Mur Ligase Online Research Meeting April 2022

[mattodd]

Date: Apr 12th
Time: 2pm UK time (other times)
Place: https://ucl.zoom.us/j/91379419977
Recording: https://youtu.be/4wkvmuuWJSA
Previous Meeting: #70

Note - updates from this meeting have been incorporated (by @mattodd) into the minutes for the subsequent meeting (#75), for simplicity.

Today's host will be @chrisdowson1, recording managed by @KatoLeonard. @mattodd sends apologies (under English Channel), @edwintse and @Yuhang-CADD might join remotely (vacations).

Agenda:

1) Elaborated Fragments Inhibiting Two Murs

• @Rebecca-Steventon to report on new inhibition or dose-response data from March and to post data either below or in Mur Ligase Online Research Meeting March 2022 #70.
• @KatoLeonard @edwintse to update on current synthetic targets related to elaboration of existing MurD/E hits derived from @danaklug elaborated fragments. Compounds were proposed here. Updates?
• Potential follow-up experiments would be to determine if these are competitive inhibitors (with ATP) - still scheduled? @Rebecca-Steventon @LauraDS1
• @LauraDS1 to update on fragment soaking and/or co-crystallisation experiments, following earlier experiments that had indicated conformational changes suggestive of binding (a partial structure had been seen for MurE and OSA749, but the molecule appeared to be binding in the wrong place).
• @KatoLeonard to report on structure determination experiments with newly-synthesised compounds in Fragment hit to lead - Round 1&2: crystallography  #68 during visit to Oxford in March.
• @chrisdowson1 and Adrian Lloyd (got Github account?) to update on status of Warwick assays. We had previously discussed the need for consistent controls, and the issue that there is currently no potent inhibitor known of MurD or MurE.
• Please can all those who present data share their decks here.

2) Atomwise Hits

• @LauraDS1 to complete SPR experiments (MurC, MurD in presence and absence of ADP/AMPPNP) on Atomwise compounds (SPR for mur ligases #55 and on wiki), following the previous crystal structures of several compounds bound (see also Last XChem EcMurE results uploaded #52).
• @LauraDS1 @Rebecca-Steventon @chrisdowson1 to obtain inhibition data with the Atomwise compounds, to verify whether binding gives inhibition.
• @LauraDS1 @LizbeK to check whether the "target site" Atomwise used included residues in the allosteric pocket where the compounds were found to bind? @LauraDS1 addressed that here and in the March meeting there was discussion that the Atomwise compounds did bind in a partial overlap with the intended binding site. <-- this has been clarified as part of Predictive Modelling Competition to Design Binders of MurD, an Antibacterial Target #69, @LauraDS1 to summarise what we know.
• Atomwise had a call with @eyermanncj @LauraDS1 @mattodd and provided a new contact - it's on @mattodd to follow up.

3) Variants of AZ Compounds

• @Yuhang-CADD to update on the synthesis of AZ5595 and the amino derivative of that compound (intended to demonstrate activity vs wild type bacteria). Some compounds have been shipped to the Rosalind Franklin Institute to obtain microED structures MicroED Experiments for MurC project #65 and this work is progressing/ongoing.
• @Rebecca-Steventon was going to assay @Yuhang-CADD 's compound here, shipped in August.
• @Yuhang-CADD compound structure (AZ cmpd 4) to be overlayed with AZ5595 (PDB 6X9N) and AZ8074 (PDB 6X9F) <-- @Yuhang-CADD to clarify what has been done and what still needs doing.
• @Yuhang-CADD to ship AZ5595 to SSGCID. This is as a control, for crystallisation work. Ideally we ship other compounds at the same time, e.g. the compounds mentioned here, if @eyermanncj can show they dock well.
• @chrisdowson1 to work on compound transfer from SSGCID to Warwick and update the group.
• "Compound W" sent to Warwick is not AZ5595, but another AZ compound, also potent. It will be useful to refer to these unambiguously by their OSA codes from now on: @Yuhang-CADD to insert the correct OSA codes here (OSA_001044), and link to where the structures are shown.
  

4) New Protein Structures

Summarised in #67. New: Pseudomonas MurD in complex with ADP. Have there been other new structures?

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. This question remains live.

There was previous discussion of full length vs truncated structures. 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

Competition launched and is live at #69. Antibiotics Research UK had kindly permitted a no-cost extension of the funding to @mattodd @chrisdowson1 and @phraenquex to the end of Jan to allow for this competition to be run before the end of June. All: please pass this competition on to any contacts in the machine learning/AI field, though note that we are specifically looking for generative methods, i.e. not large-scale docking of commercial libraries.

@jhjensen2 @cstein had proposed updated structures here, and will (?) generate new suggestions for competition.

• @Yuhang-CADD has examined these compounds for purchase/synthesisability and determine a way forward and will post the current shopping/synthesis list.

6) Other Potential Starting Points

• @loriferrins has suggested we apply to screen the Merck Open Global Health Library. Who would apply (who has the fastest contracts office?), and would this be in vitro (e.g. MurC) or vs e.g. MRSA? They provide "30 µl in a concentration of 10 mM in a solution of dimethyl sulfoxide (DMSO)". Crucially "You retain all rights to results and IP". Seems sensible that we ask warwick to screen the compounds if @chrisdowson1 can start the paperwork?
• @mattodd @chrisdowson1 to update on status of Euro Lead Factory screen, following discussion with staff at Lygature.
• @eyermanncj and @chrisdowson1 to speak to the benefits of ordering the Enamine kinase library.
• @chrisdowson1 to update on access to compounds developed/evaluated in the LifeARC project

7) Misc/AOB

Anyone else want to add anything not dicussed above?

• @edwintse to clone Open Source Malaria Logo and Poster OpenSourceMalaria/TechOps#4 for OSA so that people can "advertise" their participation.

8) Mothballs (if no actions then these need to be linked in wiki and closed)

• @Rebecca-Steventon has posted data of @LizbeK fragment screen (https://github.com/opensourceantibiotics/murligase/wiki/Pocket-binding-site - but @drc007 would like structures to be added).
• @LauraDS1 to liaise with Peter and @LizbeK to see if a soak of the @eyermanncj Enamine compounds is possible at Diamond.
• Comparative analysis was done by @ZigBu of mur ligase ATP binding sites (Comparison of the ATP Sites/Structural Similarity Between Murs #56). Any learnings?
• @ZigBu also posted (UCL Mur Ligase Local Team Meetings November 2021  #57) on a paper highlighting that mur domain movement may not be dependent on substrate binding. Again, anything we need to consider? @eyermanncj posted "As a reminder the dynamics of E. coli murD is well established. Here is a “recent” NMR study on E. coli murD."

Next Meeting

May 10th 2pm UK time

[Yuhang-CADD]

Current update:

1. AZ5595 (OSA_001051) synthetic conditions modified with a much-improved yield
   

2. Still working on WYH21-X
   

3. Synthetic routes for Jan's predictions (we only managed to secure the routes for 3 predicted structures with purchasable starting materials):
   
   

[jhjensen2]

Great. It would be interesting to know why the synthetic routes for the other molecules were not feasible. Perhaps we can modify our workflow.

[Yuhang-CADD]

Hi Jan!

Thanks for the bringing this up!
Because the rest of the predictions did not have fragments quite available to purchase or/and synthesise. Plus, the on-demand synthesis prices were really high. So we were really sorry that some of those good predictions have to be put on the waiting list.

[jhjensen2]

No worries. It's good to know that the retrosynthesis program is a bit overoptimistic wit regard to availability. I think it is possible to automatically include pricing when selecting paths and we might look at that in the future.

[Yuhang-CADD]

Yeah, me and Matt were also talking about this: whether we could possibly set up a programme like COVID moonshot, providing purchase links or prices of the fragments predicted/in database.

Thanks a lot for the effort Jan, the predictions really eased our synthetic planning process.

[jhjensen2]

Yeah, me and Matt were also talking about this: whether we could possibly set up a programme like COVID moonshot, providing purchase links or prices of the fragments predicted/in database.

That should be possible. We'd be happy to help with that.

Thanks a lot for the effort Jan, the predictions really eased our synthetic planning process.

Great, happy to hear it

[KatoLeonard]

[Niladri31005]

[Niladri31005]

My PhD work was on designing dual inhibitor targeting murD(3LK7 homology model) and murE(4C13) for s.aureus (ATCC-43300).we have designed similar type of scaffold covering the substrate,ATP and Mg++ site (lys-198, carbamylated). We checked the Mic and time kill study against s.aureus (43300) but unable to do the assay for COVID-19 lockdown. Time killing was exceptional for one series. But ADMET was poor. Can I get suggestions and l used Schrodinger.

[mattodd]

Hi @Niladri31005 - thanks for reaching out. Do you have any links to data you can share, so we can see the kinds of things you've done in more detail?
Everyone else - I'm uploading the recording (https://youtu.be/4wkvmuuWJSA) and will take a look to catch up on what happened, unless anyone wanted to flag up any big news/action items.

[Niladri31005]

Extremely sorry for delay....if you can give me some more days for uploading the data. As my PI is not convinced to disclose the structure ( applying for patent as it is government funded project)but I can sare the time-kill picture if you want. Still l will try to convince him, if you give assurance. But we have not done the enzyme assay but did the time kill using MRSA (ATCC 43300).