A Staple Diet for Peptide Mimetics

Among other things, the PPI-net conference at the Royal Society in London earlier this year gave some interesting insight into different techniques that researchers are using to synthesize stable proteomimetics. One such technique makes use of hydrocarbon ‘staples’ between amino acids along one side of an α-helical polypeptide in order to fix its conformation as well as provide some protection against proteases when in vivo.

Intramolecular bridging of α-helical peptides has been attempted in many different ways, such as lactams or disulfide bridges. In 1998, Grubbs developed the use of ring closing metathesis to form peptide ‘staples’, which were both stable and relatively easy to access synthetically. Since then, this technique has been refined so that hydrocarbon staples can be incorporated into known α-helices, allowing for relatively stable competitors for therapeutically targeted PPI’s.

TM01Scheme 1: RCM by Grubbs catalyst, to form an i, i+4 stapled peptide

 This technique was pioneered by Korsmeyer, who in 2004 published a paper detailing the use of stapled peptides to target the BH3 domain on BCL-2 family proteins. Korsmeyer was able to create peptides with nanomolar binding constants (KD = 38.8 nM), which proved to be both protease-resistant as well as exhibiting good bioavailability when tested in vivo. Other PPI’s have since been targeted by this technique, such as another ‘holy grail’ of cancer therapeutics, the p53-HDM2/HDMX interaction.


Figure 1: Binding affinity of stapled peptides to Bcl-x

More recently, some creative use of this chemistry has allowed the synthesis of stapled helices with some interesting physical properties. One such example is the work done by Rudolf Allemann at the University of Cardiff. Allemann takes advantage of the physical properties of azobenzenes, whereby they are able to undergo light induced trans-cis isomerization. By incorporating the azobenzene functionality into an intramolecular peptide bridge, this property can be transferred onto the helix. By attaching the staple in an i, i+7 conformation (or i, i+4 for shorter sequences), the helix will be stabilized when in the cis conformation, and therefore activated light energy. Alternatively, by using an i, i+11 it is the trans conformation has increased stability, and the protein is therefore deactivated by light.

TM03Scheme 2: Trans-cis isomerization of azobenzene peptide bridges

Experimentally, this technique has been shown to work using in vitro protein assays on a range of proteins. The most notable of which is probably our old friend the BH3 domain, this time using Bcl-x as the target protein. Three different photocontrollable peptides were synthesized by modification of a known Bcl-x inhibitor, using an azobenzene bridge in the i, i+7, i, i+11, and i, i+4 positions. Each three of these were shown to have activity dependent on being in either the trans or cis conformation. The i, i+7 and i, i+4 bridged peptides both showed around 20-fold increase in activity after irradiation with light, with the i, i+4 shifting from a KD [nM] of 1275 ± 139 to 55 ± 4 after isomerization.

TM04Table 1: Binding affinities of photocontrollable Bcl-x inhibitors

Although it is not immediately obvious on face value how this work could be transferred into the therapeutic environment, it provides an interesting example of how peptide staples can provide more than just stabilization of the parent α-helix.

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