Here's a good paper on the design of stapled peptides, with an emphasis on what's been learned about making them cell-penetrant. It's also a specific rebuttal to a paper from Genentech (the Okamoto one referenced below) detailing problems with earlier reported stapled peptides:
In order to maximize the potential for success in designing stapled peptides for basic research and therapeutic development, a series of important considerations must be kept in mind to avoid potential pitfalls. For example, Okamoto et al. recently reported in ACS Chemical Biology that a hydrocarbon-stapled BIM BH3 peptide (BIM SAHB) manifests neither improved binding activity nor cellular penetrance compared to an unmodified BIM BH3 peptide and thereby caution that peptide stapling does not necessarily enhance affinity or biological activity. These negative results underscore an important point about peptide stapling: insertion of any one staple at any one position into any one peptide to address any one target provides no guarantee of stapling success. In this particular case, it is also noteworthy that the Walter and Eliza Hall Institute (WEHI) and Genentech co-authors based their conclusions on a construct that we previously reported was weakened by design to accomplish a specialized NMR study of a transient ligand−protein interaction and was not used in cellular studies because of its relatively low α-helicity, weak binding activity, overall negative charge, and diminished cellular penetrance. Thus, the Okamoto et al. report provides an opportunity to reinforce key learnings regarding the design and application of stapled peptides, and the biochemical and biological activities of discrete BIM SAHB peptides.
You may be able to detect the sound of teeth gritting together in that paragraph. The authors (Loren Walensky of Dana-Farber, and colleagues from Dana-Farber, Albert Einstein, Chicago, and Yale), point out that the Genentech paper took a peptide that's about 21% helical, and used a staple modification that took it up to about 39% helical, which they say is not enough to guarantee anything. They also note that when you apply this technique, you're necessarily altering two amino acids at a minimum (to make them "stapleable"), as well as adding a new piece across the surface of the peptide helix, so these changes have to be taken into account when you compare binding profiles. Some binding partners may be unaffected, some may be enhanced, and some may be wiped out.
It's the Genentech team's report of poor cellular uptake that you can tell is the most irritating feature of their paper to these authors, and from the way they make their points, you can see why:
The authors then applied this BIM SAHBA (aa 145−164) construct in cellular studies and observed no biological activity, leading to the conclusion that “BimSAHB is not inherently cell-permeable”. However, before applying stapled peptides in cellular studies, it is very important to directly measure cellular uptake of fluorophore-labeled SAHBs by a series of approaches, including FACS analysis, confocal microscopy, and fluorescence scan of electrophoresed lysates from treated cells, as we previously reported. Indeed, we did not use the BIM SAHBA (aa 145−164) peptide in cellular studies, specifically because it has relatively low α-helicity, weakened binding activity, and overall negative charge (−2), all of which combine to make this particular BIM SAHB construct a poor candidate for probing cellular activity. As indicated in our 2008 Methods in Enzymology review, “anionic species may require sequence modification (e.g., point mutagenesis, sequence shift) to dispense with negative charge”, a strategy that emerged from our earliest studies in 2004 and 2007 to optimize the cellular penetrance of stapled BID BH3 and p53 peptides for cellular and in vivo analyses and also was applied in our 2010 study involving stapled peptides modeled after the MCL-1 BH3 domain. In our 2011 Current Protocols in Chemical Biology article, we emphasized that “based on our evaluation of many series of stapled peptides, we have observed that their propensity to be taken up by cells derives from a combination of factors, including charge, hydrophobicity, and α-helical structure, with negatively charged and less structured constructs typically requiring modification to achieve cell penetrance. . .
They go on to agree with the Genentech group that the peptide they studied has poor uptake into cells, but the tell-us-something-we-don't-know tone comes through pretty clearly, I'd say. The paper goes on to detail several other publications where these authors worked out the behavior of BIM BH3 stapled peptides, saying that "By assembling our published documentation of the explicit sequence compositions of BIM SAHBs and their distinct properties and scientific applications, as also summarized in Figure 1, we hope to resolve any confusion generated by the Okamoto et al. study".
They do note that the Genentech (Okamoto) paper did use one of their optimized peptides in a supplementary experiment, which shows that they were aware of the different possibilities. That one was apparently showed no effects on the viability of mouse fibroblasts, but this new paper says that a closer look (at either their own studies or at the published literature) would have shown them that the cells were actually taking up the peptide, but were relatively resistant to its effects, which actually helps establish something of a therapeutic window.
This is a pretty sharp response, and it'll be interesting to see if the Genentech group has anything to add in their defense. Overall, the impression is that stapled peptides can indeed work, and do have potential as therapeutic agents (and are in the clinic being tested as such), but that they need careful study along the way to make sure of their properties, their pharmacokinetics, and their selectivity. Just as small molecules do, when you get down to it.