Thanks @Wal-Ward, great comments – and I believe a kGSH assay is in the works. I have also edited my post to reflect the correct value of osimertinib Kinact. Thanks for catching that!
Regarding benchmarking the docking, wouldn’t it make more sense to (also) create an internal active/inactive/decoy set? (using internal data + for instance the DUD-e webserver)
Hey @bart.lenselink, yes that is perhaps a good idea. We still only have a few active scaffolds , but we could start to try to put it together.
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I’ve taken a look at the feasibility of linking ‘reversible’ warheads to x2646 and prepared these notes:
Using the amide nitrogen of x2646 as a synthetic handle has implications for trans/cis preferences of the amide group and there are some highly relevant measured data in this article (ref 9 in the notes):
I have submitted structures 3 and 4 from the notes as designs:
https://covid.postera.ai/covid/submissions/bbe8d7ff-23b7-4848-a830-4c2ce8fa64e9
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Hi Matt and rest of PostEra team
I think that it would be better to separate the reversible and irreversible inhibitors in data summaries. Structures of irreversibly-bound, covalent inhibitors are not especially relevant to their inhibitory activity and I would recommend generation of transition state models if the plan is to take the irreversible inhibitors forward. This could be in a number of ways and one approach would be to build a structurally-prototypical transition state and then to elaborate it structurally (i.e. add the substituents) in order to generate the transition states corresponding to the different inhibitors.
It would also be an idea to think about what the ideal inhibitor ‘profile’ with respect to k.inact and K.i would be. For example, would the profile for a kinase inhibitor be appropriate (or achievable) when targeting a catalytic cysteine residue? There seems to be an error in the unit (reciprocal micromolar) for K.i in the table.
I’ll link my ligand efficiency article again since the approach suggested for defining efficiency could also be used to normalize k.inact, K.i or k.inact/K.i values (with respect to both molecular size and lipophilicity):
I’ve posted a message on this thread about potential structural elaboration of the x2646 fragment-derived inhibitor (3-aminopyridine) and it is important to be aware that synthetic elaboration at the amide nitrogen is likely to impact on the cis/trans conformational preference of the amide. The 3-chlorophenyl group of this inhibitor occupies the S2 subsite but is something of a dead end because it does not allow access in the direction towards the S5 subsite. My guess is that it will be necessary to replace the aromatic ring with something that does allow access and it may be worth looking at the P2 substituent SAR of known peptidomimetic inhibitors.
I’m guessing that the 2-methoxyphenyl substituent in the quinolone x2910 may also need to be replaced in order to provide access in the direction of the S5 subsite and it will be more difficult to incorporate a reversible warhead than for x2646. The catalytic cysteine sulfur appears to directed at amide carbonyl carbon of x2910 and it may be worth taking a close look at the electron density to see if there is evidence for a productive interaction. The quinolone NH does not appear (at least in the modelled structure) to be donating a hydrogen bond to the protein and there are ways in which the nitrogen could be moved elsewhere in the ring.
Something that makes me a bit nervous about the quinolone is the (electron-withdrawing) amide carbonyl group at C4 (analogy with fumaramide). In general, I think that it is a good idea to check the reversibility even for non-covalent inhibitors.
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Hi Wal,
It may also be useful to think about how to use glutathione reactivity measurements might be used to evaluate reversible warheads. For example, can we measure equilibrium constants?
Is kGSH a pseudo first order rate constant or a second order rate constant?
If progressing irreversible inhibitors, we need to be thinking about how to assess selectivity with respect to other cysteine proteases and other enzymes that use cysteine catalytically. Some of the cathepsins are considered to be therapeutic targets but there will be a number of enzymes that we won’t want to hit.
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Hi Pete
The following reference gives information on using GSH reactivity in the optimisation to osimertinib. You may find it useful. They use a pseudo-first order rate constant for reaction with excess GSH. Ward, RA et al, 2013, J Med Chem 56, 7025-48.
All the best
Wal
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Thanks @pwkenny, I will get this info to the people who need it. And I will try to adjust my summaries in the future to make clear the different strands of inhibitors.
Just some notes/questions on a few of your points.
Thanks! We have seen your designs PET-UNK-bbe8d7ff, which were of interest to the team. There is a bit of a debate to as whether one starts by building more potency then attach a warhead at the end, or to attach warheads now. But I believe these designs will be tried (I have to confirm).
TRY-UNI-2eddb1ff-7 seems to be a new merge building on the 3-chlorophenyl group with a beta-lactam likely reaching towards P5. We’ll need to see a structure for sure, but this compound (4 uM IC50) did show improved potency.
Very good point, I will raise the issue with some of the people who’ve looked closely at this one.
I’ll also point them to this post for much more info outside those highlighted points. Thanks again for the detailed response.
Hi Wal,
Thanks for the reference (I recognize some of the authors). I certainly agree that it would be a good idea to measure rate constants for reaction with reduced GSH. Hopefully the data can be used to assess the viability of chloroacetamides and other irreversible warheads and to establish guidelines for design.
Looking at exposure in terms of a competition between target and GSH for the compound raises questions about how to account for the concentration and location of target within the cell. I’m guessing that intracellular concentration will be greater than Ki for some of the kinase inhibitors but will be less than Ki for the irreversible inhibitors from the Moonshot project.
While industry efforts against cathepsins have typically focused on reversible inhibition, irreversible inhibitors, such as the vinyl sulfone K777 feature in the antiparasitic literature. I believe that K777 was being readied for clinical development and there may have been some tox although I’ve not seen any details. If the team has not done so already, it might be an idea to try to make contact with people who have been involved with (at least) preclinical development of irreversible cysteine protease inhibitors. What is the team’s view with respect to potential anti-targets for irreversible cysteine protease inhibitors?
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Hi Matt,
Thanks for the update and please keep me posted on status of the designs. I’m guessing that we will ultimately need to cyclize in order to present a reversible warhead to the catalytic cysteine in a low energy conformation although the designed compounds should give us an idea about potential benefits of targeting the catalytic cysteine in this manner. This information may be transferable to other structural series. Specifically, we would get an idea of nitrile/aldehyde affinity differences (I’m currently assuming that the nitrile will be significantly less potent although I would be delighted to be proven wrong).
I believe that the substituent will need to make contact with a concave region of the molecular surface of the protein (like where a DMSO molecule binds in the pdb:6y2f crystal structure) if growing in the direction of P5 is to result useful gains in affinity. I don’t think that this will be feasible if you need to maintain the penetration of the S2 subsite by the chloro subsituent. The beta lactam ring would be regarded as potentially reactive by many medicinal chemists and I think that it would be important to have a strong rationale for using it as a molecular recognition element.
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Hi Pete
Thanks for your comments. My simplistic view is that reactivity with GSH is a very approximate surrogate for many off-target effects, including depletion of compound by reaction with intracellular GSH and other proteins (eg serum albumin), together with a crude measure of propensity to react off-target to give undesired effects.
I also am simplistic in my thinking about effects of target concentration. A typical drug has a total peak plasma concentration of around 1 uM, of which about 5% or 50 nM is free. I assume that the intracellular concentration is similar to that in plasma (not always the case, in part due to P-glycoprotein and other transporters). When the concentration of free compound in the target cytoplasmic compartment is depleted by covalent or noncovalent binding, then I assume a degree of re-equilibration occurs, so that the drug is effectively replenished to some extent from the pool of compound outside the cell. I accept that equilibration may not be complete or instantaneous and that PK effects will have significant influence, but overall, I don’t expect there to be a big issue due to depletion by binding to target in the cystoplasm. I think off target effects are much more likely to compromise efficacy by depleting compound, simply because there are more off target molecules. Of course biosynthesis of fresh target protein of could compromise efficacy and duration of action…
I hope these musings are useful. Best wishes
Wal
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To add to this PPB is an important parameter to consider (usually plasma protein binding <95% desirable) which can differ if other meds are used (highly likely for a covid patient!)… and drug drug interactions. This is very early stage, i know, but having ideas of some lead-like molecules soon in terms of CYP isoform selectivity (usually IC50>3-10 uM)) would be useful to see how these compounds would work when a very ill patient has several other drugs in their system that could also affect their concentrations? We can go on with PAMPA, efflux ratios. everything… it’s a true minefield!!!
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We have PPB in the pre-in vivo testing plan. Generally I’m with Genentech in that it’s important to understand, but not optimise against (https://pubs.acs.org/doi/abs/10.1021/jm5007935) particularly as very highly bound compounds are at risk of high inter and intra patient variability and therefore represent a toxicity risk. For the LO phase there is also the risk of issues as we move between species as very different PPB between species can lead to overdosing / underdosing and concomitant toxic effects or lack of expected PD effect.
We also have CYPs in the plan - we’ll screen for them as an issue once we have good enough leads - the consensus view amongst the medicinal chemists on the team is CYPs are always something you can optimise away from if you have an issue.
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Super, sure. Aldehyde oxidase is another one that’s poorly dealt with. We’ve been using the Baran Litmus test and it works a treat (CHF2 substitution correlates with AO susceptibility). Are triazoles A.O. substrates? Obviously to bear in mind @ a later stage.
BTW, latest J Med Chem is very useful I’m sure; it;'s on drug tox and lead optmisation strategies;
June 25, 2020 Volume 63, Issue 12
Pages 6249 - 6574
DOI: 10.1021/acs.jmedchem.9b00917.
Hi Wal,
I think that we are in broad agreement on the value and interpretation of the GSH stability assay and I agree that what happens to a compound outside cells is likely to be much more important, from the perspective of clearance, than what happens to a compound inside cells. I would guess that we should be able to use measured kGSH and typical GSH plasma levels to calculate a lower bound for the clearance of a compound that reacted irreversibly with GSH.
When I was working on cathepsins, the view was that problems associated with covalent drug-protein adducts would go away if a sufficiently low dose could be achieved. The covalent cathepsin inhibitors were reversible (nitriles) and the equilibrium constants (not measured) would have been more relevant than the rate constants that were actually measured. While I would guess that reactivity of chloroacetamides with the main protease can be improved significantly, it is likely to be more difficult to reduce the GSH reactivity of chloroacetamides since the warhead is peripheral within molecular structures that incorporate it.
My view is that one needs to have a strong rationale (e.g. essential for target engagement) to justify pursuing irreversible inhibitors given the additional complexity that these bring to design and PK/PD modelling and the general undesirability of irreversible drug-protein adduct formation for anything other than the primary targets. I would not regard the success of irreversible kinase inhibitors as adequate justification for pursuing chloroacetamides, It can be argued that the benefits of the warhead in the kinase inhibitors are more due to the recognition of the cysteine residue that to the irreversible nature of the drug with this residue. Reversible covalent bond formation might even be the better option for the kinase inhibitors (although I’d conjecture that this would be more difficult to achieve with non-catalytic cysteines).
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Hi Ed,
I would agree with monitoring PPB rather than explicitly optimizing it. My understanding is that PPB is not really an issue for drug-drug interactions unless doses are sufficiently high as to saturate the PP. Also, PPB needs to be seen in the broader context of distribution (an increase in fraction unbound from 1% to 5% is not necessarily going to lead to a 5-fold increase in unbound concentration). This article may be helpful if you’ve not seen it already:
https://doi.org/10.1038/nrd3287
In addition to the generic anti-targets (CYPs, hERG, etc), I would recommend that the team think about which cysteine proteases (and other enzymes that use catalytic cysteine) should be considered as anti-targets. This is especially important if targeting the catalytic cysteine (whether irreversibly or reversibly)
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Hi Pete
I agree that it is usually easier to optimise reversible inhibitors, which should be the first priority. However, success is not guaranteed. Covalent inhibitors could offer a useful alternative and I feel also should be pursued. As well as higher degrees of target engagement, they could give longer residence time with PD possibly extending beyond PK if clearance is rapid. If so, this may give reduced off-target toxicity.
All the best
Wal
Thanks for the reference Pete, one to add to my PPB collection, we have a cysteine protease panel lined up with Nanosyn.
Hi Wal,
Just as a point of clarification, I was not arguing against covalent inhibitors. Compounds such as odanacatib, balicatib and petesicatib delivered by industrial cathepsin inhibitor projects can be described as peptidomimetic and targeting catalytic cysteine covalently with a ‘reversible’ warhead (typically nitrile). This is the direction in which I’d be looking if I was setting up a project against a cysteine protease project. My view is that it will not be possible to (rapidly) discover a compound that combines high affinity with good physicochemical/pharmacokinetic characteristics unless the cysteine is targeted (I would be very happy to be proven wrong on this point). The two designs that I’ve submitted to the Moonshot project both seek to link a fragment-derived inhibitor to a ‘reversible’ warhead.
Arguments presented in support of irreversible inhibitors tend to parallel those presented in support of slow off-rates and this article by our former colleague Rutger may be of interest (apologies if I’ve flagged it up already):
http://dx.doi.org/10.1016/j.drudis.2017.07.016
While engagement of target by an inhibitor that is irreversible (or has slow binding kinetics) will persist during the elimination phase, the peak target engagement level will typically be lower than for an inhibitor with fast binding kinetics. I am not especially familiar with immunology although it is my understanding that there would be safety concerns about drugs binding irreversibly to extracellular proteins or remaining bound to peptide fragments after intracellular protein has been degraded.
Given the greater degree of complexity associated with design and evaluation of irreversible inhibitors, I think that it’d be a good idea to see if the GSH stability assay can be used to assess clearance.
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Hi Ed, I’d be interested in the composition of the cysteine protease panel. Something that may be of interest is that the peptidomimetic inhibitors tend to bind to SARS-CoV-2 with a binding mode that differs from the ‘cathepsin binding mode’.