eyermanncj
commented
2 years ago @mattodd @chrisdowson1 @LauraDS1 @LizbeK @loriferrins @phraenquex @drc007 @Yuhang-CADD @bartrum @Rebecca-Steventon
Better late than never maybe .... So the clone used for the Pae murC AZ x-ray structure was truncated to residues 16-322. This means the flexible domain that has been discussed in Eco and Pae murD is not included in the Pae murC AZ structure. Of course we know the AZ Pae murC inhibitor is very potent (low nanomolar) and the Pae murC AZ used in teir biochemical assay was full length Pae murC.
The dynamics of the mur ligase domain from ~300-430+ I wonder if, from a drug discovery perspective, we shoudl consider using truncated versions of murC/D/E to obtain more stable crystals for use in X-Chem and other crystallography efforts.
As I recall, I and fairly certain that all of the selections that Becca and I made were targeting the ADP site and not relying on interactions with mur ligase residues in the 300-430 range.
Maybe worth discussing making the truncated mur ligase clones and seeing if they lead to more stable crystals.
Here are the clones SSGCID used for Pae murC with truncation 4 leading to the x-ray structure 6x9n
Yuhang-CADD
mattodd
jhjensen2
Date: Mar 8th Time: 2pm UK time (other times)
Place: https://ucl.zoom.us/j/91379419977
Recording: https://youtu.be/Px2cmZKKtBE Previous Meeting: #66
From Last Time:
As mentioned in #62 the two key experiments are the first two items here:
1) Elaborated Fragments Inhibiting Two Murs
@Rebecca-Steventon had completed the dose-response experiments with the @danaklug compounds that have been found to inhibit two mur ligases, and presented the data (can be viewed in the recording), which suggested that, of the compounds, OSA759 was the most promising. Follow-ups:
@LauraDS1 presented data last month on the binding of these compounds to protein (see also #68), indicating that no structures with bound ligand have yet been found in these soaking experiments, though protein with added compounds display conformational changes suggesting binding. A partial structure was seen for MurE and OSA749, but the molecule appeared to be binding in the wrong place.
Adrian Lloyd and Anita Catherwood presented data last month on the development of a high throughput mur ligase assay and on the inhibitory values for a known AstraZeneca compound as well as the known inhibitor ADPCP. The IC50 values for the AZ compound were quite different from those published (here, 1.2 uM, in lit 8 nM vs MurC), and it will be important to compare equivalent assays. The AZ compound is clearly not a dual inhibitor, but some effectiveness vs MurE was seen, and was synergistic with ADPCP. In the March meeting this was discussed and it was agreed to ensure that suitable controls are used in future assays, though it was noted there is currently no potent inhibitor known of MurD or MurE. <-- this really ought to become a short-term target!
2) Atomwise Hits
Atomwise have reached out to @mattodd to ask whether follow-up is needed, and @mattodd has replied Yes, please can we have a new company contact.
3) Variants of AZ Compounds
4) New Protein Structures
Summarised in #67. New: Pseudomonas MurD in complex with ADP
There was previous discussion about the significance, or not, of the carbamoylation of a residue (Lys198 (-> KCX198) in a structure (7TI7). Does this influence inhibitor design? See @eyermanncj's analysis.
Also note the points (in comments, below) from @eyermanncj about truncated vs full-length protein for getting structures, of potential relevance to @LauraDS1 @LizbeK @bartrum. In the March meeting this was discussed at some length, upshots being: a) A full-length MurC structure would be useful, to see whether additional contact points were involved in the binding of AZ5595 by MurC, beyond those in the AZ (truncated structure). This had been tried in SSGCID, but it didn't give good crystals. @LauraDS1 will try it with protein she received from SSGCID. Truncated versions of MurD and MurE are feasible, but would only be possible in a few months' time.
5) De Novo Computational Modelling
@jhjensen2 had proposed updated structures here.
6) Other Potential Starting Points
7) Misc
8) Mothballs (if no actions then these need to be linked in wiki and closed)
Next Meeting
April 12th 2pm UK time (other time zones)