Topic automatically created for discussing the designs at:
https://covid.postera.ai/covid/submissions/BEN-DND-93268d01
Hi Ben/Matt @Ben_DNDi @mc-robinson BEN-DND-93268d01-8 looks very interesting given that it’s sub-micromolar and relatively polar. I’d expect the compound to be predominantly neutral at pH = 7 (the pKa for values for 1,4-dimethylpiperazine and glycine amide are each ~8). I found a couple of relevant crystal structures (HODQUP and UWUJAZ) in the Cambridge Structural Database and these point to a conformation in which the amide NH of BEN-DND-93268d01-8 eclipses the lone pair of the piperazine nitrogen. Do either of you know whether the crystallographers have looked at this inhibitor?
Hi @pwkenny, the beam-line is shutdown for another week I think, but this is definitely high priority to get into crystals! I am not sure I myself could rationalize how it it fitting in that pocket, and it is definitely a bit unexpected. Thanks for the structural data though, glad it too is pointing to something different.
Hopefully we get confirmation on this hit soon from the MS assay, biophysical data before getting too excited
Thanks for the info, Matt, and I will attempt to dock the molecule. I agree that it’s important to confirm the inhibitory activity before committing any synthetic resource and would ideally have structural data as well.
Have a look at the piperazine ring in each of the CSD structures in my earlier message from the C2-C3 ‘edge’ (so that you can see the bond vectors associated with the two ring nitrogens). If both atoms 1 and 4 in a 6-membered, saturated ring are sp3 or sp2 then the vectors will typically be parallel (but offset). In the @Ben_DNDi compound BEN-DND-93268d01-8 one of the nitrogen atoms is sp3 (pyramidal) and the other is sp2 (planar). One consequence of this is that the bond vectors associated with the ring nitrogens are not parallel.
I’ll also mention @miko_a who has proposed merging BEN-DND-93268d01-8 with PET-UNK-c9c1e0d8-3. I think this is potentially a good tactic although I’d hold off until more is known about how the individual merger components bind to the target.
Hi Joe, my current estimate that the pKa of the protonatable piperazine nitrogen is 5.5-6.0 (still working on it). I don’t know the assay pH ( @mc-robinson , is this information available?) nor do I know the relevant physiological pH. The presence of a bases with pKa below 7 can complicate interpretation of results from both enzyme inhibition and cell-based assays.
Fluorescence :pH 7.3
Compounds were seeded into assay-ready plates (Greiner 384 low volume 784900) using an Echo 555 acoustic dispenser, and DMSO was back-filled for a uniform concentration in assay plates (maximum 1%). Screening assays were performed in duplicate at 20 µM and 50 µM. Hits of greater than 50% inhibition at 50 µM were confirmed by dose response assays. Reagents for Mpro assay reagents were dispensed into the assay plate in 10 µl volumes for a final of 20 µl. Final reaction concentrations were 20 mM HEPES pH=7.3, 1mM TCEP, 50 mM NaCl, 0.01% Tween-20, 10% glycerol, 5 nM Mpro, 375 nM fluorogenic peptide substrate ([5-FAM]-AVLQSGFR-[Lys(Dabcyl)]-K-amide). Mpro was pre-incubated for 15 minutes at room temperature with compound before addition of substrate. Protease reaction was measured continuously in a BMG Pherastar FS with a 480/520 ex/em filter set.
Pete,
So likely not protonated in the fluorescence assay. Still go with the docking pose I suggested. Good to try some prospective predictions! 
Thanks for the info, Joe, and this raises the question of the question of what the physiological pH actually is. I would anticipate poor translation of enzyme inhibition to activity against infected cells if the major protonation state for the inhibitor changes going from the assay to the intracellular environment. In my experience, one needs to be wary of basic centers in cysteine protease inbibitors (lysosomal accumulation is another issue).
Hi Joe, I still want to explore potential binding modes for the neutral ligand using the OpenEye docking tools (I need to check the conformer generation) although I will take a look at how your docking compares. Particularly keen to see the results from the other assay and note of caution from @mc-robinson was particularly welcome.