Thiazolidine Reacts with Thioreactive Biomolecules
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Free Radical Biology and Medicine 104 (2017) 272–279 Contents lists available at ScienceDirect Free Radical Biology and Medicine journal homepage: www.elsevier.com/locate/freeradbiomed Original article Thiazolidine reacts with thioreactive biomolecules MARK Deyuan Sua,b,1, Yin Niana,1, Fenglei Zhanga,1, Jinsheng Hua,b, Jianmin Cuia,c, Ming Zhoua,d, ⁎ ⁎ Jian Yanga,e, , Shu Wanga,b, a Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences/Key Laboratory of Bioactive Peptides of Yunnan Province, and Ion Channel Research and Drug Development Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China b Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming 650204, China c Department of Biomedical Engineering, Center for the Investigation of Membrane Excitability Disorders, Cardiac Bioelectricity and Arrhythmia Center, Washington University, St. Louis, MO 63130, USA d Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA e Department of Biological Sciences, Columbia University, New York, NY 10027, USA ARTICLE INFO ABSTRACT Keywords: The thiazolidine ring is a biologically active chemical structure and is associated with many pharmacological Thiazolidine activities. However, the biological molecules that can interact with the thiazolidine ring are not known. We show TRPA1 channel that thiazolidine causes sustained activation of the TRPA1 channel and chemically reacts with glutathione, and Glutathione the chemical reactivity of thiazolidine ring is required for TRPA1 activation. Reducing agents reverse thiazolidine-induced TRPA1 activation, and mutagenesis studies show that nucleophilic cysteine residues in TRPA1 are critical, suggesting an activation mechanism involving thioreactive chemical reactions. In vivo studies show that thiazolidine induces acute pain and inflammation in mouse and these responses are specifically dependent on TRPA1. These results indicate that thiazolidine compounds can chemically react with biological molecules containing nucleophilic cysteines, thereby exerting biological activities. 1. Introduction TRPA1 is a non-selective, Ca2+-permeable cation channel belonging to the transient receptor potential (TRP) ion channel superfamily [7–9]. Thiazolidine, a heterocyclic organic compound, has a 5-membered It is extensively expressed in the peripheral nervous system, and its ring-like structure with a thioether group and an amine group in the 1 activation causes pain, itch and inflammatory disorders [7–9]. TRPA1 is and 3 positions, respectively (Fig. 1A). Thiazolidine compounds are of a promiscuous chemical sensor that responds to many natural and great interest due to their wide range of pharmacological effects, synthetic irritants [7–9]. Some reactive compounds, such as allyl including antimicrobial, antihypertensive and antioxidant activities isothiocyanate (AITC), cinnamaldehyde and acrolein, activate TRPA1 [1]. For example, penicillin and pioglitazone are two well known drugs through covalent modification of nucleophilic residues in the N- which contain the thiazolidine ring. Thus, the thiazolidine ring is terminus of TRPA1 [7–9]. In addition to TRPA1, many cellular recognized as an important scaffold for drug development, and the molecules or their functional groups are nucleophilic; one of the most chemistry of thiazolidine has been extensively studied [1]. The important is glutathione (GSH) [10]. GSH is an abundant tripeptide in thiazolidine ring in different compounds can undergo ring-opening plants, animals and microorganisms. GSH comprises three amino acids reaction in which an electrophilic intermediate is formed [2–6]. It was (glutamate, cysteine and glycine), and the cysteine residue provides a speculated that the electrophilic intermediate generated by thiazolidine nucleophilic thiol group that is important for the detoxification of many ring opening in penicillin may inhibit bacterial enzymes by reacting potentially toxic electrophiles [11]. with nucleophilic amino acid side chains on the enzymes [2–4]. In this study we demonstrate that thiazolidine activates TRPA1 and However, to date, there is no evidence demonstrating that the chemically reacts with GSH. TRPA1 activation depends on the chemical thiazolidine ring itself can directly interact with any biological mole- reactivity of thiazolidine compounds and nucleophilic cysteine residues cules. in TRPA1, suggesting a mechanism of covalent modification. ⁎ Corresponding authors at: Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences/Key Laboratory of Bioactive Peptides of Yunnan Province, and Ion Channel Research and Drug Development Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China, or Department of Biological Sciences, Columbia University, New York, NY 10027, USA. E-mail addresses: [email protected], [email protected] (J. Yang), [email protected] (S. Wang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.freeradbiomed.2017.01.032 Received 19 September 2016; Received in revised form 22 January 2017; Accepted 23 January 2017 Available online 24 January 2017 0891-5849/ © 2017 Elsevier Inc. All rights reserved. D. Su et al. Free Radical Biology and Medicine 104 (2017) 272–279 Fig. 1. Thiazolidine activates the TRPA1 channel. (A) Chemical structure of thiazolidine. (B) Representative fluorescence traces of intracellular Ca2+ signals in hTRPA1-expressing HEK 293 cells in response to different concentrations of thiazolidine. AITC, a TRPA1 agonist, was subsequently applied to fully activate TRPA1. RFU: relative fluorescence unit. (C) Concentration-response relationship of the thiazolidine-induced intracellular Ca2+ increase in HEK 293 cells expressing hTRPA1. Data are normalized to 100 μM AITC-induced 2+ intracellular Ca increase. The smooth curve is a fit to the Hill equation, with an EC50 of 0.91 mM and a Hill coefficient of 1.9. (n=6) (D-G) Representative fluorescence traces of intracellular Ca2+ signals in HEK 293 cells transfected with empty vector (D) or cells expressing TRPM8 (E), TRPV1 (F), or TRPC6 (G) in response to thiazolidine and subsequently applied the Ca2+ ionophore ionomycin (D), TRPM8 agonist menthol (E), TRPV1 agonist capsaicin (F), or TRPC6 agonist hyperforin (G), respectively. (H) Representative thiazolidine-induced whole-cell currents in hTRPA1-expressing HEK 293 cells. HC: HC-030031. (I) Representative intracellular thiazolidine-induced macroscopic currents in an inside-out patch from a hTRPA1-expressing HEK 293 cell. (J) Quantification of the time required to reach the peak of thiazolidine-induced currents in whole-cell or inside-out patch recording. The number of independent measurements is marked on the top of each bar. Furthermore, we show that thiazolidine can elicit biological responses 2.3. Cell culture and transfection specifically through TRPA1 activation. HEK (human embryonic kidney) 293 cells or HEK 293 cells stably expressing TRP channels were grown in DMEM (HyClone) plus 10% 2. Materials and methods fetal bovine serum (Gibco) and penicillin (100 U/ml)/streptomycin (0.1 mg/ml) (Biological Industries) with or without G418 sulfate 2.1. Chemicals (0.2 mg/ml, Gibco). HEK 293 cells were transiently transfected with wild-type or mutant TRPA1 channels with or without enhanced GFP Thiazolidine and thioproline were purchased from Tokyo Chemical plasmids using LipoD293 In Vitro DNA Transfection Reagent (SignaGen Industry. Thiazole was obtained from Adamas-beta. HC-030031, 2-APB, Laboratories) and used within 48 h. capsaicin, menthol, glutathione (GSH) and oxidized glutathione (GSSG) were obtained from Sigma-Aldrich. Ionomycin and hyperforin were 2.4. Intracellular Ca2+ imaging from Cayman Chemical. The intracellular Ca2+ imaging of HEK 293 cells was performed using a FlexStation 3 microplate reader (Molecular Devices). Cells were 2.2. Clones and mutagenesis plated in a 96-well plate and loaded with fluo-4 AM (10 μM) and Pluronic F-127 (0.2%) (Molecular Probes) at 32 ℃ for 1 h in Ca2+-free Human TRPA1 (Genbank accession number NM_007332), human imaging solution. Subsequently, real-time fluorescence changes in cells TRPV1 (NM_080706), human TRPM8 (NM_024080) and human TRPC6 upon the addition of a test compound were measured. The standard (NM_004621) were all cloned in pcDNA3.1. Site-directed mutations imaging solution contained (in mM) 145 NaCl, 5 KCl, 1 MgCl , 2 CaCl , were constructed by oligonucleotide-based mutagenesis using PCR with 2 2 and 10 HEPES, pH 7.4. Q5 polymerase (New England Biolabs) following the instruction manual of QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) and were confirmed by DNA sequencing. 2.5. HPLC analysis A high-performance liquid chromatography analysis (HPLC) was 273 D. Su et al. Free Radical Biology and Medicine 104 (2017) 272–279 performed using an Agilent 1200 Series LC System consisting of a 2.9. Statistics quaternary pump, degasser, autosampler, photodiode array detector (DAD) and thermostatted column compartment. Instrument control and Data are presented as the mean ± standard error of the mean data analysis was conducted using Agilent ChemStation software. (s.e.m.). Statistical significance was evaluated using a two-tailed t-test, Chromatography was performed on a CAPCELL PAK C18 MGⅡ column and a P-value less than 0.05 was considered statistically significant. *P (5 µm, 4.6 mm I.D.×250 mm; Shiseido, Tokyo, Japan).