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In vitro selection and characterization of HIV-1 with reduced susceptibility to PMPA
M A Wainberg1, M D Miller, Y Quan
1McGill University AIDS Center, Jewish General Hospital, Montreal, Quebec, Canada.
Abstract:
9-(2-phosphonomethoxypropyl)adenine (PMPA) has demonstrated remarkable anti-simian immunodeficiency virus (SIV) activity in macaque models of SIV infection and transmission prevention. Recently, PMPA and its oral prodrug, bis-POC PMPA, have also shown potent anti-human immunodeficiency virus type 1 (HIV-1) activity in Phase I clinical studies. In vitro experiments were performed to address the resistance properties of PMPA. After eight passages in increasing concentrations of PMPA, HIV-1IIIB was able to grow in the presence of 2 microM PMPA, fivefold above the IC50 of PMPA for wild-type parental virus. Sequence analysis of the reverse transcriptase (RT) genes from four of 15 RT clones demonstrated the presence of a K65R substitution in RT and recombinant HIV expressing the K65R RT mutation showed a threefold to fourfold increase in IC50 value for PMPA as compared to wild-type. Additional experiments demonstrated that viruses expressing other nucleoside-associated RT resistance mutations all showed wild-type or < threefold reduced susceptibility to PMPA in vitro. Interestingly, lamivudine-resistant viruses expressing the M184V RT mutation showed wild-type to slightly increased susceptibility to PMPA in vitro and addition of the M184V mutation to HIV with the K65R mutation resulted in reversion to wild-type susceptibility for PMPA. In agreement with the cell culture findings, Escherichia coli-expressed K65R RT showed fivefold reduced susceptibility to PMPA diphosphate, the active moiety of PMPA. Furthermore, in combination experiments, PMPA with hydroxyurea showed synergistic inhibition of HIV replication in vitro. The potent antiretroviral activity and favourable resistance profile of PMPA and bis-POC PMPA are being further investigated in ongoing clinical trials.
Insights
9-(2-phosphonomethoxypropyl)adenine (PMPA) shows strong anti-HIV activity and develops resistance slowly. The K65R mutation confers reduced susceptibility, but the M184V mutation can restore sensitivity, indicating a favorable resistance profile for PMPA.
Area of Science:
- Virology
- Antiviral Drug Development
- Molecular Biology
Background:
- 9-(2-phosphonomethoxypropyl)adenine (PMPA) exhibits significant anti-simian immunodeficiency virus (SIV) activity.
- PMPA and its oral prodrug, bis-POC PMPA, demonstrate potent anti-human immunodeficiency virus type 1 (HIV-1) activity in clinical studies.
Purpose of the Study:
- To investigate the in vitro resistance properties of PMPA against HIV-1.
- To characterize the impact of specific reverse transcriptase (RT) mutations on PMPA susceptibility.
Main Methods:
- HIV-1 was passaged in increasing concentrations of PMPA to select for resistant variants.
- Sequence analysis of RT genes from resistant clones.
- Recombinant HIV expressing specific RT mutations were generated and tested for susceptibility to PMPA.
- In vitro susceptibility testing of PMPA diphosphate against mutated RT enzymes.
- Combination experiments with PMPA and hydroxyurea.
Main Results:
- HIV-1IIIB developed resistance to PMPA at concentrations fivefold above the wild-type IC50.
- A K65R substitution in RT was identified in resistant clones, conferring a 3-4 fold increase in PMPA IC50.
- Other nucleoside-associated RT resistance mutations showed minimal impact on PMPA susceptibility.
- The M184V mutation conferred wild-type to slightly increased susceptibility and restored wild-type susceptibility in the presence of K65R.
- K65R RT showed a fivefold reduced susceptibility to PMPA diphosphate.
- PMPA and hydroxyurea demonstrated synergistic inhibition of HIV replication.
Conclusions:
- PMPA exhibits a favorable resistance profile, with resistance primarily associated with the K65R mutation.
- The M184V mutation may mitigate PMPA resistance, suggesting potential for combination therapy.
- PMPA demonstrates potent antiretroviral activity and is a promising candidate for further clinical investigation.