title: In vitro characterization of nonpeptide irreversible inhibitors of HIV proteases.
abstract: The irreversible inhibition of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) proteases by 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) and eight haloperidol derivatives has been studied. EPNP specifically inhibits HIV-1 and HIV-2 proteases with a stoichiometry of one EPNP molecule/dimeric enzyme. The site of modification of HIV-2 protease by EPNP has been unambiguously identified as Asp-25 using high performance tandem mass spectrometry. The haloperidol derivatives assayed consist of epoxides, ynones, and alpha,beta-unsaturated ketones. The Kinact values for these haloperidol derivatives range from 10.7 to 521 microM for HIV-1 protease and from 8.6 to 283 microM for the HIV-2 enzyme, being in some cases approximately 1000-fold more potent irreverisble inhibitors of HIV proteases than EPNP. This potency results from the haloperidol character of the compounds and the chemical reactivity of the groups capable of forming a covalent bond with the enzyme. Covalent modification of HIV-2 protease by a radiolabeled epoxide derivative of haloperidol, UCSF 84, is prevented by EPNP and the peptidomimetic transition state analog U-85548. In similar experiments, incorporation of UCSF 84 into HIV-1 protease is partially prevented by these active-site inhibitors. In contrast, a mutant HIV-1 protease, HIV-1 PR C95M, in which Cys-95 has been replaced by Met, is labeled 50% less than HIV-1 protease and is fully protected by EPNP and U-85548. These results indicate the presence of 2 reactive residues in HIV-1 protease: Cys-95 and another located in the active site of the enzyme. The alpha,beta-unsaturated ketone derivative of haloperidol, UCSF 191, which is stable over a broad pH range, was used to study the pH profile of inactivation of HIV-1 and HIV-2 proteases. Comparison of the profiles of inactivation of wild-type HIV-1 protease, HIV-1 PR C95M, and HIV-1 PR C67L as well as HIV-2 protease (which has no cysteine residues) reveals the contribution of Cys-95 to the reactivity of these irreversible inhibitors. The inhibitors UCSF 70, UCSF 84, UCSF 115, UCSF 142, and UCSF 191 reduce p55gag polyprotein processing when assayed in a mammalian cell line that produces HIV-1 viral particles lacking the envelope.
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0446.
authors.forename: R
authors.initials: R
authors.lastname: Salto
authors.forename: L M
authors.initials: LM
authors.lastname: Babé
authors.forename: J
authors.initials: J
authors.lastname: Li
authors.forename: J R
authors.initials: JR
authors.lastname: Rosé
authors.forename: Z
authors.initials: Z
authors.lastname: Yu
authors.forename: A
authors.initials: A
authors.lastname: Burlingame
authors.forename: J J
authors.initials: JJ
authors.lastname: De Voss
authors.forename: Z
authors.initials: Z
authors.lastname: Sui
authors.forename: P
authors.initials: P
authors.lastname: Ortiz de Montellano
authors.forename: C S
authors.initials: CS
authors.lastname: Craik
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D004852:Epoxy Compounds / Q000494:pharmacology; D017320:HIV Protease Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D015497:HIV-1 / Q000201:enzymology; D015498:HIV-2 / Q000201:enzymology; D006220:Haloperidol / Q000031:analogs & derivatives / Q000494:pharmacology*; D006801:Humans; D008969:Molecular Sequence Data; D009596:Nitrophenols / Q000494:pharmacology*; D009838:Oligodeoxyribonucleotides
chemical_list: D004852:Epoxy Compounds; D017320:HIV Protease Inhibitors; D009596:Nitrophenols; D009838:Oligodeoxyribonucleotides; C007606:1,2-epoxy-3-(p-nitrophenoxy)propane; D006220:Haloperidol