Mur Ligase the Preholiday Meeting Aug 2023

[Yuhang-CADD]

Date: Aug 8th 2023 Time: 2pm UK time other timezones

Place: https://ucl.zoom.us/j/91379419977

Recording: https://youtu.be/Pkvgaoe6PYs

Previous Meeting: #100 Who can come?: Anyone. No need to say anything unless you'd like to.

Apologies:

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 by SPR, but gave no activity vs wt bacteria (data on [wiki](https://github.com/opensourceantibiotics/murligase/wiki/Microbiology-evaluation-of-WYH-compounds) and a summary of data in #96. Need to complete checks of enzymatic activity of these compounds, to verify the design is OK in terms of enzyme inhibition. (Still awaiting for the IC50 values needed by Yuhang)

iii) When would the rest of the Warwick Enamine collection be scheduled for evaluation in the enzymatic assay? <-- is this still a live question?

(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. An update was given in the last meeting (#100) as follows:

11 compounds from the Enamine Library identified as MurD inhibitors at single conc. IC50s are being determined, with the first five compounds being ready: K16, M23, M17 (good curve, 149 uM), M08 (good curve, 25 uM), A19 (Hill slope < 1, 186 uM). Attention needs to be paid to the Hill coefficient (which should be around 1 for regular inhibition). For the case of high values, cooperative behaviour (usually aggregation) is implied.

Another six compounds to analyse to determine the IC50s, to be discussed at the next meeting. Will take the best compounds and test vs other ligases. Need also to search for what is commercially available and to crystallise the best compounds.

Also need to test the best compounds for ATP competitive binding.

Solubility of these compounds remains sometimes challenging because of precipitation at high concs. Also presence of electrophilic sites in several of the structures. How prevalent are these, and are there negatives containing those motifs? Adrian could repeat the assay in the presence of a thiol to see if inhibition is impacted. Adrian might need to order in fresh samples of the most promising 11 compounds, to be involved also in crystal trials by Laura.

• [ ] @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. Question: Do we have anywhere a comparison of the hits from SPR vs the hits from enzymatic screening? Would be useful. Important update: crystallisation trials for compound J06 (Z2010503124), which has in Laura's hands given: P. aeruginosa MurD IC50 = 16.6 uM; P. aeruginosa MurE IC50 = 12.4 uM; E. coli MurD IC50 = 2.5 uM; E. coli MurE IC50 = 41 uM. @eyermanncj has suggested trying crystallography with this compound with any murC isozymes SSGCID has access to, and murC and murE ADP or ATP structures would we worth trying to get. @LauraDS1 updated last time that E. coli MurD and MurE crystals were being used for soaking and co-cryst experiments. A new crystal was obtained of MurD with compound J06, which was sent to Diamond. Subsequently by email Laura said that there was no diffraction. Ultimately it was felt that E. coli is not working very well as a soaking system, with the compounds "spat out".

Crystallography at The University of Kansas: J06 (Z2010503124) had also been sent to Uni Kansas (Scott Lovell, via Bart).

• [ ] Bart to send J06 for trials (vs what?).

2) Multi-targeting Elaborated Fragments from Diamond Fragment Screen

• [ ] @LauraDS1 to update on screening (inhibition, SPR, soak) of elaborated fragments sent by @edwintse in #84.

3) Atomwise Hits

• [ ] @LauraDS1 was waiting for a new sensor type to redo SPR experiments (MurC, MurD in presence and absence of ADP/AMPPNP) on Atomwise compounds (#55 and on wiki, following the previous crystal structures of several compounds bound (see also #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 AW53 appears to inhibit MurC (Pae), MurD (Eco) and MurE (Eco), which should be checked and explored.
• [ ] @yiwei905 has sent further compounds for evaluation as mentioned below and in #100
• [ ] 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 #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 #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. @Rebecca-Steventon may also have evaluated one of @Yuhang-CADD 's compounds (which?) here.
• [ ] @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).
• [ ] @Yuhang-CADD to update on the Piramal synthesis of key synthetic intermediate.
• [ ] @eyermanncj recommended (in #100) re-testing of the AZ compounds (the ones sent by AZ) in the Warwick assay to resolve the issue of the significant (1000x) difference in potency.
• [ ] @Yuhang-CADD now making further charged derivatives, as described below. In #100 @AJLloyd105 mentioned a paper from Chris Walsh's lab re the addition of charges to improve the scope of an antibiotic. DOI?

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 to update the synthesis of molecules suggested in #85. The progress was last updated in #92.
• [ ] Last time @LauraDS1 updated on the SPR screen of the competition compounds (#85), data summarised here. 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 previous meeting, @AJLloyd105 had evaluated the compounds with a spectrophotometric assay. There is confidence in inhibition arising from OSA-001110 and an IC50 will be generated. Compound 8 also still probably good and will be pursued.
• [ ] @Yuhang-CADD to report on progress towards @jhjensen2's originally-suggested compounds (last update was here.

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.

8) CC4CARB Proposal (#93)

Following discussions between @eyermanncj @Yuhang-CADD and @mattodd a proposal is being written for CC4CARB funding to help with chemistry aimed at exploring more potential binding interactions with the Murs, specifically areas where phosphates normally bind.

• [ ] @Yuhang-CADD to finish figure that outlines the expected CC4CARB contributions as the first step. To include only exemplar chemical structures.
• [ ] Current draft proposal needs editing by Yuhang, Mat and Joe. Also need clarity on who is doing the testing!

9) Misc/AOB

Previously @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...).

10) 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

11) 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 (#56). Any learnings?
• [ ] @ZigBu also posted (#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) is a “recent” NMR study on E. coli murD."
• [ ] 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
• [ ] @chrisdowson1 to work on compound transfer from SSGCID to Warwick and update the group. Does this need an update, or can it be mothballed?

Next Meeting

Sep 12th 2pm UK time

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.

[Yuhang-CADD]

1. AZ Efflux Series Progress: 1.1 Awaiting for IC50s of AZ5595 derivative amines 1.2 Grams of starting materials made or on the way for further hit modifications.

2. CC4CARB Proposal Draft Progress: Finished the 1st draft and is getting modified. 2nd draft on the way. Arranging with Joe @eyermanncj and Matt @mattodd for further guidance.

3. Jan's De Novo Predictions Progress: Still on going, testing conditions. Aiming to ship at least 4 compounds by early September.

4. AZ Multi-targeting Modifications Progress: Will start from September.

[yiwei905]

AW53 derivatives made and sent to Warwick for evaluation + structure I'd make if I had more time.

[mattodd]

Notes from the meeting today.

1) A faulty temperature control has led to loss of crystals in Warwick, so no new results from @LauraDS1. Fingers crossed for a resolution. SPR sensor chip not yet delivered (possibility of Strep-Biotin coupling?)

2) @AJLloyd105 was evaluating 11 promising compounds from the Enamine collection. Remaining IC50s were determined, but nothing surpassed compounds M17, M08 (best), A19. These compounds have been re-ordered in Warwick. @loriferrins and @eyermanncj to order samples from Enamine to send to Uni Kansas (Scott Lovell) for crystal trials with MurD. @bartrum also to send more MurD and MurE (both Ace, Pae) to Kansas. M08 has an IC50 of 25.97 + 4.70 uM with a Hill coefficient of 0.91 + 0.13. The data very nicely fit a simple saturation model with a hyperbolic response of inhibition to [M08].

3) @AJLloyd105 testing @yiwei905 compounds - assay to match the assay that identified these as multi-targeters. @eyermanncj asked about the origin of the multi-targeting hits - these were enzymatic, from @AJLloyd105 e.g. here.

4) @AJLloyd105 mentioned some interest in diadenosine compounds that he mentioned in a previous meeting (can't remember which, can't find) that showed nice results with Pae MurE and which has led Adrian to look at other binucleotides that are commercially available.

5) @drc007 pointed out that the isatin in @yiwei905 final proposed molecule (above) is sub-optimal because of its reactivity and potential PAINS profile.

Other things that came in by email in the last month:

6) @eyermanncj had suggested profiling adenosine in Eco murD to see what potency it has and whether Eco murD crystals with it bound can be used to ‘back soak’ some of the Enamine fragments? The hope is that the Enamine fragments could be used at much lower concentrations for these experiments – maybe 125-250 uM. Adenosine is cheap.