Stapled Peptide Structure

In 1998, Grubbs and coworkers published an a-helix stabilization attempt through 0-allyl serine residues located on adjacent helical turns, via ruthenium-catalyzed ring closing metathesis (RCM) [56] unfortunately, this study did not result in enhanced a-helical structure stabilization. Subsequently, Verdine and coworkers advanced this idea by introducing two alpha-olefin substituted 

An alternative linker strategy recently employed in the synthesis of stapled peptides is through the incorporation of a triazole bridge, which was constructed through azido-acetylene click chemistry. These new stapled peptides offer enhanced chemical stability and further resistance to proteolysis [49c]. The linker length in these triazole bridge stapled peptides has a similar requirement as the hydrocarbon stapled peptide. 

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Xu, B and Irving, S.L. (2011) Design and structure of stapled peptides binding to estrogen receptors. Journal of the American Chemical Society, 133, 9696-9699. 

Although target-based methods have their limitations, they still demonstrate great potential in rationally designing new molecular entities especially for new target classes, such as PPIs. A famous example is the stapled peptide technology, which mimics a-helical structures in the PPL Short peptides can be stabilized into a-helical structures with the use of various chemical methods. These structures are almost identical to the natural folding and thus interfere with the PPI [9]. 

The a-helix features 3.6 residues per complete turn, which places the i, i -F 4, i -F 7, and i-F 11 side chains on the same face of the folded structure [58]. The stapled peptide covalent cross-links are on one face of an a-helbc, connecting two residues separated by either 1 or 2 a-helical turns (i, i -F 4 or i, i + 7 positions, respectively). Two unnatural amino acids used for the covalent linker introduces four possible combinations in chirality considering the peptide sequencing RR, SS, RS, and SR. A cartoon presentation of these chirality differences is shown in Figure 10.6. The... 

As mentioned earlier, short peptides tend to form random coils rather than an a-helix structure and not all the stapled peptides stabilize the a-helices. Circular dichroism (CD) spectroscopy is commonly used to measure the a-helix conversion enhancement of the stapled peptides. The helical content is direcdy proportional to ellipticity ([0]) or the rotation at a wavelength of 222 nm [61] therefore, the percentage of helicities can be calculated from molar ellipticities at 222 nm ([flJzzz) using —31500 (l-2.57/ ) and 0 deg cm /dmol as the values for 100 and 0% heUcity, respectively, where is the number of amino acid residues in the peptide [61]. 

Figure 9.9 3D-structural diversity of varying subclasses of hydrocarbon-stapled peptides, including double-turn, single-turn, stitch, i,i- -3 single-turn and H-bond surrogate macrocyclizations. 

Figure 9.11 Chemical structure, structure-activity and 3D biophysical properties of stapled peptide ATSP-7041, including a high resolution X-ray structure of it complexed with MDMX.

Figure 9.12 Alanine scanning and structure-activity properties of stapled peptide ATSP-3900 relative to MDM2 and MDMX binding.

Mcl-l is a recognized major resistance factor in human cancer and Mcl-l over-expression has been linked to the pathogenesis of several cancers. Its anti-apoptotic properties are mechanistically related to its neutralizing interaction with BIM, BAK, NOXA and PUMA. ° Recently, a compelling Mcl-l BH3 stapled peptide has been reported to exhibit highly specificity and affinity to bind Mcl-l as well as effectively sensitizing cancer cells to caspase-dependent apoptosis in vitroJ An X-ray structure of Mcl-l complexed with this Mcl-l BH3 stapled peptide was also determined and showed inter-molecular contact of its hydrocarbon staple moiety with Mcl-l, accounting for the specificity versus other Bcl-2 family proteins. 

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