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 "kalatakalata," 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 proteincoupled 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.yclotides are head-to-tail cyclized plant peptides containing three conserved disulfide bonds in a knotted arrangement known as a cyclic cystine-knot motif (1). This confers them high stability (2) and presumably improves their oral bioactivity relative to their linear counterparts (3). They were first discovered in a decoction of Oldenlandia affinis DC. (Rubiaceae) leaves, an herbal remedy used in traditional African medicine during childbirth (4). The observed induction of labor and shortened delivery time were later studied on isolated rat and rabbit uteri and on human uterine strips (4, 5). The peptides responsible for the contractility effects (5) raised interest because they survived boiling, presumably as a result of their unique 3D structure, which was elucidated in 1995 (6). Since then, several plant species of the coffee (Rubiaceae) (7), violet (Violaceae) (8), legume (Fabaceae) (9), potato (Solanaceae) (10) and grass (Poaceae) families (11) have been identified to produce cyclotides. Currently, ∼300 sequences have been reported (12), and the predicted number of >50,000 cyclotides in Rubiaceae alone (7) suggests them to be one of the largest peptide classes within the plant kingdom. Their high intercysteine sequence variability and structural plasticity (13), together with intrinsic bioactivities, make them interesting templates for the development of novel pharmaceuticals (14).However, five decades after the discovery of cyclotides, there still is not any information about specific molecular targe...

ABSTRACT:Cyclotides are a unique class of ribosomally synthesized cysteine-rich miniproteins characterized by a head-to-tail cyclized backbone and three conserved disulfide-bonds in a knotted arrangement. Originally they were discovered in the coffee-family plant Oldenlandia affinis (Rubiaceae) and have since been identified in several species of the violet, cucurbit, pea, potato, and grass families. However, the identification of novel cyclotide-containing plant species still is a major challenge due to the lack of a rapid and accurate analytical workflow in particular for large sam-

Antimicrobial peptides (AMPs) occur in all kingdoms of life and are integral to host defense. They have diverse structures and target a variety of organisms, both by non-specific membrane interactions or via specific targets. Here we discuss the structures of AMPs from the four main classes currently recognized, i.e. peptides with (i) α-helical; (ii) β-sheet; (iii) αβ; and (iv) non-αβ elements as well as the growing pool of complex topologies including various post-translational modifications. We propose to group these latter peptides into a fifth class of AMPs. Such peptides exhibit high stability and amenability to chemical engineering, making them of interest for the development of novel antimicrobial agents. Advances and challenges in the development of these peptides towards therapeutic leads are presented. AMPsa diverse, but unifying strategy for defense Antimicrobial resistance has been identified as a major threat to public health and without immediate and global action the world is headed for a dangerous post-antibiotic era [1]. Thus, there is an urgent need for the development of novel antibiotic drugs to treat infectious diseases. In contrast to the rising numbers of multi drug resistant pathogens the rate of discovery of novel drug candidates is dwindling [2]. In this regard antimicrobial peptides (AMP) are a promising class of bioactive compounds that have attracted increasing attention over recent years. Their broad spectrum of activities extends beyond the killing of bacteria and fungi, with several AMPs also exhibiting antiviral [3], antiparasitic [4] or anticancer activities [5]. Furthermore, their multifaceted mechanisms-of-action potentially reduce their susceptibility to suffer from microbial resistance [6]. Starting in the 1980s, at a time when the numbers of novel antibacterial agents started to drop, the field of AMP research gained momentum when several novel examples were independently discovered across different species. These included peptides such as the cecropins from insects [7], the magainins from amphibians [8] and the mammalian defensins [9] to name a few. Since then a plethora of AMPs has been identified from all kingdoms of life, from bacteria to fungi to plants and animals. It is not only evident that these peptides play an integral part of an organism's innate defense machinery, but their variety also makes them a rich source for the discovery of potential novel drug leads. Their distribution among virtually all living organisms is complemented by their structural variety and range of antimicrobial activities. In this review we highlight this vast structural diversity of antimicrobial peptides. As well as providing a brief overview of the structures of well-known classes of AMPs, we introduce the growing class of structurally complex AMP topologies, in particular, cyclic and cysteine-rich defense peptides. We discuss recent progress and challenges in the characterization and development of peptide-based antibacterial molecules. 3 Structural classes of antimicrobial peptides Th...

Cyclotides are plant-derived mini proteins. They are genetically encoded as precursor proteins that become post-translationally modified to yield circular cystine-knotted molecules. Because of this structural topology cyclotides resist enzymatic degradation in biological fluids, and hence they are considered as promising lead molecules for pharmaceutical applications. Despite ongoing efforts to discover novel cyclotides and analyze their biodiversity, it is not clear how many individual peptides a single plant specimen can express. Therefore, we investigated the transcriptome and cyclotide peptidome of Viola tricolor. Transcriptome mining enabled the characterization of cyclotide precursor architecture and processing sites important for biosynthesis of mature peptides. The cyclotide peptidome was explored by mass spectrometry and bottom-up proteomics using the extracted peptide sequences as queries for database searching. In total 164 cyclotides were discovered by nucleic acid and peptide analysis in V. tricolor. Therefore, violaceous plants at a global scale may be the source to as many as 150 000 individual cyclotides. Encompassing the diversity of V. tricolor as a combinatorial library of bioactive peptides, this commercially available medicinal herb may be a suitable starting point for future bioactivity-guided screening studies.