
the changes in chemical shifts of TAR as an MBO is titrated into
solution provides an indication of how the MBO interacts with
TAR. Upon titration of YYYAYY into TAR, the most significant chem-
ical-shift changes were observed for residues in the bulge area of
TAR (Fig. 2 and Supplementary Fig. S5), confirming that YYYAYY
specifically interacts with this bulge. Increased line broadening of
select NMR resonances is also in agreement with specific binding
of YYYAYY to the bulge of TAR (Supplementary Fig. S5), but ham-
pers high-resolution NMR structure determination of the complex.
Titration of the YYY MBO leads to similar chemical-shift changes
Characterization of Tat–TAR inhibition in a cell-based model. An
established method was used to investigate whether the in vitro
inhibition of Tat–TAR formation by MBOs could be similarly
observed in a cell-based assay, and the results guided which MBOs
would be suitable for antiviral tests.39 The assay directly probes for
inhibition of Tat–TAR complex formation relative to non-specific
binding. In this assay, HeLa cells have been modified such that
the HIV-1 Tat gene and the firefly luciferase gene (expressed on a
bicistronic mRNA), as well as an HIV-1 LTR-renilla luciferase repor-
ter gene construct are stably integrated into the HeLa cell’s DNA.
The two different luciferase proteins represent reporter signals
for HIV-1 Tat–TAR function (renilla) and non-specific toxicity (fire-
fly), and comparison of these two different signals indicates a mol-
ecule’s specificity for Tat–TAR inhibition. For instance, dose-
dependent decreases in luminescence from renilla luciferase with
no effect on firefly luciferase signal indicate that inhibition of
Tat–TAR binding occurs, while decreases in luminescence from
firefly luciferase indicate that either expression of the Tat protein
is affected (presumably due to non-specific binding) or the com-
pound is cytotoxic. The ideal inhibitor should decrease lumines-
cence from renilla luciferase without affecting firefly luciferase.
In these experiments, the IC50 is derived from the dose-dependent
decrease in renilla luciferase while the TC50 comes from changes in
0.25
H2/5-C2/5
H6/8-C6/8
H1'-C1'
G
G
G
A
U
C
30
0.20
0.15
0.10
0.05
0.00
C - G
G - C
A - U
G - C
U
U
C
40
A - U
G - C
A - U
C - G
G - C
20
G - C
5’ 3’
G17 C19 G21 U23 U25 A27 C29 U31 G33 A35 C37 C39 C41 G43 C45
TAR residues (17-45)
2
2
Figure 2. NMR chemical-shift mapping of YYYAYY binding on HIV-1 TAR. Several chemical-shift changes (
D
in ppm 0.03 ppm;
D
¼ ½ðDH
Þ
þ ð0:3 ꢁ DCÞ ꢂ1=2) were observed
after addition of 1 mM of YYYAYY to 1 mM 13C/15N-labeled TAR. Residues undergoing significant chemical-shift changes for the H10–C10, H6/8–C6/8, or H2/5–C2/5 atoms
(>0.10 ppm) are circled in grey on the secondary structure of HIV-1 TAR.

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D. Wang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6893–6897
Table 2
Since the initial discovery of Tat–TAR association as an impor-
tant part of HIV replication, there have been numerous attempts
to block formation of this protein–RNA complex with synthetic
molecules.17 To date, only a few of the currently available inhibi-
tors have the properties conducive to further development into a
drug.10,22,44–46 While research into new types of inhibitors for
Tat–TAR association is ongoing, it seems that many initial strate-
gies have been abandoned.47 In our opinion, the lack of good drug
candidates is part of a larger problem in which there exists a lack of
molecular scaffolds designed for specific targeting of RNA.
The work presented in this report provides a good starting point
to develop RNA-targeting molecules for TAR as well as other RNA
structures. In this study, fluorescence studies indicate that MBOs
bind the bulge of TAR over RRE and NMR studies corroborate the
presence of binding interactions between MBOs and the TAR bulge.
In contrast to other TAR ligands,48,49 MBOs do not induce signifi-
cant structural changes in the RNA. An interesting feature of the
MBOs is that they behave as multivalent binders of TAR that rely
on a combination of side chain and backbone interactions to
achieve their binding and specificity. As shown from the alanine
scan in Table 1, there is not a single side chain that provides the
main driving force for Tat–TAR inhibition, but rather the combina-
tion of all side chains is essential. Our results indicate that a deli-
cate balance of charge must be achieved to attain specific TAR
binding in a cell-based system. While cationic side chains can be
incorporated into an MBO to gain increased activity in vitro, the
cell-based model system shows very rapid loss in activity or spec-
ificity with modest increases in MBO charge (possibly due to in-
creased non-specific binding). The best inhibitors developed in
this study (YYYYYY and YYYAYY) maintain consistent activity
across in vitro tests, model cell-based systems, and also display
similar anti-HIV activity.
While the specificity of the MBOs synthesized in these studies
does not approach the levels seen for small molecule drugs that
target proteins, there is currently no well-defined level of specific-
ity for targeting HIV RNA. Since HIV is a highly variable and heter-
ogeneous virus, molecules that are too specific may also be
ineffective or not broadly effective. While the TAR sequence shown
in Figure 1 appears in some HIV strains, there is variability in the
sequence of the bulge from one virus clade to another.50 The obser-
vation that the MBOs listed in Table 3 display antiviral activity
against a number of different HIV clades indicates the MBOs retain
their inhibitory activity despite the natural variance among differ-
ent TAR sequences (Fig. 3). At the same time, toxicity of these MBOs
to PBMCs did not appear problematic. While specificity is an
important feature to be engineered into RNA-binding molecules,
we also feel that complete specificity may not necessarily be re-
quired or desired when targeting an RNA sequence in HIV. In par-
ticular, the propensity of HIV to mutate indicates that a highly
specific TAR-binding molecule may become rapidly ineffective
due to the development of resistance. We also note that, according
to the NMR studies, the MBO YYYAYY binds to the TAR bulge near
residues that are conserved across clades. The MBOs developed in
this study hopefully will provide a guide to future generations of
RNA-binding molecules. While significant optimization is still
Activities of MBOs against Tat and Rev function in HeLa model cell systems (errors for
IC50 values of tat function are in the Supplementary data)
MBO sequence
Tat function
Rev function
IC50
(
l
M)
TC50
(
l
M)
IC50
(
l
M)
TC50 (lM)
YYYY
YYYYY
19
30
7
>100
>100
>100
>100
>100
38
12
>100
2.4
>100
>100
34
>100
>100
—
—
60
—
>100
>100
>100
>100
>100
—
—
>100
—
YYYYYY
YYYAYY
YYYKYY
YKYKYY
YKYKWY
YYYAYYY
YYYAYYYY
6
12
35
10
16
0.9
firefly luciferase. An analogous system has also been established to
monitor Rev–RRE interactions and was used as a specificity
control.40
Results from the tests of several MBOs in the HeLa-derived
model cell systems are shown in Table 2. The results demonstrate
that the activity of the MBOs is ideal at the hexamer and heptamer
lengths. In particular, YYYYYY and YYYAYY have IC50 values for the
inhibition of Tat function in the low micromolar range with TC50
values above 100
tion, YYYYYY showed activity at 34
play any activity up to 100 M. Therefore, YYYAYY appears to be
l
M in the same assay. In the tests for Rev func-
l
M while YYYAYY did not dis-
l
highly selective. It is interesting to note that MBO hexamer vari-
ants with additional lysine sidechains or a tryptophan sidechain
actually lost potency or selectivity despite observed improvements
in the in vitro assay previously mentioned. For instance, YYYKYY is
about three times more effective at inhibiting Tat–TAR binding
than YYYYYY according to the in vitro fluorescence competition
assay (Table 1), but its IC50 value is twice as large in the Tat func-
tion assay. Incorporation of an additional lysine (as in YKYKYY)
eliminated specificity for inhibition of Tat–TAR binding compared
to non-specific inhibition (IC50 and TC50 values were about the
same). The heptamer YYYAYYY also has good activity, but it was
less specific for Tat–TAR inhibition over Rev–RRE. An octamer dis-
played considerable non-specific activity in the Tat function assay.
Antiviral activity. Based on the results of the model cell system,
three MBOs (two hexamers and a heptamer) were advanced into
tests for antiviral activity against HIV-1 infection of peripheral
blood mononuclear cells (PBMCs).41 Antiviral tests with a labora-
tory-adapted strain of HIV-1 (Ba-L) showed all three MBOs had
activity in the low micromolar range (Table 3). Based on these
results, the study was expanded to include tests against clinical
HIV-1 isolates (Clades A–O). All the MBOs displayed activity against
each HIV-1 clade that was tested. While some variation is present
between clades, the variability is not unexpected due to the hetero-
geneous nature of HIV (see discussion below). Toxicity levels of
PBMCs to the MBOs were all significantly higher than the concentra-
tions at which the molecules show activity against HIV-1.
We verified that the compounds were not preventing virus
entry using a cell-based viral entry inhibition assay in TZM-bl
cells.42,43 None of the MBOs listed in Table 3 inhibited viral entry
up to concentrations of 25 lM (Supplementary data).
Table 3
Anti-HIV activity of MBOs against infection in PBMC (all values are IC50 values in lM)
MBO sequence
HIV-1 Ba-L
A
B
C
D
E
F
G
O
UG/92/029
HT/92/599
97ZA003
UG/92/001
CMU06
BR/93/020
JV1083
BCF02
YYYYYY
YYYAYY
YYYAYYY
2
5
4
6.7
4.5
2.3
8.8
12.7
4.5
27
32
25
6.0
5.1
2.3
19.0
7.7
18.2
4.7
7.5
5.1
13.1
17.8
11.9
18.9
20.1
16.3
For the assays with HIV-1 Ba-L, the TC50 was 76, 56, 55
lM, respectively. For the assays with Clades A–O, the TC50 was >100
l
M for the hexamer MBOs and 75
lM for the
heptamer MBO (see the Supplementary data for the errors associated with the IC50 values).

D. Wang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6893–6897
15. Stevens, M.; De Clercq, E.; Balzarini, J. Med. Res. Rev. 2006, 26, 595.
6897
Site of bulge variation
U G
16. Berkhout, B.; Silverman, R. H.; Jeang, K. T. Cell 1989, 59, 273.
17. Ptak, R. G. Expert Opin. Invest. Drugs 2002, 11, 1099.
18. Turner, J. J.; Ivanova, G. D.; Verbeure, B.; Williams, D.; Arzumanov, A. A.; Abes,
S.; Lebleu, B.; Gait, M. J. Nucleic Acids Res. 2005, 33, 6837.
19. Leeper, T. C.; Athanassiou, Z.; Dias, R. L.; Robinson, J. A.; Varani, G. Biochemistry
2005, 44, 12362.
20. Chaubey, B.; Tripathi, S.; Ganguly, S.; Harris, D.; Casale, R. A.; Pandey, V. N.
Virology 2005, 331, 418.
21. Hamma, T.; Saleh, A.; Huq, I.; Rana, T. M.; Miller, P. S. Bioorg. Med. Chem. Lett.
2003, 13, 1845.
22. Litovchick, A.; Lapidot, A.; Eisenstein, M.; Kalinkovich, A.; Borkow, G.
Biochemistry 2001, 40, 15612.
23. Daelemans, D.; Schols, D.; Witvrouw, M.; Pannecouque, C.; Hatse, S.; van
Dooren, S.; Hamy, F.; Klimkait, T.; de Clercq, E.; VanDamme, A. M. Mol.
Pharmacol. 2000, 57, 116.
24. Mischiati, C.; Jeang, K. T.; Feriotto, G.; Breda, L.; Borgatti, M.; Bianchi, N.;
Gambari, R. Antisense Nucleic Acid Drug Dev. 2001, 11, 209.
25. Bueno, G. J.; Klimkait, T.; Gilbert, I. H.; Simons, C. Bioorg. Med. Chem. 2003, 11,
87.
C
G
Nucleobases that are present in other
HIV-1 clades (clade A has a single base)
C
G
G
U
A
G
A
C
U
Clade B:
G
C
C
U
Clade A
(UG/92/029):
Clade F
(BR/93/020):
-
U
U
U
A
C
U
U
U
U
C
A
G
A
C
C
Clade C
(97ZA003):
U
U
Clade G:
Clade O:
U
G
G
C
U
Clade D
(UG/92/001):
C
A
G
A
: Shaded nucleobases represent binding sites for polyamine YYYAYY.
These nucleobases are conserved across all HIV clades.
Figure 3. Points of variability within TAR compared with binding sites for YYYAYY
(HIV sequence information obtained from the HIV Database maintained by Los
Alamos National Laboratory (www.hiv.lanl.gov)).
26. Manfroni, G.; Gatto, B.; Tabarrini, O.; Sabatini, S.; Cecchetti, V.; Giaretta, G.;
Parolin, C.; Del Vecchio, C.; Calistri, A.; Palumbo, M.; Fravolini, A. Bioorg. Med.
Chem. Lett. 2009, 19, 714.
27. Tabarrini, O.; Massari, S.; Daelemans, D.; Stevens, M.; Manfroni, G.; Sabatini, S.;
Balzarini, J.; Cecchetti, V.; Pannecouque, C.; Fravolini, A. J. Med. Chem. 2008, 51,
5454.
needed for MBOs to become effective drugs, the nature of the MBO
scaffold provides significant opportunities for modification and
derivatization to target other RNA structures.
28. Mills, N. L.; Shelat, A. A.; Guy, R. K. J. Biomol. Screening 2007, 12, 946.
29. Ludwig, V.; Krebs, A.; Stoll, M.; Dietrich, U.; Ferner, J.; Schwalbe, H.; Scheffer,
U.; Durner, G.; Gobel, M. W. ChemBioChem 2007, 8, 1850.
30. Yuan, D.; He, M.; Pang, R.; Lin, S. S.; Li, Z.; Yang, M. Bioorg. Med. Chem. 2007, 15,
265.
31. Lawton, G. R.; Appella, D. H. J. Am. Chem. Soc. 2004, 126, 12762.
32. Matsumoto, C.; Hamasaki, K.; Mihara, H.; Ueno, A. Bioorg. Med. Chem. Lett.
2000, 10, 1857.
33. Moehle, K.; Athanassiou, Z.; Patora, K.; Davidson, A.; Varani, G.; Robinson, J. A.
Angew. Chem., Int. Ed. 2007, 46, 9101.
34. Mills, N. L.; Daugherty, M. D.; Frankel, A. D.; Guy, R. K. J. Am. Chem. Soc. 2006,
128, 3496.
35. Luedtke, N. W.; Baker, T. J.; Goodman, M.; Tor, Y. J. Am. Chem. Soc. 2000, 122,
12035.
36. Batey, R. T.; Inada, M.; Kujawinski, E.; Puglisi, J. D.; Williamson, J. R. Nucleic
Acids Res. 1992, 20, 4515.
37. Darfeuille, F.; Arzumanov, A.; Gait, M. J.; Di Primo, C.; Toulme, J. J. Biochemistry
2002, 41, 12186.
38. Al-Hashimi, H. M.; Gorin, A.; Majumdar, A.; Gosser, Y.; Patel, D. J. J. Mol. Biol.
2002, 318, 637.
Acknowledgements
The authors would like to thank Dr. Jim Turpin (NIAID), Dr. Lisa
Jenkins (NCI), and Mr. George Lieman (NCI) for helpful comments
on this manuscript. This research was supported in part by the
Intramural Research Programs (IRPs) of NIDDK and NIAID, and
the Intramural AIDS Targeted Antiviral Program (IATAP) at NIH.
This work was supported in part by a grant from the Canadian
Institutes for Health Research (HOP-83068 to P.L.). A.D. was sup-
ported by graduate scholarships from the Fonds de la Recherche
en Santé du Québec and the Université de Montréal. A.Y. was sup-
ported by a post-doctoral fellowship from the Université de Mont-
réal Research Group on Drug Development. P.L. holds a Canada
Research Chair in Structural Biology of RNA.
Supplementary data
39. Jeeninga, R. E.; Hoogenkamp, M.; Armand-Ugon, M.; de Baar, M.; Verhoef, K.;
Berkhout, B. J. Virol. 2000, 74, 3740.
40. Hope, T. J.; Huang, X. J.; McDonald, D.; Parslow, T. G. Proc. Natl. Acad. Sci. U.S.A.
1990, 87, 7787.
41. Buckheit, R. W., Jr.; Fliakas-Boltz, V.; Decker, W. D.; Roberson, J. L.; Stup, T. L.;
Pyle, C. A.; White, E. L.; McMahon, J. B.; Currens, M. J.; Boyd, M. R., et al Antiviral
Res. 1995, 26, 117.
Supplementary data (synthetic procedures and data for all as-
says) associated with this article can be found, in the online ver-
sion, at doi:10.1016/j.bmcl.2009.10.078.
42. Li, Y.; Svehla, K.; Mathy, N. L.; Voss, G.; Mascola, J. R.; Wyatt, R. J. Virol. 2006, 80,
1414.
References and notes
43. Li, M.; Gao, F.; Mascola, J. R.; Stamatatos, L.; Polonis, V. R.; Koutsoukos, M.;
Voss, G.; Goepfert, P.; Gilbert, P.; Greene, K. M.; Bilska, M.; Kothe, D. L.; Salazar-
Gonzalez, J. F.; Wei, X.; Decker, J. M.; Hahn, B. H.; Montefiori, D. C. J. Virol. 2005,
79, 10108.
1. Thomas, J. R.; Hergenrother, P. J. Chem. Rev. 2008, 108, 1171.
2. Foloppe, N.; Matassova, N.; Aboul-Ela, F. Drug Discovery Today 2006, 11, 1019.
3. Tor, Y. ChemBioChem 2003, 4, 998.
44. Chaubey, B.; Tripathi, S.; Pandey, V. N. Oligonucleotides 2008, 18, 9.
45. Murchie, A. I.; Davis, B.; Isel, C.; Afshar, M.; Drysdale, M. J.; Bower, J.; Potter, A.
J.; Starkey, I. D.; Swarbrick, T. M.; Mirza, S.; Prescott, C. D.; Vaglio, P.; Aboul-ela,
F.; Karn, J. J. J. Mol. Biol. 2004, 336, 625.
46. Hwang, S.; Tamilarasu, N.; Kibler, K.; Cao, H.; Ali, A.; Ping, Y. H.; Jeang, K. T.;
Rana, T. M. J. Biol. Chem. 2003, 278, 39092.
47. Haubrich, R. H.; Flexner, C.; Lederman, M. M.; Hirsch, M.; Pettinelli, C. P.;
Ginsberg, R.; Lietman, P.; Hamzeh, F. M.; Spector, S. A.; Richman, D. D. J. Infect.
Dis. 1995, 172, 1246.
48. Bardaro, M. F., Jr.; Shajani, Z.; Patora-Komisarska, K.; Robinson, J. A.; Varani, G.
Nucleic Acids Res. 2009, 37, 1529.
49. Davidson, A.; Leeper, T. C.; Athanassiou, Z.; Patora-Komisarska, K.; Karn,
J.; Robinson, J. A.; Varani, G. Proc. Natl. Acad. Sci. U.S.A. 2009, 106,
11931.
50. Spira, S.; Wainberg, M. A.; Loemba, H.; Turner, D.; Brenner, B. G. J. Antimicrob.
Chemother. 2003, 51, 229.
4. Wilson, W. D.; Li, K. Curr. Med. Chem. 2000, 7, 73.
5. Holbrook, S. R. Annu. Rev. Biophys. 2008, 37, 445.
6. Batey, R. T.; Rambo, R. P.; Doudna, J. A. Angew. Chem., Int. Ed. 1999, 38, 2326.
7. Hermann, T. Biopolymers 2003, 70, 4.
8. Krishnamurthy, M.; Schirle, N. T.; Beal, P. A. Bioorg. Med. Chem. 2008, 16, 8914.
9. Disney, M. D.; Labuda, L. P.; Paul, D. J.; Poplawski, S. G.; Pushechnikov, A.; Tran,
T.; Velagapudi, S. P.; Wu, M.; Childs-Disney, J. L. J. Am. Chem. Soc. 2008, 130,
11185.
10. Mayer, M.; Lang, P. T.; Gerber, S.; Madrid, P. B.; Pinto, I. G.; Guy, R. K.; James, T.
L. Chem. Biol. 2006, 13, 993.
11. Lind, K. E.; Du, Z.; Fujinaga, K.; Peterlin, B. M.; James, T. L. Chem. Biol. 2002, 9,
185.
12. Baumann, M.; Bischoff, H.; Schmidt, D.; Griesinger, C. J. Med. Chem. 2001, 44,
2172.
13. Aboul-ela, F.; Karn, J.; Varani, G. Nucleic Acids Res. 1996, 24, 3974.
14. Aboul-ela, F.; Karn, J.; Varani, G. J. Mol. Biol. 1995, 253, 313.