Mechanism of ORC-13661 hair cell protection
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2020 UW Oto-HNS Graduation Symposium
Posters from our graduating residents & students
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Very nice results! How long were the washes after UV irradiation? Are you planning a time-course study to see how long protection can last? It will be great to get to candidate binding proteins for the ORC compounds.
Hi Dr. Perkel, thank you for the comment! Washes were 15 min x 3. I like the idea of a time course in order to see the duration of protection. Assuming we are interacting with the MET channel, time of protection would give us an idea of how frequently channel proteins are turned over in hair cells.
Exciting results, Jo! I was interested to see that one of your pulled-down proteins (P118) seems to be expressed in the hair cell’s cuticular plate. Do you think that this binding of your protective drug to an intracellular target is functionally relevant? How will you determine which targets are ‘key’ to protection?
Hi Dr. Stone, thank you for the comments and questions! The subcellular localization of the different ORC analogues is interesting, and raises many questions about the identity of the target. It would be interesting if the target is truly intracellular, suggesting that the target is not a channel itself but rather a soluble protein. Right now we are still optimizing our labeling, so I don’t know if we can draw any specific conclusions yet.
In regards to sorting out the relevant interaction partners, we have several strategies. We will perform pull down experiments with ORC analogues, vehicle, and ORC-13661 + ORC analogues. Hopefully by comparing the MS/MS for each pulldown protein mixture, we can eliminate non-specific streptavidin interactions (vehicle) and non-specific drug interactions (ORC-13661 + ORC analogue). Additionally, we will bias our list of candidate proteins to heavily weight proteins known to be associated with MET. If there is time, I hope to start generating CRISPR KOs of candidate genes in zebrafish.