1st International Workshop on HIV Drug Resistance & Treatment Strategies 25-28 June 1997, St. Petersburg, Florida, USA

KINETIC CHARACTERIZATION OF HIV-1 REVERSE TRANSCRIPTASES EXPRESSING MUTATIONS SHOWN TO CONFER REDUCED SUSCEPTIBILITY TO PMEA (ADEFOVIR) IN VITRO

Antiviral Therapy 1997;2 (Suppl 5):16 (abstract no. 25)

AS Mulato, PD Lamy, MD Fuller, T Cihlar, MD Miller and JM Cherrington
Gilead Sciences, Foster City, CA 94404, USA

Adefovir dipivoxil (bis-POM PMEA), an oral prodrug of adefovir (PMEA), has shown potent anti-HIV and anti-HBV activity in clinical trials and is currently undergoing further clinical evaluation for the potential treatment of these diseases. Previous in vitro selection experiments showed that either a K65R or K70E mutation in HIV-1 RT could be selected for in the presence of PMEA. Recombinant viruses expressing either of these mutations showed reduced susceptibility to PMEA and lamivudine, but full susceptibility to zidovudine in vitro. Viruses expressing either the M184V or M184I mutations, shown to confer marked reduced susceptibility to lamivudine, are fully susceptible to PMEA and zidovudine in vitro. Extensive genotypic analyses of clinical samples obtained after 12 weeks (initial phase) of adefovir dipivoxil monotherapy or after 6 months (maintenance phase) during which the use of concomitant antiretroviral agents was permitted, have not revealed the K65R mutation. One patient receiving adefovir dipivoxil monotherapy during the maintenance phase developed the K70E mutation (50% mixture of K/E at codon 70) but suppression of viral replication was maintained (0.9 log decrease in viral RNA at month 6). To further investigate the molecular mechanisms involved in resistance to PMEA, we have cloned, expressed and purified HIV-1 RT enzymes from Escherichia coli carrying the K65R, K70E, or M184V mutations. The fold change in Ki values above wild-type were similar using a DNA or RNA template. Representative data are shown in the table below.

In general, the enzyme kinetic data correlated with the in vitro antiviral susceptibility data. Processivity and fidelity assays have been established for the recombinant enzymes and further experiments are underway.

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1997-06-25
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