Covering: 1988 to 2012 This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.

Cyclotides are disulfide-rich miniproteins with the unique structural features of a circular backbone and knotted arrangement of three conserved disulfide bonds. Cyclotides have been found only in two plant families: in every analyzed species of the violet family (Violaceae) and in few species of the coffee family (Rubiaceae). In this study, we analyzed >200 Rubiaceae species and confirmed the presence of cyclotides in 22 species. Additionally, we analyzed >140 species in related plant families to Rubiaceae and Violaceae and report the occurrence of cyclotides in the Apocynaceae. We further report new cyclotide sequences that provide insights into the mechanistic basis of cyclotide evolution. On the basis of the phylogeny of cyclotide-bearing plants and the analysis of cyclotide precursor gene sequences, we hypothesize that cyclotide evolution occurred independently in various plant families after the divergence of Asterids and Rosids (∼125 million years ago). This is strongly supported by recent findings on the in planta biosynthesis of cyclotides, which involves the serendipitous recruitment of ubiquitous proteolytic enzymes for cyclization. We further predict that the number of cyclotides within the Rubiaceae may exceed tens of thousands, potentially making cyclotides one of the largest protein families in the plant kingdom.

Cyclotides are plant peptides comprising a circular backbone and three conserved disulfide bonds that confer them with exceptional stability. They were originally discovered in Oldenlandia affinis based on their use in traditional African medicine to accelerate labor. Recently, cyclotides have been identified in numerous plant species of the coffee, violet, cucurbit, pea, potato, and grass families. Their unique structural topology, high stability, and tolerance to sequence variation make them promising templates for the development of peptide-based pharmaceuticals. However, the mechanisms underlying their biological activities remain largely unknown; specifically, a receptor for a native cyclotide has not been reported hitherto. Using bioactivity-guided fractionation of an herbal peptide extract known to indigenous healers as “kalata-kalata,” the cyclotide kalata B7 was found to induce strong contractility on human uterine smooth muscle cells. Radioligand displacement and second messenger-based reporter assays confirmed the oxytocin and vasopressin V_1a receptors, members of the G protein-coupled receptor family, as molecular targets for this cyclotide. Furthermore, we show that cyclotides can serve as templates for the design of selective G protein-coupled receptor ligands by generating an oxytocin-like peptide with nanomolar affinity. This nonapeptide elicited dose-dependent contractions on human myometrium. These observations provide a proof of concept for the development of cyclotide-based peptide ligands. Significance G protein-coupled receptors (GPCRs) are promising drug targets: >30% of the currently marketed drugs elicit their actions by binding to these transmembrane receptors. However, only ∼10% of all GPCRs are targeted by approved drugs. Resorting to plant-derived compounds catalogued by ethnopharmacological analyses may increase this repertoire. We provide a proof of concept by analyzing the uterotonic action of an herbal remedy used in traditional African medicine. We identified cyclic peptides, investigated the molecular mechanisms underlying their uterotonic activity, and report an oxytocic plant peptide that modulates the human oxytocin/vasopressin receptors. This naturally occurring peptide served as a template for the design of an oxytocin-like nonapeptide with enhanced receptor selectivity, highlighting the potential of cyclotides for the discovery of peptide-based GPCR ligands.

Significance: Multiple sclerosis (MS) imposes substantial economic burdens on patients, their families, and society. Until now, there are few therapies available, but often unattractive parenteral application or severe side effects are serious issues. This study highlights the use of circular peptides as orally active T-cell-specific immunosuppressive therapeutics against the MS model experimental autoimmune encephalomyelitis, without inducing major adverse effects. Our work provides a proof of principle that nature-derived cyclic peptides serve as oral active therapeutics, utilizing their intrinsic bioactivity and stable three-dimensional structure. Cyclotides are considered a combinatorial peptide library and they can be anticipated to complement the existing collections of natural products that are used in drug discovery.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) and is characterized by the destruction of myelin and axons leading to progressive disability. Peptide epitopes from CNS proteins, such as myelin oligodendrocyte glycoprotein (MOG), possess promising immunoregulatory potential for treating MS; however, their instability and poor bioavailability is a major impediment for their use clinically. To overcome this problem, we used molecular grafting to incorporate peptide sequences from the MOG_35–55 epitope onto a cyclotide, which is a macrocyclic peptide scaffold that has been shown to be intrinsically stable. Using this approach, we designed novel cyclic peptides that retained the structure and stability of the parent scaffold. One of the grafted peptides, MOG3, displayed potent ability to prevent disease development in a mouse model of MS. These results demonstrate the potential of bioengineered cyclic peptides for the treatment of MS.