PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of aacPermissionsJournals.ASM.orgJournalAAC ArticleJournal InfoAuthorsReviewers
 
Antimicrob Agents Chemother. 2008 June; 52(6): 2035–2042.
Published online 2008 April 7. doi:  10.1128/AAC.00083-08
PMCID: PMC2415781

Mechanism of Inhibition of Human Immunodeficiency Virus Type 1 Reverse Transcriptase by a Stavudine Analogue, 4′-Ethynyl Stavudine Triphosphate[down-pointing small open triangle]

Abstract

2′,3′-Didehydro-3′-deoxy-4′-ethynylthymidine (4′-Ed4T), a recently discovered nucleoside reverse transcriptase (RT) inhibitor, exhibits 5- to 10-fold-higher activity against human immunodeficiency virus type 1 (HIV-1) and less cytotoxicity than does its parental compound d4T (stavudine). Using steady-state kinetic approaches, we have previously shown that (i) 4′-ethynyl-d4T triphosphate (4′-Ed4TTP) inhibits HIV-1 RT more efficiently than d4TTP does and (ii) its inhibition efficiency toward the RT M184V mutant is threefold less than that toward wild-type (wt) RT. In this study we used pre-steady-state kinetic approaches in an attempt to understand its mechanism of inhibition. With wt and the M184V mutant RTs, 4′-Ed4TTP has three- to fivefold-lower Kd (dissociation constant) values than d4TTP, while d4TTP has up to eightfold-higher Kd values than dTTP. Inhibition is more effective in DNA replication with RNA template than with DNA template. In general, the M184V mutant exhibits poorer binding for all three nucleoside triphosphates than does wt RT. The structural basis for the lower binding affinity of d4TTP than of dTTP could be the lack of hydrogen bonds from the missing 3′-hydroxyl group in d4TTP to the backbone amide of Y115 and also to the side chain of Q151. The structural basis for the higher binding affinity of 4′-Ed4TTP than of d4TTP could be the additional binding of the 4′-ethynyl group in a preformed hydrophobic pocket by A114, Y115, M184, F160, and part of D185.

Human immunodeficiency virus type 1 (HIV-1) uses its own reverse transcriptase (RT) to convert its single-stranded RNA genome into a double-stranded DNA copy. Nucleoside RT inhibitors, including zidovudine, didanosine, lamivudine (3TC), and stavudine (d4T), constitute the most important class of antiviral compounds for the treatment of HIV-1 infection (9, 21, 22, 24). However, the application of these compounds is clinically limited due to their cytotoxicity through inhibition of the host DNA polymerases (4, 5, 23) and the rapid emergence of drug-resistant viral mutants. Therefore, developing new compounds with reduced cytotoxicity and improved antiviral potency, especially against drug-resistant viral strains, becomes an urgent therapeutic objective.

Our laboratory recently discovered a novel derivative of d4T, namely, 2′,3′-didehydro-3′-deoxy-4′-ethynylthymidine (4′-Ed4T) (Fig. (Fig.1)1) (6, 27). Compared with its parental compound d4T, 4′-Ed4T is fivefold more potent against HIV-1 replication (6, 27). It also showed much less cytotoxicity than d4T in cell culture studies (6) because 4′-Ed4TTP had no or only a weak inhibitory effect on major host DNA polymerases (41). Moreover, 4′-Ed4T was found to be active against many drug-resistant HIV-1 strains (27). Drug susceptibility studies showed that HIV-1 strains with the M184V single mutation and the P119S/T165A/M184V triple mutations in RT conferred three- to fivefold and 130-fold resistance to 4′-Ed4T, respectively (27).

FIG. 1.
Chemical structures of dT, d4T, and 4′-Ed4T.

Like other nucleoside RT inhibitors, 4′-Ed4T can be phosphorylated in vivo stepwise into its mono-, di-, and triphosphate metabolites by host cell kinases (11, 30). We showed in steady-state enzymatic analyses that 4′-Ed4TTP, the triphosphate metabolite of 4′-Ed4T, was a substrate of HIV-1 RT (41). 4′-Ed4TTP inhibited the DNA polymerase activity of RT more efficiently than d4TTP did. We also showed that the inhibition was more effective on DNA replication with RNA template than on that with DNA template. Furthermore, 4′-Ed4TTP was found to inhibit the M184V mutant with threefold-less efficiency than wild-type (wt) RT, consistent with the drug susceptibility studies (27).

Steady-state kinetic analysis showed that 4′-Ed4TTP had a sevenfold-lower Ki value than that of d4TTP, implying the stronger binding of 4′-Ed4TTP to RT. However, steady-state kinetic analysis provides only mechanistic insight into enzyme inhibition that is related to the rate-limiting step. In the case of RT, the slowest step being examined under steady-state conditions is the dissociation of the elongated DNA product from the enzyme (17). Therefore, this approach is not informative about the detailed interactions of the compound with the RT active site. On the other hand, the pre-steady-state kinetic analysis allows direct examination of the individual steps in the kinetic pathway including binding events, polymerase conformational changes, and the chemical step (14, 15).

In the present study, in order to understand the structure-activity relationship for 4′-Ed4TTP, especially the role of its 4′-ethynyl moiety, the pre-steady-state kinetic parameters for 4′-Ed4TMP incorporation by wt RT during DNA- and RNA-dependent DNA polymerization were determined and compared with those of dTMP and d4TMP incorporation. The 3TC-resistant RT mutant M184V was also included in our pre-steady-state kinetic analysis because (i) the structure of RT-primer/template (P/T)-dTTP ternary complex indicated that Met184 constituted part of the nascent base pairing pocket and could affect incoming nucleotide binding (12); (ii) the M184V viral strain conferred three- to fivefold resistance to 4′-Ed4T (27); and (iii) more importantly, M184V was the first mutation that emerged in the experiment for selection of resistant virus and perhaps is critical for the development of an additional resistance mutation(s) (27). Based on these kinetic results and the existing crystal structures, an inhibition mechanism of 4′-Ed4TTP toward RT is proposed.

MATERIALS AND METHODS

Materials.

4′-Ed4T was synthesized as previously described (10); d4T was purchased from Sigma-Aldrich (St. Louis, MO). The mono-, di-, and triphosphate forms of 4′-Ed4T and d4T were synthesized and purified following published protocols (20). The purity of these compounds was verified by high-pressure liquid chromatography analysis. [γ-32P]ATP was purchased from NEN Life Sciences Company (Boston, MA). Deoxynucleoside triphosphates (dNTPs) were purchased from Amersham/Pharmacia (Piscataway, NJ). All other chemicals used were of analytical grade.

The DNA oligonucleotides (23- and 36-mer, with sequences corresponding to HIV-1 5′ untranslated region [Table [Table1])1]) were synthesized and gel purified by the Keck Facility at Yale University. The 36-mer RNA oligonucleotide was synthesized and gel purified by New England Biolabs (Ipswich, MA). T4 polynucleotide kinase was purchased from New England Biolabs. The plasmid for expression of the wt RT p66/p51 heterodimer was generously provided by Stephen Hughes (National Cancer Institute, Frederick, MD). The M184V mutant was constructed using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). HIV-1 wt and the M184V mutant RT proteins were purified according to a published method (25).

TABLE 1.
Sequences of oligonucleotide used in this studya

Preparation of DNA/RNA and DNA/DNA duplexes.

For radioactively labeled DNA/RNA and DNA/DNA P/T, the primer was first labeled at the 5′ end with [γ-32P]ATP by T4 polynucleotide kinase and then annealed with the template in a molar ratio of 1:1.3 at 80°C for 4 min and then 50°C for 30 min.

Pre-steady-state burst and single-turnover experiments.

Pre-steady-state rapid chemical quench experiments were performed with the KinTek quench-flow apparatus (model RQF-3; KinTek Corp., University Park, PA). Unless noted otherwise, all components of the reaction mixtures are reported as final concentrations after mixing.

Burst assays were carried out under the conditions in which the P/T concentration was three times greater than the enzyme concentration. The reaction was carried out at 25°C by mixing equal volumes of buffer A (50 mM Tris-HCl, pH 7.8, 50 mM NaCl, containing the preincubated complex of 600 nM 5′-32P-labeled P/T and 200 nM wt RT or the M184V mutant RT) with buffer B (50 mM Tris-HCl, pH 7.8, containing 50 mM NaCl, 20 mM MgSO4, and 2 mM dTTP) to give final concentrations of 300 nM P/T, 100 nM enzyme, and 1 mM dTTP. The polymerization reaction was quenched with 0.5 M EDTA at defined time intervals. Products were analyzed by gel electrophoresis (20% polyacrylamide-50% urea) and quantified by phosphorimaging (Molecular Dynamics). Single-turnover assays were performed in a manner similar to that described above for the burst assays except that the enzyme (250 nM) was used in excess of the P/T (50 nM), and concentrations of dTTP (or d4TTP or 4′-Ed4TTP) were varied in order to determine Kd (dissociation constant) and kpol (maximum rate of catalysis) values.

Data analysis.

Data from burst assays were fitted to the burst equation [product] = A[1 − exp(kobt)] + ksst, where A represents the amplitude of the burst which provides an estimate of the concentration of enzyme active sites, kob is the observed first-order rate constant for deoxynucleoside monophosphate (dNMP) incorporation, and kss is the observed steady-state rate constant.

As previously reported (31, 37, 40), complex kinetics (an exponential phase followed by a linear phase) were observed with RT in single-turnover experiments. This biphasic kinetic was also observed in all cases in our study. In order to determine Kd, the dissociation constant for the incoming dNTP binding to the E-P/T complex, the data were fitted into the equation kobs = (kpol[S])/(Kd + [S]), where kpol is the maximum rate of dNMP incorporation and [S] is the concentration of incoming dNTP.

RESULTS

Burst assays.

Burst experiments are usually used to determine the active-site concentration of enzyme, the initial rate of dNMP incorporation, and the steady-state rate (kss) for the nucleotidyl transfer reaction under saturating concentrations of incoming nucleotides. In this type of experiment, the amount of the DNA/DNA or DNA/RNA P/T substrate is in slight excess over the amount of enzyme such that the first enzyme turnover as well as the subsequent turnovers can be examined. A burst formation of elongated P/T product followed by a linear phase indicates that the overall rate-limiting step for the incorporation of dNMPs by RT occurs after chemistry, which is the release of the elongated P/T product from RT. Previous studies have shown biphasic curves with dTTP and d4TTP, suggesting that the P/T release step after chemistry is rate limiting during both dTMP and d4TMP incorporation by RT (35, 38).

In this study, burst experiments were conducted using the DNA/DNA as well as the DNA/RNA P/Ts to examine the DNA- and RNA-dependent incorporation of 4′-Ed4TMP by wt RT or the M184V mutant. The results were compared with those of dTMP and d4TMP incorporation. In all cases, the incorporation of 4′-Ed4TMP into either DNA/DNA or DNA/RNA P/T by wt RT or the M184V mutant showed a biphasic burst curve, suggesting that the reaction pathway for incorporation of the three nucleotides had not changed, in which the release of elongated P/T product remained the rate-limiting step. Representative pre-steady-state burst experiments for dTMP, d4TMP, and 4′-Ed4TMP incorporation into the DNA/DNA P/T by wt RT are shown in Fig. Fig.2.2. Notably, the rates of the rate-limiting step in all cases including dTMP, d4TMP, and 4′-Ed4TMP incorporation by either wt RT or the M184V mutant remained relatively unchanged within a narrow range from 0.05 to 0.16 s−1 (Table (Table22).

FIG. 2.
Pre-steady-state burst assays of incorporation of various nucleotides into a 23-/36-mer DNA/DNA P/T by wt RT. The kinetics were measured by mixing a preincubated solution of wt RT (200 nM) and 23-/36-mer DNA/DNA P/T (600 nM) with 2 mM nucleoside triphosphate. ...
TABLE 2.
Rate of dissociation of elongated P/T (kss) after dTMP, d4TMP, and 4′-Ed4TMP incorporation by wt RT and the M184V mutanta

Single-turnover incorporation of dTMP, d4TMP, and 4′-Ed4TMP by wt RT.

Single-turnover experiments are usually performed under conditions in which the concentration of the enzyme is in excess of that of P/T, so that the dissociation constant Kd for incoming dNTP binding to the RT-P/T complex as well as the maximum rate (kpol) for the nucleotidyl transfer reaction can be determined. Under these conditions, the formation of an elongated P/T would be expected to fit a single exponential curve, as all of the prebound P/T substrate is converted to product in the first turnover step. However, complex kinetic behavior was always observed with RT (31, 37, 40). In most cases, the kinetics occurred with an exponential burst followed by a linear phase. Further, the amplitude of the first phase is always lower with the DNA/DNA P/T (less than 45% of active-site concentration of RT) than with the DNA/RNA P/T (more than 80% of active-site concentration of RT) (40). It was suggested that depending on the P/T, a certain fraction of P/T could bind to the enzyme in an incorrect orientation or configuration that does not allow nucleotide incorporation (31, 37, 40). In any case, the rate of incorporation observed during the first phase likely represents the maximum rate of catalysis and therefore was used for the determination of Kd and kpol values in this study.

To establish the inhibition mechanism of 4′-Ed4TTP toward RT, the pre-steady-state Kd and kpol values for 4′-Ed4TMP incorporation by wt RT into the DNA/DNA and the DNA/RNA P/Ts were determined and compared with those of dTMP and d4TMP incorporation. Figure Figure33 shows the concentration-dependent curve of dTMP, d4TMP, and 4′-Ed4TMP incorporation by wt RT with DNA/DNA P/T, and the results are summarized in Table Table3.3. The incorporation efficiencies (kpol/Kd) determined with each nucleotide were used to calculate the selectivity factor, which is the efficiency of dTTP divided by the efficiency of d4TTP or 4′-Ed4TTP.

FIG. 3.
Single-turnover assays for determination of Kd and kpol of dTMP, d4TMP, and 4′-Ed4TMP incorporation by wt RT. (A) The observed rates of dTMP incorporation into the 23-/36-mer DNA/DNA P/T by wt RT were plotted against dTTP concentration to give ...
TABLE 3.
Pre-steady-state kinetic parameters for dTMP, d4TMP, and 4′-Ed4TMP incorporation by wt RT and the M184V mutant with DNA/DNA and DNA/RNA P/Ts

For the DNA/DNA P/T, the values for Kd (15.4 μM) and kpol (22.6 s−1) for dTTP with wt RT gave an overall efficiency of dTMP incorporation at 1.47 μM−1 s−1, consistent with the previously reported values (18, 35). The kpol value for d4TTP is 16 s−1, similar to that for dTTP, while the Kd value for d4TTP is 48.0 μM, about threefold higher than that for dTTP. Hence, the efficiency of d4TMP incorporation is 0.33 μM−1 s−1, which is about fourfold lower than that of dTTP. On the other hand, the Kd value for 4′-Ed4TTP is 15.8 μM, the same as that for dTTP, but threefold lower than that for its parental compound d4TTP. The kpol value for 4′-Ed4TTP is 12 s−1, slightly less than those for dTTP and d4TTP. The efficiency of 4′-Ed4TMP incorporation is 0.77 μM−1 s−1, which is about twofold less than that of dTMP incorporation but about twofold higher than that of d4TMP incorporation.

For the DNA/RNA P/T, the Kd value for dTTP by wt RT is 67.1 μM, 4.5-fold higher than that for the DNA/DNA P/T, while the kpol value also increased by threefold to 65.0 s−1. This result is consistent with the previously published results, indicating that polymerization usually happens faster with the DNA/RNA P/T than with the DNA/DNA P/T (7, 17, 18). The Kd value for d4TTP is 40.8 μM, slightly lower than that for dTTP, while the kpol value for d4TTP is 18.4 s−1, 3.5-fold less than that for dTTP, resulting in the overall efficiency of d4TMP incorporation being about 50% of that of dTMP incorporation. On the other hand, the Kd value for 4′-Ed4TTP is 11.4 μM, which is sixfold less than that for dTTP, suggesting an even tighter binding to the RT-DNA/RNA complex. In spite of the low kpol value for 4′-Ed4TTP compared with that for dTTP, the overall efficiency of 4′-Ed4TMP incorporation is 1.0 μM−1 s−1, the same as that of dTMP, and twofold higher than that of d4TMP.

Single-turnover incorporation of dTMP, d4TMP, and 4′-Ed4TMP by the RT M184V mutant.

Similarly, the pre-steady-state kinetic constants for dTMP, d4TMP, and 4′-Ed4TMP incorporation by the RT M184V mutant were determined under the single-turnover conditions.

For the DNA/DNA P/T, the Kd value for dTTP with the M184V mutant is fivefold higher than that with wt RT, while the kpol value with the M184V mutant is the same as that with wt RT. The Kd value for d4TTP is eightfold higher than that for dTTP, which makes the efficiency of d4TMP incorporation about sixfold less than that of dTMP incorporation. The Kd for 4′-Ed4TTP is 168.1 μM, which is only about threefold lower than that for d4TTP, and the efficiency of 4′-Ed4TMP incorporation is twofold higher than that of d4TMP incorporation but threefold lower than that of dTMP incorporation.

For the DNA/RNA P/T, the Kd for dTTP with the M184V mutant is 143.9 μM, which is twofold higher than that for the DNA/DNA P/T and also twofold higher than the Kd value for dTTP with wt RT and the DNA/RNA P/T. The Kd value for d4TTP with the M184V mutant is 232.3 μM, which is about 1.7-fold higher than that for dTTP, while the kpol value for d4TTP is slightly lower than that for dTTP. For 4′-Ed4TTP, the Kd value is 43.4 μM, which is fivefold lower than that for d4TTP and threefold lower than that for dTTP. The kpol value for 4′-Ed4TTP is three- to fourfold lower than that for dTTP and d4TTP. The efficiency for 4′-Ed4TMP incorporation is 0.22 μM−1 s−1, which is twofold higher than that for d4TMP incorporation and similar to that for dTMP incorporation.

Computer modeling.

The crystal structure of the HIV-1 RT-P/T-dTTP ternary complex (with DNA/DNA P/T) has revealed active-site residues that are involved in the formation of the nucleotide-binding pocket (12). With the aid of computer modeling, this structure provides a framework for predicting possible interactions of HIV-1 RT with d4TTP and 4′-Ed4TTP.

To explain the observed kinetic behaviors of 4′-Ed4TTP, we modeled an ethynyl group into the 4′ position of d4TTP according to its known geometry arranged in the crystal structure of the HIV-1 RT-DNA-dTTP ternary complex (12). We observed an additional binding of the 4′-ethynyl group at a hydrophobic pocket, which is preformed by the side chains of A114, Y115, M184, F160, and D185 (Fig. (Fig.4A).4A). The estimated distances from the C-2 atom of the 4′-ethynyl group to Cβ of A114, to Cδ2 of F160, and to Sδ of M184 are 3.7 Å, 3.3 Å, and 3.4 Å, respectively, all of which fall into the ideal range for Van de Waals interactions.

FIG. 4.
Computer modeling of the 4′-Ed4TTP onto the wt RT-DNA/DNA-dTTP ternary complex (A) and the in silico mutated M184V-DNA/DNA-dTTP ternary complex (B). The modified nucleotide with the double bond formed between C-2 and C-3 was taken from the structure ...

To validate our model about the 4′-Ed4TTP binding to RT, we introduced mutations to the binding pocket, namely, A114M, A114L, Y115Q, F160A, F160L, M184A, M184F, and M184V. We predicted that these mutants would have lower binding affinity for 4′-Ed4TTP if the structural modifications destroy the binding pocket by filling or opening it. Unfortunately, all mutants except M184V and M184A exhibited very poor enzyme activity, making further investigation impossible.

DISCUSSION

The studies presented herein address the mechanistic basis for the inhibition of HIV-1 RT by 4′-Ed4TTP, highlighting the specific contribution from its 4′-ethynyl moiety.

4′-Ed4TTP has higher binding affinity for RT than does d4TTP.

Studies with 4′-substituted 2′-deoxynucleosides have demonstrated the superior potency of the 4′-ethynyl substitution against HIV-1 (10, 19, 26, 28). Our previous steady-state study showed that 4′-Ed4TTP inhibited the DNA polymerase activity of HIV-1 RT more efficiently than did d4TTP (41). Its low steady-state Ki values imply the strong binding to HIV-1 RT (41). Here we used transient kinetic analysis to show that 4′-Ed4TTP displayed three- to fivefold-lower Kd values than its parental compound, d4TTP, in the context of both wt RT and the M184V mutant, regardless of DNA/DNA or DNA/RNA P/T. This result suggests a tighter binding of RT with 4′-Ed4TTP than with d4TTP. The efficiency of 4′-Ed4TMP incorporation (kpol/Kd) is twofold higher than that of d4TMP incorporation, mostly due to the lower Kd values for 4′-Ed4TTP. Notably, 4′-Ed4TMP can be incorporated into DNA/RNA P/T by wt RT as efficiently as the natural nucleotide dTMP due to its much-reduced Kd.

The selectivity for dTTP over 4′-Ed4TTP is 0.97 with DNA/RNA P/T, twofold lower than with DNA/DNA P/T, suggesting that 4′-Ed4TTP inhibits RT more efficiently in DNA replication with RNA template than with DNA template. This is consistent with our previous steady-state measurements (41).

Incorporation of 4′-Ed4TMP by the M184V mutant.

Previously using drug susceptibility assays we showed that 4′-Ed4T inhibits HIV-1 harboring the M184V mutation three to five times less efficiently than wt HIV-1 (27), a finding that is consistent with our steady-state analysis, which showed that 4′-Ed4TTP inhibits the M184V mutant two- to threefold less efficiently than wt RT (41). In the present study, we employed transient kinetic analysis to focus on drug resistance during the first turnover of DNA polymerization.

Our results showed that the efficiency of dTMP, d4TMP, and 4′-Ed4TMP incorporation by the M184V mutant RT decreased three- to sevenfold compared with wt RT. The change is mainly due to the weaker binding affinity of these nucleotides for the M184V mutant than for wt RT (Table (Table3).3). The drug-resistant M184V mutant RT has previously been subject to extensive steady-state and pre-steady-state kinetic studies. However, resulting kinetic parameters varied widely with the experimental approach, the sequence of the P/T duplex, and the nature of both the template strand and the base pairing of the incoming nucleotides (1-3, 8, 31, 39). Notably, a pre-steady-state study indicated that the overall efficiency of d4GMP incorporation by the M184V mutant was the same as the efficiency of that by wt RT, and for both wt RT and the M184V mutant, the selectivity for dGTP over d4GTP was 2 for both the DNA/DNA and DNA/RNA P/Ts (31).

The sequence of P/T used in our study came from the HIV-1 5′ untranslated region in order to mimic the initiation of viral DNA replication in vivo. We observed that the efficiency of d4TMP incorporation by the M184V mutant decreased six- and threefold for DNA/DNA and DNA/RNA P/Ts, respectively, compared with wt RT. The corresponding selectivity for dTTP over d4TTP with M184V is 6.2 and 2.2 for the two P/Ts. In comparison, the selectivity associated with wt RT in the context of DNA/DNA and DNA/RNA P/Ts is 4.5 and 2.2, respectively. The M184V mutant is not expected to exhibit drug resistance toward d4TTP or d4GTP, in agreement with the drug susceptibility studies involving d4G or d4T treatment of HIV-1 bearing the M184V mutation (27, 31).

wt RT showed a selectivity of 1.9 for dTTP over 4′-Ed4TTP with the DNA/DNA P/T and 0.97 with the DNA/RNA P/T, whereas the M184V mutant showed a selectivity of 2.8 with the DNA/DNA P/T and 1.3 with the DNA/RNA P/T, respectively. Although the M184V mutant always shows selectivity factors slightly higher than those of wt RT, the difference is not significant enough to demonstrate the resistance of the M184V mutant to 4′-Ed4TTP (Table (Table3).3). However, about threefold resistance was observed in both a steady-state kinetic study (41) and a drug susceptibility study (27). One plausible explanation for this difference is that the threefold resistance observed in the steady-state measurement was a result of amplification after multiple turnovers of enzyme reaction, which was unlikely to be detected in our pre-steady-state single-turnover experiment. A similar amplification effect was also observed in the pre-steady-state kinetic study of 3TC-TP, where the selectivity for dCTP over 3TC-TP by the M184V mutant increased by 34-fold in DNA-dependent polymerization and 140-fold in RNA-dependent polymerization compared with wt RT (8), which led to an over-1,000-fold resistance in cell culture and clinic studies (33, 34).

The structural basis for the increased binding affinity of 4′-Ed4TTP to RT compared with d4TTP.

The crystal structure of the HIV-1 RT-P/T-dTTP ternary complex shows that the 3′-hydroxyl group of dTTP forms one hydrogen bond with the backbone amide of the sugar gate Y115 and another bond with the side chain of Q151 (12, 13). This structural feature suggests that in general DNA chain-terminating nucleotide analogues lacking the 3′-hydroxyl group do not bind well at the polymerase active site (12, 13) unless the loss of bonding is compensated elsewhere.

Our kinetic parameters for d4TMP incorporation are consistent with structural predictions. There are two differences between d4TTP and dTTP: (i) a double bond between C-2′-C-3′ bond and (ii) the missing 3′-hydroxyl group at C-3′ rendering d4TTP the chain terminator property. The absence of the 3′-hydroxyl group results in the loss of two hydrogen bonds as the crystal structure predicts, which accounts for the lower binding affinity for d4TTP than for dTTP as observed in our study. The contribution from the new double bond between C-2′ and C-3′ atoms is not expected to be substantial, because there is only a slight change in the sugar configuration between dTMP and d4TMP that binds to human thymidylate kinase (29). This model should predict the same kinetic behaviors not only for d4TTP and dTTP but also for other chain-terminating nucleotide analogues and their natural parent nucleoside triphosphates, including d4GTP and dGTP. However, we are aware that d4GTP and dGTP were reported to have the same binding affinity for wt RT (31), which cannot be simply explained by our computer modeling.

Using computer modeling, we observed additional binding of the 4′-ethynyl group at a hydrophobic pocket of RT, which could increase the binding affinity of 4′-Ed4TTP for RT compared with that of d4TTP (Fig. (Fig.4A).4A). This prediction is supported by our observation of a threefold-lower Kd value of 4′-Ed4TTP than of d4TTP (Table (Table33).

Consistent with our prediction that the size and shape of this pocket in the M184V mutant will be different from those in wt RT (Fig. (Fig.4B),4B), we showed that indeed the M184V mutation had caused a 4- and 10-fold decrease in the binding affinity of 4′-Ed4TTP for RNA and DNA template, respectively.

We have also found a fivefold reduction in the binding of dTTP to the M184V mutant (Table (Table3).3). This observation is unexpected from existing crystal structures or from the above modeling, because Met184 is not in direct contact with dTTP. A possible explanation for the lower binding affinity of incoming nucleotides for the M184V mutant is that Met184 constitutes part of the nascent base-pairing pocket (12), and the M184V mutation apparently created a gap between the polymerase and the minor groove of the nascent base pair, which could indirectly affect the binding of the incoming dTTP. Similar indirect effects have been observed in RB69 DNA polymerases with the L561A and Y567A mutations (42), which are analogous to the M184V mutation in HIV-1 RT.

Inhibition of HIV-1 RT by another 4′-ethynyl-substituted nucleoside analogue, 4′-EddCTP, has been studied by Siddiqui et al. (36). The d-enantiomer of this compound strongly inhibited both wt RT and the M184V mutant. On the other hand, its l-enantiomer inhibited only wt RT, but not the M184V mutant RT. With the solved crystal structure of 4′-EddCTP, both of its d- and l-enantiomers were docked into the active site of the RT-DNA/DNA-dNTP ternary complex (36). According to the computer modeling study, the 4′-ethynyl group of both the d- and l-enantiomers was close to the Met184 residue in wt RT. In the model of the M184V mutant, the 4′-ethynyl group of the d-enantiomer showed some negative steric interaction with Val184. When the l-enantiomer was docked into the M184V mutant, there was a steric clash between the ethynyl group and Val184, which could explain the lack of inhibition of the M184V mutant by the l-enantiomer (36). Here we can make the same modeling for 4′-EddCTP as for 4′-Ed4TTP by changing the nature of the base and the enantiomer of the sugar. Our modeling also predicts that both the d- and l-enantiomers should bind wt RT, thereby terminating the replication upon incorporation of the compound. In the l-enantiomer of 4′-EddCTP, our modeling shows that the branched Val184 side chain of the M184V mutant would clash with the compound at its branching point Cγ1 or Cγ2, but this branched Val184 side chain would not clash with the d-enantiomer (Fig. (Fig.4B).4B). Therefore, our model is consistent with the corresponding kinetic data for 4′-EddCTP.

Previously we found that 4′-Ed4TTP exhibited poor inhibitory effects on five major human DNA polymerases, α, β, γ, δ, and epsilon, in general (41). Particularly, we showed that human DNA polymerase β was selectively inhibited by d4TTP, but not by 4′-Ed4TTP (with a more-than-100-fold difference in 50% inhibitory concentration) (41). Because the crystal structures of polymerase β are available (32), we could extend computer modeling techniques to this polymerase. By adopting modeling approaches similar to what we have done for HIV-1 RT, we found that the 4′-ethynyl group of 4′-Ed4TTP could not fit into the ternary complex due to a steric clash with the F272 side chain, which could explain its apparent lower toxicity in the cell (data not shown).

In conclusion, our pre-steady-state kinetic study together with computer modeling illustrated the fact that 4′-Ed4TTP is a better RT inhibitor than d4TTP due to the additional binding of the 4′-ethynyl group at a preformed hydrophobic pocket in the RT active site. Inhibition of RT was greater in cells treated with 4′-Ed4T than in those treated with d4T, and 4′-Ed4T treatment showed a unique resistance profile (27), much less cytotoxicity (6), and superior persistence of antiviral activity (30). All these features make 4′-Ed4T a promising candidate for HIV-1 chemotherapy.

Acknowledgments

We thank William Konigsberg for the access to the KinTek Rapid Quench apparatus in his laboratory and carefully reading the manuscript. We thank Stephen Hughes for providing us with the wt RT plasmid.

This work was supported by Public Health Service grant AI-38204 from the National Institutes of Health to Y.-C.C. Y.-C.C. is a fellow of the National Foundation for Cancer Research.

Footnotes

[down-pointing small open triangle]Published ahead of print on 7 April 2008.

REFERENCES

1. Back, N. K., M. Nijhuis, W. Keulen, C. A. Boucher, B. O. Oude Essink, A. B. van Kuilenburg, A. H. van Gennip, and B. Berkhout. 1996. Reduced replication of 3TC-resistant HIV-1 variants in primary cells due to a processivity defect of the reverse transcriptase enzyme. EMBO J. 15:4040-4049. [PubMed]
2. Boyer, P. L., and S. H. Hughes. 1995. Analysis of mutations at position 184 in reverse transcriptase of human immunodeficiency virus type 1. Antimicrob. Agents Chemother. 39:1624-1628. [PMC free article] [PubMed]
3. Chao, S. F., V. L. Chan, P. Juranka, A. H. Kaplan, R. Swanstrom, and C. A. Hutchison III. 1995. Mutational sensitivity patterns define critical residues in the palm subdomain of the reverse transcriptase of human immunodeficiency virus type 1. Nucleic Acids Res. 23:803-810. [PMC free article] [PubMed]
4. Chen, C. H., and Y. C. Cheng. 1989. Delayed cytotoxicity and selective loss of mitochondrial DNA in cells treated with the anti-human immunodeficiency virus compound 2′,3′-dideoxycytidine. J. Biol. Chem. 264:11934-11937. [PubMed]
5. Chen, C. H., M. Vazquez-Padua, and Y. C. Cheng. 1991. Effect of anti-human immunodeficiency virus nucleoside analogs on mitochondrial DNA and its implication for delayed toxicity. Mol. Pharmacol. 39:625-628. [PubMed]
6. Dutschman, G. E., S. P. Grill, E. A. Gullen, K. Haraguchi, S. Takeda, H. Tanaka, M. Baba, and Y. C. Cheng. 2004. Novel 4′-substituted stavudine analog with improved anti-human immunodeficiency virus activity and decreased cytotoxicity. Antimicrob. Agents Chemother. 48:1640-1646. [PMC free article] [PubMed]
7. Feng, J. Y., and K. S. Anderson. 1999. Mechanistic studies comparing the incorporation of (+) and (−) isomers of 3TCTP by HIV-1 reverse transcriptase. Biochemistry 38:55-63. [PubMed]
8. Feng, J. Y., and K. S. Anderson. 1999. Mechanistic studies examining the efficiency and fidelity of DNA synthesis by the 3TC-resistant mutant (184V) of HIV-1 reverse transcriptase. Biochemistry 38:9440-9448. [PubMed]
9. Hamamoto, Y., H. Nakashima, T. Matsui, A. Matsuda, T. Ueda, and N. Yamamoto. 1987. Inhibitory effect of 2′,3′-didehydro-2′,3′-dideoxynucleosides on infectivity, cytopathic effects, and replication of human immunodeficiency virus. Antimicrob. Agents Chemother. 31:907-910. [PMC free article] [PubMed]
10. Haraguchi, K., S. Takeda, H. Tanaka, T. Nitanda, M. Baba, G. E. Dutschman, and Y. C. Cheng. 2003. Synthesis of a highly active new anti-HIV agent 2′,3′-didehydro-3′-deoxy-4′-ethynylthymidine. Bioorg. Med. Chem. Lett. 13:3775-3777. [PubMed]
11. Hsu, C. H., R. Hu, G. E. Dutschman, G. Yang, P. Krishnan, H. Tanaka, M. Baba, and Y. C. Cheng. 2007. Comparison of the phosphorylation of 4′-ethynyl 2′,3′-dihydro-3′-deoxythymidine with that of other anti-human immunodeficiency virus thymidine analogs. Antimicrob. Agents Chemother. 51:1687-1693. [PMC free article] [PubMed]
12. Huang, H., R. Chopra, G. L. Verdine, and S. C. Harrison. 1998. Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistance. Science 282:1669-1675. [PubMed]
13. Jamburuthugoda, V. K., D. Guo, J. E. Wedekind, and B. Kim. 2005. Kinetic evidence for interaction of human immunodeficiency virus type 1 reverse transcriptase with the 3′-OH of the incoming dTTP substrate. Biochemistry 44:10635-10643. [PubMed]
14. Johnson, K. A. 1993. Conformational coupling in DNA polymerase fidelity. Annu. Rev. Biochem. 62:685-713. [PubMed]
15. Johnson, K. J., J. Varani, and J. E. Smolen. 1992. Neutrophil activation and function in health and disease. Immunol. Ser. 57:1-46.
16. Jones, T. A., J. Y. Zou, S. W. Cowan, and M. Kjeldgaard. 1991. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47:110-119. [PubMed]
17. Kati, W. M., K. A. Johnson, L. F. Jerva, and K. S. Anderson. 1992. Mechanism and fidelity of HIV reverse transcriptase. J. Biol. Chem. 267:25988-25997. [PubMed]
18. Kerr, S. G., and K. S. Anderson. 1997. Pre-steady-state kinetic characterization of wild type and 3′-azido-3′-deoxythymidine (AZT) resistant human immunodeficiency virus type 1 reverse transcriptase: implication of RNA directed DNA polymerization in the mechanism of AZT resistance. Biochemistry 36:14064-14070. [PubMed]
19. Kodama, E. I., S. Kohgo, K. Kitano, H. Machida, H. Gatanaga, S. Shigeta, M. Matsuoka, H. Ohrui, and H. Mitsuya. 2001. 4′-Ethynyl nucleoside analogs: potent inhibitors of multidrug-resistant human immunodeficiency virus variants in vitro. Antimicrob. Agents Chemother. 45:1539-1546. [PMC free article] [PubMed]
20. Krishnan, P., J. Y. Liou, and Y. C. Cheng. 2002. Phosphorylation of pyrimidine L-deoxynucleoside analog diphosphates. Kinetics of phosphorylation and dephosphorylation of nucleoside analog diphosphates and triphosphates by 3-phosphoglycerate kinase. J. Biol. Chem. 277:31593-31600. [PubMed]
21. Lin, T. S., R. F. Schinazi, M. S. Chen, E. Kinney-Thomas, and W. H. Prusoff. 1987. Antiviral activity of 2′,3′-dideoxycytidin-2′-ene (2′,3′-dideoxy-2′,3′-didehydrocytidine) against human immunodeficiency virus in vitro. Biochem. Pharmacol. 36:311-316. [PubMed]
22. Lin, T. S., R. F. Schinazi, and W. H. Prusoff. 1987. Potent and selective in vitro activity of 3′-deoxythymidin-2′-ene (3′-deoxy-2′,3′-didehydrothymidine) against human immunodeficiency virus. Biochem. Pharmacol. 36:2713-2718. [PubMed]
23. Medina, D. J., C. H. Tsai, G. D. Hsiung, and Y. C. Cheng. 1994. Comparison of mitochondrial morphology, mitochondrial DNA content, and cell viability in cultured cells treated with three anti-human immunodeficiency virus dideoxynucleosides. Antimicrob. Agents Chemother. 38:1824-1828. [PMC free article] [PubMed]
24. Mitsuya, H., R. Yarchoan, and S. Broder. 1990. Molecular targets for AIDS therapy. Science 249:1533-1544. [PubMed]
25. Murakami, E., A. S. Ray, R. F. Schinazi, and K. S. Anderson. 2004. Investigating the effects of stereochemistry on incorporation and removal of 5-fluorocytidine analogs by mitochondrial DNA polymerase gamma: comparison of D- and L-D4FC-TP. Antivir. Res. 62:57-64. [PubMed]
26. Nakata, H., M. Amano, Y. Koh, E. Kodama, G. Yang, C. M. Bailey, S. Kohgo, H. Hayakawa, M. Matsuoka, K. S. Anderson, Y. C. Cheng, and H. Mitsuya. 2007. Activity against human immunodeficiency virus type 1, intracellular metabolism, and effects on human DNA polymerases of 4′-ethynyl-2-fluoro-2′-deoxyadenosine. Antimicrob. Agents Chemother. 51:2701-2708. [PMC free article] [PubMed]
27. Nitanda, T., X. Wang, H. Kumamoto, K. Haraguchi, H. Tanaka, Y. C. Cheng, and M. Baba. 2005. Anti-human immunodeficiency virus type 1 activity and resistance profile of 2′,3′-didehydro-3′-deoxy-4′-ethynylthymidine in vitro. Antimicrob. Agents Chemother. 49:3355-3360. [PMC free article] [PubMed]
28. Ohrui, H., S. Kohgo, K. Kitano, S. Sakata, E. Kodama, K. Yoshimura, M. Matsuoka, S. Shigeta, and H. Mitsuya. 2000. Syntheses of 4′-C-ethynyl-beta-D-arabino- and 4′-C-ethynyl-2′-deoxy-beta-D-ribo-pentofuranosylpyrimidines and -purines and evaluation of their anti-HIV activity. J. Med. Chem. 43:4516-4525. [PubMed]
29. Ostermann, N., D. Segura-Pena, C. Meier, T. Veit, C. Monnerjahn, M. Konrad, and A. Lavie. 2003. Structures of human thymidylate kinase in complex with prodrugs: implications for the structure-based design of novel compounds. Biochemistry 42:2568-2577. [PubMed]
30. Paintsil, E., G. E. Dutschman, R. Hu, S. P. Grill, W. Lam, M. Baba, H. Tanaka, and Y. C. Cheng. 2007. Intracellular metabolism and persistence of the anti-human immunodeficiency virus activity of 2′,3′-didehydro-3′-deoxy-4′-ethynylthymidine, a novel thymidine analog. Antimicrob. Agents Chemother. 51:3870-3879. [PMC free article] [PubMed]
31. Ray, A. S., Z. Yang, J. Shi, A. Hobbs, R. F. Schinazi, C. K. Chu, and K. S. Anderson. 2002. Insights into the molecular mechanism of inhibition and drug resistance for HIV-1 RT with carbovir triphosphate. Biochemistry 41:5150-5162. [PubMed]
32. Sawaya, M. R., R. Prasad, S. H. Wilson, J. Kraut, and H. Pelletier. 1997. Crystal structures of human DNA polymerase beta complexed with gapped and nicked DNA: evidence for an induced fit mechanism. Biochemistry 36:11205-11215. [PubMed]
33. Schinazi, R. F., R. M. Lloyd, Jr., M. H. Nguyen, D. L. Cannon, A. McMillan, N. Ilksoy, C. K. Chu, D. C. Liotta, H. Z. Bazmi, and J. W. Mellors. 1993. Characterization of human immunodeficiency viruses resistant to oxathiolane-cytosine nucleosides. Antimicrob. Agents Chemother. 37:875-881. [PMC free article] [PubMed]
34. Schuurman, R., M. Nijhuis, R. van Leeuwen, P. Schipper, D. de Jong, P. Collis, S. A. Danner, J. Mulder, C. Loveday, C. Christopherson, et al. 1995. Rapid changes in human immunodeficiency virus type 1 RNA load and appearance of drug-resistant virus populations in persons treated with lamivudine (3TC). J. Infect. Dis. 171:1411-1419. [PubMed]
35. Selmi, B., J. Boretto, J. M. Navarro, J. Sire, S. Longhi, C. Guerreiro, L. Mulard, S. Sarfati, and B. Canard. 2001. The valine-to-threonine 75 substitution in human immunodeficiency virus type 1 reverse transcriptase and its relation with stavudine resistance. J. Biol. Chem. 276:13965-13974. [PubMed]
36. Siddiqui, M. A., S. H. Hughes, P. L. Boyer, H. Mitsuya, Q. N. Van, C. George, S. G. Sarafinanos, and V. E. Marquez. 2004. A 4′-C-ethynyl-2′,3′-dideoxynucleoside analogue highlights the role of the 3′-OH in anti-HIV active 4′-C-ethynyl-2′-deoxy nucleosides. J. Med. Chem. 47:5041-5048. [PubMed]
37. Thrall, S. H., R. Krebs, B. M. Wohrl, L. Cellai, R. S. Goody, and T. Restle. 1998. Pre-steady-state kinetic characterization of RNA-primed initiation of transcription by HIV-1 reverse transcriptase and analysis of the transition to a processive DNA-primed polymerization mode. Biochemistry 37:13349-13358. [PubMed]
38. Vaccaro, J. A., K. M. Parnell, S. A. Terezakis, and K. S. Anderson. 2000. Mechanism of inhibition of the human immunodeficiency virus type 1 reverse transcriptase by d4TTP: an equivalent incorporation efficiency relative to the natural substrate dTTP. Antimicrob. Agents Chemother. 44:217-221. [PMC free article] [PubMed]
39. Wakefield, J. K., S. A. Jablonski, and C. D. Morrow. 1992. In vitro enzymatic activity of human immunodeficiency virus type 1 reverse transcriptase mutants in the highly conserved YMDD amino acid motif correlates with the infectious potential of the proviral genome. J. Virol. 66:6806-6812. [PMC free article] [PubMed]
40. Wohrl, B. M., R. Krebs, R. S. Goody, and T. Restle. 1999. Refined model for primer/template binding by HIV-1 reverse transcriptase: pre-steady-state kinetic analyses of primer/template binding and nucleotide incorporation events distinguish between different binding modes depending on the nature of the nucleic acid substrate. J. Mol. Biol. 292:333-344. [PubMed]
41. Yang, G., G. E. Dutschman, C. J. Wang, H. Tanaka, M. Baba, K. S. Anderson, and Y. C. Cheng. 2007. Highly selective action of triphosphate metabolite of 4′-ethynyl D4T: a novel anti-HIV compound against HIV-1 RT. Antivir. Res. 73:185-191. [PubMed]
42. Zhang, H., C. Rhee, A. Bebenek, J. W. Drake, J. Wang, and W. Konigsberg. 2006. The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Biochemistry 45:2211-2220. [PubMed]

Articles from Antimicrobial Agents and Chemotherapy are provided here courtesy of American Society for Microbiology (ASM)