Mechanism of inhibition of HIV-1 reverse transcriptase by 4'-Ethynyl-2-fluoro-2'-deoxyadenosine triphosphate, a translocation-defective reverse transcriptase inhibitor

Abstract

Nucleoside reverse transcriptase inhibitors (NRTIs) are employed in first line therapies for the treatment of human immunodeficiency virus (HIV) infection. They generally lack a 3'-hydroxyl group, and thus when incorporated into the nascent DNA they prevent further elongation. In this report we show that 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA), a nucleoside analog that retains a 3'-hydroxyl moiety, inhibited HIV-1 replication in activated peripheral blood mononuclear cells with an EC(50) of 0.05 nm, a potency several orders of magnitude better than any of the current clinically used NRTIs. This exceptional antiviral activity stems in part from a mechanism of action that is different from approved NRTIs. Reverse transcriptase (RT) can use EFdA-5'-triphosphate (EFdA-TP) as a substrate more efficiently than the natural substrate, dATP. Importantly, despite the presence of a 3'-hydroxyl, the incorporated EFdA monophosphate (EFdA-MP) acted mainly as a de facto terminator of further RT-catalyzed DNA synthesis because of the difficulty of RT translocation on the nucleic acid primer possessing 3'-terminal EFdA-MP. EFdA-TP is thus a translocation-defective RT inhibitor (TDRTI). This diminished translocation kept the primer 3'-terminal EFdA-MP ideally located to undergo phosphorolytic excision. However, net phosphorolysis was not substantially increased, because of the apparently facile reincorporation of the newly excised EFdA-TP. Our molecular modeling studies suggest that the 4'-ethynyl fits into a hydrophobic pocket defined by RT residues Ala-114, Tyr-115, Phe-160, and Met-184 and the aliphatic chain of Asp-185. These interactions, which contribute to both enhanced RT utilization of EFdA-TP and difficulty in the translocation of 3'-terminal EFdA-MP primers, underlie the mechanism of action of this potent antiviral nucleoside.

FIGURE 1.

HIV RT inhibition by EFdA-TP and other NRTIs. A, structure of EFdA. B, primer extension by HIV-1 RT was observed in the presence of fixed concentrations of 4 dNTPs, T_d100/P_d18, and MgCl₂ and increasing concentrations of EFdA-TP, ddATP, AZTTP, TFV-DP, or ddCTP. The reactions were carried out for 15 min. The arrows denote stops of the elongating DNA chain where adenosine analogs (EFdA-TP, ddATP, or TFV-DP) were expected to be incorporated. The first lane is a negative control, where no MgCl₂ was added; it shows the length of the 18-mer primer. C, the 100-mer products synthesized by HIV-1 RT were quantified and plotted against increasing concentrations of various inhibitors. The data points were fitted by GraphPad Prism 4. D, IC₅₀ values of the nucleotide analogs were determined by quantifying the percent of full extension and fitting the data points to GraphPad Prism 4 using one-site competition nonlinear regression.

FIGURE 2.

Inhibition of DNA- and RNA-dependent DNA synthesis by EFdA-TP. A, T_d31/P_d18 was incubated with HIV-1 RT for 15 min in the presence of 1 μm dNTPs and MgCl₂ and increasing concentrations of EFdA-TP (0–1000 nm). The first lane (ddATP) shows the inhibition of primer extension by ddATP to identify points of adenosine analog (ddATP or EFdA-TP) incorporation (arrows: +1, +6, and +10). B, the primer extension under the same conditions with an RNA/DNA substrate containing an RNA template annealed to a DNA primer (T_r31/P_d18).

FIGURE 3.

Incorporation of dNTP on EFdA-terminated template/primer (T/P_EFdA-MP). EFdA-TP was first incorporated at T_d31/P_d18 by HIV-1 RT and purified as described under “Experimental Procedures.” The incorporation of the next incoming nucleotide on T/P_EFdA-MP was examined in the presence of HIV-1 RT and MgCl₂ and increasing concentrations of dTTP. All other dNTPs were at a concentration of 1 μm. The reactions were stopped after 15 min (A) and 60 min (B).

FIGURE 4.

Effect of ddA or EFdA on formation of binary and ternary complexes. A, formation of a binary complex between RT and T/P_ddAMP or T/P_EFdA-MP. Purified T/P_ddAMP or T/P_EFdA-MP (20 nm) was incubated with HIV-1 RT at the indicated molar ratios and resolved by nondenaturing gel electrophoresis. B, formation of a ternary complex between RT and T/P_ddAMP or T/P_EFdA-MP and incoming dTTP. The stability of the ternary complexes was analyzed by incubating 100 nm RT and 9 nm T/P_ddAMP or T/P_EFdA-MP in the presence of increasing dTTP concentrations and heparin, which acted as an enzyme trap. In the absence of dTTP, the T/P·RT binary complex is unstable (lane 0), as RT dissociates from the T/P and is trapped by heparin.

FIGURE 5.

Determination of the translocation state of RT bound to T/P_ddAMP and T/P_EFdA-MP template/primers. A, the translocation state of RT after EFdA-MP incorporation was determined using site-specific Fe²⁺ footprinting. T/P_ddAMP or T/P_EFdA-MP (100 nm) with 5′-Cy3 label on the DNA template (see Fig. 5B) was incubated with HIV-1 RT (600 nm) and various concentrations of the next incoming nucleotide (dTTP) (as indicated). The complexes were treated for 5 min with ammonium iron sulfate (1 mm) and resolved on a polyacrylamide 7 m urea gel. An excision at position −18 indicates a pre-translocation complex, whereas the excision at position −17 represents a post-translocation complex. The scheme below the gel images indicates that in the absence of incoming dNTP, T/P_ddAMP is bound mostly in a post-translocation state, whereas T/P_EFdA-MP is bound in a pre-translocation state, with EFdA-MP positioned at the N-site. B, schematic of the excision assay. Depending on whether the 3′-primer terminus is positioned at the pre-translocation (N-site) or post-translocation (P-site) site, cleavage is observed on the 5′-labeled template strand at positions −18 or −17, respectively. The addition of varying levels of the incoming complementary dNTP serves to force the 3′-primer terminus from the N-site to the P-site.

FIGURE 6.

PP_i and ATP-dependent unblocking of ddAMP and EFdA-MP terminated primers. A, PP_i-dependent unblocking of T/P_ddAMP and T/P_EFdA-MP. Purified T/P_ddAMP or T/P_EFdA-MP was incubated with HIV-1 RT in the presence of 6 mm MgCl₂ and 150 μm PP_i at 37 °C. Aliquots were removed and reactions stopped at the indicated time points (0–30 min). Cleavage sites are indicated with arrows in the schemes below the gels. B, schematic representation of PP_i- and ATP-dependent rescue assay. The excision products of PP_i- and ATP-dependent excision of EFdA-MP are EFdA-TP and the EFdA-MP-ATP dinucleoside tetraphosphate, respectively. C, PP_i-dependent rescue of T/P_ddAMP and T/P_EFdA-MP. Purified T/P_EFdA-MP or T/P_ddAMP was incubated with HIV-1 RT in the presence of various amounts of PP_i (0–150 μm), dATP (100 μm), dTTP (0.5 μm), or ddGTP (10 μm) and 10 mm MgCl₂ at 37 °C. Aliquots of the reaction were stopped after 10 min. D, ATP-dependent rescue of T/P_ddAMP or T/P_EFdA-MP. Purified T/P_EFdA-MP or T/P_ddAMP was incubated with HIV-1 RT in the presence of ATP (3.5 mm), dATP (100 μm), dTTP (0.5 μm), or ddGTP (10 μm) and 10 mm MgCl₂ at 37 °C. Aliquots of the reaction were stopped at the indicated time points (0–90 min).

FIGURE 7.

Molecular models representing intermediates of the DNA polymerization reaction. A, molecular model of a ternary complex among RT, DNA, and EFdA-TP. The primer is bound at the P-site, and the incoming EFdA-TP is bound at the N-site. The 4′-ethynyl group of EFdA-TP is bound at a hydrophobic pocket (shown by a yellow arrow) defined by residues Met-184, Ala-114, Tyr-115, and Phe-160 and the aliphatic chain of Asp-185. For purposes of clarity the p66 fingers subdomain is not shown. B, molecular model of RT bound to EFdA-MP-terminated T/P immediately after incorporation of the inhibitor at the primer terminus and before translocation. The EFdA-MP of the 3′-primer terminus is positioned at the N-site. C, schematic representation of RT inhibition by EFdA. After incorporation of EFdA-MP at the 3′-primer terminus RT remains bound to T/P_EFdA mostly in a pre-translocation binding mode (top). In that binding mode the EFdA-MP at the 3′-primer terminus blocks binding of the incoming dNTP, thus inhibiting DNA polymerization.