Exploiting drug-resistant enzymes as tools to identify thienopyrimidinone inhibitors of human immunodeficiency virus reverse transcriptase-associated ribonuclease H

Article abstract of DOI:10.1021/jm400405z

The thienopyrimidinone 5,6-dimethyl-2-(4-nitrophenyl)thieno[2,3-d] pyrimidin-4(3H)-one (DNTP) occupies the interface between the p66 ribonuclease H (RNase H) domain and p51 thumb of human immunodeficiency virus reverse transcriptase (HIV RT), thereby indu

Full text of DOI:10.1021/jm400405z

Article
pubs.acs.org/jmc
Exploiting Drug-Resistant Enzymes as Tools To Identify
Thienopyrimidinone Inhibitors of Human Immunodeficiency Virus
Reverse Transcriptase-Associated Ribonuclease H
Takashi Masaoka,† Suhman Chung,† Pierluigi Caboni,‡ Jason W. Rausch,† Jennifer A. Wilson,§
Humeyra Taskent-Sezgin,† John A. Beutler,§ Graziella Tocco,‡ and Stuart F. J. Le Grice*,†
†RT Biochemistry Section, HIV Drug Resistance Program, National Cancer Institute, Frederick, Maryland 21702, United States
‡Unit of Drug Sciences, Department of Life and Environmental Sciences, University of Cagliari, Via Ospedale 72, 09124 Cagliari, Italy
§Molecular Targets Laboratory, National Cancer Institute, Frederick, Maryland 21702, United States
S
* Supporting Information
ABSTRACT: The thienopyrimidinone 5,6-dimethyl-2-(4-nitrophenyl)thieno[2,3-d]pyrimidin-
4(3H)-one (DNTP) occupies the interface between the p66 ribonuclease H (RNase H) domain
and p51 thumb of human immunodeficiency virus reverse transcriptase (HIV RT), thereby
inducing a conformational change incompatible with catalysis. Here, we combined biochemical
characterization of 39 DNTP derivatives with antiviral testing of selected compounds. In
addition to wild-type HIV-1 RT, derivatives were evaluated with rationally designed, p66/p51
heterodimers exhibiting high-level DNTP sensitivity or resistance. This strategy identified 3′,4′-
dihydroxyphenyl (catechol) substituted thienopyrimidinones with submicromolar in vitro
activity against both wild type HIV-1 RT and drug-resistant variants. Thermal shift analysis
indicates that, in contrast to active site RNase H inhibitors, these thienopyrimidinones destabilize
the enzyme, in some instances reducing the Tm by 5 °C. Importantly, catechol-containing
thienopyrimidinones also inhibit HIV-1 replication in cells. Our data strengthen the case for
allosteric inhibition of HIV RNase H activity, providing a platform for designing improved
antagonists for use in combination antiviral therapy.
thienyl]-2-furancarboxamide (NSC727448) were identified as
modestly potent HIV-1 RNase H inhibitors. In the absence of
crystallographic data, protein footprinting by mass spectrom-
etry implicated p51 thumb residues Cys280 and Lys281 in
inhibitor binding.10 Since Cys280-Thr290 of the p51 thumb
and Pro537-Glu546 of the p66 RNase H domain constitute
approximately 30% of the buried surface interface,11,12 these
compounds, which are equally effective against the enzyme and
the enzyme/substrate complex,13,14 represent allosteric inhib-
itors that affect RNase H activity by destabilizing the subunit
interface. A limited structure−activity study subsequently
identified the thienopyrimidinone 5,6-dimethyl-2-(4-
nitrophenyl)thieno[2,3-d]pyrimidin-4(3H)-one (DNTP), a
cyclized derivative of NSC727448, as a lead candidate for
further inhibitor optimization. Alanine scanning mutagenesis
between p51 residues Lys275 and Thr286 and a vertical scan of
Cys28014 identified selectively mutated RT heterodimers
displaying either significantly increased DNTP sensitivity
(Val276Ala, Arg284Ala) or high-level resistance (Cys280Ala,
Thr286Ala), strengthening the contention that the p51 thumb
is the target site. More importantly this scanning mutagenesis
approach provided a unique collection of mutant HIV-1
INTRODUCTION
■
For approximately 15 years, nonnucleoside reverse transcriptase
inhibitors (NNRTIs) have constituted a critical component of
combination HIV antiretroviral therapy,1 acting allosterically by
creating and occupying a hydrophobic pocket of the p66 thumb
subdomain of the p66/p51 reverse transcriptase (RT)
heterodimer2 to compromize enzyme conformational dynam-
ics3 and the chemical step of DNA synthesis.4 The possibility of
allosteric inhibition of additional HIV enzymes has recently
been demonstrated by the report that the 2-(quinolin-3-
yl)acetic acid derivatives LEDGIN-6 and BI-1001 are allosteric
inhibitors of HIV-1 integrase,5 while Perryman et al. have used
a fragment-based approach to highlight proposed non active
site allosteric inhibitors of HIV-1 protease.6 With respect to
RT-associated ribonuclease H (RNase H) activity, encoded by
the C-terminal domain of the p66 subunit and essential for
virus replication,7,8 we demonstrated that α-hydroxytropolones,
while effective inhibitors at submicromolar concentrations,
were efficiently displaced from the RNase active site by nucleic
acid.9 In light of this limitation, an alternative strategy might
therefore be to identify allosteric inhibitors that bind close to
the RNase H active site and compromise catalysis by affecting
subdomain and/or subunit dynamics.
As a first step in this direction, the vinylogous ureas 2-amino-
5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxamide
(NSC727447) and N-[3-(aminocarbonyl)-4,5-dimethyl-2-
Received: March 19, 2013
© XXXX American Chemical Society
A
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enzymes that could be exploited to screen for DNTP
derivatives with broad-spectrum activity.
In this communication, we report the synthesis, activity, and
antiviral properties of 39 novel thienopyrimidinones bearing
substitutions on the thiophene or the C2 position of the
pyrimidinone ring (Figure 1). Exploiting the panel of selectively
element in inhibition, although its role remained to be
established. We therefore elected to preserve the pyrimidinone
core and introduce substituents on the phenyl ring. Table 1
summarizes the effect of these substitutions on inhibitor
potency.
The effect of meta- and para-substituted phenyl rings was
first evaluated. The absence of any substitution (compound 1)
decreased the IC50 approximately 15-fold (11.3 μM vs 0.85 μM
for DNTP). Para-substitution of the phenyl ring appeared to be
more beneficial, as the IC50 of compounds 2, 3, and 4 were
0.79, 1.3, and 1.6 μM, respectively, while meta-substituted
compounds 7 and 8 showed no inhibition up to 50 μM. As
previously shown with DNTP, the electron withdrawing effect
of the substituent also contributes to inhibitory potency, as 3
(−CF3, 1.3 μM) and 4 (−CO2H, 1.6 μM) were 5- to 6-fold
more active than compound 5 (−OCH3, 9.1 μM). The
exception is compound 2 (−OH, 0.79 μM), which is likely to
be involved in hydrogen bonding with residues at the p51−p66
dimer interface. Introducing an additional hydroxyl at the 3′
position of compound 2 (compound 9) resulted in a 3-fold
increase in activity (IC50 = 0.26 μM), suggesting that additional
contacts provided by this substitution stabilize inhibitor
binding. This notion was strengthened by the observation
that replacing the 3′-OH with either −F (compound 13) or
−OCH3 (compound 15) reduced the IC50 almost 200-fold.
Other disubstituted thienopyrimidinones were generally
inactive (compounds 11, and 12) or displayed significantly
reduced IC50 values (compounds 10 and 14).
Table 2 shows that substituting the phenyl ring with other
heteroaromatic rings (compounds 16, 17, and 19) does not
significantly affect the activity compared to compound 1. The
exception to this was the indole-substituted pyrimidinone,
compound 18, for which an IC50 of 0.48 μM was determined.
Despite the apparently favorable properties of compound 18,
data of a later section indicate that it failed to inhibit
thienopyrimidinone-resistant HIV-1 RT mutants.
C5,C6-Cycloheptene Ring Substitutions. Since previous
work with NSC727447 suggested that a hydrophobic pocket in
the immediate vicinity of the binding site could accommodate
its cycloheptene ring but not other cycloalkenes,13 we next
evaluated thienopyrimidinones containing a cycloheptene ring
(compounds 20−23). These compounds displayed IC50 values
in the range 1.9−4.1 μM (Table 3), which is slightly higher
than values of derivatives containing a 5,6-dimethyl substitution
of the thiophene ring. This result suggests that substituting a
5,6-dimethyl with a cycloheptene ring does not significantly
affect binding affinity, although we cannot rule out the
possibility that the binding mode of these compounds differs
from that of 5,6-dimethyl derivatives as was previously
suggested.13
Figure 1. Structure of the thienopyrimidinone 5,6-dimethyl-2-(4-
nitrophenyl)thieno[2,3-d]pyrimidin-4(3H)-one (DNTP).
mutated HIV-1 RT mutants allowed us to identify four groups
of molecules, based on their resistance/sensitivity profiles.
Among these, compounds with a 3′,4′-dihydroxyphenyl
(catechol) substitution displayed activity against both wild
type and drug-resistant RT variants at submicromolar
concentrations and, importantly, inhibited HIV-1 replication
in cells. Differential scanning fluorimetry indicated that these
compounds, in contrast to α-hydroxytropolone-derived RNase
H inhibitors,15 destabilized the RT heterodimer, in some
instances lowering the Tm by almost 5 °C. Collectively, our data
provide an important structural platform for the continued
development of thienopyrimidinone-based RNase H inhibitors
and highlight the value of genetically engineered HIV-1 RT
variants with altered inhibitor sensitivity profiles for secondary
screening.
RESULTS
■
Synthesis of Thienopyrimidinones. All thienopyrimidi-
none derivatives tested were synthesized according to Bakavoli
et al.16 Briefly, 2-amino-4′,5′-disubstituted-thiophene-3-carbox-
amide intermediates were obtained from condensation of the
appropriate ketone and α-cyanoacetamide in the presence of
elemental sulfur and base.17 This intermediate was then treated
with the aromatic aldehyde and molecular iodine to produce
the corresponding thienopyrimidinone (Scheme 1).16 Addi-
tional details are provided in the Supporting Information.
C2 Aromatic Substitutions of the Thienopyrimidi-
none. DNTP, identified recently as a submicromolar HIV-1
RNase H inhibitor,13 comprises a thiophene-fused pyrimidi-
none ring containing a C2 4′-nitrophenyl substitution (Figure
1). Previous work13 suggested that the 4′-NO2 group was a key
Di- or Trihydroxyphenyl-Containing Thienopyrimidi-
none. In an effort to improve thienopyrimidinone activity, we
focused on compound 9 for further modification. We first
investigated how C5 and C6 substitutions affected activity.
Table 4 shows that replacing a C5 −CH3 with a bulkier alkyl
group, such as n-propyl (compound 24) and n-butyl
(compound 25) or replacing a C6 −CH3 with ethyl (compound
26) is tolerated, resulting in IC50 values of 0.49, 0.42, and 0.59
μM respectively. Substituting 5,6-dimethyl with an aliphatic
ring also does not affect the activity, regardless of the ring size,
suggesting that the bulkier C5 and C6 alkyl groups can be
accommodated. This result supports our previous contention
that a role of the 3′,4′-dihydroxyphenyl component is to
Scheme 1. General Scheme for Synthesis of Modified
Thienopyrimidinones
B
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a
Table 1. Inhibition of HIV-1 RNase H Enzymatic Activity by Thienopyrimidinones Carrying C2 Aromatic Substitutions
a
IC50 values represent the average of triplicate analysis.
“anchor” thienopyrimidinones at the p51−p66 subunit inter-
face, predicting that substitutions on the thiophene ring would
minimally affect the overall binding conformation. However
any change in the position of −OH substitution (compounds
31−37) or adding an additional −OH group (compounds 38
and 39) decreased activity, indicating the necessity of hydroxyl
substitutions at 3′ and 4′ positions for optimal binding of these
inhibitors.
Screening with Engineered HIV-1 RT Mutants Identi-
fies Thienopyrimidinones with Broad Spectrum Activity.
Scanning mutagenesis of p51 α-helix I14(Figure 2) demon-
strated increased DNTP sensitivity for selectively mutated HIV-
1 RT derivatives p66/p51V276A and p66/p51R284A in addition to
decreased sensitivity for mutants p66/p51C280A and p66/
p51T286A. This observation was most pronounced for RT
mutant p66/p51C280A, which exhibited ∼50-fold resistance to
DNTP.14 A high level of resistance was also observed in a
vertical scan of p51 residue Cys280, suggesting a critical
contribution from this residue to inhibitor binding. We
therefore determined IC50 values for all thienopyrimidinones
in this study with the panel of engineered RT mutants in an
attempt to identify broad-spectrum inhibitors. The results of
this analysis are presented in Table 5.
In general, thienopyrimidinones could be classed into four
groups, depending on their activity profiles. Group I inhibitors,
comprising compounds 2, 3, 6, 18, and 21, while active against
wild type RT at concentrations ranging from 0.48 to 1.9 μM,
uniformly failed to inhibit p66/p51C280A RT at 50 μM. At the
C
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albeit at IC50 values ranging from 10.6 to 41.4 μM. Group II
compounds also displayed a similar trend with respect to
mutants p66/p51V276A (increased sensitivity), p66/p51R284A
(increased sensitivity), and p66/p51T286A (decreased sensitiv-
ity).
Table 2. Inhibition of HIV-1 RNase H Enzymatic Activity by
Thienopyrimidinones Carrying C2 Heteroaromatic
Substitutions
a
Group III and group IV inhibitors were dramatically
different. Interestingly, both groups contain a catechol moiety
(2′,3′-dihydroxy for group III and 3′,4′-dihydroxyphenyl for
group IV) and, in contrast to group I and group II inhibitors,
were effective against RT mutant p66/p51C280A. Group IV
inhibitors (IC50 = 0.26−0.59 μM) were in general 4- to 8-fold
more potent than those of group III (IC50 = 1.7−1.8 μM).
More importantly, this broad-spectrum activity was also
extended to drug-sensitive mutants p66/p51V276A and p66/
p51R284A and drug-resistant mutant p66/p51T286A. Supporting
Information Table S1 provides IC50 values for compound 9
across the entire panel of selectively mutated p51 thumb α-helix
I variants (i.e., Lys275-Arg286), indicating that within
experimental error, they are uniformly sensitive to this
thienopyrimidinone. Although such an observation cannot
exclude the possibility that group III and group IV inhibitors
might interact with p51 RT at a site slightly removed from that
previously proposed,10,13 data shown below suggest that this is
unlikely. In summary, although carrying a variety of substituents
on the thiophene ring, the catechol moiety common to group
III and group IV inhibitors appears to play a critical role in
inhibitory potency.
a
IC50 values represent the average of triplicate analysis.
Table 3. Inhibition of HIV-1 RNase H Enzymatic Activity by
Cycloheptene-Containing Thienopyrimidinones
a
Thienopyrimidinone Inhibitors Destabilize HIV-1 RT
in the Absence and Presence of Substrate. Differential
scanning fluorimetry (ThermoFluor18) is a simple, rapid, and
inexpensive means of determining protein stability in the
presence of small molecule ligands,19,20 an example of which is
the demonstration by Su et al. that naphthyridinone-based
RNase H active site inhibitors increased the Tm of HIV-1 RT by
3−4 °C.21 More recently, we have also shown that allosteric
HIV-1 integrase inhibitors (ALLINIs) stabilize the integrase
tetramer and increase its melting temperature.5 Since
computer-assisted modeling studies located the parent
thienopyrimidinone, DNTP, at the interface of the p51 and
p66 RT subunits, we investigated whether binding of the
thienopyrimidinone compounds affected the stability of the
p66/p51 RT heterodimer. The results of ThermoFluor analysis
are provided in Figure 3.
For comparative purposes, we first examined alterations to
the thermal stability of wild type HIV-1 RT induced by the
RNase H active site hydroxytropolone inhibitor β-thujaplici-
nol.15 In accordance with studies of Su et al.,21 we observed a
Tm increase of ∼2.0 °C in the presence of Mg2+ and the active
site inhibitor. In contrast, all thienopyrimidinones tested
reduced the Tm by 0.5−5.5 °C (Figure 3), although there
was no linear correlation between IC50 and ΔTm. The ability of
compounds 9 and 29 to destabilize the binary enzyme/
substrate complex was also examined. For these experiments,
we chose an RNA/DNA hybrid containing the polypurine tract
(PPT) primer of HIV-1 (+) strand DNA synthesis. In the
absence of inhibitor, binding of the PPT-containing duplex
increased the Tm by 9.3 °C, indicating significant stabilization of
HIV-1 RT (Supporting Information Figure S1). However,
compounds 9 and 29 retained their destabilizing property,
reducing the Tm of the enzyme/substrate complex by 5.8 and
5.9 °C, respectively.
a
IC50 values represent the average of triplicate analysis.
same time, these compounds showed enhanced activity against
mutant p66/p51V276A (e.g., for compound 2, IC50WT = 0.79 μM
vs IC50Mut = 0.14 μM) and reduced activity against mutant p66/
p51T286A (e.g., for compound 21, IC50WT = 1.9 μM vs IC50
=
Mut
32.0 μM). Group II inhibitors, exemplified by compounds 20,
22, and 23, were slightly less active than the parent
thienopyrimidinone, DNTP, against wild type RT with IC50
values varying from 3.1 to 4.1 μM. However, these inhibitors
These results collectively support the notion that thienopyr-
imidinones inhibit RNase H activity allosterically by binding at
compromised RNase H activity of RT mutant p66/p51C280A
,
D
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a
Table 4. Inhibition of HIV-1 RNase H Enzymatic Activity by Di- or Trihydroxyphenyl-Containing Thienopyrimidinones
a
IC50 values represent the average of triplicate analysis.
the interface of two subunits in the presence of the nucleic acid
substrate and destabilizing those interactions. However, such
changes in thermal stability cannot discriminate between
denaturation of individual RT subunits, dissociation of the
p66/p51 heterodimer, or a combination of both processes.
Inhibition of DNA Polymerase and Integrase Activ-
ities by 3′,4′-Dydroxyphenyl-Containing Thienopyrimi-
dinones. In order to examine the specificity of our
thienopyrimidinones, we determined their effect on DNA-
dependent DNA polymerase and integrase activities. Figure 4a
shows that compounds 25−28 and 30 inhibited >60% of DNA-
dependent DNA polymerase activity at 50 μM. These results
are not unexpected, since these compounds most likely bind at
the dimer interface and induce alterations of overall RT−
substrate interactions, which could affect both RNase H and
polymerase activities. These compounds are equally active
against NNRTI resistant mutants (K103N, Y181C) (data not
E
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Figure 3. Effect of thienopyrimidinone RNase H inhibitors on the
thermal stability of p66/p51 HIV-1 RT. β-TP is the RNase H active
site inhibitor β-thujaplicinol. Tm values are the average of triplicate
analysis.
Antiviral Activity of 3′,4′-Dihydroxyphenyl-Contain-
ing Thienopyrimidinones. On the basis of their favorable
properties against the panel of selectively mutated p51 RT
mutants, the ability of compounds 9, 24−26, 29, 31, and 32 to
inhibit virus replication was assessed. Data of Table 6 indicate
that dihydroxyphenyl-containing thienopyrimidinones inhibited
HIV-1 replication in the low micromolar range, with selectivity
Figure 2. Model of the interface between the p66 RNase H domain
(gold) and p51 thumb subdomain (green) of HIV-1 RT. Active site
carboxylates (D443, E478, D498, D549) and the conserved His539 of the
RNase H domain are indicated in white. Residues of the p51 thumb
subdomain indicated in red were previously shown to confer resistance
(C280, T286) or sensitivity (V276, R284) to thienopyrimidinone DNTP
when substituted with Ala.
ratios ranging from 2.3 (compound 9, EC50 = 3.8 μM, CC50
=
8.9 μM) to ∼6 (compound 24, EC50 = 1.9 μM CC50 = 11.2
μM). The exception to this was compound 28. Although active
in vitro at low micromolar concentrations, this compound failed
to elicit protection from HIV infection.
shown), suggesting that they are less likely to bind to the
NNRTI binding pocket.
It has been reported that compounds possessing catechol
moieties were active against purified HIV-1 integrase (IN).22
However, the results of our analysis presented in Figure 4b
indicate these thienopyrimidinones have minimal effect on 3′-
processing of integrase, suggesting specificity for RT.
DISCUSSION AND CONCLUSIONS
■
The demonstration that NNRTIs interrupt HIV-1 DNA
synthesis by influencing enzyme conformational dynamics3,23
has provided a novel and important platform for identification
a
Table 5. Thienopyrimidinone Inhibition of RNase H Activity of Selectively Mutated p66/p51 HIV-1 RT Heterodimers
IC50 (μM)
compd
WT
V276A
C280A
>50
R284A
T286A
I
2
0.79 0.11
1.3 0.4
0.14 0.04
0.31 0.01
0.26 0.03
0.16 0.02
0.46 0.02
0.96 0.01
0.53 0.01
0.64 0.03
4.1 0.1
0.20 0.03
0.23 0.01
0.34 0.05
0.12 0.001
0.95 0.04
1.3 0.06
0.54 0.03
0.65 0.02
3.5 0.1
3.7 0.3
3
>50
7.5 1.0
6
1.5 0.1
>50
1.5 0.2
18
21
20
22
23
31
32
9
0.48 0.06
1.9 0.03
4.1 0.2
>50
0.96 0.02
32.0 15.5
27.9 6.9
7.2 0.7
>50
II
41.4 6.5
10.6 0.5
15.8 2.0
2.9 0.1
1.6 0.03
0.32 0.01
0.37 0.001
0.49 0.005
0.50 0.01
0.26 0.01
0.29 0.01
0.26 0.02
0.23 0.004
3.1 0.9
3.2 0.2
6.6 0.3
III
IV
1.7 0.1
5.7 0.2
1.8 0.1
2.8 0.2
2.9 0.1
3.6 0.2
0.26 0.01
0.49 0.01
0.42 0.02
0.59 0.04
0.32 0.02
0.37 0.03
0.29 0.11
0.26 0.01
0.39 0.04
0.56 0.02
0.91 0.02
0.76 0.03
0.21 0.01
0.41 0.01
0.37 0.01
0.11 0.01
0.30 0.01
0.80 0.01
1.2 0.04
1.4 0.04
0.47 0.01
0.92 0.04
0.55 0.004
0.28 0.01
0.28 0.04
0.57 0.02
1.1 0.01
0.82 0.02
0.37 0.01
0.46 0.01
0.36 0.02
0.29 0.01
24
25
26
27
28
29
30
a
Alanine substitutions were introduced into the thumb subdomain of the p51 RT subunit. Values reported represent the average of triplicate analysis.
Groups I, II, III, and IV designations are defined in the text.
F
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of these compounds has been shown to possess metal chelating
properties,25 the observation that they are equally active against
our panel of engineered enzymes might be indicative of active
site inhibitors that chelate the catalytically critical Mg2+ ions.
However, thermal denaturation studies of Su et al.,21 together
with our data shown in Figure 3, demonstrate that binding of
metal-chelating RNase H inhibitors stabilizes the HIV-1 RT
heterodimer, increasing the Tm by ∼2.0 °C. In contrast,
compounds 9 and 24−32 decrease the Tm, in some instances
by more than 5 °C. In addition, Yonetani−Theorell analysis26
shows that compounds 9 and 29 are mutually exclusive with
respect to the active site inhibitor, β-thujaplicinol (Figure 5).
Figure 4. Evaluation of inhibitory activity of 3′,4′-dihydroxyphenyl-
containing thienopyrimidinones against HIV-1 DNA polymerase
activities (a) and HIV-1 integrase (b) in the presence of 50 μM
3′,4′-dihydroxyphenyl-containing thienopyrimidinones.
Table 6. Antiviral Activity of Catechol-Containing
a
Thienopyrimidinones
compd
EC50 (μM)
CC50 (μM)
sr
9
3.8
1.9
7.9
9.2
1.6
3.1
2.7
8.9
11.2
25.4
33.7
6.8
2.3
5.9
3.2
3.7
4.3
4.0
3.6
24
25
26
29
31
32
12.4
9.8
a
sr, selectivity ratio (i.e., CC50/EC50).
Figure 5. Yonetani−Theorell plot for inhibition of HIV-1 RT-RNase
H activity in the presence of β-thujaplicinol and (a) compound 9 or
(b) compound 29. The inhibitory activity (defined as V0/Vi) for each
concentration of β-thujaplicinol (β-TP: 0, 0.125, 0.25, 0.5, and 1 μM)
is plotted as a function of the concentrations of the thienopyr-
imidinone inhibitors (0, 0.625, 1.25, 2.5, and 5 μM). The assay was
performed at fixed concentrations of substrate and enzyme (250 and
17 nM, respectively).
of small molecules that impose allosteric control of critical HIV
enzymes, a notion that has been extended to HIV-1 integrase5
and proposed for HIV-1 protease.6 It is therefore not
unreasonable to consider allosteric inhibition of HIV-1 RT-
associated RNase H activity, especially in light of observations
that interactions involving p51 thumb residues Cys280-Thr290
and Pro537-Glu546 of the p66 RNase H domain constitute
∼33% of the buried surface at the subunit interface.24 Since the
p66 thumb does not participate in an equivalent intersubunit
interaction, vinylogous ureas previously described by Wendeler
et al.10 most likely interfere with RNase H activity by
perturbing the dimer interface, inducing alterations to active
site geometry that are incompatible with catalysis.10,13,14
This study evaluated 39 related and novel thienopyrimidi-
nones as HIV-1 RNase H inhibitors and, more importantly,
combined chemical synthesis with a multienzyme counter-
screen using unique genetically manipulated HIV-1 RT mutants
that display enhanced resistance or sensitivity to the parent
molecule, DNTP. This strategy identified several compounds
containing a catechol-substituted pyrimidinone ring that
inhibited both wild-type and drug-resistant HIV-1 RT in vitro
and HIV-1 replication in culture. Since the catechol component
Taken together, these observations strengthen our postulate
that compounds 9 and 24−30 compromise dynamics at the
dimer interface and as a consequence interrupt catalysis by
altering active site geometry.10 This notion is supported by our
recent success in solving the structure of p66/p51 HIV-1 RT
complexed with an NNRTI and a non-polypurine tract-
containing RNA/DNA hybrid. Among several significant
conformational changes nucleic acid binding promotes an
“expansion” of the enzyme. In this model, a ∼2.5 Å outward
movement of the p66 fingers subdomain and RNase H domain
is required to accommodate the hybrid, and as a consequence,
the p66 thumb tilts toward the duplex while the p51 thumb
moves in concert with the RNase H domain.27 A small
molecule antagonist interacting at the p51 thumb/p66 RNase
H interface could therefore inhibit or alter subdomain
G
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scan width 0.70 amu, and detector at 1450 V. For APCI the
atmospheric pressure ionization (API) housing was kept at 50 °C.
Parent compounds were dissolved in a mixture consisting of (A)
acetonitrile 90% (v/v) and (B) doubly distilled water 10% (v/v) and
infused in the source at a rate of 0.05 mL/min.
movement and modify nucleic acid trajectory or active site
geometry. Although high-resolution structural data are
presently unavailable, preliminary X-ray crystallographic anal-
ysis of HIV-1 RT containing an RNA/DNA hybrid and
compound 9 shows electron density corresponding to the
inhibitor in the immediate vicinity of p51 α-helix H (W. Yang,
personal communication).
Preparation and purification of wild type p66/p51 HIV-1 RT and
reconstituted p51 thumb mutants have been previously reported.14 All
enzymes were stored at −20 °C in a 50% glycerol-containing buffer.
General Procedure of Syntheses of 2-Amino-4,5-disubsti-
tuted-thiophene-3-carboxamides (40−47). The appropriate
ketone (14 mmol), cyanoacetamide (14 mmol), sulfur (14 mmol),
and diethylamine (15.4 mmol) in 15 mL of dry ethanol were stirred
overnight at room temperature. The reaction was quenched with 100
mL of water and extracted several times with ethyl acetate/ethanol
(3:1). The organic layer was dried over Na2SO4, and after filtration,
the solvent was evaporated under vacuum to give a brown oil or solid.
The crude product was then crystallized from ethanol to give
compounds 40−47. Characterization of all thienopyrimidinones used
in the study is provided in Supporting Information.
General Procedure of Syntheses of Thieno[2,3-d]pyrimidin-
4(3H)-ones (1−39). A mixture of 2-amino-4,5-disubstituted-thio-
phene-3-carboxamide (40− 47) (1.5 mmol), molecular iodine (1.95
mmol), and the appropriate aromatic aldehyde (1.8 mmol) in the
presence of acetonitrile as solvent (15 mL) was stirred at room
temperature for the period indicated (TLC) (1−5 h). The reaction
was quenched with 35 mL of 5% Na2S2O3 solution, and the resulting
precipitate was filtered off. The crude product was washed with
petroleum ether/ethyl acetate (1:1) and recrystallized from ethanol to
give compounds 1−39. Characterization of all thienopyrimidinones
used in the study is provided in Supporting Information. The purity of
synthetic compounds 1−39 was assessed to be ≥95% by HPLC
analysis (Agilent HP-1100).
RNase H Inhibitor Analysis. IC50 values were determined as
previously reported,13 using an 18-nt 3′-fluorescein-labeled RNA
annealed to a complementary 18-nt 5′-dabsyl-labeled DNA. To a 96-
well plate was added 1 μL of each inhibitor (in DMSO), followed by
10 μL of the appropriate RT (15−80 ng/mL) in reaction buffer.
Hydrolysis was initiated by adding 10 μL of RNA/DNA hybrid (2.5
μM). Final assay conditions were 50 mM Tris-HCl, pH 8.0, 60 mM
KCl, 10 mM MgCl2, 1% DMSO, 150−800 ng of RT, 250 nM
substrate, and increasing concentrations of inhibitor. Wells containing
only DMSO were used as negative control. Plates were incubated at 37
°C in a Spectramax Gemini EM fluorescence spectrometer for 10 min,
and fluorescence (λex = 485 nm; λem = 520 nm) was measured at 1 min
intervals such that linear initial rates could be measured in the
presence (vi) and absence (vo) of inhibitor. Percent inhibition was
calculated as 100(vo − vi)/vo, and plotted against log[I]. IC50 values
were determined using Prism5 (GraphPad Software). All assays were
performed in triplicate.
Yonetani−Theorell Analysis. To determine whether the binding
site for thienopyrimidinones overlaps with that of the active site RNase
H inhibitor β-thujaplicinol,26 RNase H activity was measured in the
presence of varying concentrations of compounds 9 and 29 at fixed
concentrations of β-thujaplicinol. The assay was conducted at fixed
concentrations of RNA/DNA substrate (250 nM) and enzyme (4
nM). Hydrolysis was initiated by adding RNA/DNA hybrid and
monitored for 60 min. Data analysis was performed with SigmaPlot
(SPSS Inc.). The following equation was used for data analysis:
Since a catechol-substituted pyrimidinone is common to
compounds 9 and 24−30, their activity differences should
reflect the consequences of their thiophene ring substitutions.
However, increasing the length of C5 (methyl, propyl, and
butyl, respectively) or C6 alkyl substitution (ethyl) does not
significantly affect activity. Moreover, introducing an aliphatic
ring also does not change the IC50, regardless of the ring size,
which is in contrast to our previous data with the 2-
aminothiophene-3-carboxamide derivatives, where only the
cycloheptene containing compound (NSC727447) was ac-
tive.13 The favorable properties of these compounds are
intriguing, since their cycloalkene substituent introduces
considerable bulk and rigidity that may drive a favorable
binding conformation. One explanation may be that the 3′,4′-
dihydroxyphenyl substitution “anchors” the compounds at the
p51−p66 subunit interface deeper than the other thienopyr-
imidinones, which would strengthen their binding affinities and
provide more room to accommodate bulky substituents on the
thiophene ring without affecting overall binding. Any change in
the position of −OH substitution or adding an additional −OH
group appears to lower this anchoring effect of the inhibitors,
thus decreasing inhibitory activity. Improving the selectivity
ratio of this class of RNase H inhibitor will permit selection of
drug-resistant variants to verify that the dimer interface is the
bona fide target. Efforts are presently underway to obtain a
cocrystal of HIV-1 RT and compound 29 and to compare this
with data for compound 9, as well as to study the effect of
structural modifications of the thiophenepyrimidinone core in
order to understand its role in binding. For example, since the
thiophene ring is susceptible to hepatic oxidation by
cytochrome P450 and sulfur itself can also undergo oxidation,
it would be beneficial to find alternative aromatic rings that may
possess better pharmacological properties in the future. Our
preliminary data have already shown that substituting the
thiophene with a benzene ring, a well-known bioisostere for a
thiophene ring, retains its inhibitory activity against our
engineered drug-resistant enzymes (data not shown). This
would open a new door to the development of next generation
inhibitors by taking advantage of a highly diverse library of
substituted benzenes.
EXPERIMENTAL SECTION
■
General Experimental. All reagents were purchased from
commercial sources and used without further purification. Reaction
progress was monitored by TLC using Aldrich silica gel 60 F254 (0.25
1
mm) with detection by UV. H and 13C NMR spectra were recorded
⎛
n0 ⎝
⎞
[J]
Kj
[I][J]
1
nij
1
[I]
Ki
on a Varian Unity Inova 500 MHz spectrometer. IR spectra were
recorded in KBr on a Perkin-Elmer 1310 spectrophotometer. Melting
points were determined on a Stuart Scientific SMP 11 melting point
apparatus. For HPLC/APCI-MS analysis, a Varian tandem mass
spectrometer (Palo Alto, CA, U.S.) 1200 L triple quadrupole mass
spectrometer was used with an APCI source. Varian MS workstation
version 6.7 software was used for data acquisition and processing.
APCI was operated in both positive and negative ion modes. The
capillary potential was 75 V, the APCI torch 450 °C, and the shield
was 600 V. Nitrogen at 48 mTorr was set at 400 °C. Full scan spectra
were obtained in the range 100−1000 amu, with scan time 0.75 amu,
⎜
⎜
⎟
⎟
=
1 +
+
+
gKiKj ⎠
where nij is the enzyme velocity in the presence of both compounds at
concentrations [I] and [J] and g is the interaction term that defines the
degree to which binding of one compound perturbs binding of the
other.
Differential Scanning Fluorimetry (ThermoFluor). Thermal
stability assays were performed according to Nettleship et al.28 To a
LightCycler 480 96-well plate (Roche) was added 1 μL of 500 μM
inhibitor in DMSO, followed by 49 μL of 300 nM HIV-1 RT in
H
dx.doi.org/10.1021/jm400405z | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
reaction buffer containing 20 mM HEPES, pH 7.5, 10 mM MgCl2, 100
mM NaCl, and a 1:1000 dilution of Sypro Orange dye (Invitrogen).
The mixture was heated from 30 to 90 °C in increments of 0.2 °C.
Fluorescence intensity was measured using excitation and emission
wavelengths of 483 and 568 nm, respectively. Changes in protein
thermal stability (ΔTm) upon inhibitor binding were analyzed by using
LightCycler 480 software. All assays were performed in triplicate.
HIV-1 IN Assay. The evaluate 3′-end processing activity assay of
HIV-1 IN, a duplex DNA substrate was prepared by annealing 20-nt
32P-labeled DNA (5′-32P-TGTGGAAAATCTCTAGCAGT-3′) with
its complementary strand (5′-ACTGCTAGAGATTTTCCACA-3′).
Recombinant HIV-1 IN (750 nM) was preincubated with inhibitor (in
DMSO) for 10 min at room temperature. 3′-Processing was initiated
by adding DNA substrate, and then the sample was incubated for an
hour at 37 °C. Final assay conditions were 50 mM MOPS, pH 7.2, 10
mM MgCl2, 50 mM NaCl, 2 mM 2-mercaptoethanol, 10% DMSO, 50
nM substrate, and 50 μM inhibitor. The reaction was stopped by
adding loading buffer containing 89 mM Tris-borate, pH 8.3, 6 M
urea, 25 mM EDTA, 0.025% bromophenol blue, and 0.025% xylene
cyanol and analyzed on a 15% denaturing polyacrylamide gel. The
fraction of substrate converted to the specific 3′-processed product was
determined by PhosphorImager analysis (Typhoon Trio+, GE
Healthcare).
DNA-Dependent DNA Polymerase Assay. DNA-dependent
DNA synthesis was measured on a fluorescently labeled duplex DNA
generated by annealing a 42-nucleoptide (nt) template (5′-TAC ATA
CCC ATA CAT AAA TCC TAA CCT TGA AGA ACT CGT CAC-
3′) to the 5′ Cy5-labeled primer 5′-ATG TAT GGG TAT GTA TTT
AGG-3′. Polymerization was initiated by adding 1 μL of 2 mM
deoxynucleoside triphosphates (dNTPs) to 9 μL of a mixture
containing 15 ng of RT, 200 nM substrate, 10 mM Tris-HCl (pH
7.8), 80 mM KCl, 10 mM MgCl2, 10% DMSO, and 50 μM inhibitor at
37 °C and was terminated after 15 min by adding an equal volume of 8
M urea. Reaction products were analyzed by 15% denaturing
polyacrylamide gel electrophoresis and fluorescent imaging (Typhoon
Trio+, GE Healthcare).
ABBREVIATIONS USED
■
DNTP, 5,6-dimethyl-2-(4-nitrophenyl)thieno[2,3-d]pyrimidin-
4(3H)-one; RNase H, ribonuclease H; NNRTI, nonnucleoside
reverse transcriptase inhibitor; RT, reverse transcriptase
REFERENCES
■
(1) Luzuriaga, K.; Bryson, Y.; Krogstad, P.; Robinson, J.;
Stechenberg, B.; Lamson, M.; Cort, S.; Sullivan, J. L. Combination
treatment with zidovudine, didanosine, and nevirapine in infants with
human immunodeficiency virus type 1 infection. N. Engl. J. Med. 1997,
336, 1343−1349.
(2) Das, K.; Lewi, P. J.; Hughes, S. H.; Arnold, E. Crystallography
and the design of anti-AIDS drugs: conformational flexibility and
positional adaptability are important in the design of non-nucleoside
HIV-1 reverse transcriptase inhibitors. Prog. Biophys. Mol. Biol. 2005,
88, 209−231.
(3) Abbondanzieri, E. A.; Bokinsky, G.; Rausch, J. W.; Zhang, J. X.;
Le Grice, S. F.; Zhuang, X. Dynamic binding orientations direct
activity of HIV reverse transcriptase. Nature 2008, 453, 184−189.
(4) Spence, R. A.; Kati, W. M.; Anderson, K. S.; Johnson, K. A.
Mechanism of inhibition of HIV-1 reverse transcriptase by nonnucleo-
side inhibitors. Science 1995, 267, 988−993.
(5) Kessl, J. J.; Jena, N.; Koh, Y.; Taskent-Sezgin, H.; Slaughter, A.;
Feng, L.; de Silva, S.; Wu, L.; Le Grice, S. F.; Engelman, A.; Fuchs, J.
R.; Kvaratskhelia, M. A multimode, cooperative mechanism of action
of allosteric HIV-1 integrase inhibitors. J. Biol. Chem. 2012, 287,
16801−16811.
(6) Perryman, A. L.; Zhang, Q.; Soutter, H. H.; Rosenfeld, R.;
McRee, D. E.; Olson, A. J.; Elder, J. E.; Stout, C. D. Fragment-based
screen against HIV protease. Chem. Biol. Drug Des. 2010, 75, 257−268.
(7) Schatz, O.; Cromme, F.; Naas, T.; Lindemann, D.; Gruninger
Leitch, F.; Mous, J.; Le Grice, S. F. J. Inactivation of the RNase H
Domain of HIV 1 Reverse Transcriptase Blocks Viral Infectivity; Portfolio
Publishing Company: Houston, TX, 1990; pp 293−303.
(8) Tisdale, M.; Schulze, T.; Larder, B. A.; Moelling, K. Mutations
within the RNase H domain of human immunodeficiency virus type 1
reverse transcriptase abolish virus infectivity. J. Gen. Virol. 1991, 72
(Part 1), 59−66.
(9) Beilhartz, G. L.; Wendeler, M.; Baichoo, N.; Rausch, J.; Le Grice,
S.; Gotte, M. HIV-1 reverse transcriptase can simultaneously engage its
DNA/RNA substrate at both DNA polymerase and RNase H active
sites: implications for RNase H inhibition. J. Mol. Biol. 2009, 388,
462−474.
(10) Wendeler, M.; Lee, H. F.; Bermingham, A.; Miller, J. T.;
Chertov, O.; Bona, M. K.; Baichoo, N. S.; Ehteshami, M.; Beutler, J.;
O’Keefe, B. R.; Gotte, M.; Kvaratskhelia, M.; Le Grice, S. Vinylogous
ureas as a novel class of inhibitors of reverse transcriptase-associated
ribonuclease H activity. ACS Chem. Biol. 2008, 3, 635−644.
(11) Srivastava, S.; Sluis-Cremer, N.; Tachedjian, G. Dimerization of
human immunodeficiency virus type 1 reverse transcriptase as an
antiviral target. Curr. Pharm. Des. 2006, 12, 1879−1894.
(12) Tachedjian, G.; Aronson, H. E.; de los Santos, M.; Seehra, J.;
McCoy, J. M.; Goff, S. P. Role of residues in the tryptophan repeat
motif for HIV-1 reverse transcriptase dimerization. J. Mol. Biol. 2003,
326, 381−396.
Antiviral Activity. The antiviral activity of selected thienopyr-
imidinones was determined via the 2,3-bis[2-methoxy-4-nitro-5-
sulfophenyl]-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide
(XTT) based cell viability assay of Weislow et al.,29 using the HIV-
1RF isolate and human T-cell line CEM-SS.
ASSOCIATED CONTENT
■
S
* Supporting Information
Spectroscopic characterization of the compounds in this paper
and details related to in vitro studies. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 1-301-846-5256. Fax: 1-301-846-6013. E-mail:
legrices@mail.nih.gov.
Notes
(13) Chung, S.; Wendeler, M.; Rausch, J. W.; Beilhartz, G.; Gotte,
M.; O’Keefe, B. R.; Bermingham, A.; Beutler, J. A.; Liu, S.; Zhuang, X.;
Le Grice, S. F. Structure−activity analysis of vinylogous urea inhibitors
of human immunodeficiency virus-encoded ribonuclease H. Anti-
microb. Agents Chemother. 2010, 54, 3913−3921.
(14) Chung, S.; Miller, J. T.; Johnson, B. C.; Hughes, S. H.; Le Grice,
S. F. Mutagenesis of human immunodeficiency virus reverse
transcriptase p51 subunit defines residues contributing to vinylogous
urea inhibition of ribonuclease H activity. J. Biol. Chem. 2011, 287,
4066−4075.
(15) Budihas, S. R.; Gorshkova, I.; Gaidamakov, S.; Wamiru, A.;
Bona, M. K.; Parniak, M. A.; Crouch, R. J.; McMahon, J. B.; Beutler, J.
A.; Le Grice, S. F. Selective inhibition of HIV-1 reverse transcriptase-
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Wei Yang, NIH, for communication of
crystallographic data on HIV-1 RT containing compound 9
prior to publication. T.M., S.C., J.A.W., H.T.-S., J.W.R., J.A.B.
and S.F.J.L.G. were supported by the Intramural Research
Program of the National Cancer Institute, National Institutes of
Health. G.T. was supported by Ministero dell’Universita,
dell’Istruzione e della Ricerca MIUR- PRIN 2008 and
Fondazione Banco di Sardegna.
̀
I
dx.doi.org/10.1021/jm400405z | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
associated ribonuclease H activity by hydroxylated tropolones. Nucleic
Acids Res. 2005, 33, 1249−1256.
(16) Bakavoli, M.; Bagherzadeh, G.; Vaseghifar, M.; Shiri, A.; Pordeli,
P. Iodine catalysed synthesis and antibacterial evaluation of thieno[2,3-
d]pyrimidine derivatives. J. Chem. Res., Synop. 2009, 653−655.
(17) Jamieson, C.; Basten, S.; Campbell, R. A.; Cumming, I. A.;
Gillen, K. J.; Gillespie, J.; Kazemier, B.; Kiczun, M.; Lamont, Y.; Lyons,
A. J.; Maclean, J. K.; Moir, E. M.; Morrow, J. A.; Papakosta, M.;
Rankovic, Z.; Smith, L. A novel series of positive modulators of the
AMPA receptor: discovery and structure based hit-to-lead studies.
Bioorg. Med. Chem. Lett. 2010, 20, 5753−5756.
(18) Pantoliano, M. W.; Petrella, E. C.; Kwasnoski, J. D.; Lobanov, V.
S.; Myslik, J.; Graf, E.; Carver, T.; Asel, E.; Springer, B. A.; Lane, P.;
Salemme, F. R. High-density miniaturized thermal shift assays as a
general strategy for drug discovery. J. Biomol. Screening 2001, 6, 429−
440.
(19) Niesen, F. H.; Berglund, H.; Vedadi, M. The use of differential
scanning fluorimetry to detect ligand interactions that promote protein
stability. Nat. Protoc. 2007, 2, 2212−2221.
(20) Vedadi, M.; Niesen, F. H.; Allali-Hassani, A.; Fedorov, O. Y.;
Finerty, P. J., Jr.; Wasney, G. A.; Yeung, R.; Arrowsmith, C.; Ball, L. J.;
Berglund, H.; Hui, R.; Marsden, B. D.; Nordlund, P.; Sundstrom, M.;
Weigelt, J.; Edwards, A. M. Chemical screening methods to identify
ligands that promote protein stability, protein crystallization, and
structure determination. Proc. Natl. Acad. Sci. U.S.A. 2006, 103,
15835−15840.
(21) Su, H. P.; Yan, Y.; Prasad, G. S.; Smith, R. F.; Daniels, C. L.;
Abeywickrema, P. D.; Reid, J. C.; Loughran, H. M.; Kornienko, M.;
Sharma, S.; Grobler, J. A.; Xu, B.; Sardana, V.; Allison, T. J.; Williams,
P. D.; Darke, P. L.; Hazuda, D. J.; Munshi, S. Structural basis for the
inhibition of RNase H activity of HIV-1 reverse transcriptase by RNase
H active site-directed inhibitors. J. Virol. 2010, 84, 7625−7633.
(22) Dubey, S.; Satyanarayana, Y. D.; Lavania, H. Development of
integrase inhibitors for treatment of AIDS: an overview. Eur. J. Med.
Chem. 2007, 42, 1159−1168.
(23) Liu, S.; Abbondanzieri, E. A.; Rausch, J. W.; Le Grice, S. F.;
Zhuang, X. Slide into action: dynamic shuttling of HIV reverse
transcriptase on nucleic acid substrates. Science 2008, 322, 1092−1097.
(24) Wang, J.; Smerdon, S. J.; Jager, J.; Kohlstaedt, L. A.; Rice, P. A.;
Friedman, J. M.; Steitz, T. A. Structural basis of asymmetry in the
human immunodeficiency virus type 1 reverse transcriptase hetero-
dimer. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 7242−7246.
(25) Tchertanov, L.; Mouscadet, J. F. Target recognition by catechols
and beta-ketoenols: potential contribution of hydrogen bonding and
Mn/Mg chelation to HIV-1 integrase inhibition. J. Med. Chem. 2007,
50, 1133−1145.
(26) Yonetani, T. The Yonetani−Theorell graphical method for
examining overlapping subsites of enzyme active centers. Methods
Enzymol. 1982, 87, 500−509.
(27) Lapkouski, M.; Tian, L.; Miller, J. T.; Le Grice, S. F.; Yang, W.
Complexes of HIV-1 RT, NNRTI and RNA/DNA hybrid reveal a
structure compatible with RNA degradation. Nat. Struct. Mol. Biol.
2013, 20, 230−236.
(28) Nettleship, J. E.; Brown, J.; Groves, M. R.; Geerlof, A. Methods
for protein characterization by mass spectrometry, thermal shift
(ThermoFluor) assay, and multiangle or static light scattering. Methods
Mol. Biol. 2008, 426, 299−318.
(29) Weislow, O. S.; Kiser, R.; Fine, D. L.; Bader, J.; Shoemaker, R.
H.; Boyd, M. R. New soluble-formazan assay for HIV-1 cytopathic
effects: application to high-flux screening of synthetic and natural
products for AIDS-antiviral activity. J. Natl. Cancer Inst. 1989, 81,
577−586.
J
dx.doi.org/10.1021/jm400405z | J. Med. Chem. XXXX, XXX, XXX−XXX