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A New Cell Assay -And What It Says About Stapled Peptides

I’m always looking out for new assays that might tell us what the heck is going on inside cells, so this paper caught my eye. The authors describe a new luciferase-based complementation assay for detecting protein-protein interactions. There are several things like this in the literature already (and for sale, too), but this one has what looks like a robust way to get the split-luciferase proteins expressed, and it seems to be picking up weaker and more transient interactions than most. For example, it’s shown to pick up a specific ubiquitin ligase PPI that had been demonstrated by yeast two-hybrid assays, but never in living cells. (Depending on the signal/noise, this sensitivity could either be a bug or a feature!)
They also used this system on interactions of p53 (which has a good number of them), and found something interesting. The only-a-mother-could-love-it small molecule Nutlin 3a is believed to be an inhibitor of the p53-MDM2 interaction, but (as the current paper points out), this hasn’t been conclusively demonstrated in living cells. This assay, though, confirmed that “. . .small-molecule PPI antagonists such as Nutlin-3a can selectively and rapidly disrupt preformed p53-Mdm2 complexes in living cells.” But another reported PPI compound, SJ-172550, failed to show activity (its mechanism had already been reported as not just straight inhibition of the protein-protein interaction). RO-5963, another compound in this space, fared a bit better, but had a noticeably different profile than Nutlin-3a, which does argue for the ability of this assay to pick up fine details.
Stapled peptides have been used to target some of these p53 interactions as well, but conflicting data exist about just how well those work in this case. And the conflict continues: this assay showed some activity for ATSP-7041, but two other stapled peptides from the literature, SAHp53-8 and sMTide-02 (from that same ACS Chem Bio paper linked above) “exhibited no detectable ability to disrupt p53-Mdm2 or p53-Mdm4 complexes in living cells.”
What they found, on further study, was that these stapled peptides seem to be cytotoxic, via some mechanism that has nothing to do with p53, and that this activity is inhibited by the presence of serum in the assay conditions. A cell-free assay system was developed, which indicated that the two problematic peptides were indeed able to disrupt the p53/MDM interactions as advertised – when they can get to it, that is. Adding serum to these assays did not disrupt things, though, which takes care of the possibility that something in serum just binds the peptides and keeps them from doing their thing.
So that leaves the serum as doing something else to keep the stapled species from actually entering the cells. What seems to be happening, from further experiments, is that the compounds are actually damaging cell membranes, which gives them a chance to get in and show activity under serum-free conditions. Adding 10% serum to the assay, though, seemed to protect the membranes from disruption (and thus makes the compounds show as inactive in the resulting cell assay). This effect was seem on plain old fibroblast cells as well, so it’s not specific to cancer cells. And it wasn’t seen with all stapled peptides, either – mutant forms of these very ones, for example, didn’t have the same effect. Nutlin-3a didn’t have it, either.
The authors suggest that this might be the source of some of the conflicting data in the literature on the effects of stapled peptide compounds, especially in this p53 area. People had noted some serum effects and cytotoxicity before, but much of this was explained via p53-dependent mechanisms. What this work shows is that the membrane damage is intrinsic to some of these peptides, and that this is going to have to be taken into account in future cell assays across the field. There are, of course, plenty of nonstapled peptides that are capable of causing membrane damage (some of them on purpose, as in natural antibiotic peptides), so this doesn’t mean that stapled peptides are universally trouble. What it does mean, though, is that measurements of their penetration into cells have just gotten more complicated.

5 comments on “A New Cell Assay -And What It Says About Stapled Peptides”

  1. lam says:

    Always suspicious of cell based data that is not reproduced in the presence of plasma.

  2. Twelve says:

    Derek – could you clarify the point here a bit? Are you saying that the problematic peptides are bound to serum proteins, or not? Obviously, there are trivial ways of independently measuring the binding to HSA, or other specific serum proteins – do we know the result for such experiments?

  3. milkshake says:

    I heard from biologists that similar swampy situation exists in gene delivery with non-viral vectors… Cell biology is hard, and there is not enough carefully designed studies with well-defined systems, studies that include every conceivable control.

  4. gippgig says:

    “had been demonstrated by yeast two-hybrid assays, but never in living cells” – yeast two-hybrid assays are done in live cells.

  5. eugene says:

    Can’t they staple a third peptide that activates cellular uptake processes without destroying the membrane? Or just put it into a virus capsule like in milkshake’s post.

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