Structural basis for activation of α-boranophosphate nucleotide analogues targeting drug-resistant reverse transcriptase

Abstract

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.

Mechanistic Insights into the Suppression of Drug Resistance by Human Immunodeficiency Virus Type 1 Reverse Transcriptase Using α-Boranophosphate Nucleoside Analogs

“…In agreement with our previous reports (6,15,17), we measure a systematic loss of discrimination upon the presence of the BH 3 group. This in vitro loss of resistance is the result of a conserved efficiency of catalysis (measured with k pol ).…”

“…In some of our assays, k pol is increased more than 150-fold, for example when BH 3 -3TCTP is used instead of the unsubstituted 3TCTP. An efficient incorporation of BH 3 -TTP is even achieved using the R72A substitution, which decreases the polymerase activity ϳ100-fold (15,31,34,35). Hence, these enzymatic results highlight the general potential given by BH 3 derivatives to circumvent resistance brought by almost any discrimination-associated mutation.…”

“…[␥-32 P]ATP was purchased from Amersham Biosciences. The ␣-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 BH 3 -3TCTP where the isomers could not be separated and were used as a racemic mixture.…”

“…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 BH 3 group on their ␣-phosphate (also named ␣-BH 3 -dNTPs or BH 3 -dNTPs) (6,(15)(16)(17). The BH 3 group replacing a nonbridging oxygen on the ␣-phosphate of a nucleotide analog also reduces the DNA repair reaction with ATP or PP i when tested on the AZT-resistant RT mutant (D67N/K70R/T215F/K219Q) (15,16).…”

“…The (Rp)-diastereoisomer form of ␣-boranophosphate analogs is the preferred isomer active on RT as a chain terminator when it is devoid of a 3Ј-OH group. In addition, an increased phosphorylation activity of the human NDP kinase toward borano nucleotides is observed with the same diastereoisomer (15,16). * This work was supported in part by the Agence Nationale de Recherche sur le SIDA (AIDS) and Ensemble Contre le SIDA.…”