No abstract

Bonding evolution theory (BET), as a combination of the electron localization function (ELF) and Thom's catastrophe theory (CT), has been coupled with quantum mechanics/molecular mechanics (QM/MM) method in order to study biochemical reaction paths. The evolution of the bond breaking/forming processes and electron pair rearrangements in an inhomogeneous dynamic environment provided by the enzyme has been elucidated. The proposed methodology is applied in an enzymatic system in order to clarify the reaction mechanism for the hydrogen abstraction of the androstenedione (ASD) substrate catalyzed by the cytochrome P450 aromatase enzyme. The use of a QM/MM Hamiltonian allows inclusion of the polarization of the charges derived from the amino acid residues in the wave function, providing a more accurate and realistic description of the chemical process. The hydrogen abstraction step is found to have five different ELF structural stability domains, whereas the C−H breaking and O−H forming bond process rearrangements are taking place in an asynchronous way.

CYP19A1 aromatase is a member of the Cytochrome P450 family of hemeproteins, and is the enzyme responsible for the final step of the androgens conversion into the corresponding estrogens, via a three‐step oxidative process. For this reason, the inhibition of this enzyme plays an important role in the treatment of hormone‐dependent breast cancer. The first catalytic subcycle, corresponding to the hydroxilation of androstenedione, has been proposed to occur through a first hydrogen abstraction and a subsequent oxygen rebound step. In present work, we have studied the mechanism of the first catalytic subcycle by means of hybrid quantum mechanics/molecular mechanics methods. The inclusion of the protein flexibility has been achieved by means of Free Energy Perturbation techniques, giving rise to a free energy of activation for the hydrogen abstraction step of 13.5 kcal/mol. The subsequent oxygen rebound step, characterized by a small free energy barrier (1.5 kcal/mol), leads to the hydroxylated products through a highly exergonic reaction. In addition, an analysis of the primary deuterium kinetic isotopic effects, calculated for the hydrogen abstraction step, reveals values (∼10) overpassing the semiclassical limit for the CH, indicating the presence of a substantial tunnel effect. Finally, a decomposition analysis of the interaction energy for the substrate and cofactor in the active site is also discussed. According to our results, the role of the enzymatic environment consists of a transition state stabilization by means of dispersive and polarization effects. © 2015 Wiley Periodicals, Inc.

Invasive lobular breast carcinoma (ILC) is characterized by proliferative indolence and long-term latency relapses. This study aimed to identify how disseminating ILC cells control the balance between quiescence and cell cycle re-entry. In the absence of anchorage, ILC cells undergo a sustained cell cycle arrest in G0/G1 while maintaining viability. From the genes that are upregulated in anchorage independent ILC cells, we selected Inhibitor of DNA binding 2 (Id2), a mediator of cell cycle progression. Using loss-of-function experiments, we demonstrate that Id2 is essential for anchorage independent survival (anoikis resistance) in vitro and lung colonization in mice. Importantly, we find that under anchorage independent conditions, E-cadherin loss promotes expression of Id2 in multiple mouse and (organotypic) human models of ILC, an event that is caused by a direct p120-catenin/Kaiso-dependent transcriptional de-repression of the canonical Kaiso binding sequence TCCTGCNA. Conversely, stable inducible restoration of E-cadherin expression in the ILC cell line SUM44PE inhibits Id2 expression and anoikis resistance. We show evidence that Id2 accumulates in the cytosol, where it induces a sustained and CDK4/6-dependent G0/G1 cell cycle arrest through interaction with hypo-phosphorylated Rb. Finally, we find that Id2 is indeed enriched in ILC when compared to other breast cancers, and confirm cytosolic Id2 protein expression in primary ILC samples. In sum, we have linked mutational inactivation of E-cadherin to direct inhibition of cell cycle progression. Our work indicates that loss of E-cadherin and subsequent expression of Id2 drive indolence and dissemination of ILC. As such, E-cadherin and Id2 are promising candidates to stratify low and intermediate grade invasive breast cancers for the use of clinical cell cycle intervention drugs.

Human aromatase (CYP19A1) aromatizes the androgens to form estrogens via a three-step oxidative process.The estrogens are necessary in humans, mainly in women, because of the role they play in the sexual and reproductive development. However, these also are involved in the development and growth of hormonedependent breast cancer. Therefore, inhibition of the enzyme aromatase, by means of drugs known as aromatase inhibitors, is the frontline therapy for these types of cancers. Exemestane is a suicidal third-generation inhibitor of aromatase, currently used in the breast cancer treatment. In this study, the hydroxylation of exemestane catalyzed by aromatase has been studied by means of hybrid QM/MM methods. The Free Energy Perturbation calculations provided a free energy of activation for the hydrogen abstraction step (rate-limiting step) of 17 kcal/mol. The results reveal that the hydroxylation of exemestane is not the inhibition stage suggesting a possible competitive mechanism between the inhibitor and the natural substrate androstenedione in the first catalytic subcycle of the enzyme. Furthermore, the analysis of the interaction energy for the substrate and the cofactor in the active site, shows that the role of the enzymatic environment during this reaction consists of a transition state stabilization by means of electrostatic effects.3

The electronic structure of iron‐oxo porphyrin π‐cation radical complex Por·+FeIVO (SH) has been studied for doublet and quartet electronic states by means of two methods of the quantum chemical topology analysis: electron localization function (ELF) η(r) and electron density ρ(r). The formation of this complex leads to essential perturbation of the topological structure of the carbon–carbon bonds in porphyrin moiety. The double CC bonds in the pyrrole anion subunits, represented by pair of bonding disynaptic basins Vi=1,2(C,C) in isolated porphyrin, are replaced by single attractor V(C,C)i=1–20 after complexation with the Fe cation. The iron–nitrogen bonds are covalent dative bonds, N→Fe, described by the disynaptic bonding basins V(Fe,N)i=1–4, where electron density is almost formed by the lone pairs of the N atoms. The nature of the iron–oxygen bond predicted by the ELF topological analysis, shows a main contribution of the electrostatic interaction, Feδ+···Oδ−, as long as no attractors between the C(Fe) and C(O) core basins were found, although there are common surfaces between the iron and oxygen basines and coupling between iron and oxygen lone pairs, that could be interpreted as a charge‐shift bond. The FeS bond, characterized by the disynaptic bonding basin V(Fe,S), is partially a dative bond with the lone pair donated from sulfur atom. The change of electronic state from the doublet (M = 2) to quartet (M = 4) leads to reorganization of spin polarization, which is observed only for the porphyrin skeleton (−0.43e to 0.50e) and SH bond (−0.55e to 0.52e). © 2012 Wiley Periodicals, Inc.

No abstract

No abstract