Peptides as therapeutics are advantageous since they are generally safe and well tolerated, with a short half-life limiting risk for off-target toxicities. However, because of their small size, most peptides do not adopt a defined structure, causing them to be chemically and physically unstable. This is not the case with Cystine-Dense Peptides (CDP), a class of peptides enriched in cysteine residues that can be found in venom toxins, plants and microbes. By establishing disulfide bonds with each other, cysteines (called cystine when in disulfides) stabilize the peptide structure, an important characteristic for increased stability and potency in vivo. Despite these promising properties, CDP are poorly studied and underutilized due to the lack of technology for large scale production.
In a recent study published in the journal Nature Structural and Molecular Biology, Drs. Jim Olson and Roland Strong and lead co-authors Dr. Colin Correnti and Dr. Mesfin Gewe (Clinical Research and Basic Sciences Divisions) developed CDP large-scale production. Dr. Olson described how they first became interested in CDP: “in 2004, we focused on a scorpion-derived peptide, chlorotoxin, for Tumor Paint. Tumor Paint uses chlorotoxin to deliver fluorescent dye to cancer so that surgeons can readily distinguish cancer from normal tissue. As Tumor Paint moved out of Fred Hutch into a spinout company (Blaze Bioscience), there was an opportunity to re-invent the lab and I decided to focus on CDP since there seemed to be so much unrealized potential for discovering and developing effective drugs to treat human diseases that are currently considered “undruggable”.”
Peptides of interest were first identified in Protein Data Bank using criteria such as a content of at least 6 cysteines residues within 13 to 81 amino acids, with a specific distribution. The presence of at least 12% of cysteines within such a short motif allowed discriminating small proteins from CDP. As such, over 700 CDP were targeted for production with the new method. Dr. Gewe added, “the analysis done on the 700 CDP resulted in a unified classification scheme that brings together previously fragmented CDP structural fold classifications.” Among the identified peptides, 100 were selected based on previously existing literature that could be used as a comparison for validation of the final product. The chosen CDP represented a wide variety of functions such as epidermal-growth factor or notch-repeat like domains.
Figure provided by Dr. Mesfin Gewe.
The CDP were produced in mammalian cells, a relevant system for future clinical applications, using the Daedalus lentivirus production system developed by Dr. Strong’s laboratory, which includes a ubiquitous chromatin opening element preventing epigenetic silencing and allowing higher protein expression. The CDP were then purified by reverse phase chromatography, an optimal method for obtaining a structurally homogenous final product. Up to 10mg of peptide per liter of culture, on a 2 liters culture scale, could be obtained. 46 CDP were produced this way that were biochemically characterized and validated for stability and resistance to extreme conditions, such as high temperatures or proteolytic cleavage. The produced CDP were generally stable, including two candidates resisting all degradation conditions. Replicates of crystallization experiments also demonstrated the extreme structural stability and homogeneity of the obtained product. Poor correlation was found between the peptide sequences and structures, limiting structure and function anticipation based on sequences alone. Functionality of purified CDP with known activity as ligands for ion channels, common to many natural venoms and toxins, was confirmed using commercial electrophysiological methods.
As explained by Drs. Strong and Gewe, “Individual CDP, both natural sequence and designed mutants, have often been studied through expensive, one-off chemical synthesis. What this platform enables is the affordable production and study of wide swaths of CDP space, and whole families of engineered mutants.” For the two researchers, “the most interesting area to explore next is to uncover the details of the molecular interactions that drive homing, be it protein receptors, membranes, or bulk properties”. This could allow to direct trafficking of the peptide to the tissue of interest. Large-scale production of cystine dense peptides unlocks a broad potential for applications of over 700 CDP that were identified as candidates of interest. For example, “the hyperstable CDP have potential to be developed into drugs that treat disorders in the gastrointestinal tract. Because they are stable in the presence of stomach acid, pepsin, trypsin, and other enzymes, it would be possible to develop drugs that are taken orally and never leave the GI tract. Such drugs could treat cancer, inflammatory diseases, infectious diseases, or digestive diseases”, concluded Dr. Olson.
Funding for this work was provided by the National Institutes of Health, Project Violet, the Wissner-Slivka Foundation, the Kismet Foundation, the Sarah M. Hughes Foundation, Strong4Sam, Yahn Bernier and Beth McCaw, Len and Norma Klorfine, Anne Croco and Pocket Full of Hope.
Correnti CE, Gewe MM, Mehlin C, Bandaranayake AD, Johnsen WA, Rupert PB, Brusniak M-Y, Clarke M, Burke SE, De Van Der Schueren W, Pilat K, Turnbaugh SM, May D, Watson A, Chan MK, Bahl CD, Olson JM, Strong RK. 2018. Screening, large-scale production and structure-based classification of cystine-dense peptides. Nature Structural and Molecular Biology. 25(3), 270-278.