High Potency of Indolyl Aryl Sulfone Nonnucleoside Inhibitors towards Drug-Resistant Human Immunodeficiency Virus Type 1 Reverse Transcriptase ...
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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 2005, p. 4546–4554 Vol. 49, No. 11
0066-4804/05/$08.00⫹0 doi:10.1128/AAC.49.11.4546–4554.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
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 Hübscher,3
Silvio Spadari,1 and Giovanni Maga1*
Istituto di Genetica Molecolare IGM-CNR, via Abbiategrasso 207, I-27100 Pavia, Italy1;
Downloaded from http://aac.asm.org/ on January 23, 2021 by guest
“Istituto Pasteur-Fondazione Cenci Bolognetti”-Dipartimento di Studi Farmaceutici,
Università di Roma “La Sapienza,” P. le Aldo Moro 5, I-00185 Rome, Italy2; and
Institute of Veterinary Biochemistry and Molecular Biology,
University of Zürich-Irchel, Winterthurerstrasse 190,
CH-8057 Zürich, 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 sented by a heteroaromatic ring bearing at one side a func-
anti-human immunodeficiency virus (HIV) drugs, the nucleo- tional group capable of donating and/or accepting hydrogen
side reverse transcriptase inhibitors (NRTIs), the nonnucleo- bonds with the main chain of Lys101 and Lys103. Finally, on
side reverse transcriptase inhibitors (NNRTIs), and the pro- the butterfly body, a hydrophobic portion fills a small pocket
tease inhibitors. More recently, enfuvirtide, a 36-amino-acid formed mainly by the side chains of Lys103, Val106, and
residue peptide acting as a viral entry inhibitor, has been li- Val179. Upon complexation, the NNBS hydrophobic pocket
censed for the treatment of HIV infection (7, 8). NRTIs, changes its own conformation, leading to the inactivation of
NNRTIs, and protease inhibitors are mixed in highly active the enzyme itself. Because of the different chemical and struc-
antiretroviral therapy, which dramatically slows down viral rep- tural features of the inhibitors and the side chain flexibility, the
lication, but they are unable to eradicate the viral infection bound NNBS adopts different conformations (28). Moreover,
(29). Moreover, the rapid development of drug resistance and mutations of some amino acids cause variation of the NNBS
toxicity problems make urgent the discovery of novel anti-HIV properties, thus decreasing affinities of most of the inhibitors
agents effective against resistant mutants and without unpleas- (12, 24, 25). In particular, the NNRTI resistance mutations
ant side effects (14). Tyr188Leu and Tyr181Ile/Cys reduce - interactions, the
NNRTI interaction with HIV-1 reverse transcriptase (RT) is Gly190Ala mutation leads to a smaller active site space be-
a highly dynamic process (6). Crystal structures of RT-NNRTI cause of a steric conflict between the methyl side chain and the
complexes (19) showed that the drugs interacted with a hydro- inhibitor, and the formation of an additional hydrogen bond
phobic pocket (nonnucleoside binding site [NNBS]) on the when amino acid 103 is mutated from Lys to Asn reduces
enzyme in a “butterfly-like” mode. One of the “wings” of this inhibitor entrance into the NNBS.
butterfly is made of a -electron-rich moiety (phenyl or allyl However, HIV-1 RT itself also undergoes a conformational
substituents) that interacts through - interactions with a reorganization upon interaction with its substrates template-
hydrophobic pocket formed mainly by the side chains of aro- primer (TP) and deoxynucleoside triphosphate (dNTP), so
matic amino acids (Tyr181, Tyr188, Phe227, Trp229, and that three structurally distinct mechanistic forms can be rec-
Tyr318). On the other hand, the other wing is normally repre- ognized in the reaction pathway catalyzed by HIV-1 RT (1,
11): the free enzyme, the binary complex of RT with the
* Corresponding author. Mailing address: Istituto di Genetica Mole-
template-primer (RT/TP), and the catalytically competent ter-
colare IGM-CNR, via Abbiategrasso 207, I-27100 Pavia, Italy. Phone: nary complex of RT with both nucleic acid and dNTP (RT/TP/
(39)0382546354. Fax: (39)0382422286. E-mail: maga@igm.cnr.it. dNTP). This means that, in principle, the NNBS might not be
4546VOL. 49, 2005 INDOLYL ARYL SULFONES TARGET DIFFERENT HIV-1 RT FORMS 4547
identical in these three mechanistic forms. Several kinetic stud- Expression and purification of recombinant HIV-1 RT forms. Recombinant
ies have shown that this is indeed the case, so that some heterodimeric wild-type RT and the Lys103Asn and Tyr181Ile variants were
expressed and purified to ⬎95% purity (as judged by sodium dodecyl sulfate-
NNRTIs selectively target one or a few of the different enzy- polyacrylamide gel electrophoresis) as described previously (12).
matic forms along the reaction pathway (5, 13, 15). This ob- HIV-1 RT RNA-dependent DNA polymerase activity assay. RNA-dependent
servation likely reflects the different spatial rearrangements DNA polymerase activity was assayed as follows. A final volume of 25 l con-
not only of the NNBS itself but also of the adjacent nucleotide 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
binding site (3, 20, 26, 27). Indeed, it has been shown that a
(0.3 M 3⬘-OH ends), 10 M [3H]dTTP (1 Ci/mmol), and 5 to 10 nM RT.
“communication” exists between the NNBS and the nucleotide Reactions were incubated for 10 min at 37°C. Aliquots (20 l) were then spotted
binding site, so that some NRTI resistance mutations can in- on glass fiber filters (GF/C filters), which were immediately immersed in 5%
fluence NNRTI binding and vice versa (2, 4, 20). Thus, under- ice-cold trichloroacetic acid. Filters were washed twice in 5% ice-cold trichloro-
standing the molecular determinants governing the selective acetic acid and once in ethanol for 5 min and dried, and acid-precipitable
radioactivity was quantitated by scintillation counting.
interaction of a drug with the three different NNBS structures Inhibition assays. Reactions for inhibition assays were performed under the
present along the RT reaction pathway will be important for conditions described for the HIV-1 RT RNA-dependent DNA polymerase ac-
the design of novel, highly selective, and potent NNRTIs.
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tivity assay. Incorporation of radioactive dTTP into poly(rA)/oligo(dT) at differ-
During extensive structure-activity relationship studies on ent concentrations of DNA or dNTP was monitored in the presence of increasing
amounts of inhibitor as indicated in the figure legends.
diarylsulfones, we identified pyrryl sulfones and the novel in-
Kinetics of inhibitor binding. Kinetics of inhibitor binding experiments were
dolyl aryl sulfones (IASs) as highly potent NNRTIs (18, 22, as described previously (12). Briefly, HIV-1 RT (20 to 40 nM) was incubated for
23). In particular, indole derivatives bearing 2-methylphenyl- 2 min at 37°C in a final volume of 4 l in the presence of buffer A, with 10 mM
sulfonyl or 3-methylphenylsulfonyl moieties were found to in- MgCl2 alone or with 100 nM 3⬘-OH ends (for the formation of the RT/TP
hibit HIV-1 at nanomolar concentrations. Furthermore, the 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
introduction of a 3,5-dimethylphenylsulfonyl moiety led to a
then added to a final volume of 5 l at a concentration at which [EI]/[E0] ⫽
compound displaying high activity and selectivity not only [1 ⫺ 1/(1 ⫹ [I]/Ki)] ⬎ 0.9. Then, 145 l of a mix containing buffer A, 10 mM
against the wild-type strain but also against the Tyr181Cys and MgCl2, and 10 M [3H]dTTP (5 Ci/mmol) was added at different time points.
Lys103Asn-Tyr181Cys viral variants and the efavirenz-resistant After an additional 10 min of incubation at 37°C, 50-l aliquots were spotted on
mutant Lys103Arg-Val179Asp-Pro225His. GF/C filters, and acid-precipitable radioactivity was measured as described for
the HIV-1 RT RNA-dependent DNA polymerase activity assay. The quantity
In light of their extremely potent activities, especially to- vt/v0, representing the normalized difference between the amount of dTTP in-
wards NNRTI-resistant mutants, we sought to investigate in corporated at the zero time point and at different time points, was then plotted
detail the mechanism of action of some selected IAS deriva- against time. The kapp values were determined by fitting the experimental data to
tives. In this work, we show that IASs do not display a unique the single-exponent equation vt/v0 ⫽ e⫺kapp t, where t is time. The kon and koff
values were calculated according to the relationships (27) kapp ⫽ kon(Ki ⫹ [I])
mode of action but rather that they can selectively bind differ-
and Kd ⫽ koff/kon.
ent mechanistic forms of the enzyme, depending on the sub- Kinetic model. The mechanism of action of IASs was found to be either fully
stituents introduced on the drug pharmacophore. These results noncompetitive or partially mixed. A schematic drawing of the different equilib-
suggest that IASs are very flexible molecules, interacting dy- ria is depicted in Fig. 1. According to the ordered mechanism of the polymer-
namically with the viral RT. 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
MATERIALS AND METHODS
system is very complex and impractical to use. For these reasons, the general
Chemicals. [3H]dTTP (40 Ci/mmol) was from Amersham, and unlabeled steady-state kinetic analysis was simplified by varying one of the substrates
dNTPs were from Boehringer. Whatman was the supplier of the GF/C filters. All (either TP or dNTP) while the other was kept constant. When the TP substrate
other reagents were of analytical grade and purchased from Merck or Fluka. was held constant at saturating concentration and the inhibition at various
Chemistry. 5-Chloro-3-(phenyl)sulfonyl-1H-indole-2-carboxamide (RS1202) concentrations of dNTPs was analyzed, at the steady state all of the input RT was
(23, 30), 5-chloro-3-[(3,5-dimethylphenyl)sulfonyl]-1H-indole-2-carboxamide in the form of the RT/TP binary complex and only two forms of the enzyme (the
(RS1588)(21),N-{3-[(3,5-dimethylphenyl)sulfonyl]-5-chloro-1H-indole-2-car- binary complex and the ternary complex with dNTP) could react with the inhib-
bonyl}-D,L-alanylamide (RS1980) (21), and 4,5-difluoro-3-[(3,5-dimethylphenyl) itor, as shown in the left part of Fig. 1B. Similarly, when the dNTP concentration
sulfonyl]-1H-indole-2-carboxamide (RS1866) (R.S., unpublished data) were ob- was kept constant at saturating levels and the inhibition at various TP concen-
tained by heating the corresponding esters with concentrated ammonium hy- trations was analyzed, RT was present either as a free enzyme or in the ternary
droxide in a sealed tube. The starting esters needed for the preparation of complex with TP and dNTP, as shown in the right part of Fig. 1B.
RS1202, RS1588, and RS1866 were obtained by oxidation of the corresponding The steady-state rate equation used for the partially mixed inhibition was the
3-arylthio-1H-indole-2-carboxylates using 3-chloroperoxybenzoic acid (MCPBA). one describing a reaction involving only two mechanistic forms of the enzyme
The ethyl ester of N-{3-[(3,5-dimethylphenyl)sulfonyl]-5-chloro-1H-indole-2-car- (according to the simplified pathways in Fig. 1B) (9)
bonyl}-D,L-alanine for the synthesis of RS1980 was obtained by lithium hydroxide
冋 册
of ethyl 5-chloro-3-[(3,5-dimethylphenyl)sulfonyl]-1H-indole-2-carboxylate and 1 ⫹ k⬘cat · I
subsequent condensation of D,L-alanine ethyl ester in the presence of O-benzo- kcat · E0 ·
kcat · K⬘i
冋 册
triazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate and tri- v⫽ (1)
Ks 共1 ⫹ I · Ki兲
ethylamine. The required 3-arylthio-1H-indole-2-carboxylates were prepared by 1⫹ ·
reaction of proper arylthiodisulfides with 1H-indole-2-carboxylic acids in the S 共1 ⫹ I · K⬘i兲
presence of sodium hydride according to the Atkinson method and subsequent
esterification of the 3-arylthio-1H-indole-2-carboxylic acids with (trimethylsilyl) where kcat is the apparent reaction rate in the absence of the inhibitor, k⬘cat is the
diazomethane. Alternatively, these esters were obtained by reaction of methyl or reaction rate at infinite inhibitor concentration, E0 is the total enzyme concen-
ethyl 1H-indole-2-carboxylates with N-(arylthio)succinimides in the presence of tration, I is the inhibitor concentration, S is the substrate concentration, and Ki
boron trifluoride diethyl etherate. and K⬘i are the equilibrium dissociation constants for the different enzyme-
Nucleic acid substrates. The homopolymer poly(rA) (Pharmacia) was mixed substrate complexes. According to Fig. 1B, when TP was held constant and only
at weight ratios in nucleotides of 10:1 to the oligomer oligo(dT)12–18 (Pharmacia) the dTTP concentration was varied, Ki ⫽ Kbini (for the binding of the inhibitor
in 20 mM Tris-HCl (pH 8.0) containing 20 mM KCl and 1 mM EDTA, heated to the binary complex RT/TP), whereas K⬘i ⫽ Kteri (for the binding of the
at 65°C for 5 min, and then slowly cooled at room temperature. inhibitor to the ternary RT/TP/dNTP complex). Conversely, when dTTP was4548 CANCIO ET AL. ANTIMICROB. AGENTS CHEMOTHER.
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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, The K⬘i and k⬘cat values were derived by computer fitting of the variation of the
whereas K⬘i ⫽ Kteri (for binding to the ternary RT/TP/dNTP complex). kcat(app) values as a function of the inhibitor concentrations, according to the
The partially mixed mode of inhibition predicts that the enzyme-inhibitor equation (9)
complex is still able to bind the substrate but with a lower affinity constant K⬘s.
Conversely, the enzyme-substrate complex binds the inhibitor but with a lower K⬘i ⫹ I
1/kcat(app) ⫽ (4)
inhibition constant K⬘i. Moreover, the enzyme-inhibitor-substrate complex can 共K⬘i · kcat兲 ⫹ 共I · k⬘cat兲
still break down to give products but with a reduced catalytic rate k⬘cat (9). Thus,
equation 1 was used to derive the apparent reaction rate, kcat(app), and affinity, For the fully noncompetitive case, k⬘cat ⫽ 0, Ks ⫽ K⬘s, and Ki ⫽ K⬘i, and thus
Ks(app), for the TP and dNTP substrates of the reaction at different inhibitor equation 1 simplifies to
concentrations. Then, Ks, Ki, K⬘i, and K⬘s were derived by computer fitting of the
variation of the Ks(app) values as a function of the inhibitor concentrations, kcat · E0
according to the equations (9)
v⫽
1⫹ 冉 I
Ki 冊
I ⫹ Ki Ks
(5)
Ks(app) ⫽ (2) 1⫹
冢 冣
Ki S
I
Ks ⫺ Similarly, equation 4 simplifies to
K⬘s
and I 1
1/kcat(app) ⫽ ⫹ (6)
Ki · kcat kcat
1⫹I
Ks(app) ⫽ Ki ·
冤冉 冊冥
(3)
I Initial velocities of the reaction were determined after 10 min of incubation at
1⫹
K⬘i 37°C, which represents the midpoint of the linear range of the reaction, as
Ks determined in separate experiments (data not shown). Each experiment wasVOL. 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
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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-
FIG. 2. Structures of the compounds used in this study. 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 (k⬘cat) was
done in triplicate, and mean values were used for the analysis. Curve fitting was
found to be 2.5 to 3% of the uninhibited reaction, indicating a
done with the computer program GraphPad Prism.
maximum of 97% to 98% inhibition. Interestingly, from the
variation of the kcat(app) values measured in the presence of var-
RESULTS ious concentrations of the poly(rA)/oligo(dT) substrate, an inhi-
IAS derivatives show high potencies of inhibition of HIV-1 bition constant (Ki) value of 0.4 nM was calculated (Fig. 3B,
wild-type RT and NNRTI-resistant mutants. The IAS deriva- circles), whereas the same analysis performed with the kcat(app)
tives RS1202, RS1588, RS1866, and RS1980 (Fig. 2) were values measured in the presence of various concentrations of
tested against HIV-1 wild-type RT and different NNRTI-resis- the dTTP substrate yielded a Ki of 2.7 nM (Fig. 3B, triangles).
tant RT mutants. As summarized in Table 1, all four deriva- According to the scheme outlined in Fig. 1B, these values
tives showed potent inhibition towards wild-type RT. In gen- represent the apparent binding constants of the inhibitor to the
eral, the inhibition activities were higher towards the mutants free enzyme and the ternary RT/TP/dNTP complex, respec-
Leu100Ile, Val106Ala, and Val179Asp than towards the two tively. Thus, even though RS1588 binding did not affect the
mutants Tyr181Ile and Tyr188Leu. Interestingly, the com- affinity of RT for the substrates, the binding of the substrates
pounds RS1202 and RS1588 showed little or no loss of potency to the enzyme reduced the apparent Ki of the inhibitor.
towards the Lys103Asn mutant, being 400- to 600-fold more RS1202 is a mixed inhibitor which binds preferentially to
potent than nevirapine and efavirenz against this mutant. the free enzyme and is partially competitive with the nucleic
Given their interesting activity profile, the mechanism of inhi- acid substrate. When the IAS derivative RS1202 was studied,
bition of these IAS derivatives was studied in more detail. a strong reduction of the apparent affinity for the nucleic acid
Increasing concentrations of either the dTTP or the poly(rA)/ substrate but not for dTTP was noted. The increase in the
oligo(dT) substrates were titrated into the reaction in the pres- Ks(app)3⬘-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) for a
Compound
wt Leu100Ile Lys103Asn Val106Ala Val179Asp Tyr181Ile Tyr188Leu
RS1202 1 (⫾0.1) 100 (⫾20) 5 (⫾1) 75 (⫾8) 35 (⫾3) 140 (⫾10) 1,000 (⫾100)
RS1588 3 (⫾0.1) 60 (⫾6) 3 (⫾0.5) 90 (⫾8) 50 (⫾5) 140 (⫾10) 200 (⫾20)
RS1866 2.2 (⫾0.1) n.d. 750 (⫾60) n.d. n.d. 1,500 (⫾100) n.d.
RS1980 3 (⫾0.2) n.d. 500 (⫾50) n.d. n.d. 130 (⫾10) n.d.
Nevirapine 400 (⫾50) 900 (⫾90) 2,000 (⫾80) 2,000 (⫾300) 300 (⫾25) 5,000 (⫾400) 5,000 (⫾400)
Efavirenz 40 (⫾0.5) n.d. 1,000 (⫾40) n.d. n.d. 400 (⫾50) 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.
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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 3⬘-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 3⬘-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 3⬘-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 (Ks3⬘-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 3⬘-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.
nevirapine; n.a., not applicable (the affinity of EFV for the RTK103N and RTK103N/TP complexes was too low to be measured).
Lys103Asn mutant RT
Wild-type RT
a
NVP
EFV
RS1588
RS1202
NVP
EFV
RS1980
RS1866
RS1588
RS1202
3C) suggested a partially competitive inhibition. In addition,
Enzyme-substrate complexes and kinetic constants are as defined in Fig. 1. Numbers are the mean values of three independent experiments and (standard deviations). n.d., not determined; EFV, efavirenz; NVP,
and inhibitor
the reciprocal of the variation of the kcat(app) values followed a
RT type
nonlinear relationship, as in the case of RS1588, indicating a
partially noncompetitive mechanism (Fig. 3D) with a maximal
TABLE 2. Association and dissociation rates for the IAS derivatives to the different enzyme-substrate complexes for wild-type RT and the Lys103Asn mutant a
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
Ki (nM)
3,300
as described in Materials and Methods. As a result, it was
n.a.
400
30
found that RS1202 interacted preferentially with the free en-
0.3
1.3
0.3
2.2
0.3
0.3
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
(0.03 ⫾ 0.005) ⫻ 104
(0.04 ⫾ 0.01) ⫻ 105
(7.1 ⫾ 0.5) ⫻ 104
(1.9 ⫾ 0.2) ⫻ 105
(0.1 ⫾ 0.02) ⫻ 105
inhibitor by fourfold, giving a Kibin/ter of 1.2 nM.
(11 ⫾ 1) ⫻ 105
(6 ⫾ 0.5) ⫻ 105
(4 ⫾ 0.6) ⫻ 105
(6 ⫾ 0.5) ⫻ 105
kon (s⫺1M⫺1)
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RS1980 is a mixed inhibitor which is partially competitive
n.a.
with the nucleotide substrate. Similar experiments were
conducted with the compound RS1980 (Fig. 3E and F). This
RT
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-
(2.5 ⫾ 0.5) ⫻ 10⫺4
(16 ⫾ 1) ⫻ 10⫺4
(1 ⫾ 0.2) ⫻ 10⫺3
(2 ⫾ 0.2) ⫻ 10⫺5
(4 ⫾ 1) ⫻ 10⫺4
(2 ⫾ 0.3) ⫻ 10⫺4
(9 ⫾ 1) ⫻ 10⫺4
(2 ⫾ 0.3) ⫻ 10⫺4
(4 ⫾ 0.5) ⫻ 10⫺4
ues as a function of increasing inhibitor concentrations (Fig.
koff (s⫺1)
3E). Again, the reciprocal of the variation of the kcat(app) val-
n.a.
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
Kibin (nM)
revealed that RS1980 binding to the ternary RT/TP/dNTP
3,300
500
n.a.
complex was reduced 10-fold with respect to either the free
3
5
0.4
2.3
2.7
1.6
.30
enzyme or the binary RT/TP complex (compare Ki and Kibin
with Kiter values).
Preferential binding of IAS derivatives to specific reaction
(0.03 ⫾ 0.005) ⫻ 104
(0.03 ⫾ 0.01) ⫻ 105
(4.5 ⫾ 0.5) ⫻ 104
(0.1 ⫾ 0.02) ⫻ 105
konbin (s⫺1M⫺1)
intermediates is mainly driven by differences in the associa-
(5 ⫾ 1) ⫻ 104
(7 ⫾ 0.5) ⫻ 105
(3 ⫾ 0.2) ⫻ 105
(2 ⫾ 0.5) ⫻ 105
(3 ⫾ 0.5) ⫻ 105
tion rates. The differences in the equilibrium dissociation con-
n.a.
stants Ki, Kibin, and Kiter of the inhibitors for the different
RT/TP
Rate for:
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
(1.5 ⫾ 0.2) ⫻ 10⫺4
(2.5 ⫾ 0.5) ⫻ 10⫺4
(15 ⫾ 1) ⫻ 10⫺4
(1 ⫾ 0.2) ⫻ 10⫺3
(4 ⫾ 1) ⫻ 10⫺4
(3 ⫾ 0.3) ⫻ 10⫺4
(7 ⫾ 1) ⫻ 10⫺4
(6 ⫾ 0.5) ⫻ 10⫺4
(5 ⫾ 0.5) ⫻ 10⫺4
association and dissociation rates of the different IAS deriva-
koffbin (s⫺1)
tives were determined for the free enzyme and the binary
n.a.
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
Kiter (nM)
well as the improved binding of RS1980 to both the free en-
200
375
n.d.
n.d.
n.d.
n.d.
zyme and the binary complex, was due to a faster association
5
4
3
1.2
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
(0.04 ⫾ 0.01) ⫻ 105
(0.6 ⫾ 0.01) ⫻ 104
(0.4 ⫾ 0.02) ⫻ 105
konter (s⫺1M⫺1)
the binary complex with respect to the free enzyme. Taken
(5 ⫾ 1) ⫻ 104
(1 ⫾ 0.3) ⫻ 105
(2 ⫾ 0.3) ⫻ 105
together, these results indicate that binding of either the nu-
RT/TP/dNTP
n.d.
n.d.
n.d.
n.d.
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
(1.2 ⫾ 0.1) ⫻ 10⫺3
(2.5 ⫾ 1) ⫻ 10⫺4
(1.6 ⫾ 0.3) ⫻ 10⫺4
(15 ⫾ 1) ⫻ 10⫺4
(3 ⫾ 0.3) ⫻ 10⫺4
(3 ⫾ 0.3) ⫻ 10⫺4
all of the IAS derivatives were superior to both reference drugs
koffter (s⫺1)
in terms of binding, showing much faster association rates,
n.d.
n.d.
n.d.
n.d.
whereas the dissociation rates were of the same order of those
of efavirenz but significantly better (i.e., slower) than those of
nevirapine.4552 CANCIO ET AL. 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
Downloaded from http://aac.asm.org/ on January 23, 2021 by guest
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 FIG. 4. Structure-activity relationships for the IAS derivatives used
in this study. The different substituents on the common IAS scaffold
sensitive to the steric barrier imposed by the Lys103Asn mu- are highlighted with circles. Arrows indicate the structural relation-
tation. 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 a partially competitive mode of action towards the dNTP sub-
of selected IAS derivatives showed that the nature of the sub- strate.
stituents on the drug pharmacophore can influence the selec- Drug binding studies showed that the main determinant for
tive interaction of the drug with different mechanistic forms of drug selectivity was the inhibitor association rate, suggesting
the viral RT. Similar properties have been already observed for that the structural reorganization of the NNBS upon formation
other NNRTIs, such as efavirenz, the pyrrolobenzoxazepinone of the binary and ternary complexes of RT with its substrates
derivative PPO464, and the carboxanilide derivatives UC38 introduced some steric and/or thermodynamic barriers which
and UC84 (10, 13, 15), suggesting that a preferential comple- were differentially “sensed” by the substituents on the drug
mentarity exists between the structure of the inhibitor mole- molecule, depending on their spatial arrangement.
cule and the different spatial conformations of the NNBS. This inherent flexibility of the IAS derivatives, and their
The structure-function relationships of our IAS derivatives consequent ability to selectively target specific enzymatic
are schematically drawn in Fig. 4. The compound RS1866 forms, also had some consequences for their interaction with
{4,5-difluoro-3-[(3,5-dimethylphenyl)sulfonyl]-1H-indole-2-car- NNRTI-resistant mutated RT enzymes, such as Lys103Asn
boxyamide} showed a fully noncompetitive mode of action, and Tyr181Ile. In fact, as can be seen from Table 1, the com-
being able to bind to all of the different reaction intermediates pounds which selectively targeted the free enzyme, that is,
(RT, RT/TP, and RT/TP/dNTP complexes) (Fig. 1A) with RS1588 and RS1202, were also the least affected by the
equal affinities. Changing the nature of the alkyl group to give Lys103Asn mutation. On the other hand, those compounds
the compound RS1588 {5-chloro-3-[(3,5-dimethylphenyl)sul- which did not discriminate between the free enzyme and the
fonyl]-1 H-indole-2-carboxyamide} also modified the mecha- binary RT/TP complex, that is, RS1866 and RS1980, showed a
nism of inhibition, rendering the drug more selective for the loss of activity of about 75- to 150-fold. The RS1866 derivative
free enzyme with respect to the binary and ternary complexes. was the most affected by the Tyr181Ile mutation, but the other
Elimination of the 3,5-methyl groups gave the compound compounds also showed significant losses of potency against
RS1202 [5-chloro-3-(phenylsulfonyl)-1H-indole-2-carboxyamide], this mutant.
which was very selective for the free enzyme, like RS1588, but also When the mechanism of inhibition of the Lys103Asn mutant
showed a partially competitive mechanism of action towards by RS1202 and RS1588 was investigated, both compounds
the nucleic acid substrate. On the other hand, introducing an alanin- were shown to selectively target the unliganded form of the
amide moiety into compound RS1588 to give its deriva- mutant RT, as in the case of the wild-type enzyme, but the
tive RS1980 (N-{3-[(3,5-dimethylphenyl)sulfonyl]-5-chloro-1H- association rates (kon) of both drugs were reduced. This is
indole-2-carbonyl}alaninamide) made the drug capable of consistent with the well-known effect of the Lys103Asn muta-
discriminating against the RT/TP/dNTP ternary complex, with tion, which introduces a steric barrier to drug binding (12). OnVOL. 49, 2005 INDOLYL ARYL SULFONES TARGET DIFFERENT HIV-1 RT FORMS 4553
the other hand, RS1202 and RS1588 showed slower dissocia- REFERENCES
tion rates (koff) from the Lys103Asn mutant than from wild- 1. Bahar, I., B. Erman, R. L. Jernigan, A. R. Atilgan, and D. G. Covell. 1999.
type RT. By comparison, the NNRTIs efavirenz and nevira- Collective motions in HIV-1 reverse transcriptase: examination of flexibility
and enzyme function. J. Mol. Biol. 285:1023–1037.
pine suffered from similar reductions in their kon values but no 2. Baldanti, F., S. Paolucci, G. Maga, N. Labo, U. Hubscher, A. Y. Skoblov, L.
compensatory decrease in their koff rates. Thus, our results Victorova, S. Spadari, L. Minoli, and G. Gerna. 2003. Nevirapine-selected
suggest that drugs selectively targeting the unliganded form of mutations Tyr181Ile/C of HIV-1 reverse transcriptase confer cross-resistance
to stavudine. AIDS 17:1568–1570.
the enzyme might efficiently overcome the steric barrier intro- 3. Basavapathruni, A., C. M. Bailey, and K. S. Anderson. 2004. Defining a
duced by the Lys103Asn mutation by decreasing their associ- molecular mechanism of synergy between nucleoside and nonnucleoside
AIDS drugs. J. Biol. Chem. 279:6221–6224. (First published 13 January
ation rates from the mutated enzyme, as we have shown in the 2004.)
case of RS1202 and RS1588. This observation might help in 4. Blanca, G., F. Baldanti, S. Paolucci, A. Y. Skoblov, L. Victorova, U. Hub-
the design of more active compounds. scher, G. Gerna, S. Spadari, and G. Maga. 2003. Nevirapine resistance
mutation at codon 181 of the HIV-1 reverse transcriptase confers stavudine
Several hypotheses have been made, based on kinetic, cross- resistance by increasing nucleotide substrate discrimination and phosphoro-
linking, and structural studies, to explain the mechanism of lytic activity. J. Biol. Chem. 278:15469–15472. (First published 24 February
inhibition of the chemical step by NNRTIs (11, 19, 26, 27). 2003.)
Downloaded from http://aac.asm.org/ on January 23, 2021 by guest
5. Buckheit, R. W., Jr., E. L. White, V. Fliakas-Boltz, J. Russell, T. L. Stup,
Binding of an NNRTI leads to displacement of the 12-13 T. L. Kinjerski, M. C. Osterling, A. Weigand, and J. P. Bader. 1999. Unique
hairpin, which has direct interaction with the nucleic acid sub- anti-human immunodeficiency virus activities of the nonnucleoside reverse
transcriptase inhibitors calanolide A, costatolide, and dihydrocostatolide.
strate. Thus, it is possible that this alteration in the position of Antimicrob. Agents Chemother. 43:1827–1834.
the nucleic acid relative to the polymerase active site is respon- 6. Campiani, G., A. Ramunno, G. Maga, V. Nacci, C. Fattorusso, B. Cata-
sible for NNRTI inhibition. Another possible mechanism can lanotti, E. Morelli, and E. Novellino. 2002. Non-nucleoside HIV-1 reverse
transcriptase (RT) inhibitors: past, present, and future perspectives. Curr.
be hypothesized from the observation that the NNBS includes Pharm. Des. 8:615–657.
residues (Tyr181 and Tyr188) in the 9-10 hairpin, which 7. De Clercq, E. 2002. Highlights in the development of new antiviral agents.
contains two of the three active-site aspartic acids (Asp185 and Mini Rev. Med. Chem. 2:163–175.
8. De Clercq, E. 2002. New anti-HIV agents and targets. Med. Res. Rev.
Asp186). There are also contacts between some NNRTIs and 22:531–565.
the 6 strand that carries the third active-site aspartate 9. Dixon, M., and E. C. Webb. 1979. Enzymes, 3rd ed. Longman, London,
(Asp110). Again, even a moderate shift in the positions of the United Kingdom.
10. Fletcher, R. S., K. Syed, S. Mithani, G. I. Dmitrienko, and M. A. Parniak.
active-site residues could interfere with the chemical step of 1995. Carboxanilide derivative non-nucleoside inhibitors of HIV-1 reverse
polymerization. Finally, it has been suggested that the fingers transcriptase interact with different mechanistic forms of the enzyme. Bio-
subdomain of RT may not adopt a fully closed conformation if chemistry 34:4346–4353.
11. Madrid, M., J. A. Lukin, J. D. Madura, J. Ding, and E. Arnold. 2001.
both a dNTP and an NNRTI are bound to the enzyme. Since Molecular dynamics of HIV-1 reverse transcriptase indicates increased flex-
proper closure of the fingers is important for the positioning of ibility upon DNA binding. Proteins 45:176–182.
12. Maga, G., M. Amacker, N. Ruel, U. Hubscher, and S. Spadari. 1997. Resis-
the dNTP and the primer relative to the polymerase active site, tance to nevirapine of HIV-1 reverse transcriptase mutants: loss of stabiliz-
preventing the proper closure of the fingers subdomain would ing interactions and thermodynamic or steric barriers are induced by differ-
interfere with the chemical step of DNA synthesis. 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.
Different NNRTIs might act through one or more of these Baldanti, S. Paolucci, M. Zavattoni, A. Bergamini, B. Galletti, S. Muck, U.
mechanisms; however, all of the NNRTIs studied so far do not Hubscher, G. Giorgi, G. Guiso, S. Caccia, and G. Campiani. 2001. The
interfere with the binding of either the dNTP or the nucleic stereoselective targeting of a specific enzyme-substrate complex is the mo-
lecular mechanism for the synergic inhibition of HIV-1 reverse transcriptase
acid substrate. On the other hand, the IAS derivatives de- by (R)-(-)-PPO464: a novel generation of nonnucleoside inhibitors. J. Biol.
scribed here showed different mechanisms of action, being Chem. 276:44653–44662. (First published 25 September 2001.)
able to discriminate between different mechanistic forms of 14. Maga, G., and S. Spadari. 2002. Combinations against combinations: asso-
ciations of anti-HIV 1 reverse transcriptase drugs challenged by constella-
the viral RT and being partially competitive towards either the tions of drug resistance mutations. Curr. Drug Metab. 3:73–95.
nucleic acid or the dNTP substrate. These observations further 15. Maga, G., D. Ubiali, R. Salvetti, M. Pregnolato, and S. Spadari. 2000.
Selective interaction of the human immunodeficiency virus type 1 reverse
reinforce the notion that the interaction of IASs with the transcriptase nonnucleoside inhibitor efavirenz and its thio-substituted ana-
NNBS of HIV-1 RT is novel and different from the other log with different enzyme-substrate complexes. Antimicrob. Agents Che-
NNRTIs (18). mother. 44:1186–1194.
16. Reference deleted.
It thus appears that the IAS derivatives shown here are very 17. Reference deleted.
sensitive to the structural modifications occurring at the NNBS 18. Ragno, R., M. Artico, G. De Martino, G. La Regina, A. Coluccia, A. Di
upon complexation of the RT with its substrates and that their Pasquali, and R. Silvestri. 2005. Docking and 3-D QSAR studies on
indolyl aryl sulfones. Binding mode exploration at the HIV-1 reverse
sensitivity can be modulated through small modifications of the transcriptase non-nucleoside binding site and design of highly active
drug molecule. 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.
ACKNOWLEDGMENTS 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.
We thank S. H. Hughes (NCI-Frederick Cancer Research and De- 2:293–302.
velopment Center) for the coexpression vectors pUC12N/p66(His)/p51 20. Selmi, B., J. Deval, K. Alvarez, J. Boretto, S. Sarfati, C. Guerreiro, and B.
with the wild-type or the mutant forms of HIV-1 RT. Canard. 2003. The Y181C substitution in 3⬘-azido-3⬘-deoxythymidine-resis-
This work was supported by the Italian Ministero della Salute, tant human immunodeficiency virus, type 1, reverse transcriptase suppresses
Istituto Superiore di Sanità, Fourth National Research Program on the ATP-mediated repair of the 3⬘-azido-3⬘-deoxythymidine 5⬘-monophos-
phate-terminated primer. J. Biol. Chem. 278:40464–40472.
AIDS (R.S., M.A., and R.R.), Fifth National Research Program on
21. Silvestri, R., M. Artico, G. De Martino, G. La Regina, R. Loddo, M. La Colla,
AIDS (grant 40F.78 to S.S.), Italian MIUR-Cofin 2002 and 2004 (R.S. M. Mura, and P. La Colla. 2004. Simple, short peptide derivatives of a
and M.A.), Istituto Pasteur-Fondazione Cenci Bolognetti (R.S.), and sulfonylindolecarboxamide (L-737,126) active in vitro against HIV-1 wild
the EU FP6 Research Project LSHB-CT-2003-503480-TRIoH (G.M.). type and variants carrying non-nucleoside reverse transcriptase inhibitor
U.H. is supported by the University of Zürich. resistance mutations. J. Med. Chem. 47:3892–3896.4554 CANCIO ET AL. ANTIMICROB. AGENTS CHEMOTHER.
22. Silvestri, R., M. Artico, G. La Regina, G. De Martino, M. La Colla, R. Loddo, with nonnucleoside inhibitors using Monte Carlo simulations in combination
and P. La Colla. 2004. Anti-HIV-1 activity of pyrryl aryl sulfone (PAS) with a linear response method. Drug Des. Discov. 18:151–163.
derivatives: synthesis and SAR studies of novel esters and amides at the 26. Spence, R., K. S. Anderson, and K. A. Johnson. 1996. HIV-1 reverse tran-
position 2 of the pyrrole nucleus. Farmaco 59:201–210. scriptase resistance to nonnucleoside inhibitors. Biochemistry 35:1054–1063.
23. Silvestri, R., G. De Martino, G. La Regina, M. Artico, S. Massa, L. Vargiu, 27. Spence, R. A., W. M. Kati, K. S. Anderson, and K. A. Johnson. 1995.
M. Mura, A. G. Loi, T. Marceddu, and P. La Colla. 2003. Novel indolyl aryl Mechanism of inhibition of HIV-1 reverse transcriptase by nonnucleoside
sulfones active against HIV-1 carrying NNRTI resistance mutations: synthe- inhibitors. Science 267:988–993.
sis and SAR studies. J. Med. Chem. 46:2482–2493.
28. Temiz, N. A., and I. Bahar. 2002. Inhibitor binding alters the directions of
24. Smith, M. B., M. L. Lamb, J. Tirado-Rives, W. L. Jorgensen, C. J. Michejda,
domain motions in HIV-1 reverse transcriptase. Proteins 49:61–70.
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 29. Vella, S., and L. Palmisano. 2000. Antiretroviral therapy: state of the
TIBO: contribution of the L100I and Y181C variants to protein stability and HAART. Antivir. Res. 45:1–7.
biological activity. Protein Eng. 13:413–421. 30. Williams, T. M., T. M. Ciccarone, S. C. MacTough, C. S. Rooney, S. K.
25. Smith, M. B., S. Ruby, S. Horouzhenko, B. Buckingham, J. Richardson, I. Balani, J. H. Condra, E. A. Emini, M. E. Goldman, W. J. Greenlee, L. R.
Puleri, E. Potts, W. L. Jorgensen, E. Arnold, W. Zhang, S. H. Hughes, C. J. Kauffman, et al. 1993. 5-Chloro-3-(phenylsulfonyl)indole-2-carboxamide: a
Michejda, and R. H. Smith, Jr. 2003. HIV-1 reverse transcriptase variants: novel, non-nucleoside inhibitor of HIV-1 reverse transcriptase. J. Med.
molecular modeling of Y181C, V106A, L100I, and Lys103Asn mutations Chem. 36:1291–1294.
Downloaded from http://aac.asm.org/ on January 23, 2021 by guestYou can also read
