Mur Ligase Late Summer Campfire Meeting September 2022

[mattodd]

Date: Sept 13th 2022
Time: 2pm UK time (other times)
Place: https://ucl.zoom.us/j/91379419977
Recording: https://youtu.be/vmSDHKJGgzU
Previous Meeting: #87

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) News from Enamine library screening
ii) Shipment of samples from UCL to Warwick
(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. No update in today's meeting.
• @eyermanncj @LauraDS1 to update on any additional near-neighbour compounds that have consequently been ordered/evaluated. No update in today's meeting.
• @LauraDS1 to update on any soaking and/or co-crystallisation experiments with these compounds (last update here). No update in today's meeting.

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. No update in today's meeting.
• @LauraDS1 @LizbeK to update on any further fragment soaking and/or co-crystallisation experiments. No update in today's meeting.

3) 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). In meeting: these data need to be cross-checked with the new inhibition data.
• @LauraDS1 @Rebecca-Steventon @chrisdowson1 @AJLloyd105 to obtain inhibition data with the Atomwise compounds, to verify whether binding gives inhibition. In meeting: @AJLloyd105 presented the data shown in this slide deck showing promising inhibition values for a number of compounds, including multi-mur inhibition. These data will be examined (in concert with the SPR and X-ray data) separately.
• 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.
• @mattodd to follow up with Denzil Bernard about potential additional suggestions based on what has been found to date. <-- might make sense to wait on inhibition/SPR data? We're being asked for "I have attached your data reporting form that will help us interpret your results. The form is organized by plate location and tube barcode, so please check to ensure that the tube barcode matches the reported results. Feel free to modify the form to fit your experiment" where the relevant spreadsheet is here, screenshot below.

• @AJLloyd105 and team will evaluate Atomwise compounds vs. Pae MurF. Also will look at impact of ATP, to see if competitive (i.e. an assessment of possible mechanism of action/site of binding).

(incidentally @AJLloyd105 reported on an improved method that has been developed for data analysis from the screening experiments, making it faster to generate inhibition numbers. No solubility issues were found when using the Atomwise molecules at these high concentrations.)

4) Variants of AZ Compounds

• @Yuhang-CADD to update on the shipping of AZ5595 and the amino derivative of that compound (OSA_001095). In meeting Yuhang to ship compounds (below) to Warwick, to see if any inhibition, and then to SSGCID if there is justification. (Need a few mg only for in vitro enzymatic assays, 10 mg for MICs and mutagenesis, 3 mg for MICs only, 5-6 mg for assays and initial crystallisation if needed. Enzymatic assays to be checked before any microbiology.)
• @Yuhang-CADD to consider which proteins we should ask SSGCID to crystallise with these compounds, if activity is found at Warwick - involves checking whether the compounds have given any inhibition vs Murs other than MurC, and what we want to know with these compounds. Before shipment of these compounds to SSGCID, they will be examined at Warwick for which might interact/inhibit. Note that Nano DSF is no longer available at SSGCID (this was courtesy of UCB).
• @Yuhang-CADD to prioritise the completion of the synthesis of the amino derivative of AZ5595.
• @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).
• @Rebecca-Steventon to report on the assay @Yuhang-CADD 's compound (which?) 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. Overlay provided below, just needs addition of 2D structures for clarity.
• @chrisdowson1 to work on compound transfer from SSGCID to Warwick and update the group. Does this need an update, or can it be mothballed? No update provided in meeting.

5) New Protein Structures

In meeting Jan Abendroth updated us that new structures have been deposited:

Pseudomonas MurC with AZ13644908, PDB code 8egm
Pseudomonas MurC with AZ13643701, PDB code 8egn

Recent structure deposited for Pae MurD with UMA bound, PDB ID: 8DP2 needs collating together with other structures in wiki page (maybe this one?) or in #67. Volunteer to collect all structures? Also add the new structure of Pae MurC with OSA_001044 bound, PDB ID: 8DOF (https://doi.org/10.2210/pdb8DOF/pdb).

A discussion of the possible value of truncated structures was had as part of the Soak-In at #80. <-- conclusions we can summarise?

6) De Novo Computational Modelling

• @edwintse @danielgedder to complete synthesis/procurement of molecules suggested in Predictive Modelling Competition July Summary #85.
• @LauraDS1 to book an 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)

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.
• 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) Misc/AOB

• @mattodd to post GRC conference slides somewhere (28 MB...).
• @edwintse to clone Open Source Malaria Logo and Poster OpenSourceMalaria/TechOps#4 for OSA so that people can "advertise" their participation. Done here. @mattodd has added this to the front pages of both OSA projects. People should fee free to print out and use!
• We need a volunteer to reach out to CC4CARB to assess whether we are suitable for a proposal.

9) 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.

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.
• 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?

Next Meeting

Oct 11th 2pm UK time. The meeting is the Halloweener

[Yuhang-CADD]

I. Compounds to Warwick

Will be shipped together with the competition compounds (Ed and Daniel) by the end of this week

1. AZ5595 and its enantio isomer are ready to be shipped by the end of this week , and the amino-derivative (OSA_001095) to be shipped soon (estimating next week)

2. Urea protease inhibitors are ready to be shipped by the end of this week

II. Compounds to Seatle

Ship by today

AZ5595 and its enantio isomer and the two urea protease inhibitors

III. PDB overlay

6X9F (AZ5595), 6X9N (AZ8074), and 8DOF (AZcmpd4, WYH9-2-P, OSA_001044)

Interactions of the three AZ compounds with Pae MurC are basically the same; the three key H-bondings between asparagine (ASN 292/285), tyrosin (TYR 246/239), and the pyrazole moieties of the AZ compounds. The Pi-Pi stacking interactions vary a little bit (AZ5595 interacts with the Histidine via the benzene moiety while the other two via the pyrazole moiety). But the general positioning of these three AZ compounds shares a high similarity:

Calculated in PyMOL, aligning 8DOF against 6X9N and 6X9F gives an RMSD value of 0.27 and 0.29 respectively. Also, aligning AZcmpd4 (WYH9-2-P, OSA_001044) against AZ5595 and AZ8074 gives an RMSD value of 3.19 and 2.62 respectively.

[mattodd]

Interesting overlay @Yuhang-CADD - could you please add to the above (panel underneath, can be separate) the chemical structures of the small molecules you're mentioning?

[Yuhang-CADD]

Interesting overlay @Yuhang-CADD - could you please add to the above (panel underneath, can be separate) the chemical structures of the small molecules you're mentioning?

Sorry for missing that part, have already updated the three structures in the picture above.

Here is the enlarged version.

[rhanson1046]

These look like kinase inhibitors. What is the assay for this series of compounds? R. Hanson Department of Chemistry and Chemical Biology Northeastern University Boston, MA US

…

________________________________ From: Yuhang Wang ***@***.***> Sent: Tuesday, September 13, 2022 12:31 PM To: opensourceantibiotics/murligase ***@***.***> Cc: Hanson, Robert ***@***.***>; Team mention ***@***.***> Subject: Re: [opensourceantibiotics/murligase] Mur Ligase Late Summer Campfire Meeting September 2022 (Issue #88) Interesting overlay @Yuhang-CADD<https://nam12.safelinks.protection.outlook.com/?url=https%3A%2F%2Fgithub.com%2FYuhang-CADD&data=05%7C01%7Cr.hanson%40northeastern.edu%7C9c3593e9972a404b4c4608da95a56541%7Ca8eec281aaa34daeac9b9a398b9215e7%7C0%7C0%7C637986834849465438%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000%7C%7C%7C&sdata=88gHPIL5MFCxWqodpfcpLEyNUzGdSeXTf1OxT0IY9%2FU%3D&reserved=0> - could you please add to the above (panel underneath, can be separate) the chemical structures of the small molecules you're mentioning? Sorry for missing that part, have already updated the there structures in the picture above. Here is the enlarged version. [Structures of binders]<https://nam12.safelinks.protection.outlook.com/?url=https%3A%2F%2Fuser-images.githubusercontent.com%2F58538740%2F189956706-ce8fd20f-6c4d-4233-acf1-b152df7697c0.png&data=05%7C01%7Cr.hanson%40northeastern.edu%7C9c3593e9972a404b4c4608da95a56541%7Ca8eec281aaa34daeac9b9a398b9215e7%7C0%7C0%7C637986834849465438%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000%7C%7C%7C&sdata=2nGi9%2FPcaswImsuCF9cY07FzVljEAeD2SF2oWM9P0S8%3D&reserved=0> — Reply to this email directly, view it on GitHub<https://nam12.safelinks.protection.outlook.com/?url=https%3A%2F%2Fgithub.com%2Fopensourceantibiotics%2Fmurligase%2Fissues%2F88%23issuecomment-1245659852&data=05%7C01%7Cr.hanson%40northeastern.edu%7C9c3593e9972a404b4c4608da95a56541%7Ca8eec281aaa34daeac9b9a398b9215e7%7C0%7C0%7C637986834849621671%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000%7C%7C%7C&sdata=mXoB8vFK%2BLyNW%2Ft5J8pwafPhgnq1g4ktZEloZdEa5hg%3D&reserved=0>, or unsubscribe<https://nam12.safelinks.protection.outlook.com/?url=https%3A%2F%2Fgithub.com%2Fnotifications%2Funsubscribe-auth%2FANGWX5MRLMQON5Y7B7V7XNLV6CT5TANCNFSM56CH5XJA&data=05%7C01%7Cr.hanson%40northeastern.edu%7C9c3593e9972a404b4c4608da95a56541%7Ca8eec281aaa34daeac9b9a398b9215e7%7C0%7C0%7C637986834849621671%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000%7C%7C%7C&sdata=A%2Bd072DgKl5srbRE243ysyyNS4LBNX%2F8IiU4ld5y9W0%3D&reserved=0>. You are receiving this because you are on a team that was mentioned.Message ID: ***@***.***>

[edwintse]

As discussed in the meeting, Yuhang's compounds above and the finished competition compounds (#85) have now been shipped to Warwick for testing.
Warwick 140922.xlsx

[jhjensen2]

Thanks for the feedback regarding this compound. Can I get a bit more detail on what the trouble is @edwintse @drc007

[drc007]

@jhjensen2 The aminal N-C-N is often unstable. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5238614/