Metabolismo Xenobiótico

The main objective of our group is the study of genetic variability in drug (xenobiotic) metabolism and its role in susceptibility to therapeutic drugs and environmental agents. This variability is a major determining factor in the beneficial and adverse effects inchemical exposure.

Research concerns functional and mechanistic studies of drug metabolism enzyme variants to obtain insight in molecular mechanism of function of these enzymes, allowing the rationalization of their effect in exposure to agents and in specific disease states. We focus mainly on human cytochrome P450, one of the most important enzyme families in drug metabolism, using various in vitro models, and a pleiotropy of genetic and biochemical techniques.

Graphical Abstract

logo PRO-EURO DILI Network_Michel Kranendonk

  • Prospective European Drug-Induced Liver Injury Network
    The project regards an EU funded Cost Action which addresses a serious health problem in Europe namely drug induced liver injury or DILI. The objectives of the PRO-EURO-DILI-NET Cost Action are to create a unique, co-operative, interdisciplinary European-based DILI network of stakeholders to co-ordinate efforts in DILI, to facilitate bi-directional exchange of discovered knowledge and generated hypotheses among different disciplines, and to promote clinically impactful knowledge discovery and its translation into clinical practice.

This Action will:

    • harmonize efforts for in-depth DILI phenotyping and bio-sample repository and coordinate pre-funded database/repository studies to aggregate a large number of DILI cases in a standardized manner (WG1).
    • Establish a strategy for development, validation and performance of DILI novel biomarkers and explore multifactorial DILI risk modifiers in clinical data sets using novel approaches for future precision medicine (WG2).
    • Facilitate clinically impactful knowledge discovery by introducing biological variations and the complexity (i.e., multi-cellular/multiorgan systems) into toxicological experiments to assess hepatotoxicity to guide future drug safety testing (WG3).
    • Define criteria and establish endpoints to measure efficacy on novel interventions in DILI (WG4).
    • Draft policy recommendations about near-patient testing tools.

The network will promote and coordinate a highly translational and innovative research program in Europe and beyond with the ultimate goal to pre-empt and prevent DILI, develop innovative therapeutic approaches that could improve clinical outcomes and enhance public awareness, while developing a forum for knowledge exchange and training of young European researches.
Michel Kranendonk, the head of the Xenobiotic Metabolism lab, is member of the Management Committee of this Cost Action as well as member of the Workgroup 3.

Website: https://proeurodilinet.eu/

Open-access publication:
ADVANCED PRECLINICAL MODELS FOR EVALUATION OF DRUG INDUCED LIVER INJURY - CONSENSUS STATEMENT BY THE EUROPEAN DRUG-INDUCED LIVER INJURY NETWORK [PRO-EURO-DILI-NET] Checa et al. (2021) J Hepatol https://doi.org/10.1016/j.jhep.2021.06.021

Logo DODYCOEL_Michel Kranendonk

  • Domain Dynamics and Control of Electron flux (DODYCOEL)
    NADPH cytochrome P450 oxidoreductase (CPR) is a diflavin protein which is the obligatory electron donor of CYP and non-CYP enzymes, which are involved in many physiological important processes. CPR, like all diflavin electron transfer (ET) enzymes, is a multidomain protein which undergoes large conformational changes in its ET function. CPR exists in a conformational equilibrium, between a closed (electron acceptor (NADPH) conformation and an open electron donation (interaction with redox partner) conformation. At the start of the project there were indications that both intrinsic (CPR determinants) and extrinsic (membrane environment and redox partners) control CPR’s open-closed dynamics, however several of these critical parameters were not analyzed.

The project had as goal to address these issues by:

    • Characterizing the different conformational states of CPR populated during its interaction with various natural electron acceptors (CYP)
    • Understanding and identifying how CYP interaction controls and modulates the different steps of the CPR enzymatic cycle, ultimately defining a perfect coupling scheme between the two enzymes;
    • Describing the CPR molecular determinants that change the structural organization and conformational equilibrium to formalize how critical structural elements affect CPR function.

The collaborative project between Lisbon and Toulouse (INSA) addressed these issues using both soluble purified (Partner 1, Toulouse) and membrane bound CPR forms (Partner 2 Lisbon).
Several critical residues in a specific segment of the CPR protein, the hinge region could be identified in the open-closed dynamics of CPR, in which ionic strength seems to play a role. This extensive protein dynamics is responsible for the formation of an ensemble of open CPR conformers, by which CPR can interact with so many structurally different redox partners. Moreover, it was demonstrated that each redox partner (i.e. CYPs) interact with CPR via the use of specific biding-motifs of the interaction surface, the FMN domain, in an isoform specific manner. Additionally, we found that the substrate bound by CYP influences this interaction.

Team Members:
Bernardo Brito Palma, Bruno Costa Gomes, Francisco Esteves, Diana Campelo

Esteves F, et al. Interaction Modes of Microsomal Cytochrome P450s with Its Reductase and the Role of Substrate Binding. Int. J. Mol. Sci. 2020 Sep 11;21(18):6669. doi: 10.3390/ijms21186669

Esteves F, etal. The Role of the FMN-Domain of Human Cytochrome P450 Oxidoreductase in Its Promiscuous Interactions with Structurally Diverse Redox Partners. Frontiers Pharmacol. 2020 Mar 18;11:299. doi: 10.3389/fphar.2020.00299

Campelo D, et al. Probing the Role of the Hinge Segment of Cytochrome P450 Oxidoreductase in the Interaction with Cytochrome P450. Int. J. Mol. Sci. 2018 Dec 6;19(12):3914. doi: 10.3390/ijms19123914

Quast RB. et al., Accurate Determination of Human CPR Conformational Equilibrium by smFRET Using Dual Orthogonal Noncanonical Amino Acid Labeling. Chembiochem. 2019 Mar 1;20(5):659-666. doi: 10.1002/cbic.201800607

Esteves F, et al. Human cytochrome P450 expression in bacteria: Whole-cell high-throughput activity assay for CYP1A2, 2A6 and 3A4. Biochem Pharmacol. 2018 Dec;158:134-140. doi: 10.1016/j.bcp.2018.10.006

Campelo D, et al. The Hinge Segment of Human NADPH-Cytochrome P450 Reductase in Conformational Switching: The Critical Role of Ionic Strength. Frontiers Pharmacol. 2017 Oct 30;8:755. doi: 10.3389/fphar.2017.00755

Esteves F, et al. The Central Role of Cytochrome P450 in Xenobiotic Metabolism—A Brief Review on a Fascinating Enzyme Family. J.Xenobiot. 2021, 11, 94–114. https://doi.org/10.3390/jox11030007 

 

foto Dodecyl with text_Michel Kranendonk

 

  • “Prospective European Drug-Induced Liver Injury Network”, COST ACTION 15112 Funding by the EU; period: 16/10/2018- 15/10/2022
  •  “Domain Dynamics and Control of Electron flux"; funding by the Portuguese Foundation for Science and Technology (FCT-ANR/MHC-CED/0002/2013); Period: 01/05/2014-31/04/2018; in collaboration with the INSA Toulouse, France
  • “Domain Dynamics and Control of Electron flux"; funding by the Portuguese Foundation for Science and Technology (FCT-ANR/MHC-CED/0002/2013); Period: 01/05/2014-31/04/2018; in collaboration with the INSA Toulouse, France
    3. “Molecular and Cellular Effects of Human Mutations in Cytochrome P450 Reductase”; Funding by the National Institute of General Medical Sciences US (GM81568-01-04); Period: 04/01/2013-02/31/2017; in a collaboration with the UT Health Science Center at San Antonio (US)
  • “Molecular and Cellular Effects of Human Mutations in Cytochrome P450 Reductase”; Funding by the National Institute of General Medical Sciences US (GM81568-01-04); Period: 04/01/2008-03/31/2012; in a collaboration with the UT Health Science Center at San Antonio (US).
  • “The study of Antley-Bixler Syndrome related mutations of cytochrome P450 oxidoreductase: CYPOR polymorphism and the human cytochrome P450 enzyme complex"; Funding by the Portuguese Foundation for Science and Technology PTDC/SAU-GMG/71911/2006); Period: 03/01/2008-02/29/2012
  • “Bioactivation routes of the anti-HIV drug Nevirapine: identification of reactive metabolites and mutagenic potential; Funding by the Portuguese Foundation for Science and Technology (PTDC/QUI-QUI/113910/2009); Period: 01/02/2011-02/29/2013; in a collaboration with Institute Superior Tecnico, Universidade de Lisboa.

Fernandez-Checa et al., ADVANCED PRECLINICAL MODELS FOR EVALUATION OF DRUG INDUCED LIVER INJURY - CONSENSUS STATEMENT BY THE EUROPEAN DRUG-INDUCED LIVER INJURY NETWORK [PRO-EURO-DILI-NET] J Hepatol. 2021; S0168-8278(21)00441-4. doi: 10.1016/j.jhep.2021.06.021 (open access)

Esteves F, Rueff J, Kranendonk M. The Central Role of Cytochrome P450 in Xenobiotic Metabolism-A Brief Review on a Fascinating Enzyme Family. J. Xenobiot. 2021; 11(3):94-114. doi: 10.3390/jox11030007 (open access)

Silveira, C.M., Rodrigues, P.R., Ghach, W., Pereira, S.A., Esteves, F., Kranendonk, M., Etienne, M., Almeida, M.G. Electrochemical Activity of Cytochrome P450 1A2: The Relevance of O2 Control and the Natural Electron Donor ChemElectroChem 2021, 8, 500–507. doi: 10.1002/celc.202001255

Esteves F, Urban P, Rueff J, Truan G, Kranendonk M. Interaction Modes of Microsomal Cytochrome P450s with Its Reductase and the Role of Substrate Binding. Int. J. Mol. Sci. 2020; 21(18):6669. doi: 10.3390/ijms21186669 (open access)

Esteves F, Campelo D, Gomes BC, Urban P, Bozonnet S, Lautier T, Rueff J, Truan G, Kranendonk M. The Role of the FMN-Domain of Human Cytochrome P450 Oxidoreductase in Its Promiscuous Interactions with Structurally Diverse Redox Partners. Frontiers Pharmacol. 2020; 299. doi: 10.3389/fphar.2020.00299 (open access)

Rueff J, Rodrigues AS, Kranendonk M. A personally guided tour on some of our data with the Ames assay-A tribute to Professor Bruce Ames. Mutat. Res. 2019 Oct;846:503094. doi: 10.1016/j.mrgentox.2019.503094.

Quast RB, Fatemi F, Kranendonk M, Margeat E, Truan G. Accurate Determination of Human CPR Conformational Equilibrium by smFRET Using Dual Orthogonal Noncanonical Amino Acid Labeling. Chembiochem. 2019;20(5):659-666. doi: 10.1002/cbic.201800607.

Esteves F, Campelo D, Urban P, Bozonnet S, Lautier T, Rueff J, Truan G, Kranendonk M. Human cytochrome P450 expression in bacteria: Whole-cell high-throughput activity assay for CYP1A2, 2A6 and 3A4. Biochem. Pharmacol. 2018, 158: 134-140. doi: 10.1016/j.bcp.2018.10.006

Amit V. Pandey, Colin J. Henderson, Yuji Ishii, Michel Kranendonk, Wayne L. Backes, Ulrich M. Zanger, Role of Protein-Protein Interactions in Metabolism: Genetics, Structure, Function. Frontiers Research Topic ebook published in: Frontiers in Pharmacology (2018), by Frontiers Media SA (https://www.frontiersin.org/research-topics/4658/role-of-protein-protein-interactions-in-metabolism-genetics-structure-function) (open access)

Campelo D, Esteves F, Brito Palma B, Costa Gomes B, Rueff J, Lautier T, Urban P, Truan G, Kranendonk M. Probing the Role of the Hinge Segment of Cytochrome P450 Oxidoreductase in the Interaction with Cytochrome P450. Int. J. Mol. Sci. 2018; 19(12):3914. doi: 10.3390/ijms19123914 (open access)

Campelo D, Lautier T, Urban P, Esteves F, Bozonnet S, Truan G, Kranendonk M. The Hinge Segment of Human NADPH-Cytochrome P450 Reductase in Conformational Switching: The Critical Role of Ionic Strength. Frontiers Pharmacol. 2017; 8:755. doi: 10.3389/fphar.2017.00755 (open access)

McCammon KM, Panda SP, Xia C, Kim JJ, Moutinho D, Kranendonk M, Auchus RJ, Lafer EM, Ghosh D, Martasek P, Kar R, Masters BS, Roman LJ. Instability of the Human Cytochrome P450 Reductase A287P Variant Is the Major Contributor to Its Antley-Bixler Syndrome-like Phenotype. J. Biol. Chem. 2016;291(39):20487-502. doi: 10.1074/jbc.M116.716019.

Palma BB, Moutinho D, Urban P, Rueff J, Kranendonk M. Cytochrome P450 expression system for high-throughput real-time detection of genotoxicity: Application to the study of human CYP1A2 variants. Mutat Res Genet Toxicol Environ Mutagen. 2016 806:24-33. doi: 10.1016/j.mrgentox.2016.06.004

Palma BB, Fisher CW, Rueff J, Kranendonk M. Prototype Systems Containing Human Cytochrome P450 for High-Throughput Real-Time Detection of DNA Damage by Compounds That Form DNA-Reactive Metabolites. Chem Res Toxicol. 2016; 29(5):747-56. doi: 10.1021/acs.chemrestox.5b00455.

Kranendonk M, Alves M, Antunes P, Rueff J. Human sulfotransferase 1A1-dependent mutagenicity of 12-hydroxy-nevirapine: the missing link? Chem Res Toxicol. 2014; 27(11):1967-71. doi: 10.1021/tx5003113.

Palma BB, Silva E Sousa M, Urban P, Rueff J, Kranendonk M. Functional characterization of eight human CYP1A2 variants: the role of cytochrome b5. Pharmacogenet Genomics. 2013; 23(2):41-52. doi: 10.1097/FPC.0b013e32835c2ddf

Moutinho D, Marohnic CC, Panda SP, Rueff J, Masters BS, Kranendonk M. Altered human CYP3A4 activity caused by Antley-Bixler syndrome-related variants of NADPH-cytochrome P450 oxidoreductase measured in a robust in vitro system. Drug Metab. Dispos. 2012;40(4):754-60. doi: 10.1124/dmd.111.042820.

Gómez-Bombarelli R, Palma BB, Martins C, Kranendonk M, Rodrigues AS, Calle E, Rueff J, Casado J Alkylating potential of oxetanes. . Chem Res Toxicol. 2010; 23(7):1275-81. doi: 10.1021/tx100153w

Palma BB, Silva E Sousa M, Vosmeer CR, Lastdrager J, Rueff J, Vermeulen NP, Kranendonk M. Functional characterization of eight human cytochrome P450 1A2 gene variants by recombinant protein expression. Pharmacogenomics J. 2010; 10(6):478-88. doi: 10.1038/tpj.2010.2.

Marohnic CC, Panda SP, McCammon K, Rueff J, Masters BS, Kranendonk M. Human cytochrome P450 oxidoreductase deficiency caused by the Y181D mutation: molecular consequences and rescue of defect. Drug Metab Dispos. 2010; 38(2):332-40. doi: 10.1124/dmd.109.030445.

Kranendonk M, Marohnic CC, Panda SP, Duarte MP, Oliveira JS, Masters BS, Rueff J. Impairment of human CYP1A2-mediated xenobiotic metabolism by Antley-Bixler syndrome variants of cytochrome P450 oxidoreductase. Arch Biochem Biophys. 2008; 475(2):93-9. doi: 10.1016/j.abb.2008.04.014.

  • 2007 - Prize for the best proposed project in the program “Promoção de Projectos de Investigação Clínica na Rede de Hospitais Articulados com a NOVA Medical School, Universidade Nova de Lisboa
  • Gilles Truan and Philippe Urban, TBI, Université de Toulouse, CNRS, INRAE, INSA, Toulouse
  • Leonard Nelson, Institute for Bioengineering (IBioE), School of Engineering, Faraday Building, The University of Edinburgh, Edinburgh.
  • César Mendes, CEDOC, Chronic Diseases Research Centre, NOVA Medical School, NOVA, Universidade de Lisboa
  • Gabriela Almeida, UCIBIO, REQUIMTE, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa,
  • Cristina Almeida, Departamento de Ciências Farmacêuticas e do Medicamento, Faculty of Pharmacy, Universidade de Lisboa
  • Satya Panda, Linda Roman and Bettie Sue Masters, University of Texas Health Science Center at San Antonio, TX, US

Investigador Principal

Michel Kranendonk

Equipa

Francisco Esteves
Isabel Barata
MSc student
Inês Saldanha
guest student
Bernardo Brito Palma
Post-doc
Bruno Costa Gomes
Post-doc
Diana Campelo
PhD student
Carolina Ramos
MSc Student
Daniel Crispim
MSc Student
Marisa Cardoso
MSc student
Ana Rita Bernardes
BSc student