By Cameron Cummings
Mentor: Joshua Kritzer, Chemistry; funding source: Provost’s Officecummingscameronj_26134_2253845_Summer-scholars-2020-poster
Proteins, large molecules made up of amino acids, carry out countless functions in almost all organisms. Interactions between proteins (protein-protein interactions or PPIs) are essential for cellular function and communication, and their dysregulation characterizes several diseases, such as cancer, neurodegeneration, and heart disease. Therefore, PPI-modulating therapeutics are a hot topic in research. Unfortunately, targeting PPIs is difficult, especially when they occur within the cell.
Traditional small-molecule drugs can enter the cell but are ineffective at binding the shallow, wide surfaces over which PPIs often occur and can have high off-target toxicity. Macromolecules like antibodies can tightly and specifically bind cell-surface proteins but cannot enter the cell by themselves and are often attacked by the immune system.
Fortunately, there is a way to effectively modulate PPIs: constrained peptides. Peptides, short chains of amino acids, can recognize and bind to wide protein surfaces with high specificity and are convenient starting points for therapeutic development because they can be easily designed, synthesized, modified, and screened. However, they need to adopt a specific structure in order to bind, and they often do not adopt this structure in solution, instead existing in a flexible form. In this unstructured conformation, they are easily degraded by proteins called proteases, have trouble entering the cell, and have do not bind their targets as well. Through a strategy called conformational constraint, however, peptides can be forced into their rigid, bioactive conformation, granting them resistance to proteases, better cell penetration, and improved binding affinity, making them effective PPI modulators. The question that prompted this project is that of how to constrain short peptides into a shape called an alpha-helix. The alpha-helix is the secondary structure most commonly involved in PPIs, but peptides that are short enough to be viable therapeutics rarely adopt it in nature. Stabilizing alpha-helices in short peptides is thus a tantalizing target for peptide therapeutic development.
When a peptide folds into an alpha helix, it begins at the end called the N-terminus. The twisting of this N-terminal end into a helix is the most energetically difficult step of helix formation, so
strategies that constrain the N-terminus can have a disproportionate benefit towards helix formation relative to strategies that constrain other areas of the peptide. My project starts with a parent peptide that inhibits the action of MCL-1, a protein that can promote cancer when overactive. To bind to MCL- 1, the peptide needs to be in the alpha-helical shape, so it serves as good starting point with which to test my N-terminal constraints. Incorporating two unusual kinds of amino acids, D-cysteine and 4-
mercaptoproline, into a type of constraint strategy called stapling, I constrain the N-terminal regions of these MCL-1 inhibitor peptides in hopes of increasing helicity and making them bind MCL-1 more tightly.
This project is not finished, and it has the potential to produce well-structured, potent inhibitors of MCL-1 that could be developed into clinical anticancer agents. No matter how the data looks, however, I foresee the experiments providing valuable contributions to the field of helix stabilization in peptides. D-amino acids like D-cysteine are particularly resistant to proteases, 4-mercaptoproline has a unique structure that gives it inherent helix-initiating propensity, and both can serve as anchors for stapling, so creating staples with these amino acids that effectively induce helicity would have exciting implications for their future incorporation into peptide therapeutics.