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 In contrast, a mutant HIV-1 protease, HIV-1 PR C95M, in which Cys-95 has been replaced by Met, is labeled 50% less than HIV-1 protease and is fully protected by EPNP and U-85548.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.
Highlights
The irreversible inhibition of human immunodefi- UCSF 142,and UCSF 191reduce p5WWpolyprotein procciency virus type 1(HIV-1)and type 2 (HIV-2)proteases essing when assayedin a mammalian cellline that proby 1,2-epoxy-3-(p-nitrophenoxy)propane(EPNP)and duces human immunodeficiency virus type 1 (HIV-1) viral particles lacking the envelope
The human immunodeficiency virus type 1 (HIV-1)’ is the etiologic agent of AIDS, while infection by HIV-2 virus is responsible for immune suppression in some parts of western
HIV-1 sults from the haloperidol character of the compounds viral genomes containing a mutant protease withAsn in place and the chemical reactivity of the groups capable of of the active-sitAe sp-25 produce noninfectious virionsthat conforming acovalentbond with the enzyme.Covalent tain completely unprocessed p558”8 protein precursor [5].Pepmodification ofHIV-2 protease by a radiolabeled epoxide derivative of haloperidol, UCSF 84, is prevented by EPNP and the peptidomimetic transition stateanalog
Summary
(Received forpublication, November 16, 1993, and in revised form, January 12, 1994). Rafael SaltoSs, Lilia M. The values for these haloperidol derivatives range from 10.7 to 521 p~ for HIV-1 protease and from 8.6 to 283 p~ for the HIV-2 enzyme,being in some cases -1000-fold more potent irreversible in-. By combining the specificity of haloperidol binding and the ability to reactcovalently with the enzyme, an affinity label for HIV-1 protease can be developed. Because of their active site-directed, irreversible nature, these inhibitomrsay prove to be less sensitive to mutations in the prottehaastecould lead to. Inhibitionstudies were carried out oHn IV-2 protease (which lacks cysteine residues) as well as on variant forms of HIV-1 protease Out by the method of Schagger and Von Jagow
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