Irreversible Inhibition of the HIV-1 Protease: Targeting Alkylating Agents to the Catalytic Aspartate Groups

Irreversible inhibition of the HIV-1 protease by agents that specifically alkylate its catalytic aspartate residues is a potentially useful approach for circumventing the evolution of HIV strains that are resistant to protease inhibitors. Five haloperidol- and two FMOC-based epoxides of differing reactivities have been synthesized and tested as irreversible inhibitors of the HIV-1 protease (HIV-1 PR). Of these, two trisubstituted epoxides, a cis-1,2-disubstituted epoxide, a 1,1-disubstituted epoxide, and a monosubstituted epoxide function as irreversible inhibitors, but two trans-1,2-disubstituted epoxides do not. The most effective of the epoxides (6) inactivates HIV-1 PR with Kinact = 65 μM and Vinact = 0.009 min-1. 1,2-Epoxy-3-(p-nitrophenoxy)propane (EPNP), a nonspecific inactivating agent for aspartyl proteases, has been used to validate a protocol for establishing the stoichiometry and site of protein alkylation. Mass spectrometric analysis of the inactivated enzyme shows that one molecule of either...