En l’absence de structures tridimensionnelles expérimentales des cytochromes P450 CYP26A1, CYP26B1 et CYP26C1, la caractérisation de leur substrats et ligands s’est basée sur l’analyse des modèles structuraux obtenus par modélisation par homologie avec la structure expérimentale du cytochrome P450 CYP120. La justesse des modèles a été validée par l’amarrage de l’acide rétinoïque all-trans dans des configurations compatibles avec les métabolites attendus. L’amarrage d’agonistes et d’antagonistes des récepteurs nucléaires RARs prédirent l’acide tazaroténique (TA) et l’adapalène comme des substrats potentiels. Les expériences in vitro confirmèrent la métabolisation de ces 2 médicaments par les CYP26s. L’analyse de la cinétique de sulfoxidation du TA par CYP26A1 and CYP26B1 a permis d’établir le TA comme la référence contrôle de l’activité de ces enzymes. Puis, la comparaison des modèles des CYP26s avec la structure cristalline de CYP2C8 a permis d’identifier des similarités structurales de leurs inhibiteurs. Une corrélation entre l’inhibition de CYP26A1 et de CYP2C8 par des inhibiteurs connus de CYP2C8 a été démontrée après détermination de leurs IC50 pour CYP26A1 et CYP26B1 en utilisant le TA comme substrat de référence. La mesure de l’inhibition in vitro fut ensuite utilisée pour évaluer la possibilité que les CYP26s soient impliquées dans des interactions médicamenteuses observées pour certaines molécules. Cette thèse caractérise et appuie le rôle encore mal connu des CYP26s dans la métabolisation in vivo de certains xénobiotiques ainsi que l’effet potentiel de leur inhibition qui favoriserait la survenue d'effets indésirables. / Without crystal structures to study the CYP26 family of drug metabolizing enzymes, homology models were used to characterize CYP26A1, CYP26B1 and CYP26C1 and to identify substrates and inhibitors of the enzymes. Computational models of each isoform based on structural homology to CYP120 were validated by docking all-trans retinoic acid, an endogenous ligand of CYP26. Docking of retinoic acid receptor agonists and antagonists suggested that tazarotenic acid (TA) and adapalene may be metabolic substrates for CYP26, data which was confirmed using in vitro metabolite identification assays. Phenotyping experiments determined that CYP26s played a major role in the metabolism of these compounds in vitro. The kinetics of TA sulfoxidation by CYP26A1 and CYP26B1 were characterized and the compound was proposed as an in vitro probe of CYP26 activity in single enzyme expression systems. Structural characterization efforts identified similarities between the CYP26 homology models and the known crystal structure of CYP2C8, in agreement with previously published reports. Using TA as a probe, the IC50’s of known CYP2C8 inhibitors was measured against CYP26A1 and CYP26B1, with a statistically significant correlation observed between CYP26A1 and CYP2C8. Additional in vitro and computational experiments were used to characterize the inhibition mechanism for the most potent inhibitors. The observed in vitro inhibition was then used to predict the likelihood of CYP26 inhibition being involved in clinically relevant drug interactions. As a whole, the results presented support the role of the CYP26s in the metabolism of xenobiotic compounds as well as in potential in vivo drug interactions.

Proper embryonic patterning requires precise spatio-temporal regulation of retinoic acid (RA) activity. Morphogenesis can be regulated at the level of RA distribution, mainly via its synthesis and catabolism by the RALDH and CYP26 enzymes respectively, and at the level of RA-mediated transcription through activation of its cognate nuclear receptor, the retinoic acid receptors (RARs) α, β, and γ. Loss of Cyp26b1 leads to increased local levels of RA in tissues such as the limb and craniofacial structures, and results in neonatal lethality. Visible gross phenotypic defects in neonates include phocomelia (shortening of the limbs), adactyly (missing digits), micrognathia (shortened lower jaw), and open eyes at birth. In addition, these embryos exhibit cleft palate and have a paucity of vibrissal (whisker) and pelage (hair) follicles. We have previously shown that ablating the gene encoding RARγ in a Cyp26a1-null background was able to rescue the caudal abnormalities associated with improper RA exposure in these embryos by limiting aberrant RA signalling, and thus rescuing expression domains of target genes involved in caudal development. I show here that ablating Rarg in a Cyp26b1-null background is able to partially rescue the defects associated with loss of CYP26B1. These include a reduction in the severity of limb defects, rescued vibrissae, fused eyelids, and recovered aspects of axial skeletal development. This compound-null murine model illustrates that RARγ plays a specific role in transducing the RA signal within tissues that are affected by the loss of CYP26B1. Further molecular analysis of the pathways responsible for directing limb bud outgrowth and eyelid fusion provided insight into pathways regulated by RARγ in these rescued tissues. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2010-04-01 15:38:52.05

The aim of the studies included in the thesis is to better understand the interplay between neuronal activity-dependent gene regulation and the bioactive vitamin A metabolite all-trans-retinoic acid (RA) during postnatal development, refinement and maintenance of precise neuronal connectivity using the olfactory sensory neuron (OSN) in the olfactory epithelium (OE) of genetically modified mice as a model. We show that: Inhibition of RA receptor (RAR)-mediated transcription in OSNs reduces expression of the olfactory cyclic nucleotide-gated (CNG) ion channel, which is required for odorant receptor (OR)-mediated stimulus transduction. This, results in increased OSN death and errors in precise connectivity. The increased cell death may be a consequence of reduced intrinsic excitability and/or reduced influx of Ca2+ ions while the errors in connectivity may be due to altered OR-dependent expression of axonal guidance proteins, such as Kirrel-2 and Neuropilin-1. Expression of the RA catabolic enzyme Cyp26B1 in OSNs is positively regulated by RAR-mediated transcription as well as sensory stimulation in a CNG channel-dependent manner. This shows that neuronal activity and local vitamin A metabolism are parts of novel regulatory feedback loop controlling precise connectivity and neuronal survival. The feedback loop may be a form of homeostatic plasticity in response to global changes in neuronal activity. BACE1, an enzyme is implicated in Alzheimer´s disease, and Cyp26B1 are inversely regulated by CNG channel-dependent sensory stimulation. Cyp26B1 expression is switched on at birth, forms a topographic expression gradient in OE and inhibits BACE1 expression into an inverse counter gradient. Taken together these results reveal a novel neuronal activity-dependent mechanism by which sensory stimuli can shape spatial gene expression via altered RA bioavailability. Increased Cyp26B1 expression stimulates turnover of OSNs during adult neurogenesis by a non-cell-autonomous mechanism. The gradient of Cyp26B1 expression correlates with spatially-regulated diversification of OSNs into subpopulations that express different subsets of OR genes. Cyp26B1 expression influences spatial OR diversification of OSNs by two different mechanisms. In the ventrolateral OE, Cyp26B1 inhibits OR expression by blocking OSN differentiation at a stage that may be associated with the cell intrinsic mechanism regulating OR gene choice. In the dorsomedial OE the expression frequency of some ORs is unaltered while other increases, presumably as a consequence of neuronal activity-dependent competition. A probable function of graded and activity-dependent Cyp26B1 expression is to form a topographic partitioning of the olfactory sensory map into functional domains, which gradually differ from each other with regard to experience-driven plasticity and neurogenic potential along the dorsomedial-ventrolateral axis of OE.