Non-Natural Linker Configuration in 2,6-Dipeptidyl-Anthraquinones Enhances the Inhibition of TAR RNA Binding/Annealing Activities by HIV-1 NC and Tat Proteins

Article abstract of DOI:10.1021/acs.bioconjchem.8b00104

The HIV-1 nucleocapsid (NC) protein represents an excellent molecular target for the development of anti-retrovirals by virtue of its well-characterized chaperone activities, which play pivotal roles in essential steps of the viral life cycle. Our ongoing search for candidates able to impair NC binding/annealing activities led to the identification of peptidyl-anthraquinones as a promising class of nucleic acid ligands. Seeking to elucidate the inhibition determinants and increase the potency of this class of compounds, we have now explored the effects of chirality in the linker connecting the planar nucleus to the basic side chains. We show here that the non-natural linker configuration imparted unexpected TAR RNA targeting properties to the 2,6-peptidyl-anthraquinones and significantly enhanced their potency. Even if the new compounds were able to interact directly with the NC protein, they manifested a consistently higher affinity for the TAR RNA substrate and their TAR-binding properties mirrored their ability to interfere with NC-TAR interactions. Based on these findings, we propose that the viral Tat protein, sharing the same RNA substrate but acting in distinct phases of the viral life cycle, constitutes an additional druggable target for this class of peptidyl-anthraquinones. The inhibition of Tat-TAR interaction for the test compounds correlated again with their TAR-binding properties, while simultaneously failing to demonstrate any direct Tat-binding capabilities. These considerations highlighted the importance of TAR RNA in the elucidation of their inhibition mechanism, rather than direct protein inhibition. We have therefore identified anti-TAR compounds with dual in vitro inhibitory activity on different viral proteins, demonstrating that it is possible to develop multitarget compounds capable of interfering with processes mediated by the interactions of this essential RNA domain of HIV-1 genome with NC and Tat proteins.

Full text of DOI:10.1021/acs.bioconjchem.8b00104

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Article
Non-Natural Linker Configuration in 2,6-Dipeptidyl-
Anthraquinones Enhances the Inhibition of TAR RNA
Binding/Annealing Activities by HIV-1 NC and Tat Proteins
Alice Sosic, Irene Saccone, Caterina Carraro, Thomas Kenderdine, Elia Gamba, Giuseppe Caliendo,
Angela Corvino, Ferdinando Fiorino, Paola DiVaio, Elisa Magli, Elisa Perissutti, Vincenzo Santagada,
Beatrice Severino, Valentina Spada, Daniele Fabris, Francesco Frecentese, and Barbara Gatto
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/
acs.bioconjchem.8b00104 • Publication Date (Web): 23 May 2018
Downloaded from http://pubs.acs.org on May 25, 2018
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TITLE PAGE
Non-Natural Linker Configuration in 2,6-Dipeptidyl-Anthraquinones Enhances
the Inhibition of TAR RNA Binding/Annealing Activities by HIV-1 NC and Tat
Proteins
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a, †
b, †
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c
a
Alice Sosic , Irene Saccone , Caterina Carraro , Thomas Kenderdine , Elia Gamba , Giuseppe
b
b
b
b
b
b
Caliendo , Angela Corvino , Paola Di Vaio , Ferdinando Fiorino , Elisa Magli , Elisa Perissutti ,
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,b
Vincenzo Santagada , Beatrice Severino , Valentina Spada , Dan Fabris , Francesco Frecentese * ,
,a
Barbara Gatto*
a
Dipartimento di Scienze del Farmaco, Università di Padova, via Marzolo 5, 35131 Padova, Italy
b
Dipartimento di Farmacia, Università degli Studi di Napoli “Federico II”, Via D. Montesano 49,
8
c
0131 Napoli, Italy
The RNA Institute and Department of Chemistry, State University of New York, 1400 Washington
Avenue, Albany, New York 12222, United States
†
These two authors equally contributed to the paper.
*F.F.: phone, +39081679829; fax, +39081678649; E-mail, frecente@unina.it
*
B.G.: phone, +390498275717; fax, +390498275366; E-mail, barbara.gatto@unipd.it
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Abstractꢀ
The HIV-1 nucleocapsid (NC) protein represents an excellent molecular target for the development
of antiretrovirals by virtue of its well-characterized chaperone activities, which play pivotal roles in
essential steps of the viral life cycle. Our ongoing search for candidates able to impair NC
binding/annealing activities led to the identification of peptidyl-anthraquinones as a promising class
of nucleic acid ligands. Seeking to elucidate the inhibition determinants and increase the potency of
this class of compounds, we have now explored the effects of chirality in the linker connecting the
planar nucleus to the basic side chains. We show here that the non-natural linker configuration
imparted unexpected TAR RNA targeting properties to the 2,6-peptidyl-anthraquinones and
significantly enhanced their potency. Even if the new compounds were able to interact directly with
the NC protein, they manifested a consistent higher affinity for the TAR RNA substrate and their
TAR-binding properties mirrored their ability to interfere with NC-TAR interactions. Based on
these findings, we propose that the viral Tat protein, sharing the same RNA substrate but acting in
distinct phases of the viral life cycle, constitutes an additional druggable target for this class of
peptidyl-anthraquinones. The inhibition of Tat-TAR interaction for the test compounds correlated
again with their TAR-binding properties, while simultaneously failing to demonstrate any direct
Tat-binding capabilities. These considerations highlighted the importance of TAR RNA in the
elucidation of their inhibition mechanism, rather than direct protein inhibition. We have therefore
identified anti-TAR compounds with dual in vitro inhibitory activity on different viral proteins,
demonstrating that it is possible to develop multi-target compounds capable of interfering with
processes mediated by the interactions of this essential RNA domain of HIV-1 genome with NC
and Tat proteins.
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Introduction
Current anti-HIV therapeutics tend to exhibit desirable potency and selectivity, as well as
undesirable clinical limitations, which stem from putative toxic effects associated with long-term
treatments and the relentless emergence of resistant strains. As a consequence, there is the urgent
need to develop new therapeutic agents against essential viral determinants that are not currently
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targeted by available drugs. The HIV-1 nucleocapsid (NC) protein represents an attractive and
promising target by virtue of its highly conserved nature among viral clades and its vital roles in a
4
range of processes of the viral lifecycle. This relatively small, basic protein is characterized by two
CCHC-type zinc-finger domains that are highly conserved and confer the ability to directly interact
4
with viral nucleic acids and to catalyze essential structural rearrangements. In particular, its
interaction with susceptible substrates can induce transient melting of base-pairing, which makes
previously
paired
strands
available for reannealing in
different patterns conducive to
more thermodynamically stable
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-10
conformations.
An example of
this activity is provided by the
rearrangement
transactivation
of
the
Figure 1. Constructs replicating TAR RNA (blue) and
cTAR DNA (green) employed in the study. The scheme
describes a key step of the reverse transcription of viral
RNA, in which NC destabilizes these stem-loop structures
to promote their annealing into a stable TAR/cTAR
heteroduplex.
responsive
element (TAR), an essential
domain of the viral genome,
which is characterized by a relatively stable bulge–loop structure (Figure 1). Annealing of TAR
RNA to a reverse-transcribed complementary DNA (cTAR, Figure 1) is an obligatory step of the
reverse transcription process of viral genome. This operation is mediated by NC, which is
responsible for melting the secondary structures of TAR and cTAR and promoting the formation of
the more stable heteroduplex TAR/cTAR (Figure 1), thus allowing for the minus strand-transfer
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and reverse transcription processes to proceed.
These essential chaperone properties make NC a potential target for ligands capable of either
preventing its interaction with the target substrate, or stabilizing the substrate structure to forestall
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the expected rearrangements. Based on this hypothesis, we identified 2,6-dipeptidyl anthraquinones
12-14
(
2,6-AQ) as a class of potent anti-NC agents in vitro.
The chemical structure of these
compounds is characterized by an anthraquinone nucleus bearing aminoacyl linkers at positions 2
and 6, which connect to terminal anchor groups containing a set of different positively charged
moieties (e.g., linear or cyclic alkyl chains, as well as heterocyclic or aminophenyl chains, Figure
2
). Acting as threading intercalators, we proved that these compounds interfere with NC activity by
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stabilizing the rather dynamic structures of TAR and cTAR.
ꢀ
Figure 2. Newly synthesized 2,6-dipeptidyl anthraquinones considered in this study (i.e., 2,6-
AQ-D-Ala-X, D-4a-i). For reference purpose, the chemical structures of compounds
12
investigated earlier are also provided (i.e., 2,6-AQ-L-Ala-X, L-4a-i).
A thorough structure-activity relationship (SAR) analysis of related series of 2,6-dipeptidyl
anthraquinones, which bore a wide variety of aminoacyl linkers, identified key structural
12-13
requirements necessary for the development of putative NC inhibitors.
The studies underscored
the importance of the length of the highly basic terminal residues and the flexibility of the linear
linkers connecting them to the aromatic nucleus to achieve the desired binding and stabilization of
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the cognate nucleic acid substrates. However, when we compared Gly and L-Ala linkers in side
chains of fixed optimal length, we observed a sizable loss of inhibitory activity in spite of their
similar nucleic acid binding and stabilizing properties. This observation suggested that either the
linker configuration, or the presence of a branching methyl group in Ala, could represent important
factors in determining inhibition.
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In this report, we tested the significance of linker chirality by synthesizing a new complete
series of 2,6-disubstituted anthraquinones, which contained Ala linker residues in the non-natural D
configuration (i.e., 2,6-AQ-D-Ala-X, D-4a-i, Figure 2). We then evaluated their activity in vitro by
employing the same assays developed earlier to test the analogous series with opposite linker
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configuration (i.e., 2,6-AQ-L-Ala-X, L-4a-i, Figure 2). This modus operandi enabled direct
comparisons with previously reported results, which allowed us to detect the sometimes subtle
effects of chirality. The experiments revealed unexpected properties that suggested additional
mechanisms of action for this class of compounds and, based on these findings, pointed toward
alternative potential targets. The results were discussed in the context of our SAR analysis of
peptidyl-anthraquinones and used to glean a possible outlook for the development of actual
therapeutics from these types of structures.
Chemistry
Synthesis of 2,6-AQ-D-Ala-X anthraquinone derivatives
Compounds D-4a-i (Figure 2) were obtained according to the synthetic strategy summarized in
Scheme 1, which included appropriate modifications of a previous procedure reported for 2,6-
1
2
disubstituted anthraquinones. Briefly, the general strategy is based on the condensation of the 2,6-
diaminoanthraquinone nucleus 1 with Fmoc-protected acyl chloride 2 in the presence of pyridine in
DMF. Each reaction mixture was submitted to overnight stirring at room temperature. The resulting
material was then dried and treated with 33% diethylamine in THF to remove the Fmoc protecting
2
group. The material was successively reacted with trifluoroacetic acid in H O (9:1; v/v) to obtain
intermediate 3, which was in turn coupled with the protected aminoacid of choice in the presence of
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HBTU and DMAP in DMF. The Fmoc and Boc protecting groups were removed by using 33%
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diethylamine in THF and trifluoroacetic acid in H O (9:1; v/v), respectively, to produce the desired
compounds 4a-i.
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Scheme 1. General synthetic pathway for the preparation of the anthraquinone derivatives
included in the study. Reagents and conditions: (i) Pyridine, DMF; (ii) 33% Diethylamine, THF;
(
2 2
iii) TFA/H O; (iv) HBTU, DMAP, DMF; (v) 33% Diethylamine, THF; (vi) TFA/H O.
Fmoc-D-Ala-Cl (2) was prepared from commercially available Fmoc-D-Ala-OH, which was
treated with an excess of thionyl chloride in CH
2
Cl
2
, and then heated by microwave irradiation in a
sealed vessel for 8 min
(
Scheme 2). We found
that increasing
temperature, reaction time,
Scheme 2. Preparation of intermediate 2.
or microwave power did not increase the product yield, but rather induced reagent decomposition.
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The final compounds were purified by preparative RP-HPLC and fully characterized by mass
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spectrometry, H-NMR and C-NMR. The data recorded for all final compounds were consistent
with their proposed structures.
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ꢀ
Results and Discussion
Effects of 2,6-dipeptidyl anthraquinones on NC-mediated melting of individual TAR and
cTAR structures
The effects of the new series of 2,6-AQ-D-Ala-X derivatives (D-4a-i in Figure 2) on the NC-
induced destabilization of individual TAR and cTAR structures were evaluated by using an
1
2-15
adaptation of a high throughput screening (HTS) approach described earlier.
Briefly, the assay
employed nucleic acid substrates consisting of 29-mer constructs that replicated the apical portion
of the TAR domain of viral RNA and its complementary DNA sequence (i.e., TAR and cTAR,
Figure 1). The 5’ and 3’-ends of each construct were appropriately labeled with either a
fluorophore or a quencher, which enabled the determination of the stem-melting activity of NC
from the observed fluorescence emission. In particular, the emission recorded in the absence of
ligand provided a measure of the baseline activity of NC, which minimized quenching by
dissociating the double-stranded stem and increasing the distance between fluorophore and
quencher. In the presence of ligand, instead, decreasing fluorescent emission was caused by
inhibition of the NC’s melting activity, which left a higher proportion of substrate in the stem-loop
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form conducive to quenching.
In addition to initial baseline determinations, control
experiments were also carried out to ensure that the compounds of interest were incapable of
inducing direct quenching in the absence of NC (see Experimental). A series of determinations
were completed with increasing amounts of ligand to determine the concentration that induced 50%
1
2-14, 16
reduction of the baseline activity (expressed as IC50).
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Table 1. Inhibition of NC-mediated melting of individual TAR and cTAR structures
a
a
Compound
D-4a
IC50 TAR (µM)
IC50 cTAR (µM)
39.7±3.99
8.79±0.11
51.3±5.01
33.7±2.05
10.5±1.50
9.24±0.49
3.27±0.44
3.35±0.51
47.4±1.34
24.9±2.20
12.8±1.09
32.1±2.76
19.9±1.58
13.8±1.50
6.52±0.47
4.76±0.68
2.16±0.37
20.2±0.78
D-4b
D-4c
D-4d
D-4e
D-4f
D-4g
D-4h
D-4i
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values are the mean of data obtained from three experiments, each performed in triplicate.
ꢀ
The data obtained from the D-4a-i anthraquinones revealed that derivatives bearing doubly-
charged linear-alkyl side chains possessed potent inhibitory properties (e.g., D-4e-h in Table 1). In
particular, the lowest IC50 values were exhibited in both TAR and cTAR determinations by the D-
4g and D-4h compounds, with the latter providing 3.35 and 2.16 µM on the respective assay.
Compound D-4b with a mono-charged cyclic alkyl side chain showed somewhat intermediate
potency with preferential activity on TAR, whereas all other compounds bearing cyclic aliphatic or
aromatic moieties displayed lower potency. A close examination of these results suggested that
increasing the length of the linear side-chain may increase the potency according to a D-4h > D-4g
>
D-4f > D-4e relative scale, which agreed with the results of our previous SAR study on
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A
direct comparison between
corresponding IC50 values observed for
the analogous 2,6-AQ-D-Ala-X and
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,6-AQ-L-Ala-X series (Figure 3)
revealed that compounds bearing linear
terminal alkyl residues (i.e., 4d-h),
were generally more potent when the
linker was in the non-natural D
configuration rather than their L-
configuration counterparts. This was
Figure 3. IC50 values observed for the effects of 2,6-AQ-
D-Ala-X (purple, this study) and 2,6-AQ-L-Ala-X
12
remarkable in particular when TAR (yellow, reference ) compounds on the NC-induced helix
destabilization of TAR structure (see Experimental).
was assayed rather than cTAR (Figure
S1 in Supporting Information). 2,6-AQ-D-Ala-X compounds bearing cyclic or heterocyclic side
chains (4a, 4c and 4i) displayed in all cases weaker activity than the corresponding L-Ala-X
counterparts, with the exception of the aminomethyl-ciclohexyl D-4b, whose behavior on TAR in
this assay clusters within the flexible linear-alkyl category. These differences between the activity
of the L-Ala and the D-Ala series of compounds highlight the significant impact of the
stereochemistry of the chiral amino acidic linker on the 2,6-dipeptidyl anthraquinones structure-
activity relationship, therefore answering our initial question on the importance of the configuration
of the connecting Ala moiety in NC inhibition.
Effects of 2,6-dipeptidyl anthraquinones on NC-mediated annealing of TAR and cTAR
constructs
The nucleic acid chaperone activities of NC are substantiated by its ability to destabilize double-
stranded regions of RNA structure and enable their re-annealing into more stable pairing
arrangements. In the TAR/cTAR system, initial melting of the intra-strand pairs that define their
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individual stem-loop structures can lead to inter-strand annealing with formation of a stable
1
1, 17
TAR/cTAR heteroduplex.
The ability to monitor this process provided us with the opportunity
to evaluate the effects of the 2,6-AQ-D-Ala-X anthraquinones on the annealing activity of NC in
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vitro. We employed the nucleocapsid annealing mediated electrophoresis (NAME) assay described
12, 18
earlier,
which was carried out according to alternative protocols involving pre-incubation of
ligand with either the NC protein or the nucleic acid substrates (see Experimental). Our tests
revealed that only compounds D-4e-h possessed detectable inhibitory activity in these experiments
(see Figure S2 of Supporting Information). Furthermore, D-Ala compounds were more active
upon nucleic acid pre-incubation, in analogy with previous observations obtained from other series
1
2, 18
of peptidyl anthraquinones.
Tests were repeated using a wider range of ligand concentrations to
obtain IC50 values as a measure of their ability to interfere with annealing activity under the selected
experimental conditions (see representative gel for D-4h in Figure S3 of Supporting Information
and Table 2).
Table 2. Inhibition of NC-mediated annealing of TAR/cTAR assessed by NAME assay
ꢀ
a
Compound
D-4a
IC50 NAME (µM)
>100
D-4b
>100
D-4c
>100
D-4d
>100
D-4e
76.7±8.61
78.8±10.8
37.5±13.6
28.9±4.59
>100
D-4f
D-4g
D-4h
D-4i
a
Values represent the mean and standard error of the mean (SEM) obtained from experiments performed in triplicate.
ꢀ
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The results showed that linear anthraquinones D-4e-h possessed inhibitory activities
consistent with a D-4h > D-4g > D-4f ≈ D-4e relative scale of potency. Not surprisingly, this scale
matched that observed for the potency of inhibition of NC-induced melting of TAR and cTAR,
which was also correlated with the length of the doubly charged side chain. Compound D-4h was
the most potent inhibitor of both melting and annealing activities with respective IC50s of 3.35 and
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8.9 µM (Table 1 and 2). This was not the case for its 2,6-AQ-L-Ala-X analog L-4h, which was
significantly less active both in the melting and in the annealing assay (i.e., IC50 of 6.61 and 80.4
12
µM). The outcome of these experiments further indicates that the non-natural D configuration of
Ala-linkers is a strong inhibition determinant of 2,6-dipeptidyl-anthraquinone compounds.
Binding modes of 2,6-dipeptidyl anthraquinones to individual TAR and cTAR structures
In the formerly analyzed L-Ala linker series, the NC inhibitory properties mirrored their nucleic
acid binding capabilities, providing strong evidence that the mechanism of action involve
stabilization of TAR and cTAR secondary structure through intercalation of their anthraquinone
12-13
systems.
With the new D-Ala series, we therefore evaluated the effects of varying the linker
configuration on the binding features on TAR and cTAR, employing electrospray ionization mass
spectrometry (ESI-MS) analysis under non-denaturing conditions as performed for the L-Ala series
1
2-13, 19-21
(
see Experimental).
Initial determinations were carried out in the absence of ligand to verify the experimental
masses of the TAR and cTAR constructs, which matched very closely those calculated from the
respective sequences (i.e., 9286.2 u experimental versus 9286.3 Da monoisotopic mass calculated
for TAR; 8884.5 u versus 8884.5 Da for cTAR). Subsequent analyses were conducted on sample
solutions that contained respectively 1 and 10 µM concentrations of nucleic acid substrate and 2,6-
dipeptidyl anthraquinone ligand (i.e., a 1:10 molar ratio). Representative ESI-MS spectra obtained
from mixtures of D-4h with either TAR or cTAR are provided in Figure 4, which displays only the
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regions containing the 6- or the 5- charge states for the sake of clarity. Experimental and calculated
monoisotopic masses for all the detected species are provided in Table S1 and S2 of Supporting
Information.
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Figure 4. Representative ESI-MS spectra of samples containing a 1:10 molar ratio of either
TAR A) or cTAR B) and D-4h ( ) compound in 150 mM ammonium acetate (see
Experimental). Symbol corresponds to TAR RNA substrate and to cTAR DNA substrate.
The stoichiometries of the observed complexes are indicated in each spectrum. Signals of lower
intensity detected near free/bound species consist of typical sodium and ammonium adducts. C)
Histograms displaying the percentages of bound substrate observed in A. and B. spectra and
from Figure S4 for D-4b and D-4h. The indicated percentages of bound substrate provide a
measure of the relative affinities of D-analogues for either nucleic acid substrate.
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Consistent with the ability of ESI-MS to observe intact noncovalent complexes between
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nucleic acids and peptidyl-anthraquinones,
all spectra contained signals corresponding to free
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unbound substrates, as well as stable complexes with different stoichiometries. The observed
binding patterns did not reveal significant cooperativity for either substrate (Figures 4 and S4). In
all cases, the coexistence of free TAR or cTAR with complexes of different stoichiometries
indicated that binding to the second site was initiated before saturation of the first was complete,
consistent with the presence of independent binding sites with equivalent affinities on either
construct. The results revealed a clear correlation between binding stoichiometry and the inhibition
of annealing activity revealed by the NAME assays. In particular, the D-4g and D-4h compounds,
which possessed the most pronounced inhibitory properties, produced TAR complexes containing
up to 3 and 4 equivalents of ligand respectively. Differently, the D-4b compound with the least
pronounced inhibitory properties produced only 1:1 complexes with TAR under the same
experimental conditions. In the case of the cTAR substrate, D-4g and D-4h produced complexes
with up to 4:1 stoichiometries, whereas D-4b produced again only 1:1 complexes.
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These results are quite consistent to what previously reported for the L-Ala series. We then
proceeded to calculate the relative binding affinities of the various compounds for the different
substrates, employing the percentage of bound substrate observed in each spectrum (see
12
Experimental). The representative histograms in Figure 4C revealed that D-4g and D-4h
possessed comparable affinities that were much greater than those afforded by D-4b under the same
experimental conditions (i.e., D-4g ≈ D-4h >> D-4b relative scale). Surprisingly, D-4g and D-4h
displayed a slightly greater affinity for the RNA than for the DNA substrate, which contrasted with
the behavior manifested by the corresponding L-series analogues. In fact, the histograms obtained
1
2-13
from the data generated in our previous studies
with L-Ala linker (named L-4g, L-4h and L-4a) possessed greater affinities for cTAR than for TAR
Figure S5 of Supporting Information).
clearly demonstrated that analogue compounds
(
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The unexpected but detectable selectivity for the RNA rather than for the DNA construct
manifested by the D-analogues was never observed before and suggested further investigation. We
evaluated under the same experimental conditions the binding properties of the strong TAR binder
D-4h and its L-counterpart L-4h to different RNA substrates. We selected the stem-loop SL3
sequence, a RNA domain of the HIV-1 genome packaging signal, owing to the presence of an
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9-20, 22
apical loop,
and a random 16-mer double stranded RNA (dsRNA) sequence. As performed in
the presence of TAR, ESI-MS analyses were conducted on sample solutions that contained
respectively 1 and 10 µM concentrations of nucleic acid substrate and 2,6-dipeptidyl anthraquinone
ligand (not shown). We calculated the relative binding affinities of the compounds for the different
substrates, employing the percentage of bound substrate observed in each spectrum (see
Experimental). The representative histograms in Figure S6 (see Supporting Information) compare
the binding affinities of selected compounds for TAR (Figure 4C) with those observed for SL3 and
for the dsRNA. Once again the D-analogue displayed a slightly greater affinity for all the three
RNA substrates compared to its L-counterpart, corroborating our hypothesis that the non-natural
conformation of the linker in the side chains is important to achieve RNA binding. Noteworthy, the
data clearly revealed that the test compounds possess a remarkable greater binding affinities for the
bulge-loop TAR structure than for both the stem-loop SL3 sequence and the double stranded RNA,
highlighting their preferential binding to dynamic bulged regions of RNA structures.
The increased selectivity toward TAR induced by the D-Ala linker series suggests a possible
strategy for designing RNA-specific ligands, which deserves further investigation. In addition, it
entails the potential inhibition of other factors that interact with this region of viral genome during
the HIV-1 lifecycle, as we will discuss in the final paragraph.
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The multifaceted aspects of 2,6-dipeptidyl anthraquinones inhibition
Although the results obtained to this
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point revealed excellent correlation
between efficient inhibition of NC-
mediated reactions and nucleic acids
binding, they do not satisfactorily
explain the discrepancy between the
different potencies in NC inhibition by
the D- and L-Ala series. For this
reason, we tested whether direct
binding to NC protein by 2,6-AQ-D-
Ala-X ligands could account for
additional inhibitory effects. The
experiments were carried out by
comparing samples that contained full-
length NC and selected analogues of
the D- or L-series (see Experimental).
Also in this case, ESI-MS analyses
were carried out under non-denaturing
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Figure 5. Representative ESI-MS spectra from
samples containing full-length NC and ligand D-4b
(
A), D-4g (B), and D-4h (C), each in 1:10 substrate to
ligand ratio (see Experimental). Weaker signals near
the main peak are typical sodium and ammonium
adducts. Only the region containing the 4+ charge
state of complexes is reported for clarity. (D)
Histograms comparing the fractional occupancy of D-
analogues obtained from the above spectra with that of
their L-counterparts obtained from the data in Figure
S5 (Supporting Information).
conditions to enable the detection of intact complexes with fully metallated NC (see
2
3
Experimental). Control experiments in the absence of ligand afforded a mass of 6489.1 u, which
matched very closely the monoisotopic value of 6488.9 Da calculated from the sequence and
including two Zn(II) ions. Representative spectra obtained in the presence of selected 2,6-AQ-D-
Ala-X anthraquinones are shown in Figure 5, while those obtained in the presence of their 2,6-AQ-
L-Ala-X counterparts are reported in Figure S7 (Supporting Information). For the sake of clarity,
only the regions corresponding to the 4+ charge states were plotted on the same intensity scale to
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enable a direct comparison of the abundances of the respective complexes. Observed and expected
masses for all the detected species are reported in Table S3 (Supporting Information).
The results clearly showed that the ligands were capable of binding to full-length NC to form
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stable 1:1 complexes, with those containing compound D-4g and D-4h displaying greater
19, 24
abundances than those containing D-4b.
The masses observed for these complexes were
consistent with the presence of two Zn(II) ions, which demonstrated that ligand binding did not
interfere at all with metal coordination (Table S3). The fact that these species were detected with
the same charge state allowed us to estimate the partitioning between unbound and bound species in
1
9, 24
the sample from their respective signal intensities (see Experimental).
This treatment revealed
that D-4g and D-4h produced fractional occupancies of 14.4 ± 0.93 and 15.2 ± 0.60 respectively,
whereas D-4b provided only 3.57 ± 0.08. Under the same conditions, the corresponding L-4g and
L-4h (L-Ala linker series) analogues afforded occupancy values of 13.3 ± 0.60 and 6.14 ± 0.79,
which were decidedly lower (Figure 5C). The relative abundances observed for the complexes of
NC with D-Ala and L-Ala compounds (Figure 5 and S5, respectively) provided a putative D-4h
>
D-4g > L-4g > L-4h relative scale of binding affinities for NC protein, which accurately matched
the ranking of the IC50s for annealing inhibition provided by the NAME assays (see Table 2 and
1
2
ref. ). The fact that corresponding D-Ala and L-Ala analogues were consistently at the opposite
ends of these putative scales suggested direct correlations between linker stereochemistry and NC
binding, leading to enhanced inhibitory activities.
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Inhibition of the NC binding to TAR by 2,6-dipeptidyl anthraquinones
NC is a TAR binding protein: the ability of
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selected 2,6-dipeptidyl anthraquinones to
bind directly to NC or to TAR raises
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several possibilities on the mechanism of
NC inhibition by these ligands. To gain
more understanding on the mechanism of
action of D-Ala derivatives, we evaluated
these scenarios in systematic fashion by
Scheme 3. Schematic representation of the analyzing samples in which preformed i.
experimental procedures employed to evaluate the
inhibition of NC binding to TAR by selected NC-TAR, ii. ligand-NC, or iii. ligand-TAR
ligands. The red oval represents the ligand. i.
Displacement of NCŸTAR complex was analyzed complexes were challenged by addition of
by adding ligand to the preformed protein-RNA
complex; ii. interaction of NC with TAR substrate the remaining component (Scheme 3). This
was assayed after preincubation of the protein with
ligand; iii. NC protein was added to preformed set of binding experiments was initially
TAR-ligand complexes.
performed in the presence of the strongest
NC and TAR binder, D-4h. The outcome of the experiments was determined by performing ESI-
MS under the same conditions employed previously to evaluate ligand binding, which enabled the
unambiguous identification of all species present at equilibrium in solution.
Data in Figure 6 were obtained from a sample corresponding to case i. in Scheme 3, which
was prepared by incubating equimolar amounts of NC and TAR (i.e., 6 µM concentration of each)
for 15 min, and then added of a 1:10 molar ratio of D-4h ligand (i.e., final 60 µM concentration).
The signal observed for the NCŸTAR complex upon ligand addition in Figure 6 was significantly
weaker than that obtained for the same species in a control experiment carried out in the absence of
ligand (data not shown). At the same time, strong signals were detected for the D-4hŸTAR species
formed by displacement of the initial NC-TAR complex. It is clear that upon NC displacement the
ligand D-4h forms several D-4hŸTAR complexes, with the same stoichiometries found in Figure
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A. Interestingly, we
observe for the first time
the formation of ternary
complexes between NC
protein, TAR RNA and
D-4h ligand at different
stoichiometries, with the
notable exception of the
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Figure 6. ESI-MS spectrum of samples obtained by adding ligand
D-4h to the preformed TARŸNC complex (case i. in Scheme 3).
Lower intensity signals near free/bound species consist of typical
sodium and ammonium adducts. Symbol
corresponds to NC
ternary
complex
4 ligand
protein; to the TAR RNA substrate, and to the D-4h ligand.
For the sake of clarity only the 6- and 7- charge states of the binary
containing
molecules.
(
RNA-ligand) and ternary (RNA-ligand-protein) complexes are
labelled in the spectrum.
We
can
therefore assume that at least one of the four ligand-TAR binding site competes with NC binding
site to TAR.
In analogous fashion, we analyzed the outcome of experiments obtained by treating
preformed D-4hŸNC complex with TAR (case ii. in Scheme 3); again, this led to the detection of
abundant D-4hŸTAR formed by complex displacement. Finally, treating preformed D-4hŸTAR
with NC (case iii. in Scheme 3) provided signals corresponding to the initial species, with intensity
matching that observed in control experiments in the absence of NC. Taken together, these results
revealed that D-4hŸTAR was the most favorable complex under each scenario, consistent with the
relatively higher affinity of this ligand for the TAR substrate.
This is true also for the other strong TAR-binder D-4g. Figure 7 reports a representative histogram
comparing the results from samples of D-4h, D-4g and D-4b, a poor TAR binder. The relative
abundance of the NCŸTAR complexes in the various samples corresponding to case i. in Scheme 3
was utilized to compare the inhibition effects manifested by the ligands (see Experimental).
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g >> D-4b, which precisely mirrored
the ranking for annealing inhibition
provided by the NAME assays (Table
1
2
Figure 7. Representative histogram comparing the
percentage of NC protein bound to TAR RNA (NCŸTAR
complexes) in the presence of ligand D-4h with those of
D-4g and D-4b. Data were obtained from ESI-MS
analyses of samples prepared following the procedures i.
shown in Scheme 3 (see Experimental).
2 and ref. ), once more revealing the
correlation between TAR binding and
NC-mediated annealing inhibition.
2
,6-dipeptidyl anthraquinones effects on Tat/TAR interactions
The observed ability of 2,6-AQ-D-Ala-X compounds to establish specific interactions with the TAR
structure suggested that these ligands might be capable of interfering with other functions enacted
by this domain in the viral lifecycle. We tested this hypothesis by examining their putative effects
on the specific binding interactions between TAR RNA and a peptide mimicking the HIV-1 trans-
activator of transcription (Tat) factor. This protein can promote the efficient transcription of
2
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integrated proviral genome,
as well as support the annealing activities of NC during reverse
2
9-30
transcription, thus earning itself the definition of nucleic acids annealer.
For this reason, we
evaluated the effects of selected D-Ala analogues on the formation of the TatŸTAR complex by
using a fluorescence-quenching approach. The assay relied on a TAR construct labeled with an
appropriate quencher and a short stretch (aa 48−57) of the Tat protein corresponding to the RNA
3
1-32
14, 33-34
binding region,
which was instead labeled with a fluorescent dye (see Experimental).
In
this way, the formation of the binding complex produced effective quenching, whereas its ligand-
induced inhibition enabled the detection of fluorescence emission. As reported in Table 3,
anthraquinones D-4e-h bearing linear alkyl side-chains strongly inhibited the formation of Tat-TAR
complex with a potency ranking that matched that of NC annealing inhibition, i.e. D-4h > D-4g >
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D-4f > D-4e. In particular, the strongest Tat-TAR inhibitors were again the D-4g and D-4h
compounds, which were also the most active TAR binders and NC inhibitors.
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Table 3. Inhibition of Tat-TAR complex formation
a
Compound
D-4b
K
i
(µM)
1.31±0.18
0.79±0.18
0.54±0.08
0.14±0.01
0.11±0.01
D-4e
D-4f
D-4g
D-4h
a
Values are the mean ± SEM of data obtained by three experiments performed in triplicate.
ꢀ
Inspired by the results obtained from the investigation of NC, we tested the possibility that the
ligands may be capable of binding the Tat peptide, as observed for NC. Following the same
strategy, the experiments were carried out mixing Tat with selected analogues of the D-series, and
then analyzing the samples by ESI-MS under non-denaturing conditions. The results showed
unambiguously that the test ligands were not capable of binding directly to the Tat peptide (data not
shown), thus indicating that the ability of D-analogues to interfere with the Tat-TAR complex was
based exclusively on their TAR-binding properties.
ꢀ
Conclusions
This study was prompted by an earlier observation that the activity of 2,6-disubstituted
anthraquinones significantly benefited from replacing L-Ala with Gly residues in the linkers
1
2
between anthraquinone nucleus and charged side-chain groups. We hypothesized that the methyl
group of L-Ala or its configuration was responsible for decreasing the ability to inhibit NC. To test
this hypothesis we synthesized and analyzed a series of 2,6-AQ-D-Ala-X compounds containing
non-natural D-Ala residues. The results demonstrated that this modification restored the inhibitory
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properties, with D-Ala compounds displaying the same type of correlation with side-chain length
observed for the potent Gly analogues. Indeed, as the length of the linear alkyl side-chain increased
in the 2,6-AQ-D-Ala-X series, inhibition increased according to a D-4h > D-4g > D-4f > D-4e
relative scale of potency. In all assays, D-4h proved to be the most potent inhibitor of the series by
achieving inhibition of NC activities in vitro at concentrations comparable to those observed in
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earlier studies for the most promising hits of the 2,6-AQ-ß-Ala-Orn and 2,6-AQ-Gly-Lys series.
In addition to providing new insights into the structure-activity relationships of 2,6-dipeptidyl
anthraquinones, this study gave us the opportunity to further investigate the putative mechanism of
action of this class of compounds. At first sight, the inhibitory properties of ligands acting as
threading intercalators could be ascribed to their marked binding to both TAR and cTAR secondary
structures. In contrast, the detectable selectivity for the TAR substrate manifested by the D-
analogues was never observed before and highlighted the importance of this RNA structure to
elucidate the NC inhibition mechanism. RNA-binding small molecules are an important and highly
3
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challenging area of therapeutic research. Several classes of RNA binders have been reported,
49
but many of them display non-specificity in RNA binding. The presence of D-Ala linker in 2,6-
dipeptidyl anthraquinones leading to RNA recognition and TAR selectivity therefore constitutes a
new possible strategy for the development of RNA-specific ligands, which deserves further
investigation. Pursuing by NMR study and docking experiments the high-resolution structural
determination of ligand-RNA complexes will be necessary to understand at the molecular level the
steric effects introduced by the non-natural stereochemical configuration.
Although the compounds tested in the study demonstrated the ability to interact directly with the
NC protein, their binding affinities for the protein were consistently inferior to those manifested by
the same compounds for the TAR construct. Indeed, the detection of ternary complexes containing
protein, RNA, and ligands was consistent with the possible presence of distinct binding sites onto
the TAR structure. The exception is represented by D-4h, which might share at least a putative
binding site with the NC protein (Figure 6). Considering that this compound was also the most
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potent inhibitor in the series (Table 1 and 2), it would be tempting to attribute its enhanced activity
to the contribution of a putative competition mechanism.
Additionally, the TAR-binding agents displayed the same potency scale observed for NC
when tested with the Tat-TAR system, while simultaneously failing to demonstrate any direct Tat-
binding capabilities. These considerations confirmed that the mechanism of 2,6-dipeptidyl
anthraquinones is based on their nucleic acid tropism, rather than protein inhibition. For this reason,
they should be more appropriately defined as TAR-targeted candidates capable of interfering with
processes mediated by the interactions of this essential domain of HIV-1 genome with NC and Tat
proteins. Since Tat and NC are multifunctional auxiliary proteins of HIV-1, both acting through
interaction with the TAR sequence, we believe that agents capable of interfering with those critical
steps are expected to block HIV-1 replication at multiple levels. Dual targeting through the same
RNA substrate opens the possibility to consider D-Ala-peptidyl-anthraquinones as pleiotropic
inhibitors, able to interfere not only with NC-mediated reactions during reverse transcription, but
also with the Tat mediated processes. The concomitant inhibition of different targets within the
same pathway would possibly increase the genetic barrier required to gain resistance towards these
putative drugs.
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At the end, the absence of binding with the Tat protein represented a glaring but important
contrast with the observed NC-binding properties. Although our results are a very small sampling,
the selectivity toward the NC protein could constitute the basis for further developing the D-series
as “true” anti-NC compounds. In this direction, additional studies are currently underway to
evaluate the effects of new terminal residues to improve the direct interaction of 2,6-dipeptidyl
anthraquinones with the hydrophobic plateau of NC protein, thus combining in the same molecules
different mechanisms contributing to the overall NC inhibition.
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Experimental Section
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Materials and Methods. For compound synthesis, purification (preparative HPLC) and chemical
characterization (analytical HPLC, ESI-MS and NMR) we used the same procedures, conditions,
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reactants and reagents reported in our previous paper. Microwave reactions were performed using
a microwave oven (ETHOS 1600, Milestone) especially designed for organic synthesis.
TAR is the 29-mer RNA sequence 5′-GGCAGAUCUGAGCCUGGGAGCUCUCUGCC-3′ and
cTAR is its DNA complementary sequence 5′-GGCAGAGAGCTCCCAGGCTCAGATCTGCC-3′.
22
SL3 is the 20-mer sequence 5′-GGACUAGCGGAGGCUAGUCC-3′. Duplex construct dsRNA
was obtained by annealing the 16-mer complementary sequences 5′-UAGGGGGAAGCUUGG-3′
and 5′-CCAAAGCUUCCCCCUA-3′. All RNA and DNA oligonucleotides were purchased from
Metabion International AG (Martinsried, Germany). The full-length recombinant NC protein was
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obtained in house as previously reported.
(
R)-(9H-fluoren-9-yl)methyl 1-chloro-1-oxopropan-2-ylcarbamate (2; Fmoc-D-Ala-Cl)
Commercially available Fmoc-D-Ala-OH (Aldrich, 2.5g, 8.03 mmol) was dissolved in anhydrous
dichloromethane (40 mL) in a two-neck flask. Excess SOCl (5 mL; 68.54mmol) was added
dropwise. The mixture was transferred into a sealed vessel and heated by microwave irradiation at
0°C (300W power) for 8 minutes (30s ramping step; 7 min and 30s holding step). Solvent was
2
5
evaporated under reduced pressure and the residue was purified by crystallization from n-hexane
affording 2.60 g of pure 2a as a white solid. Yield: 98%.
N,N'-(9,10-dioxo-9,10-dihydroanthracene-2,6-diyl)bis(2-aminoacetamide)-bistrifluoroacetate
(3)
Fmoc-D-Ala-Cl (2a, 6,39 g, 19,4 mmol) and pyridine (1.27 mL, 15.8 mmol) were slowly added to a
solution of 2,6-diaminoanthraquinone (1, 350 mg, 1.94 mmol) in DMF (80 mL). The reaction
mixture was stirred at room temperature for 24 hours. Solvent was removed by reduced pressure
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distillation. The residue was purified by crystallization from ethyl acetate, thus obtaining bis((9H-
fluoren-9-yl)methyl) ((2R,2'R)-((9,10-dioxo-9,10-dihydroanthracene-2,6-diyl)bis(azanediyl))bis(1-
oxopropane-2,1-diyl))dicarbamate (2,6-Fmoc-D-Ala-anthraquinone) as an orange powder (1.47 g,
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.78 mmol, yield 92%), which was used in the next step without any further purification. This
compound was reacted with a 33% diethylamine solution in THF (60 mL) for 2 hours. Solvent was
again distilled off under reduced pressure and the residue was finally reacted with a solution of
trifluoroacetic acid in water (9:1, v/v, 20 mL) for 1 hour. Reaction mixture was then added with
diethyl ether (60 mL). The precipitate was collected by centrifugation and dried to obtain 862 mg of
1
intermediate 3a as an intense orange solid. Yield 73% (overall). H-NMR (DMSO-d6): δ 11.28 (bs,
1
3
2H), 8.49 (d, 2H), 8.30 - 8.20 (m, 8H), 8.06 (dd, 2H), 2.69 (d, 6H), 2.50 (m, 2H). C NMR
DMSO- d6): δ 181.2, 165.6, 143.6, 134.2, 128.4, 128.2, 123.4, 115.5, 49.4, 16.6. ESI-MS:
80.15[M + H]+; 191,18[M + 2H]++.
(
3
N,N'-(2S,2'S)-1,1'-(9,10-dioxo-9,10-dihydroanthracene-2,6-diyl)bis(azanediyl)-bis(1-
oxopropane-2,1-diyl)dipiperidine-4-carboxamide-bis-trifluoroacetate (D-4a)
Intermediate 3a (250 mg, 0.42 mmol) was dissolved in dry DMF (18.6 mL), and DMAP (545.56
mg, 3.24 mmol), 1-Fmoc-piperidine-4-carboxylic acid (Fmoc-Inp-OH, 1.00 g, 3.20 mmol), and
HBTU (1.36 g, 3.6 mmol) were added. The resulting solution was stirred at room temperature for
2
2 2
4 hours, then poured into Et O and centrifuged. The solid obtained was washed with Et O and the
final product was dried in vacuo and used in the next step without any further purification. In order
to remove the Fmoc- protecting group, the solid obtained was reacted with a 33% diethylamine
solution in THF (30 mL) for 2 hours, poured into Et
washed with Et O and water and then dried in vacuo. Subsequently, the obtained solid was reacted
with a solution of trifluoroacetic acid in water (9:1, v/v, 20 mL) for 1 hour and then poured into
Et O. The resulting suspension was centrifuged. The solid obtained was washed with Et O and
dried in vacuo. Purification by preparative RP-HPLC afforded the pure compound D-4a as an
2
O and centrifuged. The solid obtained was
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intense orange solid. Yield: 76%. Orange solid. K’ (HPLC): 5,5. H-NMR (DMSO-d
6
, 400MHz): δ
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
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0.71 (s, 2H), 8.71 (t, 2H), 8.44 (bs, 4H), 8.33 (d, 2H), 8.14 (bs, 4H), 8.03 (dd, 2H), 4.37 (m, 2H),
1
3
6
3.24 (m, 4H), 2.84 (m, 4H), 1.83 (m, 4H), 1.68 (m, 4H), 1.31 (d, 6H). C-NMR (DMSO-d ,
0
1
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4
5
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00MHz): δ 181.79, 172.07, 168.73, 144.89, 134.82, 129.04, 128.68, 124.11, 116.44, 51.90, 50.02,
0.59, 38.72, 28.68, 23.02, 18.31. ESI-MS: 603.3 [M + H]+; 302.1 [M + 2H]++.
(1R,1'R,4S,4'S)-N,N'-((2S,2'S)-1,1'-(9,10-dioxo-9,10-dihydroanthracene-2,6-diyl)-bis-
azanediyl)bis(1oxopropane2,1diyl))bis-(4(aminomethyl)cyclohexane-carboxamide)-bis-
(
trifluoroacetate (D-4b)
We followed the synthetic procedures reported for D-4a, starting from intermediate 3a and 4-
(Fmoc-aminomethyl)cyclohexanecarboxylic acid (N-Fmoc-tranexamic acid). Yield: 65%. Orange
1
solid. K’ (HPLC): 6.4. H-NMR (DMSO-d
6
, 400MHz): δ 10.69 (s, 2H), 8.47 (t, 2H), 8.18 (d, 2H),
8.16 (d, 2H), 8.05 (dd, 2H), 7.77 (bs, 6H), 4.36 (m, 2H), 2.85 (m, 4H), 2.64 (m, 2H), 1.76 (m, 2H),
1
3
1
1
1
6
.67 (m, 8H), 1.31 (d, 6H), 0.88 (m, 8H). C-NMR (DMSO-d , 400MHz): δ 182.02, 172.43,
71.83, 144.69, 135.03, 129.18, 128.52, 124.11, 116.41, 51.82, 43.97, 38.02, 35.67 29.66, 29.13,
7.95. ESI-MS: 659.1 [M + H]+; 330.2 [M + 2H]++.
N,N'-(1,1'-(9,10-dioxo-9,10-dihydroanthracene-2,6-diyl)bis(azanediyl)bis(1-oxo-propane-2,1-
diyl))bis(4-aminopiperidine-4-carboxamide)-tetra-trifluoroacetate (D-4c)
We followed the synthetic procedures reported for D-4a, starting from intermediate 3a and 1-Fmoc-
4
-(Fmoc-amino)-piperidine-4-carboxylic acid (Fmoc-Pip(Fmoc)-OH). Yield: 63%. Orange solid. K’
1
(HPLC): 4.7. H-NMR (DMSO-d
6
, 400MHz): δ 10.69 (s, 2H), 8.46 (t, 2H), 8.31 (d, 2H), 8.18 (bs,
4H), 8.15 (d, 2H), 8.03 (dd, 2H), 7.90 (bs, 6H), 4.34 (m, 2H), 3.58 (m, 4H), 3.14 (m, 4H), 2.85 (m,
1
3
4
1
H), 2.71 (m, 4H), 1.31 (d, 6H). C-NMR (DMSO-d
6
, 400MHz): δ 182.04, 171.37, 169.08, 145.22,
35.00, 129.21, 128.59, 124.19, 116.45, 56.87, 51.23, 35.72, 28.96, 17.94. ESI-MS: 633.4 [M +
H]+; 317.2 [M + 2H]++.
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N,N'-(2S,2'S)-1,1'-(9,10-dioxo-9,10-dihydroanthracene-2,6diyl)bis(azanediyl)bis-(1-
oxopropane-2,1-diyl)bis(2,4-diaminobutanamide)-tetra-trifluoroacetate (D-4d)
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We followed the synthetic procedures reported for D-4a, starting from the intermediate 3c and
1
Fmoc-β-Alanine-OH (Fmoc-β-Ala-OH). Yield: 81%. Yellow solid. K’ (HPLC): 4.1. H-NMR
(
DMSO-d
(bs, 6H), 4.54 (m, 2H), 2.82 (m, 4H), 1.75 (m, 4H), 1.38 (d, 6H). C-NMR (DMSO-d
81.99, 171.16, 169.46, 144.96, 134.87, 129.25, 128.61, 124.07, 116.55, 51.33, 36.02, 33.03, 18.13.
ESI-MS: 523.5 [M + H]+; 261.2 [M + 2H]++.
6
, 400MHz): δ 10.85 (s, 2H), 8.91 (t, 2H), 8.48 (d, 2H), 8.18 (d, 2H), 8.07 (dd,, 2H), 7.78
1
3
6
, 400MHz): δ
1
N,N'-(2S,2'S)-1,1'-(9,10-dioxo-9,10-dihydroanthracene-2,6diyl)bis(azanediyl)-bis(1-
oxopropane-2,1-diyl)bis(2,3-diaminopropanamide)-tetra-trifluoroacetate (D-4e)
α
We followed the synthetic procedures reported for D-4a, starting from intermediate 3a and N -
β
Fmoc-N -Boc-L-diaminopropionic acid (Fmoc-Dap(Boc)-OH). Yield: 86%. Orange solid. K’
1
(
HPLC): 4.8. H-NMR (DMSO-d
6
, 400MHz): δ 10.92 (s, 2H), 9.00 (t, 2H), 8.47 (bs, 6H), 8.19 (bs,
6
H), 8.18 (d, 2H), 8.06 (d, 2H), 8.04 (dd, 2H), 4.55 (m, 2H), 4.21 (m, 2H), 3.15 (m, 4H), 1.43 (d,
1
3
6H). C-NMR (DMSO-d
6
, 400MHz): δ 181.81, 172.169, 158.77, 144.65, 134.81, 129.07, 128.85,
1
24.26, 116.61, 50.82, 50.36, 40.15, 18.30. ESI-MS: 553.5 [M + H]+; 277.2 [M + 2H]++.
N,N'-(2S,2'S)-1,1'-(9,10-dioxo-9,10-dihydroanthracene-2,6-diyl)bis(azanediyl)-bis(1-
oxopropane-2,1-diyl)bis(2,4-diaminobutanamide)-tetra-trifluoroacetate (D-4f)
α
We followed the synthetic procedures reported for D-4a, starting from intermediate 3a and N -
γ
Fmoc-N -Boc-L-diaminobutyric acid (Fmoc-Dab(Boc)-OH). Yield: 79%. Orange solid. K’ (HPLC):
1
4
.8. H-NMR (DMSO-d
6
, 400MHz): δ 10.81 (s, 2H), 8.87 (t, 2H), 8.45(bs, 6H), 8.17 (d, 2H), 8.16
(d, 2H), 8.04 (dd, 2H), 7.78 (bs, 6H), 4.53 (m, 2H), 3.82 (m, 2H), 2.84 (m, 4H), 1.75 (m, 4H), 1.41
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(
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d, 6H). C-NMR (DMSO-d , 400MHz): δ 181.88, 172.10, 168.69 , 145.33, 134.87, 129.11,
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28.73, 124.24, 116.55, 50.86, 50.31, 35.41, 29.48, 18.13. ESI-MS: 581.4 [M + H]+; 291.3 [M +
H]++.
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N,N'-(1,1'-(9,10-dioxo-9,10-dihydroanthracene-2,6-diyl)bis(azanediyl)bis(1-oxo-propane-2,1-
diyl))bis(2,5-diaminopentanamide)-tetra-trifluoroacetate (D-4g)
α
We followed the synthetic procedures reported for D-4a, starting from intermediate 3a and N -
δ
1
Fmoc-N -Boc-L-ornithine (Fmoc-Orn(Boc)-OH). Yield: 81%. Orange solid. K’ (HPLC): 4.8. H-
NMR (DMSO-d , 400MHz): δ 10.88 (s, 2H), 8.86 (t, 2H), 8.46 (bs, 6H), 8.18 (d, 2H), 8.16 (d, 2H),
.06 (dd, 2H), 7.85 (bs, 6H), 4.51 (m, 2H), 3.85 (m, 2H), 3.16 (m, 4H), 2.83 (m, 4H), 1.77 (m, 4H),
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1.41 (d, 6H). C-NMR (DMSO-d
28.69, 124.24, 116.49, 52.13, 50.24, 38.84, 28.44, 22.79, 18.18. ESI-MS: 609.4 [M + H]+; 305.4
M + 2H]++.
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, 400MHz): δ 181.99, 169.77, 168.13, 144.74, 134.24, 129.09,
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[
N,N'-(2S,2'S)-1,1'-(9,10-dioxo-9,10-dihydro-anthracene-2,6diyl)bis(azanediyl)-bis(1-
oxopropane-2,1-diyl)bis-(2,6-diaminohexanamide)-tetra-trifluoroacetate (D-4h)
α
We followed the synthetic procedures reported for D-4a, starting from intermediate 3a and N -
ε
1
Fmoc-N -Boc-L-Lysine (Fmoc-Lys(Boc)-OH. Yield: 83%. Orange solid. K’ (HPLC): 4.9. H-NMR
(DMSO-d
6
, 400MHz): δ 10.86 (s, 2H), 8.86 (t, 2H), 8.44 (bs, 6H), 8.18 (d, 2H), 8.16 (d, 2H), 8.06
(dd, 2H), 7.78 (bs, 6H), 4.50 (q, 2H), 3.85 (m, 2H), 2.75 (m, 4H), 1.72 (m, 4H), 1.54 (m, 4H), 1.40
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(d, 6H), 1.33 (m, 4H). C-NMR (DMSO-d
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, 400MHz): δ 181.87, 172.19, 168.90, 158.75, 144.93,
34.84, 129.09, 128.67, 124.08, 116.44, 52.22, 50.04, 38.97, 30.88, 26.93, 21.48, 18.12. ESI-MS:
36.9 [M + H]+; 319.1 [M + 2H]++.
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N,N'-(2S,2'S)-1,1'-(9,10-dioxo-9,10-dihydroanthracene-2,6diyl)bis(azanediyl)bis-(1-
oxopropane-2,1-diyl)bis(2-amino-3-(4-aminophenyl)propanamide)-tetra-trifluoroacetate(D-4i)
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We followed the synthetic procedures reported for D-4a, starting from intermediate 3a and Fmoc-4-
(Boc-amino)-L-phenylalanine (Fmoc-4-(NH-Boc)Phe-OH). Yield: 69%. Orange solid. K’ (HPLC):
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5.1. H-NMR (DMSO-d
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, 400MHz): δ 10.74 (s, 2H), 8.74 (t, 2H), 8.44 (bs, 6H), 8.18 (d, 2H), 8.15
0
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(d, 2H), 8.02 (dd, 2H), 7.01 (d, 4H), 6.94 (d, 4H), 6.72 (bs, 6H), 4.44 (m, 2H), 4.00 (m, 2H), 2.88
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(
m, 4H), 1.22 (d, 6H). C-NMR (DMSO-d
34.95, 130.94, 129.03, 128.84, 128.58, 123.93, 118.84, 116.52, 53.93, 50.31, 36.87, 18.03. ESI-
MS: 705.2 [M + H]+; 353.1 [M + 2H]++.
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, 400MHz): δ 182.05, 169.72, 168.36, 158.32, 144.45,
1
Inhibition of NC-mediated destabilization of TAR and cTAR stem. The identification of
inhibitors able to impair the NC chaperone activity on TAR and cTAR was performed by means of
1
3-14, 16
the high throughput screening (HTS) previously described.
Inhibition of NC-mediated TAR/cTAR annealing. We investigated the ability of the newly
synthesized 2,6-AQ-D-Ala-X anthraquinones to impair the annealing activity of full-length NC
protein activity monitoring the annealing of TAR with cTAR through the nucleocapsid annealing
1
2, 18
mediated electrophoresis (NAME) assay described earlier,
which was carried out according to
alternative protocols involving pre-incubation of ligand with either the NC protein or the nucleic
12
acid substrates. Selected compounds were further analyzed using different sets of concentrations
in the oligo-preincubation mode to accurately determine their potency in inhibiting NC-mediated
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annealing activity.
Direct binding of ligands to individual TAR and cTAR. We evaluated the binding properties of
selected 2,6-AQ-D-Ala-X anthraquinones to either TAR or cTAR employing electrospray
ionization mass spectrometry (ESI-MS) analysis under non-denaturing conditions as previously
12-13, 19-21
reported for the L-Ala series.
The determination of free and bound RNA or DNA,
necessary to evaluate the binding affinity of compounds to TAR and cTAR, was assessed from the
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relative abundances, expressed as percentage and compared. The binding properties of selected
compounds were evaluated also towards the stem-loop SL3 and the double stranded RNA. SL3
construct was heated to 95 °C for 5 min and then ice-cooled in order to assume the proper hairpin
structure. The dsRNA was also heated and then slowly cool to room temperature in order to form
the RNA duplex. ESI-MS experiments were performed under the same experimental conditions
used in the presence of TAR.
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Direct binding of ligands to NC. The direct binding of selected 2,6-AQ-D-Ala-X anthraquinones
to the full-length NC protein was assessed by ESI-MS in positive ion mode via direct infusion
nanospray ionization on a Synapt G2 HDMS travelling-wave ion mobility spectrometry (IMS) mass
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spectrometer (Waters, Manchester, UK). Typical final mixtures contained up to 10:1 2,6-
dipeptidyl-anthraquinone:NC molar ratio in 150 mM ammonium acetate (pH 7.5). Mass Lynx
(v4.1, SCN781) software was used to process data. To evaluate the binding affinity of compounds
to the full-length NC, free and complexed protein abundances in each experiment were calculated
as above reported for TAR and cTAR, and were finally expressed as percentage and compared.
Inhibition of NC binding to TAR. Possible effects induced by 2,6-dipeptidyl anthraquinones on
the specific binding of NC protein to TAR substrate were evaluated by analyzing samples in which
preformed i. NC-TAR, ii. ligand-NC, or iii. ligand-TAR complexes where challenged by addition of
the remaining component as exemplified in Scheme 3. Keeping constant the concentration of each
component in the three different procedures, only the order incubation changed to evaluate all the
possible scenarios. Samples were prepared by incubating equimolar amounts of NC and TAR (i.e.,
6 µM concentration of each), and a 1:10 molar ratio of each ligand (i.e., final 60µM concentration)
in 150 mM ammonium acetate (pH 7.5). ESI-MS performed under non-denaturing conditions was
applied to unambiguously identify all species present at equilibrium in solution. In order to
characterize all the reaction products, all samples were analyzed both in positive and in negative ion
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