Open Source SARS Nsp13 Site 3 Monthly Meeting #13: 17:00-18:00 BST, Monday 9th Oct 2023

[mattodd]

Date: Monday 9th Oct 2023 Time: 5pm BST (other times) Place: https://ucl.zoom.us/j/97172937586 Previous meeting recordings: https://www.youtube.com/@MedChemCoreD-UCLSoP Previous Meeting Issue: https://github.com/StructuralGenomicsConsortium/CNP4-Nsp13-C-terminus-B/issues/40

Who can come?: Anyone. No need to say anything unless you'd like to. If you'd like to contribute to these meeting issues, please join Github.

Attending: Team can be seen at https://github.com/orgs/StructuralGenomicsConsortium/teams/cnp4-nsp13

Slide decks: Please https://github.com/orgs/StructuralGenomicsConsortium/teams/cnp4-nsp13 remember that if you share slides/info, drag and drop those into a comment on this page, below. Very easy and saves @mattodd having to pester you.

Key Things This Month (please add if you'd like to focus on something

1) Finalising targets from the "Fixed Core" project - @mattodd to raise in-meeting 2) Update on https://www.biorxiv.org/content/10.1101/2023.09.25.559391v1 from @tmw20653

Agenda and Actions Arising:

Synthetic Chemistry

• [ ] @TomkUCL to update with one slide on current status of his chemistry.

• [ ] @TomkUCL to ship samples of all UCL compounds to UNC for evaluation, requiring RA codes. Did this shipment take place over the summer?

• [ ] @ahsgc to maintain contact with Piramal re building block completion and delivery to UCL <-- all complete?

Assays

Is ATP-periodate compound still the best control we have?

@SumeraMalik123 examined 50 Nsp13 binders in triplicate by ATPase assay and SPR, and these were additionally filtered for experimental solubility and aggregation assays. This has led to 3 compounds being identified as promising, derived from the focussed library programs. @ahsgc has provided the structures of these compounds below.

Are there any CACHE competition icompounds (designed vs the RNA-binding site) that can be used as a comparator, or as a source of potential control compounds? Or as a source of a machine-learning model that could be used to predict better binders in a second round?

• [ ] @TomkUCL has used UCL's Creoptix wave to evaluate all local UCL compounds. Data arising? (No control protein was being used. Toronto has used WDR5, UCL could use carbonic anhydrase, etc. Alignment could help publishing the work!

Predictions/Modelling

• [ ] @kipUNC to forward new predictions, which will include some new purchaseable compounds as well as some compounds that will use the existing cores and vary the capping groups. <-- will be covered in meeting
• [ ] @mattodd to chase Alex Tropsha (and @toluene44) re the enumeration of Enamine-able libraries using current building blocks as cores. <-- will be covered in meeting
• [ ] @TomkUCL to update on progress with MD simulations in collaboration with Geoff Wells to identify cryptic pockets deeper than the existing Site 3 pocket, or modifications of the Site 3 pocket.

Structures

• [ ] Joe Newman to update on compound soaking. The compounds sent by @ahsgc were soaked, but nothing was observed bound. Joe was going to try with ATP-periodate and the original fragments. Joe to update.

AOB

Indole Compounds: @jamesday100 previously highlighted a conference poster on nsp13 inhibitors identified by group in Rome. Alvaro in the UCL lab is now making two representatives of this series and will report at a future meeting. Paper now available at https://www.sciencedirect.com/science/article/pii/S0166354223001754

NMR: @TomkUCL to update on discussions with Chris Waudby (UCL) on design of 19F ligand-protein NMR assay with Joe’s fragments as controls. Question: should we buy a 500-member 19F frag library? We're getting quotes.

@jamesday100 raised the question of whether we should go back and examine the other sites highlighted in the original fragment screen. @kipUNC mentioned that compounds were designed, purchased and evaluated vs ATP- and RNA-binding sites. @kipUNC to dig up the data.

Next Meeting

Date: Monday 13th November 2023 Time: 5pm GMT (other times) Place: https://ucl.zoom.us/j/97172937586

[mattodd]

Update from Geoff Wells by email 9/10/23 "I ran 1 microsecond of MD for Tom’s first cpd and he sent a further 4 to me when I was on holiday, so I am catching up with these now. I'm setting these up and will get some initial data this week. We should be able to look at binding energies, RMSD vs time and H-bond occupancy, as well as the bound conformation(s)."

[mattodd]

Data on the current Nsp13 hits that have "made it" thus far. Courtesy of @ahsgc (thank you):

Top NSP13 Hits.pptx

[TomkUCL]

SLIDES: Presentation (3).pdf

• Robust routes to some synthesised nitro-N-oxide predicted (GLIDE) compounds:

AMBER20 MD analysis (Geoff Wells @UCLSoP) and VINA docking (@TomkUCL) suggest that these compounds remain bound over a 1 microsecond simulation under physiological conditions, but the ligand (red) is highly flexible compared to a known binder (ATP, blue), and that the site 3 pocket shape changes over the simulation run time.

MD analysis (VMD) indicates that the N-oxide hydrogen bonding is not a key interaction here; This Nsp13-ATP-ssRNA model (PDB 7KRO and Geoff Wells) revealed that the ligand remained bound at site three over the entire length of the 1 ms simulation. However, out of the 6000 frames, NO···H hydrogen bonding with a length of less than 3 Angstroms involving the PNO motif was only visible in five frames: 356.8 ns (amide NH···O GLU591), 373.0 ns / 535.2 ns (N-oxide NO···HN ILE592), and 384.3ns / 437.9ns (nitro-O···HN ILE592). This corresponds to a period of only 0.2 ns where N-oxide NO···H bonding forms, and 0.2 ns where nitro- N-O···HN bonding forms, respectively.

Instead, charge interactions seem to dominate the interactions, offering an alternate binding mode;

• We gave @kipUNC some fixed cores (X-axis) and asked him to diversify the R1 group. The best scorers are still N-oxides and are based on the pyrrolidine cores, however all non N-oxide compounds are predicted to be weaker binders than their terminal pyridine N-oxide analouges.

[TomkUCL]

- @jamesday100 suggested prioritising based on molecular weight - more reason to pursue the benzoazepine core series.

- A route to the core has now been identified but requires scale-up and scope expansion to include functionalised benzoazepines predicted by @kipUNC. Work on this will continue unless the consensus is otherwise..

- Again, the decorated scaffolds that are biased to not include the N-oxide give lower docking scores and look synthetically trickier to make....

.... so work will continue on the original benzoazepine series once the cores have been scaled up.

[TomkUCL]

FAXS assay

Competitive FAXS assay could offer way to validate two fluorinated fragments (PDB 5RM3 and 5RM9) found at site three and check whether our fluorinated compounds are competitive..

n-FABS Screening Assay using Fluorinated Nucleotide Analogues

• Biochemical studies have demonstrated that a fluorinated ATP analogue, 2-fluoro-ATP, can serve as a substrate for several ATPase enzyme subclasses, including kinases, transferases, hydrolases, and ligases. https://pubs.acs.org/doi/full/10.1021/ja801588u https://doi.org/10.1139/o83-017
• 2-fluoro-ATP has also been demonstrated to act as a substrate for the RNA binding protein, RNA polymerase, https://doi.org/10.1021/ja047556x however it is yet to be tested on helicases.
• 2-fluoroATP synthesis ref: Altman, Michael D.; et al World Intellectual Property Organization, WO2017027646 A1 2017-02-16, page 181-182
• @tmw20653 suggested that we need to look into the kinetics of nsp13 without RNA/DNA present: Joe Newman suggested that nsp13 has lower (~10/min) turnover than when RNA is present (~100/min). Why not just include 2F-ATP and RNA oligomer in the assay?

[TomkUCL]

Proposed actions:

• Pursue a series that can be diversified based on two cores: para-nitro pyridinium-N-oxides and benzoazepines.
• These can include those based on the "diversified cores"...

... plus some that resemble the original fragments (PDB 5RM3 and 5RM9) and that can be tested by 19F NMR assay.

• Here are some options for accessing non nitro-N-oxide analogues...

• Geoff Wells is running a benzoazepine-nsp13 AMBER simulation this week so we can prioritise this series with slightly more confidence..

• He will also run an ATPase-tested compound, Kangping's compound, and fragment 5RM3 as a comparison..