In vitro characterization of nonpeptide irreversible inhibitors of HIV proteases.

Salto, Rafael; Babé, Lilia M.; Li, Jia; Rose, Jim; Yu, Zhonghua; Burlingame, Alma L.; Voss, James J. De; Sui, Zhihua; Montellano, Paul Ortiz de; Craik, Charles S.

doi:10.1016/s0021-9258(17)34114-5

Cited by 36 publications

(26 citation statements)

References 34 publications

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“…25,26 Another strategy for minimizing drug resistance is to develop irreversible rather than reversible inhibitors of HIV-1 PR. [27][28][29][30][31][32][33][34][35][36][37] Irreversible inhibitors are less sensitive to mutations because even a low degree of active-site occupancy can lead in time to complete inactivation of the protein. The catalytic aspartate residues are the ideal target for such irreversible inhibitors, because their mutation results in the complete loss of catalytic activity.…”

Section: Introductionmentioning

confidence: 99%

“…27 EPNP is a nonspecific and relatively weak irreversible inhibitor of HIV-1 PR, HIV-2 PR and simian immunodeficiency virus protease (SIV PR) (K inact = 6.7-9.9 mM, V inact = 48-60 × 10 −3 min −1 ). 35,41 In an effort to develop epoxidecontaining irreversible inhibitors with high potency, non-peptide 27 and peptidomimetic inhibitors of HIV-1 PR containing a cisepoxide were designed, synthesized and kinetically characterized (Fig. 1).…”

Section: Introductionmentioning

confidence: 99%

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Theoretical study on the mechanism of a ring-opening reaction of oxirane by the active-site aspartic dyad of HIV-1 protease

Kóňa¹

2008

Org. Biomol. Chem.

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Two possible mechanisms of the irreversible inhibition of HIV-1 protease by epoxide inhibitors are investigated on an enzymatic model using ab initio (MP2) and density functional theory (DFT) methods (B3LYP, MPW1K and M05-2X). The calculations predict the inhibition as a general acid-catalyzed nucleophilic substitution reaction proceeding by a concerted SN2 mechanism with a reaction barrier of ca. 15-21 kcal mol(-1). The irreversible nature of the inhibition is characterized by a large negative reaction energy of ca. -17-(-24) kcal mol(-1). A mechanism with a direct proton transfer from an aspartic acid residue of the active site onto the epoxide ring has been shown to be preferred compared to one with the proton transfer from the acid catalyst facilitated by a bridging catalytic water molecule. Based on the geometry of the transition state, structural data important for the design of irreversible epoxide inhibitors of HIV-1 protease were defined. Here we also briefly discuss differences between the epoxide ring-opening reaction in HIV-1 protease and epoxide hydrolase, and the accuracy of the DFT method used.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Theoretical study on the mechanism of a ring-opening reaction of oxirane by the active-site aspartic dyad of HIV-1 protease

Kóňa¹

2008

Org. Biomol. Chem.

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“…The kinetic values for the inactivation of HIV-1 PR by EPNP and conjugated ketones 8 − 10 were determined in an earlier study and are as follows: EPNP, K inact = 9900 μM, V inact = 0.06 min -1 ; 8 , K inact = 62 μM, V inact = 0.380 min -1 ; 9 , K inact = 10.7 μM, V inact = 0.057 min -1 ; 10 , K inact = 57 μM, V inact = 0.232 min -1 . These results show that the K inact value for 6 , the best epoxide, is comparable to those for 8 and 10 but somewhat worse than that for 9 .…”

Section: Resultsmentioning

confidence: 99%

“…Irreversible inhibition of HIV-1 PR was first achieved with 1,2-epoxy-3-( p -nitrophenoxy)propane (EPNP), a small epoxide molecule that is a general irreversible inhibitor of aspartyl proteases . EPNP (Figure ) is a nonspecific and relatively weak irreversible inhibitor of both HIV-1 PR ( K inact = 9.9 mM, V inact = 0.060 min -1 ) and the HIV-2 protease (HIV-2 PR; K inact = 6.7 mM, V inact = 0.048 min -1 ) . An analysis of the binding of EPNP to HIV-2 PR 19 and a crystal structure of the adduct of EPNP with the simian immunodeficiency virus (SIV) protease indicate that EPNP is bound to a catalytic aspartate residue in both of these proteins.…”

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confidence: 99%

“…Our search for irreversible inhibitors of the HIV proteases has concentrated on derivatives of haloperidol, a lead structure identified as a reversible inhibitor through computer-assisted inhibitor design methods. , We have shown in earlier work that haloperidol analogues with a 1,1-disubstituted epoxide or an α,β-unsaturated carbonyl group as the reactive center (Figure ) irreversibly inhibit HIV-1 PR and HIV-2 PR. , The generally higher activity of these compounds as inactivators of HIV-1 PR than HIV-2 PR suggests that they bind covalently to several amino acids, but the actual nature of the irreversible inhibition was not established. As part of an effort to identify the key features required to target irreversible inhibitors to the active site aspartyl residues of the HIV proteases, we have (a) synthesized seven additional epoxides with a range of chemical reactivities and evaluated their activities as irreversible inhibitors of HIV-1 PR and (b) undertaken mass spectrometric analyses of HIV-1 PR inactivated by five different agents to define the stoichiometry of the inactivation process and identify the amino acid(s) alkylated by each agent.…”

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

Caldera

McPhee

et al. 1996

J. Am. Chem. Soc.

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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 K inact = 65 μM and V inact = 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 EPNP or the cyclic 1,2-disubstituted epoxide 6 is covalently bound per HIV-1 PR dimer. Mass spectrometric sequencing of labeled proteolytic peptides shows that both inhibitors are covalently bound to a catalytic aspartate residue. The covalent binding of three α,β-unsaturated ketone derivatives of haloperidol has been similarly examined. Analysis of HIV-1 PR inactivated by these agents establishes that they bind covalently to the two cysteines and the N-terminal amino group but not detectably to the catalytic aspartate residues. The results indicate that aspartate-targeted inactivation of HIV-1 PR depends on (a) matching the reactivity of the alkylating functionality to that of the aspartates, preferably by exploiting the two-aspartate catalytic motif of the protease to activate the alkylating agent, and (b) appropriate positioning of the alkylating functionality within the active site. These requirements are readily met by a monosubstituted, 1,1-disubstituted, or cyclic cis-1,2-disubstituted epoxide but not by trans-1,2-disubstituted epoxides or α,β-unsaturated ketones.

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Cis‐Configured Aziridines Are New Pseudo‐Irreversible Dual‐Mode Inhibitors of Candida albicans Secreted Aspartic Protease 2

Degel¹,

Staib²,

Rohrer³

et al. 2008

ChemMedChem

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A series of cis-configured epoxides and aziridines containing hydrophobic moieties and amino acid esters were synthesized as new potential inhibitors of the secreted aspartic protease 2 (SAP2) of Candida albicans. Enzyme assays revealed the N-benzyl-3-phenyl-substituted aziridines 11 and 17 as the most potent inhibitors, with second-order inhibition rate constants (k(2)) between 56,000 and 121,000 M(-1) min(-1). The compounds were shown to be pseudo-irreversible dual-mode inhibitors: the intermediate esterified enzyme resulting from nucleophilic ring opening was hydrolyzed and yielded amino alcohols as transition-state-mimetic reversible inhibitors. The results of docking studies with the ring-closed aziridine forms of the inhibitors suggest binding modes mainly dominated by hydrophobic interactions with the S1, S1', S2, and S2' subsites of the protease, and docking studies with the processed amino alcohol forms predict additional hydrogen bonds of the new hydroxy group to the active site Asp residues. C. albicans growth assays showed the compounds to decrease SAP2-dependent growth while not affecting SAP2-independent growth.

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In vitro characterization of nonpeptide irreversible inhibitors of HIV proteases.

Cited by 36 publications

References 34 publications

Theoretical study on the mechanism of a ring-opening reaction of oxirane by the active-site aspartic dyad of HIV-1 protease

Theoretical study on the mechanism of a ring-opening reaction of oxirane by the active-site aspartic dyad of HIV-1 protease

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

Cis‐Configured Aziridines Are New Pseudo‐Irreversible Dual‐Mode Inhibitors of Candida albicans Secreted Aspartic Protease 2

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