Boxue Tian

Design of Reversible, Cysteine-Targeted Michael Acceptors Guided by Kinetic and Computational Analysis

Electrophilic probes that covalently modify a cysteine thiol often show enhanced pharmacological potency and selectivity. Although reversible Michael acceptors have been reported, the structural requirements for reversibility are poorly understood. Here, we report a novel class of acrylonitrile-based Michael acceptors, activated by aryl or heteroaryl electron-withdrawing groups. We demonstrate that thiol adducts of these acrylonitriles undergo β-elimination at rates that span more than 3 orders of magnitude. These rates correlate inversely with the computed proton affinity of the corresponding carbanions, enabling the intrinsic reversibility of the thiol-Michael reaction to be tuned in a predictable manner. We apply these principles to the design of new reversible covalent kinase inhibitors with improved properties. A cocrystal structure of one such inhibitor reveals specific noncovalent interactions between the 1,2,4-triazole activating group and the kinase. Our experimental and computational study enables the design of new Michael acceptors, expanding the palette of reversible, cysteine-targeted electrophiles.

Can Range-Separated and Hybrid DFT Functionals Predict Low-Lying Excitations? A Tookad Case Study

The spectral properties of Tookad (Pd-bacteriopheophorbide, Pd-BPheid), an effective photosensitizer that targets mainly prostate tumors, and metal-free BPheid have been studied using time-dependent density functional theory (TD-DFT). The well-established B3LYP functional, which is known to overestimate excitation energies, was included in the study along with recently introduced range-separated and meta hybrid DFT functionals CAM-B3LYP, M06, M06-2X, M06HF, ωB97XD, ωB97X, ωB97, LC-ωPBE, and PBE0 (PBE1PBE). The main focus is the performance of the new functionals in predicting low-lying excitations (>600 nm), to explore their potential roles in drug development for photodynamic therapy. The data suggests that ωB97XD overall performs best for the Qy transition band (the red-most absorption), followed by CAM-B3LYP. LC-ωPBE, ωB97, B3LYP, and PBE1PBE all generated the Qy band far from the experimental position. The error in absorption energy for the Qy band was found to be at most 0.05 eV for ωB97XD, compared to 0.15-0.19 eV for B3LYP. The use of different basis sets used in excited-state calculations was shown to be of less importance as was the use of either B3LYP or M06 in geometry optimizations.

Catalytic mechanism and roles of Arg197 and Thr183 in the Staphylococcus aureus sortase A enzyme.

The sortase A enzyme, which catalyzes the peptidoglycan cell wall anchoring reaction of LPXTG surface proteins, has been proposed to be a universal target for therapeutic agents against Gram-positive bacteria. The catalytic mechanism of the Staphylococcus aureus sortase A enzyme has been systematically studied using molecular dynamics simulations, ONIOM(DFT:MM) calculations, and QM/MM charge deletion analysis. The catalytic roles of Arg197 and Thr183 were analyzed. Our calculations show that Arg197 has several important roles in the mechanism. It is crucial for substrate binding, and is capable of reversible shift of its hydrogen bonds between the LP and TG carbonyls of the LPXTG substrate motif, depending on the protonation state of the catalytic Cys184-His120 dyad. Arg197 stabilizes the catalytic dyad in the active ion pair form but at the same time raises the barrier to acylation by approximately 8 kcal/mol. Thr183 is also essential for the catalytic reaction in that it correspondingly lowers the barrier by the same amount via electrostatic interactions. The catalytic mechanism proceeds via proton transfer from His120, followed by nucleophilic attack from the thiolate anion of Cys184. The data thus supports the proposed reverse protonation mechanism, and disproves the hypothesis of the Arg197 generating an oxyanion hole to stabilize the tetrahedral intermediate of the reaction.

Leveraging structure for enzyme function prediction: methods, opportunities, and challenges.

The rapid growth of the number of protein sequences that can be inferred from sequenced genomes presents challenges for function assignment, because only a small fraction (currently <1%) has been experimentally characterized. Bioinformatics tools are commonly used to predict functions of uncharacterized proteins. Recently, there has been significant progress in using protein structures as an additional source of information to infer aspects of enzyme function, which is the focus of this review. Successful application of these approaches has led to the identification of novel metabolites, enzyme activities, and biochemical pathways. We discuss opportunities to elucidate systematically protein domains of unknown function, orphan enzyme activities, dead-end metabolites, and pathways in secondary metabolism.

Catalytic roles of active-site residues in 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase : an ONIOM/DFT study

The catalytic mechanism of 2-methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) oxygenase (MHPCO) has been systematically studied using DFT and ONIOM(DFT:MM) methods. MHPCO catalyzes the hydroxylation and subsequent ring-opening of the aromatic substrate MHPC to give the aliphatic product α-(N-acetylaminomethylene)succinic acid (AAMS). Our calculations show that the active-site residues Arg211 and Tyr223 have a minor effect on the reaction, while the peptide bond of Pro295-Ala296, the side chain of Tyr82 and several crystal water molecules affect the reaction energy profile considerably. Both DFT and ONIOM calculations show that the ring-opening pathway B, in which an epoxy transition state is formed, is more favored than the direct C2-C3 cleavage pathway A. Different QM/MM partitioning schemes have been used to study the enzymatic reaction, and the results show that both the reaction barriers for the hydroxylation and the ring-opening pathways are sensitive to the QM/MM partitioning.

Catalysts or Initiators? Beckmann Rearrangement Revisited

The catalytic mechanism of the organo-mediated Beckmann rearrangement has been modeled using DFT calculations. Five representative promoters were shown to be initiators rather than catalysts. A self-propagating mechanism is shown to be energetically much more favored than the previously proposed mechanisms involving a Meisenheimer complex.

Catalytic mechanism and product specificity of oxidosqualene-lanosterol cyclase: a QM/MM study.

Oxidosqualene-lanosterol cyclase (OSC) is a key enzyme in the biosynthesis of cholesterol. The catalytic mechanism and the product specificity of OSC have herein been studied using QM/MM calculations. According to our calculations, the protonation of the epoxide ring of oxidosqualene is rate-limiting. Wild-type OSC (which generates lanosterol), and the mutants H232S (which generates parkeol) and H232T (which generates protosta-12,24-dien-3-β-ol) were modeled, in order to explain the product specificity thereof. We show that the product specificity of OSC at the hydride/methyl-shifting stage is unlikely to be achieved by the stabilization of the cationic intermediates, as the precursor of lanosterol is in fact not the most stable cationic intermediate for wild-type OSC. The energy barriers for the product-determining conversions are instead found to be related to the product specificity of different OSC mutants, and we thus suggest that the product specificity of OSC is likely to be controlled by kinetics, rather than thermodynamics.

Mechanistic insight into self-propagation of organo-mediated Beckmann rearrangement: a combined experimental and computational study.

Organo-mediated Beckmann rearrangement in the liquid phase, which has the advantage of high efficiency and straightforward experimental procedures, plays an important role in the synthesis of amides from oximes. However, the catalytic mechanisms of these organic-based promoters are still not well understood. In this work, we report a combined experimental and computational study on the mechanism of Beckmann rearrangement mediated by organic-based promoters, using TsCl as an example. A novel self-propagating cycle is proposed, and key intermediates of this self-propagating cycle are confirmed by both experiments and DFT calculations. In addition, the reason why cyclohexanone oxime is not a good substrate of the organo-mediated Beckmann rearrangement is discussed, and a strategy for improving the yield is proposed.

Predicting enzyme-substrate specificity with QM/MM methods: a case study of the stereospecificity of (D)-glucarate dehydratase.

The stereospecificity of d-glucarate dehydratase (GlucD) is explored by QM/MM calculations. Both the substrate binding and the chemical steps of GlucD contribute to substrate specificity. Although the identification of transition states remains computationally intensive, we suggest that QM/MM computations on ground states or intermediates can capture aspects of specificity that cannot be obtained using docking or molecular mechanics methods.

Structural changes of Listeria monocytogenes Sortase A: a key to understanding the catalytic mechanism.

Listeria monocytogenes is one of the most virulent foodborne pathogens. L. monocytogenes Sortase A (SrtA) enzyme, which catalyzes the cell wall anchoring reaction of the leucine, proline, X, threonine, and glycine proteins (LPXTG, where X is any amino acid), is a target for the development of antilisteriosis drugs. In this study, the structure of the L. monocytogenes SrtA enzyme‐substrate complex was obtained using homology modeling, molecular docking and molecular dynamics simulations. Explicit enzyme‐substrate interactions in the inactive and active forms of the enzyme were compared, based on 30ns simulations on each system. The active site arginine (Arg 197) was found to be able change its hydrogen donor interactions from the LP backbone carbonyl groups of the LPXTG substrate in the inactive form, to the TG backbone carbonyls in the active form, which could be of importance for holding the substrate in position for the catalytic process. Proteins 2011;