Partial selective inhibition of HIV-1 reverse transcriptase and human DNA polymerases γ and β by thiated 3′-fluorothymidine analogue 5′-triphosphates

Abstract

3′-Deoxy-3′-fluorothymidine (FLT, alovudine^®) belongs to the most potent agents inhibiting HIV-1 replication. Its 5′-triphosphate (FLTTP) is a potent inhibitor of HIV-1 reverse transcriptase (HIV RT). Unfortunately, FLT exerts substantial hematologic toxicity both in vitro and in vivo. It was suggested that this toxicity may be related to inhibition of human DNA polymerases, especially mitochondrial DNA polymerase γ, by nucleoside analogue 5′-triphosphates leading to termination of DNA synthesis and mitochondrial dysfunction. To decrease the toxicity of FLT, its thiated analogues, 4-SFLT and 2-SFLT, were previously synthesized and shown to be potent inhibitors of HIV-1 with low in vitro cytotoxicity. To explain this phenomenon in the present study the synthesis of 5′-triphosphates of thiated FLT analogues was undertaken and their interaction with recombinant HIV-1 RT and human DNA polymerases γ (pol γ) and β (pol β) was investigated. It was shown that 3′-deoxy-3′-fluoro-4-thiothymidine 5′-triphosphate (4-SFLTTP) and 3′-deoxy-3′-fluoro-2-thiothymidine 5′-triphosphate (2-SFLTTP) were, similarly to FLTTP, potent competitive inhibitors of HIV-1 RT, with $K_{\text{i}}^{\text{app}}$ values of 0.091 and 0.022 μM respectively. It is of interest that 2-SFLTTP, a compound in an unusual syn conformation around the glycosidic bond was an uncompetitive inhibitor of human mitochondrial DNA pol γ with $K_{\text{i}}^{\text{app}}$ of 0.174 μM, while 4-SFLTTP in anti conformation inhibited this enzyme similarly to FLTTP, i.e., non-competitively, with $K_{\text{i}}^{\text{app}}$ of 0.055 μM. Both 4-SFLTTP and 2-SFLTTP were competitive inhibitors of human DNA pol β, with $K_{\text{i}}^{\text{app}}$ values of 16.84 and 4.04 μM, respectively. The results point to partially selective inhibition of HIV RT by thiated 3′-fluorothymidine 5′-triphosphate analogues. Of special interest is that 2-SFLTTP, showing syn conformation, is a less potent inhibitor of human mitochondrial pol γ than 4-SFLTTP and FLTTP, both in the anti conformation, and has a higher inhibitory activity against HIV-1 RT than 4-SFLTTP. Moreover, the parent nucleoside 2-SFLT possessing the syn conformation shows a more potent anti-HIV-1 activity and a better selectivity index than its 4-thio isomer in the anti conformation (Matthes et al., 1989, Poopeiko et al., 1995), 2-SFLT is a potent and selective anti-HIV-1 agent with the selectivity index 4-fold higher than that of FLT.

Findings regarding the mechanisms of antiviral and cytotoxic activities of FLT and its thioanalogues are discussed.

Introduction

Multidrug, highly active antiretroviral therapy (HAART) employed in the treatment of human immunodeficiency virus (HIV) infection uses as its main components nucleoside inhibitors of HIV reverse transcriptase (NRTIs). It was suggested that long term therapy with the NRTIs may be associated with mitochondrial DNA polymerase γ (pol γ) inhibition and subsequent depletion of mitochondrial DNA (Kaguni, 2004, Kakuda, 2000). However, the DNA pol γ hypothesis by itself fails to explain the entire array of metabolic deficiencies associated with NRTI-induced disorders. Direct effects of NRTI's on various mitochondrial targets must also be taken into account (Lund and Wallace, 2004).

3′-Deoxy-3′-fluorothymidine (FLT, alovudine^®) belongs to the most potent agents inhibiting HIV-1 replication. In addition, it is highly active against HIV-1 multidrug resistant (MDR) strains. Its 5′-triphosphate (FLTTP) is a potent inhibitor of HIV-1 reverse transcriptase (HIV RT, RNA-directed DNA polymerase, EC 2.7.7.49) (Cheng et al., 1987). Unfortunately, FLT displays high cytotoxicity against host cells which limits its clinical applicability (Balzarini et al., 1988). It was suggested that this toxicity may be related to inhibition of human DNA polymerases (EC 2.7.7.7), especially mitochondrial DNA polymerase γ, by FLT metabolite—5′-triphosphate, leading to termination of DNA synthesis and mitochondrial dysfunction (Kaguni, 2004). To decrease the cellular toxicity of FLT, its thiated analogues 4-SFLT and 2-SFLT were previously prepared and shown to be potent in vitro inhibitors of HIV-1 with low toxicity (Matthes et al., 1989, Poopeiko et al., 1995). To establish whether inhibition of pol γ exerted by thiated FLTTP analogues is correlated with development of in vitro cytotoxicity in cell cultures, we decided to synthesise 2-SFLTTP, 4-SFLTTP as well as FLTTP and to investigate their interactions with recombinant HIV-1 RT and human polymerases γ and β.