Evolutionary interplay between sister cytochrome P450 genes shapes plasticity in plant metabolism

International audienceExpansion of the cytochrome P450 gene family is often proposed to have a critical role in the evolution of metabolic complexity, in particular in microorganisms, insects and plants. However, the molecular mechanisms underlying the evolution of this complexity are poorly understood. Here we describe the evolutionary history of a plant P450 retrogene, which emerged and underwent fixation in the common ancestor of Brassicales, before undergoing tandem duplication in the ancestor of Brassicaceae. Duplication leads first to gain of dual functions in one of the copies. Both sister genes are retained through subsequent speciation but eventually return to a single copy in two of three diverging lineages. In the lineage in which both copies are maintained, the ancestral functions are split between paralogs and a novel function arises in the copy under relaxed selection. Our work illustrates how retrotransposition and gene duplication can favour the emergence of novel metabolic functions

Structure-function and expression studies of mouse Cyp4f14 and Cyp4f39 and analysis of odor leakage from human remains containment units for use in space

The availability of both mouse and human genome sequences along with the fact that mouse is a good model organism to study mammals, especially humans, has prompted comparisons between the two species at multiple levels. Cytochrome P450 4F is a relatively new CYP subfamily that has gained a great deal of attention due to its involvement in inflammation and lung cancer. Mouse Cyp4f14 and Cyp4f39 show significant sequence similarity to human CYP4F2, CYP4F3 and CYP4F22 and are involved in limiting neuroinflammation and autosomal recessive congenital ichthyosis, respectively. Nevertheless, there are not many studies done on the structure and substrate binding by these enzymes. The first part of this thesis focuses on the mouse cytochrome P450 4F subfamily enzymes Cyp4f14 and Cyp4f39, their structure, substrate binding and expression by using a combination of experimental and computational chemistry approaches. Here we report initial expression studies of Cyp4f14 that were carried out by subcloning mouse Cyp4f14 (from Origene #MR208397) into pEx-N-His-GST vector and optimizing expression in E. coli as a function of OD600 at different isopropyl β-D-1-thiogalactopyranoside (IPTG) levels. Based on p-nitrocarboxylic acid (pNCA) oxidation assays and spectroscopy carried out with crude enzyme, induction at an OD600 of 0.6 with 0.5-0.8 mM of IPTG seems to be optimal for expression in the presence of cytochrome P450 reductase. Analysis of sequence alignments and preliminary work with homology models yielded insights into how mouse Cyp4f14 interacts with its substrate. Molecular docking studies revealed the possible binding sites and modes for substrates such as leukotriene B4, 8-pNCA, 11-pNCA and arachidonic acid. It is hoped that these results will provide insights into enzyme-substrate interactions and form a basis for future research. This study also reports a detailed structural analysis of mouse Cyp4f39 which is the ortholog of human CYP4F22. Our goal is to provide a strong basis to help future studies of these important enzymes. The second part of this thesis involves testing human remains containment units (HRCUs), or ‘body bags’. Volatile organic compounds emitted by decomposing cadavers were analyzed by polydimethylsiloxane/divinylbenzene solid phase microextraction (PDMS/DVB SPME) fibers and Gas Chromatography/Mass Spectrometry (GC/MS). These HRCUs will be used in the International Space Station (ISS) and potentially in colonies on the moon or Mars to store cadavers in the situation of a death (including return of the cadaver to Earth). The ability of bags containing non-refrigerated cadavers to contain volatiles was tested over time, with particular focus on volatiles causing human odor sensation. Different types of bags were found to have very different breakthrough times for odorous volatiles

Assessing the in vitro efficacy of in silico designed compounds targeting the malarial Qi site of cytochrome bc1

Plasmodium falciparum is the causative agent of the most commonly fatal form of malaria in Africa with annual deaths of more than 300 000. The rapid development and spread of antimalarial drug resistance by the parasite have stimulated research into the development of new drug classes. Target-based drug discovery have been used as a prominent and efficient tool to identify lead drugs. Reports suggest that selectively inhibiting the parasite mitochondrial electron transport could be a potential treatment effective at multi-stages of the parasite life cycle. Inhibitors of cytochrome bc1 (Cyt bc1), an essential inner mitochondrial membrane protein that drives ATP synthesis in the mitochondria are claimed to be lethal to apicomplexan species including Plasmodium. The emergence of resistance to atovaquone, a Cyt bc1 complex Qo site inhibitor, casts doubt over the long-term efficacy of new drugs targeting these mitochondrial proteins. Many aspects of potential drugs must be investigated to assess the suitability of new emerging drugs targeting the mitochondrion. In silico target-based drug design methods using Autodock vina were used to design compounds that would theoretically bind to and inhibit the Qi site of Cyt bc1 of the P. falciparum. The potential candidate compounds were selected from compounds defined by Gamo et al., (2010) and tested using in silico docking experiments. Homology models were developed and modified to improve their drug-likeness according to the Lipinski rule, QED parameters and synthesised by Wuxi App Tec. This study assessed the antiproliferative activity of six candidate compounds on P. falciparum parasites in vitro following in silico compound docking and drug likeness assessment. Initial in vitro screening data was obtained for the test compounds at 1 and 5 ?M over 96 h and full dose-response curves was performed for compounds showing >70% proliferation inhibition at 1 ?M against the 3D7 strain. Four of the test compounds, EE1, EE3, EE5 and EE7 gave IC50 values of 89 nM, 664 nM, 64 nM and 249 nM, respectively. The candidate compounds had a marginal >2-fold selectivity towards malaria parasites but did not show cross resistance, with resistance indices of >120. In conclusion, in silico docking using software programs could be utilised as a potential tool for rapidly identifying feasible target-based antimalarial compounds while avoiding high throughput screening. Other possible target sites on the mitochondrion can be used to design new chemotypes. All the designed compounds showed significant antimalarial activity against the asexual stages tested on 3D7 strain with a significant resistance index. However, these compounds showed minimal activity on the gametocyte stage. Finally, compound EE5 showed to be the most potent, more selective and with higher resistance index, hence this can be further optimised for preclinical studies.Dissertation (MSc)--University of Pretoria, 2018.PharmacologyMS

Development and Application of Pseudoreceptor Modeling

Quantitative Structure-Activity Relationship (QSAR) methods are a commonly used tool in the drug discovery process. These methods attempt to form a statistical model that relates descriptor properties of a ligand to the activity of that ligand compound towards a specific desired physiological response. QSAR methods are known as a ligand-based method, as they specifically use information from ligands and not protein structural data. However, a derivation of QSAR methods are pseudoreceptor methods. Pseudoreceptor methods go beyond standard QSAR by building a model representation of the protein pocket. However, the ability of pseudoreceptors to accurately replicate natural protein surfaces has not been studied. The goal of this thesis work is to investigate the necessary descriptors to map a protein binding pocket and a method to accurately recreate the 3-D spatial structure of the binding pocket. In addition, additional applications of existing pseudoreceptor methods are explored

Computational prediction of metabolism: sites, products, SAR, P450 enzyme dynamics, and mechanisms.

Metabolism of xenobiotics remains a central challenge for the discovery and development of drugs, cosmetics, nutritional supplements, and agrochemicals. Metabolic transformations are frequently related to the incidence of toxic effects that may result from the emergence of reactive species, the systemic accumulation of metabolites, or by induction of metabolic pathways. Experimental investigation of the metabolism of small organic molecules is particularly resource demanding; hence, computational methods are of considerable interest to complement experimental approaches. This review provides a broad overview of structure- and ligand-based computational methods for the prediction of xenobiotic metabolism. Current computational approaches to address xenobiotic metabolism are discussed from three major perspectives: (i) prediction of sites of metabolism (SOMs), (ii) elucidation of potential metabolites and their chemical structures, and (iii) prediction of direct and indirect effects of xenobiotics on metabolizing enzymes, where the focus is on the cytochrome P450 (CYP) superfamily of enzymes, the cardinal xenobiotics metabolizing enzymes. For each of these domains, a variety of approaches and their applications are systematically reviewed, including expert systems, data mining approaches, quantitative structure-activity relationships (QSARs), and machine learning-based methods, pharmacophore-based algorithms, shape-focused techniques, molecular interaction fields (MIFs), reactivity-focused techniques, protein-ligand docking, molecular dynamics (MD) simulations, and combinations of methods. Predictive metabolism is a developing area, and there is still enormous potential for improvement. However, it is clear that the combination of rapidly increasing amounts of available ligand- and structure-related experimental data (in particular, quantitative data) with novel and diverse simulation and modeling approaches is accelerating the development of effective tools for prediction of in vivo metabolism, which is reflected by the diverse and comprehensive data sources and methods for metabolism prediction reviewed here. This review attempts to survey the range and scope of computational methods applied to metabolism prediction and also to compare and contrast their applicability and performance.JK, MJW, JT, PJB, AB and RCG thank Unilever for funding

Computer modeling of dapsone-mediated heteroactivation of flurbiprofen metabolism by CYP2C9

The occurrence of atypical kinetics in cytochrome P450 reactions can confound in vitro determinations of a drug\u27s kinetic parameters. During drug development, inaccurate kinetic parameter estimates can lead to incorrect decisions about a lead compound\u27s potential for success. It has become widely accepted that in certain CYP subfamilies more than one molecule can occupy the active site simultaneously, in some cases resulting in enhanced substrate turnover (heteroactivation). However, the specific mechanism(s) by which dual-compound binding results in heteroactivation remain unclear. It is known that orientation of the substrate in the active site, as dictated by interactions with active site residues, plays a large role in metabolic outcome. Effector compounds have been shown in vitro to alter substrate position in the active site. Here, data obtained via in silico methods including docking, molecular dynamics, semi-empirical and ab initio quantum mechanics indicate that direct interaction between effector and substrate can play a role in stabilizing the substrate in an alternative conformation conducive to oxidation. In this study a high-throughput screening computer model of heteroactivation of flurbiprofen metabolism by CYP2C9 has been developed for the purpose of elucidating key interactions between substrate, effector, and enzyme responsible for heteroactivation in this system, as well as to predict as yet unknown activators

Deciphering The Tetrad Of Epigenetic Cytosine Modifications

A tetrad of epigenetic cytosine modifications imbues the DNA code with complex, dynamic meaning. DNA methyltransferase enzymes deposit methyl marks on the 5-carbon of cytosine, forming 5-methylcytosine (mC), which generally mediates long-term, locus-specific transcriptional repression during development and reprogramming. Ten-eleven translocation (TET) family enzymes oxidize the methyl group in three steps, forming predominantly 5-hydroxymethylcytosine (hmC) but also low levels of 5-formylcytosine (fC) and 5-carboxylcytosine (caC). These additional bases likely provide pathways for erasing methylation, but they may also harbor epigenetic functions in their own right. Questions regarding how each base forms and functions drive at the fundamental biology of the epigenome. In this thesis, I chronicle our lab’s efforts to probe the epigenome at its source—by deciphering and manipulating TET enzyme mechanisms. In particular, we aim to understand how and why TET enzymes generate rare fC and caC bases rather than hmC alone. Following a review of the field, I describe in Chapter 2 the methods that we developed to study rare cytosine modifications with high sensitivity. In Chapter 3, we applied these techniques to a rigorous kinetic study of how mouse Tet2 establishes and maintains oxidized cytosines. We found that Tet2 is capable of iterative oxidation, staying on a DNA strand to catalyze multiple rounds of oxidation and thereby enabling efficient generation of fC and caC under certain circumstances. In Chapter 4, we asked what structure-function determinants could allow for the generation of fC and caC. We discovered a conserved active site scaffold in human TET2 that specifically supports the formation of all three oxidized bases, not just hmC. By mutating the active site, we could alter the interactions between key residues to achieve stalling of oxidation at hmC. These mutants have now paved the way for applications in model systems to examine the function of hmC independently of fC and caC, which will allow us to dissect whether the rare, highly oxidized bases are truly critical for epigenetic processes. I describe our progress to date in Chapter 5, along with further mechanistic explorations of the dynamic epigenome

Pharmacogenetic modeling of human cytochrome P450 2D6; On the force of variation in inducing toxicity

Understanding the way in which drugs are metabolized by 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 research described in this PhD thesis has been performed to provide novel fundamental insights regarding the metabolic activity of CYP2D6 wild-type and several polymorphs using various state-of-the-art in silico techniques. The results of the CYP2D6-focused studies enhance our knowledge regarding the enzyme particularities, and can be used to accelerate the development of CYP2D6 modeling tools with more accurate and reliable predictions

Etude biochimique d'un cytochrome P450 de cerveau humain (le CYP2U1)

Parmi les 57 cytochromes P450 identifiés lors du séquençage complet du génome humain, on en dénombre environ 15 dont on ne connaît pratiquement rien de leurs rôles physiologiques, de leurs substrats, et de leurs structures, d où le nom de P450 orphelins . Le CYP2U1 est l un des cytochromes P450 les plus fortement exprimé au niveau du cerveau et du cervelet mais c est aussi l un des plus conservé parmi les différentes espèces du règne animal. Ce travail de thèse a tout d abord consisté à optimiser les conditions d expression du CYP2U1 sous une forme active. Un premier système d expression dans la levure Saccharomyces Cerevisiae a permis une production d un complexe CYP2U1-P450 réductase catalytiquement actif permettant des études de recherche de substrat. Un second système d expression dans Escherichia Coli devrait permettre d obtenir de plus grandes quantités d enzyme soluble destinée à des études structurales. Dans un second temps, une recherche de substrats a été effectuée à l aide d analyse d incubats par chromatographie liquide couplée à une détection par spectrométrie de masse. A ce jour, un screening dirigé de plus de soixante-dix molécules, substrats de P450s de la famille 2, a permis d identifier les premiers substrats exogènes du CYP2U1, les analogues de terfénadone et la débrisoquine. D autre part, une étude par modélisation moléculaire de la structure du CYP2U1 a été effectuée. Cette étude montre que le CYP2U1 diffère de tous les autres P450s par la présence d un insert très spécifique dans son domaine N-terminal. Des modèles par homologie basés sur les structures cristallographiques des P450s de la famille 2 ont été construits. Ces modèles ont été validés par dynamique moléculaire et ont permis de proposer un mode d interaction avec la membrane, d identifier la position des canaux d accès ainsi que de déterminer la topologie du site actif. Enfin, un docking des premiers substrats exogènes au sein du site actif du CYP2U1 a permis de confirmer la régioselectivité des hydroxylations catalysées par le CYP2U1.Among the 57 human cytochrome P450 genes that have been identified; substrates, structure and physiologic role of 15 of them is practically unknown. They are called orphan. One of them, CYP2U1 is one of the most expressed cytochrome P450 in the brain and in the cerebellum but also one of the most conserved isoform in the all animal kingdom. This manuscript first describes the optimization of the heterologous expression of an active form of CYP2U1. Expression in a eukaryotic host, yeast Saccharomyces Cerevisiae first allows the production of a catalytic active CYP2U1-P450 reductase complex needed for substrate screening. Another expression system in a prokaryote host Escherichia Coli will allow higher production rate of a truncated and soluble form of the protein which will permit structural studies. Then a directed substrate screening was performed with the liquid chromatography mass spectrometry analysis of CYP2U1 incubations. To date, 70 molecules, CYP2 family substrates, were tested that allow the identification of the two first exogenous CYP2U1 substrates: débrisoquine and terfenadone analogs. A structural study was achieved using a homology tridimensional model of the enzyme. We have found that CYP2U1 is longer than the other human CYPs, with an N-terminal 20 amino acids insertion, located after the helical membrane spanning domain. Structural models were built using six crystallized human CYP2s as templates. Molecular dynamics experiments in membrane suggested a specific interaction with the membrane. The active site topology and the access channels were also determined and a docking of the two first exogenous CYP2U1 substrates was performed in order to confirm the regioselective hydroxylation activities observed in vitro.PARIS5-Bibliotheque electronique (751069902) / SudocPARIS-BIUM-Bib. électronique (751069903) / SudocSudocFranceF