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

IN VITRO SELECTION AND CHARACTERIZATION OF HIV-1 VARIANTS WITH REDUCED SUSCEPTIBILITY TO PMPA

Antiviral Therapy 1997;2 (Suppl 5):17 (abstract no. 26)

JM Cherrington¹, R Chandok², AS Mulato¹, PD Lamy¹, H Mitsuya³, and M Wainberg^2
1Gilead Sciences, Foster City, CA 94404, USA; ²McGill University, Montreal, Canada; and ³National Cancer Institute, Frederick, Maryland, USA

9-(2-phosphonomethoxypropyl)adenine (PMPA), an acyclic nucleoside phosphonate analogue, has demonstrated remarkable anti-SIV activity in in vivo models of chronic and acute infection as well as in prevention of transmission of SIV infection. Recently PMPA has also shown potent anti-HIV activity in a Phase I clinical trial. In vitro experiments were performed to address the resistance properties of PMPA. HIV-1 (IIIb) was passaged weekly in MT-2 cells in the presence of gradually increasing concentrations of PMPA. After seven passes, virus was selected for growth in the presence of 2µM PMPA, a concentration that represents a fivefold increase above the IC₅₀ of PMPA against the wild-type parental virus, IIIb. PCR was used to clone and sequence the RT gene of the in vitro-selected PMPA-resistant viruses. Sequence analysis of RT genes from three of seven clones demonstrated the presence of a K65R substitution in RT. No other RT amino acid substitutions were identified in a significant number of clones. The K65R recombinant virus showed a 3.5-fold increase in IC₅₀ value for PMPA as compared to IIIb. Additional experiments demonstrated that zidovudine-resistant viruses (expressing M41L and T215Y) as well as multi-drug resistant viruses (expressing Q151M, A62V, F77L, V75I, F116Y) showed less than a twofold increase in IC₅₀ value for PMPA in vitro. Lamivudine-resistant viruses (M184V or M184I) also showed complete susceptibility to PMPA in vitro. A recombinant virus expressing the K70E mutation, selected for in vitro in the presence of a related molecule, PMEA, showed wild-type susceptibility to PMPA. In agreement with the cell culture findings, recombinant expressed K65R RT showed a fivefold increase in K_i value for PMPA diphosphate (PMPApp), the active moiety of PMPA, whereas the K70E and M184V enzymes had wild-type K_i values for PMPApp. Previous animal studies showed that the K65R mutation developed in RT genes from SIV-infected macaques treated with PMPA, and conferred a fourfold increase in IC₅₀ value for PMPA in vitro. Despite this mutation, the animals maintained suppression of viral replication and remain healthy at approximately 2 years post-infection. The potent antiretrovirus activity and favourable resistance profile of PMPA are being further investigated in ongoing clinical trials.

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1997-06-25
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