Inhibition of Human Immunodeficiency Virus Type 1 Reverse Transcriptase, RNase H, and Integrase Activities by Hydroxytropolones

Human immunodeficiency virus type I reverse transcriptase (RT)possesses distinct DNA polymerase and RNase H sites, whereasintegrase (IN) uses the same active site to perform 3'-end processingand strand transfer of the proviral DNA. These four enzymaticactivities are essential for viral replication and require metalions. Two Mg²⁺ ions are present in the RT polymerase site, andone or two Mg²⁺ ions are required for the catalytic activitiesof RNase H and IN. We tested the possibility of inhibition ofthe RT polymerase and RNase H as well as the IN 3'-end processingand transfer activities of purified enzymes by a series of 3,7-dihydroxytropolonesdesigned to target two Mg²⁺ ions separated by $~$ 3.7 Å. TheRT polymerase and IN 3' processing and strand transfer activitieswere inhibited at submicromolar concentrations, while the RNaseH activity was inhibited in the low micromolar range. In allcases, the lack of inhibition by tropolones and O-methylated3,7-dihydroxytropolones was consistent with the active moleculesbinding the metal ions in the active site. In addition, inhibitionof the DNA polymerase activity was shown to depend on the Mg²⁺concentration. Furthermore, selective inhibitors were identifiedfor several of the activities tested, leaving some potentialfor design of improved inhibitors. However, all tested compoundsexhibited cellular toxicity that presently limits their applications.

INTRODUCTION

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Introduction

Human immunodeficiency virus type 1 (HIV-1) is the causativeagent of AIDS. Synthesis and integration of the HIV-1 proviralDNA into the host cell genome are the result of four catalyticactivities. The virally encoded reverse transcriptase (RT) containspolymerase and RNase H active sites (1, 15, 22), whereas theintegrase (IN) possesses 3'-end processing and strand transferactivities achieved by the same catalytic site (6, 18). Allfour activities are essential for HIV-1 replication, but onlythe polymerase is currently targeted by FDA-approved inhibitors.Several drugs inhibiting the IN strand transfer activity haveproceeded to clinical trials (10).

Combinations of RT and protease inhibitors offer highly effective,durable treatment options (10). However, the high incidenceof resistance in therapy-experienced and newly infected patientsunderscores the need for new antiretroviral agents (11, 17,32, 51). Increasing the number of anti-AIDS drugs would alsofacilitate the management of their side effects (37).

One strategy to minimize the emergence of drug resistance maybe the design of compounds that would interact with amino acidsor cofactors that are essential for catalysis. A common featureof the RT and IN catalytic activities is the requirement ofa metal cofactor, which is most likely Mg²⁺ in vivo. Two Mg²⁺ions separated by 3.57 Å have been observed in the polymeraseactive site of RT in complex with a primer/template DNA duplexand an incoming nucleotide (27). The three aspartate residuesthat bind Mg²⁺ and the metal ions themselves are essential forDNA synthesis (28, 40).

Divalent metal ions, such as Mg²⁺ or Mn²⁺, are also essentialfor HIV-1 RNase H activity (7), but the number of ions involvedin the RNA cleavage reaction is still unclear. Two Mn²⁺ ionsseparated by 4 Å and coordinated to D443, E478, D498,and D549 have been observed in the isolated HIV-1 RNase H domain(8), whereas only one Mg²⁺ ion, bound to D443 and D549, wasseen in the RNase H domain of RT bound to a DNA duplex, in thepresence of an incoming deoxynucleoside triphosphate (dNTP)(27).

Similarly, IN activities require divalent metal ions (4, 30,49), but the number of metal ions involved in catalysis is stillsubject to debate (23). Structural studies of IN revealed asingle binding site for Mg²⁺ and Mn²⁺ (20, 35), while two Cd²⁺or Zn²⁺ ions separated by 3.6 to 3.7 Å were observed (4,5, 49). Indeed, it is believed that a second Mg²⁺ ion is carriedinto the integrase active site by the substrate (31).

In this study, we tested the possibility of inhibition of theRT polymerase, RNase H, and the IN 3'-end processing and transferactivities by compounds designed to target two Mg²⁺ ions separatedby $~$ 3.7 Å. To this aim, we used 3,7-dihydroxytropolones(Fig. 1), which have been shown to inhibit inositol monophosphataseby binding the Mg²⁺ ions of the catalytic site, thus preventingbinding of the substrate (41, 42). To our knowledge, this isthe first time that this strategy has been used to inhibit thepolymerase activity of HIV-1 RT, and there has been only onepublished rational attempt to simultaneously target two metalions in the RNase H active site (29). While this work was inprogress, it became increasingly likely that several IN inhibitorfamilies identified by "blind" or focused screening bind magnesiumin the IN active site (12, 23, 39). However, it is not completelyclear whether these inhibitors bind one or two metal ions (19,23). Recently, a rationally designed IN inhibitor that can bindtwo Mg²⁺ ions has been described (33).

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FIG. 1. Structure of 3,7-dihydroxytropolone and schematic drawing of its interaction with two Mg²⁺ ions separated by 3.7 Å (modified from reference 42 with permission of the publisher).

In this study, we showed that the RT polymerase and IN 3' processingand strand transfer activities were inhibited at submicromolarconcentrations, while 3,7-dihydroxytropolones inhibited theRNase H activity in the low micromolar range. In all cases,the lack of inhibition by modified tropolones and O-methylated3,7-dihydroxytropolones is consistent with the active moleculesbinding the metal ions in the active site. In line with thismechanism, inhibition of the RT polymerase by 3,7-dihydroxytropolonesand acetylated derivatives was very sensitive to the magnesiumconcentration. Furthermore, selective inhibitors were identifiedfor several of the activities tested. Even though all testedcompounds were toxic to cells, the numerous possibilities forderivatization of the dihydroxytropolone core potentially offerthe prospect for increase in selectivity and decrease in toxicityof these inhibitors.

MATERIALS AND METHODS

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Materials and Methods

Oligodeoxyribonucleotides. The sequences of the oligodeoxyribonucleotides used in thisstudy are as follows: for ODN, 5'-GTC CCT GTT CGG GCG GGA-3';ODN35, 5'-TTA GCC AGA GAG GCT CCC AGG CTC AGA TCT GGT CT-3';LE18, 5'-TAC GAA TTC TAA TAC GA CGA CTC ACT ATA GGT CTC TCTGGT TAG ACC AGA TCT GAG C-3'; U5B, 5'-GTG TGG AAA ATC TCT AGCAGT-3'; U5B-2, 5'-GTG TGG AAA ATC TCT AGC A-3'; and U5A, 5'-ACTGCT AGA GAT TTT CCA CAC-3'. All oligodeoxyribonucleotides werepurchased from Eurogentec (Belgium).

Templates, primers, and enzymes. RNA_1-311, encompassing nucleotides (nt) 1 to 311 of HIV genomicRNA (MAL isolate), was used as the template for the RT assays.It was synthesized by in vitro transcription and purified aspreviously described (36).

The template used for the RNase H assays was obtained by PCRwith two partially complementary oligodeoxyribonucleotidesgenerating a double-stranded DNA containing the T7 RNA polymerasepromoter upstream of the region coding for nucleotides 1 to47 of HIV genomic RNA (RNA_1-47) (HXB2 isolate). Ten amplificationcycles (30 s at 94°C, 30 s at 38°C, and 30 s at 72°C)were performed with 100 pmol of primers ODN35 and LE18 and 4U DyNAzyme. The PCR product was purified on a 3% low-melting-pointagarose gel containing ethidium bromide. The band containingthe transcription template was cut out under UV light and heatedfor 20 min at 50°C in the presence of phenol. The extractedDNA was ethanol precipitated and resuspended in water. RNA_1-47was in vitro transcribed and purified as described previously(36). RNA_1-47 (2 µg) was dephosphorylated with 2 U calfintestine phosphatase for 1 h at 37°C in 100 mM NaCl, 50mM Tris-HCl (pH 7.9), 10 mM MgCl₂, and 1 mM dithioerythritol.After a phenol-chloroform extraction, RNA_1-47 was ethanol precipitatedand resuspended in water.

RNA_1-47 and oligodeoxyribonucleotides ODN, U5B, and U5B-2 were5' end labeled with [ ${gamma}$ -³²P]ATP (3,000 Ci/mmol) and phage T4 polynucleotidekinase. ODN and RNA_1-47 were purified on 8% denaturing polyacrylamidegels; U5B and U5B-2 were purified with a MicroSpin G-25 column(Amersham Bioscience).

In order to form the primer/template complexes, RNA_1-311 andODN or RNA_1-47 and ODN35 were first denatured in water for 2min at 90°C and chilled on ice. Nucleic acids were annealedat 70°C for 20 min in 100 mM NaCl and cooled on ice for30 min. To prepare the substrates for integrase, U5A was annealedto either U5B or U5B-2 in 100 mM NaCl by being heated at 80°C,followed by a slow cooling.

An RNA_1-47 ladder was prepared by incubating RNA_1-47 (15,000cpm) and 1 µg Saccharomyces cerevisiae tRNA for 15 minat 90°C in 0.1 M NaHCO₃-Na₂CO₃. The RNA_1-47 fragments wereprecipitated by addition of 0.2 M LiClO₄, and the pellet wasresuspended in water.

Plasmid expressing wild-type HIV-1 RT was kindly provided tous by Torsten Unge (Uppsala, Sweden), together with the protocolsfor protein overexpression and purification. RNase H^–RT was obtained by introducing the E478Q mutation in this plasmid(44). Wild-type IN was expressed and purified as described previously(30).

The 3,7-dihydroxytropolones, mono- or disubstituted at positions4 or 4,6 (referred to as the SP compounds [Fig. 2]), were synthesizedas previously described (41). Stock solutions (20 mM) were preparedin dimethyl sulfoxide. We checked that this solvent did notaffect any of the tested enzymatic activities at the maximumconcentration used in this study.

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FIG. 2. Structures of the tropolones and dihydroxytropolones used in this study. OAc, acetate.

RT assays. (i) Minus-strand "strong-stop" DNA synthesis. ODN/RNA_1-311 (primer/template) complex (10 nM) was preincubatedat 37°C for 4 min with 30nM HIV-1 RNase H^– RT in 50mM Tris-HCl (pH 8.3), 50 mM KCl, 6 mM MgCl₂, and 1 mM dithioerythritol.Reverse transcription was initiated by addition of dNTPs (50µM each) in the presence or absence of SP compounds. Reactionswere stopped at times ranging from 30 s to 30 min by additionof 1 volume of formamide containing 50 mM EDTA, and thereaction products were analyzed on 8% denaturing polyacrylamidegels. Radioactivity was quantified with a BioImager BAS 2000(Fuji), using the MacBAS 2.5 (Fuji) software.

(ii) Polymerase-dependent RNase H cleavage. ODN35/RNA_1-47 complex (10 nM) was added to 10 nM wild-type HIV-1RT in the presence or absence of SP compounds. RNase H assayswere carried out for 15 s to 30 min, and reactions were stoppedby mixing an equal volume of formamide containing 50 mM EDTA.Cleavage products were resolved using denaturing 15% polyacrylamidegels and analyzed as described above.

3'-end processing and strand transfer for IN assays. Processing and strand transfer reactions were performed using1.25 nM U5A/U5B and 6.25 nM U5A/U5B-2 DNA/DNA complexes, respectively,in a buffer containing 20 mM HEPES (pH 7.2), 1 mM dithiothreitol,and 10 mM MgCl₂ or MnCl₂. The reactions were initiated by additionof 100 nM IN, and the mixtures were incubated at 37°C forup to 1 h in the absence or presence of SP compounds. Reactionswere stopped by phenol-chloroform extraction. The DNA productswere precipitated with ethanol, resuspended in water, and separatedon denaturing 16% polyacrylamide gels. Gels were analyzed witha STORM 840 PhosphorImager (Molecular Dynamics) and quantifiedwith Image Quant software.

Inhibition of viral replication and cytotoxicity assays. The origin of viruses and the techniques used for measuringinhibition of virus replication have been previously described(43). Briefly, with MT-4 cells, determination of antiviral activityof the SP compounds was based on a reduction of HIV-1 strainIIIB-induced cytopathogenicity, the metabolic activity of thecell being measured by the property of mitochondrial dehydrogenaseto reduce yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT) to blue formazan. For CEM-SS cells, the productionof HIV-1 strain LAI was measured by quantification of the RTactivity associated with the virus particles released in theculture supernatant (38). Infected MT-4 and CEM-SS cells werecultured in the presence of different concentrations of SP compoundsfor 5 days before virus production determination. The 50% inhibitoryconcentration (IC₅₀) was derived from the computer-generatedmedian effect plot of the dose/effect data. In parallel experiments,cytotoxicity of the SP compounds was measured by the MTT testwith uninfected cells after a 5-day incubation. The 50% cytotoxicconcentration (CC₅₀) is the concentration at which the opticaldensity at 540 nm was reduced by 50%.

RESULTS

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Results

Polymerase activity. The effect of the SP compounds (Fig. 2) on HIV-1 RT activitywas tested with in vitro minus-strand "strong-stop" DNA synthesis.This DNA is the "runoff" reverse transcription product of aprimer annealed to the primer binding site. In this study, weused a labeled 18-mer DNA primer and RNA_1-311, which correspondsto nt 1 to 311 of HIV-1 MAL genomic RNA, as the template. Inorder to avoid complications due to concomitant inhibition ofRNase H by some of the SP compounds (see below), an RNase H^–RT was used for this assay. Twenty molecules (Fig. 2) were testedat different concentrations, and minus-strand "strong-stop"DNA, which corresponds to a 178-nucleotide primer extensionin the MAL isolate, was quantified after separation of the productsby denaturing polyacrylamide gel electrophoresis.

Figure 3 shows an example of gels obtained with the SP2 andSP12 compounds. In the absence of SP compounds, minus-strand"strong-stop" DNA synthesis was efficient. When adding SP2,minus-strand "strong-stop" DNA synthesis was already inhibitedby >50% at 1 µM, drastically reduced at 10 µM,and undetectable at 100 µM (Fig. 3). These experimentsrevealed that inhibition of DNA synthesis was sequence independent.Synthesis of intermediate products was also inhibited, withthe longer products disappearing first, at the expense of theshorter ones. At 100 µM SP2, only very short productswere synthesized, and the product pattern was reminiscent ofdistributive polymerization.

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FIG. 3. Inhibition of minus-strand "strong-stop" DNA synthesis by SP2 and SP12. Radiolabeled ODN/RNA_1-311 complex (10 nM) was extended with 30 nM HIV-1 RNase H^– RT. DNA synthesis was performed in the absence (no inhibitor [inh.]) or presence of 1 µM, 10 µM, or 100 µM of SP 2 or SP12 for 0 or 30 s or 1, 5, 10, 20, or 30 min. The minus-strand (–) strong-stop product is labeled, and the pausing sites are indicated by asterisks.

The IC₅₀ for each compound was determined by using the 30-mindata set (Table 1). The parental SP15 compound was not the bestinhibitor of the polymerase activity. Four monosubstituted compounds,SP2, SP3, SP5, and SP46, inhibited minus-strand "strong-stop"DNA synthesis at low micromolar or submicromolar concentrations,indicating that some R1 groups favor interaction of 3,7-dihydroxytropoloneswith RT. The monosubstituted compounds were generally more potentinhibitors of minus-strand "strong-stop" DNA synthesis thanthe disubstituted compounds (Table 1). Five pairs of mono- anddisubstituted compounds with identical substituents could bedirectly compared. In three pairs (SP47/SP7, SP4/SP8, and SP5/SP45),the monosubstituted compound was 20- to 60-fold more efficientthan its disubstituted counterpart, while for the other pairs(SP46/SP9 and SP2/SP48) the difference was only two- to fivefold.

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TABLE 1. IC₅₀s of the SP compounds on the polymerase and RNase H activities of HIV-1 RT and on the 3' processing and transfer activities of HIV-1 IN

Several compounds were inactive or weakly active against thepolymerase activity of HIV-1 RT. Notably, the C-7-modified tropolones(SP10 and SP11), which have only two adjacent oxygens on theseven-membered ring, had no activity at 1 mM, and the O-methylated3,7-dihydroxytropolones (SP13 and SP14) were only weakly active.These results were in keeping with our hypothesis that hydroxytropolonesmight inhibit the polymerase activity by interfering with theMg²⁺ ions in the active site.

SP12 and SP20 inhibited minus-strand "strong-stop" DNA synthesis,even though they were less potent inhibitors than SP15 (Fig.3 and Table 1). For instance, SP20 was 10-fold less efficientthan the corresponding nonacetylated compound, SP15. As forSP2, a sequence-independent inhibition pattern was observed(Fig. 3). Inhibition of the RT polymerase activity by SP12 andSP20 was unexpected, as the hydroxyl groups that are supposedto chelate the magnesium ions are acetylated in these compounds(Fig. 2). The first pK_a of SP15 is 6.7, and deprotonation ofa hydroxyl group results in increased chelating potency (42).Accordingly, acetylated hydroxytropolones were inactive againstinositol monophosphatase (41, 42). Thus, our results raisedthe possibility that some or all SP compounds inhibit the RTpolymerase activity by an unpredicted mechanism.

To address this question and to gain insight into the role ofMg²⁺ chelation in the inhibition process, we studied the inhibitionof minus-strand "strong-stop" DNA synthesis by SP12 and SP15at two different magnesium concentrations (Fig. 4). At 6 mMMg²⁺, the IC₅₀ of SP15 was 3.2 µM, while SP12 was abouttwofold less active (IC₅₀ of 7.6 µM). Interestingly, inhibitionpotency of these compounds dramatically decreased at low magnesiumconcentration: at 0.3 mM Mg²⁺, the IC₅₀ of SP15 was 19 ±1 µM, while inhibition of minus-strand "strong-stop" DNAsynthesis by SP12 was hardly detectable (Fig. 4). These resultssuggest that the SP compounds can bind the RT polymerase activesite only in the presence of Mg²⁺ ions, in agreement with aninhibition mechanism involving chelation of the catalytic ions.

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FIG. 4. Effect of Mg²⁺ concentration on inhibition of minus-strand "strong-stop" DNA synthesis by SP15 and SP12. Minus-strand "strong-stop" DNA synthesis was performed for 30 min in the presence of 6 mM or 0.3 mM Mg²⁺, in the absence or presence of increasing concentrations of SP12 or SP15.

RNase H activity. Two modes of RNase H cleavage have been described: a polymerase-dependentcleavage (21), directed by the 3' end of the DNA strand, anda polymerase-independent mechanism guided by the 5' end of theRNA template (48). The first mode of cleavage is thought tooccur in concert with DNA polymerization to degrade the genomicRNA during minus-strand DNA synthesis, while the second maycontribute to the degradation of larger genomic RNA fragmentsleft after DNA 3'-end-directed cleavage and may also play arole in the formation and removal of the polypurine tract. Duringpolymerase-dependent cleavage, the polymerase active site ofthe enzyme is positioned at the recessed 3' end of the DNA strand(Fig. 5a). The RNase H active site is positioned approximately17 nt from the polymerase active site, and its position fromthe 3' end of the DNA determines the position of the primarycleavage. RT then repositions and makes a secondary cleavage7 to 9 nt from the recessed 3' end of the DNA (Fig. 5a). Wetested this mode of cleavage using RNA_1-47 as viral RNA andODN35 as DNA.

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FIG. 5. Inhibition of RNase H activity by 3,7-dihydroxytropolones. (a) Experimental design and expected products of polymerase-dependent RNase H activity. In this mode of cleavage, the polymerase active site is positioned at the 3' end of the DNA primer (ODN35) and determines the position of the cleavage 17 nt downstream, yielding RNA₂₉. RT is also able to cut 9 nt from the 3' end of the DNA strand, producing RNA₂₁. The ³²P-labeled 5' end of RNA_1-47 is indicated by an asterisk. (b) Inhibition of RNase H activity by SP9. The reaction was initiated by the addition of 10 nM HIV-1 RT to 10 nM of RNA_1-47/ODN35 complex, in the absence (no inhibitor [inh.]) or presence of 1 µM, 10 µM, or 100 µM of SP9. The reaction was stopped after 0, 15, or 30 s or 1, 3, 5, 10, 20, or 30 min. Lane L corresponds to a RNA ladder that was used to determine the sizes of the products.

As shown in Fig. 5b, both the primary (RNA₂₉) and the secondary(RNA₂₁) cleavage products accumulated over time in the absenceof SP compounds. In addition, weaker primary cuts generatedfragments 28 and 32 nt in length (Fig. 5b). However, all degradationproducts progressively disappeared as the SP9 concentrationwas increased. Formation of the two major products was slightlyslower at 1 µM, strongly inhibited at 10 µM, andundetectable at 100 µM (Fig. 5b). Interestingly, the twocleavages were inhibited with the same efficiency. The SP compoundswere tested for inhibition of the polymerase-dependent RNaseH activity, and the results are summarized in Table 1.

The best inhibitors of RNase H were the monosubstituted 3,7-dihydroxytropoloneSP47 and the disubstituted compound SP9. When comparing mono-and disubstituted 3,7-dihydroxytropolones with identical substituents,the monosubstituted compounds were 3.3- to 50-fold-more-potentRNase H inhibitors, with the exception of the SP46/SP9 pair,in which the disubstituted compound was slightly more active.In addition, all SP compounds were more potent against polymerasethan against RNase H activity, except SP47 and SP7, suggestingthat the meta-nitrophenyl group may form specific interactionsin the RNase H active site. Several molecules had a rather goodspecificity for the polymerase activity: SP2, SP3, SP4, andSP46 were at least 20-fold more efficient against polymerasethan against RNase H (Table 1). Interestingly, these compoundsare all monosubstituted 3,7-dihydroxytropolones. By contrast,none of the tested compounds displayed a high specificity forthe RNase H activity, and only SP47 and SP9 were significantlymore efficient than the parent compound.

The C-7-modified tropolones SP10 and SP11 and the methylated3,7-dihydroxytropolones SP13 and SP14, which did not significantlyinhibit the RT polymerase activity, displayed little or no activityagainst RNase H at 1 mM. Interestingly, the acetylated dihydroxytropolonesSP12 and SP20, which were moderate polymerase inhibitors, wereweaker RNase H inhibitors (Table 1). In view of the resultsobtained previously with inositol monophosphatase (41, 42),these data suggest that hydroxytropolones inhibit the RT RNaseH activity by binding the Mg²⁺ ions in the active site.

Integrase activities. The structures of the HIV-1 RNase H domain and the IN core presentstrong similitude, and their enzymatic mechanisms are probablyalso closely related (16, 50). Thus, it was interesting to testthe effects of the SP compounds on the 3' processing and strandtransfer activities of HIV-1 IN. To this end, we constructeda DNA duplex corresponding to the U5 region of the 3' long terminalrepeat of the proviral DNA (Fig. 6a). Both reactions consistin a nucleophilic attack of a phosphodiester bond by a hydroxylgroup and require either Mg²⁺ or Mn²⁺ as a cofactor. Duringthe processing reaction, the enzyme removes two 3' nucleotidesat each terminus of the proviral DNA, resulting in overhangingCA ends (Fig. 6a). In the following strand transfer reaction,the 3' processed end performs a nucleophilic attack on a phosphodiesterbond of the target DNA (6). In the in vitro assay with purifiedIN, the preprocessed 3' end is randomly integrated in the targetduplex (Fig. 6a).

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FIG. 6. Inhibition of the 3'-end processing and strand transfer activities of HIV-1 IN by 3,7-dihydroxytropolones. (a) Experimental design and expected products of the 3'-end processing and strand transfer reactions. The 3'-end processing removes 2 nt at the 3' end of U5B, yielding U5B-2. During strand transfer, U5B-2 is randomly integrated into a homologous strand or into U5A, yielding high-molecular-weight products. The 5'-radiolabeled end of U5B is indicated by an asterisk. (b) Dose-response effect of SP2, SP9, and SP15 on IN 3' processing activity. The 5'-end-labeled oligonucleotide U5B annealed to U5A was incubated for 1 h at 37°C with 100 nM IN. (c) Inhibition of IN transfer activity by SP2, SP9, and SP15. The 5'-end-labeled oligonucleotide U5B-2 annealed to U5A was incubated for 1 h at 37°C with 100 nM IN. In panels b and c, lane "–IN" is a control without integrase, lane "EDTA" is a negative control containing 20 mM EDTA, and lane "DMSO" is a control with dimethyl sulfoxide but without SP compounds. Lanes 1 to 6 correspond, respectively, to 0.3 µM, 1 µM, 3 µM, 10 µM, 30 µM, and 100 µM SP2, SP9, or SP15.

3'-end processing. Figure 6b shows the results of 3'-end processing inhibitionby SP2, SP9, and SP15. Processing of the 21-mer duplex liberatesthe 3'-terminal dinucleotide GT and the processed U5B oligonucleotide(U5B-2). IN activity was assayed in the presence of increasingSP concentrations. As shown by 15% polyacrylamide gel autoradiography,the addition of increasing concentrations of SP2, SP9, and SP15led to significant inhibition of 3'-end processing (Fig. 6b),yielding IC₅₀ values of 13 µM, 3.1 µM, and 40 µM,respectively. The IC₅₀ values for inhibition of the 3'-end processingare summarized in Table 1.

There was no clear-cut difference between mono- and disubstituted3,7-dihydroxytropolones regarding the 3'-end processing activity.Two monosubstituted (SP47 and SP46) and one disubstituted (SP45)compound inhibited 3'-end processing at submicromolar concentrations.However, all disubstituted 3,7-dihydroxytropolones had IC₅₀slower than 10 µM, while the IC₅₀s of most monosubstitutedSP compounds were greater than 10 µM (Table 1). The methylateddihydroxytropolones SP13 and SP14 and the C-7-modified tropoloneSP10 were completely inactive, whereas SP11 displayed a verylow activity. The acetylated compound SP12 was a rather poorinhibitor (IC₅₀ of 90 µM) of the 3' processing activity,while SP20 was totally inactive.

In vitro, IN can use either Mg²⁺ or Mn²⁺ ions as a cofactor,even though Mg²⁺ is most likely the cation of biological relevance.Thus, we compared the levels of inhibition of 3'-end processingby two mono- and two disubstituted SP compounds in buffers containing10 mM of either MgCl₂ or MnCl₂. In all cases, the SP compoundswere 2- to 2.6-fold more potent with Mn²⁺ than with Mg²⁺ (Table1).

Strand transfer. The inhibitory effect of SP compounds on the strand transferreaction was assayed using a preprocessed duplex (U5B-2/U5A)as the IN substrate. Monosubstituted SP47 and SP46 and disubstitutedSP45 and SP9 were potent inhibitors of the strand transfer reaction(IC₅₀ of <2 µM) (Fig. 6c and Table 1). Mono- and disubstituted3,7-dihydroxytropolones with identical substituents had similarIC₅₀s, with the exception of the SP4/SP8 pair, in which themonosubstituted SP4 compound was poorly active (Table 1). SP4was an exception, as all other 3,7-dihydroxytropolones withfree hydroxyl groups had IC₅₀s smaller than 20 µM. Onthe other hand, the C-7-modified tropolones (SP10 and SP11)and the methylated 3,7-dihydroxytropolones (SP13 and SP14) didnot inhibit strand transfer (Table 1). The acetylated SP12 wassixfold less active than the parent compound, SP15.

Biochemical data and modeling indicated that the IN active siteadopts different conformations during the two catalytic stepsof the integration process (6, 18) and that it is possible tofind step-specific IN inhibitors (12, 25). Interestingly, ourdata showed that SP1 is a rather good inhibitor of the transferstep (IC₅₀ of 17 µM) but a very poor inhibitor of 3'-endprocessing (Table 1). Conversely, SP45 preferentially inhibitedthe 3'-end processing step, indicating that substituted 3,7-dihydroxytropolonescan be selective inhibitors of the 3'-end processing or of thetransfer reaction. Nevertheless, most of the SP compounds wereequally efficient with both IN activities (SP5, SP7, SP8, SP9,SP12, SP46, SP47, and SP48).

Cytotoxicity assays and inhibition of viral replication. The inhibitory and cytotoxic effects of SP molecules were studiedwith human lymphoblastoid CEM-SS and MT-4 cells infected withHIV-1 LAI and HIV-1 IIIB strains, respectively. In the HIV-1IIIB/MT-4 cell culture assay, SP compounds were toxic in themicromolar-concentration range, displaying CC₅₀ values from3 to 30 µM (Table 2). In this assay, no SP compound displayedantiviral activity at concentrations below its CC₅₀ (IC₅₀ >CC₅₀). As a control, zidovudine was active in the low nanomolarrange, without significant cytotoxicity (Table 2).

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TABLE 2. Inhibition of HIV-1 replication (IC₅₀) and cytotoxicity (CC₅₀) of selected SP compounds on CEM-SS and MT-4 cell lines

In the HIV-1 LAI/CEM-SS cell culture system, the CC₅₀ valuesof the SP compounds were slightly lower than with the MT-4 cellsand ranged from 3 to 43 µM. In addition, in this cellline, SP compounds inhibited replication of HIV-1 LAI with IC₅₀sranging from 0.3 to 40 µM. However, for most compoundsthe IC₅₀ was only two- to threefold lower than the CC₅₀ (Table2). Thus, we cannot exclude that the antiviral effects we observedwere indeed a direct consequence of the cytotoxicity of theSP compounds. For instance, the inhibition of HIV LAI replicationby SP10 (IC₅₀ of 40 µM) was likely due to its toxicity(CC₅₀), since this C-7-modified tropolone was inactive againstall RT and IN enzymatic activities. However, it remains possiblethat some of the SP compounds have a real antiviral activity,especially SP15 and SP3, which have IC₅₀ values 30- and 3.7-foldlower, respectively, than their CC₅₀s.

DISCUSSION

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Discussion

The high incidence of resistance in therapy-experienced andnewly infected patients and the adverse effects of the existingtreatments underscore the need for new HIV-1 inhibitors (11,17, 32, 51). The RT polymerase and RNase H activities as wellas the IN 3'-end processing and strand transfer activities allrequire divalent cations and are essential for viral replication.While several inhibitors of RT polymerase activity are in theclinic and inhibitors of IN strand transfer activity have beentested in clinical trials (9), RNase H and 3'-end processinginhibitors are only at the earlier steps of the drug discoverypipeline.

In this study, we tested the inhibitory properties of 3,7-dihydroxytropoloneson four enzymatic activities. Our choice was motivated by theobservation that these compounds inhibit inositol monophosphataseby binding the two Mg²⁺ ions in the catalytic site (41, 42).Two Mg²⁺ ions separated by 3.57 Å are present in the polymeraseactive site of RT in complex with a primer/template DNA duplexand an incoming nucleotide (27), and a pair of metal ions hasalso been observed in some crystal structures of the RNase Hdomain (8) and IN (4, 5, 49).

Inhibitors, including some with marked specificity, were identifiedfor each of the four enzymatic activities tested. Interestingly,the parental compound, SP15, was never the best inhibitor, indicatingthat the efficiency of 3,7-dihydroxytro- polones can be improvedby aromatic groups at positions 4 and/or 6 of the tropolonering. Some of the 3,7-dihydroxytropolones we tested inhibitedthe RT polymerase and the IN 3'-end processing and strand transferactivities at submicromolar concentrations, while these compoundswere generally less potent against RNase H. To our knowledge,this is the first rational attempt to inhibit HIV-1 RT by simultaneouslybinding the two catalytic ions. ${alpha}$ , ${gamma}$ -Diketo acids (47) and dihydroxypyrimidinecarboxylicacid (46) were recently shown to inhibit the RNA-dependent RNApolymerase from hepatitis C virus, but whether these compoundsinteract with one or both metal ions in the polymerase activesite is not clear.

The 4-monosubstituted 3,7-dihydroxytropolone SP47 and the 4,6-disubstituted3,7-dihydroxytropolone SP9 inhibited HIV-1 RT RNase H activityat low micromolar concentrations. This compares favorably withpreviously identified RNase H inhibitors such as ilimaquinone(IC₅₀ of $~$ 20 µM) (34), N-(4-tert-butylbenzoyl)-2-hydroxy-1-naphthaldehydehydrazone (BBNH) (IC₅₀ of 3.5 µM) (2), and the new RNaseH-specific thiophene diketo acid {4-[5-(benzoylamino)thien-2-yl]-2,4-dioxobutanoicacid} (IC₅₀ of 3.2 µM) (45). Interestingly, both BBNHand the thiophene diketo acid potentially inhibit RNase H activityby chelating one metal ion in this active site. Finally, a papersubmitted at the same time as this paper showed that ß-thujaplicinol,a natural monohydroxytropolone derivative, strongly inhibitsHIV-1 and HIV-2 RNase H, while it is a weak inhibitor of Escherichiacoli and human RNase H (3). This result further stresses thepotential of hydroxytropolones as HIV inhibitors.

Several of the 3,7-dihydroxytropolones we tested were potentinhibitors of the 3'-end processing and strand transfer activitiesof HIV-1 IN (IC₅₀ of <2 µM). By comparison, diketoacids inhibit IN at submicromolar concentrations (25), and styrylquinolinederivatives have IC₅₀ values between 0.5 and 5 µM (12).However, while diketo acids preferentially inhibit strand transfer,most of the tested 3,7-dihydroxytropolones equally inhibitedthe two IN activities, with the noticeable exception of SP1,which selectively inhibits strand transfer, and SP45, whichpreferentially inhibits 3'-end processing.

While some of the 3,7-dihydroxytropolones preferentially inhibitedone of the four enzymatic activities, some had little or nospecificity (e.g., SP6, SP9, and SP47). It seems, however, unlikelythat these compounds inhibit RT and IN by binding to their nucleicacid substrates. Indeed, 3,7-dihydroxytropolones are negativelycharged at physiological pH ( $~$ 1.5 net negative charge at pH 7),and the uncharged O-methylated 3,7-dihydroxytropolones, whichare more likely to bind nucleic acids, are completely inactive.In addition, by using surface plasmon resonance, Budihas etal. (3) showed that natural tropolone derivatives do not bindnucleic acids. In line with these results, we found no evidenceof 3,7-dihydroxytropolone binding to DNA/DNA and RNA/DNA duplexesby band-shift and fluorescence competition assays (J. Didierjeanet al., unpublished data).

The data presented here suggest that 3,7-dihydroxytropolonesinhibit DNA polymerase, RNase H, and IN activities by chelatingthe Mg²⁺ ions of these active sites, even though more detailedstudies will be required to prove this point. The fact thatthe C-7-modified tropolones (SP10 and SP11) and the O-methylated3,7-dihydroxytropolones (SP13 and SP14) were inactive againstthese four enzymatic activities suggests that the inhibitorssimultaneously bind two metal ions. Acetylated dihydroxytropoloneshave lower affinity for Mg²⁺ than do their unmodified counterparts(42). Accordingly, acetylated compounds (SP12 and SP20) hadno or limited activity against RNase H and IN activities andwere also inactive against inositol monophosphatase (41, 42).These compounds were fairly active against the RT polymeraseactivity, but SP20 was 10-fold less active than the correspondingparent compound (SP15). In the RNase H site, the 4-Å distancebetween the metal ions (8) is not ideal for interaction withtropolones, in contrast with the polymerase active site (27).Therefore, acetylated dihydroxytropolones might be able to interactwith the metal ions only in the polymerase active site. Alternatively,only one Mg²⁺ ion might be present in the RNase H site (27),preventing binding of the weaker ligands.

Inhibition of DNA synthesis by acetylated and unmodified 3,7-dihydroxytropolones(SP12 and SP15) was dramatically reduced at low Mg²⁺ concentrations,in keeping with the chelation mechanism. We propose that bindingof Mg²⁺ in the catalytic site is required to bind 3,7-dihydroxytropolonesand that these compounds compete with one or several oxygensof the catalytic amino acid side chains for Mg²⁺ chelation.Thus, 3,7-dihydroxytropolones should be more efficientat high (saturating) Mg²⁺ concentrations. Crystallographic studiesshowed that binding of two Mg²⁺ ions in the polymerase siterequires binding of the incoming dNTP (14, 27). One ion is boundto the ß and ${gamma}$ phosphates of dNTP and enters with itin the catalytic site; its K_D is about 0.1 mM. The second ionis bound in a subsequent step, and recent studies showed thatits K_D is in the millimolar range (13). Thus, high-millimolarMg²⁺ concentrations are required to saturate the polymeraseactive site and to reach maximal inhibition by 3,7-dihydroxytropolones.

When we compared levels of inhibition of the IN 3'-end processingactivity between MgCl₂ and MnCl₂, we observed that the SP compoundswere slightly more efficient in the presence of Mn²⁺. At firstsight, one could expect strongly decreased inhibition with MnCl₂,as the oxygen atoms and hence 3,7-dihydroxytropolones have muchweaker affinity for Mn²⁺ than for Mg²⁺. However, as mentionedabove, we think there is competition between 3,7-dihydroxytropolonesand the side chains of the catalytic amino acids of IN (D64,D116, and E152) for binding of the metal ions. As these aminoacids and the SP inhibitors both bind the metal ions via oxygenatoms, they should bind Mn²⁺ ions less tightly than Mg²⁺. However,their relative affinity for Mn²⁺ and Mg²⁺ should be the sameand we expected little difference in inhibition of the SP compoundswith MgCl₂ and MnCl₂. In fact, our results are consistent withthose obtained with diketo acids on the IN strand transfer activity.The diketo acids that bind metal ions solely via oxygen atomshave similar activities in the presence of Mg²⁺ and Mn²⁺, whilethose binding metal ions via nitrogen atoms were at least 1order of magnitude more active with MnCl₂ (23). Obviously, eventhough our results suggest that SP compounds inhibit HIV-1 RTand IN by chelation of the metal ions in their catalytic sites,additional studies will be required to demonstrate this mechanism.

Whereas the tests with purified enzymes showed that 3,7-dihydroxytropoloneshave interesting potential as HIV-1 inhibitors, cell-based assaysindicated that their cytotoxicity represents a major challenge.This toxicity is most likely due to the inhibition of cellularbimetallic enzymes. Given the number of enzymes that use metalions for catalysis, it could be argued that it will be impossibleto find 3,7-dihydroxytropolones that specifically inhibit HIV-1enzymes. In this respect, the diketo acid derivatives constitutean interesting example. These compounds were first identifiedby high-throughput screening for IN inhibitors (25). Some ofthese compounds block HIV-1 replication by specifically inhibitingthe IN strand transfer activity and exhibit low toxicity (24-26),even though they act by binding one of the metal ions in thecatalytic site (23) and could potentially interfere with a numberof cellular enzymes. Recently, other members of the diketo acidfamily have been shown to specifically inhibit HIV-1 RNase H(45) and hepatitis C virus RNA polymerase (47). These examplesshow that inhibitors that interfere with metal ions in the enzymecatalytic sites can be specific. Along these lines, 3,7-dihydroxytropoloneconstitutes a very interesting platform allowing a number ofvery different derivatives to be synthesized. In addition, modelingand structural studies of the enzyme/3,7-dihydroxytropolonecomplexes should allow rational design of more-potent inhibitorsthat might also be more specific.

ACKNOWLEDGMENTS

We thank Chantal and Bernard Ehresmann for fruitful discussionsand for their constant interest during this work and PhilippeWalter for the gift of RT.

This work was supported by grants from the Agence Nationalede Recherches sur le SIDA (ANRS) to R.M. and from Sidactionto S.R.P. F.Q. is a predoctoral fellow of the ANRS.

FOOTNOTES

* Corresponding author. Mailing address: Unité Propre de Recherche 9002 du CNRS conventionnée à l'Université Louis Pasteur, IBMC, 15 rue René Descartes, 67084 Strasbourg cedex, France. Phone: 333 88 41 70 54. Fax: 333 88 60 22 18. E-mail: r.marquet{at}ibmc.u-strasbg.fr.

REFERENCES

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