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The Arguing Over PTC124 and Duchenne Muscular Dystrophy

Does it matter how a drug works, if it works? PTC Therapeutics seems bent on giving everyone an answer to that question, because there sure seem to be a lot of questions about how ataluren (PTC124), their Duchenne Muscular Dystrophy (DMD) therapy, acts. This article at Nature Biotechnology does an excellent job explaining the details.
Premature “stop” codons in the DNA of DMD patients, particularly in the dystrophin gene, are widely thought to be one of the underlying problems in the disease. (The same mechanism is believed to operate in many other genetic-mutation-driven conditions as well. Ataluren is supposed to promote “read-through” of these to allow the needed protein to be produced anyway. That’s not a crazy idea at all – there’s been a lot of thought about ways to do that, and several aminoglycoside antibiotics have been shown to work through that mechanism. Of that class, gentamicin has been given several tries in the clinic, to ambiguous effect so far.
So screening for a better enhancer of stop codon read-through seems like it’s worth a shot for a disease with so few therapeutic options. PTC did this using a firefly luciferase (Fluc) reporter assay. As with any assay, there are plenty of opportunities to get false positives and false negatives. Firefly luciferase, as a readout, suffers from instability under some conditions. And if its signal is going to wink out on its own, then a compound that stabilizes it will look like a hit in your assay system. Unfortunately, there’s no particular market in humans for a compound that just stabilizes firefly luciferase.
That’s where the argument is with ataluren. Papers have appeared from a team at the NIH detailing trouble with the FLuc readout. That second paper (open access) goes into great detail about the mechanism, and it’s an interesting one. FLuc apparently catalyzes a reaction between PTC124 and ATP, to give a new mixed anhydride adduct that is a powerful inhibitor of the enzyme. The enzyme’s normal mechanism involves a reaction between luciferin and ATP, and since luciferin actually looks like something you’d get in a discount small-molecule screening collection, you have to be alert to something like this happening. The inhibitor-FLuc complex keeps the enzyme from degrading, but the new PTC124-derived inhibitor itself is degraded by Coenzyme A – which is present in the assay mixture, too. The end result is more luciferase signal that you expect versus the controls, which looks like a hit from your reporter gene system – but isn’t. PTC’s scientists have replied to some of these criticisms here.
Just to add more logs to the fire, other groups have reported that PTC124 seems to be effective in restoring read-through for similar nonsense mutations in other genes entirely. But now there’s another new paper, this one from a different group at Dundee, claiming that ataluren fails to work through its putative mechanism under a variety of conditions, which would seem to call these results into question as well. Gentamicin works for them, but not PTC124. Here’s the new paper’s take-away:

In 2007 a drug was developed called PTC124 (latterly known as Ataluren), which was reported to help the ribosome skip over the premature stop, restore production of functional protein, and thereby potentially treat these genetic diseases. In 2009, however, questions were raised about the initial discovery of this drug; PTC124 was shown to interfere with the assay used in its discovery in a way that might be mistaken for genuine activity. As doubts regarding PTC124’s efficacy remain unresolved, here we conducted a thorough and systematic investigation of the proposed mechanism of action of PTC124 in a wide array of cell-based assays. We found no evidence of such translational read-through activity for PTC124, suggesting that its development may indeed have been a consequence of the choice of assay used in the drug discovery process.

Now this is a mess, and it’s complicated still more by the not-so-impressive performance of PTC124 in the clinic. Here’s the Nature Biotechnology article’s summary:

In 2008, PTC secured an upfront payment of $100 million from Genzyme (now part of Paris-based Sanofi) in return for rights to the product outside the US and Canada. But the deal was terminated following lackluster data from a phase 2b trial in DMD. Subsequently, a phase 3 trial in cystic fibrosis also failed to reach statistical significance. Because the drug showed signs of efficacy in each indication, however, PTC pressed ahead. A phase 3 trial in DMD is now underway, and a second phase 3 trial in cystic fibrosis will commence shortly.

It should be noted that the read-through drug space has other players in it as well. Prosensa/GSK and Sarepta are in the clinic with competing antisense oligonucleotides targeting a particular exon/mutation combination, although this would probably taken them into other subpopulations of DMD patients than PTC is looking to treat.
If they were to see real efficacy, PTC could have the last laugh here. To get back to the first paragraph of this post, if a compound works, well, the big argument has just been won. The company has in vivo data to show that some gene function is being restored, as well they should (you don’t advance a compound to the clinic just on the basis of in vitro assay numbers, no matter how they look). It could be that the compound is a false positive in the original assay but manages to work through some other mechanism, although no one knows what that might be.
But as you can see, opinion is very much divided about whether PTC124 works at all in the real clinical world. If it doesn’t, then the various groups detailing trouble with the early assays will have a good case that this compound never should have gotten as far as it did.

28 comments on “The Arguing Over PTC124 and Duchenne Muscular Dystrophy”

  1. Anonymous says:

    This is a great and important question: Should we care how and why a drug works, or only *IF* it works?
    Unfortunately we humans like a good story, to make sense out of things, even if what we think we understand is not actually true (think of faith, religion, superstitions, conspiracy theories, magic, and all the rest) and sometimes we will ignore the most important facts just to keep believing in a good story.
    Consider all the recent clinical trial failures with beta amyloid antibodies in AD, for example. Yet some companies choose to ignore these results and continue to pursue this avenue because it makes a good story…

  2. MoBio says:

    I guess the jury will be out until we see the Phase III data.

  3. Toad says:

    Sounds eerily like the SIRT1 (Sirtris) story all over again – red herring in the in vitro assays and iffy in vivo data. Should we expect GSK to announce a purchase soon?

  4. Anonymous says:

    Phase II did not work but no matter, lets do Phase III.

  5. So, has anyone tried GFP as the reporter instead? Wouldn’t that avoid the problem with the small molecule substrate required for luciferase?

  6. Jumbo says:

    Sorry. The jury isn’t out. Look at the Ph2 data. The MoA is wrong, and it doesn’t work in the clinic. PTC are just unable/unwilling to give up and wasting time, money, and patients’ hopes.

  7. anchor says:

    PTC got its monies worth with false positives and firing away their employees!

  8. Anonymous says:

    Prosensa/GSK and Sarepta are developing DMD drugs but their DMD drugs have nothing to do with readthrough of premature stop codons

  9. Anonymous says:

    The jury might be out on Ph2 results:
    quest dot mda dot org/sites/default/files/2010%20Final%20Summary%20of%20Ataluren%20Trial.pdf
    I agree with #1 about the influence of stories or apparent reason on decision making. Read the above report on ataluren Ph2 and see if that influence is in there. I think so. Low dose has a measurable response, high dose does not. They note it is not uncommon for U shaped curves to be seen, but one dose working and one dose not is not U shaped, that is a story that the data are fit to after the fact.
    As for the value of knowing mechanism, assume for the moment that the data are not just noise, then the low dose did well for the first 12 weeks or so, then walk distance degraded at the same rate as placebo and high dose up to about week 38. Without knowing dosing targets to cover known pharmacology, what to do next? Pulsed dosing? Lower doses? These are reasonable things to try with empirically driven development, but each trial is a year of dosing, probably much more than a year to enroll and probably $5-10 million. Does knowing mechanism truly help give guidance for future trials? I think so, even if the mechanism is not understood from a molecular sense but understood from pharmacology in cell and animal systems. Working off the wrong molecular mechanistic assumptions is not helpful, except for story telling.

  10. SAR Screener says:

    I’m not an expert on cell assays, but surely there must have been an artefact assay they could have run?

  11. HTSguy says:

    @10 Yes, they could have put luc under a constitutive promoter (preferably not too strong) and check for things that increased that signal.
    Derek, low stability (i.e. short half-life) of luc is a feature, not a problem. It gives luc a larger dynamic range in gene-induction assays than reporters (e.g. GFP) with longer half-lives.

  12. sgcox says:

    #11: They were not looking on transcription activation but ribosome activity.
    Anyway, Dundee guys provide counterscreens in the last paper.

  13. ldy says:

    I agree with Jumbo:
    “PTC are just unable/unwilling to give up and wasting time, money, and patients’ hopes.”

  14. luysii says:

    “The inhibitor-FLuc complex keeps the enzyme from degrading, but the new PTC124-derived inhibitor itself is degraded by Coenzyme A – which is present in the assay mixture, too.”
    Unlikely to be correct. Coenzyme A isn’t an enzyme but a way to carry the CH3 CO (acetyl group) about (particularly into mitochondria).
    I do think a phase III trial is a good idea. I used to run a muscular dystrophy clinic, and have watched perhaps 10 or so my Duchenne boys (it’s X linked recessive) die, usually in adolescence. Most of them had pneumonia, which at least is a relatively peaceful death, but the muscle weakness affects the heart as well, and some of them die of congestive heart failure with their lungs filling up with fluid. Essentially they drown right in front of you. It isn’t pretty. So even though it’s unlikely to work, a phase III trial would clear the air if it didn’t. If it worked, even better . . .

  15. #14 luysii: Coenzyme A might be degrading the inhibitor by a simple (uncatalyzed) chemical reaction involving nucleophilic attack by the thiol. Just a guess.

  16. sgcox says:

    Yes, CoA is a part of commercial detection kit and is included at high concentration to relieve the inhibition by product.

  17. Chris Cain says:

    Nice write up Derek! The researchers we interviewed for our SciBX story last month said there was a legitimate case to be made that the different reporter constructs used by different groups could account for the discrepancies, but the jury is definitely still out. I think the most interesting question is knowing what PTC124 hits in cells. It is clearly acting differently than the aminoglycoside antibiotics in some way, and if you take at face value that it does possess read-through activity in some systems, then maybe understanding how it works could provide a rational basis for compound design and screening going forward?

  18. Hap says:

    If the degradation of enzyme is interfering with the understanding of whether PTS124 is causing read-through or not, wouldn’t an assay using something like GFP be a better output? In this case, you have an effect (somewhere), so you don’t care as much about dynamic range, but you need to know that whatever readout you get is due to an effect on read-through and not product stability.
    Of course, in P3 isn’t the time to be asking these questions.

  19. hn says:

    Is it that hard to test your lead compound with more than one kind of in vitro assay prior to pushing it down the pipeline?

  20. Anonymous says:

    There is no way that it works. The putative mechanism is totally non-specific and would lead to read through of literally thousands to millions of real and cryptic stop codons throughout the genome, which would make cells very very angry! The whole thing is totally stupid.

  21. Anonymous says: they claim that
    “So the two types of stop codons, the normal, and the premature one, are in a very different environment. And that is what makes PTC124 so specific, it is very, very selective.”

  22. rcyran says:

    This looks like a case of “perhaps the horse will sing”.* Drug did poorly in Ph II – going into Ph III gives them a long shot chance at success. Perhaps more importantly, it allowed to raise capital to keep going and gave them time – pivotal Ph III results in ataluran are in mid-2015 according to company. Should have one of their pre-clinical candidates in Ph I by then.
    *For those that don’t know the story from Arabian Nights:

  23. jackbaba says:

    This compound was spotted from libraries by solid phase synthesis years back in the beginning of last decade. As I agree on your point, biotechnology companies just wanted to push forward a ‘lead’ by dressing it up. Now that they have spent so much on it, how could they be willing to give up. Let’s go and see.

  24. jackbaba says:

    rcyran, thanks for sharing the singing horse story. It is so true how so many biotechnology settings are following the clever man.

  25. Dan Ryder says:

    Thanks for a superb and thorough review of the situation Derek.
    And thank you rcyran for sharing the story from Arabian Nights. There may be some truth to your reference but only time will tell. It will be interesting to see how things turn out.

  26. DeusEtV says:

    Muscular dystrophy drug approval ‘recommended’ in EU

  27. Leon says:

    There is another interesting method to help read through. Using small RNA guided site specific pseudouridylation to modify U of premature stop codon to pesudo-U. It can achieve certain level of read through in cell experiment. In nature at least Virus uses similar mechanism to induce read through in plant. Though virus does it by modify tRNA instead of mRNA or pre-mRNA. It seems because of abnormal base pairing happened between tRNA and mRNA when U becomes pseudo-U.

  28. Leon says:

    Is there any research lab hiring molecular biologist working on Duchenne Muscular Dystrophy? I’m interested in this.

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