Inhibition of HIV-1 Ribonuclease H by a Novel Diketo Acid, 4-[5-(Benzoylamino)thien-2-yl]-2,4-dioxobutanoic Acid
| Autor: | Cathryn A. Shaw-Reid, James L. Cole, John S. Wai, Mark Embrey, Vandna Munshi, Marc V. Witmer, David B. Olsen, Abigail Wolfe, Daria J. Hazuda, Michael D. Miller, Steven S. Carroll, Pia L. Graham, Renee Danzeisen |
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| Rok vydání: | 2003 |
| Předmět: |
RNase P
Stereochemistry Ribonuclease H Thiophenes Biochemistry RNase PH Substrate Specificity Structure-Activity Relationship chemistry.chemical_compound Enzyme Inhibitors RNase H Molecular Biology Polymerase biology Active site RNA-Directed DNA Polymerase Cell Biology Molecular biology Reverse transcriptase Butyrates Kinetics chemistry HIV-1 biology.protein Nucleic acid Reverse Transcriptase Inhibitors DNA Foscarnet |
| Zdroj: | Journal of Biological Chemistry. 278:2777-2780 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.c200621200 |
| Popis: | Human immunodeficiency virus-type 1 (HIV-1) reverse transcriptase (RT) coordinates DNA polymerization and ribonuclease H (RNase H) activities using two discrete active sites embedded within a single heterodimeric polyprotein. We have identified a novel thiophene diketo acid, 4-[5-(benzoylamino)thien-2-yl]-2,4-dioxobutanoic acid, that selectively inhibits polymerase-independent RNase H cleavage (IC(50) = 3.2 microm) but has no effect on DNA polymerization (IC(50) > 50 microm). The activity profile of the diketo acid is shown to be distinct from previously described compounds, including the polymerase inhibitor foscarnet and the putative RNase H inhibitor 4-chlorophenylhydrazone. Both foscarnet and the hydrazone inhibit RNase H cleavage and DNA polymerization activities of RT, yet neither inhibits the RNase H activity of RT containing a mutation in the polymerase active site (D185N) or an isolated HIV-1 RNase H domain chimera containing the alpha-C helix from Escherichia coli RNase HI, suggesting these compounds affect RNase H indirectly. In contrast, the diketo acid inhibits the RNase H activity of the isolated RNase H domain as well as full-length RT, and inhibition is not affected by the polymerase active site mutation. In isothermal titration calorimetry studies using the isolated RNase H domain, binding of the diketo acid is independent of nucleic acid but strictly requires Mn(2+) implying a direct interaction between the inhibitor and the RNase H active site. These studies demonstrate that inhibition of HIV-1 RNase H may occur by either direct or indirect mechanisms, and they provide a framework for identifying novel agents such as 4-[5-(benzoylamino)thien- 2-yl]-2,4-dioxobutanoic acid that specifically targets RNase H. |
| Databáze: | OpenAIRE |
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