Ligand recognition and allosteric modulation of the human MRGPRX1 receptor

Yongfeng Liu # 1 2Can Cao # 1Xi-Ping Huang 1 2Ryan H Gumpper 1Moira M Rachman 3Sheng-Luen Shih 1 2Brian E Krumm 1Shicheng Zhang 1Brian K Shoichet 3Jonathan F Fay 4 5Bryan L Roth 6 7 8


The human MAS-related G protein-coupled receptor X1 (MRGPRX1) is preferentially expressed in the small-diameter primary sensory neurons and involved in the mediation of nociception and pruritus. Central activation of MRGPRX1 by the endogenous opioid peptide fragment BAM8-22 and its positive allosteric modulator ML382 has been shown to effectively inhibit persistent pain, making MRGPRX1 a promising target for non-opioid pain treatment. However, the activation mechanism of MRGPRX1 is still largely unknown. Here we report three high-resolution cryogenic electron microscopy structures of MRGPRX1-Gαq in complex with BAM8-22 alone, with BAM8-22 and ML382 simultaneously as well as with a synthetic agonist compound-16. These structures reveal the agonist binding mode for MRGPRX1 and illuminate the structural requirements for positive allosteric modulation. Collectively, our findings provide a molecular understanding of the activation and allosteric modulation of the MRGPRX1 receptor, which could facilitate the structure-based design of non-opioid pain-relieving drugs.

Journal Club – 2023.01.27

Ursolic acid downregulates thymic stromal lymphopoietin through the blockade of intracellular calcium/caspase‐1/NF‐κB signaling cascade in HMC‐1 cells


1department of Pharmacology, college of Korean Medicine, 2center for converging Humanities, Kyung Hee University, Seoul 02447; 3department of Food Science and Technology and Research Institute for Basic Science, Hoseo University, Asan, chungnam 31499, Republic of Korea

Received december 14, 2018; Accepted March 20, 2019 dOI: 10.3892/ijmm.2019.4144

Thymic stromal lymphopoietin (TSLP) plays an important role in allergic disorders, including atopic dermatitis and asthma. Ursolic acid (UA) has various pharmacological properties, such as antioxidant, anti‐inflammatory and anti- cancer. However, the effect of UA on TSLP regulation has not been fully elucidated. The aim of the present study was to analyze how UA regulates the production of TSLP in the human mast cell line HMc-1. Enzyme-linked immunosorbent assay, quantitative polymerase chain reaction analysis, western blotting, caspase-1 assay and fluorescent measurements of intracellular calcium levels were conducted to analyze the regulatory effects of UA. The results revealed that UA inhib- ited TSLP production and mRNA expression. In addition, UA reduced the activation of nuclear factor-κB and degradation of IκBα. caspase-1 activity was increased by exposure to phorbol myristate acetate plus calcium ionophore, whereas it was reduced by UA. Finally, UA treatment prevented an increase in intracellular calcium levels. These results indicated that UA may be a useful agent for the treatment and/or prevention of atopic and inflammatory diseases, and its effects are likely mediated by TSLP downregulation.

Berbamine Reduces Chloroquine-Induced Itch in Mice through Inhibition of MrgprX1

Kunhi Ryu Yunkyung Heo Yechan Lee Hyejin Jeon Wan Namkung 


Chloroquine (CQ) is an antimalaria drug that has been widely used for decades. However, CQ-induced pruritus remains one of the major obstacles in CQ treatment for uncomplicated malaria. Recent studies have revealed that MrgprX1 plays an essential role in CQ-induced itch. To date, a few MrgprX1 antagonists have been discovered, but they are clinically unavailable or lack selectivity. Here, a cell-based high-throughput screening was performed to identify novel antagonists of MrgprX1, and the screening of 2543 compounds revealed two novel MrgprX1 inhibitors, berbamine and closantel. Notably, berbamine potently inhibited CQ-mediated MrgprX1 activation (IC50 = 1.6 μM) but did not alter the activity of other pruritogenic GPCRs. In addition, berbamine suppressed the CQ-mediated phosphorylation of ERK1/2. Interestingly, CQ-induced pruritus was significantly reduced by berbamine in a dose-dependent manner, but berbamine had no effect on histamine-induced, protease-activated receptors 2-activating peptide-induced, and deoxycholic acid-induced itch in mice. These results suggest that berbamine is a novel, potent, and selective antagonist of MrgprX1 and may be a potential drug candidate for the development of therapeutic agents to treat CQ-induced pruritus.

Keywords: Chloroquine; MrgprA3; MrgprX1; antagonist; berbamine; itch.

Presenter: Hye In Kim

Scratch-AID, a deep learning basedsystem for automatic detection of mousescratching behavior with high accuracy

Department of Neuroscience, Perelman School of Medicine, University of Pennsylvania, Philadelphia, United States; 2
Graduate Group in Biostatistics, University of California Davis, Davis, United States; 3 Howard Hughes Medical Institute,
Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Harvard Medical School, Boston, United States; 4 Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, United States; 5 Department of Molecular and Cellular Biology, University of California Davis, Davis,
United States; 6 Department of Mathematics, University of California Davis, Davis, United States


Mice are the most commonly used model animals for itch research and for development of anti-itch drugs. Most laboratories manually quantify mouse scratching behavior to assess itch intensity. This process is labor-intensive and limits large-scale genetic or drug screenings. In this study, we developed a new system, Scratch-AID (Automatic Itch Detection), which could automatically identify and quantify mouse scratching behavior with high accuracy. Our system included a custom-designed videotaping box to ensure high-quality and replicable mouse behavior recording and a convolutional recurrent neural network trained with frame-labeled mouse scratching behavior videos, induced by nape injection of chloroquine. The best trained network achieved 97.6% recall and 96.9% precision on previously unseen test videos. Remarkably, Scratch-AID could reliably identify scratching behavior in other major mouse itch models, including the acute cheek model, the histaminergic model, and a chronic itch model. Moreover, our system detected significant differences in scratching behavior between control and mice treated with an anti-itch drug. Taken together, we have established a novel deep learning-based system that could replace manual quantification for mouse scratching behavior in different itch models and for drug screening.

Presenter: Gi Baek Lee

Sodium entry through endothelial store-operated calcium entry channels: regulation by Orai1

Ningyong Xu, Donna L. Cioffi, Mikhail Alexeyev, Thomas C. Rich, and Troy Stevens


Orai1 interacts with transient receptor potential protein of the canonical subfamily (TRPC4) and contributes to calcium selectivity of the endothelial cell store-operated calcium entry current (ISOC). Orai1 silencing increases sodium permeability and decreases membraneassociated calcium, although it is not known whether Orai1 is an important determinant of cytosolic sodium transitions. We test the hypothesis that, upon activation of store-operated calcium entry channels, Orai1 is a critical determinant of cytosolic sodium transitions. Activation of store-operated calcium entry channels transiently increased cytosolic calcium and sodium, characteristic of release from an intracellular store. The sodium response occurred more abruptly and returned to baseline more rapidly than did the transient calcium
rise. Extracellular choline substitution for sodium did not inhibit the response, although 2-aminoethoxydiphenyl borate and YM-58483 reduced it by 50%. After this transient response, cytosolic sodium continued to increase due to influx through activated store-operated calcium entry channels. The magnitude of this sustained increase in cytosolic sodium was greater when experiments were conducted in low extracellular calcium and when Orai1 expression was silenced; these two interventions were not additive, suggesting a common mechanism. 2-Aminoethoxydiphenyl borate and YM-58483 inhibited the sustained increase in cytosolic sodium, only in the presence of Orai1. These studies demonstrate that sodium permeates activated store-operated calcium entry channels, resulting in an increase in cytosolic sodium; the magnitude of this response is determined by Orai1.


Novel pro-resolving lipid mediator mimetic 3-oxa-PD1n-3 DPA reduces acute and chronic itch by modulating excitatory and inhibitory synaptic transmission and astroglial secretion of lipocalin-2 in mice

Furutani, Kenta; Chen, Ouyang; McGinnis, Aidan; Wang, Yuqing; Serhan, Charles N; Hansen, Trond Vidar; Ji, Ru-Rong Ji


Specialized pro-resolving mediators (SPMs) have demonstrated potent analgesic actions in animal models of pathological pain. The actions of SPMs in acute and chronic itch are currently unknown. Recently, n-3 docosapentaenoic acid (DPA) was found to be a substrate for the biosynthesis of several novel families of SPMs; 3-oxa-PD1n-3 DPA (3-oxa-PD1) is an oxidation-resistant metabolic stable analogue of the n-3 DPA-derived protectin D1 (PD1). Herein, we demonstrate that 3-oxa-PD1 effectively reduces both acute and chronic itch in mouse models. Intrathecal injection of 3-oxa-PD1 (100 ng) reduced acute itch induced by either histamine, chloroquine, or morphine. Furthermore, intrathecal 3-oxa-PD1 effectively reduced chronic itch, induced by cutaneous T cell lymphoma (CTCL), allergic contact dermatitis with dinitrofluorobenzene, and psoriasis by imiquimod. Intratumoral injection of 3-oxa-PD1 also suppressed CTCL-induced chronic itch. Strikingly, this anti-pruritic effect lasted for several weeks after 1-week of intrathecal 3-oxa-PD1 treatment. Whole-cell recordings revealed significant increase in excitatory postsynaptic currents in spinal dorsal horn (SDH) neurons of CTCL mice, but this increase was blocked by 3-oxa-PD1. 3-oxa-PD1 further increased inhibitory postsynaptic currents in SDH neurons of CTCL mice. CTCL increased the spinal levels of lipocalin-2 (LCN2), an itch mediator produced by astrocytes. 3-oxa-PD1 suppressed LCN2 production in CTCL mice and LCN2 secretion in astrocytes. Finally, CTCL-induced anxiety was alleviated by intrathecal 3-oxa-PD1. Our findings suggest that 3-oxa-PD1 potently inhibits acute and chronic itch via regulation of excitatory/inhibitory synaptic transmission and astroglial LCN2 production. Therefore, stable SPM analogs such as 3-oxa-PD1 could be useful to treat pruritus associated with different skin injuries.

Journal Club – 2022.11.25

Transient stimulation of TRPV4-expressing keratinocytes promotes hair follicle regeneration in mice

Pu Yang 1Ping Lu 1 2Jialie Luo 1Lixia Du 1Jing Feng 1Tao Cai 1 3Yi Yuan 1Hunter Cheng 1Hongzhen Hu 1

1Department of Anesthesiology, The Center for the Study of Itch and Sensory Disorders, Washington University School of Medicine, St. Louis, Missouri, USA

2Experimental Research Center, Dermatology Hospital, Southern Medical University, Guangzhou, China

3Department of Dermatology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China

Background and Purpose: Hair follicle telogen to anagen transition results in a break in cellular quiescence of the hair follicle stem cells, which subsequently promotes hair follicle regeneration. Many critical molecules and signalling pathways are involved in hair follicle cycle progression. Transient receptor potential vanilloid 4 (TRPV4) is a polymodal sensory transducer that regulates various cutaneous functions under both normal and disease conditions. However, the role of TRPV4 in hair follicle regenera- tion in vivo remains incompletely understood.

Experimental Approach: Using adult C57BL/6J mice, keratinocyte (K14Cre; Trpv4f/f) and macrophage (Cx3cr1Cre; Trpv4f/f) Trpv4 conditional knockout (cKO) mice, Trpv4−/− mice, we investigated the effect of a single intradermal injection of GSK1016790A, a potent and selective small molecule TRPV4 activator, on hair follicle regenera- tion. Chemical cues and signal molecules involved in hair follicle cycle progression were measured by immunofluorescence staining, quantitative RT-PCR and western blotting.

Key Results: Here, we show that a single intradermal injection of GSK1016790A is sufficient to induce telogen to anagen transition and hair follicle regeneration in mice by increasing the expression of the anagen-promoting growth factors and down- regulating the expression of growth factors that inhibit anagen. The action of GSK1016790A relies largely on the function of TRPV4 in skin and involves activation of downstream ERK signalling.

Conclusion and Implications: Our results suggest that transient chemical activation of TRPV4 in the skin induces hair follicle regeneration in mice, which might provide an effective therapeutic strategy for the treatment of hair loss and alopecia.

Journal Club – 2022.11.18

Structural basis of TRPV3 inhibition by an antagonist


The TRPV3 channel plays vital roles in skin physiology. Dysfunction of TRPV3 causes skin diseases, including Olmsted syndrome. However, the lack of potent and selective inhibitors impedes the validation of TRPV3 as a therapeutic target. In this study, we identifed Trpvicin as a potent and subtype-selective inhibitor of TRPV3. Trpvicin exhibits pharmacological potential in the inhibition of itch and hair loss in mouse models. Cryogenic electron microscopy structures of TRPV3 and the pathogenic G573S mutant complexed with Trpvicin reveal detailed ligand-binding sites, suggesting that Trpvicin inhibits the TRPV3 channel by stabilizing it in a closed state. Our G573S mutant structures demonstrate that the mutation causes a dilated pore, generating constitutive opening activity. Trpvicin accesses additional binding sites inside the central cavity of the G573S mutant to remodel the channel symmetry and block the channel. Together, our results provide mechanistic insights into the inhibition of TRPV3 by Trpvicin and support TRPV3-related drug development.

Presenter: Gi Baek Lee

Journal Club – 2022.11.11

Selective and direct inhibition of TRPC3 channels underlies biological activities of a pyrazole compound


Canonical transient receptor potential (TRPC) channels control influxes of Ca2 and other cations that induce diverse cellular processes upon stimulation of plasma membrane receptors coupled to phospholipase C (PLC). Invention of subtype-specific inhibitors for TRPCs is crucial for distinction of respective TRPC channels that play particular physiological roles in native systems. Here, we identify a pyrazole compound (Pyr3), which selectively inhibits TRPC3 channels. Structure-function relationship studies of pyrazole compounds showed that the trichloroacrylic amide group is important for the TRPC3 selectivity of Pyr3. Electrophysiological and photoaffinity labeling experiments reveal a direct action of Pyr3 on the TRPC3 protein. In DT40 B lymphocytes, Pyr3 potently eliminated the Ca2 influx-dependent PLC translocation to the plasma membrane and late oscillatory phase of B cell receptorinduced Ca2 response. Moreover, Pyr3 attenuated activation of nuclear factor of activated T cells, a Ca2-dependent transcription factor, and hypertrophic growth in rat neonatal cardiomyocytes, and in vivo pressure overload-induced cardiac hypertrophy in mice. These findings on important roles of native TRPC3 channels are strikingly consistent with previous genetic studies. Thus, the TRPC3- selective inhibitor Pyr3 is a powerful tool to study in vivo function of TRPC3, suggesting a pharmaceutical potential of Pyr3 in treatments of TRPC3-related diseases such as cardiac hypertrophy.

Keywords: Ca2+ signaling, pyrazole compounds, TRPC channels, TRPC3


μ-Opioid receptors in primary sensory neurons are essential for opioid analgesic effect on acute and inflammatory pain and opioid-induced hyperalgesia

Jie Sun Shao-Rui Chen Hong Chen Hui-Lin Pan 


Key points: μ-Opioid receptors (MORs) are expressed peripherally and centrally, but the loci of MORs responsible for clinically relevant opioid analgesia are uncertain. Crossing Oprm1flox/floxand AdvillinCre/+ mice completely ablates MORs in dorsal root ganglion neurons and reduces the MOR expression level in the spinal cord. Presynaptic MORs expressed at primary afferent central terminals are essential for synaptic inhibition and potentiation of sensory input by opioids. MOR ablation in primary sensory neurons diminishes analgesic effects produced by systemic and intrathecal opioid agonists and abolishes chronic opioid treatment-induced hyperalgesia. These findings demonstrate a critical role of MORs expressed in primary sensory neurons in opioid analgesia and suggest new strategies to increase the efficacy and reduce adverse effects of opioids.

Abstract: The pain and analgesic systems are complex, and the actions of systemically administered opioids may be mediated by simultaneous activation of μ-opioid receptors (MORs, encoded by the Oprm1 gene) at multiple, interacting sites. The loci of MORs and circuits responsible for systemic opioid-induced analgesia and hyperalgesia remain unclear. Previous studies using mice in which MORs are removed from Nav1.8- or TRPV1-expressing neurons provided only an incomplete and erroneous view about the role of peripheral MORs in opioid actions in vivo. In the present study, we determined the specific role of MORs expressed in primary sensory neurons in the analgesic and hyperalgesic effects produced by systemic opioid administration. We generated Oprm1 conditional knockout (Oprm1-cKO) mice in which MOR expression is completely deleted from dorsal root ganglion neurons and substantially reduced in the spinal cord, which was confirmed by immunoblotting and immunocytochemical labelling. Both opioid-induced inhibition and potentiation of primary sensory input were abrogated in Oprm1-cKO mice. Remarkably, systemically administered morphine potently inhibited acute thermal and mechanical nociception and persistent inflammatory pain in control mice but had little effect in Oprm1-cKO mice. The analgesic effect of intrathecally administered morphine was also profoundly reduced in Oprm1-cKO mice. Additionally, chronic morphine treatment-induced hyperalgesia was absent in Oprm1-cKO mice. Our findings directly challenge the notion that clinically relevant opioid analgesia is mediated mostly by centrally expressed MORs. MORs in primary sensory neurons, particularly those expressed presynaptically at the first sensory synapse in the spinal cord, are crucial for both opioid analgesia and opioid-induced hyperalgesia.

Keywords: TRPV1; fentanyl; opiate; opioid analgesic tolerance; presynaptic inhibition; synaptic transmission.