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DBL%20Hendrix%20small.png College chemistry, 1983

Derek Lowe The 2002 Model

Dbl%20new%20portrait%20B%26W.png After 10 years of blogging. . .

Derek Lowe, an Arkansan by birth, got his BA from Hendrix College and his PhD in organic chemistry from Duke before spending time in Germany on a Humboldt Fellowship on his post-doc. He's worked for several major pharmaceutical companies since 1989 on drug discovery projects against schizophrenia, Alzheimer's, diabetes, osteoporosis and other diseases. To contact Derek email him directly: derekb.lowe@gmail.com Twitter: Dereklowe

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August 25, 2010

GSK's Response to the Sirtuin Critics

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Posted by Derek

OK, time (finally) for the latest chapter in the GSK-Sirtris saga. (This is going to get fairly geeky, so feel free to skip ahead if you're not into enzymology). You'll recall from previous installments that Amgen and Pfizer, among others, had disputed whether the reported sirtuin compounds worked the way that had originally been reported. GSK has now published a paper in the Journal of Biological Chemistry to address those questions. How well does this clear things up? Let's take things in order:

Claim 1: Resveratrol is not a direct activator of SIRT1 activity (Amgen). Building on two 2005 papers, the Amgen team said that resveratrol, the prototype SIRT1 ligand, only works in that manner when the fluorescent peptide (Fluor de Lys) was used in the assay. This is due, they found, exclusively to the fluorophore on the peptide - it's an artifact of the assay conditions. Without it, no activation was seen with protein assays in vitro, nor in cell assays. Native substrates (p53-derived peptide and PGC-1alpha) show nothing.

GSK's response: This is true. They too, found that activation of SIRT1 depends on the structure of the substrate. Without the fluorescent label, no activation is seen.

Claim 2: Not only is this true for resveratrol, it's true for SRT1720, SRT2183, and SRT 1460 (Pfizer). The Pfizer team did a similar breakdown of the assay conditions, and found (through several biophysical methods) that the fluorophore is indeed the crucial element in the activity seen in these assays. And again, since that's an artificial tag, the Fluor de Lys-based assays can have nothing to do with real in vivo activity. Native substrates (p53-derived peptide, full-length p53, and acetyl CoA synthase 1) show nothing.

GSK's response: As above, activation of SIRT1 depends on the structure of the substrate. Without the fluorescent label, no activation is seen. SRT1460 and SRT1720 do indeed bind to the fluorescent peptide, but not to the unlabeled versions. Looking over a broader range of structures, some of them interact with the fluorophore, and some don't. There's no correlation between this affinity and a compound's ability to activate SIRT1.

A screen of 5,000 compounds in this class turned up three that actually do work with nonfluorescent peptide substrates (compounds 22, 23, and 24 in the paper). None of these have been previously disclosed. They, however, that even these still don't work when the peptide substrate lacks both the fluorescent tag and a biotin tag.

What's more, when these three compounds are tested on a p53-derived 20-mer peptide substrate, they actually inhibit acetylation, instead of enhancing it. Looking closer at a range of peptide substrates, SRT1460 and other compounds can also inhibit or enhance acetylation, depending on what peptide is being used. An allosteric mechanism could explain these results. It seems more likely that there are at least two specific sites on SIRT1 that can bind these compounds - the active site and an allosteric one. Thus there are several species in equilibrium, depending on whether these sites have substrate or small molecule bound to them, and on how this binding stabilizes or destabilizes particular pathways. In the real cell, this may all be part of various protein-protein interactions.

Claim 3: SRT1720 does not lower glucose in a high-fat-fed mouse model (Pfizer). Even though exposure of the drug was as reported previously, they saw no evidence (at 30 mg/kilo) of glucose lowering or of any increased mitochondrial function. These animals showed increased food intake and weight gain. The 100 mpk dose was not well tolerated, and killed some animals.

GSK's response: not addressed in this paper. It's an enzymology study only.

Claim 4: Resveratrol, SRT 1460, SRT1720, and SRT2183 are not selective (Pfizer). A screen of over 100 targets showed all of these compounds hitting multiple targets, with resvertrol itself showing the closest thing to a clean profile. None of them, say the Pfizer team, are suitable pharmacological tools.

GSK's response: not addressed in this paper. None of the newly disclosed compounds have selectivity data of this sort attached to them, either. I'd be very curious to know how they look, and I'd be very leery of attaching much importance to their behavior in living systems until that's been done.

The take-home: On the enzymology level, this new paper seems to be solid work. But it's the sort of solid work that should have been done around the time that GSK bought Sirtris, and not something appearing in 2010 in response to major attacks in the literature. The first main claim of those attacking papers is, in fact, absolutely true: the original Fluor de Lys assay is worthless for characterizing these compounds. What we learn from this paper is that the assay is worthless for even more complicated reasons than originally thought, and that the whole series of SRT compounds behaves in ways that were not apparent from the published work, to put it lightly.

As to the selectivity and in vivo effects of these compounds, Pfizer's gauntlet is still thrown down right where they left it. The fact that these compounds are so much harder to understand than was originally thought, even in well-controlled enzyme assays, makes me wonder how easy it will be to figure out the rest of the story. . .

Comments (36) + TrackBacks (0) | Category: Aging and Lifespan | Drug Assays | Drug Development


COMMENTS

1. Dork on August 25, 2010 12:59 PM writes...

Nice piece of reporting Derek.

My takeaway is that resveratrol does directly activate SIRT1. Even though GSK seemingly wrongly got to the correct place, they still got to the correct place. This is GOOD news for humankind, not necessarily for social security and other entitlement programs!

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2. Anonymous on August 25, 2010 1:07 PM writes...

"My takeaway is that resveratrol does directly activate SIRT1."

Did you even read the post?

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3. Bill Sardi on August 25, 2010 1:31 PM writes...

Who cares? Sirtuin1 activation is not equivalent to a calorie restriction mimetic. Aging does not involve a single gene. The road to the Sirtuin1 gene started with Leonard Guarente at MIT, but he later said calorie restriction does not universally upregulate Sirtuin1 in all tissues and organis. So it can't be used as a marker for aging or longevity. Single-gene targeted drugs have been disappointing in other fields of research such as cancer. For reference, data by Weindruch and Prolla indicates life-long calorie restriction activates 832 genes in heart tissue.

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4. darwin on August 25, 2010 2:04 PM writes...

The real scientific question is "is there reservatrol in Mad Dog 20/20?"

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5. Wagonwheel on August 25, 2010 2:36 PM writes...

Citation/Link for the new GSK J Biol Chem article?

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6. weirdo on August 25, 2010 2:37 PM writes...

"#3 who cares?"

How about, anyone who gives a crap about good science and how it is conducted?
Or, anyone who cares how limited research dollars spent pursuing cures are allocated?

Just sayin'.

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7. Wagonwheel on August 25, 2010 2:49 PM writes...

@#1 Are you by any chance on the GSK acquistions and in-licensing team?

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8. StarkChoice on August 25, 2010 3:48 PM writes...

Derek,
Perhaps not a popular point of view in this forum, but this Sitris deal does exemplify the courageous stance taken by Andrew, Moncef, and Patrick in that they are not afraid of risks. They may not call it correctly all of the time, but, on balance, they call it correctly often enough to deliver the bottom line to patients and shareholders alike.

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9. ex-GSK on August 25, 2010 4:27 PM writes...

One more time from behind the scenes: before the deal with Sirtris was done, GSK enzymologists in RTP had reached most of these conclusions and they told Moncef et al. during the due diligence and were rolled over b/c they had the "not invented here" syndrome. Courage is taking risks on unknowns - it is not ignoring data to pay $720M for nothing (happy to be proven wrong - I still have some GSK stock) b/c you got your job (Moncef) by selling the board on how great external biotech opportunities were.

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10. Anonymous on August 25, 2010 4:41 PM writes...

Agree with #9. How much has all this cost? Data are data. Just because you admit your methods were wrong doesn't make it right after all the layoffs and site closures. Perhaps 750million is a worthwhile punt for what everyone else would throw away as an assay artefact?

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11. realist on August 25, 2010 5:06 PM writes...

#8: Don't know what GSK stock you own, but mine has only gone down during these people's reign. And, don't forget, Moncef's recent "promotion", an obvious exercise to essentially get him out of the way of small molecule R&D as a reward for having done so much good for the organization.

#9: This was not a demonstration of courage as much as a display of arrogance and self-perceived superiority of judgement in absence of documented data by those who pushed the deal forward. Even then, it may have been more justifiable in today's environement of looking for new, early opportunities but for the missing (but claimed) lack of properly conducted scientific data and the cost of $720m. Ten to 20% of that, with back-end milestones, and the whole deal would only be background noise within the industry, not gathering recurring headlines.

Derek: You should have pointed out that most of your well-presented points were noted within posts of your previous blog on this topic. I think it would be proper for you to point out and acknowledge the numbers of people watching, evaluating, having preceeded your comments with essentially the same take-home messages.

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12. cancer_man on August 25, 2010 6:07 PM writes...

Interesting that the only two compounds in trials (besides SRT501, which isn't really in an active trial) are SRT2104 (phase II) and SRT2379 (phase I), neither of which have been examined by Pfizer.

Does it matter?

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13. skeptic on August 25, 2010 6:34 PM writes...

#12: It is all, in the end, going to come down to specificity...on vs off target activities.

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14. Kay on August 25, 2010 6:34 PM writes...

I am a little confused. You say "[compounds 22,23,24] actually inhibit acetylation, instead of enhancing it". Why should they enhance it? From the first part of the discussion, I take that the compounds are supposed to be SIRT1 activators, and SIRT1 is a deacetylase, not an acetylase.

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15. Anonymous on August 25, 2010 7:16 PM writes...

There's some recent evidence from the model organism aging fields that punches yet more holes in the hypothesis that sirtuin activity promotes longevity. David Gems and Linda Partridge are making the case (at conferences) that overexpression of Sir2 homologues in Drosophila or C. elegans does not actually extend life span. Previous results to the contrary, they claim, were actually artifacts. Their case seems fairly strong so far, and I've only seen one weak rebuttal from the Guarente lab.

And I say this as a basic researcher who's studying a phenotype that seems to be sirtuin-dependent...

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16. David Stipp on August 25, 2010 7:17 PM writes...

To cancer_man: It seems that for competitive reasons, biotech and drug companies generally only publish structures of drugs they've put on the shelf for some reason, so Pfizer probably has no way to make and test non-shelved SRT2104 and 2379, about which, as far as I can tell, little has been made public. It would be very interesting to know what aspects of those compounds made them seem worth taking into the clinic by Glaxo/Sirtris versus the other putative STACs whose structures are now out there in the literature.
Though mechanisms are obviously very important, I for one am most interested in the in vivo effects of these drugs. And what I think is kind of missing from the discussion here is that the small, brief mouse study that Pfizer did with SRT1720 seems (at least to me) significantly less substantial than the eight cell-based and animal studies with STACs that the Glaxo/Sirtris guys cite in their latest JBC paper (this citation is something of an answer to the gauntlet thrown down by Pfizer on SRT1720's in vivo effects, if you ask me), not to mention the forthcoming paper that Nature mentioned in a news article in March, in which SRT1720 reportedly was tested in more than 1,000 mice, some for up to two years, apparently without signs of the toxicity that the Pfizer group saw. I think we can be pretty sure that Sirtris also did a lot of in vivo work with at least some of its STACs before the GSK acquisition, and the results of those experiments haven't been disclosed (competitive reasons) but may well have figured in GSK's calculus when it bought Sirtris. None of which is to say these drugs will work in people -- but if they do, one result will be to make Moncef and Patrick look prescient, esp. in light of the continuing mechanistic murk.

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17. Pallas Renatus on August 25, 2010 7:39 PM writes...

I totally read the title of this piece as "Certain Critics"... dyslexia is catching up with me.

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18. cliffintokyo on August 25, 2010 8:59 PM writes...

Lets give the Sirtuin *circus* a rest.
However much *pressure* is applied, GSK are NOT likely to tell us what IP + alpha they have gained, if any, from this research project.
This is not an academic game in the old-fashioned *scientific polemics* sense.

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19. ElGalactico on August 26, 2010 9:31 AM writes...

#8: what planet are you on, planet GSK!?
Just in case anyone thinks that the claptrap written by this guy is unrepresentative of GSK, just view some of the comical stuff posted by GSKers on linkedin, this one from a vice president no less made me laugh "Able to create and implement strategy; through setting clear and aligned objectives, developing a winning value-based culture and building fit-for-purpose organisations focused on delivery". Are these the guys really running their R&D?

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20. sgcox on August 26, 2010 9:43 AM writes...

Unless I missed something in Method section, authors did not add any detergents in assay buffer.
But it is a common knoweledge these days that any micromolar range activity of flat, aggregation prone compounds should be viewd with suspicion.

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21. Zack on August 26, 2010 1:29 PM writes...

Science magazine published an incredible letter today from Sinclair, Guarente, and others. ("Dietary restriction: Standing up for sirtuins")

http://www.sciencemag.org/cgi/content/full/329/5995/1012

Basically, they are upset that Science published a review article on dietary restriction that didn't mention sirtuins.

"The authors state that the purpose of their Review is to "consider the role of nutrient-sensing signaling pathways in mediating the beneficial effects of dietary restriction." Yet there was no mention of the sirtuins, a family of critically important nutrient-sensing proteins that promote health span from yeast to mammals, as shown by more than 1000 peer-reviewed publications from labs around the world"

The review was written by Linda Partridge, and her response is perfect.

"Although much work has been published on sirtuins, and they undoubtedly play an important role in health and disease (1, 2), no publications have experimentally demonstrated that altered sirtuin activity can increase mammalian life span."

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22. Virgil on August 26, 2010 1:41 PM writes...

@#15 anonymous: All good points. I've seen Gems/Partridge talk on this, and as you say they have a strong case. The letter in Science linked to by Zack (@#21) is classic Partridge - short and to the point, and really placing the ball in their court.

From my own perspective as a basic researcher interested in acetylation, there's another problem here...

Step 1 - try to get SRT-nnnn compounds from Sitris
Step 2 - refused, told "they're published, make 'em yourself"
Step 3 - synthesize SRT1720
Step 4 - it does nothing in my biological system.
Step 5 - back to Sirtris, as for "some of the good stuff" to confirm whether the lack of effect is real, or just because I screwed up the prep'
Step 6 - no response
Step 7 - try again 6 months later with lots of emails in between
Step 8 - no response
Step 9 - almost a year later, a response, saying "its published, synthesize it yourself"
Step 10 - go back to step 5, repeat ad nauseam.

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23. John on August 26, 2010 3:09 PM writes...

@22: The correct approach (may be too late in your case): "I have some very exciting in vivo results for SRT1720, please verify this sample of my product so I can publish with confidence." (What results? You'd rather not say, but they may be pleased.)

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24. know GSK on August 26, 2010 4:05 PM writes...

#22: This is very typical of how they interact with everyone, even with people in GSK at other GSK R&D sites. For those of you who read this blog and try to give Sirtris credibility, as hard as it is to believe, it is the way it has been, and still remains. And then there are folks like CW, MD and others occupying VP positions now in GSK. They are poison.

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25. cliffintokyo on August 26, 2010 8:14 PM writes...

RE: #18
As I rarely read med/bio chem papers these days (shame), I had no idea how timely my comment was.
Excellent update post, Derek, on the latest enzyme-level work published by GSK, which apparently could have usefully gone a bit further, in particular in addressing the important selectivity issues.
My 2 cents:
Despite the controversy, can't help feeling that something positive (i.e. some clinically useful compounds) might still emerge from all the scientific contributions in this area (perhaps even collaboration should be contemplated, now that the *air has been cleared*, so to speak?); and kudos now seem to be due to Amgen and Pfizer for publishing their results.

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26. cliffintokyo on August 26, 2010 8:17 PM writes...

RE: #25
I forgot to add that all the credit is entirely due to the medicinal/bio chemists/scientists at the companies involved. ;-)

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27. Anonymous on August 26, 2010 9:24 PM writes...

Well, have a look at this my friends. Turd polishing 101! LOL Try not to laugh!

http://www.youtube.com/watch?v=hBj6PonX14A

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28. Anonymous on August 26, 2010 9:47 PM writes...

My question is why did a number of Sirtris top managers bolt just after the whole sirtuin story broke loose? Humm.... I think this the sirtris saga will require some time to fully deconvolute!

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29. cancer_man on August 26, 2010 10:09 PM writes...

How many were there and how many left? Westphal left four months later, but I don't think it was expected that he'd stay that long.

What are we supposed to think of the eight in vivo studies mentioned in #16?

And why did Derek think this latest study should have been done two years ago? Most here think the study doesn't show anything important anyway.

Looks like the SRT2104 phase II trials are expected to end in Feb/Mar 2011.

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30. watcher on August 26, 2010 10:10 PM writes...

#28. Certainly speaks loudly!

A number have been leaving since the GSK buy-out, taking their newly found riches and moving away from the fray.

But even more, skuttlebutt around the Cambridge corridors seems to suggest that several were being pushed out.

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31. Anon 1 on August 26, 2010 10:14 PM writes...

Actually, some studies were due to compete in the fall of 2010.....

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32. cancer_man on August 26, 2010 10:29 PM writes...

While the SRT501 multiple myeloma study was suspended, the SRT501 liver/colon cancer trial concluded in Dec 2009.

Wouldn't they have published by now if they had positive results?

It was a year ago that Sinclair said several papers would be coming over the next few months. That never happened.

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33. Anonymous on August 26, 2010 10:30 PM writes...

#28 @ #30

I just got back from the ACS in Boston. It was fun and possibly profitable ....if you have a surf board or sell umbrellas LOL. I didn't get to visit Cambridge but my hotel was just across the river and i could feel the vibes. It was fun but all my clothes are still damp and i need to go to work tomorrow!!ARRRGH

Any more insight on the Sirtris business??

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34. watcher on August 27, 2010 3:20 PM writes...

#32: Yeah, exactly.

Other studies were to have completed already in 2010 and may have had their own problems with data collection and interpretation.

Another odd thing, is that the clinical studies have been cotinued under the Sirtris umbrella without necessarily a lot of input from clinical within greater GSK, and without the same evaluation mandated for other GSK compounds by GSK Safety or Protocol Review Boards. Odd, and a bit scarry, thing, all that if accurate.

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35. Anonymous on August 27, 2010 9:41 PM writes...

@7 - don't blame this on GSK's licensing team, that lies squarely on the shoulders of Slaoui and Vallance.
@18 - plenty of people IN GSK haven't figured that out either
@24 - sadly too true
@29 Unfortunately, Westphal's actually NOT gone, he's still at GSK as President of the venture capital group SR One, and still runs Sirtris' SAB.

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36. John Joseph on July 30, 2014 11:02 AM writes...

Nice post

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