March Journal Club: Iterative optimization yields Mcl-1 targeting stapled peptides with selective cytotoxicity to Mcl-1–dependent cancer cells. 

You are here

Mcl-1protein is a validated therapeutic cancer target that is involved in apoptosis inhibition. Therefore Mcl-1 plays a critical role in cancer cell survival. Furthermore, prevention of Mcl-1 functionality has been proved to be cytotoxic for some cancer cell lines. Mcl-1 interacts with Bcl-2 family proteins through a hydrophobic region in the α-helix motif BH3, which is present in Bcl-2 family proteins. Specifically, Mcl-1 is able to bind to pro-apoptotic proteins through the BH3 domain, thus avoiding programmed cell death.

Hence, mimicking the BH3 domain motif has been envisaged as a strategy to inhibit the interaction between Mcl-1 and pro-apoptotic proteins. In fact, previous studies, such as the investigation performed by Foight et al., demonstrated that BH3 peptides inhibited Mcl-1 activity.

Nevertheless, one of the principal drawbacks of using this strategy to develop a drug candidate is the fact that unmodified BH3 peptides do not have the ability to be internalized into cells. Then, peptide-derivates are required to overcome this hindrance.

The authors of the current article have based their research on the modification of previously reported Mcl-1 binding peptides (MB2 and MS1) to generate stapled peptides. The objective is to improve binding affinity as well as cell permeability.

To achieve this goal, MB2 peptide was the starting point to design a new library of stapled peptides with modifications in the i, i +4- and i, i + 7 positions. These modification points were demonstrated to increase the α-helicity for a given peptide sequence. Then, the stapled-peptide library was screened against the Mcl-1 protein and three out of 23 peptides were found to improve the binding affinity for Mcl-1 in comparison with the parental peptide, decreasing KD to less than 10 nM.

Afterwards, the successfully introduced modifications in MB2 peptide were also applied to the MS1 peptide, generating a second stapled-peptide library. From this extended library, two peptide derivates (SAH-MS1-14 and SAH-MS1-18) were the most promising compounds due to their potency, selectivity and cell uptake. Indeed, potency was not only evaluated by biophysical studies (Fluorescence Polarization assays) but also in cellular models. On the other hand, cellular uptake was specifically studied through fluorescence microscopy using FITC-labeled peptides.


Figure 1. Cell viability after 24 h incubation of the two staple-peptide derivatives (SAH-MS1-14 and SAH-MS1-18) and ABT 737 (Bcl-2/Bcl-xL inhibitor) in a selection of cancer cell lines. O-Mcl-1 and H929 are Mcl-1 dependent cell line meanwhile the other cell lines are Bcl-2/Bcl-xL dependent.


In addition, the possible membrane lysis effect of the staple peptides was assessed. In fact, this important point was taken into account during the optimization, avoiding residue substitutions that may lead to lytic peptides.

Following evaluation studies, the two candidates (SAH-MS1-14 and SAH-MS1-18) were further investigated through structural analysis.

The specific binding in the BH3 region of Mcl-1 by SAH-MS1-14 and SAH-MS1-18 was demonstrated through crystallization of the protein/peptide complexes. The structures depicted the hydrophobic interactions between the staple moiety and the protein BH3 binding groove. Indeed, these interactions drive a conformational change in Mcl-1 protein that increases the hydrophobic exposure, allowing an increment in the contact surface.



Figure 2. Crystal structure of SAH-MS-18 (ribbon, PDB ID code 5W89) in complex with Mcl-1 (surface).  . The image represents the crystal structure of SAH-MS1-18 (ribbon, PDB ID code 5W89) in complex with Mcl-1 (surface).


To conclude, A. E. Keating and coworkers present in this work how SAR can be applied to generate staple peptides with optimized properties as protein-protein interaction modulators. However, design and optimization of peptides can become a challenge, in which a slightly modification can drastically affect potency or/and cell permeability, as have been demonstrated in the present article

At Iproteos we have developed a platform, IPROTech, that specifically designs permeable peptidomimetics able to enter inside the cell without the need of further optimization. IPROTech combines computational and experimental tools to deliver potent, stable and permeable peptidomimetics.



Rezaei Araghi et al. Iterative optimization yields Mcl-1-targeting stapled peptides with selective cytotoxicity to Mcl-1-dependent cancer cells. PNAS. 2018 Jan 30;115(5):E886-E895.