Mur Ligase Online Research Meeting June 2022

[mattodd]

Date: June 14th 2022
Time: 2pm UK time (other times)
Place: https://ucl.zoom.us/j/91379419977
Recording: https://youtu.be/FeKllES0QaU
Previous Meeting: #75

Apologies:
Decks: Please remember that if you share slides/info, to drag and drop those into a comment on this page, below.

Agenda:

1) Elaborated Fragments Inhibiting Two Murs

• Adrian Lloyd to update on screening of the Warwick Enamine Collection
• @chrisdowson1 to pester Adrian to get a Github account and send the ID to @mattodd... Done! Welcome @AJLloyd105
• @edwintse to update on completion and shipping of elaborated structures derived from existing MurD/E hits shown here. Warwick to perform inhibition assays, then SPR, then crystal soaking on any relevant compounds.
• @Rebecca-Steventon @LauraDS1 Do we know if these dual inhibitors are (OSA742, OSA754, OSA759, OSA789) are ATP-competitive?
• @LauraDS1 @LizbeK to update on recent fragment soaking and/or co-crystallisation experiments (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). Update also needed on structure determination experiments with newly-synthesised compounds in Fragment hit to lead - Round 1&2: crystallography  #68 during @KatoLeonard visit to Oxford in March. Complete - see Mur Ligase Soak-In Wednesday June 8th 2022 #80
• @LauraDS1 to ship some UMA to SSGCID to see if that assists with generating good crystals.

2) Atomwise Hits

• @mattodd to follow up with Denzil Bernard about potential additional suggestions based on what has been found to date.
• @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). Done?
• @LauraDS1 @Rebecca-Steventon @chrisdowson1 to obtain inhibition data with the Atomwise compounds, to verify whether binding gives inhibition. Done?
• @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 on wiki what we know. Was this done? Very useful to have this on the wiki, if we do not yet have it.

3) Variants of AZ Compounds

• @Yuhang-CADD to update on the synthesis of amino-AZ5595 and the amino derivative of that compound.
• @Yuhang-CADD to update on structural determination using microED of several OSA compounds sent to the Rosalind Franklin Institute
• @Yuhang-CADD to consider alternative/additional strategies for improving accumulation, and suggestions are welcome.
• @Rebecca-Steventon to report on the assay @Yuhang-CADD 's compound here.
• @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. Not clear. @Yuhang-CADD to resolve with @eyermanncj and Jan Abendroth and report back in June.
• @Yuhang-CADD to ship AZ5595 to SSGCID. This is as a control, for crystallisation work. Paused: we ideally 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. Does this need an update, or can it be mothballed?

4) New Protein Structures

As per update from Jan Abendroth in #80, new structures obtained for MurD + UMA?

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, or can this be mothballed?

A discussion of the possible value of truncated structures will be had as part of the Soak-In (being scheduled by @mattodd)

5) De Novo Computational Modelling

Competition launched and is live at #69.

Note @LauraDS1 @Rebecca-Steventon there is a live question for you here from @GemmaTuron.

@Yuhang-CADD to report on progress towards @jhjensen2's originally-suggested compounds (last update was here)

6) Other Potential Starting Points

• @chrisdowson1 to report on progress towards securing the Merck Open Global Health Library.
• @eyermanncj and @chrisdowson1 to speak to the benefits of ordering the Enamine kinase library. Should this be mothballed?
• @chrisdowson1 to update on access to compounds developed/evaluated in the LifeARC project.

7) Misc/AOB

• @edwintse to clone Open Source Malaria Logo and Poster OpenSourceMalaria/TechOps#4 for OSA so that people can "advertise" their participation.
• Future involvement with SSGCID. @bartrum potentially to report.
• We need a volunteer to reach out to CC4CARB to assess whether we are suitable for a proposal.
• A Soak-In is being organised, and we will report back in this meeting.

8) List of 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.

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 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."
• @mattodd @chrisdowson1 have parked the idea of a Euro Lead Factory screen.

Next Meeting

July 12th 2pm UK time

[mattodd]

Update by email from Becca May 12th "This week I have run a competition assay with Dana’s compound 759 to try and determine the mode of inhibition it is having on the Mur ligases.

From this assay system, it appears that 759 is acting in a non-competitive manner to ATP. To get a clearer picture of whether it is still binding in the pocket adjacent to the ATP binding site, or somewhere else on the enzyme we will still need crystallography.

Hope this helps to start answering the question of where these compounds are binding. "

Very interesting, have requested data.

[AJLloyd105]

120422 corr. 130422 updated 140622-2.pptx

[AJLloyd105]

Hopefully the attached is useful.
Best Wishes,
Adrian