Protein-protein interactions (PPIs) hold humans together. A single protein or enzyme by itself is useless, but synchronize it with the right partners, and suddenly life forces appear—from the absorption of nutrients to the contraction of muscles.
Untapped targets for drug design lurk in these essential protein bonds. Researchers are employing many different approaches—such as signal transduction, quantum chemistry, and bioinformatics—to unearth the molecular crevices where a small molecule or peptide inhibitor could knock a disease off course.
To review disease arenas where PPI manipulation is showing much promise, scientists will convene at CHI’s “Protein-Protein Interactions” conference in April.
Often the first barrier to targeting a PPI is the cell membrane. For instance, antibodies are titans when it comes to recognizing PPIs, making them essential tools for epitope mapping and a bevy of research assays, such as immunofluorescence, ELISA, and chromatography. Yet antibodies have limited clinical scope because they do not easily access the intracellular compartment.
Cyclotides, a large family of plant-derived peptides, may offer a substitute, according to Julio Camarero, Ph.D., a pharmacologist at the University of Southern California. His upcoming presentation will cover how cyclotides interfere with the small binding clefts or large surfaces that define protein interactions. These peptides not only possess certain antibody-like features, they also present a bevy of advantages, including the ability to cross cell membranes.
“PPIs are a difficult target—some call them undruggable—because large surface interactions are hard to antagonize with small molecules,” says Dr. Camarero. Nonetheless, Dr. Camarero’s team intends to develop peptide-based therapeutics. In his presentation, Dr. Camarero will discuss how cyclotides are potent drug inhibitors both inside and outside the cell.
Cyclotides were initially discovered in a tea extract that pregnant African women used to induce labor, illustrating their cell-penetrating prowess, oral bioavailability, and clinical promise.
Cyclotides are medium-sized peptides (30–40 amino acids) that have a cyclic backbone, which consists of six “loop” sections interspersed with cysteine residues. With these residues connected via disulfide bonds, the loops converge on a cysteine-based “knot” at the peptide’s core. This conformation makes cyclotides exceptionally impervious to thermal, chemical, and enzymatic degradation.
Not only are cyclotides ultrastable, they are highly diverse. In fact, five of their loops are hypervariable. These properties, according to Dr. Camarero’s team, make cyclotides suitable as scaffolds for peptide-based therapeutics.
“We have immobilized the peptides onto glass to make microarrays, and even after washing them with organic solvents, they are still functional, says Dr. Camarero. “You can boil them or change pH, and they still fold because their state is kept by covalent interactions of the cysteine knot’s disulfides.”
In a recent cancer study, they took advantage of the cyclotide’s hypervariable loops to create a drug that reactivated p53 pathways in tumors. Many cancers switch off p53, a protein trigger for cell death, by overexpressing two proteins: Hdm2 or HdmX. Hdm2 is a ubquitin ligase that marks p53 for proteasomal degradation, whereas HdmX binds and sequesters p53 via protein interaction-mediated sequestration.
Using protein engineering, Dr. Camarero and his colleagues attached a p53 peptide domain to a cyclotide that could then compete for the p53 interaction with HdmX and Hdm2. The engineered cyclotide efficiently killed four cancer cell lines; its efficiency equaled that of the cancer drug Nutlin-3. The cyclotide outperformed Nutlin-3 in a mouse xenograft tumor model with HCT116-p53+/+ human colon cancer cells.
Fluorescently tagging cyclotide hypervariable regions yield nascent biomarkers, akin to fluorescently labeled antibodies, except some cyclotide-based molecules can enter cells with the same efficiency as the popular cell-penetrating protein domain Tat.
Dr. Camarero’s medical chemists have engineered other cyclotides that block HIV-1 viral entry as well as protease inhibitors. Some cyclotides naturally function as insect repellent, so team members are also working to create green pesticides from the peptide family.
“We envision this scaffold potentially playing an important role in the future development of peptide-based therapeutics,” Dr. Camarero adds.