Stapled peptides are starting to get a lot of attention for drug discovery and development by targeting protein-protein interactions.
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 Singapores 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.
Next-generation sequencing is expected to be an useful driver in the discovery of protein-protein interaction networks often referred as the interactome. Protein-protein interactions make up almost all biological processes. Hence a knowledge of these interactions under normal and disease conditions provide many attractive targets for therapeutic intervention.
Small molecule drug discovery has generally been unsuccessful in disrupting protein-protein interactions because of a lack of candidate molecules that can target shallow interacting protein faces involving many weak interactions. Despite this general fact however, the readers are encouraged to read the review by Jim Wells and Christopher McClendon in Nature for an expert’s perspective on the challenges and myths regarding protein- protein interactions in the area of small molecule drug discovery.
Despite these challenges, several lines of evidence provide hope for finding small molecules that target proteinprotein interfaces. Although these interfaces are large, mutational studies show that a small subset of the residues involved contributes most of the free energy of binding1620 (Fig. 1). Such hotspots constitute less than half of the contact surface of a protein involved in the proteinprotein interaction and are usually found at the centre of the contact interface. Proteins involved in proteinprotein interactions can be promiscuous, binding to several targets using the same hotspot region21. Structural studies show that these promiscuous contact surfaces are adaptable, allowing one protein to engage a range of structurally diverse partners. Moreover, peptides selected for binding to one of the partners in a proteinprotein pair (by using phage display) often compete with the natural protein partner for binding to the hotspot2024. Thus, there seem to be many chemical solutions for tight binding, and large contact surfaces can be engaged by more-compact structures.
Protein therapeutics like monoclonal antibodies are unable to target intracellular protein-protein interactions because of significant challenges associated with their cellular delivery.
Thus the search for the “right” chemical entities targeting protein-protein interactions continues to be a wide open and hotly pursued field in pharma, biotech and in academia. In this respect the PNAS paper last month by a team of scientists from Aileron therapeutics and Roche brings to the forefront a new class of molecules known as stapled peptides. The paper provides detailed preclinical studies for disrupting the famed wtp53-MDM2/MDMX interactions, critical molecular target for cancer.
Aileron therapeutics licensed the stapled peptide technology from the original research developed and patented by Greg Verdine’s group at Harvard (Stabilized alpha helical peptides and uses thereof; United States Patent 772 3469). So readers will find more details on these molecules and their in vitro/in vivo properties in a series of papers from the Verdine group.
[caption id=”attachment_134” align=”aligncenter” width=”300”] Reference: Figure and
part of caption taken directly from Fig 1A (Proc Natl Acad Sci U S A. 2013
September 3; 110(36): E3445E3454).
The chemical structure of ATSP-7041 is shown highlighting the R8 (blue) and S5 (red) amino acids in the stapled peptide following ring-closing metathesis (and loss of ethylene). Note that each R8 and S5 is ?-methylated and that the all-hydrocarbon linker consists of 11 carbon atoms. [/caption]
Stapled peptides are a class of conformationally restricted peptides with a hydrocarbon bridge that stabilizes their alpha-helical confirmation. Proteins use their secondary structures like beta-turns, alpha-helices and beta-sheets to engage in protein-protein interactions. So one way to disrupt such interactions will be to use small peptides which mimic these secondary structures. However in solution such secondary structures in small peptides are unstable. They unfold and face protease degradation. In case of protein- protein interactions mediated via an alpha-helix, the alpha-helical conformation in short peptides can be stabilized by crosslinking or ‘stapling together’ i and i+4 or i+7 side chains of amino acid residues which reside on the same face of the helix. The Aileron and Roche scientists used a hydrocarbon bridge , chemically synthesized by Grubb’s ruthenium-catalyzed ring closing metathesis reaction of the terminal alkene side chains of alpha- methyl amino acids at i and i+7 in a 14-mer peptide. The propensity of alpha- methyl substituted amino acids to induce helicity of a peptide backbone has been known previously.
[caption id=”attachment_138” align=”aligncenter” width=”300”] Reference :
Fig 2C of Proc Natl Acad Sci U S A. 2013 September 3; 110(36): E3445E3454.
A high-resolution X-ray structure of ATSP-7041 bound to MDMX-shows the all- hydrocarbon staple between i and i+7 positions on the alpha-helical peptide.[/caption]
The all-hydrocarbon stapled peptides display remarkable cell-penetration abilities by virtue of their increased hydrophobicity which make them useful for targeting intracellular protein-protein interactions. By the same token however they have reduced aqueous solubility. This drawback is circumvented using structure-based design for optimization of other amino acids not involved in target interactions.
For a quick overview on p53 follow this link. As reported in the PNAS paper, in many cancers with wild-type p53, and high levels of MDMX expression (melanoma, head, neck, breast, hepatocellular and retinoblastoma) it may be necessary in disrupting both the MDM2-p53 and MDMX-p53 interactions. The authors pursued the strategy of converting the p53 a-helix of the native p53-MDM2/MDMX into a potential therapeutic lead for dual inhibition of MDM2/MDMX via peptide stapling.
Here we describe the discovery of ATSP-7041 as the first highly potent and specific stapled peptide dual inhibitor of MDM2/MDMX that possesses robust drug-like properties and on-mechanism in vitro and in vivo activity. We provide the first high resolution (1.7) crystallographic structure of a stapled peptide bound to MDMX and further evaluate the biophysical, biochemical, cell penetrating and on-target cellular properties of ATSP-7041. Our results demonstrate that antagonizing both negative p53 regulators effectively suppresses the growth of human tumor xenografts overexpressing MDM2/MDMX and suggest that stapled peptides may offer a new modality for p53-activating cancer therapy.
The cocrystallized structure of ATSP-7041 and humanized zebrafish MDMX solved reveals the detailed interaction of the stapled moiety with the protein including a significant cation-pi interaction between the olefinic unit and ND His51 of MDMX.
Both confocal studies with FAM-ATSP 7041 ( a fluorophore appended version of the stapled peptide molecule) and IP studies in MCF-7 cell lysates established the cellular uptake of ATSP-7041. In case of the IP studies, in presence of 10% FBS there was significant loss in binding of MDM2 and MDMX with p53 compared to DMSO controls. Further ATSP-7041 treatment studies in MCF-7 (breast cancer cell line; ovrexpress MDMX) and SJSA-1 (bone cancer cell line; overexpress MDM2), showed dose-dependent rise in p53 as well as its downstream targets p21 and MDM2 protein levels. Nutlin3a which is a MDM2 inhibitor only showed comparable protein level increase in SJSA-1 cell line. BrdU labeling to ascertain cell-cycle arrest and Annexin V assay to determine apoptotic response in ATSP-7041 treated cell lines showed activation of p53 functions.
In human cancer xenograft models in mouse (MCF-7 and SJSA-1), iv administration of ATSP-7041 daily or every other day, showed statistically significant tumor growth inhibition and p21 expression. On MCF-7 xenograft model,
The treatment of ATSP-7041 for 23 d with 20 mg/kg or 30 mg/kg administered i.v. qod resulted in a TGI of 63% and 87%, respectively (Fig. 6B). In comparison, the selective MDM2 small-molecule inhibitor RG7112 (administered daily at 50 and 100 mg/kg p.o.) resulted in a TGI of 61% and 74%, respectively.
Too complicated? For other discussions, the press release for the stapled peptide clinical candidate (for which ATSP-7041 is a precursor) poised for Phase 1 studies by Aileron therapeutics can be accessed here. ‘In The Pipeline’ blog also covered stapled peptides extensively.
and if everything else fails, stapled peptides can dance with you at youtube :)