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Originally published In Press as doi:10.1074/jbc.M411559200 on November 17, 2004

J. Biol. Chem., Vol. 280, Issue 5, 3838-3846, February 4, 2005
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Mechanistic Insights into the Suppression of Drug Resistance by Human Immunodeficiency Virus Type 1 Reverse Transcriptase Using {alpha}-Boranophosphate Nucleoside Analogs*

Jérôme Deval{ddagger}§, Karine Alvarez{ddagger}, Boulbaba Selmi{ddagger}, Marielle Bermond{ddagger}, Joëlle Boretto{ddagger}, Catherine Guerreiro||, Laurence Mulard||, and Bruno Canard{ddagger}**

From the {ddagger}CNRS and Universités d'Aix-Marseille I et II, UMR 6098, Architecture et Fonction des Macromolécules Biologiques, ESIL-Case 925, 163 avenue de Luminy, 13288 Marseille cedex 9, and the ||Institut Pasteur, Unité de Chimie Organique, 28 rue du Dr. Roux, 75724 Paris cedex 15, France

Received for publication, October 12, 2004 , and in revised form, November 8, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A class of amino acid substitutions in drug-resistant HIV-1 reverse transcriptase (RT) is responsible for the selectively impaired incorporation of the nucleotide analog inhibitor into DNA. We have shown previously that {alpha}-boranophosphate nucleoside analogs suppress RT-mediated resistance when the catalytic rate is responsible for drug resistance such as in the case of K65R and dideoxy (dd)NTPs, and Q151M toward AZTTP and ddNTPs. Here, we extend this property to BH3-d4TTP and BH3-3TCTP toward their clinically relevant mutants Q151M and M184V, respectively. Pre-steady-state kinetics on mutants of the Q151M RT family reveal a 3–5-fold resistance to d4TTP. This resistance is suppressed using BH3-d4TTP. Likewise, resistance to 3TCTP by M184V RT (30-fold) and K65R/M184V RT (180-fold) is suppressed using BH3-3TCTP because of a 160-fold acceleration of the catalytic constant kpol. Mechanistic insights into the rate enhancement were obtained using various {alpha}-boranophosphate nucleotides. The presence of the BH3 group renders kpol independent of amino acid substitutions present in RT. Indeed, the ~100-fold decrease in polymerase activity caused by the R72A substitution is restored to wild-type levels using BH3-dTTP. Metal ion titration studies show that {alpha}-boranophosphate nucleoside analogs enhance 3–8-fold the binding of Mg2+ ions to the active site of the RT·DNA·dNTP complex and alleviate the requirement of critical amino acids involved in phosphodiester bond formation. To our knowledge, this is the first example of rescue of polymerase activity by means of a nucleotide analog.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Reverse transcriptase (RT)1 is one of the major retroviral targets in the fight against AIDS because it plays an essential role in human immunodeficiency virus type-1 (HIV-1) replication. There are now many commercially available nucleoside analogs able to block the RT-catalyzed proviral DNA synthesis such as zidovudine (azidothimidine, AZT), didanosine (ddI), zalcitabine (ddC), stavudine (d4T), lamivudine (3TC), abacavir succinate (ABC), tenofovir disoproxil (PMPA), and emtricitabine (FTC) (1). Each of these prodrugs needs to be phosphorylated to its active 5'-triphosphate form to compete with its natural dNTP counterpart for incorporation into DNA. Unfortunately, the highly adaptive capacity of HIV-1 selects variants that are no longer drug-sensitive (2, 3), leading to failure of antiretroviral drug treatment in HIV-infected patients. Resistance-associated mutations were found to be located in the vicinity of the RT active site, and the complex molecular mechanisms involved have been well described at the molecular level in many cases (4–7). One of the strategies for salvage therapy is to develop nucleoside analogs that do not select resistance-associated mutations, or analogs that are still potent against resistant HIVs.

{alpha}-Boranophosphate nucleotides and oligonucleotides exhibit interesting properties. Such oligonucleotides are resistant to exonucleases and may find applications in direct PCR product sequencing (8–11). Oligonucleotides bearing a borano (BH3) group on the intermediate linkage have also been used in various applications such as boron neutron capture therapy for cancer treatments or antisense technologies (10, 12). Boronated nucleotides were also described to display antineoplastic and hypolipidemic activities (13, 14). They also meet antiviral drug research: we have described previously the inhibition of HIV-1 RT by nucleotide analogs harboring a BH3 group on their {alpha}-phosphate (also named {alpha}-BH3-dNTPs or BH3-dNTPs) (6, 15–17). The BH3 group replacing a nonbridging oxygen on the {alpha}-phosphate of a nucleotide analog also reduces the DNA repair reaction with ATP or PPi when tested on the AZT-resistant RT mutant (D67N/K70R/T215F/K219Q) (15, 16). The (Rp)-diastereoisomer form of {alpha}-boranophosphate analogs is the preferred isomer active on RT as a chain terminator when it is devoid of a 3'-OH group. In addition, an increased phosphorylation activity of the human NDP kinase toward borano nucleotides is observed with the same diastereoisomer (15, 16).

Borano nucleotide analogs have recently shown unexpected but interesting properties toward RT variants exhibiting nucleotide analog resistance in vitro, such as the K65R substitution as well as multiple drug resistance caused by Q151M-associated substitutions (6, 17). Both K65R and Q151M RT are responsible for a decrease in the rate of incorporation of the nucleotide analogs specifically, resulting in RT-mediated drug resistance. We have discovered that this type of resistance can be suppressed in vitro by the presence of the BH3 group on the {alpha}-phosphate of ddNTPs or AZTTP. Indeed, the presence of the BH3 group does not influence the binding of the analog to the RT active site, but provides (or restores in the case of resistance) a high catalytic rate constant kpol of incorporation of the analog specifically, no matter which amino acid substitution is present at the RT active site. On a mechanistic point of view, this suppression of drug resistance provides a sound confirmation that the catalytic step is responsible for drug discrimination. It also provides an elegant way to overcome RT-mediated resistance in vitro.

The chemical basis of this unexpected rate enhancement effect has not been investigated yet. Introduction of a BH3 group on the {alpha}-phosphate of a nucleotide changes the physical and chemical properties of the nucleotide, but not its status as a substrate for HIV-1 RT. First, replacement of the nonbridging oxygen by a BH3 group on the {alpha}-phosphate has some steric and reactivity consequences. The BH3 group is slightly bulkier than the oxygen, and its space occupancy resembles that of a methyl group. Bond distances change between a P-O (1.54 Å) and a P-B (1.91 Å), as well as angles between O-P-O ({approx}100°) and O-P-B ({approx}115°) (18), as shown in Fig. 1, A and B. Boranophosphates retain the same net charge as phosphodiesters, BH3 and O being isoelectronic. However, electronegativity and distribution of charge density within the BH3 group vary relative to an oxygen (19). The BH3 group does not form classical hydrogen bonds (20, 21) nor coordinate metal ions (19). Polarity is also disturbed as they show preferences for a hydrophobic environment (18, 19). The predominant reason why the catalytic rate of RT is enhanced by the {alpha}-borano substitution is still unclear.



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  		FIG. 1.
Model for the positioning of BH3-dTTP in the active site of RT. Upper left, molecular view of the P-BH3 bond. Upper right, Molecular view of the P-O bond. Lower panel, the atomic coordinates (PDB 1RTD [PDB] ) of Huang et al. (41) were used to visualize the complex RT·DNA·BH3-dTTP, after modeling replacing the missing 3'-OH of the primer. Magnesium ions (A and B) are represented as pink spheres. One of the nonbridging oxygens pro-(Rp) of the {alpha}-phosphate from dTTP has been replaced by a BH3 group, respecting its specific geometry and bond distance.

 
In the present study, we extend this property of drug resistance suppression to two other {alpha}-boranophosphate nucleotide analogs, BH3-d4TTP and BH3-3TCTP toward corresponding clinically relevant drug resistance mutations Q151M and M184V, respectively. Then, we conduct a mechanistic study using RT mutants to contribute to a molecular explanation for the rate enhancement of the HIV-1 RT toward boranophosphate nucleotides.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
HIV-RT Plasmid Constructions, Enzyme Preparations, and Re-agents—The wild-type RT gene construct p66RTB was used to obtain mutant RT genes as described previously (22). Q151Mcomplex RT is referred to as RT containing A62V, V75I, F77L, F116Y, and Q151M substitutions. All constructs were verified by DNA sequencing. The recombinant RTs were coexpressed with HIV-1 protease in Escherichia coli to get p66/p51 heterodimers, which were later purified using affinity chromatography. All enzymes were quantified by active site titration before biochemical studies. DNA oligonucleotides were obtained from Invitrogen. Oligonucleotides were 5'-32P labeled using T4 polynucleotide kinase (New England Biolabs). [{gamma}-32P]ATP was purchased from Amersham Biosciences. The {alpha}-boranophosphate nucleotides described here were synthesized and purified as described by Meyer et al. (15). All diastereoisomers were stereochemically pure as judged by high performance liquid chromatography analysis, except in the case of BH3-3TCTP where the isomers could not be separated and were used as a racemic mixture.

Pre-Steady-state Kinetics of Single Nucleotide Incorporation into DNA by RT—Pre-steady-state kinetics were performed using natural dNTPs or nucleotide analogs in conjunction with wild-type RT or mutants. Rapid quench experiments were performed with a Kintek Instruments model RQF-3 using reaction times ranging from 10 ms to 30 s. All indicated concentrations are final. The primer DNA/DNA oligonucleotides used for the rapid reaction were a 5'-labeled 21-mer primer (5'-ATA CTT TAA CCA TAT GTA TCC-3') annealed to a 31-mer template 31T-RT (5'-TTT TTT TTT AGG ATA CAT ATG GTT AAA GTA T-3') for the incorporation of dTTP, BH3-dTTP, d4TTP and BH3-d4TTP. A 31C-RT primer (5'-TTT TTT TTT GGG ATA CAT ATG GTT AAA GTA T-3') was used for the incorporation of dCTP, 3TCTP, and BH3-3TCTP. For the sake of comparison, all of these primer-template sequences differ from a single nucleotide only, allowing faithful comparison with our previous published data. For natural nucleotides, the reaction was performed by mixing a solution containing 50 nM (active sites) HIV-1 RT bound to 100 nM primer-template in RT buffer (50 mM Tris-HCl, pH 8.0, 50 mM KCl, 0.05% Triton X-100) and a variable concentration of dNTP in 6 mM MgCl2. Reactions involving nucleotide analogs were conducted with excess concentrations of enzyme (200 nM) over primer-template duplex (100 nM). These conditions were chosen to eliminate the influence of the enzyme turnover rate (kss), which interferes in the measurements of low incorporation rates. The products of reactions were analyzed using sequencing gel electrophoresis (14% acrylamide, 8 M urea in TBE buffer) and quantified using photostimulated plates and FujiImager. The formation of product (P) over time was fitted with a burst equation

(Eq. 1)
where A is the amplitude of the burst, kapp is the apparent kinetic constant of formation of the phosphodiester bond, and kss is the enzyme turnover rate, i.e. the kinetic constant of the steady-state linear phase. The dependence of kapp on dNTP concentration is described by the hyperbolic equation

(Eq. 2)
where Kd and kpol are the equilibrium constant and the catalytic rate constant of the dNTP for RT, respectively. Kd and kpol were determined from curve fitting using Kaleidagraph (Synergy Software).

Assays of RT DNA Polymerization Rate—The rate of polymerization was measured using a 5'-labeled oligo(dT)21 primer annealed to a poly(rA) RNA template, in conjunction with K65A and R72A RT. Extension products were analyzed in a gel assay. The primer-template (25 nM) was incubated with 50 nM RT in the RT buffer at 37 °C. The reaction was initiated by the addition of 500 µM dTTP in 6 mM MgCl2 and quenched at various time by 0.3 M EDTA. Average polymerization rates were calculated from the product profile analysis using a FujiImager: the number of nucleotide mers (size in nucleotides, nt) of the most abundant band product was divided by time to yield a polymerization rate in nt·s-1.

Assays of Magnesium Binding Affinity for the RT·DNA·dNTP Complex—The dissociation constant Kd(MgCl2)C of magnesium from the RT·DNA·nucleotide complex can be defined as

(Eq. 3)
where Kd(MgCl2)C is the Mg2+ dissociation constant for the RT·DNA·dNTP complex, and [Mg2+]free is the free Mg2+ concentration. To determine Kd(MgCl2)C, one should vary the total magnesium concentration and measure [Mg2+]free in the reaction mix simultaneously with the titration of active enzyme [RT·DNA·dNTP·Mg2+]. Alternatively, one could calculate [Mg2+]free for each total (added) magnesium concentration [Mg2+]total. Indeed, nucleotides, DNA, and RT bind magnesium at one or several sites individually, decreasing the concentration of free magnesium relative to total magnesium concentration. Thus, together with the use of a magnesium chelator buffer, one could solve all equations involving magnesium and individual dissociation constants relative to RT, nucleotides, and DNA, such as described in Refs. 23 and 24 for metal ions and prokaryotic DNA polymerases. No attempts were made to determine [Mg2+]free as a function of [Mg2+]total. Instead, for the sake of simplicity of comparison between boronated and nonboronated derivatives, all magnesium-binding species (i.e. RT, DNA, and nucleotide) were kept constant in the reactions, and only [Mg2+]total was varied in nucleotide incorporation assays. Thus, apparent binding constants Kd,app(Mg2+) were determined, and their informational content is valid in the case of nucleotide differing only by the presence of the BH3 group.

Apparent dissociation constants Kd,app(Mg2+) were measured in the presence of dTTP, BH3-dTTP, AZTTP, BH3-AZTTP, d4TTP, or BH3-d4TTP. Reactions were performed under the same conditions as for pre-steady-state kinetics of single nucleotide incorporations. Briefly, 200 nM WT RT was incubated with the 21·31T-RT primer-template (50 nM) and mixed rapidly in a quench flow apparatus with an equal volume of a mix containing varying concentrations of MgCl2 (from 0.1 to 32 mM) and nucleotides at a fixed, saturating concentration. The reaction was allowed to proceed (from time zero to 0.02–15 s), quenched with EDTA, and analyzed as described above. Kd,app(Mg2+) and kapp,max were determined from hyperbolic curve fitting.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The Role of the BH3 Group in the Suppression of Drug Resistance—We have shown previously that some resistance mutations such as K65R toward ddNTPs and Q151M toward AZTTP and ddNTPs decrease their catalytic rate of incorporation relative to that of their natural nucleotide counter-parts (6, 17). However, the corresponding {alpha}-boranophosphate nucleoside analogs are not subjected to such a selective rate decrease, and thus, they suppress RT-mediated resistance. Is that a general rule? In other words, will {alpha}-boranophosphate nucleosides suppress all nucleotide drug resistance because of a specific decrease of their catalytic rate? Although it is not possible to answer this question in a simple manner, it is possible to implement the answer to existing nucleotides and their {alpha}-boranophosphate derivatives when available. Consequently, we first addressed the issue of d4TTP and 3TCTP together with their corresponding RT-mediated mechanisms of nucleotide resistance. Then, we conducted a series of experiments using mutant RTs as well as metal titration studies to gain insight into the mechanism of rate enhancement observed with these analogs.

Resistance of Q151M RT and Q151Mcomplex RT to d4TTP and BH3-d4TTP—Comparative incorporations of d4TTP and BH3-d4TTP by Q151M RT and Q151Mcomplex (A62V, V75I, F77L, F116Y, and Q151M) purified RTs were measured using pre-steady-state kinetics. The nucleotide affinity Kd is calculated as the nucleotide concentration giving half of the maximum incorporation rate kpol. The incorporation efficiency of the nucleotide into DNA (kpol/Kd) is used to calculate the selectivity factor, (kpol/Kd)dNTP/(kpol/Kd)analog. A selectivity factor greater than 1 means that the enzyme discriminates the analog over the natural nucleotide. Finally, the resistance of RT to the inhibitor is the ratio between the selectivity of the mutant over the selectivity of the wild-type enzyme.

In accordance with others (25), the incorporation of dTTP by WT RT is not significantly preferred over that of d4TTP, considering the 1.5 selectivity factor (Table I). For the Q151M RT, discrimination of d4TTP is more pronounced (Fig. 2A), essentially at the catalytic step as judged by the lower burst rate for d4TTP (kpol = 6.7 s-1). The discrimination of the nucleotide analog is higher with Q151Mcomplex RT (kpol (d4TTP) = 2.2 s-1), resulting in a 7-fold lower incorporation efficiency (0.12 s-1·µM-1), compared with dTTP (0.79 s-1·µM-1). Q151M RT and Q151Mcomplex RT display a 2.8- and 4.7-fold resistance, respectively (Fig. 2B). In the case of the BH3-d4TTP, however, the nucleotide analog is well incorporated regardless of the enzyme used. High kpol values for BH3-d4TTP keep the incorporation efficiencies between 1.4 s-1·µM-1 for Q151M RT and 0.77 s-1·µM-1 for Q151Mcomplex RT (Table I and Fig. 2A). Hence, there is no discrimination of BH3-d4TTP relative to dTTP. As a consequence, there is no resistance arising from the Q151M-associated multiple drug resistance enzymes toward the boranophosphate analog (Fig. 2B).


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  		TABLE I
Pre-steady-state kinetic constants of dTTP/d4TTP/BH3-d4TTP incorporation by WT RT, Q151M RT, and Q151Mcomplex RT mutants

 



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  		FIG. 2.
Resistance of Q151M RT and Q151Mcomplex RT to d4TTP and BH3-d4TTP. A, plot of the incorporation of dTTP, d4TTP, and BH3-d4TTP by Q151M RT, resulting in Kd and kpol values, as described under "Experimental Procedures." B, -fold resistance of Q151M RT and Q151Mcomplex RT was determined from the ratio (selectivitymutant RT/selectivityWT RT) and is shown on Table I for the incorporation of d4TTP (white) and BH3-d4TTP (gray).

 
Resistance of M184V and K65R/M184V RT to 3TCTP and BH3-3TCTP—In a similar manner, we investigated the rapid incorporation of 3TCTP by two RTs bearing clinically relevant mutations (K65R and M184V) inducing a discrimination-type resistance toward the nucleotide analog. As described before, 3TCTP is very poorly incorporated by WT RT, compared with dCTP (5, 26, 27). As shown in Table II, kpol varies from 7.3 s-1 for dCTP to 0.047 s-1 for 3TCTP, inducing a 50-fold selectivity (27). Although this discrimination increases with M184V RT through a "Kd-dependent" mechanism, the grossly impaired catalytic step mainly brings the selectivity up to 1,500-fold. When the K65R substitution is added to the M184V background, the burst rate is severely altered (kpol = 0.003 s-1) without significant change in the affinity (Kd = 89 µM) compared with M184V RT. The combination of a "Kd effect" brought by M184V and a "kpol effect" brought by K65R gives K65R/M184V RT a 180-fold level of resistance to 3TCTP. For BH3-3TCTP, the M184V substitution still induces a loss of affinity compared with dCTP, but the catalytic step is improved, as kpol(BH3-3TCTP) is brought up to 0.36–0.5 s-1 for all three enzymes (Fig. 3A, B, and C). This represents a 160-fold improvement of kpol in the case of the K65R/M184V RT mutant. Hence, there is a very limited resistance to BH3-3TCTP given by K65R and M184V substitutions (Fig. 3D), albeit the M184V mutation still increases dissociation constants of 3TCTP and BH3-3TCTP compared with dCTP. It is quite remarkable that the incorporation rate of BH3-3TCTP is >11-fold higher than that of 3TCTP even in the case of the wild-type enzyme. In conclusion, we show that the presence of the BH3 group on 3TCTP compensates for the discrimination initially brought by K65R and M184V mutations, mostly through an increase of the catalytic rate kpol. Overall, resistance because of M184V or K65R/M184V mutations is reduced 2.5–25-fold when BH3-3TCTP is used instead of 3TCTP.


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  		TABLE II
Pre-steady-state kinetic constants of dCTP/3TCTP/BH3-3TCTP incorporation by WT RT, M184V RT, and K65R/M184V RT mutants

 



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  		FIG. 3.
Resistance of M184V and K65R/M184V to 3TCTP and BH3-3TCTP. A, plot of the incorporation of dCTP ({circ}), 3TCTP ({diamond}), and BH3-3TCTP ({square}) by M184V RT. B, plot of the incorporation of dCTP ({circ}), 3TCTP ({diamond}), and BH3-3TCTP ({square}) by K65R/M184V RT. C, evolution of the incorporation rate constant (kpol) for 3TCTP or BH3-3TCTP. The percent of the initial kpol was calculated as the ratio (kpol mutant RT/kpol WT RT) multiplied by 100 for the two nucleotides 3TCTP (white) and BH3-3TCTP (gray). D, -fold resistance of M184V RT and K65R/M184V RT was determined from the ratio (selectivitymutant RT/selectivityWT RT) and is shown on Table II for the incorporation of 3TCTP (white) and BH3-3TCTP (gray).

 
We conclude that as is the case for {alpha}-boranophosphate ddNTPs and {alpha}-boranophosphate AZTTP (6, 17), the {alpha}-boranophosphate substitution suppresses drug resistance when the catalytic rate of incorporation is responsible for resistance. Until a counterexample is found, the borano modification acts as a general suppressor of resistance whenever the catalytic rate of nucleotide incorporation lies as a cause of resistance. This applies for K65R toward ddNTPs, for Q151M and derivatives toward AZTTP, d4TTP, and ddNTPs, and for M184V toward 3TCTP. Moreover, the BH3 group bonifies 3TCTP ~7-fold relative to wild-type RT.

The Determinants of Catalysis around the {alpha}-Phosphate— During DNA polymerization, the {alpha}-phosphate of the incoming nucleoside 5'-triphosphate receives a nucleophilic attack from the 3'-OH of the DNA primer. A magnesium ion plays a critical role in activating the 3'-OH nucleophile and exacerbating the electrophilic property of the {alpha}-phosphorus (P) atom of the incoming dNTP. When one nonbridging oxygen atom of the {alpha}-phosphate is replaced by a BH3 group, the P atom becomes chiral. Structural data on various DNA polymerases (28–30) have shown that one magnesium atom (Mg2+ atom A) makes contacts with one pro-chiral atom, the pro-(Sp) oxygen in the case of a BH3 substitution. The other pro-chiral atom is away form the magnesium, and thus, the (Rp)-BH3-dNTP presents its BH3 group away from the magnesium. This latter diastereoisomer is the preferred substrate relative to the Sp. As shown above and in our previous work (6, 17), the BH3 accelerates the catalytic reaction rate in the presence of amino acid substitutions known to decrease the catalytic reaction rate for natural nucleotides. It was thus of interest to know whether other substitutions or other catalytically critical amino acid residues would also be turned into catalytically irrelevant amino acids by the {alpha}-boranophosphate group. Arg72 is such an amino acid, as its mutation to alanine nearly abrogates polymerase activity (31). Lys65 is in the vicinity of the {beta}- and {gamma}-phosphates of the incoming nucleotide, and K65R-mediated drug resistance is suppressed by the BH3 substitution. It is not known precisely whether the role of these two positively charged amino acids is to participate in the activation of the {alpha}-phosphate or in the assistance of pyrophosphate release, or both. To evaluate the ability of {alpha}-boranophosphate to promote efficient catalysis without the assistance of such critical amino acids, K65A RT and R72A RT were prepared and used in conjunction with {alpha}-boranophosphate nucleotides.

Rescued Incorporation Activity of K65A and R72A Recombinant HIV-RT Mutants BH3-dTTP—A polymerization rate assay was used with either K65A and R72A RT mutants, to measure the multiple incorporation of dTTP along a poly(rA) RNA template at the first steps of the polymerization (Fig. 4). For each reaction time (from 0.2 to 20 s), we observe that K65A RT incorporates dTTP at a rate of about 10 nt·s-1, whereas BH3-dTTP is incorporated approximately twice as fast. On the other hand, R72A RT is extremely inefficient for the incorporation of dTTP, with a global rate < 1 nt·s-1. This low polymerization rate is improved dramatically using BH3-dTTP with R72A RT, up to about 12 nt·s-1. We conclude that the borano group provides the nucleotide with intrinsic chemical properties able to circumvent the presence of key catalytic amino acids.



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  		FIG. 4.
Polymerization rate assay on dTTP and BH3-dTTP. K65A RT and R72A RT were assayed in the first seconds of the primer extension reaction on a poly(rA)-oligo(dT) template (from 0.2 to 20 s) with either dTTP (T) or BH3-dTTP (B) (see "Experimental Procedures").

 
This finding was refined further using pre-steady-state kinetics with a single nucleotide incorporation (dTTP and BH3-dTTP). We tested the contribution of Kd and kpol in the different polymerization rates observed previously. The incorporation efficiency (kpol/Kd) also gives a general overview of the single incorporation efficiency of the nucleotide. As shown in Table III, WT RT displays a slightly better incorporation efficiency for dTTP (0.75 s-1·µM-1) than for BH3-dTTP (0.53 s-1·µM-1), as the result of a lower Kd for dTTP. In the case of K65A RT, the incorporation of dTTP is not as efficient (0.18 s-1·µM-1) because of both an altered kpol going from 13 s-1 (WT RT) to 7.3 s-1 (K65A RT) and a >2-fold increase in Kd. The incorporation of BH3-dTTP is very similar to that of the WT RT (0.55 s-1·µM-1), though, because the burst rates for the two enzymes are identical. A 4-fold increase in Kd for the R72A RT is consistent with the structural data and the increase in bulk of the BH3 group showing that Arg72 does contact this pro-Rp position. kpol(dTTP) for R72A RT is even lower than that of K65A: 0.094 s-1. However, once again the incorporation of the BH3-dTTP by this mutant is not altered as judged by its high burst rate (20 s-1). This represents more than a 200-fold increase in the catalytic rate. We conclude that this experiment, together with the polymerization rate assay, shows the same particular property of the borano group on the {alpha}-phosphate of a nucleotide. The BH3 group shows a capability to compensate a mutation that induces a defect in the nucleotide incorporation rate kpol. In the mechanistic understanding of the {alpha}-boranophosphate rate enhancement effect, these results are important because they show that this effect is not necessarily linked to drug resistance suppression. Rather, the {alpha}-boranophosphate group at the RT active site gives to the nucleotide properties of incorporation quite independent from the nature of catalytic amino acid side chains. Other catalytic actors at the RT active site are the magnesium ions, which interact with the pro-(Sp) oxygen of the incoming nucleotide (see Fig. 1C). Thus, the interaction of the (Rp)-{alpha}-boranophosphate nucleoside analogs at the RT active site was studied in the presence or absence of the magnesium ions.


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  		TABLE III
Pre-steady-state kinetic constants of dTTP/BH3-dTTP incorporation by WT RT, K65A RT, and R72A RT mutants

 
Magnesium Binding Affinity to RT·DNA in Complex with Nucleotides or Boranophosphate Nucleoside Analogs—We hypothesized that if the borano group induces, favors, or modifies specific interactions with the Mg2+ catalytic ions, it would be possible to monitor differences in the binding affinity of Mg2+ to the RT active site. We used our single nucleotide incorporation assay and varied MgCl2 concentration in the reaction mix, together with a fixed saturating concentration of the nucleotide or the analog. To allow an easy evaluation of the influence of the BH3 group, each magnesium binding species was kept constant as explained under "Experimental Procedures." Fig. 5A shows a typical set of data representing the single incorporation of dTTP at various concentrations of MgCl2 and was used for the determination of kapp,max and Kd,app(Mg2+) (Fig. 5B). We first compared dTTP and BH3-dTTP (Fig. 5B and Table IV). A tighter binding is observed upon the presence of the BH3 group, as the magnesium binding constant Kd,app(Mg2+) decreases 8.3-fold from 1.5 mM (dTTP) to 0.18 mM (BH3-dTTP). Similar trends are observed when AZTTP is compared with BH3-AZTTP: Kd,app(Mg2+) decreases 3-fold when a borano group is present on the {alpha}-phosphate (Fig. 5C and Table IV). These results were also found in the case of d4TTP. The binding affinity of the Mg2+ ion increases about 3-fold in the presence of a BH3 group, from Kd,app(Mg2+) = 6 mM for d4TTP to Kd,app(Mg2+) = 2.1 mM for BH3-d4TTP.



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  		FIG. 5.
Magnesium binding affinity with nucleotides and boranophosphate derivatives. A, kinetic curves of incorporation of dTTP by WT RT from 0.02 to 15 s, using 0.1 ({circ}), 0.6 ({square}), 2 ({diamond}), 6 (x), and 18 mM (+) MgCl2, resulting in kapp,max values, as described under "Experimental Procedures." B, plot of the kapp,max as a dependent of MgCl2 concentration, for dTTP and BH3-dTTP ({square}), resulting in Kd,app(Mg2+) values, as described under "Experimental Procedures." C, same as B, for AZTTP ({diamond}) and BH3-AZTTP ({square}). D, same as B, for d4TTP ({diamond}) and Rp-BH3-d4TTP ({square}).

 


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  		TABLE IV
Apparent equilibrium dissociation constants Kd,app(Mg2+) for the RT·DNA·dNTP complex

 
As a general conclusion, Mg2+ binding is enhanced when the BH3 group is present on the {alpha}-phosphate of the incoming nucleotide. This change in Mg2+ binding affinity may be responsible for the observed (Rp)-BH3-mediated catalytic rate increase. An enhanced presence (or availability) of Mg2+ at the catalytic site may render catalytic amino acids such as Arg72 less required in their assistance of phosphoryl transfer.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The {alpha}-boranophosphate nucleoside analogs represent so far the sole example of a family of compounds able to overcome RT-mediated drug resistance in vitro. These analogs possess unique properties of acceleration of the DNA polymerization rate by RT. These properties are independent of amino acid substitutions present at the RT active site. This is precisely one of the most interesting and promising observations in a perspective of anti-retroviral drug design: if a given substitution promotes drug resistance in altering the incorporation rate of a nucleotide analog, the {alpha}-boranophosphate modification will overcome drug resistance (6, 17).

Two classes of resistance mechanisms for nucleotide inhibitors have been characterized. The first class involves a selective recognition of the natural nucleotide over the analog at the RT active site. Discrimination of the resistance mechanism for nucleotide inhibitors can be achieved either through a selective decrease of its binding at the RT active site (reflected by an increase in the binding equilibrium constant Kd), or at the catalytic step of incorporation of the analog into viral DNA (reflected by a decrease of kpol value, the catalytic constant of incorporation of the nucleotide analog into DNA). These mechanisms have been typically observed with the M184V and K65R mutations, leading to a discrimination of 3TC and ddI/ddC, respectively (5, 6, 26). The other class of resistance mechanism involves repair (or unblocking) of the analog-terminated DNA chain, using PPi or a nucleoside 5'-NTP as a cosubstrate for the pyrophosphorolysis reaction (4, 7). Mutations in RT such as M41L, D67N, K70R, L210W, T215Y/F, or K219Q/E/N (referred as thymidine analog-associated mutations) increase the unblocking activity in the case of resistance to AZT and d4T (32, 33).

In this study, we describe the inhibition properties of several boranophosphate nucleotide analogs toward a panel of RTs possessing relevant discrimination-associated mutations such as M184V, Q151M, and Q151Mcomplex. In agreement with our previous reports (6, 15, 17), we measure a systematic loss of discrimination upon the presence of the BH3 group. This in vitro loss of resistance is the result of a conserved efficiency of catalysis (measured with kpol). This recovery of sensitivity is independent of the substitutions of amino acids within the active site of RT. In some of our assays, kpol is increased more than 150-fold, for example when BH3-3TCTP is used instead of the unsubstituted 3TCTP. An efficient incorporation of BH3-TTP is even achieved using the R72A substitution, which decreases the polymerase activity ~100-fold (15, 31, 34, 35). Hence, these enzymatic results highlight the general potential given by BH3 derivatives to circumvent resistance brought by almost any discrimination-associated mutation.

The second part of our study is an attempt to understand how the presence of the BH3 group on the {alpha}-phosphate of the nucleotide affects the polymerization rate independently of the otherwise catalytically critical amino acids. There are a number of chemical properties of the BH3 group that should actually penalize the nucleophilic attack at the {alpha}-BH3-substituted phosphorus. First, one would expect that an increase in bulk (18) brought by the BH3 group would indirectly misalign reactive centers in the nucleophilic attack. Second, electronegativity and distribution of charge density within the BH3 group relative to an oxygen should slow down the reaction rate (19). In addition, the BH3 group does not form classical hydrogen bonds (20, 21). The only amino acid being in the close vicinity of the BH3 group is Arg72, and our results using R72A show that Arg72 is dispensable when the BH3 group is present. Work by others (8–11) has shown that the BH3 group does not compromise the stability of the scissile {alpha},{beta}-phosphate bond. Clearly, the presence of the BH3 group does not help catalysis through an increase of the {alpha},{beta}-phosphate bond fragility but through another mechanism independent of amino acids usually involved in assisting the catalytic step. This observation prompted us to investigate the influence of the BH3 group on the presence and availability of the catalytic magnesium ions.

We measured the affinity of RT for the catalytic Mg2+ ions in the presence of different nucleotides and analogs. We observed a general increase of the affinity for Mg2+ during reactions involving borane derivatives (3–8-fold). This decrease in the apparent dissociation constant means that Mg2+ is statistically more present upon the presence of a BH3 group than upon that of an oxygen. In other words, the presence of the BH3 group makes Mg2+ more "cozy" in its binding site (or shell) delineated by the carboxylates, the primer 3'-OH, and the incoming nucleotide. This better affinity is likely responsible for the restored catalytic rate. There are two possible, and not mutually exclusive, tracks for discussion about the increased Mg2+ affinity. They are the steric effect and the electronic effect of the BH3 group. The latter effect influences both the electrophilicity of the phosphorus atom and the charge neutralization upon the nucleophilic attack.

It is indeed possible that there is a steric component in the stabilization of the nucleotide by the presence of the BH3 group. Indeed, the BH3 group is bulkier than a hydroxyl, the P-B bond is longer than a P-O bond, and this very likely restrains the possible movements of the {alpha}-phosphate. A more stable nucleotide would maintain the magnesium shell, particularly in the case of ddNTPs where the absence of a 3'-OH group is known to affect catalysis (6, 17). In other words, this restriction of movement could well compensate the decrease in catalytic efficiency observed upon the loss of the intramolecular hydrogen bond between the 3'-OH and the oxygen {beta}-phosphate (Fig. 1) when there is an incoming ddNTP positioned at the active site. Another indirect proof of the importance of steric constraints is the work by Lewis et al. (34). Theses authors have reported that the magnitude of the catalytic deficiency of the R72A mutant is highly dependent on sequence context. In some instances, the catalytic efficiency is even not altered by the alanine substitution at Arg72. It is thus difficult to imagine for Arg72 a critical role if under certain circumstances Arg72 is dispensable. Rather, one interpretation is that sequence context packs the incoming nucleotide more or less snuggly. In turn, this conditions either the nucleophilic attack at the {alpha}-phosphorus or the negative charge neutralization of the leaving pyrophosphate by a water molecule. This interpretation is consistent with our results concerning the R72A substitution showing that it is likely that Arg72 play a quite neutral role in positioning the {alpha}-phosphate before catalysis. We note, however, that this proposition (the steric hypothesis) cannot account completely for the observed results. Indeed, an {alpha}-methylphosphonate of dTTP has been synthesized and used as a substrate for RT (36). Although no kinetic constants were reported, it is clear from this work that this analog is far well less efficient as a substrate for RT than natural dTTP. Hence, electronic, not steric only, properties of the BH3 group contribute to this rate acceleration.

It is, however, more difficult to use electronic arguments to account for a better Mg2+ affinity. Indeed, the presence of the BH3 group removes the charge delocalization between the two oxygens of a regular {alpha}-phosphodiester, leaving the nonbridging oxygen of the incoming nucleotide uncharged with a P-O bond, and thus less prone to chelate both magnesium ions A and B. Presently, we do not know precisely which magnesium A, B, or both, is assayed in the active site, nor which magnesium A or B is affected by the presence of the BH3 group. A possible interpretation is that magnesium ions adopt another position, closer to the primer 3'-oxygen for magnesium A, and closer to the pyrophosphate oxygens for magnesium B. This would exacerbate both the nucleophilic potential of the primer 3'-oxygen and the good leaving group properties of the pyrophosphate. It is also puzzling to understand why the presence of the BH3 group does not compromises the electrophilicity of the phosphorus atom. In the case of unsubstituted ddNTPs, kpol values can decrease down to 0.09 s-1 (this work Table III and Refs. 6 and 17). In contrast, it seems that the BH3 group confers almost constant kpol values in a very narrow (10–28 s-1) range, regardless of the nature of the nucleotide used (dTTP, ddATP, ddCTP, d4TTP, and AZTTP). Magnesium A could indeed lower the pKa of the primer 3'-oxygen as described above and overcome the penalty given by the presence of the BH3 group. On the other hand, it has been shown that the wider angles and longer bonds of boranophosphate analogs facilitate SN2 reactions (37).

Our results are informative on the rate limiting step of the polymerase reaction. It is thought that the initial binding of the nucleotide to an open RT-nucleic acid complex is followed by a rate limiting conformational change (closed complex) followed by the chemical reaction of incorporation (38, 39). In the case of BH3 nucleotides, we do not know whether the kpol values of 10–28 s-1 represent the maximum chemistry rate, or whether these values are included into the rate limiting step defined as a conformational change preceding chemistry. The fact that the BH3 group restores a high catalytic rate argues in favor of the proposition that below 10–28 s-1, this rate represents the chemical reaction rate. Indeed, a conformational change would not be affected positively by the presence of the bulky BH3 group. Hence, for our primer-template system use here, burst rate (kpol) values below 10–28 s-1 may reflect the rate of the chemical reaction and do not exclude the possibility that for faster chemical reaction rate values, the conformational change becomes rate-limiting.

Finally, besides the spectacular improvement in rate constant values provided by the BH3 group using drug-resistant RTs, it is quite remarkable that the adjunction of the BH3 group to 3TCTP improves 7-fold the poor incorporation properties of this drug, which is of major clinical relevance even for WT RT. In the case of another type of modification of the phosphate group, such as a phosphonate in which an oxygen is replaced by a CH2 group between phosphate and ribose, RT-mediated incorporation into DNA proceeds very poorly because of a 1,800-fold lower kpol (40). It would be interesting to determine whether a BH3 substitution would restore RT-mediated incorporation into DNA, as this would open a novel field of investigation in antiretroviral drug design.


   FOOTNOTES
 
* This work was supported in part by the Agence Nationale de Recherche sur le SIDA (AIDS) and Ensemble Contre le SIDA. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Supported by a predoctoral fellowship from the Agence Nationale de Recherche sur le SIDA. Back

Supported by a postdoctoral fellowship from the Ensemble Contre le SIDA. Back

** To whom correspondence should be addressed: CNRS, Universités d'Aix-Marseille I et II, UMR 6098, ESIL-Case 925, 163 avenue de Luminy, 13288 Marseille cedex 9, France. Tel.: 33-491-82-8644; Fax: 33-491-82-8646; E-mail: bruno{at}afmb.cnrs-mrs.fr.

1 The abbreviations used are: RT, reverse transcriptase; ABC, abacavir succinate (2',3'-dideoxy-6-cyclopropylamine-guanosine; AZT, zidovudine (3'-azido 3'-deoxythymidine); AZTTP, 3'-azido 3'-deoxythymidine 5'-triphosphate; BH3, borano; d4T, stavudine (2',3'-didehydro-2',3'-dideoxythymidine); dd, dideoxy; ddC, zalcitabine (2',3'-dideoxycytidine); ddI, didanosine (2',3'-dideoxyinosine); FTC, emtricitabine (3'-thia-2',3'-dideoxy-5-fluorocytidine); HIV-1, human immunodeficiency virus type 1; nt, nucleotide(s); PMPA, tenofovir disoproxil ((R)-9-(2-phosphonylmethoxypropyl)adenine disoproxyl); PPi, inorganic pyrophosphate; 3TC, lamivudine ((-)-{beta}-L-2',3'-dideoxy-3'-L-thiacytidine); WT, wild-type. Back


   ACKNOWLEDGMENTS
 
We thank Tom Kunkel, Bill Beard, Fritz Eckstein, and Luis Menèndez-Arias for critical reading of the manuscript. We thank Simon Sarfati for continuous support and interest in this project.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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