Mur Ligase the Christmas Present Meeting December 2022

[Yuhang-CADD]

Date: Dec 13th 2022
Time: 2pm UK time other timezones
Place: https://ucl.zoom.us/j/91379419977
Recording: https://youtu.be/LsM_xhTYAtg
Previous Meeting: #91
Who can come?: Anyone. No need to say anything unless you'd like to.

Apologies: @mattodd late arrival
Decks: Please @opensourceantibiotics/murligase remember that if you share slides/info, to drag and drop those into a comment on this page, below. Very easy and saves @mattodd having to pester you.

Agenda:

Key things today are
i) Multi-targeting compounds identified from Warwick and Atomwise collections. We need a crystal structure of one or more of these multi-targeting hits to pursue grant funding.
ii) Yuhang's "efflux-proof" amine derivative of the AZ compound was found to bind. Does it inhibit MurC?
iii) Is the rest of the Warwick Enamine collection scheduled for evaluation in the enzymatic assay?

(please add if you'd like to prioritise anything)

1) Multi-targeting Compounds from the Warwick/NEU Enamine Collection

• @AJLloyd105 @chrisdowson1 to update on further enzymatic screening of the Warwick Enamine Collection, following discovery of the hits shown here.
• @LauraDS1 to update on any soaking and/or co-crystallisation experiments with Warwick Enamine Collection compounds, following (last update here. Previously @LauraDS1 reported that hits had been found for these compounds by SPR, @LauraDS1 to update. Crucial Question: is there correlation between hits observed by SPR and hits observed by enzymatic screening? See also below for comments on the use of an alternative assay for the Atomwise compounds.

2) Multi-targeting Elaborated Fragments from Diamond Fragment Screen

• @LauraDS1 to update on screening (inhibition, SPR, soak) of elaborated fragments sent by @edwintse in Compound to Warwick Inhibition Assay Jun 2022 #84.

3) Atomwise Hits

• @LauraDS1 is waiting for a new sensor type to redo 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). To Do: these data need to be cross-checked with the new inhibition data presented by @AJLloyd105 slides to see if we have any X-ray structures of these inhibitors. Note that in the initial screen by @AJLloyd105, 15 of the compounds interfered with the coupling enzymes. 14 of those also interfered with an alternative assay (which is...?), but one did not, and has been added to the list of compounds found to be inhibitors in the Atomwise set.

• @yiwei905 to synthesise AW53. Yiwei has started to pursue this compound because it has poor numbers of analogs commercially available. The compound appears to inhibit MurC (Pae), MurD (Eco) and MurE (Eco), which should be checked and explored.

• Residual item to do: @LauraDS1 @LizbeK to clarify, on the wiki here, whether the Atomwise compounds that have been co-crystallised have a binding site that overlaps with the "target site" Atomwise used @LauraDS1 addressed this 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, and all we need now is @LauraDS1 to summarise briefly on wiki what we know.
• Based on the above, and the near neighbours identified by @edwintse, @mattodd to (eventually) follow up with Denzil Bernard about potential additional suggestions from Atomwise (see Mur Ligase Firecracker Meeting November 2022 #91 for details of what Atomwise want to know).

4) Variants of AZ Compounds

• @AJLloyd105 @LauraDS1 to report on enzymatic inhibition data for amine derivatve of AZ5595, which had shown good SPR binding data here. Any promising compounds (SPR + enzymatic) --> SSGCID.
• @mattodd to reach out to groups that may be happy to run accumulation assays, both on the AZ amine and more generally on new compounds made e.g. Hergenrother lab (e.g. student Rebecca Ulrich), Helen Zgurskaya (Oklahoma).
• @Rebecca-Steventon to report on the assay @Yuhang-CADD 's compound (which?) here.
• @chrisdowson1 to work on compound transfer from SSGCID to Warwick and update the group. Does this need an update, or can it be mothballed?

5) New Protein Structures

The PDB list has been updated with the new, recent structures: 8EGM, 8EGN, 8DOF, 8DP2, 8EWA. Jan Abendroth has completed the final SSGCID X-ray collection for MurC, with the series of three AZ compounds bound to this protein. In the two protein chains, the piperidine moiety of the ligand is in two different conformations. Jan's assumption is that the conformation in chain B is the native one, since it does not involve interaction with a symmetry mate. Thank you Jan!

• @mattodd to organise new soak-in meeting to consult on structural biology learnings. This meeting was held on Nov 22nd 2022. Outcomes:

6) De Novo Computational Modelling

• @edwintse @danielgedder to complete synthesis of molecules suggested in Predictive Modelling Competition July Summary #85. (All the Enamine compounds have now been purchased by @edwintse and will be shipped to Warwick in ~ 4 weeks.) Only a few compounds remain, and we need to decide whether to pursue or abandon.
• Last time @LauraDS1 updated on the SPR screen of the competition compounds (Predictive Modelling Competition July Summary #85), data summarised [here](https://github.com/opensourceantibiotics/murligase/wiki/Generative-Modelling-Competition%3A-SPR-results). These data are complete (repeats already done). There are some hits at 100 uM, and one at about 10 uM, which is exciting. Some compounds gave non-specific binding (straight line SPR curve). Obviously no info on where the compounds are binding. Data need adding/linking to the wiki. Next: variants of this SPR-binding compound?
• @LauraDS1 to evaluate the competition compounds in a crystal soaking assay.
  @AJLloyd105 reported that the competition compounds had been screened vs. Pae MurD enzymatically. High levels of inhibition were observed, but the compounds all also interfered with the coupling enzymes used in the assay, unfortunately. Solutions: 1) reduce the concentration of the test compounds to find a window where the assay works, or 2) find a different assay. @AJLloyd105 to update. In this meeting Should be fine to not worry about off-target effects for now but should be aware that things aren't 100% specific and shouldn't assume they would be.
• @Yuhang-CADD to report on progress towards @jhjensen2's originally-suggested compounds (last update was [here](Mur Ligase Online Research Meeting May 2022 #75 (comment))

7) Other Potential Starting Points

• @chrisdowson1 to summarise status of LifeArc projects, both the previous collaboration and the more recent fragment screening campaigns (with Peter Coombs). Data for the latter has been transferred to Warwick. @mattodd to clarify that all parties are happy for the data to be released into the public domain. @mattodd awaiting reply.
• Assuming above OK, update to be presented by someone from the Warwick team on enzymatic testing of the LifeArc fragments (which were discovered via an SPR screen). No structural information yet.
• @chrisdowson1 to report on progress towards securing the [Merck Open Global Health Library](https://www.merckgroup.com/en/research/open-innovation/biopharma-open-innovation-portal/open-global-health-library.html).

8) Misc/AOB

Last time @AJLloyd105 reported on the evaluation, enzymatically, of "double-headed" compounds containing uridine and ATP binders: . These were found to be binders and will be investigated further (need to upload deck). @AJLloyd105 to update as appropriate.

• @mattodd to post GRC conference slides somewhere (28 MB...).
• We need a volunteer to reach out to [CC4CARB](https://www.cc4carb-collection.org/) to assess whether we are suitable for a proposal.

9) List of any Raw Data That Needs Posting Online

Suitable locations: ideally an electronic lab notebook, another suitable repository (Zenodo, Figshare) or Github itself.

• @Rebecca-Steventon - dose-response data for potential dual inhibitors OSA742, OSA754, OSA759, OSA789 that was summarised in [this graphic](Mur Ligase Online Research Meeting February 2022 #66 (comment))

9) 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](https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0152075) 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](https://dx.doi.org/10.1038/srep16685)](https://dx.doi.org/10.1038/srep16685) is a “recent” NMR study on E. coli murD."
• @mattodd @chrisdowson1 have parked the idea of a Euro Lead Factory screen.
• Do we need to consider the importance of the observed carbamoylation of a residue (Lys198 (-> KCX198) in a structure (7TI7). Does this influence inhibitor design? See @eyermanncj's [[analysis](https://github.com/Feb 2022 Update on Recent Mur Ligase Crystal Structures #67#issuecomment-1054777581)](Feb 2022 Update on Recent Mur Ligase Crystal Structures #67 (comment))
• @eyermanncj and @chrisdowson1 to speak to the benefits of ordering the Enamine kinase library. Should this be mothballed?

Next Meeting

Jan 10th 2pm UK time

• @mattodd to send out diary invites for 2023.
• Everyone to eat many mince pies

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 always open.

[mattodd]

Hi all - I'm going to be late to today's meeting. Please start without me @Yuhang-CADD @chrisdowson1 @LauraDS1

[Yuhang-CADD]

Progress:

Novel compounds shipped:

In progress:

The pyrazole ring (marked in yellow background) fell off when doing TFA deprotection, likely that this molecule not quite as stable as the other variant structures. The pyrazole amine came off LCMS and HPLC columns together with the product, thus unable to obtain the pure product. .

[danielgedder]

Progress: I am currently working on the synthesis of oxadiazole (conversion of the bromine to an amine using Gabriel's synthesis). Commonly, the yield obtained in this step is low and I might try other methodologies to convert the Br to NH2, for example using the reduction of the azide.

For the imidazole fragment: I needed to change the strategy to synthesize it again. The imidazole amine was very polar. I got 4 spots in TLC. All purified and analyzed by NMR, intermediate still impure. Not sure if that is the compound. I decided to start the synthesis protecting the imidazole with Boc, I purchased the amine boronic acid and I am waiting for delivery.

[danielgedder]

I've spoken with @yiwei905, and she has done some retrosynthesis searches for other sets of compounds. I will work on AW9 and J06 too.

For J06, I've found a Tetrahedron paper that might work for the synthesis of tetrazole aniline. I will try that.