GW-8248

HIV reverse transcriptase (HIV-RT) contains two distinct protein domains catalyzing DNA polymerase and RNase H activities. Non-nucleoside reverse transcriptase inhibitor (NNRTI) binding to HIV-RT can affect RNase H activity. The structurally diverse NNRTIs capravirine, efavirenz, GW8248, TMC-125, and nevirapine all inhibited 5'-RNA directed HIV RNase H activity as partial inhibitors with maximal inhibition of 40-65%. Potencies of RNase H inhibition correlated with the respective potencies of DNA polymerase inhibition. Mutations in the NNRTI binding site (K103N, Y181C, Y188L, and K103N/Y181C) reduced the potency of RNase H inhibition, similar to their effects on DNA polymerase activity. The NNRTIs did not affect the activity of the isolated HIV RNase H domain. In contrast, 3'-DNA directed RNase H activity of HIV-RT was mechanistically distinct from 5'-RNA directed RNase H activity and was stimulated rather than inhibited by NNRTI binding to HIV-RT. Therefore, NNRTI binding to the polymerase domain of HIV-RT interferes with RNase H activity through a long-range effect, which is affected by the structure of the RNA:DNA hybrid substrate, but is independent of NNRTI compound structure and nucleic acid substrate sequence.

The full scan ESI mass spectrum of GW4511 (I) showed a deprotonated mol. at m/z 543.Upon collisional activation, it yielded two major product ions at m/z 317 and 485.The ion at m/z 317 can be easily identified as a benzophenoxide anion which may result from the intramol. nucleophilic substitution (SNi) reaction.The ion at m/z 485, corresponding to a neutral loss of 58 Da from the deprotonated I, indicated the possible loss of a C₂H₂0₂ unit due to a skeletal rearrangement process (Smiles intermediate) within the deprotonated mol. prior to or during fragmentation.Similarly, the deprotonated mol. of GW4751 (II) at m/z 482 on collisional activation gave rise to two major product ions at m/z 256 and 424.Once again, the exact mass data on the rearrangement product ion at m/z 424.0534 indicated the neutral loss of II via the Smiles rearrangement.