Thermodynamic studies of a series of homologous HIV-1 TAR RNA ligands reveal that loose binders are stronger Tat competitors than tight ones

Article abstract of DOI:10.1093/nar/gkt237

RNA is a major drug target, but the design of small molecules that modulate RNA function remains a great challenge. In this context, a series of structurally homologous 'polyamide amino acids' (PAA) was studied as HIV-1 trans-activating response (TAR) RNA ligands. An extensive thermodynamic study revealed the occurence of an enthalpy-entropy compensation phenomenon resulting in very close TAR affinities for all PAA. However, their binding modes and their ability to compete with the Tat fragment strongly differ according to their structure. Surprisingly, PAA that form loose complexes with TAR were shown to be stronger Tat competitors than those forming tight ones, and thermal denaturation studies demonstrated that loose complexes are more stable than tight ones. This could be correlated to the fact that loose and tight ligands induce distinct RNA conformational changes as revealed by circular dichroism experiments, although nuclear magnetic resonance (NMR) experiments showed that the TAR binding site is the same in all cases. Finally, some loose PAA also display promising inhibitory activities on HIV-infected cells. Altogether, these results lead to a better understanding of RNA interaction modes that could be very useful for devising new ligands of relevant RNA targets.

Full text of DOI:10.1093/nar/gkt237

Published online 19 April 2013
Nucleic Acids Research, 2013, Vol. 41, No. 11 5851–5863
doi:10.1093/nar/gkt237
Thermodynamic studies of a series of homologous
HIV-1 TAR RNA ligands reveal that loose binders
are stronger Tat competitors than tight ones
1
1
1
1
´
Lise Pascale , Stephane Azoulay , Audrey Di Giorgio , Laura Zenacker ,
Marc Gaysinski1, Pascal Clayette2 and Nadia Patino1,*
1
´
Institut de Chimie de Nice UMR7272, Universite de Nice Sophia Antipolis, 06108 Nice Cedex, France and
2Neurovirology Department, Bertin Pharma, CEA, 92265 Fontenay-aux -Roses Cedex, France
Received December 21, 2012; Revised March 6, 2013; Accepted March 13, 2013
are often involved in crucial biological processes such as,
for example, in transcription and translation steps of viral
and bacterial replication cycles, through specific inter-
actions with proteins or nucleic acids. Synthetic molecules
able to bind specifically to such relevant RNA targets and
to disrupt their association with cognate partners can
modulate their biological functions. Therefore, such
RNA ligands represent very attractive tools in molecular
biology and/or pharmacological agents for treating infec-
tious diseases.
ABSTRACT
RNA is a major drug target, but the design of small
molecules that modulate RNA function remains a
great challenge. In this context, a series of structur-
ally homologous ‘polyamide amino acids’ (PAA) was
studied as HIV-1 trans-activating response (TAR)
RNA ligands. An extensive thermodynamic study
revealed the occurence of an enthalpy–entropy
compensation phenomenon resulting in very close
TAR affinities for all PAA. However, their binding
modes and their ability to compete with the Tat
fragment strongly differ according to their structure.
Surprisingly, PAA that form loose complexes with
TAR were shown to be stronger Tat competitors
than those forming tight ones, and thermal denatur-
ation studies demonstrated that loose complexes
are more stable than tight ones. This could be
correlated to the fact that loose and tight ligands
induce distinct RNA conformational changes as
revealed by circular dichroism experiments,
although nuclear magnetic resonance (NMR) experi-
ments showed that the TAR binding site is the
same in all cases. Finally, some loose PAA also
display promising inhibitory activities on HIV-
infected cells. Altogether, these results lead to a
better understanding of RNA interaction modes
that could be very useful for devising new ligands
of relevant RNA targets.
Small molecular structures targeting viral/bacterial
hairpin RNA fragments have already been identified
using standard and virtual screening approaches (1–4).
However, thus far, these molecules have met little
clinical success as compared with those targeting
proteins and, to a less extent, DNA. A deeper understand-
ing of RNA recognition processes by both cognate
partners and synthetic ligands is essential for developing
new structurally optimized molecules capable of both
binding specifically to RNA targets and competing
strongly with the cognate partners. While RNA recogni-
tion processes have been extensively studied at the struc-
tural level, only few works have been devoted to
thermodynamic studies of RNA/small ligand associations
(5–11), in contrast to protein/ligand or DNA/ligand ones.
Nevertheless, identification of the main molecular forces
that govern RNA/ligand associations would be of great
interest for a complete understanding of RNA binding
modes and for drug design (12,13). Among the great
number of potential hairpin RNA targets, extensive
research has been dedicated to the highly conserved
HIV-1 trans-activating response (TAR) RNA fragment
in view of preventing its complexation with the trans-
activator of transcription (Tat) protein and cellular
factors (1,2,14,15). This complex plays a crucial role in
the viral transcription step of HIV. We have previously
reported a new type of molecules named a- polyamide
INTRODUCTION
In the past decade, numerous studies have emphasized the
huge potential of small non-coding hairpin RNA frag-
ments as drug targets. Indeed, such small RNA elements
*To whom correspondence should be addressed. Tel: +33 4 92 07 61 46; Fax: +33 4 92 07 61 51; Email: patino@unice.fr
ß The Author(s) 2013. Published by Oxford University Press.
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5852 Nucleic Acids Research, 2013, Vol. 41, No. 11
Figure 1. Structures of the three series of PAA.
amino acids (PAA) that represents promising structures as
HIV TAR ligands (16). These molecules are constituted by
a poly-(2-aminoethylglycyl) backbone onto which are
condensed L-a-amino acids. From a small library of a-
PAA trimers, we identified several compounds able to
bind to TAR RNA and to prevent the Tat/TAR associ-
ation at a micromolar level (17). Some of them displayed
an anti-HIV activity in peripheral blood mononuclear
cells (PBMC). However, as these compounds have a
short half-life in cell culture media, structural modifica-
tions have been considered to improve stability. Thus,
starting from seven tri-a-PAA previously identified as
‘lead’ compounds (Figure 1, 1a-7a), two new series of
tri-PAA analogs modified either on the polyamide
backbone or on the amino acid moiety have been
elaborated. In the first series, the a-amino acid residues
are changed for their corresponding b-analogs (b-PAA
series, 1b–7b), whereas for the second series, the (2-
aminoethylglycyl) unit of the backbone is replaced by a
2-aminoethyl b-alanyl one (C-a-PAA series, 1c–7c).
Besides the synthesis of the newly designed PAA
analogs, we report here a detailed study on the TAR
binding of the three series of PAA, in the absence or
presence of a Tat fragment competitor. For the three
PAA series, beyond affinity and specificity criteria, each
PAA/TAR equilibrium has been characterized by an
extensive thermodynamic study, providing new insights
on the binding modes that led to ‘structure/activity’ rela-
tionships. To support our hypotheses concerning the
distinctive binding modes of PAA, the structural TAR
RNA changes induced on binding of PAA have been
investigated by circular dichroism (CD) studies, and the
TAR interaction sites of two PAA models have also been
identified through NMR experiments. Important enough,
we also report here our results about anti-HIV activity
and toxicity on infected and non-infected PBMC.
MATERIALS AND METHODS
Experimental procedures
Unless otherwise stated, all reagents and solvents were of
analytical grade and from Sigma (St Louis, MO, USA).
HEPES
[4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid] and all inorganic salts for buffers were purchased
from Calbiochem (molecular biology grade). RNA and
DNA oligonucleotides were purchased from IBA GmbH
and used without further purification. A mixture of yeast
pre- and mature tRNAs (containing >30 different species)
was purchased from Sigma (type X-SA). The labeled Tat
peptide was purchased from EZBiolab (Carmel, CA,
USA) and used without further purification. All buffers
were filtered through 0.22-mm Millipore filters (GP
ExpressPLUS membrane). A small aliquot (50–100 ml)
was first filtered and then discarded to avoid any contam-
inants that might have been leached from the filter.
Solutions used for fluorescence experiments were
prepared by diluting the concentrated stocks in Milli-Q
water and filtered again as described above.
Fluorescence binding assays
Ligand solutions were prepared as serial dilutions by an
epMotion automated pipetting system (eppendorf) in
buffer A [20 mM HEPES (pH 7.4 at 25ꢀC), 20 mM NaCl,
140 mM KCl and 3 mM MgCl2] at a concentration twice
higher than the desired final concentration that will be
reached after addition of the RNA solution. The appro-
priate ligand solution (30 ml) was then added to a well of
a non-treated black 384-well plate (Nunc 237105), in trip-
licate. Refolding of the RNA was performed using a
thermocycler (ThermoStatPlus Eppendorf) as follows:
the RNA, diluted in 1 ml of buffer A, was first denatured
by heating to 90ꢀC for 2 min and then cooled to 4ꢀC
for 10 min, followed by incubation at 20ꢀC for 15 min.

Nucleic Acids Research, 2013, Vol. 41, No. 11 5853
After refolding, the RNA was diluted to a working con-
centration of 10 nM through addition of the appropriate
amount of buffer A. The tube was mixed, and 30 ml of
the RNA solution was added to each well containing
ligand. This subsequent dilution lowered the final RNA
concentration to 5 nM. The fluorescence was measured
on a GeniosPro (Tecan) with an excitation filter of
485 10 nm and an emission filter of 535 15 nm. Each
point was measured five times with a 500-ms integration
time and averaged. Binding was allowed to proceed for at
least 30 min at room temperature to achieve equilibrium.
To study the temperature dependence, the plates were
incubated after 30-min equilibrium at different tempera-
tures ranging from 5ꢀC to 35ꢀC. The salt dependence was
studied in 20 mM HEPES (pH 7.4 at 25ꢀC), 20 mM NaCl
and 3 mM MgCl2, with the KCl concentration varying from
70 to 250 nM. For competitive experiments in the presence
of a dsDNA, a 15-mer sequence (50-CGTTTTTATTTTTG
C-3a) and its complement, annealed beforehand, were
added to buffer A to obtain a 100-fold nucleotide excess
over TAR RNA (900 nM duplex; 5 nM RNA). For com-
petitive experiments in the presence of tRNA, a mixture of
pre- and mature yeast tRNAs (containing >30 different
species from baker’s yeast (Saccharomyces cerevisiae,
Sigma, type X-SA) was added to buffer A to obtain a
100-fold nucleotide excess over TAR RNA. Stock solutions
of tRNA were prepared in water and quantified using an
extinction coefficient of 9640 cmꢁ1 Mꢁ1 per base (18).
the melting transition as determined by the maximum of
the first-derivative plot with Prism software.
CD study
CD measurements were performed with a Jasco J-810
spectropolarimeter equipped with a Jasco PTC 423S
Peltier temperature controller. Samples were prepared in
buffer C [50 mM tris buffer (pH 7.4 at 25ꢀC), 20 mM KCl
and 0.005% Tween 20]. Spectra were obtained at
3 mM RNA or PAA (for individual spectra) or a molar
ratio of 1:1, 1:5 and 1:10 RNA:PAA for the complexes
in a 1-mm path length cell after 1 h incubation time in
the case of RNA/PAA complexes. RNA samples were
heat denatured and allowed to refold as described above
before measurement. Spectra were recorded at 20ꢀC from
360 to 200 nm at 1-nm intervals, with an integration time
of 4 s and a 50-nm/min speed. CD scans were repeated five
times and then averaged and corrected by the subtraction
of buffer background.
Data analysis
Binding data (KD and FRET experiments) were
analyzed using Prism 5 (GraphPad Software) by non-
linear regression following the equation
Y¼ Bottom+ðTop ꢁ BottomÞ=ð1+10½ðLogIC50ꢁXÞꢂHillSlopeꢃÞ
KD values were converted to ÁGꢀ values as
ÁGꢀ = ꢁRTln KD.
Salt dependence of KD was analyzed by the following
equation:
Fluorescence resonance energy transfer (FRET)
displacement assays
LogðKDÞ ¼ logðKnelÞ ꢁ Zclogð½KClꢃÞ
ð1Þ
Ligand solutions and RNA (40 nM working solution)
were prepared as described above in buffer B [50 mM
tris buffer (pH 7.4 at 25ꢀC), 20 mM KCl and 0.005%
Tween 20]. Labeled Tat peptide was prepared at 40 nM
in buffer B and mixed to an equal volume of TAR RNA
for 20 min at room temperature to form the Tat/TAR
complex before adding the ligand. The appropriate
ligand solution (30 ml) was then added to a well of a
non-treated black 384-well plate, in triplicate, and 30 ml
of the Tat/TAR solution was added. This subsequent
dilution lowered the final Tat/TAR concentration to
10 nM. Fluorescence was measured as described above
after 30 min of incubation at room temperature.
where Knel is the dissociation constant at the standard
state in 1 M KCl, Z is the number of ions displaced from
the nucleic acid and c is the fractional probability of a
counterion being thermodynamically associated with each
phosphate of the RNA. Knel and Zc were treated as
fitting parameters.
For thermodynamic analysis, ÁGꢀ values were plotted
versus temperature (T). Non-linear regression using the
three-parameter fit in Prism 5 was used to fit the following
equation to the data:
ÁGTꢀ ¼ ÁHTꢀ r+ÁCpðT ꢁ TrÞ ꢁ TÁSTꢀ r ꢁ TÁCplnðT=TrÞ ð2Þ
where Tr is a constant reference temperature (in our study
Tr = 293.15 K), and the three fit parameters are as
follows: ÁHꢀTr, the change in enthalpy on binding at Tr;
Temperature-dependent UV spectroscopy (UV melting)
Thermal denaturation scans were obtained using a Cary
300 (Varian) spectrophotometer equipped with an electro-
thermal multicell holder. A quartz cell of 700 ml with 1-cm
path length was used for all the absorbance studies.
Absorbance versus temperature profiles were recorded at
260 nm. After structuration of TAR RNA and incubation
(1 h) with the corresponding ligand, the temperature
was raised from 25 to 90ꢀC, with a heating rate of
0.5ꢀC/min. Thermal denaturation studies were carried
out at 1 mM TAR RNA and PAA concentrations of
0 (TAR alone), 1 and 5 mM. The experiments were per-
ÁSꢀ the change in entropy on binding at Tr; and ÁCp,
Tr,
the change in heat capacity. ÁCp was assumed to be inde-
pendent of temperature; inclusion of a ÁCp/ÁT term in
the analysis did not improve the quality of the fits and
gave larger standard errors for the returned parameters.
ÁHꢀ and ÁSꢀ were calculated from the results
T
T
obtained from the fitting of the curve ÁGꢀ values versus
T by the Equation (2) using the following equation:
ÁHꢀT ¼ ÁHTꢀ r+ÁCpðT ꢁ TrÞ
and
ð2:1Þ
ð2:2Þ
formed in buffer
D
[10 mM sodium cacodylate,
10 mM NaCl (pH 7.5) and 0.1 mM EDTA]. The melting
temperature (Tm) value was taken as the midpoint of
ÁSꢀT ¼ ÁSꢀTr+ÁCplnðT=TrÞ

5854 Nucleic Acids Research, 2013, Vol. 41, No. 11
ꢀ
where ÁHꢀT is the change in enthalpy on binding at
acetylation of the last residue and (iii) cleavage of the pro-
tecting groups and simultaneous release of the trimeric
compounds from the resin. PAA derivatives 1b–c/7b–c
were obtained in high HPLC purity (from 83 to 98%)
and were purified by semi-preparative HPLC. Structures
ꢀꢀ
T (25ꢀC), and ÁST is the change in entropy on binding
at T (25ꢀC).
For enthalpy–entropy compensation (EEC) analysis,
TÁSꢀ values were plotted versus ÁHꢀ. The linear regres-
sion in Prism 5 was used to fit the following equation to
the data:
were
Supplementary Table S1 in supporting information).
The protected b-PAA monomer synthons 13b–15b were
prepared using two-step procedure (Figure 2B),
confirmed
by
HRMS
experiments
(see
ÁHꢀT ¼ aTÁSꢀT+b
ð3Þ
a
Chemistry, characterization (HPLC, HRMS ESI+) of
new compounds, NMR and biological assay experiments
as well as melting temperature data are given in support-
ing information.
involving the condensation of the protected b-amino
acid residues onto the N-Boc methyl or allyl ester
backbone 8 or 8’, and then carboxyl deprotection, i.e. sa-
ponification of methyl esters 10b–11b or reaction with
catalytic Pd0(PPh3)4 in the case of allyl ester 12b.
Similarly, C-a-PAA monomers 13c–15c were synthesized
starting from protected a-amino acid residues and from
N-Boc methyl or allyl ester backbone 9 or 9’ (Figure 2B).
Backbones 9–9’ were obtained in one step, following a
Michael addition of the Boc-ethylenediamine on methyl
or allyl acrylate, respectively.
RESULTS AND DISCUSSION
Chemistry
Solid-phase synthesis of the seven a-PAA hits (Figure 1,
1a–7a) was previously described (17). b-PAA (1b–7b)
and C-a-PAA (1c–7c) analogs were prepared following
the same procedure, i.e. on a b-alanine-functionalized
4-methylbenzhydrylamine (MBHA) resin, starting from
fully and orthogonally N-protected b-PAA or C-a-PAA
monomers, respectively. This procedure, illustrated in
Figure 2A, includes (i) three successive elongation/
deprotection steps involving selected monomers, (ii)
Interaction studies: affinity and specificity
Dissociation constants (KD; Table 1) associated with
TAR/PAA 1a–c/7a–c equilibria were determined by moni-
toring the fluorescence change of a TAR18–44 fragment
labeled with a fluorescent group (Alexa 488), as previously
Figure 2. Synthesis of (A) b-PAA and (B) C-a-PAA protected monomers. (i) HBTU, DIEA, DMF; (ii) for 10b–c and 11b–c: LiOH 1 N, H2O, THF;
for 12b–c: Pd(PPh3)4/DEA/DMF; and (iii) CH3CN, Á, 9 h.

Nucleic Acids Research, 2013, Vol. 41, No. 11 5855
described (16). Analysis of the TAR/a-PAA previously
revealed a one-to-one stoichiometry of the binding (17),
and representative curves of the new compounds could
perfectly be fitted with a one-to-one model using the
non-linear least-squares numerical solver–based binding
data global analysis program BIOEQS (19) (data not
shown). Whatever the series and the sequence, all PAA
derivatives strongly bind to TAR with similar
submicromolar affinities (0.2 mM < KD < 0.9 mM). In
most cases, C-a-PAA (1c–7c) and b-PAA (1b–7b) com-
pounds display slightly higher affinities than their corres-
ponding a-PAA (1a–7a) (2.5-fold maximum), and within
each series, the FKR sequence shows the weakest TAR
affinity.
To assess the ligand specificity, KD values of C-a-PAA
and b-PAA were measured in the presence of a large
excess of tRNA (K0D) and dsDNA (K00D). We have pre-
viously shown that tetra- and tri-a-PAA display a high
TAR specificity in the presence of tRNA competitors,
whereas only tetramers retain this high specificity in the
presence of dsDNA (16,17). As shown in Table 1, most
C-a-PAA and b-PAA retain a good specificity for TAR in
the presence of both tRNA and dsDNA. Nevertheless,
PAA containing F-rich sequences are less specific
than PAA containing more cationic K/R-rich sequences
(see PAA 1–4 versus PAA 5–7 in Table 1). This suggests
that cationic residues are preferentially involved in specific
interactions, presumably through hydrogen bonding
rather than ionic interactions.
FRET displacement assays
The ability of PAA to displace a Tat fragment from a
preformed TAR/Tat complex was assessed via a FRET
assay, developed from a previously described procedure
(20), using a fluoresceinated Tat peptide fragment
(amino acids 48–57) and
a
Dabcyl-labeled TAR
fragment (18–44). In the absence of PAA, association of
these two partners results in an efficient quenching of the
dyes. On addition of increasing amounts of PAA, the
fluorescence rises in all cases, demonstrating that PAA
can displace the Tat fragment from a preformed Tat/
TAR complex. IC50 values associated with each PAA
are given in Table 1. Based on these disparate values,
ranging from 0.7 to 600 mM, one can infer that the
ability to displace Tat strongly depends on both the
nature and sequence of PAA, in contrast to KD values,
which are similar for all PAA. All b-PAA emerge as
stronger Tat/TAR inhibitors than their corresponding
C-a- and a-PAA (from 3- to 12-fold and 3.5- to 69-fold,
respectively), although KD values differ, at best, by a
factor 3. For each series, derivatives containing two
phenylalanine residues [F-rich PAA: (1–4)a–c] display a
weaker activity than those containing two basic ones
[R/K-rich PAA: (5–7) a–c]. The best F-rich PAA within
each series (a-FFR 2a, b-FRF 4b and C-a-RFF 3c) is,
respectively, 25-, 27- and 13-fold less active than the best
R/K-rich PAA within the same series (a-FRR 6a, b-RRF
7b and C-a-FRR 6c), although they all display a similar
TAR affinity. These results point out that the determin-
ation of a KD value is not enough to predict the ability

5856 Nucleic Acids Research, 2013, Vol. 41, No. 11
of a TAR ligand to compete with the Tat protein. One
hypothesis would be that even if two PAA display the
same affinity for TAR, they could interact at different
sites of the hairpin RNA, in such a way that the competi-
tive binding of the Tat protein could be more or less
hampered. However, because all PAA (except FFK se-
quences) contain at least one arginine residue, it is likely
that they interact at the bulge region of TAR, as previ-
ously reported for several guanidinium-containing com-
pounds (21,22). NMR studies were therefore conducted
to identify the TAR binding site and to compare the
mode of interaction for two PAA taken as models of
weak F-rich (b-FRF 4b) and strong R/K-rich (b-FRR
6b) inhibitors.
addition of high concentrations of b-FRR (>2 eq.), which
disappears with increasing temperature and reappears
when the temperature decreases (data not shown).
Although it is impossible to identify clearly this peak
through 1D imino spectra, it could be hypothesized that
it corresponds to the imino proton of the U23 residue
involved in a base triple with A27/U38, as previously
proposed for a few TAR complexes (23–25). In contrast,
no extra signal appears around 14 ppm in the case of the
b-FRF binding. Therefore, even if the mode of interaction
is not strictly the same because distinct local environments
can be observed, these results indicate that the TAR
binding site of F-rich and K/R-rich PAA is centered
around the bulge.
NMR studies
Thermodynamic studies
Two distinct NMR experiments were implemented. A 2D-
TOCSY experiment was monitored to identify the TAR
pyrimidine residues that experience noticeable chemical
shift changes on addition of PAA 4b and 6b (C and U
residues of TAR yield distinctive H5–H6 cross-peaks in
2D-TOCSY spectrum). In parallel, the monitoring of the
imino resonance region of TAR on titration of b-PAA led
to the identification of the G and U residues involved in
the interaction. Figure 3 shows the overlaid TOCSY
spectra of the pyrimidine region of TAR in the absence
(in black) and presence (in red) of five equivalents of b-
FRR (Figure 3A1) or b-FRF (Figure 3A2), respectively.
One-dimensional imino spectra in Figure 3B show the se-
lective changes in chemical shifts as b-FRR (Figure 3B1)
or b-FRF (Figure 3B2) is added to free TAR (from 0.5 to
10 equivalents). Figure 3C underscores the residues ex-
hibiting either a significant pyrimidine H5–H6 proton
chemical shift change (in blue; see Supplementary Table
S2 in supporting information) or a significant imino res-
onance change (in green) on addition of b-PAA.
Altogether, these two NMR experiments indicate that
the binding site of both compounds is centered around
the bulge. In addition, for both b-PAA, the interaction
seems to be very specific because other residues located
either in the lower stem or the loop remained unaffected
even in the presence of a 10-fold excess of b-PAA (data
not shown for 2D-TOCSY). The main chemical shift
changes observed for the residues of the bulge region are
likely due to a folding transition in TAR as previously
reported for other ligands carrying a guanidinium group
(23–27). In free TAR RNA, a partial stacking of the three
bulge nucleotides induces a bending in the overall helix
axis. On complexation, the two helical stems coaxially
stack to form one continuous helix; the two bulge nucleo-
tides C24 and U25 become unstacked and adopt a highly
flexible looped-out conformation. Even if our global
NMR data display very similar results for both com-
pounds, a more detailed comparison between 1D imino
spectra of the two complexes reveals some differences. In
the case of the b-FRR binding, the cluster corresponding
to U42, G36 and/or G44 residues is shifted, whereas it is
not clearly disrupted in the case of the b-FRF binding
(Figure 3B). Moreover, one can notice the appearance of
an extra signal at 13.9 ppm (asterisked in Figure 3B1) on
To gain further insights on the TAR binding modes of
the three PAA series, thermodynamic binding profiles
associated with each PAA/TAR equilibrium were deter-
mined. Free energies of Gibbs (ÁGꢀ) were first calculated
from the dissociation constants (ÁGꢀ = ꢁRTlnKD) and
were found very similar whatever the PAA (from ꢁ35 to
ꢁ38 kJ/mole). The Gibbs energy can be split into two com-
ponents: the pure electrostatic (polyelectrolyte) contribu-
tion ÁGꢀel and the non-electrostatic (non-polyelectrolyte)
contribution ÁGꢀnel. While ÁGꢀel is correlated to ionic
interactions occurring between two groups of opposite
charge and is mainly an entropic effect, ÁGꢀnel reflects
non-ionic events such as the hydrophobic effect that is
principally an entropic event, and non-electrostatic inter-
actions (H bonds, Van der Waals, p-stacking, etc.).
Electrostatic and non-electrostatic contributions to
the Gibbs energy
To determine electrostatic (ÁGꢀel) and non-electrostatic
(ÁGꢀnel) contributions associated with each PAA/TAR
equilibrium, the KD dependency on the ionic strength of
the solution was studied, over a range of KCl concentra-
tion from 70 to 250 mM. In all cases, a linear relationship
following Equation (1) was obtained between log(KD) and
log[KCl], the affinities decreasing with increasing salt con-
centration, as generally observed for charged molecules.
This ionic strength dependence may be explained by the
displacement of inorganic salt ions from both the PAA
and the RNA, to form an intermolecular ‘ion pair’.
According to the polyelectrolyte theory developed to
describe DNA–polylysine interactions (28), it can be
assumed that the energetic effect of ion displacement
from polylysine (or in the present case a PAA) is negli-
gible, and that ion displacement from the nucleic acid
energetically predominates. From Equation (1), the slope
of log(KD) versus log[KCl] is equal to ZÉ, where Z is the
number of ions displaced from the nucleic acid (essentially
the number of intermolecular ion pairs) and É is the frac-
tional probability of a counterion being thermodynamic-
ally associated with each phosphate of the RNA. For all
TAR/PAA interactions, the slopes vary from 0.5 to 1.5.
Using estimates for É ranging from 0.68 to 0.89 for single-
or double-stranded nucleic acids, respectively (29),
Z ranges approximately from 1 to 2. This indicates that

Nucleic Acids Research, 2013, Vol. 41, No. 11 5857
Figure 3. A/, top: 2D-TOCSY spectra showing pyrimidine H5–H6 cross-peaks for TAR. Black: free TAR (100 mM); red: PAA/TAR complex with
b-FRR 6b (A1/) or b-FRF 4b (A2/) at a ratio 5:1. Arrows indicate chemical shift changes on PAA binding. The spectra were acquired at 35ꢀC in a
D2O buffer (50 mM NaCl, 20 mM phosphate, pH 7.4). B/, bottom: stacked plot of 1D NMR spectra of the imino region of 50 mM TAR RNA with
increasing concentration of b-FRR (B1/) or b-FRF (B2/). The spectra were collected at 286K in a H2O/D2O (90/10) buffer (20 mM phosphate and
50 mM NaCl, pH 7.4). C/, bottom: secondary structure of the 27-mer TAR RNA fragment. Residues shown in blue are those exhibiting a pyrimidine
H5–H6 proton chemical shift change (>0.1 ppm, see Table S2 in supplementary data) on addition of b-PAA 4b or 6b. Residues in green are those
experiencing an imino resonance change (>0.1 ppm).

5858 Nucleic Acids Research, 2013, Vol. 41, No. 11
Table 2. Thermodynamic parameters for PAA/TAR complexesa
PAA sequence
FFK 1
FFR 2
RFF 3
FRF 4
FKR 5
FRR 6
RRF 7
ÁGꢀ
ꢁ36.0 1.9
ꢁ54.2 1.4
ꢁ18.2 1.4
ꢁ36.1 1.4
ꢁ51.7 0.5
ꢁ15.6 0.6
ꢁ36.1 1.4
ꢁ35.0 2.5
ꢁ55.2 1.7
ꢁ20.2 1.7
ꢁ34.7 3.0
ꢁ66.4 2.1
ꢁ31.8 2.1
ꢁ36.0 1.9
ꢁ60.3 1.8
ꢁ24.3 0.5
ꢁ34.9 1.2
ꢁ54.5 1.1
ꢁ19.6 0.5
ÁHꢀ
ꢁ53.5
ꢁ17,4
1
1
TÁSꢀ
ÁCp
ÁGꢀel
ÁGꢀnel
TÁSꢀnel
ÁGꢀnel/ÁGꢀ (%)
ꢁ2.82 0.15 ꢁ2.09 0.22 ꢁ2,01 0,19 ꢁ2.11 0.21
ꢁ2.9 0.38 ꢁ2.61 0.28 ꢁ1.98 0.23
a-PAA (a)
ꢁ5.7
2
ꢁ2.4 0.8
ꢁ33.7 0.3
ꢁ17.1 0.6
0.93
ꢁ4.5 1.5
ꢁ4.6 2.5
ꢁ30.4 0.1
ꢁ24.8 1.7
0.87
ꢁ8.82 3.1
ꢁ25.84 0.7
ꢁ40.6 2.2
0.75
ꢁ4.9 1.9
ꢁ31.1 0.4
ꢁ29.2 1.9
0.86
ꢁ7.4 1.3
ꢁ27.5 0.2
ꢁ27 1.2
0.79
ꢁ30.4 0.1
ꢁ23.8 1.4
0.84
ꢁ31.6 0.2
ꢁ21.9
1
0.88
ÁGꢀ
ꢁ37.8 1.4
ꢁ31.6 1.3
6.1 0.7
ꢁ37.7 1.2
ꢁ29.3 1.1
8.4 0.5
ꢁ37.9 1.1
ꢁ26.9 0.8
10.9 0.8
ꢁ1.6 0.1
ꢁ3.78 1.1
ꢁ34.1 0.2
7.2 0.8
ꢁ37.3 1.2
ꢁ30.6 1.1
6.8 0.5
ꢁ1.66 0.16
ꢁ3.18 1.5
ꢁ34.2 0.3
3.6 1.1
ꢁ35.1 1.3
ꢁ45.9 0.9
ꢁ10.8 0.9
ꢁ37.1 2.8
ꢁ45.0 2.5
ꢁ7.9 1.3
ꢁ37.0 2.5
ꢁ37.6 2.5
ꢁ0.5 1.1
ÁHꢀ
TÁSꢀ
ÁCp
ÁGꢀel
ÁGꢀnel
TÁSꢀnel
ÁGꢀnel/ÁGꢀ (%)
ꢁ1.04 0.19 ꢁ1.35 0.16
ꢁ1.85 0.14 ꢁ1.99 0.28 ꢁ1.58 0.14
b-PAA (b)
ꢁ5.5 1.9
ꢁ32.3 0.4
0.7 1.3
0.86
ꢁ4.2 1.5
ꢁ33.4 0.2
4.2 1.1
0.89
ꢁ4.4 1.3
ꢁ30.7 0.4
ꢁ 15.2 0.9
0.88
ꢁ6.4 2.6
ꢁ30.7 0.3
ꢁ14.3 2.5
0.83
ꢁ7.04 3.3
ꢁ30 0.5
ꢁ7.6 2.3
0.81
0.90
0.92
ÁGꢀ
ꢁ36.6 1.2
ꢁ50.8 1.2
ꢁ14.2 0.3
ꢁ38.2 1.7
ꢁ33.3 1.7
4.9 0.5
ꢁ36.7 1.2
ꢁ35.7 1.1
1.0 0.6
ꢁ37.5 2.9
ꢁ36.7 2.9
0.8 0.3
ꢁ2.34 0.43
ꢁ6.46 4.1
ꢁ31 0.3
ꢁ5.7 2.9
0.83
ꢁ34.4 1.5
ꢁ65.9 1.1
ꢁ31.5 1.1
ꢁ36.8 1.4
ꢁ53.6 1.3
ꢁ16.8 0.5
ꢁ36.8 1.4
ꢁ57.8 1.3
ꢁ21.0 0.5
ÁHꢀ
TÁSꢀ
ÁCp
ÁGꢀel
ÁGꢀnel
TÁSꢀnel
ÁGꢀnel/ÁGꢀ (%)
ꢁ2.91 0.38 ꢁ1.54 0.25
ꢁ2.0 0.4
ꢁ3.07 0.16 ꢁ2.79 0.34 ꢁ2.81 0.28
C-a-PAA (c)
ꢁ2.7 1.3
ꢁ33.9 0.4
ꢁ16.9 1.2
0.93
ꢁ5.42 2.4
ꢁ32.8 0.6
ꢁ0.5 1.8
0.86
ꢁ2 1.3
ꢁ6.67 1.6
ꢁ27.8 0.6
ꢁ38.2 1.2
0.81
ꢁ2.3 1.4
ꢁ34.5 0.2
ꢁ19.1 1.3
0.94
ꢁ2.2 1.4
ꢁ34.6 0.4
ꢁ23.3 1.3
0.94
ꢁ34.7 0.4
ꢁ1.02 1.1
0.95
aÁGꢀ, ÁHꢀ, TÁSꢀ, ÁGꢀel, ÁGꢀnel and TÁSꢀnel are expressed in kJ/mol. ÁCp is expressed in kJ/mol/K.
regardless of the sequence and nature of PAA, no more
than two positive counterions are released on binding.
These results seem to demonstrate that the majority of
ammonium and guanidinium groups of PAA interact
with TAR via hydrogen bonding and/or p-cation inter-
actions rather than via ionic interactions with the phos-
phate backbone.
Non-electrostatic parts of Gibbs energies (ÁGꢀnel) were
then obtained by extrapolation of Equation (1) to an ionic
strength of [KCl] = 1 M, where electrostatic interactions
are effectively masked (Table 2). This study highlights
that, whatever the nature and sequence of the PAA, ionic
interactions with the phosphodiester backbone are not the
major driving force for the formation of PAA/TAR
complexes, as non-electrostatic interactions dominate the
binding by contributing to 75 to 95% of the total binding
free energy (cf. ÁGꢀnel/ÁGꢀ ratio, Table 2).
This is indicative of an almost perfect EEC phenomenon
for which any decrease in binding enthalpy is compensated
by a similar decrease in binding entropy, and vice versa
(see supporting information for comment on statistical
relevance). The consequence is that even if binding
enthalpies and entropies are distributed over wide inter-
vals in a given series, ÁGꢀ values along this series are very
close. For the three series, the slopes are very similar,
underscoring that from a thermodynamic point of view
all PAA are homologous molecules. Considering this,
it seems unlikely that any tri-PAA containing this set of
amino acids (a or b Phe, Arg, Lys) can bind to TAR with
higher affinities than those observed in this study.
EEC is a general feature of many biological processes
based on molecular associations implying non-covalent
interactions. Often observed in the context of protein–
ligand interactions, it is the first time, to our knowledge,
that EEC is reported for a series of RNA–small ligand
binding events. The physical origin of this EEC phenom-
enon, theoretically (30,31) and experimentally (32,33) sup-
ported, is based on the relationship opposing ‘bonding’
and ‘motion’. The stronger is an interaction at a dimeric
interface, the lower is the mobility at this interface.
Enthalpy and entropy parameters
Enthalpy (ÁHꢀ) and entropy changes (ÁSꢀ) associated
with each PAA/TAR equilibrium were determined by
Equation (2) after determination of ÁGꢀ at several tem-
T
peratures (278–308K). The results of these thermodynamic
analyses are summarized in Table 2. In all cases, the for-
mation of PAA/TAR complexes is enthalpy driven,
demonstrating that non-covalent interactions (H-bonds,
p-stacking, p-cation, etc.) dominate the binding. While
binding processes for a-PAA are highly disfavored
entropically, TÁSꢀ parameters for b- and C-a-PAA are
less unfavorable or even favorable in the case of F-rich
compounds (1b–4b and 2c–4c). For each of the three
series, ÁHꢀ and TÁSꢀ are found strongly correlated,
according to Equation (3), with correlation coefficients
higher than 0.99 (Figure 4A) and slopes ‘a’ near unity.
ꢀ-PAA and C-ꢁ-PAA versus ꢁ-PAA. As shown in
Figure 4B, b-PAA are associated with higher ÁHꢀ and
ÁSꢀ values than their corresponding a-PAA, but the
trends in ÁHꢀ (and TÁSꢀ) along PAA sequences are
roughly parallel for b-PAA and a-PAA. This likely dem-
onstrates that the structural modification that consists in
adding a methylene group into the side chains of a-amino
acids of a-PAA leads to the same effect regardless of the
PAA sequence. On average, this structural modification
has an enthalpy cost of approximately 24 kJ/mole in the

Nucleic Acids Research, 2013, Vol. 41, No. 11 5859
-40
-20
-20
0
20
A
B
20
0
TΔS° (kJ.mol-1)
α-PAA ΔH°
β-PAA ΔH°
C-α PAA ΔH°
enthalpy driven &
entropically unfavored
-40
-60
-80
-20
-40
-60
-80
α-PAA TΔS°
β-PAA TΔS°
C-α PAA TΔS°
enthalpy
driven &
entropically
favored
α-PAA
β-PAA
C-α PAA
circle: y = 0.98( 0.04) x - 37.1( 0.9), R² = 0.9897
triangle: y = 0.91( 0.03) x - 36.9( 0.2), R² = 0.9954
square: y = 0.93( 0.02) x - 37.5( 0.4), R² = 0.9974
Figure 4. A/Enthalpy/entropy compensation plot: ÁHꢀ versus TÁSꢀ for the three series of PAA/TAR equilibria. B/Variation in ÁHꢀ and TÁSꢀ with
the PAA sequence for a-PAA (circle), b-PAA (triangle) and C-a-PAA (square).
case of the four F-rich compounds and of 18 kJ/mole
for K/R-rich ones, overweighted by an entropic gain of
26 and 19 kJ/mole, respectively. Because this increase in
both ÁHꢀ and ÁSꢀ does not depend on the PAA sequence,
it can be suggested that each isosequential b-PAA/
a-PAA pair binds to TAR following the same interaction
network. In a-PAA, the high magnitude of both enthalpy
and entropy values would reveal the establishment of tight
complexes, while in contrast, the less favorable ÁHꢀ and
less unfavorable TÁSꢀ values observed for b-PAA would
be associated with the formation of looser complexes (31).
These differences could be linked to the higher flexibility
of b-PAA vs a-PAA in the unbound state. Indeed, to keep
the same interaction network than a-PAA while avoiding
a conformational entropic penalty, one can suppose that
the b-PAA/TAR complexes relax to a more stable state by
decreasing the interaction tightness at the interface (34).
Such a behavior would result both in weakening non-
covalent interactions (i.e. less favorable ÁHꢀ) and in
increasing mobility of the b-PAA side chains (i.e. less un-
favorable TÁSꢀ).
Concerning C-a-PAA structures, thermodynamic
profiles demonstrate that the introduction of three methy-
lene groups in the backbone of a given a-PAA impacts
differently according to the PAA sequence (Figure 4B).
In the case of K/R-rich C-a-PAA 5c-7c and of C-a-FFK
1c, ÁHꢀ and ÁSꢀ values are very close to those associated
with the respective a-PAA 5a–7a and 1a. In contrast, the
TAR binding of F-rich C-a-PAA 2c–4c occurs with an
average enthalpy cost of 18 kJ/mole comparatively with
their corresponding a-PAA 2a–4a. Therefore, for C-a-
PAA 2c–4c, it could be suggested that increasing the
length of the a-PAA backbone does not modify, on com-
plexation, the interaction network with TAR but rather
induces a structural loosening of the complex.
F-rich PAA versus K/R-rich PAA. Within each series,
F-rich sequences are associated with higher entropy and
enthalpy values than R/K-rich ones (except for C-a-FFK)
(Table 2 and Figure 4B). This specific behavior of F-rich
PAA is probably because they establish fewer non-electro-
static interactions with TAR. Comparatively with R/
K-rich PAA, F-rich PAA contain only one cationic
residue able to give strong H-bonds via its side chain
and two aromatic motives prone to establish weaker p-
stacking interactions. It is reasonable thereby to observe
for F-rich PAA less enthalpy changes but more favorable
entropy values.
Finally, for all PAA/TAR complexes, heat capacities
(ÁCp) were found negative, as observed for a variety of
small molecules binding to nucleic acids (35,36) (Table 1).
However, ÁCp values are significantly higher for loose b-
PAA binders than for the corresponding tight a-PAA.
Many parameters could account for this difference
(changes in solvent-accessible surface area, changes in
protonation state, ion release, etc.), but, owing to the
structural similarity of the ligands, it may result preferen-
tially from differences in RNA conformational mobility
changes arising on binding of the two kinds of PAA
(37,38). Because a-PAA are associated with more
negative ÁCp values than b-PAA, it would be expected
that they induce a more significant RNA conformational
change. However, CD data do not seem to support this
hypothesis (see vide infra).
Overall, molecular flexibility is one of the key param-
eters of ligand efficiency, but thermodynamic conse-
quences on binding are not yet well understood. Our
results are in line with a recently reported computer
modeling study that predicts that varying molecular flexi-
bility while keeping the same interaction network leads to
near-linear EEC (39). Moreover, when the receptor is

5860 Nucleic Acids Research, 2013, Vol. 41, No. 11
A
flexible, increasing ligand flexibility leads to stronger
binding affinity. Such a trend is observed in our study
because more flexible b-PAA and some C-a-PAA
display on average a better affinity for TAR than their
corresponding a-PAA.
0.0015
0.0010
0.0005
0.0000
0.40
0.38
0.36
0.34
CD and UV melting studies
0
20 40 60 80 100
T (°C)
To compare the TAR structural changes occurring on
binding of a-, b- and C-a-PAA, CD studies were imple-
mented. F-rich PAA 2a–c and K/R-rich 7a–c, representa-
tives of the three series, were chosen as models. CD
spectra of PAA alone were preliminary measured.
Negligible CD intensity in the 200- to 350-nm region
was observed, suggesting the lack of any structuration or
secondary shape. In contrast, the CD spectrum of TAR
alone is characteristic of an A-form double helix, with
strong positive and negative bands at 260 nm and
210 nm, respectively, and a weak negative band at
240 nm (40,41). While the 260-nm CD band, very sensitive
to base stacking, may vary on binding of a ligand without
affecting the general nucleic acid structure, the 210-nm
band is more related to the A-form RNA helical structure.
CD profiles (see Supplementary Figure S1 in supporting
information) associated with tight binders (a-PAA 2a and
7a and C-a-PAA 7c) differ from those associated with
loose ones (b-PAA 2b, 7b and C-a-PAA 2c), indicating
that TAR conformational changes induced on binding
of the two classes of PAA are distinct. In the case of
tight binders (2a, 7a, 7c), only a slight decrease in ellipti-
city at 260 nm was observed, whereas the intensities of the
negative bands at 210 nm and 240 nm did not vary. This
CD signature is similar to the one reported for binding of
Tat derivatives and arginine-containing compounds, the
decrease in ellipticity at 260 nm reflecting the externaliza-
tion of the C24 and U25 residues of the bulge and their
concomitant destacking (40–45). Loose binders (2b-c, 7b)
change the CD signal of TAR to a more substantial
extent. A stronger reduction in ellipticity at 260 nm was
observed together with a marked decrease in the intensity
of the negative band at 210 nm and to a less extent at
240 nm. Because such modifications may be indicative of
a destabilization of the nucleic acid structure (46,47),
melting temperature and ultraviolet absorbance spectros-
copy studies were conducted. A destabilization of the
TAR structure on binding should result in both a
decrease of the TAR melting temperature and an
increase in its absorbance at 260 nm (48). Our results
clearly refute this hypothesis. Indeed, the TAR melting
temperature was increased on loose b-PAA 7b binding
by 4.1ꢀC and 6.6ꢀC at 1:1 and 5:1 PAA/TAR ratios,
respectively, demonstrating a stabilizing effect on the
TAR structure (Figure 5; see Supplementary Figure S2
and Supplementary Table S3 in supporting information).
In addition, a decrease in absorbance at 260 nm, particu-
larly notable at 5:1 molar ratio, was observed. By com-
parison, the binding of C-a-PAA 7c increased the TAR
melting temperature by 2.5ꢀC and 4.1ꢀC at 1:1 and 5/1
PAA/TAR ratios, respectively, and had no effect on the
TAR UV spectrum. Thus, even if loose 7b binder displays
the same TAR affinity as tight 7c one (Table 1), its binding
TAR alone
TAR : 7b (1:1)
TAR : 7b (1:5)
0
20
40
60
80
100
T (°C)
B
0.0015
0.0010
0.0005
0.0000
0.40
0.38
0.36
0.34
0
20 40 60 80 100
T (°C)
TAR alone
TAR : 7c (1:1)
TAR : 7c (1:5)
0
20
40
60
80
100
T (°C)
Figure 5. UV melting curves at 260 nm of TAR RNA and its complex
with b-PAA 7b (A) or C-a-PAA 7c (B). Inset: first-derivative UV
melting curves of TAR and its complex with b-PAA 7b (A) or C-a-
PAA 7c (B).
induces distinct RNA conformational changes, leading
to a higher stabilization of the TAR structure.
Given these results, the variations observed between the
CD spectra are likely due to TAR conformational differ-
ences. While the overall A-form of the TAR helix is
globally conserved in the two cases, the substantial ellip-
ticity decrease at 210 nm and 260 nm only observed in the
case of loose PAA 7b likely indicates a decrease in the
A character of the TAR helix on binding (49). One hy-
pothesis could be to assign this change to a distorsion
of the A-form. In response to the entropic constraint
imposed by the binding of the highly flexible b-PAA struc-
ture, the b-PAA/TAR complex would relax to a more

Nucleic Acids Research, 2013, Vol. 41, No. 11 5861
Table 3. Anti-HIV activity and toxicity of C-a and b-PAA
PAA Inhibition Toxicity %Inhibition/ ED50* CC50** SI***
observed in IC50 values, indicating a dependency both
on the nature and sequence of PAA for the TAR/Tat
inhibition. Our thermodynamic study revealed the occur-
rence of an EEC phenomenon that explains the KD values’
similarity. Using the interfacial mobility model, we high-
lighted that the TAR binding of a-PAA and of K/R-rich
C-a-PAA results in the establishment of tight complexes,
while looser ones are formed with b-PAA and F-rich C-a-
PAA. In most cases, these latter are more efficient Tat/
TAR inhibitors than their corresponding a-PAA. This
could be due to a higher stabilization of the TAR structure
on binding.
Altogether, our results give new insights into the com-
prehension of RNA binding modes that open a wide field
of investigation in the design of new RNA ligands.
Finally, b-PAA and C-a-PAA constitute new lead
compounds from which structure/activity studies can be
envisaged to increase their promising antiviral activity.
(%)
(%)
%Toxicity
(mM) (mM)
FRF 4c 67
FFK 1b 80
FFR 2b 83
RFF 3b 96
FRF 4b 92
FKR 5b 85
FRR 6b 95
RRF 7b 95
5
2
4
1
4
2
1
1
29
37
38
36
18
29
53
36
2
8
2
3
7
6
5
4
>2.3
>2.2
>2.2
>2.7
>5.1
>2.9
>1.8
>2.6
869
254
130
85
56
219
60
>1000 >1.2
id
id
id
id
id
id
id
>4
>8
>12
>18
>4.5
>17
>10.5
95
*ED50: Effective Dose. Dose of PAA required to inhibit 50% of the
viral replication.
**CC50: Cytotoxic Concentration. Concentration of PAA required to
reduce the cell number by 50%.
***SI: Selectivity Index (CC50/ED50).
stable state not only by decreasing the interaction tight-
ness at the PAA/TAR interface but also by modifying
the TAR helicity. However, we have, at this stage, no
evidence for such a scenario. Complementary studies
will be envisaged to resolve the dynamics of PAA/TAR
interactions.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online:
Supplementary Tables 1–3, Supplementary Figures 1–3,
Supplementary Methods and Supplementary References
[50–52].
Antiviral activity
Finally, the antiviral activity and the cellular toxicity of C-
a and b-PAA were evaluated on PBMC, infected (or not)
by the HIV-1 LAI-strain, first at a concentration of 1 mM.
AZT was taken as a reference. When the ratio (%inhib-
ition/%toxicity) was >2, ED50, CC50 and selectivity
indexes (SI = CC50/ED50) were determined. Results are
given in Table 3.
ACKNOWLEDGEMENTS
We thank Jean-Marie Guigonis for mass analyses.
FUNDING
C-a-PAA derivatives are less efficient to inhibit HIV
replication and more toxic than b-PAA. The best C-a-
PAA inhibitor, i.e. C-a-FRF 4c, is able to inhibit at
1 mM 67% of the viral replication but with 29% of
cellular toxicity, giving a ratio (%inhibition/%toxicity]
of only 2.1 and an ED50 value of 869 mM. The three
C-a-PAA 5c–7c containing K/R-rich sequences give the
worse results (data not shown), although they are among
the best inhibitors of the Tat/TAR interaction in free-cell
assays.
SIDACTION; the Agence Nationale de Recherche sur le
SIDA and the Caisse Primaire d’Assurance Maladie des
Professions Liberales. L. Pascale is recipient of a MENRT
´
Ph.D. fellowship. Funding for open access charge:
University fund.
Conflict of interest statement. None declared.
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The b-PAA series is more promising. Indeed, all b-PAA
inhibit at 1 mM from 80 to 96% of the viral replication,
with a ratio [%inhibition/%toxicity] from 2.2 to 5.1. The
best inhibitors, b-FRF 4b and b-FRR 6b display ED50
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to 1 mM, thus leading to a promising SI values higher than
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