Oxidative Modifications of Kynostatin-272, a Potent Human Immunodeficiency Virus Type 1 Protease Inhibitor: Potential Mechanism for Altered Activity in Monocytes/Macrophages

Previous studies have indicated that human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) are less active at blocking viral replication in HIV-1 infected peripheral blood monocytes/macrophages (M/M) than in HIV-1-infected T cells. We explored the hypothesis that oxidative modification and/or metabolism of the PIs in M/M might account for this reduced potency. We first tested the susceptibility of several PIs (kynostatin-272 [KNI-272], saquinavir, indinavir, ritonavir, or JE-2147) to oxidation after exposure to hydrogen peroxide (H2O2): only KNI-272 was highly susceptible to oxidation. Treatment of KNI-272 with low millimolar concentrations of H2O2 resulted in mono-oxidation of the sulfur in the S-methyl cysteine (methioalanine) moiety, as determined by reversed-phase high-performance liquid chromatography and mass spectrometry (RP-HPLC/MS). Higher concentrations of H2O2 led to an additional oxidation of the sulfur in the thioproline moiety of KNI-272. None of the PIs were metabolized or oxidized when added to T cells and cultured for up to 12 days. However, when KNI-272 was added to M/M, the concentration of the original KNI-272 steadily decreased with a corresponding increase in the production of three KNI-272 metabolites as identified by RP-HPLC/MS. The structures of these metabolites were different from those produced by H2O2 treatment. The two major products of M/M metabolism of KNI-272 were identified as isomeric forms of KNI-272 oxidized solely on the thioproline ring. Both metabolites had reduced capacities to inhibit HIV-1 protease activity when tested in a standard HIV-1 protease assay. These studies demonstrate that antiviral compounds can be susceptible to oxidative modification in M/M and that this can affect their antiviral potency.We thank Hiroaki Mitsuya and the Japan Energy Corporation for providing the PIs used in this study. We thank Hiroaki Mitsuya for helpful discussions. Funding for this study was provided in part by a Cooperative Research and Development Agreement between the National Cancer Institute and the Japan Energy Corporation