Click Chemistry-Based Two-Component System for Efficient Inhibition of Human Immunodeficiency Virus (HIV) Reverse Transcriptase (RT)

Abstract

We synthesized two dTTP analogues for copper-free "click" chemistry-coupling in the active sites of DNA polymerases. We found that in the presence of both analogues, human immunodeficiency virus (HIV) reverse transcriptase (RT) activity was suppressed by up to 93%. This inhibitory effect was not recovered by an excess amount of primer-template unlike that for a conventional HIV RT inhibitor, azidothymidine. This finding may become the basis for the development of efficient in vivo inhibitors of HIV RT and other DNA polymerases.

Scheme 1. Design of Click-Chemistry-Based Two-Component Inhibitor (TCI) for Inactivation of HIV RT

(A) HIV RT-assisted Cu-free “click” reaction and possible HIV RT inhibition. In vivo R1 and R2 could be the residues of NRTI triphosphates or viral RNA/DNA. In this work, R1 and R2 were a dTTP analogue and a DNA primer/template, respectively. (B) Synthetic schemes for 6Az-dTTP and DBCO-dTTP starting from aadTTP. The yields were 31 and 19% for 6Az-dTTP and DBCO-dTTP, respectively. (C) Primer/template (P/T) design for “click” chemistry-based TCI. The primer was fluorescein (FAM) labeled. The protruding 3′ end of the template had only one A (red).

Figure 1

Substrate properties of 6Az-dTTP and DBCO-dTTP in primer elongation catalyzed by HIV-1 RT. All samples contained an 18 nt FAM-labeled primer/template complex (Scheme 1C) and 50 nM HIV RT in reaction buffer (50 mM Tris pH 8.0, 100 mM KCl, 8 mM MgCl₂, 100 mg/mL BSA, and 2 mM DTT). The samples contained 5 mM dTTP, 6Az-dTTP, DBCO-dTTP, and 5 mM dVTP (V is either A, C, or G) added in different orders as indicated (e.g., +₁ means first added). All samples were incubated at 37 °C for 20–30 min at each stage of dNTP addition as described in the Supporting Information followed by analysis by electrophoresis in 15% polyacrylamide gel containing 7 M urea. The product of click reaction is indicated by green arrows (lanes 8 and 9). The primer elongation products with detached DBCO groups are indicated by blue arrows in lanes 6, 10, and 11.

Figure 2

HIV-1 RT activity by TCI and its components measured by the filter binding assay. P/T (1 μM) and HIV RT (50 nM) were incubated with dTTP or first dTTP analogue and incubation for 45 min at 37 °C. Next, second dTTP analogue was added to samples 4 and 5, and all samples were further incubated for 30 min at 37 °C followed by the addition of the dNTP mixture (100 mM dATP, 100 mM dTTP, 100 mM dCTP, and 10 mM [³H]dGTP and incubation for 45 min at 37 °C. All reactions were quenched by the addition of EDTA (0.5 M) and analyzed by filter binding assay as described in the Supporting Information. The data of three independent experiments with single standard deviations are presented.

Figure 3

TCI inhibitory effect is not significantly recovered by access amount of primer–template (P/T). All reaction mixtures contained P/T and HIV RT in the reaction buffer. In addition, samples 1 and 2 contained azidothymidine (AZT), while samples 3 and 4 contained both 6Az-dTTP and DBCO-dTTP. The samples were incubated for 45 min at 37 °C. Samples 2 and 4 were then treated with 1 μM P/T (to double its concentration), while all samples were treated with the dNTP mixture (100 μM dATP, 100 μM dTTP, 100 μM dCTP, 10 μM [8-H³]-dGTP) followed by incubation for 20 min at 37 °C. All reactions were quenched by the addition of EDTA (0.5 M) and transferred to DE 81 paper for quantification by a filter binding assay (see the Supporting Information for details). The data of three independent experiments with single standard deviations are presented.