Haloperidol-Based Irreversible Inhibitors of the HIV-1 and HIV-2 Proteases

The proteases expressed by the HIV-1 and HIV-2 viruses process the polyproteins encoded by the viral genomes into the mature proteins required for virion replication and assembly. Eight analogs of haloperidol have been synthesized that cause time-dependent inactivation of the HIV-1 protease and, in six cases, HIV-2 protease. The IC values for the analogues are comparable to that of haloperidol itself. Enzyme inactivation is due to the presence of an epoxide in two of the analogues and carbonyl-conjugated double or triple bonds in the others. Irreversible inactivation is confirmed by the failure to recover activity when one of the inhibitors is removed from the medium. At pH 8.0, the agents inactivate the HIV-1 protease 4–80 times more rapidly than the HIV-2 protease. Faster inactivation of the HIV-1 protease is consistent with alkylation of cysteine residues because the HIV-1 protease has four such residues whereas the HIV-2 protease has none. Inactivation of the HIV-2 protease requires modification of non-cysteine residues. The similarities in the rates of inactivation of the HIV-2 protease by six agents that have intrinsically different reactivities toward nucleophiles suggest that the rate-limiting step in the inactivation process is not the alkylation reaction itself. At least five of the agents inhibit polyprotein processing in an ex vivo cell assay system, but they are also toxic to the cells

In vitro characterization of nonpeptide irreversible inhibitors of HIV proteases

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 α,β-unsaturated ketones. The K(inact) values for these haloperidol derivatives range from 10.7 to 521 μM for HIV-1 protease and from 8.6 to 283 μM for the HIV-2 enzyme, being in some cases ~1000-fold more potent irreversible 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 α,β-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 p55(gag) polyprotein processing when assayed in a mammalian cell line that produces HIV-1 viral particles lacking the envelope