Discovery and Structure-Based Optimization of 2-Ureidothiophene-3-carboxylic Acids as Dual Bacterial RNA Polymerase and Viral Reverse Transcriptase Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.6b00730

We are concerned with the development of novel anti-infectives with dual antibacterial and antiretroviral activities for MRSA/HIV-1 co-infection. To achieve this goal, we exploited for the first time the mechanistic function similarity between the bacterial RNA polymerase (RNAP) "switch region" and the viral non-nucleoside reverse transcriptase inhibitor (NNRTI) binding site. Starting from our previously discovered RNAP inhibitors, we managed to develop potent RT inhibitors effective against several resistant HIV-1 strains with maintained or enhanced RNAP inhibitory properties following a structure-based design approach. A quantitative structure-activity relationship (QSAR) analysis revealed distinct molecular features necessary for RT inhibition. Furthermore, mode of action (MoA) studies revealed that these compounds inhibit RT noncompetitively, through a new mechanism via closing of the RT clamp. In addition, the novel RNAP/RT inhibitors are characterized by a potent antibacterial activity against S. aureus and in cellulo antiretroviral activity against NNRTI-resistant strains. In HeLa and HEK 293 cells, the compounds showed only marginal cytotoxicity.

Full text of DOI:10.1021/acs.jmedchem.6b00730

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Article
Discovery and Structure-Based Optimization of 2-
Ureidothiophene-3-Carboxylic Acids as Dual Bacterial RNA
Polymerase and Viral Reverse Transcriptase Inhibitors
Walid A. M. Elgaher, Kamal Kant Sharma, Jörg Haupenthal, Francesco
Saladini, Manuel Pires, Eleonore Real, Yves Mély, and Rolf W. Hartmann
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00730 • Publication Date (Web): 24 Jun 2016
Downloaded from http://pubs.acs.org on July 6, 2016
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Discovery and StructureꢀBased Optimization of 2ꢀUreidothiopheneꢀ3ꢀCarboxylic Acids
as Dual Bacterial RNA Polymerase and Viral Reverse Transcriptase Inhibitors
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Walid A. M. Elgaher †
,
Kamal K. Sharma ‡
,
Jörg Haupenthal †
,
Francesco Saladini,§ Manuel
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,†
Pires,‡ Eleonore Real,‡ Yves Mély ‡
,
and Rolf W. Hartmann*
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†Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Department of Drug Design and
Optimization & Saarland University, Pharmaceutical and Medicinal Chemistry, Campus E8.1, 66123
Saarbrücken, Germany
‡
Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg,
Faculté de pharmacie, 74 route du Rhin, 67401 Illkirch, France
§Department of Medical Biotechnologies, University of Siena, Viale Mario Bracci 16, 53100 Siena,
Italy
ABSTRACT
We are concerned with the development of novel antiꢀinfectives with dual antibacterial and
antiretroviral activities for MRSA/HIVꢀ1 coꢀinfection. To achieve this goal, we exploited for the first
time the mechanistic function similarity between the bacterial RNA polymerase (RNAP) “switch
region” and the viral nonꢀnucleoside reverse transcriptase inhibitor (NNRTI) binding site. Starting
from our previously discovered RNAP inhibitors, we managed to develop potent RT inhibitors
effective against several resistant HIVꢀ1 strains with maintained or enhanced RNAP inhibitory
properties following a structureꢀbased design approach. A quantitative structure–activity relationship
(QSAR) analysis revealed distinct molecular features necessary for RT inhibition. Furthermore, mode
of action (MoA) studies revealed that these compounds inhibit RT nonꢀcompetitively, through a new
mechanism via closing of the RT clamp. In addition, the novel RNAP/RT inhibitors are characterized
by a potent antibacterial activity against S. aureus and in cellulo antiretroviral activity against NNRTIꢀ
resistant strains. Using HeLa and HEK 293 cells the compounds showed only marginal cytotoxicity.
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INTRODUCTION
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Human immunodeficiency virus type 1 (HIVꢀ1) is the causative agent of the acquired immune
deficiency syndrome (AIDS), which is one of the major lethal infectious diseases endangering
humanity. In 2014, more than 37 million people worldwide were HIVꢀinfected with a mortality of
approximately 1.2 million.1 The HIVꢀ1 retrovirus targets the CD4 cells resulting in an impairment of
the immune system. In consequence, HIV patients are a defenseless prey for bacterial infections e.g.
tuberculosis (TB) and methicillinꢀresistant Staphylococcus aureus (MRSA).2–4 MRSA coꢀinfection is
characterized by a high incidence rate.5 In addition, the emergence of multidrug resistant MRSA
markedly increased among HIV patients.6,7 Current treatment of HIV infection requires a combination
of at least three antiretroviral drugs. This highly active antiretroviral therapy (HAART) permits
efficient suppression of virus replication and inhibits disease progression. However, the evolution of
antiretroviral drug resistance is still presenting an intractable problem due to the high viral mutation
rate and nonꢀcompliance to antiretroviral therapy (ART).8–10 Although the FDA recently approved a
combined ART as one pill daily that could improve patient adherence,11 the resistance issue is not yet
solved. Treatment of MRSA/HIVꢀ1 coꢀinfection is even more challenging. In addition to the above
mentioned issues, administration of antibiotics should take into consideration the ongoing prevalence
of resistant bacteria in HIVꢀ1 populations,6,7 as well as potential drug interactions between
antiretroviral and antibacterial agents.12 Hence, there is an urgent need for a one compound therapy of
MRSA/HIVꢀ1 coꢀinfections. Combining antibacterial and antiretroviral properties in a single molecule
should be beneficial in different aspects: it will improve patient adherence to ART by reduction of
medications’ regimen,11 and consequently decrease the probability of treatment failure. Moreover,
prescribing such antiꢀinfective medication will avoid drug interactions,12 and lower the costs of care as
well.13
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HIVꢀ1 RT is a key target for ART. It reverseꢀtranscribes the singleꢀstranded viral RNA to doubleꢀ
stranded DNA, which is essential for virus replication.14 There are two main classes inhibiting RT:
nucleoside RT inhibitors (NRTIs) and NNRTIs. NRTIs are nucleotide analogues that are incorporated
into viral DNA leading to chain termination. Through binding to an allosteric binding site, NNRTIs
inhibit RT nonꢀcompetitively.15,16 In bacteria, RNAP is an essential enzyme for bacterial viability17
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and has consequently become a pivotal drug target. It is responsible for transcription by converting
doubleꢀstranded DNA to singleꢀstranded RNA, which is a prerequisite for protein synthesis.
Consequently, there is a functional relationship between bacterial RNAP and viral RT. In this context,
the clinically used RNAP inhibitor rifampicin does also inhibit viral RT.18 On the other side, HIVꢀ1
RT monoclonal antibodies were found to inhibit E. coli RNAP, indicating structural and functional
similarities between both enzymes.19 Recently, a new RNAP binding site, the “switch region”, was
discovered with a mechanistic function close to that of the NNRTI binding site.20 However, these
important insights have not yet been exploited for drug discovery.
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In previous works, we developed new classes of RNAP “switch region” inhibitors using different drug
design approaches.21,22 Their mode of action was confirmed by biophysical methods and mutation
studies.23 Beside their activity against MRSA, the compounds showed no crossꢀresistance with
rifampicin and a low rate of resistance development in E.coli TolC.22,23 Thus, our RNAP inhibitors
should be a good starting point for designing such dual acting antiꢀinfectives. For testing our
compounds for RT inhibition, we applied our recently developed Förster resonance energy transfer
(FRET) based polymerization assay.24 Nevirapine (NVP) as an NNRTI and zidovudine (AZTꢀTP) as
an NRTI were used as references. For hit optimization, we employed a structureꢀbased design
approach. Indeed, we succeeded in developing the first antiꢀinfectives with dual antibacterial and
antiretroviral activities.
RESULTS AND DISCUSSION
Design of Compounds and SAR
In order to identify the privileged scaffold for HIVꢀ1 RT inhibition among various classes of switch
region binding RNAP inhibitors, we selected eight compounds representing four regioisomeric
ureidothiophenes (Chart 1).
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Chart 1. Structures of the ureidothiophene regioisomers used for finding the privileged scaffold for
RT inhibition.
Results revealed that only compound 4 (class II) showed a high RT inhibitory activity (IC50 = 0.9 µM,
Table 1). This indicates that the introduction of an additional chlorine substituent in the 3ꢀposition of
the phenyl ring leads to a dramatic increase in potency (4 vs 3), which is more pronounced than
observed for RNAP inhibition.21,22 Comparing class II compounds which are highly similar to
compounds of class I (the only difference is the position of the S in the thiophene ring) and had shown
similar RNAP inhibition,22 strong differences regarding the inhibition of RT were observed, too. This
finding can be attributed to the different binding sites and the slight differences in the molecular
properties of the ligands. Comparing the RNAP “switch region” and the RT allosteric binding site, it
becomes apparent that both pockets are mainly hydrophobic. The RNAP “switch region” is Uꢀshaped
with a cavity volume of ~ 500 Å3.25 The RT allosteric binding site is highly flexible regarding size, i.e.
it is nonexistent in absence of NNRTIs,26,27 whereas it possesses a cavity volume of 620–720 Å3 in
presence of NNRTIs.14 Analyzing the molecular similarity between class I and II in silico by a 4ꢀpoint
pharmacophore fingerprint method28 and the Tanimoto coefficient (TC) as a similarity metric, class I
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showed partial similarity to II (TC = 0.75). This is in contrast to the results obtained recently using a 3ꢀ
point pharmacophore as a fingerprint.22 As 4 was the only compound showing a strong inhibition
against both enzymes, we focused on class II as the most appropriate scaffold for the development of
dual RNAP and RT inhibitors.
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Table 1. In Vitro Inhibitory Activities of Compounds 1–8 against HIVꢀ1 RT and E. coli RNAP
Compound % inhibition of HIVꢀ1 RT at 1 µM IC50 E. coli RNAP (µM)
12a
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> 100
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> 200
100
a% inhibition at 25 µM; bIC50 value (µM); c% inhibition at 10 µM.
In the next step, we varied the substituents at the ureido motif of 4 (compounds 9–11, Table 2). While
increasing bulkiness and lipophilicity at the ureido motif led to a potent RNAP inhibitory activity
(10),21,22 the RT inhibitory activities observed for compounds 9ꢀ11 were surprising. Exchange of the
ethyl substituent by a hydrogen (9) as well as by a sterically demanding benzyl substituent (10)
reduced activity drastically. Activity was restored by omitting the methylene spacer of the R1
substituent in 9, i.e. exchanging the benzyl group by a phenyl (11). As compound 11 shows a better
ligand efficiency29 and ligandꢀlipophilicity efficiency29 compared to 4 (LE: 0.32 vs 0.29; LLE: 2.51 vs
2.33), the subsequent structure modifications were performed based on this compound.
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Table 2. In Vitro Inhibitory Activities of Compounds 9–20 against HIVꢀ1 RT and E. coli RNAP
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Compound
R1
R2
H
IC50 HIVꢀ1 RT (µM) IC50 E. coli RNAP (µM)
0.9
64a
63a
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43
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H
H
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0.8
64a
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H
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H
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NVP
AZTꢀTP
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0.1
7.0
> 200
n.d.b
a% inhibition at 25 µM; bnot determined.
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In order to reduce the number of compounds to be synthesized, we followed a structureꢀbased design
strategy. Using a high resolution coꢀcrystal of the RT–NVP complex (PDB code 1VRT),14 we
performed an induced fit molecular docking of 11 into the RT allosteric binding site. The compound
adopted a Uꢀshape stabilized by an intramolecular hydrogen bond between the ureido NH and the
carboxylate group forming a six membered ring (Figure 1). The thiophene core is located lowermost
near the cavity entrance, while the 3,4ꢀdichlorophenyl and the Nꢀphenyl moieties are positioned
uppermost overlapping the two pyridine rings of NVP. Four hydrophobic interactions characterize the
binding of 11 to the NNRTI binding pocket. Two arene–H interactions can be observed between
Leu100 and the thiophene core as well as Leu100 and the 3,4ꢀdichlorophenyl moiety. The latter also
binds to Val106 through an arene–H interaction from the other side. The fourth interaction is an edgeꢀ
toꢀface (Tꢀshaped) arene interaction between Trp229 and the mꢀposition of the Nꢀphenyl group.
Binding is further strengthened through a hydrogen bond interaction between Lys101 and the
carboxylate C=O group.
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Figure 1. (A) Docking pose of 11 (magenta) compared to NVP (orange) in the RT allosteric binding
site: hydrophobic surface (green), polar surface (pink); (B) 2D ligand interactions of 11.
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According to the docking results, all three aromatic motifs of 11 are essential for binding. Moreover,
no π–π contacts between 11 and the frequently mutated amino acids Tyr181 and Tyr18810 were
observed. Interestingly, mutations of the amino acids in contact with 11 are known to result in high
(Trp229, Val106, Leu100) and low fitness costs (Lys101).10,30–32
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Since Trp229, Phe227 and Leu234 belong to the polymerizationꢀessential “primer grip” and are
known to show low mutation frequency,14,32,33 we further optimized our compounds for interaction
with these highly conserved residues. To achieve this goal, we exploited the space between the pꢀ
position of the Nꢀphenyl moiety and this conserved region by introduction of diverse substituents with
varying bulkiness, electronic and lipophilic properties (12–20, Table 2). Results indicate that except
for compound 14, the new modifications improve the activity up to 12ꢀfold (18). The RT inhibitory
activity of compound 18 is comparable to that of NVP and significantly higher than that of AZTꢀTP.
Inspection of the substituent constants and molecular properties of the new compounds (Table S1)
reveals that hydrophilic and hydrogen bond donor/acceptor substituents at the Nꢀphenyl group are
important for activity. Obviously, activity can be enhanced to a certain extent by increasing the
conformational flexibility of the compounds. Interestingly, introduction of aromatic moieties (phenyl
or 1,2,4ꢀtriazolyl) at the sulfonamide or the acetamide group negatively affects RT inhibitory activity
(19 vs 18, and 16 vs 15) leading to the hypothesis that too bulky substituents at the ureido motif are
not favorable for RT inhibition. These findings are in agreement with our docking model (Figure 1), as
these aryl motifs elicit a steric clash with the amino acid residues (Tyr188, Phe227, Trp229 and
Leu234) lining the deep side of the NNRTI binding pocket. As a consequence of this, they either are
shifted in the binding cavity outward or adopt another conformation leading to lower binding
interaction.
For further SAR elucidation, we calculated a QSAR model for compounds 11–20 using a multiple
regression analysis
pIC50 = 4.83 + 1.15 PC+ ꢀ 0.12 vsa_don ꢀ 0.27 SMR
(n = 10, R2 = 0.94, RMSE = 0.13)
(1)
Where pIC50 is ꢀlog IC50, PC+ is the total positive partial charge, vsa_don is the sum of van der Waals
surface areas of pure hydrogen bond donors, and SMR is molecular refractivity.
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Equation 1 reveals a role of electrostatic and steric interactions for activity. Increasing the positive
partial charge seems beneficial, whereas the number of hydrogen bond donor atoms at the Nꢀphenyl
moiety should be kept to a minimum (0–1 atoms). The inverse correlation with molecular refractivity
underlines the importance of less polarizable and nonꢀbulky substituents at the ureido motif for RT
inhibition.
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Regarding RNAP inhibition, the modifications were tolerated or even enhanced the activity (12 and
19). This could be attributed to the flexibility of this class of compounds and their ability to adopt
more than one binding mode within the RNAP “switch region”.23
Notably, as NVP shows no RNAP inhibitory activity, this stresses that the 2ꢀureidothiophenꢀ3ꢀ
carboxylic acids present a privileged structure for dual RNAP/RT inhibition. The fact that the new
inhibitors show different activity profiles against the two target enzymes, resulting in compound 10 as
the most potent RNAP inhibitor and 18 as the most active RT inhibitor, indicates specificity of the
compounds and excludes promiscuity.
Mode of Action on RT Polymerization
To characterize the mode of action through which our compounds inhibit RT, we monitored first their
effects on the binding parameters and orientation of RT on a model primer/template (p/t) duplex. To
this end, we used a FRET assay with RT labeled by Alexa488 and a p/t duplex labeled by
carboxytetramethylrhodamine (TAMRA).34 The labeled RT mutant was found to bind to the p/t duplex
with an affinity of ~3 nM (Figure S1 and Table S2), in good agreement with values reported for DNA
p/t sequences in the literature (Table S2).35 Interestingly, similar affinities as well as no change in RT
orientation on the duplex were observed in the presence of compounds 4, 11–13, and 15–20, indicating
that these compounds marginally affected the binding of RT to the p/t duplexes, as anticipated for
NNRTIs (Table S2).
In a next step, we investigated whether these compounds had the same mode of action as NNRTIs. As
shown above, the latter bind usually to the hydrophobic pocket in the palm subdomain of p66 and
inhibit RT via an allosteric mechanism,26,27,36 forcing the p66 thumb into an open extended position in
respect to the finger domain, which prevents deoxyribonucleotides (dNTPs) incorporation37 (Figure
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S2C). In order to monitor the effect of our compounds on the relative motions of the thumb and finger
domains, we developed a fluorescenceꢀbased assay with a RT double mutant where both Lys287 and
Trp24 residues were replaced by cysteine residues.
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By labeling the two Cys residues with Bodipy dyes, the intramolecular distance changes associated to
the motion of RT’s thumb and finger could be monitored by fluorescence spectroscopy. This distance
is a key parameter as the relative separation between the thumb and finger subdomains defines the
opening and closing of the clamp that holds and positions p/t duplexes at the polymerase active site
(Figure S2B). A precise positioning of the primer 3ʹꢀend with respect to the polymerase active site
(Asp110, Asp185 and Asp186) is required for the catalytic addition of a nucleotide. Due to the close
proximity of the finger and thumb in the absence of p/t (Figure S2A), the two Bodipy probes were
observed to quench each other, providing a low emission (Figure 2A).
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Figure 2. Conformational changes of bodipyꢀlabeled RT p66 W24C+K287C mutant. (A) Emission
spectra of 60 nM bodipyꢀlabeled RT in the absence (black line) or in the presence of 160 nM p/t
duplex (red line) or 10 µM NVP (green line); (B) Graph representing the ratio between the
fluorescence intensities of the 100 nM doublyꢀlabeled RT in presence of either the p/t duplex (160
nM) or NVP (10 µM) or the tested compounds (3 µM) to the fluorescence intensity of the free form.
Red line corresponds to the best fit with linear regression (y = m*x + b), with values of slope (m) =
0.38 (±0.04) µM and intercept (b) = 0.25 (±0.03). Excitation wavelength was 490 nm and ratios were
calculated at emission wavelength of 510 nm; (C) Stoppedꢀflow kinetic traces of 60 nM doubly
labeled RT in the absence (red trace) and the presence of 10 µM NVP (green trace) or compound 11
(blue trace). All traces were fitted by eq 2 and the rate constant values given in Table S2. Excitation
wavelength was 490 nm and emission was collected by using a 525/50 nm band pass emission filter.
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In contrast, a 1.6ꢀfold increase in Bodipy fluorescence was observed on binding to p/t (Figure 2A and
2B), in line with the expected increase in the distance between the thumb and finger (Figure S2B). An
even higher fluorescence increase (twoꢀfold) was observed in the presence of NVP (Fig 2A and 2B),
as anticipated from NVP’s ability to further separate the two RT subdomains and block them in this
open position (Figure S2C). Altogether, the data obtained with the Bodipyꢀlabeled RT were fully
consistent with the molecular structures of RT, validating this assay as a convenient means to
investigate the inhibitory mechanisms of the synthesized RT inhibitors. In contrast to NVP and p/t
duplex, a decrease in Bodipy fluorescence was observed with all tested compounds (Figure 2B). Since
no fluorescence quenching was observed when free Bodipy was titrated to the compounds (data not
shown), these compounds probably exerted an effect opposite to NVP, decreasing the distance
between thumb and finger subdomains. Therefore, the compounds likely exert their inhibitory activity
by blocking the RT clamp in a closed conformation.
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To get further information on the inhibition mechanism of our compounds, we monitored the changes
in fluorescence in real time by stoppedꢀflow (Figure 2C and Table S2). Using NVP as a control, a
monoꢀexponential increase of Bodipy emission was observed (Figure 2C and Table S2), suggesting
that the NVPꢀinduced opening of RT clamp occurs in a single step. The kinetic traces were fitted by
using
obs (t−t0))
I(t) = IF − (IF − I0 )e(−k
(2)
where t0 is the dead time, kobs is the observed kinetic rate constant and I0 and IF are the fluorescence
intensities before and after addition of inhibitors, respectively. The I0 value was obtained from the
steadyꢀstate fluorescence spectrum of the doubly labeled mutant and was thus fixed.
Similarly, a monoꢀexponential decrease in Bodipy emission was observed with all compounds (Figure
2C and Table S2), suggesting that they induce the closing of RT clamp, also in a single step. The
kinetic rate constant values of all compounds were found to fluctuate in a narrow range, indicating that
they exhibit similar mechanisms of RT inhibition. Interestingly, a clear correlation could be observed
between the closing of the clamp and the IC50 values (Figure 2B). This indicates that the inhibitory
activity of the compounds is related to their ability to efficiently close the RT clamp and thus, hinder
the incorporation of dNTPs.
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Antibacterial and Antiretroviral Activities
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We complemented our in vitro results by firstly evaluating the activity of the new RNAP/RT inhibitors
against S. aureus. Results revealed a high antibacterial effect for the majority of the compounds with
minimum inhibitory concentrations (MICs) between 3–13 µg/mL (Table 3). Markedly, these values
correlate with the corresponding RNAP inhibitory activities. Secondly, we applied a cellular
infectivity assay to assess the antiretroviral activity of our compounds. This assay is based on the
infection of HeLa cells by a third generation of pseudoviral particles,38 mimicking the early steps of
the HIVꢀ1 life cycle. Most of the tested compounds exhibited good antiretroviral activities in the low
micromolar range (Table 3). Noticeably, a marked difference was observed in the activity profiles
when comparing the in vitro and in cellulo results: The compounds turned out to be less active in the
cellular system. This might be due to the lipophilic nature of the compounds leading to an inefficient
permeation into the cells, or an interaction with other proteins in the antiviral assay leading to a
lowered concentration at the target enzyme.
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Next, we examined the effect of our compounds on cell viability in two different cell lines (HeLa and
HEK 293) and found them to display only marginal or no cytotoxicity (Table 3).
Table 3. Antibacterial, Antiretroviral and Cytotoxic Activities of the Dual RNAP/RT Inhibitors.
MIC S. aureus
IC50 Viral infectivity
% Inhibition of HeLa cell
LD50 HEK 293
Compound
(µg/mL)
(µM)
27
viability at 25 µM
cells (µM)
10
6
23
0
58
61
4
20
n.i.a
11
12
13
17
18
4
0
68
8
35
12
6
80
25
39
30
86
30
0
> 100
n.d.b
n.d.b
3
94
19
n.d.b
n.d.b
13
> 100
20
ano inhibition; bnot determined.
Antiretroviral Activity in NNRTI Resistant HIVꢀ1 Strains
Based on the docking study, the binding mode of our new NNRTIs could be advantageous regarding
the resistance issue. The numerous interactions between our ligands and the NNRTI binding pocket,
especially those with the highly conserved amino acids, should only be moderately affected by the
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single site mutations that result from using other NNRTIs and are responsible for resistance.9,10,30 To
verify this point and to get more insight into the binding mode of our compounds, we tested the
antiviral activity of compounds 11, 18 and 19 against the HIVꢀ1 wildꢀtype (WT) NL4ꢀ3 and a panel of
strains with multiple resistance mutations to clinically used NNRTIs in a TZMꢀbl cell line based
phenotypic assay (Table 4).
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Table 4. Antiretroviral Activity of 11, 18, 19 and Reference NNRTIs against HIVꢀ1 WT (114) and
NNRTIsꢀResistant Strains (12237, 12235, 12231, 12229).
IC50 (µM)
Fold change in susceptibilitya (biologicalb or clinicalc cutꢀoff)
12237d
(V106I, Y181C,
G190A, H221Y)e
1
12235d
(A98G, K101E,
Y181C, G190A)e
3
12231d
(K103N, V179F,
Y181C)e
> 4
12229d
(L100I, K103N,
H221Y)e
> 4
Compound
114d
NL4ꢀ3 (WT)
5
5
11
18
1.5
3.5
> 4
> 4
15
> 1.5
1
> 1.5
> 1.5
19
NVP
EFV
ETR
RPV
0.1 f
> 200 (4.5)b
26 (3)b
6.0 (2.9)c
3.5 (2)b
> 200 (4.5)b
> 200 (3)b
15 (2.9)c
22 (2)b
> 200 (4.5)b
90 (3)b
8.8 (2.9)c
2.3 (2)b
> 200 (4.5)b
> 200 (3)b
6.8 (2.9)c
6.3 (2)b
0.002 f
0.005 f
0.0009 f
aResistant virus IC50/WT virus IC50 ratio calculated with the Phenosense assay; bThe biological cutꢀoff
was determined by in vitro analysis of multiple viral variants not exposed to the drug and defined as
the mean foldꢀchange plus two standard deviations with respect to the reference viral strain. It is used
c
when a clinical cutꢀoff has not been established; The clinical cutꢀoff was determined by statistical
analysis of clinical data and is defined as the foldꢀchange value corresponding to loss of drug activity
d
e
in vivo; ID of viruses are referred to the NIH AIDS Reagent Program catalogue number; The
NNRTIs resistance mutations; fIC50 values against WT were previously determined39; EFV = efavirenz,
ETR = etravirine, RPV = rilpivirine.
Our results reveal that the tested compounds show good activity against the WT (IC50s 5–15 µM) as
well as against the mutants 12237 and 12235. In contrast, they are unable to halt the replication of
12231 and 12229 strains. At the same time, the reference drugs NVP, EFV and ETR show a dramatic
loss of potency against all resistant mutants.
These data indicate that our compounds are still able to bind to and to inhibit the RTs with mutations
K101E, V106I, Y181C and G190A, which confer highꢀlevel resistance against NNRTIs.10,30 This can
on the one side be attributed to the presence of essential contacts between our ligands and the amino
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acid residues of high fitness costs such as Leu100, Phe227, and Trp229. On the other side, due to the
conformational flexibility of our compounds they can easily adopt to the steric changes in the binding
site of the mutated proteins. Interestingly, the K103N mutation seems to have a negative effect on our
compounds’ RT inhibition and antiretroviral activity. This result does not contradict our predicted
binding mode, since this mutation affects the pocket entrance.40 In addition, the L100I mutation may
impair the binding to RT.
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These findings are consistent with the predicted binding mode of our new NNRTIs and thus further
confirm their binding site. Moreover, the fact that no crossꢀresistance was observed in some NNRTIsꢀ
resistant strains is an important advantage over the 1st (NVP and EFV) and 2nd generation (ETR and
RPV) NNRTIs.
Finally, we performed a docking study for the most active compound 18 (Figure 3). Comparing to the
binding of 11, the eastern part of the molecules binds identically to the protein. Unexpectedly, in the
western part a slight change was observed as the newly introduced methanesulfonamido group leads to
CH–π interaction with the highly conserved Trp229. As a consequence of this, the Nꢀphenyl ring is
slightly shifted downward away from Trp229. Moreover, an atypical hydrogen bond was identified
between the conserved Phe227 (as donor) and the 3,4ꢀdichlorophenyl motif (as acceptor).
Furthermore, no π–π interaction was observed between Tyr181 and the Nꢀphenyl moiety (centroid–
centroid distance > 4 Å).
Figure 3. Docking pose of 18 in the NNRTI binding site.
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Chemistry
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Synthesis of the 5ꢀarylꢀ2ꢀureidothiopheneꢀ3ꢀcarboxylic acids was accomplished through a
straightforward procedure starting with 3,4ꢀdichlorophenylacetic acid 21 (Scheme 1). Reduction with
LiAlH4 followed by mild oxidation of the produced alcohol 22 using PCC delivered the
arylacetaldehyde 23 in a good yield. Gewald reaction of 23 with ethyl cyanoacetate and elemental
sulfur under basic conditions in a oneꢀpot reaction afforded the 2ꢀaminothiopheneꢀ3ꢀcarboxylate 24.22
The ester was saponified to the corresponding acid 25, which was treated with BTC to produce the 6ꢀ
arylthiaisatoic anhydride 26. Synthesis was finalized via coupling of 26 and the appropriate amine in
water/TEA (2:1) mixture at room temperature followed by acidic workup to yield the 5ꢀarylꢀ2ꢀ
ureidothiopheneꢀ3ꢀcarboxylic acids 9‒20.
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Scheme 1. Synthesis of the 2ꢀureidothiopheneꢀ3ꢀcarboxylic acids 9–20a
aReagents and conditions: (a) LiAlH4, THF, reflux, 2 h; (b) PCC, DCM, rt, 2 h; (c) 1/8 S8,
NCCH2COOEt, TEA, ethanol, reflux, 12 h; (d) KOH, MeOH/H2O, reflux, 4 h; (e) BTC, THF, rt, 2 h;
(f) R1R2NH, H2O/TEA, rt, 2 h.
The Nꢀmonosubstitutedꢀpꢀphenylenediamines were prepared from pꢀnitroaniline 27 keeping the nitro
moiety as a hidden protected amino group (Scheme 2). Reaction of 27 with acyl chlorides or
sulfonyl chlorides in the presence of pyridine yielded the corresponding amides or sulfonamides,
respectively. The nitro derivatives were reduced by iron/ammonium chloride mixture to afford the
corresponding amines in excellent yields.
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Scheme 2. Synthesis of the intermediate aromatic aminesa
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aReagents and conditions: (a) Fe/NH4Cl, EtOH, reflux, 1 h; (b) RCOCl or RSO2Cl, pyridine/DCM, rt,
12 h; (c) 1,2,4ꢀtriazole, K2CO3/DMF, 70 °C, 1 h.
It is noteworthy that TEA is crucial for formation of the desired ureidothiophene carboxylic acids.
Modification of the previous procedures that did not employ TEA in the coupling step21,22 was
necessary to enhance the reaction regioselectivity. Since the utilized anilines in this work are weak
nucleophiles, the coupling reaction using pꢀanisidine for example in absence of TEA afforded a
mixture of the amide 42 and the ureidocarboxylic acid 13 in nearly 1:1 ratio as indicated from the 1H
NMR spectrum (Figure S3). In contrast, running the same reaction in presence of TEA produced
exclusively the ureidothiophene carboxylic acid 13 in 100% regioselectivity (Scheme 3). Moreover,
we found that the loss of amine regioselectivity and reactivity is directly proportional to the electron
deficiency of the substituted aniline, i.e. no ureidothiophene product could be obtained from the
reaction of 26 with pꢀnitroaniline 27 even when TEA was applied.
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Scheme 3. Reaction of 26 with pꢀanisidine in presence and absence of TEA.
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CONCLUSION
In this work we discovered and developed 2ꢀureidothiopheneꢀ3ꢀcarboxylic acids as dual bacterial
RNAP and HIVꢀ1 RT inhibitors for the treatment of MRSA/HIVꢀ1 coꢀinfections. This development
was based on an unprecedented exploitation of the functional and structural similarities between the
RNAP “switch region” and the NNRTI allosteric binding site. First, we identified the privileged
structure for RT inhibition by screening four regioisomeric classes of “switch region” binding RNAP
inhibitors. Next, we explored the steric volume allowed in the NNRTI binding pocket through
enlarging the bulkiness of the ureido moiety. Finally, we applied a structureꢀbased design strategy for
hit optimization. We were able to improve the RT inhibitory activity, in parallel with enhancing, or at
least maintaining, the activity against RNAP. SAR studies revealed the importance of hydrophilic nonꢀ
bulky substituents at the ureido side chain for RT inhibition. A MoA investigation revealed a nonꢀ
competitive inhibition mechanism of our compounds, which is typical for NNRTIs. Interestingly, the
compounds seem to act through a new mechanism via closing of the RT clamp. The new RNAP/RT
inhibitors displayed high potency against S. aureus as well as in cellulo antiretroviral activity
accompanied by marginal or no cytotoxicity. Furthermore, these compounds were active against
NNRTIꢀresistant strains. Thus, our work establishes the basis for the development of a single
compound therapy not only against MRSA/HIV coꢀinfection, but also against other challenging
coinfections of major public health concern especially Mtb/HIV coinfection.
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EXPERIMENTAL SECTION
Materials and Methods
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Starting materials and solvents were purchased from commercial suppliers, and used without further
purification. All chemical yields refer to purified compounds, and were not optimized. Reaction
progress was monitored using TLC Silica gel 60 F254 aluminium sheets, and visualization was
accomplished by UV at 254 nm. Flash chromatography was performed using silica gel 60 Å (40−63
ꢁm). Preparative RPꢀHPLC was carried out on a Waters Corporation setup contains a 2767 sample
manager, a 2545 binary gradient module, a 2998 PDA detector and a 3100 electron spray mass
spectrometer. Purification was performed using a Waters XBridge column (C18, 150 × 19 mm, 5 µm),
a binary solvent system A and B (A = water with 0.1% formic acid; B = MeCN with 0.1% formic
acid) as eluent, a flow rate of 20 mL/min and a gradient of 60% to 95% B in 8 min were applied. NMR
spectra were recorded either on Bruker DRXꢀ500 (1H, 500 MHz; 13C, 126 MHz), or Bruker Fourier
300 (1H, 300 MHz; 13C, 75 MHz) spectrometer at 300 K. Chemical shifts were recorded as δ values in
ppm units by reference to the hydrogenated residues of deuterated solvent as internal standard (CDCl3:
δ = 7.27, 77.00; DMSOꢀd6: δ = 2.50, 39.51). Splitting patterns describe apparent multiplicities and are
designated as s (singlet), br s (broad singlet), d (doublet), dd (doublet of doublet), t (triplet), q
(quartet), m (multiplet). Coupling constants (J) are given in Hertz (Hz). Weak or coalesced signals
were elucidated by Heteronuclear Multiple Quantum Coherence (HMQC) and Heteronuclear Multiple
Bond Coherence (HMBC) 2DꢀNMR techniques. Purity of all compounds used in biological assays
was ≥ 95% as measured by LC/MS Finnigan Surveyor MSQ Plus (Thermo Fisher Scientific, Dreieich,
Germany). The system consists of LC pump, autosampler, PDA detector, and singleꢀquadrupole MS
detector, as well as the standard software Xcalibur for operation. RP C18 Nucleodur 100ꢀ5 (125 × 3
mm) column (MachereyꢀNagel GmbH, Dühren, Germany) was used as stationary phase, and a binary
solvent system A and B (A = water with 0.1% TFA; B = MeCN with 0.1% TFA) was used as mobile
phase. In a gradient run the percentage of B was increased from an initial concentration of 0% at 0 min
to 100% at 15 min and kept at 100% for 5 min. The injection volume was 10 µL and the flow rate was
set to 800 µL/min. MS (ESI) analysis was carried out at a spray voltage of 3800 V, a capillary
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temperature of 350 °C and a source CID of 10 V. Spectra were acquired in positive mode from 100 to
1000 m/z and at 254 nm for UV tracing.
6
General
procedure
for
synthesis
of
5ꢀ(3,4ꢀdichlorophenyl)ꢀ2ꢀ[3ꢀ(4ꢀ
7
8
9
(un)substitutedphenyl)ureido]thiopheneꢀ3ꢀcarboxylic acids 11‒20
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To a stirred suspension of the thiaisatoic anhydride 26 (150 mg, 0.48 mmol) in a mixture of water (6
mL) and TEA (1 mL), the appropriate amine (1.0 mmol) in TEA (2 mL) was added. The reaction
mixture was stirred at rt overnight, then poured on iceꢀcooled 2 N HCl (40 mL), and extracted with
EtOAc/THF (1:1, 40 mL). The organic layer was washed with cold 2 N HCl/brine (1:1, 2 × 40 mL),
dried over anhydrous MgSO4, and concentrated in vacuo. The obtained crude material was suspended
in nꢀhexane/EtOAc (4:1, 50 mL), stirred in a water bath at 40 °C for 10 min, cooled, and collected by
filtration.
5ꢀ(3,4ꢀDichlorophenyl)ꢀ2ꢀ(3ꢀphenylureido)thiopheneꢀ3ꢀcarboxylic acid (11)
Yield 90%; beige solid; 1H NMR (500 MHz, DMSOꢀd6) δ 13.02 (br s, 1H), 10.61 (br s, 1H), 10.36 (br
s, 1H), 7.88 (d, J = 2.0 Hz, 1H), 7.63 (s, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.58 (dd, J = 8.0, 2.0 Hz, 1H),
7.52 (m, 2H), 7.33 (m, 2H), 7.04 (m, 1H); 13C NMR (126 MHz, DMSOꢀd6) δ 165.80, 150.98, 150.15,
138.86, 134.31, 131.87, 131.07, 129.07, 128.94 (2C), 127.72, 126.10, 124.70, 122.77, 122.27, 118.49
(2C), 112.36; m/z (ESI+) 407 [M + H]+; tR = 14.49 min.
5ꢀ(3,4ꢀDichlorophenyl)ꢀ2ꢀ(3ꢀ(4ꢀfluorophenyl)ureido)thiopheneꢀ3ꢀcarboxylic acid (12)
Yield 82%; grey solid; 1H NMR (300 MHz, DMSOꢀd6) δ 13.04 (br s, 1H), 10.60 (br s, 1H), 10.39 (br
s, 1H), 7.88 (d, J = 1.5 Hz, 1H), 7.63 (s, 1H), 7.59 (m, 2H), 7.51 (m, 2H), 7.17 (m, 2H); 13C NMR (75
MHz, DMSOꢀd6) δ 165.81, 157.84 (d, J = 239.2 Hz, 1C), 151.00, 150.12, 135.17 (d, J = 2.2 Hz, 1C),
134.28, 131.86, 131.07, 129.08, 127.75, 126.10, 124.70, 122.25, 120.27 (d, J = 7.5 Hz, 2C), 115.50 (d,
J = 22.4 Hz, 2C), 112.37; m/z (ESI+) 424 [M]+; tR = 14.96 min.
5ꢀ(3,4ꢀDichlorophenyl)ꢀ2ꢀ(3ꢀ(4ꢀmethoxyphenyl)ureido)thiopheneꢀ3ꢀcarboxylic acid (13)
Yield 86%; beige solid; 1H NMR (300 MHz, DMSOꢀd6) δ 12.66 (br s, 1H), 10.56 (br s, 1H), 10.16 (br
s, 1H), 7.88 (d, J = 1.0 Hz, 1H), 7.62 (s, 1H), 7.57 (m, 2H), 7.41 (d, J = 8.8 Hz, 2H), 6.91 (d, J = 8.8
Hz, 2H), 3.73 (s, 3H); 13C NMR (75 MHz, DMSOꢀd6) δ 165.86, 155.11, 151.04, 150.40, 134.37,
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131.86, 131.78, 131.08, 129.01, 127.52, 126.07, 124.69, 122.27, 120.31 (2C), 114.15 (2C), 112.16,
55.20; m/z (ESI+) 437 [M + H]+; tR = 14.36 min.
6
2ꢀ(3ꢀ(4ꢀAminophenyl)ureido)ꢀ5ꢀ(3,4ꢀdichlorophenyl)thiopheneꢀ3ꢀcarboxylic acid (14)
Yield 80%; pink solid; 1H NMR (300 MHz, DMSOꢀd6) δ 11.88 (br s, 1H), 10.72 (br s, 1H), 9.83 (br s,
1H), 7.84 (d, J = 1.0 Hz, 1H), 7.57 (m, 3H), 7.14 (d, J = 8.6 Hz, 2H), 6.54 (d, J = 8.6 Hz, 2H), 3.43 (br
s, 2H); 13C NMR (75 MHz, DMSOꢀd6) δ 166.19, 151.23, 150.33, 144.86, 134.61, 131.81, 131.06,
128.75, 127.51, 126.94, 125.90, 124.55, 122.55, 120.92 (2C), 114.12 (2C), 112.83; m/z (ESI+) 421
[M]+; tR = 9.78 min.
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2ꢀ(3ꢀ(4ꢀAcetamidophenyl)ureido)ꢀ5ꢀ(3,4ꢀdichlorophenyl)thiopheneꢀ3ꢀcarboxylic acid (15)
Yield 90%; beige solid; 1H NMR (500 MHz, DMSOꢀd6) δ 13.04 (br s, 1H), 10.58 (br s, 1H), 10.27 (br
s, 1H), 9.87 (br s, 1H), 7.89 (d, J = 1.9 Hz, 1H), 7.63 (s, 1H), 7.61 (d, J = 8.5 Hz, 1H), 7.58 (dd, J =
8.5, 1.9 Hz, 1H), 7.53 (d, J = 9.1 Hz, 2H), 7.42 (d, J = 9.1 Hz, 2H), 2.02 (s, 3H); 13C NMR (126 MHz,
DMSOꢀd6) δ 167.91, 165.79, 150.94, 150.27, 134.60, 134.34, 133.93, 131.85, 131.07, 129.02, 127.60,
126.09, 124.70, 122.26, 119.62 (2C), 118.95 (2C), 112.20, 23.87; m/z (ESI+) 464 [M + H]+; tR = 17.40
min.
2ꢀ(3ꢀ(4ꢀ(2ꢀ(1Hꢀ1,2,4ꢀTriazolꢀ1ꢀyl)acetamido)phenyl)ureido)ꢀ5ꢀ(3,4ꢀdichlorophenyl)thiopheneꢀ3ꢀ
carboxylic acid (16)
Yield 65%; pale grey solid; 1H NMR (500 MHz, DMSOꢀd6) δ 13.02 (br s, 1H), 10.94 (br s, 1H), 10.61
(br s, 1H), 10.52 (br s, 1H), 8.65 (s, 1H), 8.04 (s, 1H), 7.89 (d, J = 1.9 Hz, 1H), 7.63 (s, 1H), 7.61 (d, J
= 8.5 Hz, 1H), 7.60 (d, J = 9.1 Hz, 2H), 7.58 (dd, J = 8.5, 1.9 Hz, 1H), 7.47 (d, J = 9.1 Hz, 2H), 5.21
(s, 2H); 13C NMR (126 MHz, DMSOꢀd6) δ 165.66, 164.11, 150.99, 150.91, 150.13, 145.44, 134.60,
134.35, 133.78, 131.84, 131.09, 129.00, 127.60, 126.08, 124.72, 122.30, 119.83 (2C), 118.94 (2C),
112.35, 51.82; m/z (ESI+) 531 [M + H]+; tR = 17.10 min.
5ꢀ(3,4ꢀDichlorophenyl)ꢀ2ꢀ(3ꢀ(4ꢀ((methoxycarbonyl)amino)phenyl)ureido)thiopheneꢀ3ꢀcarboxylic
acid (17)
Yield 65%; white solid; 1H NMR (500 MHz, DMSOꢀd6) δ 13.01 (br s, 1H), 10.60 (br s, 1H), 10.25 (br
s, 1H), 9.55 (br s, 1H), 7.87 (d, J = 1.9 Hz, 1H), 7.62 (s, 1H), 7.60 (d, J = 8.2 Hz, 1H), 7.57 (dd, J =
8.2, 1.9 Hz, 1H), 7.42 (d, J = 8.5 Hz, 2H), 7.40 (d, J = 8.5 Hz, 2H), 3.65 (s, 3H); 13C NMR (126 MHz,
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DMSOꢀd6) δ 165.87, 154.09, 151.01, 150.28, 134.39, 134.36, 133.59, 131.89, 131.11, 129.05, 127.60,
126.10, 124.72, 122.31, 119.23 (2C), 118.91 (2C), 112.34, 51.57; m/z (ESI+) 480 [M + H]+; tR = 13.46
min.
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5ꢀ(3,4ꢀDichlorophenyl)ꢀ2ꢀ(3ꢀ(4ꢀ(methylsulfonamido)phenyl)ureido)thiopheneꢀ3ꢀcarboxylic acid
(18)
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Yield 70%; white crystals; 1H NMR (300 MHz, DMSOꢀd6) δ 13.01 (br s, 1H), 10.60 (br s, 1H), 10.35
(br s, 1H), 9.54 (br s, 1H), 7.88 (d, J = 1.0 Hz, 1H), 7.62 (s, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.56 (dd, J =
8.0, 1.0 Hz, 1H), 7.48 (d, J = 8.6 Hz, 2H), 7.19 (d, J = 8.6 Hz, 2H), 2.94 (s, 3H); 13C NMR (75 MHz,
DMSOꢀd6) δ 165.82, 150.97, 150.16, 135.49, 134.31, 133.08, 131.88, 131.08, 129.07, 127.71, 126.10,
124.72, 122.27, 121.73 (2C), 119.45 (2C), 112.36, 38.95; m/z (ESI+) 499 [M]+; tR = 12.86 min.
5ꢀ(3,4ꢀDichlorophenyl)ꢀ2ꢀ(3ꢀ(4ꢀ(phenylsulfonamido)phenyl)ureido)thiopheneꢀ3ꢀcarboxylic acid
(19)
Yield 88%; reddish solid; 1H NMR (500 MHz, DMSOꢀd6) δ 13.04 (br s, 1H), 10.56 (br s, 1H), 10.29
(br s, 1H), 10.09 (br s, 1H), 7.88 (d, J = 2.2 Hz, 1H), 7.72 (m, 2H), 7.62 (s, 1H), 7.60 (m, 2H), 7.55
(m, 3H), 7.36 (d, J = 9.1 Hz, 2H), 7.03 (d, J = 9.1 Hz, 2H); 13C NMR (126 MHz, DMSOꢀd6) δ 165.76,
150.87, 150.08, 139.46, 135.56, 134.28, 132.77, 132.27, 131.85, 131.07, 129.16 (2C), 129.06, 127.69,
126.63 (2C), 126.10, 124.70, 122.26, 121.88 (2C), 119.20 (2C), 112.33; m/z (ESI+) 561 [M]+; tR =
17.26 min.
5ꢀ(3,4ꢀDichlorophenyl)ꢀ2ꢀ(3ꢀ(4ꢀ((N,Nꢀdimethylsulfamoyl)amino)phenyl)ureido)thiopheneꢀ3ꢀ
carboxylic acid (20)
Yield 79%; beige solid; 1H NMR (500 MHz, DMSOꢀd6) δ 13.06 (br s, 1H), 10.59 (br s, 1H), 10.31 (br
s, 1H), 9.72 (br s, 1H), 7.89 (d, J = 2.2 Hz, 1H), 7.63 (s, 1H), 7.61 (d, J = 8.2 Hz, 1H), 7.58 (dd, J =
8.2, 2.2 Hz, 1H), 7.44 (d, J = 9.1 Hz, 2H), 7.18 (d, J = 9.1 Hz, 2H), 2.69 (s, 6H); 13C NMR (126 MHz,
DMSOꢀd6) δ 165.78, 150.95, 150.18, 134.78, 134.31, 133.45, 131.85, 131.07, 129.05, 127.66, 126.10,
124.70, 122.24, 120.95 (2C), 119.32 (2C), 112.27, 37.75 (2C); m/z (ESI+) 529 [M + H]+; tR = 17.20
min.
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Experimental procedures for the synthesis of all other target compounds and necessary precursors
together with their characterization and NMR spectra are described in details in the Supporting
Information.
8
Computational Chemistry All computational work was performed using Molecular Operating
Environment (MOE) version 2015.10, Chemical Computing Group Inc., 1010 Sherbrooke St. West,
Suite 910, Montreal, Quebec, H3A 2R7, Canada.
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Similarity Analysis A database containing compounds 2 and 4 was created and the fingerprint
piDAPH4 (piꢀdonorꢀacceptorꢀpolarꢀhydrophobeꢀ4 point pharmacophore) was calculated for both
entries. Compound 4 was selected as a reference structure and sent to MOE window. In the database
viewer window, similarity search was performed by setting the fingerprint system to piDAPH4, and
using the similarity metric Tanimoto coefficient (TC) to measure similarity between molecules. TC
values range from 0 (no similarity) to 1 (complete similarity).
Preparation of Ligands and Protein Structure for Docking In the database viewer window,
compounds 11 and 18 were selected and washed via compute | molecule | wash command.
Deprotonation of strong acids and protonation of strong bases options were checked in the wash panel.
Xꢀray crystal structure of the HIVꢀ1 RT in complex with NVP (PDB code 1VRT)14 was used to
perform the molecular docking study. Potential was setup to Amber10:EHT for force field and Rꢀfield
for solvation. Addition of hydrogen atoms and removal of water molecules were performed via LigX
module.
Ligand–Receptor Docking Using the induced fit docking protocol, the binding site was set to dummy
atoms which were identified by the site finder mode, and the amino acid residues were chosen where
nevirapine binds in the RT allosteric binding site. Docking placement was triangle matcher with rotate
bonds option. The 1st rescoring was ASE with force field refinement, and the 2nd rescoring was alpha
HB.
QSAR Analysis A database containing compounds 11–20 was created, and each structure was
subjected to energy minimization up to a gradient 0.01 kcal/mol/Å using the MMFF94x force field and
distance solvation model. In the database viewer window, molecular descriptors were calculated for all
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entries via activating the compute panel, choosing descriptors calculate option (Table S1). The QSAR
model was computed using partial least squares (PLS) method.
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Binding Measurements were performed at 20 °C using a Fluoromax 4 spectrofluorometer (Jobinꢀ
Yvon Horiba) by monitoring the increase of FRET efficiency, EFRET, associated to the formation of the
complex between Alexa488ꢀlabeled RT and TAMRAꢀlabeled p/t duplexes,. The FRET efficiency,
calculated by EFRET = 1 ꢀ IDA/ID (where ID and IDA are the intensities of the Alexa 488 donor in the
absence and in the presence of the TAMRA acceptor, respectively), was then plotted as a function of
the total concentration of the labeled oligonucleotide (ODN) and fitted with a rewritten Scatchard
equation considering a model with a single binding site
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



K + M + Ltot −
[
Kd + Mtot + Ltot
]
2 − 4Mtot Ltot
d
tot
EFRET = E
(3)
F 

2Kd Ltot




where EF corresponds to the FRET efficiency at saturation, Ltot and Mtot are respectively the
concentration of RT and p/t duplex and Kd is the dissociation constant. Fit parameters were recovered
from nonꢀlinear fits of eq 3 to the experimental datasets using a nonꢀlinear leastꢀsquares method and
the Levenberg–Marquardt algorithm in the Origin 8 software.
RT Inhibitory Activity of Compounds was determined as described previously.24 All measurements
were carried out using a 23ꢀmer (5′ꢀ CAG CAG TAC AAA TGG CAG TAT TC) DNAꢀprimer labeled
at the T19 position with Cyanine 5 (Cy5), annealed to a 63ꢀmer (3′ꢀTGT CGT CAT GTT TAC CGT
CAT AAG TAG GTG TTA CTA GTC CGA TTT CCC CTA GTC CGA CCC ATG)ꢀtemplate labeled
at the T2 position with TAMRA. Both TAMRA and Cy5 were covalently attached via a C6 amino link
to their respective T residues in the primer and the template. Primer and template ODN were annealed
by heating equimolar amounts in buffer at 90 °C for 2 min, followed by cooling to room temperature
over several hours. All measurements were performed at 20 °C using a Fluoromax 4
spectrofluorometer (JobinꢀYvon Horiba) or a stoppedꢀflow apparatus (SF3, Biologics). The FRET
donor, TAMRA, was excited at 540 nm, and its emission was recorded at 580 nm. Nucleotide
incorporation kinetics was triggered by addition of dNTPs in excess to a preꢀincubated mixture of
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HIVꢀRT and p/t duplexes at equimolar concentrations. The annealing kinetic traces were adequately
fitted using
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obs 2 (t−t0))
I(t) = IF − (IF − I0 )(ae(−k (t−t0)) −(1− a)e(−k
)
obs1
(4)
9
where t0 is the dead time, kobs1,2 are the observed kinetic rate constants, a is the amplitude of the fast
component, and I0 and IF are the fluorescence intensities before dNTPs addition and at completion of
the reaction, respectively. The I0 value was obtained from the steadyꢀstate fluorescence spectrum of
the doubly labeled p/t duplex in the presence of RT and was thus fixed. All fitting procedures were
carried out with Origin™ 8.6 software using nonlinear, leastꢀsquare methods and the Levenbergꢀ
Marquardt algorithm.
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HeLa Infectivity and Cytotoxicity Assay The infectivity assay was based on infection of Hela cells
by a thirdꢀgeneration of pseudoparticles38 mimicking the early steps of the HIVꢀ1 virus cycle. The
pseudoparticles contain Gag, Gagꢀpol, RRE and Vesicular Stomatitis Virus (VSV)ꢀG proteins and a
RNA encoding reporter gene for luciferase. The luciferase sequence is incorporated in the host
genome during infection by pseudoparticles which give the possibility to quantify infection. A total of
5×103 HeLa cells/well were seeded in 96 wells plate 24 h prior to infection in Dulbecco’s Modified
Eagle Medium (Life Technologies) (DMEM) complemented with 10% fetal bovine serum, penicillin
(100 UI/mL), and streptomycin (100 ꢁg/mL) and incubated in a 5% CO2 incubator. After medium
removal, 50 µL of two times concentrated solutions of compounds were added 30 min before adding
50 µL of pseudoparticles, which can infect 50% of cells in presence of polybrene (8 µg/mL). Cells
were washed, 24 h after infection, one time by PBS and lysed with Passive Lysis Buffer (Promega)
supplemented with 0.5% Triton Xꢀ100 for 30 min under constant shaking. Luciferase activity was
measured for 10 s in a luminometer (Tristar 2 Multimode reader LB 942, Berthold) after injection of
50 µL of luciferine reagent (25 mM Tricine Buffer pH = 7.8, 0.5 mM EDTA pH 7.9, 5 mM MgSO4, 5
mM DTT, 0.5 mM ATP, 1.65 mM Dꢀluciferin sodium salt and 0.325 mM Coenzyme A Sodium Salt
hydrate).
For each compound, we used as a control, cells incubated with DMEM containing the same
percentage of DMSO (maximum 0.5% v/v) as the solutions with the tested molecules. For each tested
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molecule and concentration, we calculated the percentage of inhibition (%inh) with the following
4
5
equation
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(5)
Each concentration of tested compound was tested in sextuplicate. To determine the IC50 values, we
plotted the percentage of inhibition against the inhibitor concentration (I) and fitted it with a modified
version of the doseꢀresponse effect equation
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−
A +
(A2
)
A
1
1
y =
(6)
)−log(I))*p)
+10(log(I
50
1
where, A1 and A2 represent the percentage of inhibition in the absence (0%) and in saturating
concentrations (100%) of inhibitor, respectively. I50 represents the half maximal inhibitory
concentration and p denotes hill coefficient.
Cytotoxicity of compounds was quantified by the MTT assay (3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀ
diphenyl tetrazolium bromide).41 Another plate was prepared in the same conditions as for the
infectivity assay. 24 h after infection, medium was replaced by 110 µL of a mix containing 100 µL of
DMEM and 10 µL of 12 mM MTT solution in PBS and cells were incubated during 4 h. In order to
dissolve the insoluble purple formazan reduced by living cells, 85 µL of the mix was replaced by 50
µL of DMSO and gently shaken for 10 min. The absorbance was then measured at 540 nm in a
spectrophotometer (Safas Monaco) and converted in percentage of cytotoxicity (% cyt), in reference to
the control (DMEM + DMSO).
Antiretroviral Assay against HIVꢀ1 WT and NNRTIsꢀResistant Strains The WT virus and the
NNRTIsꢀresistant strains were obtained through the NIH AIDS Reagent Program (ARP,
www.aidsreagent.org) and have been already characterized by the Phenosense Assay (Monogram
Biosciences), considered as the reference assay for phenotypic investigation of HIV drug resistance
due to its large application in clinical trials. The infectious clones were firstly transfected in 293LX
cells and then expanded in MTꢀ2 cells. The antiretroviral assay was determined in a TZMꢀbl cell line
based phenotypic assay. TZMꢀbl cells are characterized by the presence of the luciferase and β–
galactosidase reporter genes integrated in the cell genome under control of the HIVꢀ1 LTR promoter.
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Expression of the reporter genes is regulated by the viral Tat protein, which is produced following
transcription of the integrated provirus. TZMꢀbl cells were seeded at a concentration of 30,000
cells/well in a 96ꢀwell plate using DMEM medium without fetal bovine serum (FBS) and infected
with the viruses in the presence of 20, 10, 5 and 1 ꢁM of each compound. After 48 hours, cells were
lysed by adding 40 µL of Glo Lysis Buffer (Promega) to each well for 5 minutes, then 40 µL of
BrightꢀGlo Luciferase Reagent (Promega) were added to each well for counting relative luminescence
units (RLU) using the GloꢀMax Multi Detection System (Promega). RLU values from each well were
elaborated using the GraphPad v5.0 software to calculate the IC50 of each compound.
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RNAP Inhibition, Antibacterial Activity and Cytotoxicity Transcription inhibition assay,
determination of IC50 values, minimal inhibitory concentrations (MICs) and cytotoxicity in HEK 293
cells were performed as described previously.22
ASSOCIATED CONTENT
Supporting Information
This material is available free of charge via the Internet at http://pubs.acs.org.
Experimental procedures and analytical data for all other compounds; Substituent constants and
1
molecular descriptors of compounds 11–20; Binding and mechanistic studies of the compounds; H
and 13C NMR spectra (PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Author
* Eꢀmail: rolf.hartmann@helmholtzꢀhzi.de, Phone: +(49) 681 302 70300.
ACKNOWLEDGMENTS
The authors thank Jeannine Jung for technical assistance. Walid A. M. Elgaher gratefully
acknowledges a scholarship from the German Academic Exchange Service (DAAD). Kamal K.
Sharma, Francesco Saladini, Manuel Pires and part of this work were supported by the European
Project THINPAD “Targeting the HIVꢀ1 Nucleocapsid Protein to fight Antiretroviral Drug
Resistance” [FP7ꢀGrant Agreement 601969 to Y.M.].
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ABRREVIATIONS USED
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ART, antiretroviral therapy; AZTꢀTP, azidothymidine triphosphate; BTC, bis(trichloromethyl)
carbonate; DMEM, Dulbecco's modified Eagle's medium; dNTPs, deoxyribonucleotides; EFV,
efavirenz; ETR, etravirine; FBS, fetal bovine serum; HAART, highly active antiretroviral therapy;
LLE, ligandꢀlipophilicity efficiency; MoA, mode of action; Mtb, Mycobacterium tuberculosis; MTT,
3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyl tetrazolium bromide; NVP, nevirapine; ODN,
oligonucleotide; piDAPH4, piꢀdonorꢀacceptorꢀpolarꢀhydrophobeꢀ4 point pharmacophore; p/t,
primer/template; RLU, relative luminescence units; RNAP, RNA polymerase; RPV, rilpivirine; RT,
reverse transcriptase; TAMRA, carboxytetramethylrhodamine; TC, Tanimoto coefficient; TEA,
triethylamine.
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