This requires considerable explanation. This is a cascade stripped down for speed of decision making. Critically the CAT-3 labelled assays are done using live virus and therefore throughput is limited. In other circumstances the cell assay would be in the second wave - but here we need to pre-filter any cytotoxics out before getting there to (a) avoid wasting valuable slots, (b) allow accurate interpretation. The ferret in vivo models are even more valuable, therefore we have to be as confident as possible before putting compounds into that model that the compounds are safe and will have exposure. As in most LO projects, once the problems of the compounds have been identified the cascade should be reconfigured. Further toxicity screening will be added in.
Some of the discussion that’s already taken place by email:
From Jiye Shi:
- Having the cell toxicology in the first tier (and presumably as a gate-keeper assay to the later cascade?) seems over cautious and presumably expensive, unless there is easy access? I would put it lower in the cascade maybe after initial PK assessment and also maybe have a broader selectivity panel (the Cerep 44 safety targets) alongside proteases when running PK for potential tox. Perhaps the cytotox is necessary as a selectivity check for the viral cell screen? If this is the case then makes more sense.
- The cut-off for Viral EC50 at 5uM seems high to me (unless it is a whole blood assay) , that being said I am not a virologist so difficult to judge. I assume it would be a total target for coverage rather than free, so if you had low Cl and low Cmax/Cmin you would probably be OK. I wonder if it would be possible to have the viral assay higher up maybe in parallel to the cassette rat PK. Is the fact it is after reflective of screening capacity restraints? Seems to me that as the crucial potency assay that you base your human dose assumptions and safety margins on perhaps you would want it earlier?
- May not necessary to have dog and ferret heps early on, just rat and human then dog later. Not sure ferret heps are easily available and critical path. If we have decent rat exposure and human parameters I am sure we would find a way to expose ferret for efficacy studies, and we don’t want to be optimising for ferrets!
From Bobby Glen:
I guess you’ve thought about this already, but I’ll throw in a few of my own contributions for discussion. In the last week I’ve learned that everyone is trying to move at breakneck speed, so a lot of corner cutting compared to a standard DD workflow to get a solution quickly.The anti viral assays are precious so we should test compounds with a chance.
To assay in the viral cell assays we should be fairly confident of at least
binding affinity to the protease,
no cell toxicity in the dose range
- and of course for cell activity, the concentrations are typically much higher than for in-vitro studies so toxicity appears. To see activity for initially weak compounds we may have to go up to much higher that 5uM. In particular when I’ve talked to the virologists, they don’t want to get distracted with cell toxicity. So if the compound is active and soluble, gets into the cell and doesn’t be toxic and inhibits the protease that’s the fundamental steps for me initially.
I would usually say that selectivity (hence the Cerep assays) is very important, but they cost about £5K / compound, still, if the compounds are not selective, they could be doing something else or have activity at other receptors that cause toxicity (e.g. drop blood pressure). Would need to do these before animal studies definitely or after we get some active compounds and need to understand that they are only working as we think they are.
In order to accelerate the DMTA cycle can you move the primary protease IC50 assay to Enamine too so they can make and test immediately?
Thanks for updating the forum Ed!
Thanks for sharing. The test cascade certainly looks efficient - that’s great.
Are you imposing phys chem filters in addition to solubility?
Protease IC50 below 10 nM sounds like a big challenge. Could it exclude potentially useful compounds?
What about assays for selectivity & reversibility? Maybe not on critical path, but for key compounds and exemplars to profile each series?
You may want to include stability to glutathione for the covalent inhibitors.
I would echo your query about the IC50<10 nM although I would be reasonably confident that it could be achieved (there are a couple of aldehyde-based inhibitors with IC50s around 50 nM and azapeptide nitriles pack an even heavier punch than aldehydes). I think that it would be useful to try to quickly generate a couple of tool compounds (potent, soluble and permeable) for optimizing/tuning the cell-based assays and assessing the validity of the target.
I don’t think that an IC50 cutoff would be sufficient for irreversible inhibitors and reversibility is something that may need to be considered when interpreting EC50 values from the cell-based assays and assessing selectivity. The glutathione reactivity assay is routinely run when optimizing covalent cysteine protease inhibitors and I agree with your suggestion.
Thanks for your comments. I haven’t seen any IC50s in the Moonshot project. Do you know where to find them or are you referring to published values?
No doubt you’re aware of how IC50s can change according to substrate concentration, enzyme concentration, time and even substrate Km. At some point, I’d recommend it’s worth kinetic characterisation some compounds in more detail.
Stay well! All the best
I’ve not seen any IC50s yet although I understand that they have been measured and were being double-checked prior to posting. I believe that there was a plan to get SPR measurements which would yield SAR for compounds that bound too weakly to give meaningful results in the biochemical assay.
I heartily endorse your comments on the variability of IC50s and I’m assuming that irreversible inhibitors will need kinetic characterisation to be done routinely. As an aside, I believe there are published cheminformatic analyses of the variability of literature IC50 values and I’m not sure that the authors of these studies are fully aware that a given set of IC50 values may reflect genuine differences (as opposed to a set of replicates).
Hi Pete, Wal, Current thinking is that the 10nM is probably too higher hurdle for the first compounds to go into the mammalian cell viral assay to look at the drop off enzyme - cell. We’re well aware that the enzyme IC50 is a highly ‘tunable’ number, so we need to understand how our configuration of the enzyme assay relates to cellular potency. Once we’ve put a few compounds in we can set that hurdle more realistically. What is less negotiable is the cell potency which,in my view, needs to be under 5uM IC50 and preferably under 1uM. That in turn depends on the PK-PD relationship in an in vivo model - and so it goes on…