Belimumab concentration measurements using a homologous anti-idiotype immunoassay.

Monitoring belimumab concentrations in patients can be a valuable tool for assessing treatment response and for personalizing drug doses. Various assay formats may be used to measure concentrations of therapeutic monoclonal antibodies. A particularly useful format involves the use of anti-idiotype monoclonal antibodies, selected to be highly specific to the antibody of interest. Here, we describe the development of a specific, high-affinity anti-idiotype antibody to belimumab, and the application of this antibody in a homologous sandwich ELISA to measure belimumab concentrations.

Plasma interferon-alpha protein levels during pregnancy are associated with lower birth weight in systemic lupus erythematosus.

OBJECTIVES: Adverse pregnancy outcomes are more common in women with systemic lupus erythematosus (SLE) compared with healthy women, but we lack prognostic biomarkers. Plasma interferon alpha (IFNα) protein levels are elevated in a subgroup of pregnant women with SLE, but whether this is associated with pregnancy outcomes is unknown. We investigated the relationship between IFNα, adverse pregnancy outcomes and the presence of autoantibodies in SLE pregnancy. METHODS: We followed 76 women with SLE prospectively. Protein levels of IFNα were quantified in plasma collected in the 2nd and 3rd trimester with single-molecule array. Positivity for antinuclear and antiphospholipid antibodies was assessed during late pregnancy with multiplexed bead assay. Clinical outcomes included the adverse pregnancy outcomes small for gestational age (SGA), preterm birth, and preeclampsia. RESULTS: During SLE pregnancy, women with SGA infants compared with those without had higher levels of plasma IFNα protein, and IFNα positivity was associated with lower birth weight of the infant. Preterm birth was associated with autoantibodies against chromatin. IFNα protein levels associated positively with autoantibodies against chromatin, Smith/ribonucleoprotein (SmRNP) and RNP, but negatively with phospholipid antibodies. CONCLUSION: Elevated IFNα protein in plasma of women with SLE is a potential risk factor for lower birth weight of their infants. The association between IFNα and lower birth weight warrants further investigation regarding the pathophysiological role of IFNα during SLE pregnancy.

Structural insights into GABAA receptor potentiation by Quaalude.

Methaqualone, a quinazolinone marketed commercially as Quaalude, is a central nervous system depressant that was used clinically as a sedative-hypnotic, then became a notorious recreational drug in the 1960s-80s. Due to its high abuse potential, medical use of methaqualone was eventually prohibited, yet it persists as a globally abused substance. Methaqualone principally targets GABAA receptors, which are the major inhibitory neurotransmitter-gated ion channels in the brain. The restricted status and limited accessibility of methaqualone have contributed to its pharmacology being understudied. Here, we use cryo-EM to localize the GABAA receptor binding sites of methaqualone and its more potent derivative, PPTQ, to the same intersubunit transmembrane sites targeted by the general anesthetics propofol and etomidate. Both methaqualone and PPTQ insert more deeply into subunit interfaces than the previously-characterized modulators. Binding of quinazolinones to this site results in widening of the extracellular half of the ion-conducting pore, following a trend among positive allosteric modulators in destabilizing the hydrophobic activation gate in the pore as a mechanism for receptor potentiation. These insights shed light on the underexplored pharmacology of quinazolinones and further elucidate the molecular mechanisms of allosteric GABAA receptor modulation through transmembrane binding sites.

Loss-of-Function Thr347Ala Variant in the G Protein Subunit-α11 Causes Familial Hypocalciuric Hypercalcemia 2 (FHH2).

CONTEXT AND OBJECTIVES: To date, only four loss-of-function variants in the GNA11 gene encoding the G protein subunit α11 (Gα11) leading to familial hypocalciuric hypercalcemia 2 (FHH2) have been characterized. Gα11 is involved in calcium-sensing receptor (CaSR) signaling, and loss-of-function variants in GNA11 lead to reduced agonist potency at CaSR and an FHH phenotype. DESIGN AND PARTICIPANTS: We have identified a family with a heterozygous GNA11 Thr347Ala variant and characterized its impact on calcium homeostasis in FHH2 patients and the signaling properties of CaSR through the Gα11-Thr347Ala variant in vitro. MAIN OUTCOME MEASURES: The index patient and her family had clinical, biochemical, and genetic analyses performed. The expression levels of Gα11 and the cell-surface expression levels of CaSR in human embryonic kidney 293A Gq/11 knock-out cells (ΔGq/11-HEK293A) co-transfected with CaSR and Gα11 (wild type (WT) or Thr347Ala) were determined, and the functional properties exhibited by calcium at CaSR were characterized in an inositol monophosphate (IP1) accumulation assay. RESULTS: Heterozygous carriers of the GNA11 Thr347Ala variant had mild asymptomatic hypercalcemia, hypocalciuria, and inappropriately high normal parathyroid hormone (PTH) levels considering their elevated serum calcium levels. Whereas the variant did not impact Gα11 expression or CaSR cell surface expression levels, calcium displayed a moderately but significantly lower agonist potency at CaSR/Gα11-Thr347Ala-transfected cells compared with CaSR/Gα11-WT-transfected cells in the IP1 accumulation assay (EC50 values of 5.67 mM and 4.38 mM, respectively). CONCLUSIONS: This identification of a novel GNA11 variant causing FHH2 substantiates the important role of Gα11 for CaSR signaling and Ca2+ homeostasis.

Early aerial expedition photos reveal 85 years of glacier growth and stability in East Antarctica.

During the last few decades, several sectors in Antarctica have transitioned from glacial mass balance equilibrium to mass loss. In order to determine if recent trends exceed the scale of natural variability, long-term observations are vital. Here we explore the earliest, large-scale, aerial image archive of Antarctica to provide a unique record of 21 outlet glaciers along the coastline of East Antarctica since the 1930s. In Lützow-Holm Bay, our results reveal constant ice surface elevations since the 1930s, and indications of a weakening of local land-fast sea-ice conditions. Along the coastline of Kemp and Mac Robertson, and Ingrid Christensen Coast, we observe a long-term moderate thickening of the glaciers since 1937 and 1960 with periodic thinning and decadal variability. In all regions, the long-term changes in ice thickness correspond with the trends in snowfall since 1940. Our results demonstrate that the stability and growth in ice elevations observed in terrestrial basins over the past few decades are part of a trend spanning at least a century, and highlight the importance of understanding long-term changes when interpreting current dynamics.

Discovery and Structure-Activity Relationships of 2,5-Dimethoxyphenylpiperidines as Selective Serotonin 5-HT2A Receptor Agonists.

Classical psychedelics such as psilocybin, lysergic acid diethylamide (LSD), and N,N-dimethyltryptamine (DMT) are showing promising results in clinical trials for a range of psychiatric indications, including depression, anxiety, and substance abuse disorder. These compounds are characterized by broad pharmacological activity profiles, and while the acute mind-altering effects can be ascribed to their shared agonist activity at the serotonin 2A receptor (5-HT2AR), their apparent persistent therapeutic effects are yet to be decidedly linked to activity at this receptor. We report herein the discovery of 2,5-dimethoxyphenylpiperidines as a novel class of selective 5-HT2AR agonists and detail the structure-activity investigations leading to the identification of LPH-5 [analogue (S)-11] as a selective 5-HT2AR agonist with desirable drug-like properties.

Precious Metal Free Hydrogen Evolution Catalyst Design and Application.

The quest to identify precious metal free hydrogen evolution reaction catalysts has received unprecedented attention in the past decade. In this Review, we focus our attention to recent developments in precious metal free hydrogen evolution reactions in acidic and alkaline electrolyte owing to their relevance to commercial and near-commercial low-temperature electrolyzers. We provide a detailed review and critical analysis of catalyst activity and stability performance measurements and metrics commonly deployed in the literature, as well as review best practices for experimental measurements (both in half-cell three-electrode configurations and in two-electrode device testing). In particular, we discuss the transition from laboratory-scale hydrogen evolution reaction (HER) catalyst measurements to those in single cells, which is a critical aspect crucial for scaling up from laboratory to industrial settings but often overlooked. Furthermore, we review the numerous catalyst design strategies deployed across the precious metal free HER literature. Subsequently, we showcase some of the most commonly investigated families of precious metal free HER catalysts; molybdenum disulfide-based, transition metal phosphides, and transition metal carbides for acidic electrolyte; nickel molybdenum and transition metal phosphides for alkaline. This includes a comprehensive analysis comparing the HER activity between several families of materials highlighting the recent stagnation with regards to enhancing the intrinsic activity of precious metal free hydrogen evolution reaction catalysts. Finally, we summarize future directions and provide recommendations for the field in this area of electrocatalysis.

Low CD4 + T cell count is related to specific anti-nuclear antibodies, IFNα protein positivity and disease activity in systemic lupus erythematosus pregnancy.

BACKGROUND: Lymphopenia, autoantibodies and activation of the type I interferon (IFN) system are common features in systemic lupus erythematosus (SLE). We speculate whether lymphocyte subset counts are affected by pregnancy and if they relate to autoantibody profiles and/or IFNα protein in SLE pregnancy. METHODS: Repeated blood samples were collected during pregnancy from 80 women with SLE and 51 healthy controls (HC). Late postpartum samples were obtained from 19 of the women with SLE. Counts of CD4 + and CD8 + T cells, B cells and NK cells were measured by flow cytometry. Positivity for anti-nuclear antibodies (ANA) fine specificities (double-stranded DNA [dsDNA], Smith [Sm], ribonucleoprotein [RNP], chromatin, Sjögren's syndrome antigen A [SSA] and B [SSB]) and anti-phospholipid antibodies (cardiolipin [CL] and ß2 glycoprotein I [ß2GPI]) was assessed with multiplexed bead assay. IFNα protein concentration was quantified with Single molecule array (Simoa) immune assay. Clinical data were retrieved from medical records. RESULTS: Women with SLE had lower counts of all lymphocyte subsets compared to HC throughout pregnancy, but counts did not differ during pregnancy compared to postpartum. Principal component analysis revealed that low lymphocyte subset counts differentially related to autoantibody profiles, cluster one (anti-dsDNA/anti-Sm/anti-RNP/anti-Sm/RNP/anti-chromatin), cluster two (anti-SSA/anti-SSB) and cluster three (anti-CL/anti-ß2GPI), IFNα protein levels and disease activity. CD4 + T cell counts were lower in women positive to all ANA fine specificities in cluster one compared to those who were negative, and B cell numbers were lower in women positive for anti-dsDNA and anti-Sm compared to negative women. Moreover, CD4 + T cell and B cell counts were lower in women with moderate/high compared to no/low disease activity, and CD4 + T cell count was lower in IFNα protein positive relative to negative women. Finally, CD4 + T cell count was unrelated to treatment. CONCLUSION: Lymphocyte subset counts are lower in SLE compared to healthy pregnancies, which seems to be a feature of the disease per se and not affected by pregnancy. Our results also indicate that low lymphocyte subset counts relate differentially to autoantibody profiles, IFNα protein levels and disease activity, which could be due to divergent disease pathways.

Sediment discharge from Greenland's marine-terminating glaciers is linked with surface melt.

Sediment discharged from the Greenland Ice Sheet delivers nutrients to marine ecosystems around Greenland and shapes seafloor habitats. Current estimates of the total sediment flux are constrained by observations from land-terminating glaciers only. Addressing this gap, our study presents a budget derived from observations at 30 marine-margin locations. Analyzing sediment cores from nine glaciated fjords, we assess spatial deposition since 1950. A significant correlation is established between mass accumulation rates, normalized by surface runoff, and distance down-fjord. This enables calculating annual sediment flux at any fjord point based on nearby marine-terminating outlet glacier melt data. Findings reveal a total annual sediment flux of 1.324 + /- 0.79 Gt yr-1 over the period 2010-2020 from all marine-terminating glaciers to the fjords. These estimates are valuable for studies aiming to understand the basal ice sheet conditions and for studies predicting ecosystem changes in Greenland's fjords and offshore areas as the ice sheet melts and sediment discharge increase.

A survey of ficolin-3 activity in Systemic Lupus Erythematosus reveals a link to hematological disease manifestations and autoantibody profile.

The complement system plays a central role in the pathogenesis of Systemic Lupus Erythematosus (SLE), but most studies have focused on the classical pathway. Ficolin-3 is the main initiator of the lectin pathway of complement in humans, but its role in systemic autoimmune disease has not been conclusively determined. Here, we combined biochemical and genetic approaches to assess the contribution of ficolin-3 to SLE risk and disease manifestations. Ficolin-3 activity was measured by a functional assay in serum or plasma samples from Swedish SLE patients (n = 786) and controls matched for age and sex (n = 566). Genetic variants in an extended 300 kb genomic region spanning the FCN3 locus were analyzed for their association with ficolin-3 activity and SLE manifestations in a Swedish multicenter cohort (n = 985). Patients with ficolin-3 activity in the highest tertile showed a strong enrichment in an SLE cluster defined by anti-Sm/DNA/nucleosome antibodies (OR 3.0, p < 0.001) and had increased rates of hematological disease (OR 1.4, p = 0.078) and lymphopenia (OR = 1.6, p = 0.039). Genetic variants associated with low ficolin-3 activity mapped to an extended haplotype in high linkage disequilibrium upstream of the FCN3 gene. Patients carrying the lead genetic variant associated with low ficolin-3 activity had a lower frequency of hematological disease (OR 0.67, p = 0.018) and lymphopenia (OR 0.63, p = 0.031) and fewer autoantibodies (p = 0.0019). Loss-of-function variants in the FCN3 gene were not associated with SLE, but four (0.5 %) SLE patients developed acquired ficolin-3 deficiency where ficolin-3 activity in serum was depleted following diagnosis of SLE. Taken together, our results provide genetic and biochemical evidence that implicate the lectin pathway in hematological SLE manifestations. We also identify lectin pathway activation through ficolin-3 as a factor that contributes to the autoantibody response in SLE.

The Concise Guide to PHARMACOLOGY 2023/24: Ion channels.

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and over 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (https://www.guidetopharmacology.org/), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.16178. Ion channels are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

MRI BrainAGE demonstrates increased brain aging in systemic lupus erythematosus patients.

Introduction: Systemic lupus erythematosus (SLE) is an autoimmune connective tissue disease affecting multiple organs in the human body, including the central nervous system. Recently, an artificial intelligence method called BrainAGE (Brain Age Gap Estimation), defined as predicted age minus chronological age, has been developed to measure the deviation of brain aging from a healthy population using MRI. Our aim was to evaluate brain aging in SLE patients using a deep-learning BrainAGE model. Methods: Seventy female patients with a clinical diagnosis of SLE and 24 healthy age-matched control females, were included in this post-hoc analysis of prospectively acquired data. All subjects had previously undergone a 3 T MRI acquisition, a neuropsychological evaluation and a measurement of neurofilament light protein in plasma (NfL). A BrainAGE model with a 3D convolutional neural network architecture, pre-trained on the 3D-T1 images of 1,295 healthy female subjects to predict their chronological age, was applied on the images of SLE patients and controls in order to compute the BrainAGE. SLE patients were divided into 2 groups according to the BrainAGE distribution (high vs. low BrainAGE). Results: BrainAGE z-score was significantly higher in SLE patients than in controls (+0.6 [±1.1] vs. 0 [±1.0], p = 0.02). In SLE patients, high BrainAGE was associated with longer reaction times (p = 0.02), lower psychomotor speed (p = 0.001) and cognitive flexibility (p = 0.04), as well as with higher NfL after adjusting for age (p = 0.001). Conclusion: Using a deep-learning BrainAGE model, we provide evidence of increased brain aging in SLE patients, which reflected neuronal damage and cognitive impairment.

Ligand selectivity hotspots in serotonin GPCRs.

Serotonin is a neurotransmitter regulating numerous physiological processes also modulated by drugs, for example, schizophrenia, depression, migraine, and obesity. However, these drugs typically have adverse effects caused by promiscuous binding across 12 serotonin and more than 20 homologous receptors. Recently, structures of the entire serotonin receptor family uncovered molecular ligand recognition. Here, we present a map of 19 'selectivity hotspots', that is, nonconserved binding site residues governing selectivity via favorable target interactions or repulsive 'off-target' contacts. Furthermore, we review functional rationale from observed ligand-binding affinities and mutagenesis effects. Unifying knowledge underlying specific probes and drugs is critical toward the functional characterization of different receptors and alleviation of adverse effects.

Repeated low doses of psilocybin increase resilience to stress, lower compulsive actions, and strengthen cortical connections to the paraventricular thalamic nucleus in rats.

Psilocybin (a classic serotonergic psychedelic drug) has received appraisal for use in psychedelic-assisted therapy of several psychiatric disorders. A less explored topic concerns the use of repeated low doses of psychedelics, at a dose that is well below the psychedelic dose used in psychedelic-assisted therapy and often referred to as microdosing. Psilocybin microdose users frequently report increases in mental health, yet such reports are often highly biased and vulnerable to placebo effects. Here we establish and validate a psilocybin microdose-like regimen in rats with repeated low doses of psilocybin administration at a dose derived from occupancy at rat brain 5-HT2A receptors in vivo. The rats tolerated the repeated low doses of psilocybin well and did not manifest signs of anhedonia, anxiety, or altered locomotor activity. There were no deficits in pre-pulse inhibition of the startle reflex, nor did the treatment downregulate or desensitize the 5-HT2A receptors. However, the repeated low doses of psilocybin imparted resilience against the stress of multiple subcutaneous injections, and reduced the frequency of self-grooming, a proxy for human compulsive actions, while also increasing 5-HT7 receptor expression and synaptic density in the paraventricular nucleus of the thalamus. These results establish a well-validated regimen for further experiments probing the effects of repeated low doses of psilocybin. Results further substantiate anecdotal reports of the benefits of psilocybin microdosing as a therapeutic intervention, while pointing to a possible physiological mechanism.

In Vitro and In Vivo Evaluation of Pellotine: A Hypnotic Lophophora Alkaloid.

Quality of life is often reduced in patients with sleep-wake disorders. Insomnia is commonly treated with benzodiazepines, despite their well-known side effects. Pellotine (1), a Lophophora alkaloid, has been reported to have short-acting sleep-inducing properties in humans. In this study, we set out to evaluate various in vitro and in vivo properties of 1. We demonstrate that 1 undergoes slow metabolism; e.g. in mouse liver microsomes 65% remained, and in human liver microsomes virtually no metabolism was observed after 4 h. In mouse liver microsomes, two phase I metabolites were identified: 7-desmethylpellotine and pellotine-N-oxide. In mice, the two diastereomers of pellotine-O-glucuronide were additionally identified as phase II metabolites. Furthermore, we demonstrated by DESI-MSI that 1 readily enters the central nervous system of rodents. Furthermore, radioligand-displacement assays showed that 1 is selective for the serotonergic system and in particular the serotonin (5-HT)1D, 5-HT6, and 5-HT7 receptors, where it binds with affinities in the nanomolar range (117, 170, and 394 nM, respectively). Additionally, 1 was functionally characterized at 5-HT6 and 5-HT7, where it was found to be an agonist at the former (EC50 = 94 nM, Emax = 32%) and an inverse agonist at the latter (EC50 = 291 nM, Emax = -98.6). Finally, we demonstrated that 1 dose-dependently decreases locomotion in mice, inhibits REM sleep, and promotes sleep fragmentation. Thus, we suggest that pellotine itself, and not an active metabolite, is responsible for the hypnotic effects and that these effects are possibly mediated through modulation of serotonergic receptors.

Serologic immunogenicity and safety of Herpes Zoster subunit vaccine in patients with Rheumatoid Arthritis receiving Janus Kinase inhibitors.

OBJECTIVE: Patients with rheumatoid arthritis (RA) treated with Janus Kinase inhibitors (JAKi) are at increased risk of Herpes Zoster (HZ). The objective of this study was to investigate serological immunogenicity and safety of the HZ subunit (HZ/su) vaccine in RA patients treated with JAKi, for which little is known. METHODS: RA patients treated with JAKi (n = 82) at the Department of Rheumatology, Skåne University Hospital, Sweden, and healthy controls (n = 51) received two doses of the HZ/su vaccine (Shingrix). Vaccine-specific antibody responses were analysed using indirect enzyme-linked immunosorbent assay (ELISA). Post-vaccination antibody levels were compared between patients and controls using analysis of covariance. Potential predictors for vaccine response were investigated using a multivariable linear regression analysis. Self-reported adverse events (AEs) and changes in RA disease activity were analysed. RESULTS: Following vaccination, vaccine-specific antibody levels increased significantly in both patients and controls (p< 0.0001). 80.5% of patients and 98.0% of controls achieved a ≥ 4-fold increase in antibody levels. Post-vaccination antibody levels were lower in patients than controls (ratio 0.44, 95% CI 0.31-0.63), and lower in patients receiving JAKi+Methotrexate than JAKi monotherapy (ratio 0.43, 95% CI 0.24-0.79). AEs, mostly mild/moderate, were common. One patient developed HZ and six patients (6.5%) had increased RA disease activity following vaccination. CONCLUSION: The HZ/su vaccine was serologically immunogenic in most RA patients treated with JAKi. Moreover, the vaccine had an acceptable safety profile. These results support recommendations for usage of the HZ/su vaccine in this vulnerable population. TRIAL REGISTRATION: ClinicalTrials.gov, https://clinicaltrials.gov, NCT03886038.

B cell polygenic risk scores associate with anti-dsDNA antibodies and nephritis in systemic lupus erythematosus.

OBJECTIVE: B cell function and autoantibodies are important in SLE pathogenesis. In this work, we aimed to investigate the impact of cumulative SLE B cell genetics on SLE subphenotype and autoantibody profile. METHODS: Female patients with SLE (n=1248) and healthy controls (n=400) were genotyped using Illumina's Global Screening Array. Two polygenic risk scores (PRSs), one representing B cell genes and the other B cell activation genes, were calculated for each individual using risk loci for SLE in genes assigned to B cell-related pathways according to the Kyoto Encyclopedia of Genes and Genomes, Gene Ontology and Reactome Databases. RESULTS: Double-stranded DNA (dsDNA) antibodies were more prevalent among patients with a high compared with a low SLE B cell PRS (OR 1.47 (1.07 to 2.01), p=0.018), and effect sizes were augmented in patients with human leucocyte antigen (HLA) risk haplotypes HLA-DRB1*03:01 and HLA-DRB1*15:01 (DRB1*03/15 -/- (OR 0.99 (0.56 to 1.77), p=0.98; DRB1*03/15 +/- or -/+ (OR 1.64 (1.06 to 2.54), p=0.028; and DRB1*03/15 +/+ (OR 4.47 (1.21 to 16.47), p=0.024). Further, a high compared with a low B cell PRS was associated with low complement levels in DRB1*03/15 +/+ patients (OR 3.92 (1.22 to 12.64), p=0.022). The prevalence of lupus nephritis (LN) was higher in patients with a B cell activation PRS above the third quartile compared with patients below (OR 1.32 (1.00 to 1.74), p=0.048). CONCLUSIONS: High genetic burden related to B cell function is associated with dsDNA antibody development and LN. Assessing B cell PRSs may be important in order to determine immunological pathways influencing SLE and to predict clinical phenotype.

Evolutionary conservation of Zinc-Activated Channel (ZAC) functionality in mammals: a range of mammalian ZACs assemble into cell surface-expressed functional receptors.

In contrast to the other pentameric ligand-gated ion channels in the Cys-loop receptor superfamily, the ZACN gene encoding for the Zinc-Activated Channel (ZAC) is exclusively found in the mammalian genome. Human ZAC assembles into homomeric cation-selective channels gated by Zn2+, Cu2+ and H+, but the function of the receptor in human physiology is presently poorly understood. In this study, the degree of evolutionary conservation of a functional ZAC in mammals was probed by investigating the abilities of a selection of ZACs from 10 other mammalian species than human to be expressed at the protein level and assemble into cell surface-expressed functional receptors in mammalian cells and in Xenopus oocytes. In an enzyme-linked immunosorbent assay, transient transfections of tsA201 cells with cDNAs of hemagglutinin (HA)-epitope-tagged versions of these 10 ZACs resulted in robust total expression and cell surface expression levels of all proteins. Moreover, injection of cRNAs for 6 of these ZACs in oocytes resulted in the formation of functional receptors in two-electrode voltage-clamp recordings. The ZACs exhibited robust current amplitudes in response to Zn2+ (10 mM) and H+ (pH 4.0), and the concentration-response relationships displayed by Zn2+ at these channels were largely comparable to that at human ZAC. In conclusion, the findings suggest that the functionality of ZAC at the molecular level may be conserved throughout mammalian species, and that the channel thus may govern physiological functions in mammals, including humans.

Identification of 5-HT2 Serotonin Receptor Modulators through the Synthesis of a Diverse, Tropane- and Quinuclidine-alkaloid-Inspired Compound Library.

The recombination of natural product (NP) fragments in unprecedented ways has emerged as an important strategy for bioactive compound discovery. In this context, we propose that privileged primary fragments predicted to be enriched in activity against a specific target class can be coupled to diverse secondary fragments to engineer selectivity among closely related targets. Here, we report the synthesis of an alkaloid-inspired compound library enriched in spirocyclic ring fusions, comprising 58 compounds from 12 tropane- or quinuclidine-containing scaffolds, all of which can be considered pseudo-NPs. The library displays excellent predicted drug-like properties including high Fsp3 content and Lipinski's rule-of-five compliance. Targeted screening against selected members of the serotonin and dopamine G protein-coupled receptor family led to the identification of several hits that displayed significant agonist or antagonist activity against 5-HT2A and/or 5-HT2C, and subsequent optimization of one of these delivered a lead dual 5-HT2B/C antagonist with a highly promising selectivity profile.