Amiloride as a new RNA-binding scaffold with activity against HIV-1 TAR

Article abstract of DOI:10.1039/c6md00729e

Diversification of RNA-targeted scaffolds offers great promise in the search for selective ligands of therapeutically relevant RNA such as HIV-1 TAR. We herein report the establishment of amiloride as a novel RNA-binding scaffold along with synthetic routes for combinatorial C(5)- and C(6)-diversification. Iterative modifications at the C(5)- and C(6)-positions yielded derivative 24, which demonstrated a 100-fold increase in activity over the parent dimethylamiloride in peptide displacement assays. NMR chemical shift mapping was performed using the 2D SOFAST-[¹H-¹³C] HMQC NMR method, which allowed for facile and rapid evaluation of binding modes for all library members. Cheminformatic analysis revealed distinct differences between selective and non-selective ligands. In this study, we evolved dimethylamiloride from a weak TAR ligand to one of the tightest binding selective TAR ligands reported to date through a novel combination of synthetic methods and analytical techniques. We expect these methods to allow for rapid library expansion and tuning of the amiloride scaffold for a range of RNA targets and for SOFAST NMR to allow unprecedented evaluation of small molecule:RNA interactions.

Full text of DOI:10.1039/c6md00729e

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RESEARCH ARTICLE
Amiloride as a new RNA-binding scaffold with
activity against HIV-1 TAR†‡
Cite this: DOI: 10.1039/c6md00729e
Neeraj N. Patwardhan, a Laura R. Ganser,b Gary J. Kapral,a Christopher S. Eubanks,a
Janghyun Lee,§b Bharathwaj Sathyamoorthy,¶b
ab
Hashim M. Al-Hashimiab and Amanda E. Hargrove
*
Diversification of RNA-targeted scaffolds offers great promise in the search for selective ligands of thera-
peutically relevant RNA such as HIV-1 TAR. We herein report the establishment of amiloride as a novel
RNA-binding scaffold along with synthetic routes for combinatorial C(5)- and C(6)-diversification. Iterative
modifications at the C(5)- and C(6)-positions yielded derivative 24, which demonstrated a 100-fold in-
crease in activity over the parent dimethylamiloride in peptide displacement assays. NMR chemical shift
mapping was performed using the 2D SOFAST-[1H-13C] HMQC NMR method, which allowed for facile and
rapid evaluation of binding modes for all library members. Cheminformatic analysis revealed distinct differ-
ences between selective and non-selective ligands. In this study, we evolved dimethylamiloride from a
weak TAR ligand to one of the tightest binding selective TAR ligands reported to date through a novel
combination of synthetic methods and analytical techniques. We expect these methods to allow for rapid
library expansion and tuning of the amiloride scaffold for a range of RNA targets and for SOFAST NMR to
allow unprecedented evaluation of small molecule:RNA interactions.
Received 31st December 2016,
Accepted 14th March 2017
DOI: 10.1039/c6md00729e
rsc.li/medchemcomm
Small molecule chemical probes offer a unique opportu-
nity to study both the structure and function of bio-
Introduction
Although often regarded as an intercessor in the transfer of
genetic information, RNA is presently identified as a key regu-
latory element in several cellular processes and has been im-
plicated in a range of disease states.1,2 Furthermore, several
regulatory RNA molecules play significant roles in infectious
diseases.3 HIV infection, for example, relies on a single-
stranded RNA genome in which specific RNA structures and
RNA:protein interactions are recognized as crucial to HIV rep-
lication and as potential therapeutic targets.4–7 In line with
this growing appreciation for the regulatory power of RNA
comes a need for the continued advancement of methods and
tools for the elucidation of RNA structure and cellular func-
tion as well as for the therapeutic targeting of RNA.
macromolecules, including RNA.8–10 The small size and ex-
tensive tunability of small molecules have led to highly
selective chemical probes for many biological targets, particu-
larly proteins, and have been invaluable to the field of chemi-
cal biology. This potential has yet to be fully realized for
RNA, possibly due to the limited chemical functionality of
RNA that can be exploited for specific molecular recognition
and by the highly dynamic structure of RNA. Screening of
general small molecule libraries for selective ligands has
been successful for some RNA targets; however, this ap-
proach has generally yielded low hit-rates.8–20 One plausible
explanation is that current high-throughput screening librar-
ies are largely designed and optimized for protein targets.
Moreover, screens for RNA targets often identify promiscuous
ligands such as large polycationic aminoglycosides,21,22
which are well known to possess poor pharmacological prop-
erties and interact with several other cellular targets.8–10 It is
worth noting, however, that aminoglycosides and their multi-
valent analogs have featured prominently in several studies
elucidating high affinity interactions with RNA and have
demonstrated utility in studying RNA biology.23–30 Although
significant advances have recently been made towards ratio-
nally designing small molecule ligands for RNA,9,14,31–37 the
limited available knowledge of the guiding principles of
small molecule:RNA recognition has remained a significant
a Department of Chemistry, Duke University, Durham, North Carolina 27708, USA.
E-mail: amanda.hargrove@duke.edu; Tel: +1 919 660 1522
b Department of Biochemistry, Duke University Medical Center, Durham, North
Carolina 27708, USA
† The authors declare no competing interests.
‡ Electronic supplementary information (ESI) available: Synthetic procedures,
compound characterizations, biological assays and data, procedures and param-
eters for docking, and cheminformatic analysis. See DOI: 10.1039/c6md00729e
§ Current Address: Department of Chemistry, Korea Advanced Institute of Sci-
ence and Technology (KAIST), Daejeon, 34141 South Korea.
¶ Current Address: Department of Chemistry, Indian Institute of Science Educa-
tion and Research Bhopal, Bhopal 462066, India.
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hurdle. At the same time, some of the most successful
screens have been conducted on RNA-targeted libraries gen-
erated by the diversification of molecular scaffolds known to
interact with RNA, such as phenylbenzamidazoles,38–40
oxazolidinones,41–44 and diphenylfurans45–47 though the li-
brary sizes and number of scaffolds tested has been fairly
limited.40,48 Based on these results, we see an urgent need
for the identification and development of new RNA-targeted
scaffolds that can expand scientists' repertoire for probing
RNA structure and function, particularly RNA structures criti-
cal to infectious agents such as HIV.
libraries generated through rapid diversification of RNA-
binding scaffolds.
Results and discussion
DMA binds both the apical loop and bulge of TAR
We began our study by characterizing the activity and TAR-
binding properties of the parent molecule dimethylamiloride
1 (Fig. 1A). Activity screening was performed by measuring
displacement of a fluorescently labeled Tat-derived peptide
(N-AAARKKRRQRRRAAAK-C) from a truncated TAR sequence
featuring the stem-bulge-loop region.67 The competitive
dosage for 50% displacement (CD50) was calculated for
Specifically, small molecule probes targeted to discrete
HIV RNA structures would both facilitate the elucidation of
critical pathways in HIV biology and open novel therapeutic
opportunities.5,7 One of the most studied HIV RNA targets is
the HIV-1 transactivation response (TAR) element, which
binds to the Tat protein and is necessary for efficient HIV
replication.49–52 High-throughput and fragment-based
screens have yielded small molecules that bind TAR in the
micromolar range and inhibit Tat interactions.53–57 Recently,
small molecule microarrays have been employed by
Schneekloth and coworkers to discover a TAR-binding probe
that inhibits the HIV-induced cell death of T-lymphocytes
at 28 μM without measured toxicity.17 Importantly, this
thienopyridine-based probe was charge neutral and bound
TAR with a dissociation constant of 2.4 μM. Finally, branched
and cyclic peptides58–60 as well as small synthetic proteins61
have also shown promise as potent inhibitors of the TAR:Tat
interaction. In order to expedite screening and increase hit
rates, the Al-Hashimi lab recently pioneered a virtual docking
method for RNA by first generating dynamic ensembles of
TAR RNA through molecular dynamics (MD) simulations and
NMR spectroscopy refinements.62 Virtual screening of a
∼51 000 molecule library followed by experimental binding
studies led to identification of six TAR ligands, which in-
cluded aminoglycosides and intercalators as well as dimethyl-
amiloride 1 (DMA).14 Amiloride had not been previously iden-
tified among RNA ligands, though DNA duplex binding was
reported by Waring and co-workers at pH below the pKa of
amiloride (∼8.7).63,64
Although 1 bound TAR with a modest affinity (Kd = 120
μM), it demonstrated a unique predicted binding profile in
its preference for the apical loop of TAR over the more
commonly bound bulge region in docking.14,63 Further-
more, amiloride derivatives are readily accessed through
common synthetic transformations on a commercially avail-
able scaffold and offer a straightforward route to combina-
torial library design.65,66 In this study we describe the de-
velopment of an amiloride-based small molecule library
and analysis of the suitability of this library for targeting
HIV-1 TAR. We evaluated the activity of these derivatives
against the TAR:Tat interaction and explored their binding
properties via 2D NMR and docking experiments. The iden-
tification of multiple amiloride-based ligands that selec-
tively interact with TAR RNA supports the approach of iden-
tifying RNA chemical probes from targeted small molecule
Fig. 1 A) Structure of dimethylamiloride 1 (DMA). B) Displacement
assay binding curve with 1 (50 mM Tris-HCl, 50 mM KCl, 0.01% Triton-
X-100 at pH ∼ 7.5). Error bars represent standard deviation for three
independent replicates. C) SOFAST-HMQC spectra of TAR in the pres-
ence and absence of 1 (50 μM TAR, 200 μM DMA, 25 °C, buffer: 15 mM
Na2HPO4/NaH2PO4, 15 mM NaCl, 0.1 mM EDTA at pH ∼ 6.4). *C2H2
peaks were folded into the spectral window. D) Heat maps for WT-,
UUCG- and Bulgeless-TAR. Colors represent the magnitude of chemi-
cal shift changes for spins C2H2, C6H6, or C8H8 on the given residue
upon addition of 4× compound 1: <0.025 ppm (gray); 0.025–0.05 (yel-
low); 0.05–0.1 ppm (light orange); >0.1 ppm (dark orange); and peak
unassignable due to broadening or large chemical shift changes (red).
E) Vector diagrams for select residues upon DMA binding to WT-TAR
compared to DMA binding to UUCG-TAR (left) and Bulgeless-TAR
(right).
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displacement of Tat peptide induced by 1, and control experi-
ments included repeating the assay in the presence of 100-
fold excess tRNA and measuring fluorescence background in
the absence of TAR (Fig. 1B, page S107 of ESI‡). While 1 dem-
onstrated moderate inhibition with a CD50 of 540 171 μM,
activity was not diminished in the presence of the tRNA con-
trol (CD50 = 542 47 μM), which supported a selective 1:TAR
interaction as previously reported.14 Furthermore, the activity
of 1 was not diminished in the presence of a 30-mer (dA-dT)
alternating DNA duplex (see page S112 and S113 of the ESI‡),
which is consistent with the lack of intercalation observed by
Waring and co-workers for C(5)-substituted amilorides63 and
highlights the potential selectivity of this scaffold.
The binding mode of 1 to TAR was then studied using the
previously described 2D selective optimized flip angle short
transient (SOFAST) [1H-13C] HMQC NMR method,68 which
allowed for the rapid assessment of small molecule-induced
structural perturbations as observed by changes in 13C and
1H chemical shifts of signals from the aromatic residues on
TAR (Fig. 1C). To allow ready visualization, we depicted the
magnitude of each perturbation as a heat map overlay on the
TAR secondary structure (Fig. 1D). Whereas low range chemi-
cal shift perturbations <0.025 ppm (grey) were considered in-
significant, the measurably larger shifts ranging from 0.025–
0.050 ppm (yellow), 0.05–0.1 ppm (light orange) and >0.1
ppm (dark orange) were surmised to originate from interac-
tion of the small molecule with the RNA, reflecting direct li-
gand–RNA interactions and/or ligand-induced conforma-
tional changes. Lastly, peaks that could not be conclusively
identified in the bound spectrum, either due to broadening
or the magnitude of the shift, were labeled red. In the pres-
ence of 1, significant perturbations were observed in residues
21–26, as well as G17, G36, C39, and C41, which we hypothe-
size to originate from the binding of 1 to both the bulge and
apical loop regions of TAR.
tions were seen in the bulge residues, which are very similar
in magnitude and direction to the perturbations observed in
WT-TAR (Fig. 1D). Likewise, in the Bulgeless-TAR mutant, sig-
nificant perturbations were seen in the apical loop residues,
which were very similar in magnitude and direction to the
perturbations observed in WT-TAR (Fig. 1D). The similarity of
the chemical shift changes in the WT and mutant sequences
were clearly seen when plotting the 2D chemical shift
changes as vectors (Fig. 1E) and were consistent with two in-
dependent binding sites in WT-TAR, one localized in the api-
cal loop and another in the bulge.69 The small differences in
direction or magnitude of the perturbations, particularly for
G34(C8H8), may suggest that having both the bulge and api-
cal loop has a subtle effect on the binding of 1 to either site.
Accurate stoichiometry of binding, however, could not be
gleaned from these experiments. Low solubility of
1
prevented the use of traditional techniques such as isother-
mal titration calorimetry, and, due to the low affinity of
DMA, it was not possible to carry out NMR NOE experiments
to more directly assess ligand binding sites.
The selective binding of 1 to TAR in the presence of tRNA
and DNA confirmed the potential for amiloride as a promis-
ing RNA scaffold while the ability of 1 to bind both the bulge
and apical loop implies that a range of motif specificities
may be possible with further scaffold diversification. To fur-
ther explore the utility of amiloride as an RNA selective scaf-
fold, we pursued synthetic routes that allowed for diversifica-
tion of the C(5)- and C(6)-positions. We chose a variety of
motifs, including several found in reported RNA-binding
ligands38,39 and others enriched in aromatic, nitrogen-rich,
and hydrogen bonding groups. We then synthesized 28
amiloride derivatives, purchased 6 commercial amilorides,
and characterized the TAR binding properties of the com-
bined library using the Tat displacement assay and NMR
chemical shift mapping.
In the previous studies, traditional HSQC NMR chemical
shift mapping experiments revealed that 1 induced signifi-
cant perturbations to resonances belonging to the apical loop
as well as in and around bulge residues while also demon-
strating a non-linear dependence on concentration under
these conditions (50 μM TAR, 15 mM Na2HPO4/NaH2PO4, 0.1
mM EDTA, 25 mM NaCl, 10% D2O at pH ∼ 6.4 and 25 °C).14
In principle, the perturbations observed at the apical loop
could have arisen due to conformational changes that are in-
duced due to binding of 1 at the bulge or due to the presence
of two discrete binding sites. To determine whether 1 does
indeed bind the TAR apical loop as indicated by docking,14
control experiments were performed with TAR mutants where
residues of the apical loop or bulge were selectively modified.
The UUCG-TAR RNA mutant featured a truncated and com-
pact 4 nucleotide (UUCG) apical loop while maintaining the
remaining bulge and stem structural features of WT-TAR,
whereas the Bulgeless-TAR mutant replaced the three nucleo-
tide bulge residues with a fully base paired stem while
retaining the apical loop of the WT-TAR structure (Fig. 1D).
In the compact loop UUCG-TAR mutant, significant perturba-
Synthetic routes toward a library of C(5)- and C(6)-substituted
amiloride derivatives.
Amiloride derivatives have been previously synthesized and
evaluated against a variety of biological targets.65,66,70–73
Based on reported synthetic procedures for amilorides and
other analogous compounds,74–77 we designed and tested
several synthetic routes to achieve diversity at the C(5)- and
C(6)-positions (Schemes 1–5) of the core pyrazine ring.
C(5)-derivatives were achieved through a concise two-step
pathway (Scheme 1). Commercially available methyl 3-amino-
5,6-dichloropyrazine-2-carboxylate 2 was substituted with vari-
ous primary and secondary amines to obtain C(5)-substituted
intermediates 3a–n in moderate to high yields. Reactions of
the methyl esters 3a–n with guanidine at 65 °C yielded deriv-
atives 4a–n, which were isolated as the HCl salts. The acyl
guanidine was installed at the final step to avoid challenges
in purification.
To allow rapid expansion of the C(5)-derivative library out-
side of commercially available primary amines, we installed
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Scheme 1 Synthesis of C(5)-derivatives via designed two-step protocol. Reagents and conditions: a: amine, diisopropyl ethylamine (DIEA), DMF,
rt, 18 h; b: guanidine (2 M solution in methanol), THF, 65 °C, 18 h; c: HCl (2 M in Et2O), Et2O.
Scheme 2 Synthesis of ethylenediamine containing amiloride derivatives. Reagents and conditions: a: N-Boc-1,2-diaminoethane, DIEA, DMF, rt,
18 h; b: HCl (1 M) in MeOH, rt, 12 h; c: BnBr, DIEA, DMF, rt, 18 h; d: TsCl, DIEA, THF, rt, 18 h; e: guanidine 2 M solution in methanol, THF, 65 °C, 18
h; f: HCl (2 M in Et2O), Et2O; g: synthesized by guanidinylation of 6.
an ethyleneamine linker (Scheme 2). Intermediate 6 can un-
dergo a wide range of reactions, including functionalization
with benzyl or tosyl groups as achieved here to yield interme-
diates 7 and 8, respectively. In addition to the three deriva-
tives 9–11 tested in this study, it is expected that ethyl-
enediamine derivative 6 will open several possible routes to
future diversification.
To test the possibility of using click-type reactions for fur-
ther diversification, we installed an alkyne through reaction
with propargyl amine (Scheme 3). Reaction of intermediate
12 with benzyl azide yielded the 1,2,3-triazole containing
methyl ester derivative 13, which was subsequently
guanidinylated to yield product 14. As with derivative 6, ter-
minal alkyne derivative 12 opens up a variety of possibilities
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Scheme 3 Synthesis of 1,2,3-triazole containing derivative 14. Reagents and conditions: a: propargylamine, DIEA, DMF, rt, 18 h; b: benzyl azide,
CuSO4·HCl, sodium ascorbate, iPrOH : water : CH2Cl2 (1 : 1 : 1), rt, 18 h; c: guanidine 2 M solution in methanol, THF, 65 °C, 18 h; d: HCl (2 M in Et2O),
Et2O.
Scheme 4 (A) and (B): Sonogashira cross-coupling reactions and palladium catalyzed reduction for synthesis of C(6)-amiloride derivatives 17, 19,
and 20. Reagents and conditions: a: phenylacetylene, CuI (1 mol%), PdIJPPh3)2Cl2 (2.5 mol%), triethylamine (5 eq.), DMF, 60 °C, 18 h; b: guanidine (2
M in methanol), THF, 65 °C, 18 h; c: HCl (2 M in Et2O), Et2O; d: H2, 10% Pd–C (20 mol%), methanol, rt, 48 h.
Scheme 5 (A) Suzuki cross-coupling reactions of intermediate 15 for synthesis of C(6)-derivatives 22a–f. (B): Synthesis of C(5)–C(6) dual
substituted derivative 24. Reagents and conditions: a: boronic acid (1.25 eq.), PdIJPPh3)4 (5 mol%), Na2CO3 (5 eq.), THF : water (1 : 1), 60 °C, 18 h; b:
guanidine (2 M in methanol), THF, 65 °C, 18 h; c: HCl (2 M in Et2O), Et2O.
for diversification through coupling to various commercially
available azides, in addition to the derivative evaluated here.
While synthesis of C(6)-amiloride derivatives had been
previously described through chloride reduction
a
–
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bromination/iodination procedure,65 we achieved direct
palladium-catalyzed cross coupling reactions on C(6)-chloro-
amiloride derivatives (Schemes 4 and 5). Sonogashira cross-
coupling reaction was first performed on intermediate 15
(Scheme 4A), which was synthesized by the reaction of N,N-
dimethylamine with intermediate 2. Guanidinylation of inter-
mediate 16 yielded derivative 17 (Scheme 4A). Commercially
available amiloride 18, where the acyl guanidine residue is al-
ready present, was directly converted to two derivatives
(Scheme 4B): derivative 19 (right) through cross-coupling as
above, and C(6)-reduced derivative 20 (left) via a palladium-
catalyzed hydrogenation.
Suzuki cross-coupling reactions were next performed with
intermediate 15 and a series of commercially available bo-
ronic acids to generate derivatives 22a–f (Scheme 5A). Finally,
in order to test if functionalization of both C(5)- and C(6)-
positions yielded more potent binders, intermediate 3j, fea-
turing a tryptamine side-chain at the C5 position, was subject
to the Suzuki cross-coupling reaction with benzeneboronic
acid to yield bis-functionalized amiloride derivative 24
(Scheme 5B). The combination of these functionalization
methods not only allowed for the generation of 28 diverse
amiloride derivatives but also established a combination of
efficient synthetic pathways that will allow rapid expansion of
amiloride libraries in future studies.
combining the C(5)-indole motif of 4j and C(6)-phenyl motif
of 22a to design 24 led to the most active of all small mole-
cules tested (CD50 = 4.4 1.4 μM). The 7 non-selective ligands
(Group B, Fig. 2, right) include most of the amilorides with
substituents that are expected to be positively charged at pH
7.4 (4h, 9, 4n), suggesting that their binding events involve
nonspecific electrostatic interactions with the RNA backbone.
Several ligands with neutral alkyl and aromatic substituents,
however, are also included in this non-selective group. Unfor-
tunately, a subset of derivatives (Group C) displayed strong
background fluorescence in the presence of the Tat peptide
alone, presumably due to fluorescence of the small molecule
itself or through aggregation. Finally, 4c, 4g, 4i, and 14
showed no displacement of the Tat peptide (Group D).
Though no single trend was identified for this small group, it
is worth noting that 4g and 4i contain electronegative func-
tional groups, specifically an amide and sulfonamide, which
may be repulsed by the RNA phosphate backbone. The very
strong interaction of the Tat peptide with TAR RNA under
these conditions prevents calculation of a dissociation con-
stant for the TAR:Tat interaction, which in turn prohibits the
determination of discrete dissociation constants for
amiloride derivatives.
Although the displacement assay results did not point to
definitive correlations between the structural properties of
amiloride derivatives and their observed displacement activ-
ity, possibly due to the many accessible binding conforma-
tions of TAR, some broad conclusions could be derived. To
begin, all synthetic derivatives that demonstrated measurable
activity in the displacement assay were more active than the
parent compound 1. As expected, polar positively charged
subunits at the C(5)-position improved the displacement ac-
tivity but did so nonspecifically as binding was partially or
completely abrogated in the presence of the tRNA control.
Both aryl and alkynyl groups at the C(6)-position showed im-
proved affinity and selectivity compared to derivatives bear-
ing a C(6)-chloro or C(6)-hydrogen substituent. The high hit
rate of Tat peptide displacement observed with amiloride de-
rivatives (18 out of 34, 53%) supports amiloride as an effec-
tive RNA-binding scaffold while the decreased activity with
tRNA observed for 8 of the 18 active ligands highlights the in-
herent difficulties of developing selective ligands for RNA
structures. We further tested the tRNA-selective amiloride an-
alogues against DNA and found no loss in activity, similar to
that observed with analogue 1 (see pages S112 and S113 of
the ESI‡).
Screening of amiloride derivatives using a displacement
assay with fluorescently labeled Tat peptide
The 28-member synthetic amiloride library along with the 6
commercially available derivatives were screened for activity
using the Tat displacement assay previously described.67
While all of the commercially available derivatives (S1–S6,
Fig. S1‡) were found to be insoluble or interfere with the as-
say signal, several of the synthetic derivatives yielded promis-
ing results (Table 1).
The observed displacement activities amongst synthetic
amiloride derivatives can be separated into four groups: A)
clear displacement of the Tat peptide in the presence of
tRNA, i.e. selective binding; B) clear displacement of the Tat
peptide only in the absence of tRNA, i.e. non-selective bind-
ing; C) significant signal interference, presumably caused by
the fluorescence emission of the small molecule or aggrega-
tion; or D) no displacement. Group A is composed of 10 de-
rivatives that showed mild to good displacement (CD50 ∼ 4–
200 μM) and were selective for TAR in presence of tRNA as
seen from their minimally changed CD50 values in the pres-
ence of 100-fold excess tRNA (Fig. 2, left). Notably, each of
the selective ligands features aromatic substituents. While
only one of the C(5)-derivatives, 4j, fell into this category, 9 of
the 10 synthesized C(6)-derivatives, including 22a, displayed
selective interactions. These results imply that C(6)-
Evaluation of binding preferences via SOFAST-[1H-13C]
HMQC NMR and docking
To evaluate the individual binding modes of the synthesized
amiloride derivatives to HIV-1 TAR, we employed the SOFAST-
[1H-13C] HMQC NMR experiment. In general, the perturba-
tions induced by the derivatives were unique when compared
to other known TAR binders.14 This result suggests a unique
overall mode of binding, though there were significant
functionalized amilorides represent
a privileged class of
ligands for targeting TAR. The differences in C(5)- and C(6)-
derivatives likely stem from increased rigidity of the ligand
and/or the presence of extended conjugation. Gratifyingly,
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Table 1 Tat peptide displacement screening and NMR assay results for synthetic amiloride derivatives
Groupa
CD50 Tat disp. (μM)
CD50 Tat disp. w/ tRNAc (μM)
NMR assay resultsi
b
Amiloride
C(5)-substitutions (R1 = Cl)
4a
B
C
12 4.5
No disp.e
N.Af
Binds
Binds
4b
4c
Interferenced
D
B
C
No disp.d
74 26
N.Af
Binds
Binds
Binds
4d
4e
No disp.e
N.Af
Interferenced
4f
C
D
Interferenced
No disp.e
N.Af
Binds
4g
No disp.e
No binding
4h
4i
B
120 30
No disp.e
No disp.e
No disp.e
Binds
D
No binding
4j
A
33 12
42 17
Binds
4k
4l
C
B
Interferenced
47 23
N.Af
Binds
Binds
No disp.e
4m
4n
B
C
143 19
No disp.e
No disp.e
Binds
Interferenced
Bindsh
9
B
C
33 5.3
No disp.e
N.Af
Binds
Binds
10
Interferenced
11
14
C
D
Interferenced
No disp.e
N.Af
Binds
No disp.e
No binding
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Table 1 (continued)
Groupa
CD50 Tat disp. (μM)
CD50 Tat disp. w/ tRNAc (μM)
NMR assay resultsi
b
Amiloride
C(6)-substitutions
17
R1 = CC-Ph
A
A
B
A
A
A
A
A
A
54 17
86 46
48 18
No disp.e
124 43
18 3.6
31 4.6
29 8.2
57 8.9
64 8.7
Binds
Binds
Binds
Binds
Binds
Binds
Binds
Binds
Binds
R2 = NIJCH3)2
R1 = CC-Ph
R2 = NH2
R1 = H
R2 = NH2
19
40 19
20
730 223g
192 163
22a
22b
22c
22d
22e
22f
R1 = Ph
R2 = NIJCH3)2
R1 = (p-C6H4)-Ph
R2 = NIJCH3)2
R1 = (p-C6H4)-CN
R2 = NIJCH3)2
R1 = β-napthyl
R2 = NIJCH3)2
R1 = (p-C6H4)-CH3
R2 = NIJCH3)2
R1 = (p-C6H4)-F
R2 = NIJCH3)2
h
—
101 40
32 12
h
—
60 18
C(5)- and C(6)-substitutions
24
R1 = Ph
A
4.4 1.4
13 2.5
Binds
a
b
See text for description of groups. CD50: competitive dosage required for displacement of 50% of Tat peptide from preformed TAR:Tat
complex, as measured by fluorescence of FAM- and TAMRA-labeled Tat peptide at 590 nm, the emission λmax of TAMRA. Errors are standard de-
c
viation for three independent replicates. 100× tRNA (brewer's yeast, Roche) used as control to test selectivity of amiloride derivatives towards
d
e
TAR. Interference originating from the fluorescent small molecule or from aggregation of the assay mixture at 590 nm. No displacement of
f
g
Tat peptide observed. Not applicable. Curve fitting is poor due to weak binding of 20. Mean and standard deviation calculated from two
CD50 values. 22b and 22e curves could only be accurately fit in the presence of tRNA, possibly due to differences in the background (no TAR
added) signal. As seen by SOFAST NMR experiments explained below. Some derivatives that did not measurably displace Tat did demonstrate
h
i
binding by NMR.
Fig. 2 CD50 values determined for all active amiloride derivatives in the Tat-displacement assay (50 mM Tris-HCl, 50 mM KCl, 0.01% Triton-X-100
at pH ∼ 7.5) using WT-TAR alone (orange) and with 100-fold excess tRNA (blue). Error bars represent standard deviation for three independent
replicates. Ligands on the right did not demonstrate measurable activity in the presence of tRNA. Note: CD50 values could not be determined for
22b and 22e in the absence of tRNA (see Table 1).
variations among the different amiloride derivatives as well.
Similar to the study of 1 shown in Fig. 1, Fig. 3 shows
residue-specific heat maps for NMR chemical shift perturba-
tions caused by our synthetic amiloride derivatives.
As previously discussed, the perturbations in the NMR
chemical shifts caused by amiloride derivatives measured via
SOFAST NMR provided a fast and direct method to lend in-
sight into the site-selectivity of binding interactions.
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binding derivatives by NMR, confirming the importance of
multiple analytical methods. S1, S3, S6, 20, 19, 4l, 17, and 9
caused significant broadening of resonances and disappear-
ance of several peaks (Fig. S2‡). This widespread broadening
may originate from strong interactions of the small molecule
with the RNA that lead to exchange at the micro-to-
millisecond timescale and/or from other bulk events such as
aggregation. If broadening was due to aggregation and conse-
quently lower overall tumbling rates, we would expect all resi-
dues to experience similar extents of broadening. However,
we found that the loss of intensity was much more signifi-
cant for specific residues, largely in and around the bulge,
compared to residues in the helical regions (Fig. S2‡). The
non-uniform nature of the broadening suggests that it arose
due to intermediate exchange at the micro-to-millisecond
timescale between free and small molecule bound TAR states,
possibly due to tighter binding and slower off rates as com-
pared to the parent compound 1. However, we could not rule
out that the broadening was partially due to small molecule
induced aggregation. To examine this latter possibility, we
measured the dynamic light scattering (DLS) of the amiloride
derivatives alone as well as in the presence of TAR and found
that only 9 demonstrated a significant increase in particle
size over background (see Fig. S8‡). Still we cannot rule out
transient formation of larger aggregates that are invisible to
these DLS experiments and that contribute to the observed
broadening in the NMR experiments.
For a more in-depth analysis of the ligand-induced spec-
tral changes, vector diagrams were generated that depict both
the magnitude and direction of the chemical shift perturba-
tions (Fig. 4, Fig. S4–S7‡). To begin, amiloride derivatives that
caused strong perturbations in the apical loop residues (e.g.
4a, 4h, 22a, and 4n) generally caused similar chemical shift
changes as observed with 1 (Fig. 4). On the other hand, most
derivatives perturbed the bulge residue signals with the
greatest magnitude and a subset of derivatives exhibit strik-
ing differences in direction, particularly the C(6)-derivatives
(Fig. 4). For example, both 22f and 24 caused perturbations
at bulge residues U25(C6H6) and G26(C8H8) in directions
similar to each other but distinct relative to other ligands,
consistent with a specific interaction in this region. 22d,
which contains a large naphthyl group at the C(6)-position,
also caused perturbations at bulge residues A22(C2H2),
U25(C6H6), and G26(C8H8) but in unique directions. Inter-
estingly, the perturbations in the helical regions of TAR, such
as A27(C8H8), displayed lower magnitudes but the greatest
variation in direction among the ligands. This latter effect
likely reflects the subtle differences in the TAR conforma-
tions stabilized by each amiloride derivative, rather than
specific interactions with the ligands. Taken together, the
differences in perturbations among amiloride derivatives,
particularly those closely related such as 22a, 22e and 22f,
which differ by a Ph-H, Ph-CH3, and Ph-F substitution, re-
spectively, suggest that subtle changes in the shape of the
small molecule may significantly impact the bound confor-
mation of TAR.
Fig. 3 Representative chemical shift maps reflecting the interaction of
synthetic amiloride derivatives with HIV-1 TAR using the SOFAST-[1H-
13C] HMQC NMR experiment. Results represented as heat maps show-
ing the extent of perturbations to the TAR C8H8/C6H6/C2H2 chemical
shifts caused by amiloride derivatives. A) Perturbations to the apical
loop and bulge residues; B) perturbations primarily to the bulge resi-
dues; C) broadening of signals. Spectra and remaining chemical shift
maps can be found on pages S114–S122 of the ESI.‡
Compared to standard NMR HSQC experiments, the SOFAST
method allowed for rapid (<1.5 h per ligand) screening of all
derivatives using low (50 μM) concentrations of RNA.68 As
seen from Fig. 3 (also see Fig. S3 in ESI‡), the amiloride de-
rivatives show significant variation in the degree and sites of
signal perturbation, which is represented with respective
colors in the heat maps. Initial comparison of the heat maps
revealed that a small subset of derivatives (Fig. 3A) perturbed
signals in both the apical loop and bulge region, similar to
those observed with 1. A much larger subset of the library
members, however, perturbed signals almost exclusively in
the bulge region (Fig. 3B, Fig. S3‡). Notably, no perturbations
in any nucleotides of TAR were observed upon addition of de-
rivatives 4g, 4i, and 14, which was in alignment with the neg-
ative results in the Tat displacement assay. On the other
hand, 4c displayed no significant activity in the displacement
assay but caused significant NMR chemical shift perturba-
tions, presumably due to the high sensitivity of the NMR ex-
periment, which can reveal weak binding interactions. A
binding mode that does not displace the Tat peptide, how-
ever, cannot be ruled out.
Importantly, synthetic derivatives 4b, 4e, 4k, and 10 along
with commercial derivatives S1–S6, which all demonstrated
interference in the displacement assay, are identified as TAR
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to Conformer 14. It is important to note, however, that the
predicted binding analysis did not show other trends that
could be clearly correlated with the NMR or CD50 data. Re-
markably, the fact that docking often resulted in more than
one favorable binding mode for a single molecule as well
as strikingly different binding modes for very similar mole-
cules (i.e. 22a, 22e, and 22f) reinforces the previously de-
scribed lack of clearly discernible structure–activity relation-
ships with amiloride derivatives. Interestingly, a deeper
analysis of the terms in the scoring function revealed that
the selective binders (Group A) predicted higher contribu-
tions from van der Waals forces while the non-selective
binders (Group B) predicted higher contributions from hy-
drogen bonding (Fig. S9‡), suggesting a possible variation
in the binding modes of the two sets of ligands.
Cheminformatic analysis of amiloride library
In order to determine chemical features associated with each
of the three binding classes, (i.e. specific binding, non-
specific binding, and non-binding), we evaluated our
amiloride library using cheminformatic analyses. We first
performed a preliminary evaluation of library diversity80–84 by
calculating the Tanimoto coefficient (Tc) values for all possi-
ble pairs of library members (see page S126 of ESI‡).85,86
Interestingly, only two library members (4a and 9) were pre-
dicted to be similar to 1 (Tc > 0.85) (Fig. 6A).87 Despite the
presence of the amiloride scaffold in every molecule, only 8%
of the total possible pairings of synthetic derivatives were
found to be similar, and nearly half of these were within the
aryl-C(6)-derivative class.
Fig. 4 Representative vector diagrams indicating the direction and
magnitude of chemical shift perturbations induced by each synthetic
amiloride derivative binding to HIV-1 TAR measured using the SOFAST-
[1H-13C] HMQC NMR experiment. Perturbations less than 0.025 ppm
were considered negligible and excluded. Perturbations that could not
be assigned due to large chemical shifts and/or broadening are listed
in Fig. S4–S7,‡ as are the vector diagrams for all other residues.
The ability of the amiloride derivatives to adopt multiple
binding modes was reflected in docking experiments of the
synthesized library against the TAR ensemble previously
reported by the Al-Hashimi lab,78 and using the docking
program, ICM (Molsoft).79 Each derivative was docked
against each of the 20 TAR conformers (see page S124 of
ESI‡). The docking energies for each complex, as measured
by ICM's interaction score, were used to predict the popula-
tion of each TAR conformer when bound to the amiloride
derivatives assuming a Boltzmann distribution (see page
S124 of ESI‡). We examined the population of each TAR
conformer over all amiloride derivatives and found that four
of the 20 conformers were strongly preferred for amiloride:
TAR interactions (Fig. 5). These docking results confirm the
NMR-based observation that both the bulge and loop are
potential ligand binding sites for different amiloride deriva-
tives (Fig. 3 and 4). Lead derivative 24 was the strongest pre-
We further sought to evaluate whether standard
cheminformatic parameters83,84 may be able to explain the
binding patterns within our synthetic derivative library.
Given the difference in size between the group that bound
by NMR (N = 25) and the group that showed no binding (N
= 3), statistical comparisons between these groups were not
possible. Qualitatively, however, the binding group
displayed a mean decrease in the hydrogen bond acceptor
number (HBA), total polarizable surface area (tPSA) and ro-
tatable bonds (RotB) relative to the non-binding ligands,
along with a decreased number of oxygens and nitrogens
(Fig. 6B, Table S4‡). Statistical analysis was performed be-
tween the groups whose CD50 values were consistent with
selective (N = 10) or non-selective (N = 7) binding interac-
tions. Group analysis, rather than direct correlation of
cheminformatic parameters to CD50 values, was performed
due to the narrow range of CD50 values recorded. The li-
brary members that displayed interference in the displace-
ment assay were removed from this analysis. As previously
discussed, of the four library members with positively
charged side chains at pH 7.4, two were in the non-selective
binding group and two interfered with the assay. All selec-
tive binders in this library had neutral substituents. The
most strikingly different cheminformatic parameter between
the two groups was the predicted distribution coefficient
dicted binder with an interaction score (−51 kcal mol−1
)
three standard deviations above the mean (mean score =
−34 5 kcal mol−1, see Table S2‡), and exclusively demon-
strated bulge binding interactions (Fig. 5). This result is
consistent with the bulge-localized NMR chemical shift per-
turbations measured for 24. Derivative 22d yielded distinct
NMR perturbations (Fig. 4) relative to the other derivatives
and is the only derivative predicted to have strong binding
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Fig. 5 Docking predicted bound poses of amiloride derivatives to their preferred conformers of the HIV-1 TAR ensemble. The TAR loop is in blue,
the bulge in orange, and the stem in gray, while the ligands are shown in green (Group A) or yellow (Group B). Conformers are ordered based on
the docking predicted population of each, averaged over all amilorides, with the most strongly preferred conformer (20) listed first. The listed per-
centages indicate the docking predicted population of each ligand:TAR complex for the individual ligands.
(log D), a pH-dependent partition coefficient, where the non-
selective binders are predicted to be more hydrophilic
(mean log D = −0.78) while the selective binders more lipo-
binding groups and found that 99.9% of the variance could
be explained with a single linear discriminant that weighted
HBA and tPSA the most heavily, followed by O, N, RotB, log
D, and HBD (see pages S129–S130 of the ESI‡). While excel-
lent separation was observed (estimation sample 100%),
leave-one-out cross-validation analysis returned correct group
assignments with 65% accuracy, suggesting that larger sam-
ple sizes will be needed for these calculations to be predic-
tive. When the fourth group, i.e. those derivatives that inter-
fered with the displacement assay but bound by NMR, was
added to the LDA analysis, this group was found to overlap
with both the non-selective and non-binding groups
(Fig. 6C). This result may imply that these derivatives were
largely weak binders.
Finally, we further sought to compare the diversified scaf-
fold design to fragment-based screening by examining the
difference between 1, 4j, and 24. 4j demonstrated an order of
magnitude decrease in CD50 value as well as more resolved
NMR chemical shifts as compared to 1 and differs in the ad-
dition of an indole at the C(5)-position. N-Acetyltryptamine,
philic (mean log D
= 1.75). The selective binders also
displayed the lowest fraction of sp3 hybridized carbons
(Fsp3), hydrogen bond acceptor number (HBA), hydrogen
bond donor number (HBD), and relative polar surface area
(relPSA) of the three classes. These analyses, along with the
docking results, are consistent with stacking and other van
der Waals interactions, rather than electrostatics and hydro-
gen bonding, as the driving forces behind selective TAR
binding among amiloride derivatives. Interestingly, all li-
brary members were found to follow Lipinski's rules of
five88 except for 4i, 11, and 14, none of which displaced the
Tat peptide, though it is not clear whether Lipinski's rules
of five are applicable to RNA-targeted ligands. No library
members were identified as PAINS compounds.89
To determine whether a combination of parameters might
be used to predict selectivity among the amiloride derivatives,
we performed linear discriminate analysis (LDA) of the three
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Fig. 6 A) Heat map of Tanimoto coefficients for synthesized derivatives; B) representative cheminformatic parameters among binding classes
(**p-value 0.01, *p-value 0.05 between non-selective and selective binding classes); C) linear discriminant analysis (LDA) plot of
<
<
cheminformatic-based clusters of binding classes.
which mimics the C(5)-side chain in 4j, was tested in the dis-
placement assay as well as the SOFAST NMR experiments
and showed no displacement of the Tat peptide or perturba-
tions to the TAR NMR signals, suggesting that the subunit
may not have been identified as a hit in a fragment based
screen. The simultaneous combination of the amiloride and
tryptamine fragment can thus be concluded to be important
for binding activity of 4j. Expansion of 4j in 24 led to an addi-
tional order of magnitude decrease in CD50 value, consistent
with increased molecular complexity,90 which may prove an
important feature in small molecule:RNA recognition.
the binding mode of amiloride derivatives with TAR at low
concentration in a time efficient assay. Chemical shift map-
ping demonstrates that while the parent ligand 1 bound both
the apical loop and bulge, modifications at the C(5)- and par-
ticularly at the C(6)-positions allowed this binding to be
tuned to tight and specific binding in the bulge region. More
detailed docking studies are underway to pursue correlations
with the NMR perturbation data, which would prove a power-
fully predictive docking technique and, along with the
established synthetic methods, allow for the tailoring of li-
gands to bind specific TAR conformations, including those
that favor the apical loop. Cheminformatic analysis suggested
that stacking and other van der Waals interactions are the
most critical for selective binding of amilorides to TAR RNA
and that it may be possible to predict this selectivity in future
library designs. Finally, the success of the combined C(5)-
and C(6)-modifications in 24 to yield a ligand with 100× im-
proved activity relative to the parent molecule and a unique
binding mode strongly supports the pursuit of combinatorial
library designs on modifiable RNA scaffolds. Indeed, 24 is
one of the tightest non-aminoglycoside TAR ligands reported
to date, and future work will investigate the combination of
Conclusions and future directions
In summary, we have used scaffold diversification to generate
a small RNA-targeted library that establishes amiloride as a
new RNA-binding scaffold capable of highly specific interac-
tions. Expansion of this scaffold led to the discovery of 10
novel and selective ligands for HIV-1 TAR RNA that demon-
strated increased or similar affinity relative to other small
molecule ligands.9,17 Furthermore, use of the recently devel-
oped SOFAST NMR method allowed us to directly evaluate
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even more potent C(6)-derivatives. Furthermore, the use of
multiple experimental and computational methods allowed
specific insight into the molecular recognition of RNA by
these small molecules without the determination of a high-
resolution structure. Future studies will include explorations
of multivalent amiloride-based probes such as dimers and
conjugates with other RNA active molecules, as these strate-
gies have been shown to enhance activity in some
cases.24,27,91 We hope that the tools and insights developed
in this study will allow the development of much-needed
guiding principles for small molecule:RNA interactions.
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Notes
The authors declare no competing financial interests.
Abbreviations used
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14 A. C. Stelzer, A. T. Frank, J. D. Kratz, M. D. Swanson, M. J.
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and H. M. Al-Hashimi, Nat. Chem. Biol., 2011, 7, 553.
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Zhao, Z. Feng, K. K. Y. Ling, G. M. Karp, H. Qi, M. G. Woll,
G. Chen, N. Zhang, V. Gabbeta, P. Vazirani, A.
Bhattacharyya, B. Furia, N. Risher, J. Sheedy, R. Kong, J. Ma,
A. Turpoff, C.-S. Lee, X. Zhang, Y.-C. Moon, P. Trifillis, E. M.
Welch, J. M. Colacino, J. Babiak, N. G. Almstead, S. W. Peltz,
L. A. Eng, K. S. Chen, J. L. Mull, M. S. Lynes, L. L. Rubin, P.
Fontoura, L. Santarelli, D. Haehnke, K. D. McCarthy, R.
Schmucki, M. Ebeling, M. Sivaramakrishnan, C.-P. Ko, S. V.
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TAR
Trans-activation response element
Dimethylamiloride
Competitive displacement of 50% Tat peptide
Transfer RNA
DMA
CD50
tRNA
SOFAST Selective optimized flip angle short transient
HMQC Heteronuclear multiple-bond quantum correlation
HSQC
ITC
DLS
MW
HBA
HBD
RotB
tPSA
Heteronuclear single-bond quantum correlation
Isothermal titration calorimetry
Dynamic light scattering
Molecular weight
Hydrogen bond acceptor
Hydrogen bond donor
Rotatable bonds
Topical polar surface area
relPSA Relative polar surface area
LDA Linear discriminant analysis
17 J. Sztuba-Solinska, S. R. Shenoy, P. Gareiss, L. R. H. Krumpe,
S. F. J. Le Grice, B. R. O'Keefe and J. S. Schneekloth, J. Am.
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Acknowledgements
We thank the members of the Hargrove and Al-Hashimi labs
for stimulating discussions. AEH and HMA wish to acknowl-
edge financial support for this work from Duke University
and the US National Institute of Health (P50GM103297). We
also wish to thank: Dr. George R. Dubay, Director of Analyti-
cal Services, Duke University for performing the high resolu-
tion mass spectroscopy analyses; the Duke University NMR
Center for use of the 800 MHz and 600 MHz spectrometers
for 2D SOFAST NMR experiments and the 500 MHz and 400
MHz spectrometers for 1D analysis of small molecules; Prof.
Michael Therien and Dr. Melanie O'Sullivan for assistance
and access to dynamic light scattering instruments; Mr. David
Jones, Ms. Dillan Casanova, and Ms. Aline Umuhire-Juru for
assistance with synthesis of amiloride derivatives; and Mr.
Raymond Li for help with the fluorescence assays.
18 J. A. Howe, H. Wang, T. O. Fischmann, C. J. Balibar, L. Xiao,
A. M. Galgoci, J. C. Malinverni, T. Mayhood, A. Villafania, A.
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