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Watch Your Covalent Drugs Carefully

EGFR is a growth-factor receptor protein that’s well known as a cancer target, and there are a number of drugs that target its kinase activity in order to shut it down. But as is also well known, many cancer cells are rather genomically unstable, and throw off mutations constantly. One of the most common problems with EGFR kinase inhibitor therapy is the development of the T790M mutation, where a methionine gets substituted for threonine in the kinase domain’s ATP binding pocket. That’s right where the inhibitors bind, and it makes the resulting EGFR protein resistant to their action. Up to 50% of the resistant tumors that develop have this change – it’s unfortunately quite effective, which is why we see it so much.

That’s led to efforts to target the mutated receptor, of course, and in late 2015 the FDA approved AstraZeneca’s Tagrisso (osimertinib) for just this indication. It’s one of the newer wave of covalent inhibitors – there’s a Michael addition acceptor hanging off one end of the molecule, which reacts (more or less irreversibly) with a cysteine residue on the protein. Ibrutinib (targeting another protein, BTK, in Burkitt’s lymphoma) was one of the first compounds of this type, and it’s done very well indeed , as have neratinib and afatinib for their respective targets. (Update: caterinib was an early entry that fell out of the clinic, and dacomtinib is still in trials). Mechanistically, this idea has been very well validated; it’s a clear success story.

But as usual, there’s more to that story. That’s the take-away from this paper from Cravatt lab at Scripps, in collaboration with a team at Pfizer. They’re using their expertise in covalent profiling across entire proteomes (and they’re some of the best in the world at this) to look at several covalent EGFR compounds – osimertinib, Clovis’ recently abandoned compound rociletinib, and PF-06747775, which is still in clinical trials. The group prepared versions of these with acetylene reporter groups (in order to find them after they’d labeled their protein targets, which is standard chemical genomics practice and yet another reason why the whole bio-orthogonal chemistry field is a plausible candidate for a Nobel). And things aren’t as simple as you might have thought:

Our chemical proteomic studies reveal that, despite the highly engineered EGFR mutant inhibition profile achieved by all three third-generation inhibitors and their shared unsubstituted acrylamide reactive group, the inhibitors exhibited strikingly distinct proteome-wide reactivity profiles in human cancer cells. More in-depth characterization of the specific off-targets for each third-generation inhibitor revealed that inhibitor 1 (osimertinib – DBL) reacts at high stoichiometry with multiple cathepsins in cell and animal models due to lysosomal accumulation of the drug. That these off-target interactions for 1 were not observed in vitro underscores the importance of performing chemical proteomic studies on drug action directly in living systems.

That is very sound advice. Many studies by now have shown that covalent labeling is a very context-dependent thing. Proteins have different surfaces exposed (and hidden) in living cells as opposed to cell lysates, for example. And this effect is another big one – compartmentalization inside the cell. The whole point of cellular architecture is to make various regions very different from each other and to keep them separated, with defined roles and interactions, and drug molecules can find themselves barred from some of them and accumulating in others. At the whole organism level, such effects are known even to many people outside the biomedical field (the idea that some drugs get into the brain and some don’t, for example), but the same principles apply within cells as well.

These findings build on those from previous Cravatt-group papers and others, and suggest that drug targeting covalent cysteine interactions really need to be profiled carefully across the entire proteome. Cysteine is a unique and important amino acid residue, one that shows up in many binding sites and in the active sites of enzymes. Its free SH group can vary widely in reactivity depending on its environment in a given protein, from not-so-reactive to “red hot”. Which of these you react with when you give a covalent-acting compound will depend on the particular drug structure that your covalent warhead is attached to – it’s quite reasonable to assume that every one of them will have a different fingerprint, to some degree.

In this case, some of these off-targets definitely merit a closer look. One of them, CHEK2, has been variously reported as a tumor suppressor or tumor promoter, depending on the cell line, so you’d really want to see if your drug is hitting it under clinical conditions and what that might be doing. The cathepsins (mentioned in that extract above) are an important class of enzyme themselves, and hitting one of these in particular (cathepsin C) could have direct consequences for the immune system.

Importantly, if you just screen osimertinib against cathepsin C in vitro, it doesn’t look like there would be a problem at all. It’s not that reactive under normal assay conditions, but in the cell, when the compound piles up in the lysosomes, things are different. Lysosomal accumulation is a known behavior for many compounds, and can be a bug or a feature depending on the situation. Tyrosine kinase inhibitors in particular are known to accumulate in this way, an effect that is quite possibly of clinical relevance, so covalent ones need to be checked in real living cells to make sure that they’re not going to do more than you want. Covalent drug developers should take note.

35 comments on “Watch Your Covalent Drugs Carefully”

  1. R. Thomas Winters says:

    Your missing the first of these EGFR Irreversible inhibitors CI-1033 and Dacomitinib.

    https://en.wikipedia.org/wiki/Dacomitinib
    https://en.wikipedia.org/wiki/Canertinib

    Both of these originated in the former Parke-Davis research organization (assimilated by Pfizer via Warner-Lambert take over) way back in the mid to late 90’s. Wyeth also prior to their being assimilated into Pfizer was also working in this area as well around the same time.

    Actually a wide range of Michael addition acceptors where investigated some of them equally potent. Perhaps some of these should be examined in this proteomic study method. Perhaps a better profile will emerge.

    1. Derek Lowe says:

      Thanks! Just added both of those. It’s been a pretty winding road for dacomtinib, hasn’t it?

      1. R. Thomas Winters says:

        Yes – Unfortunately that is the case. I think it could have had better clinical impact had it not sat on the shelf for so long, and the clinical trial not had included folks that had already been treated with Tarceva. I also think this class of drugs should have been better investigated in Wild Type KRAS/EGFR CRC patients well where Erbitux and Vectibix have shown benefit. I think these agents would perform better than these Mabs in CRC personally.

  2. Wavefunction says:

    It seems like covalent inhibitors are going down the same road as kinase inhibitors; presumably designed to be selective but later found to be “selectively non-selective”.

  3. David Borhani says:

    Very interesting. Cathepsin C has a broad activity maximum, from pH ~4 to 6.5. http://pubs.acs.org/doi/full/10.1021/bi8007627

    ~18% of dosed osimertinib is retained in the human body 84 days after a single (radioactive) dose. http://dmd.aspetjournals.org/content/44/8/1201.long

  4. tt says:

    I’m going to nitpick…Michael addition acceptor implies that the nucleophile is a carbon. In the cases mentioned here, they are all heteroatoms (like a cysteine), hence the warheads are actually conjugate addition acceptors.

  5. a says:

    Could not disagree more with the assertion that we need to go looking for trouble using chemical proteomics. We didn’t do that for aspirin or nexium, other amazing, important drugs that happen to be covalent inhibitors. Is there unexplained tox that we are trying to nail down here?

    I think that a lot of the increased spend in R&D since the golden age comes from academic questions like these.

    1. Derek Lowe says:

      I think aspirin would have been dropped under modern conditions anyway, once the GI bleeding was noted in animal models.

      1. NJBiologist says:

        It depends… ASA was a safer alternative to salicylate. If we had salicylate on the market, and there were studies showing “baby salicylate” tablets to reduce cardiac mortality, Bayer might still be able to get an approval for ASA.

        But it would be a huge uphill climb, and I don’t think they’d get a label for pain control.

      2. tangent says:

        But those specifics aside, doesn’t the previous poster have a good question?

        Will these techniques give an earlier / cheaper / safer / better readout of eventual problems? Well, if those cases where they save you from running trials that fail late with toxicity, then yes, it’s got to be worth the cost of running the proteomic fancy stuff. How often is that, and on the other hand how many safe drugs will these screens kill by false suspicion? That’s the $1.0e+9 question.

        (How many kinase inhibitors would have been advanced if their true level of selectivity were known early on? But obviously it’s a bit of a different ballgame for covalent actors.)

  6. mallam says:

    For many of us who have been doing such work for quite a while, this is a total “duh” moment. Especially those “trained” in enzymology!

  7. Barry says:

    Well, there are covalent inhibitors and there are covalent inhibitors. The cases here rely on enforces propinquity to promote the conjugate attack of a cysteine on a weak electrophile. That can happen in a compartment that concentrates the reactants without an inter-molecular recognition event.
    But that doesn’t speak to actual suicide substrates, in which the electrophile is only made in-situ by the action of the target enzyme.

  8. Conjugate perdition says:

    Can someone suggest a good review article discussing different types of covalent warheads, particularly the cysteine targeted ones. Acrylamide and the dimethylaminomethyl version of it seem most popular, but what about other things that can be used. Not easy to find a thorough treatment. Thanks.
    Are there any in-vivo friendly lysine targeted warheads?

    1. Mark Plummer says:

      There is a recent JMC perspective on the subject that just came out that is quite good.

    2. Andy II says:

      Bioconjugate Techniques, Third Edition 3rd Edition by Greg T. Hermanson

      This book will give you an idea of functional groups that you can incorporate into your ligand (molecular tool). If it would be stable enough in the incubation solvent, goes into the binding pocket, and position in the close vicinity, it will react with a lysine residue. Some water soluble NHS-ester may work. If the lysine is nucleophilic enough, a photo affinity labeling, or a fluorescent affinity labeling of bifunctional O-nitrobenzoxadiazole may work. Good luck!

    3. GutDecipher says:

      The boronic acid aldehydes are the main ones I can think of targeting lysines, work originally coming from Lisbon but most recently reported on out of MIT. Look for a Nat Chem Biol article from 2016 and then the work it cites.

  9. Barry says:

    But if the lysosome is truly a dead-end repository for proteins that will never see the cytosol again, maybe I’m not too concerned that many of them get covalently modified?

      1. Barry says:

        Do we expect that a cancer cell would be killed by lysosome blockade faster than a wt cell?

      2. Barry says:

        “these results suggest that inhibition of lysosomal proteases, such as CtsD, could be a new therapeutic approach to reduce renal fibrosis and slow progression of CKD.”

        https://www.nature.com/articles/srep20101

  10. Crocodile Chuck says:

    Why do so many Pharma brand names end with ‘ib’?

    1. NJBiologist says:

      -ib is a standard USAN/INN suffix for kinase inhibitors (of which there are approximately a billion).

      1. Derek Freyberg says:

        And -tinib is the INN suffix for tyrosine kinase inhibitors, as I recall.

        1. David Young MD says:

          There are several website that explain drug generic name suffixes and such:

          https://druginfo.nlm.nih.gov/drugportal/jsp/drugportal/DrugNameGenericStems.jsp

    2. Druid says:

      … and it is not a brand name but a name for the drug which is suggested by the Pharma company and agreed through negotiation by authorities for everyone to use. It is often referred to as the “generic name” though it strictly only becomes that when the drug is produced off-patent. Names are chosen to be similar to others in the same class (as in “-inib” or “-tinib”) but different enough not to be confused with other drugs. This is partly because the chemical names are too difficult or long for anyone to use conveniently and safely. Unfortunately patients sometimes die because they are proscribed or dispensed the wrong drug with a similar name. This can lead to a name change down the road, but a bit late.
      There is also an official pronunciation, which you can usually find on-line if you want to look smart. Since you asked the question, I think you do!

  11. milkshaken says:

    I don’t like Osimetinib right hand portion – triamino-substituted anisole. This electron ring is begging for oxidation to electrophilic quinone-iminiums in liver.

    Another horrifying thing about the molecule is ILD as a common adverse effect (1-10%). Sure most patients won’t live long enough with advancing NSCLC, but it is ugly. Another unpleasant one is QT prolongation and very long MRT – probably due to basic sidechain and partition into organs and sequestering in lyzozomes… I think whoever designed this molecule did a quick job on DMPK and the company took a gamble on it.

    What I mean is, quite likely it is not your average covalent kinase inhibitors, but the local accumulation within lysosomes is to be expected for a kinase drug with strongly basic sidechain

    1. Anon says:

      With regard to “electrophilic quinone,” I fully agree with you that a very highly activated aryl ring is a baggage, but Tylenol does the same thing in liver and is available for at least for 50 years! FDA will force some action, if taking this Kinase inhibitors results in death. Until then, anything goes especially if it shows efficacy!

      1. Derek Lowe says:

        Tylenol/acetaminophen is also the number one cause of OTC drug poisoning in most areas, IIRC. Pretty sure that one wouldn’t have made it through in the current regulatory environment, either.

    2. GutDecipher says:

      I’d be shocked if they didn’t think of that, and to wit, how could it be generated other than o-demethylation? Also it’s a bit harder to overdose on a TCI than it is on acetaminophen.

    3. Anon says:

      The ring only appears very electron rich if you don’t pay any attention to the three dimensional conformation of the molecule.

  12. Bryan Lanning, PhD, TSRI says:

    This paper was previously published as a PhD thesis ( Lanning , TSRI, 2016 ) and lack of credit for this is highly inappropriate.

  13. Anonymous says:

    Did citation of these papers (Lanning, Nature. 2016 Jun 23;534(7608):570-4. and Lanning, Nat. Chem. Biol. 2014; 10: 760–767) not cover everything? Should you have been a co-author on this paper? Does TSRI have an ombudsman for problems like this? Well, if you feel you didn’t get proper credit, all I can say is, “Welcome to the club!”

    1. jeeze says:

      I’m not sure why you would cite that Nature paper as Lanning, Nature… when Lanning was a very middle author on it. Don’t fuel the fire.

      1. milkshaken says:

        Leaving key authors off publication, for whatever reason – for example as a form of PI vendetta – is unfortunately all to common. The journal editors and ORI does not like to deal with such a messy situation even if you have ironclad evidence. With regard to TSRI, I would not rely on their internal research integrity office – having lived through the situation when boss of Scripps FL medchem tried to screw all his coworkers off inventorship on patents (because he wanted to keep the royalty money for himself, he was licensing his group research for his own private companies and he was also paying from his private company money the patent lawyers who working for Scripps were writing his patents on his wok done at Scripps – a blatant conflict of interest if there is one. Their research integrity people slept through it.)

        Also, it happened to me recently, that my former employers published overview on research that I proposed and helped to develop. There are six patent applications as well as nearly identical ACS conference talk on it to prove it. Their level of bad faith quite convinced me to fight back. But the way to deal with a research misconduct is not to write irate internet posts.

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