Dissertation: “Structural studies of drug targets and a drug metabolizing enzyme”
- Location: Biomedicinskt centrum, BMC B41, Husargatan 3, Uppsala
- Doctoral student: Daniela Cederfelt
- About the dissertation
- Organiser: Department of Chemistry - BMC
- Contact person: Helena Danielson
Daniela Cederfelt defends her doctoral thesis entitled “Structural studies of drug targets and a drug metabolizing enzyme” within the subject of Biochemistry.
Opponent: Prof. lnari Kursula, University of Oulu, Finland, and University of Bergen, Norway.
Supervisor: Prof. Helena Danielson, Biochemistry, Department of Chemistry – BMC, Uppsala University.
The work presented in this thesis describes how structural information about a protein can be acquired, and how it can be used to answer scientific questions about proteins’ function, their dynamic behaviour and their interactions with other proteins or ligands.
The catalytic function of the pyrimidine-degrading, drug metabolizing enzyme β-ureidopropionase (βUP) is dependent on the shift between oligomeric states. Substitution of amino acids H173 and H307 in the dimer-dimer interface and E207Q in the active site revealed that these are crucial for βUP activation. Inhibition studies of substrate-and product analogues allowed for a hypothesis that the ability to interact with F205 might distinguish activators from inhibitors. The first structure of the activated higher oligomer state of human βUP was determined using cryogenic electron microscopy, and confirmed that the closed entrance loop conformations and dimer-dimer interfaces are conserved between HsβUP and DmβUP.
Interactions between the epigenetic drug target SET and MYND domain containing protein 3 (SMYD3) and possible inhibitors were investigated. A crystal structure confirmed the covalent bond of a rationally designed, targeted inhibitor to C186 in the active site of SMYD3. A new allosteric binding site was discovered using a biosensor screen with a blocked active site. Crystal structures revealed the location of the new binding site, and the binding mode of the (S)-and (R) enantiomers of the allosteric inhibitor. Lastly, a fragment based drug discovery approach was taken, co-crystallizing and soaking SMYD3 with hits from a fragment screen. This resulted in four crystal structures with weak electron density of fragments at several locations in the enzyme.
The dynamic acetylcholine binding protein (AChBP) is a homologue of a Cys-loop type ligand gated ion channel. Hits from various biosensor screens, of which some indicated conformational changes, were co-crystallized with AChBP. Seven crystal structures of AChBP in complex with hit compounds from the biophysical screens were determined. Small conformational changes in the Cys-loop were detected in several of the crystal structures, coinciding with the results from the biosensor screens.
In these studies, we explore new strategies for the investigation of the function and regulation of proteins relevant in drug discovery and optimization.