Mur Ligase Online Research Meeting August 2022

[mattodd]

Date: August 9th 2022
Time: 2pm UK time (other times)
Place: https://ucl.zoom.us/j/91379419977
Recording: https://youtu.be/eDAVZVc3p-M
Previous Meeting: #86

Apologies: @LizbeK @chrisdowson1
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 reports from Warwick on the evaluation of Enamine compounds, and any related crystallography, the evaluation of Yuhang's amine compound, new mur ligase fragment screen data from LifeArc and a new SSGCID Pae MurD structure.

1) Elaborated Fragments Inhibiting Two Murs

• @AJLloyd105 @chrisdowson1 to update on further screening of the Warwick Enamine Collection, following discovery of the hits shown here.
• @eyermanncj @LauraDS1 to update on any additional near-neighbour compounds that have consequently been ordered/evaluated. In meeting: no new compounds have been ordered, only some have been re-ordered.
• @LauraDS1 to update on screening (inhibition, SPR, soak) of elaborated fragments sent by @edwintse in Compound to Warwick Inhibition Assay Jun 2022 #84 In meeting: will be run in ATP-competition experiments.
• @LauraDS1 @LizbeK to update on any further fragment soaking and/or co-crystallisation experiments. In meeting Laura presented the slide shown below (from 12:20 in meeting recording). Summary: no structures crystallised yet with Enamine structures bound. @eyermanncj mentioned the possibility of trying a +ve control compound such as ADP. Laura will do this once more protein is obtained. @eyermanncj mentioned a possible diffusion system (using capillaries) but Jan said such a thing was quite finicky.

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). Hasn't this been done and will be uploaded @LauraDS1 ?
• @LauraDS1 @Rebecca-Steventon @chrisdowson1 to obtain inhibition data with the Atomwise compounds, to verify whether binding gives inhibition. In meeting - first screen done, but need to be re-run. To report back at next meeting. Inhibition will lead to new conversation with Atomwise.
• 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.

3) Variants of AZ Compounds

• @Yuhang-CADD to update on the shipping of AZ5595 and the amino derivative of that compound (OSA1095). In meeting Yuhang to ship ASAP. There was some discussion of how much sample is needed for microbiology, which was apparently 50-60 mg, which seemed very high. After the meeting it was clarified that what was needed was: 10 mg of compound for MICs and mutagenesis, 3 mg for MICs only, 5-6 mg for assays and initial crystallisation if needed. In any case it makes sense to check the in vitro assay first. If compounds hit, then microbiology. 5 mg plenty (to @bartrum) for soaking at SSGCID. Note: Jan's involvement ends at the end of August, but @bartrum HAS BEEN REFUNDED (WOOHOO!).
• @Yuhang-CADD to post a summary of which of his molecules have been shipped where, along with their local and OSA codes. This is important since there may be some confusion about which molecule went where, and we'd like to be sure that the molecules have been evaluated. Was done here - @Yuhang-CADD please add this info to wiki here with a little bit of explainer text and a link to previous meeting Mur Ligase Online Research Meeting July 2022 #86.
• @Yuhang-CADD to consider alternative/additional strategies for improving accumulation, and suggestions are welcome. DONE, but we should make a connection with a lab that can do accumulation assays, e.g. Hergenrother? To be discussed. In meeting: Which groups should we work with, and should we be building in accumulation earlier in the molecule design phase? e.g. Hergenrother (e.g. student Rebecca Ulrich), Helen Zgurskaya (Oklahoma). An American co-PI can help with NIH grants. Need measurements anyway for @Yuhang-CADD compounds.
• @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.
• @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? Others? Update last time was:

1. Acet MurD with UMA. Got number of crystal forms, all in apo conformation without UMA. Surprising since had worked previously for Psae. In Meeting - not taken further since no UMA bound.

2. Psae MurC - got one molecule per asymmetric unit. Goal to re-grow for soaking with AZ compounds. Was going to get those re-screened and do soaking (had seen good thermal shifts for). In meeting - presented structure that has been obtained with one AZ compound (AZ13644908) (screenshot below) and will submit for peer review. More structures coming, hopefully! Crystals melted upon soaking. Took careful titration of time and salt conc of compound. The "one molecule per asymmetric unit" was not replicated, and instead the usual 8 molecules per unit was found. Again, flexible side chain found pointing out into solvent.

New structures:

3. Pae MurD with UMA PDB ID: 8DP2
   Structure deposited on: 07/14/2022
   SSGCID PROTEIN ID: PsaeA.17938.a.B2.PS38065
   Ligand(s): UMA
   Structure of:UDP-N-acetylmuramoylalanine--D-glutamate ligase (EC 6.3.2.9) (D-glutamic acid-adding enzyme) (UDP-N-acetylmuramoyl-L-alanyl-D-glutamate synthetase) (MurD)
   From: Pseudomonas aeruginosa
   SSGCID gene/pathway page: http://apps.sbri.org/SSGCIDTargetStatus/Target/PsaeA.17938.a

Needs to be added to wiki, but we don't have a dedicated page, only an Issue #67.

4. Jan/Peter have submitted the structure of @Yuhang-CADD compound (OSA_001044) bound to Pae MurC:
   Title : Pseudomonas aeruginosa MurC with WYH9-2-P - OSA_001044
   Corresponding authors : Peter Horanyi, Jan Abendroth, Wang Yuhang, Matthew Todd, Isabelle Phan, Peter Myler, Donald Lorimer, Tom Edwards
   Your deposition D_1000267042 is assigned the following accession code(s): PDB ID 8DOF

I think this is held for release, but need not be. @mattodd to clarify with Peter Horanyi and release.

Mothball this? --> 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 was had as part of the Soak-In at #80. <-- conclusions we can summarise?

5) De Novo Computational Modelling

• @edwintse @danielgedder to complete synthesis/procurement of molecules suggested in Predictive Modelling Competition July Summary #85. In meeting: Most molecules ordered or completed. A few are still being synthesised. (Summary in recording, from 34 mins). Some updates posted below. @LauraDS1 to aim for SPR screen in the last week in August (week of 29th?).
• @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. <-- This was discussed at the recent GRC on antibiotics attended by @mattodd and sounds very promising.

7) Misc/AOB

• @mattodd presented the group's work at the Gordon Conference on new Antibiotics. Lots of interest in the science and the open model. @mattodd to follow up and to post the slides somewhere (28 MB...). From meeting: @mattodd to follow up with @chrisdowson1 on the (seemingly) two projects, and which data can be placed in the public domain (Laura has the SMILES), and to ensure written agreement is in place, e.g. with LifeArc.
• Update to be presented in next meeting by someone from the Warwick team on enzymatic testing of the LifeArc fragments (which were discovered via an SPR screen). No structural information yet.
• @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.

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

Sept 13th 2pm UK time. As per @LizbeK request, this meeting will be named. It's the Late Summer Mur Ligase Campfire.

[Yuhang-CADD]

MicroED Samples (Aug Submission)

Microbiology Samples

5 mg each for assay first. On it.

SSGC Samples

5 mg each for soaking experiments. On it.

[danielgedder]

Progress

[chrisdowson1]

Apologies all family commitments to sort out today. Hope the GRC went well Some news to share from this end next time. BW. Chris Get Outlook for iOS<https://aka.ms/o0ukef>

…

________________________________ From: Daniel Gedder ***@***.***> Sent: Tuesday, August 9, 2022 2:40:12 PM To: opensourceantibiotics/murligase ***@***.***> Cc: Dowson, Christopher ***@***.***>; Mention ***@***.***> Subject: Re: [opensourceantibiotics/murligase] Mur Ligase Online Research Meeting August 2022 (Issue #87) Progress [09-08-22-progress]<https://user-images.githubusercontent.com/88835924/183663846-29fd2198-3a26-47fa-aa16-753ae802477c.jpg> — Reply to this email directly, view it on GitHub<#87 (comment)>, or unsubscribe<https://github.com/notifications/unsubscribe-auth/ALNDKBPZXGTFTIS563T7DXTVYJNTZANCNFSM56AHB4VQ>. You are receiving this because you were mentioned.Message ID: ***@***.***>

[drc007]

Minor typo in oxadiazole chemistry, final product should be amine.

…

On 9 Aug 2022, at 14:40, Daniel Gedder ***@***.***> wrote: Progress <https://user-images.githubusercontent.com/88835924/183663846-29fd2198-3a26-47fa-aa16-753ae802477c.jpg> — Reply to this email directly, view it on GitHub <#87 (comment)>, or unsubscribe <https://github.com/notifications/unsubscribe-auth/ABWAURDZOIDTN4SGYWK55ZTVYJNTXANCNFSM56AHB4VQ>. You are receiving this because you were mentioned.

[danielgedder]

Minor typo in oxadiazole chemistry, final product should be amine.
…
On 9 Aug 2022, at 14:40, Daniel Gedder @.***> wrote: Progress https://user-images.githubusercontent.com/88835924/183663846-29fd2198-3a26-47fa-aa16-753ae802477c.jpg — Reply to this email directly, view it on GitHub <#87 (comment)>, or unsubscribe https://github.com/notifications/unsubscribe-auth/ABWAURDZOIDTN4SGYWK55ZTVYJNTXANCNFSM56AHB4VQ. You are receiving this because you were mentioned.

Fixed it, thank you!

[AJLloyd105]

Apologies currently on holiday in Dartmoor Will update when back Best Wishes Adrian

…

Sent from my iPhone On 9 Aug 2022, at 14:40, Daniel Gedder ***@***.***> wrote:  Progress [09-08-22-progress]<https://user-images.githubusercontent.com/88835924/183663846-29fd2198-3a26-47fa-aa16-753ae802477c.jpg> — Reply to this email directly, view it on GitHub<#87 (comment)>, or unsubscribe<https://github.com/notifications/unsubscribe-auth/AY5MUXG2UJE3XMZEHAIHYIDVYJNT3ANCNFSM56AHB4VQ>. You are receiving this because you were mentioned.Message ID: ***@***.***>

[mattodd]

Laura's slide from today

Jan's structure from today

[mattodd]

@danielgedder @edwintse - was just talking to @jhjensen2 (in person!) and he said that he thought that one of the reasons the diol compounds were suggested is that they were commercially available, and that we would not have to make that component ("synthesisability" is part of the in silico assessment). Is that right? Was it unavail/£££?

[danielgedder]

@mattodd, I have checked all the diol derivatives, and to buy the final compounds ready would be super expensive. Actually, a similar compound in Enamine (without the diol portion cost 248 dollars), take a look: https://enaminestore.com/catalog

I also check to buy the amine diol, or any other intermediate that I could access the diol, super expensive. However, I believe I can get these diol done, as soon as stores reopen. I have already identified the issue of the following synthesis: synthesis not working due to the volatility of the silyl intermediate, I lost the compound in the rota vapor. I have obtained a few mmols of this butene amine and I could finish synthesizing one compound.

This is the one intermediate that I got:

I have also the other cores ready or in progress, I will focus only on these intermediate from now on, I believe in a few weeks I can overcome these problems. The biggest problem this week is the store is closed and the starting material to work on the diol synthesis is locked there. I emailed Sonja asking her for help to pick up these chemicals but I have not gotten a response yet.

However, if @jhjensen2 can find any supplier that we can buy it with less cost, let us know because I could not find it.