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

Pharmacological Research 164 (2021) 105326

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Pharmacological Research

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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. 36.9 μmol, 75 % yield). H NMR (500 MHz, CDCl3) δ 8.54 (s, 1 H), 7.86 – Despite the variety of known biologically relevant functions of TASK- 7.76 (m, 9 H), 7.74 – 7.66 (m, 6 H), 7.65 – 7.60 (m, 2 H), 7.60 – 7.55 (m, 3 and the therapeutic potential of its modulation, there are only a few 2 H), 7.50 – 7.40 (m, 4 H), 7.38 – 7.32 (m, 1 H), 4.69 (br, 2 H), 4.35 (br, 2 reported examples of small molecule inhibitors [26]. These include 5,6, H), 4.12 – 3.49 (br m, 6 H), 3.18 – 2.79 (br m, 4 H), 2.60 (br, 1 H), 2.21 – 13 7,8-tetrahydropyrido[4,3-d]pyrimidine (THPP)-based compounds [27] 1.42 (br m, 6 H). C NMR (126 MHz, CDCl3) δ 174.1, 170.6, 163.8, capable of reaching intracellular membranes and molecules like DR16, 156.0, 143.1, 140.1, 135.4 (d, JCP = 2.4 Hz), 134.1, 133.8 (d, JCP =10.1 DR16.1 [28] as well as the recently described Withaferin A [29], all Hz), 130.8 (d, JCP =12.6 Hz), 129.0, 128.0, 127.6, 127.4, 127.2, 117.8 proven to inhibit TASK-3 currents. We used THPP-derivatives for our (d, JCP =86.4 Hz), 115.2, 63.4 (d, JCP =17.3 Hz), 48.0, 40.8, 28.1, 22.3 + studies in order to dissect the specific function of mitoTASK-3 and to (d, JCP = 2.4 Hz), 20.4 (d, JCP = 52.9 Hz). ESI-MS: 745 m/z [M-I] . obtain information on the cellular effects of THPP-based compounds in the context of melanoma. 2.5. Analysis of mitoIN-THPP hydrolysis

2. Materials and methods B16F10 cells were seeded in 100 mm culture dishes and allowed to grow to 80–90 % confluency.Cells were treated either for 2 h or for 24 h 2.1. Synthesis of mitoIN-THPP with 10 μM mitoIN-THPP in 8 mL phenol red- and serum-free medium. The medium was then removed, cells were washed with PBS, harvested The synthetic Scheme is shown in Fig. 1 A (Lower part). Compound 1 in 2 ml ice-cold PBS with a scraper and centrifuged at 800 g for 5 min at ◦ ◦ (IN-THPP) was synthesized as described in Coburn et al. [27]. 4 C. The medium and the cell pellet were stored at -80 C until analysis. Cell pellets were resuspended in 100 μL acetonitrile, vortexed, sonicated 2.2. Synthesis of compound 2: 1-(6-([1,1’-biphenyl]-4-carbonyl)- for 5 min and finallycentrifuged at 12000 g for 7 min. The supernatant 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-yl)piperidine-4-carboxylic was then collected and analyzed by HPLC-UV. The medium was acid (2; IN-THPP-COOH) centrifuged (12000 g, 7 min) to remove cell debris, and analyzed without further treatments. HPLC analysis was performed with a 1290 1 (305 mg, 0.648 mmol, 1 eq.) was dissolved in MeOH (6 mL) and a Infinity LC System (Agilent Technologies) equipped with a UV diode 1.0 M aqueous solution of KOH (1.94 mL, 1.94 mmol, 3 eq.) was added. array detector (190 500 nm), using a reverse phase column (Zorbax The mixture was stirred for 4.5 h at room temperature. Then it was RRHT Extend-C18, 1.8 μm, 50 × 3.0 mm i.d.; Agilent Technologies) kept ◦ diluted with a saturated solution of NH4Cl (300 mL) and extracted with at 35 C. Solvents A and B were water containing 0.1 % trifluoroacetic EtOAc (300 mL, then 3 × 100 mL). The total organic phase was dried acid (TFA) and acetonitrile, respectively. The gradient for solvent B was over Na2SO4 and concentrated under reduced pressure to afford product as follows: B 10 % for 0.5 min, then from 10 % to 100 % in 4.5 min, 100

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◦ % for 1 min. The flow rate was 0.6 mL/min. The eluate was preferen 1”) and centrifuged at 6000 g for 10 min at 4 C. The resulting pellet was tially monitored at 270 nm (corresponding to the absorbance maximum resuspended in a small volume of TES buffer (50 μL were stored at 80 ◦ for mitoIN-THPP and IN-THPP-COOH). Analytes were quantifiedusing a C as “fraction 2”) and further purified on a discontinous Percoll calibration curve correlating the peak area with the concentration of the gradient (60 %, 30 % and 18 % in TES buffer) by centrifugation at 8500 g ◦ analytes. for 10 min at 4 C. The floatingmaterial in the 18 % layer, containing ER and plasma membrane contaminants (denominated “fraction 3”), and 2.6. Cell culture material at the upper interface, containing purified mitochondria (denominated “m”), were collected and washed three times in TES ◦ B16F10 cells (kind gift of Prof. Erich Gulbins, University of Essen) buffer with centrifugation at 17000 g for 10 min at 4 C. The finalpellet ◦ were grown in Minimum Essential Medium (MEM, Gibco) supplemented was resuspended in buffer and stored at -80 C. n = 3. with 10 % (v/v) fetal bovine serum (FBS, Gibco), 100 U/mL penicillin G, 0.1 mg/mL streptomycin (Gibco), 1% non-essential amino acids (100X 2.10. Western blot solution, Gibco), 2 mM L-Glutamine (100X solution, Gibco) and 1 mM sodium pyruvate (100X solution, Gibco). MEF cells (kind gift of Prof. Purified mitochondria and the fractions from the purification steps Luca Scorrano, Padova) were cultured in DMEM (4.5 g/mL D-glucose) were quantified by the BCA assay and 25 μg from each sample were supplemented with 10 % FBS, 100 U/mL penicillin 0.1 mg/mL strep loaded onto 4–12 % Bis-Tris gels (Genscript). Proteins were blotted on tomycin, 10 mM Hepes (100X solution, Gibco) and 1% non-essential PVDF membranes with BioRad Transblot® Turbo™ standard protocols. amino acids. IPC-298 and SK-MEL-2 human melanoma cells were Membranes were blocked with 2% BSA in TBS for 1 h at room temper cultured in DMEM GlutaMAX™ (Gibco) supplemented as above. MDA- ature and incubated overnight with the following antibodies: PMCA MB-231 human triple-negative breast cancer cells were cultured in (1:2000 in TTBS, Thermofisher#MA3-914); Calnexin (1:1000 in TTBS, DMEM-F12 (Gibco) supplemented with 10 % (v/v) FBS, 100 U/mL Abcam #ab22595); TASK-3 (1:200 in TBS-2% BSA, Sigma-Aldrich penicillin G, 0.1 mg/mL streptomycin, 1% non-essential amino acids, 2 #P5247); VDAC1 (1:2000 in TTBS, Santa Cruz #sc-390996); TOM20 mM L-Glutamine and 1 mM sodium pyruvate. WM-35 cells were main (1:1000 in TTBS, Santa Cruz #sc-11415). tained as previously described [30]. All cells were used at a maximal Expression of TASK-3 in B16F10 cells and MEFs was analysed in passage number of 30 depending on the cell line. whole-cell lysates. Briefly, 106 cells were detached, washed in PBS and resuspended in lysis buffer (1 M sucrose, 100 mM TES, 100 mM EGTA, 2 2.7. MTS assay and calculation of EC 50 mM DTT, 1% NP-40, 1X protease inhibitors). The lysate was vortexed for ◦ 30 s and centrifuged at 15000 g for 10 min at 4 C. Protein concentration 7,500 cells/well were seeded in standard 96-well plates and allowed in the supernatant was quantifiedand 50 μg of each sample were loaded to grow in normal culture medium for 24 h. The growth medium was on precast gels and blotted as above. The amount of TASK-3 protein was then replaced with phenol red-free DMEM (supplemented with 10 mM determined using β-actin as loading control (1:3000 in TTBS, Chem HEPES, 100 U/mL penicillin G, 0.1 mg/mL streptomycin, 1% non- icon® #MAB-1501). n = 4. essential amino acids, 2 mM L-Glutamine and 1 mM sodium pyruvate) Expression of TASK-3 in IPC-298, SK-MEL-2 and MDA-MB-231 cells containing the desired compound. Each condition was tested in was analysed in membrane-enriched fractions, obtained using Pro quadruplicate. After treatment for 24 h, CellTiter 96® AQ eous One eous teoExtract® Native Membrane Protein Extraction Kit (Sigma Aldrich, Solution (Promega) was used to determine cell viability following the #444810) following manufacturer’s instructions. 40 μg of each sample manufacturer’s protocols. The percentage of viable cells was plotted were loaded on precast gels and blotted as above. PMCA was used as a against log of the drug concentration (expressed in μM units) and the 10 loading control. concentration at which the assay readout was reduced by 50 % was For investigation of α-AMPK activation, 200,000 B16F10 cells were calculated using the sigmoidal curve fittingfunction of OriginLab©. For seeded in normal culture medium. The next day, they were treated in all cell lines used, n≥3. phenol red-free DMEM (supplemented with 10 mM HEPES, 100 U/mL penicillin G, 0.1 mg/mL streptomycin, 1% non-essential amino acids, 2 2.8. Annexin V assay mM L-Glutamine and 1 mM sodium pyruvate) with 40 μM mitoIN-THPP or IN-THPP or the corresponding amount of DMSO (0.1 %) for 8 h, 15,000 cells/well were seeded in standard 48-well plates and washed in PBS and lysed as above. 30 μg of each sample were loaded on allowed to grow in normal culture medium for 24 h. The growth medium precast gels and blotted as above. The membrane was incubated over was then replaced with phenol red-free DMEM (supplemented with 10 night with antibodies against AMPK pT172 (1:1000 in TBS-2% BSA, Cell mM HEPES, 100 U/mL penicillin G, 0.1 mg/mL streptomycin, 1% non- Signaling Technologies #2535) or AMPK (1:1000 in TBS-2%BSA, Cell essential amino acids, 2 mM L-Glutamine and 1 mM sodium pyruvate) Signaling Technologies #5831). Vinculin (1:1000 in TTBS, EMD Milli containing the desired compound. After treatment for 24 h, 0.5 μL ◦ pore #AB6039) was used as housekeeping control and the amount of Annexin V-FITC was directly added to each well, incubated at 37 C for phosphorylated α-AMPK/total α-AMPK was calculated after densitom 30 min and the cells were visualized with a Leica DMI4000 inverted etry using Image Lab software (Biorad). n = 3. microscope. Annexin V-positive cells and total cell number were deter mined using ImageJ software and the percentage of positive cells/total cell number was calculated. For all cell lines used, n = 3 4. 2.11. Mitochondrial morphology

2.9. Isolation of mitochondria 70,000 B16F10 cells were seeded on glass coverslips in standard 6- well plates in normal culture medium and allowed to grow for 48 h. B16F10 cells were seeded in 150 mm culture dishes in normal culture The growth medium was then replaced with phenol red-free DMEM medium. About 60*106 cells at 80–90 % confluencywere harvested with (supplemented with 10 mM HEPES, 100 U/mL penicillin G, 0.1 mg/mL a scraper, washed in ice-cold PBS and resuspended in TES buffer (300 streptomycin, 1% non-essential amino acids, 2 mM L-Glutamine and 1 mM sucrose, 10 mM TES, 0.5 mM EGTA, pH 7.4). Resuspended cells mM sodium pyruvate) containing the treatment compound at the were kept on ice for 1 h, then fragmented with a Dounce homogenizer. desired concentration. After 8 h of treatment, cells were incubated with ◦ Intact cells were pelleted by centrifugation at 1000 g for 10 min at 4 C, 200 nM MitoTracker™ Green (Thermofisher) in HBSS for 30 min at 37 ◦ resuspended in TES buffer, homogenized and centrifuged again. The C and subsequently visualized with a Leica SP5 confocal microscope. n ◦ supernatants were combined (200 μL were stored at -80 C as “fraction = 3.

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2.12. Mitochondrial membrane potential and ROS production for reasons of synthetic expediency. The study of Coburn et al. [27] provided a detailed structure-activity 6,500 B16F10 cells/well were seeded in glass bottom 96-well cell relationship (SAR) elucidated from high-throughput screening of a imaging plates in standard culture medium and incubated for 24 h. 3000-membered THPP compound library. SAR elucidated that substi Then, cells were incubated with either 25 nM TMRM (Thermofisher)or tution at the C-4 position (R1 in Fig. 1A) tolerated a wide variety of ◦ 1 μM mitoSOX™ Red (Thermofisher) in HBSS for 20 min at 37 C. The substituents, while those at other positions of the THPP scaffold resulted medium was then substituted with HBSS (supplemented with 5 nM in significant changes in potency and selectivity. In IN-THPP (Fig. 1A) TMRM in the case of membrane potential determinations) and cells were the THPP scaffold is substituted at the C-4 position with ethyl 4-piperi imaged for 10 min using the Operetta® system (PerkinElmer). After 10 dinecarboxylate (ethyl IsoNipecotate) through a C–N bond and at the min, treatment compounds at the indicated concentration were added N-6 position with a p-biphenyl-4-carbonyl group. and cells were imaged every 5 min for 40 min. 5 fields/well were ana Here, we describe the synthesis and pharmacological evaluation of lysed. Each condition was tested in duplicate. Analysis was performed mitoIN-THPP (Fig. 1A), a mitochondria-targeted derivative of IN-THPP. using Harmony ® high-content analysis software. n = 3. To target the compound to mitochondria, we conjugated a lipophilic The results from Operetta analysis were confirmed also by live cell triphenylphosphonium (TPP) cation to the molecular structure of IN- imaging using a Leica SP5 microscope. Briefly, 70,000 B16F10 cells THPP. TPP-containing molecules have been reported to exploit the were seeded on glass coverslips in standard 6-well plates in normal electrical potential difference across the IMM to pass through the culture medium and allowed to grow for 48 h. Cells were incubated with phospholipid bilayers and selectively accumulate within minutes in the ◦ 25 nM TMRM or 1 μM mitoSOX™ Red in HBSS for 20 min at 37 C and mitochondria in cultured cells and in vivo (e.g. [13,31–34]). subsequently imaged using the same setting as above. 1 μM FCCP was TPP-modified compounds exhibit anti-cancer activity in different set used as a control at the end of each TMRM experiment time-course and tings [35–38]. mitoIN-THPP contains a reversible carboxyester link cells were imaged 5 min after FCCP addition. n = 2. between the TPP moiety and the remaining part of the molecule (IN-THPP-COOH in Fig. 1A). Given that modification of the R1 group 2.13. ATP measurement was shown to affect only slightly the high, sub-μM affinity for the channel [27], IN-THPP-COOH, formed by hydrolysis of the ester bond, ATP amount was measured in B16F10 cells using the ATPlite can be assumed to be an efficient inhibitor of TASK-3, like the other, Luminescence ATP Detection Assay System (PerkinElmer). 7,000 cells/ previously described THPP analogs. The other product of the hydrolysis well were seeded onto white 96-well viewplates. To measure mito is TPPP-OH (3-hydroxypropyl)triphenylphosphonium iodide), har chondrial ATP production, 48 h after seeding glycolysis was inhibited bouring the mitochondria-targeting TPP moiety. pre-incubating cells with 5.5 mM 2-deoxy-D-glucose (2-DG) for 1 h and Next, we tested if mitoIN-THPP is indeed able to release IN-THPP- then all treatments were performed in medium containing 2-DG. For COOH following administration to intact cells. After 2 or 24 h of incu total ATP content, treatments were performed in medium containing 5.5 bation with 10 μM mitoIN-THPP, cells retained both mitoIN-THPP and mM glucose. 1 μg/mL oligomycin was used as positive control. 2 h after IN-THPP-COOH (Fig. 1B). Assuming that the volume of a single cell is treatment, the assay was performed following the manufacturer’s in about 3000 femtoliters [39], and since the analyzed cell pellets contain structions. n = 3. about 4 × 106 cells, these results correspond to concentrations, in each cell, of at least 800 nM for the active molecule, IN-THPP-COOH. These 2.14. Wound scratch assay levels are 10 times higher than the concentration required for channel inhibition by IN-THPP [27]. In the culture medium the relative abun 35,000 B16F10 cells/well or 45,000 SK-MEL-2 cells/well were dance of mitoIN-THPP and IN-THPP-COOH after 24 h of incubation was seeded in standard 24-well plates in standard culture medium and quite different, with IN-THPP-COOH being as abundant as allowed to grow for 48 h. SK-MEL-2 cells were treated with 5 μg/mL mitoIN-THPP. These results can be explained considering that mitomycin C (Sigma Aldrich, #M4287) for 1 h prior to performing the mitoIN-THPP accumulates into cells/mitochondria thanks to the pres scratch. The scratch was performed with a sterile p200 pipette tip. The ence of the positively charged TPP moiety; once inside, the derivative is medium was removed and substituted, after one wash in PBS, with hydrolyzed by cellular esterases to release IN-THPP-COOH, which then phenol red- and serum-free medium in case of B16F10 cells and with can exit the cell because of the lack of the TPP. To restore the electro phenol red-free medium with 1% FBS in case of SK-MEL-2 cells, con chemical equilibrium, more mitoIN-THPP enters the cells, but it is then taining the treatment compound at the indicated concentration. Images hydrolyzed, and so forth. These events take place continuously as long as of the same fields were taken 0, 15, 20 and 24 h (B16F10) or 0, 24, 48 mitoIN-THPP is still present in the medium. In summary, these results and 72 h (SK-MEL-2) after performing the scratch with a Leica DMI4000 demonstrate that mitoIN-THPP is indeed bioreversible at least in part inverted microscope. The area of the scratch was calculated using and thus, it can release an efficientTASK-3 inhibitor at the site of action, ImageJ software. The migration rate is expressed as % of the initial gap i.e. in mitochondria. As expected, IN-THPP-COOH was able to block area. n = 3. TASK-3 activity in preliminary patch clamp experiments on mitoplasts from HaCaT cells, prepared according to [19] (not shown). 2.15. Statistical analysis The two drugs, IN-THPP and mitoIN-THPP, were compared then for their efficiency in modulating the viability of a mouse melanoma cell All statistical analyses were performed using GraphPad Prism soft line (B16F10), of three human melanoma lines (WM-35, SK-MEL-2 and ware. Statistical details can be found in the figure legends. IPC-298) as well as of a human breast cancer line (MDA-MB-231). Fig. 2A shows that both drugs decrease the B16F10 MTS (3-(4,5-dime 3. Results thylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium) readout, with different EC50’s (20 μM for mitoIN-THPP vs. The molecules that inhibit TASK-3 with highest affinity are those 40 μM for IN-THPP). Likewise, the sensitivity of SK-MEL-2 and IPC-298 based on the THPP scaffold [27] (see Fig. 1A). Of direct relevance for cells was significantlyhigher for the mitochondriotropic drug (Fig. 2A). this paper is the molecule denoted “12f” in ref. [27], which we will In the case of MDA-MB-231 cells, 40 μM mitoIN-THPP decreased the henceforth indicate as “IN-THPP” (see Fig. 1A). According to [27], MTS signal by 50 % while the non-mitochondrial version had the same IN-THPP inhibits TASK-3 with a satisfactorily low IC50 of 74 ± 9 nM. effect at around 70 μM. In light of the consistent dose-dependent PK-THPP (compound 23 in ref. [27]) has an even lower IC50, but we decrease of the MTS signal and of comparable EC50 values (Table 1) as chose IN-THPP as the basis for the new mitochondriotropic derivative well as TASK-3 expression levels (Fig. S1A) in different melanoma lines,

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Fig. 1. Synthesis and stability of mitoIN-THPP. A) Upper part: General structure of the 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine (THPP) scaffold, structure of IN-THPP, of mitoIN-THPP and of IN-THPP-COOH (see text). Lower part: main steps of the synthesis; i) MeOH, KOH 1.0 M, RT, 4.5 h, 97 % yield; ii) DMF, K2CO3, 1,3- ◦ diiodopropane, RT, 2 h, 88 % yield; iii) acetonitrile, PPh3, 80 C, 23 h, 75 % yield. B) Levels of mitoIN-THPP and its hydrolysis product (IN-THPP-COOH) in the medium and in B16F10 cells after 24 h of incubation with 10 μM mitoIN-THPP. See Materials and Methods for details. Mean values ± SEM, n = 5. we focused our subsequent work on this disease. Aiming to understand demonstrating that solely IN-THPP-COOH is responsible for the whether these drugs can diminish the MTS signal in a cancer-specific observed effects at the concentrations used in the study (20 and 40 μM). manner, we employed immortalized mouse embryonic fibroblasts These results are also in agreement with the findings that propyl-TPP (MEFs) as a control and compared TASK-3 expression in B16F10 versus does not affect proton leak and respiration [41]. MEFs using a specific antibody (Fig. 2B). In accordance with the two- Given the strong effects of mitoIN-THPP on cell survival, we inves fold higher expression of TASK-3 in the melanoma cells, they were tigated the localization of TASK-3 in the widely used B16F10, in order to significantlymore sensitive to both drugs with respect to MEFs (Fig. 2C). generalize previous findings, i.e. that TASK-3 is expressed in mito Finally, to confirm specificity of the drug’s action on the TASK-3 chan chondria in different melanoma lines. Fig. 3A shows that a specific nel, we employed human melanoma WM-35 cells, previously shown to antibody against TASK-3 gives a strong recognition of a 40 kDa band express mitoTASK-3 [40]. These cells, stably transfected either with (compatibly with the expected molecular weight of TASK-3) in the scrambled shRNA or with shRNA targeting TASK-3, resulting in an 60 % partially purified (f3) and in the highly purified (m) mitochondrial decrease of the channel protein level [40], were tested for their sensi fraction. In f3 a higher band (45 kDa) is also visible, similarly to other tivity to mitoIN-THPP (Fig. 2D and E). Both knock-down lines displayed studies and as also observed in Fig. S1A (e.g. [30]). At equal protein a greater resistance to mitoIN-THPP compared to scrambled loading, similarly to TASK-3, a progressive enrichment of the mito shRNA-transfected control cells, indicating that the action of the drug chondrial porin VDAC-1 and of TOM-20 can be observed in the mito takes place prevalently via TASK-3. chondrial fractions with respect to the whole-cell lysate (f1) and to the Measurement of mitochondrial metabolic rate using MTS indirectly membrane-enriched fractions (f2), while the ER membrane-resident reflects viable cell number, however the metabolic activity may be calnexin and the calcium-ATPase pump of the plasma membrane changed by application of drugs without affecting cell viability. There (PMCA) show the opposite tendency. These results indicate that TASK-3 fore, to assess whether a decrease in the MTS signal correlated with an is indeed present in the mitochondria of B16F10 cells. increase in cell death, the Annexin V-binding assay was employed to When assessing the effect of the two drugs on mitochondrial func visualize apoptotic cells in B16F10, MEF (Figs. 2F, G and S1B), SK-MEL- tion, a significant decrease of Δψ, and an almost complete depolariza 2 (Fig. S1C) and IPC-298 cells (Fig. S1D). Interestingly, MEF cells did not tion within 40 min (Fig. 3B and C, see also Fig. S2A) was observed only undergo apoptosis, while B16F10 cells were resistant to IN-THPP but upon incubation of the cells with mitoIN-THPP, consistent with both the underwent programmed cell death up to 60 % when incubated for 24 h localization of the channel in mitochondria and death induction only by with 40 μM mitoIN-THPP (Figs. 2F, G and S1B). These results suggest mitoIN-THPP. TPPP-OH did not exert the same effect (Fig. S2B). Inhi that both THPP derivatives are able to alter mitochondrial metabolism bition of mitochondrial potassium channels has been reported to trigger and in particular the ability of dehydrogenases converting tetrazolium mitochondrial ROS production (e.g. [42,43]), that in turn above a ring to formazan, but only mitoIN-THPP can trigger apoptosis. Impor certain threshold may lead to long-lasting opening of the permeability tantly, MEFs were resistant to both IN-THPP and mitoIN-THPP. In light transition pore with consequent mitochondrial depolarization [44]. In of a recent observation reporting toxicity of some TPP moieties [41] and our case, both IN-THPP and mitoIN-THPP triggered mitochondrial ROS to further confirm that the action of mitoIN-THPP is due to the active release with the latter drug being significantlymore effective (Figs. 3D, molecule (IN-THPP-COOH) released after hydrolysis of the ester bound E and S3A), while TPPP-OH did not change ROS levels (Fig. S3B). + and not to the released TPP moiety (TPPP-OH (TPP-propyl-OH)), we Loss of mitochondrial membrane potential is generally associated tested the cellular effects of this latter molecule. TPPP-OH itself was not with fragmentation of the mitochondrial network [45,46]. In accor able to trigger apoptosis in B16F10 cells even up to 80 μM (Fig. S1E), dance, mitoIN-THPP but not IN-THPP triggered mitochondrial fission

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Fig. 2. MitoIN-THPP induces apoptosis of melanoma cells. A) Dose-response curves for EC50 calculation and sensitivity of B16F10, SK-MEL-2, IPC-298 and MDA- MB-231 cells to IN-THPP and mitoIN-THPP. The concentration at which 50 % of cells were viable (EC50) is indicated by the dotted line. EC50 values are shown in Table 1. The statistical difference between the curves was assessed with Two-Way Anova. n = 3-5. B) Representative Western Blot showing the amount of TASK-3 dimer in MEF and B16F10 cells. 50 μg protein/lane were loaded. β-actin was used as loading control. The densitometric analysis is shown below (n = 4, mean + SEM, Welch’s t test, * p-value < 0.05). C) EC50 values (MTS assay) of IN-THPP and mitoIN-THPP for MEF and B16F10 cells, calculated as shown in A) (n = 5, mean + SEM, unpaired t test, * p-value < 0.05, *** p-value < 0.001). D) Dose-response curves showing the sensitivity of WM-35 cells (scrambled control cells and two stable TASK- 3 knock-down lines) to mitoIN-THPP. The % of viable cells is plotted against log10 of drug concentration. N = 3. The statistical difference was assessed by Two-Way Anova. E) Comparison of the sensitivity of WM-35 scrambled control cells and TASK-3 knockdown lines to mitoIN-THPP (treatment for 24 h) as assessed by MTS assay (n = 3, mean + SEM, Two-way Anova with Bonferroni’s posttest, *** p-value < 0.001). F) Representative images of an Annexin V-FITC cell death assay. B16F10 cells were treated for 24 h with the indicated compounds at the indicated concentration and apoptotic cells were stained with Annexin V-FITC. Staurosporin was used as a positive control. Bright field images, FITC fluorescence and the merged images are shown. Scale bar =75 μm. G) Quantification of Annexin V-FITC cell death assays. The percentage of FITC-positive cells amongst all cells after treatment for 24 h with the drugs at the indicated concentrations is reported for B16F10 (upper panel) and MEF cells (below) (n = 4, mean + SEM, One-Way Anova with Dunnett’s posttest, *** p-value < 0.001 with respect to the control indicated as “ref”).

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Table 1 mitochondrial membrane potential, and aldosterone production. The EC50 values of IN-THPP and mitoIN-THPP in different cell lines, tested with MTS observed tumor-promoting effect of TASK-3 has been hypothesized to be assays. EC50 values were calculated using the sigmoidal graph-fittingfunction of ascribable to mitoTASK-3 and to its role in the maintenance of mito OriginLab. The mean and standard deviation are indicated. The statistical dif chondrial potassium homeostasis. Indeed, a more than 50 % reduction in ference between IN-THPP and mitoIN-THPP in each cell line was assessed by TASK-3 protein in knockdown human melanoma cells resulted in unpaired t-test. The p-value of each comparison is indicated in the table. n = 3-5 swollen mitochondria and a significantlyreduced metabolic activity (by for each cell line. approximately 50 %) as assessed by MTS assay [51]. Mitochondria in μ μ Cell line EC50 IN-THPP ( M) EC50 mitoIN-THPP ( M) p-value TASK-3 knockdown cells were more depolarized and were fewer with B16F10 39.4 ± 6.4 20.9 ± 1.8 0.0008 respect to the organelles in the control cells. Most interestingly, 50 % of MEF 51.1 ± 9.1 39.4 ± 14.3 0.3246 the knockdown cells died by apoptosis upon induction of oxidative stress > μ ± SK-MEL-2 80 M 30.0 4.9 N.D. [30] in contrast to scrambled shRNA-transfected or non-transfected IPC-298 68.9 ± 17.5 5ì 20.8 ± 1.3 0.0015 MDA-MB-231 71.6 ± 7.4 39.6 ± 9.5 0.0100 cells, suggesting that either the lack of TASK-3 itself induces an oxida tive stress, or the channel is necessary for the anti-oxidant defence of melanoma cells. It is well known that cancer cells are characterized by a and impairment of the mitochondrial network (Fig. 4A). As the preser higher ROS level with respect to healthy cells [53] and that mitochon vation of the mitochondrial network is essential for ATP production by drial potassium channels regulate ROS production by this organelle, by mitochondria [47], we next measured cellular ATP in cells treated either modulating membrane potential and respiration (e.g. [13,43,54,55]). in the absence or the presence of 2-deoxyglucose (2-DG), an agent used Importantly, the relative contribution of mitoTASK-3 versus PM TASK-3 to block glycolysis. MitoIN-THPP caused a decrease in cellular ATP even remains to be determined. when glycolysis was not impaired, up to an extent similar to that trig Our results using mitoIN-THPP were similar to those obtained by gered by oligomycin, an inhibitor of the FOF1 ATP-synthase. When TASK-3 downregulation regarding mitochondrial depolarization and glycolysis was blocked, the ATP content was reduced to nearly zero at apoptosis induction. In addition, we show that mitoIN-THPP triggers a the higher drug concentration, indicating that mitoIN-THPP impaired massive mitochondrial fragmentation that is paralleled by complete mitochondrial function and cellular energetics (Fig. 4B). In contrast, mitochondrial ATP depletion, partial cellular ATP depletion and, as a TPPP-OH, the positively charged hydrolytic product, did not affect ATP consequence, AMPK activation. Apoptosis occurred in various human levels (Fig. S4A). IN-THPP itself was also able to decrease cellular ATP and mouse melanoma cells but MEFs, that are non-cancerous cells content by 30 % in the presence of 2-DG . A drop in the level of cellular expressing a low level of TASK-3, were resistant to mitoIN-THPP. The ATP and consequent increase in [AMP] activates the master kinase induction of apoptosis by mitoIN-THPP in cancer cells might be linked to AMPK (AMP-dependent kinase), that in turn has multiple effects within the increase in ROS level that may drive the cells above a critical the cells, including removal of cells with severely damaged mitochon oxidative threshold, but in part also to ATP depletion and subsequent dria by apoptosis and enhancement of mitochondrial fission. Indeed, AMPK activation, since treatment with 1-(3-chloro-4-((trifluoromethyl) treatment of cells with inhibitors of the electron transport chain that in thio)phenyl)-3-(4-(trifluoromethoxy)phenyl)urea (FND-4b) alone, a turn cause a decrease of the membrane potential, was able to activate novel AMPK activator, was sufficientto trigger apoptotic death in colon AMPK that in turn was required for fission [48]. In agreement with re cancer and in triple negative breast cancer cells [56]. Most likely all the sults of Fig. 4B, AMPK activation occurred upon treatment of B16F10 above events together decide cell fate upon treatment with with both IN-THPP and mitoIN-THPP, however to a greater extent with mitoIN-THPP. the latter drug. Indeed, both drugs increase phosphorylation of AMPK on As mentioned above, AMPK activation and ATP depletion are both threonine 172 of the α-subunit, resulting in its activation (Fig. 4C). linked not only to apoptosis induction, but also to an impairment of AMPK activation has been related to p53 activation as well as to invasion in melanoma cell lines [49,50]. Our observations are in impairment of invasion [49]. Likewise, inhibition of mitochondrial agreement with the assumption that the decrease in ATP levels observed respiration and depletion of ATP was shown to prevent migration of even at sublethal concentrations of mitoIN-THPP might impact the metastatic melanoma [50]. Thus, we tested, using the scratch assay, migration of B16F10 cells. IN-THPP, itself a membrane-permeant in whether the two drugs slow down migration. As illustrated in Fig. 4E hibitor of TASK-3 which may therefore reach the mitochondrial channel, (representative image) and Fig. 4F (quantification), both IN-THPP and also decreased mitochondrial membrane potential, induced ROS release mitoIN-THPP negatively affected migration of B16F10 cells. This effect and reduced mitochondrial ATP production and cellular migration, but was more evident in the case of mitoIN-THPP (50 % inhibition of to a significantly lower extent than mitoIN-THPP. The migration) than with IN-THPP (10 % inhibition). To corroborate this migration-reducing effect of the mitochondriotropic drug was confirmed finding also in a human melanoma cell line, we performed the same also in a human metastatic melanoma line (SK-MEL-2). Although these experiment on SK-MEL-2, cells of metastatic origin, and obtained com data are suggestive of a so far unrecognized, important role played by parable results taking into account the EC50 values (Figs. 4D and S4B). mitoTASK-3 versus PM TASK-3, we cannot fully exclude a contribution Altogether, these results underline the ability of mitoIN-THPP to effi of the latter channel to these processes. The role of mitoTASK-3 in the ciently impair melanoma cell migration. regulation of the above-mentioned processes has been inferred by using knock-out or knock-down approaches, which however target both PM 4. Discussion and mitoTASK-3 and cannot conclusively determine the role of mitoTASK-3. Our data thus substantially reinforce the hypothesis that In the present study we exploit a novel, mitochondria-targeted mitoTASK-3 is involved in apoptosis induction and point to novel as version of IN-THPP, a rather specific TASK-3 inhibitor, in order to pects of mitoTASK-3 modulation such as AMPK activation and regula dissect the cellular effects of the pharmacological modulation of tion of migration. mitoTASK-3 in melanoma cells. Human melanoma cell lines have been MitoIN-THPP triggers apoptosis, but at relatively high concentra extensively studied regarding the expression of mitoTASK-3, however, tions in all tested cell lines. Given that modification of IN-THPP at C-4 to our knowledge, no attempts have been carried out to pharmacologi does not profoundly affect the affinity of the drug variants for TASK-3 cally target this channel. [27], this is not a likely cause of the requirement for high dosage to The presence of TASK-3 in mitochondria has previously been induce cell death. According to a virtual screening [28], the drug/ demonstrated in human WM35 cells [51] and in A2058 melanoma cells channel interaction is mediated mainly by contacts between N3 of the 5, [30]. TASK-3 was also identified in mitochondria of zona glomerulosa 6,7,8-tetrahydropyrido[4,3-d]pyrimidine moiety and the carbonyl oxy cells [52], where it was shown to regulate mitochondrial morphology, gen, acting as hydrogen bond acceptors, and threonines in the channel

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Fig. 3. Mitochondriotropic TASK-3 inhibitors profoundly affect mitochondrial physiology. A) Representative Western Blot showing the mitochondrial localization of TASK-3 in B16F10 cells. f1: whole-cell extract; f2: membrane-enriched fraction; f3: mitochondria-enriched fraction; m: Percoll-purifiedmitochondria (see Experimental Methods). The same amount of proteins (25 μg/lane) of the different fraction were loaded onto SDS-PAGE and were decorated. Plasma membrane marker PMCA, ER marker Calnexin and mitochondrial membrane markers VDAC-1 and TOM-20 are shown. B) Representative images of mitochondrial membrane potential changes in B16F10 cells visualized by changes in TMRM fluorescenceupon addition of 40 μM mitoIN-THPP or IN-THPP at the indicated time points. FCCP was used as a positive control. Scale bar =100 μm. C) Quantification of mitochondrial membrane potential changes in B16F10 cells as determined by analyzing TMRM fluorescenceusing Operetta and Harmony software. Basal fluorescencewas monitored for 10 min prior to addition of the drugs at the indicated concentration. Images were acquired every 5 min for 40 min after addition of the compounds. FCCP was used as a positive control. Fluorescence is expressed as percentage of the initial intensity (n = 3, mean +/- SEM, Two-Way Anova with Bonferroni’s posttest, * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001 with respect to the control, or between IN-THPP and mitoIN-THPP as indicated). D) Representative images of mitochondrial ROS production, followed as changes in mitoSOX fluo rescence, upon addition of 40 μM mitoIN-THPP or IN-THPP. Scale bar =100 μm. E) Quantification of ROS production in B16F10 cells by analyzing mitoSOX fluorescenceusing Operetta and Harmony software. Basal fluorescencewas monitored for 10 min prior to addition of the drugs at the indicated concentration. Images were acquired every 5 min for 40 min after addition of the compounds. Fluorescence is expressed as percentage of the initial intensity (n = 3, mean +/- SEM, Two- Way Anova with Bonferroni’s posttest, * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001 with respect to the control, or between IN-THPP and mitoIN-THPP as indicated).

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Fig. 4. MitoIN-THPP induces mitochondrial fragmentation and ATP depletion and reduces cell migration. A) Representative confocal images showing the mitochondrial network of B16F10 cells after 8 h of treatment with 40 μM mitoIN-THPP or IN-THPP, highlighted with the MitoTracker Green probe. After mitoIN- THPP treatment, mitochondria are visibly fragmented (n = 3, scale bar =25 μm). B) Total ATP (cells treated in medium containing glucose, upper panel) and mitochondrial ATP (cells treated in medium containing 5.5 mM 2-DG, below) was measured in B16F10 cells after treating them for 2 h with the drugs at the indicated concentration. Oligomycin was used as a positive control (n = 3, mean + SEM, One-Way Anova with Dunnett’s posttest, * p-value < 0.05, ** p-value < 0.01, *** p- value < 0.001 with respect to the control indicated as “ref”). C) On the left, representative Western Blots showing the level of phosphorylated α-AMPK after 8 h of treatment of B16F10 cells with DMSO 0.2 % (DMSO), 40 μM IN-THPP or 40 μM mitoIN-THPP (mitoT.). Vinculin was used as loading control. On the right, densitometric analysis of phosphorylated α-AMPK/total α-AMPK (n = 3, mean + SEM, Welch’s t test, * p-value < 0.05, ** p-value < 0.01 with respect to the control indicated as “ref”, or between mitoIN-THPP- and IN-THPP-treated samples as indicated). D) Quantificationof the migration rate of SK-MEL-2 cells treated with DMSO 0.2 % (control), 10 μM mitoIN-THPP or IN-THPP in wound scratch assays, as shown in S4B. The gap area was measured 0, 24, 48 and 72 h after performing the scratch using ImageJ and expressed as % of the initial gap area (n = 3, mean +/- SEM, Two-Way Anova with Bonferroni’s posttest, *** p-value < 0.001 with respect to the control). E) Representative images of wound scratch assays in B16F10 cells (quantified in F). The area of the gap has been highlighted in blue for better visualization and compared with the initial scratch. Scale bar =250 μm. F) Quantificationof the migration rate of B16F10 cells treated with DMSO 0.2 % (control), 10 μM mitoIN-THPP or IN-THPP in wound scratch assays, as shown in E. The gap area was measured 0, 15, 20 and 24 h after performing the scratch using ImageJ and expressed as % of the initial gap area (n = 3, mean +/- SEM, Two-Way Anova with Bonferroni’s posttest, ** p-value < 0.01, *** p-value < 0.001 with respect to the control, or between mitoIN-THPP and IN-THPP-treated samples as indicated). selectivity filter. The phenyl ring bound to the carbonyl contributes to Appendix A. Supplementary data hydrophobic interactions. On the other hand, please note that the EC50 value of TASK-3 current inhibition was found to be 1.1 ± 2.0 μM for Supplementary material related to this article can be found, in the PK-THPP when the effect of the drug was studied in a mammary online version, at doi:https://doi.org/10.1016/j.phrs.2020.105326. epithelial line [57] rather than on TASK-3 expressed in HEK293 cells, where the EC50 value was 35 nM only. Generally, a concentration higher References than that required to inhibit a given channel in patch clamp experiments + is needed to exert a biological effect. For example, Withaferin A can [1] L.A. Pardo, W. Stuhmer, The roles of K( ) channels in cancer, Nature reviews, – ± μ μ Cancer 14 (1) (2014) 39 48. inhibit TASK-3 with an IC50 value of 17.1 3.6 M, but even 40 M [2] L. Leanza, L. Biasutto, A. Manago, E. Gulbins, M. Zoratti, I. Szabo, Intracellular ion Withaferin A decreased cell viability by only 20 %. Another example is channels and cancer, Front. Physiol. 4 (2013) 227. that of mitoKv1.3: a mitochondriotropic derivative of PAP-1, a specific [3] N. Prevarskaya, R. Skryma, Y. Shuba, Ion channels and the hallmarks of cancer, Trends Mol. Med. 16 (3) (2010) 107–121. Kv1.3 blocker, inhibited the channel with an EC50 of 31 nM, but con [4] R. Peruzzo, L. Biasutto, I. Szabo, L. Leanza, Impact of intracellular ion channels on centrations in the μM range were necessary to trigger apoptosis of cancer cancer development and progression, Eur. Biophys. J. 45 (7) (2016) 685–707. cells, even though the drug quickly reached the mitochondria due to the [5] L. Biasutto, M. Azzolini, I. Szabo, M. Zoratti, The mitochondrial permeability transition pore in AD 2016: an update, Biochimica et biophysica acta 1863 (10) TPP moiety, as expected [13]. In that work we provided evidence that (2016) 2515–2530. the mitoKv1-3-specificdrugs caused an increased ROS level, subsequent [6] S. Marchi, V.A.M. Vitto, A. Danese, M.R. Wieckowski, C. Giorgi, P. Pinton, PTP activation, swelling, loss of Δψm, loss of cytochrome c, further ROS Mitochondrial calcium uniporter complex modulation in cancerogenesis, Cell Cycle – release and finally apoptosis. The same mechanism seems to apply for (Georgetown, Tex.) 18 (10) (2019) 1068 1083. [7] S. Reina, F. Guarino, A. Magri, V. De Pinto, VDAC3 As a potential marker of mitoIN-THPP. Similarly to the mitoKv1.3 inhibitors, the action of mitochondrial status is involved in cancer and pathology, Front. Oncol. 6 (2016) mitoIN-THPP on pathological cells may depend on the synergy between 264. different factors such as high TASK-3 expression and altered basal redox [8] V. Shoshan-Barmatz, D. Ben-Hail, L. Admoni, Y. Krelin, S.S. Tripathi, The mitochondrial voltage-dependent anion channel 1 in tumor cells, Biochimica et state in B16F10 cells. We also showed that the TPP-comprising moiety biophysica acta 1848 (10 Pt B) (2015) 2547–2575. presumably accumulating within the cells following hydrolysis of [9] I. Szabo, M. Zoratti, Mitochondrial channels: ion fluxesand more, Physiol. Rev. 94 mitoIN-THPP is not responsible for the observed loss of mitochondrial (2) (2014) 519–608. [10] L. Leanza, E. Venturini, S. Kadow, A. Carpinteiro, E. Gulbins, K.A. Becker, membrane potential and for induction of apoptosis. Targeting a mitochondrial potassium channel to fight cancer, Cell Calcium 58 (2014) 131–138. 5. Conclusion [11] A. Rasola, P. Bernardi, The mitochondrial permeability transition pore and its adaptive responses in tumor cells, Cell Calcium 56 (6) (2014) 437–445. [12] L. Leanza, L. Trentin, K.A. Becker, F. Frezzato, M. Zoratti, G. Semenzato, In summary, the present work confirmsthe expression of TASK-3 in E. Gulbins, I. 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Enyedi, G. Czirjak, Molecular background of leak K+ currents: two-pore domain potassium channels, Physiol. Rev. 90 (2) (2010) 559–605. The authors declare that they have no known competing financial [15] A.J. Patel, M. Lazdunski, The 2P-domain K+ channels: role in apoptosis and tumorigenesis, Pflugers Arch. 448 (3) (2004) 261–273. interests or personal relationships that could have appeared to influence [16] A. Innamaa, L. Jackson, V. Asher, G. Van Shalkwyk, A. Warren, D. Hay, A. Bali, the work reported in this paper. H. Sowter, R. Khan, Expression and prognostic significance of the oncogenic K2P potassium channel KCNK9 (TASK-3) in ovarian carcinoma, Anticancer Res. 33 (4) (2013) 1401–1408. Acknowledgements [17] K. Pocsai, L. Kosztka, G. Bakondi, M. Gonczi, J. Fodor, B. Dienes, P. Szentesi, I. Kovacs, R. Feniger-Barish, E. Kopf, D. Zharhary, G. Szucs, L. Csernoch, The authors are grateful for financialsupport to AIRC and the Italian Z. Rusznak, Melanoma cells exhibit strong intracellular TASK-3-specific immunopositivity in both tissue sections and cell culture, Cell. Mol. Life Sci. 63 Ministry of Education (PRIN 2015795S5W) to I.S.. The research leading (19–20) (2006) 2364–2376. to these results was supported by was funding from Italian Association [18] Z. Rusznak,´ G. Bakondi, L. Kosztka, K. Pocsai, B. Dienes, J. Fodor, A. Telek, ¨ for Cancer Research (AIRC) under IG 2017 ID. 20286 to I.S. and in part M. Gonczi, G. Szucs, L. Csernoch, Mitochondrial expression of the two-pore domain – TASK-3 channels in malignantly transformed and non-malignant human cells, also by MFAG 2019 ID. 23271 project to L.L. We acknowledge Virchows Arch. 452 (4) (2008) 415–426. DeBioImaging Facility, Dept. of Biology, Padua University for support in image acquisition and analysis.

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