Mur Ligase Online Research Meeting 2pm Aug 3rd 2021

[mattodd]

Meeting follows on from July 2021 meeting at #47. When: Tue Aug 3rd 2pm London (BST). Where: https://ucl.zoom.us/j/98754959307 Recording:

Actions from Last Time

• [x] July meeting posted to Youtube.
• [x] @WieselHenson has completed the synthesis of the AZ alcohol compound. Action on Yuhang: send to @bartrum (which enzyme? @eyermanncj says acetobacter or pseudomonas MurD. Peter Horanyi says he has crystals of Pseudomonas MurC ready to soak) and to Laura at Warwick (for MurE). This will also help to confirm the compound's identity. Note that to validate the MurC activity of this compound we still require the MurC biochemical assay. @WieselHenson now pursuing a compound that can be easily turned into a primary amine, and potentially a negative control (can be useful in biochemical assays, and for machine learning models).
• [ ] @Rebecca-Steventon has posted data of @LizbeK fragment screen (https://github.com/opensourceantibiotics/murligase/wiki/Allosteric-Pocket - but @drc007 would like structures to be added).
• [ ] @Rebecca-Steventon to pursue dose-response curves for the previously-identified compounds inhibiting both MurD and MurE. @danaklug to post structures of the hits and the non-hits. These structures to be forwarded to Jan for inclusion in the modelling.
• [ ] @jhjensen2 @dkoes and @cstein to update on the predictions made by the genetic algorithm here, full previous discussion in #47. The full Enamine library has been screened vs MurC, using Entryway criteria. Gave 100 molecules for consideration. Then the genetic algorithm was deployed, also using Entryway criteria, giving 88 suggestions. A score of -9.3 is the best obtained to date. All binding at the same site as the AZ compound. Action on @jhjensen2 to show the pose overlap for these compounds and to post the csv file of the outputs. Is it possible to run algorithm vs two Mur enzymes simultaneously? Yes. Docking has now been done for murC, murD, murE but they have different ligands bound (i.e. not all the same small molecule). Suggestion from @eyermanncj was to dock AZ8074 to each structure or to use constrained docking (enforce H-bonds to the Asn that H-bonds to ADP). Done, and gave strikingly different XP docking scores, which tells us...? Not sure, since we don't know how well the AZ compound inhibits MurD and MurE. @eyermanncj cautioned against compounds with carboxylic acids, since the ionised form of the molecule can dominate the interaction. @jhjensen2 said that in the docked forms the acids were protonated, but obviously at physio pH the acids may be deprotonated. Jan can compare before and after LigPrep analysis to see if this makes a difference. Would it help to have three proteins bound to the same small molecule ligand, as a way to compare the modelling across proteins? @eyermanncj thought not, and will share some resources/cautionary tales. Jan now searching for compounds with a compulsory primary amine.
• [ ] Laura to continue crystallisation trials of MurD and MurE in Warwick, with the plan to take crystals to Diamond. Streptococcus agalactiae MurD is 1-2 months, and being difficult so this is being parked. E. coli MurE is faster and will be taken to Diamond. @chrisdowson1 says the MurC biochemical assay is not yet on the road, but is certainly coming out of the garage.
• [ ] @eyermanncj, Chris and @bartrum to pursue AZ compound shipment for crystallisation trials. @mattodd to chase @bartrum by email to see where the blockage is.
• [ ] Peter Horanyi to continue crystallisation of e.g. MurC with Enamine compounds (Structures) sent by @eyermanncj. Peter presented data on this (Action to upload slides) indicating that, when compounds were incubated with protein (murC and murD from E. coli and XXX), thermal shift destabilisation was seen, which is unexpected. No positive results yet seen from any soaks. Peter happy to continue doing NanoDSF, since it's not expensive re protein, but crystallisation trials are more expensive.
• [ ] Chris Dowson to liaise with others to establish protein needs, and what's needed from SSGCID <-- this is getting mothbally. Needed?
• [ ] Laura to liaise with Peter and Lizbe to see if a soak of the @eyermanncj Enamine compounds is possible at Diamond. <-- this is getting mothbally. Needed?

Additional items for discussion:

• Atomwise shipment. Compounds have been sent to Diamond (representative 3 compounds have passed quality control done by @danaklug), and await Laura there.
• Status of ELF Screen application. ELF are requesting extra details of Chris Dowson's screen and will re-engage with the proposal upon receipt of those details.
• @loriferrins pursuing inputs from Cyclica - prediction of actives. Not based on activity (where we do not have many data) but based on crystal structures (as for Atomwise). @eyermanncj did not think there were enough lit data (on binders at the ATP pocket) to build a good machine learning model. The Becca dose response curves may give us that. In the meantime, Peter has structures that may be use to Cyclica.

Overall Aims remain: i) Structures of two mur ligases bound to the same small molecule. ii) Identify new starting points for inhibitors.

Future logistical aim: devise platform legal agreements within OSA in order to simplify the MTA-related paperwork, which is proving problematic. (Could we transform OSA into a charity (though since none of us would work for this charity, it's not clear how much that might help)).

Actions Arising

• [ ] @Yuhang-CADD to ship AZ compound to @bartrum and Laura.
• [ ] @Yuhang-CADD to propose new target of an AZ compound modified to include a primary amine.
• [ ] Need structures added of @LizbeK fragment screen (https://github.com/opensourceantibiotics/murligase/wiki/Allosteric-Pocket) <-- need to clarify what is needed here.
• [ ] @Rebecca-Steventon to pursue dose-response curves for the previously-identified compounds inhibiting both MurD and MurE. Structures of the hits and non-hits are available on the wiki <-- need link.
• [ ] @jhjensen2 @cstein to provide pose overlap for Enamine and/or genetic algorithm compounds and to post the csv files of the outputs.
• [ ] @jhjensen2 @cstein to investigate whether LigPrep alters docking scores.
• [ ] @jhjensen2 @cstein to prioritise screening hits possessing primary amines.
• [ ] Laura to continue crystallisation trials of E. colin MurE at Diamond
• [ ] @chrisdowson1 to continue to pursue the MurC biochemical assay
• [ ] @loriferrins to invite Cyclica to next meeting and put them in touch with Peter Horanyi.
• [ ] @chrisdowson1 to maintain interaction with ELF following their interest in the assay/screen we proposed.
• [ ] @eyermanncj to post some lit cautionary tales re unsuitable conclusions from computational modelling.
• [ ] Peter Horanyi to post deck that he presented, below.
• [ ] Aug meeting posted to Youtube

Next meeting: Likely Tuesday Sept 7th at 1pm Location:

[Yuhang-CADD]

Chemistry Update:

WYH9-1-P (14 mg pure for characterisations)

LCMS: clean and right m/z 385 peak

NMR: consistent with the product data reported and the starting material data we have measured

WYH9-2-P (14 mg pure to ship)- A repetition of WYH9-1

LCMS: clean and right m/z 385 peak

Crude NMR (before further purification): peaks in line with those of WYH9-1-P

[Rebecca-Steventon]

Unfortunately I won't be able to attend the meeting today due to other commitments. Update on Dana's compounds: We produced active MurD and MurE from P.aeruginosa and have run the dual inhibitory fragments against them. Data analysis from these runs is ongoing. Hill slope dose response curves will then be carried out on any fragments that are able to target both bacteria.

[Yuhang-CADD]

Update on shipment of WYH9-2-P

6.2 mg to Seattle

6.2 mg to Warwick

[eyermanncj]

Team,

Please see attached for an informative analysis of docking program results and the important role of visual inspection of results. My own experience is that dockig scores only separate out the very bad poses and that poses with good docking scores need to be visually inspected to weed out the many flaws remaing in scoring functions.

Decision making in SBDD JMedChem 2021.pdf