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RNase H Inhibitors: Obstacles to Developments
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Reported by David Margolis, University of North Carolina
CROI, Feb 2007, Los Angeles
The RNase H function of reverse transcriptase is required for successful production of a DNA copy of the HIV genome. As HIV reverse transcriptase (RT) copies the (+) RNA into complementary (-) DNA, the (+) RNA strand must be degraded to release the (-) DNA to be used as template for synthesis of the second (+) DNA strand. The ribonuclease H (RT-RNase H) activity of HIV RT executes this function. RT-RNase H is absolutely essential for HIV replication and is therefore a logical target for antiretroviral intervention. Drug discovery efforts focusing on RT-RNase H have lagged behind those for other HIV targets, but are ongoing.
However, several presentations suggested that changes in RNase H activity can affect drug susceptibility, and some studies raised new concerns about RNAse H as an antiviral target. Studies have shown that the NRTI AZT and the NNRTI efavirenz (EFV, Sustiva) are synergistic, that is that together they inhibit RT function to a greater extent that the sum of their individual inhibitory activities. Sluis-Cremer (Abstr. 58) suggested that RT inhibition by EFV allowed the innate RNAseH activity of RT to cleave the RNA template, which, in turn, increases susceptibility to AZT. They hypothesized that EFV and AZT may be synergistic in the following scenario:
- AZT incorporates into the growing DNA chain, stopping reverse transcription unless it is excised
- In the presence of EFV, RNAse H activity of RT is enhanced, leading to destruction of the RNA template before AZT excision can efficiently occur, which can increase the apparent activity of AZT.
This led to the speculation by a questioner, M. Gotte from Montreal, that RNAse H inhibitors could decrease the antiviral activity of NRTIs.
Lennerstrand (Abstr. 595) presented other evidence that this might be so. They studied the effect on excision of AZT-5'-monophosphate (AZT-MP) from growing viral DNA chains that had been halted by changing reaction conditions such as pH, magnesium or AZT-MP concentrations. Such conditions might occur in human lymphocytes. Conditions that increased resistance to AZT also decreased the activity of RNAse H, suggesting that decreased RNAse H activity played a role in the insensitivity of RT enzymes containing typical AZT resistance mutations to AZT.
Gotte's group highlighted an obstacle to the development of RNAse H inhibitors (abstr. 89). They studied the mechanism of action of the RNase H inhibitor b-thujaplicinol. In an assay that measures cleavage of RNA by RNAse H, they found that b-thujaplicinol efficiently hacked RNA strands. However, in the context of reverse transcriptase tightly bound to the RNA substrate, i.e. a conformation that is seen during reverse transcription, b-thujaplicinol is unable to inhibit RNase H. This suggested that RNAse H inhibitors that bind directly to the RNAse H active site within RT might have difficulty accessing this site when RT is doing its job, bound to an RNA template.
These findings suggest that RNAse H inhibitors might need to be found that do not bind in the active site of RNase H within HIV RT, and if they are used in the clinic the question of antagonism with other RT inhibitors must be addressed.
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