In Silico Pharmacogenetics CYP2D6 Study Focused on the Pharmacovigilance of Herbal Antidepressants

The annual increase in depression worldwide together with an upward trend in the use of alternative medicine as treatment asks for developing reliable safety profiles of herbal based medicine. A considerable risk on adverse reactions exists when herbal remedies are combined with prescription medication. Around 25% of the drugs, including many antidepressants, depend on the activity of CYP2D6 for their metabolism and corresponding efficacy. Therefore, probing CYP2D6 inhibition by the active substances in herbal based medicine within the wild-type enzyme and clinically relevant allelic variants is crucial to avoid toxicity issues. In this in silico study several compounds with herbal origin suggested to have antidepressant activity were analyzed on their CYP2D6 wild-type and CYP2D6*53 inhibition potential using molecular docking. In addition, several pharmacokinetic properties were evaluated to assess their probability to cross the blood brain barrier and subsequently reach sufficient brain bioavailability for the modulation of central nervous system targets as well as characteristics which may hint toward potential safety issues

Deciphering Reaction Determinants of Altered-Activity CYP2D6 Variants by Well-Tempered Metadynamics Simulation and QM/MM Calculations

The xenobiotic metabolizing enzyme CYP2D6 is the P450 cytochrome family member with the highest rate of polymorphism. This causes changes in the enzyme activity and specificity, which can ultimately lead to adverse reactions during drug treatment. To avoid or lower CYP-related toxicity risks, prediction of the most likely positions within a molecule where a metabolic reaction might occur is paramount. In order to obtain accurate predictions, it is crucial to understand all phenomena within the active site of the enzyme that contribute to an efficient substrate recognition and the subsequent catalytic reaction together with their relative weight within the overall thermodynamic context. This study aims to define the weight of the driving forces upon the C-H bond activation within CYP2D6 wild-type and a clinically relevant allelic variant with increased activity (; CYP2D6*53; ) featuring two amino acid mutations in close vicinity of the heme. First, we investigated the steric and electrostatic complementarity of the substrate bufuralol using well-tempered metadynamics simulations with the aim to obtain the free energy profiles for each site of metabolism (SoM) within the different active sites. Second, the stereoelectronic complementarity was determined for each SoM within the two different active-site environments. Relying on the well-tempered metadynamics simulation energy profiles of each SoM, we identified the binding mode that was closest to the preferred transition-state geometry for efficient C-H bond activation. The binding modes were then used as starting structures for the quantum mechanics/molecular mechanics calculations performed to quantify the corresponding activation barriers. Our results show the relevance of the steric component in orienting the SoM in an energetically accessible position toward the heme. However, the corresponding intrinsic reactivity and electronic complementarity within the active site must be accurately evaluated in order to obtain a meaningful reaction prediction, from which the predominant SoM can be determined. The F120I mutation lowered the activation barrier for the major site and one of the minor SoMs. However, it had an impact neither on the CYP2D6 enantioselectivity preference of the oxidation reaction nor on the stereoselectivity from the substrate point of view

Out-compute drug side effects: Focus on cytochrome P450 2D6 modeling

Understanding the way in which drugs are metabolized by cytochrome P450 2D6 (CYP2D6) and hence the underlying mechanisms that define potential toxicity is crucial to avoid adverse reactions. The high occurrence of CYP2D6 polymorphs enhances the complexity of the toxicity assessment of a drug candidate and should be tackled from early drug discovery phase on. The recent increase in available mammalian CYP2D6 X‐ray structures opens the gateway to the development of in silico three‐dimensional CYP2D6 toxicity prediction techniques that address also the major clinically relevant allelic variants. This review presents the basic principles needed for comprehending the enzyme particularities, gives a concise overview of several clinical relevant allelic CYP2D6 variants, and explores the state‐of‐the‐art CYP2D6 research field to accelerate the development and use of such integrative in silico modeling technologies. This article is categorized under: • Structure and Mechanism > Computational Biochemistry and Biophysics • Structure and Mechanism > Molecular Structures • Software > Molecular Modelin

Molecular Dynamics Simulations Reveal Structural Differences among Allelic Variants of Membrane-Anchored Cytochrome P450 2D6

Cytochrome P450 2D6 (CYP2D6) is an enzyme that is involved in the metabolism of roughly 25% of all marketed drugs and therefore belongs to the most important enzymes in drug metabolism. CYP2D6 features a high degree of genetic polymorphism that can significantly affect the metabolic activity of an individual. In extreme cases, structural changes at the level of single amino acids can either increase its enzymatic activity abolishing the drug therapeutic effect or completely disable the enzyme and elevate drug plasma level potentially leading to adverse effects. In this study, starting from the crystal structure, we built a full-length membrane-anchored all-atom model of the wild-type CYP2D6 as well as five of its variants differing in the enzymatic activity. We validated our models with available experimental data and compared their structural properties with molecular dynamics simulations. The main focus of this study was to identify differences that could mechanistically explain the altered activity of the variants and improve our understanding of their functioning. We observed differences in the opening frequencies and minimal diameters of tunnels that connect the buried active site to the surrounding solvent environment. The variants CYP2D6*4 and CYP2D6*10 associated with missing or decreased activity showed less frequent opening of the tunnels compared to the wild-type. Both CYP2D6*10 and CYP2D6*17 showed a deprivation of an important ligand tunnel suggesting a feasible reason for their altered substrate specificity. Next, the altered fold at the N-terminal anchor region and the decreased active site volume caused by the amino acid mutations of the CYP2D6*4 variant offer an explanation for the absence of its metabolic activity. The mutations in CYP2D6*53 contributed to a significant enlargement of an important ligand tunnel and an extension of the active site cavity. This could explain the altered metabolic profile as well as the enhanced metabolic rates of this particular variant supporting its designation as a possible cause for the ultrarapid metabolizer phenotype. We believe these novel structural insights could advance the fields of personalized medicine and enzyme engineering. Furthermore, they could aid in guiding laboratory as well as computational experiments in the future

Conformational Landscape of Cytochrome P450 Reductase Interactions

Oxidative reactions catalyzed by Cytochrome P450 enzymes (CYPs), which constitute the most relevant group of drug-metabolizing enzymes, are enabled by their redox partner Cytochrome P450 reductase (CPR). Both proteins are anchored to the membrane of the endoplasmic reticulum and the CPR undergoes a conformational change in order to interact with the respective CYP and transfer electrons. Here, we conducted over 22 microseconds of molecular dynamics (MD) simulations in combination with protein-protein docking to investigate the conformational changes necessary for the formation of the CPR-CYP complex. While some structural features of the CPR and the CPR-CYP2D6 complex that we highlighted confirmed previous observations, our simulations revealed additional mechanisms for the conformational transition of the CPR. Unbiased simulations exposed a movement of the whole protein relative to the membrane, potentially to facilitate interactions with its diverse set of redox partners. Further, we present a structural mechanism for the susceptibility of the CPR to different redox states based on the flip of a glycine residue disrupting the local interaction network that maintains inter-domain proximity. Simulations of the CPR-CYP2D6 complex pointed toward an additional interaction surface of the FAD domain and the proximal side of CYP2D6. Altogether, this study provides novel structural insight into the mechanism of CPR-CYP interactions and underlying conformational changes, improving our understanding of this complex machinery Cytochrome P450 reductase; CPR; conformational; dynamicsrelevant for drug metabolism

Large-scale in silico screening of compounds contained in printing inks for food packaging materials

Providing sustainable and safe nutrition to the population is one of the main tasks of the modern food industry. While food packaging materials fulfill their invaluable protective role, certain substances can migrate from them into food. Switzerland is worldwide the only country, in which substances in printing inks for food packaging are regulated. Approx. 5000 substances are listed in Annex 10 of the Ordinance on Food Contact Materials FCM (SR 817.023. 21). Briefly, they were evaluated based on toxicity data and have a specific migration limit (part A). Alternatively, they should not be classified as CMR substances and not be detectable in food (< 10 ppb)(part B). In the order to identify potentially harmful compounds, we performed a virtual screening study along with a complex toxicokinetic characterization of chemical compounds contained in printing inks for FCM

Less is more: Coarse-grained integrative modeling of large biomolecular assemblies with HADDOCK

Predicting the 3D structure of protein interactions remains a challenge in the field of computational structural biology. This is in part due to difficulties in sampling the complex energy landscape of multiple interacting flexible polypeptide chains. Coarse-graining approaches, which reduce the number of degrees of freedom of the system, help address this limitation by smoothing the energy landscape, allowing an easier identification of the global energy minimum. They also accelerate the calculations, allowing to model larger assemblies. Here, we present the implementation of the MARTINI coarse-grained force field for proteins into HADDOCK, our integrative modelling platform. Docking and refinement are performed at the coarse-grained level and the resulting models are then converted back to atomistic resolution through a distance restraints-guided morphing procedure. Our protocol, tested on the largest complexes of the protein docking benchmark 5, shows an overall ~7-fold speed increase compared to standard all-atom calculations, while maintaining a similar accuracy and yielding substantially more near-native solutions. To showcase the potential of our method, we performed simultaneous 7 body docking to model the 1:6 KaiC-KaiB complex, integrating mutagenesis and hydrogen/deuterium exchange data from mass spectrometry with symmetry restraints, and validated the resulting models against a recently published cryo-EM structure

Less Is More: Coarse-Grained Integrative Modeling of Large Biomolecular Assemblies with HADDOCK

Predicting the 3D structure of protein interactions remains a challenge in the field of computational structural biology. This is in part due to difficulties in sampling the complex energy landscape of multiple interacting flexible polypeptide chains. Coarse-graining approaches, which reduce the number of degrees of freedom of the system, help address this limitation by smoothing the energy landscape, allowing an easier identification of the global energy minimum. They also accelerate the calculations, allowing for modeling larger assemblies. Here, we present the implementation of the MARTINI coarse-grained force field for proteins into HADDOCK, our integrative modeling platform. Docking and refinement are performed at the coarse-grained level, and the resulting models are then converted back to atomistic resolution through a distance restraints-guided morphing procedure. Our protocol, tested on the largest complexes of the protein docking benchmark 5, shows an overall ∼7-fold speed increase compared to standard all-atom calculations, while maintaining a similar accuracy and yielding substantially more near-native solutions. To showcase the potential of our method, we performed simultaneous 7 body docking to model the 1:6 KaiC-KaiB complex, integrating mutagenesis and hydrogen/deuterium exchange data from mass spectrometry with symmetry restraints, and validated the resulting models against a recently published cryo-EM structure

Microsecond MD simulations of human CYP2D6 wild-type and five allelic variants reveal mechanistic insights on the function.

Characterization of cytochrome P450 2D6 (CYP2D6) and the impact of the major identified allelic variants on the activity of one of the most dominating drug-metabolising enzymes is essential to increase drug safety and avoid adverse reactions. Microsecond molecular dynamics simulations have been performed to capture the dynamic signatures of this complex enzyme and five allelic variants with diverse enzymatic activity. In addition to the apo simulations, three substrates (bufuralol, veliparib and tamoxifen) and two inhibitors (prinomastat and quinidine) were included to explore their influence on the structure and dynamical features of the enzyme. Our results indicate that the altered enzyme activity can be attributed to changes in the hydrogen bonding network within the active site, and local structural differences in flexibility, position and shape of the binding pocket. In particular, the increased (CYP2D6*53) or the decreased (CYP2D6*17) activity seems to be related to a change in dynamics of mainly the BC loop due to a modified hydrogen bonding network around this region. In addition, the smallest active site volume was found for CYP2D6*4 (no activity). CYP2D6*2 (normal activity) showed no major differences in dynamic behaviour compared to the wild-type