Chromenone derivatives as a versatile scaffold with dual mode of inhibition of HIV-1 reverse transcriptase-associated Ribonuclease H function and integrase activity

Article abstract of DOI:10.1016/j.ejmech.2019.111617

A number of compounds targeting different processes of the Human Immunodeficiency Virus type 1 (HIV-1) life cycle have been developed in the continuing fight against AIDS. Coumarin-based molecules already proved to act as HIV-1 Protease (PR) or Integrase (IN) inhibitors and also to target HIV-1 reverse transcriptase (RT), blocking the DNA-dependent DNA-polymerase activity or the RNA-dependent DNA-polymerase activity working as common NNRTIs. In the present study, with the aim to exploit a coumarin-based scaffold to achieve the inhibition of multiple viral coded enzymatic functions, novel 4-hydroxy-2H, 5H-pyrano (3, 2-c) chromene–2, 5–dione derivatives were synthesized. The modeling studies calculated the theoretical binding affinity of the synthesized compounds on both HIV-1 IN and RT-associated Ribonuclease H (RNase H) active sites, which was confirmed by biological assays. Our results provide a basis for the identification of dual HIV-1 IN and RT RNase H inhibitors compounds.

Full text of DOI:10.1016/j.ejmech.2019.111617

European Journal of Medicinal Chemistry 182 (2019) 111617
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Chromenone derivatives as a versatile scaffold with dual mode of
inhibition of HIV-1 reverse transcriptase-associated Ribonuclease H
function and integrase activity
,
,
Francesca Esposito a 1, Francesca Alessandra Ambrosio b 1, Rita Maleddu c,
Giosue Costa d, Roberta Rocca b d, Elias Maccioni c, Raffaella Catalano b
,
b,
,
, d
ꢀ
Isabella Romeo d e, Phaedra Eleftheriou f, Denish C. Karia g, Petros Tsirides f,
,
Nilesh Godvani g, Hetal Pandya g, Angela Corona a, Stefano Alcaro b d, Anna Artese b
,
,
, d, *
Athina Geronikaki h, Enzo Tramontano a
, i, **
a Department of Life and Environmental Sciences, University of Cagliari, Cittadella Universitaria SS554, 09042, Monserrato, CA, Italy
b Department of Health Sciences, “Magna Græcia” University of Catanzaro, Campus “S. Venuta”, Viale Europa, 88100, Catanzaro, Italy
c Department of Life and Environmental Sciences, Via Ospedale 72, 09124, Cagliari, Italy
d Net4Science Acedemic Spin-Off, “Magna Græcia” University of Catanzaro, Campus “S. Venuta”, Viale Europa, Germaneto, 88100, Catanzaro, Italy
e Department of Chemistry and Chemical Technology, University of Calabria, Via Pietro Bucci, 87036, Arcavacata di Rende, Cosenza, Italy
f Department of Medical Laboratory Studies, School of Health and Medical Care, Alexander Educational Institute of Thessaloniki, 57400, Greece
g Department of Chemistry, Patel JDK Davolwala Science College, Borsad, Anand-Gujarat, India
h Department of Medicinal Chemistry, School of Pharmacy, Aristotle University of Thessaloniki, Greece
i Genetics and Biomedical Research Institute, National Research Council (CNR), Cittadella Universitaria SS554, 09042, Monserrato, CA, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A number of compounds targeting different processes of the Human Immunodeficiency Virus type 1
(HIV-1) life cycle have been developed in the continuing fight against AIDS. Coumarin-based molecules
already proved to act as HIV-1 Protease (PR) or Integrase (IN) inhibitors and also to target HIV-1 reverse
transcriptase (RT), blocking the DNA-dependent DNA-polymerase activity or the RNA-dependent DNA-
polymerase activity working as common NNRTIs. In the present study, with the aim to exploit a
coumarin-based scaffold to achieve the inhibition of multiple viral coded enzymatic functions, novel 4-
hydroxy-2H, 5H-pyrano (3, 2-c) chromenee2, 5edione derivatives were synthesized. The modeling
studies calculated the theoretical binding affinity of the synthesized compounds on both HIV-1 IN and
RT-associated Ribonuclease H (RNase H) active sites, which was confirmed by biological assays. Our
results provide a basis for the identification of dual HIV-1 IN and RT RNase H inhibitors compounds.
© 2019 Elsevier Masson SAS. All rights reserved.
Received 30 May 2019
Received in revised form
9 August 2019
Accepted 10 August 2019
Available online 12 August 2019
Keywords:
Coumarin derivatives
HIV-1
RNase H
Integrase
Dual inhibitors
LEDGF/p75 cellular cofactor
1. Introduction
three main enzymatic functions: an RNA-dependent DNA-poly-
merase (RDDP) activity, that synthesizes the RNA:DNA intermedi-
During the viral infection, the Human Immunodeficiency Virus
type 1 (HIV-1) genome is retro-transcripted by the viral enzyme
reverse transcriptase (RT), a multifunctional protein endowed with
ate, the Ribonuclease H (RNase H) activity, responsible of the
hydrolytic cleavage of the RNA strand of the RNA:DNA hybrid and
the DNA-dependent DNA-polymerase (DDDP) activity, that com-
pletes the synthesis of the integration-competent double strand
DNA [1]. After the retrotranscription process, the viral DNA is in-
tegrated into the host genome through two enzymatic reactions
accomplished by the viral enzyme integrase (IN) that operates
within a pre-integration complex (PIC), composed of viral DNA and
viral and cellular proteins [2]. Among the cellular factors involved
in the integration process there is the nuclear protein Lens-
* Corresponding author. Department of Health Sciences, University “Magna
Græcia” of Catanzaro, Viale Europa, 88100, Catanzaro, Italy.
** Corresponding author. Department of Life and Environmental Sciences, Uni-
versity of Cagliari, Cittadella Universitaria SS554, 09042, Monserrato, CA, Italy.
E-mail addresses: artese@unicz.it (A. Artese), tramon@unica.it (E. Tramontano).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.ejmech.2019.111617
0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

2
F. Esposito et al. / European Journal of Medicinal Chemistry 182 (2019) 111617
Epitelium-derived (LEDGF/p75) that establishes specific in-
teractions with chromatin and the catalytic core domain (CCD) of
the IN dimer through its IN binding domain (IBD) [3].
[30], 16 coumarin (4-hydroxypyranobenzopyran) derivatives were
synthesized and their theoretical binding affinity versus both the
enzymes were evaluated through computational studies. Finally,
the biological activity, against HIV-1 RNase H and IN functions,
were tested, thus identifying novel chemical structures with dual
HIV inhibition.
The current HIV treatment is based on the highly active anti-
retroviral therapy (HAART) that allowed a major decrease in
morbidity and mortality for AIDS patients [4]. However, in a pro-
longed therapy, the lack of compliance and the compartmentali-
zation phenomena still consent the selection of drug resistant
strains, that can affect multiple classes of drugs [5] with increased
transmission of drug-resistant viral strains detected in antiretro-
viral-treatedenaïve patients [6]. Therefore, the search for novel
inhibitors continues, aiming to achieve drugs with not only a novel
mode of action, but also lower long-term treatment toxicity. In this
respect, the inhibition of two viral functions by a single molecule
could provide a higher barrier to drug resistance selection, reduce
the number of administered drugs, and the consequent long-term
toxicity [7].
Among the different categories of anti-HIV drugs, the RT in-
hibitors are the most prescribed drugs to treat HIV infection [8]. In
fact, the RT inhibitors play a crucial role in the treatment of HIV-
infected patients and IN inhibitors (INIs) are the last innovation,
representing a good second line therapy together with protease
inhibitors. The approved RT inhibitors belong to two families: the
nucleoside/nucleotide RT inhibitors (NRTIs/NtRTIs) and the non-
nucleoside RT inhibitors (NNRTIs) [9]. The NRTIs were the first
approved anti-HIV agents, derived from former known anticancer
and antiviral agents. Unfortunately, they are characterized by heavy
side effects. With respect to the NRTIs, the NNRTIs are characterized
by a higher potency, lower toxicity, and higher selectivity and
specificity [10]. Though the approved NNRTIs have different
chemical structures, all of them bind the same site of RT, selectively
inhibiting its polymerase function. No approved inhibitor of HIV-1
RT-associated RNase H activity is currently available [11], despite
this function is essential for viral replication [12], is a well validated
target for drug development [11,13e20], and some promising hits
are currently investigated [21,22]. Among the INIs, all the approved
drugs are active-site inhibitors [1], while allosteric inhibitors are
still under development [23e27]. The IN inhibitors are unable to
bind IN alone but they bind the pre-integration complex formed by
IN and viral DNA and subsequently inhibit the strand-transfer re-
action [28]. Interestingly, some molecules, able to target both RT
and IN, have been reported recently [27,29e32], enlightening the
possibility of a dual inhibition of the two viral functions [33].
In this study, with the aim to develop novel drugs targeting
multiple enzymatic functions, we exploited the coumarin scaffold.
It has been reported that coumarin (2H-chromen-2-one; 1-
benzopyran-2-one) derivatives possess a wide range of activities.
While antibiotic and anticoagulant activities are the most common
actions [34], several coumarin derivatives, such as 4-hydroxy-3-(5-
2. Results and discussion
2.1. Chemistry
Sixteen novel coumarin derivatives were synthesized
according to Scheme 1. In the first stage 4-hydroxy coumarin de-
rivatives were prepared by reaction of phenol and malonic acid in
the presence of phosphorous oxychloride and zinc chloride. These
hydroxy coumarin derivatives were further treated with malonic
acid, phosphorous oxychloride and zinc chloride resulting in de-
rivatives of 4-hydroxy-2-methylenepyrano [3,2-c] chromene-2,5-
dione (1e5). The acetylation of substituted 4-hydroxy-2-
methylenepyrano [3,2-c] chromene-2,5-diones gave various
acetyl substituted coumarin compounds (6e9). Reaction of these
acetyl derivatives with ethyl acetate and sodium metal led to
substituted derivatives of 4-hydroxy-3-(3-oxobutanoyl)pyrano
[3,2-c]chromene-2,5-dione (10e12). Acidic hydrolysis resulted in
compound 13, while cyclization of acetoacetyl derivatives of
coumarin (10e12) with 3,4-diaminobenzophenone and phenyl
hydrazine resulted in 3-(7-benzoyl-3H-benzo [b] [1,4]diazepin-2-
yl)-4-hydroxypyrano [3,2-c]chromene-2,5-dione (14,15) and 4-
hydroxy-3-(5-methyl-1-phenyl-1H-pyrazol-3-yl)pyrano
[3,2-c]
chromene-2,5-dione (16) respectively. The corresponding reactions
proceeded smoothly and resulted in considerably good yields
(56%e82%). Structures of synthesized compounds were confirmed
by IR, 1H NMR (SI), 13C NMR (SI), EI-MS, and elemental analysis.
2.2. Physicochemical properties of selected compounds
Theoretical calculation of the physicochemical properties of
selected compounds (Table S2) was performed in order to estimate
the ability of these compounds to become orally active drugs and to
evaluate the probable association of biological activity with the
physicochemical properties of the compounds. Average logP of the
compounds was between 0.49 and 3.50. Consequently, all com-
pounds fulfil the related criterion of Lipinski and Ghose rules
concerning membrane penetration and oral adsorption. Com-
pounds have intermediate aqueous solubility with ALOGS
between ꢀ1.69 and ꢀ4.98. Topological polar surface area (TPSA)
value was between 90.6 and 131.9 that is lower than 140 Å2 that is
the upper limit of Ghose fifth criterion. Polarizability was between
27.196 and 52.73 cmꢀ3, and molar refractivity ranged from 68.60 to
133.01 cmꢀ3. Compound 14, with predicted molar refractivity equal
to 133.01 cmꢀ3, is the only one that slightly surpasses the upper
limit of 130 cmꢀ3 of Ghose second criterion. All compounds fulfil
the criterion of molecular weight of both Lipinski and Ghose rules
with MW of compounds ranging between 290.3 and 494.5. The
calculated molecular volume of the compounds ranges from 233.3
to 412.0 Å3. In general, according to theoretical calculations, all
compounds fulfil the Lipinski's rule of cell membrane penetration
by passive diffusion and all of them, but for a minor aberration at
the molar refractivity of compound 14, also fulfil all Ghose's criteria.
methyl-1-phenyl-1H-pyrazol-3-yl)pyrano
[3,2-c]chromene-2,5-
dione derivatives [35], as khellactone derivatives [36], dihy-
droseselins [37] and 2,3-dimethyl-4-chromanol derivatives [38],
were associated to anti-HIV activity. This activity has been referred
in many cases to the inhibition of PR [39] and IN activities [40e43].
On the contrary, RT inhibition has not reported for many of these
compounds, but for khellactone derivatives [34]. Distinct modes of
action include interaction with the HIV-1 encoded proteins Tat [44]
and Vpr [45]. Differently form the majority of the known NNRTIs,
(þ)-Calanolide was found to be an NNRTI and interestingly, 30,40-di-
O-(ꢀ)-camphanoyl-(þ)-cis-khellactone derivatives were found to
target RT, inhibiting selectively its DDDP activity but not its RDDP
activity [36,43]. Starting from structural and physicochemical
characteristics of known active compounds against HIV-1 RT
[35,41] and IN enzymes and given the structural similarities be-
tween the viral RT-associated RNase H domain and the IN domain
2.3. Molecular modeling results
The docking analysis was performed by using two metal sites as
constrain [46] in order to better understand the theoretical affinity
and the binding modes of the studied compounds versus both HIV-

F. Esposito et al. / European Journal of Medicinal Chemistry 182 (2019) 111617
3
Scheme 1. Synthesis and 2D structure of compounds 1e16.
1 RT-associated RNase H and IN activities. RDS1643 [47] and Ral-
tegravir were used as reference compounds for the molecular
recognition analysis, respectively of the HIV-1 RT-associated RNase
H function and IN enzymes. The HIV-1 RNase H active site contains
a highly conserved and essential DDE motif, comprising the
carboxylate residues Asp443, Glu478 and Asp498, which
coordinates two divalent Mg2þ cations [46]. Our docking results
suggested that most of the synthesized coumarin derivatives were
well accommodated into the RNase H active site and were able to
chelate the metal cofactor ions, thanks to the chelating core present
in their scaffold. The Docking score values (D-score), for each
studied compound, are reported in Table 1.

4
F. Esposito et al. / European Journal of Medicinal Chemistry 182 (2019) 111617
Table 1
Ligands with the lowest docking scores are predicted to have a
better theoretical binding affinity towards the protein. Specifically,
compounds 7, 5, 6, 3 and 1 were the best predicted within the active
site of RNase H due to the interactions between their chelating core
and the two Mg2þ ions, which are coordinated to the active site acid
residues Asp443, Glu478, Asp498 and Asp549, crucial for the RT-
RNase H activity [48].
Additionally, compound 7 was involved in hydrophobic in-
teractions with His539, Gly444 and Asn474. Compound 9 was well
stabilized into the enzyme binding pocket through the coordina-
tion with the two metal cofactors, hydrophobic interactions with
Docking score values of all the studied coumarin derivatives versus the HIV-1 RNase
H active site and the PFV IN catalytic binding site in the presence of LEDGF cofactor,
respectively. D-score values are expressed in kcal/mol.
Compounds
Docking score value
RT RNASE H
PFV IN WITH LEDGF COFACTOR
7
6
8
9
12
4
3
10
2
1
5
11
ꢀ6.22
ꢀ6.29
ꢀ3.04
ꢀ6.37
ꢀ4.71
ꢀ5.52
ꢀ6.38
ꢀ4.92
ꢀ4.07
ꢀ6.31
ꢀ6.74
ꢀ5.01
ꢀ5.08
e
ꢀ9.96
ꢀ9.93
ꢀ9.90
ꢀ9.91
ꢀ8.83
ꢀ8.69
ꢀ8.46
ꢀ8.46
ꢀ8.36
ꢀ8.35
ꢀ8.29
ꢀ8.21
ꢀ7.83
ꢀ7.82
ꢀ6.75
ꢀ6.43
ꢀ6.12
ꢀ5.65
e
Gln500 and Asp498 and a pivotal p-p interaction with His539. The
predicted binding mode of best pose for compounds 3, 1, 6 and 7
within the RT RNase H active site are shown in Fig. 1, while the
binding mode of the best poses of the other compounds are re-
ported in Fig. S1 of the Supplementary Material. Compounds 10 and
12 showed a similar binding mode characterized by the chelation of
the two metal cofactors and by the interaction with the catalytic
triad, while their benzene rings were exposed outside from the
active pocket. Due to their highly similar structure, also compound
14 and 15 are associated to the same binding mode, whereas 16
showed a slightly different binding mode. Compounds 2 and 8 have
a disadvantaged theoretical binding affinity (D-Score values) for the
RT RNase H active site, due to the loss of the interaction between
14
RAL
13R
13S
15
16
RDS1643
ꢀ4.25
ꢀ4.30
ꢀ4.94
ꢀ4.94
ꢀ5.82
Fig. 1. 3D representation of the best docking poses of compounds A) 3; B) 1; C) 6 and D) 7 into the HIV-1 RT RNase H active site. The ligands are shown, respectively, as yellow,
green, dark-green and gold carbon sticks, while the receptor and the Mg2þ ions are reported as light blue cartoon and pink spheres, respectively. Amino acids involved in ligand
binding are highlighted as slate carbon sticks. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

F. Esposito et al. / European Journal of Medicinal Chemistry 182 (2019) 111617
5
the chelating core of the compounds and the two Mg2þ ions.
HIV-1 IN and RT-RNase H share similar active site configura-
tions, thus inhibitors of these enzymes functions exhibit over-
lapping pharmacophore features [46]. Therefore, at the same time
we performed molecular recognition studies of the same com-
pounds versus the PFV IN, in the presence of the LEDGF cofactor, in
order to identify dual-acting inhibitors as new approach in HIV-1
drug development. After Molecular Dynamics simulations (MDs),
the best receptor structure, selected according to the Boltzmann
population and Prime energy parameters [57], was adopted for
further docking studies of the synthetized compounds. Our docking
results highlighted that most of the analysed compounds were able
to bind the PFV IN catalytic site in the presence of the LEDGF
cofactor, establishing interactions with the three acidic residues
Asp128, Asp185 and Glu211 (corresponding to Asp64, Asp116 and
Glu152 in HIV-1 IN) of the IN active site and by coordinating the
essential Mg2þ metal cations [49]. In Table 1 the theoretical binding
affinities of the compounds versus the PFV IN active pocket, in the
presence of the LEDGF cofactor, are reported.
Pro214 and Tyr212 (corresponding to Pro145 and Tyr143, respec-
tively in HIV-1 IN). Compounds 3, 10, 11, 13R and 14 were found to
chelate the metal cofactors and showed their aromatic rings ori-
ented toward the viral DNA, thus establishing a staking interaction
with the nucleobase C16 of the viral DNA. Compounds 1, 2, 4 and 5
shared the same arrangement into the PFV IN catalytic site, while 1
and 4 were better stabilized by extra hydrophobic interactions with
Tyr212 and Gln186 (corresponding to Tyr143 and Gln117 in HIV-1
IN). The predicted binding modes of the best poses of compounds
7, 6, 3 and 1 within the PFV IN catalytic site are shown in Fig. 2,
while the binding modes of the other compounds best poses are
reported in Fig. S2 of the Supplementary Material. For compounds
15 and 16 our simulations highlighted that both compounds were
involved in hydrophobic contacts with the tyrosine at position 212.
2.4. Effect of coumarin derivatives on HIV-1 RT-associated RNase H
activity
It has been reported that coumarin derivatives are able to target
HIV-1 RT polymerase activity, and a moderate affection of E. coli
RNase H function was once reported [50], even if the activity of this
class of molecules, on the HIV-1 RT-associated RNase H function,
was never deeply investigated. In addition, some compounds, re-
ported to allosterically inhibit the RDDP function, were also able to
interfere with the RNase H activity [51]. Since the RT-associated
Compounds 7, 6, 8, 9 and 12 were associated to the best theo-
retical binding affinity versus the active site of PFV IN thanks to the
interactions established between their chelating core and the two
Mg2þ ions, coordinated to the crucial active site acid residues such
as Asp128, Asp185, Glu221 (corresponding to Asp64, Asp116 and
Glu152 in HIV-1 IN), and additional hydrophobic contacts with
Fig. 2. 3D representation of the best docking poses of compounds A) 7; B) 6; C) 3 and D) 1 into the PFV IN catalytic site. The ligands are shown, respectively, as slate, gray, cyan and
chocolate carbon sticks, while the receptor and the vDNA are showed as green and orange cartoon, respectively. Amino acids involved in ligand binding are highlighted as green
carbon sticks. The Mg2þ ions are showed as pink spheres.

6
F. Esposito et al. / European Journal of Medicinal Chemistry 182 (2019) 111617
RNase H function is a validated target for the development of
promising drugs, to confirm the docking prediction, that these
coumarin derivatives interact with the catalytic site of HIV-1 RNase
H, we tested the series of 16 coumarin derivatives for their ability to
inhibit HIV-1 RNase H function in a biochemical assay, using the
diketoacid RDS1643 as a positive control [47]. As shown in Table 2,
the different position of methyl group in the phenyl ring of com-
pounds 1, 2 and 3 consistently changed the potency of inhibition, in
fact, differently from compound 2, compounds 1 and 3 were able to
inhibit HIV-1 RNase H activity with IC50 values around 16 and 30
HIV-1 IN catalytic activity inhibition [52]. Our docking results
predicted that coumarin derivatives 1e16 were able to bind the
HIV-1 IN taking interactions in the active site. In order to support
these results, we tested compounds 1e16 for their ability to inhibit
HIV-1 IN in presence of LEDGF/p75 cofactor, using the strand
transfer inhibitor Raltegravir as a positive control (Table 2). Results
show that the different substitutions on coumarin derivatives led to
compounds that inhibit HIV-1 IN in presence of LEDGF/p75 with a
different potency of inhibition.
Similarly to what found for the RNase H activity, compounds
that presented a methyl group in position 2 on the phenyl ring were
more active if compared with their isomers, with compound 1
m
M, respectively. The introduction of an additional phenyl ring on
compounds 4 and 5 did not improve the potency of inhibition (IC50
values of 61 and 25 M, respectively). Then, we explored if the
m
showing IC50 value of 9.5
41.5 M and 24 M, respectively). A similar behaviour was found in
the presence of the acetyl substituent, with compound 7 showing
an IC50 value of 6.4 M, being more potent than compounds 6, 8 and
9 (IC50 values of 8.5, 22.4 and 18.5 M, respectively). Differently
mM, being more potent than 2 and 3 (with
introduction of an acetyl substituent, as on compounds 1, 2 and 3,
might improve the potency of these derivatives. Results showed
that when the phenyl ring was unsubstituted (compound 6) the
m
m
m
potency of inhibition was not improved (IC50 value of 26
mM). Also
m
compound 9 showed an IC50 value similar to that of compound 3,
while compound 8 was not active, similarly to 2. Differently, the
insertion of acetyl group in compound 1, led to compound 7 that
from the HIV-1 RNase H activity, the introduction of an additional
phenyl ring led to compounds 4 and 5 that retained a good HIV-1 IN
inhibition (IC50 values of 21.5 mM and 7.5 mM, respectively). The
potently inhibited the HIV-1 RNase H function (IC50 around 7
m
M).
elongation of the lateral chain of these two derivatives with
insertion of an acetylacetone group had a detrimental effect on the
potency of inhibition, obtaining compounds 10, 11 and 12, that
inhibit the HIV-1 IN activity with an IC50 values of 42, 47.5 and
Then, we further increased the length of the chain with insertion of
an acetylacetone (compounds 10, 11 and 12) and obtained an in-
crease in potency only in the case of compound 11 that inhibits the
HIV-1 RNase H activity with an IC50 value of 13.8
mM. On the con-
11 mM, respectively. The insertion of an extra ring on compound 6,
trary, the potency of 10 and 12 was negatively affected with respect
to their acetyl substituted counterparts. Instead, the insertion of an
additional pyran ring on compound 6 led to compound 13 that
exhibited a slightly improved HIV-1 RNase H inhibitory activity
(IC50 value of 16.8 mM). Finally, the introduction of additional het-
erocyclic groups (compounds 14, 15 and 16) led to derivatives that
led to compound 13 which decreased the HIV-1 IN inhibitory ac-
tivity by two folds. Finally, the introduction of an additional het-
erocyclic ring condensed with a benzophenone moiety, led to
compound 14 that slightly inhibited the HIV-1 IN activity. On the
contrary the corresponding de-hydroxylated compound 15 and the
phenyl pyrazole derivative 16 were not active.
slightly inhibited the HIV-1 RNase H activity, with an IC50 value
ranging between 38 and 52 mM.
2.6. Characterization of the mode of in inhibition by coumarin
derivatives
2.5. Effect of coumarin derivatives on HIV-1 in activity in presence
of LEDGF/p75 cellular cofactor
It has been previously reported that compounds binding to the
LEDGINs binding pocket can allosterically modulate the dynamic
interplay between IN subunits, inhibiting the IN subunit exchange,
It has been reported that coumarin-based compounds present
Table 2
Coumarine derivatives effects on the HIV-1 RT-associated RNase H and HIV-1 Integrase activities.
Compounds aIC50 HIV-1 RNase H cIC50 IN LEDGF-dependent activity dIC50 IN-IN subunit exchange
eMI50 IN-multimerization
M)
fIC50 IN-LEDGF binding
M)
(
m
M) M) M)
(
m
(
m
(
m
(m
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
16.5 0.76
>100 (89%)b
29.8 2.12
61.3 3.17
24.7 2.02
26.31 0.92
6.75 0.51
>100 (63%)
25.5 2.29
85.8 3.77
13.8 0.91
51.2 0.57
16.8 2.08
38.4 6.02
42.9 1.78
51.8 6.68
e
9.5 1.5
69
3
>100
ND
ND
ND
>100
ND
>100
ND
ND
ND
ND
>100 (55%)
ND
ND
ND
>100 (53%)
ND
>100 (52%)
ND
ND
ND
ND
41.5 8.5
24.0 4.0
21.5 2.5
7.5 0.5
ND
ND
ND
57.0 10
ND
56.5 6.5
ND
ND
ND
ND
85
>100 (78%)
>100 (85%)
ND
8.5 1.5
6.45 0.45
22,35 3,65
18.5 5.5
42.0 2.0
47.5 8.5
11.0 2.0
14.0 3.0
20.0 3.0
>100 (78%)
>100 (61%)
9.0 2.0
7
>100
>100
>100
ND
>100 (73%)
>100 (56%)
>100 (81%)
ND
ND
ND
ND
LEDGIN-6
RAL
RDS1643
>100 (100%)
>100 (100%)
e
10
1
13
3
e
0.058 0.01
e
>100 (100%)
e
>100 (100%)
e
11.2 2.4
a
Compound concentration required to inhibit the HIV-1 reverse transcriptase RNase H activity by 50%.
b
c
d
e
f
Percentage of control activity measured in the presence of 100 mM concentration.
Compound concentration required to inhibit the HIV-1 IN catalytic activities by 50% in the presence of LEDGF.
Compound concentration required to inhibit the HIV-1 IN-IN subunit exchange by 50%.
Compound concentration required to inhibit the multimerization increase by 50%.
Compound concentration required to inhibit the HIV-1 IN-LEDGF interaction by 50%. -f e

F. Esposito et al. / European Journal of Medicinal Chemistry 182 (2019) 111617
7
promoting and stabilizing the multimerization form of IN Ref. [53].
To further support the computational studies, we verify if also
coumarin derivatives were able to bind into the LEDGINs binding
pocket, testing them in a HTRF IN subunit exchange assay, using the
allosteric inhibitor LEDGIN-6 as control [53] (Table 2). In this assay,
when a compound inhibits the IN subunit exchange the HTRF signal
decreases, while when a compound promotes the IN multi-
merization, the HTRF signal increases. Our findings showed that
compounds 1, 5, 7 and 12 were able to weakly inhibit the IN-IN
LigPrep tools at pH 7.4 and after submitted to 10000 steps of energy
minimization, using OPLS_2005 as force field [55] (software:
MacroModel, Schrodinger, LLC, New York, NY, 2018).
For the modeling studies on the RT RNase H active site, we used
the crystallographic structure of an N-hydroxythienopyrimidine-
2,4-dione RNase H active site inhibitor with multiple binding
modes to HIV-1 RT retrieved from the Protein Data Bank (PDB) with
the code 6AOC [48]. This model was chosen for its recently release
(2017) and its better X-ray resolution (1.8 Å), with respect to the
other RT PDB models.
subunit exchange (IC50 value range between 56 and 85 mM), but,
differently from the control LEDGIN-6, none was able to affect IN
multimerization, showing that IN inhibition through interactions
with the catalytic site reveals a profile of inhibition different from
the one of the already reported allosteric inhibitors. To evaluate if
also coumarin derivatives possesses the same LEDGINs inhibitory
mode of action, we tested them for their ability to inhibit IN/LEDGF/
p75 binding, using LEDGIN-6 as a positive control (Table 2). Results
showed that compounds 1, 5, 7 and 13 weakly inhibited the IN/
In order to evaluate the reliability of our docking calculations,
we used the Standard Protocol (SP) Glide algorithm that was able to
reproduce the experimentally determined binding modes, since we
obtained a Root Mean Square Deviation (RMSD) value between the
best docking pose and the ligand co-crystallized into the RNase H
active site equal to 0.992 Å. The receptor was prepared by means of
the Protein Preparation Wizard implemented in Maestro, using
OLPS_2005 as force field [56]. Residual crystallographic buffer
components were removed, missing side chains were built using
the Prime module [57], hydrogen atoms were added, zero-order
bonds to metals were created followed by the generation of
metal binding states and side chains protonation states at pH 7.4
were assigned. The structure was submitted to 10000 minimization
steps using OPLS_2005 as force field [55] (software: MacroModel,
Schrodinger, LLC, New York, NY, 2018). For the grid generation, a
box of 26 Å ꢁ 26 Å X 26 Å, centered on the active site Mg2þ ions, was
created. The docking studies were carried out by means of Glide
software v. 6.7 [54] by using SP algorithm and generating 100 poses
for ligands.
LEDGF/p75 binding (IC50 values around 110 mM), supporting the
consideration that, by binding to the catalytic site the correct
binding between IN and LEDGF/p75 cofactor was disturbed. All the
other derivatives were not able to inhibit LEDGF/p75-IN binding.
Otherwise from LEDGIN-6, coumarin derivatives can allosterically
modulate the dynamic interplay between the IN subunits without
stabilize the IN multimeric form, suggesting that these compounds
do not interact with IN at the LEDGF binding site. Unfortunately,
when tested in cell-based assays these coumarin derivatives were
not active (data not shown).
3. Conclusion
For the IN studies, we generated structure theoretical model in
order to elucidate the binding mode and the interactions between
the compounds and the IN catalytic core domain in the presence of
the LEDGF/p75 cofactor.
In the optimization of the coumarin scaffold to achieve com-
pounds able to inhibit multiple HIV-1 enzymatic functions, we
performed docking analysis of coumarin derivatives on HIV-1 IN
and RNase H active sites. Most of the synthesized coumarin de-
rivatives were well accommodated into both the investigated active
sites. Mode-of-action and docking studies revealed compounds 1, 3,
6 and 7 as promising HIV-1 IN and RT RNase H dual inhibitors.
Overall, compound 7 resulted as the most interesting derivative
since it inhibits both HIV-1 IN and RNase H activities in the low
micro-molar range, thus paving the way for a future rational opti-
mization process for dual HIV inhibitors.
The Prototype Foamy Virus (PFV) IN-LEDGF complex was con-
structed by assembling two crystallographic structures, the crystal
structure of the PFV intasome in complex with magnesium and
Raltegravir at 2.65 Å resolution (PDB code 3OYA) [58], and the
experimental structure of the LEDGF/p75 cofactor (PDB code 2B4J)
[59]. We used the PFV IN model due to the high level of conser-
vation between retroviral INs, especially in their active site [49,60].
The obtained receptor model was submitted to 10000 steps of
energy minimization, carried out using OLPS_2005 as force field,
(software: MacroModel, Schrodinger, LLC, New York, NY, 2018) and
further 100ns of Molecular Dynamics simulations (MDs) were
carried out. MDs were run using Desmond package v. 3.8 at 300 K
temperature and ensemble NPT class; the system was put in an
orthorhombic box of TIP3P water molecules, extending at least 10 Å
from the protein, and counter ions were added to neutralize the
system charge [61]. The resulting trajectory was clustered with
respect to the RMSD, calculated on all atoms of the enzyme, thus
obtaining ten representative structures. In detail, for the further
molecular recognition studies, we selected the most populated
structure, according to the Boltzmann population value, which also
corresponded to the Prime lowest-energy structure [57]. For the
grid generation, a box of 40 Å ꢁ 40 Å X 40 Å, centered on the active
site Mg2þ ions, was created. The docking studies were carried out
by means of Glide software v. 6.7 [54] by using SP algorithm and
generating 100 poses for ligands.
4. Experimental section
4.1. Chemistry
The synthesis of coumarin derivatives and their characterization
are reported in the Supplementary Material section. All melting
points were determined in open glass capillaries in a liquid-
paraffin-bath and are uncorrected. Purity of the compounds was
checked by TLC using silica gel-G as adsorbent and visualization
was accomplished by UV light or iodine. IR spectra were recorded
on FT-IR spectrophotometer and NMR spectra in CDCl3 on a
BRUKER AC (300 MHz or 400 MHz) FT NMR spectrometer using
TMS as internal standard (chemical shifts in
d ppm). Elemental
analyses were carried out in the Saurashtra University, Rajkot on
PerkinElmer Elemental Analyzer. The theoretical calculation of
physicochemical properties of the coumarin derivatives are re-
ported in Table S2 of the Supplementary Material.
4.3. Expression and purification of recombinant HIV-1 RT, INs and
LEDGFs
4.2. Molecular modeling studies
His-tagged p66/p51 HIV-1 RTs were expressed in E. coli strain
M15 and purified as described [21,62]. Full-length IN and LEDGF
proteins were expressed in E. coli BL21 (DE3) and purified as
The molecular modeling analysis was performed by means of
Schrodinger package [54]. The ligands were prepared by means of

8
F. Esposito et al. / European Journal of Medicinal Chemistry 182 (2019) 111617
described [3,63,64].
Abbreviations
Human Immunodeficiency Virus type 1 (HIV-1), Protease (PR),
4.4. HTRF LEDGF-dependent assay
Integrase (IN), Reverse Transcriptase (RT), Ribonuclease H (RNase
H), RNA-dependent DNA-polymerase (RDDP), DNA-dependent
DNA-polymerase (DDDP), Preintegration complex (PIC), Lens-
Epitelium-derived (LEDGF/p75), IN binding domain (IBD), Highly
active antiretroviral therapy (HAART), IN inhibitors (INIs), nucleo-
side/nucleotide RT inhibitors (NRTIs /NtRTIs), non-nucleoside RT
inhibitors (NNRTIs), coumarin (2H-chromen-2-one; 1-benzopyran-
2-one), coumarin (4-hydroxyepyranobenzopyran), Molecular Dy-
namics simulations (MDs), Protein Data Bank (PDB), Root Mean
Square Deviation (RMSD), Prototype Foamy Virus (PFV).
The IN LEDGF/p75-dependent assay measure the inhibition of
30-processing and strand-transfer IN reactions in the presence of
recombinant LEDGF/p75 protein [30,65,66]. 50 nM IN was pre-
incubated with increasing concentration of compounds for 1 h at
room temperature in reaction buffer containing 20 mM HEPES pH
7.5, 1 mM DTT, 1% Glycerol, 20 mM MgCl2, 0.05% Brij-35 and 0.1 mg/
ml BSA. To this mixture, 9 nM DNA donor substrate (50-ACAGGCC-
TAGCACGCGTCG-Biotin-30 annealed with 50-CGACGCGTGG-
TAGGCCTGT-Biotin30) and 50 nM DNA acceptor substrate (50-Cy5-
ATGTGGAAAATCTCTAGCAGT-30 annealed with 50-Cy5- TGAGCTC-
GAGATTTTCCACAT-30) and 50 nM LEDGF/p75 protein (or without
LEDGF/p75 protein) were added and incubated at 37 ꢂC for 90 min.
After the incubation, 4 nM of Europium-Streptavidine were added
at the reaction mixture and the HTRF signal was recorded using a
PerkinElmer Victor 3 plate reader using a 314 nm for excitation
wavelength and 668 and 620 nm for the wavelength of the acceptor
and the donor substrates emission, respectively.
Declaration of interest statement
The authors report no conflict of interest.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2019.111617.
4.5. HTRF-based Integrase-LEDGF interaction assay
References
His-IN was pre-incubated with different concentrations of
compound in a buffer containing 150 mM NaCl, 2 mM MgCl2, 0.1%
Nonidet P-40, 1 mg/ml BSA, 25 mM Tris (pH 7.4) for 30 min at room
temperature.3 Then, FLAG-LEDGF was added to the reaction and a
mixture of anti-His6-XL665 and anti-FLAG-EuCryptate antibodies
were then added to the reaction. After 4 h at 4 ꢂC, the HTRF signal
was recorded using a PerkinElmer Victor 3 plate reader using
314 nm for excitation wavelength and 668 and 620 nm for the
wavelength of the acceptor and donor emission, respectively. The
HTRF signal is defined as the emission ratio 665 nm/620 nm
multiplied by 10,000.
[1] F. Esposito, E. Tramontano, Past and future. Current drugs targeting HIV-1
integrase and reverse transcriptase-associated ribonuclease H activity: sin-
gle and dual active site inhibitors, Antivir. Chem. Chemother. 23 (2013)
129e144, https://doi.org/10.3851/IMP2690.
[2] E.M. Poeschla, Integrase, LEDGF/p75 and HIV replication, Cell. Mol. Life Sci. 65
(2008) 1403e1424, https://doi.org/10.1007/s00018-008-7540-5.
[3] F. Esposito, C. Tintori, R. Martini, F. Christ, Z. Debyser, R. Ferrarese, G. Cabiddu,
A. Corona, E.R. Ceresola, A. Calcaterra, V. Iovne, B. Botta, M. Clementi,
F. Canducci, M. Botta, E. Tramontano, Kuwanon-l as a new allosteric HIV-1
integrase inhibitor: molecular modeling and biological evaluation, Chem-
biochem 16 (2015) 2507e2512, https://doi.org/10.1002/cbic.201500385.
[4] H.B. Andrade, C.R. Shinotsuka, I.R.F. Da Silva, C.S. Donini, H. Yeh Li, F.B. De
Carvalho, P.E.A. Americano do Brasil, F.A. Bozza, A. Miguel Japiassu, Highly
active antiretroviral therapy for critically ill HIV patients: a systematic review
and meta-analysis, PLoS One 12 (2017), e0186968, https://doi.org/10.1371/
journal.pone.0186968.
4.6. HTRF-based in subunit exchange assay
€
[5] A. Schneider, A. Corona, I. Sporing, M. Jordan, B. Buchholz, E. Maccioni, R. Di
€
Santo, J. Bodem, E. Tramontano, B.M. Wohrl, Biochemical characterization of a
His and FLAG-tagged INs were mixed in 25 mM Tris (pH 7.4)
buffer containing 150 mM NaCl, 2 mM MgCl2, 0.1% Nonidet P-40,
1 mg/ml BSA [67]. Test compounds were then added to the mixture
and incubated for 2.5 h at room temperature. A mixture of anti-
His6-XL665 and anti-FLAG-EuCryptate antibodies were then added
to the reaction and incubated at room temperature for 3 h. The
HTRF signal was recorded as above.
multi-drug resistant HIV-1 subtype AG reverse transcriptase: antagonism of
AZT discrimination and excision pathways and sensitivity to RNase H in-
hibitors, Nucleic Acids Res. 44 (2016) 2310e2322, https://doi.org/10.1093/
nar/gkw060.
[6] N.A. Margot, P. Wong, R. Kulkarni, K. White, D. Porter, M.E. Abram,
C. Callebaut, M.D. Miller, Commonly transmitted HIV-1 drug resistance mu-
tations
in
reverse-transcriptase
and
protease
in
antiretroviral
treatmentenaive patients and response to regimens containing tenofovir
disoproxil fumarate or tenofovir alafenamide, J. Infect. Dis. 215 (2017)
920e927, https://doi.org/10.1093/infdis/jix015.
[7] S. Distinto, E. Maccioni, R. Meleddu, A. Corona, S. Alcaro, E. Tramontano,
Molecular aspects of the RT/drug interactions. Perspective of dual inhibitors,
Curr. Pharmaceut. Des. 19 (2013) 1850e1859.
4.7. RT-associated RNase H polymerase-independent cleavage
assay
[8] L. Pouga, M.M. Santoro, C. Charpentier, D. Di Carlo, I. Romeo, A. Artese,
S. Alcaro, A. Antinori, M. Wirden, C.F. Perno, F.A. Ambrosio, V. Calvez,
D. Descamps, A.C. Marcelin, F. Ceccherini-Silberstein, S. Lambert-Niclot, New
resistance mutations to nucleoside reverse transcriptase inhibitors at codon
184 of HIV-1 reverse transcriptase (M184L and M184T), Chem. Biol. Drug Des.
93 (2019) 50e59, https://doi.org/10.1111/cbdd.13378.
[9] F. Esposito, A. Corona, E. Tramontano, HIV-1 reverse transcriptase still remains
a new drug target: structure, function, classical inhibitors, and new inhibitors
with innovative mechanisms of actions, Mol. Biol. Int. 2012 (2012) 586401,
https://doi.org/10.1155/2012/586401.
[10] G. Costa, R. Rocca, A. Corona, N. Grandi, F. Moraca, I. Romeo, C. Talarico,
M.G. Gagliardi, F.A. Ambrosio, F. Ortuso, S. Alcaro, S. Distinto, E. Maccioni,
E. Tramontano, A. Artese, Novel natural non-nucleoside inhibitors of HIV-1
reverse transcriptase identified by shape- and structure-based virtual
screening techniques, Eur. J. Med. Chem. 161 (2019) 1e10, https://doi.org/
10.1016/j.ejmech.2018.10.029.
The HIV-1 RT-associated RNase H activity was measured as
described [68e71] in 100
Tris HCl pH 7.8, 6 mM MgCl2, 1 mM DTT, 80 mM KCl, hybrid RNA/
DNA
(50-GTTTTCTTTTCCCCCCTGAC-30-Fluorescein, 50-CAAAA-
mL reaction volume containing 50 mM
GAAAAGGGGGGACUG-30-Dabcyl) and 3.8 nM RT. The reaction
mixture was incubated for 1 h at 37 ꢂC, the reaction was stopped by
addition of EDTA and products were measured with a Victor 3
(Perkin) equipped with excitation/emission filters of 490/528 nm.
Author contributions
[11] A. Corona, T. Masaoka, G. Tocco, E. Tramontano, S.F. Le Grice, E. Tramontano,
Active site and allosteric inhibitors of the ribonuclease H activity of HIV
The manuscript was written through contributions of all au-
thors. All authors have given approval to the final version of the
manuscript. Francesca Esposito and Francesca Alessandra Ambrosio
contributed equally to this work.
reverse transcriptase, Future Med. Chem.
doi.org/10.4155/fmc.13.178.
5 (2013) 2127e2139, https://
[12] M. Tisdale, T. Schulze, B.A. Larder, K. Moelling, Mutations within the RNase H

F. Esposito et al. / European Journal of Medicinal Chemistry 182 (2019) 111617
9
domain of human immunodeficiency virus type
abolish virus infectivity, J. Gen. Virol. 72 (1991) 59e66.
1
reverse transcriptase
[29] A. Corona, S.F. di Leva, G. Rigogliuso, L. Pescatori, N.V. Madia, F. Subra,
O. Delelis, F. Esposito, M. Cadeddu, R. Costi, S. Cosconati, E. Novellino, R. di
Santo, E. Tramontano, New insights into the interaction between pyrrolyl
diketoacids and HIV-1 integrase active site and comparison with RNase H,
Antivir. Res. 134 (2016) 236e243, https://doi.org/10.1016/j.antiviral.2016.
09.008.
[13] A. Corona, F. Esposito, E. Tramontano, Can the ever-promising target HIV
reverse transcriptase-associated RNase H become a success story for drug
development ? 9 (2014) 445e448.
ꢁ
[14] X. Wang, P. Gao, L. Menendez-Arias, X. Liu, P. Zhan, Update on recent de-
velopments in small molecular HIV-1 RNase H inhibitors (2013-2016): op-
portunities and challenges, Curr. Med. Chem. (2017) 1e21, https://doi.org/
10.2174/0929867324666170113.
[30] M. Carcelli, D. Rogolino, A. Gatti, N. Pala, A. Corona, A. Caredda, E. Tramontano,
C. Pannecouque, L. Naesens, F. Esposito, Chelation motifs affecting metal-
dependent viral enzymes: N'-acylhydrazone ligands as dual target inhibitors
of HIV-1 integrase and reverse transcriptase ribonuclease H domain, Front.
Microbiol. 8 (2017) 1e10, https://doi.org/10.3389/fmicb.2017.00440.
[15] F. Esposito, T. Kharlamova, S. Distinto, L. Zinzula, Y.C. Cheng, G. Dutschman,
G. Floris, P. Markt, A. Corona, E. Tramontano, Alizarine derivatives as new dual
inhibitors of the HIV-1 reverse transcriptase-associated DNA polymerase and
RNase H activities effective also on the RNase H activity of non-nucleoside
resistant reverse transcriptases, FEBS J. 278 (2011) 1444e1457, https://
doi.org/10.1111/j.1742-4658.2011.08057.x.
[16] V. Suchaud, F. Bailly, C. Lion, E. Tramontano, F. Esposito, A. Corona, F. Christ,
Z. Debyser, P. Cotelle, Development of a series of 3-hydroxyquinolin-2(1H)-
ones as selective inhibitors of HIV-1 reverse transcriptase associated RNase H
activity, Bioorg. Med. Chem. Lett 22 (2012) 3988e3992, https://doi.org/
10.1016/j.bmcl.2012.04.096.
ꢁ
[31] G. Cuzzucoli Crucitti, M. Metifiot, L. Pescatori, A. Messore, V.N. Madia, G. Pupo,
F. Saccoliti, L. Scipione, S. Tortorella, F. Esposito, A. Corona, M. Cadeddu,
C.Y. Marchand, Pommier, E. Tramontano, R. Costi, R. Di, Santo Structure-
activity relationship of pyrrolyl diketo acid derivatives as dual inhibitors of
HIV-1 integrase and reverse transcriptase ribonuclease H domain, J. Med.
Chem. 58 (2015) 1915e1928, https://doi.org/10.1021/jm501799k.
ꢁ
[32] R. Costi, M. Metifiot, F. Esposito, G. Cuzzucoli Crucitti, L. Pescatori, A. Messore,
L. Scipione, S. Tortorella, L. Zinzula, E. Novellino, Y. Pommier, E. Tramontano,
C. Marchand, R. Di Santo, 6-(1- benzyl-1H-pyrrol-2-yl)-2, 4-dioxo-5-hexenoic
acids as dual inhibitors of recombinant HIV-1 integrase and ribonuclease H,
[17] F. Esposito, A. Corona, L. Zinzula, T. Kharlamova, E. Tramontano, New
anthraquinone derivatives as inhibitors of the HIV-1 reverse transcriptase-
synthesized by
a parallel synthesis approach, J. Med. Chem. 56 (2013)
associated ribonuclease
H
function, Chemotherapy 58 (2012) 299e307,
8588e8598, https://doi.org/10.1021/jm401040b.
https://doi.org/10.1159/000343101.
[33] S.X. Gu, P. Xue, X.L. Ju, Y.Y. Zhu, Advances in rationally designed dual in-
hibitors of HIV-1 reverse transcriptase and integrase, Bioorg. Med. Chem. 24
(2016) 5007e5016.
[18] A. Corona, R. Meleddu, F. Esposito, S. Distinto, G. Bianco, T. Masaoka,
ꢁ
E. Maccioni, L. Menendez-Arias, S. Alcaro, S.F.J. Le Grice, E. Tramontano,
Ribonuclease H/DNA polymerase HIV-1 reverse transcriptase dual inhibitor:
mechanistic studies on the allosteric mode of action of isatin-based com-
pound RMNC6, PLoS One 11 (2016) 1e18, https://doi.org/10.1371/
journal.pone.0147225.
[34] H. Kawaguchi, T. Naito, H. Tsukiura, Studies on coumermycin. A new anti-
biotic. II. Structure of coumermycin A1, J. Antibiot. 18 (1965) 11e25.
[35] D.C. Karia, H.K. Pandya, N.K. Godvani, A. Shah, Synthesis, Characterization and
anti-HIV Activity of 4-Hydroxy-3-(5-methyl-1-phenyl-1H-pyrazol-3-yl) pyr-
ano [3, 2-c] chromene-2, 5-dione, Elixir Org. Chem. 47 (2012) 8797e8799.
[36] Y. Takeuchi, L. Xie, L.M. Cosentino, K.H. Lee, Anti-AIDS agents-XXVIII. 1 Syn-
thesis and Anti-HIV activity of methoxy substituted 3’,4’-Di-O-(ꢀ)-campha-
noyl-(þ)-cis-khellactone (DCK) analogues, Bioorg. Med. Chem. Lett 7 (1997)
2573e2578, https://doi.org/10.1016/S0960-894X(97)10050-6.
[19] N. Pala, F. Esposito, D. Rogolino, M. Carcelli, V. Sanna, M. Palomba, L. Naesens,
A. Corona, N. Grandi, E. Tramontano, M. Sechi, Inhibitory effect of 2,3,5,6-
tetrafluoro-4-[4-(Aryl)-1H-1,2,3-triazol-1-yl]benzenesulfonamide derivatives
on HIV reverse transcriptase associated rnase H activities, Int. J. Mol. Sci. 17
(2016) 1371, https://doi.org/10.3390/ijms17081371.
[20] T. Kharlamova, F. Esposito, L. Zinzula, G. Floris, Y.C. Cheng, G.E. Dutschman,
E. Tramontano, Inhibition of HIV-1 ribonuclease H activity by novel frangula-
emodine derivatives, Med. Chem. 5 (2009) 398e410. MC-Abs-13 [pii].
[21] A. Corona, F.S. Di Leva, S. Thierry, L. Pescatori, G. Cuzzucoli Crucitti, F. Subra,
O. Delelis, F. Esposito, G. Rigogliuso, R. Costi, S. Cosconati, E. Novellino, R. Di
Santo, E. Tramontano, Identification of highly conserved residues involved in
inhibition of HIV-1 RNase H function by diketo acid derivatives, Antimicrob.
Agents Chemother. 58 (2014) 6101e6110, https://doi.org/10.1128/aac.03605-
14.
[22] S.K.V. Vernekar, J. Tang, B. Wu, A.D. Huber, M.C. Casey, N.S. Myshakina,
D.J. Wilson, J. Kankanala, K.A. Kirby, M.A. Parniak, S.G. Sarafianos, Z. Wang,
S. Kumar, V. Vernekar, J. Tang, B. Wu, A.D. Huber, M.C. Casey, N.S. Myshakina,
D.J. Wilson, J. Kankanala, K.A. Kirby, M.A. Parniak, S.G. Sarafianos, Z. Wang,
Double-winged 3-Hydroxypyrimidine-2,4-diones: potent and selective inhi-
bition against HIV-1 RNase H with significant antiviral activity, J. Med. Chem.
60 (2017) 5045e5046, https://doi.org/10.1021/acs.jmedchem.7b00440.
[23] N. Deng, A. Hoyte, Y.E. Mansour, M.S. Mohamed, J.R. Fuchs, A.N. Engelman,
M. Kvaratskhelia, R. Levy, Allosteric HIV-1 integrase inhibitors promote
aberrant protein multimerization by directly mediating inter-subunit in-
teractions: structural and thermodynamic modeling studies, Protein Sci. 25
(2016) 1911e1917, https://doi.org/10.1002/pro.2997.
[37] L. Huang, Y. Kashiwada, L.M. Cosentino, S. Fan, C.H. Chen, T. McPhail,
T. Fujioka, K. Mihashi, K.H. Lee, Anti-aids agents. 15. Synthesis and anti-HIV
activity of dihydroseselins and related analogs, J. Med. Chem. 37 (1994)
3947e3955, https://doi.org/10.1021/jm00049a014.
[38] N. Thomas, S.M. Zachariah, Pharmacological activities of chromene de-
rivatives: an overview, Asian J. Pharmaceut. Clin. Res. 6 (2013) 11e15.
[39] T.O. Olomola, R. Klein, N. Mautsa, Y. Sayed, P.T. Kaye, Synthesis and evaluation
of coumarin derivatives as potential dual-action HIV-1 protease and reverse
transcriptase inhibitors, Bioorg. Med. Chem. 21 (2013) 1964e1971, https://
doi.org/10.1016/j.bmc.2013.01.025.
[40] T.R. Burke, M.R. Fesen, A. Mazumder, J. Wang, A.M. Carothers, D. Grunberger,
J. Driscoll, K. Kohn, Y. Pommier, Hydroxylated aromatic inhibitors of HIV-1
integrase, J. Med. Chem. 38 (1995) 4171e4178, https://doi.org/10.1021/
jm00021a006.
[41] M. Faisal, A. Saeed, D. Shahzad, T.A. Fattah, B. Lal, P.A. Channar, J. Mahar,
S. Saeed, P.A. Mahesar, F.A. Larik, Enzyme inhibitory activities an insight into
the structureeActivity relationship of biscoumarin derivatives, Eur. J. Med.
Chem. 141 (2017) 386e403, https://doi.org/10.1016/J.EJMECH.2017.10.009.
[42] P. Wadhwa, P. Jain, H.R. Jadhav, S. Rudrawar, Quinoline, coumarin and other
heterocyclic analogues based HIV-1 integrase inhibitors, Curr. Drug Discov.
Technol. 15 (2018) 2e19, https://doi.org/10.2174/15701638146661705311
15452.
[24] C. Burlein, C. Wang, M. Xu, T. Bhatt, M. Stahlhut, Y. Ou, G.C. Adam, J. Heath,
D.J. Klein, J. Sanders, K. Narayan, P. Abeywickrema, R. Heo, S.S. Carroll,
J.A. Grobler, S. Sharma, T.L. Diamond, A. Converso, D.J. Krosky, Discovery of a
distinct chemical and mechanistic class of allosteric HIV-1 integrase inhibitors
with antiretroviral activity, ACS Chem. Biol. 12 (2017) 2858e2865, https://
doi.org/10.1021/acschembio.7b00550.
[43] D. Yu, M. Suzuki, L. Xie, S.L. Morris-Natschke, K.H. Lee, Recent progress in the
development of coumarin derivatives as potent anti-HIV agents, Med. Res.
Rev. 23 (2003) 322e345, https://doi.org/10.1002/med.10034.
[44] P.H. Lin, Y.Y. Ke, C.T. Su, H.Y. Shiao, H.P. Hsieh, Y.K. Chao, C.N. Lee, C.L. Kao,
Y.S. Chao, S.Y. Chang, Inhibition of HIV-1 tat-mediated transcription by a
coumarin derivative, BPRHIV001, through the akt pathway, J. Virol. 85 (2011)
9114e9126, https://doi.org/10.1128/JVI.00175-11.
[25] L.D. Fader, E. Malenfant, M. Parisien, R. Carson, F. Bilodeau, S. Landry,
M. Pesant, C. Brochu, S. Morin, C. Chabot, T. Halmos, Y. Bousquet, M.D. Bailey,
S.H. Kawai, R. Coulombe, S. La Plante, A. Jakalian, P.K. Bhardwaj, D. Wernic,
P. Schroeder, M. Amad, P. Edwards, M. Garneau, J. Duan, M. Cordingley,
[45] E.B.B. Ong, N. Watanabe, A. Saito, Y. Futamura, K.H. Abd El Galil, A. Koito,
N. Najimudin, H. Osada, Vipirinin, a coumarin-based HIV-1 Vpr inhibitor, in-
€
R. Bethell, S.W. Mason, M. Bos, P. Bonneau, M.A. Poupart, A.M. Faucher,
teracts with a hydrophobic region of Vpr, J. Biol. Chem. 286 (2011)
B. Simoneau, C. Fenwick, C. Yoakim, Y. Tsantrizos, Discovery of BI 224436, a
noncatalytic site integrase inhibitor (NCINI) of HIV-1, ACS Med. Chem. Lett. 5
(2014) 422e427, https://doi.org/10.1021/ml500002n.
14049e14056, https://doi.org/10.1074/jbc.M110.185397.
[46] J. Kankanala, K.A. Kirby, F. Liu, L. Miller, E. Nagy, D.J. Wilson, M.A. Parniak,
S.G. Sara, Z. Wang, Z. Design, Synthesis, and biological evaluations of
hydroxypyridonecarboxylic acids as inhibitors of HIV reverse transcriptase
associated RNase H, J. Med. Chem. 59 (2016) 5051e5062, https://doi.org/
10.1021/acs.jmedchem.6b00465.
[47] E. Tramontano, F. Esposito, R. Badas, R. Di Santo, R. Costi, P. La Colla, 6-[1-(4-
Fluorophenyl)methyl-1H-pyrrol-2-yl)]-2,4-dioxo-5-hexenoic acid ethyl ester
a novel diketo acid derivative which selectively inhibits the HIV-1 viral
replication in cell culture and the Ribonuclease H activity in vitro, Antivir. Res.
65 (2005) 117e124, https://doi.org/10.1016/j.antiviral.2004.11.002.
[48] J. Kankanala, K.A. Kirby, A.D. Huber, M.C. Casey, D.J. Wilson, S.G. Sarafianos,
Z. Wang, Z. Design, Synthesis and biological evaluations of N-hydroxy thie-
nopyrimidine-2,4-diones as inhibitors of HIV reverse transcriptase-associated
[26] F. Esposito, I. Carli, C. Del Vecchio, L. Xu, A. Corona, N. Grandi, D. Piano,
E. Maccioni, S. Distinto, C. Parolin, E. Tramontano, Sennoside A, derived from
the traditional Chinese medicine plant Rheum L., is a new dual HIV-1 inhibitor
effective on HIV-1 replication, Phytomedicine 23 (2016) 1383e1391, https://
doi.org/10.1016/j.phymed.2016.08.001.
[27] R. Martini, F. Esposito, A. Corona, R. Ferrarese, E.R. Ceresola, L. Visconti,
C. Tintori, A. Barbieri, A. Calcaterra, V. Iovine, F. Canducci, E. Tramontano,
M. Botta, Natural product kuwanon-L inhibits HIV-1 replication through
multiple target binding, Chembiochem 18 (2017) 374e377, https://doi.org/
10.1002/cbic.201600592.
[28] I. Malet, F.A. Ambrosio, F. Subra, B. Herrmann, H. Leh, M.C. Bouger, A. Artese,
C. Katlama, C. Talarico, I. Romeo, S. Alcaro, G. Costa, E. Deprez, V. Calvez,
A.G. Marcelin, O. Delelis, Pathway involving the N155H mutation in HIV-1
integrase leads to dolutegravir resistance, J. Antimicrob. Chemother. 75
(2018) 1158e1166, https://doi.org/10.1093/jac/dkx529.
RNase H, Eur. J. Med. Chem.
j.ejmech.2017.09.054.Design.
1 (2018) 149e161, https://doi.org/10.1016/
[49] S. Hare, A.M. Vos, R.F. Clayton, J.W. Thuring, M.D. Cummings, P. Cherepanov,
Molecular mechanisms of retroviral integrase inhibition and the evolution of

10
F. Esposito et al. / European Journal of Medicinal Chemistry 182 (2019) 111617
viral resistance, Proc. Natl. Acad. Sci. U.S.A. 107 (2010) 20057e20062, https://
doi.org/10.1073/pnas.1010246107.
[50] T. Pengsuparp, M. Serit, S.H. Hughes, D.D. Soejarto, J.M. Pezzuto, Specific in-
hibition of human immunodeficiency virus type reverse transcriptase
Lupeol mode of action, Pathog. Dis. 75 (2017), https://doi.org/10.1093/
femspd/ftx065 ftx065.
[63] C. Tintori, F. Esposito, F. Morreale, R. Martini, E. Tramontano, M. Botta,
Investigation on the sucrose binding pocket of HIV-1 Integrase by molecular
dynamics and synergy experiments, Bioorg. Med. Chem. Lett 25 (2015)
3013e3016, https://doi.org/10.1016/j.bmcl.2015.05.011.
1
mediated by soulattrolide, a coumarin isolated from the latex of Chalophillum
teysmannii, J. Nat. Prod. 59 (1996) 839e842.
[51] A. Corona, V. Onnis, A. Deplano, G. Bianco, M. Demurtas, S. Distinto,
Y.C. Cheng, S. Alcaro, F. Esposito, E. Tramontano, Design, synthesis and anti-
viral evaluation of novel heteroarylcarbothioamide derivatives as dual in-
[64] A. Virgilio, T. Amato, L. Petraccone, F. Esposito, N. Grandi, E. Tramontano,
R. Romero, S.H. Der, I. Gomez-monterrey, E. Novellino, L. Mayol, V. Esposito,
A. Galeone, Improvement of the activity of the anti-HIV-1 integrase aptamer
T30175 by introducing a modified thymidine into the loops, Sci. Rep. (2018),
https://doi.org/10.1038/s41598-018-25720-1.
[65] C. Sanna, D. Rigano, P. Cortis, A. Corona, M. Ballero, C. Parolin, C. Del Vecchio,
G. Chianese, E. Saccon, C. Formisano, E. Tramontano, F. Esposito, Onopordum
illyricum L., a Mediterranean plant, as a source of anti HIV-1 compounds, Plant
Biosyst. (2018), https://doi.org/10.1080/11263504.2018.1439118, 1274-1271.
[66] C. Sanna, M. Scognamiglio, A. Fiorentino, A. Corona, V. Graziani, A. Caredda,
P. Cortis, M. Montisci, R. Ceresola, F. Canducci, F. Poli, E. Tramontano,
F. Esposito, Prenylated phloroglucinols from Hypericum scruglii , an endemic
species of Sardinia (Italy), as new dual HIV-1 inhibitors effective on HIV-1
replication, PLoS One 13 (2018) 1e19, https://doi.org/10.1371/journal.pone.
0195168.
hibitors of HIV-1 Reverse transcriptase-associated RNase
H and RDDP
functions, Pathog. Dis. 75 (2017), https://doi.org/10.1093/femspd/ftx078
ftx078.
[52] R.B. Patil, S.D. Sawant, 4D-QSAR studies of coumarin derivatives as HIV-1
integrase 30-processing inhibitors, Med. Chem. Res. 24 (2015) 3062e3076,
https://doi.org/10.1007/s00044-015-1359-z.
[53] J.J. Kessl, N. Jena, Y. Koh, H. Taskent-Sezgin, A. Slaughter, L. Feng, S. De Silva,
L. Wu, S.F.J. Le Grice, A. Engelman, J.R. Fuchs, M. Kvaratskhelia, Multimode,
cooperative mechanism of action of allosteric HIV-1 integrase inhibitors,
J. Biol. Chem. 287 (2012) 16801e16811, https://doi.org/10.1074/jbc.M112.
354373.
€
[54] Schrodinger, LLC, New York, NY, 2017.
[55] D. Shivakumar, E. Harder, W. Damm, R.A. Friesner, W. Sherman, Improving the
prediction of absolute solvation free energies using the next generation OPLS
force field, J. Chem. Theory Comput. 8 (2012) 2553e2558, https://doi.org/
10.1021/ct300203w.
[67] M. Sala, A. Spensiero, F. Esposito, M.C. Scala, E. Vernieri, A. Bertamino,
M. Manfra, A. Carotenuto, P. Grieco, E. Novellino, M. Cadeddu, E. Tramontano,
D. Schols, P. Campiglia, I.M. Gomez-Monterrey, Development and identifica-
tion of a novel anti-HIV-1 peptide derived by modification of the N-terminal
domain of HIV-1 integrase, Front. Microbiol. 7 (2016) 845, https://doi.org/
10.3389/fmicb.2016.00845.
€
€
[56] Schrodinger Suite 2016-3 Protein Preparation Wizard, Epik, Schrodinger, LLC,
€
New York, NY, 2016. Impact, Schrodinger, LLC, New York, NY, 2016; Prime,
€
Schrodinger, LLC, New York, NY. 2017.
[68] F. Esposito, C. Sanna, C. Del Vecchio, V. Cannas, A. Venditti, A. Corona,
A. Bianco, A.M. Serrilli, L. Guarcini, C. Parolin, M. Ballero, E. Tramontano,
Hypericum hircinum L. Components as new single-molecule inhibitors of
both HIV-1 reverse transcriptase-associated DNA polymerase and ribonu-
clease H activities, Pathog. Dis. 68 (2013) 116e124, https://doi.org/10.1111/
2049-632X.12051.
€
[57] Prime, Schrodinger, LLC, New York, NY. 2017.
[58] S. Hare, A.M. Vos, R.F. Clayton, J.M. Thuring, M. D: Cummings, P. Cherepanov,
Molecular Mechanisms of Retroviral Integrase Inhibition and the Evolution of
Viral Resistance, vol. 107, 2010, pp. 20057e20062, https://doi.org/10.1073/
pnas.1010246107.
[59] P. Cherepanov, A.L.B. Ambrosio, S. Rahman, T. Ellenberger, A. Engelman,
Structural basis for the recognition between HIV-1 integrase and transcrip-
tional coactivator p75, Proc. Natl. Acad. Sci. U.S.A. 102 (2005) 17308e17313,
https://doi.org/10.1073/pnas.0506924102.
ꢀ
[69] D. Spano, F. Pintus, F. Esposito, D. Loche, G. Floris, R. Medda, Euphorbia
characias latex: micromorphology of rubber particles and rubber transferase
activity, Plant Physiol. Biochem. 87 (2015) 26e34, https://doi.org/10.1016/
j.plaphy.2014.12.008.
[60] S. Hare, S.S. Gupta, E. Valkov, A. Engelman, P. Cherepanov, Retroviral intasome
assembly and inhibition of DNA strand transfer, Nature 464 (2010) 232e236,
https://doi.org/10.1038/nature08784.Retroviral.
[61] Desmond Molecular Dynamics System, D. E. Shaw Research, New York, NY,
2017.
[70] C. Tintori, A. Corona, F. Esposito, A. Brai, N. Grandi, E.R. Ceresola, M. Clementi,
F. Canducci, E. Tramontano, M. Botta, Inhibition of HIV-1 reverse transcriptase
dimerization by small molecules, Chembiochem 17 (2016) 683e688, https://
doi.org/10.1002/cbic.201500668.
ꢀ
[71] M.B. Pisano, S. Cosentino, S. Viale, D. Spano, A. Corona, F. Esposito,
[62] F. Esposito, M. Mandrone, C. Del Vecchio, I. Carli, S. Distinto, A. Corona,
M. Lianza, D. Piano, M. Tacchini, E. Maccioni, F. Cottiglia, E. Saccon, F. Poli,
C. Parolin, E. Tramontano, Multi-target activity of Hemidesmus indicus
decoction against innovative HIV-1 drug targets and characterization of
E. Tramontano, P. Montoro, C.I.G. Tuberoso, R. Medda, F. Pintus, Biological
activities of aerial parts extracts of euphorbia characias, BioMed Res. Int.
(2016), https://doi.org/10.1155/2016/1538703.