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Quinine’s Target

Every “history of pharmaceuticals” article ever written probably mentions quinine, and well they should. (I certainly reserved an entry for it while writing my own chemical history book). It’s a classic example of a natural product drug, one that was not known to the classical Mediterranean world but was only appreciated by Europeans after they came into contact with the New World civilizations. Malaria was most certainly known to them – it was a disease thought to be cause by harmful vapors (mal aria) particularly in warm, humid areas, but there was no effective treatment in the Old World pharmacopeia. The bark of the cinchona tree, though, was something new. It had been used by natives of the Inca Empire to treat shivering, and since that was a notable feature of malaria cases, the Spanish Jesuits who brought the bark to Europe tried it for that with notable success.

And as the idea of active natural products began to take hold, quinine itself was isolated as a pure substance in 1820. Later in the 19th century, with the Plasmodium parasites were identified as the cause of the disease, it was clear that quinine was affecting the organisms directly. But how? It may come as a surprise, but despite a great deal of work that’s still been unclear – if you’re looking for one, it’s a pretty good example of how tricky drug research can be. The mode of action of any of the antimalarial drugs (of any class) has been hard to pin down, for that matter, and it’s not for lack of trying. Chloroquine is perhaps the best worked-out of the bunch, but quinine doesn’t quite seem to share its proposed mode of action.

Here, though, is a new paper with what might be an answer. A Singapore-Karolinska collaboration tackled the problem with one of the best target ID technologies available, MS-CETSA. The Nordlund lab, which spans both locations, was the source of the first CETSA paper, and they’ve continued to develop the technique. The idea is that when a ligand binds to a protein target, it stabilizes it – the complex has to be lower-energy overall, or it wouldn’t form in the first place. And if you heat up a protein with and without such a ligand, you can see the effect on its melting and denaturation (that’s the DSF assay, differential scanning fluorimetry).

Wildly, this even works in living cells, which is the CETSA assay. Heat is a real stress situation on them, since proteins get misfolded/denatured under those conditions and tend to turn insoluble. If you lyse the cells, with and without compound treatment, and look for how much soluble protein target is left, it’s a measure of how much a compound stabilized it under the heat. Combining this with modern mass spec proteomics, you can do this trick and then monitor the whole proteome (OK, a lot of it) to see which proteins were stabilized, and thus identify targets from scratch: MS-CETSA. No labels needed, real cells, whatever dosages and time points you want to try.

Doing that with Plasmodium falciparum (and with infected red blood cells) after treatment with both quinine and mefloquine identified the organism’s purine nucleoside phosphorylase enzyme (PfPNP) as a target of both drugs. These assays weren’t trivial to set up, but after a good deal of optimization they’ve provided a new look at the proteomes of both the malaria parasites and of erythrocytes. That will be an excellent baseline for further CETSA work in either system. The troubleshooting and validation took place with pyrimethamine (famous these days as Daraprim), which is largely useless in the field these days against malaria, but whose target (dihydrofolate reductase) is well characterized.

Interestingly, resistance to mefloquine isn’t mediated by mutations in that enzyme; that’s instead via a changed transporter protein that becomes available to pump them out of the organism’s cytosol. And it turns out that mefloquine is a much weaker binder than quinine itself (40 micromolar versus 30 nanomolar, in an SPR assay), so the PNP enzyme is probably mainly relevant just for quinine’s effects. A DSF screen  showed that quinidine (a quinine stereoisomer) is an even weaker binder, while chloroquine, primaquine and another antimalarial (lumefantrine) don’t even seem to bind to PfPNP at all. No wonder it’s been taking so long to unravel all this.

The team was able to obtain a very nice X-ray structure of quinine bound to PfPNP itself, and that picture alone (quinine bound to its target!) is something that many people in the field wondered when (and if) they’d ever see. As it turns out, though, it isn’t a new target itself. PNP inhibitors have already been pursued as antimalarials from first principles, with the idea of targeting the parasite’s purine salvage pathway. But no one realized that the first antimalarial of them all was working through the same mechanism! Quinine itself may still have some other modes of action (all of these compounds are a mess, and if you think this is bad you should see the MOA work on artemisinin). But this certainly appears to be the main one, and I think that case can finally be closed, after hundreds of years.

18 comments on “Quinine’s Target”

  1. Chad Irby says:

    I did suffer some bad side effects from taking quinine once.

    Or maybe it was the gin.

    Does eight gin and tonics count as an overdose?

    1. Orvan Taurus says:

      If the dosing wasn’t properly spaced, yes.

    2. Fredbo says:

      You joke, but making your own tonic has become quite the hipster thing to do. At a recent Bartending Conference in Nee Orleans several attendees were hospitalized and the unifying factor is that they were “tasting” Gin&Tonics with some of these home made (more bitter than thou) tinctures.

  2. John Adams says:

    Not to be picky, but MS-CETSA cannot interrogate the “entire proteome”….but a hell of a big chunk of it !!!

  3. RandomWok says:

    Another quick factoid about quinine’s contribution to the world’s pharmacopoeias. Apparently quinine tablets made by The Upjohn Company were provided to Panama Canal workers as an anti-malarial, but were found to be ineffective. An investigation showed that they wouldn’t dissolve (aka bedpan bullets), which led to the development of dissolution testing, pharmaceutics, pharmacokinetics , and bioavailability down the road…

  4. Barry says:

    Long before the 1945 synthesis by Woodward and Doering, Perkins attempted to make quinine. It didn’t work, but he did make a fortune selling mauve as the dyestuff he did make came to be known.

  5. J says:

    I’m going to express a very small pet peeve here, about the shifting of units in journalism.

    See, I know the difference between ‘micromolar’ and ‘nanomolar’, as does the rest of the audience here, but it takes a moment. Wouldn’t the result have more impact if you wrote:

    “40,000 nanomolar vs. 30 nanomolar” – leaving the units the same?

    Humans aren’t real good at big numbers. “5 million” is a big number. “5 billion” is a big number. “5,000 million” sounds like a lot bigger number, but it’s exactly the same as the previous one. Yeah, with a little thought that’s obvious, but the emotional impact is different. Yeah, all journalists do that – shifting units – and the readers can do it, but they shouldn’t have to.

    Anyhow, that’s my pet peeve. Do with it what you will.

    1. Isidore says:

      For most people who work in pharma or in life science-related departments in academia or in medical institutions and who, I suspect, comprise the vast majority of Derek’s readers, micromolar and nanomolar have immediate connotations: the former is something that’s not useable and would have to be improved significantly, the latter is really, really interesting.

    2. doc says:

      Second that idea. Consistency clarifies and creates comprehension. The objective is to communicate! The more work- even trivial work- a reader must do, the worse the comprehension. I learned to write and present at one of the world’s most rigorous institutions, Mayo, in a group that was notable even there for its impact. The effort spent personally and institutionally (Mayo had a large department of scientific editors) was astonishing. A three paragraph abstract routinely took a new resident two weeks to meet the standard required. To this day I can spot a Mayo trained presenter. The point about consistent units is exactly the kind of small thing that in aggregate results in excellence.

      I too find myself mentally making units consistent as I read anything. Doing so often reveals embarrassing flaws in what sounds plausible.

    3. Jorden says:

      I agree with Isadore. Since I use these units all day if I were to see “40,000 nanomolar vs. 30 nanomolar” I would have to translate it to “40 micromolar vs 30 nanomolar”. Because nobody who uses these units would ever say 40,000 nanomolar

    4. Nick K says:

      “40,000 nanomolar vs. 30 nanomolar” – leaving the units the same?

      Had I read that, I would have mentally converted the 40 000 nanomolar to 40 micromolar.

      1. I was being educated in Canada during their conversion from Imperial to Metric units, and one of the pitches for metric was that you could conveniently always use the nearest prefix. So 40,000 nanomolar would in fact be incorrect usage. I believe we had worksheets in which we would “correct” such usage, in a manner similar to reducing fractions.

        Also, you’d never say 12 liters, you’d say 1.2 decaliters, and other such conveniences.

        Why on earth this was supposed to be a good thing was never explained.

        1. Orv says:

          As someone who lives in a non-metric country, I do sometimes wonder why the decimeter isn’t used more. It seems like it would be useful for things like human height, where centimeters are too small and meters are too large.

    5. Anonymous says:

      Others have replied but I will add, “Play to your audience.” Much of the Pipeline audience knows the common way to present such data. I, for one, see the integer part (30 or 40 = same thing; ignore) and the exponent part (milli or nano) and I immediately know that the exponent is the important part and that, in this case, the difference is large (1000x; in a first reading, some might bother with 40000/30 = 1333x). If I were reading “30 nM vs 40 nM” I, for one, would start with “close enough, same thing – within experimental error.” I find 40,000 vs 30 more difficult to “see” quickly compared to mM vs nM.

      If your audience is a non-science group, it is probably important to emphasize the large difference in some other way to make sure they understand it better. (Philip Morrison (MIT physicist) had a nice little book on “Powers of Ten” written for non-scientists.)

    6. Surfactrant says:

      Agree … but using uM as the common unit would be better. as it gives smaller numbers.
      40uM vs 0.03uM is more immediately readable

    7. Sig fig newtons says:

      I prefer having it in uM and nM. At some point you would have to count how many zeros there are to figure out what the units are. Putting it in uM and nM is also like putting it in scientific notation with the correct significant figures. 40,000 is more ambiguous about the number of significant figures.

      My only suggestion would be to list it as “WITH A Kd of 40 micromolar versus 30 nanomolar, in an SPR assay” so that it is clear what measured quantity the units refer to.

  6. Scott says:

    “MS-CETSA. No labels needed, real cells, whatever dosages and time points you want to try.”

    That sounds like the ‘killer app’ for answering the question “where is our drug candidate actually working?”

    But why do I have a horrible feeling that it is obscenely expensive to do?

  7. Professor Electron says:

    A quick thought before I read the paper… If quinine has such a simple mode of action, why is resistance so difficult to generate? Despite centuries of use, quinine resistance has only become significant in the last 30 or so years. Is the binding site on PNP impossible to mutate?

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