Irreversible Inhibition of the HIV-1 Protease: Targeting Alkylating Agents to the Catalytic Aspartate Groups
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- 作者：Zhonghua Yu ; Patricia Caldera ; Fiona McPhee ; James J. De Voss ; Patrick R. Jones ; Alma L. Burlingame ; Irwin D. Kuntz ; Charles S. Craik ; and Paul R. Ortiz de Montellano
- 刊名：Journal of the American Chemical Society
- 出版年：1996
- 出版时间：June 26, 1996
- 年：1996
- 卷：118
- 期：25
- 页码：5846 - 5856
- 全文大小：323K
- 年卷期：v.118,no.25(June 26, 1996)
- ISSN：1520-5126
文摘
Irreversible inhibition of the HIV-1 protease by agents thatspecifically alkylate its catalytic aspartateresidues is a potentially useful approach for circumventing theevolution of HIV strains that are resistant to proteaseinhibitors. Five haloperidol- and two FMOC-based epoxides ofdiffering reactivities have been synthesized andtested as irreversible inhibitors of the HIV-1 protease (HIV-1 PR).Of these, two trisubstituted epoxides, acis-1,2-disubstituted epoxide, a 1,1-disubstituted epoxide, and amonosubstituted epoxide function as irreversibleinhibitors,but two trans-1,2-disubstituted epoxides do not. Themost effective of the epoxides (6) inactivates HIV-1 PRwithKinact = 65 
M andVinact = 0.009min-1.1,2-Epoxy-3-(p-nitrophenoxy)propane (EPNP), anonspecific inactivatingagent for aspartyl proteases, has been used to validate a protocol forestablishing the stoichiometry and site of proteinalkylation. Mass spectrometric analysis of the inactivated enzymeshows that one molecule of either EPNP or thecyclic 1,2-disubstituted epoxide 6 is covalently bound perHIV-1 PR dimer. Mass spectrometric sequencing oflabeled proteolytic peptides shows that both inhibitors are covalentlybound to a catalytic aspartate residue. Thecovalent binding of three 
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ddle">-unsaturated ketone derivatives ofhaloperidol has been similarly examined. Analysisof HIV-1 PR inactivated by these agents establishes that they bindcovalently to the two cysteines and the N-terminalamino group but not detectably to the catalytic aspartate residues.The results indicate that aspartate-targetedinactivation of HIV-1 PR depends on (a) matching the reactivity of thealkylating functionality to that of the aspartates,preferably by exploiting the two-aspartate catalytic motif of theprotease to activate the alkylating agent, and (b)appropriate positioning of the alkylating functionality within theactive site. These requirements are readily met bya monosubstituted, 1,1-disubstituted, or cycliccis-1,2-disubstituted epoxide but not bytrans-1,2-disubstituted epoxidesor ,ddle">-unsaturated ketones.
M andVinact = 0.009min-1.1,2-Epoxy-3-(p-nitrophenoxy)propane (EPNP), anonspecific inactivatingagent for aspartyl proteases, has been used to validate a protocol forestablishing the stoichiometry and site of proteinalkylation. Mass spectrometric analysis of the inactivated enzymeshows that one molecule of either EPNP or thecyclic 1,2-disubstituted epoxide 6 is covalently bound perHIV-1 PR dimer. Mass spectrometric sequencing oflabeled proteolytic peptides shows that both inhibitors are covalentlybound to a catalytic aspartate residue. Thecovalent binding of three ,ddle">-unsaturated ketone derivatives ofhaloperidol has been similarly examined. Analysisof HIV-1 PR inactivated by these agents establishes that they bindcovalently to the two cysteines and the N-terminalamino group but not detectably to the catalytic aspartate residues.The results indicate that aspartate-targetedinactivation of HIV-1 PR depends on (a) matching the reactivity of thealkylating functionality to that of the aspartates,preferably by exploiting the two-aspartate catalytic motif of theprotease to activate the alkylating agent, and (b)appropriate positioning of the alkylating functionality within theactive site. These requirements are readily met bya monosubstituted, 1,1-disubstituted, or cycliccis-1,2-disubstituted epoxide but not bytrans-1,2-disubstituted epoxidesor ,ddle">-unsaturated ketones.