Mur Ligase Online Research Meeting May 2022

[mattodd]

Date: May 10th
Time: 1pm UK time (other times)
Place: https://ucl.zoom.us/j/91379419977
Recording: https://youtu.be/MGMRfm4wzyg
Previous Meeting: #71

Apologies: @jhjensen2
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

• @Rebecca-Steventon to post data on dose-response data from March (for potential dual inhibitors OSA742, OSA754, OSA759, OSA789) and to post data either below or in Mur Ligase Online Research Meeting March 2022 #70. Update: The data have been summarised in this graphic. The image has been copied over to the wiki here. Ideally we have access to the raw data behind the image @Rebecca-Steventon, is that available?
• @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 and there is an update below. @edwintse aiming to complete the purification in the next week for shipping to Warwick for inhibition assays, then SPR, then crystal soaking on any relevant compounds.
• Potential follow-up experiments would be to determine if these (the dual inhibitors that have shown enzymatic inhibition, OSA742, OSA754, OSA759, OSA789) are competitive inhibitors (with ATP). These are 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). May update: this is ongoing. Attempts are being made with UMA and ADP, since these are included in the known PDB crystal structures. Crystals/protein have been obtained with E. coli MurD and MurE.
• @LauraDS1 to ship some UMA to SSGCID to see if that assists with generating good crystals.
• @KatoLeonard @LizbeK 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. May update: @LauraDS1 will upload a brief report on what was found, but no useful structures obtained.
• @chrisdowson1 and Adrian Lloyd (Action: get 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. The Enamine library (Warwick Enamine Collection, designed by @Rebecca-Steventon and @eyermanncj) is being evaluated, and there are some compounds giving dual inhibition and even tri-inhibition. In April, Adrian reported on the good data for compound LO6 and JO6 (see recording). Adrian to report at the next meeting.

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). Has this now been 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 what we know. Was this done?
• Atomwise had a call with @eyermanncj @LauraDS1 @mattodd and provided a new contact (Denzil Bernard) - 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. May update is provided below. The amino-AZ5595 has been obtained as the Boc-protected derivative, and this is now being deprotected. Other amines can be explored in this position. The aim with this molecule is to improve accumulation of the compound by virtue of the primary amine. @Yuhang-CADD is also considering alternative strategies for improving accumulation, and suggestions are welcome.
• @Rebecca-Steventon was going to assay @Yuhang-CADD 's compound here, shipped in August. May update: this is still scheduled.
• @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.
• @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.

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 under consideration, with no clear answer yet.

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. First entries have been coming in from @vandan-revanur @finlayiainmaclean. 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 has generated new suggestions for competition. (<- corrected after @jhjensen2 comment below).
@Yuhang-CADD has examined @jhjensen2's original compounds for purchase/synthesisability and has determined a way forward and will post the current shopping/synthesis list. May update: several of the starting materials have been ordered and have arrived at UCL. These will be checked and then the syntheses begin by @Yuhang-CADD.

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? May update: @chrisdowson1 is now in touch with the Merck group and is securing the library.

@mattodd @chrisdowson1 have parked the idea of a 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.

New participants e.g. @Niladri31005 always welcome to join these meetings.

There was a discussion of compound numbering conventions. It was felt that there was a need for a systematic numbering system (this already exists and is being used) as well as a more colloquial system that maintains the provenance of molecules. For the latter, any "bespoke" code can be used in the Master List (column J), and there are other columns that may be used too. Maintaining a strong link between molecule and contributor is core to the idea of open source science.

Future of SSGCID - this is currently being decided. Jan Abendroth's participation will cease to be funded at the end of August, so further participation after that date will be voluntary. If SSGCID is funded, then OSA will continue to be an affiliated project without the need for resubmission.

Funding. @eyermanncj suggests NIH R21 leading to R01. Carb-X seen as being further downstream. @chrisdowson1 and @mattodd to consider UK/EU based options, but we will need decent MIC values/dual inhibitors for that. We need also to investigate the potential for resources from CC4CARB - anyone have a connection there who might be willing to discuss?

A Soak-In. A separate meeting towards the end of May to discuss ways towards new crystallography data on OSA molecules was thought to be a good idea. @mattodd to organise. @LauraDS1 and others to (very briefly) summarise what has been attempted, so that gaps in approaches/systems could be identified. Are there others who might want to come along?

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

June 14th 2pm UK time

[jhjensen2]

To clarify regarding 5) De Novo Computational Modelling.:

@cstein and I have submitted entries to the competition here issue 72

The updated structures referred to above (issue 46) are for the AZ8074 binding pocket of MurC.

I am a little unsure what this means "and will (?) generate new suggestions for competition" but we are happy to suggest even more compounds based on feedback on our submissions so far.

[KatoLeonard]

Since my work on the project is coming to an end, I wanted to give a quick overview of what I've accomplished over the past few months. I designed 22 compounds that are intended to bind the allosteric pocket of at least two Mur ligases.

Compounds OSA_1054 - 1061 of Elaboration Round 2a were based on original MurD fragment hit 373 (or OSA_2) and the SAR analysis of Hit-to-Lead elaboration round 1. An attempt to detect binding by X-ray crystallography has already been made, whereby the compounds were soaked with E. coli MurD and MurE crystals, but unfortunately without success (big thanks to @LizbeK and @LauraDS1 for their wonderful support and guidance!).

In the meantime, dose-response curves were obtained for 4 of the dual inhibitors, carried out by @Rebecca-Steventon. OSA_759 was found to have a promising IC50 value of 21.6 μM. This was the starting point for the design of compounds OSA_1062 - 1070 and OSA_1088 - 1092 as part of Elaboration Round 2b.

Compounds in green are fully characterised and ready for crystallography (co-crystals and soaking, if not already done), enzymatic assay and SPR. Compounds in orange need further purification. Compounds in grey are being synthesised by @edwintse.

[Yuhang-CADD]

1. AZ5595 and its amine derivative

1.1 AZ5595/OSA_001051 (optimum conditions obtained)

1.2 AZ5595 derivative (1st step, optimum conditions obtained)

2. AZ cpmd 4 (WYH9-2-P, OSA_001044) to be overlayed with AZ5595 (PDB: 6X9N) and AZ8074 (PDB: 6X9F)

2.1 Peter was on this project, and he managed to visualise the position of AZ cpmd 4 in the Pae MurC binding pocket. Need help with visualisation of this compound overlaying with AZ5595 (PDB: 6X9N) and AZ8074 (PDB: 6X9F) @dr. Jan Abendroth.

3. Need help from @eyermanncj about docking the following two compounds into Pae MurC binding pocket to see if there is a potential to build up a new virtual hit library.

4. WIll ship AZ5595 (10 mg per vial) together with OSA_001042 and OSA_001049 (10 mg per vial) to Dr. Jan Abendroth and Laura respectively this week.

5. Shopping/synthesis list for @jhjensen2 predictions:

[Yuhang-CADD]

Just an update on the crystallography progress for AZ cmpd 4 (also referred as WYH9-2-P, OSA_001044):

1. We were expecting this AZ cmpd 4 to interact with Pae. MurC binding pocket in a similar manner as AZ5595 (6X9N) and AZ8074 (PDB: 6X9F) do.
2. Peter will help deposit the PDB structure of Pae. MurC with this AZ cmpd 4 this week.