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Bromopyruvate Revealed

3-bromopyruvate is an interesting and controversial compound. It’s been reported to be an active chemotherapy agent, apparently acting via covalent inhibition of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and subsequent metabolic effects via loss of pyruvate itself. Several years ago, you could come across numerous web pages touting it as a wonderful anticancer agent that Big Pharma was keeping from patients, and although that seems to have calmed down a little bit, such stories never really go away. The deaths of three patients in Germany a couple of years ago might have something to do with that.

Any chemist who looks at that molecule, though, will tell you that there’s surely more going on than just the GAPDH story. It’s such a small species, and has a reactive primary halide in it; it has to be hitting other targets. Indeed, it turns out that cells treated with the compound cannot be rescued by giving them back excess pyruvate, suggesting that this isn’t the only pathway that’s being messed up. Here’s a new paper that does just what needs to be done in this case: a full proteomics workup.

The authors do that in straightforward fashion – they synthesized a version of the compound with an acetylene tag on it and showed that it seems to recapitulate the effects of 3-bromopyruvate in cell assays. This was done by making a substituted oxime of the keto-carbonyl; the group refers to that as the amino-oxy (AO) probe. They then exposed cells to this version, lyse them, and use the alkyne as a way to functionalize the now-covalently-modified targets with either a fluorescent tag (to see them in a gel) or a biotin tag (to immobilize them on a solid support for later analysis). There are several controls that you want to run with these sorts of proteomics experiments – for example, competition with an unmodified ligand – but all of this is in order.

When you combine that with mass-spec based proteomic identification, you can assemble a plot like the one on the right. Chemical biology types will immediately recognize the “volcano plot” format, which you see a lot in proteomic and RNA-seq experiments. Along the X-axis you plot the change in the amount of protein that you captured in the AO probe experiments versus the ones where that were being competed with plain 3-bromopyruvate, and along the Y axis you plot the significance of that change. The stuff you truly don’t care about is down there in the caldera (not much change, not much significance), while the things of interest get sprayed up towards the upper left and upper right corners of the plot.

GAPDH is definitely one of those, which is a good sign that the experiment worked the way that it was supposed to. But there are plenty of others. This is, in fact, a fairly sprayed-out plot for its type, and the authors identify over 60 proteins that are affected by the bromopyruvate group in general (the significance-bearing upper part of the plot). The authors refer to that reactivity as “rather broad”, and they have a point. The proteins highlighted in that plot are already known to have reactive cysteine residues in them, and these are almost certainly what are being picked up by the covalent compound (alpha-halo ketones being well known for just that sort of thing). And if you wanted to bear down on it, you could probably find more – the paper shows some results of competition experiments versus both 3-bromopyruvate and an oxime derivative that doesn’t have the acetylene tag, and while they’re broadly similar, they don’t overlap perfectly. So a suite of different competitive reagents could provide an even larger picture.

What this says to me is that while GAPDH inhibition might well be a metabolic target for some tumor types, bromopyruvic acid is a pretty crude tool to accomplish this. You would worry that a molecule this small and this reactive would be accompanied by severe toxicity, and that’s just what you get. The volcano plot above is a first step towards understanding how that toxicity develops, but it’s also enough to serve as a warning. Giving this drug to patients was. . .a borderline call, to be sure, considering that targeting the Warburg effect by itself might well not be curative, rat studies aside.

On the other hand, nitrogen mustards are still chemotherapy agents, and it would be quite interesting to see what some of those volcano plots would look like. They are known to target DNA, but surely they hit a chunk of the proteome as well (and in fact, take a number of nuclear proteins and crosslink them to DNA). But in 2018, I have to think that “not necessarily worse than nitrogen mustard agents” is not a strong case. I can see the potential in topical application, but systemic seems a bit much. You’re going to want a better GAPDH inhibitor, and that may be in the works.

12 comments on “Bromopyruvate Revealed”

  1. Thomas says:

    Last link: Koningic acid – Koning is King in Dutch – Aqua Regia in Dutch is Koningswater – King’s water.
    Talk about reactive small molecules. Luckily it is something else, but the thought alone…

  2. Anonymous says:

    Paywall, so I can’t read the whole paper. Wasn’t there a recent Pipeline discussion of Open Access? Like, yesterday? There was also a recent Pipeline (Med-Chem Labs, What’s Changed? 27 August 2018) in which someone mentioned the former New England Nuclear site and the decreasing use of radiolabeled compounds in research.

    Since the bromopyruvate folks were tracking covalently modified proteins, radiolabeled bromopyruvate would have obviated the need for a lot of the work to validate the oxime analog.

    3-(C-14) pyruvate is commercially available from Bio-Trend. The location of the tag is unambiguous. It would even track the proteins in the event of metabolic loss of C1 (CO2H) and C2 (CO), assuming covalent linkage via the 3-Br C-14.

    Doing 100x more work to validate the oxime was probably easier than getting approval to use and dispose of the radioactive material.

    1. john adams says:

      As a “dinosaur” who also favors radiolabeled reagents for many applications, I’m pretty convinced that 14C-labeling wouldn’t be hot enough for the application(s) you have in mind. Also, tagging with biotin to “collect” the modified proteins is a quite useful “trick”….

    2. loupgarous says:

      You’re right, NRC and Agreement State regulations can be so onerous as to price use of radionuclides in research out of reach for many.

      But I’ve worked in and around hospitals and laboratories that used radionuclides almost all my professional life, and I remember the bad old days when thin polyethylene bags full of patient radwaste (a biohazard and radiation safety issue) were left in patient-accessible hallways for a week or so until someone got around to stowing the stuff under the hospital in the crawl space (this was the state-owned Earl K. Long hospital in Baton Rouge, built on land where the water table rose above the ground in rainy months).

      The worst instance I remember was at the LSU Nuclear Science center where several drums of tritiated waste (someone didn’t calculate the heat build-up within a wax target bombarded by the center’s linear accelerator, and it “underwent rapid disassembly”, painting floor, ceiling, walls and equipment with tritiated wax) were placed right next to a busy sidewalk where students moved between classes every hour of a typical class day. Containing tritium in 55-gallon drums in hot Louisiana weather isn’t a trivial problem, and those drums were out there “cooling off” for years.

      The people who didn’t want to spend money on protecting innocent bystanders from incidental radiation releases are the folks to thank for the straitjacket of regulations that all researchers have to deal with. If radiation safety had been first priority for every lab that used radionuclided, reasonable measures could have been worked out, and the few folks who didn’t want to take those measures handled according to the law.

  3. jbosch says:

    Running your hot probe through the mass spec is probably the biggest hurdle.

    1. Anonymous says:

      When I worked with radioactive isotopes, the most expensive piece of exposed hardware was the ultracentrifuge and that was just a matter of wiping down (and scintillation counting the wipes) the rotors and surfaces. Glassware, etc. for radioactive materials was labeled and segregated; disposables (plastics; wipes) went into the radioactive waste drum.

      I don’t know the current regs, but you’re right in that an instrument used with hot materials remains stigmatized for at least 10 half lives if not forever.

      I wonder if there are any CROs that have dedicated hot labs and equipment for clients to use for experiments like this bromopyruvate case.

  4. AC says:

    Now I’m not one to be presumptuous, but… did anyone really think that an alpha-bromoketone was going to be remotely selective?

    1. AC says:

      Sorry I should specify, an alpha-bromoketone with just *three* carbons.

      1. Druid says:

        If dimethylfumarate can do some good, why not try it?

  5. lynn says:

    Fosfomycin is pretty darn specific – with 3 carbons and an epoxide.

  6. anon says:

    In an update of the German clinic incident from the ongoing multiyear investigation it has been determined that the patients received a very substantial overdose of 3-BP. Characterizing the nature of their response to this overdose has significant relevance.

    The formulation of 3-BP that the FDA approved for immediate clinical trials several years ago was designed to address some of the issues noted in the thread. Would 3-(C-14) pyruvate
    be a suitable way to screen patients for 3-BP treatment? Prospectively knowing which patients would respond to 3-BP treatment (and to what extent) would also help to manage risk.

    1. loupgarous says:

      That’s the same basic strategy as a test for neuroendocrine tumors, the “octreotate scan”, where a tagged form of somatostatin/octreotide (until recently, In-111, but Ga-68 tagged octreotate has just been approved because of the much higher sensitivity of PET/CT scanners to its radiation) measures uptake, thus probable success of various therapies directed at a given patient’s tumors.

      I was a test subject in the Ga-68 octreotate clinical trials, and benefited greatly from that difference. .

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