Development of a V5-tag-directed nanobody and its implementation as an intracellular biosensor of GPCR signaling.

Protein-protein interactions (PPIs) form the foundation of any cell signaling network. Considering that PPIs are highly dynamic processes, cellular assays are often essential for their study because they closely mimic the biological complexities of cellular environments. However, incongruity may be observed across different PPI assays when investigating a protein partner of interest; these discrepancies can be partially attributed to the fusion of different large functional moieties, such as fluorescent proteins or enzymes, which can yield disparate perturbations to the protein's stability, subcellular localization, and interaction partners depending on the given cellular assay.

Profiling of basal and ligand-dependent GPCR activities by means of a polyvalent cell-based high-throughput platform.

Representing the most attractive and successful druggable receptors of the proteome, GPCRs regulate a myriad of physiological and pathophysiological functions. Although over half of present pharmaceuticals target GPCRs, the advancement of drug discovery is hampered by a lack of adequate screening tools, the majority of which are limited to probing agonist-induced G-protein and β-arrestin-2-mediated events as a measure of receptor activation. Here, we develop Tango-Trio, a comprehensive cell-based high-throughput platform comprising cumate-inducible expression of transducers, capable of the parallelized profiling of both basal and agonist-dependent GPCR activities.

Identification and differential usage of a host metalloproteinase entry pathway by SARS-CoV-2 Delta and Omicron.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike glycoprotein (S) binds to angiotensin-converting enzyme 2 (ACE2) to mediate membrane fusion via two distinct pathways: 1) a surface, serine protease-dependent or 2) an endosomal, cysteine protease-dependent pathway. In this study, we found that SARS-CoV-2 S has a wider protease usage and can also be activated by TMPRSS13 and matrix metalloproteinases (MMPs). We found that MMP-2 and MMP-9 played roles in SARS-CoV-2 S cell-cell fusion and TMPRSS2- and cathepsin-independent viral entry in cells expressing high MMP levels.

Arsenal of nanobodies shows broad-spectrum neutralization against SARS-CoV-2 variants of concern in vitro and in vivo in hamster models.

Nanobodies offer several potential advantages over mAbs for the control of SARS-CoV-2. Their ability to access cryptic epitopes conserved across SARS-CoV-2 variants of concern (VoCs) and feasibility to engineer modular, multimeric designs, make these antibody fragments ideal candidates for developing broad-spectrum therapeutics against current and continually emerging SARS-CoV-2 VoCs. Here we describe a diverse collection of 37 anti-SARS-CoV-2 spike glycoprotein nanobodies extensively characterized as both monovalent and IgG Fc-fused bivalent modalities.

Distinct functional roles for the M4 α-helix from each homologous subunit in the heteropentameric ligand-gated ion channel nAChR.

The outermost lipid-exposed α-helix (M4) in each of the homologous α, β, δ, and γ/ε subunits of the muscle nicotinic acetylcholine receptor (nAChR) has previously been proposed to act as a lipid sensor. However, the mechanism by which this sensor would function is not clear. To explore how the M4 α-helix from each subunit in human adult muscle nAChR influences function, and thus explore its putative role in lipid sensing, we functionally characterized alanine mutations at every residue in αM4, βM4, δM4, and εM4, along with both alanine and deletion mutations in the post-M4 region of each subunit.

Functional Characterization of Sodium Channel Inhibitors at the Delta-Opioid Receptor.

Existing pharmacotherapies acting on the opioid receptor system have been extensively used to treat chronic pain and addictive disorders. Nevertheless, the adverse side effects associated with opioid therapy underscore the need for concerted measures to develop safer analgesics. A promising avenue of research stems from the characterization of a sodium-dependent allosteric regulation site housed within the delta-opioid receptor and several other G protein-coupled receptors (GPCRs), thereby revealing the presence of a cluster of sodium and water molecules lodged in a cavity thought to be present only in the inactive conformation of the receptor.

Structural analysis of Atopobium parvulum SufS cysteine desulfurase linked to Crohn's disease.

Crohn's disease (CD) is characterized by the chronic inflammation of the gastrointestinal tract. A dysbiotic microbiome and a defective immune system are linked to CD, where hydrogen sulfide (H S) microbial producers positively correlate with the severity of the disease. Atopobium parvulum is a key H S producer from the microbiome of CD patients.

Relative Ratios of Human Seasonal Coronavirus Antibodies Predict the Efficiency of Cross-Neutralization of SARS-CoV-2 Spike Binding to ACE2.

Background: Antibodies raised against human seasonal coronaviruses (sCoVs), which are responsible for the common cold, are known to cross-react with SARS-CoV-2 antigens. This prompts questions about their protective role against SARS-CoV-2 infections and COVID-19 severity. However, the relationship between sCoVs exposure and SARS-CoV-2 correlates of protection are not clearly identified.

Identification of a High-Frequency Intrahost SARS-CoV-2 Spike Variant with Enhanced Cytopathic and Fusogenic Effects.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a virus that is continuously evolving. Although its RNA-dependent RNA polymerase exhibits some exonuclease proofreading activity, viral sequence diversity can be produced by replication errors and host factors. A diversity of genetic variants can be observed in the intrahost viral population structure of infected individuals.

Ebola virus triggers receptor tyrosine kinase-dependent signaling to promote the delivery of viral particles to entry-conducive intracellular compartments.

Filoviruses, such as the Ebola virus (EBOV) and Marburg virus (MARV), are causative agents of sporadic outbreaks of hemorrhagic fevers in humans. To infect cells, filoviruses are internalized via macropinocytosis and traffic through the endosomal pathway where host cathepsin-dependent cleavage of the viral glycoproteins occurs. Subsequently, the cleaved viral glycoprotein interacts with the late endosome/lysosome resident host protein, Niemann-Pick C1 (NPC1).

Parallel Interrogation of β-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay.

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent one of the most promising targets for the pharmaceutical industry. Ergo, the design, implementation, and optimization of GPCR ligand screening assays is crucial, as they represent remote-control tools for drug discovery and for manipulating GPCR pharmacology and outcomes. In the past, G-protein dependent assays typified this area of research, detecting ligand-induced events and quantifying the generation of secondary messengers.

Molecular aspects of delta opioid receptors.

The delta opioid receptor (DOP) belongs to the Class A, rhodopsin-like family of G protein-coupled receptors. Although this receptor has a high level of similarity with the other opioid receptors, it displays unique aspects and functions. Indeed, as opposed to most membrane receptors, DOP is poorly addressed to the plasma membrane.

Measurement of β-Arrestin Recruitment at GPCRs Using the Tango Assay.

Intracellular signal transduced by G protein-coupled receptors (GPCRs) is tightly controlled by a guanine nucleotide-binding complex made of G protein Gα, Gβ, and Gγ subunits, as well as a growing array of regulatory and accessory proteins such as arrestins. G protein-independent β-arrestin recruitment at GPCRs is universally accepted as the canonical interactor system and it has been found to be a powerful tracker of most GPCRs activation. Pharmacological concepts have evolved remarkably after the finding that different ligands, binding at the same receptor, can selectively activate specific subsets of signaling pathways among all pathways activated by balanced ligands.

Propagation of the Allosteric Modulation Induced by Sodium in the δ-Opioid Receptor.

Allosteric sodium in the helix bundle of a G protein-coupled receptor (GPCR) can modulate the receptor activation on the intracellular side. This phenomenon has confounded the GPCR community for decades. In this work, we present a theoretical model that reveals the mechanism of the allosteric modulation induced by sodium in the δ-opioid receptor.

Design and Discovery of Functionally Selective Serotonin 2C (5-HT) Receptor Agonists.

On the basis of the structural similarity of our previous 5-HT agonists with the melatonin receptor agonist tasimelteon and the putative biological cross-talk between serotonergic and melatonergic systems, a series of new (2,3-dihydro)benzofuran-based compounds were designed and synthesized. The compounds were evaluated for their selectivity toward 5-HT, 5-HT, and 5-HT receptors in the calcium flux assay with the ultimate goal to generate selective 5-HT agonists. Selected compounds were studied for their functional selectivity by comparing their transduction efficiency at the G protein signaling pathway versus β-arrestin recruitment.

Structure-based discovery of opioid analgesics with reduced side effects.

Morphine is an alkaloid from the opium poppy used to treat pain. The potentially lethal side effects of morphine and related opioids-which include fatal respiratory depression-are thought to be mediated by μ-opioid-receptor (μOR) signalling through the β-arrestin pathway or by actions at other receptors. Conversely, G-protein μOR signalling is thought to confer analgesia.

Further Advances in Optimizing (2-Phenylcyclopropyl)methylamines as Novel Serotonin 2C Agonists: Effects on Hyperlocomotion, Prepulse Inhibition, and Cognition Models.

A series of novel compounds with two halogen substituents have been designed and synthesized to further optimize the 2-phenylcyclopropylmethylamine scaffold in the quest for drug-like 5-HT2C agonists. Compound (+)-22a was identified as a potent 5-HT2C receptor agonist, with good selectivity against the 5-HT2B and the 5-HT2A receptors. ADMET assays showed that compound (+)-22a possessed desirable properties in terms of its microsomal stability, and CYP and hERG inhibition, along with an excellent brain penetration profile.

Design and Synthesis of (2-(5-Chloro-2,2-dimethyl-2,3-dihydrobenzofuran-7-yl)cyclopropyl)methanamine as a Selective Serotonin 2C Agonist.

A conformationally restricted analog of a selective cyclopropane-bearing serotonin 2C agonist was designed and synthesized. A 2,2-dimethyl-2,3-dihydrobenzofuran scaffold was investigated as a constrained variant of a biologically active isopropyl phenyl ether. Construction of the required dimethyl-2,3-dihydrobenzofuran intermediate began using a procedure that relied on a microwave-assisted alkylation reaction.

A Non-Canonical Function of Gβ as a Subunit of E3 Ligase in Targeting GRK2 Ubiquitylation.

G protein-coupled receptors (GPCRs) comprise the largest family of cell surface receptors, regulate a wide range of physiological processes, and are the major targets of pharmaceutical drugs. Canonical signaling from GPCRs is relayed to intracellular effector proteins by trimeric G proteins, composed of α, β, and γ subunits (Gαβγ). Here, we report that G protein β subunits (Gβ) bind to DDB1 and that Gβ2 targets GRK2 for ubiquitylation by the DDB1-CUL4A-ROC1 ubiquitin ligase.

A New DREADD Facilitates the Multiplexed Chemogenetic Interrogation of Behavior.

DREADDs are chemogenetic tools widely used to remotely control cellular signaling, neuronal activity, and behavior. Here we used a structure-based approach to develop a new Gi-coupled DREADD using the kappa-opioid receptor as a template (KORD) that is activated by the pharmacologically inert ligand salvinorin B (SALB). Activation of virally expressed KORD in several neuronal contexts robustly attenuated neuronal activity and modified behaviors.

PRESTO-Tango as an open-source resource for interrogation of the druggable human GPCRome.

G protein-coupled receptors (GPCRs) are essential mediators of cellular signaling and are important targets of drug action. Of the approximately 350 nonolfactory human GPCRs, more than 100 are still considered to be 'orphans' because their endogenous ligands remain unknown. Here, we describe a unique open-source resource that allows interrogation of the druggable human GPCRome via a G protein-independent β-arrestin-recruitment assay.

Optimization of 2-phenylcyclopropylmethylamines as selective serotonin 2C receptor agonists and their evaluation as potential antipsychotic agents.

The discovery of a new series of compounds that are potent, selective 5-HT2C receptor agonists is described herein as we continue our efforts to optimize the 2-phenylcyclopropylmethylamine scaffold. Modifications focused on the alkoxyl substituent present on the aromatic ring led to the identification of improved ligands with better potency at the 5-HT2C receptor and excellent selectivity against the 5-HT2A and 5-HT2B receptors. ADMET studies coupled with a behavioral test using the amphetamine-induced hyperactivity model identified four compounds possessing drug-like profiles and having antipsychotic properties.

G Protein signaling modulator-3 inhibits the inflammasome activity of NLRP3.

Inflammasomes are multi-protein complexes that regulate maturation of the interleukin 1β-related cytokines IL-1β and IL-18 through activation of the cysteine proteinase caspase-1. NOD-like receptor family, pyrin domain containing 3 (NLRP3) protein is a key component of inflammasomes that assemble in response to a wide variety of endogenous and pathogen-derived danger signals. Activation of the NLRP3-inflammasome and subsequent secretion of IL-1β is highly regulated by at least three processes: transcriptional activation of both NLRP3 and pro-IL-1β genes, non-transcriptional priming of NLRP3, and final activation of NLRP3.