Labelling of Voltage Gated Sodium Channels

Sammanfattning

DNA Encoded Library (DEL) screening has emerged as a transformative technology in pharmaceutical research, enabling the high-throughput screening of billions of compounds. While DEL screening has been successful in identifying small molecule ligands for various protein targets, challenges remain in targeting specific classes of proteins, such as membrane proteins, which are challenging to purify and study due to their complex structure and cellular localization. This master's thesis investigates the adaptation of DEL screening to target membrane proteins directly in living cells. Initial experiments focused on validating the expression of the NaV1.7 sodium ion channel, a prominent membrane protein target that was used as a model system in this thesis. Immunofluorescence and western blot analysis confirmed the expression of NaV1.7 in the HEK293-NaV1.7 cells, paving the way for subsequent DEL experiments. Labelling of NaV1.7 channel in live cells was conducted using a single-stranded DNA probe conjugated with ligand for the NaV1.7 channel. Despite initial promise, results from labelling experiments without photo-crosslinking revealed inconsistent binding patterns, indicating the need for further optimization. Attempts to optimize the photo-crosslinking protocol for labelling with UV photo-crosslinking were inconclusive. Further investigations using Western blot analysis were undertaken to improve the specificity of labelling of NaV1.7 results. Despite these efforts, challenges in achieving specific binding of DEL probes to NaV1.7 persisted, highlighting the complexities involved in targeting membrane proteins using DEL technology. In conclusion, this report provides valuable insights into the feasibility and challenges of conducting DEL screening on membrane proteins in living cells. Future research efforts should focus on refining experimental protocols and addressing technical hurdles to unlock the full potential of DEL technology for drug discovery targeting membrane proteins.