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Derek Lowe The 2002 Model

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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: Twitter: Dereklowe

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December 17, 2012

Stapled Peptides Take a Torpedo

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Posted by Derek

I wrote here about "stapled peptides", which are small modified helical proteins. They've had their helices stabilized by good ol' organic synthesis, with artificial molecular bridging between the loops. There are several ways to do this, but they all seem to be directed towards the same end.

That end is something that acts like the original protein at its binding site, but acts more like a small molecule in absorption, metabolism, and distribution. Bridging those two worlds is a very worthwhile goal indeed. We know of hordes of useful proteins, ranging from small hormones to large growth factors, that would be useful drugs if we could dose them without their being cleared quickly (or not making it into the bloodstream in the first place). Oral dosing is the hardest thing to arrange. The gut is a very hostile place for proteins - there's a lot of very highly developed machinery in there devoted to ripping everything apart. Your intestines will not distinguish the live-saving protein ligand you just took from the protein in a burrito, and will act accordingly. And even if you give things intravenously, as is done with the protein drugs that have actually made it to clinical use (insulin, EPO, etc.), getting their half-lives up to standard can be a real challenge.

So the field of chemically modified peptides and proteins is a big one, because the stakes are high. Finding small molecules that modulate protein-protein interactions is quite painful; if we could just skip that part, we'd be having a better time of it in this industry. There's an entire company (Aileron, just down the road from me) working on this idea, and many others besides. So, how's it going?

Well, this new paper will cause you to wonder about that. It's from groups in Australia and at Genentech, (Note: edited for proper credit here) and they get right down to it in the first paragraph:

Stabilized helical peptides are designed to mimic an α-helical structure through a constraint imposed by covalently linking two residues on the same helical face (e.g., residue i with i + 4). “Stapling” the peptide into a preformed helix might be expected to lower the energy barrier for binding by reducing entropic costs, with a concomitant increase in binding affinity. Additionally, stabilizing the peptide may reduce degradation by proteases and, in the case of hydrocarbon linkages, reportedly enhance transport into cells, thereby improving bioavailability and their potential as therapeutic agents. The findings we present here for the stapled BH3 peptide (BimSAHB), however, do not support these claims, particularly in regards to affinity and cell permeability.

They go on to detail their lack of cellular assay success with the reported stapled peptide, and suggest that this is due to lack of cell permeability. And since the non-stapled peptide control was just as effective on artificially permeabilized cells, they did more studies to try to figure out what the point of the whole business is. A detailed binding study showed that the stapled peptide had lower affinity for its targets, with slower on-rates and faster off-rates. X-ray crystallography suggested that the modifying the peptide disrupted several important interactions.

Update: After reading the comments so far, I want to emphasize that this paper, as far as I can see, is using the exact same stapled peptide as was used in the previous work. So this isn't just a case of a new system behaving differently; this seems to be the same system not behaving the way that it was reported to.

The entire "staple a peptide to make it a better version of itself" idea comes in for some criticism, too:

Our findings recapitulate earlier observations that stapling of peptides to enforce helicity does not necessarily impart enhanced binding affinity for target proteins and support the notion that interactions between the staple and target protein may be required for high affinity interactions in some circumstances.19 Thus, the design of stapled peptides should consider how the staple might interact with both the target and the rest of the peptide, and particularly in the latter case whether its introduction might disrupt otherwise stabilizing interactions.

That would be more in line with my own intuition, for what it's worth, which is that making such changes to a peptide helix would turn it into another molecule entirely, rather than (necessarily) making it into an enhanced version of what it was before. Unfortunately, at least in this case, this new molecule doesn't seem to have any advantages over the original, at least in the hands of the Genentech group. This is, as they say, very much in contrast to the earlier reports. How to resolve the discrepancies? And how to factor in that Roche has a deal with Aileron for stapled-peptide technology, and this very article is (partly) from Genentech, now a part of Roche? A great deal of dust has just been stirred up; watching it settle will be interesting. . .

Comments (29) + TrackBacks (0) | Category: Cancer | Chemical Biology | Pharmacokinetics


1. ScientistSailor on December 17, 2012 12:22 PM writes...

If any of you were still wondering why Genentech scientists poke at Roche...Roche paid $25MM for a bag of air that GNE already knew didn't work.

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2. barry on December 17, 2012 12:23 PM writes...

it's a great piece of research and a great sample of science writing. Pity that it makes no money for the company and doesn't lead perceptibly to an advance in healthcare

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3. Anon on December 17, 2012 12:28 PM writes...

Genentech routinely reports negative results to maybe dissuade competition. It's really not surprising a new molecular entity doesn't work the way you expect. It would be more surprising if it did work.

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4. Anonymous on December 17, 2012 12:38 PM writes...

Doesn't this reiterate a common theme in drug design, that: what may work for some family of targets may not necessarily work on another?

I can show you 100 examples of an additional methyl group being critical for affinity or permeability, and another 100 that show the opposite. Would we not expect the same for stapled peptides?

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5. Teddy Z on December 17, 2012 12:52 PM writes...

Just a point of science. Insulin and EPO are delivered sub-Q, or IM, not IV.

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6. Rafael Najmanovich on December 17, 2012 1:03 PM writes...

"Thus, the design of stapled peptides should consider how the staple might interact with both the target and the rest of the peptide, and particularly in the latter case whether its introduction might disrupt otherwise stabilizing interactions"

This is so obvious that I can't believe it is what the authors conclude from the whole study. Any modification, in a ligand, in the target, no matter how small may have drastic results. Without small differences sometimes causing drastic effects we wouldn't have evolution. In any case, it is nice to see industry publishing negative results.

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7. Harry on December 17, 2012 1:10 PM writes...

This stapled peptide idea is interesting. One area where it might prove useful is allowing peptide endorphins to be orally dosed (at least from my layman's perspective it is).

I welcome input from those with greater expertise.

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8. John on December 17, 2012 1:37 PM writes...

The first author and two of the three corresponding authors are from 'The Walter and Eliza Hall Institute of Medical Research' in Australia... probably not fair to call it Genentech's work.

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9. Derek Lowe on December 17, 2012 1:50 PM writes...

#8, John - that's an excellent point, and I've corrected to the post to reflect it. Thanks!

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10. LeeH on December 17, 2012 3:32 PM writes...

It's not too surprising that the stapled peptides show poor membrane permeability. The stapling groups do nothing to mask the hydrogen bond forming groups on the peptide, so the desolvation energy should be about the same, give or take. Too many carbonyls (say, about 4, total), and you're in doubtful territory.

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11. imatter on December 17, 2012 3:58 PM writes...

@#7. The peptides involved in your interest might have too few residues to get the benefit from the alpha-helical stabilizing properties that stapling is said to have. A couple of labs are looking into this hoping that it will be different from just regular cyclic lactams.

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12. MoMo on December 17, 2012 4:13 PM writes...

Stapled peptides, scotch-taped proteins, glued amino-acids, velcro-ed domains---Do I discern a pattern here?

It emerged, it crawled, it prospered, and did not evolve and now it's time go extinct.

Bye, Bye, stapled peptides!

Who gets to keep the stapler?

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13. anonymous2 on December 17, 2012 6:06 PM writes...

What does this say about the original sensationalist papers from Harvard? Maybe someone at Harvard should have a very close look at the records of the data used to produce those papers. Maybe the journals that published these papers should ask the authors for clarification or retraction?

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14. Diver dude on December 17, 2012 9:55 PM writes...

Stapled proteins just scream "hapten" to me. 20 years ago, I worked on a compound designed to stabilize a certain conformation of a critical protein and when we got to the clinic, surprise, immune reactions all over the place.

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15. TheDoc on December 18, 2012 3:46 AM writes...

@Teddy Z:
As a matter of fact, Insulin may be given IV under certain indications, i.e. for bringing blood sugar down immediately, or if the patient is hyperkaliemic.

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16. Boghog on December 18, 2012 11:23 AM writes...

It looks like the design rationale for this particular stapled peptide was faulty. The E151/R154/E158 sidechain interaction in the wild-type peptide is effectively double stapled through non-covalent electrostatic attraction. Without the staple, the peptide is already 20% alpha-helical and introducing a staple only doubles the alpha helicity. Hence there is not much entropic advantage to introducing a staple in this particular peptide. Staples have a much more dramatic effect when the alpha helicity of the unstapled peptide is only a few percent. In these cases, stapling can increase the affinity by 10-100 fold.

The lack of cell permeability of the stapled peptide is far more disturbing/surprising.

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17. marcello on December 18, 2012 1:37 PM writes...

Pfizer (St. Louis site) has been evaluating Aileron's technology internally and somehow has passed up...

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18. Stapler on December 18, 2012 7:45 PM writes...

Now come on! there are a lot of people sitting in corner offices depending on Roche paying big money for their staples, shhhhhh

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19. David Lane on December 19, 2012 4:02 AM writes...

This is an excellent paper mostly indeed from the WEHI group. It raises important questions.
It is important though that the "baby is not thrown out with the bathwater". We have had excellent results with a stapled peptide that we designed to block the p53 Mdm2/Mdm4 interaction. This clearly goes in to cells and is highly specific and active. This is now published. Aileron has reported at recent international meeting similar success and indeed in vivo efficacy against the same target. The issue seems to be all around cell entry as it is relatively easy to get stapled peptides that bind their target proteins well but not so easy to get peptides that combine that property with good cell entry activity. The peptide activity can be greatly effected by the serum used in the tissue culture medium . Our peptide works in 10%FCS but many of the published ones appear only to work in very low serum conditions. The basic concept therefore has merit but as with other small molecules intense and detailed chemical development will be needed to develop molecules that will work in the clinic.

Stapled Peptides with Improved Potency and Specificity That Activate p53.
Brown CJ, Quah ST, Jong J, Goh AM, Chiam PC, Khoo KH, Choong ML, Lee MA, Yurlova L, Zolghadr K, Joseph TL, Verma CS, Lane DP.
ACS Chem Biol. 2012 Dec 18. [Epub ahead of print]
PMID: 23214419 [PubMed - as supplied by publisher

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20. InterestedPeptideChemist on December 19, 2012 11:08 AM writes...

The work that David Lane referred to shows that stapled peptides can work. However, the ability of stapled peptides to enter cells is dependent on the sequence of the peptide and not the staple per se. Aileron have done some excellent work on optimizing the affinity, cell penetration and in vivo stability of the p53-helix and the peptide sequence clearly (obviously, some might say) influences these properties. The staple itself does not induce peptides to be cell permeable, as suggested in the original papers.

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21. Cellbio on December 19, 2012 1:02 PM writes...


How about activity in whole blood? Is your construct better, but still greatly limited by interaction with serum components? Not sure if I interpret figure 2 correctly in the context of F2H assay and 'much smaller difference', but looks like activity is better in serum free conditions? What is the magnitude of the shift, and would it go much further south in whole blood.


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22. querty on December 19, 2012 2:34 PM writes...

Anyone notice that this paper (Czabotar) was under review for more than a year? Would love to see the review/revision history to this one...

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23. Arnold Satterthwait on December 20, 2012 7:10 PM writes...

We warned about the steric problems in our review in Houben Weyl Methods in Organic Chemistry| Synthesis of Peptidomimetics VolE22c titled "synthesis of peptides incorporating helix Mimetics inducers". As one blogger wrote, they are obvious. An alternative approach is to append a helix nucleation site to the N-terminus of the peptide, exterior to the binding site and replace helix destabilizing amino acids with Ala if possible as described in considerable detail in the review. Using our hydrogen bond mimic to form a helix nucleation site by solid phase synthesis (JACS 121: 3862) we were able to improve the helical content and affinity of the Bad BH3 peptide for Bcl-2 by about 5 fold. And, contrary to a claim in another JACS paper which was made without any support!, this type of helical peptide is easy to make as demonstrated by several post-docs if one can follow and understand the given protocol.

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24. Jonathan David on December 21, 2012 12:35 AM writes...

Something did not smell right to me about this paper and it's sensationalist title. If you go back and look at the original paper, the peptide they used was actually one that was purposefully designed to have relatively low affinity for the target in order to facilitate NMR studies. It's a shame that the WEHI have allowed themselves to be sucked into the South San Francisco reality distortion field.

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25. Justin on December 23, 2012 6:16 PM writes...

"Thus, the design of stapled peptides should consider how the staple might interact with both the target and the rest of the peptide, and particularly in the latter case whether its introduction might disrupt otherwise stabilizing interactions."

Well, as a former early employee at Aileron that modeled and designed quite a few stapled peptides, I can say that this was fairly obvious to us.

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26. Healthy on December 29, 2012 6:58 PM writes...

Certainly peptides have strong mobility advantages over proteins, but if you remove the rest of the protein you are introduccing changes, if you remove the chaperone processing you are adding more changes and then you chemically modify it to obtain a secondary structure??? Ok, may worth trying but may very likely fail. Still may have some applications.
For funding, research and peer finding please refer to the non-profit Aging Portfolio.

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27. Arnold Satterthwait on January 2, 2013 4:20 PM writes...

One can stabilize peptides as alpha helices as was shown by several workers more than 20 years ago including my lab. We reviewed the rapid progress in this area for Houben Weyl Methods in Organic Chemistry (2002): Synthesis of Peptidomimetics, Vol E22C as referenced in my note above. Initially, many were surprised that one could stabilize such a complex peptide structure but a rationale followed as day follows night. Namely, by reducing entropy using covalent linkages of many types, the natural tendency of amino acids sequences to form secondary structures win out and the peptide begins to fold. This happens not only for alpha helices but also even more strikingly for other more complex structures including complex loops. Thus cyclizing a peptide to form a loop will even stabilize adjacent reverse turns that also appear in protein crystal structures of the sequence. We spent considerable time and effort developing a solid phase synthesis for introducting a hydrazone hydrogen bond mimic into peptides demonstrating that it can be used to stabilize short peptides as full length alpha helix in (JACS 121:3862) as well as medium sized peptides as complex loop structures (Biochem 39:14377) both in water at 25oC. THus alpha helices could be stabilized by replacing the main chain i, i+4 hydrogen bond which is characteristic of alpha helices while loop structures were stabilized by replacing main chain hydrogen bonds found at greater sequence lengths. There are many additional examples and methods for stabilizing peptide structures presented in the Houben Weyl volumes on peptidomimetics. The Houben Weyl series is a standard reference work found in many chemical libraries.

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28. Peptides on June 5, 2013 3:08 AM writes...

Any update on this article? seems to be a bit out of date. Great read regardless. Peptide research has always struck me as a fun read.

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29. Anonymous on January 6, 2014 9:55 AM writes...

Distinct BimBH3 (BimSAHB) Stapled Peptides for Structural and Cellular Studies- new Walensky paper commenting on Genentech rebuckle-

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