Open-Source Coronavirus Monthly Update Meeting #1: Monday 4th April 2022.

[TomkUCL]

Meeting Zoom link: https://ucl.zoom.us/j/99880132263

Topic: Nsp13 Monthly Meeting UCL/UNC/Astex.

Time: Apr 4, 2022 05:00 PM London (BST), UNC 12:00 noon.

Chair: Tom Knight

Minute taker: UCL

The meeting agenda can be found here: nsp13 monthly meeting agenda.docx

The meeting recording link can be found here:

Minutes can be found below.

[TomkUCL]

Today's agenda for the first meeting 4th April 2022:

[tmw20653]

nsp13 helicases.pptx

[TomkUCL]

Hi all,

Thanks for all your input during today's meeting. The meeting minutes and the recording will be uploaded here shortly.

@tmw20653 below are some videos on the GCI (Creoptix WAVE method) discussed towards the end for some background, including a case study from Domainex (@ 20:00 mins), a UCL/ICR spin-out drug discovery company based in the UK.

Here are some useful videos in case this could be worth exploring further:

Challenges and opportunities in Drug Screening https://www.youtube.com/watch?v=DCIY4gQk4pU

Sensitivity with Grating-Coupled Interferometry (GCI) vs. Surface Plasmon Resonance (SPR) https://www.youtube.com/watch?v=dcr4FHmPPOQ

[TomkUCL]

See below the Nsp13 Meeting No. 1: Monday 4th April 2022 recording link: https://ucl.zoom.us/rec/share/aLWR_UBo482zYBD1-l-KXce0sHe053iO119LkOreartNrOJfR5hFmxLVb2eetnPX._ZEKSz1hZR029_tq?startTime=1649088230000 (Passcode: W5TmMF#p)

Actions:

• Tom; finish the synthesis of the non-purchasable fragments from Kosta's first list of top 30 scoring compounds and send a list of the purchasable compounds.

• Mat / Tom; follow up with John Diffley (Francis Crick Institute) about getting hold of enough nsp13 protein to set up an SPR assay (Dave Selwood, UCL).

• Tom; perform a structural similarity (cluster) analysis in Data Warrior of Kosta's Enamine REAL space 40BN library "top 100 virtual hits" to find common scaffolds (overlay with the top 30 scoring compounds from the previous virtual screen?).

• Tim; updates on the synthesis of nsp13 protein and the use of UNC's SPR method for the next meeting.

SUMMARY:

• Surface plasmonic resonance (SPR) seems the likely the primary screening method once fragments are ready.

• James suggests that ITC may be a better indicator for lower potency fragments than SPR.

[mattodd]

Good meeting the other day! Very good to have such a nice description of the background from @tmw20653. Some misc notes:

1) Is Nsp13 a validated target? We assume so, but so far we (the scientific community) have no inhibitors, right? I guess "validation" of the target comes from efforts vs. Nsp13 equivalents in other viruses?

2) James (not on Github yet, right?) mentioned the wisdom of securing potency measurements on the original fragments, which is not something we've done. He suggested doing some optimisation of the frags before growing them. Interesting approach that we're not taking (yet), but maybe we should.

3) James also advocated for isothermal titration calorimetry (ITC) as a go-to method to assess binding. Again, not something we're currently doing, but could.

4) From @kipUNC talk, there was good overlap between fragalysis and FtMap in terms of the key binding interactions. Helps to validate the FTMap approach. I'd not previously clocked that.

5) Good to see @Daren-fearon at the meeting. Daren please shout if we're missing anything. These meetings will be monthly if you're interested.

[H-agha]

NSP13- Monthly meeting- 4-4-2022.pptx

[jamesday100]

It was great to join the team and put faces to names. Thanks for the minutes. Few additional comments on the points above.

1) To me the current pocket (Site 3) looks shallow and surface exposed. There is a slight risk there will not be enough potency to be found. Measuring the potency/LE of the starting fragment hits will suggest what the baseline is and could support the validity of this site. If you find that LE drops off as you grow the fragments hits and are seeing flat SAR, this will help guide decision making. Looking for a biophysical technique here to help you cross this bridge and validate the site and fragment hits would be good. Techniques like SPR would need compounds to be in the uM range and therefore there could be a risk of not seeing anything if there is not potency to be had in this pocket. ITC could be the assay you need here as it can measure mM potency.

2) ITC could be used as a "bridging assay" to get compounds on scale on the bioassay. As the assay is protein hungry as soon as compounds are reliably on scale in the bioassay (~100 uM), ITC can be phased out.

3) In FBDD you want to squeeze as much potency per heavy atom as possible. So ideally you want to optimise the fragment starting point before growing. Tracking the changes in LE as you attempt to optimise lets you know if the changes you are making are going in the right direction.

4) In the hit validation phase where we are currently, you want to eliminate dead ends quickly. Not all fragment hits can be optimised. Typically, you see this in flat SAR and failure to improve LE. You can only know this if you know the LE of your starting point and subsequent fragment follow ups. Knowing when to stop a fragment hit is as important as whether you are making progress.

[TomkUCL]

Thanks for these points @jamesday100. On the point of pocket depth, the following 2022 molecular dynamics paper claims to have identified four potential allosteric binding pockets between domains 1A and 2A, in a region away from the ATP and the nucleic acid active sites.

Ref; Molecular dynamics simulations of the flexibility and inhibition of SARS-CoV-2 NSP 13 helicase B.A. Raubenolt et al. Journal of Molecular Graphics and Modelling 112 (2022) 108122. https://doi.org/10.1016/j.jmgm.2022.108122

To my eye, those pockets look more buried. @H-agha @mattodd Could these be other potential targets for a non-competitive inhibitor of nsp13 by limiting the flexibility of domain 2A?

I have summarised this paper in the GH Wiki > 'What is known' section, so feel free to read it and let me know your thoughts.

Notably, the x-ray crystallographic fragment screen performed by F. von Delft and J. Newman (Nat. Commun. 12 (2021) 4848) identified a possible allosteric pocket (structures 5RMB and 5RMF) with residues in a similar region to one of the binding pockets identified from the molecular dynamics simulations from this more recent study (pocket 3).

Lastly a quote for the top 100 scoring compounds from @kipUNC 's ENAMINE REAL 40BN library virtual screen has come in, and can be found in issue #3 .