Identification of a ß-arrestin-biased negative allosteric modulator for the ß2-adrenergic receptor.

Catecholamine-stimulated ß2-adrenergic receptor (ß2AR) signaling via the canonical Gs-adenylyl cyclase-cAMP-PKA pathway regulates numerous physiological functions, including the therapeutic effects of exogenous ß-agonists in the treatment of airway disease. ß2AR signaling is tightly regulated by GRKs and ß-arrestins, which together promote ß2AR desensitization and internalization as well as downstream signaling, often antithetical to the canonical pathway. Thus, the ability to bias ß2AR signaling toward the Gs pathway while avoiding ß-arrestin-mediated effects may provide a strategy to improve the functional consequences of ß2AR activation. Since attempts to develop Gs-biased agonists and allosteric modulators for the ß2AR have been largely unsuccessful, here we screened small molecule libraries for allosteric modulators that selectively inhibit ß-arrestin recruitment to the receptor. This screen identified several compounds that met this profile, and, of these, a difluorophenyl quinazoline (DFPQ) derivative was found to be a selective negative allosteric modulator of ß-arrestin recruitment to the ß2AR while having no effect on ß2AR coupling to Gs. DFPQ effectively inhibits agonist-promoted phosphorylation and internalization of the ß2AR and protects against the functional desensitization of ß-agonist mediated regulation in cell and tissue models. The effects of DFPQ were also specific to the ß2AR with minimal effects on the ß1AR. Modeling, mutagenesis, and medicinal chemistry studies support DFPQ derivatives binding to an intracellular membrane-facing region of the ß2AR, including residues within transmembrane domains 3 and 4 and intracellular loop 2. DFPQ thus represents a class of biased allosteric modulators that targets an allosteric site of the ß2AR.

Phenylalanine 193 in Extracellular Loop 2 of the ß 2-Adrenergic Receptor Coordinates ß-Arrestin Interaction.

G protein-coupled receptors (GPCRs) transduce a diverse variety of extracellular stimuli into intracellular signaling. These receptors are the most clinically productive drug targets at present. Despite decades of research on the signaling consequences of molecule-receptor interactions, conformational components of receptor-effector interactions remain incompletely described. The ß 2-adrenergic receptor (ß 2AR) is a prototypical and extensively studied GPCR that can provide insight into this aspect of GPCR signaling thanks to robust structural data and rich pharmacopeia. Using bioluminescence resonance energy transfer -based biosensors, second messenger assays, and biochemical techniques, we characterize the properties of ß 2AR-F193A. This single point mutation in extracellular loop 2 of the ß 2AR is sufficient to intrinsically bias the ß 2AR away from ß-arrestin interaction and demonstrates altered regulatory outcomes downstream of this functional selectivity. This study highlights the importance of extracellular control of intracellular response to stimuli and suggests a previously undescribed role for the extracellular loops of the receptor and the extracellular pocket formed by transmembrane domains 2, 3, and 7 in GPCR regulation that may contribute to biased signaling at GPCRs. SIGNIFICANCE STATEMENT: The role of extracellular G protein-coupled receptor (GPCR) domains in mediating intracellular interactions is poorly understood. We characterized the effects of extracellular loop mutations on agonist-promoted interactions of GPCRs with G protein and ß-arrestin. Our studies reveal that F193 in extracellular loop 2 in the ß2-adrenergic receptor mediates interactions with G protein and ß-arrestin with a biased loss of ß-arrestin binding. These results provide new insights on the role of the extracellular domain in differentially modulating intracellular interactions with GPCRs.

Biased signaling of the proton-sensing receptor OGR1 by benzodiazepines.

GPCRs have diverse signaling capabilities, based on their ability to assume various conformations. Moreover, it is now appreciated that certain ligands can promote distinct receptor conformations and thereby bias signaling toward a specific pathway to differentially affect cell function. The recently deorphanized G protein-coupled receptor OGR1 [ovarian cancer G protein-coupled receptor 1 ( GPR68)] exhibits diverse signaling events when stimulated by reductions in extracellular pH. We recently demonstrated airway smooth muscle cells transduce multiple signaling events, reflecting a diverse capacity to couple to multiple G proteins. Moreover, we recently discovered that the benzodiazepine lorazepam, more commonly recognized as an agonist of the γ-aminobutyric acid A (GABAA) receptor, can function as an allosteric modulator of OGR1 and, similarly, can promote multiple signaling events. In this study, we demonstrated that different benzodiazepines exhibit a range of biases for OGR1, with sulazepam selectively activating the canonical Gs of the G protein signaling pathway, in heterologous expression systems, as well as in several primary cell types. These findings highlight the potential power of biased ligand pharmacology for manipulating receptor signaling qualitatively, to preferentially activate pathways that are therapeutically beneficial.-Pera, T., Deshpande, D. A., Ippolito, M., Wang, B., Gavrila, A., Michael, J. V., Nayak, A. P., Tompkins, E., Farrell, E., Kroeze, W. K., Roth, B. L., Panettieri, R. A. Jr Benovic, J. L., An, S. S., Dulin, N. O., Penn, R. B. Biased signaling of the proton-sensing receptor OGR1 by benzodiazepines.

Knockdown siRNA Targeting GPR55 Reveals Significant Differences Between the Anti-inflammatory Actions of KLS-13019 and Cannabidiol.

KLS-13019 was reported previously to reverse paclitaxel-induced mechanical allodynia in a mouse model of chemotherapy-induced peripheral neuropathy (CIPN). Recent studies demonstrated that paclitaxel-induced increases in inflammatory markers (GPR55, NLRP3, and IL-1ß) of dorsal root ganglion (DRG) cultures were shown to be reversed by KLS-13019 treatment. The mechanism of action for KLS-13019-mediated reversal of paclitaxel-induced neuroinflammation now has been explored using GPR55 siRNA. Pre-treatment of DRG cultures with GPR55 siRNA produced a 21% decrease of immunoreactive (IR) area for GPR55 in cell bodies and a 59% decrease in neuritic IR area, as determined by high-content imaging. Using a 24-h reversal treatment paradigm, paclitaxel-induced increases in the inflammatory markers were reversed back to control levels after KLS-3019 treatment. Decreases in these inflammatory markers produced by KLS-13019 were significantly attenuated by GPR55 siRNA co-treatment, with mean IR area responses being attenuated by 56% in neurites and 53% in cell bodies. These data indicate that the percentage decreases in siRNA-mediated attenuation of KLS-13019-related efficacy on the inflammatory markers were similar to the percentage knockdown observed for neuritic GPR55 IR area. Similar studies conducted with cannabidiol (CBD), the parent compound of KLS-13019, produced low efficacy (25%) reversal of all inflammatory markers that were poorly attenuated (29%) by GPR55 siRNA. CBD was shown previously to be ineffective in reversing paclitaxel-induced mechanical allodynia. The present studies indicated significant differences between the anti-inflammatory properties of KLS-13019 and CBD which may play a role in their observed differences in the reversibility of mechanical allodynia in a mouse model of CIPN.

Knockdown siRNA targeting GPR55 reveals significant differences between the anti-inflammatory actions of KLS-13019 and cannabidiol.

KLS-13019 was reported previously to reverse paclitaxel-induced mechanical allodynia in a mouse model of chemotherapy-induced peripheral neuropathy (CIPN). Recent studies demonstrated that paclitaxel-induced increases in inflammatory markers (GPR55, NLRP3 and IL-1b) of dorsal root ganglion (DRG) cultures were shown to be reversed by KLS-13019 treatment. The mechanism of action for KLS-13019-mediated reversal of paclitaxel-induced neuroinflammation now has been explored using GPR55 siRNA. Pretreatment of DRG cultures with GPR55 siRNA produced a 21% decrease of immunoreactive (IR) area for GPR55 in cell bodies and a 59% decrease in neuritic IR area, as determined by high content imaging. Using a 24-hour reversal treatment paradigm, paclitaxel-induced increases in the inflammatory markers were reversed back to control levels after KLS-3019 treatment. Decreases in these inflammatory markers produced by KLS-13019 were significantly attenuated by GPR55 siRNA co-treatment, with mean IR area responses being attenuated by 56% in neurites and 53% in cell bodies. These data indicate that the percentage decreases in siRNA-mediated attenuation of KLS-13019-related efficacy on the inflammatory markers were similar to the percentage knockdown observed for neuritic GPR55 IR area. Similar studies conducted with cannabidiol (CBD), the parent compound of KLS-13019, produced low efficacy (25%) reversal of all inflammatory markers that were poorly attenuated (29%) by GPR55 siRNA. CBD was shown previously to be ineffective in reversing paclitaxel-induced mechanical allodynia. The present studies indicated significant differences between the anti-inflammatory properties of KLS-13019 and CBD which may play a role in their observed differences in the reversibility of mechanical allodynia in a mouse model of CIPN.

ß2 -Adrenoceptor agonist profiling reveals biased signalling phenotypes for the ß2 -adrenoceptor with possible implications for the treatment of asthma.

BACKGROUND AND PURPOSE: ß-Adrenoceptor agonists relieve airflow obstruction by activating ß2 -adrenoceptors, which are G protein-coupled receptors (GPCRs) expressed on human airway smooth muscle (HASM) cells. The currently available ß-adrenoceptor agonists are balanced agonists, however, and signal through both the stimulatory G protein (Gs )- and ß-arrestin-mediated pathways. While Gs signalling is beneficial and promotes HASM relaxation, ß-arrestin activation is associated with reduced Gs efficacy. In this context, biased ligands that selectively promote ß2 -adrenoceptor coupling to Gs signalling represent a promising strategy to treat asthma. Here, we examined several ß-adrenoceptor agonists to identify Gs -biased ligands devoid of ß-arrestin-mediated effects. EXPERIMENTAL APPROACH: Gs -biased ligands for the ß2 -adrenoceptor were identified by high-throughput screening and then evaluated for Gs interaction, Gi interaction, cAMP production, ß-arrestin interaction, GPCR kinase (GRK) phosphorylation of the receptor, receptor trafficking, ERK activation, and functional desensitization of the ß2 -adrenoceptor. KEY RESULTS: We identified ractopamine, dobutamine, and higenamine as Gs -biased agonists that activate the Gs /cAMP pathway upon ß2 -adrenoceptor stimulation while showing minimal Gi or ß-arrestin interaction. Furthermore, these compounds did not induce any receptor trafficking and had reduced GRK5-mediated phosphorylation of the ß2 -adrenoceptor. Finally, we observed minimal physiological desensitization of the ß2 -adrenoceptor in primary HASM cells upon treatment with biased agonists. CONCLUSION AND IMPLICATIONS: Our work demonstrates that Gs -biased signalling through the ß2 -adrenoceptor may prove to be an effective strategy to promote HASM relaxation in the treatment of asthma. Such biased compounds may also be useful in identifying the molecular mechanisms that determine biased signalling and in design of safer drugs.

Biased agonism at ß-adrenergic receptors.

The ß-adrenergic receptors (ßARs) include three subtypes, ß1, ß2 and ß3. These receptors are widely expressed and regulate numerous physiological processes including cardiovascular and metabolic functions and airway tone. The ßARs are also important targets in the treatment of many diseases including hypertension, heart failure and asthma. In some cases, the use of current ßAR ligands to treat a disease is suboptimal and can lead to severe side effects. One strategy to potentially improve such treatments is the development of biased agonists that selectively regulate a subset of ßAR signaling pathways and responses. Here we discuss the compounds identified to date that preferentially activate a Gs- or ß-arrestin-mediated signaling pathway through ßARs. Mechanistic insight on how these compounds bias signaling sheds light on the potential development of even more selective compounds that should have increased utility in treating disease.

Geographic variation in diapause incidence, life-history traits, and climatic adaptation in Drosophila melanogaster.

In Drosophila melanogaster, exposure of females to low temperature and shortened photoperiod can induce the expression of reproductive quiescence or diapause. Diapause expression is highly variable within and among natural populations and has significant effects on life-history profiles, including patterns of longevity, fecundity, and stress resistance. We hypothesized that if diapause expression is associated with overwintering mechanisms and adaptation to temperate environments, the frequency of diapause incidence would exhibit a latitudinal cline among natural populations. Because stress resistance and reproductive traits are also clinal in this species, we also examined how patterns of fecundity and longevity varied with geography and how stress resistance and associated traits differed constitutively between diapause and nondiapause lines. Diapause incidence was shown to vary predictably with latitude, ranging from 35% to 90% among natural populations in the eastern United States Survivorship under starvation stress differed between diapause and nondiapause lines; diapause phenotypes were also distinct for total body triglyceride content and the developmental distribution of oocytes in the ovary following stress exposure. Patterns of longevity, fecundity, and ovariole number also varied with geography. The data suggest that, for North American populations, diapause expression is functionally associated with overwintering mechanisms and may be an integral life-history component in natural populations.