Fragments from the extracellular matrix proteins laminin and osteopontin and a sequence from VEGF have potent proangiogenic activity despite their small size (< 10 residues). However, these linear peptides have limited potential as drug candidates for therapeutic angiogenesis because of their poor stability. In the present study, we show that the therapeutic potential of these peptides can be significantly improved by "grafting" them into cyclic peptide scaffolds. Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II) and sunflower trypsin inhibitor-1 (SFTI-1), naturally occurring, plant-derived cyclic peptides of 34 and 14 residues, respectively, were used as scaffolds in this study. Using this approach, we have designed a peptide that, in contrast to the small peptide fragments, is stable in human serum and at nanomolar concentration induces angiogenesis in vivo. This is the first report of using these scaffolds to improve the activity and stability of angiogenic peptide sequences and is a promising approach for promoting angiogenesis for therapeutic uses. (Blood. 2011;118(25):6709-6717)

Cyclotides are plant derived mini-proteins with compact folded structures and exceptional stability. Their stability derives from a head-to-tail cyclized backbone coupled with a cystine knot arrangement of three-conserved disulfide bonds. Taking advantage of this stable framework we developed novel VEGF-A antagonists by grafting a peptide epitope involved in VEGF-A antagonism onto the stable cyclotide framework. Antagonists of this kind have potential therapeutic applications in diseases where angiogenesis is an important component of disease progression, including cancer and rheumatoid arthritis. A grafted analogue showed biological activity in an in vitro VEGF-A antagonism assay at low micromolar concentration and the in vitro stability of the target epitope was markedly increased using this approach. In general, the stabilization of bioactive peptide epitopes is a significant problem in medicinal chemistry and in the current study we have provided insight into one approach to stabilize these peptides in a biological environment.

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...

In recent years, the cyclotides have emerged as the largest family of naturally cyclized proteins. Cyclotides display potent cytotoxic activity that varies with the structure of the proteins, and combined with their unique structure, they represent novel cytotoxic agents. However, their mechanism of action is yet unknown. In this work we show that disruption of cell membranes plays a crucial role in the cytotoxic effect of the cyclotide cycloviolacin O2 (1), which has been isolated from Viola odorata. Cell viability and morphology studies on the human lymphoma cell line U-937 GTB showed that cells exposed to 1 displayed disintegrated cell membranes within 5 min. Functional studies on calcein-loaded HeLa cells and on liposomes showed rapid concentration-dependent release of their respective internal contents. The present results show that cyclotides have specific membrane-disrupting activity.

Cyclotides stand out as the largest family of circular proteins of plant origin hitherto known, with more than 280 sequences isolated at peptide level and many more predicted from gene sequences. Their unusual stability resulting from the signature cyclic cystine knot (CCK) motif has triggered a broad interest in these molecules for potential therapeutic and agricultural applications. Since the time of the first cyclotide discovery, our laboratory in Uppsala has been engaged in cyclotide discovery as well as the development of protocols to isolate and characterize these seamless peptides. We have also developed methods to chemically synthesize cyclotides by Fmoc-SPPS, which are useful in protein grafting applications. In this review, experience in cyclotide research over two decades and the recent literature related to their structures, synthesis, and folding as well the recent proof-of-concept findings on their use as "epitope" stabilizing scaffolds are summarized.