Design, synthesis, and biological evaluation of 1,3-diarylpropenones as dual inhibitors of HIV-1 reverse transcriptase

Article abstract of DOI:10.1002/cmdc.201402015

A small library of 1,3-diarylpropenones was designed and synthesized as dual inhibitors of both HIV-1 reverse transcriptase (RT) DNA polymerase (DP) and ribonuclease H (RNase H) associated functions. Compounds were assayed on these enzyme activities, which highlighted dual inhibition properties in the low-micromolar range. Interestingly, mutations in the non-nucleoside RT inhibitor binding pocket strongly affected RNase H inhibition by the propenone derivatives without decreasing their capacity to inhibit DP activity, which suggests long-range RT structural effects. Biochemical and computational studies indicated that the propenone derivatives bind two different interdependent allosteric pockets.

Full text of DOI:10.1002/cmdc.201402015

CHEMMEDCHEM
FULL PAPERS
DOI: 10.1002/cmdc.201402015
Design, Synthesis, and Biological Evaluation of 1,3-
Diarylpropenones as Dual Inhibitors of HIV-1 Reverse
Transcriptase
Rita Meleddu,[a] Valeria Cannas,[b] Simona Distinto,*[a] Giorgia Sarais,[a] Claudia Del Vecchio,[c]
Francesca Esposito,[b] Giulia Bianco,[a] Angela Corona,[b] Filippo Cottiglia,[a] Stefano Alcaro,[d]
Cristina Parolin,[e] Anna Artese,[d] Daniela Scalise,[d] Massimo Fresta,[d] Antonella Arridu,[a]
Francesco Ortuso,[d] Elias Maccioni,*[a] and Enzo Tramontano[b]
A small library of 1,3-diarylpropenones was designed and syn-
thesized as dual inhibitors of both HIV-1 reverse transcriptase
(RT) DNA polymerase (DP) and ribonuclease H (RNase H) associ-
ated functions. Compounds were assayed on these enzyme ac-
tivities, which highlighted dual inhibition properties in the
low-micromolar range. Interestingly, mutations in the non-nu-
cleoside RT inhibitor binding pocket strongly affected RNase H
inhibition by the propenone derivatives without decreasing
their capacity to inhibit DP activity, which suggests long-range
RT structural effects. Biochemical and computational studies in-
dicated that the propenone derivatives bind two different in-
terdependent allosteric pockets.
Introduction
The design of multiple-acting ligands has become a fascinating
challenge for the therapy of diseases with multifarious patho-
logical mechanisms such as human immunodeficiency virus
(HIV) and acquired immunodeficiency syndrome (AIDS).[1] The
inhibition of multiple targets with a single molecule could im-
prove patient compliance and decrease the occurrence of drug
resistance.[2]
transcriptase (RT), which is responsible for retrotranscription.
This process converts the viral single-stranded RNA genome
into integration-competent double-stranded DNA through the
formation of an RNA/DNA hybrid intermediate. RT consists of
two subunits of different length, p66 and p51, which are com-
bined in a stable asymmetric heterodimer.[4]
Currently, two classes of RT inhibitors (RTIs) are included in
approved combination treatments used for HIV-1 handling,
namely, nucleoside/nucleotide RT inhibitors (NRTIs/NtRTIs) and
non-nucleoside RT inhibitors (NNRTIs).[3,5] Notably, despite its
critical relevance for the HIV life cycle,[6] no drugs are clinically
available for the inhibition of the RT-associated RNase H func-
tion, even though some RNase H inhibitors have recently been
designed and studied.[7] Most of the RNase H inhibitors identi-
fied so far chelate divalent metal ions [magnesium(II), MgII]
that are coordinated in the active site by the catalytic residues
D443, E478, D498, and D549. These compounds, however,
show toxicity as a result of the lack of specific binding.[8] Inter-
estingly, it was recently reported that some hydrazones,[9]
naphthyridinone,[8c] and anthraquinone derivatives[10] inhibit
the HIV-1 RNase H function by recognizing an allosteric pocket
located between the polymerase catalytic region and the
NNRTI binding pocket (NNRTIBP), which is 50 ꢀ away from the
RNase H catalytic site, and by directly communicating with the
NNRTIBP.[9]
Since the identification of HIV-1 as the causative agent of
AIDS, more than 20 antiretroviral drugs targeting different
steps of the HIV replication cycle have been approved for the
clinical treatment of HIV-infected patients.[3] Among these, one
of the most attractive and explored targets is HIV-1 reverse
[a] Dr. R. Meleddu, Dr. S. Distinto, Dr. G. Sarais, Dr. G. Bianco, Dr. F. Cottiglia,
Dr. A. Arridu, Prof. E. Maccioni
Department of Life and Environmental Sciences
University of Cagliari, Via Ospedale 72, 09124 Cagliari (Italy)
E-mail: s.distinto@unica.it
maccione@unica.it
[b] Dr. V. Cannas, Dr. F. Esposito, Dr. A. Corona, Prof. E. Tramontano
Department of Life and Environmental Sciences
University of Cagliari, Cittadella Universitaria di Monserrato, SS554
09042 Monserrato, Cagliari (Italy)
[c] Dr. C. Del Vecchio
Department of Molecular Medicine
University of Padua, Via Gabelli, 63, 35121 Padua (Italy)
[d] Prof. S. Alcaro, Dr. A. Artese, Dr. D. Scalise, Prof. M. Fresta, Dr. F. Ortuso
Dipartimento di Scienze della Salute
Clearly, the development of compounds that inhibit both
RT-associated RNA-dependent DNA polymerase (RDDP) and
RNase H activities would have several advantages that would
lead to a complete block of RT functions, new favorable drug-
resistance profiles, a decrease in the use of drug combinations,
and a reduction in toxic side effects. However, almost all
classes of RTIs are selective toward one of the two main RT-as-
Universitꢀ Magna Graecia di Catanzaro, Campus “S. Venuta”
Viale Europa, 88100 Catanzaro (Italy)
[e] Prof. C. Parolin
Department of Biology
University of Padua, via U. Bassi 58/b, 35121 Padua (Italy)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201402015.
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 12
&
1
&
ÞÞ
These are not the final page numbers!

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
sociated activities[3,5,7d] and only
a few are active on both of
them.[9–11]
Recently, we reported the
identification of HIV-1 RT single-
site dual inhibitors (SSDIs) by
a combined shape-, 2D-finger-
print-, and pharmacophore-
based virtual screening ap-
proach.[12] Pursuing the strategy of developing new anti-HIV in-
hibitors, we designed and synthesized a series of 3-(1-methox-
ynaphthalen-2-yl)-1-arylprop-2-en-1-one derivatives, evaluated
their activity against both HIV-1 RT-associated functions, and
characterized their mechanism of action.
Scheme 2. General synthetic pathway to compounds of this study.
carbonyl feature.[15] As expected, the entire series had the E
configuration according to the coupling constants for the
proton on the C=C bond. All compounds were synthesized by
Claisen–Schmidt condensation (Scheme 2). Briefly, in a general
procedure, the appropriate methyl aryl ketone (1 equiv) was
dissolved in ethanol and a solution of 10% aqueous NaOH was
added dropwise. 1-Methoxy-2-naphtaldehyde (2 equiv) was
added to the basic solution under vigorous stirring at room
temperature. The mixture was stirred for 24 h, and the formed
solid was filtered, washed with water, and crystallized from
water/ethanol. The structures of all the derivatives were further
confirmed by mass spectrometry (Figure S1, Supporting Infor-
mation). All compounds exhibited a similar fragmentation pat-
tern, which led to common sets of characteristic and well-de-
tectable fragment ions (Table 1).
Results and Discussion
Design and synthesis of new compounds
On the basis of compound 46 (compound numbering in
Ref. [12]) as a hit compound (HC) for the dual inhibition of
both associated HIV-1 RTs, we applied bioisosteric substitutions
for the identification of novel compounds. Bioisosterism dem-
onstrated to be a valid approach to navigate the chemical
space to optimize the biological performance of
small molecules.[13] The HC should have a completely
well-known chemical structure and possess an
equally well-known mechanism of action, if possible
at the level of topographic interaction with the re-
ceptor, including knowledge of its complete phar-
macophore model. Docking analysis of 46 was per-
formed not only toward the wild-type (WT) enzyme,
but also versus the most common mutants (i.e.,
Y181C, K103N). Interestingly, a common binding fea-
ture in all of the theoretical ligand–enzyme com-
plexes is the formation of a p–p interaction between
the indolinone ring and W229, a highly conserved
residue.[14] The main HC structural features are an ar-
omatic portion (A ring), a hydrazine spacer (B), and
Table 1. Representative fragment ions of compounds EMAC2000–2005 with their rela-
tive abundance.
Compd
Molecular and fragment ions a–d [m/z](%)[a]
[M+]
a
b
c
d
EMAC2000
EMAC2001
EMAC2002
EMAC2003
EMAC2004
EMAC2005
367(100)
307(83.7)
319(100)
323(100)
334(80)
335(57.7)
285(100)
287(79.3)
291(77.1)
302(100)
333(65.9)
285(57.7)
275(36.7)
285(14.9)
285(37.1)
285(17.2))
285(34.1)
241(46.2)
241(61.2)
241(6.9)
285(37.1)
241(16.0)
241(27.3)
211(19.2)
211(10.4)
211(26.4)
211(14.3)
211(8.0)
365(100)
211(18.2)
[a] See Supporting Information Figure S1.
Evaluation of 1,3-diarylpropenones on the functions of
HIV-1 RT
The efficacy of the synthesized 1,3-diarylpropenone derivatives
on both of the RT-associated functions was measured in bio-
chemical assays by using RDS1643[8b] and efavirenz as positive
controls (Table 2). Interestingly, the most potent inhibitors
were EMAC2005 and EMAC2002.
Preliminary structure–activity relationship analysis showed
that although the RDDP activity was not affected by variation
of the substituent at the 4-position of the D ring, the RNase H
activity was strongly influenced. In particular, bulky and strong-
ly/weakly activating groups (e.g., methoxy and phenyl) were
preferred with respect to deactivating substituents (e.g., halo-
gens). The introduction of a nitro group at the 3-position of
the D ring was slightly more tolerated, probably because of its
minor conjugative electron-withdrawing effect.
Scheme 1. Schematic representation of HC 46 and new derivatives.
a thiazole ring (C) bearing a second aromatic ring (D) at the 4-
position (Scheme 1).
3-(1-Methoxynaphthalen-2-yl)-1-arylprop-2-en-1-one deriva-
tives were designed according to this pharmacophoric
scheme. The indolinone ring was replaced by the 1-methoxy-
naphtalene moiety, the hydrazine spacer was substituted by
a vinyl group, and the thiazole was replaced by the bioisosteric
Next, the activity of the compounds was tested on the repli-
cation ability of HIV-1 in a single round of infection in Jurkat
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 12
&
2
&
ÞÞ
These are not the final page numbers!

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
site, which is essential for enzyme function. To verify the possi-
bility that 1-(4-biphenyl)-3-(1-methoxynaphthalen-2-yl)prop-2-
en-1-one (EMAC2005) could chelate the divalent ions in the
RNase H catalytic site, we determined its absorbance spectra in
the absence and presence of MgCl2, but we did not observe
any differences (data not shown).
Table 2. HIV-1 RT-associated activity inhibition by the 1,3-diarylprope-
none derivatives.
Subsequently, we further investigated if EMAC2005 and the
diketo acid derivative RDS1643, an RNase H catalytic site inhibi-
tor,[8b] were able to simultaneously bind to the RT. Such an
evaluation was performed by means of the Yonetani-revised
Yonetani–Theorell model, which allows the competition be-
tween two inhibitors of a certain enzyme for the same binding
site or two non-overlapping binding sites to be determined. In
this revised model, the plot of the inverse of the reaction ve-
locity (1/V) observed in the presence of various concentrations
of the first inhibitor, in the absence or in the contemporaneous
presence of the second inhibitor, leads to a series of lines that
are parallel if the two inhibitors compete for the same binding
site or a series of lines that intersect if the inhibitors bind to
different enzyme sites.[16]
Compd
R
IC50 [mm]
RNase H[a]
RDDP[b]
EMAC2000
EMAC2001
EMAC2002
EMAC2003
EMAC2004
EMAC2005
RDS1643
4-Br
4-F
4-OCH3
4-Cl
3-NO2
4-Ph
47ꢀ1
23ꢀ3
9ꢀ2
6ꢀ1
5ꢀ1
6ꢀ2
76ꢀ11
31ꢀ4
6ꢀ2
5 ꢀ1
5ꢀ1
4ꢀ1
13
>10
13ꢀ3
>10
efavirenz
[a] Compound concentration required to inhibit HIV-1 RT-associated
RNase H activity by 50%. [b] Compound concentration required to inhibit
HIV-1 RT-associated RDDP activity by 50%.
Therefore, the HIV-1 RT RNase H activity was measured in
the presence of increasing concentrations of both EMAC2005
and RDS1643 and analyzed with the Yonetani–Theorell plot
(Figure 2). The results showed that the slope of the plots of 1/
V versus the EMAC2005 concentration decreased at increasing
RDS1643 concentrations, which confirmed that the two com-
pounds were not kinetically mutually exclusive. Overall, these
data support the hypothesis that EMAC2005 does not bind to
the HIV-1 RNase H catalytic site.
cells. Given that the CC50 values (cytotoxic concentration for
50% of cells) ranged between 3 and 15 mm, compound con-
centrations were set at 10 mm for EMAC2000; 5, 0.5, and
0.05 mm for EMAC2000 and EMAC2003; and 0.5 and 0.05 mm
for EMAC2004 and EMAC2005. All compounds were not able
to inhibit HIV-1 replication within these experimental condi-
tions (Figure 1). Thus, we performed in vitro permeability
assays to asses if a reduced or absent transmembrane permea-
tion could explain these results.
Next, we evaluated the effects of EMAC2005 on K103N- and
Y181C-mutated RTs involved in NNRTI resistance (Table 3). The
results showed that upon testing K103N RT, EMAC2005 was
10-fold less potent on the RNase H. On the contrary, no influ-
ence of K103N mutation on the RDDP activity was observed. In
the case of Y181C mutation, a more dramatic effect relative to
that observed for K103N was observed: the activity toward
Biochemical characterization of the mechanism of HIV-1 RT
inhibition by EMAC2005
Several classes of HIV-1 RNase H have been reported, and in
general, they act by chelating the MgII ions within the active
Figure 2. Yonetani–Theorell plots of the interaction between EMAC2005 and
*
*
Figure 1. Inhibition of HIV-1 RT-associated RDDP ( ) and RNase H ( ) func-
tions by EMAC2005; data represent mean values ꢀSD from three independ-
ent determinations.
RDS1643 on HIV-1 RNase H activity. HIV-1 RT was incubated in the presence
*
of various concentrations of EMAC2005 and in the absence ( ) or in the
!
~
&
presence of 2.5 ( ), 5 ( ), and 10 mm ( ) RDS1643.
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 12
&
3
&
ÞÞ
These are not the final page numbers!

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
binding into two different sites[17] or by its binding into
a single site.[7d,8c,9,10c,12] Therefore, we investigated both possi-
bilities.
Table 3. Inhibition of drug-resistant HIV-1 mutated RT-associated func-
tions by EMAC2005.
Compd
IC50 [mm]
The very diverse group of NNRTIs bind allosterically in the
hydrophobic NNRTIBP and lock the enzyme into an inactive
form. Owing to the flexibility of the target and to the different
shapes of the known inhibitors (Figure 3a), we decided to per-
form ensemble docking experiments.[18] The major conforma-
tional changes in the NNRTIBP were taken into account to per-
form a clustering of the available RT-NNRTI complexes. In par-
ticular, the orientation of Y181, Y188, Y183, and primer grip
b12–b13 hairpin were considered (Figure 3b).[19] A representa-
tive of each different cluster was picked, and the 3D structure
of HIV RT was retrieved from the RCSB Protein Data Bank
(PDB).[20]
Y181C RT
RNase H[a] RDDP[b]
K103N RT
RNase H[a] RDDP[b]
EMAC2005
efavirenz
>100
–
8ꢀ3
0.40ꢀ0.03
59ꢀ8
3ꢀ1
0.68ꢀ0.05
–
[a] Compound concentration required to inhibit HIV-1 RT-associated
RNase H activity by 50%. [b] Compound concentration required to inhibit
HIV-1 RT-associated RDDP activity by 50%.
RNase H activity was almost suppressed, whereas the activity
toward the RT-associated RDDP was only slightly affected.
This behavior might be explained either by the binding of
EMAC2005 to a single site close to Y181, the hydrazones
pocket,[9] or the NNRTIBP, or by the interaction of the com-
pound with two interdependent pockets, the conformations of
which are affected by the Y181C mutation.
QM (quantum mechanical) polarized docking was per-
formed.[21] This recognition workflow combines docking with
semi-empirical methods to calculate charges within the protein
environment. This methodology performs significantly better
than classical molecular mechanics approaches.[22] We validated
the protocol for this target by performing re-docking experi-
ments (data not shown). The obtained [EMAC2005–RT] com-
plexes were subjected to a post-docking procedure based on
energy minimization and successive binding free energy calcu-
lations. The binding free energies (DGbind) were obtained by
applying molecular mechanics and continuum solvation
models by using the molecular mechanics generalized Born/
surface area (MMGBSA) method.[23] As reported in Table 4, by
comparing the DGbind MMGBSA values, we could assert that
the most probable binding mode in the NNRTIBP was obtained
by docking the compound into the RT conformation model re-
ported in PDB ID 1TV6.[24]
In silico modeling of the interaction of EMAC2005 with
HIV-1 RT
A computational study based on molecular docking experi-
ments in tandem with molecular dynamics simulations was
performed to understand the possible mechanism of inhibi-
tion of this series of diarylpropenone derivatives. The studies
were focused on the most active compound only, that is,
EMAC2005.
Molecular docking approaches have become very useful and
largely widespread for the prediction of feasible binding
modes of a ligand, the target site of which is either known or
unknown (blind docking). According to the available literature,
dual inhibitory activity could be achieved either by inhibitor
The best docking pose of EMAC2005 and its comparison
with respect to the relative co-crystallized compounds are re-
ported in the Supporting Information (Figures S2–S7). To evalu-
Figure 3. a) Structures and PDB IDs of co-crystallized NNRTIs selected for the ensemble docking procedure. b) Superimposition of the primer grip region and
of residues 181, 183, and 188 of employed RT PDB structures for ensemble docking experiments.
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 12
&
4
&
ÞÞ
These are not the final page numbers!

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
Table 4. Ensemble docking scores and binding free energies of EMAC2005–RT complexes.
Docking site and (box center)
NNRTIBP (center on W229)
PDB ID
Co-crystallized ligand
G score
DGbind MMGBSA
1VRT[25]
2ZD1[26]
1EP4[27]
3QO9[28]
1RTI[25]
nevirapine
rilpivirine
capravirine
TSAO-T
HEPT
CP-94,707
ꢁ4.18
ꢁ8.43
ꢁ8.26
ꢁ9.21
ꢁ11.04
ꢁ9.53
ꢁ30.6
ꢁ30.7
ꢁ38.8
ꢁ35.0
ꢁ34.1
ꢁ48.0
1TV6[24]
RNase H domain (center on Q500)
1TV6
–
ꢁ7.50
ꢁ5.58
ꢁ38.6
ꢁ24.6
EN37[17]
MK3
RNase H allosteric pocket (center on W229)
3LP2[8c]
ꢁ6.19
ꢁ38.4
ate the stability of the complex and the interactions in
EMAC2005, we ran a molecular dynamics (MD) simulation up
to 6 ns by using Desmond Molecular Dynamics System (ver-
sion 2.4)[29] and allowed the whole enzyme free to move into
the explicit solvent water environment. Docking and MD simu-
lations were also performed on the mutated enzyme com-
plexes by applying the same protocol described above (dock-
ing, energy minimization, DGbind MMGBSA calculations, and MD
simulations). The best binding mode is depicted in Figure S8.
The interaction energy values of the [EMAC2005–RT] com-
plexes are reported in Table 5, whereas their variations, sam-
pled at regular intervals during the simulations over the entire
MD trajectory, are illustrated in Figure S9. Furthermore, analysis
of the root-mean-square deviation (RMSD), computed onto the
RT heavy atoms for the wild-type and mutated enzymes
during the MD, showed that the system was structurally stable
inhibitors that bind at a Q507 centered cleft was reported.[17]
Probably, these compounds induce an RNase H domain confor-
mation that prevent this function.
Hence, to include the whole RNase H domain for investiga-
tion, in our docking experiments the binding site was defined
by a regular box of 97336 ꢀ3 centered on residue Q500. The
RT conformational structures adopted for docking experiments
were the 1TV6 X-ray structure and the crystallographic model
reported in a recent study published by Felts et al., not yet
available in the PDB.[17] In Table 4, the DGbind values for the
best poses are reported, and in Figure S10, the favorite binding
mode is depicted. RMSD and total interaction energy fluctua-
tions during the MD simulations are reported in Figure S11.
Simultaneously, we also investigated whether the RNase H
inhibitory activity could depend on the binding into the same
allosteric site occupied by hydrazone and naphthyridinone de-
rivatives, described by Himmel.[8c,9] We found that
EMAC2005 showed a similar affinity if bound into the
two allosteric sites (Table 4). Furthermore, it was al-
Table 5. XP docking G scores, DGbind MMGBSA values, and MD total interaction
energy (Eint) terms for EMAC2005 all expressed in kcalmolꢁ1
ready reported that some compounds that occupy
the latter pocket are dual inhibitors. Therefore, we
analyzed if binding into this pocket would better ex-
plain the dual inhibitory activity of EMAC2005 (Fig-
ure S12). In particular, we ran MD simulations by ap-
plying the same protocol described above (Fig-
ure S13).
.
Complex
G score
DGbind MMGBSA
Eint [kcalmolꢁ1
]
NNRTIBP-WT
NNRTIBP-Y181C
NNRTIBP-K103N
allosteric RNaseHIBP-WT
allosteric RNaseHIBP-Y181C
allosteric RNaseHIBP-K103N
SiteQ500
ꢁ9.53
ꢁ9.90
ꢁ9.59
ꢁ6.19
ꢁ5.40
ꢁ6.06
ꢁ7.50
ꢁ48.00
ꢁ44.30
ꢁ45.50
ꢁ38.40
ꢁ37.10
ꢁ36.40
ꢁ38.60
ꢁ52.01
ꢁ48.03
ꢁ50.78
ꢁ40.37
ꢁ38.79
ꢁ39.06
ꢁ42.40
According to analysis of the energies of the com-
plexes with the ligand bound in the allosteric binding
pocket of RNase H (Table 5 and Figure S14), the ob-
tained interaction energies of EMAC2005 in the wild-
type RT and mutated complexes are similar.
during the simulation (Figure S9).
Thus, the binding in this allosteric pocket cannot explain the
loss of inhibitory activity toward RNase H if Y181 is mutated in
cysteine or the decrease in activity if K103 is mutated in aspar-
agine. Consequently, we concluded that the binding of
EMAC2005 in this pocket is not the most favored.
To assess which binding pocket was responsible for the
RNase H inhibitory activity, we considered the described bind-
ing pockets: one was located in the catalytic domain and the
other was an allosteric site described by Himmel as a hydra-
zone site[8c] that was already considered in our previous stud-
ies.[10c,12] The biochemical assay that was directed at verifying
the ability of this series of compounds to coordinate the metal
ions indicated that the chelation mechanism could be exclud-
ed. However, we could not ignore the possibility of a binding
site close to the RNase H catalytic residues. Recently, RNase H
Hence, we supposed that polymerase inhibition was due to
the binding of EMAC2005 into the NNRTI pocket (Figure 4a).
This hypothesis is also supported by the similar behavior of
EMAC2005 and CP-94,707 upon testing in the mutated en-
zymes. In fact, CP-94,707 also acts in the same manner, and
both compounds interestingly retain their RDDP activity.[24] Dif-
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 12
&
5
&
ÞÞ
These are not the final page numbers!

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
Figure 4. Superimposed structures of 6 ns MD simulation frames of the [EMAC2005–RT] complex: initial, final, and intermediate structure snapshots. a) Overall
structure of the HIV-1 RT heterodimer with the NNRTIBP occupied. b) Close-up view of the binding cavity. d) Overall structure of the HIV-1 RT heterodimer
with the Q500 site occupied. e) Close-up view of the binding cavity. c,f) Residues involved in the complex stabilization sorted by number of contacts between
ligand and receptor. Interacting and catalytic residues are represented as stick structures.
ferently, both biochemical and modeling studies seemed to
confirm that binding into an allosteric site close to RNase H
catalytic residues was responsible for RNase H inhibitory activi-
ty (Figure 4b). To corroborate this hypothesis further, we evalu-
ated the activity of EMAC2005 on A502F RT.[30] As we expected,
this mutation did not affect the RDDP activity. On the contrary,
the activity of EMAC2005 on the RNase H function was fivefold
lower with respect to that of the wild-type RT, which indicated
that residue A502 was involved in the formation of an RNase H
allosteric binding pocket in which EMAC2005 could be accom-
modated.
understood, but they likely involve changes in the positioning
of the RNA/DNA duplex nucleic acid.[31] Therefore, we exam-
ined long-range effects of both mutations by checking the
fluctuations of the residues [root-mean-square fluctuation
(RMSF)] during MD of the wild-type and mutated enzymes
(Figure 5a). We noticed that although the wild-type RT did not
show relevant fluctuations in the Q500 site involved in the
binding of EMAC2005, some residues in the mutated RTs
showed a huge fluctuation that might have disturbed the
binding of EMAC2005 (Figure 5b).
Finally, we evaluated the stability of the ternary enzyme–
EMAC2005 complex bound in the two allosteric sites
(Figure 6). Plots for potential energy and RMSD fluctuations re-
lated to the complex are depicted in Figure 6b,c. Analysis
showed that the structure reached equilibrium and that the
low fluctuations supported the stability of the intermolecular
interactions.
Still, it remained to be understood why the EMAC2005 inhib-
itory activity toward RNase H was modified in the mutated en-
zymes. In this regard, there is considerable evidence that the
binding of NNRTIs as well as mutations in the allosteric pocket
in the RT DNA polymerase domain affect the activity of the
spatially remote RNase H domain. The mechanisms involved in
this long-range alteration of RNase H activity are not entirely
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 12
&
6
&
ÞÞ
These are not the final page numbers!

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
Figure 7. 1. In vitro transmembrane permeation through a biological mem-
brane model of EMAC2005. The experiments were performed at room tem-
perature. Any unapparent error bars are hidden behind the data point;
values represent the mean ꢀSD of five independent experiments.
EMAC2005 pass through the lipophilic layer, which thus leads
to a delay in transmembrane permeation (prolonged lag time).
Considering that one of the main factors governing the passive
permeation through a biological membrane is the physico-
chemical feature of the drug, the elevated hydrophobicity of
EMAC2005 could be the reason for the unsuitable transmem-
brane permeation through the biological membrane model. In
fact, the first interaction between the hydrophobic molecule of
EMAC2005 and the hydrophilic layer of the biomembrane
model may represent the limiting step for easy biological
membrane diffusion. This finding seems to be in agreement
with other observations[32] reporting the need for a suitable
balance between hydrophilic and hydrophobic features of
a molecule to have suitable biological membranes and biologi-
cal barriers (e.g., blood–brain barrier) passage.
Figure 5. RMSF of subunit p66 during MD. a) Entire wild-type and mutated
RT. b) Close-up view of site Q500 residues (boxed region in panel a).
In vitro permeability study of EMAC2005
An in vitro experiment of transmembrane permeation through
a biological membrane model was performed to predict the
possible interaction of EMAC2005 with a biological membrane,
which would thus mimic drug adsorption through a passive
diffusion. As shown in Figure 7, the transmembrane permea-
tion profile of EMAC2005 was characterized by a prolonged lag
time followed by gradual permeation. This finding was proba-
bly due to the fact that the hydrophobic drug has first to dif-
fuse through the aqueous medium and then to interact with
the outer hydrophilic layer of the biomembrane model. Only
after interaction with the outer biomembrane layer can
Figure 6. Superimposed structures of 6 ns MD simulation frames of the EMAC2005–RT ternary complex: initial, final, and intermediate structure snapshots.
a) Overall structure of the HIV-1 RT heterodimer. b) Potential energy of the complex during the MD simulation. c) RMSD fluctuations during trajectory.
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 12
&
7
&
ÞÞ
These are not the final page numbers!

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
multiplier voltage was set to 1700 V. Elemental analyses were ob-
tained with a PerkinElmer 240 B microanalyzer. Analytical data of
the synthesized compounds are in agreement with the theoretical
data. TLC chromatography was performed by using silica gel plates
(Merck F254), and spots were visualized by UV light.
Conclusions
With the aim to obtain dual inhibitors of RT-associated func-
tions, a small series of 1,3-diarylpropenones were designed,
synthesized, and tested. The activity of some compounds and
the profile toward mutated enzymes was remarkable and sug-
gestive of further modifications and studies. Moreover, investi-
gating the possible mechanism of action of the most-promis-
ing compound, that is, EMAC2005, we found that its inhibitory
activity could be addressed to the binding at two different
enzyme clefts: the NNRTIBP site and an allosteric site close to
the RNase H catalytic DEDD motif (site Q500). We highlighted
that the compound was better accommodated in a pocket
with Y181 and Y188 in close conformation (PDB ID: 1TV6) than
in the open conformation of most NNRTIs. This facilitates
enzyme recognition if common mutations, such as Y181C and
K103N, occur, and therefore, RDDP activity is not impaired. The
EMAC2005 binding mode confirms the known key role of
W229 and Y188 in the stabilization of the complex. Other inter-
acting residues, namely, L100, P225, L234, Y318, V106, and
P236, highlighted the importance of hydrophobic contacts. In-
stead, most likely, the loss and decrease in RNase H inhibitory
potency is due to the improbable entrance of EMAC2005 into
the Q500 site if Y181C and K103N mutations occur. This hy-
pothesis was further confirmed by a single-point mutation ex-
periment on the A502 residue. In fact, whereas the inhibition
potency of EMAC2005 toward the RDDP function of HIV-1 RT
A502F was almost not modified (IC50 increased 1.5-fold), the in-
hibition of the RNase function was remarkably affected with
a fivefold decrease in potency. Thus, EMAC2005 most likely be-
haves as a dual-site dual-function inhibitor.
Synthetic procedures: 1,3-Diarylpropenones were synthesized ac-
cording to a slightly modified Claisen–Schmidt reaction. Analysis
by NMR spectroscopy supported the E configuration according to
the coupling constants of the double-bond protons that range
from 15 to 16 Hz.
Preparation of (E)-1-(4-bromophenyl)-3-(1-methoxynaphthalen-
2-yl)prop-2-en-1-one (EMAC2000) as a representative procedure:
1-(4-Bromophenyl)ethanone (0.9 g, 4.5 mmol) was dissolved in eth-
anol (15 mL) and a solution of 10% NaOH was added dropwise at
RT. The mixture was stirred for 10 min, and then 1-methoxy-2-
naphthaldehyde (1 g, 5.4 mmol) in ethanol solution (15 mL) was
added. After 24 h, the reaction was complete (as monitored by
TLC, n-hexane/ethyl acetate=2:1), and the pale yellow crystalline
solid was filtered, washed with water, crystallized with a mixture of
water/ethanol, and characterized. Yellow crystals; yield: 67%; mp:
1
110–1128C; H NMR (300 MHz, CDCl3): d=4.06 (s, 3H, OCH3), 7.3 (d,
J=9.1 Hz, 1H, ArꢁCH), 7.41 (t, J=7.5 Hz, 1H, ArꢁCH), 7.55 (t, J=
7.5 Hz, 1H, ArꢁCH), 7.65 (d, J=8.3 Hz, 2H, ArꢁCH), 7.82 (d, J=
7.5 Hz, 1H, ArꢁCH), 7.85 (d, J=15.6 Hz, 1H, ꢁCH=), 7.9 (d, J=
9,0 Hz, 1H, ArꢁCH), 7.93 (d, J=8.3 Hz, 2H, ArꢁCH), 8.25 (d, J=
8.6 Hz, 1H, ArꢁCH), 8.51 ppm (d, J=15.6 Hz, 1H, ꢁCH=); 13C NMR
(100 MHz, CDCl3): d=56.1 112.7, 117.1, 123.3, 124.1, 126.6, 127.6,
128.7, 129.0, 130.0, 130.1 (2C), 131.8 (2C), 132.1, 133.1, 137.3,
138.3, 157.2, 190.2 ppm.
(E)-1-(4-Fluorophenyl)-3-(1-methoxynaphthalen-2-yl)prop-2-en-1-
one (EMAC2001): Yellow crystals; yield: 81%; mp: 93–958C;
1H NMR (300 MHz, CDCl3): d=4.07 (s, 3H, OCH3), 7.11 (t, J=8.5 Hz,
1H, ArꢁCH), 7.26 (d, J=9.1 Hz, 2H, ArꢁCH), 7.42 (d, J=7.1 Hz, 1H,
ArꢁCH), 7.50 (t, J=8.5 Hz, 1H, ArꢁCH), 7.57 (d, J=7.1 Hz, 1H, Arꢁ
CH), 7.78 (d, J=15.9 Hz, 1H, ꢁCH=), 7.93 (d, J=8.6 Hz, 1H, ArꢁCH),
8.09 (t, JH,H =9.1 Hz, JH,F =9.3 Hz, 2H, ArꢁCH), 8.18 (d, J=8.5 Hz, 1H,
ArꢁCH), 8.43 ppm (d, 1H, J=15.9 Hz, ꢁCH=); 13C NMR (100 MHz,
CDCl3): d=56.5, 112.7, 115.7, 123.3, 124.0, 126.8, 127.6, 128.6 (2C),
129.0, 130.0 (2C), 131.1, 131.3, 132.0, 133.0, 136.2, 138.3, 157.2,
190.0 ppm.
Experimental Section
Chemistry
Materials and methods: Starting materials and reagents were ob-
tained from commercial suppliers and were used without purifica-
tion. All melting points were determined by the capillary method
with a Stuart SMP11 melting point apparatus or a Bꢂchi-540 capil-
lary melting points apparatus. Melting points, yields of reactions,
and the analytical data of the derivatives are reported in Tables S1
(E)-1-(4-Methoxyphenyl)-3-(1-methoxynaphthalen-2-ylprop-2-en-
1-one (EMAC2002): Yellow crystals; yield: 83%; mp: 137–1398C;
1H NMR (300 MHz, CDCl3): d=3.9 (s, 3H, OCH3), 4.05 (s, 3H, OCH3),
7.00 (d, J=8.9 Hz, 1H, ArꢁCH), 7.33 (d, J=9.0 Hz, 2H, ArꢁCH), 7.70
(t, J=8 Hz, 1H, ArꢁCH), 7.47 (d, J=16.0 Hz, 1H, ꢁCH=), 7.55 (t, J=
8 Hz, 1H, ArꢁCH), 7.82 (d, J=8.0 Hz, 1H, ArꢁCH), 7.89 (d, J=9.0 Hz,
1H, ArꢁCH), 8.08 (d, J=9.0 Hz, 1H, ArꢁCH), 8.28 (d, J=9.0 Hz, 2H,
ArꢁCH), 8.45 (d, J=16.0 Hz, 1H, ꢁCH=); 13C NMR (100 MHz, CDCl3):
d=55.4, 56.5, 112.7, 113.5, 117.7, 123.5, 124.1, 127.2, 127.6, 128.6,
129.1, 130.9 (2C), 131.5 (2C), 131.7, 133.1, 137.0, 156.9, 163.3,
189.5 ppm.
1
and S2. The H NMR spectra of all samples were measured in CDCl3
at 278.1 K with a Varian Unity 300 spectrometer. In the signal as-
signments, the chemical shifts of the proton were referenced to
the solvent (1H: d=7.24 ppm). 13C NMR were recorded with
a Varian Unity 500 spectrometer by using CDCl3 as the solvent at
278.1 K. Mass spectra were recorded by using an HPLC–MS/MS
Varian (Varian, Palo Alto, CA, USA) system fitted with a 1200 L triple
quadrupole mass spectrometer equipped with an electrospray ioni-
zation (ESI) source. A Varian MS workstation version 6.8 software
was used for data acquisition and processing. Rapid identification
was achieved with direct infusion of the purified molecule, dis-
solved in methanol, on the mass spectrometer source. The ESI
mass spectrometer was operated in the positive ion mode. The
system was optimized as follows: the electrospray capillary poten-
tial was set to 65 V, whereas the shield was set at 725 V. Nitrogen
was used as the desolvation solvent gas. The atmospheric pressure
ionization (API) housing and drying gas temperatures were kept at
54 and 3758C, respectively. The scan time was 1 s, and the detector
(E)-1-(4-Chlorophenyl)-3-(1-methoxynaphthalen-2-yl)prop-2-en-
1-one (EMAC2003): Yellow crystals; yield: 53%; mp: 108–1098C;
1H NMR (300 MHz, CDCl3): d=4.06 (s, 3H, OCH3), 7.33 (d, J=9.1 Hz,
1H, ArꢁCH), 7.41 (t, J=7.8 Hz, 1H, ArꢁCH), 7.49 (d, J=8.4 Hz, 2H,
ArꢁCH), 7.53 (d, J=15.6 Hz, 1H, ꢁCH=), 7.55 (t, J=7.8 Hz, 1H, Arꢁ
CH), 7.74 (d, J=8.1 Hz, 1H, ArꢁCH), 7.90 (d, J=9.6 Hz, 1H, ArꢁCH),
8.01 (d, J=8.3 Hz, 2H, ArꢁCH), 8.25 (d, J=8.6 Hz, 1H, ArꢁCH),
8.51 ppm (d, J=15.6 Hz, 1H, ꢁCH=); 13C NMR (500 MHz, CDCl3): d=
56.3, 112.7, 117,1, 123.3, 124.0, 126.7, 127.6, 128.6, 128.8 (2C),
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 12
&
8
&
ÞÞ
These are not the final page numbers!

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
129.0, 130.0 (2C), 132.1, 133.1, 136.9, 138.3, 139.0, 157.2,
190.0 ppm.
Cell lines and virus: The human embryonic c kidney cells 293T and
the human T-lymphoid Jurkat cell line (clone E6-1) were from the
American Type Culture Collection and maintained in DMEM or
RPMI medium (Invitrogen), respectively, containing 10% fetal
bovine serum (FBS, Invitrogen), at 378C under a humidified 5%
CO2 atmosphere. Recombinant viral stock was produced by transi-
ent transfection of 293T cells as previously described[34] and used
to transduce Jurkat cells. In this context, an env-defective provirus
encoding the bacterial chloramphenicol acetyltransferase (CAT)
gene was complemented in trans by the envelope glycoprotein
derived from the laboratory-adapted T-cell-tropic strain HXBc2. The
level of CAT expression in the infected cells reflects the efficiency
of a single round of the retroviral infection cycle.
(E)-1-(3-Nitrophenyl)-3-(1-methoxynaphthalen-2-yl)prop-2-en-1-
one (EMAC2004): Pale orange crystals; yield: 64%; mp: 143–
1458C; 1H NMR (300 MHz, CDCl3): d=4.10 (s, 3H, OCH3), 7.35 (d,
J=8.9 Hz, 1H, ArꢁCH), 7.42 (t, J=7.8 Hz, 1H, ArꢁCH), 7.58 (t, J=
8.5 Hz, 1H, ArꢁCH), 7.72 (t, J=7.8 Hz, 1H, ArꢁCH), 7.83 (d, J=
8.0 Hz, 1H, ArꢁCH), 7.93 (d, J=8.5 Hz, 1H, ArꢁCH), 7.94 (d, J=
15.6 Hz, 1H, ꢁCH=), 8.25 (d, J=8.5 Hz, 1H, ArꢁCH), 8.39 (d, J=
8.5 Hz, 1H, ArꢁCH), 8.44 (d, J=8.0 Hz, 1H, ArꢁCH), 8.60 (d, J=
15.6 Hz, <1H, C->CH=), 8.89 ppm (s, 1H, ArꢁCH); 13C NMR
(500 MHz, CDCl3): d=56.3, 112.6, 116.6, 123.1, 123.4, 124.1, 125.7,
126.8, 127.8, 128.8, 129.0, 129.8, 132.7, 133.1, 134.1, 139.5, 140.0,
148.3, 157.6, 189.0 ppm.
Cytotoxicity assay: For cytotoxicity assays, cell lines were seeded in
96-well plates (Falcon) at an initial density of 105 cells per 100 mL in
medium containing 10% FBS, in the absence or presence of serial
dilutions of test compounds. Plates were incubated for 72 h at
378C in under a humidified 5% CO2 atmosphere. Cell viability was
determined by using Cell Proliferation Kit I (MTT) (Roche).
(E)-1-(4-Biphenyl)-3-(1-methoxynaphthalen-2-yl)prop-2-en-1-one
1
(EMAC2005): Yellow crystals; yield: 87%; mp: 104–1058C; H NMR
(300 MHz, CDCl3): d=4.07 (s, 3H, OCH3), 7.34–7.50 (m, 2H, ArꢁCH),
7.56 (t, J=8.1 Hz, 1H, ArꢁCH), 7.69 (d, J=7.8 Hz, 2H, ArꢁCH), 7.75
(d, J=7.8 Hz, 2H, ArꢁCH), 7.83(d, J=8.8 Hz, 1H, ArꢁCH), 7.88 (d, J=
8.0 Hz, 1H, ArꢁCH), 7.91 (d, J=8.2 Hz, 2H, ArꢁCH), 7.95 (d, J=
15.9 Hz, 1H, ꢁCH=), 8.04 (d, J=8.2 Hz, 2H, ArꢁCH), 8.19 (d, J=
8.8 Hz, 1H, ArꢁCH), 8.29 (d, J=8.1 Hz, 1H, ArꢁCH), 8.54 ppm (d, J=
15.9 Hz, 1H, ꢁCH=); 13C NMR (100 MHz, CDCl3): d=56.4, 112.8,
117.4, 123.5, 124.0, 127.2 (3C), 127.3 (3C), 127.5, 128.1, 128.6, 128.9
(2C), 129.1, 129.2 (2C), 131.8, 137.3, 137.7, 140.1, 145.3, 157.1,
190.7 ppm.
Molecular modeling
Ligand preparation: Theoretical 3D models of the most active de-
rivative EMAC2005 was built by means of Maestro.[35] The inhibitor
structure was optimized by means of an energy minimization per-
formed by using the MMFFs force field,[36] the GB/SA[37] water im-
plicit solvation model, and the Polak–Ribier Conjugate Gradient
(PRCG) method by converging on gradient with a threshold of
Biology
0.05 kJ(molꢀ)ꢁ1
.
Protein preparation: The coordinates for reverse transcriptase en-
zymes were taken from the RCSB Protein Data Bank[20] (PDB IDs:
1VRT,[25] 2ZD1,[26] 1EP4,[27] 3QO9,[28] 1RTI,[25] 1TV6,[24] and 3LP2).[8c]
The proteins were prepared by using the Maestro Protein Prepara-
tion Wizard. Original water molecules were removed and termini
were capped. The bond orders and formal charges were added for
hetero groups, and all the hydrogen atoms were added in the
structure. Missing atoms and residues were included. After prepa-
ration, the structures were refined to optimize the hydrogen-bond
network by using OPLS_2005[38] force field. The minimization was
terminated once the energy converged or the RMSD reached
a maximum cutoff of 0.30 ꢀ.
Protein expression and purification: The recombinant HIV-1 RT pro-
tein, the coding gene of which was subcloned in the p6HRT_prot
plasmid, was expressed in E. coli strain M15.[33] The bacteria cells
were grown up to an optical density (at 600 nm) of 0.8 and in-
duced with 1.7 mm isopropyl b-d-1-thiogalactopyranoside (IPTG)
for 5 h. HIV-1 RT purification was performed as described.[8h] Briefly,
cell pellets were re-suspended in lysis buffer (20 mm HEPES,
pH 7.5; 0.5m NaCl; 5 mm b-mercaptoethanol; 5 mm imidazole;
0.4 mgmLꢁ1 lysozyme), incubated on ice for 20 min, sonicated, and
centrifuged at 30000 g for 1 h. The supernatant was applied to
a His-binding resin column and washed thoroughly with wash
buffer (20 mm HEPES, pH 7.5; 0.3m NaCl; 5 mm b-mercaptoetha-
nol; 60 mm imidazole; 10% glycerol). RT was eluted by imidazole
gradient, and the enzyme-containing fractions were pooled and di-
alyzed and aliquots were stored at ꢁ808C.
Docking protocol: Molecular docking studies were performed by
using the QMPL workflow protocol.[21] Grids were defined around
the refined structure by centering on the residue indicated in
Table 3 and fixing the box volume at 97336 ꢀ3. The extra precision
(XP) docking algorithm was applied for scoring theoretical poses.
The other settings were left as default. The same protocol was ap-
plied for all the simulations indicated in Tables 3 and 4.
RNase H polymerase-independent cleavage assay: The HIV-1 RT-asso-
ciated RNase H activity was measured as described[12] in 100 mL re-
action volume containing 50 mm Tris HCl, pH 7.8; 6 mm MgCl2;
1 mm dithiothreitol (DTT); 80 mm KCl; hybrid RNA/DNA (5’-GTT
TTC TTT TCC CCC CTG AC-3’-fluorescein; 5’-CAA AAG AAA AGG
GGG GAC UG-3’-dabcyl) and 3.8 nm RT. The reaction mixture was
incubated for 1 h at 378C. The enzymatic reaction was stopped
with the addition of ethylenediaminetetraacetic acid (EDTA) and
measured with a Victor3 instrument (Perkin) at 490/528 nm.
Post-docking protocol: A total of 10000 steps of the Polak–Ribier
conjugate gradient (PRCG) minimization method were conducted
on the top-ranked theoretical complexes by using the OPLS_2005
force field. The optimization process was performed up to the de-
rivative convergence criterion equal to 0.01 kJ(molꢀ)ꢁ1. The bind-
ing free energies (DGbind) were computed by applying molecular
mechanics and continuum solvation models with the molecular
mechanics generalized Born/surface area (MMGBSA) method.[23]
DNA polymerase assay: The HIV-1 RT-associated (RDDP) activity was
measured by using an Invitrogen EnzCheck Reverse Transcriptase
Assay Kit, in 50 mL volume containing 60 mm Tris HCl, pH 8.1;
8 mm MgCl2; 60 mm KCl; 13 mm DTT; 100 mm dTTP; 2 nm HIV-1 RT;
poly(A)-oligo(dT). The reaction mixture was incubated for 30 min at
378C. The enzymatic reaction was stopped with the addition of
EDTA and measured with Victor3 (Perkin) at 502/523 nm after the
addition of picogreen.
Best docking complexes were submitted to 6 ns of MD by using
Desmond (version 2.4).[39] The complexes were solvated with
a TIP3P (transferable intermolecular potential 3-Point)[40] box of
water and counter ions were added to neutralize the system net
charge. The solvated models were optimized, and subsequently
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 12
&
9
&
ÞÞ
These are not the final page numbers!

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
the MTK_NPT (Martyna–Tobias–Klein with constant number of par-
ticles, pressure and temperature) ensemble was employed.[41] The
default stages in the relaxation process for the NPT ensemble in-
cluded two energy minimizations and four simulation steps.
During the energy minimizations, two runs of 2000 iteration were
processed by using the steepest descent method: during the first
run, the protein structure was fixed by a force restraint constant of
50 kcal(molꢀ)ꢁ1, and in the second run all restraints were removed.
With the first simulation, at NVT (constant number of particles,
volume, and temperature) ensemble, the system reached a temper-
ature of 10 K. In the following three simulations in the NPT ensem-
ble, the system was heated up to 300 K and the pressure was kept
constant at 100 kPa by using the Berendsen thermostat–barostat.
During the production phase, temperature and pressure were kept
constant by using the Nosꢃ–Hoover thermostat–barostat. The
energy and trajectory were recorded every 1.2 and 4.8 ps, respec-
tively. For multiple time step integration, a RESPA (reversible refer-
ence system propagator algorithm)[42] was applied to integrate the
equation of motion with Fourier-space electrostatics computed
every 6 fs, and all remaining interactions computed every 2 fs. All
chemical bond lengths involving hydrogen atoms were fixed with
SHAKE.[43] The short-range cutoff was set to 9 ꢀ, and the smooth
particle mesh Ewald method (PME)[44] was used for long-range elec-
trostatic interactions. The resulting seven trajectories were ana-
lyzed in terms of interaction energies and geometries. The same
protocol was applied for the EMAC2005–RT ternary complex. Mo-
lecular modeling figures were depicted by LigandScout[45] and
VMD (version 1.8.7).[46]
carbonate membrane (50 nm pore size) was presoaked in pH 7.4
isotonic phosphate buffer for 3 h and layered on a synthetic cellu-
lose nitrate membrane (molecular weight cutoff=10000 Da),
which was previously impregnated with a liquid paraffin/lauryl al-
cohol (2.1:10 w/w) mixture up to the doubling of the weight. Flow
Franz diffusion cells were used for the transmembrane permeation
of EMAC2005, and they were characterized by a surface area of
0.75 cm2 and a nominal receiving volume of 4.75 mL. The model of
biological membrane was placed horizontally between the donor
and receptor compartments. An ethanol/water mixture (50:50 v/v)
was used to fill the receptor compartment. The same mixture
(200 mL) was used to suspend the drug. This suspension was
placed into the donor compartments. The receptor fluid was con-
stantly stirred at 600 rpm during the experiments by means of
a magnetic anchor and warmed (GR 150 thermostat, Grant Instru-
ments Ltd, Cambridge, UK) to 378C. These conditions were main-
tained throughout the experiments. At predetermined times,
400 mL of the receptor compartment was withdrawn by using
a Minipuls 3 peristaltic pump [Gilson Italia S.r.l., Cinisello Balsamo
(MI), Italy] connected to a FC 204 fraction collector [Gilson Italia
S.r.l., Cinisello Balsamo (MI), Italy] and immediately replaced with
the same volume of fresh solution. The amount of EMAC2005 that
permeated through the membranes was immediately analyzed by
HPLC. Experiments were performed in triplicate, and results are the
mean ꢀSD of five different experiments.
Acknowledgements
This work was supported by RAS (Regione Autonoma della Sarde-
gna) grant LR 7/2007 CRP2 450, CRP-24915 and Istituto Superi-
ore di Sanitꢀ grant RF-PAD-2009-1304305 n.40H98. The PhD
grant of R.M. was funded by Fondazione Banco di Sardegna. F.E.
was supported by RAS fellowships, co-financed with funds from
PO Sardinia FSE 2007–2013 and of LR 7/2007, project CRP2 683.
In vitro membrane permeation studies
HPLC determination of EMAC2005: The sensitive HPLC method
with UV detection was developed for the quantitative determina-
tion of EMAC2005. The chromatographic system was a HPLC Jasco
model PU-1580 (Tokyo, Japan) with a 20 mL loop injection valve.
The chromatographic system was equipped with a Jasco MD 1510
diode array detector, which was set at lmax =296 for EMAC2005.
The separation was performed by using a C18 reverse-phase Phe-
nomenex column (Jupiter 250ꢄ4.60 mm, 5 mm particle size), which
was maintained at room temperature. The mobile phase was pH 9
water (eluent A) and acetonitrile (eluent B) and it was delivered at
a flow rate of 1 mLminꢁ1. Solvents were degased by sonication for
15 min. A gradient elution method was applied for the determina-
tion of EMAC2005. The gradient was set as follows: eluent A/
eluent B=70:30, 0–7 min; linear increase of eluent B to 60%, 7–
9 min; linear increase of eluent B to 70%, 9–12 min; eluent A/
eluent B=30:70, 12–19 min; linear decrease of eluent B to 50%,
19–21 min; and then the system was linearly returned to the origi-
nal conditions, 21–25 min (see the Supporting Information for fur-
ther details). HPLC data were processed by using the Borwin chro-
matography software (version 1.5) from Jasco. A pure ethanol solu-
tion of EMAC2005 was prepared (1 mgmLꢁ1) and used as a stock
solution for the calibration curve. EMAC2005 quantification was
performed by using a calibration curve in a linear concentration in-
terval ranging from 0.1 to100 mgmLꢁ1, according to Equation (1):
Keywords: antiviral agents · dual inhibitors · enzymes · HIV-1 ·
molecular modeling · RNase H
[1] a) P. Zhan, X. Liu, Z. Li, C. Pannecouque, E. De Clercq, Curr. Med. Chem.
2009, 16, 3903–3917; b) R. Morphy, Z. Rankovic, J. Med. Chem. 2005, 48,
6523–6543.
[2] B. L. Roth, D. J. Sheffler, W. K. Kroeze, Nat. Rev. Drug Discovery 2004, 3,
353–359.
[3] Y. Mehellou, E. De Clercq, J. Med. Chem. 2010, 53, 521–538.
[4] G. N. Nikolenko, S. Palmer, F. Maldarelli, J. W. Mellors, J. M. Coffin, V. K.
Pathak, Proc. Natl. Acad. Sci. USA 2005, 102, 2093–2098.
[5] F. Esposito, A. Corona, E. Tramontano, Mol. Biol. Int. 2012, 586401.
[6] O. Schatz, F. V. Cromme, F. Grꢂninger-Leitch, S. F. J. Le Grice, FEBS Lett.
1989, 257, 311–314.
[7] a) K. Klumpp, T. Mirzadegan, Curr. Pharm. Des. 2006, 12, 1909–1922;
b) E. Tramontano, Mini-Rev. Med. Chem. 2006, 6, 727–737; c) E. Tramon-
tano, R. Di Santo, Curr. Med. Chem. 2010, 17, 2837–2853; d) S. Distinto,
E. Maccioni, R. Meleddu, A. Corona, S. Alcaro, E. Tramontano, Curr.
Pharm. Des. 2013, 19, 1850–1859.
[8] a) J. Q. Hang, S. Rajendran, Y. Yang, Y. Li, P. W. K. In, H. Overton, K. E. B.
Parkes, N. Cammack, J. A. Martin, K. Klumpp, Biochem. Biophys. Res.
Commun. 2004, 317, 321–329; b) E. Tramontano, F. Esposito, R. Badas,
R. Di Santo, R. Costi, P. La Colla, Antiviral Res. 2005, 65, 117–124;
c) D. M. Himmel, K. A. Maegley, T. A. Pauly, J. D. Bauman, K. Das, C.
Dharia, A. D. Clark, Jr., K. Ryan, M. J. Hickey, R. A. Love, S. H. Hughes, S.
Bergqvist, E. Arnold, Structure 2009, 17, 1625–1635; d) T. A. Kirschberg,
M. Balakrishnan, N. H. Squires, T. Barnes, K. M. Brendza, X. Chen, E. J. Ei-
senberg, W. Jin, N. Kutty, S. Leavitt, A. Liclican, Q. Liu, X. Liu, J. Mak, J. K.
Perry, M. Wang, W. J. Watkins, E. B. Lansdon, J. Med. Chem. 2009, 52,
AUC ¼ 0:5598xꢁ1:94010
ð1Þ
in which AUC is the area under the curve (mAuꢄmin) and x is the
drug concentration (mgmLꢁ1).
In vitro transmembrane permeation: A model of biological mem-
brane was prepared as elsewhere reported.[47] Briefly, a poly-
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 12
&10
&
ÞÞ
These are not the final page numbers!

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
5781–5784; e) H.-P. Su, Y. Yan, G. S. Prasad, R. F. Smith, C. L. Daniels, P. D.
Abeywickrema, J. C. Reid, H. M. Loughran, M. Kornienko, S. Sharma, J. A.
Grobler, B. Xu, V. Sardana, T. J. Allison, P. D. Williams, P. L. Darke, D. J.
Hazuda, S. Munshi, J. Virol. 2010, 84, 7625–7633; f) S. Chung, D. M.
Himmel, J.-K. Jiang, K. Wojtak, J. D. Bauman, J. W. Rausch, J. A. Wilson,
J. A. Beutler, C. J. Thomas, E. Arnold, S. F. J. Le Grice, J. Med. Chem. 2011,
54, 4462–4473; g) E. B. Lansdon, Q. Liu, S. A. Leavitt, M. Balakrishnan,
J. K. Perry, C. Lancaster-Moyer, N. Kutty, X. Liu, N. H. Squires, W. J. Wat-
kins, T. A. Kirschberg, Antimicrob. Agents Chemother. 2011, 55, 2905–
2915; h) V. Suchaud, F. Bailly, C. Lion, E. Tramontano, F. Esposito, A.
Corona, F. Christ, Z. Debyser, P. Cotelle, Bioorg. Med. Chem. Lett. 2012,
22, 3988–3992.
[26] K. Das, J. D. Bauman, A. D. Clark, Y. V. Frenkel, P. J. Lewi, A. J. Shatkin,
S. H. Hughes, E. Arnold, Proc. Natl. Acad. Sci. USA 2008, 105, 1466–
1471.
[27] J. S. Ren, C. Nichols, L. E. Bird, T. Fujiwara, H. Sugimoto, D. I. Stuart, D. K.
Stammers, J. Biol. Chem. 2000, 275, 14316–14320.
[28] K. Das, J. D. Bauman, A. S. Rim, C. Dharia, A. D. Clark, M.-J. Camarasa, J.
Balzarini, E. Arnold, J. Med. Chem. 2011, 54, 2727–2737.
[29] K. J. Bowers, E. Chow, H. Xu, R. O. Dror, M. P. Eastwood, B. A. Gregersen,
J. L. Klepeis, I. Kolossvary, M. A. Moraes, F. D. Sacerdoti, J. K. Salmon, Y.
Shan, D. E. Shaw in Proceedings SC06 ACM/IEEE Conference, Tampa, FL
(USA), 2006, p. 84.
[30] Q. Gong, L. Menon, T. Ilina, L. G. Miller, J. Ahn, M. A. Parniak, R. Ishima,
Chem. Biol. Drug Des. 2011, 77, 39–47.
[31] a) J. M. Seckler, M. D. Barkley, P. L. Wintrode, Biophys. J. 2011, 100, 144–
153; b) T. Ilina, K. LaBarge, S. G. Sarafianos, R. Ishima, M. A. Parniak, Biol-
ogy 2012, 1, 521–541.
[9] D. M. Himmel, S. G. Sarafianos, S. Dharmasena, M. M. Hossain, K. McCoy-
Simandle, T. Ilina, A. D. Clark, J. L. Knight, J. G. Julias, P. K. Clark, K.
Krogh-Jespersen, R. M. Levy, S. H. Hughes, M. A. Parniak, E. Arnold, ACS
Chem. Biol. 2006, 1, 702–712.
[10] a) T. Kharlamova, F. Esposito, L. Zinzula, G. Floris, Y.-C. Cheng, G. Dutsch-
man, E. Tramontano, Med. Chem. 2009, 5, 398–410; b) E. Tramontano, T.
Kharlamova, L. Zinzula, F. Esposito, J. Chemother. 2011, 23, 273–276;
c) F. Esposito, T. Kharlamova, S. Distinto, L. Zinzula, Y.-C. Cheng, G.
Dutschman, G. Floris, P. Markt, A. Corona, E. Tramontano, FEBS J. 2011,
278, 1444–1457.
[32] C. Celia, D. Cosco, D. Paolino, M. Fresta, Med. Res. Rev. 2011, 31, 716–
756.
[33] a) E. Tramontano, Y. c. Cheng, Biochem. Pharmacol. 1992, 43, 1371–
1376; b) J. W. Mellors, G. J. Im, E. Tramontano, S. R. Winkler, D. J. Medina,
G. E. Dutschman, H. Z. Bazmi, G. Piras, C. J. Gonzalez, Y. C. Cheng, Mol.
Pharmacol. 1993, 43, 11–16.
[11] F. Esposito, A. Corona, L. Zinzula, T. Kharlamova, E. Tramontano, Chemo-
therapy 2012, 58, 299–307.
[12] S. Distinto, F. Esposito, J. Kirchmair, M. C. Cardia, M. Gaspari, E. Maccioni,
S. Alcaro, P. Markt, G. Wolber, L. Zinzula, E. Tramontano, Eur. J. Med.
Chem. 2012, 50, 216–229.
[13] L. M. A. Lima, E. J. Barreiro, Curr. Med. Chem. 2005, 12, 23–49.
[14] H. Pelemans, R. Esnouf, E. De Clercq, J. Balzarini, Mol. Pharmacol. 2000,
57, 954–960.
[15] T. Rosen, A. A. Nagel, J. P. Rizzi, J. L. Ives, J. B. Daffeh, A. H. Ganong, K.
Guarino, J. Heym, S. McLean, J. Med. Chem. 1990, 33, 2715–2720.
[16] T. Yonetani, Methods Enzymol. 1982, 87, 500–509.
[34] C. Parolin, B. Gatto, C. Del Vecchio, T. Pecere, E. Tramontano, V. Cecchet-
ti, A. Fravolini, S. Masiero, M. Palumbo, G. Palꢅ, Antimicrob. Agents Che-
mother. 2003, 47, 889–896.
[35] Schrçdinger LLC, Maestro GUI, New York, NY, USA, 2012.
[36] T. Halgren, J. Comput. Chem. 1996, 17, 520–552.
[37] W. Hasel, T. F. Hendrickson, W. C. Still, Tetrahedron Comput. Methodol.
1988, 1, 103–116.
[38] G. A. Kaminski, R. A. Friesner, J. Tirado-Rives, W. L. Jorgensen, J. Phys.
Chem. B 2001, 105, 6474–6487.
[39] K. J. Bowers, R. O. Dror, D. E. Shaw, J. Chem. Phys. 2006, 124, 184109–
184111.
[17] A. K. Felts, K. LaBarge, J. D. Bauman, D. V. Patel, D. M. Himmel, E. Arnold,
M. A. Parniak, R. M. Levy, J. Chem. Inf. Model. 2011, 51, 1986–1998.
[18] S.-Y. Huang, X. Zou, Proteins: Struct. Funct. Bioinf. 2007, 66, 399–421.
[19] K. A. Paris, O. Haq, A. K. Felts, K. Das, E. Arnold, R. M. Levy, J. Med. Chem.
2009, 52, 6413–6420.
[20] H. M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T. N. Bhat, H. Weissig,
I. N. Shindyalov, P. E. Bourne, Nucleic Acids Res. 2000, 28, 235–242.
[21] Schrçdinger LLC, “QM Polarized Protocol” in Schrçdinger Suite, New
York, NY, USA.
[40] W. L. Jorgensen, J. Chandrasekhar, J. D. Madura, R. W. Impey, M. L. Klein,
J. Chem. Phys. 1983, 79, 926–935.
[41] G. J. Martyna, D. J. Tobias, M. L. Klein, J. Chem. Phys. 1994, 101, 4177–
4189.
[42] D. A. Gibson, E. A. Carter, J. Phys. Chem. 1993, 97, 13429–13434.
[43] J.-P. Ryckaert, G. Ciccotti, H. Berendsen, J. Comp. Physiol. 1977, 23, 327–
341.
[44] T. Darden, D. York, L. Pedersen, J. Chem. Phys. 1993, 98, 10089–10092.
[45] a) InteLigand Software GmbH, LigandScout 3.0, Maria Enzersdorf, Aus-
tria, 2009; b) G. Wolber, T. Langer, J. Chem. Inf. Model. 2005, 45, 160–
169.
[22] J. Y. Chung, J.-M. Hah, A. E. Cho, J. Chem. Inf. Model. 2009, 49, 2382–
2387.
[23] P. A. Kollman, I. Massova, C. Reyes, B. Kuhn, S. Huo, L. Chong, M. Lee, T.
Lee, Y. Duan, W. Wang, O. Donini, P. Cieplak, J. Srinivasan, D. A. Case,
T. E. Cheatham, Acc. Chem. Res. 2000, 33, 889–897.
[46] W. Humphrey, A. Dalke, K. Schulten, J. Mol. Graphics 1996, 14, 33–38.
[47] G. Cavallaro, M. Fresta, G. Giammona, G. Puglisi, A. Villari, Int. J. Pharm.
1994, 111, 31–41.
[24] J. D. Pata, W. G. Stirtan, S. W. Goldstein, T. A. Steitz, Proc. Natl. Acad. Sci.
USA 2004, 101, 10548–10553.
[25] J. S. Ren, R. Esnouf, E. Garman, D. Somers, C. Ross, I. Kirby, J. Keeling, G.
Darby, Y. Jones, D. Stuart, D. Stammers, Nat. Struct. Biol. 1995, 2, 293–
302.
Received: February 6, 2014
Published online on && &&, 0000
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 12
&
11
&
ÞÞ
These are not the final page numbers!

FULL PAPERS
R. Meleddu, V. Cannas, S. Distinto,*
G. Sarais, C. Del Vecchio, F. Esposito,
G. Bianco, A. Corona, F. Cottiglia,
S. Alcaro, C. Parolin, A. Artese, D. Scalise,
M. Fresta, A. Arridu, F. Ortuso,
1,3-Diarylpropenones were synthesized
and assayed for their activity toward
HIV-1 reverse transcriptase (RT). Dual in-
hibition properties in the low-micromo-
lar range were observed, and mutations
in the RT binding pocket were found to
have an interesting effect on inhibition.
Biochemical and computational ap-
proaches were applied with an aim to
understand the possible mechanism of
action.
E. Maccioni,* E. Tramontano
&& – &&
Design, Synthesis, and Biological
Evaluation of 1,3-Diarylpropenones as
Dual Inhibitors of HIV-1 Reverse
Transcriptase
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 12
&12
&
ÞÞ
These are not the final page numbers!