Optimization of Potent and Selective Cyclohexyl Acid ERAP1 Inhibitors Using Structure- and Property-Based Drug Design.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) cleaves the -terminal amino acids of peptides, which can then bind onto major histocompatibility class I (MHC-I) molecules for presentation onto the cell surface, driving the activation of adaptive immune responses. In cancer, overtrimming of mature antigenic peptides can reduce cytotoxic T-cell responses, and ERAP1 can generate self-antigenic peptides which contribute to autoimmune cellular responses. Therefore, modulation of ERAP1 activity has potential therapeutic indications for cancer immunotherapy and in autoimmune disease.

Structure-based design of targeted covalent inhibitors.

Covalent inhibition is a rapidly growing discipline within drug discovery. Many historical covalent inhibitors were discovered by serendipity, with such a mechanism of action often regarded as undesirable due to potential toxicity issues. Recent progress has seen a major shift in this outlook, as covalent inhibition shows promise for targets where previous efforts to identify non-covalent small molecule inhibitors have failed.

Methodology Development in Directed Evolution: Exploring Options when Applying Triple-Code Saturation Mutagenesis.

Directed evolution of stereo- or regioselective enzymes as catalysts in asymmetric transformations is of particular interest in organic synthesis. Upon evolving these biocatalysts, screening is the bottleneck. To beat the numbers problem most effectively, methods and strategies for building "small but smart" mutant libraries have been developed.

Structural and Computational Insight into the Catalytic Mechanism of Limonene Epoxide Hydrolase Mutants in Stereoselective Transformations.

Directed evolution of limonene epoxide hydrolase (LEH), which catalyzes the hydrolytic desymmetrization reactions of cyclopentene oxide and cyclohexene oxide, results in (R,R)- and (S,S)-selective mutants. Their crystal structures combined with extensive theoretical computations shed light on the mechanistic intricacies of this widely used enzyme. From the computed activation energies of various pathways, we discover the underlying stereochemistry for favorable reactions.

Expanding the Armory: Predicting and Tuning Covalent Warhead Reactivity.

Targeted covalent inhibition is an established approach for increasing the potency and selectivity of potential drug candidates, as well as identifying potent and selective tool compounds for target validation studies. It is evident that identification of reversible recognition elements is essential for selective covalent inhibition, but this must also be achieved with the appropriate level of inherent reactivity of the reactive functionality (or "warhead"). Structural changes that increase or decrease warhead reactivity, guided by methods to predict the effect of those changes, have the potential to tune warhead reactivity and negate issues related to potency and/or toxicity.

Correction: Simultaneous engineering of an enzyme's entrance tunnel and active site: the case of monoamine oxidase MAO-N.

[This corrects the article DOI: 10.1039/C6SC05381E.].

Simultaneous engineering of an enzyme's entrance tunnel and active site: the case of monoamine oxidase MAO-N.

A new directed evolution approach is presented to enhance the activity of an enzyme and to manipulate stereoselectivity by focusing iterative saturation mutagenesis (ISM) simultaneously on residues lining the entrance tunnel and the binding pocket. This combined mutagenesis strategy was applied successfully to the monoamine oxidase from (MAO-N) in the reaction of sterically demanding substrates which are of interest in the synthesis of chiral pharmaceuticals based on the benzo-piperidine scaffold. Reversal of enantioselectivity of Turner-type deracemization was achieved in the synthesis of ()-1,2,3,4-tetrahydro-1-methyl-isoquinoline, ()-1,2,3,4-tetrahydro-1-ethylisoquinoline and ()-1,2,3,4-tetrahydro-1-isopropylisoquinoline.

Whole-Cell-Catalyzed Multiple Regio- and Stereoselective Functionalizations in Cascade Reactions Enabled by Directed Evolution.

Biocatalytic cascade reactions using isolated stereoselective enzymes or whole cells in one-pot processes lead to value-added chiral products in a single workup. The concept has been restricted mainly to starting materials and intermediate products that are accepted by the respective wild-type enzymes. In the present study, we exploited directed evolution as a means to create E.

Comparing Different Strategies in Directed Evolution of Enzyme Stereoselectivity: Single- versus Double-Code Saturation Mutagenesis.

Saturation mutagenesis at sites lining the binding pockets of enzymes constitutes a viable protein engineering technique for enhancing or inverting stereoselectivity. Statistical analysis shows that oversampling in the screening step (the bottleneck) increases astronomically as the number of residues in the randomization site increases, which is the reason why reduced amino acid alphabets have been employed, in addition to splitting large sites into smaller ones. Limonene epoxide hydrolase (LEH) has previously served as the experimental platform in these methodological efforts, enabling comparisons between single-code saturation mutagenesis (SCSM) and triple-code saturation mutagenesis (TCSM); these employ either only one or three amino acids, respectively, as building blocks.

Exploring substrate scope and stereoselectivity of P450 peroxygenase OleTJE in olefin-forming oxidative decarboxylation.

As recently reported, the olefin-forming oxidative decarboxylation of straight-chain C4-C22 carboxylic acids catalyzed by P450 peroxygenase OleTJE constitutes a mild alkene synthesis. The present study shows that structurally different carboxylic acids are also accepted, including those that generate di-substituted olefins. Exploratory mutational experiments lead to increased trans-selectivity.

Quantum Mechanics/Molecular Mechanics Modeling of Drug Metabolism: Mexiletine N-Hydroxylation by Cytochrome P450 1A2.

The mechanism of cytochrome P450(CYP)-catalyzed hydroxylation of primary amines is currently unclear and is relevant to drug metabolism; previous small model calculations have suggested two possible mechanisms: direct N-oxidation and H-abstraction/rebound. We have modeled the N-hydroxylation of (R)-mexiletine in CYP1A2 with hybrid quantum mechanics/molecular mechanics (QM/MM) methods, providing a more detailed and realistic model. Multiple reaction barriers have been calculated at the QM(B3LYP-D)/MM(CHARMM27) level for the direct N-oxidation and H-abstraction/rebound mechanisms.

High-level QM/MM calculations support the concerted mechanism for Michael addition and covalent complex formation in thymidylate synthase.

Thymidylate synthase (TS) is a promising cancer target, due to its crucial function in thymine synthesis. It performs the reductive methylation of 2'-deoxyuridine-5'-phosphate (dUMP) to thymidine-5'-phosphate (dTMP), using N-5,10-methylene-5,6,7,8-tetrahydrofolate (mTHF) as a cofactor. After the formation of the dUMP/mTHF/TS noncovalent complex, and subsequent conformational activation, this complex has been proposed to react via nucleophilic attack (Michael addition) by Cys146, followed by methylene-bridge formation to generate the ternary covalent intermediate.

Reduction of α,β-Unsaturated Ketones by Old Yellow Enzymes: Mechanistic Insights from Quantum Mechanics/Molecular Mechanics Calculations.

Enoate reductases catalyze the reduction of activated C═C bonds with high enantioselectivity. The oxidative half-reaction, which involves the addition of a hydride and a proton to opposite faces of the C═C bond, has been studied for the first time by hybrid quantum mechanics/molecular mechanics (QM/MM). The reduction of 2-cyclohexen-1-one by YqjM from Bacillus subtilis was selected as the model system.

Bioorthogonal Enzymatic Activation of Caged Compounds.

Engineered cytochrome P450 monooxygenase variants are reported as highly active and selective catalysts for the bioorthogonal uncaging of propargylic and benzylic ether protected substrates, including uncaging in living E. coli. observed selectivity is supported by induced-fit docking and molecular dynamics simulations.

Large-Scale Density Functional Theory Transition State Searching in Enzymes.

Linear-scaling quantum mechanical density functional theory calculations have been applied to study the rearrangement of chorismate to prephenate in large-scale models of the Bacillus subtilis chorismate mutase enzyme. By treating up to 2000 atoms at a consistent quantum mechanical level of theory, we obtain an unbiased, almost parameter-free description of the transition state geometry and energetics. The activation energy barrier is calculated to be lowered by 10.

Reshaping an enzyme binding pocket for enhanced and inverted stereoselectivity: use of smallest amino acid alphabets in directed evolution.

Directed evolution based on saturation mutagenesis at sites lining the binding pocket is a commonly practiced strategy for enhancing or inverting the stereoselectivity of enzymes for use in organic chemistry or biotechnology. However, as the number of residues in a randomization site increases to five or more, the screening effort for 95 % library coverage increases astronomically until it is no longer feasible. We propose the use of a single amino acid for saturation mutagenesis at superlarge randomization sites comprising 10 or more residues.

In pursuit of an accurate spatial and temporal model of biomolecules at the atomistic level: a perspective on computer simulation.

Despite huge advances in the computational techniques available for simulating biomolecules at the quantum-mechanical, atomistic and coarse-grained levels, there is still a widespread perception amongst the experimental community that these calculations are highly specialist and are not generally applicable by researchers outside the theoretical community. In this article, the successes and limitations of biomolecular simulation and the further developments that are likely in the near future are discussed. A brief overview is also provided of the experimental biophysical methods that are commonly used to probe biomolecular structure and dynamics, and the accuracy of the information that can be obtained from each is compared with that from modelling.

Biocatalytic route to chiral acyloins: P450-catalyzed regio- and enantioselective α-hydroxylation of ketones.

P450-BM3 and mutants of this monooxygenase generated by directed evolution are excellent catalysts for the oxidative α-hydroxylation of ketones with formation of chiral acyloins with high regioselectivity (up to 99%) and enantioselectivity (up to 99% ee). This constitutes a new route to a class of chiral compounds that are useful intermediates in the synthesis of many kinds of biologically active compounds.

Extreme synergistic mutational effects in the directed evolution of a baeyer-villiger monooxygenase as catalyst for asymmetric sulfoxidation.

Structure-based directed evolution utilizing iterative saturation mutagenesis (ISM) has been applied to phenyl acetone monooxygenase (PAMO), a thermally robust Baeyer-Villiger monooxygenase, in the quest to access a mutant which displays reversed enantioselectivity in the asymmetric sulfoxidation of prochiral thioethers. Whereas WT PAMO leads to 90% ee in the sulfoxidation of p-methylbenzyl methyl thioether with preference for the (S)-sulfoxide, the evolved mutant I67Q/P440F/A442N/L443I is 95% (R)-selective in the reaction of this and other thioethers. Partial deconvolution of the (R)-selective mutant with generation of the respective four single mutants shows that all of them are (S)-selective, which points to pronounced synergism (cooperative nonadditivity) when they interact in concert.

CH-activating oxidative hydroxylation of 1-tetralones and related compounds with high regio- and stereoselectivity.

Mutants of P450-BM3 evolved by directed evolution are excellent catalysts in the CH-activating oxidative hydroxylation of 1-tetralone derivatives and of indanone, with unusually high regio- and enantioselectivity being observed. Similar results were achieved in the oxidative hydroxylation of tetralin and indane. The products are useful building blocks in the synthesis of a number of biologically active compounds.

A multiscale approach to modelling drug metabolism by membrane-bound cytochrome P450 enzymes.

Cytochrome P450 enzymes are found in all life forms. P450s play an important role in drug metabolism, and have potential uses as biocatalysts. Human P450s are membrane-bound proteins.

Trends in predicted chemoselectivity of cytochrome P450 oxidation: B3LYP barrier heights for epoxidation and hydroxylation reactions.

Prediction of epoxide formation in drug metabolism is a difficult but important task, as epoxide formation is linked to drug toxicity. A comparison of the energy barriers for cytochrome P450 mediated epoxidation of alkenes to the barriers for the hydroxylation of an aliphatic carbon atom next to a double bond has been performed using B3LYP and B3LYP-D3. Relevant experimental data on oxidation selectivity has also been assessed.

QM/MM modelling of drug-metabolizing enzymes.

Making reliable predictions of drug metabolites requires detailed knowledge of the chemical reactivity of drug metabolizing enzymes. Cytochrome P450 enzymes (P450s) play an important role in drug metabolism. Numerous adverse drug reactions have been identified that occur as a result of interactions with P450s.

Quantum mechanics/molecular mechanics modeling of regioselectivity of drug metabolism in cytochrome P450 2C9.

Cytochrome P450 enzymes (P450s) are important in drug metabolism and have been linked to adverse drug reactions. P450s display broad substrate reactivity, and prediction of metabolites is complex. QM/MM studies of P450 reactivity have provided insight into important details of the reaction mechanisms and have the potential to make predictions of metabolite formation.

Effects of Dispersion in Density Functional Based Quantum Mechanical/Molecular Mechanical Calculations on Cytochrome P450 Catalyzed Reactions.

Density functional theory (DFT) based quantum mechanical/molecular mechanical (QM/MM) calculations have provided valuable insight into the reactivity of the cytochrome P450 family of enzymes (P450s). A failure of commonly used DFT methods, such as B3LYP, is the neglect of dispersion interactions. An empirical dispersion correction has been shown to improve the accuracy of gas phase DFT calculations of P450s.