Fixed conformation nucleoside analogs effectively inhibit excision-proficient HIV-1 reverse transcriptases.

An important mechanism of resistance to nucleoside analogs is the enhanced excision of the analog after it has been incorporated. Excision requires that the analog be located at the 3' terminus of the primer. We have developed nucleoside analogs that do not block DNA synthesis at the point of incorporation, but only after additional normal dNTPs have been added to the DNA. Such "delayed chain terminators" should be relatively resistant to excision and therefore effective against drug-resistant HIV-1 reverse transcriptases (RTs) that are proficient at excision. We tested a class of nucleoside analogs in which a pseudosugar ring is locked in either the North or the South conformation. These analogs have a 3' OH present on the pseudosugar ring, which allows extension of the primer strand after the analog is incorporated. We asked whether these analogs would inhibit polymerization by HIV-1 RT in assays using purified HIV-1 RT and in cell-based assays. HIV-1 RT did not effectively incorporate the analogs in which the pseudosugar is in the South conformation. The North conformation analogs are readily incorporated into the primer; the primer can be extended for two or three additional nucleotides before extension is inhibited. This block to polymerization is not complete; larger extension products are detectable at longer incubation times. Experiments with purified excision-proficient HIV-1 RT mutants suggest that the North conformation analogs are relatively resistant to excision. These analogs can also block the replication of viruses containing excision-proficient RTs. Although the fixed-conformation nucleotides are probably not suitable for development as drugs, other nucleoside analogs that cause delayed chain termination may complement the nucleoside analogs already approved for HIV-1 therapy.