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Synthesis and Cellular Effects of a Mitochondria-Targeted Inhibitor of the Two-Pore Potassium Channel TASK-3

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Synthesis and Cellular Effects of a Mitochondria-Targeted Inhibitor of the Two-Pore Potassium Channel TASK-3 Pharmacological Research 164 (2021) 105326 Contents lists available at ScienceDirect Pharmacological Research journal homepage: www.elsevier.com/locate/yphrs Synthesis and cellular effects of a mitochondria-targeted inhibitor of the two-pore potassium channel TASK-3 Magdalena Bachmann a, Andrea Rossa b, Giuseppe Antoniazzi b, Lucia Biasutto c,d, Andrea Carrer a,d, Monica Campagnaro a, Luigi Leanza a, Monika Gonczi e, Laszlo Csernoch e, Cristina Paradisi b, Andrea Mattarei f, Mario Zoratti c,d, Ildiko Szabo a,c,* a Department of Biology, University of Padua, Italy b Department of Chemical Sciences, University of Padua, Italy c CNR Institute of Neuroscience, Padua, Italy d Department of Biomedical Sciences, University of Padua, Italy e Department of Physiology, Faculty of Medicine, University of Debrecen, Hungary f Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Italy ARTICLE INFO ABSTRACT Keywords: The two-pore potassium channel TASK-3 has been shown to localize to both the plasma membrane and the Mitochondria mitochondrial inner membrane. TASK-3 is highly expressed in melanoma and breast cancer cells and has been TASK-3 potassium channel proposed to promote tumor formation. Here we investigated whether pharmacological modulation of TASK-3, Pharmacological targeting and specifically of mitochondrial TASK-3 (mitoTASK-3), had any effect on cancer cell survival and mitochon­ Melanoma drial physiology. A novel, mitochondriotropic version of the specific TASK-3 inhibitor IN-THPP has been syn­ Chemical compounds studied in this article: thesized by addition of a positively charged triphenylphosphonium moiety. While IN-THPP was unable to induce Staurosporin (PubChem CID: 44259) FCCP (PubChem CID: 3330) apoptosis, mitoIN-THPP decreased survival of breast cancer cells and efficientlykilled melanoma lines, which we Antimycin A (PubChem CID: 16218979) show to express mitoTASK-3. Cell death was accompanied by mitochondrial membrane depolarization and fragmentation of the mitochondrial network, suggesting a role of the channel in the maintenance of the correct function of this organelle. In accordance, cells treated with mitoIN-THPP became rapidly depleted of mito­ chondrial ATP which resulted in activation of the AMP-dependent kinase AMPK. Importantly, cell survival was not affected in mouse embryonic fibroblasts and the effect of mitoIN-THPP was less pronounced in human melanoma cells stably knocked down for TASK-3 expression, indicating a certain degree of selectivity of the drug both for pathological cells and for the channel. In addition, mitoIN-THPP inhibited cancer cell migration to a higher extent than IN-THPP in two melanoma cell lines. In summary, our results point to the importance of mitoTASK-3 for melanoma cell survival and migration. 1. Introduction mitochondrial counterpart of the voltage-gated potassium channel Kv1.3 (mitoKv1.3), the calcium uniporter (MCU) - and of the perme­ A variety of channel types – in the plasma membrane (PM) as well as ability transition pore (MTPT) has indeed been shown to affect cancer in intracellular organelles – are involved in neoplastic progression and cell survival, metastatic potential and apoptosis [5–11]. Importantly, contribute considerably to the acquisition of some hallmarks of cancer pharmacological modulators of inner mitochondrial membrane (IMM) [1–4]. Mitochondrial ion channels/transporters are interesting targets channels can lead to cell death bypassing the upstream players of since they play a crucial role in setting the physiological parameters in intrinsic apoptosis (for example p53, Bax/Bak/Bcl-2) (e.g. [12,13]). an organelle that is fundamental not only for modulating the metabolic Therefore, this approach can be useful also in the case of pathological state of cancer cells but also for their removal by apoptosis. Pharma­ cells that are resistant to chemotherapeutics due to loss of p53 function cological or genetic manipulation of some of the ion channels in this or to overexpression of anti-apoptotic proteins such as Bcl-2. organelle - for example the voltage-gated anion channel VDAC, the Given the documented advantages of targeting mitochondrial ion * Corresponding author at: Department of Biology, University of Padua, Italy. E-mail address: [email protected] (I. Szabo). https://doi.org/10.1016/j.phrs.2020.105326 Received 8 August 2020; Received in revised form 3 November 2020; Accepted 23 November 2020 Available online 15 December 2020 1043-6618/© 2020 Elsevier Ltd. All rights reserved. M. Bachmann et al. Pharmacological Research 164 (2021) 105326 channels, and the presence of TASK-3 (Twik-related Acid-Sensitive K 2 (278 mg, 0.628 mmol, 97 % yield). 1H NMR (500 MHz, DMSO) δ 12.19 channel-3) (also called KCNK9, K2P9) in the mitochondria of several (br, 1 H), 8.51 (s, 1 H), 7.82 – 7.61 (m, 4 H), 7.60 – 7.43 (m, 4 H), 7.43 – types of cancer cells, our aim was to examine whether specific phar­ 7.35 (m, 1 H), 4.83 – 4.37 (br m, 2 H), 4.10 – 3.36 (br m, 4 H), 3.16 – macological targeting of this channel (mitoTASK-3) might affect mito­ 2.69 (br m, 4 H), 2.63 – 2.31 (br m, 1 H), 2.07 – 1.41 (br m, 4 H). 13C chondrial physiology and cell survival. TASK-3 is normally expressed NMR (126 MHz, DMSO) δ 175.8, 169.1, 162.0, 155.4, 141.6, 139.3, mainly in the central nervous system, is voltage-independent, is 134.4, 129.0, 127.9, 127.6, 126.9, 126.8, 115.1, 47.4, 40.1, 29.0, 28.0. + responsible for the so-called basal leak potassium current across the ESI-MS: 443 m/z [M+H] . plasma membrane and is involved in oxygen sensing as well as in regulation of the action potential. This protein is a two-pore channel 2.3. Synthesis of intermediate 3: 3-iodopropyl 1-(6-([1,1’-biphenyl]-4- [14], emerging as an important participant in tumor progression, dis­ carbonyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-yl)piperidine-4- playing oncogenic potential [15]. carboxylate (3) In particular, TASK-3 is overexpressed 5–100 fold in 44 % of breast tumors, in 35 % of lung cancers, and in over 90 % of ovarian cancers 2 (25.0 mg, 56.5 μmol, 1 eq.) was dissolved in anhydrous DMF (1.5 [16]. The channel is highly expressed also in melanoma [17–19]. Its mL), K2CO3 (15.6 mg, 113 μmol, 2 eq.) was added and the mixture was + overexpression promotes tumor formation depending on its K channel stirred for 15 min at room temperature. 1,3-diiodopropane (65 μL, 0.57 function [20] and confers resistance to hypoxia in vitro, suggesting one mmol, 10 eq.) was added and the mixture was stirred at room temper­ pathologically important role in cancer [15]. In vitro, knockdown of ature for 2 h. Then it was diluted with EtOAc (50 mL) and washed with KNCK9 has been associated with cell cycle arrest, reduced proliferation, water (50 mL). The organic phase was dried over Na2SO4 and concen­ induction of cellular senescence, increased apoptosis and/or reduced trated under reduced pressure. The crude product was purifiedby flash migration in different cancer cell models [21,22]. In accordance with silica gel column chromatography (DCM/acetone 6:4) to afford product 1 these data, a monoclonal anti-TASK-3 antibody raised against the 3 (30.2 mg, 49.5 μmol, 88 % yield). H NMR (500 MHz, CDCl3) δ 8.59 (s, extracellular domain of the channel and shown to inhibit PM TASK-3 1 H), 7.68 – 7.62 (m, 2 H), 7.62 – 7.56 (m, 2 H), 7.52 – 7.42 (m, 4 H), activity was able to inhibit human lung cancer xenograft growth and 7.40 – 7.35 (m, 1 H), 4.68 (br, 2 H), 4.32 – 3.53 (br m, 6 H), 3.30 – 2.78 breast cancer metastasis in vivo [23]. Furthermore, KCNK9 has been (br m, 6 H), 2.66 – 2.40 (br m, 1 H), 2.23 – 1.65 (br m, 6 H). 13C NMR identified as one of the genes enriched in lung metastasis of (126 MHz, CDCl3) δ 174.1, 170.7, 163.8, 156.2, 143.2, 140.2, 134.1, triple-negative breast cancer patients [24] and, along with other TASK 129.0, 128.0, 127.6, 127.4, 127.3, 115.3, 64.3, 48.0, 41.0, 32.3, 29.7, + channels, as diagnostic and prognostic marker in hepatocellular carci­ 29.4, 28.1, 1.4. ESI-MS: 611 m/z [M+H] . noma [25]. In addition to the roles of PM TASK-3, an important contribution of 2.4. Synthesis of compound 4: (3-((1-(6-([1,1’-biphenyl]-4-carbonyl)- mitoTASK-3 to the regulation of cell survival has been proposed as 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-yl)piperidine-4-carbonyl) pointed out by multiple lines of evidence. TASK-3 has been identifiedfor oxy)propyl)triphenylphosphonium iodide (4; mitoIN-THPP) the first time in intracellular membranes by immunohistochemistry of melanoma tissues [17] and was later on identified specifically in the 3 (30.2 mg, 49.5 μmol, 1 eq.) and PPh3 (51.9 mg, 198 μmol, 4 eq.) mitochondria of melanoma cells [18]. TASK-3 channel activity was also were dissolved in acetonitrile (1.0 mL) and the mixture was stirred for ◦ recorded directly by patch clamping the mitochondrial inner membrane 23 h at 80 C in a sealed vial. The solvent was then removed under [19]. Other TASK channels are not known to be located in mitochondria, reduced pressure and the residue was purifiedby flashsilica gel column and to the best of our knowledge their expression in melanoma cell lines chromatography (DCM/MeOH 95:5) to afford mitoIN-THPP (32.2 mg, 1 has not been reported.
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