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J Biol Chem. 2014 Jun 6;289(23):16270-7. doi: 10.1074/jbc.M114.569707. Epub 2014 Apr 9.

Inhibition of the ribonuclease H activity of HIV-1 reverse transcriptase by GSK5750 correlates with slow enzyme-inhibitor dissociation.

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From the Department of Microbiology and Immunology, McGill University, Montreal, Quebec H3A 2B4, Canada.
the Departments of Medicinal Chemistry.
Computational Chemistry, and.
Virology, Infectious Diseases Therapeutic Area Unit, GlaxoSmithKline, Research Triangle Park, North Carolina 27709.
From the Department of Microbiology and Immunology, McGill University, Montreal, Quebec H3A 2B4, Canada, the Department of Biochemistry, McGill University, Montreal, Quebec H3G1Y6, Canada, and the Department of Medicine, Division of Experimental Medicine, McGill University, Montreal, Quebec H3A 1A3, Canada


Compounds that efficiently inhibit the ribonuclease (RNase) H activity of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have yet to be developed. Here, we demonstrate that GSK5750, a 1-hydroxy-pyridopyrimidinone analog, binds to the enzyme with an equilibrium dissociation constant (K(d)) of ~400 nM. Inhibition of HIV-1 RNase H is specific, as DNA synthesis is not affected. Moreover, GSK5750 does not inhibit the activity of Escherichia coli RNase H. Order-of-addition experiments show that GSK5750 binds to the free enzyme in an Mg(2+)-dependent fashion. However, as reported for other active site inhibitors, binding of GSK5750 to a preformed enzyme-substrate complex is severely compromised. The bound nucleic acid prevents access to the RNase H active site, which represents a possible biochemical hurdle in the development of potent RNase H inhibitors. Previous studies suggested that formation of a complex with the prototypic RNase H inhibitor β-thujaplicinol is slow, and, once formed, it dissociates rapidly. This unfavorable kinetic behavior can limit the potency of RNase H active site inhibitors. Although the association kinetics of GSK5750 remains slow, our data show that this compound forms a long lasting complex with HIV-1 RT. We conclude that slow dissociation of the inhibitor and HIV-1 RT improves RNase H active site inhibitors and may circumvent the obstacle posed by the inability of these compounds to bind to a preformed enzyme-substrate complex.


Drug Development; HIV-1; Retrovirus; Reverse Transcription; Ribonuclease

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