Table1.PDF

<p>The combined quantum mechanical (QM) and molecular mechanical (MM) approach (QM/MM) is a popular method to study reactions in biochemical macromolecules. Even if the general procedure of using QM for a small, but interesting part of the system and MM for the rest is common to all approaches, the details of the implementations vary extensively, especially the treatment of the interface between the two systems. For example, QM/MM can use either additive or subtractive schemes, of which the former is often said to be preferable, although the two schemes are often mixed up with mechanical and electrostatic embedding. In this article, we clarify the similarities and differences of the two approaches. We show that inherently, the two approaches should be identical and in practice require the same sets of parameters. However, the subtractive scheme provides an opportunity to correct errors introduced by the truncation of the QM system, i.e., the link atoms, but such corrections require additional MM parameters for the QM system. We describe and test three types of link-atom correction, viz. for van der Waals, electrostatic, and bonded interactions. The calculations show that electrostatic and bonded link-atom corrections often give rise to problems in the geometries and energies. The van der Waals link-atom corrections are quite small and give results similar to a pure additive QM/MM scheme. Therefore, both approaches can be recommended.</p

Protonation States of Homocitrate and Nearby Residues in Nitrogenase Studied by Computational Methods and Quantum Refinement

Nitrogenase is the only enzyme that can break the triple bond in N₂ to form two molecules of ammonia. The enzyme has been thoroughly studied with both experimental and computational methods, but there is still no consensus regarding the atomic details of the reaction mechanism. In the most common form, the active site is a MoFe₇S₉C­(homocitrate) cluster. The homocitrate ligand contains one alcohol and three carboxylate groups. In water solution, the triply deprotonated form dominates, but because the alcohol (and one of the carboxylate groups) coordinate to the Mo ion, this may change in the enzyme. We have performed a series of computational calculations with molecular dynamics (MD), quantum mechanical (QM) cluster, combined QM and molecular mechanics (QM/MM), QM/MM with Poisson–Boltzmann and surface area solvation, QM/MM thermodynamic cycle perturbations, and quantum refinement methods to settle the most probable protonation state of the homocitrate ligand in nitrogenase. The results quite conclusively point out a triply deprotonated form (net charge −3) with a proton shared between the alcohol and one of the carboxylate groups as the most stable at pH 7. Moreover, we have studied eight ionizable protein residues close to the active site with MD simulations and determined the most likely protonation states

Reaction Mechanism of Cobalt-Substituted Homoprotocatechuate 2,3-Dioxygenase: A QM/MM Study

The reaction mechanisms of cobalt-substituted homoprotocatechuate 2,3-dioxygenase (Co-HPCD) with electron-rich substrate homoprotocatechuate (HPCA) and electron-poor substrate 4-nitrocatechol (4NC) were investigated by quantum mechanical/molecular mechanical (QM/MM) calculations. Our results demonstrated that the Co-O₂ adducts has doublet ground state with a Co^III-O₂^•– character when 4NC was used as the substrate, in good agreement with the EPR spectroscopic experiment. The reactive oxygen species is the doublet Co^III-O₂^•– for Co-HPCD/4NC and the quartet SQ^•↑-Co^II-O₂^•–↓ species for Co-HPCD/HPCA, indicating that the substrate plays important roles in the dioxygen activation by Co-HPCD. B3LYP was found to overestimate the rate-limiting barriers in Co-HPCD. TPSSh predicts barriers of 21.5 versus 12.0 kcal/mol for Co-HPCD/4NC versus Co-HPCD/HPCA, which is consistent with the fact that the rate of the reaction is decreased when the substrate was changed from HPCA to 4NC

Concomitant inhibition of GSK3β and the Wnt/β-catenin pathway in 3MCIC-treated HepG2 cells.

<p><b>(A)</b> Western blotting of GSK3β-Ser9, total GSK3β and β-catenin, respectively. <b>(B)</b> Quantification of 3MCIC-induced changes in the Wnt/β-catenin-GSK3β pathway. Normalized relative band-intensity ratios of pathway component/calnexin in 3MCIC-treated groups over controls were plotted. *P<0.05, **P<0.01 and ***P<0.001 vs control (n = 3).</p

Cell viability assay.

<p>Dose-response curves of five cancer cell lines and the fetal liver L02 cells treated with different concentrations of 3MCIC for 48 h. The bars are means ± SEM (n = 6 or 8).</p

A Hybrid Chalcone Combining the Trimethoxyphenyl and Isatinyl Groups Targets Multiple Oncogenic Proteins and Pathways in Hepatocellular Carcinoma Cells

<div><p>Small molecule inhibitors that can simultaneously inhibit multiple oncogenic proteins in essential pathways are promising therapeutic chemicals for hepatocellular carcinoma (HCC). To combine the anticancer effects of combretastatins, chalcones and isatins, we synthesized a novel hybrid molecule 3’,4’,5’-trimethoxy-5-chloro-isatinylchalcone (3MCIC). 3MCIC inhibited proliferation of cultured HepG2 cells, causing rounding-up of the cells and massive vacuole accumulation in the cytoplasm. Paxillin and focal adhesion plaques were downregulated by 3MCIC. Surprisingly, unlike the microtubule (MT)-targeting agent CA-4 that inhibits tubulin polymerization, 3MCIC stabilized tubulin polymers both in living cells and in cell lysates. 3MCIC treatment reduced cyclin B1, CDK1, p-CDK1/2, and Rb, but increased p53 and p21. Moreover, 3MCIC caused GSK3β degradation by promoting GSK3β-Ser9 phosphorylation. Nevertheless, 3MCIC inhibited the Wnt/β-catenin pathway by downregulating β-catenin, c-Myc, cyclin D1 and E2F1. 3MCIC treatment not only activated the caspase-3-dependent apoptotic pathway, but also caused massive autophagy evidenced by rapid and drastic changes of LC3 and p62. 3MCIC also promoted cleavage and maturation of the lysosomal protease cathepsin D. Using ligand-affinity chromatography (LAC), target proteins captured onto the Sephacryl S1000-C₁₂-3MCIC resins were isolated and analyzed by mass spectrometry (MS). Some of the LAC-MS identified targets, i.e., septin-2, vimentin, pan-cytokeratin, nucleolin, EF1α1/2, EBP1 (PA2G4), cyclin B1 and GSK3β, were further detected by Western blotting. Moreover, both septin-2 and HIF-1α decreased drastically in 3MCIC-treated HepG2 cells. Our data suggest that 3MCIC is a promising anticancer lead compound with novel targeting mechanisms, and also demonstrate the efficiency of LAC-MS based target identification in anticancer drug development.</p></div

Activation of cell death pathways in 3MCIC-treated HepG2 cells.

<p><b>(A)</b> Western blots showing 3MCIC-induced PARP, caspase-3 and vimentin cleavages, CatD maturation, LC3 I & II upregulation, and p62 downregulation. <b>(B)</b> The TUNEL assay after 3MCIC treatment for 12 h. <b>(C)</b> Normalized relative band-intensity ratios of mature/pro-CatD in 3MCIC-treated groups over controls were plotted. <b>(D)</b> Immunofluorescence of HepG2 cells treated with 3MCIC for 3 h, and stained with p62 mAb (green), phalloillin (red) and Hoechst 33342. <b>(E)</b> The early-stage alternations of LC3 under 12 μg/ml 3MCIC treatment. Normalized relative band-intensity ratios of LC3 II/LC3 I in 3MCIC-treated groups over controls were plotted. In <b>C</b> & <b>E</b>, *P<0.05, **P<0.01 and ***P<0.001 vs control (n = 3). <b>(F)</b> LC3 immunofluorescence of HepG2 cells treated with 12 μg/ml 3MCIC at the indicated times. <b>(G)</b> A HepG2 cell was observed either by a light microscope (LM) to show the vacuoles, or by immunofluorescence (IF) with a LC3 mAb, after incubation with 12 μg/ml 3MCIC for 3 h.</p

Influence of tubulin polymerization by 3MCIC.

<p>HepG2 cells were incubated at 37°C with 3MCIC, paclitaxel or colchicine, and assayed with α-tubulin mAb by immunofluorescence <b>(A)</b> and Western blotting <b>(B)</b> after separation of soluble (supernatant, S) and polymerized (pellet, P) tubulins. <b>(C)</b> HepG2 cell lysates were incubated at 25°C for 10 or 30 min with 3MCIC or paclitaxel respectively, and analyzed by Western blotting. In both <b>B</b> & <b>C</b>, normalized changes of the band-intensity ratios of P/S-tubulin in drug-treated groups over controls were plotted. *P<0.05, **P<0.01 and ***P<0.001 vs control (n = 3).</p

Diminish of paxillin and focal adhesion plaques in 3MCIC-treated HepG2 cells.

<p><b>(A)</b> Cells were treated with 3MCIC and assayed by Western blotting. Calnexin was used as loading control. <b>(B)</b> Immunofluorescence of HepG2 cells stained with paxillin mAb (green), phalloidin (red) and Hoechst 33342, after incubation with 8 or 12 μg/ml 3MCIC for 3 h.</p

Changes of cell-cycle control proteins in 3MCIC-treated HepG2 cells.

<p>Cells were treated with 8 or 12 μg/ml 3MCIC at indicated times respectively. Calnexin was used as loading control. <b>(A)</b> Western blotting of cyclins and CDKs. <b>(B)</b> Western blotting of c-Myc, E2F1, Ser567 phosphorylated p-Rb, Rb, p53 and p21.</p