Neomycin-acridine conjugate: A potent inhibitor of Rev-RRE binding [16]

Full text of DOI:10.1021/ja993387i

980
J. Am. Chem. Soc. 2000, 122, 980-981
Neomycin-Acridine Conjugate: A Potent Inhibitor
of Rev-RRE Binding
Sarah R. Kirk, Nathan W. Luedtke, and Yitzhak Tor*
Department of Chemistry and Biochemistry
UniVersity of California, San Diego
La Jolla, California 92093-0358
ReceiVed September 20, 1999
The successful replication of many retroviruses requires an
ordered pattern of viral gene expression.1 Small organic molecules
that target viral RNA sites and prevent the formation of key
RNA-protein complexes are promising candidates for drug
discovery.2,3 Regulatory proteins that act posttranscriptionally are
particularly attractive since the RNA sequences they recognize
are likely to also serve as coding sequences for other essential
viral proteins.4 The HIV-1 Rev-Response-Element (RRE) serves
as the Rev-binding site responsible for the active export of
unspliced HIV genomic RNA from the nucleus as well as part of
the envelope-protein open reading frame.5 Because of this dual
function, the evolution of resistant variants may be prevented or
impeded. Neomycin B and other aminoglycoside antibiotics have
been shown to competitively block the binding of the Rev protein
to its RNA recognition element (RRE), thus providing an
important precedent for the utilization of small molecules to target
viral RNA sites.6,7 The natural antibiotics, however, are rather
promiscuous RNA binders and bind the RRE with relatively low
affinity.3a We hypothesize that by combining structural elements
essential for RNA binding with structure-specific determinants,
new small molecules with high affinity to the RRE site can be
identified.
Figure 1. The proposed secondary structure of the 67-nt RRE within
domain II of the HIV-1 RNA and the arginine-rich RNA-binding domain
of the Rev protein (Rev34-50). The RRE sequence is shown with a
schematic summary of the enzymatic protection experiments (see text).
Footprinting in the presence of Rev (open half-circle) and neo-acridine
(filled half-circle) as well as enhanced cleavage in the presence of Rev
(open triangle) and neo-acridine (filled triangle) are shown.
Scheme 1. Synthesisa and Structure of Neo-acridine (2)12
The high-affinity Rev binding domain within the RRE consists
of a stem-bulge-stem structure (Figure 1).8 The arginine-rich
fragment, Rev34-50, binds the RRE with a dissociation constant
comparable to that of the full-length Rev protein (Figure 1).9 The
NMR structure of a Rev-RRE complex reveals that binding of
the Rev peptide to the RRE initiates G48-G71 and G47-A73 base
pairing and forces U72 to bulge out.10 Since single base bulges
are potential high-affinity sites for intercalating agents,11 we
hypothesized that the combination of strong ionic interactions with
intercalation ability may lead to potent inhibitors of Rev-RRE
binding. A neomycin-acridine conjugate (2) has, therefore, been
aReagents and conditions: (a) (Boc)2O, DMF, H2O, Et3N, 60 °C, 2 h,
72%; (b) 2,4,6-triisopropylbenzenesulfonyl chloride, pyridine, room
temperature, 20 h, 75%; (c) H2NCH2CH2SH, NaOEt/EtOH, room
temperature, 4.5 h, 80%; (d) 9-phenoxyacridine, phenol, 60-75 °C, 1 h,
84%; (e) 4 M HCl/dioxane, HSCH2CH2SH, room temperature, 5 min,
80%.
synthesized by covalently linking neomycin B (1) to 9-amino-
acridine via a short spacer (Scheme 1).12 We report that neo-
acridine (2) is a potent inhibitor of Rev-RRE binding. Its affinity
to the RRE is two orders of magnitude higher than that of the
parent neomycin B (1) and approaches that of the Rev peptide.
Gel-shift mobility assays have been employed to qualitatively
study the interactions of Rev34-50 and neo-acridine (2) with the
RRE.12,13 As illustrated in Figure 2, neo-acridine (2) displaces
Rev from the Rev-RRE complex much more effectively than
neomycin B (1). Inhibition curves yield IC50 values of 5.9 ( 1.9
and 0.65 ( 0.1 µM for neomycin B and neo-acridine, respectively.
While not observed for neomycin B, a binary neo-acridine-RRE
complex is formed at ca. 2.5 µM 2. At this concentration the
Rev-RRE complex formation is completely inhibited. This
suggests the presence of a single high affinity site that is
responsible for disrupting the RNA-peptide complex.14 Gel shift
experiments demonstrate that the relative affinity of neo-acridine
(2) to the RRE is 2-fold lower than that of the Rev peptide.12
(1) Fields, B. N.; Knipe, D. M.; Howley, P. M., Eds. Fundamental Virology;
Lippincott-Raven: Philadelphia, 1996.
(2) Tor, Y. Angew. Chem., Int. Ed. 1999, 38, 1579-1582.
(3) (a) Michael, K.; Tor, Y. Chem. Eur. J. 1998, 4, 2091-2098. (b) Ecker,
D. J.; Griffey, R. H. Drug DiscoVery Today 1999, 4, 420-429. (c) For a
comprehensive review of RNA-ligand interactions, see: Chow, C. S.; Bogdan,
F. M. Chem. ReV. 1997, 97, 1489-1513.
(4) Coffin, J. M. in ref 1, pp 763-843.
(5) Vaishnav, Y. N.; Wong-Staal., F. Annu. ReV. Biochem. 1991, 60, 577-
630. Frankel, A. D.; Young, J. A. T. Annu. ReV. Biochem. 1998, 67, 1-25.
Pollard, V. W.; Malim, M. H. Annu. ReV. Microbiol. 1998, 52, 491-532.
Hope, T. J. ArchiV. Biochem. Biophys. 1999, 365, 186-191.
(6) Zapp, M. L.; Stern, S.; Green, M. R. Cell 1993, 74, 969-978.
(7) See: Zapp, M. L.; Young, D. W.; Kumar, A.; Singh, R.; Boykin, D.
W.; Wilson, W. D.; Green, M. R. Bioorg. Med. Chem. 1997, 5, 1149-1155.
Tok, J. B.-H.; Cho, J.; Rando, R. R. Tetrahedron 1999, 55, 5741-5758. Park,
W. K. C.; Auer, M.; Jaksche, H.; Wong, C.-H. J. Am. Chem. Soc. 1996, 118,
10150-10155.
(8) Holland, S. M.; Chavez, M.; Gerstberger, S.; Venkatesan, S. J. Virol.
1992, 66, 3699-3706. Tilley, L. S.; Malim, M. H.; Tewary, H. K.; Stockley,
P. G.; Cullen, B. R. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 758-762.
(9) Kjems, J.; Canlan; B. J.; Frankel, A. D.; Sharp, P. A. EMBO J. 1992,
11, 1119-1129.
(10) Battiste, J. L.; Mao, H.; Rao, N. S.; Tan, R.; Muhandiram, D. R.;
Kay, L. E.; Frankel, A. D.; Williamson, J. R. Science 1996, 273, 1547-1551.
(11) Wilson, W. B.; Ratmeyer, L.; Cegla, M. T.; Spychala, J.; Boykin, D.;
Demeunynyck, M.; Lhomme, J.; Krishnan, G.; Kennedy, D.; Vinayak, R.;
Zon, G. New J. Chem. 1994, 18, 419-423.
(12) See Supporting Information for experimental details.
(13) Modified Rev34-50 peptides of the sequence sucTRQARRNRRRRWR-
ERQRAAAACam have been utilized for all experiments. Succinylation of the
N-terminus and addition of four alanine residues to the C-terminus have been
shown by Frankel to enhance the R-helicity of the peptide and, consequently,
increase its affinity and specificity to the RRE (see: Tan, R.; Chen, L.;
Buettner, J. A.; Hudson, D.; Frankel, A. D. Cell 1993, 73, 1031-1040). The
cys residue at the C-terminus is used for fluorescent tagging. In the unlabeled
peptide, this residue is blocked as an acetamide SCH2CONH2 derivative.
10.1021/ja993387i CCC: $19.00 © 2000 American Chemical Society
Published on Web 01/21/2000

Communications to the Editor
J. Am. Chem. Soc., Vol. 122, No. 5, 2000 981
located around the G46-G48 bulge region (Figure 1). At low
concentrations (0.5-2 µM), both the Rev peptide and neo-acridine
(2) show protection at nucleotides U45, G46, G47, G48, G67,
and U72. Neo-acridine (2) and Rev are, therefore, competing for
the same site on the RRE. Intriguing differences between the
cleavage patterns observed for neo-acridine (2) and the Rev
peptide are localized around the bulged-out U72 (Figure 1). In
the presence of the Rev peptide, G70 and G71 are protected and
enhanced cleavage is observed at C69. Protection by neo-acridine
(2) at nucleotides A44 and C74 is not observed with the Rev
peptide. Importantly, enhanced cleavage appears at nucleotides
C49, G70, and G71 in the presence of neo-acridine. These data
suggest that the acridine moiety of neo-acridine (2) interacts near
or at the bulged U72 while the neomycin B portion flanks the
nucleotides A44-G47.14 The enhanced RNase T1 cleavage
observed at G71 in the presence of neo-acridine contrasts the
protection observed for the Rev peptide at the same site. The
increased single-stranded nature of the G71 suggests that neo-
acridine may disrupt the G48-G71 base pair.15 These results,
together with gel-shift and fluorescence anisotropy data, suggest
neo-acridine competitively inhibits Rev-RRE complex formation
by binding at the same region of RRE as the Rev peptide.16
To the best of our knowledge, neo-acridine (2) is the strongest
competitive inhibitor of Rev-RRE binding. It binds the RRE with
an inhibition constant of 1.5 nM and effectively disrupts the Rev-
RRE complex. The affinity of neo-acridine to the RRE is only
2-fold lower than that of the Rev peptide. Our results demonstrate
that (a) small molecules can effectively interfere with protein-
RNA interactions, (b) synthetic ligands can achieve very high
RNA affinity, approaching that of the natural RNA-binding
domains on proteins, and (c) the combination of different binding
modes (e.g., ionic and intercalation) is a powerful approach for
enhancing the RNA affinity of synthetic binders. Small molecules
that target viral RNA sites and prevent the formation of pivotal
regulatory RNA-protein complexes are promising candidates for
antiretroviral drug discovery.
Figure 2. Gel mobility shift of the Rev-RRE complex in the presence
of increasing concentrations of neomycin B (1) or neo-acridine (2).12 All
lanes contained 25 nM RRE with trace 5′-32P labeled RRE and lanes
2-19 contain 2 µM Rev. Lane 1, RRE alone; lanes 2 and 19, RRE and
Rev; lanes 3-10, 0.5 µM, 1 µM, 1.25 µM, 2.5 µM, 3.75 µM, 5 µM, 7.5
µM, and 10 µM neomycin B, respectively; lanes 11-18, 25 nM, 50 nM,
100 nM, 200 nM, 500 nM, 750 nM, 1.25 µM, and 2.5 µM neo-acridine,
respectively.
Figure 3. Inhibition of Rev-Fl binding to the RRE by Rev (4), neo-
acridine (b), neomycin B (9), and 9-aminoacridine (0) as determined
by fluorescence anisotropy displacement measurements.12
Fluorescence anisotropy has been employed to quantitatively
determine the RRE binding affinity of Rev-RRE inhibitors.12
Inhibition is measured by the displacement of a fluorescein-labeled
Rev34-50 peptide (Rev-Fl) off the RRE construct (Figure 1).12,13
Competition experiments yield IC50 values of 24 ( 1 µM for
9-aminoacridine, 0.8 ( 0.2 µM for neomycin B, 15 ( 1 nM for
neo-acridine (2), and 12 ( 5 nM for the Rev peptide (Figure 3).
Since gel-shift assays suggest a single high-affinity site for both
high-affinity ligands, Ki values of 1.0 ( 0.8 and 1.5 ( 0.3 nM
can be calculated for the Rev peptide and neo-acridine (2),
respectively.12 In agreement with the gel-shift data, the affinity
of neo-acridine (2) to the RRE is only 2-fold lower than that of
the Rev peptide.
Acknowledgment. This paper is dedicated to the memory of Professor
Paul Saltman, an outstanding scientist, educator, and human being. We
thank the National Institutes of Health (AI 40315) and the Universitywide
AIDS Research Program, University of California (R97-SD-036), for
partial support. N.L. was supported by a National Institutes of Health
Molecular Biophysics Training Grant (GM 08326). We also thank Dr.
Georg Schlectingen and Professor Murray Goodman for their assistance
with peptide synthesis.
Supporting Information Available: Synthetic procedures and ana-
lytical data for all new derivatives as well as procedures and data for
gel-shift assays, fluorescence anisotropy measurements, calculations of
Kd and Ki values, and enzymatic protection reactions (PDF). This material
is available free of charge via the Internet at http://pubs.acs.org.
To locate the binding sites of Rev34-50 and neo-acridine (2) on
the RRE, enzymatic protection experiments with three ribo-
nucleases, RNase T1, RNase A, and RNase V1, have been
conducted.12 Footprinting for both Rev and neo-acridine (2) is
JA993387I
(15) A possible explanation is the formation of a G48‚U72 base pair and
expulsion of G71 into solution upon intercalation of the acridine moiety.
(16) The Rev peptide has been shown to have the same RNA binding
domain as the full length Rev protein. See ref 9 and: Daly, T. J.; Rennert, P.;
Lynch, P.; Barry, J. K.; Dundas, M.; Rusche, J. R.; Doten, R. C.; Auer, M.;
Farrington, G. K. Biochemistry 1993, 32, 8945-8954.
(14) A second lower affinity RRE binding site is filled at higher neo-acridine
concentrations (5-10 µM). Footprinting data at these concentrations suggest
a lower affinity neo-acridine binding site located around A68, another bulged
nucleotide. See ref 12.