Stapled Peptides: Targeting the “Undruggable”

May 31, 2013 | SINGAPORE—Sir David Lane kicked off the 2013 Bio-IT World Asia conference this week by describing an alternative paradigm for drug discovery and development: using stapled peptides to target protein-protein interactions.

“The problem that we face in drug discovery is the limited range of targets due to intrinsic features of the molecules that we are trying to use,” declared Lane, the Chief Scientist of Singapore’s Agency for Science, Technology and Research. Even though inhibiting their function genetically may have proven therapeutic benefits, a vast majority of proteins are considered “undruggable”. 

However, disrupting a protein’s interaction with key binding partners could achieve the same therapeutic effect as targeting it directly. In fact, such interactions often involve extended domains that could serve as targets for drug discovery. 

 

Two small molecules currently in clinical trials provide a promising precedent for the success of this approach with respect to cancer therapy. Nutlin (Roche) interferes with the binding between the tumor suppressor p53 and its key negative regulator MDM2, thereby activating p53 and its anti-cancer function. ABT-737 (Abbott) blocks interactions with the BCL2 protein and thus induces cell death.  

Lane claimed, “It is relatively easy to find exquisitely specific and very tight-binding peptides that block the binding of these molecules and block protein-protein interactions.”

The real challenge, Lane said, is to turn these peptides, which look like good drugs in a test tube, into drugs that actually work in cells, animals, and humans. 

Peptides do not make good drugs because they have an unstable conformation, do not readily enter cells and are easily destroyed by proteolysis. To overcome these problems, one can use cross-links or cyclization to give peptides stable helical conformations. 

Greg Verdine of Harvard University, one of Lane’s collaborators, combined cross-links and cyclization to invent stapled peptide technology. Within each peptide, two amino acids are substituted with others containing side chains that allow cross-link formation across the peptide, thereby introducing an all-hydrocarbon “staple” into the peptide’s linear backbone. 

These “stapled” peptides have a higher affinity for their targets, enter cells more easily and are less readily degraded. However, controversy flared late last year with the publication of a report that a particular stapled peptide did not work as previously described. 

To validate stapled peptides, Lane and his team designed one to interfere with the p53-MDM2 interaction. 

“The key process for us has been realizing that we shouldn’t just use the native sequence of the peptide that binds the protein target, but that we should use every method possible to enhance the affinity of the peptide,” said Lane. Indeed, they achieved a dramatic 1740-fold increase in the binding affinity of their initial linear peptide. But it was not cell-permeable.  

Lane’s team used computational modeling to derive an optimized stapled peptide and variants for testing. Molecular dynamic modeling was particularly important, because binding partners have conformational flexibility and their interaction is dynamic, not rigid or static. The in silico experiments enabled these studies to be “inexpensive compared to conventional drug discovery”.

The stapled peptide was validated in biophysical and cell-based assays. The researchers demonstrated that it enters every cell and activates p53 in a highly dynamic manner, switching p53 on and off rapidly upon its introduction and removal, respectively. 

Furthermore, the stapled peptide, “achieved a higher level of p53 activation than that seen before”, without the nonspecific toxicity that they observed with nutlin. It was also “exquisitely specific” because a single change in its sequence abolished its activity in the cell-based assays. 

Lane enthused, “Stapled peptides can be quite remarkable molecules, in that they can efficiently go into cells, they can activate target genes and they really do have a number of very desirable and drug-like properties.”

Stapled peptide technology was first described in 2000 but remains in its nascent stages. “It is clear that one thing that has held this field back is access to the technology,” Lane said, bemoaning the ten-year delay in progress. “To have patents that protect technologies is a mistake. Those technologies then don’t get used, they get bypassed and they don’t bring benefit to the community.”

Stapled peptides can be powerful tools for cell biologists to validate and interrogate drug targets. With the technology now available for the large-scale production of these molecules, they themselves can be fully developed as drugs. Lane predicted, “I think we should have a lot more activity in this field.”

Lane is optimistic that improved assays and technologies combined with more focused efforts will fuel progress in the development of stapled peptides as drugs. Not even “undruggable” targets will be safe from this new addition to our arsenal of therapies.