Dunge, Andreas2025-04-022025-04-022025-04-02978-91-8115-210-4 (tryckt)978-91-8115-211-1 (PDF)https://hdl.handle.net/2077/85703This thesis explores the application of serial synchrotron crystallography (SSX) in drug discovery, focusing on the structural studies of soluble epoxide hydrolase (sEH) and cytochrome P450 3A4 (CYP3A4). Utilizing SSX, a relatively novel technique that collects data from a large number of small crystals at room temperature (RT), this research aims to investigate the structural dynamics and ligand interactions of these enzymatic targets. A workflow is developed that enables the transition from macrocrystals to microcrystals, and this study incorporates ligand soaking to explore protein-ligand interactions in their near-native states. We successfully established a method for obtaining microcrystals and soaking compounds, which facilitated a comprehensive fragment study. This study led to the identification of 40 active site-binding fragments out of 384 tested for sEH, showcasing the method's effectiveness. Additionally, the RT structures revealed conformational nuances of F497 in sEH, with inward movement observed in response to potent inhibitors. For CYP3A4, an RT structure provided valuable comparisons to cryogenic analyses, emphasizing structural differences. The thesis highlights SSX's capability to capture structural information that may be obscured under traditional cryogenic conditions, demonstrating its utility alongside conventional cryogenic macromolecular crystallography to enhance understanding of protein-ligand complexes. Additionally, the work addresses the logistical and technical challenges inherent in SSX and proposes strategies to optimize experimental conditions effectively. By focusing on these targeted studies, this research highlights the capability of SSX to advance drug discovery efforts. SSX offers a novel approach for detailed structural analysis of proteins, enabling the identification of interactions which could be hidden at cryogenic temperature which can be useful for designing effective therapeutic agents.engDrug discoverySSXsEHCYP3A4Serial synchrotron crystallographyFragmentsX-ray crystallographyProtein crystallographyMicrocrystalsText