Thiazolidine Reacts with Thioreactive Biomolecules

Free Radical Biology and Medicine 104 (2017) 272–279

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Free Radical Biology and Medicine

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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). The column < 0.05, **P < 0.01, and ***P < 0.001. temperature was maintained at 30 ℃. The mobile phase consisted of a mixture of 10% acetonitrile and 90% water, and the flow rate was kept 3. Results at 0.8 ml/min. The injection volume was 10 μl. By careful analysis of the chromatograms at different wavelengths at a scale of 210–280 nm, 3.1. Thiazolidine activates TRPA1 the detection wavelength was set at 210 nm. Because of the critical role of TRPA1 in chemical sensing, we first examined whether TRPA1 was a target of thiazolidine. Thiazolidine 2.6. Liquid chromatography and mass spectrometry increased intracellular Ca2+ in HEK 293 cells expressing recombinant human TRPA1 (hTRPA1) in a dose-dependent manner (Fig. 1B), with an The chromatographic separation was carried out using an Agilent EC of 0.91 mM (Fig. 1C). This effect did not occur in mock-transfected fi 50 1290 in nity series UHPLC instrument (Agilent Technologies, USA) cells (Fig. 1D) or in cells expressing TRPM8, TRPV1 or TRPC6 (Fig. 1E, equipped with a quaternary pump (G4204A, USA), a de-gasser, a diode- F, G), which are subtypes of TRP channels that are structurally related array detector (G4212B, USA), an autosampler (G4226A, USA) and a to TRPA1. Consistent with results from the calcium imaging study, column compartment (G1316C, USA). The chromatographic separation thiazolidine also elicited whole-cell currents in hTRPA1-expressing HEK Ⅱ was achieved on a CAPCELL PAK C18 MG column (5 µm, 4.6 mm 293 cells, which were completely inhibited by the TRPA1-specific I.D.×250 mm; Shiseido, Tokyo, Japan) using a mixture of 10% antagonist HC-030031 (Fig. 1H). In addition, direct application of fl methanol and 90% water as mobile phase at a ow rate of 0.5 ml/ thiazolidine to the intracellular side of inside-out membrane patches min. The column temperature and injection volume were 30 °C and from hTRPA1-expressing cells elicited HC-030031-sensitive macro- μ 5 l, respectively. Mass spectrometric studies were carried out on a scopic currents (Fig. 1I). The thiazolidine-induced currents in inside- fl quadrupole time-of- ight (Q-TOF) high-resolution mass spectrometer out patches developed more rapidly than did whole-cell currents (Q-TOF LC/MS 6540 series, Agilent Technologies, Santa Clara, CA, (compare Fig. 1H and I), and the time-to-peak of the currents in USA) coupled with electrospray ionization (ESI). The data was acquired inside-out patches was significantly less than that of the whole-cell using MassHunter Workstation software. The detection was performed currents (Fig. 1J). These results suggest that thiazolidine activates in positive ESI mode. The MS parameters were optimized as follows: the TRPA1 from the intracellular side. fragmentor voltage was set at 135 V; the capillary was set at 3500 V; the skimmer was set at 65 V; and nitrogen was used as the drying 3.2. Reducing agents reverse sustained TRPA1 activation caused by (350 °C, 7 L/min) and nebulizing (30 psi) gas. thiazolidine

2.7. Electrophysiology We then sought to understand how thiazolidine activates TRPA1. TRPA1 is activated by reactive compounds through covalent modifica- All experiments were performed at room temperature (~22 ℃). tion of nucleophilic cysteine residues or by non-reactive compounds via – Pipettes were fabricated and fire-polished to resistances of 1–2MΩ for a ligand-binding mechanism [7 9]. In our study, thiazolidine-induced inside-out patch recording and 2–3MΩ for whole-cell recording. TRPA1 currents were not easily reversible by washout in both whole- Macroscopic currents were elicited by 500-ms voltage ramps from cell and inside-out patch-clamp recording (Fig. 2A and B), suggesting a fi −100 to +100 mV at a frequency of 0.5 Hz with a holding potential of more likely mechanism of covalent modi cation in channel activation. 0 mV. Currents were amplified by Axopatch 200B and digitized by Dithiothreitol (DTT), a reducing agent, completely reversed thiazoli- Digidata 1440 A (Molecular Devices). Currents were low-pass filtered at dine-induced sustained TRPA1 activation in both whole-cell and inside- fi 1 kHz and sampled at 10 kHz. pCLAMP software (Molecular Devices) out recording (Fig. 2C and D). In contrast, DTT did not signi cantly ff was used for data acquisition and analysis. The standard extracellular a ect TRPA1 currents elicited by a non-reactive TRPA1 agonist, menthol (Fig. 2E). Similarly, the endogenous reducing agent GSH also solution contained (in mM) 150 NaCl, 1 MgCl2, and 10 HEPES, pH 7.4. The pH was adjusted with NaOH. The standard intracellular solution reversed thiazolidine-induced TRPA1 activation (Fig. S1). These results fi contained (in mM) 150 KCl, 5 EGTA, and 10 HEPES, pH 7.2. The pH suggest that thiazolidine may induce covalent modi cation of cysteine was adjusted with KOH. For inside-out patch recording, 5 mM sodium residues in TRPA1 to open the channel. triphosphate was included in the intracellular solution. 3.3. Thiazolidine chemically reacts with GSH

2.8. Mouse model Paradoxically, it has been reported that thiazolidine is stable in cell culture medium and in rat blood [12], and it has never been shown that Seven- to nine-week-old TRPA1-/- and wild-type littermates were thiazolidine can chemically react with cysteine residues. GSH is an used. Before the assessment of pain behavior, the mice were acclimated endogenous tripeptide, and it contains a nucleophilic cysteine residue to a Plexiglas chamber for 30 min. The 10 mM thiazolidine solution was that reacts with many toxic electrophiles [10,11]. If thiazolidine does prepared in a 0.9% saline solution. Thiazolidine solution or control chemically react with nucleophilic cysteine residues in TRPA1 to open saline in a 20 μl volume was injected into the plantar surface of mouse the channel, it should react with GSH. Thus, we mixed thiazolidine and hindpaws, and the mice were immediately returned to the Plexiglas GSH in solution and then performed a chemical composition analysis of chamber and recorded with a digital video camera. The time that the the mixture using high-performance liquid chromatography (HPLC). mice spent licking or lifting the injected paw was counted for 5 min Representative HPLC chromatograms of GSH (50 mM) or thiazolidine after injection. The thickness of the paw was measured using a digital (50 mM) are shown in Fig. 3A and B, respectively. GSH gave a major thickness gauge (Mitutoyo Corp.), which has a resolution of 10 µm. Paw chromatographic peak at 2.9 min and a minor peak at 2.6 min (Fig. 3A). swelling was calculated as (paw thickness at 15 min)–(paw thickness at The minor peak at 2.6 min (named as peak2.6) may reﬂect an unknown 0 min). impurity or a background oxidation of GSH. Indeed, HPLC chromato-

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Fig. 2. Dithiothreitol (DTT) reverses thiazolidine-induced sustained activation of TRPA1. (A) Representative whole-cell currents in hTRPA1-expressing HEK 293 cells in response to a consecutive application of thiazolidine, bath solution and HC-030031(HC). (B) Representative macroscopic currents in an inside-out patch from a hTRPA1-expressing HEK 293 cell in response to a consecutive application of thiazolidine, bath solution and HC. (C) Representative whole-cell currents in hTRPA1-expressing cells in response to a consecutive application of thiazolidine, bath solution and DTT. (D) Representative macroscopic currents in an inside-out patch from an hTRPA1-expressing cell in response to a consecutive application of thiazolidine, bath solution and DTT. (E) Representative whole-cell currents in hTRPA1-expressing cells in response to menthol in the absence or presence of DTT.

Fig. 4A and B). Both the mixture and GSH gave a TIC peak at 5.2 min (Fig. 4A and B), suggesting that this peak represents GSH. Consistently, the corresponding ESI spectra at 5.2 min of both GSH and the mixture showed a molecular ion peak at m/z 330 [GSH+Na]+(Fig. 4C and D). The TIC peaks at 4.4, 4.7, 5.5, and 5.9 min of the mixture may represent reaction products (Fig. 4A). The ESI spectrum of the mixture at 4.1– 4.5 min gave molecular ion peaks at m/z 635 and 651 (Fig. 4E). GSSG has a molecular weight of 612 (Fig. 4F). The ion peaks at m/z 635 and 651 should correspond to [GSSG+Na]+ and [GSSG+K]+, respectively. This is consistent with the data from HPLC analysis which showed that thiazolidine promoted GSSG production. In Fig. 4G, the ESI spectrum of the mixture at 5.8–5.9 min gave [M+H]+ and [M+Na]+ ion peaks at m/z 383 and 405, respectively, which indicates a chemical product with a molecular weight of 382. The deduced chemical structure is shown in Fig. 4H. The ESI spectra of the GSH-thiazolidine mixture at 4.6–4.7 and 5.5–5.6 min gave ion peaks at m/z 330, 352, and 374 (Fig. S3), which may represent different salt forms of GSH, such as [GSH+Na]+, + + [GSHNa+Na] , and [GSHNa2+Na] . We also tested other thiazolidine compounds. Thioproline (thiazolidine-4-carboxylic acid) (Fig. 5A) is a common metabolite in brain Fig. 3. New chemical products are generated in a GSH-thiazolidine mixture. (A-C) produced through the catabolism of 5-hydroxytryptamine [13]. HPLC Representative HPLC chromatograms of GSH (A), thiazolidine (B) and the GSH- analysis showed that the chromatographic peaks of GSH and thiopro- thiazolidine mixture (C). The detection wavelength: 210 nm. mAU: milli Absorption line partly overlapped and peak changed only moderately (Fig. S4); Units. (D) Quantification of the area of the peak at 2.6 min (peak2.6) in the chromatogram 2.6 of GSH (A) or of the mixture (C) (n=3). the area of peak2.6 of the mixture was only 32 ± 13% larger than that of GSH alone (Fig. S4 and Fig. 5B, compare with Fig. 3D). As expected, gram of oxidized GSH (GSSG) also gave a peak at 2.6 min (Fig. S2), thioproline induced an intracellular Ca2+ increase in a dose-dependent suggesting that peak2.6 represents GSSG. However, the HPLC chroma- manner in HEK 293 cells expressing hTRPA1; however, its potency was togram of GSH significantly changed after being mixed with thiazoli- significantly lower than thiazolidine, with an EC50 of 18 mM (Fig. 5C dine, and new peaks were observed (Fig. 3C). Notably, the area of and D). Thioproline had no effects on mock-transfected cells or cells peak2.6 of the GSH-thiazolidine mixture was 304 ± 75% larger than expressing TRPM8 (Fig. 5E and F). On the other hand, thiazole that of GSH alone (Fig. 3A, C, D), suggesting the production of GSSG. (Fig. 5G), a heterocyclic compound similar to thiazolidine, did not These results suggest that new chemical products other than GSH and significantly change the chromatogram of GSH (Fig. S5), and the area of thiazolidine are formed in the solution of the GSH-thiazolidine mixture. peak2.6 of a GSH-thiazole mixture was not different from that of GSH To further determine the reaction products, we performed liquid alone (Fig. S5 and Fig. 5H). Accordantly, thiazole did not significantly chromatography-mass spectrometry (LC-MS) analysis of the GSH- affect intracellular Ca2+ in cells expressing hTRPA1(Fig. 5I). thiazolidine mixture. The MS total ion chromatogram (TIC) of GSH Taken together, these results indicate that thiazolidine can chemi- was significantly changed after being mixed with thiazolidine (compare cally react with GSH, and the chemical reactivity of thiazolidine

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Fig. 4. MS-TIC and ESI spectra of GSH-thiazolidine mixture and GSH alone. (A, B) MS-TIC chromatograms of a GSH-thiazolidine mixture (A) or GSH alone (B). (C) Corresponding ESI spectrum of the GSH-thiazolidine mixture at 5.2 min (D) Corresponding ESI spectrum of GSH alone at 5.2 min (E) Corresponding ESI spectra of the mixture at 4.1–4.5 min (F) Chemical structure of GSSG. (G) Corresponding ESI spectra of the mixture at 5.8–5.9 min (H) Chemical structure of the reaction product deduced from ion peaks at m/z 383 and 405 in (G). compound is required for TRPA1 activation. Substituents in the electrophilic compounds or hyperoxia [14–16] (Fig. 6A). We mutated thiazolidine ring and the electronic structure of the ring itself can these cysteine residues to serine or lysine residues to arginine in affect the chemical reactivity of thiazolidine or thiazolidine-like different combinations and studied the mutant channels in HEK 293 compounds. cells. Both the single lysine mutation K710R and the double cysteine mutation C421S/C856S significantly diminished the intracellular Ca2+ 3.4. Cysteine residues are involved in thiazolidine-induced TRPA1 increase induced by 3 mM thiazolidine (Fig. 6B, C, E). In contract, both fi ff 2+ activation mutations had no signi cant e ect on the intracellular Ca increase induced by 2-APB (Fig. 6B and C). The triple mutation C421S/K710R/ If thiazolidine also chemically reacts with TRPA1 to open the C856S further diminished the channel response to thiazolidine (Fig. 6D ffi fi channel, nucleophilic cysteine residues are likely to be involved. We and E). The e cacy and/or potency of thiazolidine were signi cantly therefore searched for structural elements critical for TRPA1 activation decreased in these mutant channels (Fig. 6F). by thiazolidine. Intracellular cysteine residues, including C421 and We further performed patch-clamp recordings of the C421S/K710R/ C856, and lysine residue K710 are important for TRPA1 activation by C856S mutant channel. Although thiazolidine elicited robust currents

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Fig. 5. The effects of thioproline and thiazole on GSH and TRPA1. (A) Chemical structure of thioproline. (B) Quantification of the area of the peak at 2.6 min (peak2.6) in the chromatogram of GSH alone or of the GSH-thioproline mixture (n=4). (C) Representative fluorescence traces of intracellular Ca2+ signals in hTRPA1-expressing HEK 293 cells in response to different concentrations of thioproline. (D) Concentration-response relationship of thioproline-induced intracellular Ca2+ increase in hTRPA1-expressing cells. Data are normalized to 100 μM AITC-induced intracellular Ca2+ increases. The smooth curve is a fit to the Hill equation. (n=9) (E, F) Representative fluorescence traces of intracellular Ca2+ signals in HEK 293 cells transfected with empty vector (E) or cells expressing TRPM8 (F) in response to thioproline and subsequently applied ionomycin (E) or TRPM8 agonist menthol

(F), respectively. (G) Chemical structure of thiazole. (H) Quantification of the area of the peak at 2.6 min (peak2.6) in the chromatogram of GSH alone or of the GSH-thiazole mixture (n=4). (I) Representative fluorescence trace of the intracellular Ca2+ signal in hTRPA1-expressing cells in response to 100 mM thiazole. that were comparable in amplitude to currents induced by 2-APB in 4. Discussion cells expressing wild-type (WT) hTRPA1 (Fig. 6G), it produced little currents in cells expressing the C421S/K710R/C856S mutant channel The LC-MS analysis indicates that two disulfides including GSSG (Fig. 6H), even though the mutant channel responded robustly to 2-APB and a cysteamine-adduct of GSH are formed in the GSH-thiazolidine (Fig. 6H). This result further confirmed the Ca2+ imaging result that mixture (Fig. 4F and H). Theoretically, thiazolidine can be hydrolyzed nucleophilic cysteines in TRPA1 play an important role in thiazolidine- under controlled conditions of chemical reaction [1,5,6] to generate induced channel activation, supporting a mechanism of covalent cysteamine and formaldehyde. If that were the case, ambient oxygen- modification in thiazolidine-induced TRPA1 activation. induced oxidation may cause disulfide formation between cysteamine and/or GSH. However, in parallel control experiments, GSH was not significantly oxidized to GSSG in the presence of ambient oxygen 3.5. Thiazolidine causes pain and inflammation through TRPA1 in vivo (Fig. 3A and Fig. 4B). Thus, even if ambient oxygen can cause disulfide bond formation in our experiments, it would be very slow, which TRPA1 activation in mammals causes pain and inflammation [7–9]; contradicts with the experimental observation that disulfide formation therefore, we examined whether thiazolidine could cause pain and is robust in the GSH-thiazolidine mixture (Fig. 3C and Fig. 4A). inflammation in vivo. Intraplantar injection of 10 mM thiazolidine (in Meanwhile, formaldehyde, being a good electrophile, would be ex- 20 μl volume) into a mouse hindpaw produced marked nociceptive pected to react with GSH more easily and quickly [17,18]; but, behavior within 5 min, including licking and lifting of the injected paradoxically, the hydroxymethyl adduct of GSH was not observed in hindpaw (Fig. 7A). Meanwhile, the thiazolidine injection also caused our LC-MS analysis. Furthermore, it has been reported that no hydro- acute inflammation within 15 min as measured by paw swelling lysis of thiazolidine occurred in rat blood in 30 min at room tempera- (Fig. 7B). By contrast, in TRPA1-/- mice, thiazolidine-induced nocicep- ture [12]. For the LC-MS experiments in our study, thiazolidine was tive responses and paw swelling were attenuated by ~89% and ~82%, dissolved immediately before the experiments and used within 30 min. respectively (Fig. 7A and B), indicating that TRPA1 mainly mediated Thus, the formation of the cysteamine-adduct of GSH is more likely thiazolidine-induced acute pathological reactions in vivo.

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Fig. 6. Mutagenesis study identified amino acid residues critical for thiazolidine-induced TRPA1 activation. (A) Schematic depiction of the TRPA1 structure and the critical amino acid residues. (B-D) Representative intracellular Ca2+ signals in response to different concentrations of thiazolidine in HEK 293 cells expressing mutant TRPA1 channel K710R (B), C421S/ C856S (C) or C421S/K710R/C856S (D). (E) Normalized intracellular Ca2+ increase in response to 3 mM thiazolidine in HEK 293 cells expressing wild-type (WT) hTRPA1 or hTRPA1 mutants. (n≥4) (F) Concentration-response relationships of the thiazolidine-induced intracellular Ca2+ increase in cells expressing WT or mutant TRPA1 channels. Smooth curves are fit to the Hill equation. n≥4 for each construct at each concentration. In (E) and (F), the data are normalized to 200 μM 2-APB-induced intracellular Ca2+ increases. (G, H) Representative 2- APB- and subsequent thiazolidine-induced whole-cell currents in HEK 293 cells expressing WT hTRPA1 (G) or the mutant channel (C421S/K710R/C856S) (H).

(RSSR'). This reaction is moderately fast and occurs reversibly at room temperature in water at physiological pH [20]. Thus, it is conceivable that GSH first reacts with thiazolidine to produce the cysteamine- adduct of GSH, and then GSSG is formed from thiol-disulfide interchange reactions. Our findings that the amino acids C421, K710 and C856 are important for thiazolidine-induced sustained TRPA1 activation and the activation is reversed by DTT suggest a gating mechanism of disulfide formation. A recent electron cryomicroscopy (cryo-EM) structure reveals the first atomic-level view of TRPA1 [21]. C856 is located in the S4-S5 linker, which interacts with the TRP-like domain. This domain is directly attached to the gate-forming S6 helix and acts as a nexus for communication between the gate and other domains [21]. Amino acids in the S4-S5 linker, like C856, are thus well situated to detect intracellular chemical signals and alter channel gating. Fig. 7. TRPA1 mediates thiazolidine-induced pain and inflammation in mice. (A) Consistent with this notion, a point mutation of an asparagine residue fi Quanti cation of the nociceptive responses within 5 min after intraplantar injection of (N855S) adjacent to C856 was recently found to underlie a familial control saline in wild-type (WT) mice (n=7) or of 10 mM thiazolidine in WT (n=6) and episodic pain syndrome, and the mutant channel showed significantly TRPA1-/- mice (n=11). (B) Quantification of paw swelling at 15 min after intraplantar injection of control saline in WT mice (n=9) or of 10 mM thiazolidine in WT (n=13) and altered biophysical properties [22]. C421 and K710 are both located in TRPA1-/- mice (n=11). the N-terminal region. K710 is in the pre-S1 region, close to the TRP- like domain [21]. This location provides a plausible link between K710 through a mechanism other than thiazolidine hydrolysis. This mechan- and channel gating. C421 is in the eleventh ankyrin repeat (AR11), but ism remains unclear. The catalytic effect of thiazolidine on GSSG because AR11 is unresolved in the cryo-EM structure [21], it is too formation may come from unsymmetrical thiol-disulfide interchange speculative to explain how C421 may influence channel gating. reactions [19]. Thiol-disulfide interchange is the reaction of a thiol Thiazolidine compounds are important in biochemistry and phar- (RSH) with a disulfide (R'SSR'), with the formation of a new disulfide macology. Identifying biological molecules such as TRPA1 and glu-

278 D. Su et al. Free Radical Biology and Medicine 104 (2017) 272–279 tathione as the targets of thiazolidine and elucidating its action Springer, Berlin, 2006. [2] A.M. Davis, M.I. Page, Opening of the thiazolidine ring of penicillin derivatives, J. mechanisms provide useful information on the biochemical activity of Chem. Soc. Chem. Commun. 23 (1985) 1702–1704. thiazolidine compounds. Through such activity, thiazolidine may [3] A.M. Davis, M. Jones, M.I. Page, Thiazolidine ring-opening in penicillin derivatives. produce diverse physiologic and pathologic effects in vivo. Part 1. Imine formation, J. Chem. Soc. Perkin Trans. 2 (8) (1991) 1219–1223. [4] A.M. Davis, N.J. Layland, M.I. Page, F. Martin, R.M. Oferrall, Thiazolidine ring- opening in penicillin derivatives. Part 2. Enamine formation, J. Chem. Soc. Perkin 5. Conclusion Trans. 2 (8) (1991) 1225–1229. [5] R. Luhowy, F. Meneghini, Mechanism of alkaline-hydrolysis of thiazolidines, J. 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