Labelling of Voltage Gated Sodium Channels
(2024) MOBN03 20232Degree Projects in Molecular Biology
- Abstract
- 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... (More) - 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. (Less) - Popular Abstract
- Labelling of Voltage Gated Sodium Channels
DNA-encoded library (DEL) screening has emerged as a powerful hit finding approach, in which a huge DNA barcoded combinatorial library of small molecules can be screened for binding to a target of interest. Typically, DNA encoded library screening requires high quality purified protein targets for highest chances of successful hit finding, for membrane proteins this has been a challenge and only few reports exist of successful DEL campaigns against membrane proteins. A novel approach has been described that applies DNA encoded library screening on live cells with the membrane protein in their native environment, using local enrichment of the library near the target of interest to get... (More) - Labelling of Voltage Gated Sodium Channels
DNA-encoded library (DEL) screening has emerged as a powerful hit finding approach, in which a huge DNA barcoded combinatorial library of small molecules can be screened for binding to a target of interest. Typically, DNA encoded library screening requires high quality purified protein targets for highest chances of successful hit finding, for membrane proteins this has been a challenge and only few reports exist of successful DEL campaigns against membrane proteins. A novel approach has been described that applies DNA encoded library screening on live cells with the membrane protein in their native environment, using local enrichment of the library near the target of interest to get specificity. This has been described predominantly for folate receptor as model system, and G-protein coupled receptors.
In my thesis project, I explored the novel approach of using DEL probe to label voltage-gated sodium ion channel, which are a group of proteins with significant pharmaceutical interest due to their involvement in pain pathways. Initial experiments validated the expression of NaV1.7 in HEK293 cells using immunofluorescence and western blot, setting the stage for labelling with DEL probes. The core of the project involved labeling the NaV1.7 channel in live cells using a DNA probe conjugated with a ligand specific to NaV1.7 allowing the ion channel to remain in its native cellular environment. The major techniques employed to achieve the labelling of the protein of interest (POI) were photo-crosslinking, confocal microscopy, and western blot. Despite the initial promise, the results highlighted the complexities of achieving consistent and specific binding, underscoring the need for further optimizations.
In conclusion, my thesis sheds light on the potential and challenges of using DEL screening for membrane proteins in live cells. While specific binding of DEL probes to NaV1.7 proved challenging, the insights gained from this work pave the way for future research to refine these methods. The ultimate goal is to harness DEL technology more effectively in drug discovery, particularly for targets as complex and therapeutically relevant as membrane proteins.
Figure: Method to label membrane proteins with a DNA tag. L, a known ligand; tag, fluorophore, biotin or other. The BP/CP duplex is pre-formed and then added to the cell. After the BP/CP-mediated affinity labelling, the BP is removed via toehold displacement. This image is reproduced from (Huang et al., 2020).
Master’s Degree Project in Molecular Biology 60 credits 2023-24
Department of Biology, Lund University
Supervisor: Nils-Olov Hermansson; Co-Supervisors: Arjan Snijder, Yinan Song
Discovery Biology, Discovery Sciences, R&D, AstraZeneca, Gothenburg (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9176980
- author
- Soni, Aditya
- supervisor
- organization
- course
- MOBN03 20232
- year
- 2024
- type
- H2 - Master's Degree (Two Years)
- subject
- language
- English
- id
- 9176980
- date added to LUP
- 2024-10-23 14:21:17
- date last changed
- 2024-10-23 14:21:17
@misc{9176980,
abstract = {{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.}},
author = {{Soni, Aditya}},
language = {{eng}},
note = {{Student Paper}},
title = {{Labelling of Voltage Gated Sodium Channels}},
year = {{2024}},
}