Vicia sativa subsp. sativa native to the Middle East comprises Pea Albumin1 b-like homologs: A promising natural biopesticide.

The extensive and indiscriminate use of chemical pesticides in agriculture has led to adverse effects on human health, environmental pollution, and the emergence of pesticide-resistant pests. To mitigate these challenges, the development of environmentally friendly alternatives is crucial, with biopesticides emerging as promising solutions such as peptides. Legume seeds naturally contain diverse insecticidal peptides or proteins to combat pest attacks. One such peptide is PA1b (Pea Albumin 1, subunit b), a 37 amino acid extracted from pea seeds (Pisum sativum). PA1b has shown significant potential in controlling cereal weevils (Sitophilus spp.), a major pest of stored cereals. Here, we screened PA1b-like peptides in five wild seeds of vetches (Vicia sativa subsp. sativa) from the Middle East. Using a comprehensive set of biochemical, biological, and molecular techniques, we characterized different PA1b homologs and assessed their toxicity and expression profiles. Our results reveal that PA1b homolog from Vicia sativa subsp. sativa originating from turkey displays outstanding insecticidal activity against Sitophilus oryzae through binding to the receptor site found in the midgut of the insect. Moreover, it exhibits a strong cytotoxic effect against Sf9 cells. This cysteine-rich peptide shows sequence identity and the same hydrophobic pole as AG41, a tenfold more toxic isoform of PA1b from Medicago truncatula. Such observations pave the way for the development of bioinsecticides, with PA1b-like peptides as lead compounds.

Recombinant Production, NMR Solution Structure, and Membrane Interaction of the Phα1ß Toxin, a TRPA1 Modulator from the Brazilian Armed Spider Phoneutria nigriventer.

Phα1ß (PnTx3-6) is a neurotoxin from the spider Phoneutria nigriventer venom, originally identified as an antagonist of two ion channels involved in nociception: N-type voltage-gated calcium channel (CaV2.2) and TRPA1. In animal models, Phα1ß administration reduces both acute and chronic pain. Here, we report the efficient bacterial expression system for the recombinant production of Phα1ß and its 15N-labeled analogue. Spatial structure and dynamics of Phα1ß were determined via NMR spectroscopy. The N-terminal domain (Ala1-Ala40) contains the inhibitor cystine knot (ICK or knottin) motif, which is common to spider neurotoxins. The C-terminal α-helix (Asn41-Cys52) stapled to ICK by two disulfides exhibits the µs-ms time-scale fluctuations. The Phα1ß structure with the disulfide bond patterns Cys1-5, Cys2-7, Cys3-12, Cys4-10, Cys6-11, Cys8-9 is the first spider knottin with six disulfide bridges in one ICK domain, and is a good reference to other toxins from the ctenitoxin family. Phα1ß has a large hydrophobic region on its surface and demonstrates a moderate affinity for partially anionic lipid vesicles at low salt conditions. Surprisingly, 10 µM Phα1ß significantly increases the amplitude of diclofenac-evoked currents and does not affect the allyl isothiocyanate (AITC)-evoked currents through the rat TRPA1 channel expressed in Xenopus oocytes. Targeting several unrelated ion channels, membrane binding, and the modulation of TRPA1 channel activity allow for considering Phα1ß as a gating modifier toxin, probably interacting with S1-S4 gating domains from a membrane-bound state.

Residues of Legume AG41 Peptide Crucial to Its Bio-Insecticidal Activity.

Currently, crop protection relies heavily on chemical treatments, which ultimately leads to environmental contamination and pest resistance. Societal and public policy considerations urge the need for new eco-friendly solutions. In this perspective, biopesticides are effective alternatives to chemical insecticides for the control of various insect pests. Legumes contain numerous insecticidal proteins aimed at protecting their high nitrogen content from animal/insect predation. Investigating one such protein family at genome scale, we discovered a unique diversity of the albumin 1 family in the (model) barrel medic genome. Only some members retained very high insecticidal activity. We uncovered that AG41 peptide from the alfalfa roots displays an outstanding insecticidal activity against several pests such as aphids and weevils. Here we report the 3D structure and activity of AG41 peptide. Significant insights into the structural/functional relationships explained AG41 high insecticidal activity. Such observations pave the way for the development of bio-insecticides, with AG41 peptide as the lead compound.

SPECT/CT Imaging, Biodistribution and Radiation Dosimetry of a 177Lu-DOTA-Integrin αvß6 Cystine Knot Peptide in a Pancreatic Cancer Xenograft Model.

INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignant neoplasms, as many cases go undetected until they reach an advanced stage. Integrin αvß6 is a cell surface receptor overexpressed in PDAC. Consequently, it may serve as a target for the development of probes for imaging diagnosis and radioligand therapy. Engineered cystine knottin peptides specific for integrin αvß6 have recently been developed showing high affinity and stability. This study aimed to evaluate an integrin αvß6-specific knottin molecular probe containing the therapeutic radionuclide 177Lu for targeting of PDAC. METHODS: The expression of integrin αvß6 in PDAC cell lines BxPC-3 and Capan-2 was analyzed using RT-qPCR and immunofluorescence. In vitro competition and saturation radioligand binding assays were performed to calculate the binding affinity of the DOTA-coupled tracer loaded with and without lutetium to BxPC-3 and Capan-2 cell lines as well as the maximum number of binding sites in these cell lines. To evaluate tracer accumulation in the tumor and organs, SPECT/CT, biodistribution and dosimetry projections were carried out using a Capan-2 xenograft tumor mouse model. RESULTS: RT-qPCR and immunofluorescence results showed high expression of integrin αvß6 in BxPC-3 and Capan-2 cells. A competition binding assay revealed high affinity of the tracer with IC50 values of 1.69 nM and 9.46 nM for BxPC-3 and Capan-2, respectively. SPECT/CT and biodistribution analysis of the conjugate 177Lu-DOTA-integrin αvß6 knottin demonstrated accumulation in Capan-2 xenograft tumors (3.13 ± 0.63%IA/g at day 1 post injection) with kidney uptake at 19.2 ± 2.5 %IA/g, declining much more rapidly than in tumors. CONCLUSION: 177Lu-DOTA-integrin αvß6 knottin was found to be a high-affinity tracer for PDAC tumors with considerable tumor accumulation and moderate, rapidly declining kidney uptake. These promising results warrant a preclinical treatment study to establish therapeutic efficacy.

Tying pest insects in knots: the deployment of spider-venom-derived knottins as bioinsecticides.

Spider venoms are complex chemical arsenals that contain a rich variety of insecticidal toxins. However, the major toxin class in many spider venoms is disulfide-rich peptides known as knottins. The knotted three-dimensional fold of these mini-proteins provides them with exceptional chemical and thermal stability as well as resistance to proteases. In contrast with other bioinsecticides, which are often slow-acting, spider knottins are fast-acting neurotoxins. In addition to being potently insecticidal, some knottins have exceptional taxonomic selectivity, being lethal to key agricultural pests but innocuous to vertebrates and beneficial insects such as bees. The intrinsic oral activity of these peptides, combined with the ability of aerosolized knottins to penetrate insect spiracles, has enabled them to be developed commercially as eco-friendly bioinsecticides. Moreover, it has been demonstrated that spider-knottin transgenes can be used to engineer faster-acting entomopathogens and insect-resistant crops. © 2019 Society of Chemical Industry.

Simultaneous extraction and analysis of multiple cystine-dense peptides by µSPE and microflow-MS/MS from plasma.

Aim: Develop a universal extraction and liquid chromatography-mass spectrometer method to simultaneously analyze cystine-dense peptide (CDP) miniproteins from rat and human plasma. The results of the analysis will be used to assist selection of therapeutic drug candidates from the vast CDP library. Methods & results: A micro-elution solid-phase extraction method was developed for the sample preparation of the CDP peptides in rat and human plasma followed by analysis by microflow liquid chromatography MS/MS. The methods developed for drug discovery were found to be accurate (±≤15.2% from nominal concentrations) and precise (≤13.4% CV), with a dynamic range of 1.00-500 ng/ml and extraction recoveries of 47.2-99.0%. Conclusion: This bioanalytical method can be utilized to screen CDP proteins and other miniproteins for drug discovery, candidate selection and further drug development.