Unveiling RNA-Binding Properties of Verapamil and Preparation of New Derivatives as Inhibitors of HIV-1 Tat-TAR Interaction

Article abstract of DOI:10.1002/cplu.201900650

Targeting RNA using small molecules is now established as a very promising strategy for many therapeutic applications since coding and non-coding RNAs bear a pivotal role both in viral and bacterial infections as well as in diseases such as cancer. Here, we focused on HIV-1 TAR RNA as a promising target for the development of new anti-HIV therapies but also as an ideal model to validate the discovery of original RNA ligands. First, we performed an initial screening of a library of compounds against TAR that led to the discovery of verapamil, a marketed calcium-channel blocker, as a promising chemical structure for the development of new RNA ligands. The synthesis of a series of analogs of verapamil led to promising structure activity relationships and to the discovery of a conjugate between verapamil and an indole fragment, as an efficient and selective TAR binder able to inhibit Tat/TAR interaction with an IC₅₀ of 18.8 μM. This work supports the potential of library screening for the discovery of original and selective RNA ligands and illustrates how existing drugs directed against protein targets still need to be studied for RNA binding as a promising strategy in the field of RNA targeting by small molecules.

Full text of DOI:10.1002/cplu.201900650

Accepted Article
Title: Unveiling RNA Binding Properties of Verapamil and Preparation
of New Derivatives as Inhibitors of HIV-1 Tat-TAR Interaction
Authors: Céline Martin, Serena De Piccoli, Marc Gaysinski, Cécile
Becquart, Stéphane Azoulay, Audrey Di Giorgio, and Maria
Duca
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10.1002/cplu.201900650
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Unveiling RNA Binding Properties of Verapamil and Preparation of New Derivatives as
Inhibitors of HIV-1 Tat-TAR Interaction
Céline Martin, Serena De Piccoli, Marc Gaysinski, Cécile Becquart, Stéphane Azoulay, Audrey Di
Giorgio and Maria Duca*[a]
[a] C. Martin, S. De Piccoli, Dr. M. Gaysinski, C. Becquart, Dr. S. Azoulay, Dr. A. Di Giorgio, Dr. M.
Duca
Université Côte d’Azur, Institute of Chemistry of Nice (ICN), 28 avenue Valrose, 06100 Nice, France
Corresponding author: maria.duca@univ-cotedazur.fr
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Abstract
Targeting RNA using small molecules is now established as a very promising strategy for many
therapeutic applications since coding and non-coding RNAs bear a pivotal role both in viral and bacterial
infections as well as in human pathologies such as cancer. Here, we focused on the targeting of HIV-1
TAR RNA as a promising target for the development of new anti-HIV therapies but also as an ideal
model to validate the discovery of original RNA ligands. First, we performed an initial screening of a
library of compounds against TAR that led to the discovery of verapamil, a marketed calcium-channel
blocker, as a promising chemical structure for the development of new RNA ligands. The synthesis of
a series of analogs of verapamil led to promising structure activity relationships and to the discovery of
compound 2h, a conjugate between verapamil and indole fragment, as an efficient and selective TAR
binder able to inhibit Tat/TAR interaction with an IC50 of 18.8 µM. This work supports the potential of
library screening for the discovery of original and selective RNA ligands and illustrates how existing
drugs directed against protein targets still need to be studied for RNA binding as a promising strategy
in the field of RNA targeting by small molecules.
2
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Introduction
Targeting biologically relevant RNAs using small-molecule drugs is becoming an important challenge
for current medicinal chemistry.[1] RNA, for a long time reduced to an intermediate in the transmission
of the genetic information, is now largely recognized as a major actor of important biological processes
especially in the regulation of gene expression.[2] Various prokaryotic, eukaryotic and viral non-coding
RNAs represent relevant targets for small-molecule ligands, peptides or oligonucleotides and recent
successful examples in this field showed the feasibility of this approach.[1b, 3] While peptides and
oligonucleotides may represent efficient and specific targeting tools, they often lack favorable
pharmacodynamics/pharmacokinetic properties for further therapeutic applications. Targeting RNA
using small molecules is thus emerging as a promising and efficient approach thanks to the fact that
biologically relevant RNAs bear a three-dimensional structure offering specific binding pockets thus
being much more favorable for small-molecule specific interaction compared to double helical DNA.[1d]
Indeed, RNA is a major target for various classes of antimicrobial drugs, such as aminoglycosides,
tetracyclines or oxazolidinones that are able to selectively bind to prokaryotic ribosomal RNA thus
impairing protein synthesis in bacteria.[4] Its role in RNA viruses is also largely recognized thus
rendering viral RNAs potential targets for innovative therapies.[5]
In this context, a potential target for the development of new antiviral therapies is represented by the
HIV-1 transactivation response (TAR) element. This RNA fragment binds to the Tat protein and is
essential for efficient human immunodeficiency virus (HIV) replication (Figure 1A).[5-6] This RNA is
constituted by 59 nucleosides located in the 3'-UTR of each viral transcript and spontaneously generates
a stem-loop structure.[7] TAR RNA interacts with the viral protein Tat and with positive transcription
elongation factor b (P-TEFb) itself constituted by cyclin T1 and cyclin-dependent kinase 9 (CDK9), thus
controlling transcription. The interaction between Tat and TAR is mediated by an arginine-rich region
that recognizes the bulged region of TAR.[8] The inhibition of Tat/TAR interaction should thus induce
the inhibition of viral replication and a various RNA binders able to block this interaction have been
reported.[6] TAR RNA thus represents particularly relevant model system to study ligand binding to
RNA as well as a promising target for the discovery of compounds with anti-HIV activity.[9]
During recent years, various small-molecule RNA binders able to target biologically relevant RNAs
have been identified.[1a, 1c, 10] One major example is represented by RNA-interacting compounds acting
as pre-mRNA splicing modifiers of SMN2 (survival motor neuron 2 protein). Some of these ligands
recently found clinical application in the treatment of Spinal Muscular Atrophy (SMA).[11] Very specific
RNA binders targeting the production of oncogenic microRNAs in cancer cells and in vivo have also
been reported.[12] In this case, an approach called InfoRNA was developed to identify lead binders using
a statistical prediction of compounds affinity and selectivity based on a previous screening of a large
library of compounds against a library of RNA motifs.[13] This allowed for the discovery of compounds
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bearing an exceptional specificity for miR-96 precursor or for miR-210 precursor both in cells and in
animals models.[10a, 12c]
We also discovered various RNA ligands upon combination of different RNA binding domains carefully
selected to bring both affinity and selectivity for the target.[14] For example, we conjugated artificial
nucleobases, that are able to interact selectively with RNA base pairs, with basic amino acids that can
bring affinity for an efficient targeting of TAR RNA in vitro and in infected cells.[15] A similar approach
was used for the preparation of C-nucleosides targeting prokaryotic A-site RNA.[16] We also conjugated
known antibiotics, such as aminoglycosides, with artificial nucleobases and amino acids leading to
multimodal RNA ligands able to selectively target oncogenic miRNAs in vitro and in cancer cells
overexpressing these miRNAs.[14a] Concomitantly to the focused design of new RNA ligands, we
performed screening of larger libraries of compounds against oncogenic miRNAs.[17] This led to the
identification of an array of chemical scaffolds bearing favorable properties in terms of binding and
selectivity in vitro but also inside cancer cells.
Here, we describe the screening of an in-house library of commercial compounds that allowed for the
identification of verapamil, a phenylalkylamine calcium channel blocker currently in clinical use as an
antiarrhythmic compound (Figure 1B), as a potential scaffold for the preparation of new RNA ligands.
As mentioned above, the conjugation of RNA ligands with other RNA binding domains led to promising
and selective RNA binders. We thus decided to prepare a reduced form of verapamil and to conjugate it
with fragments identified as RNA binders during the screening of the library and with known RNA-
binding moieties led to the preparation of a series of verapamil analogs and to the improvement of TAR
RNA affinity and selectivity. Noteworthy, the conjugation of verapamil to an indole fragment through
reductive amination led to a compound (2h) which is able to efficiently inhibit Tat/TAR interaction in
vitro with 18.8 µM IC50. This represents a very promising inhibition activity compared to small-
molecule RNA ligands already reported in the literature.[9, 18] The detailed study of the molecular
mechanism of interaction of 2h with TAR RNA led to important insights about the chemical features
that are relevant for binding and for the design of future efficient TAR binders and potential antiviral
compounds. Verapamil has never been considered for RNA binding and this work represents the first
example of verapamil chemical modification applied to RNA targeting thus opening new perspectives
in the RNA targeting field.
Results and discussion
Library screening. The library of compounds and fragments chosen for this study belongs to an in-
house collection of commercial compounds and contains 188 molecules (see Table S1 in the Supporting
Information for the complete list) whose 90% can be considered as fragments (MW < 300, ClogP, H-
bond donors and acceptors and n° of rotatable bonds < 3).[19] The remaining 10% are molecules bearing
higher molecular weight but that could present an interest as scaffolds for the preparation of new RNA
binders. We also decided to add to this library some antibiotics that are efficient and non-selective RNA
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binders, such as aminoglycosides and tetracyclines that could function as positive controls. The aim of
the initial screening was to identify compounds bearing even modest affinity for TAR RNA in order to
later improve their binding and biological activity upon chemical modification as usually performed in
a fragment-based approach. Hits growing (addition of substituents) and linking (combination with other
hits) could lead indeed to relevant improvements of affinity and of selectivity allowing for the discovery
of new scaffolds and for the design of RNA ligands with potential antiviral activity.
First, we screened the library measuring the affinity toward a 27-mer TAR fragment labeled in 5′
position with Alexa488. This kind of fluorescence-based assay relies on the fact that binding of an
efficient ligand to the labeled target induces a change in fluorescence intensity that is dependent on the
concentration of ligand and it has been largely validated in the literature.[20] We screened fragments at
10 µM and 100 µM leading to the identification of 9 compounds showing affinity (at least 20%
fluorescence variation) at both concentrations (guanine, thioguanine, phenothiazine, neomycin,
oxytetracycline, 1,6-dibromo-2-hydroxynaphthalene-3-carboxylic acid, cinchonidine, verapamil and
apramycin, Figure S1) and 9 compounds showing some affinity only at 100 µM concentration (2-
aminobenzimidazole, 5-aminomethylindole, 2-aminothiazole, 3,5-dibromosalycilic acid, 3,5-
dichlorosalycilic acid, 3-acetylindole, 4-dimethylamino benzaldehyde, 2,6-diaminopurine and
puromycin, Figure S2). Among the strongest binders, neomycin, oxytetracycline, apramycin and
puromycin are known RNA binders able to bind prokaryotic ribosomal RNA and impair protein
synthesis in bacteria. Phenothiazine, cinchonidine and naphthalene derivative are known intercalating
agents and have been previously studied for RNA binding.[21] Purines, such as guanine and thioguanine
have also been studied as RNA binding motifs.[14c, 22] The remaining compound, verapamil, revealed to
have interesting affinity for TAR and this pharmacophore has never been explored for RNA binding. It
thus seemed an appropriate starting point for the development of new and original RNA ligands. Indeed,
verapamil is a calcium channel blocker currently used clinically for the treatment of arrhythmia and
other cardiovascular pathologies.[23] We thus decided to take advantage of verapamil to synthesize
conjugates with other scaffolds identified during the initial screening (namely phenothiazine,
naphthalene, quinolone, benzimidazole, indole, thiazole, 3,5-dibromo-salicilic acid and 4-
dimethylaminobenzaldehyde) as well as with known RNA binding domains (such as benzothiazole,
phenylthiazole, imidazole and amino acids lysine, histidine, phenylalanine and arginine).
Synthesis of a new series of verapamil analogs. In order to conjugate verapamil to other RNA-binding
motifs, we first chose to reduce verapamil nitrile group using LiAlH4 in THF to obtain desired amine-
containing compound 1 in 96% yield as previously reported in the literature (Scheme 1).[23] Starting
from this compound, we chose three different functionalization routes: (i) reductive amination upon
reaction with aldehydes (Scheme 1), (ii) amide formation upon reaction with carboxylic acids (Scheme
2) and (iii) introduction of a carboxyl group on verapamil using succinic anhydride followed by amide
formation upon reaction with amines (Scheme 3).
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Regarding the reductive amination reaction, we chose to react 1 with aldehyde-containing compounds
that demonstrated to have some affinity during fragment screening (Scheme 1): 4-
dimethylaminobenzaldehyde (as in 2a), 1-naphthaldehyde (as in 2b), 3,5-dibromo-2-
hydroxybenzaldehyde (as in 2c), 4-quinolinecarboxaldehyde (as in 2d), N-Boc-3-formyl-1H-indole-1-
carboxylate (as in 2'h). Beside these five aldehyde compounds, we also chose four different heterocyclic
moieties containing an aldehyde group and known as interesting moieties for RNA targeting[10c]
:
benzothiazole (as in 2e), phenylthiazole (as in 2f), thiazole (as in 2g) and N-Boc-indoline (as in 2'i).
Reductive amination in the presence of these aldehydes was performed using classical conditions
(NaBH3CN in CH2Cl2/MeOH 50:50) and led to the formation of compounds 2a-g and 2'h-2'i in 52-68%
yields. Compounds 2'h and 2'i containing a Boc protecting group were further deprotected in the
presence of TFA in CH2Cl2 leading to 2h and 2i in 94% and 78% yields, respectively.
For the preparation of analogs bearing an amide linkage between verapamil and the modifying moiety,
we chose two different strategies. The first one is the direct coupling of carboxylic acids on the amino
group of compound 1 (Scheme 2). In this case, we also took advantage of chemical moieties identified
during the screening process by coupling 4-dimethylaminobenzoic acid (as in 3a), 10H-phenothiazine-
10-carboxylic acid (as in 3b), benzoic acid (as in 3c), 2-thiophenecarboxylic acid (as in 3d) and 4-N-
tert-butoxycarbonyl-aminobenzoic acid (as in 3'e). We also conjugated 1 with N,N-di-Boc-L-lysine
(as in 3'f), N,Nim-di-Boc-L-histidine (as in 3'g), N-Boc-L-phenylalanine (as in 3'h) and N-Boc-N-
(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine (as in 3'i). Amino acids are indeed RNA
binding agents as they constitute natural RNA ligands, i.e. peptides. Peptide coupling between
compound 1 and these carboxylic acids in the presence of HOBt, EDC and Et3N in CH2Cl2 led to the
formation of compounds 3a-d and 3'e-3'i in 56-86% yields. Compounds 3'e-3'h containing a Boc group
were deprotected in the presence of TFA in CH2Cl2 leading to desired products 3e-3h in 79-95% yields.
Compound 3'i containing a Mtr and a Boc protecting groups was first deprotected on the N in the
presence of TFA in CH2Cl2 then treated with a solution of phenol/TFA 5% w/w to remove the Mtr group
of N. These two steps led to 3i in 95% yield.
Finally, compound 1 was modified with the introduction of a succinamic acid side chain that was
introduced upon reaction of 1 with succinic anhydride in CH2Cl2 leading to compound 4 in 95% yield
(Scheme 3). 4 was then coupled with compound 1 to form a verapamil dimer (as in compound 5a) as
well as with 2-aminothiazole (as in compound 5b) in the presence of HOBt, EDC, Et3N in CH2Cl2
leading to desired compounds 5a and 5b and 5'c in 27% and 25% yield, respectively. Compound 4 was
also coupled with N-Boc-2-aminobenzimidazole (as in compound 5'c) in the presence of HBTU and
DIPEA in CH2Cl2 leading to a mixture of 5'c and 5c. Addition of TFA in CH2Cl2 led to desired
compound 5c in 25% yield over two steps.
All synthesized compounds were characterized by NMR, HRMS and ultimately purity was verified by
HPLC before their biological evaluation.
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Evaluation of binding affinity and selectivity of the newly synthesized verapamil analogs. All
synthesized compounds 2a-i, 3a-i and 5a-c were evaluated as TAR RNA binders using the same
fluorescence-based assay that was employed for the initial screening but performing this assay over a
range of ligands concentrations. Compounds 1 (reduced verapamil) and 4 (verapamil conjugated to the
succinamic acid linker) as well as all moieties that were employed for conjugates synthesis were used
as controls. The measurement of fluorescence variation as a function of compounds concentration led
to the calculation of dissociation constants (KD) as reported in Figure 2. First of all, while verapamil
showed a KD > 100 µM that could not be measured precisely, compound 1 bears a KD of 3.2 µM
demonstrating a strong improvement of the affinity for the target once the nitrile group is reduced.
Reductive amination products 2a-2i were the most promising TAR binders with KD in the low
micromolar range from 0.66 µM for 2e, 0.71 and 0.72 µM for 2i and 2h, respectively, until 22.1 µM for
2c. Amide formation products 3a-3i were globally less strong binders except 3f, 3g and 3i that showed
a submicromolar KD (0.41 µM, 0.45 µM and 0.63 µM, respectively. Finally, amide formation products
5a-5c showed weak affinity with 12.9 µM KD for 5a and no measurable binding for 5b and 5c. These
results demonstrated that benzothiazole, indole and indoline as in 2e, 2h, 2i as well as basic amino acids
lysine, histidine and arginine as in 3f, 3g and 3i are more favorable for binding once conjugated to
verapamil with KD values in the submicromolar range. 4-dimethylaminophenyl, naphthalene, quinolein
and phenylthiazole moieties as in 2a, 2b, 2d, 2f and 3h keep affinity for the target with KD in the low
micromolar range but globally loose affinity compared to the first group of ligands. Compounds 2g, 3b,
3c and 3d bearing thiazole, phenothiazine, phenyl and thiophene substituents, respectively, showed only
low affinity with KD over 10 µM. Finally, compounds 2g, 3a, 3e, 5b and 5c bearing thiazole (conjugated
via amide formation as in 2g and 5b), 4-dimethylaminophenyl (conjugated via amide formation), 4-
aminophenyl and benzimidazole showed no affinity for the target. It is important to note that all moieties
tested without conjugation with verapamil showed very low affinity for the target and a KD value could
not be measured in the tested range of concentrations.
Affinity measurements alone are not representative of the potential of an RNA ligand, since a lack of
selectivity could hamper the biological activity. In order to appreciate the selectivity of each compound
we decided to measure KD values in the presence of a large excess of competitors such as tRNA and
DNA (100 eq. of each competitor). As illustrated in Figure 2, these experiments allowed us to
discriminate the most selective compounds. While 1, 2a-b, 2e-f, 2h-i, 3f-i and 5a retained affinity in the
presence of tRNA, only compounds 1, 2h, 3f and 3i retained TAR RNA affinity in the presence of DNA.
This suggested that these latter were much more favorable for future studies keeping in mind that 2h, 3f
and 3i are also the strongest binders.
Based on these results, we analyzed the thermodynamic features of the interactions of the most
promising compounds 2h, 3f and 3i in comparison with parent compound 1 as well as compound 2a
both being good but non-selective ligands and the results are as summarized in Table 1.[24] Free energies
of Gibbs (ΔG°) were first calculated from the dissociation constants (ΔG° = −RT ln KD) for all
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compounds and found to be very similar (from −30.0 to −36.5 kJ/mol). To complete the thermodynamic
binding profiles, the enthalpic (ΔH°) and the entropic (−TΔS°) energy contributions were determined
after the determination of ΔG°T at several temperatures (278−308 K). In all cases, the interaction is
driven by the enthalpy suggesting the formation of specific interactions, such as hydrogen bonds, with
the RNA target. In compounds 2h and 3f, the entropic contribution is close to zero while for compounds
2a and 3i its relevance is more important suggesting that electrostatic interactions and desolvation
probably contribute to binding of these latter compounds. Furthermore, the total energy represented by
the G° value is composed of two components: (i) the first one reflects the non-electrostatic interactions
(nonionic hydrophobic effects driven by entropy, specific interactions, including H-bonds, van der
Waals interactions, and -stacking) that contribute to the total free nergy (G°nel value) and ii) the pure
electrostatic contribution given by ionic interactions occurring between two groups of opposite charge
(G°el value) that can be highly dependent on the salt concentration. As suggested by the enthalpy
values, G°nel is over 90% for all compounds with 2h showing 99% of non-electrostatic interactions
formed. Altogether, binding studies demonstrated that various compounds are good RNA binders, but
only few conjugates are selective for TAR and that compound 2h, conjugate between verapamil and
indole using reductive amination, is the best and most selective ligand.
Inhibition of TAT/Tar interaction. Once identified the strongest and most selective binders, namely
compound 2h followed by 1, 3f and 3i, we wondered if these ligands could be able to inhibit Tat/TAR
interaction in vitro and we compared their efficacy with compound 2a. The ability of these compounds
to displace a Tat fragment from a preformed TAR/Tat complex was assessed via a FRET assay,
developed from a previously described procedure,[25] using a fluoresceinated Tat peptide fragment
(amino acids 48–57) and a Dabcyl-labeled TAR fragment (nucleotides 18–44). In the absence of ligand,
association of these two partners results in an efficient quenching of the dyes. Upon addition of
increasing amounts of compounds 1, 2a, 2h, 3f and 3i, the fluorescence rises in almost all cases,
demonstrating that these ligands can displace the Tat fragment from a preformed Tat/TAR complex.
IC50 values associated with each compound are given in Table 1. These experiments showed that reduced
verapamil (1) and compound 2a were not efficient inhibitors of Tat/TAR interaction with IC50 of 1.87
mM for 1 and no inhibition for 2a and that all other ligands were able to inhibit the formation of the
complex with micromolar IC50. Compounds 3f and 3i showed an IC50 of 302 µM and 404 µM,
respectively, further confirming the potential of basic amino acids lysine and arginine for the preparation
of RNA binders as previously reported. The best TAR ligand 2h showed an IC50 of 18.8 µM thus
demonstrating to be not only the strongest and most selective ligand but also the most efficient inhibitor
of Tat/TAR interaction. This results further underlines how inhibition studies are essential for the
identification of the most promising binders since dissociation constants alone do not allow the
discrimination between equally efficient ligands. All in vitro studies converged to the demonstration
that reduced verapamil conjugated to an indole via reductive amination, as in compound 2h, led to the
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best binding features and in vitro activity thus suggesting that the mode of binding and the site of
interaction of 2h is particularly favorable for inhibition and deserved to be studied more in detail.
Evaluation of the molecular mechanism of interaction. In order to study more in detail the molecular
mechanism of interaction of compound 2h with TAR, we decided to identify which residues were
affected upon binding in comparison to non-modified parent compound 1. NMR spectroscopy was thus
employed to examine the structure of HIV-1 TAR RNA in both ligand-bound and free states. Therefore,
following the changes in chemical shifts of TAR as a ligand is titrated into solution provides an
indication of how the ligand interacts with TAR. Upon titration of 1 into TAR, the most significant
chemical shifts changes were observed for residues in the bulge area of TAR (Figure 3), confirming that
verapamil specifically interacts with this bulge. The most affected residues are located between U23 and
G26 as well as between U38 and U40. Titration of 2h leads to similar chemical-shift changes but two
important differences were observed: (i) the interaction with TAR is extended to more residues since
the strongest variations are observed from U23 to G26 and between C37 and U42 and (ii) G26 chemical
shift is particularly modified by 2h with 0.3 ppm variation versus 0.05 ppm for 1. This demonstrates
that the binding site of 1 and 2h remains the same before and after conjugation but that further
interactions are formed with the target by the additional moiety thus reinforcing the interaction and
rendering it more efficient in terms of selectivity and inhibition activity. Interestingly, other compounds
of the series that showed good affinity do not show the same level of inhibition. As an example,
compound 2a, containing a dimethylaminophenyl substituent conjugated upon reductive amination,
bears a KD of 1.3 µM but no inhibition of Tat/TAR interaction. Indeed, the NMR study of its site of
interaction (Figure S7) confirmed that this kind of chemical modification induces a different binding
site compared to 1 and 2h. In fact, G26 is not affected by 2a while residues from U38 to U42 are only
slightly shifted.
Confirmation of these results came from molecular docking that we performed using AutoDock 4. We
employed a flexible ligand and TAR receptor to calculate independent docking events utilizing a
Lamarckian genetic algorithm and selected the 10 docking poses bearing the lowest energy in the
presence of compounds 1 and 2h. As illustrated in Figure 4, docking suggested that the interaction of
both compounds occurs at the level of the bulge site. The obtained results suggested the formation of H-
bonds and electrostatic interactions with phosphates and bases located in the region between G21 and
U25 as well as between C41 and U42 in the case of compound 1 (Figure 4A). Molecular docking is also
consistent with the formation of more specific hydrophobic interactions between the indole moiety and
G26 as well as various H-bonds with U26-A27 and U40-C41 in the case of 2h (Figure 4B). The
comparison of these results with the molecular docking of compound 2a (Figure S8) suggested that this
latter interacts more specifically with the stem region with residues G17 and C19 as well as with residues
U38, C39, U40 and U42 as suggested by NMR studies. 2a thus seems to interact in a very different
manner compared to 1 and 2h further demonstrating that the site of interaction is extremely important
for an efficient inhibition of RNA interaction with its biological partners. i.e. TAR interaction with Tat
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peptide. Indole moiety forms additional hydrophobic interactions with residue G26 as well as hydrogen
bonds with U40 and C41 thus increasing the strength and the number of interactions and improving the
selectivity and the inhibition activity. Efficient and selective binding at a particular site of the target is
thus essential to have good inhibition activity as demonstrated by the comparison of 2h with 2a.
Conclusion. The screening of a library of commercially available compounds for their ability to bind
HIV-1 TAR RNA led to the discovery of a small number of hits and in particular to the identification of
verapamil, a phenylalkylamine calcium channel blocker currently in clinical use as an antiarrhythmic
compound, as a promising scaffold for the preparation of new RNA binders. Verapamil has never been
studied as an RNA ligand and we chose it for the synthesis of a new series of RNA ligands obtained
upon conjugation of verapamil to other hits identified during screening or other known RNA-binding
motifs. This led to the synthesis of 21 new compounds. Some of these compounds demonstrated to be
very good TAR binders and ligands 2h, 3f and 3i also showed selectivity in the presence of a large
excess of both tRNA and DNA. 2h, where indole fragment was coupled to verapamil, was the most
efficient inhibitor of Tat/TAR interaction with an IC50 of 18.8 µM and the detailed study of its molecular
mechanism of interaction demonstrated that this compound binds to the bulge site of TAR efficiently
and selectively thus being a promising hit for future optimization.
Drug discovery in the field of RNA targeting is an interesting challenge for medicinal chemistry with
great potential for future therapies. Library screening showed to be an efficient approach for the
discovery of new RNA ligands bearing biological activity. Here, we thus described a screening that led
to the discovery of verapamil, a drug currently employed in the treatment of some cardiovascular
diseases, as an efficient TAR RNA binding agent. The chemical modification of this marketed drug
showed that the biological activity can be greatly improved thus leading to strong and selective binders
of HIV-1 TAR RNA. Even if the most active and selective compound 2h needs further optimization
before antiviral studies in infected cells, this work demonstrated that the study of existing drugs as
scaffolds for the preparation of efficient RNA ligands could be an interesting strategy toward original
ligands in the field of RNA targeting by small molecules.
Experimental section
Chemistry
Materials. Reagents and solvents were purchased from Merck and Carlo Erba Reagents and used
without further purification. All reactions involving air- or moisture-sensitive reagents or intermediates
were performed under an argon atmosphere. Flash column chromatography was carried out on silica gel
columns (Interchim Puriflash silica HP 15 µm) on a Puriflash XS420 system (Interchim). Analytical
thin-layer chromatography (TLC) was conducted on Sigma Aldrich precoated silica gel and compounds
were visualized by irradiation (254 nm) and/or by staining with ninhydrin and phosphomolybdic acid.
HPLC was performed using a Water Alliance 2695 pump coupled with Waters 996 photodiode array
detector and a Waters XSELECTTM CSHTM Fluoro-Phenyl (2.5 µm, 4.6x150 mm ColumnXP for
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analytical HPLC and 10 x 150 mm for semipreparative HPLC). All HPLC analyses were run at room
temperature using a gradient of CH3CN containing 0.1% TFA (eluent B) and water containing 0.1%
TFA (eluent A) from 5% to 100% of B in 30 min at a flow rate of 1 mL/min for the analytical column
1
and 3.5 mL/min for the semipreparative column. H and 13C NMR spectra were recorded on a Bruker
AC 200 MHz or a Bruker AC 400 MHz spectrometer. Chemical shifts are reported in parts per million
(ppm, δ) referenced to the residual 1H resonance of the solvent (CDCl3, δ 7.26; CD3OD δ 3.31; DMSO-
d6 δ 2.50). Splitting patterns are designated as follows: s (singlet), d (doublet), t (triplet), m (multiplet),
br (broad). Coupling constants (J values) are listed in hertz(Hz). Low resolution mass spectra (MS) were
obtained with a Brucker Daltonics Esquire 3000+ electronspray spectrometer equipped with API
ionization source. High resolution mass spectra (HRMS) were obtained with a LTQ Orbitrap hybrid
mass spectrometer with an electronspray ionization probe (Thermoscientific, San Jose, CA) by direct
infusion from a pump syringe to confirm correct molar mass and high purity of compounds.
General procedure for reductive amination (General procedure A). To a solution of 1.3 eq. of the
appropriate aldehyde (0.15 mmol) in CH2Cl2 or CH2Cl2/MeOH (1:1) (4 mL) is added 1 eq. of compound
1 (50 mg, 0.11 mmol). When necessary the pH is adjusted to pH=5-6 by addition of acetic acid. Then
NaBH3CN (14 mg, 0.22 mmol, 2 eq.) is added and the reaction mixture is stirred at room temperature
overnight. The solvent is then removed under reduced pressure. CH2Cl2 is added to the crude product
and the organic phase is washed with a solution of NaHCO3 (10%), water and brine. After drying over
MgSO4 and filtration, the solvent is evaporated under reduced pressure and the residue is purified by
flash chromatography on a silica gel column using a gradient of CH2Cl2/MeOH 9:1 as the eluent.
(±)-5-[(3,4-Dimethoxyphenethyl)methylamino]-2-(3,4-dimethoxyphenyl)-2-isopropylpentyl-N1-
(tert-butyl-1H-indolecarboxylate-3-methyl)amine (2'h).
Compound 2'h was prepared following general procedure A in the presence of tert-butyl 3-formyl-1H-
indole-1-carboxylate (36 mg). Compound 2'h was obtained as a yellow solid: 44 mg (59%); Rf = 0.25
(CH2Cl2/MeOH 95:5); 1H NMR (400 MHz, CD3OD) δ = 8.09 (d, J=8.2 Hz, 1H), 7.59 (d, J=7.7 Hz, 1H),
7.56 (s, 1H), 7.31-7.15 (m, 2H), 6.86-6.76 (m, 4H), 6.74 (d, J=1.9 Hz, 1H), 6.66 (dd, J=8.1, 1.9 Hz, 1H),
3.94 (d, J=13.7 Hz, 1H), 3.89 (d, J=14.1 Hz, 1H), 3.80-3.74 (m, 9H), 3.68 (s, 3H), 3.07-2.97 (m, 2H),
2.71-2.58 (m, 4H), 2.48 (t, J=7.6 Hz, 2H), 2.26 (s, 3H), 1.99-1.91 (m, 1H), 1.86-1.77 (m, 2H), 1.65 (s,
9H), 1.46-1.26 (m, 2H), 0.76, 0.71 (2d, J=6.8 Hz, 6H); 13C NMR (101 MHz, CD3OD) δ = 151.0, 150.5,
149.8, 149.0, 148.8, 137.1, 136.7, 133.6, 131.6, 125.5, 125.1, 123.7, 122.0, 121.7, 120.6, 120.4, 116.2,
113.7, 113.5, 113.2, 112.3, 84.9, 59.9, 58.8, 56.6, 56.5, 56.4, 56.4, 53.7, 47.8, 45.6, 42.3, 35.3, 33.0,
32.2, 28.4, 22.1, 18.5, 18.4; MS (ESI): m/z 688.27 (M+H)+ (theoretical 688.42).
(±)-5-[(3,4-Dimethoxyphenethyl)methylamino]-2-(3,4-dimethoxyphenyl)-2-isopropylpentyl-N1-
(1H-indole-3-methyl)amine (2h).
Compound 2h was dissolved in CH2Cl2 (3 mL) and TFA (195 µL; 2.55 mmol, 40 eq.) was added. The
reaction mixture was stirred at 35 °C for 3 hours. The solvent was removed under reduce pressure. The
crude product was then triturated with Et2O and after filtration, 5b was obtained as a brown solid: 35
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mg (94%); Rf = 0.3 (CH2Cl2/MeOH 93:7); 1H NMR (400 MHz, CD3OD) δ = 7.79 (d, J=7.8 Hz, 1H, h),
7.55 (s, 1H), 7.47 (d, J=8.0 Hz, 1H), 7.25-7.13 (m, 2H), 6.92-6.85 (m, 2H), 6.82-6.72 (m, 2H), 6.55-
6.46 (m, 2H), 4.56 (d, J=14.0 Hz, 1H), 4.49 (d, J=13.9 Hz, 1H), 3.84-3.73 (m, 9H), 3.63-3.51 (m, 5H),
3.32-3.05 (m, 4H), 3.04-2.87 (m, 2H), 2.79 (s, 3H), 2.08-1.73 (m, 3H), 1.61-1.43 (m, 2H), 0.71, 0.55
(2d, J=6.7 Hz, 3H); 13C NMR (101 MHz, CD3OD) δ = 150.8, 150.2, 149.8, 149.8, 138.0, 131.6, 130.1,
129.4, 128.8, 123.6, 122.3, 121.6, 121.5, 119.0, 113.7, 113.4, 113.2, 112.8, 112.6, 104.89, 58.4, 57.2,
56.5, 56.5, 56.3, 50.0, 46.9, 44.0, 40.5, 34.3, 31.0, 30.3, 19.8, 17.9, 17.7; HRMS (ESI): m/z 588.37988
(M+H)+ (C36H50N3O4 requires 588.37958).
NMR experiments
Materials and equipment
NMR experiments were conducted as described in a previous work and detailed below.[26] High
resolution NMR experiments were recorder on a BRUKER AVANCE Ultra shield DRX 500
spectrometer operating at 500.13 MHz for 1H, equipped with a temperature control unit (BCU 6.0, BVT
3000), and an inverse probe head (5mm PHTXI 1H-13C/15N Z-GRD). Proton chemical shift was
referenced internally by setting the carrier frequency on water at the center of the spectrum (4.71ppm at
13°C and 4.70ppm at 35°C). Chemical shifts (δ) are expressed in parts per million (ppm). All NMR
experiments were carried out using standard pulse sequences supplied by the spectrometer manufacturer
(BRUKER). 1D and 2D spectra were processed using TOPSPIN 2.1 NMR Software (BRUKER).
For the preparation of all NMR samples, a folding of TAR RNA (100 μM) was performed in the
appropriate buffer (vide infra) as described in material and methods section of the paper. After refolding,
the NMR sample (alone or with the appropriate amount of ligand) is incorporated into a Shigemi NMR
tube.
1H NMR imino proton spectra were recorded in a H2O/D20 (90/10) buffer containing 20 mM phosphate
and 50 mM NaCl at 286°K (13°C) by using a WATERGATE 3-9-19 water suppression. Each proton
NMR spectrum was acquired using 10.964 KHz Spectral Width (SW), 64K complex data point,
acquisition time (aq) of 2.98 s, relaxation delay (D1) of 1s, number of scan (ns) between 1000 and 2000,
number of dummy scan (ds) 4 and a 90° flip angle pulse width. Water suppression was achieved using
WATERGATE pulse sequence. Gradient pulse were sine shape (SINE.100), 1.5 ms long (P16) with 100
μs gradient recovery delay (D16) and strengths set to 8.44 Gauss.cm-1 (20%). A 45.6 μs delay (D19)
was used for binomial water suppression. Prior to Fourier transformation, the fids were multiplied by an
exponential line broadening function of 3 Hz.
gs-TOCSY Phase sensitive (States – TPPI mode) experiments using MLEV 17 pulse sequence for spin
lock were recorded in a D20 buffer (50 mM NaCl, 20 mM phosphate, pH 7.4) at 308K (35°C) by using
a WATERGATE 3-9-19 water suppression. Each TOCSY 2D NMR spectrum was acquired with a
spectral width of 5 KHz in both dimension, 2K complex data point in F2, 256 t1 increments (between
32 and 64 scans by increment ) in F1, 0.20 s for aq and D1 of 2 s. MLEV 17 pulse sequence for spin
lock was set to 60 ms. Water suppression was achieved using WATERGATE pulse sequence. Gradient
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pulse were sine shape (SINE.100), 1.5 ms long (P16) with 100 μs gradient recovery delay (D16) and
strengths set to 8.44 Gauss.cm-1 (20%). A 100 μs delay (D19) was used for binomial water suppression.
Prior to Fourier transformation a QSINE window function (SSB =2) was applied in both dimension and
the data were zero filled and linear predicted (NC=32) to 1K data points in F1.
Molecular Modeling and Docking. The experimental structure of RNA TAR complexed with ligand
rbt158 (pdb code 1UUI) was used as input for our calculations. As described in our previous work,[27]
for docking with AutoDock, polar hydrogen atoms, Kollman united charges and solvent parameters
were
applied
to
the
RNA
using
pmol2q
script
(http://www.sourcefiles.org/Scientific/Biology/Proteins/pmol2q_2.3.0.tar.gz). This script converts the
.pdb file format to of the RNA template to the .pdbqt file format that is compatible with AutoDock
program version 4 (http://autodock.scripps.edu/). The compounds .pdbqt files compatible with
Autodock program version 4 were prepared from the .pdb files obtained from http://ligand-
expo.rcsb.org/ (NMY).
RNA-ligand molecular dockings were conducted using AutoDock program version 4. The rotational
bonds of the ligand were treated as flexible, whereas the receptor was kept rigid. Grid box was fixed in
order to include the entire RNA sequence. RNA-ligand interactions were analyzed and visualized using
Discovery Studio Visualizer version 4.1 (http://accelrys.com/products/discovery-studio/).
Biochemistry
Unless otherwise stated, all reagents and solvents were of analytical grade and from Sigma (St Louis,
U.S.A.). HEPES and all inorganic salts for buffers were purchased from Merck (molecular biology
grade). RNA and DNA oligonucleotides were purchased from IBA GmbH and used without further
purification.
Oligonucleotide
sequences:
TAR
(5'-Alexa488-
CCAGGUCUGAGCCUGGGAGCUCCCUGG-3'), DNA (5'-CGTTTTTATTTTTGC-3' and 5'-
GCAAAAATAAAAACG-3'). A mixture of pre- and mature yeast tRNAs (containing over 30 different
species) was purchased from Sigma (type X-SA). Stocks of tRNAmix can be quantified in its native
form (without base hydrolysis) using an extinction coefficient of 9640 cm-1 M-1 per base.
Buffers: All buffers were filtered through 0.22 μm Millipore filters (GP ExpressPLUS membrane). A
small aliquot (100 mL) was first filtered and then discarded to avoid any contaminants that might be
leached from the filter. The solutions to be used in the fluorescence experiments were prepared by
diluting the concentrated stocks in Milli-Q water and filtered again as described above. All standard
fluorescence measurements were performed in buffer A (20 mM HEPES (pH 7.4 at 25°C), 20 mM NaCl,
140 mM KCl and 3 mM MgCl2). FRET experiments were performed in buffer B (50mM tris buffer (pH
7.4 at 25°C), 20 mM KCl and 0.005% Tween 20). For competitive experiments in the presence of a
tRNA, a mixture of pre- and mature yeast tRNAs (containing over 30 different species from baker's
yeast (S. cerevisiae, Sigma, type X-SA)) was added to buffer A to obtain a 100-fold nucleotide excess
regarding TAR RNA.
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Inhibition of Tat/TAR interaction. As previously described,[28] Ligand solutions and RNA (40 nM
working solution) were prepared as described above in buffer B (50mM tris buffer (pH 7.4 at 25°C), 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 µL) was then added to a well of a non-treated
black 384-well plate, in triplicate, and 30 µL 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.
Binding studies: Ligand solutions were prepared as serial dilutions in buffer A at a concentration four
times higher than the desired final concentration to allow for the subsequent dilution during the addition
of the RNA solution. Binding experiments were performed in 384-well plates (Greiner bio-one) in a
final volume of 60 µL using a 5070 EpMotion automated pipetting system (Eppendorf). Each
experiment was performed in duplicate. 10 nM TAR beacon was used in each well. Each ligand was
added in 15 dilutions (from 0.030 nM to 0.5 µM) and the fluorescence increase measured after 4 hours.
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 10 times with a 500 μs integration time and
averaged. Binding data were analyzed using Graphpad Prism 5 software. Unless otherwise stated,
binding profiles were well modeled using a simple model assuming the one to one stoichiometry.
Refolding of the RNA was performed using a thermocycler (ThermoStat Plus Eppendorf) as follow: the
RNA, diluted in 1 mL of buffer A at a concentration of 200 nM, was first denatured by heating to 90°C
for 2 min, then cooled to 4°C for 10 min followed by incubation at 20°C for 15 min. After refolding, the
RNA was diluted to a working concentration of 10 nM through addition of the appropriate amount of
buffer A. The tube was mixed and 50 μL of the RNA solution was added to each well containing ligand.
This subsequent dilution lowered the final RNA concentration to 5 nM. To study the temperature
dependence, the plates were incubated after overnight equilibrium at different temperature ranging from
5°C to 35°C.
Data analysis: Binding data (KD and FRET experiments) were analyzed using Prism 5 (GraphPad
Software) by nonlinear regression following the equation:
Y = Bottom + (Top – Bottom)/(1 + 10[(LogIC50-X)xHillSlope])
KD values were converted to G° values as G°= -RTln KD. Binding data were analyzed using the
nonlinear least squares numerical solver-based binding data global analysis program GraphPad, in which
the calculated binding surface is obtained using a numerical constrained optimization chemical
equilibrium solver. Unless otherwise stated, binding profiles were well modeled using a simple model
assuming the one to one stoichiometry. A higher initial fluorescence value is observed in the presence
of tRNA, which is consistent with the modification of the polarity of the solvent and a small fluorescence
of the tRNA mixture.
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For thermodynamic analysis, G° values were plotted versus T for the three-parameter fit5. Nonlinear
regression in Prism 4 (GraphPad Software) was used to fit the following equation to the data:
G°T = H°Tr + CP (T - Tr) – TS°Tr - TCP ln (T/Tr)
where Tr is a constant reference temperature (in our study Tr = 293.15 K), and the three fit parameters
are H°Tr, the change in enthalpy upon binding at Tr; S°Tr, the change in entropy upon binding at Tr;
and CP, the change in heat capacity. Starting values for the three parameters did not affect the final
values. CP was assumed to be independent of temperature; inclusion of a CP/T term in the analysis
did not improve the quality of the fits and gave larger standard errors for the returned parameters.
H°T and S°T were calculated by using:
H°T = H°Tr + CP (T - Tr) and S°T = S°Tr + CP ln (T/Tr).
Salt dependence of KD was analyzed by the following equation:
log(KD) = log(Knel) - Zψ log [KCl]
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 (essentially the number of intermolecular ion pairs) and ψ is the fractional
probability of a counterion being thermodynamically associated with each phosphate of the RNA
number of cations. Knel and Zψ were treated as fitting parameters.
Acknowledgments
Thanks are due to J. M. Guigonis (Plateforme Bernard Rossi, CEA TIRO, Nice, France) for HRMS
analyses. PhD fellowship (to C.M.) is supported by grant from Agence Nationale de la Recherche (ANR-
17-CE18-0009).
Keywords
RNA ligands, TAR RNA, Verapamil, inhibition, RNA binding
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Figures and Schemes
Figure 1. A) Primary and secondary structure of HIV-1 TAR RNA in complex with Tat, Cyclin T1 and
CDK9. Residues 18-44 that were used in this study are indicated; B) chemical structure of Verapamil.
Figure 2. KD values () of compounds 2a-i, 3a-i and 5a-c against HIV1 TAR RNA 27-mer fragment
labeled at the 5'-end with Alexa488. Selectivity for TAR in the presence of 100 eq. of tRNA () and of
DNA () is reported. KD values were determined from duplicates performed over three independent
experiments using the change in fluorescence intensity. Error bars represent the standard deviation
determined from three independent experiments.
Figure 3. A) and B) Stacked plot of 1D NMR spectra of the imino region of 50 µM TAR RNA with
increasing concentration of 1 (A) and 2h (B). The spectra were collected at 286K in a H2O/D2O (90/10)
buffer (20mM phosphate and 50mM NaCl, pH 7.4). C) and D) 2D-TOCSY spectra showing pyrimidine
H5–H6 cross-peaks for TAR. Black: free TAR (100 µM); red: 1 (C) or 2h (D)/TAR complex at a ratio
5:1. Arrows indicate chemical shift changes on ligand binding. The spectra were acquired at 308K in a
D2O buffer (50mM NaCl, 20mM phosphate, pH 7.4); E) and F) 27-mer TAR RNA primary and
secondary structure illustrating the nucleotides that are the most affected by the presence of 1 (E) or 2h
(F).
Figure 4. Docking of 1 (A) and 2h (B) with the 27-mer TAR hairpin loop performed by using autodock
4, in which the grid boxes were fixed on the entire RNA sequence.
Scheme 1. Synthetic procedure for the preparation of reduced verapamil 1 as well as of reductive
amination products 2a-i. Reagents: a) LiAlH4, THF, 60°C, overnight; b) aldehyde, NaBH3CN, CH2Cl2,
r.t., overnight; c) TFA, CH2Cl2, r.t., overnight.
Scheme 2. Synthesis of amide-linked conjugates 3a-i. Reagents: a) carboxylic acid, HBTU, DIPEA,
CH2Cl2, r.t., overnight; b) TFA, CH2Cl2, r.t., overnight.
Scheme 3. Synthetic procedure for the synthesis of compound 4 bearing a succinamic acid linker and
of amide-linked derivatives 5a-c. Reagents: a) succinic anhydride, CH2Cl2, r.t., overnight; b) amine,
HOBt, EDC, Et3N, CH2Cl2, r.t., overnight; c) TFA, CH2Cl2, r.t., overnight.
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Figure 1
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Figure 2.
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Figure 3.
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Figure 4.
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Scheme 1.
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Scheme 2.
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Scheme 3.
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Table 1. Thermodynamic parameters for ligands/TAR RNA interaction and IC50 for the inhibition of
Tat/TAR interaction.
G° (kJmol-1) H°[a] (kJmol-1) TS°[a] (kJmol-1) G°nel[b] (kJmol-1)
C50 (µM)
1870 ± 12
no inhibition
18.8 ± 1.1
302 ± 5
ID
-32.1 ± 2.7
-32.6 ± 1.8
-34.8 ± 3.1
-36.5 ± 3.1
-36.0 ± 3.2
-50.5 ± 4.7
-16.1 ± 1.2
-33.3 ± 1.6
-35.1 ± 2.9
-24.2 ± 2.1
-18.4 ± 1.2
16.5 ± 1.5
1.5 ± 1.0
-28.8 (90%)
not determined
-34.4 (99%)
-32.8 (90%)
-35.0 (97%)
1
2a
2h
3f
3i
1.4 ± 1.0
11.8 ± 1.2
404 ± 7
[a] Determined by temperature effect experiments by using the equation G°T = H°Tr + CP-(T-Tr)-TS°Tr-TCPln(T/Tr).
See the Supporting Information for definitions and further details. [b] Determined by salt effect experiments by using the
equation log(KD)=log(Knel)-ZYlog[KCl]. See the Supporting Information for definitions and further details. The percentage
of non-electrostatic interactions (G°nel/G°) is given in parentheses.
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