PLCz1 Open Online Meeting - Tue Nov 12th

[AnnaGirtle]

Date: Tue Nov 12th 2024

Time: 2pm UK time (other times)

Place: Zoom link
Previous Meeting: issue #21

Who can come?: Anyone. No need to say anything unless you'd like to. If you'd like to contribute, please join Github.

Attendees:

Decks: Eve's presentation

Agenda:

• Update from Madison Edwards and @EveCarter

In meeting:

Madison sent her avi-tagged chPLCz1 (see last update) to Eve who has confirmed its activity. She is currently working on finding a construct suitable for crystallography by testing expression levels of a chPLCz1 with mutated threonine (single mutant) which comes into the active site as well as a version with the mutated threonine and nearby serines (triple mutant). She found that the single mutant shows good expression, while the triple mutant seems to express at a lower level. Madison plans to try purifying the single mutant which she will send to Eve to determine activity and will try co-crystallize with IP3. She also now has constructs with a cleavable avi-tag and sumo-tag for use in future experiments.

Madison provided chPLCz1 protein for the AS-MS screen, but it has not been completed yet. She is also waiting on approval from World Courier and HitGen to send proteins for DEL screen (chPLCz1, hPLCd, hPLCg1, hPLCz1), but the proteins are prepared for when that comes through.

Madison ran HDX-MS last week but didn’t see any binding with 1:10 ratio of protein:compound. This seemed odd because the protein has such a low IC50, so she set up another round with 1:40 ratio. She will analyze these results and if there is still no binding, she may need to adjust the conditions of the assay or verify that the batch of Nuvisan compound is functional. Matilda shared that her lab has done HDX-MS in the past with another PLC and found that IP3 did not show up in the assay despite evidence of binding from crystal structures. It seems that HDX-MS can work but does not always.

Eve has been screening the SGC library of donated chemical probes and performed dose-response experiments to get IC50s for the best hits. The best 4 hits were:

• BAY-277 METAP2 degrader probe (Bayer)
• BAY-8805 METAP2 degrader control (Bayer)
• FM-743 MAPK14 control (SGC Frankfurt)
• PROTAC SGK3 degrader probe (Dundee)

Nico has agreed to screen these compounds in Nuvisan’s assays to follow up on these findings, and Eve will be looking for fragments of these large molecules that could be provided, purchased or synthesized. She hopes to identify the specific part of each molecule that is contributing to the inhibitory effects. Madison commented that it is a good sign you are getting constant IC50s for BAY-277 and BAY-8805. Eve noted that the first 3 hits were fine solubility wise, but the PROTAC molecule is less soluble so required more DMSO. Madison highlighted the importance of adding the same amount of DMSO across concentrations because it can effect activity, and that high concentrations of DMSO can start denaturing the protein. Opher commented that the fact that the MAPK14 control works but the associated probe does not is promising because it may be removing the worry of kinases as off-targets. He also suggested perhaps performing a degradation assay to see if the degrader molecules are just inhibiting function or in fact degrading the protein as a whole. He also suggested looking at turbidity to see if PLCz1 is aggregating upon introduction of the test compounds. Karl commented that he has luciferase tagged PLCz1 which could detect degradation and give insight into protein stability. Claudia commented that it might be the long lipophilic linker regions that are binding PLCz1, and that Eve may have already tested smaller components of the Bayer compounds as they might have been in the probe library. Eve plans to go back and check if any of the other probes she tested are indeed components of the best hits, which may give insight into the important regions for inhibition. Karl and Madison discussed that it may be useful to include another protein in the Aldol assays to assess non-specific binding or overall effects of the probes on the experimental system.

On a side note, Opher has human-chicken chimera constructs that he will be testing with hopes of making anti-human antibodies for use in future experiments.

To do:

• Madison to purify PLCz1 with a mutated threonine
• Eve to research fragments of DCP screen hits

L'esprit de l'escalier
If you'd like to follow up after the meeting, please comment below. You can also email, but please be clear if anything in the email should not be public domain - the default is open.

Next meeting: 7th January 2025

[mattodd]

Hi there - I need to be an apology today. This meeting clashes with one happening over on Open Source Antibiotics and I ought to be there this month. If we could avoid this 2pm slot, that'd be super. e.g. 1pm or 3pm. Or shift by a week? So, not 2pm Dec 10th.
It'd be very useful if @EveCarter could run people past latest results and we think how we can most quickly follow up on those. Also whether Karl might be interested in running the three best probe hits?

[EveCarter]

Sorry about the clashes. The meeting was originally scheduled for 2pm on the first Tuesday of each month, but there have been lots of clashes recently so it has been on the second Tuesday. I think that 1pm UK time is a bit early for Toronto, so how about we try 3pm for next month? We can aim for the first Tuesday of the month then shift a week later if needed. I will bring this up in today's meeting and see if that would work.