10.1128/AAC.44.5.1186-1194.2000.

2000, 44(5):1186. DOI:Antimicrob. Agents Chemother. Pregnolato and Silvio SpadariGiovanni Maga, Daniela Ubiali, Raul Salvetti, Massimo Complexeswith Different Enzyme-SubstrateEfavirenz and Its Thio-Substituted Analog Transcriptase Nonnucleoside InhibitorImmunodeficiency Virus Type 1 Reverse Selective Interaction of the Human

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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY,0066-4804/00/$04.0010

May 2000, p. 1186–1194 Vol. 44, No. 5

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Selective Interaction of the Human Immunodeficiency Virus Type 1Reverse Transcriptase Nonnucleoside Inhibitor Efavirenz and Its

Thio-Substituted Analog with Different Enzyme-Substrate ComplexesGIOVANNI MAGA,1 DANIELA UBIALI,2 RAUL SALVETTI,2 MASSIMO PREGNOLATO,2*

AND SILVIO SPADARI1

Istituto di Genetica Biochimica ed Evoluzionistica IGBE-C.N.R.1 and Dipartimento di Chimica Farmaceutica,Universita degli Studi,2 I-27100 Pavia, Italy

Received 16 August 1999/Returned for modification 24 November 1999/Accepted 27 December 1999

Accumulating data have brought the nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) into theforefront of antiretroviral therapy. Among the emerging compounds in this class, a particularly attractive oneis efavirenz (Sustiva), recently approved for clinical use by the U.S. Food and Drug Administration. In thepresent study, the equilibrium dissociation constants for efavirenz binding to the different catalytic forms ofhuman immunodeficiency virus type 1 RT as well as the association and dissociation rates have been deter-mined using a steady-state kinetic approach. In addition, the same enzymological analysis has been extendedto the thio-substituted analog, sefavirenz, which showed comparable activity in vitro against RT. Both com-pounds have been found to act as purely uncompetitive inhibitors at low drug concentrations (5 to 50 nM) andas mixed noncompetitive inhibitors at higher doses (50 to 500 nM). This behavior can be interpreted in termsof the relative affinities for the different catalytic forms of the enzyme. Both efavirenz and sefavirenz showedincreasing affinities for the different forms of RT in the following order: free enzyme < (i.e., bound with loweraffinity) binary RT–template-primer (TP) complex < ternary RT-TP-deoxynucleoside triphosphate (dNTP)complex. The rate of binding of the two inhibitors to the different enzyme-substrate complexes was well belowthe diffusion limit (on the order of 104 M21 s21); however, both inhibitors, when bound to the ternaryRT-TP-dNTP complex, showed very low dissociation rates, on the order of 1024 s21 for both compounds, typicalof tightly binding inhibitors. Thus, efavirenz and its thio-substituted derivative sefavirenz appear to be peculiarin their mechanism of action, being selective tightly binding inhibitors of the ternary RT-TP-dNTP complex.Efavirenz is the first clinically approved NNRTI to show this property.

The virus-encoded human immunodeficiency virus type 1(HIV-1) reverse transcriptase (RT) is essential for the viralreplication cycle and therefore represents a logical target forantiviral chemotherapy (11, 15). Recently, a class of inhibitorstargeted to the viral RT, the so-called nonnucleoside RT in-hibitors (NNRTIs), have gained a definitive place in the treat-ment of HIV-1 infections along with nucleoside RT inhibitors(NRTIs) and protease inhibitors (PIs) (5). These compounds,in spite of their different chemical structures, are highly specificfor HIV-1 RT and bind to the enzyme at the same allostericsite, close to but distinct from the catalytic site, behaving astypically noncompetitive inhibitors with respect to the differentsubstrates of the polymerization reaction (7, 28, 30). There arecurrently three NNRTIs approved for clinical use, nevirapine(Viramune), delavirdine (Rescriptor), and the most recentlylicensed, efavirenz (Sustiva). When used in monotherapy reg-imens, NNRTIs have rapidly selected for resistance (19, 20, 21,22, 26, 27, 30), but the results of clinical trials with NNRTIs ascomponents of highly effective antiretroviral therapy regimensin combination with NRTIs and/or PIs have been impressive(http://www.medscape.com/Medscape/HIV/TreatmentUpdate/1999/tu04/tu04-03html). In general, NNRTIs often show syn-ergistic (or at least additive) effects in combination withNRTIs, as well as positive pharmacokinetic properties. In con-trast to NRTIs or PIs, NNRTIs are characterized by less severeadverse effects for patients (4). There now exists a large

amount of data justifying the use of NNRTIs plus NRTIs asinitial therapy as well as in the treatment of individuals whohave very advanced disease or who have already failed multipleNRTI or NRTI-PI combination therapies.

Among the emerging compounds in this class, a particularlyattractive one is efavirenz. Efavirenz had very promising re-sults in clinical trials aimed at evaluating its effect in associa-tion with NRTIs, NNRTIs, and PIs under a variety of clinicalscenarios. It was particularly effective both in treatment-expe-rienced individuals switched to the new therapy and in salvageregimens for patients not responding to standard NRTI-PIcombinations. Like the other NNRTIs, however, efavirenz alsoselects for genotypic drug resistance, in particular, for theK103N mutation in the drug-binding site of HIV-1 RT (1, 9,32, 33). This mutation was also the most frequently observed insamples from patients experiencing postvirological treatmentfailure and was already known to confer cross-resistance toother NNRTIs (30). These observations highlight the need forextended-spectrum efavirenz derivatives that may be activeagainst the K103N mutant. A detailed understanding of themechanism of action of efavirenz is an obligatory step in de-veloping new molecules with a better profile of activity againstdrug-resistant mutants.

In the present study, the equilibrium dissociation constantsfor efavirenz binding to the different catalytic forms of HIV-1RT as well as the association and dissociation rates have beendetermined using a steady-state kinetic approach. In order toevaluate how minor conformational changes in the structure ofefavirenz could affect its binding to HIV-1 RT, a derivativebearing an oxocarbonyl-thiocarbonyl substitution has been syn-

* Corresponding author. Mailing address: Dipartimento di ChimicaFarmaceutica, via Taramelli 12, I-27100 Pavia, Italy. Phone: 39-0382-507583. Fax: 39-0382-422975. E-mail: maxp@pbl.unipv.it.

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thesized and called sefavirenz. While sefavirenz displayed com-parable activity in in vitro RT assays, the results indicated thatthe compounds bound with different affinities to the variouscatalytic forms of the enzyme-substrate complex.

MATERIALS AND METHODS

Chemicals. [3H]dTTP (40 Ci/mmol) was from Amersham, and unlabeled de-oxynucleoside triphosphates (dNTPs) were from Boehringer. Whatman was thesupplier of the GF/C filters. All other reagents were of analytical grade and werepurchased from Merck or Fluka.

Synthesis of compounds. Melting points were measured using a Kofler hot-stage apparatus and are uncorrected. 1H and 13C nuclear magnetic resonance(NMR) spectra were recorded in CDCl3 at 300 and 75.46 MHz, respectively,using a Bruker ACE-300 spectrometer. 1H chemical shifts (d) were reported withMe4Si (d 5 0.00 ppm) as an internal standard. 13C chemical shifts (d) werereported with CDCl3 (central peak, d 5 77.00 ppm) as an internal standard. Thefollowing abbreviations are used: br, broad; s, singlet; d, doublet; dd, doubledoublet; and m, multiplet. Mass spectra were obtained on a Finnigan MAT 8222spectrometer via the direct inlet. Electron ionization was performed at 70 eV and0.5 mA with a source temperature of 250°C. Elemental analyses indicated by thesymbols were within 60.4% of the theoretical values and were performed on aCarlo Erba 1106 Elemental Analyzer. All reactions were monitored by thin-layerchromatography carried out on 0.25-mm Merck silica gel (60 F254) and visualizedby UV light (d 5 264 or 365 nm); flash chromatography was performed with silicagel 60 (60 to 200 mm; Merck). High-pressure liquid chromatography (HPLC)analyses were run on a Merck-Hitachi L-7100 instrument equipped with anL-7400 UV detector and an L-7300 column oven. The column was an RP 18Lichrospher 100 5-mm column (Merck). The conditions were as follows: eluent,CH3CN-H2O (65:35); flow rate, 1.1 ml/min; UV detector wavelength, 247 nm;and temperature, 25°C.

(i) 6-Chloro-4-cyclopropylethynyl-4-trifluoromethyl-1,4-dihydro-2H-3,1-ben-zoxazin-2-one (efavirenz). The product was synthesized as described by Tan et al.(29); the mp was 133 to 136°C (hexane-toluene). HPLC analysis: retention time(RT), 6.57 min. 1H NMR (CDCl3): 0.85 (m, 2H), 0.94 (m, 2H), 1.40 (m, 1H), 6.81(d, J 5 8.5 Hz, 1H), 7.37 (dd, J 5 2.5, 8.5 Hz, 1H), 7.49 (d, J 5 2.5 Hz, 1H), 8.71(br s, 1H). 13C NMR: 148.0, 133.2, 131.6, 129.0, 127.8, 127.7, 123.9, 120.1, 116.3,115.7, 95.8, 77.3, 77.1, 76.9, 76.5, 55.0, 8.9, 20.7. Mass spectrometry: m/z (ra[relative abundance] %): 315 (M1, 30), 248 (23), 246 (100), 243 (33), 182 (13),180 (36), 167 (12). Analysis calculated for C14H9NO2ClF3: C, 53.27; H, 2.87; N,4.44. Found: C, 52.90; H, 2.92; N, 4.77.

(ii) 6-Chloro-4-cyclopropylethynyl-4-trifluoromethyl-1,4-dihydro-2H-3,1-ben-zoxazin-2-thione (Sefavirenz). To a solution of efavirenz (506 mg, 1.602 mmol)in 15 ml of anhydrous toluene, Lawesson’s reagent (324 mg, 0.801 mmol) wasadded, and the mixture was refluxed for 3.5 h. After the mixture was cooled toroom temperature, the solid residue was removed by filtration. The solvent wasdistilled under reduced pressure, and the residue was purified by flash chroma-tography (hexane-ethyl acetate, 75:25). Crystallization from hexane gave whitecrystals; the mp was 147 to 148°C. HPLC analysis: RT, 7.29 min. 1H NMR(CDCl3): 0.85 (m, 2H), 0.94 (m, 2H), 1.39 (m, 1H), 6.80 (d, J 5 8.5 Hz, 1H), 7.49(dd, J 5 2.5, 8.5 Hz, 1H), 7.52 (d, J 5 2.5 Hz, 1H), 9.33 (br s, 1H). 13C NMR:180.1, 131.8, 130.6, 130.5, 127.8, 127.4, 123.6, 118.7, 115.9, 115.3, 96.8, 79.6, 79.2,78.6, 78.2, 55.5, 8.8, 20.7. Mass spectrometry: m/z (ra %): 331 (M1, 70), 296 (19),262 (100), 243 (75), 234 (13), 224 (25), 167 (29), 87 (24). Analysis calculated forC14H9NOSClF3: C, 50.6; H, 2.73; N, 4.22. Found: C, 50.86; H, 2.77; N, 4.14.

Molecular modeling. The calculations and simulation were performed on anO2 R10000 SGI workstation by using the software modules Discover and Builderof the Biosym/MSI software package. The structure of nevirapine was obtainedfrom the atomic coordinates of the crystal structure of the HIV-1 RT–nevirapinecomplex (protein data bank file RVO). The structure of efavirenz was modeledusing the Biosym/MSI software package, further subjected to the steepest de-scendent minimization for 1,000 steps, and then minimized with conjugate gra-dients for 10,000 steps. The superimposition of efavirenz and nevirapine gave aroot mean square deviation value in the aligned position of 0.095.

Nucleic acid substrates. The homopolymer poly(rA) (Pharmacia) was mixedat weight ratios (in nucleotides) of 10:1 with the oligomer oligo(dT)12–18 (Phar-macia) in 20 mM Tris-HCl (pH 8.0) containing 20 mM KCl and 1 mM EDTA;the mixture was heated at 65°C for 5 min and then slowly cooled at roomtemperature.

Expression and purification of recombinant HIV-1 RT forms. RecombinantRT was expressed and purified to .95% purity as described previously (20). Ithad a specific activity on poly(rA)-oligo(dT) (see below) of 75,670 U/mg; 1 U ofDNA polymerase activity corresponds to the incorporation of 1 nmol of dNMPinto acid-precipitable material in 60 min at 37°C.

HIV-1 RT RNA-dependent DNA polymerase activity assay. RNA-dependentDNA polymerase activity was assayed as follows. A final volume of 25 ml con-tained buffer A (50 mM Tris-HCl [pH 7.5], 1 mM dithiothreitol, 0.2 mg of bovineserum albumin per ml, 4% glycerol), 10 mM MgCl2, 0.5 mg of poly(rA)-oligo(dT)(10:1) (0.3 mM 39-OH ends), 10 mM [3H]dTTP (1 Ci/mmol), and 2 to 4 nM RT.Reaction mixtures were incubated for 10 min at 37°C. Aliquots (20 ml) were thenspotted on GF/C glass fiber 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-precipitableradioactivity was quantitated by scintillation counting.

Inhibition assays. Inhibition assay reactions were performed under the con-ditions described for the HIV-1 RT RNA-dependent DNA polymerase activityassay. Incorporation of radioactive dTTP into poly(rA)-oligo(dT) at differentconcentrations of DNA or dNTPs was monitored in the presence of increasingamounts of inhibitor. Data were then plotted according to Dixon (6).

Kinetics of inhibitor binding. HIV-1 RT (20 to 40 nM) was incubated for 2min at 37°C in a final volume of 4 ml in the presence of buffer A, 10 mM MgCl2,and 100 nM 39-OH ends (for the formation of the RT–template-primer [TP]complex) or in the same mixture complemented with 10 mM unlabeled dTTP (forthe formation of the RT-TP-dNTP complex). The inhibitor to be tested was thenadded to a final volume of 5 ml, at a concentration at which [EI]/[E0] 5 1 2{1/[(1 1 [I])/Ki]} . 0.9, where [E0] is free enzyme at the beginning of thereaction and [EI] is the enzyme-inhibitor complex. Then, 145 ml of a mixturecontaining buffer A, 10 mM MgCl2, and 10 mM [3H]dTTP (5 Ci/mmol) wasadded at different time points. After an additional 10 min of incubation at 37°C,50-ml aliquots were spotted on GF/C filters, and acid-precipitable radioactivitywas measured as described for the HIV-1 RT RNA-dependent DNA polymeraseactivity assay. The quantity (vt/v0) representing the normalized difference be-tween the amount of dTTP incorporated at the zero time point and at thedifferent time points was then plotted against time.

Kinetic parameter calculation. All values were calculated by non-least-squarescomputer fitting of the experimental data to the appropriate rate equations. Kdvalues for the various reaction intermediates (Fig. 1A) were calculated accordingto the equations for uncompetitive inhibition and for mixed noncompetitiveinhibition, respectively (6):

v 5 Vm/@~Km/@S#! 1 ~1 1 @I#/Kd(ter))] (1)

v 5 $Vm/@~1 1 @I#!/Kd!#}/@1 1 ~Km/@S#!$@~1 1 @I#!/Kd!#@~1 1 @I#!Kd9!#} (2)

where Kd is Kd(bin) under the assay conditions specified in Fig. 1B, left panel, orKd is Kd(E) under the conditions specified in Fig. 1B, right panel. Kd9 equalledKd(ter) in both cases.

Apparent rate of binding (kapp) values were determined by fitting the exper-imental data to the single-exponent equation (vt/v0) 5 e2kappt, where t is time.

Determination of synergy. Two approaches were used to determine synergy.The first was based on the median-effect method of Chou and Talalay as mod-ified by Villahermosa et al. (31). Dose-response curves for the interaction be-tween 39-azido-29, 39-dideoxythymidine triphosphate (AZTTP) and efavirenz orsefavirenz were generated by fitting the experimental data to the equation

[EI] 5 [EI]max/[11(D50/[I])m] (3)

where [EI] is the fraction of inhibited enzyme, which was expressed as percentinhibition with respect to the control reaction without the inhibitor, and D50 wasthe concentration of inhibitor giving 50% inhibition. Accordingly, [EI]max wasassumed to be 100% at infinite inhibitor concentrations. The parameter m is thesigmoidicity term.

The interaction index (I) was calculated according to the relationship I 5(d1/D1) 1 (d2/D2), where d1 and d2 were the doses of the inhibitor giving 50%inhibition when tested in the combination (d1 1 d2) and D1 and D2 are the D50values of the corresponding inhibitor when tested alone. The I parameter isequivalent to the combin. index of Chou and Talalay. I of ,1 indicates synergy,I of .1 indicates antagonism, and I of 1 indicates additivity, according to themutually exclusive model for additivity of Chou and Talalay.

The second method was based on the Lowe additivity model as modified byGreco et al. (10). Dose-response curves for the interaction between AZTTP andefavirenz or sefavirenz were assumed to follow Hill’s model and were generatedby fitting the experimental data to the equation

E 5 @Econ([I]/D50)m#/@1 1 ~D50/[I])m# (4)

where E is the observed effect (percent activity), Econ is the control effect (activityin the absence of the inhibitor), and all the other parameters are as definedabove. Effective inhibitor concentrations at different fractional inhibition levelswere calculated from the parameters D50, [EI]con, and m according to theequation

Dx 5 D50[E/~Econ 2 E!]1/m (5)

where Dx is the dose of drug giving a particular percent inhibition. I was thencalculated according to Greco et al. (10) with the equation

I 5 ~D1/Dx1! 1 ~D2/Dx2! (6)

where D1 and D2 are the concentrations of the drugs in combination and Dx1 andDx2 are the predicted inhibitory concentration of each drug giving the observedeffect of the combination D1 1 D2. I of ,1 indicates synergy, I of .1 indicatesantagonism, and I of 1 indicates additivity, according to the Lowe additivitymodel (10).

All the analyses were based on the results of three independent experiments

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for each drug combination, and the standard deviations (SD) for each parameterestimate are indicated.

RESULTS

Synthesis. Efavirenz was synthesized as previously described(29). The thio-substituted analog sefavirenz was obtained bydirect sulfuration (16) of efavirenz with Lawesson’s reagent(Fig. 2). Although the sulfuration of carbamates has beenreported (16), this is the first example of sulfuration of ben-zoxazin-2-ones with this reagent. The reaction yield is poor;however, after several recyclings of the unreacted product, thethio-substituted derivative can be obtained with a yield of.95%.

Kinetic analysis. A schematic reaction pathway for the in-hibition of HIV-1 RT-catalyzed RNA-dependent DNA synthe-sis by efavirenz and/or sefavirenz is depicted in Fig. 1A. Thedata were analyzed according to the steady-state assumptionthat shortly after the initiation of the reaction, the enzyme-substrate complex is formed at the same rate as it dissociates.According to the ordered mechanism of the polymerizationreaction, whereby TP binds first, followed by the addition ofdNTP, the HIV-1 RT can be present in three different catalyticforms: as a free enzyme, in a binary complex with TP, and in aternary complex with TP and dNTP (12, 17, 23, 24). Accord-ingly, it was assumed that the inhibitor could bind to any ofthese different forms and at the corresponding equilibria re-ported in Fig. 1A. The assay conditions used allowed proces-

FIG. 1. Simplified steady-state reaction scheme for interactions between HIV-1 RT and the inhibitors. (A) Steady-state reaction scheme for HIV-1 RT. I, inhibitor;Km, apparent Michaelis-Menten constant for the various enzyme-substrate complexes; Km9, apparent Michaelis-Menten constant for the various enzyme-substrate-inhibitor complexes; Kd(E), equilibrium dissociation constant for the RT-I complex; Kd(bin), equilibrium dissociation constant for the RT-I-TP complex; Kd(ter),equilibrium dissociation constant for the RT-I-TP-dNTP complex. kon and koff represent the binding and dissociation rates, respectively, for the correspondingequilibrium constants. (B) Simplified kinetic pathways for the inhibition of HIV-1 RT by efavirenz and sefavirenz, depending on various assay conditions. Abbreviationsare as defined in panel A.

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sive synthesis by RT; thus, the complex of the enzyme with itsproducts does not differ from the RT-TP complex, in the sensethat the former shuttles back to the RT-TP state followingincorporation and translocation along the template at a rateequal to the turnover number, kcat. The resulting rate equationfor such a system is very complex and too impractical to beused. For these reasons, the general steady-state kinetic anal-ysis was simplified by varying one of the substrates (either TPor dNTP) while the other was kept constant, as outlined in Fig.1B. Because of the ordered mechanism of the two-substratereaction catalyzed by HIV-1 RT, when the TP concentrationwas kept constant at a saturating level (50-fold over its Km) andinhibition was analyzed with various concentrations of dNTPs,at the steady state all the input RT was in the form of theRT-TP binary complex and only two forms of the enzyme (thebinary complex and the ternary complex with dNTP) couldreact with the inhibitor, as shown in the left panel of Fig. 1B.Similarly, when the dNTP concentration was kept constant ata saturating level (fivefold over its Km) and inhibition wasanalyzed with various TP concentrations, RT was present ei-ther as a free enzyme or in the ternary complex with TP anddNTP, as shown in the right panel of Fig. 1B. Complex forma-tion between RT and its substrates was assumed to occur withrapid equilibrium kinetics, so in the presence of saturatingdNTP, conversion of the binary RT-TP complex into a ternarycomplex was assumed to occur at a much higher rate thaninhibitor binding. Thus, in both cases, simple steady-state ki-netic analysis could be used for the determination of the equi-librium dissociation constants of the different enzyme-inhibitorcomplexes.

Efavirenz and sefavirenz bind with different affinities to thebinary RT-TP and ternary RT-TP-dNTP complexes. The effectof increasing concentrations of efavirenz on the RNA-depen-dent DNA synthesis catalyzed by HIV-1 RT on poly(rA)-oli-go(dT) was tested with saturating TP and in the presence oftwo different dTTP concentrations. Under these conditions,only the binary RT-TP and the ternary RT-TP-dNTP com-plexes were available for inhibitor binding (Fig. 1B). The re-sults are shown in Fig. 3A in the form of Dixon plots. When arange of concentrations of inhibitor from 5 to 180 nM wastested, the resulting inhibition displayed nonlinear kinetics,with a change in the slope of the curves. Inspection of thecurves at low efavirenz concentrations (5 to 20 nM) showed auncompetitive mechanism of inhibition. Fitting of the data toequation 1 for fully uncompetitive inhibition (see Materialsand Methods) gave a value for the equilibrium dissociationconstant for the ternary complex [Kd(ter)] of 4 nM, consistentwith the reported Ki for efavirenz against HIV-1 RT. When theinhibitor was tested at higher concentrations, however, theresulting Dixon plot was diagnostic of a mixed noncompetitive

mechanism of inhibition (Fig. 3A, inset). Fitting of the data toequation 2 gave a value for the equilibrium dissociation con-stant for the binary complex [Kd(bin)] of 30 nM. The two linesintersected below the x axis, according to the relationshipKd(bin) . Kd(ter). When sefavirenz was tested under the sameconditions, a similar behavior was observed (Fig. 3B), withuncompetitive inhibition at low inhibitor concentrations and acalculated Kd(ter) value of 8 nM and mixed noncompetitiveinhibition at higher concentrations (Fig. 3B, inset). The calcu-lated Kd(bin) was 230 nM, indicating a reduced affinity of sefa-virenz for the binary RT-TP complex with respect to efavirenz.Again, the two lines intersected below the x axis, according tothe relationship Kd(bin) . Kd(ter). It should be noted that theobserved mechanism is indicated with the term “uncompeti-tive” according to the standard nomenclature (6), in order toindicate the preferential binding of the inhibitor to the en-zyme-substrate complex, a behavior which makes it distinctfrom other noncompetitive types of inhibition.

Efavirenz and sefavirenz show different affinities for bindingto free RT. The effect of increasing concentrations of efavirenzon the RNA-dependent DNA synthesis catalyzed by HIV-1 RTwith saturating dNTP was tested in the presence of two differ-ent TP (39-OH primer ends) concentrations. According to Fig.1B, right panel, the inhibitor could interact only with the freeenzyme or the ternary RT-TP-dNTP complex. The results areshown in Fig. 4A. Dixon plots of the experimental data showednonlinear kinetics, with uncompetitive inhibition at efavirenzconcentrations of 5 to 20 nM (Fig. 4A) and mixed noncompet-itive inhibition at higher concentrations (20 to 180 nM) (Fig.4A, inset). The Kd(ter) value calculated according to the un-competitive pathway was 4.5 nM, in good agreement with thevalue derived in the previous experiments (Fig. 3A). On theother hand, the equilibrium dissociation constant calculatedfor the free enzyme [Kd(E)] according to the mixed noncom-petitive mechanism was 170 nM, indicating a poor affinity ofefavirenz for the free enzyme. When sefavirenz was tested, asimilar concentration dependence of the mechanism of inhibi-tion was observed, with an uncompetitive Kd(ter) value of 7.5nM (Fig. 4B). However, sefavirenz showed a significantly loweraffinity for the free enzyme than efavirenz, with a Kd(E) derivedaccording to the mixed noncompetitive mechanism of 750 nM.In both cases, the curves obtained at high concentrations ofinhibitor intersected below the x axis, in accordance with therelationship Kd(E) . Kd(ter) (6).

Determination of the rates for the formation and dissocia-tion of the different RT-substrate-inhibitor complexes withefavirenz and sefavirenz. Both efavirenz and sefavirenzshowed increasing affinities for the different catalytic forms ofRT. However, given that Kd 5 koff/kon, the observed differ-ences in the equilibrium dissociation constants could reflect

FIG. 2. Drug structures.

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various combinations of the association and dissociation rateskon and koff, respectively, for the different enzyme intermedi-ates. In order to address this point, the apparent rate of bind-ing (kapp) of the inhibitor to the binary RT-TP and ternaryRT-TP-dNTP complexes was measured and koff an kon valueswere derived from the relationships kapp 5 kon(Kd 1 [I]) andKd 5 koff/kon (20, 28). The experiment was done as describedin Materials and Methods. Since the amount of dTTP incor-porated at zero time (v0, uninhibited reaction) was propor-tional to the amount of RT-TP or RT-TP-dNTP present at thebeginning of the reaction, v0 was also proportional to [E]0. Theincorporation measured at subsequent time points (vt) wasdirectly related to the amount of uninhibited enzyme ([E]t 5[E]0 2 [EI]t). Thus, since [E]0 decreased at a rate equal to theformation of the [EI] complex, the analysis of the dependenceof vt/v0 on time allowed estimation of the kapp value.

Parallel reactions were run for 10 min at 37°C with enzyme,substrates, and inhibitors at the same concentrations as in thediluted mixture but without any preincubation. The incorpo-ration observed in these control reactions was typically be-tween 5 and 10% of v0 and was substracted as background. Asshown in Fig. 5, the data points fitted the simple exponentialrelationship vt/v0 5 e2kapp t. The calculated kapp, kon, and koffvalues for efavirenz and sefavirenz are listed in Table 1, alongwith the corresponding Kd values. In both cases, formation ofthe RT-inhibitor-TP complex was characterized by lower asso-ciation and higher dissociation rates with respect to the RT-inhibitor-TP-dNTP complex. Similar experiments were per-formed with nevirapine, another NNRTI included forcomparison. Under the different experimental conditionstested, nevirapine, contrary to efavirenz and sefavirenz,showed a pure noncompetitive mechanism of action, as well asidentical kon and koff values for binding to the different enzy-matic forms of RT.

Efavirenz and sefavirenz show synergistic inhibition ofHIV-1 RT in combination with AZTTP. The effect of variouscombinations of efavirenz and AZTTP on the RNA-dependentDNA synthetic activity of HIV-1 RT was tested as described byVillahermosa et al. (31) and Greco et al. (10) (see Materialsand Methods). Briefly, dose-response curves for each inhibitoralone were obtained within a wide range of concentrations andcompared to inhibition curves obtained with combinations ofthe inhibitors at a fixed molar ratio. This ratio was determinedaccording to the different potencies of the compounds, ensur-ing in this way that both inhibitors significantly contributed tothe inhibition observed. Data were fitted to the correspondingequations (see Materials and Methods), and the calculatedparameters for both efavirenz and sefavirenz combinationswith AZTTP are listed in Table 2. Efavirenz was found to besignificantly synergistic with AZTTP in its inhibition of HIV-1RT at fractional inhibition levels of 20 to 90%. This result wasin agreement with previous observations indicating a synergis-tic action of efavirenz in combination with NRTIs in in vitroRT inhibition assays. The thio-substituted analog sefavirenzdisplayed a similar behavior, but its synergistic effect wasslightly reduced, as reflected by higher indexes estimated forthe different fractional inhibition levels. The reduced I forsefavirenz can be explained in terms of the observed reducedaffinity of the thio-substituted analog for the RT-TP-dNTPcomplex (Table 1).

A modeled tridimensional structure of efavirenz shows anal-ogy to nevirapine. As shown in Fig. 6, the energy-minimizedtridimensional structure of efavirenz showed a conformationsimilar to the one assumed by the NNRTI nevirapine in theRT-nevirapine complex, as revealed by crystal structure deter-mination. In particular, the oxygen substituent at position C-2

FIG. 3. Inhibition of HIV-1 RT by efavirenz and sefavirenz, depending onvariable dNTP concentrations. Reactions were carried out as described in Ma-terials and Methods. (A) Dixon plot of RT inhibition by efavirenz in the presenceof 2 mM dTTP (circles) or 10 mM dTTP (triangles) at inhibitor (I) concentrationsof 5 to 25 nM or 25 to 160 nM (inset). (B) Dixon plot of RT inhibition bysefavirenz in the presence of 2 mM dTTP (circles) or 10 mM dTTP (triangles) atinhibitor concentrations of 5 to 20 nM or 20 to 160 nM (inset). Error bars showSD.

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of efavirenz is perfectly superimposable and coplanar with theoxygen at position C-6 of nevirapine. For nevirapine, this po-sition has been proposed to make important contacts withdifferent residues of the NNRTI-binding pocket, including Phe227 and Val 106. It is conceivable that similar contacts are alsoimportant for the stabilization of efavirenz. Thus, substitutionof the oxygen with a sulfur atom at this position in sefavirenzcould impair some of these interactions.

DISCUSSION

A detailed understanding of the mechanism of action ofefavirenz is an obligatory step in the development of newderivatives with a better activity profile against drug-resistantmutants. In the present study, the equilibrium dissociationconstants for efavirenz binding to the different catalytic formsof HIV-1 RT as well as the association and dissociation rateshave been determined using a steady-state kinetic approach. Inaddition, the same enzymological analysis has been extendedto its thio-substituted analog, sefavirenz, which showed com-parable activity in vitro against RT. Both compounds displayednonlinear kinetics of inhibition (Fig. 3 and 4). They acted aspurely uncompetitive inhibitors at low drug concentrations (5to 50 nM) and as mixed noncompetitive inhibitors at higherdoses (50 to 500 nM). According to the reaction scheme illus-trated in Fig. 1A and to the equilibrium dissociation constantsfor inhibitor binding listed in Table 1, this behavior can beinterpreted in terms of the relative affinities for the differentcatalytic forms of the enzyme. Both efavirenz and sefavirenzshowed increasing affinities for the different forms of RT in thefollowing order: free enzyme , (i.e., bound with lower affinity)binary RT-TP complex , ternary RT-TP-dNTP complex.Thus, when only the binary RT-TP and the ternary RT-TP-dNTP complexes were available for inhibitor binding (Fig. 1B,left panel), at low drug concentrations the inhibitor interactedonly with the ternary complex, the affinity for the binary com-plex being too low. In such a situation, the inhibition followed

FIG. 4. Inhibition of HIV-1 RT by Efavirenz and Sefavirenz, depending onvariable TP [poly(rA)-oligo(dT)] concentrations. Reactions were carried out asdescribed in Materials and Methods. (A) Dixon plot of RT inhibition by efa-virenz in the presence of 0.03 mM TP (as 39-OH ends) (triangles) or 0.15 mM TP(circles) at inhibitor concentrations of 5 to 25 nM or 25 to 160 nM (inset). (B)Dixon plot of RT inhibition by sefavirenz in the presence of 0.03 mM TP (as39-OH ends) (triangles) or 0.15 mM TP (circles) at inhibitor concentrations of 20to 160 nM or 80 to 800 nM (inset). Error bars show SD.

FIG. 5. Determination of the apparent rate of inhibitor binding to the dif-ferent enzyme-substrate complexes. Reactions were carried out as described inMaterials and Methods. Computer fitting of the data to a simple exponentialequation was used to generate progress curves for efavirenz (open symbols) orsefavirenz (filled symbols) binding to the RT-TP (circles) or RT-TP-dNTP (tri-angles) complexes. t, time. Error bars show SD.

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a fully uncompetitive mechanism according to equation 1 (seeMaterials and Methods). When the inhibitor concentrationswere raised, a ternary complex with the inhibitor (RT-inhibi-tor-TP) could also be formed; thus, the inhibition was gov-erned by equation 2, describing a mixed noncompetitive mech-anism. The same was true for the example illustrated in Fig.1B, right panel. Under these conditions, at low efavirenz andsefavirenz concentrations, only the quaternary complex withthe inhibitor (RT-inhibitor-TP-dNTP) was formed, revealingpurely uncompetitive inhibition. At higher drug doses, an RT-inhibitor complex started to be formed, resulting in mixednoncompetitive inhibition.

Both efavirenz and sefavirenz displayed the lowest affinitytoward the free enzyme, but with a different selectivity ratio,Kd(E)/Kd(ter), which was 42.5 for efavirenz but which was in-creased to 93.7 for Sefavirenz. The greatest difference betweenefavirenz and sefavirenz was seen in the selectivity for theternary complex versus the binary complex, since the Kd(bin)/Kd(ter) value of 7.5 for efavirenz was increased to 28.5 forsefavirenz.

Determination of the binding (kon) and dissociation (koff)rates for the interaction of the two inhibitors with the differentcatalytic forms of HIV-1 RT showed that the increases in thekoff value for the conversion of the RT-inhibitor-TP-dNTPcomplex into the RT-inhibitor-TP complex were similar forboth inhibitors (2- and 2.5-fold, respectively), whereas the konvalue for drug binding to the RT-inhibitor-TP complex was

reduced 10-fold for sefavirenz but only 2.5-fold for efavirenz.The rates of binding of the two inhibitors to the differentenzyme-substrate complexes were well below the diffusionlimit (on the order of 104 M21 s21) but substantially higherthan those for nevirapine (another clinically used NNRTI)(20). In comparison, the estimated rate for RT-TP complexformation was shown to be on the order of 106 M21 s21, thusjustifying the assumption of faster equilibrium kinetics forbinding of the enzyme to its substrates than to the inhibitor(12, 23, 24). The quaternary complex RT-inhibitor-TP-dNTPshowed very low koff values, on the order of 1024 s21 for bothcompounds (Tables 1 and 2), typical of tightly binding inhibi-tors.

Both efavirenz and sefavirenz showed significant synergy incombination with AZTTP. Comparison of the observed D50

values with the corresponding d1 and d2 values (Table 2)showed an apparent 6- to 12-fold increase in the potency ofAZTTP when combined with efavirenz or sefavirenz. Sincethese inhibitors have a greater affinity for the RT-TP-dNTPcomplex (Table 1), it is unlikely that this increase was due to aneffect on the kon for complex formation between the RT-inhibitor-TP intermediate and AZTTP. The observed synergywas most likely due to the accumulation of the chain-termi-nated complex RT-TP-AZTMP, which in turn could be con-verted to a dead-end RT-inhibitor-TP-AZTMP quaternarycomplex with a very low dissociation rate (koff), thus account-

TABLE 1. Kinetic parameters for binding of efavirenz, sefavirenz, and nevirapine to different catalytic forms of HIV-1 RT

Drug Catalytic formMean 6 SD

Kd (nM) kapp (s21) kon (s21 M21)a koff (s21)b

Efavirenz RT 170 6 5 NAc NA NART-TPd 30 6 2 (6 6 1) 3 1023 (1.3 6 0.2) 3 104 (4 6 1) 3 1024

RT-TP-dNTPe 4 6 0.5 (7 6 1) 3 1023 (3.4 6 0.2) 3 104 (1.6 6 0.5 3 1024

Sefavirenz RT 750 6 15 NA NA NART-TPd 230 6 20 (4 6 1) 3 1023 (0.5 6 0.1) 3 104 (1 6 0.5) 3 1023

RT-TP-dNTPe 8 6 0.5 (6 6 1) 3 1023 (5.5 6 0.5) 3 104 (4 6 1) 3 1024

Nevirapine RT 400 6 15 NA NA NART-TPd 400 6 20 (5.7 6 1) 3 1023 (0.15 6 0.03) 3 104 (6 6 0.5) 3 1024

RT-TP-dNTPe 450 6 25 (6 6 1) 3 1023 (0.2 6 0.05) 3 104 (6 6 1) 3 1024

a Calculated from kapp 5 kon (Ki 1 [I]).b Calculated from Kd 5 koff/kon.c NA, not applicable.

TABLE 2. Calculated interaction parameters for combinations of AZTTP with efavirenz or sefavirenza

Drug(s) Reference

Mean 6 SD

D50(nM)

d1(nM) d2 (nM) d1 1 d2

(nM)

I

50 20 90

Efavirenz (d1) 31 11 6 2 NA NA NA NA10 13 6 2 NA NA NA NA NA NA

Sefavirenz (d1) 31 25 6 1 NA NA NA NA10 30 6 1 NA NA NA NA NA NA

AZTTP (d2) 31 19 6 2 NA NA NA NA10 21 6 2 NA NA NA NA NA NA

Efavirenz 1 AZTTP 31 NA 5.9 6 0.3 1.4 6 0.2 7.3 6 0.3 0.6 6 0.210 NA 5.6 6 0.3 1.5 6 0.2 7.1 6 0.3 0.5 6 0.2 0.58 6 0.05 0.4 6 0.1

Sefavirenz 1 AZTTP 31 NA 11.2 6 0.2 2.8 6 0.3 14 6 1 0.68 6 0.0510 NA 13.6 6 0.2 3.4 6 0.3 17 6 1 0.61 6 0.05 0.64 6 0.04 0.55 6 0.1

a See the text for details. NA, not applicable. I50, I20, and I90, Berenbaum’s I for 50, 20, and 90% inhibition, respectively.

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ing for the observed effect on the equilibrium constant forAZTTP inhibition.

Other classes of NNRTIs have been reported to follow com-plex kinetics of inhibition (2, 3, 8). For example, carboxanilideUC38 interacted specifically either with the binary RT-TPcomplex or with the ternary RT-TP-dNTP complex; however,the equilibrium dissociation constants were 100- to 1,000-foldhigher than the corresponding constants for efavirenz, thussuggesting much lower binding and/or higher dissociationrates. The thiocarboxanilide derivative UC781 showed a pref-erence for the different catalytic forms of RT similar to that ofefavirenz, but the dissociation rate for the RT-inhibitor-TP-dNTP complex was 10-fold higher than the corresponding ratefor efavirenz, resulting an even more tightly binding inhibitor.Thus, efavirenz and its thio-substituted derivative sefavirenzappear to be unique in their mechanism of action, being se-lective tightly binding inhibitors of the ternary RT-TP-dNTPcomplex. Efavirenz is thus the first clinically approved NNRTIto show this property. Preliminary in vitro characterization ofRT inhibition by efavirenz showed either noncompetitive ormixed but not uncompetitive inhibition. This difference couldbe attributable either to the TP utilized or to the range ofinhibitor concentrations used for the analysis.

We found that there was a reduced affinity of sefavirenz forboth free RT and the RT-TP complex. This selectivity for theternary complex can be explained by the crystallographic struc-tures of different RT-substrate-inhibitor complexes (13, 14, 18,25). The specific effect seen for the substitution of a sulfuratom in place of an oxygen atom in reducing the affinity ofsefavirenz for both free RT and the RT-TP binary complexcould reflect the different structures of unbound RT with re-spect to the binary or ternary complexes with its substrates. Infact, in the ternary complex, there are large-scale structuraldifferences as well as local conformational changes in compar-ison with both the free enzyme and the RT-TP complex. Thus,it is possible that the structure of the ternary complex displayedthe optimal side-chain conformation for inhibitor binding andthat the thio substitution at position C-6 of sefavirenz madethis compound more sensitive to the structural differences be-tween the different catalytic forms of the enzyme-substratecomplexes.

ACKNOWLEDGMENTS

This work was supported by an ISS-AIDS fellowship (to G.M.); bythe CNR Target Project on Biotechnology (to S.S.); by the ISS II AIDSResearch National Program, project 2.1.3, research proposal 133 (toS.S.); and by TMR grant ERBMRXCT 970125 (to S.S.). FAR 1999(Universita degli Studi, Pavia, Italy) is gratefully acknowledged.

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