title: Inhibition of HIV-1 ribonuclease H by a novel diketo acid, 4-[5-(benzoylamino)thien-2-yl]-2,4-dioxobutanoic acid.
abstract: 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.
authors.affiliation: Department of Biological Chemistry, Merck Research Laboratories, West Point, Pennsylvania 19486-0004, USA. cathryn_shawred@merck.com
authors.forename: Cathryn A
authors.initials: CA
authors.lastname: Shaw-Reid
authors.forename: Vandna
authors.initials: V
authors.lastname: Munshi
authors.forename: Pia
authors.initials: P
authors.lastname: Graham
authors.forename: Abigail
authors.initials: A
authors.lastname: Wolfe
authors.forename: Marc
authors.initials: M
authors.lastname: Witmer
authors.forename: Renee
authors.initials: R
authors.lastname: Danzeisen
authors.forename: David B
authors.initials: DB
authors.lastname: Olsen
authors.forename: Steven S
authors.initials: SS
authors.lastname: Carroll
authors.forename: Mark
authors.initials: M
authors.lastname: Embrey
authors.forename: John S
authors.initials: JS
authors.lastname: Wai
authors.forename: Michael D
authors.initials: MD
authors.lastname: Miller
authors.forename: James L
authors.initials: JL
authors.lastname: Cole
authors.forename: Daria J
authors.initials: DJ
authors.lastname: Hazuda
mesh_terms: D002087:Butyrates / Q000138:chemical synthesis / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000494:pharmacology*; D017245:Foscarnet / Q000494:pharmacology; D015497:HIV-1 / Q000201:enzymology*; D007700:Kinetics; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology; D016914:Ribonuclease H / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013876:Thiophenes / Q000138:chemical synthesis / Q000494:pharmacology*
chemical_list: C472557:4-(5-(benzoylamino)thien-2-yl)-2,4-dioxobutanoic acid; D002087:Butyrates; D004791:Enzyme Inhibitors; D018894:Reverse Transcriptase Inhibitors; D013876:Thiophenes; D017245:Foscarnet; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H