1-(ß-D-Dioxolane) Thymine Is Effective against HIV-1-containing TAM and M184V
Johan Lennerstrand*1, G Bluemling1, M Ruckstuhl1, M Bennett1, C Chu2, and R Schinazi1
1Emory Univ and VAMC, Decatur, GA, US and 2Coll of Pharmacy, Univ of Georgia, Athens, US
thymine (DOT) is a potent inhibitor of HIV-1 with excellent pharmacological
properties including good oral bioavailability in rats and monkeys, rapid
intracellular phosphorylation to form DOT-TP, and low
toxicity. In culture, DOT has shown selective activity against viruses
containing thymidine analog
mutations (TAM) and M184V. To confirm this and to further characterize its resistance
profile, DOT-TP was evaluated against a large panel of purified HIV-1 reverse transcriptases (RT) with multi-nucleoside resistance
mutations. Since ATP-dependent excision may also influence the clinical efficacy of FDA-approved
nucleoside analogs, enzymatic studies were also designed to explore the
mechanism of resistance.
Methods: Site-directed RT mutants were constructed
containing M41L, D67N, K70R, L210W, T215Y (TAM-mutant), M41L, T69S-SG, L210W,
T215Y (69-mutant), V75I, F77L, F116Y, Q151M (151-mutant), or K65R (65-mutant)—the
last 2 representing a non-excision mechanism of resistance, i.e., binding
discrimination. The M184V mutation was studied separately and also added to the
TAM, 69- and 151-mutant. A non-radioactive HIV-RT assay was used to study the ATP-mediated
excision of DOT-MP, zidovudine (AZT)-MP, carbovir-MP, and tenofovir (TFV),.
Results: DOT-TP had a Ki value of 0.011 µM for wild
type RT (HXB2), which was similar to the Ki for AZT-TP (0.015 µM). The 69-mutant RT demonstrated
ATP-mediated excision, whereas, the 151- and the 65-mutant showed
non-ATP-dependent decreased incorporation rates for all the inhibitors. The TAM-mutant
RT demonstrated ATP-mediated resistance for
AZT-TP and TFV-DP, whereas the M184V mutation, either by itself or in added
form, had no significant effect on DOT-TP resistance. The IC50 of
the various RT mutants were determined for the TAM-mutant; 69-mutant; 151-mutant; and
65-mutant RT, respectively, in the presence of 5 mM
ATP. The level of resistance as mutant IC50 /wild type IC50 were: DOT-TP (1.1; 4.1; 18; 3.9), AZT-TP (5.0; 9.6; 20; 2.0), carbovir-TP (ND; 3.4; 7.2; 6.1),
and TFV-DP (2.5; 7.3; 3.3; 18), respectively.
Conclusions: Compared with
other RT inhibitors, DOT-TP
was overall more effective against RT-containing TAM, M184V, and K65R. The 69-mutant
demonstrated a lower level of resistance to DOT-TP than AZT-TP and TFV-DP.
However, as expected for dioxolane nucleosides, an increased non-ATP-dependent
resistance was found with the 151-mutant. These enzymatic studies indicate that DOT resistance mainly
involves binding discrimination and only modest ATP-dependent excision.