N-(4-tert-Butylbenzoyl)-2-hydroxynaphthaldehyde hydrazone (BBNH) is a potent inhibitor of the ribonuclease H (RNase H) activity of human immunodeficiency virus (HIV)-1 reverse transcriptase (RT). Molecular modeling predicted that BBNH binds to the HIV-1 RT RNase H active site via two major interactions, coordination to the metal ion cofactor (Mg2+ or Mn2+) in the enzyme active site and aromatic ring-stacking interaction between the naphthyl ring of BBNH and amino acid Tyr-501. The latter residue equivalent is conserved in virtually all RNases H, suggesting the need for an aromatic or π-stacking interaction in this region. To assess the importance of Tyr-501 in the binding of BBNH for the inhibition of RT RNase H activity, we used site-specific mutagenesis to generate RT with a variety of substitutions at this position. Most substitutions resulted virtually in a complete loss of RNase H activity. However, three mutants, Y501F, Y501W, and Y501R, possessed RNase H activities comparable with wild-type enzyme. Whereas BBNH inhibited Y501F RT RNase H activity with potency equivalent to wild-type RT, the Y501W mutant showed a 6-fold resistance to inhibition by BBNH, and the Y501R mutant was completely resistant to inhibition by BBNH. The replication “fitness” of HIV molecular clones with the Y501W or Y510R mutation was significantly compromised compared with wild-type virus. Importantly, BBNH was an effective inhibitor of the DNA polymerase activity of all Y501X mutants tested. Our results highlight the importance of Tyr-501 in RT RNase H activity and inN-acylhydrazone inhibitor binding and suggest that drugs that target critical residues in HIV-1 proteins may be a useful approach in new antiviral development.

# EXPERIMENTAL PROCEDURES

### Materials

BBNH andN-(4-tert-butylbenzoyl)-2-hydroxy-1-salicylylhydrazone (BBSH) were synthesized by standard methods (14). 1H NMR and elemental analyses of these compounds were entirely consistent with the expected chemical structures. [3H]TTP, [γ-32P]ATP, and the homopolymeric template/primer poly(rA)-oligo(dT)12–18 were purchased from Amersham Biosciences, Inc. All other reagents were of the highest quality available and were used without further purification.

### Molecular Modeling

A model for the interaction of BBNH with HIV-1 RT was constructed using the x-ray crystallographic coordinates for HIV-1 RT complexed with nevirapine (15) (Protein Data Bank accession number 3HVT). This structure was chosen because of the presence of magnesium atoms bound in the RNase H active site. The initial conformation of BBNH was derived from its x-ray crystal structure (16). Docking and energy minimization experiments were carried out with the anneal function of Sybyl 6.5 (Tripos Inc., St Louis, MO). Charges were calculated by the Gasteiger-Huckel method, and iterative minimization was carried out with the Tripos force field until the energy difference between iterations was <0.01 kcal/mol/Å.

### Cloning, Site-directed Mutagenesis, and Purification of WT and Tyr-501 Mutant HIV-1 RT

The p66- and p51-kDa subunits of HIV-1 RT were cloned into a pBAD/HisB prokaryotic expression vector (Invitrogen) between the XhoI and HindIII restriction endonuclease cleavage sites. Mutations at position 501 were introduced into the gene encoding for the p66-kDa subunit of HIV-1 RT by site-directed mutagenesis using either the SculptorTM
                              in vitro mutagenesis (Amersham Biosciences, Inc.) or QuikChangeTM site-directed mutagenesis (Stratagene) systems. All clones were sequenced for the desired mutation using a T7-sequencing kit (Amersham Biosciences, Inc.). WT and mutant RT p66/p51 heterodimers were purified as described previously (17).

### Assay of RT-catalyzed RNA-dependent DNA Polymerase Activity

The specific activities of WT and mutant RT were determined by measuring the RNA-dependent DNA polymerase activity of each enzyme using 0.2 units/ml of poly(rA)-oligo(dT)12–18, 20 μm[3H]dTTP, and 8.5 nm purified RT p66/p51 heterodimer in 50 mm Tris-HCl, pH 7.9, 60 mmKCl, 10 mm MgCl2. Samples were incubated for 10 min at 37 °C and then quenched by the addition of ice-cold 10% trichloroacetic acid containing 20 mm sodium pyrophosphate. After a 20-min incubation on ice, samples were filtered using a 1.2-μm glass fiber type C filter multi-well plates (Millipore) and washed sequentially with 10% trichloroacetic acid and ethanol. The extent of radionucleotide incorporation was then determined by liquid scintillation spectrometry. Assays for the inhibition of RT DNA polymerase activity by BBNH were carried out as described previously (11).

More detailed inhibition kinetic analysis of RT RNA-dependent DNA polymerase activity employed a heteropolymeric template/primer prepared as previously described (18) using the T7 polymerase RNA transcript fromAccI-linearized plasmid pHIV-PBS as template and a synthetic 18-nucleotide deoxyoligonucleotide as DNA synthesis initiation primer. Reaction assays comprised 0.5 units/ml of heteropolymeric template/primer, variable concentrations of a mixture of equimolar [3H]dATP, [3H]dCTP, [3H]dGTP, [3H]TTP (ranging from 1–20 μm total dNTP concentration), and 10 nmpurified RT p66/p51 heterodimer in 50 mm Tris-HCl, pH 7.9, 60 mm KCl, 10 mm MgCl2. Samples were incubated and processed as described above. Inhibition data were analyzed by Dixon plot analysis.

### Assay of RT-catalyzed RNase H Activity

RNase H activity was assayed using an 18-nucleotide 5′-32P-heteropolymeric RNA template (5′-GAUCUGAGCCUGGGAGCU-3′) annealed to a complementary 18-nucleotide DNA oligomer (5′-AGCTCCCAGGCTCAGATC-3′). 14 nm 5′-[32P]RNA-DNA duplex was incubated with 5 nm RT heterodimer in 50 mm Tris-HCl, pH 7.9, 60 mm KCl, 2.5 mm MgCl2 in 10 μl of final volume. Samples were incubated for 10 min at 37 °C and then quenched with an equal volume of gel loading dye (98% deionized formamide, 10 mm EDTA, 1 mg/ml bromphenol blue, and 1 mg/ml xylene cyanol). RNase H-generated degradation products were resolved by electrophoresis using 14% acrylamide/7 m urea sequencing gels visualized by autoradiography and quantified by densitometry.

For assay of BBNH or BBSH inhibition of RT RNase H activity, WT or mutant RT (30 nm) was preincubated with BBNH or BBSH (dissolved in Me2SO) in 50 mm Tris-HCl, pH 7.9, 60 mm KCl, 2.5 mm MgCl2, 0.2 mg/ml of ovalbumin in a final volume of 50 μl for 10 min. The final concentration of Me2SO never exceeded 2%. 10-μl aliquots of the preincubation mixture was then added to 40 μl of an assay mixture containing 14 nm RNA-DNA duplex and assayed as described above. Inhibition data were calculated from Dixon and Lineweaver-Burk plots.

### HIV-1 Molecular Clone Preparation and Virus Culture

Plasmid SVC21-BH10 encodes an infectious molecular clone of the IIIB strain of HIV-1 and carries an SV40 origin of replication for expression in COS cells (19). Mutations in the region corresponding to residue 501 of the RT gene were introduced using the QuikChangeTMsite-directed mutagenesis kit (Stratagene). Virus was prepared by transfection of COS-7 cells using a standardized calcium phosphate coprecipitation method (20). Cell-free supernatants were collected 48 h following transfection, and the amount of virus in these supernatants was quantified by analyzing the levels of HIV-1 p24 antigen. Aliquots of these supernatants, containing equal amounts of viral p24, were then added to cultures of CD4+ MT2 lymphoblastoid cells. Cells were examined daily for the appearance of HIV-1-induced syncytium formation. In addition, aliquots of cell-free supernatants were reserved for the assessment of viral p24 levels.

# RESULTS

### Molecular Model for the Interaction of BBNH with HIV-1 RT RNase H

The model for the binding of BBNH to the RNase H domain of HIV-1 RT is shown in Fig. 2
                              . Crystallographic analysis shows BBNH to be a flat planar molecule (16). Our docking studies indicate a good fit between the inhibitor and a region close to the RNase H active site that is open and accessible to ligand binding (Fig. 2
                              A). Residues within 4 Å of the bound inhibitor include Asn-474, Gln-475, Arg-448, Glu-478, Ser-499, His-539, and Tyr-501 (Fig. 2
                              B).

Two major binding determinants were identified. The first involves the coordination of one of the hydrazone-linkage nitrogen atoms and the hydrazone carbonyloxygen to the RNase H active site metal. This interaction is consistent with our observations that BBNH inhibition of RT RNase H activity is a metal-dependent process (i.e. metal coordination is essential), and that BBNH is a competitive inhibitor of RT RNase H activity (11).

The second major interaction identified is an aromatic-stacking interaction between the phenyl ring of Tyr-501 and the second naphthyl ring of BBNH (Fig. 2
                              A). To confirm this possibility, we examined the inhibitory potency of a BBNH analog,N-(4-tert-butylbenzoyl)-2-hydroxy-1-salicylylhydrazone (Fig. 1), in which a single aromatic ring replaces the naphthyl double ring system of BBNH. The salicylyl single ring is not positioned to participate in aromatic stacking with Tyr-501, and in accordance with our model BBSH, was an exceptionally weak inhibitor of RT RNase H activity (Table I
                              ). Nevertheless, BBSH retained significant inhibitory potency against RT DNA polymerase activity.

### Characterization of HIV-1 RT Y501XSubstitution Mutants

To test the interaction between Tyr-501 and BBNH, we generated a number of mutant enzymes with various substitutions at position 501, including Y501F, Y501W, Y501R, Y501E, Y501Q, Y501G, Y501A, Y501L, Y501S, and Y501H. Most of these mutant enzymes had significantly impaired RNase H activity (Fig. 3
                              , TableII
                              ), and some also showed attenuated RNA-dependent DNA polymerase activity (Table II) compared with the WT enzyme. Only the Y501F, Y501W, and Y501R mutants retained appreciable RNase H activity.

### Ability of BBNH to Inhibit RNase H-active Tyr-501 RT Mutants

BBNH inhibited the RNase H activity of Y501F RT as effectively as WT RT (Table III
                              ). However, the Y501W mutant showed a 6-fold decreased sensitivity to inhibition of its RNase H activity by BBNH, and the RNase H activity of the Y501R mutant enzyme was completely resistant to BBNH inhibition. Interestingly, the DNA polymerase activity of all Y501Xmutants tested remained fully sensitive to inhibition by BBNH (TableIII).

### Replication Fitness of HIV-1 with Y501X Substitutions in RT

Molecular clones of HIV-1 with various substitutions at RT residue 501 were constructed and examined for their ability to infect and replicate in lymphoblastoid MT2 cells. Only the Y501F and Y501W mutant viruses were able to replicate as the Y501R could not (Fig. 4
                              , TableIV
                              ). However, the replication-competent mutants showed substantially reduced replication fitness relative to WT virus (Table IV).

# DISCUSSION

We previously suggested that the ability of BBNH to inhibit multiple RT activities might be because of the binding of the inhibitor to two different sites on the enzyme based on biochemical and kinetic data (11). Our hypothesis was that binding to a site at or near the NNIBP in the polymerase domain of RT results in the inhibition of the DNA polymerase activity of the enzyme, whereas binding to the second site in the RNase H domain provides inhibition of the RNase H activity of the enzyme. However, it was still possible, although unlikely, that the inhibition of both activities might arise from single site binding of the inhibitor. Indeed, certain non-nucleoside reverse transcriptase inhibitors have been shown to affect RT RNase H activity despite binding only to the NNIBP (21).

Molecular modeling showed that BBNH can be readily docked into the RT RNase H domain in a manner consistent with previously reported data (Fig. 2
                     A). For example, a major interaction involves the coordination of the metal-chelating moieties of BBNH with the active site metal in the RNase H domain. This is consistent with the metal dependence for BBNH inhibition of RT RNase H activity as well as the competitive nature of this inhibition (11). Despite considerable effort, very few potent small molecule inhibitors of HIV-1 RT RNase H have been identified. This may partly be attributed to the fact that the RNase H active site has a rather open structure (as might be expected for an active site that must accommodate the large RNA-DNA duplex substrate), and initially it appears that the region possesses insufficient potential contacts to allow for reasonable binding interactions with small molecules. However, BBNH is a flat molecule with an extended planar structure (16) and possesses metal-binding properties that allow interaction with the active site metal. Interestingly, the only other potent inhibitor of RT RNase H, the phenylhydrazone PD029687, was recently shown to also be capable of metal binding. Thus, metal binding may be an important parameter for the identification of RNase H inhibitors.

The molecular model also predicts that aromatic ring-stacking interactions between the phenol ring of Tyr-501 and the second ring of the naphthyl moiety of BBNH is important for the non-metal binding interactions of the inhibitor with the RT RNase H active site. The observation that BBSH, a BBNH analog with a reduced ring system, is unable to inhibit RT RNase H is also consistent with this model. Furthermore, BBSH remains a potent inhibitor of RT DNA polymerase activity, implying that this analog can still bind to the RT polymerase domain presumably at or near the NNIBP. This observation provides further evidence that the inhibition of multiple RT activities by BBNH arises from the binding of the inhibitor to multiple sites on RT.

Mutational analyses of Tyr-501 also support the prediction that this residue is crucial for the binding of BBNH. In the absence of bound nucleic acid duplex substrate, Tyr-501 is completely exposed to solvent, and the phenol ring is free to “flip” around its Cβ–Cγ bond. This conformational flexibility enables the phenol side chain of Tyr-501 to readily assume an orientation to interact with the naphthyl ring system of BBNH, thereby providing the crucial stacking interaction. The Y501F substitution would not affect the side chain conformational flexibility. Thus, the RNase H activity of Y501F RT is fully sensitive to BBNH inhibition. The increased side chain bulk of the Y501W mutant could decrease the rotational freedom about the Cβ–Cγ bond. This decreased mobility would restrict the side chain from adopting the appropriate orientation to facilitate BBNH binding, consistent with the decreased sensitivity of the Y501W mutant to BBNH inhibition. Whereas arginine is capable of π-bonding interactions, the size and non-planar nature of the Y501R mutation is such that interactions with BBNH would be difficult. Indeed, the RNase H activity of the Y501R mutant RT is completely insensitive to BBNH inhibition. Importantly, the DNA polymerase activity of the Y501F, Y501W, and Y501R mutants remains fully sensitive to inhibition by BBNH, additional evidence that the inhibition of multiple RT activities of WT enzyme arises from multiple site binding of the inhibitor.

Our mutational analyses of Tyr-501 also provided the surprising observation that most substitutions at this position resulted in significant losses of RNase H activity, suggesting an essential role for Tyr-501 in RT RNase H activity. In this context, it is interesting to note that virtually all RNases H for which sequence information is available have a tyrosine residue in the position corresponding to that of Tyr-501 in the HIV-1 RT RNase H domain (22, 23). The functional role of Tyr-501 is unclear at present, however, it is interesting to note that each of the amino acid substitutions at Tyr-501, which allowed retention of enzyme activity, namely phenylalanine, tryptophan, and arginine, are capable of participating in π-stacking interactions. One possibility is that Tyr-501 participates in stacking interactions with base moieties of the RNA-DNA nucleic acid duplex substrate, thereby assisting in positioning the substrate in the RNase H active site. Indeed, crystal structures of the binary and ternary complexes of HIV-1 RT bound with double-stranded DNA suggest that Tyr-501 contacts the bound nucleic acid duplex (24, 25). However, further structural information is needed to clarify the role of Tyr-501 in RT RNase H and the equivalent residue in other RNases H.

Although the Y501F, Y501W, and Y501R mutant RT retained WT levels of DNA polymerase and RNase H activity in in vitro assays, molecular clones of HIV-1 containing these same mutations showed varying decreases in viral replication fitness. Whereas the Y501F mutant virus was only slightly decreased in replication efficiency, the Y501W mutant virus showed a 5-fold decrease in replication compared with WT virus, and the Y501R virus was unable to replicate. These observations provide additional evidence of the essential nature of Tyr-501 and also suggest that Tyr-501 may be “immutable” in the context of viral fitness. The development of antiviral drug resistance is a major problem in the therapy of HIV infection, and it may be advantageous to identify drugs that “target” essential protein residues such as Tyr-501. Although it is certainly likely that HIV-1 will develop resistance to such compounds, the development of this resistance may be delayed relative to some other therapeutics, thereby providing prolonged clinical utility.

Whereas BBNH is one of the few relatively potent inhibitors of HIV-1 RT RNase H so far identified, its inhibitory potency is significantly less than other antivirals in clinical use, and as such it should be considered mainly as an interesting lead compound for the development of more potent RNase H inhibitors. Our molecular model suggests that the bound BBNH may be within contact distance of several other RT residues, namely Arg-448, Asn-474, Gln-475, Ser-499, and His-539 (Fig.2
                     B). BBNH analogs that possess structural moieties, which can take advantage of these potential additional contacts, might demonstrate significant improvements in inhibitory potency. Such studies are underway in our laboratory.