Margaret O’Brien

Ollscoil na Gaillimhe – University of Galway

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Christian W. Gruber

Medical University of Vienna

Johannes Koehbach

ARC Centre of Excellence for Innovations in Peptide and Protein Science

Terry Smith

Ollscoil na Gaillimhe – University of Galway

Michael Freissmuth

Medical University of Vienna

Markus Muttenthaler

University of Vienna

David J. Craik

University of Queensland

Susan Wray

University of Liverpool

Marion Miazzo

Medical University of Vienna

Sarah Arrowsmith

University of Liverpool

Muharrem Akcan

Dumlupinar University

Cooperative Institutions

University of Queensland

Brown University

Medical University of Vienna

Research Institute for Bioscience and Biotechnology

Australian Research Council

Brisbane School of Theology

John Brown University

ARC Centre of Excellence for Innovations in Peptide and Protein Science

QIMR Berghofer Medical Research Institute

Australian Institute of Tropical Health and Medicine

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Cyclotides as templates for peptide GPCR ligand design - discovery of the target receptor of the oxytocic plant peptide kalata B7

Johannes Koehbach Margaret O’Brien Markus Muttenthaler Marion Miazzo Muharrem Akcan

Publication Information Koehbach, J,O'Brien, M,Muttenthaler, M,Miazzo, M,Akcan, M,Elliott, AG,Daly, NL,Harvey, PJ,Arrowsmith, S,Gunasekera, S,Smith, TJ,Wray, S,Goransson, U,Dawson, PE,Craik, DJ,Freissmuth, M,Gruber, CW (2013) 'Oxytocic plant cyclotides as templates for peptide G protein-coupled receptor ligand design'. Proceedings Of The National Academy Of Sciences Of The United States Of America, 110 (52):21183-21188.

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10.2210/pdb2m9o/pdb

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Uterotonic Plants and their Bioactive Constituents

Planta Medica (2010)

Christian W. Gruber Margaret O’Brien

Abnormalities in the process of uterine muscle contractility during pregnancy and birth can have major clinical implications, including preterm labour, which is the single largest cause of maternal and prenatal mortality in the Western world and a major contributor to childhood developmental problems. In contrast, induction of labour may be necessary in certain conditions. Currently used interventional therapies to suppress (tocolytic agents) or to induce (uterotonic agents) uterine contractions lack potency and/or selectivity and can have harmful side effects for mother and baby. Nature's diversity has always been, and still is, one of the biggest resources of therapeutic lead compounds. Many natural products exhibit biological activity against unrelated targets, thus providing researchers with starting points for drug development. In this review we will provide an overview of uterine muscle physiology, describe currently available biological screening procedures for testing of uterotonic plant compounds and will summarise traditionally-used uterotonic plants, their active components and their mechanisms, primarily focusing on uterotonic active circular plant peptides called cyclotides. Finally we will comment on the discovery of novel cyclotide-producing plant species and the possibility for the development of novel plant-derived uterotonic and tocolytic drugs.

Uterotonic

10.1055/s-0030-1250317

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Peptide GPCRs as molecular targets for plant cyclotides

Biopolymers (2013)

Johannes Koehbach Margaret O’Brien Markus Muttenthaler Muharrem Akcan Theresa J. Smith

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Circular plant peptides as templates for GPCR drug discovery?

BMC Pharmacology (2011)

Johannes Koehbach Margaret O’Brien Marion Miazzo David J. Craik Michael Freissmuth

Background Cyclotides are disulfide-rich miniproteins with the unique structural features of a circular backbone and knotted arrangement of three conserved disulfide bonds. These features make them exceptionally stable and they have applications as plant defense (insecticidal) agents and stable drug frameworks. So far they have been found mainly in two plant families, including in every species of the violet family (Violaceae) so far examined, and in a few species of the coffee family (Rubiaceae).

Violaceae

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Structural motif

10.1186/1471-2210-11-s2-a16

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Modulation of Myometrial Smooth Muscle Cell Contractility by Plant Cyclotides

Marie Kinsella Johannes Koehbach Terry Smith Christian W. Gruber Margaret O’Brien

Contractility

Modulation (music)

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Researchin the human myometrium

Margaret O’Brien Padraig Earley John J. Morrison Terry Smith

Background: Ghrelin is a 28-amino acid octanolyated peptide, synthesised primarily in the stomach. It stimulates growth hormone release, food intake and exhibits many other diverse effects. Our group have previously determined that ghrelin inhibited human contractility in vitro. The aim of this study therefore, was to investigate the expression of ghrelin, its receptor, the growth hormone secretagogue receptor type 1 (GHS-R1), ghrelin O-acyltransferase (GOAT) which catalyses ghrelin octanoylation, prohormone convertase 1/3 (PC1/3) responsible for pro-ghrelin processing, in human myometrium, during pregnancy prior to labour, during labour and in the non-pregnant state. Modulation of ghrelin and ghrelin receptor expression in cultured myometrial cells was also investigated. Methods: mRNA and protein were isolated from human myometrium and the myometrial smooth muscle cell line hTERT-HM; and real-time fluorescence RT-PCR, western blotting and fluorescence microscopy performed. The effects of β-Estradiol and bacterial lipopolysaccharide (LPS) on hTERT-HM gene expression were evaluated by western blotting. Results: We have reported for the first time the expression and processing of ghrelin, GHS-R1, GOAT and PC1/3 expression in human myometrium, and also the down-regulation of ghrelin mRNA and protein expression during labour. Furthermore, GHS-R1 protein expression significantly decreased at labour. Myometrial GOAT expression significantly increased during term non-labouring pregnancy in comparison to both non-pregnant and labouring myometrium. Mature PC1/3 protein expression was significantly decreased at term pregnancy and labour in comparison to non-pregnant myometrium. Ghrelin, GHS-R1, GOAT and PC1/3 mRNA and protein expression was also detected in the hTERT-HM cells. Ghrelin protein expression decreased upon LPS treatment in these cells while βEstradiol treatment increased GHS-R1 expression. Conclusions: Ghrelin processing occurred in the human myometrium at term pregnancy and in the non-pregnant state. GOAT expression which increased during term non-labouring pregnancy demonstrating a similar expression pattern to prepro-ghrelin and GHS-R1, decreased at labour, signifying possible myometrial ghrelin acylation. Moreover, the presence of PC1/3 may contribute to pro-ghrelin processing. These results along with the previous in vitro data suggest that myometrially-produced and processed ghrelin plays a significant autocrine or paracrine role in the maintenance of relaxation in this tissue during pregnancy. Furthermore, the significant uterine modulators LPS and βEstradiol are involved in the regulation of ghrelin and ghrelin receptor expression respectively, in the human myometrium.

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Peptidomics screening for the discovery of uterotonic plant peptides

BMC Pharmacology and Toxicology (2012)

Johannes Koehbach Alfred F. Attah Margaret O’Brien Michael Freissmuth Christian W. Gruber

Background Drug discovery from natural products is still one of the biggest sources of novel lead compounds. In particular, plant cyclotides, disulfide-rich peptides comprising three conserved disulfide bonds in a knotted arrangement, known as cyclic cystine knot motif, and a head-to-tail cyclization, have been extensively investigated over the last four decades for their use as scaffolds in drug development. However, their distribution among flowering plants still remains limited to few species of the families of Rubiaceae (coffee), Violaceae (violet), Cucurbitaceae (cucurbit), Fabaceae (bean) and recently Solanaceae (potato family), but it is very likely that cyclotides are more widely distributed since their predicted number in Rubiaceae alone is ~50.000. Additionally, the pharmacological validation of plants used in traditional medicines may trigger the discovery of novel uterotonic compounds [1].

Violaceae

10.1186/2050-6511-13-s1-a89

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Uterine contractility of plants used to facilitate childbirth in Nigerian ethnomedicine

Journal of Ethnopharmacology (2012)

Alfred F. Attah Margaret O’Brien Johannes Koehbach Mubo Adeola Sonibare J. O. Moody

Pregnant women in Nigeria use plant preparations to facilitate childbirth and to reduce associated pain. The rationale for this is not known and requires pharmacological validation.Obtain primary information regarding the traditional use of plants and analyze their uterine contractility at cellular level.Semi-structured, open interviews using questionnaires of traditional healthcare professionals and other informants triggered the collection and identification of medicinal plant species. The relative traditional importance of each medicinal plant was determined by its use-mention index. Extracts of these plants were analyzed for their uterotonic properties on an in vitro human uterine cell collagen model.The plants Calotropis procera, Commelina africana, Duranta repens, Hyptis suaveolens, Ocimum gratissimum, Saba comorensis, Sclerocarya birrea, Sida corymbosa and Vernonia amygdalina were documented and characterized. Aqueous extracts from these nine plants induced significant sustained increases in human myometrial smooth muscle cell contractility, with varying efficiencies, depending upon time and dose of exposure.The folkloric use of several plant species during childbirth in Nigeria has been validated. Seven plants were for the first time characterized to have contractile properties on uterine myometrial cells. The results serve as ideal starting points in the search for safe, longer lasting, effective and tolerable uterotonic drug leads.

Uterotonic

10.1016/j.jep.2012.06.042

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

Proceedings of the National Academy of Sciences (2013)

Johannes Koehbach Margaret O’Brien Markus Muttenthaler Marion Miazzo Muharrem Akcan

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 V1a 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.

10.1073/pnas.1311183110

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Elevated levels of intracellular RNA lariats suppress the antiviral response

Chaorui Duan Luke Buerer Cory Bowers Allison J. Taggart Margaret O’Brien

Recent studies report the genetic loss of the lariat debranching enzyme (DBR1) activity increases susceptibility to viral infection. Here, we show that more than 25% of human introns contain large hairpin structures created by the folding of two Alu elements inserted in opposite orientation. In wildtype cells, this large reservoir of endogenous dsRNA is efficiently degraded. In DBR1-null cells, lariats accumulate in the cytosol and dsRNA becomes enriched. We demonstrate how the chronic exposure to these lariats attenuates the dsRNA sensors, reducing the response of the MDA5, RIG-I, RNase L and PKR sensing pathways. We observe evidence for both attenuation and endogenous dsRNA in anti-viral response and viral evasion. Lariats are transiently elevated during infection (e.g. HSV-1, influenza, KSHV). The HSV-1 genome expresses multiple, stable lariats that may attenuate dsRNA sensors during latency.

10.1101/2024.12.07.627371

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