Design, synthesis and biological evaluation of small molecules for controlling cellular development

Cellular differentiation is a process directed by a wide range of controlling signaling molecules and pathways. All-trans-retinoic acid (ATRA) is one such compound that shows a wide range of biological activity. The endogenous effects of ATRA have the potential to be translated into many in vitro and in vivo applications; however, its administration is associated with many drawbacks. Consequently, a large group of synthetic analogues known as synthetic retinoids - that are structurally similar to ATRA have been prepared and tested in vitro in the search for higher stability and more potency. A small library of stable synthetic retinoids known as EC and GZ derivatives were prepared and their biological activity investigated using TERA2.cl.SP12 human embryonal carcinoma (EC) stem cells and SHSY5Y neuroblastoma cells. Two compounds, EC23 and GZ25 were found to inhibit cellular proliferation and induce neural differentiation in both cell lines. EC50s showed higher binding affinity of these two analogues to all RAR types and was confirmed by how they fit into the binding pocket of the different RARs. They bind into the binding pocket through a hydrophilic network of carboxylate group with Arg (salt bridge) and Ser (two hydrogen bonds) residues similar to ATRA. These effects were thoroughly characterized and quantified by monitoring the phenotypic changes of both cell lines and the gene expression markers such as RAR-β, PAX6, NeuroD1 which showed higher order of efficacy for induction of neuronal differentiation.In this study, the combined use of calculated chemical structures, molecular docking tools with receptor binding assays and biological characterization was useful to probe, and hence, understand the biological activity of certain synthetic retinoids with the ultimate goal of designing more specific synthetic retinoic acid derivatives

Dicarba-closo-dodecarborane-containing half-sandwich complexes of ruthenium, osmium, rhodium and iridium : biological relevance and synthetic strategies

This review describes how the incorporation of dicarba-closo-dodecarboranes into half-sandwich complexes of ruthenium, osmium, rhodium and iridium might lead to the development of a new class of compounds with applications in medicine. Such a combination not only has unexplored potential in traditional areas such as Boron Neutron Capture Therapy agents, but also as pharmacophores for the targeting of biologically important proteins and the development of targeted drugs. The synthetic pathways used for the syntheses of dicarba-closo-dodecarboranes-containing half-sandwich complexes of ruthenium, osmium, rhodium and iridium are also reviewed. Complexes with a wide variety of geometries and characteristics can be prepared. Examples of addition reactions on the metal centre, B–H activation, transmetalation reactions and/or direct formation of metal–metal bonds are discussed (103 references)

Biological Role of Aldo–Keto Reductases in Retinoic Acid Biosynthesis and Signaling

Several aldo–keto reductase (AKR) enzymes from subfamilies 1B and 1C show retinaldehyde reductase activity, having low Km and kcat values. Only AKR1B10 and 1B12, with all-trans-retinaldehyde, and AKR1C3, with 9-cis-retinaldehyde, display high catalytic efficiency. Major structural determinants for retinaldehyde isomer specificity are located in the external loops (A and C for AKR1B10, and B for AKR1C3), as assessed by site-directed mutagenesis and molecular dynamics. Cellular models have shown that AKR1B and 1C enzymes are well suited to work in vivo as retinaldehyde reductases and to regulate retinoic acid (RA) biosynthesis at hormone pre-receptor level. An additional physiological role for the retinaldehyde reductase activity of these enzymes, consistent with their tissue localization, is their participation in β-carotene absorption. Retinaldehyde metabolism may be subjected to subcellular compartmentalization, based on enzyme localization. While retinaldehyde oxidation to RA takes place in the cytosol, reduction to retinol could take place in the cytosol by AKRs or in the membranes of endoplasmic reticulum by microsomal retinaldehyde reductases. Upregulation of some AKR1 enzymes in different cancer types may be linked to their induction by oxidative stress and to their participation in different signaling pathways related to cell proliferation. AKR1B10 and AKR1C3, through their retinaldehyde reductase activity, trigger a decrease in the RA biosynthesis flow, resulting in RA deprivation and consequently lower differentiation, with an increased cancer risk in target tissues. Rational design of selective AKR inhibitors could lead to development of novel drugs for cancer treatment as well as reduction of chemotherapeutic drug resistance

Peroxisome Proliferator-Activated Receptor Alpha: Insight into the Structure, Function and Energy Homeostasis

Peroxisome proliferator-activated receptor alpha (PPAR alpha) belongs to the family of ligand-activated nuclear transcription factors and serves as a lipid sensor to regulate nutrient metabolism and energy homeostasis. The transcriptional activity of PPAR alpha is thought to be regulated by the binding of exogenous ligands (example, fenofibrate, TriCor), as well as endogenous ligands including fatty acids and their derivatives. Although long-chain fatty acids (LCFA) and their thioesters (long-chain fatty acyl-CoA; LCFA-CoA) have been shown to activate PPAR alpha of several species, the true identity of high-affinity endogenous ligands for human PPAR alpha (hPPAR alpha) has been more elusive. This two part dissertation is a structural and functional evaluation of human and mouse PPAR alpha binding to LCFA and LCFA-CoA using biophysical and biochemical approaches of spectrofluorometry, circular dichroism spectroscopy, mutagenesis, molecular modelling and transactivation assays. The first goal of this dissertation was to determine whether LCFA and LCFA-CoA constitute high-affinity endogenous ligands for full-length hPPAR alpha. Data from spectrofluorometry suggests that LCFA and LCFA-CoA serve as physiologically relevant endogenous ligands of hPPAR alpha. These ligands bind hPPAR alpha and induce strong secondary structural changes in the circular dichroic spectra, consistent with the binding of ligand to nuclear receptors. Ligand binding is also associated with activation of hPPAR alpha, as observed in transactivation assays. The second goal of this dissertation was to determine whether there exist species differences for ligand specificity and affinity between hPPAR alpha and mouse PPAR alpha (mPPAR alpha). This is important because despite high amino acid sequence identity (\u3e90 precent), marked differences in PPAR alpha ligand binding, activation and gene regulation have been noted across species. Similar to previous observations with synthetic agonists, we reported differences in ligand affinities and extent of activation between hPPAR alpha and mPPAR alpha in response to saturated long chain fatty acids. In order to determine if structural alterations between the two proteins could account for these differences, we performed in silico molecular modeling and docking simulations. Modeling suggested that polymorphisms at amino acid position 272 and 279 are likely to be responsible for differences in saturated LCFA binding to hPPAR alpha and mPPAR alpha. To confirm these results experimentally, spectrofluorometry based-binding assays, circular dichroism, and transactivation studies were performed using a F272I mutant form of mPPAR alpha. Experimental data correlated with in silico docking simulations, further confirming the importance of amino acid 272 in LCFA binding. Although the driving force for evolution of species differences at this position are yet unidentified, this study enhances our understanding of ligand-induced regulation by PPAR alpha. Apart from demonstrating significant structure activity relationships explaining species differences in ligand binding, data in this dissertation identifies endogenous ligands for hPPAR alpha which will further help delineate the role of PPAR alpha as a nutrient sensor in regulating energy homeostasis

Peroxisome Proliferator-Activated Receptor Alpha: Insight into the Structure, Function and Energy Homeostasis

Peroxisome proliferator-activated receptor alpha (PPAR alpha) belongs to the family of ligand-activated nuclear transcription factors and serves as a lipid sensor to regulate nutrient metabolism and energy homeostasis. The transcriptional activity of PPAR alpha is thought to be regulated by the binding of exogenous ligands (example, fenofibrate, TriCor), as well as endogenous ligands including fatty acids and their derivatives. Although long-chain fatty acids (LCFA) and their thioesters (long-chain fatty acyl-CoA; LCFA-CoA) have been shown to activate PPAR alpha of several species, the true identity of high-affinity endogenous ligands for human PPAR alpha (hPPAR alpha) has been more elusive. This two part dissertation is a structural and functional evaluation of human and mouse PPAR alpha binding to LCFA and LCFA-CoA using biophysical and biochemical approaches of spectrofluorometry, circular dichroism spectroscopy, mutagenesis, molecular modelling and transactivation assays. The first goal of this dissertation was to determine whether LCFA and LCFA-CoA constitute high-affinity endogenous ligands for full-length hPPAR alpha. Data from spectrofluorometry suggests that LCFA and LCFA-CoA serve as physiologically relevant endogenous ligands of hPPAR alpha. These ligands bind hPPAR alpha and induce strong secondary structural changes in the circular dichroic spectra, consistent with the binding of ligand to nuclear receptors. Ligand binding is also associated with activation of hPPAR alpha, as observed in transactivation assays. The second goal of this dissertation was to determine whether there exist species differences for ligand specificity and affinity between hPPAR alpha and mouse PPAR alpha (mPPAR alpha). This is important because despite high amino acid sequence identity (\u3e90 precent), marked differences in PPAR alpha ligand binding, activation and gene regulation have been noted across species. Similar to previous observations with synthetic agonists, we reported differences in ligand affinities and extent of activation between hPPAR alpha and mPPAR alpha in response to saturated long chain fatty acids. In order to determine if structural alterations between the two proteins could account for these differences, we performed in silico molecular modeling and docking simulations. Modeling suggested that polymorphisms at amino acid position 272 and 279 are likely to be responsible for differences in saturated LCFA binding to hPPAR alpha and mPPAR alpha. To confirm these results experimentally, spectrofluorometry based-binding assays, circular dichroism, and transactivation studies were performed using a F272I mutant form of mPPAR alpha. Experimental data correlated with in silico docking simulations, further confirming the importance of amino acid 272 in LCFA binding. Although the driving force for evolution of species differences at this position are yet unidentified, this study enhances our understanding of ligand-induced regulation by PPAR alpha. Apart from demonstrating significant structure activity relationships explaining species differences in ligand binding, data in this dissertation identifies endogenous ligands for hPPAR alpha which will further help delineate the role of PPAR alpha as a nutrient sensor in regulating energy homeostasis

Caractérisation des substrats xénobiotiques et des inhibiteurs des cytochromes CYP26A1, CYP26B1 et CYP26C1 par modélisation moléculaire et études in vitro

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.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

An isochroman analog of CD3254 and allyl-, isochroman-analogs of NEt-TMN prove to be more potent retinoid-X-receptor (RXR) selective agonists than bexarotene

Bexarotene is an FDA-approved drug for the treatment of cutaneous T-cell lymphoma (CTCL); however, its use provokes or disrupts other retinoid-X-receptor (RXR)-dependent nuclear receptor pathways and thereby incites side effects including hypothyroidism and raised triglycerides. Two novel bexarotene analogs, as well as three unique CD3254 analogs and thirteen novel NEt-TMN analogs, were synthesized and characterized for their ability to induce RXR agonism in comparison to bexarotene

The Role of Retinoic Acid in the Formation and Modulation of Invertebrate Electrical Synapses

Communication between cells in the nervous system is dependent upon structures known as synapses. Synapses are broadly characterized as either chemical or electrical in nature, owing to the type of signals that are transmitted across them. Factors that can affect chemical synapses have been extensively studied. However, the factors that can influence the formation and modulation of electrical synapses are poorly understood. Retinoic acid, a vitamin A metabolite, is a known regulator of chemical synapses, yet its capacity to regulate electrical synapses is not as well established. Preliminary evidence from the central neurons of both invertebrates and vertebrates suggests that it is also capable of regulating the strength of electrical synapses. In this study, I provide further insights into how retinoic acid can act as a neuromodulator of electrical synapses. My findings suggest that retinoic acid is capable of rapidly altering the strength of electrical synapses in a dose- and isomer-dependent manner. Further, I provide evidence that this acute effect might be independent of either the retinoid receptors or a protein kinase. In addition, I provide novel findings to suggest retinoic acid is also capable of regulating the formation of electrical synapses. Long term exposure to two isomers of retinoic acid, all-trans-retinoic acid and 9-cis-retinoic acid, reduces both the proportion of cell pairs, and the average synaptic strength between cells that form electrical synapses. In summary, these investigations provide novel insights into the role that retinoids play in the both the formation and modulation of electrical synapses in the CNS

Activation of Atlantic cod (Gadus morhua) retinoid X receptors by organic tin compounds

Masteroppgave i biologiBIO399MAMN-HAVSJMAMN-BI