Biology subjects

van Rijn, R. M.

Publications and source records attributed to van Rijn, R. M..

6 recordsLinked to original sources

bioRxiv2022

ClickArr: a novel, high throughput assay for evaluating β-arrestin isoform recruitment

Modern methods for quantifying signaling bias at GPCRs rely on using a single {beta}-arrestin isoform. However, it is increasingly appreciated that the two {beta}-arrestin isoforms have unique roles, requiring the ability to assess {beta}-arrestin isoform preference. Herein, we present ClickArr, a live-cell assay that simultaneously reports recruitment of both {beta}-arrestin isoforms as they compete for interaction with GPCRs. We demonstrate that an agonist can have {beta}-arrestin isoform bias, potentially opening up a new dimension for drug development.

pharmacology and toxicology↗

bioRxiv2021

A novel mitragynine analog with low efficacy mu-opioid receptor agonism displays antinociception with attenuated adverse effects

Dried kratom leaves are anecdotally used for the treatment of pain, opioid dependence, and alcohol use disorder. We have previously shown that kratoms natural products (mitragynine) and semi-synthetic analogs (7-hydroxy mitragynine (7OH) and mitragynine pseudoindoxyl) are mu opioid receptor (MOR) agonists that show minimal {beta}-arrestin2 recruitment. To further investigate the structure activity relationships of G-protein potency, efficacy, and {beta}-arrestin2 recruitment, we diversified the mitragynine/7OH templates at the C9, -10 and -12 positions of the aromatic ring of the indole moiety. Three lead C9 analogs, synthesized by swapping the 9-methoxy group with varied substituents, namely phenyl (SC11), methyl (SC12), 3-furanyl (SC13), were further characterized using a panel of in vitro and ex vivo electrophysiology assays. All three compounds were partial agonists with lower efficacy than both DAMGO and morphine in heterologous G-protein assays and synaptic physiology. SC11-13 also showed lower recruitment of both {beta}-arrestin subtypes compared to DAMGO, and in assays with limited MOR receptor reserve, the G-protein efficacy of SC11, SC12 and SC13 was comparable to buprenorphine. In mouse models, at equianalgesic doses SC13 showed MOR-dependent analgesia with potency similar to morphine without respiratory depression, hyperlocomotion, constipation, or place conditioning. Taken together, these results suggest that MOR agonists with a G-protein efficacy profile similar to buprenorphine can be developed into opioids that are effective analgesics with greatly reduced liabilities.

pharmacology and toxicology↗

bioRxiv2019

β-arrestin-dependent ERK signaling positively correlates with reduced anxiety-like and conditioned fear-related behavior in mice

Exposure to anxiety- or fear-invoking stimuli initiates a convergence of executive actions orchestrated by multiple proteins and neurotransmitters across the brain. Dozens of G protein-coupled receptors (GPCRs) have been linked to regulation of fear and anxiety. GPCR signaling involves canonical G protein pathways but may also engage downstream kinases and effectors through {beta}-arrestin scaffolds. Here, we investigate whether {beta}-arrestin signaling can regulate anxiety-like and fear-related behavior. Using the {delta}-opioid receptor ({delta}OR) as a model GPCR, we found that {beta}-arrestin 2-dependent activation of extracellular signal-regulated kinases (ERK1/2) in the dorsal hippocampus and the amygdala are critical for {delta}OR agonist-induced anxiolytic-like effects. In contrast, G protein-mediated {delta}OR signaling was associated with decreased ERK1/2 activity and increased fear-related behavior. Our results also indicate unique contributions for {beta}-arrestin isoforms in modulation of anxiety-like and fear-related behavior. Overall, our findings highlight the significance of non-canonical {beta}-arrestin signaling in the regulation of emotions. One sentence summaryUsing pharmacological and genetic strategies, we reveal the importance of non-canonical {beta}-arrestin-mediated G protein-coupled receptor signaling in anxiety-like behaviors.

neuroscience↗

bioRxiv2019

The Meta-Position of Phe4 in Leu-enkephalin Regulates Potency, Selectivity, Functional Activity, and Signaling Bias at the Delta and Mu Opioid Receptors

As tool compounds to study cardiac ischemia, the endogenous {delta}-opioid receptors ({delta}OR) agonist Leu5-enkephalin and the more metabolically stable synthetic peptide [D-Ala2, D-Leu5]-enkephalin are frequently employed. However, both peptides have similar pharmacological profiles that restrict detailed investigation of the cellular mechanism of the {delta}ORs protective role during ischemic events. Thus, a need remains for {delta}OR peptides with improved selectivity and unique signaling properties for investigating the specific roles for {delta}OR signaling in cardiac ischemia. To this end, we explored substitution at the Phe4 position of Leu5-enkephalin for its ability to modulate receptor function and selectivity. Peptides were assessed for their affinity to bind to {delta}ORs and -opioid receptors (ORs) and potency to inhibit cAMP signaling and to recruit {beta}-arrestin 2. Additionally, peptide stability was measured in rat plasma. Substitution of the meta-position of Phe4 of Leu5-enkephalin provided high-affinity ligands with varying levels of selectivity and bias at both the {delta}OR and OR and improved peptide stability, while substitution with picoline derivatives produced lower-affinity ligands with G protein biases at both receptors. Overall, these favorable substitutions at the meta-position of Phe4 may be combined with other modifications to Leu5-enkephalin to deliver improved agonists with finely tuned potency, selectivity, bias and drug-like properties. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=51 SRC="FIGDIR/small/750794v2_ufig1.gif" ALT="Figure 1"> View larger version (9K): org.highwire.dtl.DTLVardef@143ee98org.highwire.dtl.DTLVardef@e55c94org.highwire.dtl.DTLVardef@2114b4org.highwire.dtl.DTLVardef@1d63ba4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOTOC FIGUREC_FLOATNO C_FIG

pharmacology and toxicology↗

bioRxiv2018

Rubsicolins are naturally occurring G-protein-biased delta opioid receptor peptides

The impact that {beta}-arrestin proteins have on G-protein-coupled receptor trafficking, signaling and physiological behavior has gained much appreciation over the past decade. A number of studies have attributed the side effects associated with the use of naturally occurring and synthetic opioids, such as respiratory depression and constipation, to excessive recruitment of {beta}-arrestin. These findings have led to the development of biased opioid small molecule agonists that do not recruit {beta}-arrestin, activating only the canonical G-protein pathway. Similar G-protein biased small molecule opioids have been found to occur in nature, particularly within kratom, and opioids within salvia have served as a template for the synthesis of other G-protein-biased opioids. Here, we present the first report of naturally occurring peptides that selectively activate G-protein signaling pathways with minimal {beta}-arrestin recruitment. We find that rubiscolin peptides, which are produced as cleavage products of the plant protein rubisco, bind to and activate G-protein signaling at {delta} opioid receptors. However, unlike the naturally occurring {delta} opioid peptides leu-enkephalin and deltorphin II, the rubiscolin peptides only very weakly recruit {beta}-arrestin 2 and have undectable recruitment of {beta}-arrestin 1 at the {delta} opioid receptor.

pharmacology and toxicology↗

bioRxiv2017

Functional histamine H3 and adenosine A2A receptor heteromers in recombinant cells and rat striatum

In the striatum, histamine H3 receptors (H3Rs) are co-expressed with adenosine A2A receptors (A2ARs) in the cortico-striatal glutamatergic afferents and the GABAergic medium-sized spiny neurons that originate the indirect pathway of the basal ganglia. This location allows H3Rs and A2ARs to regulate the striatal GABAergic and glutamatergic transmission. However, whether these receptors interact to modulate the intra-striatal synaptic transmission has not yet been assessed. To test this hypothesis a heteromer-selective in vitro assay was used to detect functional complementation between a chimeric A2AR302-Gqi4 and wild-type H3Rs in transfected HEK-293 cells. H3R activation with the agonist RAMH resulted in Ca2+ mobilization (pEC50 7.31 {+/-} 0.23; maximal stimulation, Emax 449 {+/-} 25 % of basal) indicative of receptor heterodimerization. This response was not observed with histamine, suggesting a RAMH bias for heteromers. Functional A2AR-H3R heteromers were confirmed by co-immunoprecipitation and observations of differential cAMP signaling when both receptors were co-expressed in the same cell. In membranes from rat striatal synaptosomes, H3R activation decreased A2AR affinity for the agonist CGS-21680 (pKi values 8.10 {+/-} 0.04 and 7.70 {+/-} 0.04). Moreover, H3Rs and A2ARs co-immunoprecipitated in protein extracts from striatal synaptosomes. These results support the existence of a H3R/A2AR heteromer, and reveal a new mechanism by which these receptors may modulate the unction of the striatum and the basal ganglia.

neuroscience↗