Development of high-throughput fluorescent-based screens to accelerate discovery of P2X inhibitors from animal venoms

Animal venoms can play an important role in drug discovery, as they are a rich source of evolutionarily tuned compounds that target a variety of ion channels and receptors. To date, there are six FDA-approved drugs derived from animal venoms, with recent work using high-throughput platforms providing a variety of new therapeutic candidates. However, high-throughput methods for screening animal venoms against purinoceptors, one of the oldest signaling receptor families, have not been reported. Here, we describe a variety of quantitative fluorescent-based high-throughput screening (HTS) cell-based assays for screening animal venoms against ligand-gated P2X receptors. A diverse selection of 180 venoms from arachnids, centipedes, hymenopterans, and cone snails were screened, analyzed, and validated, both analytically and pharmacologically. Using this approach, we performed screens against human P2X3, P2X4, and P2X7 using three different fluorescent-based dyes on stable cell lines and isolated the active venom components. Our HTS assays are performed in 96-well format and allow simultaneous screening of multiple venoms on multiple targets, improving testing characteristics while minimizing costs, specimen material, and testing time. Moreover, utilizing our assays and applying them to the other natural product libraries, rather than venoms, might yield other novel natural products that modulate P2X activity

Spider venoms and chronic pain – developing novel pharmacological tools from the spider venoms to target P2X4 in microglia

Today, one in five adults experience chronic pain and this figure increases for those over 65 years old. However, frustration is mounting over the inadequate treatment for chronic neuropathic pain since its symptoms are challenging to treat and often resistant to opioids. Processing of pain signals relies on the activities of ion channels with the microglial P2X4 receptor being an important player. Animal venoms play an essential role in drug discovery as they contain a rich source of bioactive molecules evolutionarily fine-tuned to target ion channels such as P2X receptors. First, we have established and validated several fluorescent-based high throughput screening assays for assessing the activity of venom toxins at P2X receptors. Second, a diverse selection of 180 crude venoms has been screened against human P2X4 in HEK293 and 1321N21 cells, resulting in several venoms containing inhibitors against hP2X4. Two of them, LK-601 and LK-729, were confirmed to be structurally uncharacterized acylpolyamines, which potently inhibited hP2X4 with the apparent IC50 values between 1.1 – 4.5 μM, however only LK-601 showed a relatively high level of selectivity over hP2X3, hP2X7 and NMDA 1a/2a. Species differences were evident with no effect at rat P2X4, however, blocking the mouse P2X4. Using LK-601 as a structural guide, the fragment-based screening was carried out and five smaller toxin analogues chemically synthesized. One of them, LA-3, was found to block the hP2X4 (IC50 of 9.7 – 18.6 μM) and showed selectivity to hP2X4 over hP2X3, hP2X7 and rP2X4 with a modest inhibition at mP2X4. Due to the differential sensitivity of LA-3 to block P2X4 orthologues, the potential binding site were identified, and the validation showed that two crucial amino acid residues, D220 and N238, might be involved in LA-3 binding to hP2X4; however, more experiments are needed to confirm that effect fully. In summary, we discovered a novel toxin from a spider venom with inhibitory activity at human P2X4 ion channels that shows selectivity at hP2X4 over other P2X receptors. Further characterization and validation are required to understand whether these novel compounds could be useful as analgesics

Inspirationen und Impulse auf der WiTa

Die 16. Ausgabe der Wissenschaftstagung Ökologischer Landbau (WiTa) fand nach vier Jahren Corona-Pause von 8. bis 10. März unter dem Motto „One Step Ahead – einen Schritt voraus!“ auf dem neuen Campus des Forschungsinstituts für biologischen Landbau (FiBL) statt und regte spannende Diskussionen zu aktuellen, aberauch künftigen Fragen rund um die ökologische Land- und Lebensmittelwirtschaft an. Am 8. März begrüßte das FiBL über 300 Gäste zur WiTa, die als bedeutendste Plattform für den Austausch von Forschungsergebnissen der Ökolandwirtschaft im deutschsprachigen Raum gilt. Unter der Trägerschaft der Stiftung Ökologie & Landbau (SÖL) und des FiBL e.V. findet die Tagung in der Regel alle zwei Jahre an wechselnden Standorten statt. Die 16. Ausgabe wurde vom FiBL Schweiz gemeinsam mit der FiBL Projekte GmbH veranstaltet. In den rund dreieinhalb Tagen fanden sieben Exkursionen statt, außerdem konnten die Teilnehmenden 154 Vorträgen lauschen, 102 Poster-Präsentationen begutachten und 14 Workshops sowie sechs Science Pitches besuchen

Radiative MRI coil design using parasitic scatterers: MRI Yagi

Conventionally, radiofrequency (RF) coils used for magnetic resonance imaging (MRI) are electrically small and designed for nearfield operation. Therefore, existing antenna design techniques are mostly irrelevant for RF coils. However, the use of higher frequencies in ultrahigh field (UHF) MRI allows for antenna design techniques to be adapted to RF coil designs. This study proposes the use of parasitic scatterers to improve the performance of an existing 7T MRI coil called the single-sided adapted dipole (SSAD) antenna. The results reveal that scatterers arranged in a Yagi fashion can be applied to reduce local specific absorption rate (SAR) maxima of a reference SSAD by 40% with only a 6% decrease in the propagated B1+ field at the tissue depth of 15 cm. The higher directivity of the proposed design also decreasing the coupling with additional elements, making this antenna suitable for use in high density arrays. These findings show the potential of parasitic scatterers as an effective method to improve the performance of existing radiative MRI coils

Design and development of stapled transmembrane peptides that disrupt the activity of G-protein coupled receptor oligomers

Membrane proteins can associate into larger complexes. Examples include receptor tyrosine complexes, ion channels, transporters and G-protein coupled receptors (GPCRs). For the latter, there is abundant evidence indicating that GPCRs, assemble into complexes, through both homo or heterodimerization. However, the tools for studying and disrupting these complexes, GPCR or otherwise, are limited. Here we have developed stabilized interference peptides for this purpose. We have previously reported that tetrahydrocannabinol-mediated cognitive impairment arises from homo- or hetero-oligomerization between the GPCRs cannabinoid receptor type 1 (CB1R) and 5-hydroxytryptamine 2A (5-HT2AR) receptors. Here, to disrupt this interaction through targeting CB1–5-HT2A receptor heteromers in HEK293 cells and using an array of biochemical techniques, including calcium and cAMP measurements, bimolecular fluorescence complementation assays, and CD-based helicity assessments, we developed a NanoLuc binary technology (NanoBiT)-based reporter assay to screen a small library of aryl-carbon–stapled transmembrane mimicking peptides produced by solid-phase peptide synthesis. We found that these stapling peptides have increased α-helicity and improved proteolytic resistance without any loss of disrupting activity in vitro, suggesting that this approach may also have utility in vivo. In summary, our results provide proof of concept for using NanoBiT to study membrane protein complexes and for stabilizing disrupting peptides to target such membrane complexes through hydrocarbon-mediated stapling. We propose that these peptides could be developed to target previously un-druggable GPCR heteromers

Bug Off Pain: An Educational Virtual Reality Game on Spider Venoms and Chronic Pain for Public Engagement

Virtual reality (VR) technology has been capturing the public imagination for decades. VR software applications that allow for interactive immersion are emerging as a renowned medium in many areas, including educating the public in biochemistry-related subjects via public engagement events. This report provides information about an immersive, interactive and educational virtual reality (VR) game named Bug Off Pain that increases scientific literacy about chronic pain and spider venoms among the public and high school students. Here, VR was shown to be an innovative and fun approach to learning and public engagement in biochemistry. Bug Off Pain places the viewer inside the brain and shows the molecular system that allows people to sense pain. After securing three (learning) points via the multimedia-based clips, this experience translates to the interactive game. Here, a player has to choose a venom that shuts down the pain until that results in “pain over”. Bug Off Pain can be played (free of charge) on two different VR platforms: Oculus Rift and Android devices

(68)Ga-labeled superparamagnetic iron oxide nanoparticles (SPIONs) for multi-modality PET/MR/Cherenkov luminescence imaging of sentinel lymph nodes.

The aim of this study was to develop (68)Ga-SPIONs for use as a single contrast agent for dynamic, quantitative and high resolution PET/MR imaging of Sentinel Lymph Node (SLN). In addition (68)Ga enables Cherenkov light emission which can be used for optical guidance during resection of SLN. SPIONs were labeled with (68)Ga in ammonium acetate buffer, pH 5.5. The labeling yield and stability in human serum were determined using instant thin layer chromatography. An amount of 0.07-0.1 mL (~5-10 MBq, 0.13 mg Fe) of (68)Ga-SPIONs was subcutaneously injected in the hind paw of rats. The animals were imaged at 0-3 h and 25 h post injection with PET/CT, 9.4 T MR and CCDbased Cherenkov optical systems. A biodistribution study was performed by dissecting and measuring the radioactivity in lymph nodes, kidneys, spleen, liver and the injection site. The labeling yield was 97.3 ± 0.05% after 15 min and the (68)Ga-SPIONs were stable in human serum. PET, MR and Cherenkov luminescence imaging clearly visualized the SLN. Biodistribution confirmed a high uptake of the (68)Ga-SPIONs within the SLN. We conclude that generator produced (68)Ga can be labeled to SPIONs. Subcutaneously injected (68)Ga-SPIONs can enhance the identification of the SLNs by combining sensitive PET and high resolution MR imaging. Clinically, hybrid PET/MR cameras are already in use and (68)Ga-SPIONs have a great potential as a single-dose, tri-modality agent for diagnostic imaging and potential Cherenkov luminescent guided resection of SLN

Impact of the COVID-19 lockdown on patients and families with Dravet syndrome

We explored the impact of coronavirus virus 2019 (COVID-19) pandemic on patients with Dravet syndrome (DS) and their family. With European patient advocacy groups (PAGs), we developed an online survey in 10 languages to question health status, behavior, personal protection, and health services before and after lockdown. Approximately 538 European PAG members received electronic invitations. Survey ran from April 14, to May 17, 2020, with 219 answers; median age 9year 10months. Protection against infection was highly used prior to COVID-19, but 88% added facemask-use according to pandemic recommendations. Only one patient was tested positive for COVID-19. Most had stable epilepsy during lockdown, and few families (4%) needed emergency care during lockdown. However, behavior disorder worsened in over one-third of patients, regardless of epilepsy changes. Half of appointments scheduled prior to lockdown were postponed; 12 patients (11%) had appointments fulfilled; and 39 (36%) had remote consultations. Responders welcomed remote consultations. Half of responders were unsatisfied with psychological remote support as only few (21 families) received this support. None of the five of patient in clinical trials stopped investigational treatment. Prior adoption of protective measures against general infection might have contributed to avoiding COVID-19 infections. Protocols for the favored remote contact ought to now be prepared

PREGLED REZULTATA KISELOSTI SIROVOG MLEKA NA TERITORIJI OPŠTINE SJENICA

The aim of the work is to determine the acidity (pH) of milk produced on family farms in the Municipality of Sjenica depending on the season and month of production. It found an analysis of 3,226 milk samples that manufacturers and processors brought voluntarily to the lab. The process of receiving and analyzing the samples was done according to the Rulebook on the Quality of Raw Milk and ISO/IEC 17025:2017. The number of samples of raw milk in winter (529) is lower than in summer (1094). The average pH is the highest in the month of December (6.70), and the lowest in March (6.52). On the territory of the Municipality of Sjenica there was a steady acidity of milk per month in 2019. and moved within the boundaries envisioned in the Regulation on the Quality of Raw Milk.Publishe