Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/OrgLett
Synthetic Anion Transporters as Endoplasmic Reticulum (ER) Stress Inducers Adil S. Aslam,† Ahmed Fuwad,∥ Hyunil Ryu,‡ Baskar Selvaraj,§ Jae-Won Song,† Dae Won Kim,⊥ Sun Min Kim,∥ Jae Wook Lee,*,§ Tae-Joon Jeon,*,‡ and Dong-Gyu Cho*,† †
Department of Chemistry and Chemical Engineering, Inha University, Incheon 22212, Republic of Korea Department of Biological Engineering, Inha University, Incheon 22212, Republic of Korea § Natural Product Research Center, Korea Institute of Science and Technology, Gangneung Institute, Gangneung 25451, Republic of Korea ∥ Department of Mechanical Engineering, Inha University, Incheon 22212, Republic of Korea ⊥ Department of Biochemistry and Molecular Biology, Research Institute of Oral Science, College of Dentistry, Gangneung Wonju National University, Gangneung 25457, Republic of Korea
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‡
S Supporting Information *
ABSTRACT: Cl−-ion transporters (2a−2h) were synthesized based on the binding motifs of prodigiosin. Transporter 2e clearly displays Cl−-ion transportation activity across both model and live cell membranes. Furthermore, 2e can disrupt Ca2+ homeostasis and increase the intracellular concentration of Ca2+ in the DLD-1 cell. This disruption can lead to Caspase-dependent apoptosis supported by CHOP expression (a marker of ER stress) and the appearance of the cleaved forms of Caspase 3 and PARP.
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membranes. Our experiments suggest that 2g can interact with the model membrane and loosen its structure, which allows ions to pass through it. Thus, 2e could be the first Cl−-ion transporter that induces a certain level of ER stress leading to apoptosis, whereas 2g may transfer ions nonselectively. Our design principle relies on the Cl−-ion binding motifs of prodigiosin (1). The current designed structures (2) eliminate the possibility of protonated imminic pyrrole in 1 under cellular conditions and mimic three hydrogen bond donors of protonated prodigiosin using two NHs and one CH motif of 2 for Cl−-anion binding. To synthesize the target compounds (2a−2h) shown in Scheme 1, the requisite pyrrole connected benzoic acids (3) were synthesized by following the literature procedure.5 The obtained carboxylic acids were coupled with various amines to give the target transporters. The abilities of 2a−2h to transport Cl− ions were measured using a model cell membrane (large unilamellar vesicles, LUVs) prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) (see Figure 2). The efflux of Cl− ions was monitored using a chloride-selective electrode for 600 s after adding 2a−2h (4 mol %, relative to POPC) dissolved in dimethylsulfoxide (DMSO). 10% Triton-X was added to rupture the vesicles to obtain the final concentration of Cl−. The normalized Cl− efflux values for each compound were obtained using the Cl−ion-selective electrode. As summarized
rodigiosin (1) and its derivatives have been studied as Cl−-ion transporters (see Figure 1).1 It has also been
Figure 1. Prodigiosin (1) and rationally designed synthetic anion transporters (2), based on the Cl− ion binding motifs of prodigiosin.
reported that their anticancer activities originate from disrupting the intracellular chloride concentration.2,3 Recently, the disruption of autophagic processes and apoptosis were independently observed on a cellular level when monosquaramide was treated as an ion transporter.4 Furthermore, we speculated that disturbed chloride ion concentration induced by an ion transporter in a cellular level affects other ion transportation machineries that cause a certain level of stress to a cellular organelle and lead to apoptosis. Herein, we report two synthetic ion transporters that can induce endoplasmic reticulum (ER) stress by increasing Ca2+ concentration and lead to apoptosis. This possibility is proposed and demonstrated by 2e and 2g for the first time. Specifically, molecule 2e acts as a typical anion transporter, while 2g neither relies on typical transporters nor ion channel mechanisms in model © XXXX American Chemical Society
Received: August 9, 2019
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DOI: 10.1021/acs.orglett.9b02823 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters Scheme 1. Synthetic Scheme of Cl−-Ion Transporters (2a− 2h)
meta to the amide group, compounds 2h and 2g showed an ∼2-fold increase in chloride efflux, when compared to 2c. In contrast, when F or Cl groups were introduced ortho to the amide group, the activities of 2f and 2e decreased dramatically to 6% and 10%, respectively. We also performed an Tl+/ANTS assay7 to exclude the possibility that the synthesized compounds may interfere with membranes and rupture vesicles, resulting in the leakage of ions/molecules from the vesicles during the chloride transport assay.6 In this assay, POPC vesicles were prepared with ANTS and the ANTS present outside the vesicles was removed using a PD-10 desalting column. When DMSO was used as a negative control, the normalized fluorescence value was reduced accordingly, because of the unremoved residual ANTS outside the vesicles (see Figure S10 in the Electronic Supporting Information (ESI)). The vesicles were lysed upon adding Triton-X to the solution, resulting in the release of all the entrapped ANTS within the vesicles. The released ANTS was further quenched (by Tl+) in the presence of TlNO3. As shown in Figures S11−S18 in the ESI, the solutions of 2a and 2b and those of 2e and 2f show similar fluorescence intensities to DMSO (negative control) in the presence of Tl+. Thus, these experiments prove that the solution of 2a and 2b and that of 2e and 2f do not make Tl+ pass through vesicles and ruled out nonselective chloride transportation. On the other hand, the fluorescence intensities of the solutions of 2c and d and those of 2g and 2h decreased in the presence of Tl+, when compared to DMSO. This experiment showed that any ions, including Tl+, can pass through the vesicles in the presence of 2c and 2d and in the presence of 2g and 2h without any ion selectivity. The Cl− binding affinity of the transporters was investigated in CD 3CN, using 1 H NMR titration experiments by monitoring the chemical shift of the aromatic CH group (see Table S1 and Figures S1−S5 in the ESI). Higher binding affinities of 2g (KCl− = 248 M−1) and 2h (KCl− = 220 M−1) were obtained because the presence of the electronegative halogen substituents increased the acidity of the two NH and CH groups. The compound containing no halogen substituent (2c, KCl− = 112 M−1) was placed next, and a lower binding affinity was observed for 2e (KCl− = 54 M−1) and 2f (KCl− = 37 M−1), even though they contain a halogen group. The difference in the binding ability of 2e and 2g was taken into consideration using DFT calculations. The optimized energies of 2g and 2g·Cl− were lower than those of 2e and 2e·Cl−, respectively. The higher energy of 2e and 2e·Cl− may reflect that the steric repulsions ((o-Cl vs CO) of 2e·Cl− and (NH vs o-Cl) of 2e) are more dominant than the stabilizing intramolecular hydrogen bond (NH···o-Cl) of 2e (see Figures S6−S9 in the ESI). Most Cl−-ion transporters reported in the literature show antiproliferative activity toward cancer cells.8 Thus, the antiproliferative effect was studied using several cancer cell lines in the presence of 50 μM of the as-synthesized ion transporters (2a−2h) for 48 h. The cell viabilities were measured using an MTT assay.9 Among these Cl− ion transporters, compounds 2d−2h showed cell viabilities of 2d > 2b > 2a). The chloride efflux observed for 2c was 57%, which was 2-fold higher than that observed for 2a. Thus, the octyl group was fixed and the structure of 2c was further optimized by introducing halogen substituents onto the phenyl ring. A similar strategy has been applied to other transporters to increase their lipophilicity and Cl− binding activity.6 When F or Cl groups were introduced B
DOI: 10.1021/acs.orglett.9b02823 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters
Figure 4. Comparison of the (A) HT29 and (B) DLD-1 cell viability in Cl−-ion-containing and Cl−-ion-free HBSS buffer upon the dosedependent treatment of 2e after 6 h. Figure 3. Cell viability obtained from the single-point screening of ion transporters (2a−2h), using an MTT assay. The cell viability was obtained upon treating various cells with each ion transporter for 48 h.
(IC50 = 15.9 μM), and MIA PaCa-2 (IC50 = 58.2 μM) (see Figures S21−S25 and Table S6 in the ESI). To test whether compound 2e and 2g form a Cl−-ion channel, a patch clamp assay was performed using gramicidin A (gA) as a positive control. Any signal representing ion channel activity or damaged membranes was not observed as 2e and 2g were treated, although an irregular (nonspecific) type of Cl−-ion transport was observed for only 2g at high concentrations of 0.2 mM (see Figure S20 in the ESI). Obviously, the addition of gA (0.2 nM) to the solution resulted in ion channel activity. All these experiments indicate that 2g can make any ion, including Cl−, pass out of the model membrane without any ion selectivity. Cl− ion efflux activity can be also affected by other ionic transport machineries under cellular conditions. Therefore, the effect of valinomycin as a selective K+ ionophore was investigated. Compound 2e (4 mol %) was added with or without 125 nM of valinomycin. The activity of 2e was significantly enhanced from 17% to 72% in the presence of valinomycin, while any significant synergic effect with 2g was not detected under the same conditions (see Figure 2, as well as Figure S19 in the ESI).10 When 1 mol % of 2g was used with or without the same amount of valinomycin, the activity increased from 66% to 81% over 600 s. Upon comparing the synergic effect of 2e (4 mol %) to 2g (1, 2, and 4 mol %) in the presence of the same amount of valinomycin (125 nM), compound 2e displayed a higher synergic effect than 2g. We further analyzed the cell viability of 2e and 2g to observe the relationship between cytotoxicity and Cl− in cells. HT29 and DLD-1 cells were treated with a serial dilution of 2e and 2g in the presence of Cl− and in the absence of Cl−. The cell viability was measured using an MTT cell proliferation assay kit. In this experiment, the cell viability of both HT29 and DLD-1 cells show that 2e was more cytotoxic in the presence of Cl− ions after incubation for 6 h (see Figure 4)11 and the difference was not dose-dependent after 17 h (see Figure S29 in the ESI). However, 2g-treated cells show no significant difference in the presence and absence of Cl− ions, especially at a low concentration of 2g after 6 h (see Figure S30 in the ESI). This result indicates that the observed cytotoxicity of 2e may result from Cl− efflux activity in both HT29 and DLD-1 cells. In addition, we also tested whether 2e and 2g induced a change in the intracellular Ca2+ concentration. DLD-1 cells were incubated with 50 and 100 μM of 2e and 2g for 12 h, respectively, and then treated with a Ca2+-sensitive fluorescent probe (Fluo-3 AM) for 30 min (Figure 5).12 In this study,
Figure 5. Live cell imaging of DLD-1 cells upon treatment with (A) 0, (B) 50 μM of 2e, (C) 100 μM of 2e, (D) 50 μM of 2g, and (E) 100 μM of 2g, for 24 h, followed by staining with Fluo-3 AM dye. (F) Fluorescent change observed upon treatment with 2e and 2g.
treatment with 2e increased the intracellular Ca2+ concentration in a dose-dependent manner. However, treatment with 2g does not increase the intracellular Ca2+ concentration as much as 2e and does not show a clear dose dependency. These experiments, coupled with the increased Cl− activity in the presence of valinomycin shown in Figure 2, imply that a Cl−ion-selective transporter (2e) can perturb Ca2+ homeostasis in the cell lines studied. Typically, the concentration of Ca2+ is controlled in the endoplasmic reticulum (ER), where proteins are produced.13 Perturbation of the Ca2+ concentration in the ER (ER stress) can cause an accumulation of misfolded or unfolded proteins.14 Ultimately, prolonged protein folding can lead to the activation of apoptosis.15 The pro-apoptotic gene, C/EBP-homologous protein (CHOP), is usually activated upon ER stress.16 To investigate whether 2e and 2g induce ER stress, human pancreatic cells (MIA PaCa-2) were transiently transfected with CHOP-LUC plasmid and incubated with 100 μM of 2e and 2g for 4, 8, 16, and 24 h, respectively (Figure 6). Under these conditions, the cellular luciferase signals start to increase at 8 h and reach a maximum at 24 h. In addition, the cellular luciferase signals increased in a dose-dependent manner as 2e and 2g were treated. To determine the level of CHOP, the cells were incubated with 100 μM of 2e and 2g for 0, 4, 8, 16, and 24 h for Western blot analysis (Figures 7C and 7D). The results of the Western blot analysis provide further confirmation that CHOP C
DOI: 10.1021/acs.orglett.9b02823 Org. Lett. XXXX, XXX, XXX−XXX
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Organic Letters
In conclusion, we propose that transporter 2e disrupts Ca2+ homeostasis via its Cl− transportation activity coupled with the Ca2+ transportation machinery present in cells. The induced ER stress can lead to Caspase-dependent apoptosis, which was supported by CHOP expression and the appearance of the cleaved form of Caspase 3 and PARP. In contrast, 2g does not induce as large a change in the Ca2+ concentration, when compared to 2e, while its cytotoxicity also relies on Caspasedependent apoptosis. In addition, the Cl− efflux activity of 2e increased when coupled with a K+ selective ionophore (valinomycin) in the model membrane. However, the higher Cl− efflux activity of 2g was a result of a nonspecific ion transportation mechanism, which was confirmed by an Tl+/ ANTS assay and patch clamp experiment.
Figure 6. (A) CHOP expression obtained from single-point compound treatment (100 μM) with time (4, 8, 16, and 24 h). (B) CHOP expression obtained from serial dilution compound treatment after incubation for 24 h.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.9b02823.
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Figure 7. Western blot analysis of the increased cellular level of apoptosis markers (c-Caspase 3 and c-PARP) and CHOP protein in MIA PaCa-2 cells upon treatment with (A and C) 100 μM of 2e and (B and D) 100 μM of 2g at different incubation times.
Experimental procedures, biological evaluation data, theoretical calculations, and 1H, and 13C NMR spectra for all new compounds (PDF)
AUTHOR INFORMATION
Corresponding Authors
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expression was increased from 8 h and reached a maximum at 24 h. This result indicates that 2e and 2g gradually increases apoptosis by inducing CHOP expression. To know whether 2e and 2g induce apoptosis, HeLa cells and DLD-1 cells were incubated with 50 and 100 μM of 2e and 2g for 48 h, respectively, and then treated with a mixture of fluorescein conjugated annexin V and propidium iodide (PI). The result of the FACS (fluorescence activated cell sorting) analysis revealed that 100 μM of 2e induced apoptosis with 80.9% apoptotic and dead cells. 2g showed 19.2% apoptotic and dead cells (see Figure S27 in the ESI). In addition, to confirm whether the observed cytotoxicity of 2e was caused by apoptotic cell death, the levels of cleaved Caspase 3 and cleaved PARP were assessed by Western blotting analysis in MIA PaCa-2 cells (Figures 7A and 7B). Caspase-3 has essential roles in initiating apoptotic signaling and executing the final stage of cell death, because it is responsible for the proteolytic cleavage of many key proteins, such as the nuclear enzyme poly(ADP-ribose) polymerase (PARP). Under normal conditions, Caspase 3 exists as an inactive form of the enzyme. However, under severe ER stress conditions, Caspase 3 undergoes proteolytic cleavage to become an active enzyme. In turn, active Caspase 3 cleaves PARP.17 Therefore, the appearance of the cleaved form of Caspase 3 and PARP is an indication of Caspase-dependent apoptosis induced by compounds 2e and 2g. In addition, the apoptosis inducing factor (AIF)18 is typically translocated into the nucleus during Caspase-independent apoptosis. The level of AIF in the nucleus was examined after incubating MIA PaCa-2 cells with 2e and 2g. Any detectable translocation of AIF into the nucleus was not observed with both compounds (see Figure S26 in the ESI). These results ruled out Caspaseindependent apoptosis.
ORCID
Sun Min Kim: 0000-0001-8420-637X Jae Wook Lee: 0000-0002-0171-2160 Tae-Joon Jeon: 0000-0002-4882-9040 Dong-Gyu Cho: 0000-0002-3272-0745 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology (Grant Nos. NRF-2016R1D1A1A09917824, NRF-2019R1F1A1058712, and NRF-2017R1A2B4002523).
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REFERENCES
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DOI: 10.1021/acs.orglett.9b02823 Org. Lett. XXXX, XXX, XXX−XXX
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Organic Letters (3) Ko, S.-K.; Kim, S. K.; Share, A.; Lynch, V. M.; Park, J.; Namkung, W.; Van Rossom, W.; Busschaert, N.; Gale, P. A.; Sessler, J. L.; Shin, I. Nat. Chem. 2014, 6, 885. (4) Busschaert, N.; Park, S.-H.; Baek, K.-H.; Choi, Y. P.; Park, J.; Howe, E. N. W.; Hiscock, J. R.; Karagiannidis, L. E.; Marques, I.; Felix, V.; Namkung, W.; Sessler, J. L.; Gale, P. A.; Shin, I. Nat. Chem. 2017, 9, 667. (5) Maeda, H.; Kinoshita, K.; Naritani, K.; Bando, Y. Chem. Commun. 2011, 47, 8241−8243. (6) (a) Lee, E.-B.; Ryu, H.; Lee, I.; Choi, S.; Hong, J.-H.; Kim, S. M.; Jeon, T.-J.; Cho, D.-G. Chem. Commun. 2015, 51, 9339. (b) Busschaert, N.; Wenzel, M.; Light, M. E.; Iglesias-Hernandez, P.; Perez-Tomas, R.; Gale, P. A. J. Am. Chem. Soc. 2011, 133, 14136. (7) ANTS (8-amino-1,3,6-naphthalenetrisulfonic acid disodium salt) has excitation and emission at 355 and 512 nm as a quencher for Tl+. (8) For recent reported nonprodigiosin Cl−-ion transporters, see: (a) Akhtar, N.; Saha, A.; Kumar, V.; Pradhan, N.; Panda, S.; Morla, S.; Kumar, S.; Manna, D. ACS Appl. Mater. Interfaces 2018, 10 (40), 33803. (b) Saha, T.; Hossain, M. S.; Saha, D.; Lahiri, M.; Talukdar, P. J. Am. Chem. Soc. 2016, 138, 7558. (9) MTT = 3-(4,5-diemthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. (10) The approximate EC50 value of 2g is 8.3 μM (Figure S31 in the ESI), although 2g relies on nonspecific chloride ion transport mechanism. Thus, the EC50 value of 2g does not have significant meaning. However, the Cl− efflux activity of 2e (10%, measured at pH 7.0) is apparently lower than the activity of synthetic prodigiosins (81%, measured at pH 7.4), unless 2e is used with valinomycin.1d (11) Please note that the incubation time difference between Figures 4 and 5. (12) Fluo-3 AM = 4-(6-acetoxymethoxy-2,7-dichloro-3-oxo-9xanthenyl)-4′-methyl-2,2′(ethylenedioxy)dianiline-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl) ester. (13) (a) Braakman, I.; Bulleid, N. Annu. Rev. Biochem. 2011, 80, 71. (b) Krebs, J.; Agellon, L. B.; Michalak, M. Biochem. Biophys. Res. Commun. 2015, 460, 114. (14) (a) Pinton, P.; Giorgi, C.; Siviero, R.; Zecchini, E.; Rizzuto, R. Oncogene 2008, 27, 6407. (b) Corazzari, M.; Gagliardi, M.; Fimia, G. M.; Piacentini, M. Front. Oncol. 2017, 7, 78. (15) (a) Zuppini, A.; Navazio, L.; Mariani, P. J. Cell Sci. 2004, 117, 2591. (b) Sano, R.; Reed, J. C. Biochim. Biophys. Acta, Mol. Cell Res. 2013, 1833, 3460. (16) (a) Fonseca, S. G.; Gromada, J.; Urano, F. Trends Endocrinol. Metab. 2011, 22, 266. (b) Papa, F. R. Cold Spring Harbor Perspect. Med. 2012, 2, a007666. (c) Eizirik, D. L.; Cardozo, A. K.; Cnop, M. Endocr. Rev. 2008, 29, 42. (d) Wang, S.; Kaufman, R. J. J. Cell Biol. 2012, 197, 857. (17) (a) Slee, E. A.; Harte, M. T.; Kluck, R. M.; Wolf, B. B.; Casiano, C. A.; Newmeyer, D. D.; Wang, H. G.; Reed, J. C.; Nicholson, D. W.; Alnemri, E. S.; Green, D. R.; Martin, S. J. J. Cell Biol. 1999, 144, 281. (b) Herceg, Z.; Wang, Z. Q. Mutat. Res., Fundam. Mol. Mech. Mutagen. 2001, 477, 97. (18) Candé, C.; Vahsen, N.; Garrido, C.; Kroemer, G. Cell Death Differ. 2004, 11, 591.
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DOI: 10.1021/acs.orglett.9b02823 Org. Lett. XXXX, XXX, XXX−XXX
