Mur Ligase the Early Summer Tune Meeting June 2023

[Yuhang-CADD]

Date: June 13th 2023
Time: 2pm UK time other timezones

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

Recording: https://www.youtube.com/watch?v=O5lk-p7UiJA&list=PL0eLxnHhou_k1Upbn5X1mdHBwKMYkRpLH&index=1

Previous Meeting: #97
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 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)

iii) When would the rest of the Warwick Enamine collection be 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. 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 Compound to Warwick Inhibition Assay Jun 2022 #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 (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 AW53 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](Mur Ligase Online Research Meeting February 2022 #66 (comment)) 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. @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.

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 Predictive Modelling Competition July Summary #85. The progress was last updated in Mur Ligase the Christmas Present Meeting December 2022 #92.
• Last time @LauraDS1 updated on the SPR screen of the competition compounds (Predictive Modelling Competition July Summary #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.

9) 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 to assess whether we are suitable for a proposal.

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 (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.
• 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
• @eyermanncj and @chrisdowson1 to speak to the benefits of ordering the Enamine kinase library. Should this be mothballed?
• - [ ] @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

July 11th 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]

#93 has been updated with a summary picture, need guide from @eyermanncj and @mattodd.

[mattodd]

Re Enamine compound screening, @AJLloyd105 very kindly shared the attached deck by email on 2/6/23.

020623.pptx

@eyermanncj asked the following:

Q. Are the percent inhibition data at 500uM?
A. Inhibition (%) in the presence of 0.5 mM compound measured relative to a DMSO control.

Q. And you want low percent inhibition of PNP and high percent inhibition of MurD in the PNP/MESG assay?
A. Crudely, yes. However its’s a bit more nuanced than that. In the stopped PNP/XO/HRP 10 ul assay, there appeared to be little tolerance for inhibition of the coupling system, where impact here impacted the assay. Therefore by this measure, all 11 compounds would be discarded. In the continuous PNP/XO/H|RP assay and MESG continuous assay which I subsequently used, I could lose half of the activity of the PNP coupler without losing signal in the MurD assay. So, on the summary table, by virtue of the PNP/MESG assay, for the compounds therein, PNP was either insufficiently inhibited or even activated, and therefore the diagnosis of MurD inhibition held.

Q. Is there any significance to the structures drawn in blue?
A. Only from an organizational point of view: The Enamine compounds are divided between two sets of plates *01 and *02, where there are three of each, A01, B01 and C01 and A02, B02, C02. Blue structures are of compounds from the *02 plate, black ones from *01.

@AJLloyd105 added: I will be looking to do more to reduce the fragment concentration in assays by offsetting this reduction through decreasing the affinity for the competing substrate by what ever means possible.

[Yuhang-CADD]

Jan's Prediction Progress:

We did a lot of redesign because those reagents in the predicted procedures were not commercial available while some reaction types did not work (used alternatives). @jhjensen2

Green represents achieved.
Blue means in-progress.