Date: Tue June 11th 2024
Time: 2pm UK time (other times)
Place:Zoom linkPrevious Meeting: issue #18
Who can come?: Anyone. No need to say anything unless you'd like to. If you'd like to contribute, please join Github.
Attendees: Dr Eve Carter (UCL), Prof Matilda Katan (UCL), Prof Karl Swann (Uni Cardiff), Dr Claudia Tredup (Goethe-University Frankfurt), Dr Nico Braeuer (Nuvisan), Prof Al Edwards (Uni Toronto), Anna Girtle (UCL)
Eve has been furthering her work with the Aldol 518 assay. She tested 7 of Nuvisan’s compounds and 4 of Matilda’s compounds with PLCγ1 and chicken PLCζ1. As before, she found that all of Nuvisan’s compounds inhibited PLCζ1. She took the 4 best compounds and ran dose response experiments. The resulting IC50s seem to correlate with the results from Nuvisan’s IP1 and XY69 assays, but it should be noted that there was a small sample size. Eve also tested 250µM of Nuvisan’s compounds against PLCζ1 and PLCγ1 in Aldol 518 assays and found that they appear to selectively inhibit PLCζ1. The same tests using Matilda’s compounds appear to selectively inhibit PLCγ1, though one compound had some activity against PLCζ1 at 250µM. However, Aled warns that 250µM is a high concentration to expect any biological relevance, and recommends not using that concentration in future experiments.
Madison has been furthering her work on the crystallization of PLCz1. She has successfully solved the crystal structure of chicken PLCz1 with calcium, and it is published on PDB (9BCZ). Her future plans include reproducing previous conditions and sending them to the synchrotron, to optimize screens of successful conditions, and running co-crystallization trials with 4 compounds. She will be trying to get crystal structures of human or chicken PLCz1 with or without compounds bound.
Eve has been working with Nuclera on cell-free protein production. They tried 3 constructs of human PLCz1, but purification yields were very poor and it is not recommended that we try scaling up.
Matilda has said she can provide more PLCγ1 if needed, and mentioned that the 2 Nuvisan compounds that affected PLCγ1 might be candidates for a pan-PLC inhibitor. She also has a cell assay for PLCγ1 and PLCd1 which could be used to test the compounds for general inhibition. Karl has said he can do mouse egg injections with the top compounds to test effect on PLCz1. Aled outlined how to use the nanoBRET method to test specificity of the compounds in another cell-based assay.
To do:
[x] Madison to continue crystallography work as described
[ ] DEL screen
[ ] ASMS screen.
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.
Date: Tue June 11th 2024 Time: 2pm UK time (other times) Place: Zoom link Previous Meeting: issue #18 Who can come?: Anyone. No need to say anything unless you'd like to. If you'd like to contribute, please join Github.
Attendees: Dr Eve Carter (UCL), Prof Matilda Katan (UCL), Prof Karl Swann (Uni Cardiff), Dr Claudia Tredup (Goethe-University Frankfurt), Dr Nico Braeuer (Nuvisan), Prof Al Edwards (Uni Toronto), Anna Girtle (UCL)
Decks: Eve's presentation
Agenda:
In meeting:
Eve has been furthering her work with the Aldol 518 assay. She tested 7 of Nuvisan’s compounds and 4 of Matilda’s compounds with PLCγ1 and chicken PLCζ1. As before, she found that all of Nuvisan’s compounds inhibited PLCζ1. She took the 4 best compounds and ran dose response experiments. The resulting IC50s seem to correlate with the results from Nuvisan’s IP1 and XY69 assays, but it should be noted that there was a small sample size. Eve also tested 250µM of Nuvisan’s compounds against PLCζ1 and PLCγ1 in Aldol 518 assays and found that they appear to selectively inhibit PLCζ1. The same tests using Matilda’s compounds appear to selectively inhibit PLCγ1, though one compound had some activity against PLCζ1 at 250µM. However, Aled warns that 250µM is a high concentration to expect any biological relevance, and recommends not using that concentration in future experiments.
Madison has been furthering her work on the crystallization of PLCz1. She has successfully solved the crystal structure of chicken PLCz1 with calcium, and it is published on PDB (9BCZ). Her future plans include reproducing previous conditions and sending them to the synchrotron, to optimize screens of successful conditions, and running co-crystallization trials with 4 compounds. She will be trying to get crystal structures of human or chicken PLCz1 with or without compounds bound.
Eve has been working with Nuclera on cell-free protein production. They tried 3 constructs of human PLCz1, but purification yields were very poor and it is not recommended that we try scaling up.
Matilda has said she can provide more PLCγ1 if needed, and mentioned that the 2 Nuvisan compounds that affected PLCγ1 might be candidates for a pan-PLC inhibitor. She also has a cell assay for PLCγ1 and PLCd1 which could be used to test the compounds for general inhibition. Karl has said he can do mouse egg injections with the top compounds to test effect on PLCz1. Aled outlined how to use the nanoBRET method to test specificity of the compounds in another cell-based assay.
To do:
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: Tue 13th Aug, 2pm UK time