High potency of indolyl aryl sulfone nonnucleoside inhibitors towards drug-resistant human immunodeficiency virus type 1 reverse transcriptase mutants is due to selective targeting of different mechanistic forms of the enzyme

Article abstract of DOI:10.1128/AAC.49.11.4546-4554.2005

Indolyl aryl sulfone (IAS) nonnucleoside inhibitors have been shown to potently inhibit the growth of wild-type and drug-resistant human immunodeficiency virus type 1 (HIV-1), but their exact mechanism of action has not been elucidated yet. Here, we descr

Full text of DOI:10.1128/AAC.49.11.4546-4554.2005

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 2005, p. 4546–4554
0066-4804/05/$08.00ϩ0 doi:10.1128/AAC.49.11.4546–4554.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 49, No. 11
High Potency of Indolyl Aryl Sulfone Nonnucleoside Inhibitors towards
Drug-Resistant Human Immunodeficiency Virus Type 1 Reverse
Transcriptase Mutants Is Due to Selective Targeting of
Different Mechanistic Forms of the Enzyme
Reynel Cancio,1 Romano Silvestri,2 Rino Ragno,2 Marino Artico,2 Gabriella De Martino,2
Giuseppe La Regina,2 Emmanuele Crespan,1 Samantha Zanoli,1 Ulrich Hu¨bscher,3
Silvio Spadari,1 and Giovanni Maga1*
Istituto di Genetica Molecolare IGM-CNR, via Abbiategrasso 207, I-27100 Pavia, Italy1;
“Istituto Pasteur-Fondazione Cenci Bolognetti”-Dipartimento di Studi Farmaceutici,
Universita` di Roma “La Sapienza,” P. le Aldo Moro 5, I-00185 Rome, Italy2; and
Institute of Veterinary Biochemistry and Molecular Biology,
University of Zu¨rich-Irchel, Winterthurerstrasse 190,
CH-8057 Zu¨rich, Switzerland3
Received 28 May 2005/Returned for modification 28 July 2005/Accepted 29 August 2005
Indolyl aryl sulfone (IAS) nonnucleoside inhibitors have been shown to potently inhibit the growth of
wild-type and drug-resistant human immunodeficiency virus type 1 (HIV-1), but their exact mechanism of
action has not been elucidated yet. Here, we describe the mechanism of inhibition of HIV-1 reverse transcrip-
tase (RT) by selected IAS derivatives. Our results showed that, depending on the substitutions introduced in
the IAS common pharmacophore, these compounds can be made selective for different enzyme-substrate
complexes. Moreover, we showed that the molecular basis for this selectivity was a different association rate of
the drug to a particular enzymatic form along the reaction pathway. By comparing the activities of the different
compounds against wild-type RT and the nonnucleoside reverse transcriptase inhibitor-resistant mutant
Lys103Asn, it was possible to hypothesize, on the basis of their mechanism of action, a rationale for the design
of drugs which could overcome the steric barrier imposed by the Lys103Asn mutation.
Anti-AIDS therapy is actually based on three classes of
anti-human immunodeficiency virus (HIV) drugs, the nucleo-
side reverse transcriptase inhibitors (NRTIs), the nonnucleo-
side reverse transcriptase inhibitors (NNRTIs), and the pro-
tease inhibitors. More recently, enfuvirtide, a 36-amino-acid
residue peptide acting as a viral entry inhibitor, has been li-
censed for the treatment of HIV infection (7, 8). NRTIs,
NNRTIs, and protease inhibitors are mixed in highly active
antiretroviral therapy, which dramatically slows down viral rep-
lication, but they are unable to eradicate the viral infection
(29). Moreover, the rapid development of drug resistance and
toxicity problems make urgent the discovery of novel anti-HIV
agents effective against resistant mutants and without unpleas-
ant side effects (14).
NNRTI interaction with HIV-1 reverse transcriptase (RT) is
a highly dynamic process (6). Crystal structures of RT-NNRTI
complexes (19) showed that the drugs interacted with a hydro-
phobic pocket (nonnucleoside binding site [NNBS]) on the
enzyme in a “butterfly-like” mode. One of the “wings” of this
butterfly is made of a ␲-electron-rich moiety (phenyl or allyl
substituents) that interacts through ␲-␲ interactions with a
hydrophobic pocket formed mainly by the side chains of aro-
matic amino acids (Tyr181, Tyr188, Phe227, Trp229, and
Tyr318). On the other hand, the other wing is normally repre-
sented by a heteroaromatic ring bearing at one side a func-
tional group capable of donating and/or accepting hydrogen
bonds with the main chain of Lys101 and Lys103. Finally, on
the butterfly body, a hydrophobic portion fills a small pocket
formed mainly by the side chains of Lys103, Val106, and
Val179. Upon complexation, the NNBS hydrophobic pocket
changes its own conformation, leading to the inactivation of
the enzyme itself. Because of the different chemical and struc-
tural features of the inhibitors and the side chain flexibility, the
bound NNBS adopts different conformations (28). Moreover,
mutations of some amino acids cause variation of the NNBS
properties, thus decreasing affinities of most of the inhibitors
(12, 24, 25). In particular, the NNRTI resistance mutations
Tyr188Leu and Tyr181Ile/Cys reduce ␲-␲ interactions, the
Gly190Ala mutation leads to a smaller active site space be-
cause of a steric conflict between the methyl side chain and the
inhibitor, and the formation of an additional hydrogen bond
when amino acid 103 is mutated from Lys to Asn reduces
inhibitor entrance into the NNBS.
However, HIV-1 RT itself also undergoes a conformational
reorganization upon interaction with its substrates template-
primer (TP) and deoxynucleoside triphosphate (dNTP), so
that three structurally distinct mechanistic forms can be rec-
ognized in the reaction pathway catalyzed by HIV-1 RT (1,
11): the free enzyme, the binary complex of RT with the
template-primer (RT/TP), and the catalytically competent ter-
nary complex of RT with both nucleic acid and dNTP (RT/TP/
dNTP). This means that, in principle, the NNBS might not be
* Corresponding author. Mailing address: Istituto di Genetica Mole-
colare IGM-CNR, via Abbiategrasso 207, I-27100 Pavia, Italy. Phone:
(39)0382546354. Fax: (39)0382422286. E-mail: maga@igm.cnr.it.
4546

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Expression and purification of recombinant HIV-1 RT forms. Recombinant
heterodimeric wild-type RT and the Lys103Asn and Tyr181Ile variants were
expressed and purified to Ͼ95% purity (as judged by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis) as described previously (12).
identical in these three mechanistic forms. Several kinetic stud-
ies have shown that this is indeed the case, so that some
NNRTIs selectively target one or a few of the different enzy-
matic forms along the reaction pathway (5, 13, 15). This ob-
servation likely reflects the different spatial rearrangements
not only of the NNBS itself but also of the adjacent nucleotide
binding site (3, 20, 26, 27). Indeed, it has been shown that a
“communication” exists between the NNBS and the nucleotide
binding site, so that some NRTI resistance mutations can in-
fluence NNRTI binding and vice versa (2, 4, 20). Thus, under-
standing the molecular determinants governing the selective
interaction of a drug with the three different NNBS structures
present along the RT reaction pathway will be important for
the design of novel, highly selective, and potent NNRTIs.
During extensive structure-activity relationship studies on
diarylsulfones, we identified pyrryl sulfones and the novel in-
dolyl aryl sulfones (IASs) as highly potent NNRTIs (18, 22,
23). In particular, indole derivatives bearing 2-methylphenyl-
sulfonyl or 3-methylphenylsulfonyl moieties were found to in-
hibit HIV-1 at nanomolar concentrations. Furthermore, the
introduction of a 3,5-dimethylphenylsulfonyl moiety led to a
compound displaying high activity and selectivity not only
against the wild-type strain but also against the Tyr181Cys and
Lys103Asn-Tyr181Cys viral variants and the efavirenz-resistant
mutant Lys103Arg-Val179Asp-Pro225His.
HIV-1 RT RNA-dependent DNA polymerase activity assay. RNA-dependent
DNA polymerase activity was assayed as follows. A final volume of 25 ␮l con-
tained buffer A (50 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, 0.2 mg/ml bovine
serum albumin, 4% glycerol), 10 mM MgCl2, 0.5 ␮g of poly(rA)/oligo(dT)10:1
(0.3 ␮M 3Ј-OH ends), 10 ␮M [3H]dTTP (1 Ci/mmol), and 5 to 10 nM RT.
Reactions were incubated for 10 min at 37°C. Aliquots (20 ␮l) were then spotted
on glass fiber filters (GF/C filters), which were immediately immersed in 5%
ice-cold trichloroacetic acid. Filters were washed twice in 5% ice-cold trichloro-
acetic acid and once in ethanol for 5 min and dried, and acid-precipitable
radioactivity was quantitated by scintillation counting.
Inhibition assays. Reactions for inhibition assays were performed under the
conditions described for the HIV-1 RT RNA-dependent DNA polymerase ac-
tivity assay. Incorporation of radioactive dTTP into poly(rA)/oligo(dT) at differ-
ent concentrations of DNA or dNTP was monitored in the presence of increasing
amounts of inhibitor as indicated in the figure legends.
Kinetics of inhibitor binding. Kinetics of inhibitor binding experiments were
as described previously (12). Briefly, HIV-1 RT (20 to 40 nM) was incubated for
2 min at 37°C in a final volume of 4 ␮l in the presence of buffer A, with 10 mM
MgCl2 alone or with 100 nM 3Ј-OH ends (for the formation of the RT/TP
complex), or in the same mixture complemented with 10 ␮M unlabeled dTTP
(for the formation of the RT/TP/dNTP complex). The inhibitor to be tested was
then added to a final volume of 5 ␮l at a concentration at which [EI]/[E0] ϭ
[1 Ϫ 1/(1 ϩ [I]/Ki)] Ͼ 0.9. Then, 145 ␮l of a mix containing buffer A, 10 mM
MgCl2, and 10 ␮M [3H]dTTP (5 Ci/mmol) was added at different time points.
After an additional 10 min of incubation at 37°C, 50-␮l aliquots were spotted on
GF/C filters, and acid-precipitable radioactivity was measured as described for
the HIV-1 RT RNA-dependent DNA polymerase activity assay. The quantity
vt/v0, representing the normalized difference between the amount of dTTP in-
corporated at the zero time point and at different time points, was then plotted
against time. The kapp values were determined by fitting the experimental data to
the single-exponent equation vt/v0 ϭ eϪkappt, where t is time. The kon and koff
values were calculated according to the relationships (27) kapp ϭ kon(Ki ϩ [I])
In light of their extremely potent activities, especially to-
wards NNRTI-resistant mutants, we sought to investigate in
detail the mechanism of action of some selected IAS deriva-
tives. In this work, we show that IASs do not display a unique
mode of action but rather that they can selectively bind differ-
ent mechanistic forms of the enzyme, depending on the sub-
stituents introduced on the drug pharmacophore. These results
suggest that IASs are very flexible molecules, interacting dy-
namically with the viral RT.
and Kd ϭ koff/kon
.
Kinetic model. The mechanism of action of IASs was found to be either fully
noncompetitive or partially mixed. A schematic drawing of the different equilib-
ria is depicted in Fig. 1. According to the ordered mechanism of the polymer-
ization reaction, whereby template-primer (TP) binds first followed by the ad-
dition of dNTP, HIV-1 RT can be present in three different catalytic forms as
reported in Fig. 1A: as a free enzyme, in a binary complex with the TP, and in a
ternary complex with TP and dNTP. The resulting rate equation for such a
system is very complex and impractical to use. For these reasons, the general
steady-state kinetic analysis was simplified by varying one of the substrates
(either TP or dNTP) while the other was kept constant. When the TP substrate
was held constant at saturating concentration and the inhibition at various
concentrations of dNTPs was analyzed, at the steady state all of the input RT was
in the form of the RT/TP binary complex and only two forms of the enzyme (the
binary complex and the ternary complex with dNTP) could react with the inhib-
itor, as shown in the left part of Fig. 1B. Similarly, when the dNTP concentration
was kept constant at saturating levels and the inhibition at various TP concen-
trations was analyzed, RT was present either as a free enzyme or in the ternary
complex with TP and dNTP, as shown in the right part of Fig. 1B.
MATERIALS AND METHODS
Chemicals. [3H]dTTP (40 Ci/mmol) was from Amersham, and unlabeled
dNTPs were from Boehringer. Whatman was the supplier of the GF/C filters. All
other reagents were of analytical grade and purchased from Merck or Fluka.
Chemistry. 5-Chloro-3-(phenyl)sulfonyl-1H-indole-2-carboxamide (RS1202)
(23, 30), 5-chloro-3-[(3,5-dimethylphenyl)sulfonyl]-1H-indole-2-carboxamide
(RS1588)(21),N-{3-[(3,5-dimethylphenyl)sulfonyl]-5-chloro-1H-indole-2-car-
bonyl}-D,L-alanylamide (RS1980) (21), and 4,5-difluoro-3-[(3,5-dimethylphenyl)
sulfonyl]-1H-indole-2-carboxamide (RS1866) (R.S., unpublished data) were ob-
tained by heating the corresponding esters with concentrated ammonium hy-
droxide in a sealed tube. The starting esters needed for the preparation of
RS1202, RS1588, and RS1866 were obtained by oxidation of the corresponding
3-arylthio-1H-indole-2-carboxylates using 3-chloroperoxybenzoic acid (MCPBA).
The ethyl ester of N-{3-[(3,5-dimethylphenyl)sulfonyl]-5-chloro-1H-indole-2-car-
bonyl}-D,L-alanine for the synthesis of RS1980 was obtained by lithium hydroxide
of ethyl 5-chloro-3-[(3,5-dimethylphenyl)sulfonyl]-1H-indole-2-carboxylate and
subsequent condensation of D,L-alanine ethyl ester in the presence of O-benzo-
triazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate and tri-
ethylamine. The required 3-arylthio-1H-indole-2-carboxylates were prepared by
reaction of proper arylthiodisulfides with 1H-indole-2-carboxylic acids in the
presence of sodium hydride according to the Atkinson method and subsequent
esterification of the 3-arylthio-1H-indole-2-carboxylic acids with (trimethylsilyl)
diazomethane. Alternatively, these esters were obtained by reaction of methyl or
ethyl 1H-indole-2-carboxylates with N-(arylthio)succinimides in the presence of
boron trifluoride diethyl etherate.
The steady-state rate equation used for the partially mixed inhibition was the
one describing a reaction involving only two mechanistic forms of the enzyme
(according to the simplified pathways in Fig. 1B) (9)
1 ϩ kЈcat · I
kcat · E0 ·
ͫ ͬ
kcat · KЈi
v ϭ
(1)
Ks ͑1 ϩ I · Ki͒
1 ϩ
·
ͫ
ͬ
S
͑1 ϩ I · KЈi͒
where kcat is the apparent reaction rate in the absence of the inhibitor, kЈcat is the
reaction rate at infinite inhibitor concentration, E0 is the total enzyme concen-
tration, I is the inhibitor concentration, S is the substrate concentration, and Ki
and KЈi are the equilibrium dissociation constants for the different enzyme-
substrate complexes. According to Fig. 1B, when TP was held constant and only
the dTTP concentration was varied, Ki ϭ Kbini (for the binding of the inhibitor
to the binary complex RT/TP), whereas KЈi ϭ Kteri (for the binding of the
inhibitor to the ternary RT/TP/dNTP complex). Conversely, when dTTP was
Nucleic acid substrates. The homopolymer poly(rA) (Pharmacia) was mixed
at weight ratios in nucleotides of 10:1 to the oligomer oligo(dT)12–18 (Pharmacia)
in 20 mM Tris-HCl (pH 8.0) containing 20 mM KCl and 1 mM EDTA, heated
at 65°C for 5 min, and then slowly cooled at room temperature.

4548
CANCIO ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 1. Schematic diagram of the HIV-1 reverse transcriptase RNA-dependent DNA polymerization reaction pathway. (A) Full reaction
pathway. (B) The general steady-state kinetic analysis was simplified by varying one of the substrates (either TP or dNTP) while the other was kept
constant. When the TP substrate was held constant at saturating concentration and the inhibition at various concentrations of dNTPs was analyzed,
at the steady state all of the input RT was in the form of the RT/TP binary complex and only two forms of the enzyme (the binary complex and
the ternary complex with dNTP) could react with the inhibitor (left part of the panel). Similarly, when the dNTP concentration was kept constant
at saturating levels and the inhibition at various TP concentrations was analyzed, RT was present either as a free enzyme or in the ternary complex
with TP and dNTP (right part of the panel). For details, see Materials and Methods.
held constant, Ki referred to the binding of the inhibitor to the free enzyme,
whereas KЈi ϭ Kteri (for binding to the ternary RT/TP/dNTP complex).
The partially mixed mode of inhibition predicts that the enzyme-inhibitor
complex is still able to bind the substrate but with a lower affinity constant KЈs.
Conversely, the enzyme-substrate complex binds the inhibitor but with a lower
inhibition constant KЈi. Moreover, the enzyme-inhibitor-substrate complex can
still break down to give products but with a reduced catalytic rate kЈcat (9). Thus,
equation 1 was used to derive the apparent reaction rate, kcat(app), and affinity,
The KЈi and kЈcat values were derived by computer fitting of the variation of the
kcat(app) values as a function of the inhibitor concentrations, according to the
equation (9)
KЈi ϩ I
͑KЈi · kcat͒ ϩ ͑I · kЈcat
1/kcat(app) ϭ
(4)
͒
For the fully noncompetitive case, kЈcat ϭ 0, Ks ϭ KЈs, and Ki ϭ KЈi, and thus
equation 1 simplifies to
K
s(app), for the TP and dNTP substrates of the reaction at different inhibitor
concentrations. Then, Ks, Ki, KЈi, and KЈs were derived by computer fitting of the
variation of the Ks(app) values as a function of the inhibitor concentrations,
according to the equations (9)
kcat · E0
I
Ki
Ks
S
1 ϩ
ͩ ͪ
v ϭ
(5)
I ϩ Ki
K
s(app) ϭ
(2)
(3)
1 ϩ
Ki
I
KЈs
΂
Ks Ϫ
΃
Similarly, equation 4 simplifies to
and
I
1
1/kcat(app) ϭ
ϩ
(6)
Ki · kcat kcat
1 ϩ I
K
s(app) ϭ Ki ·
I
Initial velocities of the reaction were determined after 10 min of incubation at
37°C, which represents the midpoint of the linear range of the reaction, as
determined in separate experiments (data not shown). Each experiment was
1 ϩ
ͩ ͪ
KЈi
΄ ΅
Ks

VOL. 49, 2005
INDOLYL ARYL SULFONES TARGET DIFFERENT HIV-1 RT FORMS
4549
ence of increasing fixed amounts of inhibitors. From the vari-
ation of the apparent affinity of the enzyme for its substrates,
Ks(app), and the apparent reaction rate, kcat(app), as a function
of the inhibitor concentration, all of the kinetic parameters for
drug binding were determined, as explained in Materials and
Methods and in Fig. 1.
RS1866 is a fully noncompetitive inhibitor of HIV-1 RT. As
shown in Fig. 3A, the IAS derivative RS1866 showed a fully
noncompetitive mechanism of action, since it did not influence
the affinity of the enzyme for the substrates, affecting only the
rate of the reaction. Accordingly, the reciprocals of the kcat(app)
values followed linear relationships as a function of the inhib-
itor concentrations, as described by equation 6 in Materials
and Methods.
RS1588 is a partially noncompetitive inhibitor of HIV-1 RT
which shows preferential binding to the free enzyme. The same
kind of analysis was applied to the compound RS1588. As
shown in Fig. 3B, this inhibitor did not influence the affinity of
wild-type RT for the reaction substrates, affecting only the
reaction rate. However, the reciprocals of the kcat(app) values
did not follow a linear relationship with the inhibitor concen-
trations, as expected for a fully noncompetitive inhibitor, but
instead followed the nonlinear relationship described by equa-
tion 4 of Materials and Methods, diagnostic of a partially
noncompetitive type of inhibition (Fig. 3B). Thus, the reaction
rate extrapolated at infinite inhibitor concentration (kЈcat) was
found to be 2.5 to 3% of the uninhibited reaction, indicating a
maximum of 97% to 98% inhibition. Interestingly, from the
variation of the kcat(app) values measured in the presence of var-
ious concentrations of the poly(rA)/oligo(dT) substrate, an inhi-
bition constant (Ki) value of 0.4 nM was calculated (Fig. 3B,
circles), whereas the same analysis performed with the kcat(app)
values measured in the presence of various concentrations of
the dTTP substrate yielded a Ki of 2.7 nM (Fig. 3B, triangles).
According to the scheme outlined in Fig. 1B, these values
represent the apparent binding constants of the inhibitor to the
free enzyme and the ternary RT/TP/dNTP complex, respec-
tively. Thus, even though RS1588 binding did not affect the
affinity of RT for the substrates, the binding of the substrates
to the enzyme reduced the apparent Ki of the inhibitor.
RS1202 is a mixed inhibitor which binds preferentially to
the free enzyme and is partially competitive with the nucleic
acid substrate. When the IAS derivative RS1202 was studied,
a strong reduction of the apparent affinity for the nucleic acid
substrate but not for dTTP was noted. The increase in the
FIG. 2. Structures of the compounds used in this study.
done in triplicate, and mean values were used for the analysis. Curve fitting was
done with the computer program GraphPad Prism.
RESULTS
IAS derivatives show high potencies of inhibition of HIV-1
wild-type RT and NNRTI-resistant mutants. The IAS deriva-
tives RS1202, RS1588, RS1866, and RS1980 (Fig. 2) were
tested against HIV-1 wild-type RT and different NNRTI-resis-
tant RT mutants. As summarized in Table 1, all four deriva-
tives showed potent inhibition towards wild-type RT. In gen-
eral, the inhibition activities were higher towards the mutants
Leu100Ile, Val106Ala, and Val179Asp than towards the two
mutants Tyr181Ile and Tyr188Leu. Interestingly, the com-
pounds RS1202 and RS1588 showed little or no loss of potency
towards the Lys103Asn mutant, being 400- to 600-fold more
potent than nevirapine and efavirenz against this mutant.
Given their interesting activity profile, the mechanism of inhi-
bition of these IAS derivatives was studied in more detail.
Increasing concentrations of either the dTTP or the poly(rA)/
oligo(dT) substrates were titrated into the reaction in the pres-
Ks(app)3Ј-OH (apparent affinity for the nucleic acid substrate)
TABLE 1. Inhibition potencies of IAS derivatives against HIV-1 wild-type RT and NNRTI-resistant mutants
ID50 (nM) fora
Compound
wt
Leu100Ile
Lys103Asn
Val106Ala
Val179Asp
Tyr181Ile
Tyr188Leu
RS1202
1 (Ϯ0.1)
3 (Ϯ0.1)
2.2 (Ϯ0.1)
3 (Ϯ0.2)
400 (Ϯ50)
40 (Ϯ0.5)
100 (Ϯ20)
60 (Ϯ6)
n.d.
5 (Ϯ1)
3 (Ϯ0.5)
750 (Ϯ60)
500 (Ϯ50)
2,000 (Ϯ80)
1,000 (Ϯ40)
75 (Ϯ8)
90 (Ϯ8)
n.d.
35 (Ϯ3)
50 (Ϯ5)
n.d.
140 (Ϯ10)
140 (Ϯ10)
1,500 (Ϯ100)
130 (Ϯ10)
5,000 (Ϯ400)
400 (Ϯ50)
1,000 (Ϯ100)
200 (Ϯ20)
n.d.
RS1588
RS1866
RS1980
Nevirapine
Efavirenz
n.d.
n.d.
n.d.
n.d.
900 (Ϯ90)
n.d.
2,000 (Ϯ300)
n.d.
300 (Ϯ25)
n.d.
5,000 (Ϯ400)
n.d.
a ID50, concentration of the inhibitor that reduced the in vitro activity of HIV-1 RT by 50% under the conditions described in Materials and Methods. Numbers are
the mean values of three independent experiments (standard deviations are in brackets). wt, wild type; n.d., not determined.

4550
CANCIO ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 3. Mechanisms of inhibition of HIV-1 RT by the IAS derivatives. (A) Increasing concentrations of RS1866 were titrated in the presence
of 5 nM RT and either 2 ␮M, 4 ␮M, 10 ␮M, or 20 ␮M dTTP or 0.04 ␮M, 0.08 ␮M, 0.2 ␮M, and 0.4 ␮M (as 3Ј-OH ends) poly(rA)/oligo(dT) under
the conditions described in Materials and Methods. Initial velocities of the reaction were plotted as a function of the substrate concentration. The
variations of the kcat(app) values for the reaction derived from these experiments were plotted as a function of the RS1866 concentrations. Data
were fitted to equation 6 as described in Materials and Methods. (B) Increasing concentrations of RS1588 were titrated in the presence of 5 nM
RT and 2 ␮M, 4 ␮M, 10 ␮M, and 20 ␮M dTTP or 0.04 ␮M, 0.08 ␮M, 0.2 ␮M, and 0.4 ␮M (as 3Ј-OH ends) of poly(rA)/oligo(dT) under the
conditions described in Materials and Methods. Initial velocities of the reaction were plotted as a function of the substrate concentration. The
variation of the kcat(app) values for the reaction derived from these experiments was plotted as a function of the RS1588 concentrations. Data were
fitted to equation 4 as described in Materials and Methods. (C) Increasing concentrations of RS1202 were titrated in the presence of 5 nM RT
and 2 ␮M, 4 ␮M, 10 ␮M, and 20 ␮M dTTP or 0.04 ␮M, 0.08 ␮M, 0.2 ␮M, and 0.4 ␮M (as 3Ј-OH ends) poly(rA)/oligo(dT) under the conditions
described in Materials and Methods. Initial velocities of the reaction were plotted as a function of the substrate concentration. The values of the
apparent affinity for the nucleic acid substrate (Ks3Ј-OH) derived from these experiments were plotted as a function of the inhibitor concentration.
Data were fitted to both equation 2 and equation 3, as described in Materials and Methods. (D) The variation of the kcat(app) values for the reaction
derived as described for panel C was plotted as a function of the RS1202 concentrations. Data were fitted to equation 4 as described in Materials
and Methods. (E) Increasing concentrations of RS1980 were titrated in the presence of 5 nM RT and 2 ␮M, 4 ␮M, 10 ␮M, and 20 ␮M dTTP or
0.04 ␮M, 0.08 ␮M, 0.2 ␮M, and 0.4 ␮M (as 3Ј-OH ends) poly(rA)/oligo(dT) under the conditions described in Materials and Methods. Initial
velocities of the reaction were plotted as a function of the substrate concentration. The values of the apparent affinity for the nucleotide (KsTTP,
circles) derived from these experiments were plotted as a function of the inhibitor concentration. Data were fitted to both equation 2 and equation
3, as described in Materials and Methods. (F) The variation of the kcat(app) values for the reaction derived as described for panel E was plotted
as a function of the RS1980 concentrations. Data were fitted to equation 4 as described in Materials and Methods.

VOL. 49, 2005
INDOLYL ARYL SULFONES TARGET DIFFERENT HIV-1 RT FORMS
4551
values as a function of increasing inhibitor concentration (Fig.
3C) suggested a partially competitive inhibition. In addition,
the reciprocal of the variation of the kcat(app) values followed a
nonlinear relationship, as in the case of RS1588, indicating a
partially noncompetitive mechanism (Fig. 3D) with a maximal
reduction of the reaction rate of 95%. Thus, RS1202 behaved
as a mixed inhibitor, and according to the kinetic model out-
lined in Fig. 1B, all of the kinetic parameters were determined
as described in Materials and Methods. As a result, it was
found that RS1202 interacted preferentially with the free en-
zyme (similarly to RS1588), with a Ki of 0.3 nM. Binding of the
nucleic acid substrate to the enzyme reduced the affinity of the
inhibitor by fourfold, giving a Kibin/ter of 1.2 nM.
RS1980 is a mixed inhibitor which is partially competitive
with the nucleotide substrate. Similar experiments were
conducted with the compound RS1980 (Fig. 3E and F). This
inhibitor showed a partially competitive mechanism with
the nucleotide substrate, as shown by the increase in the
Ks(app)TTP (apparent affinity for the nucleotide substrate) val-
ues as a function of increasing inhibitor concentrations (Fig.
3E). Again, the reciprocal of the variation of the kcat(app) val-
ues followed a nonlinear relationship, as in the case of RS1588,
indicating a partially noncompetitive mechanism (Fig. 3F) with
a maximal inhibition of 92%. Determination of the kinetic
constant according to the reaction scheme outlined in Fig. 1
revealed that RS1980 binding to the ternary RT/TP/dNTP
complex was reduced 10-fold with respect to either the free
enzyme or the binary RT/TP complex (compare Ki and Kibin
with Kiter values).
Preferential binding of IAS derivatives to specific reaction
intermediates is mainly driven by differences in the associa-
tion rates. The differences in the equilibrium dissociation con-
stants Ki, Kibin, and Kiter of the inhibitors for the different
reaction intermediates could reflect either slower association
(kon) or faster dissociation (koff) rates or both. In order to more
quantitatively address which step of inhibitor binding was af-
fected by the nucleic acid or the nucleotide substrates, the
association and dissociation rates of the different IAS deriva-
tives were determined for the free enzyme and the binary
RT/TP and ternary RT/TP/dNTP complexes. Experiments
were performed as described in Materials and Methods, and
the calculated rate constants are summarized in Table 2. As
can be seen, the preference of RS1202 for the free enzyme, as
well as the improved binding of RS1980 to both the free en-
zyme and the binary complex, was due to a faster association
rate, whereas the dissociation step was not significantly af-
fected. Notably, RS1588 showed a threefold reduction of its
kon and a concomitant threefold increase in its koff values for
the binary complex with respect to the free enzyme. Taken
together, these results indicate that binding of either the nu-
cleic acid or the nucleotide substrate to HIV-1 RT imposed
some steric and/or thermodynamic barrier to subsequent in-
hibitor binding. As a comparison, the association and dissoci-
ation rates were also determined for the two clinically ap-
proved NNRTIs nevirapine and efavirenz. It can be seen that
all of the IAS derivatives were superior to both reference drugs
in terms of binding, showing much faster association rates,
whereas the dissociation rates were of the same order of those
of efavirenz but significantly better (i.e., slower) than those of
nevirapine.

4552
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ANTIMICROB. AGENTS CHEMOTHER.
A high potency of inhibition towards Lys103Asn mutant RT
correlates with preferential association of IASs with the unli-
ganded form of the enzyme. As reported in Table 1, both
RS1202 and RS1588 retained high activity against the
Lys103Asn mutant RT. Remarkably, RS1588 inhibited the mu-
tated enzyme with the same potency as the wild type. Thus, the
mechanism of inhibition by RS1202 and RS1588 towards the
Lys103Asn mutant was investigated. The calculated kinetic
parameters for the different enzymatic forms are listed in
Table 2. Both RS1202 and RS1588 preferentially bound to the
unliganded form of the Lys103Asn mutant, as in the case of
wild-type RT. However, their association rates (kon) were de-
creased by the Lys103Asn mutation, in accord with previous
structural and enzymatic studies which showed that this muta-
tion increased the thermodynamic barrier for drug binding.
Remarkably, RS1202 and RS1588 showed a slower dissocia-
tion rate (koff) from the mutant enzyme with respect to wild-
type RT, thus explaining their high inhibition potencies (Ki).
Compared with the NNRTI nevirapine, which does not dis-
criminate among the different enzymatic forms, or efavirenz,
which selectively targets the ternary RT/TP/dNTP complex, it
could be seen that both IASs showed more favorable interac-
tion parameters. These results suggested that drugs which se-
lectively target the unliganded form of the RT might be less
sensitive to the steric barrier imposed by the Lys103Asn mu-
tation.
FIG. 4. Structure-activity relationships for the IAS derivatives used
in this study. The different substituents on the common IAS scaffold
are highlighted with circles. Arrows indicate the structural relation-
ships among the different compounds. For each compound, the mech-
anism of action is indicated below its structure. For details, see the
Discussion.
DISCUSSION
The results of our investigation of the mechanism of action
of selected IAS derivatives showed that the nature of the sub-
stituents on the drug pharmacophore can influence the selec-
tive interaction of the drug with different mechanistic forms of
the viral RT. Similar properties have been already observed for
other NNRTIs, such as efavirenz, the pyrrolobenzoxazepinone
derivative PPO464, and the carboxanilide derivatives UC38
and UC84 (10, 13, 15), suggesting that a preferential comple-
mentarity exists between the structure of the inhibitor mole-
cule and the different spatial conformations of the NNBS.
The structure-function relationships of our IAS derivatives
are schematically drawn in Fig. 4. The compound RS1866
{4,5-difluoro-3-[(3,5-dimethylphenyl)sulfonyl]-1H-indole-2-car-
boxyamide} showed a fully noncompetitive mode of action,
being able to bind to all of the different reaction intermediates
(RT, RT/TP, and RT/TP/dNTP complexes) (Fig. 1A) with
equal affinities. Changing the nature of the alkyl group to give
the compound RS1588 {5-chloro-3-[(3,5-dimethylphenyl)sul-
fonyl]-1 H-indole-2-carboxyamide} also modified the mecha-
nism of inhibition, rendering the drug more selective for the
free enzyme with respect to the binary and ternary complexes.
Elimination of the 3,5-methyl groups gave the compound
RS1202 [5-chloro-3-(phenylsulfonyl)-1H-indole-2-carboxyamide],
which was very selective for the free enzyme, like RS1588, but also
showed a partially competitive mechanism of action towards
thenucleicacidsubstrate.Ontheotherhand,introducinganalanin-
amide moiety into compound RS1588 to give its deriva-
tive RS1980 (N-{3-[(3,5-dimethylphenyl)sulfonyl]-5-chloro-1H-
indole-2-carbonyl}alaninamide) made the drug capable of
discriminating against the RT/TP/dNTP ternary complex, with
a partially competitive mode of action towards the dNTP sub-
strate.
Drug binding studies showed that the main determinant for
drug selectivity was the inhibitor association rate, suggesting
that the structural reorganization of the NNBS upon formation
of the binary and ternary complexes of RT with its substrates
introduced some steric and/or thermodynamic barriers which
were differentially “sensed” by the substituents on the drug
molecule, depending on their spatial arrangement.
This inherent flexibility of the IAS derivatives, and their
consequent ability to selectively target specific enzymatic
forms, also had some consequences for their interaction with
NNRTI-resistant mutated RT enzymes, such as Lys103Asn
and Tyr181Ile. In fact, as can be seen from Table 1, the com-
pounds which selectively targeted the free enzyme, that is,
RS1588 and RS1202, were also the least affected by the
Lys103Asn mutation. On the other hand, those compounds
which did not discriminate between the free enzyme and the
binary RT/TP complex, that is, RS1866 and RS1980, showed a
loss of activity of about 75- to 150-fold. The RS1866 derivative
was the most affected by the Tyr181Ile mutation, but the other
compounds also showed significant losses of potency against
this mutant.
When the mechanism of inhibition of the Lys103Asn mutant
by RS1202 and RS1588 was investigated, both compounds
were shown to selectively target the unliganded form of the
mutant RT, as in the case of the wild-type enzyme, but the
association rates (kon) of both drugs were reduced. This is
consistent with the well-known effect of the Lys103Asn muta-
tion, which introduces a steric barrier to drug binding (12). On

VOL. 49, 2005
INDOLYL ARYL SULFONES TARGET DIFFERENT HIV-1 RT FORMS
4553
REFERENCES
the other hand, RS1202 and RS1588 showed slower dissocia-
tion rates (koff) from the Lys103Asn mutant than from wild-
type RT. By comparison, the NNRTIs efavirenz and nevira-
pine suffered from similar reductions in their kon values but no
compensatory decrease in their koff rates. Thus, our results
suggest that drugs selectively targeting the unliganded form of
the enzyme might efficiently overcome the steric barrier intro-
duced by the Lys103Asn mutation by decreasing their associ-
ation rates from the mutated enzyme, as we have shown in the
case of RS1202 and RS1588. This observation might help in
the design of more active compounds.
Several hypotheses have been made, based on kinetic, cross-
linking, and structural studies, to explain the mechanism of
inhibition of the chemical step by NNRTIs (11, 19, 26, 27).
Binding of an NNRTI leads to displacement of the ␤12-␤13
hairpin, which has direct interaction with the nucleic acid sub-
strate. Thus, it is possible that this alteration in the position of
the nucleic acid relative to the polymerase active site is respon-
sible for NNRTI inhibition. Another possible mechanism can
be hypothesized from the observation that the NNBS includes
residues (Tyr181 and Tyr188) in the ␤9-␤10 hairpin, which
contains two of the three active-site aspartic acids (Asp185 and
Asp186). There are also contacts between some NNRTIs and
the ␤6 strand that carries the third active-site aspartate
(Asp110). Again, even a moderate shift in the positions of the
active-site residues could interfere with the chemical step of
polymerization. Finally, it has been suggested that the fingers
subdomain of RT may not adopt a fully closed conformation if
both a dNTP and an NNRTI are bound to the enzyme. Since
proper closure of the fingers is important for the positioning of
the dNTP and the primer relative to the polymerase active site,
preventing the proper closure of the fingers subdomain would
interfere with the chemical step of DNA synthesis.
Different NNRTIs might act through one or more of these
mechanisms; however, all of the NNRTIs studied so far do not
interfere with the binding of either the dNTP or the nucleic
acid substrate. On the other hand, the IAS derivatives de-
scribed here showed different mechanisms of action, being
able to discriminate between different mechanistic forms of
the viral RT and being partially competitive towards either the
nucleic acid or the dNTP substrate. These observations further
reinforce the notion that the interaction of IASs with the
NNBS of HIV-1 RT is novel and different from the other
NNRTIs (18).
It thus appears that the IAS derivatives shown here are very
sensitive to the structural modifications occurring at the NNBS
upon complexation of the RT with its substrates and that their
sensitivity can be modulated through small modifications of the
drug molecule.
1. Bahar, I., B. Erman, R. L. Jernigan, A. R. Atilgan, and D. G. Covell. 1999.
Collective motions in HIV-1 reverse transcriptase: examination of flexibility
and enzyme function. J. Mol. Biol. 285:1023–1037.
2. Baldanti, F., S. Paolucci, G. Maga, N. Labo, U. Hubscher, A. Y. Skoblov, L.
Victorova, S. Spadari, L. Minoli, and G. Gerna. 2003. Nevirapine-selected
mutations Tyr181Ile/C of HIV-1 reverse transcriptase confer cross-resistance
to stavudine. AIDS 17:1568–1570.
3. Basavapathruni, A., C. M. Bailey, and K. S. Anderson. 2004. Defining a
molecular mechanism of synergy between nucleoside and nonnucleoside
AIDS drugs. J. Biol. Chem. 279:6221–6224. (First published 13 January
2004.)
4. Blanca, G., F. Baldanti, S. Paolucci, A. Y. Skoblov, L. Victorova, U. Hub-
scher, G. Gerna, S. Spadari, and G. Maga. 2003. Nevirapine resistance
mutation at codon 181 of the HIV-1 reverse transcriptase confers stavudine
resistance by increasing nucleotide substrate discrimination and phosphoro-
lytic activity. J. Biol. Chem. 278:15469–15472. (First published 24 February
2003.)
5. Buckheit, R. W., Jr., E. L. White, V. Fliakas-Boltz, J. Russell, T. L. Stup,
T. L. Kinjerski, M. C. Osterling, A. Weigand, and J. P. Bader. 1999. Unique
anti-human immunodeficiency virus activities of the nonnucleoside reverse
transcriptase inhibitors calanolide A, costatolide, and dihydrocostatolide.
Antimicrob. Agents Chemother. 43:1827–1834.
6. Campiani, G., A. Ramunno, G. Maga, V. Nacci, C. Fattorusso, B. Cata-
lanotti, E. Morelli, and E. Novellino. 2002. Non-nucleoside HIV-1 reverse
transcriptase (RT) inhibitors: past, present, and future perspectives. Curr.
Pharm. Des. 8:615–657.
7. De Clercq, E. 2002. Highlights in the development of new antiviral agents.
Mini Rev. Med. Chem. 2:163–175.
8. De Clercq, E. 2002. New anti-HIV agents and targets. Med. Res. Rev.
22:531–565.
9. Dixon, M., and E. C. Webb. 1979. Enzymes, 3rd ed. Longman, London,
United Kingdom.
10. Fletcher, R. S., K. Syed, S. Mithani, G. I. Dmitrienko, and M. A. Parniak.
1995. Carboxanilide derivative non-nucleoside inhibitors of HIV-1 reverse
transcriptase interact with different mechanistic forms of the enzyme. Bio-
chemistry 34:4346–4353.
11. Madrid, M., J. A. Lukin, J. D. Madura, J. Ding, and E. Arnold. 2001.
Molecular dynamics of HIV-1 reverse transcriptase indicates increased flex-
ibility upon DNA binding. Proteins 45:176–182.
12. Maga, G., M. Amacker, N. Ruel, U. Hubscher, and S. Spadari. 1997. Resis-
tance to nevirapine of HIV-1 reverse transcriptase mutants: loss of stabiliz-
ing interactions and thermodynamic or steric barriers are induced by differ-
ent single amino acid substitutions. J. Mol. Biol. 274:738–747.
13. Maga, G., A. Ramunno, V. Nacci, G. A. Locatelli, S. Spadari, I. Fiorini, F.
Baldanti, S. Paolucci, M. Zavattoni, A. Bergamini, B. Galletti, S. Muck, U.
Hubscher, G. Giorgi, G. Guiso, S. Caccia, and G. Campiani. 2001. The
stereoselective targeting of a specific enzyme-substrate complex is the mo-
lecular mechanism for the synergic inhibition of HIV-1 reverse transcriptase
by (R)-(-)-PPO464: a novel generation of nonnucleoside inhibitors. J. Biol.
Chem. 276:44653–44662. (First published 25 September 2001.)
14. Maga, G., and S. Spadari. 2002. Combinations against combinations: asso-
ciations of anti-HIV 1 reverse transcriptase drugs challenged by constella-
tions of drug resistance mutations. Curr. Drug Metab. 3:73–95.
15. Maga, G., D. Ubiali, R. Salvetti, M. Pregnolato, and S. Spadari. 2000.
Selective interaction of the human immunodeficiency virus type 1 reverse
transcriptase nonnucleoside inhibitor efavirenz and its thio-substituted ana-
log with different enzyme-substrate complexes. Antimicrob. Agents Che-
mother. 44:1186–1194.
16. Reference deleted.
17. Reference deleted.
18. Ragno, R., M. Artico, G. De Martino, G. La Regina, A. Coluccia, A. Di
Pasquali, and R. Silvestri. 2005. Docking and 3-D QSAR studies on
indolyl aryl sulfones. Binding mode exploration at the HIV-1 reverse
transcriptase non-nucleoside binding site and design of highly active
N-(2-hydroxyethyl)carboxamide and N-(2-hydroxyethyl)carbohydrazide
derivatives. J. Med. Chem. 48:213–223.
19. Ren, J., R. Esnouf, E. Garman, D. Somers, C. Ross, I. Kirby, J. Keeling, G.
Darby, Y. Jones, D. Stuart, and D. Stammers. 1995. High resolution struc-
tures of HIV-1 RT from four RT-inhibitor complexes. Nat. Struct. Biol.
2:293–302.
20. Selmi, B., J. Deval, K. Alvarez, J. Boretto, S. Sarfati, C. Guerreiro, and B.
Canard. 2003. The Y181C substitution in 3Ј-azido-3Ј-deoxythymidine-resis-
tant human immunodeficiency virus, type 1, reverse transcriptase suppresses
the ATP-mediated repair of the 3Ј-azido-3Ј-deoxythymidine 5Ј-monophos-
phate-terminated primer. J. Biol. Chem. 278:40464–40472.
21. Silvestri, R., M. Artico, G. De Martino, G. La Regina, R. Loddo, M. La Colla,
M. Mura, and P. La Colla. 2004. Simple, short peptide derivatives of a
sulfonylindolecarboxamide (L-737,126) active in vitro against HIV-1 wild
type and variants carrying non-nucleoside reverse transcriptase inhibitor
resistance mutations. J. Med. Chem. 47:3892–3896.
ACKNOWLEDGMENTS
We thank S. H. Hughes (NCI-Frederick Cancer Research and De-
velopment Center) for the coexpression vectors pUC12N/p66(His)/p51
with the wild-type or the mutant forms of HIV-1 RT.
This work was supported by the Italian Ministero della Salute,
Istituto Superiore di Sanit`a, Fourth National Research Program on
AIDS (R.S., M.A., and R.R.), Fifth National Research Program on
AIDS (grant 40F.78 to S.S.), Italian MIUR-Cofin 2002 and 2004 (R.S.
and M.A.), Istituto Pasteur-Fondazione Cenci Bolognetti (R.S.), and
the EU FP6 Research Project LSHB-CT-2003-503480-TRIoH (G.M.).
U.H. is supported by the University of Zu¨rich.

4554
CANCIO ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
22. Silvestri, R., M. Artico, G. La Regina, G. De Martino, M. La Colla, R. Loddo,
and P. La Colla. 2004. Anti-HIV-1 activity of pyrryl aryl sulfone (PAS)
derivatives: synthesis and SAR studies of novel esters and amides at the
position 2 of the pyrrole nucleus. Farmaco 59:201–210.
23. Silvestri, R., G. De Martino, G. La Regina, M. Artico, S. Massa, L. Vargiu,
M. Mura, A. G. Loi, T. Marceddu, and P. La Colla. 2003. Novel indolyl aryl
sulfones active against HIV-1 carrying NNRTI resistance mutations: synthe-
sis and SAR studies. J. Med. Chem. 46:2482–2493.
24. Smith, M. B., M. L. Lamb, J. Tirado-Rives, W. L. Jorgensen, C. J. Michejda,
S. K. Ruby, and R. H. Smith, Jr. 2000. Monte Carlo calculations on HIV-1
reverse transcriptase complexed with the non-nucleoside inhibitor 8-Cl
TIBO: contribution of the L100I and Y181C variants to protein stability and
biological activity. Protein Eng. 13:413–421.
25. Smith, M. B., S. Ruby, S. Horouzhenko, B. Buckingham, J. Richardson, I.
Puleri, E. Potts, W. L. Jorgensen, E. Arnold, W. Zhang, S. H. Hughes, C. J.
Michejda, and R. H. Smith, Jr. 2003. HIV-1 reverse transcriptase variants:
molecular modeling of Y181C, V106A, L100I, and Lys103Asn mutations
with nonnucleoside inhibitors using Monte Carlo simulations in combination
with a linear response method. Drug Des. Discov. 18:151–163.
26. Spence, R., K. S. Anderson, and K. A. Johnson. 1996. HIV-1 reverse tran-
scriptase resistance to nonnucleoside inhibitors. Biochemistry 35:1054–1063.
27. Spence, R. A., W. M. Kati, K. S. Anderson, and K. A. Johnson. 1995.
Mechanism of inhibition of HIV-1 reverse transcriptase by nonnucleoside
inhibitors. Science 267:988–993.
28. Temiz, N. A., and I. Bahar. 2002. Inhibitor binding alters the directions of
domain motions in HIV-1 reverse transcriptase. Proteins 49:61–70.
29. Vella, S., and L. Palmisano. 2000. Antiretroviral therapy: state of the
HAART. Antivir. Res. 45:1–7.
30. Williams, T. M., T. M. Ciccarone, S. C. MacTough, C. S. Rooney, S. K.
Balani, J. H. Condra, E. A. Emini, M. E. Goldman, W. J. Greenlee, L. R.
Kauffman, et al. 1993. 5-Chloro-3-(phenylsulfonyl)indole-2-carboxamide: a
novel, non-nucleoside inhibitor of HIV-1 reverse transcriptase. J. Med.
Chem. 36:1291–1294.