SARS-CoV-2 Nsp13 DEL screen hits for CRO synthesis and testing.

[TomkUCL]

• Convincing active hits against SARS-CoV-2 helicase remain elusive despite attempts from many groups and organisations.

• Zhou et al. (2024) https://doi.org/10.1038/s41557-024-01442-y published reported several small-molecule hits against SARS-CoV-2 helicase (nsp13) using a protein-templated DEL-screen approach.

• Several hits were reported in the low micromolar range, however, crystal structures are currently unavailable and compounds have only so far been tested in the Nsp13 helicase ATPase activity assay.

• Compounds N-1 and N-2 are most interesting due to their relative synthetic simplicity and the stereospecific mode of inhibition. These are not commercially available, so we are attempting to outsource to CRO (Piramal) for resynthesis. Compound S-11 is more complex in terms of synthesis and is therefore lower priority, however, will not be excluded if a fast synthetic route can be found.

• If obtained, Joe Newman is interested in soaking these compounds for co-crystallography, in addition to further testing in his group's FRET DNA unwindase assay. We would also like to test these compounds in-house using an established grating-coupled interferometry (GCI) assay.

• One possible route has been suggested, however a route that minimises the use of copper would be preferred due to copper toxicity towards the protein resulting from contamination.

[TomkUCL]

Follow up to previous conversations on this DEL paper https://doi.org/10.1038/s41557-024-01442-y, there is characterisation for the low micromolar Nsp13 hits, N-1 / N-2, which they refer to as compound 73-1-143 in the SI (page 130).

• The authors don't specify which route was used for this compound (Supplementary Figure 18, and page 86 onwards).

• A plausible route could look like the one shown below. Note: the 2-fluoropyridinyl analogue is available as a building block from Enamine for 1485 USD/g for 19F NMR screening.

• “Off-DNA” validation of the NSP13-templated reactions, where the alkyne/azide fragments of N-1 without the DNA tags were incubated with NSP13 helicase, showed product formation monitored with LC-MS at different time durations (Supplementary Figure 33). This suggests that the Click reaction is catalysed without the DNA tags present, suggesting binding the two fragments at the same site.

• Docking studies with AutoDock Vina suggest that N-1 and N-2 are ATP competitors (Supplementary Figure 30), binding between the two RecA helicase domains (1A and 2A).

• @tmw20653 is not convinced of making any of this compound due to the lack of follow-up or orthogonal studies, however, given the diastereospecific nature of N1/N2 and the apparent difference in activity by ATPase assay, there could be something.

• Joe Newman is eager to co-crystallise this compound with Nsp13.

• Overall, given the lack of any hits against Nsp13, it is worth getting a quote from Piramal for these two compounds for screening in the ATPase, unwindase, GCI, and 19F NMR assay.

Actions to follow up:

I have requested quotes from two external CROs (Charnwood and Enamine).

@rahmanszsaleem or @ahsgc can you please share these structures with Piramal and ask them for a proposed synthetic scheme and time required for 10-100 mg synthesis?

SMILES: O=C(N1CSC[C@H]1C(OC)=O)C2=CN(N=N2)C@HCC3=CC=NC=C3 O=C(N1CSC[C@H]1C(OC)=O)C2=CN(N=N2)C@HCC3=CC=NC(F)=C3

For the proposed synthetic route:

• We are looking to ensure low levels of copper contamination as this was flagged as a possible point of concern in the paper leading to false positives.

• Obtain both the ester and carboxylic acid forms of these compounds.

• Initially, we only need the non-fluorinated analogue (should be cheaper), then if active we will want fluorinated analogues of these compounds for the 19F NMR screen, either with CF3 groups or a single fluoro on the aromatics, like that shown above.