ABSTRACT
Optimization studies using an HIV RNase H active site inhibitor containing a 1-hydroxy-1,8-naphthyridin-2(1H)-one core identified 4-position substituents that provided several potent and selective inhibitors. The best compound was potent and selective in biochemical assays (IC(50)=0.045 µM, HIV RT RNase H; 13 µM, HIV RT-polymerase; 24 µM, HIV integrase) and showed antiviral efficacy in a single-cycle viral replication assay in P4-2 cells (IC(50)=0.19 µM) with a modest window with respect to cytotoxicity (CC(50)=3.3 µM).
Subject(s)
Anti-HIV Agents/pharmacology , Enzyme Inhibitors/pharmacology , HIV-1/enzymology , Ribonuclease H/antagonists & inhibitors , Anti-HIV Agents/chemistry , Enzyme Inhibitors/chemistry , HeLa Cells , Humans , Naphthyridines/chemistry , Naphthyridines/pharmacologyABSTRACT
Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are highly specific and potent allosteric inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. NNRTIs inhibit reverse transcription in a substrate length-dependent manner in biochemical assays and in cell-based HIV-1 replication assays, suggesting a stochastic inhibitory mechanism. Surprisingly, we observed that NNRTIs potently inhibited plus-strand initiation in vitro under conditions in which little or no inhibition of minus-strand DNA synthesis was observed. In assays that recapitulated the initiation of plus-strand DNA synthesis, greater inhibition was observed with an RNA PPT primer than with a DNA primer of corresponding sequence and with wild-type reverse transcriptase but not with NNRTI-resistant enzymes. Structural elements that dictate sensitivity to NNRTIs were revealed using modified plus-strand initiation substrates. The data presented here suggest that specific inhibition of plus-strand initiation may be an important mechanism by which NNRTIs block HIV-1 replication.
Subject(s)
Drug Resistance, Viral , HIV Reverse Transcriptase/metabolism , Reverse Transcriptase Inhibitors/pharmacology , Alkynes , Anti-HIV Agents/pharmacology , Benzoxazines , Benzoxazoles/pharmacology , Cyclopropanes , DNA/chemistry , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , In Vitro Techniques , Inhibitory Concentration 50 , Ligands , Models, Genetic , Oxazines/pharmacology , Pyridones/pharmacology , Stochastic Processes , Transcription, Genetic , Virus ReplicationABSTRACT
Highly active antiretroviral therapy (HAART) has led to significant changes in mortality and morbidity in the human immunodeficiency virus type 1 (HIV-1) epidemic. Nevertheless, because of molecular mechanisms of viral persistence, HAART does not eradicate HIV-1. Didanosine and hydroxyurea were added to the antiretroviral regimens of 3 HIV-1-infected men who were receiving stable HAART and who had HIV-1 RNA levels <50 copies/mL at the initiation of the study protocol, as a novel intensification to attack cryptic viral replication; low-dose OKT3 was then administered, followed by a course of interleukin-2, to stimulate latent provirus. Replication-competent virus was undetectable after treatment, and plasma viral RNA was either undetectable or <5 copies/mL. In trial periods during which no antiretroviral therapy was administered, the patients developed plasma viral rebound. This translational approach combines novel intensification and stimulation therapy to deplete residual HIV-1 reservoirs. Additional experimental approaches must be developed if HIV-1 eradication is to become possible in patients receiving virally suppressive HAART.
