Structural basis for activation of α-boranophosphate nucleotide analogues targeting drug-resistant reverse transcriptase
2000; Springer Nature; Volume: 19; Issue: 14 Linguagem: Inglês
10.1093/emboj/19.14.3520
ISSN1460-2075
AutoresPhilippe Meyer, Benoı̂t Schneider, Simon Sarfati, Dominique Deville‐Bonne, Catherine Guerreiro, Joëlle Boretto, Joël Janin, Michel Véron, Bruno Canard,
Tópico(s)Ferrocene Chemistry and Applications
ResumoArticle17 July 2000free access Structural basis for activation of α-boranophosphate nucleotide analogues targeting drug-resistant reverse transcriptase Philippe Meyer Philippe Meyer Laboratoire d'Enzymologie et Biochimie Structurales, UPR-9063 CNRS, 91198 Gif-sur-Yvette, France Search for more papers by this author Benoît Schneider Benoît Schneider Unité de Régulation Enzymatique des Activités Cellulaires, CNRS URA 1773, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, Cedex 15, France Search for more papers by this author Simon Sarfati Simon Sarfati Unité de Chimie Organique, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris, Cedex 15, France Search for more papers by this author Dominique Deville-Bonne Dominique Deville-Bonne Unité de Régulation Enzymatique des Activités Cellulaires, CNRS URA 1773, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, Cedex 15, France Search for more papers by this author Catherine Guerreiro Catherine Guerreiro Unité de Chimie Organique, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris, Cedex 15, France Search for more papers by this author Joëlle Boretto Joëlle Boretto Laboratoire d'Architecture et Fonction des Macromolécules Biologiques, UPR-9039 CNRS, Ecole Supérieure d'Ingénieurs de Luminy, Campus de Luminy, 13288 Marseille, Cedex 09, France Search for more papers by this author Joël Janin Joël Janin Laboratoire d'Enzymologie et Biochimie Structurales, UPR-9063 CNRS, 91198 Gif-sur-Yvette, France Search for more papers by this author Michel Véron Michel Véron Unité de Régulation Enzymatique des Activités Cellulaires, CNRS URA 1773, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, Cedex 15, France Search for more papers by this author Bruno Canard Corresponding Author Bruno Canard Laboratoire d'Architecture et Fonction des Macromolécules Biologiques, UPR-9039 CNRS, Ecole Supérieure d'Ingénieurs de Luminy, Campus de Luminy, 13288 Marseille, Cedex 09, France Search for more papers by this author Philippe Meyer Philippe Meyer Laboratoire d'Enzymologie et Biochimie Structurales, UPR-9063 CNRS, 91198 Gif-sur-Yvette, France Search for more papers by this author Benoît Schneider Benoît Schneider Unité de Régulation Enzymatique des Activités Cellulaires, CNRS URA 1773, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, Cedex 15, France Search for more papers by this author Simon Sarfati Simon Sarfati Unité de Chimie Organique, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris, Cedex 15, France Search for more papers by this author Dominique Deville-Bonne Dominique Deville-Bonne Unité de Régulation Enzymatique des Activités Cellulaires, CNRS URA 1773, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, Cedex 15, France Search for more papers by this author Catherine Guerreiro Catherine Guerreiro Unité de Chimie Organique, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris, Cedex 15, France Search for more papers by this author Joëlle Boretto Joëlle Boretto Laboratoire d'Architecture et Fonction des Macromolécules Biologiques, UPR-9039 CNRS, Ecole Supérieure d'Ingénieurs de Luminy, Campus de Luminy, 13288 Marseille, Cedex 09, France Search for more papers by this author Joël Janin Joël Janin Laboratoire d'Enzymologie et Biochimie Structurales, UPR-9063 CNRS, 91198 Gif-sur-Yvette, France Search for more papers by this author Michel Véron Michel Véron Unité de Régulation Enzymatique des Activités Cellulaires, CNRS URA 1773, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, Cedex 15, France Search for more papers by this author Bruno Canard Corresponding Author Bruno Canard Laboratoire d'Architecture et Fonction des Macromolécules Biologiques, UPR-9039 CNRS, Ecole Supérieure d'Ingénieurs de Luminy, Campus de Luminy, 13288 Marseille, Cedex 09, France Search for more papers by this author Author Information Philippe Meyer1, Benoît Schneider2, Simon Sarfati3, Dominique Deville-Bonne2, Catherine Guerreiro3, Joëlle Boretto4, Joël Janin1, Michel Véron2 and Bruno Canard 4 1Laboratoire d'Enzymologie et Biochimie Structurales, UPR-9063 CNRS, 91198 Gif-sur-Yvette, France 2Unité de Régulation Enzymatique des Activités Cellulaires, CNRS URA 1773, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, Cedex 15, France 3Unité de Chimie Organique, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris, Cedex 15, France 4Laboratoire d'Architecture et Fonction des Macromolécules Biologiques, UPR-9039 CNRS, Ecole Supérieure d'Ingénieurs de Luminy, Campus de Luminy, 13288 Marseille, Cedex 09, France *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:3520-3529https://doi.org/10.1093/emboj/19.14.3520 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info AIDS chemotherapy is limited by inadequate intracellular concentrations of the active triphosphate form of nucleoside analogues, leading to incomplete inhibition of viral replication and the appearance of drug-resistant virus. Drug activation by nucleoside diphosphate kinase and inhibition of HIV-1 reverse transcriptase were studied comparatively. We synthesized analogues with a borano (BH3−) group on the α-phosphate, and found that they are substrates for both enzymes. X-ray structures of complexes with nucleotide diphosphate kinase provided a structural basis for their activation. The complex with d4T triphosphate displayed an intramolecular CH…O bond contributing to catalysis, and the Rp diastereoisomer of thymidine α-boranotriphosphate bound like a normal substrate. Using α-(Rp)-boranophosphate derivatives of the clinically relevant compounds AZT and d4T, the presence of the α-borano group improved both phosphorylation by nucleotide diphosphate kinase and inhibition of reverse transcription. Moreover, repair of blocked DNA chains by pyrophosphorolysis was reduced significantly in variant reverse transcriptases bearing substitutions found in drug-resistant viruses. Thus, the α-borano modification of analogues targeting reverse transcriptase may be of generic value in fighting viral drug resistance. Introduction The fight against human immunodeficiency virus (HIV) relies on the association of drugs directed towards two viral enzymes, the reverse transcriptase (Lightfoote et al., 1986) and the protease. Most drugs targeting reverse transcriptase are nucleoside analogues, such as 3′-azido 3′-deoxythymidine (AZT, Zidovudine) and 2′,3′-didehydro 2′,3′-dideoxythymidine (d4T, Stavudine). Antiviral nucleoside analogues per se are neither substrates nor inhibitors of reverse transcriptase. They must first be converted into triphosphates and compete with natural 2′-deoxynucleotides (dNTPs) for incorporation into viral DNA by reverse transcriptase. Because they lack a 3′-OH group, DNA chain elongation terminates, which accounts for the observed antiviral effect (Mitsuya et al., 1985; Larder, 1992; Balzarini, 1999). Their potency as a drug depends on their uptake by the infected cell, their phosphorylation by cellular kinases and their ability to block viral DNA synthesis. A single enzyme, nucleoside diphosphate kinase (NDPK), produces the triphosphate derivatives. In vitro, NDPK is 104 and 103 times less efficient on AZT and d4T diphosphate, respectively, than on thymidine diphosphate (Bourdais et al., 1996; Schneider et al., 1998, 2000). The efficacy of incorporation is limited by the low concentration of the triphos phate analogues relative to dNTPs in the infected cell. The poor activation of the drugs has a dramatic consequence: incomplete suppression of viral DNA synthesis allows the selection of resistance mutations (Larder, 1992). The emergence of variant viruses is the most serious threat against the efficacy of currently used combinations of three anti-retroviral drugs (tritherapies). The advent of second-generation drugs is urgent: ∼20 and 3% of HIV-1 isolates in western urban areas have nucleoside- and multidrug-resistant phenotypes, respectively (Wainberg and Friedland, 1998; Boden et al., 1999). We conducted a structural and biochemical study of nucleoside analogue activation, which gives a basis to the rational design of novel drugs overcoming these limitations. Here we consider analogues where an oxygen of the α-phosphate is replaced with a borano (BH3−) group yielding α-(Rp)-boranophosphate. X-ray structures at 1.9 Å resolution show that the analogues bind NDPK like normal substrates. Biochemical data demonstrate that their activation by NDPK is enhanced, and that they provide reverse transcription inhibitors of increased efficiency. Moreover, repair of the analogue-terminated DNA chain by pyrophosphorolysis in drug-resistant reverse transcriptase mutants is greatly impeded. Results Synthesis of nucleotide analogues We synthesized nucleotide analogues of thymidine, AZT and d4T containing a borano group (BH3−) on the α-phosphate. The borano group is isoionic to oxygen, and it generates a chiral centre when present on the α-phosphate of a dNTP (Figure 1). The two diastereo isomers were separated by reverse-phase high-performance liquid chromatography (HPLC). The same single diastereoisomer of unknown absolute configuration acted as an efficient substrate for both NDPK and HIV-1 reverse transcriptase. Figure 1.Chemical formula of the α-(Rp)-borano-d4T triphosphate diastereoisomer. Download figure Download PowerPoint Stereochemistry of α-borano TDP in complex with NDPK To determine the absolute configuration of the active diastereoisomer, we crystallized a complex of α-borano-thymidine diphosphate (TDP) with NDPK. NDPK transfers the γ-phosphate of a nucleoside triphosphate onto a nucleoside diphosphate via a phospho-histidine intermediate. Eukaryotic NDPKs are closely related. Human NDPK has a very similar structure to the Dictyostelium enzyme, and essentially the same active site (Dumas et al., 1992; Moréra et al., 1995; Webb et al., 1995). The crystals of Dictyostelium NDPK in complex with α-borano-TDP were isomorphous to a previously determined complex with ADP and aluminium fluoride, where AlF3 mimicks the γ-phosphate of ATP undergoing transfer onto the catalytic histidine (Xu et al., 1997b). The 1.92 Å resolution X-ray structure (Table I) demonstrates that the α-borano analogue binds like ADP and makes the same interactions (Figure 2A). The presence of boron with five electrons instead of oxygen with eight electrons is apparent in the electron density, which is consistently weaker at the Rp than the Sp position of the α-phosphate. Moreover, a Mg2+ ion ligates the α- and β-phosphates of the analogue in the same way as for natural nucleotides. As only the Rp diastereoisomer could be crystallized and the α-borano group does not interact with the protein, metal ligation by oxygen atoms of the phosphates is likely to determine the stereochemistry of recognition of the α-borano group by NDPK. Figure 2.Nucleotide analogues bound to NDPK. (A) α-(Rp)-borano thymidine diphosphate bound to the wild-type Dictyostelium enzyme. The 2Fo – Fc electron density map at 1.92 Å resolution is contoured at 1σ. The borano group (BH3− in green) points towards the reader. An Mg2+ ion ligates the α- and the β-phosphate, and four water molecules (red spheres) complete the octahedral geometry. The thymine base is sandwiched between F64 and V116. The geometry of the boranophosphate group was taken from the crystal structure of a dimethyl ester (Summers et al., 1998) where the P–B and P–O bond lengths are 1.90 and 1.51 Å, respectively. (B) d4T triphosphate bound to the H122G variant. The 2Fo – Fc electron density map at 1.85 Å resolution is contoured at 1σ. The Mg2+ ion ligates all three phosphates. (C) Comparison with the natural substrate in the ADP–AlF3 complex (PDB file 1KDN; Xu et al., 1997b). Bonds in d4T triphosphate are atom-coloured, the ADP–AlF3 complex is in blue. AlF3 mimics the γ-phosphate undergoing transfer to His122, on top of the figure. In ADP, a hydrogen bond links the 3′-OH of the ribose to oxygen O7 of the β-phosphate. In d4T, the 3′-CH group makes a short (3.2 Å) contact with the equivalent oxygen, which can be interpreted as a CH…O bond (dashes). The superposition is based on all Cα positions. The figure was made with TURBO (Roussel and Cambillau, 1991). Download figure Download PowerPoint Table 1. Summary of crystallographic data collection and refinement statistics Complex d4T triphosphate α-borano-TDP Resolution (Å) 1.85 1.92 Unique reflections 37 405 33 469 Average redundancy 5.0 5.6 Rsym (%)a 5.6 (32.8) 10.5 (32.7) Completeness 99.3 97.1 Range for refinement (Å) 30–1.85 30–1.92 Total reflections used 35 535 32 052 Protein atoms 3446 3446 Solvent molecules 346 400 Ligand atoms 84 75 Average B-factor (Å2) 24.3 28 R.m.s.d. bond lengths (Å) 0.012 0.010 R.m.s.d. bond angles (°) 1.7 1.5 Rcryst (%)b 20.9 22.8 Rfree (%) 24.2 27.4 a Rsym = Σhi |I(h)i − |/Σhi I(h)i. Outer shell of resolution in parentheses. b Rcrys = Σh ‖Fo| − |Fc‖/Σh |Fo| calculated with no I/σ cut-off; Rfree is derived from 4% of the data set excluded from refinement. A CH…O bond in d4T triphosphate bound to NDPK To check whether this structural model is valid for antiviral nucleoside analogues used in AIDS therapy, we prepared a complex of d4T triphosphate with NDPK and determined its crystal structure at 1.85 Å resolution. A similar analysis has been performed on AZT derivatives bound to thymidylate kinase (Lavie et al., 1997) and to NDPK (Xu et al., 1997a). The present study makes use of the H122G mutant of the Dictyostelium enzyme. This mutant lacks the catalytic histidine and cannot autophos phorylate, but it binds ATP and transfers its γ-phosphate efficiently onto externally supplied imidazole (Admiraal et al., 1999). Again, the complex with d4T triphosphate was isomorphous with the ADP–AlF3 complex. The analogue binds like ADP, the additional γ-phosphate occupying the AlF3 position, and the Mg2+ ion interacts with all three phosphate groups (Figure 2B and C). Interactions with the protein are the same as for ADP–AlF3, with an important exception. In a natural substrate, the 3′-OH of ribose or deoxyribose receives two hydrogen bonds from protein groups, and donates one to the oxygen atom that is labelled O7 in Figure 2C and bridges the β- and γ-phosphates. The latter bond is particularly important in catalysis (Xu et al., 1997b; Gonin et al., 1999). Instead of a 3′-OH, d4T carries a double bond in the sugar ring. We find that polar protein groups pack around the double bond making short contacts (3.2–3.4 Å) with the C2′ and C3′ atoms. The shortest contact is between C3′ and O7, the oxygen that bridges the β- and γ-phosphates. The comparison of our structural analysis with NDPK bound to AZT triphosphate suggests a reason why d4T derivatives are better substrates of NDPK than AZT and other analogues lacking a 3′-OH group. In AZT, the bulky 3′-azido substituent interferes with the proper positioning of a lysine side chain (K16 in Figure 2C) that is involved in catalysis (Xu et al., 1997a). With d4T triphosphate, this side chain and all other protein catalytic groups have the same position as in complexes with natural substrates such as ADP, GDP or TDP (Cherfils et al., 1994; Moréra et al., 1995). Also, we noted the short distance between the modified sugar ring and the bridging oxygen O7. This oxygen is the leaving group when the γ-phosphate is transferred. In a natural substrate, the hydrogen bond to the sugar 3′-OH activates it (Figure 2C). In d4T triphosphate, the 3′-CH group points towards O7 in the plane of the double bond. The distance and geometry of the contact are consistent with a CH…O bond. CH…O bonds are observed in small molecule crystals, in proteins and in DNA (Neidle and Taylor, 1977; Derewenda et al., 1995; Mandel-Gutfreund et al., 1998). Although much weaker than the bond with the 3′-OH of a natural substrate, the CH…O7 bond may explain why d4T diphosphate is phosphorylated 10-fold faster than dideoxyTDP (Schneider et al., 1998, 2000). The dideoxy sugar has methylene groups instead of a double bond and their hydrogens are not oriented properly for an interaction with the bridging oxygen. Activation of α-borano nucleotide analogues by NDPK We examined pre-steady-state kinetic parameters of the phosphorylation of the α-borano diphosphate derivatives of AZT and d4T by Dictyostelium NDPK. Protein fluorescence is quenched upon phosphorylation of the protein by an NTP substrate and enhanced upon its dephosphorylation by NDP (Deville-Bonne et al., 1996). With AZT diphosphate as the phosphate acceptor, the reaction is 104-fold slower than with TDP (Schneider et al., 1998). When α-borano-AZT diphosphate is tested in this way, the Sp isomer is inactive but the Rp isomer is a better phosphate acceptor than AZT diphosphate (Figure 3A). Its phosphorylation follows an exponential time course with a rate constant kobs that increases with nucleotide concentration. The initial slope yields the catalytic efficiency of the reaction (Schneider et al., 1998), which is 10-fold higher for the α-borano compound (Figure 3B). A 10-fold enhancement of catalytic efficiency is also observed for the Rp isomer of α-borano TDP relative to TDP (Table II). As α-borano-d4T diphosphate was not available, we used α-borano-d4T triphosphate to phosphorylate NDPK and study phosphate transfer in the reverse direction. Again, the presence of the α-borano group in the Rp position results in an enhancement of the catalytic efficiency of the reaction relative to d4T triphosphate (Figure 3C; Table II). Figure 3.Enhancement of AZT and d4T activation by NDPK in the presence of the Rp α-borano group. (A) Kinetics of phosphorylation of AZT diphosphate and α-borano-AZT diphosphate by phosphorylated human NDPK A. The phosphorylated enzyme (1 μM), prepared as described (Deville-Bonne et al., 1996), is mixed with 400 μM AZT diphosphate or α-borano-AZT diphosphate in 50 mM Tris–HCl pH 7.5, 5 mM MgCl2, 75 mM KCl, 1 mM DTT and 5% glycerol (final concentrations) at 20°C. The decrease in fluorescence is monitored with a Hi-Tech SF-61DX2 stopped flow device (λexc = 304 nm, excitation slit = 2 nm, emission filter <320 nm). The solid lines represent the best fit of each curve to a monoexponential. (B) Concentration dependence of the pseudo-first order phosphorylation rate constant kobs: α-borano-AZT diphosphate (circles) and AZT diphosphate (squares). Data were analysed according to the classical model of a fast binding reaction followed by a slow, rate-limiting phosphotransfer (Schneider et al., 1998). The catalytic efficiency is kmax/KD = 2000 M−1s−1 for α-borano-AZT diphosphate and 200 M−1s−1 for AZT diphosphate. As no saturation is reached, the maximum rate of phosphotransfer, kmax, is not measurable in this experiment. (C) Concentration dependence of the pseudo-first order rate constant kobs for NDPK A phosphorylation by α-borano-d4T triphosphate (diamonds) and d4T triphosphate (triangles). The catalytic efficiency is kmax/KD = 6000 M−1s−1 for α-borano-d4T triphosphate and 800 M−1s−1 for d4T triphosphate. Download figure Download PowerPoint Table 2. NDPK binding and activation of α-(Rp)-borano thymidine nucleotide analogues Nucleotide Catalytic efficiency (M−1s−1)a KD (μM)b TTP – 1 × 106 1.2 AZT triphosphate – 80 30.0 α-borano 375 2.0 d4T triphosphate – 800 2.0 α-borano 6000 0.25 TDP – 2 × 106 – α-borano 27 × 106 – AZT diphosphate – 200 – α-borano 2000 – d4T diphosphate – 2600 – α-borano ND – a The phosphotransfer reaction between human NDPK A and thymidine derivatives was studied in both directions as described in Figure 3. Note that the α-borano group increases the catalytic efficiency 4- to 10-fold. b The F64W-H122G variant of Dictyostelium NDPK is designed to measure NTP binding in the absence of phosphotransfer. Protein fluorescence (λexc = 310 nm, λem = 330 nm) increases by 50% upon binding saturating amounts of NTP at 20°C in 50 mM Tris–HCl, 5 mM MgCl2 and 75 mM KCl pH 7.5. KD values are estimated by fitting the binding curve to a quadratic equation. The binding affinity for the analogues was measured using a variant of Dictyostelium NDPK that lacks the catalytic histidine and where a tryptophan replaces the phenylalanine stacking on the base in the active site (F64 in Figure 2B). In this variant, protein fluorescence changes upon nucleotide binding whilst no phosphorylation takes place (Deville-Bonne et al., 1996; Schneider et al., 2000). Binding isotherms show a 10-fold decrease in KD for the α-borano analogue relative to d4T triphosphate (not shown). Similar experiments with TTP, AZT triphosphate and their α-borano analogues also indicate a 10-fold decrease in KD when the borano group is present in the Rp position (Table II). We conclude that the increase in catalytic efficiency of α-boranophosphate derivatives results from an improvement in the affinity for the enzyme. Inhibition of reverse transcription by α-borano nucleotide analogues Examination of crystal structures of nucleotide substrates bound either to NDPK (this work) or to DNA polymerases such as reverse transcriptase (Huang et al., 1998) and bacteriophage T7 DNA polymerase (Doublié et al., 1998) shows that their NDP moieties have similar conformations. This is illustrated in Figure 4 by comparing d4T triphosphate from Figure 2B with dideoxyGTP bound to T7 DNA polymerase (Figure 4A; Doublié et al., 1998) and with deoxyTTP bound to reverse transcriptase (Figure 4B; Huang et al., 1998). Common features are the conformation of the α- and β-phosphates and the position of one of the bound Mg2+ ions. The similarity is especially striking in the case of T7 polymerase, which is a higher resolution (2 Å) structure than for reverse transcriptase, making details of the nucleotide conformation and the Mg2+ binding mode more reliable. In all three enzymes, the same oxygen of the α-phosphate ligates the metal, explaining why the other α-phosphate oxygen can be modified without interfering with activation by NDPK or incorporation in DNA by reverse transcriptase. Figure 4.Conformation of the nucleotide substrate in NDPK, T7 DNA polymerase and HIV reverse transcriptase. d4T triphosphate from the NDPK complex in Figure 2B is shown in atom-type coloured bonds superimposed onto (A) dideoxyGTP in the ternary complex with bacteriophage T7 DNA polymerase–DNA (PDB file 1T7P) and (B) deoxyTTP in the ternary complex with HIV reverse transcriptase–DNA (PDB file 1RTD). Least-square fitting was performed on atom N1 of the base and common atoms in the sugar and the α-phosphate. In T7 polymerase and reverse transcriptase, relevant active site residues, DNA and the ligand are in blue bonds, and blue spheres represent two Mg2+ ions bound. In NDPK, the red sphere is the single Mg2+ ion bound to d4T triphosphate. It is located 0.98. b KD and kpol were obtained as described in Figure 5 (Kati et al., 1992). Standard deviations were <16%. c Determinations carried out at 25°C and taken from Reardon and Miller (1990). Targeting drug-resistant reverse transcriptase using α-borano analogues In the crystal structure of reverse transcriptase in complex with DNA and deoxyTTP, the positive charges of R72 and K65 side chains interact with α- and γ-phosphate groups of the nucleotide, respectively (Figure 4B). As these side chains would be closest to the borano group in an Rp α-borano substrate analogue, the R72A and K65A substitutions were made in order to characterize putative interactions with the α-borano group. Unexpectedly and unlike wild-type reverse transcriptase, R72A reverse transcriptase discriminates against AZT triphosphate and d4T triphosphate 350- and 13-fold, respectively (Table IV). The activity of R72A reverse transcriptase is at least 25-fold lower than that of wild type (Table IV; Sarafianos et al., 1995), and mutations at position 72 are never observed in viral isolates. K65A reverse transcriptase also discriminates 6- to 7-fold against analogues. Therefore, both R72A and K65A confer resistance to AZT triphosphate and d4T triphosphate in vitro. In contrast, neither variant reverse transcriptase discriminates against α-borano analogues (Table IV). We conclude that an intricate pattern of interactions between residues 72 and 65, the phosphate groups and the sugar 3′ position of the substrate nucleotide mediates the AZT triphosphate and d4T triphosphate resistance observed here in vitro. Table 4. Inhibition of wild-type and drug-resistant HIV-1 reverse transcriptase by nucleotide analogues Reverse transcriptase Wild type R72A K65A K65R Relative catalytic efficiencya 1 0.04 0.46 0.96 AZT triphosphate Ki (nM)b 21.8 6500 125 20.5 α-borano Ki (nM)b 8.25 32.5 9 9 Ratio 2.6 200 13.9 2.2 d4T triphosphate Ki (nM)b 49.2 500 275 60 α-borano Ki (nM)b 20.3 5 27.5 19.5 Ratio 2.4 100 10 3 a Oligo(dT)12–18–poly(rA) primer–template, TTP as the nucleotide substrate (Ueno et al., 1995). The catalytic efficiency is measured relative to wild-type reverse transcriptase (0.63 μM−1s−1). b These Ki values were determined as described (Ueno et al., 1995), from three a
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