The amino acid change K65R in human immunodeficiency virus type 1-reverse transcriptase (RT) confers viral resistance to various 2,3-dideoxynucleoside drugs in vivo. Using pre-steady state kinetic methods, we found that K65R-reverse transcriptase is 3.2-14-fold resistant to 2,3-dideoxynucleotides in vitro relative to wild-type reverse transcriptase, in agreement with resistance levels observed in vivo. A decreased catalytic rate constant k pol mostly accounts for the lower incorporation efficiency observed for 2,3-dideoxynucleotides. Examination of the crystal structure of the RT⅐DNA⅐dNTP complex suggested that both the charge at position 65 and the 3-OH of the incoming nucleotide act in synergy during the creation of the phosphodiester bond, resulting in a more pronounced decreased catalytic rate constant for 2,3-dideoxynucleotides than for dNTPs. This type of intramolecular activation of the leaving phosphate by the 3-OH group appears to be conserved in several nucleotide phosphotransferases. These data were used to design dideoxynucleotide analogues targeting K65R RT specifically. ␣-Boranophosphate ddATP was found to be a 2-fold better substrate than dATP and inhibited DNA synthesis by K65R RT 153-fold better than ddATP. This complete suppression of drug resistance at the nucleotide level could serve for other reverse transcriptases for which drug resistance is achieved at the catalytic step.

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 (BH(3)(-)) group on the alpha-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 R(p) diastereoisomer of thymidine alpha-boranotriphosphate bound like a normal substrate. Using alpha-(R(p))-boranophosphate derivatives of the clinically relevant compounds AZT and d4T, the presence of the alpha-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 alpha-borano modification of analogues targeting reverse transcriptase may be of generic value in fighting viral drug resistance.

Nucleoside analogues are currently used to treat human immunodeficiency virus infections. The appearance of up to five substitutions (A62V, V75I, F77L, F116Y, and Q151M) in the viral reverse transcriptase promotes resistance to these drugs, and reduces efficiency of the antiretroviral chemotherapy. Using pre-steady state kinetics, we show that Q151M and A62V/V75I/F77L/F116Y/ Q151M substitutions confer to reverse transcriptase (RT) the ability to discriminate an analogue relative to its natural counterpart, and have no effect on repair of the analogue-terminated DNA primer. Discrimination results from a selective decrease of the catalytic rate constant k pol : 18-fold (from 7 to 0.3 s ؊1 ), 13-fold (from 1.9 to 0.14 s ؊1 ), and 12-fold (from 13 to 1 s ؊1 ) in the case of ddATP, ddCTP, and 3-azido-3-deoxythymidine 5-triphosphate (AZTTP), respectively. The binding affinities of the triphosphate analogues for RT remain unchanged. Molecular modeling explains drug resistance by a selective loss of electrostatic interactions between the analogue and RT. Resistance was overcome using ␣-boranophosphate nucleotide analogues. Using A62V/V75I/ F77L/F116Y/Q151M RT, k pol increases up to 70-and 13-fold using ␣-boranophosphate-ddATP and ␣-boranophosphate AZTTP, respectively. These results highlight the general capacity of such analogues to circumvent multidrug resistance when RT-mediated nucleotide resistance originates from the selective decrease of the catalytic rate constant k pol . The human immunodeficiency virus (HIV)1 infects more than 40 million individuals in the world. 3Ј-Azido-3Ј-deoxythymidine (AZT, zidovudine) was the first antiretroviral drug to receive approval from the FDA in 1987 to treat HIV-1-infected patients. AZT is a nucleoside analogue acting on viral replication. It is metabolically activated by cellular kinases of the host cell to its corresponding triphosphate form AZTTP before reaching its target, reverse transcriptase (RT). RT is an essential viral DNA polymerase responsible for viral DNA synthesis. AZTTP is a poor substrate for cellular DNA polymerases, but is incorporated into the nascent viral DNA strand by RT with the same efficiency as its natural nucleotide counterpart dTTP. Because AZT lacks a 3Ј-hydroxyl group (3Ј-OH) on its ribose moiety, AZTMP is incorporated into DNA and viral DNA synthesis is terminated.The prolonged use of AZT as the sole drug in the clinic has resulted in the emergence of AZT-resistant viruses (1). A set of six specific substitutions on RT (M41L, D67N, K70R, T215Y or F, L210W, and K219E or Q) gives rise to high level AZT resistance (2), the appearance of T215F or Y being the most important substitution. A long awaited mechanism of AZT resistance because of these mutations has been proposed, based on biochemical studies using purified reverse transcriptase: AZTresistant RT is able to catalyze a primer-unblocking reaction related to pyrophosphorolysis (3, 4) to remove the chain-terminating AZTMP. This "repair" reaction allows the RT to resume elongation of the primer DNA.

Nucleotide analogs are widely used in antiviral therapy and particularly against AIDS. Delivered to the cell as nucleosides, they are phosphorylated into their active triphospho derivative form by cellular kinases from the host. The last step in this series of phosphorylations is performed by nucleoside diphosphate (NDP) kinase, an enzyme that can use both purine or pyrimidine and oxy- or deoxynucleotides as substrates. Using pure recombinant human NDP kinase type B (product of the gene nm23-H2), we have characterized the kinetic parameters of several nucleotide analogs for this enzyme. Contrary to what is generally assumed, diphospho- and triphospho- derivatives of azidothymidine as well as of dideoxyadenosine and dideoxythymidine are very poor substrates for NDP kinase. The rate of phosphorylation of these analogs varies between 0.05% and 0.5%, as compared to the corresponding natural nucleotide, a result that is not due to the inability of the analogs to bind to the enzyme. Using the data from the high resolution crystal structure of NDP kinase, we provide an interpretation of these results based on the crucial role played by the 3'-OH moiety of the nucleotide in catalysis.

Human immunodeficiency virus type 1 is resistant to 3-azido-3-deoxythymidine (AZT) when four amino acid substitutions (D67N, K70R, T215F, and K219Q) are present simultaneously in its reverse transcriptase. Wildtype and AZT-resistant reverse transcriptases show identical binding to a 3-azido-3-deoxythymidine 5-monophosphate (AZTMP)-terminated primer/RNA template. On DNA templates, the equilibrium dissociation constant (K D ) for primer/template and AZT-resistant reverse transcriptase (RT) (K D ‫؍‬ 4.1 nM) is similar to that of the wild-type enzyme (K D ‫؍‬ 6.2 nM). However, k off is 4 -25-fold lower for the AZT-resistant enzyme than for the wild-type enzyme, depending on the nucleotide and the template. The kinetic decay of a wild-type RT/primer/AZTMP-terminated DNA template complex is biphasic. Seventy percent of the initial complex decays with a rate constant greater than 0.05 s ؊1 , and 30% with a rate constant of 0.0017 s ؊1. Decay of an AZT-resistant RT/ AZTMP-terminated primer/DNA template complex is monophasic, with a rate constant of 0.0018 s ؊1 . The last two nucleotides at the 3 end of the AZTMP-terminated DNA primer in complex with AZT-resistant RT, but not wild-type RT, and a DNA template are protected from exonuclease digestion, suggesting that enhanced binding of the 3 end of the AZTMP-terminated DNA primer to reverse transcriptase is involved in the mechanism of AZT resistance by human immunodeficiency virus type 1. Reverse transcriptase (RT)1 of the human immunodeficiency virus type 1 (HIV-1) is a DNA-and RNA-dependent DNA polymerase that synthesizes a double-stranded DNA from the viral (ϩ) RNA genome (1). Most of the replicative steps of the HIV-1 genome are dependent on the RT encoded by the viral pol gene. This dependence of the replication cycle on RT has directed much of the anti-HIV-1 chemotherapy strategy toward this enzyme (2). Nucleoside analogues were among the first RT inhibitors described (3, 4). Once taken up by the infected eukaryotic cell, most nucleoside analogues require phosphorylation of their 5Ј-hydroxyl group by cellular kinases to convert them to the 5Ј-triphosphate, substrates for RT. Most of these analogues do not possess a free 3Ј-hydroxyl group, and chain termination occurs once they are added to the nascent viral DNA. Termination is believed to account for the observed inhibition of viral growth. It is not known whether incorporation of the analogue in vivo occurs during DNA-or RNA-dependent DNA synthesis, or both.3Ј-Azido-3Ј-deoxythymidine (AZT) is a prototype of the chain-terminating analogues (4). 3Ј-Azido-3Ј-deoxythymidine 5Ј-triphosphate (AZTTP) is over a thousandfold more efficient as a substrate for RT than for human polymerases (5-7), and is as good a substrate as the natural substrate dTTP for RT. Since AZTTP and dTTP pools are comparable in cultured cells (8), one would expect that replication and hence viral growth would be inhibited completely (9). However, decreased sensitivity to AZT appears gradually over time together with the rapid emergence of mutations...