A Ganoderma-Derived Compound Exerts Inhibitory Effect Through Formyl Peptide Receptor 2

ORIGINAL RESEARCH published: 24 March 2020 doi: 10.3389/fphar.2020.00337 A Ganoderma-Derived Compound Exerts Inhibitory Effect Through Formyl Peptide Receptor 2 Huirong Wang 1,2, Xingrong Peng 3, Yunjun Ge 1, Shuo Zhang 4, Zhenyi Wang 5, Yu Fan 1, Wei Huang 2, Minghua Qiu 3* and Richard D. Ye 1,6* 1 Institute of Chinese Medical Sciences, State Key Laboratory of Quality Research in Chinese Medicine, University of Macau, Macau, Macau, 2 Department of Biology, Southern University of Science and Technology, Shenzhen, China, 3 Kunming Institute of Botany, Chinese Academy of Science, Kunming, China, 4 School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China, 5 Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China, Key Laboratory of Structural Biology, Chinese Academy of Sciences, Hefei, China, 6 Kobilka Institute of Innovative Drug Discovery, School of Life and Health Sciences, The Chinese University of Hong Kong, Shenzhen, China Edited by: Anna Karolina Kiss, Medical University of Warsaw, Poland Reviewed by: Claes Dahlgren, University of Gothenburg, Sweden Igor Schepetkin, Montana State University, United States Vincenzo Brancaleone, University of Basilicata, Italy *Correspondence: Minghua Qiu Richard D. Ye Specialty section: This article was submitted to Ethnopharmacology, a section of the journal Frontiers in Pharmacology Received: 07 December 2019 Accepted: 06 March 2020 Published: 24 March 2020 Citation: Wang H, Peng X, Ge Y, Zhang S, Wang Z, Fan Y, Huang W, Qiu M and Ye RD (2020) A Ganoderma-Derived Compound Exerts Inhibitory Effect Through Formyl Peptide Receptor 2. Front. Pharmacol. 11:337. doi: 10.3389/fphar.2020.00337 Formyl peptide receptors (FPRs) are G protein-coupled receptors (GPCRs) widely expressed in neutrophils and other phagocytes. FPRs play important roles in host defense, inflammation, and the pathogenesis of infectious and inflammatory diseases. Because of these functions, FPRs are potential targets for anti-inflammatory therapies. In order to search for potentially novel anti-inflammatory agents, we examined Ganoderma (Lingzhi), a Chinese medicinal herbs known for its anti-inflammatory effects, and found that compound 18 (C18) derived from Ganoderma cochlear could limit the inflammatory response through FPR-related signaling pathways. Further studies showed that C18 could bind to FPR2 and induce conformation change of the receptor that differed from the conformational change induced by the pan-agonist, WKYMVm. C18 inhibited at the receptor level and blocked WKYMVm signaling through FPR2, resulting in reduced superoxide production and compromised cell chemotaxis. These results identified for the first time that a Ganoderma-derived component with inhibitory effects that acts through a G protein-coupled receptor FPR2. Considering its less than optimal IC50 value, further optimization of C18 would be necessary for future applications. Keywords: formyl peptide receptors, Ganoderma, anti-inflammatory, chemotaxis, superoxide, fluorescence resonance energy transfer INTRODUCTION Formyl peptide receptors (FPRs) are cell surface pattern recognition receptors (PRRs) that belong to the evolutionarily conserved family of G protein-coupled receptors (GPCRs). They are widely expressed in circulating blood granulocytes (de Paulis et al., 2004; Migeotte et al., 2006; Svensson et al., 2007), especially neutrophils, the most abundant type of circulating leukocytes (Dorward et al., 2015). Although three Abbreviations: FPRs, Formyl peptide receptors; GPCRs, G protein-coupled receptors; fMLF, N-Formyl-Met-Leu-Phe; PMA, phorbol-12-myristate-13-acetate; C5a, complement component 5a; MFI, mean fluorescent intensity; FRET, FLAsH-based Fluorescence resonance energy transfer; dHL60, differential HL-60; PKC, protein kinase C; DAG, diacylglycerol; BSA, Bovine Serum Albumin; ECFP, Enhanced Cyan Fluorescent Protein; GMs, Ganoderma meroterpenoids. Frontiers in Pharmacology | www.frontiersin.org 1 March 2020 | Volume 11 | Article 337 Wang et al. Ganoderma-Derived Inhibitory Compound antagonists, based on its long-term medical practice (Yuan et al., 2016). In this study, Ganoderma (Lingzhi) was chosen because it has been used extensively in Asian countries for more than 2,000 years, due to its various pharmacological effects, including immunomodulation, antibacterial, anticancer, antioxidant, and antiviral activities (Gao et al., 2003; Sliva, 2004; Yuen and Gohel, 2005; Joo et al., 2008; Sanodiya et al., 2009; Ma et al., 2011; Xu et al., 2011). Ganoderma is rich in active compounds, including triterpenoids, fatty acids, polysaccharides, peptides, and other chemicals (Sanodiya et al., 2009; Peng and Qiu, 2018), and that has led to the possibility of identifying FPR agonists and antagnosits. In this study, 34 Ganoderma-derived compounds that were available in our collection were subjected to initial screening using FPR2-dependent superoxide generation assay and degranulation assay. Among these triterpenoids and meroterpenoids, C18 was identified to have strong inhibitory activities. C18 and 5 other structurally similar compounds (Figure 1), all Ganoderma meroterpenoids (GMs) (Peng and Qiu, 2018), were selected for further studies. (Figure 1). C18, was found to display significant inhibition in several FPR-mediated functional assays, but had no effect on C5a receptor and PKC-mediated signaling pathways. To assess the structure-activity relationship, FLAsH-based fluorescence resonance energy transfer (FRET) detection and molecular docking analysis were performed. The results demonstrated that C18 could inhibit FPR-mediated pro-inflammatory response by targeting FPR2. In short, our work demonstrated the in vitro inhibitory effects of a novel Ganoderma-derived compound through FPR2, further revealing its detailed mechanism with competitive binding assay and FRET detection assay, and finally show its interaction with FPR2 by molecular docking analysis. These results suggest that C18 may be a naturally active component and exert its inhibitory effects through FPR2. subtypes of FPRs have been identified in human (FPR1, FPR2, and FPR3), only FPR1 and FPR2 are expressed on neutrophils and play a vital role in innate immunity (Murphy et al., 1992; Ye et al., 2009). The primary role of the FPRs is to recognize N-formylated peptides of protein fragments from bacteria and mitochondria, and induce proinflammatory responses such as chemotaxis, superoxide generation, and degranulation. These bactericidal functions contribute to the clearance of invading microbes and removal of tissue debris (Ye et al., 2009). In addition, FPRs also play important roles in various inflammatory diseases (Weiss and Kretschmer, 2018). For example, inhibition of mouse FPRs attenuates obesity-linked inflammation and leads to increased glucose tolerance and insulin levels in obese mice (Claria et al., 2012; Wollam et al., 2019). Activat (...truncated)