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Download the Book of Abstracts for Posters Book of poster abstracts 1A Probing the P450 3A4 allosteric site via bioconjugation of ligand analogues Julie Ducharme1, Vanja Polic1, Karine Auclair1 1 Department of Chemistry, McGill University, Montreal, Canada. Introduction. P450 3A4 is the most abundant human P450 and is well- known for its wide substrate promiscuity, making it the most important drug-metabolizing enzyme. This enzyme has the particularity of binding multiple ligands simultaneously, which is associated with heterotropic or homotropic, positive or negative, cooperativity (1). Solving the kinetics of such complex systems remains challenging, and so is identifying the binding pockets involved. Many substrates are also known to be allosteric activators of P450 3A4. For instance, progesterone (PRG) is an activator of P450 3A4-catalyzed 7-benzyloxy-4-trifluoromethylcoumarin (BFC) debenzylation (2). To our knowledge, the location of the allosteric site is still debated and is also likely to depend on the specific effector involved. Aims. The aims are to define the location of the P450 3A4 PRG allosteric site and investigate how sensitive the allosteric activation is to the binding orientation of the effector. Methods. To probe the location of the allosteric site, a progesterone analogue (PGM) was covalently attached, separately at several locations near a peripheral binding pocket (3). The impact of the PGM label was evaluated by monitoring the changes in enzyme kinetics before and after labeling in the presence and absence of PRG effector. Results. A total of six different PGM bioconjugates were successfully generated. The kinetics studies of those bioconjugates, indicate that two PGM-labeled mutants are efficiently mimicking PRG allosteric activation. Interestingly, PGM-bioconjugate which better mimicked the PRG molecule in one of the crystal structure of P450 3A4 gave rise to higher permanent activation than other bioconjugates. This suggests that the orientation of the PRG effector in the allosteric site matters but is not crucial for activation. Discussion. Our method allowed us to narrow down the location of the P450 3A4 allosteric site. PGM labeling at positions that were not mimicking allostery led to different, yet still interesting results. In one case, the PGM labeled showed an antagonistic behavior while in other cases, it activated the BFC metabolism without occupying the allosteric site, suggesting that different mechanisms of activation exist. Conclusion. This work creates further opportunities to study other systems showing heterotropic activation. Our results are of considerable interest not only in the fields of biocatalysis and enzymology, but also in the area of drug metabolism and for the prediction of drug interactions. 1. Guengerich, F. P. (1999) Cytochrome P-450 3A4: regulation and role in drug metabolism. Annu. Rev. Pharmacol. Toxicol., 39, 1-17. 2. Domanski, T. L., He, Y.-A., et al. (2001) Phenylalanine and tryptophan scanning mutagenesis of CYP3A4 substrate recognition site residues and effect on substrate oxidation and cooperativity. Biochem., 40, 10150-10160. 3. Williams, P. A., Cosme, J., et al. (2004). Crystal structure of human cytochrome P450 3A4 bound to metyrapone and progesterone. Science, 305, 683-686 Book of poster abstracts 2B Tryptophan-75 is a potential gating residue of cytochrome P450 2D6 Laura Lowe Furge1, Kevin D. McCarty1 1Department of Chemistry, Kalamazoo College, Kalamazoo, USA. Introduction. The active site of CYP2D6 is buried, and both access to the active site and resulting metabolism are influenced by amino acid side chains, a phenomenon referred to as tunnel gating. This investigation hypothesizes that tunnel gating exists in CYP2D6, and that it is mediated by tryptophan-75 residue. Aims. This investigation aims to determine how tryptophan-75 might serve as a gating residue and impact the kinetics of CYP2D6, to gain a deeper understanding of tunnel gating in CYP2D6, and to understand the role of plasticity in enzyme mechanisms. Methods. To study the influence of tryptophan-75 on ligand metabolism rates, a tryptophan-75 to alanine mutant (CYP2D6*W75A) along with Figure 1. Tryptophan-75 (in yellow, at CYP2D6*1 were expressed and purified in E. coli host cells. All left) visualized in ball-and-stick form in two conformations observed in the P450 experimentation conducted with the 2D6*W75A mutant was replicated crystal structure. with CYP2D6*1 for comparison. Results. The interaction of CYP2D6*W75A and the *1 control with the substrates dextromethorphan and bufuralol was characterized in terms of spectral binding properties and Michaelis Menten kinetics. Discussion. Molecular Dynamics studies have shown that tryptophan-75 has the ability to swing out from the 2b channel to discharge a ligand. Also, visualization of 2D6 crystal structures by molecular imaging software showed the presence of several distinct conformations of the residue, some of which were observed to obstruct the opening of the 2b tunnel and thus inhibit access and egress of substrates from the active site. Based on the mobility of the residue in 3D space, we theorize that a gating mechanism of tryptophan-75 impacts ligand metabolism rates of CYP2D6. Conclusion. This investigation provides greater understanding of gating mechanisms in CYP2D6. Understanding of channel gating in CYPs has clinical importance as many questions remain as to its role in the process of drug metabolism. (Support: NIH 2R15GM086767-03). Book of poster abstracts 3C The effect of ageing and tryptophan hydroxylase 2 (TPH2) deficit on the CYP2D activity in rat brain and liver Anna Haduch1, Natalia Alenina2, Agnieszka Nikiforuk3, Piotr Popik3, Michael Bader2, Władysława A Daniel1 1Department of Pharmacokinetics and Drug Metabolism, Institute of Pharmacology, Polish Academy of Sciences, Kraków, Poland; 2Max-Delbrück-Center for Molecular Medicine, Berlin, Germany; 3Department of Behavioral Neuroscience and Drug Development, Institute of Pharmacology, Polish Academy of Sciences, Kraków, Poland. Introduction. Liver cytochrome P450 2D contributes to the metabolism of drugs, carcinogens and neurotoxins, while brain CYP2D plays an important role in the local metabolism of drugs and endogenous neuroactive substrates. Studies in rodents indicate that brain CYP2D mediates 5-methoxytryptamine O- demethylation to serotonin. This alternative pathway can gain significance in deficit of the main pathway of serotonin synthesis. Recent studies indicate a decreased CYP2D activity in aging rats and an increased CYP2D6 expression in human brain (the frontal cortex, substantia nigra, cerebellum) in elderly. But the activity of brain CYP2D has not been investigated. Aims. The aim of the present study was to ascertain whether the level of CYP2D activity changes with increasing age and in conditions of serotonin deficiency in the rat brain. Methods. The experiment was carried out on male Dark Agouti and Wistar Han rats. Livers and selected brain structures (the frontal cortex, hippocampus, hypothalamus, thalamus, brain stem, cortex, striatum and cerebellum) were isolated. Kinetic parameters of 5-methoxytryptamine O-demethylation (HPLC) were estimated in liver and brain microsomes from both strains. The activity of CYP2D was studied in Dark Agouti wild type (wt) rats (mature 3-month-old and senescent 21-month-old rats) and in tryptophan hydroxylase 2 (TPH2) deficient senescent rats by measuring the rate of bufuralol 1’-hydroxylation in liver or brain microsomes (HPLC). Results. Liver microsomes of Dark Agouti rats catalyzed the O-demethylation of 5-methoxytryptamine to serotonin with lower efficiency than those of Wistar Han. However, brain microsomes of both strains showed a similar efficiency of catalyzing this reaction, though lower than did liver microsomes. The activity of CYP2D in liver microsomes was significantly lower in senescent Dark Agouti wt rats than in the mature animals and further decreased in senescent TPH2-deficient rats. The CYP2D activity in the frontal cortex decreased in senescent wt rats, but increased in senescent TPH2-deficient rats (compared to senescent wt). However, the CYP2D activity in the hippocampus, hypothalamus and striatum increased with ageing in Dark Agouti wt rats and was not changed in senescent TPH2-deficient rats (compared to senescent wt). Discussion. The obtained results indicate that ageing negatively affects the liver CYP2D activity which can lead to the inhibition of drug metabolism. The brain metabolism of CYP2D substrates may be changed in regio-dependent way: elevated in the hippocampus, hypothalamus and striatum and diminished in the frontal cortex. The increased CYP2D activity in the frontal cortex at TPH2 deficit suggest activation of the alternative pathway of serotonin synthesis via CYP2D. Conclusion. Ageing and tryptophan hydroxylase deficit affect liver and brain CYP2D, which may have an impact on the metabolism of endogenous substrates and drugs catalyzed by this enzyme. (This study was financially supported by the Grant ERA-NET Neuron II JTC 2015 Respond and statutory funds from the Institute of Pharmacology, PAS, Kraków, Poland.) Book of poster abstracts 4A Determination of the distal ligand coordination to resting state cytochrome P450 CYP199A4 and its correlation to activity Joshua S. Harbort1, Tom Coleman2, Matthew N. Podgorski2, Rebecca R. Chao2, Jeanette E. Stok3, John B. Brunning4, James J. De Voss3, Jeffrey R. Harmer1, Stephen G. Bell2 1Centre for Advanced Imaging, University of Queensland, St Lucia, Australia; 2Department of Chemistry,
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