Bioorganic & Medicinal Chemistry Letters
Recognition of HIV-TAR RNA using neomycin–benzimidazole
conjugates
Nihar Ranjan a, Sunil Kumar a, , Derrick Watkins b, Deyun Wang c, Daniel H. Appella c, Dev P. Arya a,b,
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a Laboratory of Medicinal Chemistry, Department of Chemistry, Clemson University, Clemson, SC 29634, United States
b NUBAD LLC, 900 B West Faris Road, Greenville, SC 29630, United States
c Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis of a novel class of compounds and their biophysical studies with TAR-RNA are presented. The
synthesis of these compounds was achieved by conjugating neomycin, an aminoglycoside, with benzim-
idazoles modeled from a B-DNA minor groove binder, Hoechst 33258. The neomycin–benzimidazole con-
jugates have varying linkers that connect the benzimidazole and neomycin units. The linkers of varying
length (5–23 atoms) in these conjugates contain one to three triazole units. The UV thermal denaturation
experiments showed that the conjugates resulted in greater stabilization of the TAR-RNA than either neo-
mycin or benzimidazole used in the synthesis of conjugates. These results were corroborated by the FID
displacement and tat-TAR inhibition assays. The binding of ligands to the TAR-RNA is affected by the
length and composition of the linker. Our results show that increasing the number of triazole groups
and the linker length in these compounds have diminishing effect on the binding to TAR-RNA. Com-
pounds that have shorter linker length and fewer triazole units in the linker displayed increased affinity
towards the TAR RNA.
Received 25 May 2013
Revised 30 July 2013
Accepted 5 August 2013
Available online 14 August 2013
Keywords:
TAR
Neomycin
Aminoglycosides
tat-TAR inhibition
Benzimidazole
Ó 2013 Elsevier Ltd. All rights reserved.
Since the first reports of HIV-AIDS in the United States in the
early 1980s, research towards its cure have identified an RNA di-
rected strategy which targets the interactions of tat protein with
the Trans Activating Region (TAR) of the viral RNA.1 A 29-mer oli-
gonucleotide, which is a model of a full 59 mer TAR region of the
viral RNA, contains two of the most commonly found structural
features in the RNA-namely the hairpin loop and the short trinu-
cleotide bulge (Fig. 1a). The trinucleotide bulge region has a wide
major groove that is accessed by the tat protein for viral replication
and thus inhibition of this interaction has developed into a viable
approach to stop viral growth.2,3
Several DNA and RNA binders have been investigated to inhibit
tat-TAR interactions. These binders include polyamines (arginina-
mide),3 polyamides,4 peptides,5,6 peptidomimetics,7 intercalators,8
quinoline derivatives,9,10 quinolones,11 DNA minor groove bind-
ers,2 aminoglycosides12–14 and their derivatives.15–17 Neomycin is
an aminosugar (Fig. 1b) that has been known for decades for its
RNA binding.18,19 Neomycin has been shown to inhibit the tat-
TAR interaction by binding to the trinucleotide pyrimidine bulge
of the TAR-RNA14 and has the highest affinity among the selected
aminoglycosides studied.12 Neomycin has been observed to bind
with the TAR-RNA making contacts in the minor groove present
in the lower stem using its ring III and IV while rings I and II have
been found to interact with the trinucleotide bulge (Fig. 1a and b).
The bis-benzimidazole Hoechst 33258, a known B-DNA minor
groove binding molecule, has also been shown to interact with
the TAR-RNA at a site opposite to the bulge region where it recog-
nizes the helical region below the hairpin loop (Fig. 1a). RNAase A
footprinting analysis has suggested that Hoechst 33258 binds to
GCUCU bases of the TAR RNA in the upper stem.2
Aminoglycosides have emerged as versatile nucleic acid binders
over the past decade.20–26 Several aminoglycoside conjugates have
been synthesized27–30 and shown to have enhanced binding to
variety of DNA,31–34 RNA,35 and DNA:RNA hybrid36 structures.
We have recently reported recognition of TAR-RNA using a series
of dimeric neomycin conjugates.37,38 The neomycin dimers have
shown significant enhancement in the protection of MT-2 cells
from the cytopathic effects of HIV infection in comparison to
neomycin alone.37 These studies have opened new avenues for
multi-valent approaches for the recognition of TAR RNA by amino-
glycoside based small molecules.
Abbreviations: UV, ultra violet; FID, fluorescent intercalators displacement; CD,
circular dichroism; TAR, trans activating region; NMR, nuclear magnetic resonance;
MALDI-TOF, matrix assisted laser desorption and ionization-time of flight; NA, not
available; ND, not determined.
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Corresponding author. Tel.: +1 (864) 656 1106; fax: +1 (864) 656 6613.
Present address: Department of Molecular Biophysics and Biochemistry, Yale
University, CT 06520, United States.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.