Fig. 3
Biochemical examination of the dual mechanism of clofarabine. a Direct clofarabine-TP incorporation by HIV-1 RT. A 5′ 32P-labeled 23-mer DNA primer (P) annealed to a 24-mer DNA template with a single T overhang was incubated with HIV-1 RT and increasing concentrations of clofarabine-triphosphate (Clof-TP). E, Extended product; +, 50 μM dATP positive control; −, no dATP control. b Effect of clofarabine-TP incorporation on DNA synthesis. A 5′ 32P-labeled 17-mer DNA primer (P) annealed to a 38-mer RNA template was extended by HIV-1 RT with fixed dNTP concentrations found in either activated T cells (T cell, 5 μM) or monocyte-derived macrophages (MDM, 50 nM) with increasing concentrations of clofarabine-TP (two-fold dilutions starting at 125 μM). F, Fully extended product; +, 50 μM dNTP positive control; −, no dNTP control. Blue asterisks (*) indicates the clofarabine-TP incorporation site followed by kinetic pauses (]). c Clofarabine inhibition of NRTI-resistant RT mutants. MAGI cells were treated with increasing concentrations of clofarabine for 2 h prior to infection with Vsvg-pseudotyped HIV-1 containing mutations in RT. Flow cytometry analysis for infected cells was conducted at 48–72 h post-infection. IC50 values and 95 % confidence intervals are shown. NL4-3 MIG: wild-type HIV-1 RT, Q151: A62V, V75I, F77L, F116Y and Q151M, T69: M41L, A62V, T69S, K70R, T215Y and serine–serine insertion between 69 and 70. d Biochemical simulation of HIV-1 RT activity at dNTP concentrations found in cells with and without clofarabine treatment. A 5′ 32P-labeled 17-mer DNA primer (P) annealed to a 38-mer RNA template (shown in box) was extended using an equal amount of purified HIV-1 RT protein with dNTP concentrations found in T cells (T, 5 μM), macrophages (M, 50 nM) or macrophages treated with 300 nM clofarabine (M/C, 10 nM dATP, 28 nM dCTP, 28 nM dGTP, 40 nM TTP). Blue stars (*) indicate kinetic pause sites, F, fully extended 38 bp product; −, no dNTP control