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 ß-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. We capture the functional diversity of GPCRs, reporting ß-arrestin-1/2 couplings, selectivities, and receptor internalization signatures across the GPCRome. Moreover, we present the construction of cumate-induced basal activation curves at approximately 200 receptors, including over 50 orphans. Overall, Tango-Trio's robustness is well-suited for the functional characterization and screening of GPCRs, especially for parallel interrogation, and is a valuable addition to the pharmacological toolbox.

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. Owing to their smaller size, epitope tags may exhibit a diminished susceptibility to instigate such perturbations. However, while they have been widely used for detecting or manipulating proteins in vitro, epitope tags lack the in vivo traceability and functionality needed for intracellular biosensors. Herein, we develop NbV5, an intracellular nanobody binding the V5-tag, which is suitable for use in cellular assays commonly used to study PPIs such as BRET, NanoBiT, and Tango. The NbV5:V5 tag system has been applied to interrogate G protein-coupled receptor signaling, specifically by replacing larger functional moieties attached to the protein interactors, such as fluorescent or luminescent proteins (∼30 kDa), by the significantly smaller V5-tag peptide (1.4 kDa), and for microscopy imaging which is successfully detected by NbV5-based biosensors. Therefore, the NbV5:V5 tag system presents itself as a versatile tool for live-cell imaging and a befitting adaptation to existing cellular assays dedicated to probing PPIs.

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. MMP-dependent viral entry required cleavage at the S1/S2 junction in viral producer cells, and differential processing of variants of concern S dictated its usage; the efficiently processed Delta S preferred metalloproteinase-dependent entry when available, and less processed Omicron S was unable to us metalloproteinases for entry. As MMP-2/9 are released during inflammation, they may play roles in S-mediated cytopathic effects, tropism, and disease outcome.

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. The nanobodies were collectively shown to have high intrinsic affinity; high thermal, thermodynamic and aerosolization stability; broad subunit/domain specificity and cross-reactivity across existing VoCs; wide-ranging epitopic and mechanistic diversity and high and broad in vitro neutralization potencies. A select set of Fc-fused nanobodies showed high neutralization efficacies in hamster models of SARS-CoV-2 infection, reducing viral burden by up to six orders of magnitude to below detectable levels. In vivo protection was demonstrated with anti-RBD and previously unreported anti-NTD and anti-S2 nanobodies. This collection of nanobodies provides a potential therapeutic toolbox from which various cocktails or multi-paratopic formats could be built to combat multiple SARS-CoV-2 variants.

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. Studies into the structure-function relationship of said pocket demonstrated its critical involvement in the functional control of GPCR signaling. While the sodium pocket has been proposed to be present in the majority of class A GPCRs, the shape of this allosteric cavity appears to have significant structural variation among crystallographically solved GPCRs, making this site optimal for the design of new allosteric modulators that will be selective for opioid receptors. The size of the sodium pocket supports the accommodation of small molecules, and it has been speculated that promiscuous amiloride and 5'-substituted amiloride-related derivatives could target this cavity within many GPCRs, including opioid receptors. Using pharmacological approaches, we have described the selectivities of 5'-substituted amiloride-related derivatives, as well as the hitherto undescribed activity of the NHE1 inhibitor zoniporide toward class A GPCRs. Our investigations into the structural features of the delta-opioid receptor and its ensuing signaling activities suggest a bitopic mode of overlapping interactions involving the orthosteric site and the juxtaposed Na+ pocket, but only at the active or partially active opioid receptor.

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, ß, 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, ßM4, δM4, and εM4, along with both alanine and deletion mutations in the post-M4 region of each subunit. Although no critical interactions involving residues on M4 or in post-M4 were identified, we found that numerous mutations at the M4-M1/M3 interface altered the agonist-induced response. In addition, homologous mutations in M4 in different subunits were found to have different effects on channel function. The functional effects of multiple mutations either along M4 in one subunit or at homologous positions of M4 in different subunits were also found to be additive. Finally, when characterized in both Xenopus oocytes and human embryonic kidney 293T cells, select αM4 mutations displayed cell-specific phenotypes, possibly because of the different membrane lipid environments. Collectively, our data suggest different functional roles for the M4 α-helix in each heteromeric nAChR subunit and predict that lipid sensing involving M4 occurs primarily through the cumulative interactions at the M4-M1/M3 interface, as opposed to the alteration of specific interactions that are critical to channel function.

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 (H2 S) microbial producers positively correlate with the severity of the disease. Atopobium parvulum is a key H2 S producer from the microbiome of CD patients. In this study, the biochemical characterization of two Atopobium parvulum cysteine desulfurases, ApSufS and ApCsdB, shows that the enzymes are allosterically regulated. Structural analyses reveal that ApSufS forms a dimer with conserved characteristics observed in type II cysteine desulfurases. Four residues surrounding the active site are essential to catalyse cysteine desulfurylation, and a segment of short-chain residues grant access for substrate binding. A better understanding of ApSufS will help future avenues for CD treatment.

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. METHODS: We performed a cross-sectional analysis of cross-reactivity and cross-neutralization to SARS-CoV-2 antigens (S-RBD, S-trimer, N) using pre-pandemic sera from four different groups: pediatrics and adolescents, individuals 21 to 70 years of age, older than 70 years of age, and individuals living with HCV or HIV. Data was then further analysed using machine learning to identify predictive patterns of neutralization based on sCoVs serology. FINDINGS: Antibody cross-reactivity to SARS-CoV-2 antigens varied between 1.6% and 15.3% depending on the cohort and the isotype-antigen pair analyzed. We also show a range of neutralizing activity (0-45%) with median inhibition ranging from 17.6 % to 23.3 % in serum that interferes with SARS-CoV-2 spike attachment to ACE2 independently of age group. While the abundance of sCoV antibodies did not directly correlate with neutralization, we show that neutralizing activity is rather dependent on relative ratios of IgGs in sera directed to all four sCoV spike proteins. More specifically, we identified antibodies to NL63 and OC43 as being the most important predictors of neutralization. INTERPRETATION: Our data support the concept that exposure to sCoVs triggers antibody responses that influence the efficiency of SARS-CoV-2 spike binding to ACE2, which may potentially impact COVID-19 disease severity through other latent variables. FUNDING: This study was supported by a grant by the CIHR (VR2 -172722) and by a grant supplement by the CITF, and by a NRC Collaborative R&D Initiative Grant (PR031-1).

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. Most mutations will follow a neutral molecular evolution and will not make significant contributions to variations within and between infected hosts. Herein, we profiled the intrasample genetic diversity of SARS-CoV-2 variants, also known as quasispecies, using high-throughput sequencing data sets from 15,289 infected individuals and infected cell lines. Despite high mutational background, we identified recurrent intragenetic variable positions in the samples analyzed, including several positions at the end of the gene encoding the viral spike (S) protein. Strikingly, we observed a high frequency of C→A missense mutations resulting in the S protein lacking the last 20 amino acids (SΔ20). We found that this truncated S protein undergoes increased processing and increased syncytium formation, presumably due to escaping M protein retention in intracellular compartments. Our findings suggest the emergence of a high-frequency viral sublineage that is not horizontally transmitted but potentially involved in intrahost disease cytopathic effects. IMPORTANCE The mutation rate and evolution of RNA viruses correlate with viral adaptation. While most mutations do not make significant contributions to viral molecular evolution, some are naturally selected and produce variants through positive selection. Many SARS-CoV-2 variants have been recently described and show phenotypic selection toward more infectious viruses. Our study describes another type of variant that does not contribute to interhost heterogeneity but rather phenotypic selection toward variants that might have increased cytopathic effects. We identified that a C-terminal truncation of the spike protein removes an important endoplasmic reticulum (ER) retention signal, which consequently results in a spike variant that easily travels through the Golgi complex toward the plasma membrane in a preactivated conformation, leading to increased syncytium formation.

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). This interaction is hypothesized to trigger viral and host membrane fusion, which results in the delivery of the viral genome into the cytoplasm and subsequent initiation of replication. Some studies suggest that EBOV viral particles activate signaling cascades and host-trafficking factors to promote their localization with host factors that are essential for entry. However, the mechanism through which these activating signals are initiated remains unknown. By screening a kinase inhibitor library, we found that receptor tyrosine kinase inhibitors potently block EBOV and MARV GP-dependent viral entry. Inhibitors of epidermal growth factor receptor (EGFR), tyrosine protein kinase Met (c-Met), and the insulin receptor (InsR)/insulin like growth factor 1 receptor (IGF1R) blocked filoviral GP-mediated entry and prevented growth of replicative EBOV in Vero cells. Furthermore, inhibitors of c-Met and InsR/IGF1R also blocked viral entry in macrophages, the primary targets of EBOV infection. Interestingly, while the c-Met and InsR/IGF1R inhibitors interfered with EBOV trafficking to NPC1, virus delivery to the receptor was not impaired in the presence of the EGFR inhibitor. Instead, we observed that the NPC1 positive compartments were phenotypically altered and rendered incompetent to permit viral entry. Despite their different mechanisms of action, all three RTK inhibitors tested inhibited virus-induced Akt activation, providing a possible explanation for how EBOV may activate signaling pathways during entry. In sum, these studies strongly suggest that receptor tyrosine kinases initiate signaling cascades essential for efficient post-internalization entry steps.

Parallel Interrogation of ß-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. However, since the advent of functional selectivity, as well as an increased awareness of several other G protein-independent pathways and the limitations associated with G-protein dependent assays, there is a greater push towards the creation of alternative GPCR ligand screening assays. Towards this endeavor, we describe the application of one such resource, the PRESTO-Tango platform, a luciferase reporter-based system that enables the parallel and simultaneous interrogation of the human GPCR-ome, a feat which was previously considered technically and economically unfeasible. Based on a G-protein independent ß-arrestin2 recruitment assay, the universality of ß-arrestin2-mediated trafficking and signaling at GPCRs makes PRESTO-TANGO an apt tool for studying approximately 300 non-olfactory human GPCRs, including approximately 100 orphan receptors. PRESTO-Tango's sensitivity and robustness make it suitable for primary high-throughput screens using compound libraries, employed to uncover new GPCR targets for known drugs or to discover new ligands for orphan receptors.

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. In this chapter, we first review the molecular and cellular mechanisms regulating the expression and the cellular trafficking/sorting of DOP. We then summarize the structural insights of this receptor through the analysis of the existing crystal structures, with a particular focus on the role of the sodium binding site. Finally, we review the current signaling mechanisms mediating receptor function and desensitization.

Measurement of ß-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, Gß, and Gγ subunits, as well as a growing array of regulatory and accessory proteins such as arrestins. G protein-independent ß-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. This new paradigm referred to as functional selectivity or biased signaling, has opened new avenues for the design of tailored drugs with enhanced therapeutic efficacies and reduced side effects. Here, we describe a unique platform for the interrogation of GPCR using a transcriptional-based assay to measure transient ß-arrestin recruitment called Tango.

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. We found that the allosteric sodium ion exploits a distinct conformation of the key residue Trp2746.48 to propagate the modulation to helices 5 and 6, which further transmits along the helices and regulates their positions on the intracellular side. This mechanism is supported by subsequent functional assays. Remarkably, our results highlight the contrast between the allosteric effects towards two GPCR partners, the G protein and ß-arrestin, as indicated by the fact that the allosteric modulation initiated by the sodium ion significantly affects the ß-arrestin recruitment, while it alters the G protein signaling only moderately. We believe that the mechanism revealed in this work can be used to explain allosteric effects initiated by sodium in other GPCRs since the allosteric sodium is highly conserved across GPCRs.

Design and Discovery of Functionally Selective Serotonin 2C (5-HT2C) Receptor Agonists.

On the basis of the structural similarity of our previous 5-HT2C 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-HT2A, 5-HT2B, and 5-HT2C receptors in the calcium flux assay with the ultimate goal to generate selective 5-HT2C agonists. Selected compounds were studied for their functional selectivity by comparing their transduction efficiency at the G protein signaling pathway versus ß-arrestin recruitment. The most functionally selective compound (+)-7e produced weak ß-arrestin recruitment and also demonstrated less receptor desensitization compared to serotonin in both calcium flux and phosphoinositide (PI) hydrolysis assays. We report for the first time that selective 5-HT2C agonists possessing weak ß-arrestin recruitment can produce distinct receptor desensitization properties.

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 ß-arrestin pathway or by actions at other receptors. Conversely, G-protein µOR signalling is thought to confer analgesia. Here we computationally dock over 3 million molecules against the µOR structure and identify new scaffolds unrelated to known opioids. Structure-based optimization yields PZM21-a potent Gi activator with exceptional selectivity for µOR and minimal ß-arrestin-2 recruitment. Unlike morphine, PZM21 is more efficacious for the affective component of analgesia versus the reflexive component and is devoid of both respiratory depression and morphine-like reinforcing activity in mice at equi-analgesic doses. PZM21 thus serves as both a probe to disentangle µOR signalling and a therapeutic lead that is devoid of many of the side effects of current opioids.

In Vitro and In Vivo Characterization of the Alkaloid Nuciferine.

RATIONALE: The sacred lotus (Nelumbo nucifera) contains many phytochemicals and has a history of human use. To determine which compounds may be responsible for reported psychotropic effects, we used in silico predictions of the identified phytochemicals. Nuciferine, an alkaloid component of Nelumbo nucifera and Nymphaea caerulea, had a predicted molecular profile similar to antipsychotic compounds. Our study characterizes nuciferine using in vitro and in vivo pharmacological assays. METHODS: Nuciferine was first characterized in silico using the similarity ensemble approach, and was followed by further characterization and validation using the Psychoactive Drug Screening Program of the National Institute of Mental Health. Nuciferine was then tested in vivo in the head-twitch response, pre-pulse inhibition, hyperlocomotor activity, and drug discrimination paradigms. RESULTS: Nuciferine shares a receptor profile similar to aripiprazole-like antipsychotic drugs. Nuciferine was an antagonist at 5-HT2A, 5-HT2C, and 5-HT2B, an inverse agonist at 5-HT7, a partial agonist at D2, D5 and 5-HT6, an agonist at 5-HT1A and D4 receptors, and inhibited the dopamine transporter. In rodent models relevant to antipsychotic drug action, nuciferine blocked head-twitch responses and discriminative stimulus effects of a 5-HT2A agonist, substituted for clozapine discriminative stimulus, enhanced amphetamine induced locomotor activity, inhibited phencyclidine (PCP)-induced locomotor activity, and rescued PCP-induced disruption of prepulse inhibition without induction of catalepsy. CONCLUSIONS: The molecular profile of nuciferine was similar but not identical to that shared with several approved antipsychotic drugs suggesting that nuciferine has atypical antipsychotic-like actions.

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. Evaluation of (+)-22a in animal models of schizophrenia-related behaviors revealed that it had a desirable activity profile, as it reduced d-amphetamine-stimulated hyperlocomotion in the open field test, it restored d-amphetamine-disrupted prepulse inhibition, it induced cognitive improvements in the novel object recognition memory test in NR1-KD animals, and it produced very little catalepsy relative to haloperidol. These data support the further development of (+)-22a as a drug candidate for the treatment of schizophrenia.

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. The synthesis of the designed compound as its HCl salt is reported in a total of 12 steps and 17% overall yield. Biological evaluation revealed the constrained analog to be a selective serotonin 2C agonist with modest potency.