Skip to Content

Drug Assays

Tecfidera Explained

One of the more unusual drugs on the market is Tecfidera (dimethyl fumarate). I went into its history a bit in this post, if you’re wondering how a molecule that small and unfunctionalized became a multiple sclerosis drug. As that shows, it went into trials for the disease with quite a bit of clinical rationale, but no clear molecular mechanism. So how does it work?

This new paper in Science has what could be the solution. It’s long been suspected that there’s a covalent mechanism at work, since the compound is an unsaturated small methyl ester (and thus potentially reactive with nucleophiles like cysteine). The history of allergic reactions in some patients fits in with that idea as well – you’d have to imagine that if it’s reacting with Cys residues that it’s capable of reacting with quite a few of them. One suspect had been a protein called KEAP1, which (along with its partner Nrf2) is a key player in inflammation, but that alone doesn’t seem to account for the drug’s effects. But now it looks like a more mechanistically important one has been tracked down: Cys150 of the enzyme GAPDH (glyceraldehyde 3-phosphate dehydrogenase.

That one, as many will recognize, is responsible for a step in the glycolysis pathway. Why should messing with metabolism help out MS patients (and psoriasis patients as well, the drug’s original market)? Glycolysis is actually important for immune cell function: if you block it, macrophages cannot activate. The cell types affected after Tecfidera treatment are just those that are the most glycolitic (such as effector T cells), while Treg cells (among others) are left alone. Overall, the population of immune cells gets shifted around in ways that benefit autoimmune disease.

The paper demonstrates this in a number of convincing ways: if you incubate the enzyme with dimethyl or monomethyl fumarate, you can see the new mass adduct. And you see the same modified protein if you isolate GAPDH from the blood mononuclear cells of patients who have been taking Tecfidera for months as well. You get the same results from cell cultures and by dosing mice. The isolated enzyme shows dose- and time-dependent inhibition on treatment with either fumarate in vitro. Looking more closely at the cells, the compound shows its effects on glycolysis in activated macrophages and lymphocytes, but not in resting cells, as shown by several downstream markers. This treatment also abrogates their immune functions (as shown by their response to the classic LPS antigen), and you can reproduce all these effects with a known GAPDH inhibitor (heptelidic acid). So yeah, this appears to be pretty nailed down.

It’s also noted that fumarate is actually a downstream product of glycolysis, so it may be that dimethyl fumarate is just a prodrug for getting product inhibition of the pathway as well. None of this rules out other enzymes being modified by it, though – in fact, given its size and simplicity, other enzymes surely are. And the GAPDH-driven effects in other cells may also be important (it’s already been suggested that it has metabolic effects in oligodendrocytes, although the mechanism wasn’t figured out). So what we have here is a small, simple, promiscuous covalent-inhibitor molecule that inactivates a ubiquitous enzyme that’s crucial to metabolic activity. And it’s a beneficial drug with a useful therapeutic index. Go figure!

Update: here’s a recent chemical proteomics effort on dimethyl fumarate, which identified a number of potential binding sites – but not this one on GAPDH! That’s worth thinking about (from both directions) and these sorts of disconnects may turn into a future blog post. . .

 

28 comments on “Tecfidera Explained”

  1. Paul Brookes says:

    First, fumarate is a very weak electrophile and its modification of thiols is highly pH dependent (see early work of Norma Frizzel on this topic: https://www.ncbi.nlm.nih.gov/pubmed/16624247). The amount of fumarate required to inactivate a significant proportion of GAPDH (one of the most abundant enzymes in a cell – that’s why it is used as a housekeeping marker) would therefore appear to be quite large. This is where site occupancy/stoichiometry of PTMs becomes a major issue – you need to modify 50% of the cysteines in the whole population of the protein in order to cause a 50% decrease in enzyme activity.

    Second, it’s surprising (?) to me that a study showing a well-known electrophile modifies a thiol on GAPDH can get into a journal such as this? Again Frizzell’s work has shown much of this already (https://www.ncbi.nlm.nih.gov/pubmed/20964553, https://www.ncbi.nlm.nih.gov/pubmed/17934141).

    For anyone who’s followed redox biology field for a while (cue Lane Simonian and “peroxynitrates” in 3.. 2…1…) the real question is whether there’s anything that doesn’t modify GAPDH? It’s quite literally the most studied thiol in all of biology, and the definite go to low-hanging fruit when you want to show something hits thiols. Whether it has any significant regulatory role in glycolysis (versus say PFK) is not clear either.

    TL/DR: Not very novel, and of questionabe relevance.

    1. anon1 says:

      ^^ sounds kinda convincing…

    2. sgcox says:

      The dosage is 240 mg twice a day. Given the MW=144 it might get to 50 uM in blood. Should be enough to deactivate most of GAPDH. But I do not know its PK so might be much lower concentration.

    3. NMH says:

      Solomon Snyder being the corresponding author is why it got into Science. And the lucky first author will now have a shot of a decent job. The system is rigged.

    4. YogiNinja says:

      Since you seem to know a lot about the drug’s chemistry, can you explain why it access the gastrointestinal system? I took the drug for 3 months, after I was misdiagnosed with RRMS and it has destroyed my digestive system.

  2. luysii says:

    Some interesting chemistry here. This is NOT the displacement of the methyl ester with formation of a thioester, but the addition of the SH group across the double bond forming a thioether.

    1. anon says:

      How is that interesting?

    2. Useless Molecule says:

      Which chemistry is interesting here??

    3. Michael says:

      Quite the addition!

  3. E-phile says:

    Who could have imagined such a thing?
    oh wait
    https://www.ncbi.nlm.nih.gov/pubmed/16682416

  4. MrXYZ says:

    FYI, it’s published in Science, not Science Translational Medicine.

    1. Derek Lowe says:

      Not sure how I made that mistake, but it’s fixed – thanks.

  5. Synthon says:

    It would be interesting to know what percentage of MS patients treated with dimethyl fumarate get an allergic reaction. Nine years ago it was totally banned in Europe as an anti-fungal agent in furniture because of this. https://www.chemistryworld.com/news/massive-payout-for-toxic-sofa-victims/3002429.article.

    Something which has puzzled me for years though. I have never seen any studies on dimethyl maleate, which on the face of it should be more reactive. Is it isomerised too rapidly to the fumarate? Is not selective enough?

  6. tlp says:

    It looks like this study was designed with the conclusion in mind, whereas Cravatt’s approach measures relative ratio of reactivity of Cys with DMF vs. iodoacetamide. Also protein abundance may play a role in a way that for a highly abundant protein it’d be difficult to reach 90-95% of labeling by DMF to rank high in Cravatt’s hit list, especially taking into account that majority of it will probably end up reacting with glutathione.

  7. Mach4 says:

    Its amazing that it costs 55K$ per year for this simple drug and as a reagent its 59$ per 100 grams from Sigma. Whats not amazing is that it has pleiotropic activity against cells and mitochondria, and everyone is focusing on the glutathione depletion as its only activity.

    I don’t think anyone should be describing a singular mechanism of action in any one cell type with this drug, and that we are all naïve when it comes to mitochondria and the effect of small molecules on their dynamics in MS, inflammation and in general.

  8. LiqC says:

    Such a fertile ground for research as well as speculation! I think that organism-wide GSH depletion and downstream Nrf2/Keap1 business bringing about systemic antioxidant response is a more important factor and you won’t get Tecfidera’s effects from, say, only selectively hitting GAPDH.

    The cool thing about Michael acceptors is that they will deplete GSH without oxidative stress from the “natural” means of doing that – through ROS. GSH depletion induces enzymes – catalase and SOD, that can take care of ROS far more effectively (catalysis!) Thus, paradoxically, compounds that kill off one of your primary antioxidants, end up bringing on a far stronger antioxidant response. The simpler the electrophile – the better, you want most of your active electrophiles mopped up by GSH rather than going after some vital proteins. GAPDH reactivity (and abundance), like people above noted, is not news, but the effects on immune system are very interesting. I’m sure there will be more to the story.

    Now, consider the plethora of natural substances that the magazines at the register will tell you are the new anti-inflammatory superfood. In many cases you’ll find promiscuous Michael acceptors there. Nrf activation was the “scientific basis” of several nutraceutical companies, perhaps best known among them is the company that employed Joe McCord of the SOD fame (he’s cut ties with them after some clinical failures).

    Synthon: Dimethyl maleate is not flat and for that reason is not “doubly activated” like the trans-isomer. Similar factors at play but to a different extent than in cycloadditions due to TS geometry.

    1. Orthogon says:

      This paper is worth a look: “Dimethyl fumarate treatment induces adaptive and innate immune modulation independent of Nrf2”

      http://www.pnas.org/content/113/17/4777

      1. LiqC says:

        Thank you!

    2. Synthon says:

      Dimethyl maleate looks pretty flat to me. However in a study regarding treatment of psoriasis with dimethyl fumarates there was a marked effect on granulocytes with dimethyl fumarate and monomethyl fumarate but not dimethyl maleate. As an aside they mentioned that ethyl fumarate had no or little effect either, suggesting size and shape are critically important.

      1. LiqC says:

        It can’t be flat, the ester groups would bump into each other. One has to rotate out.

        Also, once one ester is hydrolyzed, the second one may fall off rapidly via maleic anhydride.

        1. Synthon says:

          Yes, I see now it is not flat. Whether this is the key factor I do not know.

  9. Some idiot says:

    I agree that there could be some interesting chemistry here. Reason for that is yes, it is a Michael acceptor, but… Yes, it is covalent, but especially for Michael acceptors, covalent is in no way the same as irreversible. The direction of this equilibrium will be a function of many factors, not the least the the shape of the electron density environment/pH around where it is acting… I have no personal knowledge in this area, but it is conceivable that with certain sites, the backwards reaction would also be fast, leading to little/no effective occupation.

    As regards monomethyl vs dimethyl esters, it could be that the diester is too hindered to react effectively, or puts the product in a conformation which activated the reverse reaction. Just a thought…!

    (On something different: it could have been quite interesting to send that message accepting all the autocorrects that came in… many of them, but “diester” to “divesting” springs rapidly to mind…! 🙂 )

  10. Isidore says:

    I thought dimethyl fumarate (I wish people would stop abbreviating to DMF) reacted preferentially via Michael addition with cysteines that are oxidized to sulfenic acid, like dimedone, which is used to label monooxidized cysteines in redox proteins. If so it should not be especially promiscuous since only a relatively small percentage of all protein cysteines are oxidized to sulfenic acids at any given moment.

  11. festus says:

    I can’t see the papers, does anyone know if their assay’s are run in the presence of HSA?

    1. Bravo says:

      According to the methods, the enzyme assays were run in sodium pyrophosphate buffer, with no mention of HSA or anything else. Of course they also do assays on lysates, which will have a ton of random protein…

      BTW the supplementary materials, with the methods, aren’t behind the paywall:
      http://science.sciencemag.org/content/sci/suppl/2018/03/28/science.aan4665.DC1/aan4665_Kornberg_SM.pdf

  12. Bla says:

    Given how common the use of GAPDH as a housekeeping gene/protein is, I hope they were good and ran multiple housekeepers, or they would have been fumbling in the dark for some time before they worked this one out!

  13. Isaidit says:

    Can I do the same think with Tylenol and get a science paper because it will do the samething (eg react with GAPDH and reduce inflammation). Then repeat with aspirin?

    Wow didn’t know it was so easy….if you have the right name

  14. Bart Whitehead says:

    My sister was prescribed Tecfidera and after approximately 6 months was taken off after lab test results were positive for JC virus. The interesting long term side effect for my sister is if she isn’t on Bactrim DS daily as prophet, in 8-12 weeks, she suffers from altered mental state and is hospitalized. She returns to “normal” after 3 days of IV Rocephin. No physician has been able to help her and we are desperate for answers. A full battery of lab tests have been completed, all negative. Please help.

Leave a Reply

Your email address will not be published. Required fields are marked *

Time limit is exhausted. Please reload CAPTCHA.