Biochemical assay kinetics

Hi all,
I’ve been looking through the posted activity data (very nice and clear presentation), and I’ve seen IC50 data. Since there are quite a few covalent compounds in there, I was wondering about kinact/KI values, are you planning to post these as well? I’m not sure about the assay setup, but if it’s a continuous protease assay, you should be able to extract these.
Sorry if this is redundant, I’m new here and happy to provide some input if needed!

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Hi @Lukilu, thanks for the comment. I was just looking over @Wal-Ward detailed comments here Data Release: 2020-05-10, and the need for understanding K_{inact} has been apparent to many on the team for some time. I am not well versed in assays, but I was under the impression that you have to do different kinetic studies to extract the ratio, is that wrong?

We should have more covalent hits coming in, so we might begin with “warhead scans” on these compounds quite soon. Kinetic studies are hopefully being setup also.

Hi mc-robinson, great to see the team is aware of this!
To extract the Ki/kinact vaues, you can run kinetic studies at different inhibitor concentrations.
Assuming you are running a fluorescent peptide cleavage assay to measure MPro activity, this is quiet straightforward, as you have a continuous assay and don’t need detection reagents or the like. Instead of picking a timepoint to readout, as I assume you are doing now, you can start measuring immidately for maybe 30-60 min. We’ve run MPro assay in such a format and that works beautifully and I’m sure your assay team does this routinely anyway. I really like this reference for more on mechanistic considerations / fitting etc…
I would see value in already collecting this data for the initial set of covalent compounds, as the differences in IC50 might stem from either Ki or kinact, so it might be helpfull to have for future rounds of design (but that’s something the medchemists can correct me on).


Actually, I’d be happy to support this experimentally as well, let me know if there’s need!


I would be very interested in hearing more about your efforts as we are keen on going after a reversible covalent inhibitor as part of the Moonshot program.

Is there a way to connect with you offline?


Hi @Joe, just making sure that was intended for @Lukilu, correct? Just want to make sure I am not following behind on email (somewhat inevitable at this point).

Yes, was hoping to engage with @Lukilu

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Hi Joe,
happy to connect offline, I’ve tried to email using the above address, but that bounced…
Is there a typo in there maybe?


My bad. I have too many variations and email addresses. I should have given


Activity comparison

I see there is reasonable agreement between pIC50 in fluorescence & rapid fire assays, with close to the expected consistency in ranking

The fluoresence pIC50 generally is a little lower than rapid fire, which could be due to differences in protocol.

Please can someone point me to the protocol for rapid fire?

Thanks. Stay well!


Hi @Wal-Ward,

Sorry I am just seeing this! I actually don’t have a nicely written up version of the rapid fire protocol on hand. @frankvondelft might be able to help though. I know I have an initial version, but I want to make sure it is reflecting the way the assay is currently being run.

Chloroacetamide dose-response curves

These data indirectly suggest reversible inhibition, please see my post

where I suggest possible reasons why compounds with covalent warheads do not appear to be giving irreversible inhibition.

Stay well everyone!

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Irreversible inhibitors - measurement of kinact/Ki

@Lukilu @Joe The procedures described in the linked reference (Schwartz PA et al, 2014, PNAS 111, 173) are quite complicated and seem to require Dynafit software, together with precise estimates of the concentration of active enzyme and inhibitor. These methods may be required if the inhibitor is very potent (IC50 close to enzyme concentration).

The Covid Moonshot project is not yet at this level of potency, so that more straightforward approaches can be used. I am happy to advise on how to measure kinact/Ki and some of the possible complexities (eg inhibitor reacts with reducing agent, co-operativity, inhibitor acting as substrate, or IC50 close to enzyme concentration). If you wish to discuss this further, then please contact me or refer to the text book by Robert Copeland (Copeland, RA, 2005, Evaluation of enzyme inhibitors in drug discovery, Wiley Interscience, Hoboken New Jersey).

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Hi Wal,
Thanks for the feedback!
Your very right, the procedure in the pnas paper is more elaborate than would be required here, but i think it’s a nice open access example for such profiling (not everybody might have copelands book available). I think it’d be nice to connect offline, once these measurements get more concrete. We routinely measure KI kinact values, but it’s always good to share experiences!

Hi Lukilu
I have a document on calculating kinact and Ki for the covalent MPro inhibitors. I hope you find it interesting and useful. Contact me if you have any questions
Take care
Analysis of Mpro time course data.pdf (631.7 KB)


Thanks Wal, i’ll have a look!

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Hi Wal,
thanks for the detailed document, this is great to have as a reference.
The MPro data you use in the examples is beautiful, when working with more noisy data a global fit, plugging the kobs equation (2) into equation (1) might be beneficial. We’ve started doing that for noisy data when characterising slow non-covalent inhibitors and, at least based on MC simulations, it increases accuracy and precision!