Periodic Reporting for period 1 - metabolicomp (Computational dynamics studies of drug metabolism by P450 enzymes)
Berichtszeitraum: 2017-06-21 bis 2019-06-20
Iron-containing enzymes, such as the cytochrome P450s (mainly found in livers), play key roles in the metabolism of drugs and small-molecules in human body, and also in biotechnology as they have shown efficiency and specificity in substrate functionalization. Understanding metabolism and drug-drug interactions is crucial in the development of safe and efficacious pharmaceutical agents. For instance, ethylcarbamate reacting with P450 mainly results in hydroxylation product; however, in the competing pathway of desaturation, it could lead to an alkene product, which can serve as precursor to epoxide, a carcinogen. Due to the difficulty in capturing reactive intermediates, recent computational developments have helped fill in a more complete picture of P450’s activities and selectivity, focusing on the importance of their various spin states in catalytic roles.
Basic mechanisms of desaturation and hydroxylation of ethylcarbamate by P450 model active site have been studied computationally; however, non-statistical dynamics had not been explored. After elucidating the stationary points along the PES, we carried out dynamics study with ethylcarbamate substrate to examine how the flat potential energy surfaces following C-H abstraction may play a role in product distributions. The aims of the project are to develop an understanding for alcohol/alkene product selectivity for ethylcarbamate using quasi-classical dynamics simulations, and to also understand the difference between heme and non-heme Fe-containing active site in product selectivity. In an extended effort to study reactive intermediates and natural metabolites, we collaborated with Burton group in Oxford to study oxonium ions and their roles in natural product biosyntheses.
Basic mechanisms of desaturation and hydroxylation of ethylcarbamate by P450 model active site have been studied computationally; however, non-statistical dynamics had not been explored. After elucidating the stationary points along the PES, we carried out dynamics study with ethylcarbamate substrate to examine how the flat potential energy surfaces following C-H abstraction may play a role in product distributions. The aims of the project are to develop an understanding for alcohol/alkene product selectivity for ethylcarbamate using quasi-classical dynamics simulations, and to also understand the difference between heme and non-heme Fe-containing active site in product selectivity. In an extended effort to study reactive intermediates and natural metabolites, we collaborated with Burton group in Oxford to study oxonium ions and their roles in natural product biosyntheses.
Firstly, for the reaction of ethylcarbamate in simplified heme active site, we computed all the minima and transition state structures along the potential energy surface with Gaussian09. The potential energy surface was elucidated for both the doublet and quartet spin states of the iron compound. For both spin states, the energy barriers were around 20 kcal/mol, which was in line with previous literature. After the first hydrogen abstraction transition state, the resulting intermediate can undergo either isomerization to attain the productive conformation for either the hydroxylation or desaturation step without having to bypass another transition state – a common feature of a bifurcation. Thus, traditional DFT calculations could not help explaining product distribution by simply comparing which transition state was more energetically feasible, or which product was more thermodynamically stable. Herein, we employed quasi-classical dynamics simulations to understand product distribution. 100 dynamics trajectories were simulated from the first transition state structure. The OH-rebound pathway toward hydroxylation product was found to dominate. This result was in line with previous literature reporting that hydroxylation product was the main product. Our results also suggested that the pathway toward desaturation product was less efficient as it required more time for intermediate reorganization.
To understand the difference between heme and non-heme enzymes, we needed to further examine the reactions catalyzed by non-heme active site. Potential energy surfaces were elucidated for the stepwise N-demethylation reactions for KMe3 and KMe3 methylated lysine with (AcO)2(imidazole)2Fe=O. Transition state structures and corresponding minima were computed for quintet and triplet surfaces, using the same method as above. Quasi-classical dynamics were then simulated for both substrates, starting from the hydrogen abstraction transition state. 100 trajectories were simulated for each system. Comparing the KMe3 and KMe2 system, an additional side product was observed for the KMe2 substrate due to the presence of the NH proton. This prompted reexamination of the potential energy surface with DFT for the formation of this side product. The results are discussed in more details in the upcoming publication.
Following the idea of site selectivity in enzyme active site and how that would affect product distribution, we collaborated with an experimental group in the same department to investigate the role of oxonium ions as reactive intermediates in several natural product biosynthesis. I performed quantum mechanical calculations to help confirm the identity of the oxonium ion intermediates by sampling various conformations of oxonium ions, computing chemical shifts for the ensemble of energetically relevant conformers, and then comparing the computational NMR data to their experimental data. This workflow was repeated for all 5 oxonium ions. Transition state structures (TSSs) were calculated for 1,2-hydride shifts to help understand the decomposition of the reactive oxonium ion. TSSs were also calculated for the reactions of forming oxonium ions and for the reactions between oxonium ions and nucleophiles. Though there are three possible sites on the oxonium ions for nucleophilic attack, the observed product distribution in earlier experimental study suggested that one site was more favorable. Distortion-interaction analyses revealed that reaction at this site involved the least structural reorganization of the substrate in enzyme active site. Manuscript with detailed discussion of these results are under reviews for publication.
To understand the difference between heme and non-heme enzymes, we needed to further examine the reactions catalyzed by non-heme active site. Potential energy surfaces were elucidated for the stepwise N-demethylation reactions for KMe3 and KMe3 methylated lysine with (AcO)2(imidazole)2Fe=O. Transition state structures and corresponding minima were computed for quintet and triplet surfaces, using the same method as above. Quasi-classical dynamics were then simulated for both substrates, starting from the hydrogen abstraction transition state. 100 trajectories were simulated for each system. Comparing the KMe3 and KMe2 system, an additional side product was observed for the KMe2 substrate due to the presence of the NH proton. This prompted reexamination of the potential energy surface with DFT for the formation of this side product. The results are discussed in more details in the upcoming publication.
Following the idea of site selectivity in enzyme active site and how that would affect product distribution, we collaborated with an experimental group in the same department to investigate the role of oxonium ions as reactive intermediates in several natural product biosynthesis. I performed quantum mechanical calculations to help confirm the identity of the oxonium ion intermediates by sampling various conformations of oxonium ions, computing chemical shifts for the ensemble of energetically relevant conformers, and then comparing the computational NMR data to their experimental data. This workflow was repeated for all 5 oxonium ions. Transition state structures (TSSs) were calculated for 1,2-hydride shifts to help understand the decomposition of the reactive oxonium ion. TSSs were also calculated for the reactions of forming oxonium ions and for the reactions between oxonium ions and nucleophiles. Though there are three possible sites on the oxonium ions for nucleophilic attack, the observed product distribution in earlier experimental study suggested that one site was more favorable. Distortion-interaction analyses revealed that reaction at this site involved the least structural reorganization of the substrate in enzyme active site. Manuscript with detailed discussion of these results are under reviews for publication.
In the case of heme and non-heme enzyme, the mechanisms involved in substrate metabolism had not been examined by quasi-classical dynamics until now. The dynamics results collected in this study will help understand small molecule metabolism that the more common DFT method failed to explain, as well as simulate further research to better understanding P450 chemistry and development of safe and effective therapeutics. In the case of oxonium ions, our research finding provided the first direct evidence for the existence of biosynthetically relevant, complex tricyclic oxonium ions and providing insight into the connections between these oxonium ions and a variety of natural products through quenching with nucleophiles. Across all these systems, dynamics and DFT results suggested that enzymes didn’t lower the transition state barriers to favor one product over the other. But rather, product selectivity seemed to be driven by conformation of the preceding intermediate, favoring whichever pathway required the least conformational reorganization. Our work expands the general knowledge of reactive intermediates and will stimulate further studies of these compounds.