114 RNAse H: Can Selective Inhibitors Be Identified? Michael A Parniak Univ of Pittsburgh, PA, USA

HIV carries its genome as (+)RNA, but must replicate through a double strand DNA intermediate. All steps in the conversion of viral (+)RNA into dsDNA are catalyzed by the viral enzyme, reverse transcriptase (RT), and this requires RT to be multifunctional. RT RNA-dependent DNA polymerase (RDDP) activity copies the (+)RNA into complementary (-)DNA. RT DNA-dependent DNA polymerase (DDDP) activity copies the newly formed (-)DNA to form the complementary (+)DNA strand, thereby completing viral dsDNA synthesis. However, in order to transition from RDDP to DDDP activity, the (+)RNA strand must be degraded to release the (-)DNA to be used as template for synthesis of the (+)DNA strand. This degradation is carried out by RT-associated ribonuclease H (RT-RNase H). RNase H specifically degrades the RNA component of an RNA-DNA hybrid duplex. RNase H is ubiquitous, found in all cells including human. Nonetheless, RT-RNase H is absolutely essential for HIV replication since the cellular enzyme cannot complement for a lack of the viral activity. HIV RT-RNase H is therefore a logical target for antiretroviral intervention. Drug discovery efforts focusing on RT-RNase H have lagged behind those for other HIV targets such as integrase, due to factors such as the lack of a suitable high throughput screening assay for RT-RNase H and the perception that identification of compounds that inhibit HIV RT-RNase H specifically, without affecting the human enzyme, may be difficult if not impossible. Accordingly, very few inhibitors of RT-RNase H have been described. Recent advances in the field suggest that RT-RNase H may indeed represent an excellent target for antiretroviral development. A novel fluorescence-based screening assay has led to the identification of new inhibitors of RNase H, some of which are quite specific for HIV RT-RNase H and show little or no inhibition of the human enzyme. Furthermore, the crystal structure of one of our most potent compounds complexed with HIV RT and the location of mutations arising in HIV resistance to this compound indicate binding interactions that are unique to HIV RT and would not be possible in human RNase H. This suggests that specific inhibitors of HIV RT-RNase H can be identified and further validates this viral enzyme activity as an important target for anti-HIV drug development.