title: Studies on the mechanism of inactivation of the HIV-1 nucleocapsid protein NCp7 with 2-mercaptobenzamide thioesters.
abstract: The HIV-1 nucleocapsid protein (NCp7) is a small basic protein with two CysCysHisCys zinc-binding domains that specifically recognizes the Psi-site of the viral RNA. NCp7 plays a number of crucial roles in the viral lifecycle, including reverse transcription and RNA encapsidation. Several classes of potential anti-HIV compounds have been designed to inactivate NCp7 through zinc ejection, including a special class of thioester compounds. We have investigated the mechanism of action of two N-substituted-S-acyl-2-mercaptobenzamide compounds (compounds 1 and 2) that target NCp7. UV/Visible spectroscopy studies demonstrated that both thioesters were able to eject metal from NCp7. NMR and mass spectroscopy studies showed that the thioester compounds specifically ejected zinc from the carboxyl-terminal zinc-binding domain of NCp7 by covalent modification of Cys(39). Exposure of NCp7 to compounds 1 and 2 destroyed its ability to specifically bind RNA, whereas NCp7 already bound to RNA was protected from zinc ejection by the thioesters. The thiol component of the thioesters (compound 3, 2-mercaptobenzoyl-beta-alaninamide) did not eject zinc from NCp7, but when compound 3 was incubated with acetyl CoA prior to incubation with NCp7, we observed extensive metal ejection. Thus, the thiol released by the reaction of compounds 1 and 2 could be re-acylated in vivo by acyl CoA to form a new thioester compound that is able to react with NCp7. These studies provide a better understanding of the mechanism of action of thioester compounds, which is important for future design of anti-HIV-1 compounds that target NCp7.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA.
authors.forename: Lisa M Miller
authors.initials: LM
authors.lastname: Jenkins
authors.forename: J Calvin
authors.initials: JC
authors.lastname: Byrd
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Hara
authors.forename: Pratibha
authors.initials: P
authors.lastname: Srivastava
authors.forename: Sharlyn J
authors.initials: SJ
authors.lastname: Mazur
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Stahl
authors.forename: John K
authors.initials: JK
authors.lastname: Inman
authors.forename: Ettore
authors.initials: E
authors.lastname: Appella
authors.forename: James G
authors.initials: JG
authors.lastname: Omichinski
authors.forename: Pascale
authors.initials: P
authors.lastname: Legault
mesh_terms: D000595:Amino Acid Sequence; D019380:Anti-HIV Agents / Q000138:chemical synthesis / Q000737:chemistry*; D001549:Benzamides / Q000138:chemical synthesis / Q000737:chemistry*; D036022:Capsid Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D024202:Electrophoretic Mobility Shift Assay; D015683:Gene Products, gag / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D013053:Spectrophotometry / Q000379:methods; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014764:Viral Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D015032:Zinc / Q000737:chemistry; D015091:beta-Alanine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry*; D054301:gag Gene Products, Human Immunodeficiency Virus
chemical_list: D019380:Anti-HIV Agents; D001549:Benzamides; D036022:Capsid Proteins; D015683:Gene Products, gag; C500618:N-(2-(5-pyridiniovaleroylthio)benzoyl)-beta-alaninamide; C500619:N-(2-(nicotinoylthio)benzoyl)-beta-alaninamide; C071884:NCP7 protein, Human immunodeficiency virus 1; D013438:Sulfhydryl Compounds; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; D015091:beta-Alanine; D015032:Zinc; D003545:Cysteine