Sure @krisbirchall, but just for clarification, is this what you are looking for?
https://covid.postera.ai/covid/structures
Its good to have such a list, but I was more thinking about in the compound tracker and individual compound pages, otherwise people will have to keep cross-checking between pages
Another useful feature would be to have a tag for compounds that have been attempted in crystallography and what the outcome was - it would help us to see if there are new compounds that have not gone into crystallography but would be worthwhile to try. In fact on that note it would be good to provide a list of ‘closest to crystallised’ for each design to help identify the most suitable frameworks for building on the design (or if the compound is sufficiently novel to warrant crystallography).
Sorry for all the requests - there’s so many things to be done so of course it’s a question of time and priority
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Good point!
Yes, I think adding more info about what crystallized and what didn’t is a great idea. Obviously, we have a long list of things to start building… but we are getting there!
I’ll have this more automatic in a day or two, but if you want to start exploring the data. Here is all of the activity data so far. CSV also includes all of the unique designs without datamoonshot_initial_activity_data.csv (307.5 KB)
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Great to see these data being here and will certainly take a look. Will the next wave also contain alternative warheads to the chloroacetamide, as well as some testing of previously-reported inhibitors? Will be nice to know where we stand there. Congrats to the team!
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Hi @JoostU, Great questions!
Yes, we definitely expect to explore alternative warheads in coming rounds of synthesis. Actually, quite a lot of acrylamides have been ordered as a part of Ugi hit expansions such as here https://covid.postera.ai/covid/submissions/adc59df6-a3dd-4d89-ae76-550f19fbfbe3 Last I checked, the feeling is that those hits may still be mostly K_{inact} driven, and some tuning needs to be done.
And I expect that is the feeling with many of the hits showing lower IC50 too.
As for past inhibitors, quite a few are being made! If memory serves, I believe we have at least 6 synthetic chemists on past inhibitors for the next two weeks. There is a lot of interest in getting them into structures as soon as we can. And I have also put through orders for the Real Space compounds you so kindly helped me look into here A brief exploration of past SARS small-molecule inhibitors
I just wanted to flag up a couple of points.
These certainly look interesting. One problem with the ligand efficiency metric is that perception of efficiency varies with the concentration unit in which affinity (or potency) is expressed. The nature of ligand efficiency discusses the problem and suggests a solution (which could also be applied to other measures of activity such as k_inact/K_i):
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I’ve been taking a look at the moonshot_initial_acitivity_data.csv list. Why are MAK-UNK-6435e6c2-3 and MAK-UNK-6435e6c2-2 missing from the list of IC50 measurements? The AVG % inhibition at 20 and 50 µM is over 50…
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Hi, @vvoelz, thanks for pointing this out. These compounds with that high inhibition are actually the corresponding chloroacetamides see below, and they were mislabelled with the wrong Id when they came back from synthesis. I apologize that they made there way into the system still (will correct that soon), but the IC50 data below should be correct.
Thanks @pwkenny, yes, the computational work is mostly on predicting “hot” warheads in order to know what to make next, while of course some real assays need to be done in the future. My understanding is that thiol reactivity assay should at least give us some idea how generally reactive these things are.
And thanks, I’ll look into the paper. Though, I don’t think anybody is relying on it too much here. I was using it pretty crudely in a “small thing bind not bad” kind of way.
-Matt
I thought I’d best cross-post. I posted a modification of the submission data from this release, but with combinations of different warheads both in reacted and unreacted forms here: Covalent attachments with "*"
Very nice work, thank you! 
A small suggestion from me on data visualisation, if I may take a stab at the “wishlist” (like @krisbirchall said, there are many other things to be done, and this is just something I thought of while playing with the data) 
:
To indicate in a column as to whether a compound is a reversible inhibitor or irreversible inhibitor. My guess is that most chemists currently determine this by checking for the warhead; for instance, chloroacetamides might count as irreversible inhibitors.
In the same vein, compounds could be differentiated by whether they are competitive or non-competitive, so we can quickly see whether they all bind (or are predicted to bind) to the active site. Or, in non-competitive cases, we can also see which binding sites they are binding to.
Thanks for the suggestion @Zhang-He, yes the covalent/non-covalent issue is one we are constantly dealing with. We are currently doing thiol reactivity screens to understand the reactivity, but it is still a “murky” issue. Obviously, the chloroacetamides are mostly very reactive; however, the acrylamides are kind of on the edge of irreversible/reversible which is an issue in need of elucidation on our end.
As for competitive / non-competitive, I believe most everything in this project is aimed at targeting the active site (though a few of the crystals do indeed show lurking into other sites)
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If labeling inhibitors as reversible/irreversible then it would be necessary to say whether the label refers to a warhead-based classification (assumption) or the result from a reversibility assay (measurement).
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Many congratulations on this great work! The results clearly resolve between compounds with different potencies and there are IC50s as low as 1.8 uM. I am only an enzymologist, but to me several compounds look very interesting with good ligand efficiencies and clogP values. Unfortunately, I have been unable to determine whether there are crystal structures for complexes with these compounds.
Seventeen out of the 18 dose-response curves give slopes greater than 1, possibly because there is some positive co-operativity.
Why do compounds with covalent warheads appear to give reversible inhibition?
Several of these inhibitors have covalent warheads. However, the kinetic data strongly suggest reversible inhibition (because IC50s are well above the 5 nM enzyme concentration and the data don’t show a very steep dose-response which is characteristic of irreversible inhibition). Possible reasons for these compounds not giving irreversible inhibition are:
- The warheads are not positioned correctly relative to Cys145, or have insufficient reactivity.
- Covalent inhibition occurs, but is reversible.
- The compounds react with something (maybe TCEP?) and inhibition is by a product.
For med chem progress, it seems important to elucidate what is happening. Crystal structures will show if there is covalent bond formation.
I agree with Pete Kenny that direct kinetic studies are needed to characterise any irreversible inhibitors.
Experimental protocols
When using recombinant enzyme, it is important to be sure the assay is following the correct activity, rather than a contaminant. I assume these assays do follow MPro, but it would be re-assuring to confirm that SAR from IC50s is in agreement with crystal structures.
For MPro from SARS-Cov, valid kinetics require a full-length construct with authentic N- & C-termini (Xue et al, 2007, J Mol Biol 366, 965). Given the 96% sequence identity, I expect MPro from SARS-Cov2 has similar requirements. The MPro construct used for these IC50s is not stated.
A similar substrate against a similar enzyme gave a Km of 40 uM (MPro from SARS-Cov, Xue et al 2007), and the data from Nir London & Haim Barr used 375 nM substrate. (So not many turnovers for 5 nM enzyme, especially given rate tends away from linear after using around 10% of the substrate.) I often recommend using substrate at its Km value to balance sensitivity between competitive, noncompetitive and uncompetitive inhibitors. This assay, therefore, probably disfavours uncompetitive compounds. The same bias occurs when screening the free enzyme by crystallography and mass spec. This may be acceptable because uncompetitive inhibitors probably are rare.
Choice of reducing agent
It can be difficult to optimise assays for enzymes involving a catalytic Cys residue, balancing the need to prevent oxidation with the possibility of reducing agents perturbing the results. Using “papain-like” protease from SARS-Cov, Lee et al (2012, Analyt Biochem 423, 46) determined kinetic parameters, hit rate and IC50 values to compare no reducing agent with the physiological reduced glutathione (GSH), DTT and TCEP. It was recommended to use 5 mM GSH. I have been involved with a project, which found similar discrepancies with a cathepsin. I usually recommend use of 5 mM GSH, but it needs to be a fresh solution because it is limited stability. A stronger reducing agent may be required to store the stock enzyme solution.
The studies of Nir London & Haim Barr use 1 mM TCEP, which could affect potency of some inhibitors.
GSH stability data would be informative
It is advisable to measure stability in GSH, to ensure active compound is not depleted excessively inside cells. Poor stability in GSH (or TCEP) also could compromise isolated enzyme data, because IC50 measurements assume that inhibitor concentration is constant throughout the assay.
Suggestions for future work
I feel uncomfortable recommending further work, because I know everyone has so much to do and has already completed. Some of these suggestions have already come from other people:
• In the database, link IC50 and crystal structure on a compound by compound basis.
• If not already studied by crystallography, med chem may be helped by directly investigating whether active compounds with covalent warheads actually do give reversible inhibition (eg by dilution) or covalent inhibition (eg by mass spec).
• Stability in GSH should be measured for active compounds that contain covalent warheads.
• Kinetic characterisation (measurement of Ki and kinact) for compounds giving covalent inhibition.
• Consider whether the team are comfortable with the prospect of missing uncompetitive inhibitors.
• If any apparent inconsistencies develop with other data (eg activity in cells), it may be worth testing compounds using GSH rather than TCEP.
Once again, thanks for performing and sharing this excellent work.
Wal
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@mc-robinson
We are doing some Mpro screening in the US DOE national labs, and started out using Bachem’s EDANS/Dabcyl substrate but think your fluorophores look better. We are looking for clarification on your fluorogenic peptide [5-FAM]-AVLQSGFR-[Lys(Dabcyl)]-K-amide - is it K(Dabcyl) and the another N-terminal K, or is K(Dabcyl) the terminal residue?
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Thanks @Wal-Ward, really appreciate the incredibly detailed writeup. I will make sure this gets passed on to everyone on the assay teams. I don’t feel qualified to comment on all of the issues myself, but recognize their importance from what you and others have voiced in this thread.
I can comment on a few issues though:
Yes, great idea. We are working on it! And to some of your other points, we do have crystal structures of some of the amino-pyridine non-covalent hits which are quite ligand efficient. We also have a very new crystal structure (should be released this week) on one of the <10 uM quinolone hits; no covalent bonding is seen in the structure.
And if you still have trouble finding structures you can either look here https://covid.postera.ai/covid/structures#new_structures
or go directly to fragalysis https://fragalysis.diamond.ac.uk/viewer/react/preview/target/Mpro
Hi @sgalanie, thanks for your message. I am the wrong person to be answering this, but perhaps @londonir, and his team can help.
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Hi,
Yes - it is K(Dabcyl) and then another K as the terminal residue. (so two K’s).
Please let us know if you need any more details.
Good luck,
Nir.
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These quinolones (2-hydroxyquinolines) look like molecules with strong fluorescence, is it checked that this does not mess up the read-out of the fluorescence based affinity assays?