Mechanism of Inhibition of HIV-1 Integrase by G-tetrad-forming Oligonucleotides in Vitro

Abstract

The G-tetrad-forming oligonucleotides and have been identified as potent inhibitors of human immunodeficiency virus type 1 integrase (HIV-1 IN) activity (Rando, R. F., Ojwang, J., Elbaggari, A., Reyes, G. R., Tinder, R., McGrath, M. S., and Hogan, M. E. (1995) J. Biol. Chem. 270, 1754-1760; Mazumder, A., Neamati, N., Ojwang, J. O., Sunder, S., Rando, R. F., and Pommier, Y. (1996) Biochemistry 35, 13762-13771; Jing, N., and Hogan, M. E. (1998) J. Biol. Chem. 273, 34992-34999). To understand the inhibition of HIV-1 IN activity by the G-quartet inhibitors, we have designed the oligonucleotides and, composed of three and four G-quartets with stem lengths of 19 and 24 A, respectively. The fact that increasing the G-quartet stem length from 15 to 24 A kept inhibition of HIV-1 IN activity unchanged suggests that the binding interaction occurs between a GTGT loop domain of the G-quartet inhibitors and a catalytic site of HIV-1 IN, referred to as a face-to-face interaction. Docking the NMR structure of (Jing and Hogan (1998)) into the x-ray structure of the core domain of HIV-1 IN, HIV-1 IN-(51-209) (Maignan, S., Guilloteau, J.-P. , Qing, Z.-L., Clement-Mella, C., and Mikol, V. (1998) J. Mol. Biol. 282, 359-368), was performed using the GRAMM program. The statistical distributions of hydrogen bonding between HIV-1 IN and were obtained from the analyses of 1000 random docking structures. The docking results show a high probability of interaction between the GTGT loop residues of the G-quartet inhibitors and the catalytic site of HIV-1 IN, in agreement with the experimental observation.

Full text

Stability-Activity Relationships of a Family of G-tetrad Forming
Oligonucleotides as Potent HIV Inhibitors
A BASIS FOR ANTI-HIV DRUG DESIGN*
(Received for publication, July 23, 1999, and in revised form, October 6, 1999)
Naijie Jing‡§, Erik De Clercq¶, Robert F. Rando储, Luke Pallansch**, Carol Lackman-Smith**,
Sandy Lee‡, and Michael E. Hogan‡
From the ‡Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030,
the ¶Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven,
Belgium, the 储Biochem Pharma, Laval, Que´bec H7V 4A7, Canada, and the **Southern Research Institute,
Frederick, Maryland 21701
Recently, we have demonstrated that T30695, a G-tet-
rad-forming oligonucleotide, is a potent inhibitor of hu-
man immunodeficiency virus, type I (HIV-1) integrase
and the K
ⴙ
-induced loop folding of T30695 plays a key
role in the inhibition of HIV-1 integrase (Jing, N., and
Hogan, M. E. (1998) J. Biol. Chem. 273, 34992–34999).
Here we have modified T30695 by introducing a hydro-
phobic bulky group, propynyl dU, or a positively
charged group, 5-amino dU, into the bases of T residues
of the loops, and by substitution of the T-G loops by T-T
loops. Physical measurements have demonstrated that
the substitution of propynyl dU or 5-amino dU for T in
the T residues of the loops did not alter the structure of
T30695, and these derivatives also formed an intramo-
lecular G-quartet structure, which is an essential re-
quirement for anti-HIV activity. Measured IC
50
and EC
50
values show that these substitutions did not induce an
apparent decrease in the ability to inhibit HIV-1 inte-
grase activity and in the inhibition of HIV-1 replication
in cell culture. However, the substitution of T-T loops for
T-G loops induced a substantial decrease in both ther-
mal stability and anti-HIV activity. The data analysis of
T30695 and the 21 derivatives shows a significant, func-
tional correlation between thermal stability of the G-
tetrad structure and the capacity to inhibit HIV-1 inte-
grase activity and between thermal stability of the
G-tetrad structure and the capacity to inhibit HIV-1 rep-
lication, as assessed with the virus strains HIV-1 RF,
IIIB, and MN in cell culture. This relationship between
thermostability and activity provides a basis for improv-
ing the efficacy of these compounds to inhibit HIV-1
integrase activity and HIV-1 replication in cell culture.
Anti-HIV chemotherapy has been studied intensively for
over a decade. To date, most compounds that have been ap-
proved for the treatment of HIV infection belong to the class of
2⬘,3⬘-dideoxynucleoside analogues (1), such as AZT (zidovu-
dine) (2), DDC (zalcitabine), DDI (didanosine) (3, 4), D4T
(stavudine) (5), and 3TC (lamivudine) (6). These 2⬘,3⬘-
dideoxynucleoside analogues act as competitive inhibitors of
the reverse transcriptase, thus stopping the viral replication
cycle at the reverse transcription step. Although the combina-
tion therapy, which uses two or more drugs simultaneously to
inhibit HIV
1
activity, can reduce HIV load to undetectable
levels in the blood of many HIV-positive patients, the viruses in
T cells are still capable of replicating and infecting other cells
(7–9). The results from the assay for integrated and total HIV-1
DNA (7) demonstrated that integrated HIV-1 DNA in resting
CD4
⫹
T cells from patients receiving combination treatment is
not significantly decreased, and resting CD4
⫹
T cells seem to
be a stable reservoir for integrated HIV-1 DNA. However,
unintegrated HIV-1 DNA seems to be relatively unstable in
vitro with a short half-life in vivo. Thus, development of new
drugs against the HIV enzyme called integrase could be a
major advance in the treatment of HIV infection because it may
eliminate HIV-1 from intracellular sites. Integrase is the only
enzyme that catalyzes the integration of the HIV-1 proviral
DNA into a host chromosome, which is an essential step in
HIV-1 viral replication. The recently reported candidates for
pharmaceutical inhibition of HIV-1 integrase had IC
50
values
in the micromolar range for inhibition of HIV-1 integrase
activity (10).
A family of G-tetrad-forming oligonucleotides was recently
developed as potential anti-HIV therapeutic drugs (11–13).
These compounds have shown a strong interaction with HIV-1
integrase in vitro, and to inhibit the integration of viral DNA
into host DNA. In previous studies (12, 13), the most potent
inhibitors of HIV-1 integrase were found to be T30695, 5⬘-
g*ggtgggtgggtggg*t-3⬘, and T30177, 5⬘-g*tggtgggtgggtggg*t-3⬘.
IC
50
values of inhibition for HIV-1 integrase 3⬘processing and
strand transfer, obtained from a gel-based method, were 47 and
24 nMfor T30695 and 79 and 49 nMfor T30177. Compared with
T30177, T30695 forms an even more stable and orderly G-
quartet fold. Our NMR and kinetic data demonstrated that in
response to K
⫹
binding, T30695 folded into a stable and sym-
metric G-tetrad complex (13, 14). The folding has been shown
to be a two-step process, which is dependent on the nature of
the alkaline metal ion. The first step of the process involves the
coordination of one K
⫹
ion, which competes with a Li
⫹
ion to
bind within the core of two G-quartets. The second step in-
volves the binding of two additional K
⫹
ions to the loop do-
mains. NMR and optical analysis have shown that the second
binding step is associated with substantial ordering of the
oligonucleotide fold. NMR data and molecular modeling have
determined (14, 15) that T30695 in the absence of K
⫹
(with Li
⫹
ions) forms an intramolecular G-quartet structure with the
* This work was supported by National Institutes of Health Grants
GM60153 (to N. J.) and CA74173 (to M. E. H.). The costs of publication
of this article were defrayed in part by the payment of page charges.
This article must therefore be hereby marked “advertisement”inac-
cordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 713-798-3685;
Fax: 713-798-6033; E-mail: njing@bcm.tmc.edu.
1
The abbreviations used are: HIV, human immunodeficiency virus;
SPA, scintillation proximity assay; TBA, thrombin-binding aptamer;
PT, phosphorothioate.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 5, Issue of February 4, pp. 3421–3430, 2000
© 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org 3421

opened loop structures. Upon coordination with three K
⫹
ions,
the loop structure is rearranged, and the bases of loops are
folded onto the underlying G-quartets. The structure of T30695
in the presence of K
⫹
becomes symmetric and compact. The
inhibition of HIV-1 integrase activity was found to greatly
increase upon K
⫹
binding to the loops. Thus, the folding of the
loop domains of these oligonucleotides plays an important role
in the function of G-tetrad-forming oligonucleotides.
To investigate the structure and activity of these tetrad-
forming oligonucleotides and to improve inhibition of HIV-1
integrase activity and/or inhibition of HIV-1 replication in cell
culture, we have designed derivatives by adding positively
charged or large hydrophobic groups into T30695. The deriva-
tives were designed to replace T residues in the loop domains
with 5-amino dU or with 5-propynyl dU or to substitute G in
the loop domains with T. The derivatives were monitored for
melting temperature (T
m
), inhibition of HIV-1 integrase activ-
ity (IC
50
), and the inhibition of HIV-1 replication in cell culture
(EC
50
). Based upon these measurements, we propose a rela-
tionship between thermal stability of the G-quartet structure
and its ability to inhibit the activity of HIV-1 in cell culture,
which could be useful as the basis for improvement of these
oligonucleotides as anti-HIV drug candidates.
EXPERIMENTAL PROCEDURES
Oligonucleotide Synthesis—Oligonucleotides used in this study were
synthesized on an Applied Biosystems Inc. DNA synthesizer, model 380D
or 394, using standard phosphoramidite chemistry or fast deblocking
expedite chemistry on a Milligen synthesizer as described previously (11,
13). Purification was accomplished by preparative anion exchange high
pressure liquid chromatography on Q-Sepharose, followed by pressure
filtration in H
2
O (Amicon) to remove metal ions. Product purity was
confirmed by analytical Q-Sepharose chromatography and by denaturing
electrophoresis of
32
P-labeled oligomers on a 20% polyacrylamide/bis-
acrylamide (19/1), 7 Murea gel matrix. Oligomer folding was monitored by
native gel electrophoresis on a 15% acrylamide (19:1) matrix in Tris-
borate EDTA. 5-Propynyl dU was obtained from Glenn Research and was
used in oligonucleotide synthesis.
Thermal Denaturation—Oligonucleotides at 7
␮
Min strand equiva-
lents (20 mMLi
3
PO
4
, pH 7) were heated to 90 °C for 5 min and then
incubated for1hat37°Cinthepresence of KCl at 0.1, 0.5, and 1.0 mM.
Subsequent to the incubation step, thermal denaturation profiles of the
oligonucleotides were obtained at a rate of 1.25 °C/min over a range of
20–90 °C. Absorbance was measured at 240 nm by an HP8452A
(Hewlett-Packard) diode array spectrophotometer using an HP 89090A
temperature regulator.
The thermal denaturation curves of the oligonucleotides were ana-
lyzed by an intramolecular folding equilibrium (16) as shown in the
following equations.
A共T兲⫽共1⫺
␣
兲Arc ⫹
␣
Ast (Eq. 1)
FIG.1.A, intramolecular G-tetrad folding model of T30695. The for-
mation of G-quartet is indicated by dashed lines.B, two-step kinetic
model for ion-induced folding of T30695. C, the T-G-T-G loop base
alignment formed in T30695 in the presence of K
⫹
ions as calculated
from NMR and modeling (15). A plausible T-G-T-G loop base alignment
which can be formed in T30929 (D) and in T40106 (E) when the two
thymidines are substituted with 5-propynyl dU and 5-amino dU, re-
spectively (see text for details).
FIG.2.The molecular structure of T30695 in the absence of K
ⴙ
,
referred to as the Li
ⴙ
form, and in the presence of K
ⴙ
, referred
to as the K
ⴙ
form. The two molecular structures were calculated based
upon NMR constraints (15). Comparing the two structures with each
other, Li
⫹
form structure is an intramolecular G-quartet with twisted
G-quartet plates and opened loops. K
⫹
form structure shows a more
symmetric and compact G-tetrad with about 15 Å width and 15 Å
length.
TABLE I
The oligonucleotides with 0.1 mMKCl in 20 mMLi
3
PO
4
,pH7
*, PT linkages; T, propynyl dU.
Oligomer Sequence T
m
⌬H° ⌬S° ⌬G°(T⫽295K) Fitting coefficient
(°C) kcal/mol (cal/K 䡠mol) (kcal/mol)
T30695 5⬘-g*ggtgggtgggtggg*t-3⬘46.3 ⫺53.29 ⫺166.90 ⫺4.06 0.9995
T30925 5⬘-g*ggTgggtgggtggg*t-3⬘51.0 ⫺64.49 ⫺199.02 ⫺5.77 0.9995
T30926 5⬘-g*ggtgggTgggtggg*t-3⬘51.1 ⫺64.17 ⫺197.98 ⫺5.76 0.9992
T30927 5⬘-g*ggtgggtgggTggg*t-3⬘51.6 ⫺64.64 ⫺199.15 ⫺5.89 0.9997
T30928 5⬘-g*ggtgggtgggtggg*T-3⬘53.7 ⫺70.25 ⫺215.01 ⫺6.82 0.9991
T30929 5⬘-g*ggTgggtgggTggg*t-3⬘48.6 ⫺62.79 ⫺195.24 ⫺5.19 0.9996
T30924 5⬘-g*ggTgggTgggTggg*t-3⬘48.7 ⫺45.59 ⫺141.72 ⫺3.78 0.9994
Stability-Activity Relationships of G-tetrad Oligos3422

␣
⫽共0.5 ⫹0.5 共Keq ⫺1兲/关共1⫺Keq兲2⫹4
␴
Keq兴1/2兲(Eq. 2)
Keq ⫽exp关共 ⫺⌬H°⫹T⌬S°兲/RT兴(Eq. 3)
where K
eq
is the constant for the random coils to folded oligonucleotide
equilibrium,
␣
is the fraction of folded strands, 1 ⫺
␣
is the fraction of
random coils, A(T) is the absorbance at temperature T, A
rc
is the
absorbance when all strands are random coils, A
st
is the absorbance
when all strands are folded, and
␴
is the cooperativity of the melting
transition, which is referred to the helix interruption constant and
␴
⫽
exp(⌬S
i
/R) where ⌬S
i
is in units/mol of interruption. In our analysis
study, the values of
␴
are in the range of 0.9–0.999, determined by a
optimized fitting program. Values for T
m
and ⌬Gwere obtained on the
basis of the fitting procedure, which inputs the values of ⌬H°, ⌬S°, A
rc
,
and A
st
estimated from the experimental measurements and then uses
an optimized fitting program to search for the best fit.
Gel Electrophoresis—The G-rich oligonucleotides, T30695 and its
derivatives, plus 10 mMKCl in 20 mMLi
3
PO
4
, pH 7, were labeled by
32
P,
using 5⬘-end labeling procedure and purified using Microspin G-25
columns. The oligonucleotide solution was heated 90 °C for 5 min and
cooled at 4 °C for 30 min. 20% polyacrylamide gels (with 10 ⫻Tris-
borate EDTA, 10% ammonium persulfate, and 30
␮
lofN,N,N⬘,N⬘-
tetramethylethylenediamine) in 1⫻TBA buffer was precooled in a 4 °C
cold room for an hour. Then the gels with loaded samples were run for
4–6 h in the 4 °C cold room.
Assays for Inhibition of HIV-1 Integrase Activity in Vitro and for
Inhibition of HIV-1 Replication in Cell Culture—Anti-HIV integrase
activity was determined utilizing a 96-well scintillation proximity assay
(SPA) according to the manufacturer’s protocol (Amersham Pharmacia
Biotech). Briefly, each test reaction contained 1) tritiated oligonucleo-
tide substrate in assay buffer supplemented with 50 mMMnCl
2
(pH was
adjusted according to the experimental protocol), 2) diluted test mate-
rial, and 3) diluted integrase enzyme (final concentration, ⬃50 nM)ina
total of 100
␮
l; the total mixture contained a final concentration of 20
mMHEPES, pH 7.5, 10 mMdithiothreitol, 0.05% (w/v) Nonidet P-40,
and 0.05% (w/v) sodium azide. Following incubation for1hat31–33 °C,
the reaction was stopped with 50 mMEDTA, pH 8, and 110
␮
lofSPA
FIG.3.A stereo view of the top of the molecular structure of T30695 in the K
ⴙ
form. The structure shows that each of the T methyl
groups of the two thymidines (green) is pointed out from the folded surface.
TABLE II
The oligonucleotides with 0.5 mMKCl in 20 mMLi
3
PO
4
,pH7
*, PT linkages; T, propynyl dU.
Oligomer Sequence T
m
⌬H° ⌬S° ⌬G°
(T⫽295K) Fitting coefficient
°C kcal/mol (cal/K 䡠mol) (kcal/mol)
T30695 5⬘-g*ggtgggtgggtggg*t-3⬘59.7 ⫺68.88 ⫺207.07 ⫺7.80 0.9993
T30925 5⬘-g*ggTgggtgggtggg*t-3⬘61.8 ⫺76.80 ⫺229.36 ⫺9.14 0.9997
T30926 5⬘-g*ggtgggTgggtggg*t-3⬘61.5 ⫺77.60 ⫺230.38 ⫺9.09 0.9994
T30927 5⬘-g*ggtgggtgggTggg*t-3⬘62.2 ⫺74.55 ⫺222.39 ⫺8.94 0.9996
T30928 5⬘-g*ggtgggtgggtggg*T-3⬘63.2 ⫺89.47 ⫺266.08 ⫺10.97 0.9919
T30929 5⬘-g*ggTgggtgggTggg*t-3⬘59.1 ⫺81.74 ⫺246.13 ⫺9.13 0.9991
T30924 5⬘-g*ggTgggTgggTggg*t-3⬘57.6 ⫺64.3 ⫺194.50 ⫺6.92 0.9994
TABLE III
The oligonucleotides with 1.0 mMKCl in 20 mMLi
3
PO
4
,pH7
*, PT linkages; T, propynyl dU.
Oligomer Sequence T
m
⌬H° ⌬S° ⌬G°
(T⫽295K) Fitting coefficient
°C kcal/mol (cal/K 䡠mol) (kcal/mol)
T30695 5⬘-g*ggtgggtgggtggg*t-3⬘69.8 ⫺66.97 ⫺195.32 ⫺9.35 0.9963
T30925 5⬘-g*ggTgggtgggtggg*t-3⬘68.8 ⫺77.90 ⫺227.92 ⫺10.66 0.9997
T30926 5⬘-g*ggtgggTgggtggg*t-3⬘69.1 ⫺68.27 ⫺199.60 ⫺9.39 0.9991
T30927 5⬘-g*ggtgggtgggTggg*t-3⬘68.7 ⫺79.40 ⫺232.38 ⫺10.85 0.9996
T30928 5⬘-g*ggtgggtgggtggg*T-3⬘71.1 ⫺74.95 ⫺217.83 ⫺10.69 0.9996
T30929 5⬘-g*ggTgggtgggTggg*t-3⬘66.4 ⫺80.41 ⫺236.90 ⫺10.53 0.9994
T30924 5⬘-g*ggTgggTgggTggg*t-3⬘65.0 ⫺77.81 ⫺230.23 ⫺9.89 0.9984
Stability-Activity Relationships of G-tetrad Oligos 3423

bead/denaturing reagent was added to each well and mixed gently.
Plates were sealed and incubated at room temperature for 30 min; all
compounds were tested in duplicate. The degree of integrase-catalyzed
oligonucleotide strand transfer was then quantified with a Wallac Mi-
croBeta scintillation counter, and the resulting data were utilized to
calculate the IC
50
value for each test compound.
Virus culture assays for inhibition of the HIV-1 cytopathicity induced
by the three HIV-1 virus strains, IIIB, MN, and RF, in MT-4 cells were
carried out as described elsewhere (17).
RESULTS
Molecular Structure and Thermal Stability—T30695, 5⬘-
g*ggtgggtgggtggg*t-3⬘, forms an extremely stable intramolec-
ular G-quartet structure in the presence of K
⫹
via a two-step
process that involves the binding of one K
⫹
ion to a central pair
of G-quartets and two additional K
⫹
ions to loops (Fig. 1, Aand
B). NMR data and molecular modeling (14, 15) have demon-
strated that T30695 in the presence of Li
⫹
forms an asymmet-
ric, less stable G-quartet structure with twisted G-quartet
plates and opened loops, referred to as the Li
⫹
form structure
(Fig. 2). Upon coordination with three K
⫹
ion equivalents, the
structure of T30695 becomes symmetric and compact (15 Å
width and 15 Å length), referred to as the K
⫹
form structure
(Fig. 2). This coordination greatly increases the thermal stabil-
ity of the molecular structure of T30695 and its activity on
HIV-1 integrase inhibition. As seen in Tables I—III, the T
m
of
T30695 increased as a function of K
⫹
concentration, e.g. 46.3 °C
at 0.1 mMKCl, 59.7 °C at 0.5 mMKCl and 69.8 °C at 1.0 mM
KCl. The free energy, ⌬G°, of the molecular structure of T30695
decreased from ⫺4.06 kcal/mol at 0.1 mMKCl to ⫺9.35 kcal/mol
at 1.0 mMKCl.
The NMR and molecular modeling observations (14, 15) sug-
gest that several features of the structure of T30695 should be
considered when attempting to rationalize the inhibition of
HIV-1 integrase, even though the molecular structure of the
integrase-T30695 complex is not available yet. The K
⫹
form
structure of T30695 lacks a groove, and its two ends are nearly
planar. This cylindrical symmetry and large surface area may
increase the probability of T30695 binding to HIV-1 integrase.
The base planes of all G and T residues of T30695 are coordi-
nated with K
⫹
ions, and the coordination greatly increases the
thermal stability of the structure while greatly decreasing its
capacity for dimers or higher aggregate formation. The higher
stability and lowered capacity for aggregation may enhance the
ability to resist nuclease degradation and for its efficient deliv-
ery into cells. Three unhydrated K
⫹
ions are bound to T30695,
creating a cylinder with positive charges inside and negative
charges on the surface, which also could be a factor for en-
hanced interaction between T30695 and HIV-1 integrase.
NMR data also showed that upon binding K
⫹
ions, the bases
of T-G-T-G loop domains of T30695 folded into an approximate
plane aligned with the G-quartets. The formation of the hydro-
gen bonds in the loop structure greatly increased the thermal
stability of G-quartet structure of T30695. When the bases of
T-G-T-G loop domains fold in an approximate plane, each of the
FIG.4.A, one-dimensional Ising model employed to fit the melting
curve of T30927 in 1.0 mMKCl in 20 mMLi
3
PO
4
buffer (pH 7), obtained
by UV absorption at the wavelength of 240 nm. The function used to fit
the data is that of Longfellow et al. (16): A(T)⫽(1 ⫺
␣
)A
rc
⫹
␣
A
st
;
␣
⫽
0.5 ⫹0.5(K
eq
⫺1)/[(1 ⫺K
eq
)
2
⫹4
␴
K
eq
]1⁄2; and K
eq
⫽exp[(⫺⌬H⫹
T⌬S°)/RT] (see text for details). B, the calculated ⌬G° values of T30924,
T30695, T30929, and T30925 were plotted versus Log
10
[KCl]. These
data were fitted to a straight line yielding a slope (⌬⌬G°/⌬Log
10
[K
⫹
]) of
5.8, 5.3, 5.3, and 4.9 for T30924, T30695, T30929, and T30925, respec-
tively. According to the simple model of the transition between the
folded state and unfolded state for intramolecular tetrad (13), the
values of the released K
⫹
equivalents, ⌬n(⫽⌬⌬G°/2.3RT⌬Log
10
[K
⫹
]), of
T30924, T30695, T30929, and T30925 are about 4.2, 3.8, 3.8, and 3.5,
respectively, which correspond to the range of releasing three K
⫹
ion
equivalents.
TABLE IV
The oligonucleotides with 1.0 mMKCl in 20 mMLi
3
PO
4
,pH7
I, 5-amino du(3C).
Oligomer Sequence T
m
⌬H° ⌬S° ⌬G°
(T⫽295K) Fitting coefficient
°C kcal/mol (cal/K 䡠mol) (kcal/mol)
T40101 5⬘-gggIgggtgggtgggt-3⬘69.2 ⫺73.37 ⫺214.43 ⫺10.12 0.993
T40102 5⬘-gggtgggIgggtgggt-3⬘69.5 ⫺64.11 ⫺187.18 ⫺8.89 0.998
T40103 5⬘-gggtgggtgggIgggt-3⬘69.3 ⫺59.33 ⫺173.33 ⫺8.20 0.996
T40104 5⬘-gggIgggIgggtgggt-3⬘69.6 ⫺71.74 ⫺209.42 ⫺9.96 0.997
T40105 5⬘-gggtgggIgggIgggt-3⬘67.2 ⫺82.68 ⫺243.04 ⫺10.99 0.996
T40106 5⬘-gggIgggtgggIgggt-3⬘65.9 ⫺50.80 ⫺149.89 ⫺6.58 0.995
T40107 5⬘-gggIgggIgggIgggt-3⬘69.2 ⫺49.30 ⫺144.88 ⫺6.69 0.993
Stability-Activity Relationships of G-tetrad Oligos3424

T methyl groups (CH
3
) is pointed out of the folded plane and
faces the solution (Fig. 3). That special orientation for the eight
T methyl groups of T30695 provides potential sites for the
substitution of other chemical groups, such as bulky groups or
charged groups, without disruption of loop structure.
T30924 –T30929 —In this study, a set of T30695 derivatives
were synthesized to investigate the relationship between se-
quence and thermal stability and the relation between the
thermal stability of the oligomers and their anti-HIV activity.
One oligonucleotide set (T30925–T30928) was derived from
T30695 by replacing one T of the loop domains with a propynyl
dU, whereas T30929 and T30924 were derived by the substi-
tuting two and three propynyl dUs (Tables I–III and Fig. 1D).
Thermal denaturation of these oligonucleotides was measured
optically at three K
⫹
ion concentrations (0.1, 0.5, and 1.0 mM
KCl), and the resulting absorbance versus temperature curves
were analyzed by curve fitting, using a two-step formalism for
the folding equilibrium proposed by Longfellow et al. (16). Fig.
4Ashows a representative analysis of T
m
for T30927 in 1.0 mM
KCl. The data points are UV absorbance values at 240 nm, and
the solid line is a curve fitting to the data points with a fitting
coefficient of 0.9996, based upon the relationships derived by
Longfellow et al. (16). Coefficients of the other members of the
set were also in the 0.99 or higher range. The free energy of
T30927 folding in 1.0 mMKCl at 295 K, ⌬G°⫽⫺10.85 (kcal/
mol), was obtained from this analysis (Table III), as were
equivalent values for the other oligonucleotides of this family.
As seen from these data, the substitution of propynyl dU in
the loops does not induce a substantial change in the thermal
stability of the G-quartet based folding when compared with
T30695 (Tables I–III). The average T
m
and ⌬Gof these oligo-
nucleotides were 50.1 ⫾3.8 °C and ⫺5.32 ⫾1.54 kcal/mol) in
0.1 mMKCl (Table I), 60.9 ⫾3.3 °C and ⫺8.86 ⫾2.11 kcal/mol
in 0.5 mMKCl (Table II), and 67.9 ⫾3.2 °C and ⫺10.24 ⫾0.89
kcal/mol in 1.0 mMKCl (Table III). The slope of a linear regres-
sion of ⌬G°versus log[K
⫹
] for T30695 and its derivatives was
5.3 ⫾0.5 (independent of propynyl substitution) with fitting
coefficients of 0.96 to 1.0 (Fig. 4B). The ⌬n(⫽⌬⌬G°/
2.3RT⌬Log
10
[K
⫹
]) values of T30695, T30924, T30925, and
T30929 (3.8, 4.2, 3.5, and 3.8, respectively) demonstrate that
three or more K
⫹
equivalents are released from the folded
structures upon the melting of T30695 and each of the propynyl
dU derivatives. The K
⫹
-induced loop folding of T30695 by bind-
ing three K
⫹
ions was previously confirmed by NMR titration
study (13), and the loop folding of T30695 plays a key role in
structure stability and in inhibition of HIV integrase activity
(15). The plots in Fig. 4Bshow that both thermal stability and
K
⫹
-induced loop folding are not significantly affected by the
propynyl substitution. Interestingly, T
m
values of T30928 sug-
gest that propynyl dU at the 3⬘-end may lead to a higher
thermal stability (Tables I–III).
T40101–T40107—This set of T30695 derivatives was de-
signed to investigate the inhibition of HIV-1 integrase activity
by a G-tetrad structure with added positive charge in the loop
domains, using the substitution of 5-amino dU for T 5-methyl
(CH
3
). T40101–T40103 possess one such substitution, so that
the loop with a 5-amino dU will carry one positive charge.
T40104 and T40105 have two such substitutions in the two
T-G-T-G loop domains (top and bottom). T40106 has two posi-
tive charges in a single T-G-T-G loop domain (Fig. 1E). T40107
was designed by substituting three T methyls with three 5-a-
mino dUs. The thermal denaturation measurements in Table
IV show that in 1.0 mMKCl, T
m
values of T40101–T40107 are
in the range of 66–70 °C, the same as T
m
of T30695. The ⌬G°
values of T40101–T40105 are also in the same range of ⌬G°of
T30695. T40106 and T40107 have a slight lower ⌬G°, which
could result from the two substitutions of 5-animo dU in a
single T-G-T-G loop domain.
To confirm the obtained T
m
values of T30695 and its deriv-
atives, we determined the influence on T
m
by employing a
different heating rate in the melting studies. We found that
upon alteration of heating rate from 0.5 to 2.5 °C/min, the T
m
values of these G-quartet structures shifted less than 1 °C
when T
m
was in the range of 60–80 °C (data not shown). Our
results suggest that the T
m
values of these G-tetrad-forming
oligonucleotides are independent of the heating rate in the
range of 0.5–2.5 °C/min. The previous kinetic studies of T30695
and TBA demonstrated that in the presence of K
⫹
the first
folding step, forming a self-associated G-quartet structure, is
FIG.5.Electrophoresis of T30695, TBA, and derivatives in the
presence of 10.0 mMKCl in nondenaturating gels (see text for
details). A, the oligos used are T30928, T30929, T30924, TBA, and
T30695 from left to right.B, the oligos used are T40105, T40102,
T40103, TBA, T30695, T40104, and T40106 from left to right.
TABLE V
Oligomer Sequence T
m
(1 mMKCl) ⌬G
(T⫽295K)
EC
50
HIV-1 RF HIV-1 IIIB HIV-1 MN
°C kcal/mol nMnMnM
T30177 5⬘-g*tggtgggtgggtggg*t-3⬘54.0 ⫺6.19 82 ⫾10 170 ⫾50 480 ⫾100
T30695 5⬘-g*ggtgggtgggtggg*t-3⬘69.8 ⫺9.35 8.2 ⫾0.6 14 ⫾355⫾20
T30916 5⬘-ggg ttg gtg ggt ggg-3⬘45.4 ⫺3.56 280 ⫾100 400 ⫾280 340 ⫾120
T30917 5⬘-ggg ttg gtg ggt ggg t-3⬘42.6 ⫺2.90 550 ⫾320 450 ⫾50 1100 ⫾300
T30918 5⬘-ggg tgg gtt ggt ggg-3⬘47.2 ⫺3.09 450 ⫾280 400 ⫾220 530 ⫾50
T30919 5⬘-ggg tgg gtt ggt ggg t-3⬘47.3 ⫺2.26 720 ⫾120 350 ⫾130 740 ⫾130
T30920 5⬘-ggg tgg gtg ggt tgg-3⬘46.4 ⫺2.77 420 ⫾40 180 ⫾80 680 ⫾20
T30921 5⬘-ggg tgg gtg ggt tgg t-3⬘47.3 ⫺1.83 450 ⫾50 250 ⫾100 540 ⫾90
T30922 5⬘-ggg tgg gtg ggt ggg-3⬘65.1 ⫺11.08 37 ⫾14 18 ⫾3 150 ⫾50
T30923 5⬘-ggg tgg gtg ggt ggg t-3⬘65.2 ⫺9.68 68 ⫾28 26 ⫾5 212 ⫾45
Stability-Activity Relationships of G-tetrad Oligos 3425

very fast (15). In melting measurements UV absorption at
240-nm wavelength mainly monitors the disordered and reor-
dered G-quartet structures of these oligonucleotides. The fact
that T
m
values of T30695, TBA, and derivatives are independ-
ent of heating rate in the range of 0.5–2.5 °C/min could result
from a fast forming G-quartet structure.
The Structure of T30695 Derivatives—The structural stabil-
ity of T30695 derivatives, T30924–T30929, and T40101–
T40107, were thoroughly measured, and T
m
values of these
derivatives did not show significantly different from that of
T30695 (Tables I–IV). T30924–T30929 were also estimated to
have the same ion binding equivalents with T30695 (Fig. 4B).
Hence it was suggested that T30695 derivatives T30924–
T30929 and T40101–T40107 form an intramolecular G-quartet
structure the same as that of T30695. Further evidence to
support the suggestion was provided by running nondenatur-
ating gels at 4 °C. T30695 and TBA were used as controls
because the structures of T30695 and TBA have been deter-
mined to form an intramolecular G-quartets with two G-quar-
tets in central by NMR (15, 20, 21). In the presence of K
⫹
,
T30928, T30929, and T30924, substituted T methyls by one,
two, and three 5-propynyl dUs, respectively, have the bands at
the same position with those of T30695 and TBA (Fig. 5A). As
seen in Fig. 5B, all the bands of T40102–T40106, with substi-
tutions of T methyls by 5-amino dUs at different T residues,
also have the same migration with those of T30695 and TBA.
The rate of migration of an oligonucleotide in nondenaturating
gels depends on the size of its molecular structure. The same
migrational rates indicate that these oligonucleotides have the
same structural size. These results show clearly that T30924–
T30929 and T40101–T40107 form the same molecular confor-
mation as that of T30695 in the presence of K
⫹
, and the
substitution of 5-propynyl dU or 5-amino dU for the loop T
methyl does not disrupt the formation of an intramolecular
G-quartet structure.
T30916 –T30923—In previous studies (13, 15), it has been
shown that K
⫹
-induced loop folding of T30695 plays a key role
in inhibition of HIV-1 integrase activity. The substitution of
T-T loops for T-G loops significantly decreases anti-HIV inte-
grase activity. To further investigate the capacity to inhibit
HIV-1 replication in cell culture by substituting T-T loops for
T-G loops and the resistance of degradation using phosphoro-
thioate (PT) linkages, we designed a set of oligonucleotides
(T30916–T30923) as listed in Table V. This oligonucleotide set
was generated by three type of modifications of T30695: 1)
terminal PT linkages were replaced by phosphodiester linkages
in the G1 and G15 positions (T30923); 2) one of the three T-G
loops of the G-quartet structure of T30695 was substituted by
a T-T loop (T30916–T30921); and 3) the thymine at the 3⬘-end
of T30695 was eliminated in the sequences of T30916, T30918,
T30920, and T30922.
T
m
values of T30916 to T30923 were measured in 1.0 mMKCl
(Table V). T30923 had the close T
m
and ⌬G° to T30695, thereby
confirming that themodynamic stability is not altered by PT
substitution. Elimination of the thymine at the 3⬘-end had no
apparent influence on the stability of the G-quartet structures
because matched pairs of the oligonucleotides, T30916 and
T30917, T30918 and T30919, T30920 and T30921, and T30922
and T30923, had similar T
m
values and ⌬G° values. However,
T
m
values of T30916–T30921 were about 46.0 ⫾3.4 °C to
nearly 20 °C lower than that of T30695. In each of those in-
stances, the observed decrease in thermal stability of the G-
quartet structures was caused by the conversion of a single T-G
loop to form a T-T loop, in general agreement with previous
studies (14). The studies have proven that loop folding is cru-
cial to overall stability of T30695 derivatives.
Inhibition of HIV-1 Integrase Activity and Inhibition of
HIV-1 Replication in Culture—Previous studies (13, 15) have
shown that T30695 is a potent HIV-1 inhibitor and has sug-
gested that the intramolecular G-tetrad fold might be a re-
quirement for the inhibition of HIV integrase activity. To con-
firm that hypothesis, we monitored the inhibitory capacity of
the entire set of 22 T30695 derivatives in this study. Because
the members of this set are very similar in overall sequence
structure but show systematic variation in the free energy of
folding, we have used this set to verify whether the capacity to
inhibit integrase is a direct function of the stability of the
intramolecular G-tetrad fold. To acquire quantitative data for
this analysis, the measurements of the IC
50
values of T30695
and its derivatives were carried out in a 96-well-based HIV-1
SPA.
The data in Table VI were obtained with gel-based methods
(12, 13) or by the SPA assay (Fig. 6A) and revealed a relatively
small error for all IC
50
values. T30695 and T30177 show strong
inhibition of HIV-1 integrase. However, the derivatives of
T30695 or T30177 with the substitutions of T-T or T-G-T loops
for T-G loops, such as the TBA and T30676–T30679, gave
poorer IC
50
values. As seen in Fig. 7, the plots of Log (IC
50
)
versus T
m
revealed an apparent linear correlation between the
inhibition of HIV-1 integrase activity and structural stability in
both assay systems. The correlation coeffiecients for a linear
fitting of the data points in Fig. 7Afor group A, Fig. 7Bfor 3⬘
processing of group B and Fig. 7Cfor strand transfer of group
B are 0.73, 0.55, and 0.74, respectively. Thus, the data support
a correlation between activity as an integrase inhibitor and the
stability of the intramolecular tetrad fold within members of
the T30695 family.
Previous work has also demonstrated (15) that T30695 and
T30177 can inhibit HIV-1 integrase in the K
⫹
form structure
but not in the Li
⫹
form structure. TBA, which cannot form
orderly loop structures, has high IC
50
values in the presence
and absence of K
⫹
ions. Compared with the matched pairs of
TABLE VI
IC
50
values of group A were obtained in this study; IC
50
values of group B were obtained by Pommier’s lab at the National Cancer Institute (10,
13).
Oligomer Sequence T
m
(1 mMKCl) IC
50
of group A IC
50
of group B
3⬘processing Strand transfer
°C nMnM
T30695 5⬘-g*ggtgggtgggtggg*t-3⬘69 31 43 ⫾17 24 ⫾4
T30177 5⬘-g*tggtgggtgggtggg*t-3⬘54 26 79 ⫾24 49 ⫾5
TBA 5⬘-ggttggtgtggttgg-3⬘25 470 870 750
T30676 5⬘-gtggtgggtgtggtgggt-3⬘33 238 148 ⫾26 134 ⫾16
T30677 5⬘-gtggttggtgggttggt-3⬘25 165 725 620
T30678 5⬘-gtggtggggtgggttggt-3⬘27 229 98 ⫾13 120 ⫾50
T39679 5⬘-gtggttggtggggtgggt-3⬘26 364 159 ⫾28 156 ⫾28
T30918 5⬘-gggtgggttggtgggg-3⬘48 60 — —
T30923 5⬘-gggtgggtgggtgggt-3⬘65 90 — —
“—” means that no IC
50
data was obtained in that group.
Stability-Activity Relationships of G-tetrad Oligos3426

T30695 derivatives (Table V), elimination of the T residue at
the 3⬘-end had no apparent influence on both T
m
and EC
50
values. However, the substitution of a T-G loop by a T-T loop in
the sequence caused a marked decrease in both the thermal
stability and the inhibition of HIV-1 replication in cell culture
(Table V). The decrease in the thermal stability of the G-
quartet structure substituted with a T-T loop has been shown
to be due to loss of a K
⫹
binding site between the loops and
G-quartets (15). The corresponding decrease in IC
50
is ascribed
the requirement that T30695 loops must be folded to inhibit the
HIV-1 integrase activity (15).
IC
50
and EC
50
Values of T30924–T30929 —As seen in Table
VII and Fig. 6B,IC
50
values of the derivatives with propynyl T,
T30924–T30929, were in the range of 50–200 nM. As men-
tioned in last section, the substitution of the T methyl by a
propynyl dU in the loop residues did not disrupted the K
⫹
form
structure of T30695. T30929 with two propynyl dUs in a single
T-G-T-G loop domain had an IC
50
close to that of T30695. The
IC
50
values of T30924 and T30926 seem to show that the
substitution of the T methyl group by a hydrophobic bulky
group in residue 8 of the sequence only causes a minor decrease
in the inhibition of HIV-1 integrase activity. The residue, T8,
TABLE VII
T, propynyl dU.
Oligomer Sequence T
m
(1 mM
KCl) IC
50
°C nM
T30924 5⬘-g*ggTgggTgggTggg*t-3⬘65.0 199
T30925 5⬘-g*ggTgggtgggtggg*t-3⬘68.8 136
T30926 5⬘-g*ggtgggTgggtggg*t-3⬘69.1 171
T30927 5⬘-g*ggtgggtgggTggg*t-3⬘68.7 50
T30928 5⬘-g*ggtgggtgggtggg*T-3⬘71.1 141
T30929 5⬘-g*ggTgggtgggTggg*t-3⬘66.4 40
FIG.6.The plots show that the IC
50
values of T30695 and its
derivatives obtained from the SPA HIV integrase assay were
presented as percentage of activity of HIV-1 integrase versus
concentration of these oligonucleotides. Plots A,B, and Cwere
obtained based upon Tables VI, VII, and IX, respectively.
FIG.7. The plots of log (IC
50
)versus T
m
for T30695 and its
derivatives, A, B and C, were obtained based upon the data in
Table VI, groups A and B, respectively. The mean square coeffi-
cients for fitting the data points for plots A,B, and Care 0.73, 0.55, and
0.74, and the slopes of the linear regressions are ⫺0.022, ⫺0.022, and
⫺0.028, respectively. These plots (A–C) demonstrate a statistically sig-
nificant relationship between integrase inhibition and thermal stability
of the folded tertiary structure of these oligonucleotides, which appears
to be independent of the methods used to assay integrase activity.
Stability-Activity Relationships of G-tetrad Oligos 3427

folds into a T-G-T-G loop plane with a pseudo T-G loop when
binding a K
⫹
ion with a G-quartet (Fig. 1). As seen in Table
VIII, T30924–T30929 have the same EC
50
as T30695, appar-
ently showing that the substitution of a hydrophobic bulky
group in the loop structure has no an apparent effect on the
inhibition of HIV-1 replication in cell culture.
The IC
50
and EC
50
values of T30924–T30929, for the inhibi-
tion of HIV-1 integrase activity and of HIV-1 replication in cell
culture respectively, were about the same as those of T30695
(Tables VII and VIII). Thus, the conclusion to be drawn from
these derivatives is that a substitution of a hydrophobic bulky
group for a T methyl group does not alter the structure and
thermostability of the T30695 and also does not disrupt the
interaction between T30695 and HIV-1 integrase, keeping IC
50
and EC
50
values unchanged.
IC
50
Values of T40101–T40107—Although T40101–T40107
form a same G-quartet structure with T30695, the IC
50
values
of T40101–T40107 were decreased to 6–9-fold compared with
that of T30695 (Table IX and Fig. 6C). We tentatively postulate
that the substitution of a positively charged group for a T
methyl group weakens the interaction between T30695 and
HIV-1 integrase, whereas the substitution of a hydrophobic
bulky group does not. Based upon a computed model of the
T30695-integrase complex,
2
T30695 appears to be bound into
the binding site of HIV-1 integrase, nearby many residues with
positively charged side chains, such as Lys
156
, Lys
159
, and
Lys
160
. Thus, the decrease in the inhibition of HIV integrase
activity for T40101–T40107 may be caused by the charge-
charge interaction between the positively charged loops of
T30695 derivatives and the positive charges of the lysine resi-
dues in the binding site of HIV-1 integrase.
Therapeutic Index—The therapeutic index of T30695 and its
derivatives, shown as a ratio of CC
50
(50% cytotoxic concentra-
tion) to EC
50
, were obtained from the measurements of cyto-
toxity and anti-HIV activity with three virus strains: HIV-1
RF, IIIB, and MN (Table X). The method of the measurements
was described elsewhere (17). T30695 is seen to have a thera-
peutic index in the range of 200 for the virus strains HIV-1 RF
and MN and in the range of 50 for HIV-1 IIIB. Similar values
were observed for T30177. Compared with T30695, the thera-
peutic index of T30923 was markedly decreased for all three
virus strains. The substitution of phosphodiester linkages for
PT linkages at G1 and G15 seems to have a strong influence on
the therapeutic indexes for HIV-1 IIIB and MN. From the
ratios of CC
50
to EC
50
of T30924–T30929, it seems that there is
no influence on the therapeutic index following substituting
hydrophobic bulky groups in the T residues.
Relationship between Thermal Stability and Anti-HIV-1 Ac-
tivity—Our previous results have shown that the T
m
of T30695
homologues is correlated with the IC
50
for integrase inhibitors
(13) and have suggested that the structure of the intramolec-
ular G-quartet might be required for anti-HIV integrase activ-
ity. Here we analyzed results obtained from many derivatives
to confirm whether the capacity to inhibit HIV-1 integrase is
direct function of the stability of the intramolecular G-tetrad
fold. As seen in Fig. 7, the three plots of log(IC
50
)versus T
m
of
T30695 were obtained based on the data from Table VI. The
correlation coefficients for fitting the data points in plots A, B,
and C were 0.73, 0.55, and 0.74. The slopes of linear regression
for plots A, B, and C were ⫺0.022, ⫺0.022, and ⫺0.028, respec-
tively. Fig. 8 shows three related plots of log(EC
50
)versus T
m
of
T30695 derivatives for the inhibition of the three HIV-1 viral
strains RF, IIIB, and MN, respectively. These data were ob-
tained from the data in Tables V and VIII. The mean square
coefficients of plots in Fig. 8 are 0.90 (A), 0.93 (B), and 0.86 (C).
The Pearson correlation coefficients (Pvalues) of the plots
between Aand B, between Aand C, and between Band Cwere
0.88, 0.91, and 0.92, respectively, as obtained from the SAS
computational system.
A few important concepts can be drawn from these analyses.
The high correlation coefficients for the data demonstrate a
significant, functional relationship between the thermal stabil-
ity and anti-HIV activity of the folded G-quartet structures,
which further confirms our previous observations (13). The
relationships between T
m
and IC
50
and between T
m
and EC
50
appear to be independent of the methods used to test anti-HIV
activity because the data were obtained from several different
assays. The plots for Figs. 7 and 8 demonstrate that the rela-
tionship between T
m
and IC
50
is surprisingly similar to that
between T
m
and EC
50
, which suggests that the inhibition of
HIV-1 integrase and the inhibition of HIV-1 replication in cell
culture may be depending on closely related structural features
of the compounds. Additional observations in cell culture as-
says have demonstrated that compounds such as T30695 also
interfere with virus adsorption and entry into the cell (19), and
thus it would be worth further exploring the relationship be-
tween T
m
and IC
50
for inhibition of virus adsorption. The
relationship between the thermal stability and anti-HIV activ-
ity for the tetrad-forming oligonucleotides provides critical in-
formation for improving the ability of these compounds to in-
hibit HIV-1 integrase activity, virus-cell binding, and HIV-1
replication. This should greatly help in the design of anti-HIV
therapeutic drugs.
2
R. Mitra, S. Lee, M. E. Hogan, and M. Pettitt, submitted for
publication.
TABLE VIII
g with an asterisk is a PT guanine, and T is a propynyl dU.
Oligomer Sequence T
m
(1 mM
KCl) ⌬G(T⫽
295)
EC
50
HIV-1 RF HIV-1 IIIB HIV-1 MN
°C kcal/mol nM
T30924 5⬘-g*ggTgggTgggTggg*t-3⬘65.0 ⫺9.89 10.5 ⫾414⫾10 54 ⫾3
T30925 5⬘-g*ggTgggtgggtggg*t-3⬘68.8 ⫺10.66 10.0 ⫾215⫾785⫾35
T30926 5⬘-g*ggtgggTgggtggg*t-3⬘69.1 ⫺9.39 12.0 ⫾229⫾962⫾21
T30927 5⬘-g*ggtgggtgggTggg*t-3⬘68.7 ⫺10.85 6.5 ⫾218⫾945⫾25
T30928 5⬘-g*ggtgggtgggtggg*T-3⬘71.1 ⫺10.69 14.0 ⫾620⫾876⫾34
T30929 5⬘-g*ggTgggtgggTggg*t-3⬘66.4 ⫺10.53 10.0 ⫾214⫾270⫾30
TABLE IX
I, 5-amino dU(3C).
Oligomer Sequence T
m
(1 mM
KCl) IC
50
°C nM
T40101 5⬘-gggIgggtgggtgggt-3⬘69.2 264
T40102 5⬘-gggtgggIgggtgggt-3⬘69.5 326
T40103 5⬘-gggtgggtgggIgggt-3⬘69.3 243
T40104 5⬘-gggIgggIgggtgggt-3⬘69.6 278
T40105 5⬘-gggtgggIgggIgggt-3⬘67.2 227
T40106 5⬘-gggIgggtgggIgggt-3⬘65.9 191
T40107 5⬘-gggIgggIgggIgggt-3⬘68.2 216
Stability-Activity Relationships of G-tetrad Oligos3428

DISCUSSION
Integration of viral DNA into host cell chromosome is an
essential step for HIV-1 replication. Based upon recent studies
mentioned in the Introduction (7–9), HIV-1 IN as a target for
anti-HIV therapy has attracted more attention. The major
steps for the integration involved by HIV-1 IN are 1) process-
ing: nicking of 3⬘-ends of viral DNA adjacent to highly con-
served CA dinucleotides and 2) joining: insertion of the pre-
cleaved viral DNA 3⬘-ends into both strands of host DNA (22–
24). HIV-1 IN is composed of three functional and structural
domains: N-terminal, central core, and C-terminal. Although
all three domains are required for 3⬘processing and strand
transfer (25), the central domain is directly involved in catal-
ysis for the strand transfer reaction, demonstrated by a disin-
tegration assay (26). However, no crystal structure has been
determined for a full integrase, although structures of all three
domains of HIV-1 IN have been identified individually (27–30).
The precise functions of the N- and C-terminal domains in the
overall integrase are not clear yet. Therefore, it is difficult to
design a highly effective anti-HIV IN inhibitor based upon the
known structure-activity information of HIV-1 IN.
A recent review reported that a large number of HIV-1 IN
inhibitors have been identified to data (18). Most of the inhib-
itors have IC
50
values in the range of 5–100
␮
M, and very few
inhibitors have IC
50
values in the nanomole range. Also more
than 50% of the reported inhibitors have no antiviral activity in
cell culture. A G-quartet oligonucleotide, T30177 (T30695 ho-
mologue), as an HIV-1 IN inhibitor was also reported in the
review with IC
50
in the range of 50 nMand with antiviral
activity in cell culture. The inhibition of HIV-1 IN in vitro by
the G-quartet oligonucleotides was identified with IC
50
in the
nanomole range based upon disintegration reaction (10). The
results demonstrated that T30695 homologues require a coor-
dination of the enzyme zinc finger region in the N-terminal
domain for inhibitory activity and suggested that the zinc fin-
ger assists to stabilize the binding interaction between the
G-quartet inhibitor and the catalytic domain of HIV-1 IN. The
inhibition of HIV-1 virus activity in cell culture by the G-
quartet oligonucleotides was also observed previously (19).
Based upon DNA sequence analysis, the G-quartet inhibitor
was proposed to target the envelope glycoprotein gp120 in cell
culture. In the reported HIV-1 IN inhibitors (18), the strong
ability to inhibit HIV-1 activity in vitro and in cell culture leads
the G-quartet oligonucleotides to be a useful tool to understand
the enzymology of HIV-1 IN and to develop a highly effective
anti-HIV therapeutic drugs.
The structure-activity of the G-quartet inhibitors has been
studied (13, 15). We have found that the ability to inhibit HIV-1
IN activity in vitro strongly depends on the thermostability and
conformation of the G-quartet oligonucleotides. Here we fur-
ther investigated the relationship between structural stability
and anti-HIV ability in vitro and in cells, using 22 T30695
derivatives. The results show clearly that the inhibition of
FIG.8.Plots of log (EC
50
)versus T
m
for T30695 and its deriva-
tives for three HIV-1 viral strains, RF (A), IIIB (B), and MN (C)
(Tables V and VIII). The slopes of linear regressions to the data are
0.070, 0.057, and 0.042, respectively. The mean square fitting coeffi-
cients of the data points are 0.90 (A), 0.93 (B), and 0.86 (C). The Pearson
correlation coefficients (Pvalues) of the plots between Aand B, between
Aand C, and between Band Care 0.88, 0.91, and 0.92, respectively,
obtained from the SAS computational system. All three cellular assays
suggest a statistically significant relationship between anti-HIV-1 ac-
tivity and thermal stability of the folded tertiary structure of these
oligonucleotides.
TABLE X
g with an asterisk is a PT guanine, and T is a propynyl dU.
Oligomer Sequence Therapeutic index (CC
50
/EC
50
)
HIV-1 RF HIV-1 IIIB HIV-1 MN
T30695 5⬘-g*ggtgggtgggtggg*t-3⬘152 ⫾32 44 ⫾17 247 ⫾20
T30923 5⬘-ggg tgg gtg ggt ggg t-3⬘134 ⫾100 5 ⫾118⫾7
T30177 5⬘-g*tggtgggtgggtggg*t-3⬘195 ⫾80 56 ⫾15 308 ⫾30
T30924 5⬘-g*ggTgggTgggTggg*t-3⬘108 ⫾35 19 ⫾1 117 ⫾50
T30925 5⬘-g*ggTgggtgggtggg*t-3⬘126 ⫾35 28 ⫾12 201 ⫾23
T30926 5⬘-g*ggtgggTgggtggg*t-3⬘156 ⫾50 18 ⫾685⫾10
T30927 5⬘-g*ggtgggtgggTggg*t-3⬘175 ⫾80 125 ⫾80 340 ⫾80
T30928 5⬘-g*ggtgggtgggtggg*T-3⬘118 ⫾60 33 ⫾14 185 ⫾80
T30929 5⬘-g*ggTgggtgggTggg*t-3⬘64 ⫾518⫾7 107 ⫾30
Stability-Activity Relationships of G-tetrad Oligos 3429

HIV-1 IN activity in vitro largely decreases when a modifica-
tion in loop domains of T30695 induces a decrease in thermo-
stability of the G-quartet, in agreement with the previous ob-
servation in which the conformation of loop domains of T30695
plays a key role in inhibition of HIV-1 IN activity (15). We also
have obtained a linear correlation between thermostability of
the G-quartet oligonucleotides and their anti-HIV replication
in cell culture, identified with three viral strains, and using 22
T30695 derivatives. Whether the inhibition of HIV-1 replica-
tion by the T30695 derivatives in cell culture is due to inhibi-
tion of integrase and/or virus adsorption, the high correlation
between the T
m
and EC
50
values demonstrates that the struc-
tural stability of the G-quartet oligonucleotides is a strong
determinant for inhibition of HIV-1 replication in cell culture.
This stability-activity correlation provides critical information
for new drug design, so that the search for a highly thermo-
stable structure for the G-quartet oligonucleotides will be next
priority. Moreover, the correlations between T
m
and IC
50
val-
ues and between T
m
and EC
50
values also can be used for a
rapid screen of newly designed candidates. Based upon the T
m
value of a new candidate, we can quickly make a judgement of
whether it is worthwhile to put the candidate through the
anti-HIV assay in vitro or virus replication in cells.
Here we also report the EC
50
values for inhibition of HIV-1
replication for a large number of G-quartet inhibitors in cell
culture, using three virus stains: RF, IIIB, and MN. The
greater inhibitory potency of T30695 and its derivatives sug-
gests that the G-tetrad-forming oligonucleotides could be novel
anti-HIV therapeutic drugs, in accord with previous sugges-
tions (10). To the best of our knowledge, no single integrase
inhibitor has so far been shown to owe its anti-HIV activity in
cells to intracellular inhibition of HIV-1 IN. The close relation-
ship between T
m
and IC
50
for inhibition of HIV-1 IN in vitro
and between T
m
and EC
50
for inhibition of HIV replication
points to the importance of T
m
determinations in structure-
activity studies on T30695, a G-tetrad-forming oligonucleotide.
Acknowledgments—We thank Gemini Biotech, Ltd. (The Woodlands,
TX) for the synthesis of T40101–T40107 and Yongli Guan for obtaining
Fig. 5.
REFERENCES
1. De Clercq, E. (1995) J. Med. Chem. 38, 2491–2517
2. Yarchoan, R., Klecker, R. W., Weinhold, K. J., Markham, P. D., Lyerly, H. K.,
Durack, D. T., Gelmann, E., Nusinoff-Lehrman, S., Blum, R. M., Barry,
D. W., Shearer, G. M., Fishl, M. A., Mitsuya, H., Gallo, R. C., Collin, J. M.,
Bolognesi, D. P., Myers, C. E., and Broder, S. (1986) Lancet 1, 575–580
3. Mitsuya, H., and Broder, S. (1986) Proc. Natl. Acad. Sci. U. S. A. 83,
1911–1915
4. Yarchoan, R., Perno, C. F., Thomas, R. V., Klecker, R. W., Allain, J.-P., Wills,
R. J., McAtee, N., Fischl, M. A., Dubinsky, R., McNeely, M. C., Mitsuya, H.,
Pluda, J. M., Lawley, T. J., Leuther, M., Safai, B., Collins, J. M., Myers,
C. E., and Broder, S. (1988) Lancet 1, 76–81
5. Browne, M. J., Mayer, K. H., Chafee, S. B. D., Dudely, M. N., Posner, M. R.,
Steiberg, S. M., Graham, K. K., Geletko, S. M., Zinner, S. H., Denman, S. L.,
Dunkle, L. M., Kaul, S., McLaren, C., Skowron, G., Kouttab, N. M.,
Kennedy, T. A., Wettberg, A. B., and Curt, G. A. (1993) J. Infect. Dis. 167,
21–29
6. Collier, A. C., Bozzette, S., Coombs, R. W., Causey, D. M., Schoenfeld, D. A.,
Spector, S. A., Pettinelli, C. B., Davies, G., Richman, D. D., Leedom, J. M.,
Kidd, P., and Corey, L. (1990) N. Engl. J. Med. 323, 1015–1021
7. Chun, T. W., Stuyver, L., Mizell, S. B., Enler, L. A., Mican, J. A. M., Baseler,
M., Lloyd, A. L., Nowak, M. A., and Fauci, A. S. (1997) Proc. Natl. Acad. Sci.
U. S. A. 94, 13193–13197
8. Wong, J. K., Hezareh, M., Gunthard, H. F., Havlir, D. V., Ignacio, C. C., Spina,
C. A., and Richman, D. D. (1997) Science 278, 1291–1295
9. Finzi, D., Hermankova, M., Pierson, T., Carruth, L. M., Buck, C., Chaisson,
R. E., Quinn, T. C., Chadwick, K., Margolick, J., Btookmeyer, R., Gallant, J.,
Markowitz, M., Ho., D. D., Richman, D. D., and Siliciano, R. (1997) Science
278, 1295–1300
10. Mazumder, A., Neamati, N., Sommadossi, J.-P., Gosselin, G., Schinazi, R. F.,
Imbach, J.-L., and Pommier, Y. (1996) Mol. Pharmacol. 49, 621–628
11. Rando, R. F., Ojwang, J., Elbaggari, A., Reyes, G. R., Tinder, R., McGranth,
M. S., and Hogan, M. E. (1995) J. Biol. Chem. 270, 1754–1760
12. Mazumder, A., Neamati, N., Ojwang, J. O., Sunder, S., Rando, R. F., and
Pommier, Y. (1996) Biochemistry 35, 13762–13771
13. Jing, N., Rando, R. F., Pommier, Y., and Hogan, M. E. (1997) Biochemistry 36,
12498–12505
14. Jing, N., Gao, X., Rando, R. F., and Hogan, M. E. (1997) J. Biomol. Struct.
Dynamics 15, 573–585
15. Jing, N., and Hogan, M. E. (1998) J. Biol. Chem. 273, 34992–34999
16. Longfellow, C. E., Kierzek, R., and Turner, D. H., (1990) Biochemistry 29,
278–285
17. De Clercq, E., Yamamoto, N., Pauwels, R., Baba, M., Schols, D., Nakashima,
H., Balzarini, J., Debyser, Z., Murrer, B. A., Schwatz, D., Thornton, D.,
Bridger, G., Fricker, S., Henson, G., Abrams, M., and Picker, D. (1992) Proc.
Natl. Acad. Sci. U. S. A. 89, 5286–5290
18. Pommier, Y., and Neamati N. (1998) Adv. Virus Res. 52, 427–457
19. Este, J. A., Cabrera, C., Schols, D., Cherepanov, P., Gutierrez, A., Witvrouw,
M., Pannecouque, C., Debyser, Z., Rando, R, F., Clotet, B., Desmyter, J., and
De Clercq, E. (1998) Mol. Pharmacol. 53, 340–345
20. Wang, K. Y., McCurdy, S., Shea, S. G., Swaminathan, S., and Bolton, P. H.
(1993) Biochemistry 32 1899–1904
21. Schultze, P., Macaya, R. F., and Feigan, J. (1994) J. Mol. Biol. 235, 1532–1547
22. Katz, R. A., and Skalka, A. M. (1994) Annu. Rev. Biochem. 63, 133–173
23. Engelman, A., Mizuch, K., and Craigei, R. (1991) Cell 67, 1211–1221
24. Katzman, M., and Katz, R. A. (1998) Adv. Virus Res. 52, 371–395
25. Bushman, F. D., Engelman, A., Palmer, I., Wingfield, P. T., and Craigie, R.
(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3428–3432
26. Hickman, A. B., Palmer, I., Engelman, A., Craigie, R., and Wingfield, P. (1994)
J. Biol. Chem. 269, 29279–29287
27. Dyda, F., Hickman, A. B., Jenkins, T. M., Engelman, A., Craigie, R., and
Davies, D. R. (1994) Science 266, 1981–1986
28. Cai, M., Zheng, R., Caffry, M., Craigie, R., Clore, G. M., and Gronenborn, A. M.
(1997) Nat. Struct. Biol. 4, 567–577
29. Lodi, P. J., Ernst, J. A., Kuszewski, J., Hickman, A. B., Engleman, A., Craigie,
R., clore, G. M., and Gronenborn, A. M. (1995) Biochemistry 34, 9826–9833
30. Eijkelenboom, A. P. A. M., Lutzke, R. A. P., Boelens, R., Plasterk, R. H. A.,
Kaptein, R., and Hard, K. (1995) Nat. Struct. Biol. 2, 807–810
Stability-Activity Relationships of G-tetrad Oligos3430