J Med Chem. 2022 Feb 24;65(4):3218-3228. doi: 10.1021/acs.jmedchem.1c01709.Epub 2022 Feb 4.

Thieno[2,3- d]pyrimidine-Based Positive Allosteric Modulators of Human Mas-Related G Protein-Coupled Receptor X1 (MRGPRX1)

Ilyas Berhane, Niyada Hin, Ajit G Thomas, Qian Huang, Chi Zhang, Vijayabhaskar Veeravalli, Ying Wu, Justin Ng, Jesse Alt, Camilo Rojas, Hiroe Hihara ¹, Mika Aoki ¹, Kyoko Yoshizawa ¹, Tomoki Nishioka ¹, Shuichi Suzuki ¹, Shao-Qiu He, Qi Peng, Yun Guan, Xinzhong Dong, Srinivasa N Raja, Barbara S Slusher, Rana Rais, Takashi Tsukamoto

• PMID: 35119273
• DOI: 10.1021/acs.jmedchem.1c01709

Abstract

Mas-related G protein-coupled receptor X1 (MRGPRX1) is a human sensory neuron-specific receptor and potential target for the treatment of pain. Positive allosteric modulators (PAMs) of MRGPRX1 have the potential to preferentially activate the receptors at the central terminals of primary sensory neurons and minimize itch side effects caused by peripheral activation. Using a high-throughput screening (HTS) hit, a series of thieno[2,3-d]pyrimidine-based molecules were synthesized and evaluated as human MRGPRX1 PAMs in HEK293 cells stably transfected with human MrgprX1 gene. An iterative process to improve potency and metabolic stability led to the discovery of orally available 6-(tert-butyl)-5-(3,4-dichlorophenyl)-4-(2-(trifluoromethoxy)phenoxy)thieno[2,3-d]pyrimidine (1t), which can be distributed to the spinal cord, the presumed site of action, following oral administration. In a neuropathic pain model induced by sciatic nerve chronic constriction injury (CCI), compound 1t(100 mg/kg, po) reduced behavioral heat hypersensitivity in humanized MRGPRX1 mice, demonstrating the therapeutic potential of MRGPRX1 PAMs in treating neuropathic pain.

Exploring neuronal mechanisms involved in the scratching behavior of a mouse model of allergic contact dermatitis by transcriptomics

Boyu Liu, Ruixiang Chen, Jie Wang, Yuanyuan Li, Chengyu Yin, Yan Tai, Huimin Nie, Danyi Zeng, Junfan Fang, Junying Du, Yi Liang, Xiaomei Shao, Jianqiao Fang , Boyi Liu 

Abstract

Background: Allergic contact dermatitis (ACD) is a common skin condition characterized by contact hypersensitivity to allergens, accompanied with skin inflammation and a mixed itch and pain sensation. The itch and pain dramatically affects patients’ quality of life. However, still little is known about the mechanisms triggering pain and itch sensations in ACD.

Methods: We established a mouse model of ACD by sensitization and repetitive challenge with the hapten oxazolone. Skin pathological analysis, transcriptome RNA sequencing (RNA-seq), qPCR, Ca²⁺ imaging, immunostaining, and behavioral assay were used for identifying gene expression changes in dorsal root ganglion innervating the inflamed skin of ACD model mice and for further functional validations.

Results: The model mice developed typical ACD symptoms, including skin dryness, erythema, excoriation, edema, epidermal hyperplasia, inflammatory cell infiltration, and scratching behavior, accompanied with development of eczematous lesions. Transcriptome RNA-seq revealed a number of differentially expressed genes (DEGs), including 1436-DEG mRNAs and 374-DEG-long noncoding RNAs (lncRNAs). We identified a number of DEGs specifically related to sensory neuron signal transduction, pain, itch, and neuroinflammation. Comparison of our dataset with another published dataset of atopic dermatitis mouse model identified a core set of genes in peripheral sensory neurons that are exclusively affected by local skin inflammation. We further found that the expression of the pain and itch receptor MrgprD was functionally upregulated in dorsal root ganglia (DRG) neurons innervating the inflamed skin of ACD model mice. MrgprD activation induced by its agonist β-alanine resulted in exaggerated scratching responses in ACD model mice compared with naïve mice.

Conclusions: We identified the molecular changes and cellular pathways in peripheral sensory ganglia during ACD that might participate in neurogenic inflammation, pain, and itch. We further revealed that the pain and itch receptor MrgprD is functionally upregulated in DRG neurons, which might contribute to peripheral pain and itch sensitization during ACD. Thus, targeting MrgprD may be an effective method for alleviating itch and pain in ACD.

Keywords: Allergic contact dermatitis; Itch; Pain; RNA-seq; Sensory neurons.

The online version contains supplementary material available at https://cmbl.biomedcentral.com/articles/10.1186/s11658-022-00316-w

Anoctamin 1/TMEM16A in pruritoceptors is essential for Mas-related G protein receptor-dependent itch

Kim, Hyesu^1,3,7; Kim, Hyungsup^1,7; Cho, Hawon²; Lee, Byeongjun¹; Lu, Huan-Jun¹; Kim, Kyungmin¹; Chung, Sooyoung¹; Shim, Won-Sik⁴; Shin, Young Kee³; Dong, Xinzhong⁵; Wood, John N⁶; Oh, Uhtaek^1,3,*Author InformationPAIN: February 08, 2022 – Volume – Issue –doi: 10.1097/j.pain.0000000000002611

Abstract

Itch is an unpleasant sensation that evokes a desire to scratch. Pathologic conditions such as allergy or atopic dermatitis produce severe itching sensation. Mas-related G protein receptors (Mrgprs) are receptors for many endogenous pruritogens. However, signaling pathways downstream to these receptors in dorsal root ganglion (DRG) neurons are not yet understood. We found that Anoctamin 1 (ANO1), a Ca²⁺-activated chloride channel, is a transduction channel mediating Mrgprs-dependent itch signals. Genetic ablation of Ano1 in DRG neurons displayed a significant reduction in scratching behaviors in response to acute and chronic Mrgprs-dependent itch models and the epidermal hyperplasia induced by dry skin. In-vivo Ca²⁺ imaging and electrophysiological recording revealed that chloroquine and other agonists of Mrgpr receptors excited DRG neurons via ANO1. More importantly, the overexpression of Ano1 in DRG neurons of Ano1-deficient mice rescued the impaired itching observed in Ano1-deficient mice. These results demonstrate that ANO1 mediates the Mrgprs-dependent itch signaling in pruriceptors and provides clues to treating pathologic itch syndromes.

Cell. 2021 Jul 8;184(14):3762-3773.e10. doi: 10.1016/j.cell.2021.05.017. Epub 2021 Jun 15.

Sneezing reflex is mediated by a peptidergic pathway from nose to brainstem

Fengxian Li ¹, Haowu Jiang ¹, Xiaolei Shen ¹, Weishan Yang ¹, Changxiong Guo ¹, Zhiyao Wang ¹, Maolei Xiao ¹, Lian Cui ², Wenqin Luo ², Brian S Kim ³, Zhoufeng Chen ⁴, Andrew J W Huang ⁵, Qin Liu ⁶

• PMID: 34133943
• DOI: 10.1016/j.cell.2021.05.017

Abstract

Sneezing is a vital respiratory reflex frequently associated with allergic rhinitis and viral respiratory infections. However, its neural circuit remains largely unknown. A sneeze-evoking region was discovered in both cat and human brainstems, corresponding anatomically to the central recipient zone of nasal sensory neurons. Therefore, we hypothesized that a neuronal population postsynaptic to nasal sensory neurons mediates sneezing in this region. By screening major presynaptic neurotransmitters/neuropeptides released by nasal sensory neurons, we found that neuromedin B (NMB) peptide is essential for signaling sneezing. Ablation of NMB-sensitive postsynaptic neurons in the sneeze-evoking region or deficiency in NMB receptor abolished the sneezing reflex. Remarkably, NMB-sensitive neurons further project to the caudal ventral respiratory group (cVRG). Chemical activation of NMB-sensitive neurons elicits action potentials in cVRG neurons and leads to sneezing behavior. Our study delineates a peptidergic pathway mediating sneezing, providing molecular insights into the sneezing reflex arc.

Keywords: caudal ventral respiratory group; nasal sensory neurons; neuropeptide; sneeze; sneeze-evoking region.

mMrgprA3/mMrgprC11/hMrgprX1: potential therapeutic targets for allergic contact dermatitis induced pruritus in mice and human

Fengxian Li , Changming Wang , Danyou Hu , Xinyu Zhang , Ran Shen , Yuan Zhou , Yan Yang , Chan Zhu , Zongxiang Tang , Guang Yu 

•Department of Anesthesiology, Zhujiang Hospital of Southern Medical University, Guangzhou, Guangdong, China •School of Medicine & Holistic Integrative Medicine, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China •Key Laboratory for Chinese Medicine of Prevention and Treatment in Neurological Diseases, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China

Abstract

Background: Although the Mas-related G-protein-coupled receptors (Mrgprs) play essential roles in itch detection, their contribution to allergic contact dermatitis (ACD) associated itch remains unclear.

Objectives: To investigate whether Mrgprs are involved in ACD and whether Mrgprs can be identified as potential therapeutic targets.

Methods: Mrgpr-clusterΔ^-/- mice and human MrgprX1 (hMrgprX1) transgenic mice were applied to evalute the function of Mrgprs in oxazolone-induced ACD.

Results: Utilizing ACD model, we find that Mrgpr-clusterΔ^-/- mice display significantly reduced pruritus. Among 12 Mrgprs deleted in Mrgpr-clusterΔ^-/- mice, the expression of MrgprC11 and MrgprA3 are significantly increased in ACD model, which also innervate the skin and spinal cord at higher-than-normal densities, the proportions of dorsal root ganglia neurons responding to bovine adrenal medulla peptide 8-22 and chloroquine are also remarkably increased in ACD model and result in enhancing itch behavior. To study the function of human Mrgprs in ACD-induced itch, we utilize hMrgprX1 transgenic mice, which rescues the severe itch defect of Mrgpr-clusterΔ^-/- mice in ACD model. Remarkably, pharmacological blockade of hMrgprX1 significantly attenuates ACD itch in hMrgprX1 transgenic mouse.

Conclusions: Our study provides the first evidence that Mrgprs are involved in ACD induced chronic itch, which provides new avenues for itch management in ACD. This article is protected by copyright. All rights reserved.

Keywords: Allergic contact dermatitis; Mrgprs; pruritus; sensory neurons.

MrgprB4 in trigeminal neurons expressing TRPA1 modulates unpleasant sensations

Shota Tobori a, 1, Haruka Hiyama a, 1, Takahito Miyake a, b, Yuichi Yano a, Kazuki Nagayasu a, Hisashi Shirakawa a, *, Takayuki Nakagawa c, Yasuo Mori d, Shuji Kaneko a

a Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan
b Department of Systems Biology, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-Shimoadachi-cho, Sakyo-ku, Kyoto 606- 8501, Japan

c Department of Clinical Pharmacology and Therapeutics, Kyoto University Hospital, 54 Shogoin -Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
d Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Building A4, Katsura Campus, Nishikyo-ku, Kyoto 615-8510, Japan

ABSTRACT

Gentle touch such as stroking of the skin produces a pleasant feeling, which is detected by a rare subset of sensory neurons that express Mas-related G protein-coupled receptor B4 (MrgprB4) in mice. We examined small populations of MrgprB4-positive neurons in the trigeminal ganglion and the dorsal root ganglion, and most of these were sensitive to transient receptor potential ankyrin 1 (TRPA1) agonist but not TRPV1, TRPM8, or TRPV4 agonists. Deficiency of MrgprB4 did not affect noxious pain or itch be- haviors in the hairless plantar and hairy cheek. Although behavior related to acetone-induced cold sensing in the hind paw was not changed, unpleasant sensory behaviors in response to acetone appli- cation or sucrose splash to the cheek were significantly enhanced in Mrgprb4-knockout mice as well as in TRPA1-knockout mice. These results suggest that MrgprB4 in the trigeminal neurons produces pleasant sensations in cooperation with TRPA1, rather than noxious or cold sensations. Pleasant sensa- tions may modulate unpleasant sensations on the cheek via MrgprB4.

© 2021 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).

MRGPR-mediated activation of local mast cells clears cutaneous bacterial infection and protects against reinfection

Mohammad Arifuzzaman, Yuvon R. Mobley, HAE WOONG CHOI,

PRADEEP BISTCRISTINA A. SALINASZACHARY D. BROWNSWAINE L. CHEN, HERMAN F. STAATS, SOMAN N. ABRAHAM 

Published in Science advances, 2 Jan 2019

Abstract

Mast cells (MCs) are strategically distributed at barrier sites and prestore various immunocyte-recruiting cytokines, making them ideal targets for selective activation to treat peripheral infections. Here, we report that topical treatment with mastoparan, a peptide MC activator (MCA), enhances clearance of Staphylococcus aureus from infected mouse skins and accelerates healing of dermonecrotic lesions. Mastoparan functions by activating connective tissue MCs (CTMCs) via the MRGPRX2 (Mas-related G protein-coupled receptor member X2) receptor. Peripheral CTMC activation, in turn, enhances recruitment of bacteria-clearing neutrophils and wound-healing CD301b⁺ dendritic cells. Consistent with MCs playing a master coordinating role, MC activation also augmented migration of various antigen-presenting dendritic cells to draining lymph nodes, leading to stronger protection against a second infection challenge. MCAs therefore orchestrate both the innate and adaptive immune arms, which could potentially be applied to combat peripheral infections by a broad range of pathogens.

ZBTB20 in nociceptive neurons of the trigeminal ganglia regulates pruritus

Xin Jia, Meng-Han Dai, An-Jing Ren, Ting-Ting Wang, Weiping J. Zhang and Ling Zhang

Published in :-Frontiers of Medicine (2021.Mar.04) Impact factor:-5.02

Abstract

Recent studies have shown that ZBTB20, a zinc-finger protein containing transcription factor, is highly expressed in small-diameter primary sensory neurons in mice, and modulates pain through regulating TRP channels. However, whether ZBTB20 regulates itch sensation has not been demonstrated. In this study, small-diameter primary sensory
neuron-specific ZBTB20 knockout (PN-ZB20KO) mice were used to investigate the role of ZBTB20 in the regulation of itch sensation. First, both histamine-dependent and non-histamine-dependent itch behaviors induced by injection of histamine and chloroquine (CQ) into the cheek were significantly diminished in PN-ZB20KO mice. Second, double immunohistochemistry showed that ZBTB20 was mainly expressed in CGRP-labeled small peptidergic neurons and was expressed at low levels in IB4-labeled small non-peptidergic and NF200-labeled large neurons in the trigeminal ganglia (TG). ZBTB20 was also expressed in most TRPV1+ and TRPA1+ neurons and to a lesser extent in TRPM8+ neurons in the TG. Furthermore, cheek injection of histamine and CQ enhanced the mRNA expression of TRPV1 and TRPA1 but not TRPM8 in the TG. Moreover, TRPV1 and TRPA1 knockout (KO) mice exhibited attenuation of itch behavior induced by histamine and CQ, respectively. Finally, silencing endogenous ZBTB20 with recombinant lentivirus expressing a short hairpin RNA against ZBTB20 (LV-shZBTB20) in TG neurons attenuated histamine- and non-histamine-induced itch and downregulated TRP channels in the TG. Our study suggests that ZBTB20 plays an important role in mediating itch in small primary sensory neurons.

Keywords: itch, TRPA1, TRPV1, ZBTB20, trigeminal ganglia, pain, pruritus

Histamine enhances ATP-induced itching and responsiveness to ATP in keratinocytes

Yoshihiro Inami, Miki Fukushima, Toshiaki Kume, Daisuke Uta

Abstract

Mechanical stimulation of cultured keratinocytes and a living epidermis increases intracellular calcium ion concentrations ([Ca²⁺]_i) in stimulated cells. This action propagates a Ca²⁺ wave to neighboring keratinocytes via ATP/P2Y₂ receptors. Recent behavioral, pharmacological studies revealed that exogenous ATP induces itching via P2X₃ receptors in mice. We previously showed that alloknesis occurs when an external stimulus is applied to the skin with increased epidermal histamine in the absence of spontaneous pruritus. Based on these results, we investigated the effects of histamine at a concentration that does not cause itching on ATP-induced itching. The mean number of scratching events induced by the mixture of ATP and histamine increased by 28% over the sum of that induced by histamine alone or ATP alone. A317491, a P2X₃ receptor antagonist, suppressed the mixture-induced scratching more often than the ATP-induced scratching. Next, we examined the ATP-induced [Ca²⁺]_i change before and after histamine stimulation using normal human epidermal keratinocytes. Some cells did not respond to ATP before histamine stimulation but responded to ATP afterward, the phenomenon suppressed by chlorpheniramine maleate. These findings suggest that histamine enhances ATP-induced itching and that a potential mechanism could involve increased responsiveness to ATP in keratinocytes.

Keywords: ATP, Histamine, Keratinocytes, Itch, Calcium

Tentonin 3/TMEM150C regulates glucose-stimulated insulin secretion in pancreatic b-cells

Jungwon Wee,1,4,5 Sungmin Pak,2,4,5 Tahnbee Kim,4 Gyu-Sang Hong,4 Ji Seon Lee,3 Jinyan Nan,3 Hyungsup Kim,4 Mi-Ock Lee,2 Kyong Soo Park,1,3,* and Uhtaek Oh1,4,6,*
1Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul National University, Seoul 03080, Korea

2College of Pharmacy, Seoul National University, Seoul 08826, Korea
3Department of Internal Medicine, College of Medicine, Seoul National University, Seoul 03080, Korea 4Brain Science Institute, Korea Institute of Science and Technology (KIST), Seoul 02792, Korea 5These authors contributed equally
6Lead contact
*Correspondence: kspark@snu.ac.kr (K.S.P.), utoh@kist.re.kr (U.O.)

SUMMARY

Glucose homeostasis is initially regulated by the pancreatic hormone insulin. Glucose-stimulated insulin secretion in b-cells is composed of two cellular mechanisms: a high glucose concentration not only depolar- izes the membrane potential of the b-cells by ATP-sensitive K+ channels but also induces cell inflation, which is sufficient to release insulin granules. However, the molecular identity of the stretch-activated cation chan- nel responsible for the latter pathway remains unknown. Here, we demonstrate that Tentonin 3/TMEM150C (TTN3), a mechanosensitive channel, contributes to glucose-stimulated insulin secretion by mediating cation influx. TTN3 is expressed specifically in b-cells and mediates cation currents to glucose and hypotonic stim- ulations. The glucose-induced depolarization, firing activity, and Ca2+ influx of b-cells were significantly lower in Ttn3/ mice. More importantly, Ttn3/ mice show impaired glucose tolerance with decreased insulin secretion in vivo. We propose that TTN3, as a stretch-activated cation channel, contributes to glucose-stim- ulated insulin secretion.