Anne Bjørnskov Jensen
PhD student
Curriculum Vitae
Education and employment
2011-2014:
PhD student at the Department of Medicinal Chemistry, Faculty of PharmaceuticalSciences, University of Copenhagen
2009-2011:
M.Sc. Medicinal Chemistry, Department of Chemistry, University of Aarhus
2010-2011:
Master’s student in the Molecular Pharmacology group, Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen
2009/2010:
Exchange student at Strathclyde University, Glasgow, Scotland
2006-2009:
B.Sc. Medicinal Chemistry, Department of Chemistry, University of Aarhus
Previous scientific projects
2010-2011
Master’s project: Molecular Pharmacology group, Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen
Title: “Molecular determinants for in vitro expression of functional α6* nicotinic acetylcholine receptors"
2010
Molecular biology project: Innate Immunity group, Department of Molecular Biology, University of Aarhus
Title: ”Enzymatic properties of 2’-5’-Oligoadenylate Synthetase 3”
2009
Bachelor project: Centre of DNA nanotechnology, Department of Chemistry, University of Aarhus
Title: ”Synthesis of a serotonin derivative”
Project
“Human alpha 6* nicotinic acetylcholine receptors: Molecular pharmacology and therapeutic prospects”
Background
Nicotinic acetylcholine receptors (nAChRs) are widely distributed in the central nervous system as well as in the peripheral nervous system, where they mediate the rapid ‘phasic’ signaling of ACh [1]. They are protein complexes assembled from five subunits and can be either homomeric (assembled from a single type of subunit) or heteromeric (assembled from several different subunits) [2]. The neuronal nAChRs assemble from eight α (α2-α7 and α9-α10) and three β (β2-β4) subunits resulting in formation of a large variety of receptors within this family, each with different functional and pharmacological profiles [1].
Activation of these receptors typically results in enhanced release of various key neurotransmitters, including dopamine, serotonin, glutamate and γ-aminobutyric acid (GABA) [1]. Because of their great modulatory effect on other neurotransmitter systems, the nAChRs have been proposed as therapeutic targets for a wide range of neurodegenerative and psychiatric disorders such as Alzheimer’s disease, Parkinson’s disease, schizophrenia, depression and anxiety [3]. Furthermore, the considerable heterogeneity of the native nAChRs in the CNS presents the possibility of developing drugs with minimum side effects by specifically targeting a single or a few nAChR subtypes.
The nAChRs containing α6 subunits (α6* nAChRs, the asterisk representing the presence of other subunits) are located on dopaminergic neurons in the midbrain, where they modulate release of dopamine [4]. The α6* nAChRs have been proposed as potential targets for treatment in both Parkinson’s disease and nicotine addiction [5]. Unfortunately it has proved to be very difficult to efficiently express functional α6* nAChRs in recombinant expression systems despite the fact that functional α6* nAChRs are observed in vivo [6-8]. Furthermore, the insights into and understanding of the physiological functions and pathophysiological potential in the α6* nAChRs has been limited by the lack of truly specific ligands.
One approach used in several laboratories to enable functional expression of α6* nAChRs is the use of chimeric subunits in which the extracellular domain (ECD) of the α6 subunit is fused with the transmembrane domain (TMD) and the intracellular domain (IND) of the α3 or α4 subunits [6, 8-10]. Co-expression of these α6/α3 or α6/α4 chimeras with β2, β3 and β4 subunits in both mammalian cell lines and in oocytes have resulted in formation of functional receptors. In contrast, co-expression of the reverse chimera (consisting of the ECD of α3 or α4 and the TMD and IND of α6) with β2 does not result in the formation of functional nAChRs in Xenopus oocytes [6, 10]. This strongly suggests that the ‘problem regions’ for functional expression of α6* nAChRs are situated in the TMD and/or IND of α6. Further chimeric studies of these two domains performed in our lab indicates that the large second intracellular loop of the α6 subunit impair functional expression of functional receptors (unpublished data).
Objectives
Studies of the molecular and neuronal determinants of expression of functional α6* nAChRs
The identification of the large second intracellular loop of the α6 subunit as a ‘problem region’ for expression of functional receptors forms the basis for this part of the project. By applying the Yeast-Two-Hybrid technique a search for intracellular scaffold/chaperone proteins of importance for α6* nAChR function will be carried out. This approach has previously been used to identify several intracellular proteins interacting with α4* and α3* nAChRs [11, 12].
Yeast-Two-Hybrid screening enables the identification of ‘prey’ proteins (generated from plasmids containing cDNAs from libraries isolated from a native tissue) interacting with a chosen ‘bait’ (a receptor/protein region). A cDNA library derived from human midbrain as preys and the large second intracellular loop of the α6 subunit.
Proteins identified as “binders” to the bait in the screenings will subsequently be verified in co-immunoprecipitation studies using rat brain tissue homogenate and in Fluorescence Resonance Energy Transfer (FRET) studies using nAChR subunits and the candidate proteins tagged with fluorescent probes [13]. Furthermore, the influence of the respective proteins on cell surface expression, assembly and functional properties of α6* nAChRs in mammalian cell lines will be investigated by confocal microscopy, ELISA techniques, a [3H]epibatidine-binding assay, and in TEVC recordings at α6* nAChRs and the proteins co-expressed in Xenopus oocytes.
Identification of novel allosteric modulators with α6* nAChR activity
Considering the importance of a better understanding of the roles of and the therapeutic potential in α6* nAChRs, surprisingly few medicinal chemistry efforts have been focused on these receptors. A couple of α-conotoxins have been found to be relatively selective antagonists of α6* nAChRs over other nAChRs [14, 15], but with one notable exception [16] these do not appear to discriminate between the many different subtypes containing α6. Furthermore, no agonists or positive allosteric modulators with selective α6* activity have been reported.
In this part of the project the FLIPR membrane potential (FMP) blue assay will be used for the screening of a compound library at a functional α6/α3* receptor in the search for novel ligands with activity on α6* nAChRs. The lab has a small commercial compound library of 7000 compounds that will be used for this.
Following identification of putative modulators, a series of analogs will be purchased from commercial companies and characterized functionally at different nAChR subtypes, including α6α4β2β3, α6β2β3 and α6β4 nAChRs. Through this iterative process, we will attempt to increase the potency and the α6* nAChR-selectivity of the original hits.
References
- Taly, A., et al. (2009) Nicotinic receptors: allosteric transitions and therapeutic targets in the nervous system. Nature reviews. Drug discovery 8(9):733-50.
- Sine, S.M. and A.G. Engel (2006) Recent advances in Cys-loop receptor structure and function. Nature 440(7083):448-55.
- Zouridakis, M., et al. (2009) Recent advances in understanding the structure of nicotinic acetylcholine receptors. IUBMB life 61(4):407-23.
- Yang, K.C., G.Z. Jin, and J. Wu (2009) Mysterious alpha6-containing nAChRs: function, pharmacology, and pathophysiology. Acta pharmacologica Sinica 30(6):740-51.
- Quik, M. and J.M. McIntosh (2006) Striatal alpha6* nicotinic acetylcholine receptors: potential targets for Parkinson's disease therapy. The Journal of pharmacology and experimental therapeutics 316(2):481-9.
- Kuryatov, A., et al. (2000) Human alpha6 AChR subtypes: subunit composition, assembly, and pharmacological responses. Neuropharmacology 39(13):2570-90.
- Gerzanich, V., et al. (1997) "Orphan" alpha6 nicotinic AChR subunit can form a functional heteromeric acetylcholine receptor. Molecular pharmacology 51(2):320-7.
- Evans, N.M., et al. (2003) Expression and functional characterisation of a human chimeric nicotinic receptor with alpha6beta4 properties. European journal of pharmacology 466(1-2):31-9.
- Kuryatov, A. and J. Lindstrom (2011) Expression of functional human alpha6beta2beta3* acetylcholine receptors in Xenopus laevis oocytes achieved through subunit chimeras and concatamers. Molecular pharmacology 79(1):126-40.
- Papke, R.L., et al. (2008) Extending the analysis of nicotinic receptor antagonists with the study of alpha6 nicotinic receptor subunit chimeras. Neuropharmacology 54(8):1189-200.
- Lin, L., et al. (2002) The calcium sensor protein visinin-like protein-1 modulates the surface expression and agonist sensitivity of the alpha 4beta 2 nicotinic acetylcholine receptor. The Journal of biological chemistry 277(44):41872-8.
- Rezvani, K., et al. (2009) UBXD4, a UBX-containing protein, regulates the cell surface number and stability of alpha3-containing nicotinic acetylcholine receptors. The Journal of neuroscience : the official journal of the Society for Neuroscience 29(21):6883-96.
- Masi, A., et al. (2010) Optical methods in the study of protein-protein interactions. Advances in experimental medicine and biology 674:33-42.
- Dowell, C., et al. (2003) Alpha-conotoxin PIA is selective for alpha6 subunit-containing nicotinic acetylcholine receptors. The Journal of neuroscience : the official journal of the Society for Neuroscience 23(24):8445-52.
- McIntosh, J.M., et al. (2004) Analogs of alpha-conotoxin MII are selective for alpha6-containing nicotinic acetylcholine receptors. Molecular pharmacology 65(4):944-52.
- Azam, L., et al. (2010) alpha-Conotoxin BuIA[T5A;P6O]: a novel ligand that discriminates between alpha6ss4 and alpha6ss2 nicotinic acetylcholine receptors and blocks nicotine-stimulated norepinephrine release. The FASEB journal : official publication of the Federation of American Societies for Experimental Biology 24(12):5113-23.
Financial support
H. Lundbeck A/S
Contact
Department of Medicinal Chemistry
Faculty of Pharmaceutical Sciences
Fruebjergvej 3, room 115
2100 Copenhagen
Denmark
Phone: 39179653
E-mail: aje(at)farma.ku.dk
