Atomistic Insights into the Inhibition of Cysteine Proteases: First QM/MM Calculations Clarifying the Stereoselectivity of Epoxide-Based Inhibitors

Mladenović, Milena; Ansorg, Kay; Fink, Reinhold F.; Thiel, Walter; Schirmeister, Tanja; Engels, Bernd

doi:10.1021/jp803895f

Cited by 42 publications

(55 citation statements)

References 48 publications

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“…The increase of this angle has been shown to correlate with the regioselectivity and reactivity in QM/MM studies of inhibition of cysteine protease by epoxide. 72,73 The regioselectivity for GSH attack is thus mainly distortion-controlled. The preference for C1 over C2 attack is further enhanced, from 4.8 to 8.9 kcal/mol, by including the MM environment, as can be seen from the differences in the barriers computed at the QM and QM/MM levels ( Table 3).…”

Section: Qm Regionmentioning

confidence: 99%

Determinants of Regioselectivity and Chemoselectivity in Fosfomycin Resistance Protein FosA from QM/MM Calculations

Liao

Thiel

2013

J. Phys. Chem. B

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FosA is a manganese-dependent enzyme that utilizes a Mn 2+ ion to catalyze the inactivation of the fosfomycin antibiotic by glutathione (GSH) addition. We report a theoretical study on the catalytic mechanism and the factors governing the regioselectivity and chemoselectivity of FosA. Density functional theory (DFT) calculations on the uncatalyzed reaction give high barriers and almost no regioselectivity even when adding two water molecules to assist the proton transfer. According to quantum mechanics/molecular mechanics (QM/MM) calculations on the full solvated protein, the enzyme-catalyzed glutathione addition reaction involves two major chemical steps that both proceed in the sextet state: proton transfer from the GSH thiol group to the Tyr39 anion and nucleophilic attack by the GSH thiolate leading to epoxide ring-opening. The second step is rate-limiting and is facilitated by the presence of the high-spin Mn 2+ ion that functions as a Lewis acid and stabilizes the leaving oxyanion through direct coordination. The barrier for C1 attack is computed to be 8.9 kcal/mol lower than that for C2 attack, in agreement with the experimentally observed regioselectivity of the enzyme. Further QM/MM calculations on the alternative water attack predict a concerted mechanism for this reaction, where the deprotonation of water, nucleophilic attack, and epoxide ring-opening take place via the same transition state. The calculated barrier is 8.3 kcal/mol higher than that for GSH attack, in line with the observed chemoselectivity of the enzyme, which manages to catalyze the addition of GSH in the presence of water molecules around its active site. The catalytic efficiency, regioselectivity, and chemoselectivity of FosA are rationalized in terms of the influence of the active-site protein environment and the different stabilization of the distorted substrates in the relevant transition states.

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Section: Qm Regionmentioning

confidence: 99%

Determinants of Regioselectivity and Chemoselectivity in Fosfomycin Resistance Protein FosA from QM/MM Calculations

Liao

Thiel

2013

J. Phys. Chem. B

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“…Several groups have studied the catalytic mechanism of cysteine protease with computational techniques, including quantum mechanics/molecular mechanics (QM/MM) (Helten et al, 2004; Ma et al, 2007; Mladenovic et al, 2008a,b,c) and density functional theory (DFT) model complexes and focused on cathepsin B proteases (Shokhen et al, 2009; Shankar et al, 2010). These studies implicated strong influences of the hydrogen bonding network in the substrate/inhibitor binding pocket and led to increased stability of the HisH + —Cys − ion pair as compared to its neutral form, although this may change after substrate binding.…”

Section: Introductionmentioning

confidence: 99%

Cysteine protease inhibition by nitrile-based inhibitors: a computational study

2013

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Cysteine protease enzymes are important for human physiology and catalyze key protein degradation pathways. These enzymes react via a nucleophilic reaction mechanism that involves a cysteine residue and the proton of a proximal histidine. Particularly efficient inhibitors of these enzymes are nitrile-based, however, the details of the catalytic reaction mechanism currently are poorly understood. To gain further insight into the inhibition of these molecules, we have performed a combined density functional theory and quantum mechanics/molecular mechanics study on the reaction of a nitrile-based inhibitor with the enzyme active site amino acids. We show here that small perturbations to the inhibitor structure can have dramatic effects on the catalysis and inhibition processes. Thus, we investigated a range of inhibitor templates and show that specific structural changes reduce the inhibitory efficiency by several orders of magnitude. Moreover, as the reaction takes place on a polar surface, we find strong differences between the DFT and QM/MM calculated energetics. In particular, the DFT model led to dramatic distortions from the starting structure and the convergence to a structure that would not fit the enzyme active site. In the subsequent QM/MM study we investigated the use of mechanical vs. electronic embedding on the kinetics, thermodynamics and geometries along the reaction mechanism. We find minor effects on the kinetics of the reaction but large geometric and thermodynamics differences as a result of inclusion of electronic embedding corrections. The work here highlights the importance of model choice in the investigation of this biochemical reaction mechanism.

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“…In two instances, 21a-b and 22a-b, the importance of the S , S epoxide stereochemistry was confirmed. 28, 29 …”

Section: Resultsmentioning

confidence: 99%

Design, Synthesis, and Optimization of Novel Epoxide Incorporating Peptidomimetics as Selective Calpain Inhibitors

et al. 2013

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Hyperactivation of the calcium-dependent cysteine protease, calpain-1 (Cal1), is implicated as a primary or secondary pathological event in a wide range of illnesses, and in neurodegenerative states, including Alzheimer’s disease (AD). E-64 is an epoxide-containing natural product identified as a potent non-selective, calpain inhibitor, with demonstrated efficacy in animal models of AD. Using E-64 as a lead, three successive generations of calpain inhibitors were developed using computationally assisted design to increase selectivity for Cal1. First generation analogs were potent inhibitors, effecting covalent modification of recombinant Cal1 catalytic domain (Cal1cat), demonstrated using LC-MS/MS. Refinement yielded 2nd generation inhibitors with improved selectivity. Further library expansion and ligand refinement gave three Cal1 inhibitors, one of which was designed as an activity-based protein profiling probe. These were determined to be irreversible and selective inhibitors by kinetic studies comparing full length Cal1 with the general cysteine protease, papain.

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Atomistic Insights into the Inhibition of Cysteine Proteases: First QM/MM Calculations Clarifying the Stereoselectivity of Epoxide-Based Inhibitors

Cited by 42 publications

References 48 publications

Determinants of Regioselectivity and Chemoselectivity in Fosfomycin Resistance Protein FosA from QM/MM Calculations

Determinants of Regioselectivity and Chemoselectivity in Fosfomycin Resistance Protein FosA from QM/MM Calculations

Cysteine protease inhibition by nitrile-based inhibitors: a computational study

Design, Synthesis, and Optimization of Novel Epoxide Incorporating Peptidomimetics as Selective Calpain Inhibitors

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