Main objective is the study of genetic variability in drug 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 in chemical 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 specific disease states. We focus mainly on human cytochrome P450, one of the most important enzyme families in drug metabolism.
Functional studies of human cytochrome P450 enzymes variants
Genetic variants of the major P450 isoforms involved in xenobiotic metabolism are studied to understand the effect of their structural deviations on enzyme activity. These effects are interpreted by protein dynamics and protein:protein interactions, and can assist in the clarification of the function of the facultative protein cytochrome b5 in the enzyme complex.
Palma et al. Pharmacogenetics & Genomics (2013) and Mutation Research (2016)
Functional and Mechanistic studies of human cytochrome P450 reductase (CPR)
1. Protein dynamics of P450 reductase (CPR) in electron-transfer
CPR is a multi-domain, redox-protein which sustains microsomal P450 activity by electron-transfer adapting specific conformations, transitioning between closed (electron loading, NADPH) and open structures (electron-donation, CPR:P450 interaction). The protein dynamics and structural requisites for the open-closed transformation in its gated electron donation are subjects of this research line.
Role of Protein-Protein Interactions in Metabolism: Genetics, Structure, Function. eBook (2018) Front. Pharmacol. Publisher: Frontiers Media. Editors: Pandey A, Henderson CJ, Ishii Y, Kranendonk M, Backes W, Zanger U. (Free download)
2. Rare genetic disease – Antley-Bixler Syndrome (ABS)
Specific forms of ABS are thought to be due to deficient steroidogenesis and have been related to mutations in the human POR gene, encoding the cytochrome P450 reductase (CPR). Functional studies of these ABS-related CPR-proteins and their perturbed activity, allows not only to understand the underlying mechanism of this disease which is clinically relevant but also to gain insight in the molecular requisites of this protein in sustaining microsomal P450s in their activity.
Moutinho et al. (2012) Drug Metabolism and Disposition
3. Degenerared interaction of CPR with its redox partners
The supply of electrons is provided by a single CPR to all microsomal P450s. However, the variant nature of the interface of these P450s implies that the interacting between the two proteins is degenerated: each P450 interacts in an isoform specific manner. The structural features of CPR for this degenerated interaction are the subject of this research-topic.
Campelo et al. (2018) Int J Mol Sci (free access)
Whole Cell bacterial bioreactor, Human biotransformation and Bioactivation
CYP catalysis assessments are routinely made to identify metabolic profile of compounds, potential drug-drug interactions, protein-protein interactions in the CYP enzyme complex and the role of polymorphic enzymes. We developed a bacterial whole-cells high-throughput method for the activity evaluation of human P450s.
Esteves et al. (2018), Biochem. Pharmacol.
Adverse drug reactions (ADRs) are a major complication of drug therapy. The formation of reactive metabolites through biotransformation (bioactivation) is considered to be the major culprit in ADRs. This research-line concerns development of in vitro cell models, competent in human biotransformation, notably human P450, and their application in the detection (e.g. HT-screening) of reactive metabolites, in particular genotoxic or hard electrophilic intermediates.
Palma et al. (2016) Mutat Res.
As PI / Coordinator:
“Domain Dynamics and Control of Electron flux”; Agency Portuguese Foundation for Science and Technology (Fundação para a Ciência e a Tecnologia; FCT-ANR/MHC-CED/0002/2013); Period: 01/05/2014-31/04/2018
“The study of Antley-Bixler Syndrome related mutations of cytochrome P450 oxidoreductase: CYPOR polymorphism and the human cytochrome P450 enzyme complex”; Agency: Fundação para a Ciência e a Tecnologia; PTDC/SAU-GMG/71911/2006); Period: 03/01/2008-02/29/2012
As Integrated Member (2004-2017)
“Molecular and Cellular Effects of Human Mutations in Cytochrome P450 Reductase”; Agency: National Institute of General Medical Sciences; Type: 1-R01 (GM81568-01-04); Period: 04/01/2013-02/31/2017
“Bioactivation routes of the anti-HIV drug Nevirapine: identification of reactive metabolites and mutagenic potential; Agency: Fundação para a Ciência e a Tecnologia; (PTDC/QUI-QUI/113910/2009); Period: 01/02/2011-02/29/2013
“Molecular and Cellular Effects of Human Mutations in Cytochrome P450 Reductase”; Agency: National Institute of General Medical Sciences; Type: 1-R01 (GM81568-01-04); Period: 04/01/2008-03/31/2012.
Esteves F, Urban P, Rueff J, Truan G, Kranendonk M. (2020)
Interaction Modes of Microsomal Cytochrome P450s with Its Reductase and the Role of Substrate Binding.
Int J Mol Sci. 21(18):E6669. [Published 2020 Sep 11]. doi:10.3390/ijms21186669
Esteves F,Campelo D, Gomes BC, et al. (2020)
The Role of the FMN-Domain of Human Cytochrome P450 Oxidoreductase in Its Promiscuous Interactions With Structurally Diverse Redox Partners.
Front Pharmacol. 11:299. [published correction appears in Front Pharmacol. 2020 May 21;11:773].
Rueff J, Rodrigues AS, Kranendonk M (2019)
A personally guided tour on some of our data with the Ames assay-A tribute to Professor Bruce Ames.
Mutat. Res.846:503094; doi: 10.1016/j.mrgentox.2019.503094.
Quast RB, Fatemi F, Kranendonk M, Margeat E,Truan G (2019)
Accurate Determination of Human CPR Conformational Equilibrium by smFRET Using Dual Orthogonal Noncanonical Amino Acid Labeling.
Chembiochem. 20:659-666; doi: 10.1002/cbic.201800607.
Campelo D, Esteves F, Palma BB, Gomes BC, Rueff J, Lautier T, Urban P, Truan G, Kranendonk M (2018)
Probing the Role of the Hinge Segment of Cytochrome P450 Oxidoreductase in the Interaction with Cytochrome P450.
Int. J. Mol. Sci. 19: 3914; doi:10.3390/ijms19123914.
Esteves F, Campelo D, Urban P, Bozonnet S, Lautier T, Rueff J, Truan G, Kranendonk M (2018)
Human cytochrome P450 expression in bacteria: Whole-cell high-throughput activity assay for CYP1A2, 2A6 and 3A4.
Biochem Pharmacol. 158:134-140. doi: 10.1016/j.bcp.2018.10.006.
Role of Protein-Protein Interactions in Metabolism: Genetics, Structure, Function. (2018)
Front. Pharmacol. Publisher: Frontiers Media. Editors: Pandey A, Henderson CJ, Ishii Y, Kranendonk M, Backes W, Zanger U.
E-book, free download.
Pandey A, Henderson CJ, Ishii Y, Kranendonk M, Backes W, Zanger U. (2017)
Editorial: Role of Protein-Protein Interactions in Metabolism: Genetics, Structure, Function.
Front. Pharmacol. 8:811. doi: 10.3389/fphar.2017.00881.
Campelo D, Lautier T, Urban P, Esteves F, Bozonnet S, Truan G, Kranendonk M. (2017)
The hinge segment of human NADPH-cytochrome P450 reductase in conformational switching: the critical role of ionic strength.
Front. Pharmacol. 8:755. doi: 10.3389/fphar.2017.00755.
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 (2016)
Instability of the human cytochrome P450 reductase A287P variant is the major contributor to its Antley-Bixler Syndrome-like phenotype. Journal of Biological Chemistry, 291: 20487-502.
Palma BB, Moutinho D, Urban P, Rueff J, Kranendonk M. (2016)
Cytochrome P450 expression system for high-throughput real-time detection of genotoxicity: Application to the study of human CYP1A2 variants.
Mutation Research, 806: 24-33.
B. Brito Palma, CW. Fisher, J. Rueff, M. Kranendonk (2016)
Prototype Systems Containing Human Cytochrome P450 for High-Throughput Real-Time Detection of DNA Damage by Compounds That Form DNA-Reactive Metabolites.
Chemical Research in Toxicology, 29: 747-56.
Kranendonk, M. Alves, M., Antune, P. and Rueff, J. (2014)
Human sulfotransferase 1A1-dependent mutagenicity of 12-hydroxy-nevirapine: the missing link?
Chemical Research in Toxicology, 27:1967-71.
Palma, B.B., Silva e Sousa, M. Urban, P. Rueff,J. Kranendonk M. (2013)
Functional characterization of eight human CYP1A2 variants: the role of cytochrome b5.
Pharmacogenetics & Genomics, 23:41-52.
D. Moutinho, CC. Marohnic, SP Panda, J Rueff, BS Masters, M Kranendonk. (2012)
Altered human CYP3A4 activity caused by Antley-Bixler syndrome-related variants of NADPH-cytochrome P450 oxidoreductase measured in a robust in vitro system.
Drug Metabolism and Disposition, 40:754-60
• Satya Panda, Linda Roman and Bettie Sue Masters, University of Texas Health Science Center at San Antonio, TX, US
• Gilles Truan and Phillipe Urban, National Institute of Applied Sciences, Toulouse, France
• Jeroen Kool and Nico Vermeulen, Vrije Universiteit Amsterdam, Netherlands