Quality assessment and harmonization of laboratories across Europe for multiple SARS-CoV-2 serology assays.

BACKGROUND AND OBJECTIVES: There is a need for conversion of SARS-CoV-2 serology data from different laboratories to a harmonized international unit. We aimed to compare the performance of multiple SARS-CoV-2 antibody serology assays among 25 laboratories across 12 European countries. MATERIALS AND METHODS: To investigate this we have distributed to all participating laboratories a panel of 15 SARS-CoV-2 plasma samples and a single batch of pooled plasma calibrated to the WHO IS 20/136 standard. RESULTS: All assays showed excellent discrimination between SARS-CoV-2 seronegative plasma samples and pre-vaccinated seropositive plasma samples but differed substantially in raw antibody titres. Titres could be harmonized to binding antibody units per millilitre by calibration in relation to a reference reagent. CONCLUSION: The standardization of antibody quantification is of paramount importance to allow interpretation and comparison of serology data reported in clinical trials in order to identify donor cohorts from whom the most effective convalescent plasma can be collected.

Intranasal administration of convalescent plasma protects against SARS-CoV-2 infection in hamsters.

BACKGROUND: Convalescent plasma (CP) transfusion is an early option for treating infections with pandemic potential, often preceding vaccine or antiviral drug rollout. Heterogenous findings from randomized clinical trials on transfusion of COVID-19 CP (CCP) have been reported. However, meta-analysis suggests that transfusion of high titer CCP is associated with a mortality benefit for COVID-19 outpatients or inpatients treated within 5 days after symptom onset, indicating the importance of early administration. METHODS: We tested if CCP is an effective prophylactic against SARS-CoV-2 infection by the intranasal administration of 25 µL CCP/nostril (i.e. 0.01-0.06 mg anti-RBD antibodies/kg) in hamsters exposed to infected littermates. FINDINGS: In this model, 40% of CCP treated hamsters were fully protected and 40% had significantly reduced viral loads, the remaining 20% was not protected. The effect seems dose-dependent because high-titer CCP from a vaccinated donor was more effective than low-titer CCP from a donation prior to vaccine rollout. Intranasal administration of human CCP resulted in a reactive (immune) response in hamster lungs, however this was not observed upon administration of hamster CCP. INTERPRETATION: We conclude that CCP is an effective prophylactic when used directly at the site of primary infection. This option should be considered in future prepandemic preparedness plans. FUNDING: Flanders Innovation & Entrepreneurship (VLAIO) and the Foundation for Scientific Research of the Belgian Red Cross Flanders.

Corrigendum: Striatal dopamine D2-muscarinic acetylcholine M1 receptor-receptor interaction in a model of movement disorders.

[This corrects the article DOI: 10.3389/fphar.2020.00194.].

A novel competition ELISA for the rapid quantification of SARS-CoV-2 neutralizing antibodies in convalescent plasma.

BACKGROUND: COVID-19 convalescent plasma (CCP) ideally contains high titers of (neutralizing) anti-SARS-CoV-2 antibodies. Several scalable immunoassays for CCP selection have been developed. We designed an enzyme-linked immunosorbent assay (ELISA) that measures neutralizing antibodies (of all isotypes) in plasma by determining the level of competition between CCP and a mouse neutralizing antibody for binding to the receptor binding domain (RBD) of SARS-CoV-2. METHODS: Plasma was collected from 72 convalescent individuals and inhibition of viral infection was determined by plaque reduction neutralization (PRNT50). The level of neutralizing antibodies was measured in the novel competition ELISA and in a commercially available ELISA that measures inhibition of recombinant ACE2 binding to immobilized RBD. These results were compared with a high throughput chemiluminescent microparticle immunoassay (CMIA). RESULTS: The results from both ELISAs were correlating, in particular for high titer CCP (PRNT50 ≥ 1:160) (Spearman r = .73, p < .001). Moderate correlation was found between the competition ELISA and CMIA (r = .57 for high titer and r = .62 for low titer CCP, p < .001). Receiver operator characteristic analysis showed that the competition ELISA selected CCP with a sensitivity and specificity of 61% and 100%, respectively. However, discrimination between low and high titer CCP had a lower resolution (sensitivity: 34% and specificity: 89%). CONCLUSION: The competition ELISA screens for neutralizing antibodies in CCP by competition for just a single epitope. It exerts a sensitivity of 61% with no false identifications. These ELISA designs can be used for epitope mapping or for selection of CCP.

SARS-CoV-2 neutralising antibody testing in Europe: towards harmonisation of neutralising antibody titres for better use of convalescent plasma and comparability of trial data.

We compared the performance of SARS-CoV-2 neutralising antibody testing between 12 European laboratories involved in convalescent plasma trials. Raw titres differed almost 100-fold differences between laboratories when blind-testing 15 plasma samples. Calibration of titres in relation to the reference reagent and standard curve obtained by testing a dilution series reduced the inter-laboratory variability ca 10-fold. The harmonisation of neutralising antibody quantification is a vital step towards determining the protective and therapeutic levels of neutralising antibodies.

Discovery of a true bivalent dopamine D2 receptor agonist.

Employing two different alkyne-modified dopamine agonists to construct bivalent compounds via click chemistry resulted in the identification of a bivalent ligand (11c) for dopamine D2 receptor homodimer, which, compared to its parent monomeric alkyne, showed a 16-fold higher binding affinity for the dopamine D2 receptor and a 5-fold higher potency in a cAMP assay in HEK 293T cells stably expressing D2R. Molecular modeling revealed that 11c can indeed bridge the orthosteric binding sites of a D2R homodimer in a relaxed conformation via the TM5-TM6 interface and allows to largely rationalize the results of the receptor assays.

Striatal Dopamine D2-Muscarinic Acetylcholine M1 Receptor-Receptor Interaction in a Model of Movement Disorders.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor control deficits, which is associated with the loss of striatal dopaminergic neurons from the substantia nigra. In parallel to dopaminergic denervation, there is an increase of acetylcholine within the striatum, resulting in a striatal dopaminergic-cholinergic neurotransmission imbalance. Currently, available PD pharmacotherapy (e.g., prodopaminergic drugs) does not reinstate the altered dopaminergic-cholinergic balance. In addition, it can eventually elicit cholinergic-related adverse effects. Here, we investigated the interplay between dopaminergic and cholinergic systems by assessing the physical and functional interaction of dopamine D2 and muscarinic acetylcholine M1 receptors (D2R and M1R, respectively), both expressed at striatopallidal medium spiny neurons. First, we provided evidence for the existence of D2R-M1R complexes via biochemical (i.e., co-immunoprecipitation) and biophysical (i.e., BRET1 and NanoBiT®) assays, performed in transiently transfected HEK293T cells. Subsequently, a D2R-M1R co-distribution in the mouse striatum was observed through double-immunofluorescence staining and AlphaLISA® immunoassay. Finally, we evaluated the functional interplay between both receptors via behavioral studies, by implementing the classical acute reserpine pharmacological animal model of experimental parkinsonism. Reserpinized mice were administered with a D2R-selective agonist (sumanirole) and/or an M1R-selective antagonist (VU0255035), and alterations in PD-related behavioral tasks (i.e., locomotor activity) were evaluated. Importantly, VU0255035 (10 mg/kg) potentiated the antiparkinsonian-like effects (i.e., increased locomotor activity and decreased catalepsy) of an ineffective sumanirole dose (3 mg/kg). Altogether, our data suggest the existence of putative striatal D2R/M1R heteromers, which might be a relevant target to manage PD motor impairments with fewer adverse effects.

Assessment of Biased Agonism among Distinct Synthetic Cannabinoid Receptor Agonist Scaffolds.

Cannabinoid receptor 1 (CB1) is a key drug target for a number of diseases, including metabolic syndromes and neuropathic pain. Most of the typical cannabinoid ligands provoke psychotropic side effects that impair their therapeutic utility. As of today, it is not yet clearly known which structural features of cannabinoid ligands determine a preference toward specific signaling pathways. Distinct bioassays are typically used to elucidate signaling preferences. However, these are often based on different cell lines and use different principles and/or read-outs, which makes straightforward assessment of "ligand bias" difficult. Within this context, this study is the first to investigate ligand bias among synthetic cannabinoid receptor agonists (SCRAs) in as closely analogous conditions as possible, by applying a new functional complementation-based assay panel to assess the recruitment of Gαi protein or ß-arrestin2 to CB1. In a panel of 21 SCRAs, chosen to cover a broad diversity in chemical structures, distinct, although often subtle, preferences toward specific signaling pathways were observed. Relative to CP55940, here considered as a "balanced" reference agonist, most of the selected SCRAs (e.g., 5F-APINACA, CUMYL-PEGACLONE, among others) displayed preferred signaling through the ß-arrestin2 pathway, whereas MMB-CHMICA could serve as a potential "balanced" agonist. Interestingly, EG-018 was the only SCRA showing a significant (10-fold) preference toward G protein over ß-arrestin2 recruitment. While it is currently unclear what this exactly means in terms of abuse potential and/or toxicity, the approach proposed here may allow construction of a knowledge base that in the end may allow better insight into the structure-"functional" activity relationship of these compounds. This may aid the development of new therapeutics with less unwanted psychoactive effects.

Assessment of structure-activity relationships and biased agonism at the Mu opioid receptor of novel synthetic opioids using a novel, stable bio-assay platform.

Fentanyl and morphine are agonists of the Mu opioid receptor (MOR), which is a member of the GPCR family. Their analgesic effects are associated with unwanted side effects. On a signaling level downstream from MOR, it has been hypothesized that analgesia may be mediated through the G protein pathway, whereas the undesirable effects of opioids have been linked to the ß-arrestin (ßarr) pathway. Despite being an increasingly debated subject, little is known about a potential 'bias' (i.e. the preferential activation of one pathway over the other) of the novel synthetic opioids (NSO) - including fentanyl analogs - that have emerged on the illegal drug market. We have therefore developed and applied a novel, robust bio-assay platform to study the activity of 21 NSO, to evaluate to what extent these MOR agonists show biased agonism and to investigate the potential correlation with their structure. In addition, we evaluated the functional selectivity of TRV130, a purported G protein-biased agonist. We applied newly established stable bio-assays in HEK293T cells, based on the principle of functional complementation of a split nanoluciferase, to assess MOR activation via recruitment of a mini-Gi protein (GTPase domain of Gαi subunit) or ßarr2. All but two of the tested NSO demonstrated a concentration-dependent response at MOR in both bio-assays. The developed bio-assays allow to gain insight into the ßarr2 or G protein recruitment potential of NSO, which may eventually help to better understand why certain opioids are associated with higher toxicity. Adding to the recent discussion about the relevance of the biased agonism concept for opioids, we did not observe a significant bias for any of the evaluated compounds, including TRV130.

Insights into biased signaling at cannabinoid receptors: synthetic cannabinoid receptor agonists.

Cannabinoid receptors type 1 (CB1) and type 2 (CB2) are promising targets for a number of diseases, including obesity, neuropathic pain, and multiple sclerosis, among others. Upon ligand-mediated activation of these receptors, multiple receptor conformations could be stabilized, resulting in a complex pattern of possible intracellular effects. Although numerous compounds have been developed and widely used to target cannabinoid receptors, their mode of action and signaling properties are often only poorly characterized. From a drug development point of view, unraveling the underlying complex signaling mechanism could offer the possibility to generate medicines with the desired therapeutic profile. Recently, an increased interest has emerged for the development of agonists that are signaling pathway-selective and thereby do not evoke on-target adverse effects. This phenomenon, in which specific pathways are preferred upon receptor activation by certain ligands, is also known as 'biased signaling'. For a particular group of cannabinoid receptor ligands (i.e. CB1/CB2 agonists), namely the synthetic cannabinoid receptor agonists (SCRAs), the research on biased signaling is still in its infancy and interesting outcomes are only recently being revealed. Therefore, this review aims at providing insights into the recent knowledge about biased agonism mediated by SCRAs so far. In addition, as these outcomes are obtained using a distinct panel of functional assays, the accompanying difficulties and challenges when comparing functional outcomes are critically discussed. Finally, some guidance on the conceptualization of ideal in vitro assays for the detection of SCRA-mediated biased agonism, which is also relevant for compounds belonging to other chemical classes, is provided.

Luminescence- and Fluorescence-Based Complementation Assays to Screen for GPCR Oligomerization: Current State of the Art.

G protein-coupled receptors (GPCRs) have the propensity to form homo- and heterodimers. Dysfunction of these dimers has been associated with multiple diseases, e.g., pre-eclampsia, schizophrenia, and depression, among others. Over the past two decades, considerable efforts have been made towards the development of screening assays for studying these GPCR dimer complexes in living cells. As a first step, a robust in vitro assay in an overexpression system is essential to identify and characterize specific GPCR-GPCR interactions, followed by methodologies to demonstrate association at endogenous levels and eventually in vivo. This review focuses on protein complementation assays (PCAs) which have been utilized to study GPCR oligomerization. These approaches are typically fluorescence- and luminescence-based, making identification and localization of protein-protein interactions feasible. The GPCRs of interest are fused to complementary fluorescent or luminescent fragments that, upon GPCR di- or oligomerization, may reconstitute to a functional reporter, of which the activity can be measured. Various protein complementation assays have the disadvantage that the interaction between the reconstituted split fragments is irreversible, which can lead to false positive read-outs. Reversible systems offer several advantages, as they do not only allow to follow the kinetics of GPCR-GPCR interactions, but also allow evaluation of receptor complex modulation by ligands (either agonists or antagonists). Protein complementation assays may be used for high throughput screenings as well, which is highly relevant given the growing interest and effort to identify small molecule drugs that could potentially target disease-relevant dimers. In addition to providing an overview on how PCAs have allowed to gain better insights into GPCR-GPCR interactions, this review also aims at providing practical guidance on how to perform PCA-based assays.

Distinct Dopamine D2 Receptor Antagonists Differentially Impact D2 Receptor Oligomerization.

Dopamine D2 receptors (D2R) are known to form transient homodimer complexes, of which the increased formation has already been associated with development of schizophrenia. Pharmacological targeting and modulation of the equilibrium of these receptor homodimers might lead to a better understanding of the critical role played by these complexes in physiological and pathological conditions. Whereas agonist addition has shown to prolong the D2R dimer lifetime and increase the level of dimer formation, the possible influence of D2R antagonists on dimerization has remained rather unexplored. Here, using a live-cell reporter assay based on the functional complementation of a split Nanoluciferase, a panel of six D2R antagonists were screened for their ability to modulate the level of D2LR dimer formation. Incubation with the D2R antagonist spiperone decreased the level of D2LR dimer formation significantly by 40-60% in real-time and after long-term (≥16 h) incubations. The fact that dimer formation of the well-studied A2a-D2LR dimer was not altered following incubation with spiperone supports the specificity of this observation. Other D2R antagonists, such as clozapine, risperidone, and droperidol did not significantly evoke this dissociation event. Furthermore, molecular modeling reveals that spiperone presents specific Tyr1995.48 and Phe3906.52 conformations, compared to clozapine, which may determine D2R homodimerization.

Functional evaluation of carboxy metabolites of synthetic cannabinoid receptor agonists featuring scaffolds based on L-valine or L-tert-leucine.

Indole- and indazole-based synthetic cannabinoid receptor agonists (SCRAs), featuring valine or tert-leucine substituents, are commonly abused new psychoactive substances (NPS). A major metabolic pathway for these SCRAs is hydrolysis of the terminal amide or methylester functionalities. Although these hydrolysis products were already detected as main ingredients in some "legal highs," these metabolites are often poorly characterized. Here, we report a systematic investigation of the activity of 7 common hydrolysis metabolites of 15 SCRAs featuring scaffolds based on L-valine or L-tert-leucine in direct comparison to their parent compounds. An activity-based cannabinoid receptor 1 (CB1 ) bio-assay was used for activity profiling of SCRAs and their metabolites in a stable HEK293T cell system. The recruitment of ß-arrestin2 to the activated CB1 (each fused to one part of a split Nanoluciferase) was provoked by adding the (putative) SCRAs. Luminescence of the functionally complemented luciferase was monitored by a 96-well plate-reader. The major hydrolysis metabolites of 5F-AB-PINACA, ADB-CHMICA, ADB-CHMINACA, ADB-FUBICA, and their methyl- and ethylester derivatives showed no detectable CB1 activation at concentrations up to 1 µM. On the other hand, metabolites of 5F-ADB-PINACA, AB-CHMINACA, and ADB-FUBINACA did retain activity, although significantly reduced as compared to the parent compounds (EC50 values >100 nM). Activity-based characterization of SCRAs and their metabolites at CB1 may not only allow a better insight into the complex interplay between SCRAs and their metabolites in intoxications, but may also allow application of the concept of "activity equivalents" present in biological fluids or, alternatively, in confiscated materials.

The validation of Short Interspersed Nuclear Elements (SINEs) as a RT-qPCR normalization strategy in a rodent model for temporal lobe epilepsy.

BACKGROUND: In gene expression studies via RT-qPCR many conclusions are inferred by using reference genes. However, it is generally known that also reference genes could be differentially expressed between various tissue types, experimental conditions and animal models. An increasing amount of studies have been performed to validate the stability of reference genes. In this study, two rodent-specific Short Interspersed Nuclear Elements (SINEs), which are located throughout the transcriptome, were validated and assessed against nine reference genes in a model of Temporal Lobe Epilepsy (TLE). Two different brain regions (i.e. hippocampus and cortex) and two different disease stages (i.e. acute phase and chronic phase) of the systemic kainic acid rat model for TLE were analyzed by performing expression analyses with the geNorm and NormFinder algorithms. Finally, we performed a rank aggregation analysis and validated the reference genes and the rodent-specific SINEs (i.e. B elements) individually via Gfap gene expression. RESULTS: GeNorm ranked Hprt1, Pgk1 and Ywhaz as the most stable genes in the acute phase, while Gusb and B2m were ranked as the most unstable, being significantly upregulated. The two B elements were ranked as most stable for both brain regions in the chronic phase by geNorm. In contrast, NormFinder ranked the B1 element only once as second best in cortical tissue for the chronic phase. Interestingly, using only one of the two algorithms would have led to skewed conclusions. Finally, the rank aggregation method indicated the use of the B1 element as the best option to normalize target genes, independent of the disease progression and brain region. This result was supported by the expression profile of Gfap. CONCLUSION: In this study, we demonstrate the potential of implementing SINEs -notably the B1 element- as a stable normalization factor in a rodent model of TLE, independent of brain region or disease progression.

Synthesis toward Bivalent Ligands for the Dopamine D2 and Metabotropic Glutamate 5 Receptors.

In this study, we designed and synthesized heterobivalent ligands targeting heteromers consisting of the metabotropic glutamate 5 receptor (mGluR5) and the dopamine D2 receptor (D2R). Bivalent ligand 22a with a linker consisting of 20 atoms showed 4-fold increase in affinity for cells coexpressing D2R and mGluR5 compared to cells solely expressing D2R. Likewise, the affinity of 22a for mGluR5 increased 2-fold in the coexpressing cells. Additionally, 22a exhibited a 5-fold higher mGluR5 affinity than its monovalent precursor 21a in cells coexpressing D2R and mGluR5. These results indicate that 22a is able to bridge binding sites on both receptors constituting the heterodimer. Likewise, cAMP assays revealed that 22a had a 4-fold higher potency in stable D2R and mGluR5 coexpressing cell lines than 1. Furthermore, molecular modeling reveals that 22a is able to simultaneously bind both receptors by passing between the TM5-TM6 interface and establishing six protein-ligand H-bonds.