Mur Ligase Online Research Meeting Oct 2021

[mattodd]

Meeting time: Tue Oct 5th 2pm London
Location: https://ucl.zoom.us/j/98356058106
Recording: https://youtu.be/6hqsapouJEU
Meeting follows on from #49 and #50

Mur ligase minutes

1. Laura XChem screen of Atomwise compounds vs EcMurE. In progress, reported in the wiki. Binding of several to an allosteric site. Does this site overlay with the fragment hits? Yes, some of them.
   Next:

• 2D maps of how the molecules are interacting with the protein (@LauraDS1)

Chris/Laura have done overlay with same protein with ADP bound (no inhibitors). Show: helix moves inward when ADP binds. So Q: in presence of ADP, is the pocket still available for inhibitor binding? This may of course influence subsequent compound prediction by Atomwise. The soaking experiment still provides enough flexibility for such changes (helix motion) to take place.
Next:

• @LauraDS1 to do SPR binding assays with MurC, MurD in presence and absence of ADP/AMPPNP (SPR for mur ligases #55)

• @Rebecca-Steventon to run activity assays.
  Laura has enough compound for follow-ups (with the actives? Would be good to use all, if possible, since there could be inhibitors that have not been shown to bind).

• Action on @mattodd: Contact Atomwise: are these binding to the same site that Atomwise designed for? Atomwise original proposal location: ?? Lizbe mentions that the allosteric site was only revealed after we asked Atomwise to predict molecules, so would be odd if they were designed to hit that pocket.

2. Becca assays on original fragments found to bind in “allosteric” site. The compounds giving inhibition don't have crystal structures (these are needed), and the compounds with crystal structures don't inhibit. So there is the possibility that the "allosteric site" it not relevant to inhibition. This needs resolving.

3. Dana/Kato analysis of MurD/MurE dual inhibitors. Early SAR analysis based on MurD % activity results: see Kato slides below. Chris D noted that highly lipophilic compounds do not historically get into bacteria, something to keep in mind for future design.
   Next:

• @Rebecca-Steventon dose response experiments in progress.
• @danaklug to post structures of the hits and the non-hits. These structures to be forwarded to Jan for inclusion in the modelling.
• @LauraDS1 purifying E coli MurD to use in further experiments.

4. Yuhang compounds. AZ compound 4: bound to MurC. Crystal structure resolved (P. aeruginosa MurC, 2.6 A) and final structure coming. SGC received three compounds from AZ (structures in publication/proposal).
   Next:

• @Yuhang-CADD compound structure to be overlayed with AZ5595 (PDB 6X9N) and AZ8074 (PDB 6X9F).
• Peter Horanyi progressing with Acinetobacter and/or Pseudomonas MurD (nano DSF + crystallization).
• @LauraDS1 to share her SPR optimization when in hand.

AZ5595: New route to synthesize correct pyrazole isomer looks to have been successful. To be confirmed.
Next:

• @Yuhang-CADD to ship to SGC

• @Yuhang-CADD to synthesize amine derivative; see slides below.

• Action on @Yuhang-CADD: Give his structures an OSA compound code.

• Action on @chrisdowson1 to work on compound transfer from SGC to Warwick and update the group.

5. Jan Jensen docking (more discussion at Docking studies on MurC ligase #46). Extra precision results coming for Enamine Hit Locator library (have now been posted?)
   Next:

• @jhjensen2 to get docking poses on top scorers.

AOB: @mattodd - ANTRUK proposal has been funded! 9-12 months bridging funding. @chrisdowson1 - Waiting on response from AZ legal.

Actions from last time

• @Rebecca-Steventon has posted data of @LizbeK fragment screen (https://github.com/opensourceantibiotics/murligase/wiki/Allosteric-Pocket - but @drc007 would like structures to be added).
• @LauraDS1 to continue crystallisation trials of MurD and MurE in Warwick, with the plan to take crystals to Diamond. Streptococcus agalactiae MurD is 1-2 months, and being difficult so this is being parked. E. coli MurE is faster and will be taken to Diamond. @chrisdowson1 says the MurC biochemical assay is not yet on the road, but is certainly coming out of the garage.
• @chrisdowson1 to liaise with others to establish protein needs, and what's needed from SSGCID <-- this is getting mothbally. Needed?
• @LauraDS1 to liaise with Peter and Lizbe to see if a soak of the Joe Enamine compounds is possible at Diamond. <-- this is getting mothbally. Needed?
• @loriferrins pursuing inputs from Cyclica - prediction of actives. Not based on activity (where we do not have many data) but based on crystal structures (as for Atomwise). @eyermanncj did not think there were enough lit data (on binders at the ATP pocket) to build a good machine learning model. The Becca dose response curves may give us that. In the meantime, Peter has structures that may be use to Cyclica. Lori to invite Cyclica to next meeting and put them in touch with Peter Horanyi.
• @chrisdowson1 to maintain interaction with ELF following their interest in the assay/screen we proposed.
• Peter Horanyi to post deck that he presented in Aug. meeting.

Next meeting: Tuesday Nov 9th at 4pm UK
Location: https://ucl.zoom.us/j/93738560190

[KatoLeonard]

Hi everyone,
In the run up to tomorrow's meeting, I made structure-activity relationship diagrams of the murD % activity as a visualisation tool. I based them on the 6 compounds tested by Becca against both murD and murE. I will definitely go over these tomorrow, but you can take a look in advance if you want.

[Yuhang-CADD]

Update on Synthesis:

AZ Compound4 (WYH9-2-P)

Sent by August, Peter has the crystallography data. Needs to be overlayed with AZ5595 in 6x9n as well as AZ8074 in 6x9f (in Pae. MurC)

AZ5595

1. WYH5-X series proven by WYH6-2 not making the right structure of the five-membered group:

2. A different route: protection-methylation-deprotection

3. Ready to make and ship the AZ5595 in two weeks (hopefully)

Future focus: AZ5595 amine derivative

[Yuhang-CADD]

@eyermanncj Further to the conversation, shall we have the information of the 3 structures we ordered from the AZ company? Many thanks!

[eyermanncj]

AZ murC selections.pdf
Compound numbers from AZ paper

[mattodd]

@Yuhang-CADD can you please quickly generate a picture of these structures and paste below?

[Yuhang-CADD]

Just screenshot it from Joe’s pdf...

from Joe:

CN1C(C(C)(C)C)=CC(NC2=C3C(NN=C3)=NC(N[C@H]4C@@HCC5=C4C=CC=C5)=N2)=N1 AZ_13

CN1C(C(C)(C)C)=CC(NC2=C3C(NN=C3)=NC(NC@HCOC(NCCN)=O)=N2)=N1 AZ_22

CN1C(C(C)(C)C)=CC(NC2=C3C(NN=C3)=NC(NC@HCOC(N5CC@@HOCC5)=O)=N2)=N1 AZ_26
(those "at" symbols may be messing that up - need to insert as plain text, if anyone knows how to do that...

[Yuhang-CADD]

@mattodd We were talking about testing the AZ compound 4 (WYH9-2-P),

the compound we sent to Warwick for Pae MurC inhibition (positive control) around August, against two Murligases (MurC and the other one preferably from the same bacterial species: Pseudomonas aeruginosa)

And thanks to @LauraDS1 for offering to do SPR assay for this idea.

@mattodd Could you please confirm that's all we wanna do right now?

[mattodd]

How best to use the compound? Well, we're always aiming to establish activity vs two mur ligases, where "activity" means crystallographic binding or enzyme inhibition (better than micromolar). We'll soon be receiving the Xstal structure of Yuhang's compound bound to enzyme (which one, again, @Yuhang-CADD?) from Peter Horanyi. Supplementing that with biochemical data would be fantastic. In an ideal world, we'd be able to verify data vs MurC (since this compound was reported in 10.1021/cb500360c as active (compound 4, above)) and secure new data vs another Mur of the same species, to make maximal use of the sample. I don't think this compound 4 has been evaluated vs another Mur, right?
So @LauraDS1 what's possible for you here with the sample that you already have? Biochemical assay vs MurD/E (on the understanding that the AZ team already did MurC), or SPR/Cryst using this compound, again vs more than one mur?
Pinging @eyermanncj just FYI.

[Yuhang-CADD]

@mattodd Peter is currently modifying the crystal stucture of Pseudomonas aeruginosa MurC with AZ Compound 4 (WYH9-2-P) in it.