Oxytocic plant cyclotides as templates for peptide G protein-coupled receptor ligand design

Abstract

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...

Oral activity of a nature-derived cyclic peptide for the treatment of multiple sclerosis

“…This grafting introduced peptide sequences into cyclotide loops and resulted in a chimeric molecule that was orally active as bradykinin B1 receptor antagonist in the treatment of chronic inflammatory pain (18). Cyclotides represent a natural combinatorial peptide library and they probe a chemical space that is difficult to target by using small organic molecules (17). Thus, at the very least, they can be anticipated to complement the existing collections of natural products or synthetic molecules that are used in drug discovery.…”

“…Historically, cyclosporine A was instrumental to the development of modern immunopharmacology and it is still in clinical use today (29,36). Despite the limitations of such comparisons, we believe that the rich diversity of cyclotides (17,43) justifies their position as a treasure trove for drug discovery.…”

“…This unique 3D fold confers them intrinsic stability to resist chemical, enzymatic, and thermal degradation (14). Therefore, cyclotides have become attractive tools in chemical biology and drug development (15), for instance as templates for molecular grafting applications (16) as well as for receptor ligand design (17), because they presumably exhibit activity following oral administration (18).…”

T20K: An Immunomodulatory Cyclotide on Its Way to the Clinic

“…This unique three-dimensional fold confers them intrinsic stability to resist chemical, enzymatic and thermal degradation (Colgrave and Craik 2004). Therefore cyclotides have become attractive tools in chemical biology and drug development (Huang et al 2015), for instance as template for molecular grafting applications (Craik et al 2012) and for receptor ligand design (Koehbach et al 2013), since they exhibit activity following oral administration (Wong et al 2012).…”

Effects of combined progesterone and 17β-estradiol treatment on the transcriptome of cultured human myometrial smooth muscle cells

“…These findings, therefore, provide the basis for future investigations in human myometrial tissue ex vivo and in vivo. The use of hUtSMCs to assess the effects of a broad range of natural biological compounds, including steroid hormones, and potential tocolytic agents on uterine smooth muscle cells has been reported in previous publications by ourselves and by others (4,14,27,39). Confirmed expression of the oxytocin receptor (OXTR), calponin, and smoothelin genes (71) (data not shown) and immunofluorescence staining of smooth muscle ␣-actin (55) attested to the smooth muscle origin of the cells.…”