Click dimers to target HIV TAR RNA conformation

Article abstract of DOI:10.1021/bi201657k

A series of neomycin dimers have been synthesized using "click chemistry" with varying functionality and length in the linker region to target the human immunodeficiency virus type 1 (HIV-1) TAR RNA region of the HIV virus. The TAR (Trans-Activation Respo

Full text of DOI:10.1021/bi201657k

Article
pubs.acs.org/biochemistry
Click Dimers To Target HIV TAR RNA Conformation
Sunil Kumar,† Patrick Kellish,† W. Edward Robinson, Jr.,‡ Deyun Wang,§ Daniel H. Appella,§
and Dev P. Arya*,†
†Laboratory of Medicinal Chemistry, Department of Chemistry, Clemson University, Clemson, South Carolina 29634, United States
‡Department of Pathology and Laboratory Medicine, University of California, Irvine, California 92697, United States
§Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health,
Bethesda, Maryland 20892, United States
S
* Supporting Information
ABSTRACT: A series of neomycin dimers have been syn-
thesized using “click chemistry” with varying functionality and
length in the linker region to target the human immunode-
ficiency virus type 1 (HIV-1) TAR RNA region of the HIV
virus. The TAR (Trans-Activation Responsive) RNA region, a
59 bp stem−loop structure located at the 5′-end of all nascent
viral transcripts, interacts with its target, a key regulatory protein,
Tat, and necessitates the replication of HIV-1. Neomycin, an
aminosugar, has been shown to exhibit multiple binding sites on
TAR RNA. This observation prompted us to design and
synthesize a library of triazole-linked neomycin dimers using
click chemistry. The binding between neomycin dimers and TAR RNA was characterized using spectroscopic techniques, including
FID (fluorescent intercalator displacement), a FRET (fluorescence resonance energy transfer) competitive assay, circular dichroism
(CD), and UV thermal denaturation. UV thermal denaturation studies demonstrate that binding of neomycin dimers increases the
melting temperature (Tm) of the HIV TAR RNA up to 10 °C. Ethidium bromide displacement (FID) and a FRET competition
assay revealed nanomolar binding affinity between neomycin dimers and HIV TAR RNA, while in case of neomycin, only weak
binding was detected. More importantly, most of the dimers exhibited lower IC50 values toward HIV TAR RNA, when compared to
the fluorescent Tat peptide, and show increased selectivity over mutant TAR RNA. Cytopathic effects investigated using MT-2 cells
indicate a number of the dimers with high affinity toward TAR show promising anti-HIV activity.
ibonucleic acid−protein interactions are essential for regu-
Tat protein and a guanine base in the major groove of TAR
RNA.10 Disruption of the TAR RNA−Tat interaction therefore
represents an attractive strategy for inhibiting viral replication. A
number of molecules have been investigated with this strategy in
mind.11,12 These include intercalators12 (ethidium bromide13
and proflavine), DNA minor groove binders14 (Hoechst 33258
and DAPI), phenothiazine,15 argininamide,16 peptides,17
peptidomimetics,18 aminoglycosides,19 and cyclic polypeptides.20
Aminoglycosides are naturally occurring aminosugars that
bind to a wide variety of RNA structures.21 In the past few years,
a number of aminoglycoside conjugates have been synthesized
to achieve higher binding affinity and specificity toward
RNA21−26 and DNA-based targets27−44 such as duplex,45 tri-
plex,46−48 and quadruplex structures.29,30 In an attempt to
achieve higher binding affinity and explore multiple bind-
ing sites on RNA targets, the homo- and heterodimeric units
of aminoglycosides49,50 (tobramycin, neamine, neomycin B,
and kanamycin A) have been synthesized with various linker
lengths and functionalities through disulfide bond formation.
R
lation of many important biological processes such as
translation, RNA splicing, and transcription.1−3 An important
example of such an interaction is involved in the regulation of
human immunodeficiency virus type 1 (HIV-1). HIV TAR
RNA (trans-activation responsive region), a 59 bp stem−loop
structure located at the 5′-end of the nascent viral transcripts,
interacts with Tat protein (an 86-amino acid protein) and
regulates the level of transcription of HIV.4,5 The cooperative
interaction of Tat protein along with its cellular cofactor,
transactivating elongation factor-b (TEFb), with TAR RNA
recruits and activates the CDK9 kinase that phosphorylates
RNA polymerase II (RNAP II) and significantly enhances the
processivity of RNAP II.3,6,7
HIV transcription in virus-infected cells is strongly triggered
by the interaction between Tat protein and its cognate TAR
RNA. TAR RNA structure is comprised of two stems (upper
and lower), a three-nucleotide bulge region, and a hairpin. An
arginine rich domain of Tat protein interacts with the trinuc-
leotide bulge (U23, C24, and U25) of TAR RNA1,8,9 and causes
a substantial enhancement in the transcript level (∼100-fold).2
Nuclear magnetic resonance (NMR) studies show that the com-
plexation takes place specifically between the arginine residue of
Received: November 2, 2011
Revised: February 10, 2012
Published: February 17, 2012
© 2012 American Chemical Society
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These aminoglycosides exhibit higher binding affinity toward
the dimerized A-site 16S construct, RRE RNA, than their
corresponding monomeric aminoglycoside units. Also, amino-
glycoside dimers exhibit 20−12000-fold stronger inhibitory
effects toward the catalytic function of the Tetrahymena ribo-
zyme than the monomeric units.50 Neamine dimers have been
shown to exhibit remarkable antibiotic effects and resistance to
aminoglycoside-modifying enzymes.51
path length were used for all the absorbance studies. Spec-
trophotometer stability and λ alignment were checked prior to
initiation of each melting point experiment. Fluorescence spectra
were recorded on a Photon Technology International instrument
(Lawrenceville, NJ). The fluorescence measurements in 96-well
plates were taken on a Genios Multi-Detection Microplate Reader,
TECAN, with Magellan software.
UV Thermal Denaturation Experiment. The UV thermal
denaturation samples (1 mL) of TAR RNA (1 μM/strand)
were mixed with a ligand (neomycin dimers) with rdr values of
1 and 2 (rdr is the ratio of drug to HIV TAR RNA concen-
tration) in 100 mM KCl, 10 mM SC, and 0.5 mM EDTA
(pH 6.8) and incubated for 4 h at 4 °C before the experiment
was started. The UV thermal denaturation spectra of the
samples in 1 cm path length quartz cuvettes were recorded at
260 nm as a function of temperature (10−95 °C, heating rate of
0.3 °C/min). First-derivative plots were used to determine the
denaturation temperature.
TAR RNA Synthesis. HIV TAR RNA was purchased from
Amersham Biosciences (now GE Healthcare Life Sciences,
Piscataway, NJ). Before being used, HIV TAR RNA (5′-GGC
AGA UCU GAG CCU GGG AGC UCU CUG CC-3′) in a
10 μM batch in sterile water was heated to 95 °C for 4.5 min
and then cooled rapidly in an ice bath for 5 min. This snap-
cooling causes the RNA to adopt the kinetically favored hairpin
rather than the thermodynamically favored duplexes.
FRET-Mediated Competition Binding Assay. The
relative affinity of each dimer for HIV-1 TAR RNA was deter-
mined using a FRET-based competitive binding assay with a
fluorescein-labeled HIV-1 Tat peptide as described previ-
ously.53 The fluorescence experiments were performed with a
Spectra Max fluorimeter (Molecular Devices) at 25 °C, with
excitation and emission wavelengths of 495 and 570 nm,
respectively. All samples were prepared in 96-well plates in 1×
TK buffer [50 mM Tris and 20 mM KCl (pH 7.4)] with 0.1%
Triton X-100 (Sigma). The binding affinity (IC50) values reported
for each dimer are the averages of three to five individual
measurements and were determined by fitting the experimental
data to a sigmoidal dose−response nonlinear regression model
on GraphPad Prism 4.0. Prior to the competition experiments,
the affinity of the fluorescein-labeled Tat peptide for HIV-1
TAR RNA was determined by monitoring fluorescence inten-
sity changes of the fluorescent probe upon addition of HIV-1
TAR RNA. Addition of an increasing concentration (from 0 to
1000 nM) of HIV-1 TAR RNA to a 100 nM solution of
fluorescein-labeled Tat peptide in TK buffer at 25 °C afforded
a saturation binding curve. The IC50 value obtained from this
binding curve was 86 nM.
Competition FRET Assay. To a solution of 100 nM HIV-1
TAR RNA and 100 nM fluorescein-labeled HIV-1 Tat peptide
were added appropriate concentrations (0 nM to 100 μM) of
the dimer antagonists at 25 °C; the total volume of the incu-
bation solution was 80 μL. After 60 min, fluorescence changes
of the sample solution were determined with the Spectra Max
fluorimeter detector. The experimental dose−response data for
a given polyamine were fit to a sigmoidal dose−response non-
linear regression model on GraphPad Prism 4.0 to afford the
IC50 values for each dimer.
Among all the aminoglycosides targeted toward TAR bind-
ing, neomycin has exhibited the strongest inhibitory effect (<1 μM).19
Electrospray ionization mass spectrometry (ESI-MS)52 and
ribonuclease protection experiments22 have suggested that the
binding site of neomycin is the stem region just below the tri-
nucleotide bulge in TAR RNA. Further ESI-MS experiments and
gel shift assays have revealed the existence of three neomycin
binding sites on HIV TAR RNA.52 These sites do not overlap
with the Tat binding site, and thus, neomycin shows a weak
ability to allosterically compete with protein binding, leading to
weak HIV inhibition. To achieve improved binding and speci-
ficity profiles, we have explored neomycin’s multiple binding sites
on HIV TAR RNA and designed a series of neomycin dimers
using click chemistry. Even though these dimers are not expected
to directly compete with Tat binding, their binding is expected to
lock the conformation of RNA such that Tat−TAR binding is
weakened through an allosteric mechanism. We synthesized neo-
mycin dimers using click chemistry with various linker lengths
and functionalities to optimize the RNA binding affinity. Our
results show that neomycin dimers display nanomolar affinity for
HIV TAR RNA. Spectroscopic techniques, UV thermal denatur-
ation, a FID (fluorescent intercalator displacement) assay, and
a FRET (fluorescence resonance energy transfer) assay were
utilized to study the binding between neomycin dimers and TAR
RNA. In this report, we present our work detailing a simple and
efficient route toward the synthesis of triazole-linked aminosugar
dimers, their binding to TAR RNA, their ability to effectively
inhibit the tat−TAR interaction, and their cytopathic effects on
HIV in MT-2 cells.
EXPERIMENTAL PROCEDURES
■
Materials. All of the chemicals were purchased from com-
mercial suppliers and used without further purification.
Neomycin B trisulfate was purchased from MP Biomedicals
(Solon, OH). Di-tert-butyl dicarbonate (Boc anhydride) was
purchased from Advanced ChemTech (Louisville, KY). SC
(sodium cacodylate), EDTA (ethylenediaminetetraacetic acid),
KCl, and sodium phosphate (mono and di) salts were purchased
from Fisher Scientific. TPS-Cl (2,4,6-triisopropylbenzenesulfo-
nyl chloride) and 4 M HCl/dioxane were purchased from Sigma
Aldrich. Silica gel for flash column chromatography was pur-
chased from Sorbent Technologies (Atlanta, GA) as silica gel
standard grade (particle size of 40−63 μm). All solvents were
purchased from VWR. Reaction solvents were distilled over calcium
hydride [toluene, pyridine, and DCM (dichloromethane)]. EtOH
(ethanol) was first distilled with sodium metal and then redistilled
over magnesium turnings. Reactions were conducted under N2
using dry solvent, unless otherwise noted.
1
Instrumentation. H NMR spectra were recorded on a
Bruker 500 MHz FT-NMR spectrometer. MS [matrix-assisted
laser desorption ionization time-of-flight (MALDI-TOF)]
spectra were recorded using a Bruker Omniflex MALDI-TOF
mass spectrometer. All UV spectra were recorded on a Cary
100 Bio UV−vis spectrophotometer equipped with a thermo-
electrically controlled 12-cell holder. Quartz cells with a 1 cm
Ethidium Bromide Displacement Titration. A solu-
tion of ethidium bromide (1.25 μM, 1800 μL) was excited at
545 nm, and its fluorescence emission was monitored from 560
to 620 nm before and after the addition of HIV TAR RNA. The
concentration of HIV TAR RNA was 50 nM/strand. A small
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fraction of ethidium bromide is bound (<20%) under these
conditions. Buffer conditions were 100 mM KCl, 10 mM SC,
and 0.5 mM EDTA (pH 6.8).
(0.05 mmol) in dry toluene (5 mL) was added dialkyne linker
(0.025 mmol, 0.50 equiv) followed by the addition of CuI (4.76 mg,
0.025 mmol) and DIPEA (6.46 mg, 0.05 mmol). The reaction
mixture was stirred at 90 °C for 18 h in an atmosphere of
argon. The progress of the reaction was monitored by
TLC. The volatiles were rotoevaporated in vacuo. Purification
by flash column chromatography (Rf = 0.38−0.44, 0 to 10%
ethanol in CH2Cl2) afforded the desired product(s) as a white
solid (percent yields are reported for individual compounds in
Table 1) [Rf = 0.38−0.44, 10% ethanol in CH2Cl2 (v/v)].
General Procedure for the Deprotection of N-Boc DPA51−
DPA65. To a solution of neomycin dimer (0.012 mmol) in
dioxane (3 mL) was added a 4 M HCl/dioxane solution (1 mL),
and the mixture was stirred at room temperature. A white pre-
cipitate formed after 15 min. The reaction mixture was centri-
fuged and the solid collected. The solid was washed with a
diethyl ether/hexane solution [3 × 5 mL, 1:1 (v/v)]. The solid
was dissolved in water and lyophilized to afford the desired
product(s) as a powder (percent yields are reported for indi-
vidual compounds in Table 1).
Ethidium Bromide Displacement Titration To Deter-
mine the Binding Constant via Scatchard Analysis.
A solution of ethidium bromide (5.00 μM, 1800 μL) was
excited at 545 nm, and its fluorescence emission was monitored
from 560 to 620 nm before and after the addition of HIV TAR
RNA. The concentration of HIV TAR RNA was 200 nM/
strand. A small fraction of ethidium bromide is bound (<20%)
under these conditions. Buffer conditions were 100 mM KCl,
10 mM SC, and 0.5 mM EDTA (pH 6.8).
Assay for Inhibition Activity toward HIV Antigen
Synthesis in Treated Cells. Cells (500000) were pretreated
with each compound for 1 h. Next, cells were inoculated with
approximately 50000 infectious particles of NL4-3 (an
infectious molecular clone of HIV). Infections were performed
in triplicate. Every 2 days cells were pooled and stained for HIV
antigen synthesis using an antibody to all HIV antigens (HIV
immune globulin).
In Vitro Cell Culture To Determine the Amount of
Reverse Transcriptase (RT) Activity Release. Culture
supernatants were precipitated for RT release. In all assays,
MT-2 cells were used, which are highly susceptible to HIV and
are completely lysed by HIV. Anti-HIV activities can be rou-
tinely checked by measuring the percentage of cells positive for
HIV antigens using an immunofluorescence assay (IFA) and
for the release of pelletable reverse transcriptase (RT) into the
supernatant. For these assays, 500000 MT-2 cells in 1 mL of
medium were added to the wells of a 24-well tissue culture
plate. Next, 0.5 mL of 4× concentrated compound was added
to triplicate wells of the plate. The cells and compounds were
incubated for 1 h at 37 °C. Finally, 0.5 mL of HIVNL4‑3, pro-
duced in H9 cells (a CD4+ lymphoblastoid cell line), 100000 cpm
of RT activity per well, was added to each well. Virus control
wells contained no compounds. This inoculum is at a multiplicity
of infection of <1. On days 2, 4, and 6, 0.75 mL of supernatants
was removed and placed into individual microfuge tubes for
the RT assay. The supernatants from each well were precipitated
at 4 °C overnight in a solution that included 30% polyethylene
glycol. After precipitation, the precipitate was lysed in a solu-
tion containing Triton X-100, Tris buffer, and DTT.
The RT activity of each aliquot was determined as incorporation
of [3H]dTTP into a poly(rA)-oligo(dT) template. After a 1 h
assay at 37 °C, the incorporated dTTP was precipitated onto a
ZetaProbe (BioRad) membrane. Slots were excised and placed
into liquid scintillation cocktail. After overnight incubation, the
counts per minute for each sample were determined on a β
counter. Results were calculated as counts per minute per milli-
liter of original culture supernatant. Next, cells were resuspended
in the medium, and 0.5 mL of the total remaining volume was
removed for IFA. Cells were combined from their triplicate
infections and pelleted, and the enriched cells were air-dried onto
glass slides. The dried cells were fixed in an acetone/methanol
mixture (50:50). After being fixed, cells were stained with HIV
immunoglobulin, washed in PBS, and counterstained with FITC-
conjugated goat and human IgG. Slides were washed in PBS, and
the percentage of HIV-positive cells was determined by epifluo-
rescence. To the remaining cells in culture was added 1.25 mL of
medium, and the cultures were maintained at 37 °C.
DPA51: IR (neat, cm−1) 3421 (br, OH), 1686, 1524, 1366;
1H NMR (500 MHz, D2O) δ 8.02 (s, 2 H, triazole), 5.92 (d, J =
3.94 Hz, 2 H, H1II), 5.28 (d, J = 3.16 Hz, 2 H, H1III), 5.17 (s,
2 H, H1IV), 4.41 (m, 2 H, H4III), 4.34 (d, J = 5.20 Hz, 2 H,
H
2III), 4.18 (t, J = 4.73 Hz, 2 H, H4IV), 4.10 (d, J = 2.84 Hz,
2 H, H4I), 3.96 (t, J = 9.62 Hz, 2 H, H6II), 3.90 (t, J = 9.93 Hz,
2 H, H5I), 3.86−3.76 (m, 8 H, H2IV, H4II, H5IV, H3III), 3.70 (d,
J = 1.89 Hz, 2 H, H6I), 3.54 (t, J = 9.61 Hz, 2 H, H3II), 3.49−
3.45 (m, 4 H, H3I, H5II), 3.44−3.32 (m, 2 H, H1I), 3.30−3.20
(m, 8 H, H5III and propargyl ether protons), 2.39−2.32 (dt, J1 =
3.94 Hz, J2 = 4.25 Hz, 2 H, H2Ieq), 1.72−1.82 (q, J = 12.45 Hz,
2 H, H2Iax); 13C NMR (125 MHz, D2O) δ 162−162 (q, CF3-
COOH), 127, 125, 115, 110, 95, 85, 79, 77, 75, 73, 72, 70, 69, 68, 67,
63, 62, 53, 52, 50, 49, 48, 40.5, 40.1, 28; MS (MALDI-TOF) m/z calcd
for C52H96N18O25 1391.44, found 1392.66 [M + H2O]+.
DPA52: IR (neat, cm−1) 3375 (br, OH), 2979, 2932, 2108,
1
1727, 1686, 1522, 1457; H NMR (500 MHz, D2O) δ 8.06
(s, 2 H, triazole), 5.95 (d, J = 3.94 Hz, 2 H, H1II), 5.32
(d, J = 3.15 Hz, 2 H, H1III), 5.17 (d, J = 1.26 Hz, 2 H, H1IV),
4.47 (d, J = 1.26 Hz, 2 H), 4.48−4.42 (m, 2 H, H4III), 4.40−4.36
(t, J = 5.20 Hz, 2 H, H2III), 4.24−4.20 (t, J = 4.41 Hz, 2 H, H4IV),
4.14−4.11 (t, J = 2.99 Hz, 2 H, H4I), 4.03−3.98 (t, J = 9.62 Hz, 2
H, H6II), 3.96−3.92 (m, 2 H, H5I), 3.92−3.80 (m, 8 H, H2IV, H4II,
H5IV, H3III), 3.74−3.72 (m, 2 H, H6I), 3.61−3.55 (t, J = 9.14 Hz,
2 H, H3II), 3.54−3.48 (m, 4 H, H3I, H5II), 3.48−3.40 (m, 4 H),
3.38−3.20 (m, 8 H), 2.42−2.35 (dt, J1 = 3.47 Hz, J2 = 4.25 Hz, 2
H, H2Ieq), 1.85−1.75 (q, J = 12.61 Hz, 2 H, H2Iax); MS (MALDI-
TOF) m/z calcd for C53H98N18O24 1389.45, found 1389.66 [M +
H2O]+. Anal. Calcd for C53H110N18O24Cl12: C, 35.19; H, 6.13; Cl,
23.52; N, 13.94; O, 21.23. Found: C, 34.89; H, 6.21; N, 13.71.
1
DPA53: IR (neat, cm−1) 3368 (br, OH), 2090, 1642; H
NMR (500 MHz, D2O) δ 8.51 (s, 2 H, triazole), 7.91 (s, 4 H,
Ar), 6.02 (d, J = 3.60 Hz, 2 H, H1II), 5.39 (d, J = 3.00 Hz, 2 H,
H
1III), 5.24 (s, 2 H, H1IV), 4.27 (t, J = 4.70 Hz, 2 H), 4.20−4.05
(m, 6 H, H5I, H6II), 4.00−3.80 (m, 6 H), 3.73 (m, 6 H), 3.65
(m, 12 H), 3.55 (m, 8 H), 3.45−3.20 (m, 10 H), 3.18−3.10 (m,
2 H), 2.43−2.35 (dt, J1 = 4.15 Hz, J2 = 4.28 Hz, 2 H, H2Ieq),
1.82−1.72 (q, J = 12.45 Hz, 2 H, H2Iax); MS (MALDI-TOF)
m/z calcd for C56H96N18O24 1423.47, found 1424.41 [M +
H2O]+; UV (water) λmax = 275 nm. Anal. Calcd for C56H108
-
Synthesis and Characterization of DPA51−DPA65
Dimers. General Procedure for the Synthesis of N-Boc
DPA51−DPA65. To a solution of neomycin-Boc-5″-azide
N18O24Cl12: C, 36.49; H, 5.91; Cl, 23.08; N, 13.68; O, 20.83.
Found: C, 36.04; H, 5.79; N, 13.31.
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Table 1. Linker Structures and Percent Yields (for two steps) for the Synthesis of Dimeric Neomycins
DPA54: 1H NMR (500 MHz, D2O) δ 7.86 (s, 2 H, triazole),
5.79 (s, 2 H, H1II), 5.30 (s, 2 H, H1III), 5.20 (s, 2 H, H1IV), 4.47
(d, J = 1.26 Hz, 2 H), 4.48−4.42 (m, 2 H, H4III), 4.38 (t, J =
5.20 Hz, 2 H, H2III), 4.46 (s, 8 H), 4.25 (s, 4 H), 4.22 (s, 4 H),
4.12 (m, 4 H), 3.97−3.90 (m, 6 H), 3.75−3.65 (m, 10 H),
3.55−3.45 (m, 6 H), 3.40−3.20 (m, 12 H), 3.40−3.23 (m, 12 H),
3.20−3.05 (m, 8 H), 2.38 (d, J = 12.14 Hz, 2 H, H2Ieq.), 1.70−
1.50 (m, 2 H, H2Iax.); MS (MALDI-TOF) m/z calcd for
C54H100N18O24 [M + H2O]+ 1405.48, found 1404.64; UV (water)
λmax = 225 nm.
H1III), 5.21 (s, 2 H, H1IV), 4.53−4.47 (m, 4 H, H4III), 4.45 (t,
J = 5.05 Hz, 2 H), 4.23 (t, J = 5.36 Hz, 4 H), 4.17−4.11 (m,
6 H), 3.97 (t, J = 9.77 Hz, 4 H, H5I, H6II), 3.93−3.83 (m, 8 H,
H
2IV, H4II, H5IV, H3III), 3.75−3.70 (m, 6 H), 3.69−3.59 (m,
10 H), 3.55−3.59 (m, 2 H), 3.54 (s, 4 H), 3.50−3.53 (m,
4 H), 3.43−3.34 (m, 8 H), 3.34−3.24 (m, 8 H), 3.22−3.15
(m, 8 H), 2.64−2.58 (m, 4 H), 2.40−2.33 (m, 2 H, H2Ieq),
1.96 (m, 6 H, H2Iax and protons from linker), 1.55 (m, 4 H,
protons from linker); MS (MALDI-TOF) m/z calcd for
C56H104N18O24 [M + Na]+ 1435.53, found 1434.83; UV
(water) λmax = 227 nm. Anal. Calcd for C56H116N18O24Cl12: C,
36.34; H, 6.32; Cl, 22.98; N, 13.62; O, 20.74. Found: C,
36.24; H, 6.11; N, 13.41.
DPA55: 1H NMR (500 MHz, D2O) δ 7.82 (s, 2 H, triazole),
5.95 (s, 2 H, H1II), 5.33 (s, 2 H, H1III), 5.22 (s, 2 H, H1IV),
4.55−4.43 (br, 4 H, H4III), 4.24 (m, 2 H), 4.13 (s, 2 H), 3.99
(m, 2 H), 3.95−3.80 (m, 8 H), 3.78−3.70 (4 H), 3.66−3.58
(m, 4 H), 3.56 (m, 2 H), 3.54 (s, 2 H), 3.51 (s, 2 H), 3.45−3.35
(m, 6 H), 3.34−3.30 (s, 2 H), 3.30−3.19 (m, 6 H), 2.40−2.31
(m, 6 H), 2.26 (m, 2 H, H2Ieq), 1.81−1.72 (m, 2 H, H2Iax.); MS
(MALDI-TOF) m/z calcd for C58H106N18O24 [M + H2O]+
1456.77, found 1457.90; UV (water) λmax = 227 nm.
DPA58: 1H NMR (500 MHz, D2O) δ 8.06 (s, 2 H, triazole),
5.97 (d, J = 3.47 Hz, 2 H, H1II), 5.33 (d, J = 3.00 Hz, 2 H,
H
1III), 5.22 (s, 2 H, H1IV), 4.80−4.74 (m, 2 H), 4.51−4.43
(m, 4 H, H4III), 4.24 (t, J = 4.42 Hz, 2 H), 4.13 (t, J = 2.37 Hz,
2 H), 3.97 (t, J = 9.61 Hz, 2 H, H6II), 3.93 (t, J = 10.40 Hz,
2 H), 3.90−3.78 (m, 6 H), 3.76−3.70 (m, 4 H), 3.69−3.58
(m, 8 H), 3.57−3.53 (m, 6 H), 3.52−3.47 (m, 6 H), 3.44
(t, J = 9.14 Hz, 4 H), 3.41−3.34 (m, 4 H), 3.32 (t, J = 6.14 Hz,
DPA56: 1H NMR (500 MHz, D2O) δ 7.80 (s, 2 H, triazole),
6.00 (d, J = 3.63 Hz, 2 H, H1II), 5.36 (d, J = 2.84 Hz, 2 H,
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2 H), 3.30−3.16 (m, 6 H), 2.41−2.33 (dd, J1 = 3.94 Hz, J2 =
3.62 Hz, 2 H), 1.84−1.72 (q, J = 13.24 Hz, 2 H, H2Iax.), 1.53−
1.43 (m, 4 H, linker protons), 1.25−1.14 (m, 8 H, linker
protons), 1.13−1.04 (t, J = 6.62 Hz, 4 H, linker protons); MS
(MALDI-TOF) m/z calcd for C60H112N18O26 [M + 2H2O]+
1537.64, found 1538.11; UV (water) λmax = 229 nm.
Scheme 1. Structures of Small Molecule Ligands (neomycin
and neomycin dimer) and TAR RNA Used in This Study
DPA60: 1H NMR (500 MHz, D2O) δ 8.05 (s, 2 H, triazole),
5.97 (d, J = 3.78 Hz, 2 H, H1II), 5.34 (d, J = 3.31 Hz, 2 H,
H
1III), 5.22 (s, 2 H, H1IV), 4.55 (s, 4 H), 4.50−4.46 (m, 2 H,
H4III), 4.43 (t, J = 4.57 Hz, 2 H), 4.23 (t, J = 4.42 Hz, 2 H),
4.19−4.15 (m, 2 H), 3.94 (t, J = 2.53 Hz, 2 H), (N 4.05 (t,
J = 9.93 Hz, 2 H), 3.96−3.90 (t, J = 10.25 Hz, 2 H), 3.90−
3.79 (m, 8 H), 3.76−3.69 (m, 6 H), 3.70−3.58 (m, 12 H),
3.55−3.42 (m, 12 H), 3.42−3.38 (m, 2 H), 3.38−3.35 (m,
2 H), 3.34−3.30 (m, 4 H), 3.30−3.22 (m, 6 H), 2.38 (dd, J1 =
4.10 Hz, J2 = 4.73 Hz, 2 H), 1.85−1.75 (q, J = 11.98 Hz,
2 H, H2Iax.), 1.54−1.41 (m, 4 H, linker protons), 1.24−1.10 (m,
18 H, linker protons); MS (MALDI-TOF) m/z calcd for
C64H120N18O26 [M + H2O]+, found 1576.11; UV (water) λmax
=
228 nm.
DPA65: IR (neat, cm−1) 3434 (br, OH), 2086 (weak), 1637;
1H NMR (500 MHz, D2O) δ 8.52 (s, 2 H, triazole), 8.24 (s,
1 H, Ar), 7.80 (d, J = 7.72 Hz, 2 H, Ar), 7.58 (t, J = 7.88 Hz,
1 H, Ar), 5.94 (d, J = 3.62 Hz, 2 H, H1II), 5.38 (d, J = 3.00 Hz,
2 H, H1III), 5.25 (d, J = 1.10 Hz, 2 H, H1IV), 4.90−4.83 (m,
2 H, H4III), 4.81−4.76 (m, 2 H), 4.64−4.56 (m, 6 H), 4.24 (t,
J = 4.73 Hz, 2 H), 4.16 (t, J = 4.10 Hz, 2 H), 4.13 (t, J =
2.99 Hz, 2 H), 4.05 (t, J = 9.77 Hz, 4 H, H5I, H6II), 3.95−3.85
(m, 6 H), 3.85−3.79 (m, 2 H), 3.74−3.71 (m, 2 H), 3.67−3.59
(m, 4 H), 3.58−3.49 (m, 8 H), 3.49−3.38 (m, 4 H), 3.47−3.34
(m, 2 H), 3.47−3.31 (m, 4 H), 3.31−3.29 (m, 4 H), 3.29−3.22
(m, 6 H), 3.07−3.01 (m, 2 H), 2.37 (dd, J1 = 4.10 Hz, J2 =
4.25 Hz, 2 H, H2Ieq.), 1.82−1.72 (q, 2 H, H2Iax); MS (MALDI-
TOF) m/z calcd for C56H96N18O24 (M + Na+) 1423.47, found
1424.41 [M + H2O]+; UV (water) λmax = 236 nm. Anal. Calcd
for C56H108N18O24Cl12: C, 36.49; H, 5.91; Cl, 23.08; N, 13.68;
O, 20.83. Found: C, 36.22; H, 5.81; N, 13.48.
a
Scheme 2.
RESULTS AND DISCUSSION
■
Design and Synthesis of Triazole-Linked Neomycin
Dimers. Previous ESI-MS data have shown that neomycin has
at least two binding sites on HIV TAR RNA.52 One binding site
has been reported to be below the trinucleotide bulge region
(see Scheme 1 for structures), and the second binding site lies
in the upper stem or hairpin region. To take advantage of these
multiple sites and to improve ligand affinity and selectivity, we
synthesized a series of neomycin dimers. The neomycin dimers
were synthesized using a high-yielding, inert, and robust syn-
thetic “click chemistry” approach as shown in Scheme 2.54−56
The length and functionality of the linker that tethers the two
neomycin units have been varied to investigate the optimal
binding interaction between neomycin dimers and HIV TAR
RNA. Azide 2 was synthesized in three steps from commercially
available neomycin 157 and then reacted with terminal dialkynes
in a 2:1 ratio to yield the traizole-linked dimers (Scheme 2).
Deprotection of tert-butoxycarbonyl (Boc) groups using 4 N
HCl in dioxane yielded the dimer hydrochloride salts58−61
DPA52−DPA65 in excellent yields.
a
Neomycin Dimers Significantly Enhance the Thermal
Stability of HIV TAR RNA. UV thermal denaturation studies
were conducted with the dimers and HIV 1 TAR RNA
(Figure 1 and Table 2; see section S3 of the Supporting
Information for UV denaturation plots). Neomycin dimers
Reagents and conditions: (a) (Boc)2O, DMF, H2O, Et3N, 60 °C, 5 h,
60%; (b) TPS-Cl, pyridine, room temperature, 40 h, 50%; (c) NaN3,
DMF/H2O (10:1), 12 h, 90 °C, 90%; (d) toluene, CuI, DIPEA, 90 °C;
(e) 4 N HCl in dioxane, room temperature, 5 min. Yield for steps
d and e of 82−90%.
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Figure 1. Concentration dependent UV thermal denaturation profile of HIV TAR RNA with neomycin dimer DPA52. UV thermal denaturation data
of HIV TAR RNA with no DPA52, and 4 μM (4 mol equiv) of DPA52 (A). Plot showing the change in thermal stabilization (ΔTm) as a function of
molar equivalents of DPA52 (B). Buffer conditions: 100 mM KCl, 10 mM SC, 0.5 mM EDTA, pH 6.8. [HIV TAR RNA] = 1 μM/strand. The
heating rate was 0.3 °C.
Table 2. UV Thermal Denaturation Data of HIV TAR RNA
in the Presence of Increasing Concentrations of Neomycin
Dimer DPA52
when going from a 0:1 to a 1:1 ratio (neomycin dimer:HIV
TAR RNA). (2) The UV thermal denaturation of HIV TAR
RNA with an increase in the amount of all the neomycin dimers
from 1 to 2 molar equiv shows the same change in Tm (2−3 °C).
(3) There was a thermal stabilization of 12.4 °C observed for
HIV TAR RNA in the presence of 4 molar equiv of DPA52 in
comparison to a value of 9.3 °C at 1 molar equiv. The thermal
denaturation data suggest that in all likelihood (1) a specific
binding interaction takes place between neomycin dimers and
HIV TAR RNA at a binding stoichiometry of 1:1 (neomycin
dimer:HIV TAR RNA). The concentration-dependent study
suggests that binding of the neomycin dimer to HIV TAR RNA
is not simply a result of charge interactions but involves poten-
tial and shape complementarity to the RNA target. (2) A higher
molar ratio of the neomycin dimer does not enhance the UV
thermal stability significantly. It is indicative of a 1:1 binding
ratio between neomycin dimer and HIV TAR RNA. (3) Addition
of a large excess of neomycin to TAR does not have any effect on
the thermal stability of HIV TAR RNA, suggesting that the sum of
the monomer parts clearly leads to a much improved ligand with
enhanced RNA affinity.
CD Spectroscopy Studies for Characterizing the Bind-
ing between the Neomycin Dimer and HIV TAR RNA. CD
spectroscopy is a powerful tool for monitoring and character-
izing the binding interaction between macromolecules (nucleic
acids and proteins) and ligands.32,33 CD spectroscopy can pro-
vide useful insight into the change in the conformation of the
macromolecule during the binding reaction.32 CD spectroscopy
was used to investigate the binding interaction between the
neomycin dimer and HIV TAR RNA. The CD spectrum of
HIV TAR RNA consists of a strong positive peak at 265 nm
and an equally strong peak with a negative magnitude at 211 nm.
Additionally, there is a negative peak observed at ∼240 nm. This
CD spectrum is indicative of an A-form conformation. Earlier
CD studies of the Tat−HIV TAR RNA complex revealed a
substantial change in the structure of HIV TAR RNA induced
by Tat protein. During the Tat−HIV TAR RNA binding inter-
action, there is a slight red shift observed at 265 nm. Additionally,
the CD intensity of HIV TAR RNA at 265 nm decreases by
15%.62 The CD peak at 265 nm is a signature peak for the
a
DPA52:TAR RNA ratio
Tm (°C)
ΔTm (°C)
TAR RNA
68.9
78.2
80.4
81.3
−
9.3
1
2
4
11.5
12.4
a
Buffer conditions: 100 mM KCl, 10 mM SC, 0.5 mM EDTA, pH 6.8.
The HIV TAR RNA concentration was 1 μM/strand. The heating rate
was 0.3 °C.
significantly enhance the thermal stability of HIV TAR RNA
[ΔTm = 3.3−10.2 °C (Table 2)]. The level of thermal stabi-
lization of HIV TAR RNA by neomycin dimers decreases with
increasing linker length between the two neomycin units. The
UV thermal denaturation data indicate that the probable
binding sites of two neomycin units are in the proximity of each
other. Additionally, neomycin displayed no thermal stabiliza-
tion of TAR RNA, as opposed to the neomycin dimers. There
was very little thermal stabilization observed (ΔTm = 0.5 °C) of
HIV TAR RNA even when the concentration of neomycin was
increased above a 1:1 ratio.
The UV denaturation data suggest that the thermal stabi-
lization of HIV TAR RNA by neomycin dimers is a result of
specific interaction as opposed to a nonspecific electrostatic
cation (neomycin dimers)−anion (HIV TAR RNA) interaction.
To further validate our assumption, a concentration-dependent
thermal denaturation study was performed in which the con-
centration of the dimer was varied at a fixed RNA concentration.
UV thermal denaturation of HIV TAR RNA was monitored at
2 molar equiv of neomycin dimers, and the results are summari-
zed in Table 3. Additionally, a concentration-dependent UV
thermal denaturation study was conducted with HIV TAR RNA
and DPA52 [up to 4 molar equiv of DPA52 (Figure 1)], and
the results are also summarized in Table 2.
There are few important points to be noted. (1) There was
only 2−3 °C additional thermal stabilization observed in going
from 1 to 2 molar equiv of neomycin dimers. This is in contrast
to the UV thermal stabilization of HIV TAR RNA of ∼11 °C,
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formed63 (ethidium bromide as an intercalator) (Figure 2C).
The change in fluorescence was plotted as a function of the
number of molar equivalents of the neomycin dimer, and the
inflection point also allows us to estimate the neomycin
dimer:HIV TAR RNA binding stoichiometry. Both CD and
FID titration data reveal a 1:1 neomycin dimer:HIV TAR
RNA binding stoichiometry.
Table 3. Representative UV Thermal Denaturation Data of
Ligand−HIV TAR RNA Complexes at rdr Values of 1 and 2
a
Tm
ΔTm (°C)
(at rdr = 1)
ΔΔTm (°C)
(°C)
(rdr = 2 − rdr = 1)
b
b
HIV 1 TAR
RNA
68.9
NA
NA
DPA51
DPA52
DPA65
DPA53
DPA54
DPA55
DPA56
DPA58
DPA60
neomycin
79.1
78.2
78.2
78.5
77.1
76.4
74.9
74.3
72.1
69.1
10.2
9.3
9.3
9.6
8.2
7.6
6.1
5.4
3.3
0.2
2.8
2.5
2.1
2.9
2.2
2.8
3.2
2.9
3.0
0.4
Ethidium Bromide Displacement and FRET Competi-
tion Binding Assay for Characterizing the Binding of
Neomycin Dimers to HIV TAR RNA. To further investigate
the binding between HIV TAR RNA and neomycin dimers,
ethidium bromide displacements63 were performed, and
the results are summarized in Table 4 (see section S4 of the
Supporting Information for FID assay plots). The affinities of
neomycin dimers are directly proportional to the amount of
ethidium bromide displaced from HIV TAR RNA and reflected
in the IC50 (the concentration of ligand required to displace
50% ethidium bromide from HIV TAR RNA) values. Ethidium
bromide displacement experiments indicate that in general,
neomycin dimers with shorter linker lengths have a higher
affinity for HIV TAR RNA than neomycin dimers with a longer
linker. The IC50 values for neomycin dimers fall within a range
of 36−99 nM. In comparison to neomycin dimers, neomycin
has a very low affinity for HIV TAR RNA. The IC50 value of
neomycin toward HIV TAR RNA is 417 115 nM. The trend
of linker length versus IC50 values observed here is the same as
that observed for linker length versus δTm values from UV
thermal denaturation experiments. These results also indicate
that the two neomycin binding sites on HIV TAR RNA are
very close to each other. Hence, the neomycin dimers with
shorter linkers bind tightly, as reflected in the lower IC50 and
stronger thermal stabilization toward HIV TAR RNA.
The CD titration and UV denaturation data show that the
dimers bind with a stoichiometry of 1:1 with TAR RNA. We
therefore performed ethidium bromide displacement titrations
between HIV TAR RNA and the neomycin dimer to determine
the association constant using Scatchard analysis63 (Figure 3A−D).
The plot between the change in fluorescence and the number of
molar equivalents of neomycin dimer(s) to TAR RNA was used
(as described above) to determine the binding stoichiometry of
neomycin dimer toward HIV TAR RNA (Figure 3B). The data
from ethidium bromide (EtBr) displacement titrations were
analyzed and transformed using Scatchard analysis (Figure 3C).
The slope of the plot allows us to determine the association
constant of neomycin dimer(s) with HIV TAR RNA (Figure 3D).
FID titrations were performed with all the neomycin dimers
and the neomycin monomer (see section S6 of the Supporting
Information for FID titrations) with TAR RNA, and the results
are summarized in Table 5. The binding constant for binding of
neomycin to TAR RNA could not be determined using this
analysis, because of the weak affinity of neomycin for HIV TAR
RNA (as indicated by the small displacement of EtBr). This is
consistent with the earlier observations (UV melt and FID assay)
that show a weak affinity of neomycin for HIV TAR RNA.
A comparison was then made between the linker lengths of
neomycin dimers and the binding constants derived from
Scatchard analysis (Figure 4). The binding constant decreases with
an increase in the length of the linker. Neomycin dimer DPA56
(linker length of 10) was an exception, as it showed a higher affinity
than DPA54 with a linker length of 8, but still much lower affinity
than dimers with linker lengths of 7 or 8. The trend in binding
constant versus Ka was compared with the data from the UV
thermal denaturation experiment (Figure 4). The trends are similar
a
rdr is the ratio of neomycin dimer to HIV TAR RNA. Buffer
conditions: 100 mM KCl, 10 mM SC, 0.5 mM EDTA, pH 6.8. The
HIV TAR RNA concentration was 1 μM/strand. The neomycin dimer
concentrations were 1 and 2 μM. The neomycin concentrations were
b
1 and 2 μM. Not applicable.
trinucleotide bulge region (UCU) and has been attributed to the
arrangement of base stacking in the bulge region.22 During the
Tat−HIV TAR RNA interaction, the Tat protein binds at its
primary binding site, which is the trinucleotide region, and
modifies the arrangement of base stacking in the bulge region.
The binding interaction was monitored by a decrease in the CD
intensity at 265 nm. There is a marginal red shift observed at
265 nm, indicative of the small distortion of the A-form structure.
A CD titration was performed with the neomycin dimer and
HIV TAR RNA by continuous addition of the dimer to an HIV
TAR RNA solution (Figure 2A). The overall shapes of the CD
spectra in the absence and presence of the dimer are similar,
suggesting that the overall conformation of HIV TAR RNA is
conserved. There are some changes observed in the spectrum
of HIV TAR RNA upon addition of the dimer. The addition
of the neomycin dimer causes a gradual decrease in the mag-
nitudes of the peaks at 210 and 240 nm. Contrary to Tat pro-
tein binding, there was no change in the CD intensity of the
HIV TAR RNA−dimer complex at 265 nm; however, a red
shift was observed. This observation suggests that the dimer
does not interact with the trinucleotide bulge region (Figure 2 A).
However, the interaction of the neomycin dimer causes a slight
distortion of the A-form conformation of HIV TAR RNA and
pushes it toward B-form. All these observations collectively
provide insight into the probable binding site of the neomycin
dimer on HIV TAR RNA. The primary binding site of neomycin
is just below the trinucleotide bulge region (see Scheme 1);
hence, one unit of neomycin likely binds to the lower stem region
of HIV TAR RNA, and the second neomycin unit binds to the
upper stem or the hairpin region. Further structural studies aim to
evaluate the effect of dimer binding on RNA conformation and
will be reported in due course.
CD spectroscopy is useful in identifying global changes in the
macromolecule structure, but it also can provide valuable infor-
mation about the binding stoichiometry of ligands involved in
the study.33 The change in CD intensity was plotted as a func-
tion of neomycin dimer:HIV TAR RNA molar ratio, and the
plot results in an inflection point (Figure 2 B) suggesting that
the binding stoichiometry is 1:1 (neomycin dimer:HIV TAR
RNA). To verify the results of CD spectroscopy, an FID (fluo-
rescent intercalator displacement) titration was also per-
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Figure 2. Comparison of CD titration and FID titration for determining the binding stoichiometry between HIV TAR RNA and DPA65. (A) CD
titration of HIV TAR RNA with increasing concentrations of the neomycin dimer. This figure represents the molar ellipticity for CD titration of HIV
TAR RNA with the neomycin dimer. The continuous changes in the CD spectra correspond to the incremental amount of neomycin dimer ranging
from an rdr of 0 to an rdr of 3. (B) Plot of normalized molar ellipticity vs rdr for CD titration of HIV TAR RNA with the neomycin dimer. The solid
lines reflect the linear least-squares fit of each apparent linear domain of the experimental data (red square) before and after the apparent inflection
point. (C) Raw fluorescence emission spectra in the presence of increasing concentrations of DPA65. (D) The plot of the normalized fluorescence
intensity (at 605 nm) of the HIV TAR RNA−ethidium bromide complex as a function of DPA65 concentration results in a saturating binding plot.
For CD, the molar ellipticity is per molecule of HIV TAR RNA, and rdr is the drug:HIV TAR RNA ratio. The HIV TAR RNA concentration was
4 μM/strand. For FID titration, the HIV TAR RNA concentration was 100 nM/strand.
Table 4. Representative Data from an Ethidium Bromide
Displacement Assay
Salt-Dependent Studies of the Neomycin Dimer with
TAR RNA. We then studied the effect of salt on the binding
between the neomycin dimer and HIV TAR RNA. FID titrations
were conducted at three different salt concentrations (Figure 5),
and the data was analyzed and fit using Scatchard analysis to
determine the binding constants. The binding constants at
three different salt concentrations are summarized in Table 6.
The binding affinity of neomycin dimer DPA52 for HIV
TAR RNA continuously decreases with an increase in the KCl
concentration from 50 to 150 mM. The salt-dependent binding
parameters were used to calculate the number of ion pairs
formed during the association of the neomycin dimer and HIV
TAR RNA, as described by Record.64 In the case of HIV TAR
RNA−neomycin dimer interaction, there are two or three ion
pairs between the ligand and RNA. The free energy of the
electrolytic contribution was then calculated using eq 1.64,65
a
ligand
linker length
IC50 (nM)
DPA51
DPA52
DPA65
DPA53
DPA54
DPA55
DPA56
DPA58
DPA60
neomycin
7
7
56
52
36
67
81
99
97
67
74
417
7
8
8
10
10
16
20
b
NA
a
Buffer conditions: 100 mM KCl, 10 mM SC, 0.5 mM EDTA, pH 6.8.
b
The HIV TAR RNA concentration was 50 nM/strand. Not
applicable.
ΔG = −ZφRT ln[KCl]
pe
(1)
if not identical. The binding constant decreases with an increase in
linker length; similarly, the UV thermal stabilization of HIV TAR
decreases with an increase in neomycin dimer linker length.
where Z denotes the charge on the ligand and ϕ is the number
of the counterions associated with each phosphate group on
nucleic acid that normally varies for nucleic acids. The value of
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Figure 3. FID titration of DPA65 with HIV TAR RNA. (A) Raw fluorescence emission spectra in the presence of increasing concentrations of
DPA65. (B) The decrease in fluorescence intensity (at 605 nm) of the HIV TAR RNA−EtBr complex with an increasing concentration of DPA65
results in a saturating binding plot. (C) The plot between normalized fluorescence intensity (at 605 nm) of the HIV TAR RNA−EtBr complex as a
function of DPA65 concentration results in a saturating binding plot. (D) Scatchard plot analysis of DPA65 with HIV TAR RNA. Buffer conditions:
100 mM KCl, 10 mM SC, 0.5 mM EDTA, pH 6.8. The HIV TAR RNA concentration was 200 nM/strand. The EtBr concentration was 5 μM.
Table 5. Representative Binding Constants Derived from
Scatchard Analysis of the FID Assay (ethidium bromide as
an intercalator) between the Neomycin Dimer and HIV TAR
TAR RNA contains hairpin, bulge, and duplex regions;
therefore, it is likely that the value of ϕ falls within the range
of these two values, and hence, the average of these two values
was used in the analysis. The free energy of the electrolytic
contribution was calculated by inserting the experimental values
into eq 1. The electrolytic contribution to the free energy
(ΔGpe) obtained is −3.45 kcal/mol at 100 mM KCl, and the
total free energy of interaction (ΔGobs) is −10.69 kcal/mol.
The free energy contribution of nonelectrolytic energy was
calculated using eq 2 as −7.24 kcal/mol (Figure 6).
a
RNA
linker length
K (M−1
)
DPA51
DPA52
DPA65
DPA53
DPA54
DPA55
DPA56
DPA58
DPA60
neomycin
7
7
1.17 × 108
7.08 × 107
1.39 × 108
1.46 × 108
2.61 × 107
1.06 × 107
6.60 × 107
7.58 × 106
2.53 × 107
−
7
8
8
ΔG = −RT ln K = ΔG + ΔG
non‐pe
10
10
16
20
obs
pe
(2)
Figure 6 shows that the major contribution to the binding
event between the neomycin dimer and HIV TAR RNA is
nonelectrolytic. This observation provides further insight into
the mode of binding of the neomycin dimer to HIV TAR RNA.
Though the dimer is a highly positively charged ligand (12
aliphatic amines, 10 of which are expected to be protonated at
physiological pH), the free energy of the electrolytic contri-
bution does not play a major part in the binding to RNA. The
observation suggests that the shape complementarity of the
b
NA
a
Buffer conditions: 100 mM KCl, 10 mM SC, 0.5 mM EDTA, pH 6.8.
The HIV TAR RNA concentration was 200 nM/strand. The EtBr
b
concentration was 5 μM. Not applicable.
ϕ for the A-form RNA duplex [poly(rA)·poly(rU)] is 0.89,
while that for single-stranded RNA [poly(rA)] is 0.78. HIV
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Figure 4. Bar graph showing the comparison of binding constants derived from Scatchard analysis of the FID titrations (ethidium bromide as an
intercalator) (left) and UV thermal denaturation profile of neomycin dimers with HIV TAR RNA (right). Buffer conditions: 100 mM KCl, 10 mM
SC, 0.5 mM EDTA, pH 6.8. For FID titration, the HIV TAR RNA concentration was 200 nM/strand. For the UV thermal denaturation experiment,
the HIV TAR RNA concentration was 1 μM/strand. The neomycin dimer concentration was 1 μM. The neomycin concentration was 1 μM.
Figure 5. Salt-dependent studies between neomycin dimer DPA52 and HIV TAR RNA. The binding constants were determined from the FID
titration using Scatchard plot analysis at (A) 50, (B) 100, and (C) 150 mM KCl. (D) Plot of log(K) as a function of log[KCl] fit with linear
regression. The solid line reflects the linear fit that results in a slope that reflects the number of ion pairs forming between DPA52 and HIV TAR
RNA. Buffer conditions: 10 mM SC, 0.5 mM EDTA, pH 6.8. The HIV TAR RNA concentration was 200 nM/strand. The EtBr concentration was 5 μM.
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among an analogue of Tat protein, a fluorescently labeled Tat
peptide, and the ligands used in the study. In this assay, the
Tat49−57 peptide was functionalized using fluorescein dye at its
N-terminus and tetramethyl rhodamine dye at the C-terminus
to collectively form a FRET system.53
Table 6. Representative Binding Constants Derived from
Scatchard Analysis Showing the Salt Dependence of Binding
between DPA52 and HIV TAR RNA
a
[KCl] (mM)
K (M−1
)
50
100
150
3.18 × 108
7.08 × 107
1.87 × 107
The fluorescein-labeled Tat49−57 peptide was first titrated
against HIV TAR RNA to determine its affinity (Figure 7). The
fluorescence increases with an increase in the concentration of
HIV TAR RNA in a fluorescein-labeled Tat49−57 peptide solution
and reaches a constant value at a saturating concentration of HIV
TAR RNA. Dimers were then titrated against the fluorescein-
labeled Tat49−57 peptide with an HIV TAR RNA solution (Figure 8;
see section S5 of the Supporting Information for FRET assays). The
binding affinity of ligands is directly related to the decrease in the
fluorescence. The FRET assay was performed with neomycin
dimers and neomycin, and the results are summarized in Table 7.
The IC50 value of neomycin is almost 1 order of magnitude
higher than that of the neomycin dimers, suggesting much higher
affinities for the dimers toward HIV TAR RNA than the
monomer neomycin. The IC50 value of fluorescein-labeled
a
Buffer conditions: 10 mM SC, 0.5 mM EDTA, pH 6.8. The HIV
TAR RNA concentration was 200 nM/strand. The EtBr concentration
was 5 μM.
neomycin dimer to the TAR RNA conformation is largely re-
sponsible for the high binding affinity of this highly positively
charged pharmacophore toward HIV TAR RNA.
Fluorescence Resonance Energy Transfer-Mediated
Competition Binding Assay. A FRET-mediated fluoremetric
competition binding assay was performed to characterize the
binding interaction of neomycin dimers with HIV TAR RNA.
The assay developed by Hamashaki and co-workers53 is very
useful in defining the binding interaction of ligands with HIV
TAR RNA. This particular assay is different from the ethidium
bromide displacement assay. The assay gives direct competition
Tat49−57 peptide is 86
9 nM. Neomycin dimers show IC50
values in the range of 47−80 nM. In general, neomycin dimers
exhibit higher binding affinities for HIV TAR RNA than
neomycin or the Tat49−57 peptide does. The FRET results for
dimer−TAR RNA binding are consistent with the data obtained
from the aforementioned techniques such as UV thermal
denaturation and ethidium bromide displacement.
The results of the FRET-mediated assay showed that neo-
mycin dimers exhibit nanomolar affinity toward HIV TAR
RNA. A comparison of binding affinity can be made with the
earlier reported neamine dimers (Figure 9). Previously reported
neamine dimers showed IC50 values in the range of 150−400 nM
(pH 7.4 in 50 mM Tris-HCl and 20 mM KCl) using a FRET-
mediated competition binding assay and have much lower
affinities compared to those of neomycin dimers reported here
(47−128 nM at pH 7.4 in 50 mM Tris-HCl and 20 mM KCl).
The results clearly show a 3−4-fold preference of neomycin
dimers versus neamine dimers for HIV TAR RNA.51 These
observations acknowledge the contribution of rings 3 and 4 in
neomycin−HIV TAR RNA binding.
The affinity of neomycin dimers for HIV TAR RNA was
compared using FRET and ethidium bromide FID assays
Figure 6. Bar graph showing the dissection of the free energy into its
components, including electrolytic and nonelectrolytic contributions.
Figure 7. (A) Fluorescein-labeled Tat49−57 peptide used for the FRET-mediated fluoremetric competition binding assay. (B) Saturating binding
curve of fluorescein-labeled Tat49−57 peptide with HIV TAR RNA at 25 °C. The binding affinity (IC50) reported is the average of three to five
individual measurements and was determined by fitting the experimental data to a sigmoidal dose−response nonlinear regression model via
GraphPad Prism 4.0.
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Figure 8. Determination of IC50 values using the FRET competition binding assay. Titration curves are shown for the IC50 values of (A) neomycin
and (B) DPA53. The binding affinity (IC50) values reported for each ligand are the averages of three to five individual measurements and were
determined by fitting the experimental data to a sigmoidal dose−response nonlinear regression model via GraphPad Prism 4.0.
Table 7. IC50 Values of Ligands Determined by a FRET-Mediated Competition Binding Assay
(Figure 10). The results from both assays were comparable as
observed from the trends in IC50 values as a function of linker
length. The binding affinity of shorter and rigid linkers is higher
and reflected in the lower IC50 value, as compared to the higher
IC50 obtained with longer linkers. The lowest IC50 was dis-
played by DPA53 and DPA65 with HIV TAR RNA. Dimers
DPA65 and DPA53 are the most rigid structures in the triazole
library reported here. They have a phenyl ring between two
neomycin units in addition to the two triazole rings leading to
an extended aromatic system that enhances the linker rigidity of
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Figure 9. Chemical structure of neomycin and neamine dimers.
Figure 11. Structure of TAR RNA and mutant tetraloop TAR used in
this study.
may manifest themselves as a perceived anti-HIV effect. CT5 is
a nontoxic concentration at which 95% of the cells are viable.
The maximal concentrations of the neomycin conjugates used
are listed in Table 9. For all compounds, this is the concen-
tration of ligand at which no more than 5% cell death was
observed (CT5). For comparison, water was used as control.
We next assessed the anti-HIV activities of the compounds.
This assay was performed in triplicate wells of a 96-well plate.
It utilized Finter’s neutral red dye, a vital dye, to assess the ability of
any given antibody or compound to inhibit the HIV-induced cyto-
pathic effect. As indicated in Table 8, neomycin dimers showed
much improved protection from the cytopathic effect as opposed
to neomycin and water (control). Neomycin dimer DPA52
showed little protection from the cytopathic effect, suggesting
that minor variations in linker length can significantly affect the
pharmacodynamic and/or pharmacokinetic properties of these
ligands. Neomycin dimers DPA53−DPA56 showed 20−33% pro-
tection from the cytopathic effect in the concentration range of 4−
17 μM. Overall, the compounds showed modest anti-HIV activity
in this assay with a maximal 33% suppression of cell death.
We then confirmed the anti-HIV activity by two additional
assays. These include the spread of HIV through culture, as
measured by the immunofluorescence assay (IFA), and the
release of reverse transcriptase (RT) into supernatant fluids. In
this experiment, nontoxic concentrations of each of five neo-
mycin dimers, with some anti-HIV activity (Table 8), were
selected. We pretreated 500000 cells with each compound for
1 h. Next, cells were inoculated with approximately 50000
infectious particles of HIVNL4‑3. Infections were performed in
triplicate. Every 2 days cells were collected and stained for HIV
antigen synthesis. Culture supernatants were analyzed for RT
release. As shown in Table 9, the compounds had a more
robust effect on HIV antigen synthesis than was apparent in the
Figure 10. Bar graph showing the comparison of IC50 values of
neomycin dimers with HIV TAR RNA using a FRET-mediated
competition binding assay and an ethidium bromide displacement
assay.
these dimers in comparison to other more flexible dimers such
as DPA52 and DPA54.
To investigate the specificity of the tight binders, we then
studied the binding of DPA65 with a TAR mutant (tetraloop
TAR), as shown in Figure 11. FID titrations were conducted
between DPA65 and mutant TAR (Figure 12), and Scatchard
analysis of the data yielded a Ka of ∼107 M−1. When compared
to its affinity for wild-type HIV TAR RNA, DPA65 binds to
mutant HIV TAR RNA with an affinity that is 1 order of
magnitude lower, suggesting that linker length variations can
lead to RNA binders with higher affinities and selectivity
than monomeric aminoglycosides.
In Vitro Assay To Study the Effect of Neomycin
Dimers in MT-2 Cells. An important step in the life cycle of
HIV is the death of the infected cell population. The HIV
envelope uses the CD4 cell as a receptor. The infection caused
by HIV through CD4 results in an immense enhancement in
the number of infected cells, including cell−cell fusion and cell
death, a phenomenon called the cytopathic effect (CPE).66
Neomycin dimers and neomycin were monitored for their
ability to protect MT-2 cells from the cytopathic effect caused
by HIV (Table 9). We first assessed toxicity against MT-2
cells, a CD4+, lymphoblastoid cell line. We utilized the highly
restrictive definition of 5% cytotoxicity (CT5), rather than the
more commonly reported 50% cytotoxicity (CT50), as HIV is
an obligate intracellular parasite. Any toxic effects on the cell
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Figure 12. FID titration of DPA65 with a tetraloop TAR RNA mutant. (A) Raw fluorescence emission spectra in the presence of increasing
concentrations of DPA65. (B) Decrease in fluorescence intensity (at 610 nm) of the tetraloop TAR RNA−EtBr complex with an increasing DPA65
concentration. (C) Plot between normalized fluorescence intensity (at 610 nm) of the tetraloop TAR RNA−EtBr complex as a function of DPA65
concentration. (D) Scatchard analysis of DPA65 with tetraloop TAR RNA. Buffer conditions: 100 mM KCl, 10 mM SC, 0.5 mM EDTA, pH 6.8.
The tetraloop TAR RNA concentration was 200 nM/strand. The EtBr concentration was 5 μM.
Table 8. Five Percent Toxicity Concentrations of Neomycin
Dimers and Neomycin Showing the Maximal Protection
Obtained from HIV Cytopathic Effects
Table 9. Inhibition by Neomycin Dimers of HIV Antigen
Synthesis in Treated Cells
percent of HIV antigen synthesis
5% toxicity
maximal protection [concentration achieved
concn (μM)
day 2
day 4
day 6
(μM)
(μM)]
DPA52
25
9
15
2
100
40
100
100
100
100
100
100
neomycin
DPA52
DPA53
DPA54
DPA55
DPA56
water
>206
>138
17
9% (206)
1% (9)
DPA53
DPA54
17
4
3−5
3−5
5−7
15
80
33% (8)
31% (17)
20% (4)
33% (17)
2%
DPA55
30
69
DPA56
8
40
8
a
virus control
NA
100
34
a
Not applicable.
none
RNA as borne out by the FRET-mediated competition binding
assay, the ethidium bromide assay, and the UV thermal dena-
turation experiment.
Neomycin dimers also reduced the amount of reverse tran-
scriptase (RT) released into the cell culture supernatant as
opposed to the virus control.67 HIV replication was suppressed
by an even greater amount in this assay. For example, DPA56
suppressed RT release by 90% at day 4 and at least 20% at day
6 (Table 10). A couple of important observations need to be
cytopathicity assay. Indeed, several compounds inhibited HIV
replication by as much as 70% (day 4).
The neomycin dimers inhibit HIV antigen synthesis effec-
tively up to 4 days postinoculation. Neomycin dimers DPA53,
DPA55, and DPA56 inhibit HIV antigen synthesis from 60 to
70% up to the fourth day as opposed to other neomycin dimers.
The virus control contains the infected cells and water solvent.
Dimer DPA53 has one of the highest affinities for HIV TAR
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a
Table 10. Release of Reverse Transcriptase into the Culture Supernatant (counts per minute per milliliter)
concn (μM)
day 2 (cpm/mL)
day 4 (cpm/mL)
day 6 (cpm/mL)
DPA52
25
9
21485 (4574)
8880 (1373)
14805 (1309)
20072 (3748)
15989 (2523)
46029 (6651)
347845 (10680)
45539 (32831)
165301 (22967)
107933 (17963)
105704 (15948)
268357 (57519)
221445 (20678)
427475 (49583)
305277 (59913)
412475 (19612)
1078741 (188673)
DPA53
DPA54
17
4
DPA55
DPA56
8
b
virus control
NA
928112 (126547)
a
b
Values are means of triplicate infections, and values in parentheses are one standard deviation. Not applicable.
noted. (1) All the neomycin dimers inhibit the release of RT.
(2) Neomycin dimer DPA53 is the most effective among all the
neomycin dimers studied, which is reflected by the maximal
inhibition (Table 10) at moderate concentrations. (3) As opposed
to the HIV antigen inhibition assay, neomycin dimers inhibit the
release of RT even after 6 days.
with a stoichiometry of 1:1 with HIV TAR RNA. (2) The CD
spectra in the presence of a neomycin dimer suggest that it does
not interact with the trinucleotide bulge region, which is the
binding site of Tat protein. Additionally, neomycin dimer
binding pushes the conformation of HIV TAR RNA away from
an A-form. (3) The ethidium bromide displacement titration
between neomycin dimers and HIV TAR RNA showed a
binding stoichiometry of 1:1, validating the results from CD
and UV thermal denaturation experiments. (4) Scatchard analysis
of FID titrations results in a binding constant of 107−108 M−1
between neomycin dimers and HIV TAR RNA depending on
linker length. The trends in binding affinity obtained are similar to
the trends in UV thermal denaturation stabilization. The neomycin
dimers with shorter linker lengths show the highest affinity for
HIV TAR RNA. (5) Salt-dependent studies show that despite the
highly charged nature of the dimeric ligands, the major contributor
to the free energy of binding comes from nonelectrolytic terms.
(6) The FRET-mediated competition binding assay confirms the
high affinity of neomycin dimers for HIV TAR RNA. The patterns
of TAR RNA−dimer binding affinity are similar to those obtained
from UV thermal denaturation and the FID assay. The binding
affinity of neomycin is much weaker than those of all the
neomycin dimers. (7) In vitro assays show that neomycin
dimers are effective inhibitors of HIV. Some of the neomycin
dimers effectively inhibit the release of reverse transcriptase at
low concentration. Our studies outline the growing potential of
click chemistry in efficiently generating multivalent ligands to
improve binding of ligands to therapeutic targets. Further
studies are ongoing to explore SAR of modified aminosugars,
structural studies of binding, and the details of mechanisms of
HIV inhibition by these dimers. These studies will be reported
in due course.
These findings were somewhat surprising, as we do not
usually see a significant anti-HIV effect in confirmatory assays
that is stronger than the anti-HIV activity seen in the cytopathic
effect assay (Table 8). A much more significant anti-HIV effect
of neomycin dimers was seen in the confirmatory assays.
Because DPA65 was one of the most potent binding agents,
two separate experiments were performed to determine the
anti-HIV activities of this compound. As with the other
compounds, DPA65 was inactive to weakly active in the 96-well
plate screening assay. When the anti-HIV properties of the
compound as measured by the release of RT into the culture
media were determined, they were highly reproducible over a
period of months and over many replicates. The compound was
tested three separate times, in triplicate each time. On day 2
post-inoculation, it protected cells by 40−67%. On day 4, it
protected cells by 68−73%, and on day 6, it protected cells by
24−27%. The lower levels of protection on day 6 were due to
lysis of the virus control infections on day 4, and thus, there was
little additional release of virus into the culture media from day
4 to day 6. To ascertain whether the compounds were acting at
Tat−TAR interactions within cells, we attempted to raise
inhibitor-resistant virus by culturing HIV in increasing concen-
trations of neomycin dimers. We were not able to raise a
resistant virus, indicating that the combination of anti-HIV
activity with the likelihood of deleterious mutations within Tat
or TAR precluded the selection of viable, inhibitor-resistant
HIV. Nevertheless, the unusual finding of weaker activity
against the HIV-induced cytopathic effect but a more potent
inhibition of antigen synthesis is consistent with an effect on
HIV transcription.
ASSOCIATED CONTENT
■
S
* Supporting Information
CONCLUSION
■
Characterization of triazole-linked neomycin dimers using click
chemistry, UV thermal denaturation profiles, ethidium bromide
displacement assay plots, FRET-mediated competition binding
assay plots, and ethidium bromide displacement titration to
determine the binding constant plots. This material is available
free of charge via the Internet at http://pubs.acs.org.
A series of neomycin dimers were synthesized via click chemistry
in high yields. The dimers were then studied with HIV TAR
RNA using various analytical techniques: UV thermal denatura-
tion, CD titration, FID assay, a FRET-mediated competition
binding assay, and in vitro cellular assays for HIV inhibition.
There are a number of conclusions that can be drawn from the
studies. (1) UV thermal denaturation studies show that neo-
mycin dimers enhance the melting temperature of HIV TAR
RNA up to 10.3 °C. The monomer neomycin does not show
any effect on the thermal stabilization of HIV TAR RNA. The
neomycin dimers with the shortest linker lengths displayed the
highest thermal stabilization, which decreases with an increase
in linker length. The concentration-dependent thermal
denaturation studies indicate that the neomycin dimer binds
AUTHOR INFORMATION
■
Corresponding Author
*Telephone: (864) 656-1106. Fax: (864) 656-6613. E-mail:
dparya@clemson.edu.
Notes
The authors declare no competing financial interest.
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Funding
Article
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We thank the National Science Foundation (CHE/MCB-
0134972) and the National Institutes of Health (R15CA125724)
for financial support.
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