Rapanone, a naturally occurring benzoquinone, inhibits mitochondrial respiration and induces HepG2 cell death

Journal Pre-proof Rapanone, a naturally occurring benzoquinone, inhibits mitochondrial respiration and induces HepG2 cell death

Gilberto L. Pardo Andreu, Felipe Zuccolotto Dos Reis, Michael González-Durruthy, René Delgado Hernández, Richard F. D'Vries, Wim Vanden Berghe, Luciane C. Alberici PII:

S0887-2333(19)30681-2

DOI:

https://doi.org/10.1016/j.tiv.2019.104737

Reference:

TIV 104737

To appear in:

Toxicology in Vitro

Received date:

31 August 2019

Revised date:

15 November 2019

Accepted date:

18 November 2019

Please cite this article as: G.L. Pardo Andreu, F.Z.D. Reis, M. González-Durruthy, et al., Rapanone, a naturally occurring benzoquinone, inhibits mitochondrial respiration and induces HepG2 cell death, Toxicology in Vitro(2018), https://doi.org/10.1016/ j.tiv.2019.104737

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© 2018 Published by Elsevier.

Journal Pre-proof Rapanone, a naturally occurring benzoquinone, inhibits mitochondrial respiration and induces HepG2 cell death. Gilberto L. Pardo Andreua*, Felipe Zuccolotto Dos Reisb, Michael González-Durruthyc,d, René Delgado Hernándeza, Richard F. D'Vriese, Wim Vanden Berghef, Luciane C. Albericib

Center for Research and Biological Evaluations, Institute of Pharmaceutical and Foods

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a

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Sciences, University of Havana (UH). Av. 23 # 2317 b/ 214 and 222, La Coronela, La

Department of Physics and Chemistry. Faculty of Pharmaceutical Sciences of Ribeirao

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b

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Lisa, PO 13600, Havana, Cuba.

Preto, University of Sao Paulo (USP), Brazil.

LAQV-REQUIMTE of Chemistry and Biochemistry, Faculty of Sciences, University of

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Porto, 4169-007 Porto, Portugal.

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Soft Matter and Molecular Biophysics Group, Department of Applied Physics,

Facultad de Ciencias Básicas, Universidad Santiago de Cali, Calle 5 # 62-00, Cali,

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e

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University of Santiago de Compostela, 15782 Santiago de Compostela, Spain

Valle del Cauca, Colombia. f

Laboratory of Protein Chemistry, Proteomics and Epigenetic Signaling (PPES),

Department of Biomedical Sciences, University of Antwerp (UA), Belgium. * Corresponding author at: Center for Research and Biological Evaluations, Institute of Pharmaceutical and Foods Sciences, University of Havana (UH). Av. 23 # 2317 b/ 214 and 222, La Coronela, La Lisa, PO 13600, Havana, Cuba. E-mail address: [email protected] (G.L. Pardo-Andreu).

Journal Pre-proof Abstract Rapanone is a natural occurring benzoquinone with several biological effects including unclear cytotoxic mechanisms. Here we addressed if mitochondria are involved in the cytotoxicity of rapanone towards cancer cells by employing hepatic carcinoma (HepG2) cells and isolated rat liver mitochondria. In the HepG2, rapanone (20 – 40 μM) induced a concentration-dependent mitochondrial membrane potential dissipation, ATP

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depletion, hydrogen peroxide generation and, phosphatidyl serine externalization; the

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latter being indicative of apoptosis induction. Rapanone toxicity towards primary rats

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hepatocytes (IC50 = 35.58 ± 1.50 µM) was lower than that found for HepG2 cells (IC50 =

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27.89 ± 0.75 µM). Loading of isolated mitochondria with rapanone (5 – 20 μM) caused a concentration-dependent inhibition of phosphorylating and uncoupled respirations

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supported by complex I (glutamate and malate) or the complex II (succinate) substrates,

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being the latter eliminated by complex IV substrate (TMPD/ascorbate). Rapanone also dissipated mitochondrial membrane potential, depleted ATP content, released Ca2+

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from Ca2+-loaded mitochondria, increased ROS generation, cytochrome c release and

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membrane fluidity. Further analysis demonstrated that rapanone prevented the cytochrome c reduction in the presence of decylbenzilquinol, identifying complex III as the site of its inhibitory action. Computational docking results of rapanone to cytochrome bc1 (Cyt bc1) complex from the human sources found spontaneous thermodynamic processes for the quinone-Qo and Qi binding interactions, supporting the experimental in vitro assays. Collectively, these observations suggest that rapanone impairs mitochondrial respiration by inhibiting electron transport chain at Complex III and

Journal Pre-proof promotes mitochondrial dysfunction. This property is potentially involved in rapanone toxicity on cancer cells.

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Keywords: rapanone, mitochondria, complex III, cytotoxicity, HepG2, anticancer

Journal Pre-proof 1. Introduction Cancer is an unsolved health problem considered as a leading cause of morbidity and mortality worldwide. The combination of aging with population growing result in an estimated increase of 11 million new cancer cases by 2040 (Wild, 2019). The rise will be even more evident in low-income countries, which are least able to meet the growing cancer burden. Therefore, there is an increasing demand to develop new, more potent

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and effective anticancer drugs. Searching for new bioactive compounds to treat human

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cancer has rested largely on the screening of natural products and their analogs. Thus,

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the search for improved cytotoxic agents from natural sources continues to be an

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important line in the discovery of modern anticancer drugs (Shahabipour et al., 2017; da Rocha et al., 2001). Particularly, the anti-tumoral effects of naturally occurring quinones

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have been demonstrated in different cancer types (Lu et al., 2013; Colucci et al., 2008).

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Rapanone (2,5-dihydroxy-3-tridecyl-1,4-benzoquinone) is a natural benzoquinone, early synthesized in 1948 (Fieser and Chamberlin, 1948), having a broad spectrum of

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biological actions, highlighting its antioxidant (de la Vega-Hernández et al., 2017), anti-

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inflammatory (Ospina et al., 2001), antibacterial (Omosa et al., 2016), antiparasitic (Shah et al., 1987; da Costa et al., 2014) and cytotoxic activities (Cordero et al., 2004; Kuete et al., 2016). Rapanone anticancer potential has been addressed recently against a panel of human tumor cells line including HT-29 (colorectal cancer), MCF-7 (breast cancer), HEp-2 (larynx cancer) and MKN-45 (gastric cancer), A549 (non-small cell lung cancer), SPC212 (human mesothelioma), DLD-1 (colorectal adenocarcinoma), Caco2 (colorectal adenocarcinoma), and HepG2 (hepatocarcinoma cells) (Cordero et al., 2004; Kuete et al., 2016). These studies arrived at the conclusion that rapanone is a potent

Journal Pre-proof cytotoxic compound that deserves more investigations towards the development of a novel antitumor agent. Mitochondrial-mediated events such as reactive oxygen species generation, transmembrane potential dissipation, calcium dynamic dysregulation, and release of caspase-activating proteins are implicated in cell death by apoptosis or necrosis (Kroemer and Reed, 2000). Therefore, compounds capable to reach the mitochondrial

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membrane could induce cell death by mitochondrial mechanisms. Rapanone seems to

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meet this criterion because of its high theoretical Log P value (6.5) and structural

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similarity with coenzyme Q (Fig. 1), characteristics that could warrant its access to

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mitochondrial membranes. In this context, the present work addressed the capacity of rapanone to interfere with mitochondrial function in hepatocarcinoma cells (HepG2) and

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potential anticancer effects.

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in isolated rat liver mitochondria as a plausible mechanism to explain its cytotoxicity and

2. Materials and methods

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2.1. Compounds and reagents

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All reagents were obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA). Stock solutions of rapanone were prepared in dimethyl sulfoxide (DMSO) and added to the cell culture or mitochondrial reaction media at 1/1000 (v/v) dilution. Control experiments contained DMSO at 1/1000 dilution. 2.2. Rapanone isolation Rapanone was obtained from Connarus venezuelanus Baill stem bark as previously reported (de la Vega-Hernández et al., 2017). 2.3. Culture of HepG2 cells

Journal Pre-proof HepG2 cells were obtained from the American Type Culture Collection, No. HB 8065. The cell line was cultured in Dulbecco's medium with 10% defined supplement fetal bovine fetal serum plus penicillin G (100 IU/ml), streptomycin (100 mg/ml) and amphotericin (1 μg/ml). Cells were seeded into 12-well plates (Nunc, Roskilde, Denmark), with 1×105 cells/well in 1 ml of culture medium at 37 °C, flushed with 5% CO 2 in the air for 24 hours. After the incubation period, the cells were rinsed with a buffered

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saline solution.

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2.4. Isolation of rat hepatocytes

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Hepatocytes were isolated from adult male Wistar rats, weighting 150–180 g, by

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collagenase perfusion of the liver (Bracht et al., 2003). The hepatocytes were cultured in Dulbecco’s medium with 10% defined supplement fetal bovine serum plus 100 mg/ml

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streptomycin, 100 IU/ml penicillin G, 250 lg/ml amphotericin B and 2 mg/ml

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ciprofloxacin. The cells were seeded as described for HepG2 cell culture. 2.5. Assessment of HepG2 cells and primary hepatocytes viability by annexin-

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V/propidium iodide double staining

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Cells were seeded into a 12-well plate (Falcon, USA) at a density of 1 × 10 5 cells/well in 1 ml of culture medium. After the 24-hr, the cells were rinsed with phosphate-buffered saline (PBS, pH 7.4), and then incubated with fresh medium for 24 hours in the absence (control) or presence of rapanone (10, 20, 30, and 40 μM) or CCCP (25 μM). The cells were trypsinized for 10 min (incubated at 5% CO 2 air, at 37 °C), and centrifuged at 1000 rpm for 5 min. The pellet was washed with ice-cold PBS pH 7.4, centrifuged again, and suspended in 0.1 ml Annexin-V binding buffer (10 mM 4(2-hydroxyethyl)-1-piperazine ethane sulfonic acid (HEPES)/NaOH, 2.5 mM CaCl 2, 140

Journal Pre-proof mM NaCl). The cells were then incubated with FITC-conjugated Annexin-V (1:100 dilution, BD Bioscience, CA, USA) for 15 min, at room temperature, in darkness. Propidium iodide (PI, 1 μg/ml, BD Bioscience, CA, USA) was added to the samples immediately prior to flow cytometry analysis. The cells were analyzed using a BD FACSCANTO™ flow cytometer (BD Bioscience, CA, USA); 10.000 cells were counted per sample. Data were analyzed using the BD FACSDIVA software (BD Biosciences,

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CA, USA). The results were expressed as the percentage in relation to the non-treated

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control.

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2.6. Determination of mitochondrial membrane potential, ATP and ROS generation in HepG2 cells

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The mitochondrial membrane potential (ΔΨ) was estimated using the TMRE probe

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(tetramethylrhodamine ethyl ester, Molecular Probes Inc., Invitrogen, USA). The cells (1

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×105 cells/well in 96-well cell culture plates) were treated for 24 hours in the absence (control) or presence of rapanone (10, 20, 30, and 40 μM) or 25 μM CCCP. After

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washing with PBS pH 7.4, cells were incubated with 25 nM TMRE in fresh serum-free

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medium for 30 min, at 37 °C in dark. Excess TMRE was washed with PBS pH 7.4. The fluorescence intensities of 10,000 cells were analyzed using a BD FACSCANTO™ flow cytometer (BD Bioscience, CA, USA). Data were analyzed using the BD FACSDIVA software (BD Biosciences, CA, USA). Cellular ATP was determined using the firefly luciferin-luciferase assay system. Cells (1 ×105/well in 24-well plates) were incubated as described for the TMRE assay. Bioluminescence was measured in the supernatant using the Sigma-Aldrich assay kit according to the manufacturer's instructions (Product No. FLAA) and an Auto Lumat LB953 Luminescence photometer (Perkin–Elmer Life

Journal Pre-proof Sciences, Wilbad, Germany). The results were expressed as the percentage in relation to the non-treated control. Intracellular oxidation of dichlorodihydrofluorescein diacetate (H2DCFDA) to 2,7-dichlorofluorescein (DCF) by reactive oxygen species (ROS) was monitored through fluorescence increase. HepG2 cells were seeded in a 12-well plate at a density of 1×105 cells/well and incubated as for the cell viability assay. The cells were washed with PBS pH 7.4 and incubated in serum-free medium with 100 μl/well of

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10 μM H2DCFDA. After incubations, the well plates were washed with PBS and the

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intracellular fluorescence was measured in a model F-4500 Hitachi fluorescence

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spectrophotometer (Tokyo, Japan) at the 503/529 nm excitation/emission wavelength

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pair (slits 5/10 nm) (Halliwell and Whiteman, 2004). 2.7. Animals

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Male Wistar rats (200 g) were obtained from CENPALAB (Havana, Cuba) and for

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acclimation, housed in the animal care facility for 1 week prior to experiments. Rats were housed in a temperature-controlled environment (22-24°C) with a 12-hour

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light/dark cycle and had access to food and water ad libitum. Animal housing and care

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and all experimental procedures were in accordance with the Principles of Laboratory Animal Care as adopted and promulgated by the National Institutes of Health, USA, and institutional guidelines under approved animal protocols (Animal Care Committee of Institute of Pharmaceutical and Foods Sciences, University of Havana, Cuba, and the Faculty of Pharmacy of Ribeirao Preto, University of Sao Paulo, Brazil). 2.8. Isolation of rat liver mitochondria and submitochondrial particles (SMP) Mitochondria were isolated by differential centrifugation (Pedersen et al., 1978). Male Wistar rats weighing approximately 200 g were euthanized by decapitation using a

Journal Pre-proof rodent guillotine, and their livers were immediately removed, sliced, and washed three times in 50 ml of an ice-cold medium composed of sucrose (250 mM), ethylene glycolbis (b-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA) (1 mM), and HEPES–KOH (10 mM) at pH 7.4. The sliced liver tissue was transferred into a Potter–Elvehjem homogenizer and homogenized three times for 15 s at 1-min intervals, in an ice bath. Homogenates were centrifuged at 4 °C (580 ×g, 5 min) in a Hitachi RT15A5 rotor

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(Japan), and the resulting supernatant was further centrifuged at 10300 ×g for 10 min,

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at 4 °C. Pellets were suspended in a medium (10 ml) consisting of sucrose (250 mM),

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EGTA (0.3 mM), and HEPES–KOH (10 mM) at pH 7.2, and centrifuged again at 4 °C

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(3400 ×g, 15 min). The final mitochondrial pellet was suspended in a medium (1 ml) consisting of sucrose (250 mM) and HEPES–KOH (10 mM) at pH 7.4, and used within 3

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h (Marín Prida et al., 2017). Submitochondrial particles (SMPs) were obtained by

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freezing and thawing (three cycles) of isolated mitochondria (20 mg/ml) as previously described for rat heart mitoplast (Uyemura and Curti, 1992). The protein concentration

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was determined using the method described by Lowry et al. (1951) with bovine serum

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albumin as a standard.

2.9. Functional assays in isolated mitochondria Mitochondria were energized with glutamate (10 mM) plus malate (3 mM) to provide electrons to complex I, potassium succinate (5 mM) plus rotenone (2.5 μM) to provide electrons to complex II, or 0.25 mM N,N,N,N-tetramethyl-pphenylenediamine/3 mM ascorbate (TMPD/asc) plus antimycin A (2.5 μM) that provide electrons to cytochrome c, in a standard incubation medium consisting of sucrose (125 mM), KCl (65 mM), potassium phosphate (K2HPO4) (2 mM), and HEPES–KOH (10 mM)

Journal Pre-proof at pH 7.4, at 30 °C. Mitochondrial respiration was monitored using a Clark-type oxygen electrode (Hansatech Instruments, oxytherm electrode unit, UK). ΔΨ was estimated spectrofluorimetrically using safranine O (10 μM) as a probe (Zanotti and Azzone, 1980), at the 495/586 nm (ex/em) wavelength pair; these two assays were performed in the presence of EGTA (0.1 mM). The Ca2+ efflux was monitored spectrofluorimetrically using Calcium Green 5 N (150 nM, Molecular Probes, OR, USA) as a probe, at the

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506/531 nm (ex/em) wavelength pair (Rajdev and Reynolds, 1993). ROS were

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monitored spectrofluorimetrically using 1 µM Amplex red (Molecular Probes, OR, USA)

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and 1 IU/ml horseradish peroxidase (Votyakova and Reynolds, 2001).

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Spectrofluorescence was monitored in a Model F-4500 Hitachi fluorescence spectrophotometer (Tokyo, Japan). Mitochondrial swelling was estimated

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spectrophotometrically from the absorbance decrease at 540 nm, using a Model U-2910

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Hitachi spectrophotometer (Japan). For the cytochrome c estimation, isolated rat liver mitochondria (2 mg/ml) were incubated (10 min) in standard medium (125 mM sucrose,

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65 mM KCl, 10 mM HEPES–KOH, pH 7.4) supplemented with succinate (5 mM),

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rotenone (2.5 mM) and inorganic phosphate (2 mM). Mitochondria were sedimented by high-speed centrifugation, and the differential absorption spectra (reduced with dithionite/oxidized) of the supernatants were recorded in a 96-well plate. The extinction coefficient for cytochrome c absorption (A550 -A540 nm) was assumed to be 19.1 mM –1 cm –1 (Kruglov et al., 2008). 2.10. ATP assay in isolated mitochondria Mitochondrial ATP was assessed via the firefly luciferin-luciferase assay system (Lemasters and Hackenbrock, 1976). After 10 min treatment with rapanone, the

Journal Pre-proof mitochondrial suspension (0.5 - 1 mg protein/ml) was centrifuged at 9000×g for 5 min at 4 °C, and the pellet was treated with 1 ml of ice-cold 1 M HClO4. After centrifugation at 14000 × g for 5 min at 4 °C, 100 µl aliquots were drawn and processed according to the manufacturer's instructions. 2.11. Complex III inhibition assay To measure the inhibition of complex III, 75 μM decylbenzylquinol (dUQH2) and

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100 μM cyt c3+ were used as substrates.

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dUQH2 + Cytochrome c (oxidized) = dUQ + Cytochrome c (reduced).

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Complex III-specific activity was estimated by the increase in absorbance due to

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the reduction of cytochrome c at 550 nm (extinction coefficient for cytochrome c = 18.7 mM/cm) (Müllebner et al., 2010). The residual dUQH2:Cyt c3+ oxidoreductase activity of

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the non-inhibited rates (100%).

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SMPs in the presence of test compounds was obtained and expressed in percentage of

2.12. Evaluation of mitochondrial membrane fluidity

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Mitochondrial membrane fluidity was evaluated by fluorescence anisotropy (r) as

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previously reported (Praet et al., 1986). 2.13. Molecular Docking of rapanone to Cyt bc1 complex In order to study the interaction between rapanone and the human mitochondrial respiratory complex III, an in silico mechanistic study based on molecular docking was performed. This allowed a deeper analysis of the molecular mechanisms involved in rapanone’s in vitro cytotoxicity. In this analysis, the first step consists of preparing the protein receptor file (i.e., mitochondrial respiratory complex III, which was obtained from the RCSB Protein Data Bank (PDB) x-ray structures like Cryo-EM structure of human

Journal Pre-proof respiratory complex III (Cyt bc1 complex) with code PDB ID: 5XTE with resolution of 3.4 Å. Thenceforth we proceeded to edit the protein just considering the Q o and Qi sites using open-source Pymol 1.7.x. to finally get a modified PDB ID: 5XTE, which represent the transmembrane region composed by the chains J, H, P, U, V, where the heme groups of Cyt bc1 (heme bl and heme bh) from the Qo and Qi binding sites are located the (Supplementary Material SM1). The box simulation was setted at: Qo site grid box

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size with dimensions of X=30 Å, Y= 30 Å, Z= 30 Å and Q o site grid box center X= 236.7

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Å, Y= 281.3 Å, Z= 225.9 Å and 2) like Qi site grid box size with dimensions of X= 30 Å,

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Y= 30 Å, Z= 30 Å and Qi site grid box center X= 228.7 Å, Y= 269.3 Å, Z= 239.9 Å (Trott

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and Olson, 2010). An exhaustiveness (average accuracy) option set to 100 was used. Due to the complexity of the protein structure with several chains, the structure was

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edited to retain only the structural protein model. The ligands rapanone, stigmatellin,

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antimycin, and coenzyme Q10 were selected directly from the ZINC database to obtain the ligand structural files (Irwin and Shoichet, 2005). Both of the target protein-ligands

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are energy minimized by AutoDock Vina. For all the docking runs, the target protein (Cyt

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bc1) was considered as a rigid moiety and the ligands as relatively rotatable due to the high success rates for small ligands with less than ten flexible rotatable bonds. Docking attempts were made at the known binding sites with stigmatellin interaction amino acids in the crystal structure (D-271, I-146, M-124) of Cyt bc1 complex using AutoDock Vina (Trott and Olson, 2010). All docking images were designed using open-source Pymol 1.7.x. Additional details concerning AutoDock Vina scoring function can be found at Supplementary Material (SM equation 1). 2.14. Statistical analysis

Journal Pre-proof Statistical analyses were performed using two-way ANOVA, assuming equality of variance with Student–Newman–Keuls posthoc test for pairwise comparisons. Results with P ≤ 0.05 were considered to be statistically significant. The IC50 values were estimated using a non-linear regression algorithm. 3. Results

generation, and increases ROS levels in HepG2 cells

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3.1. Rapanone reduces viability, disrupts mitochondrial membrane potential and ATP

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The incubation of HepG2 cells with rapanone for 24 hours promoted a dose-

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dependent cell viability decrease (Fig. 2A), as assessed by Annexin-V/PI double

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staining. At 40 μM rapanone, cell death reached ~95% characterized by increased percentages of Annexin V-positive cells, indicating increased early apoptosis. CCCP (25

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µM), the classical mitochondrial membrane dissipating agent, here used as positive

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Control, induced 50% of cell death, predominantly by apoptosis, but also with contributions of necrosis (PI-positive cells) and late apoptosis (Annexin V and PI-

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positive cells). Interesting, the exposition of isolated rat hepatocytes (Fig. 2B) or primary

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mouse macrophages (results not shown) to rapanone resulted in a slightly reduction of cytotoxicity (IC50 = 35.58 ± 1.50 µM and 32.46 ± 1.92, respectively) in relation to that found in HepG2 cancer cells (IC50 = 27.89 ± 0.75 µM), opening perspectives for future studies on the toxicity of this molecule on different non-cancer and cancer cell lines. The effect of rapanone on mitochondrial membrane potential in HepG2 cells was estimated with the mitochondrion-specific dye, TMRE. As shown in Fig. 2C, rapanone promoted an extensive mitochondrial membrane potential depolarization, evidence by an increased count of cells with low TMRE fluorescence, reaching 81% of HepG2 cells

Journal Pre-proof at 40 µM. At this concentration, rapanone also induced ~two-fold ROS levels increase (Fig. 2D), as well as ATP depletion in HepG2 cells after 24-hr incubation (Fig. 2E). The concentration-response pattern for all above rapanone effects was closely similar, suggesting a correlation between them. Therefore, we used isolated rat-liver mitochondria to characterize the rapanone effects in mitochondrial metabolism. 3.2. Rapanone reduces the respiration rates in isolated mitochondria.

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Figure 3 shows the effects of rapanone on glutamate/malate (complex I

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substrates) or succinate (complex II substrate) supported respiration of isolated rat liver

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mitochondria regarding phosphorylating (V3), resting (V4) and CCCP-induced uncoupling (VCCCP) states. Figure 3A and 3B show that the treatment of mitochondrial

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suspension with 2.5-20 μM rapanone results in a concentration-dependent decrease of

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V3 and VCCCP. Succinate-supported respiration inhibition was suppressed by the

complex III inhibition.

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complex IV substrate TMPD/ascorbate (Figure 3C), indicating that rapanone promotes

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3.3. Rapanone increments mitochondrial swelling, ROS generation, Ca2+ and

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cytochrome c release, and decreases ΔΨ and ATP level in isolated mitochondria Suspensions of rat liver mitochondria treated with rapanone (2.5 to 20 µM) and in the presence of 20 µM Ca2+, presented a decrease in apparent absorbance in relation to controls, consistent with large amplitude swelling (Fig. 4A), that were accompanied by membrane depolarization (Fig. 4B), release of Ca 2+ (Fig. 4C), increased H2O2 generation (Fig. 4D), reduced levels of ATP (Fig. 4E), and increased release of cytochrome c (Fig. 4F). The addition of TMPD/ascorbate re-established normal ATP levels, since no differences were observed when compared to the Control group (Fig.

Journal Pre-proof 4E). The swelling of the organelles and the cytochrome c release were almost completely inhibited by the classical mitochondrial permeability transition inhibitors CsA (1 µM) plus EGTA (100 µM) (Fig. 4A, line f, and Fig. 4F). 3.4. Rapanone inhibits complex III activity. Complex III activity was assayed as the antimycin A-sensitive electron transport from the reduced CoQ analog, dUQH2, to cytochrome c in SMPs from rat liver

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mitochondria. Figure 5 shows that rapanone inhibited the complex III oxidoreductase

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activity, evidenced by a dose-dependent diminution in the absorbance at 550 nm due to

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cytochrome c2+ formation. The IC50 value, estimated by a non-linear regression

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algorithm was 12.25 ± 1.81 μM (Inset).

3.5. Rapanone increases membrane fluidity in isolated mitochondria

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Rapanone interacted with mitochondrial membranes and increased its fluidity,

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which was evidenced by the decreased fluorescence anisotropy (R) (Fig. 6), reaching its maximal effect at 20 μM.

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3.6. Binding mode of rapanone to Cyt bc1 subunit

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The docking of rapanone to known inhibitor sites at Cyt bc1 resulted in the binding affinity at the Qo site with a free energy of binding (FEB) of -7.0 kcal/mol compared with the high-affinity binding of coenzyme Q10 with a measured FEB of -6.5 kcal/mol. Stigmatellin FEB also found to be -6.5 kcal/mol (Fig. 7). At Qi site, the FEB of Rapanone was -7.0 kcal/mol (Fig. 8). Antimycin showed higher FEB than rapanone (-9.1 kcal/mol), envisaging more potency of the classical inhibitor at this site. Docking trails using AutoDock tolls and AutoDock Vina suggests that the binding mode of the rapanone is quite similar to that of coenzyme Q10, pointing its potential competitive inhibitory effects.

Journal Pre-proof It is important to note that, in order to avoid false positives in the docking experiments, we carried out both the structural characterization and crystallographic validation of the protein (Cyt bc1). Results on these analyses can be found at Supplementary Materials, Figures SM1 and SM2, respectively. 4. Discussion Rapanone is a natural occurring benzoquinone with well-documented cytotoxic

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effects and interesting anti-tumor potential due to higher levels of selectivity index (the

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IC50 ratios of normal fibroblasts to cancer cell lines) (Kuete et al., 2016). Herein, using

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isolated mitochondria, submitochondrial particles and cultured cells, we provide for the

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first time, a potential mechanism involved in rapanone-induced cytotoxicity: a strong inhibition of mitochondrial electron transport, at complex III. Rapanone inhibits

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respiration, leading to dissipation of mitochondrial membrane potential, Ca 2+efflux, ATP

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level decrease, stimulation of ROS generation, swelling, and cytochrome c release. The events observed in isolated mitochondria are in apparent association with the

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mitochondrial manifestations observed in HepG2 cells: rapanone caused significant

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dose-dependent cell death predominantly by apoptosis, in close association to a membrane potential dissipation, ATP depletion and increased ROS generation. This pattern is partially in agreement with previous reports regarding other quinonesmediated cells death like adaphostin (Le et al., 2007, Long et al., 2007), RH1 (Park et al., 2011), embelin (Wang et al., 2013; Zhu et al., 2015), ardisianone (Yu et al., 2013), and menadione derivative (Teixeira et al., 2018). In this sense, the mitochondrial electron transport inhibition has been identified as an apoptosis-inducing mechanism prompted by an increase in mitochondrial-derived reactive oxygen species generation

Journal Pre-proof and permeability transition pores occurrence (Li et al., 2003; Lanju et al., 2014; Pelicano et al., 2003). Particularly, the inhibition of mitochondrial complex III seems to be a common pathway for the apoptosis induction of different compounds like ceramide (Gudz et al., 1997), benzyl isothiocyanate (Xiao et al., 2008), the alkaloid lamellarin D (Ballot et al., 2010), withaferin A (Hahm et al., 2011), and several quinones derivatives (Le et al., 2007; Fiorillo et al., 2016; Kapur et al., 2018; Vyssokikh et al., 2013). The

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mitochondrial complex III inhibition also activates the tumor suppressor p53 pathway

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triggered by the impairment of the de novo pyrimidine biosynthesis due to the

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suppression of the mitochondrial enzyme dihydroorotate dehydrogenase (Khutornenko

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et al., 2014). After a mitochondrial function impairment, it could be expected not only an energetic failure but also a reduction in the anaplerotic supply of precursors for

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biosynthetic pathways like aspartate, for pyrimidine biosynthesis (Molina et al., 2018).

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Considering all these background, it can be proposed that the mechanisms for the rapanone cytotoxic action on HepG2 cells may include a mitochondrial impairment,

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which could, in turn, lead to apoptosis by the intrinsic pathway. Nevertheless, further

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studies should be done to clarify whether mitochondria impairment is the primary target of rapanone toxicity towards HepG2 cells. The modulation of mitochondrial signaling and physiology may also bypass the drug-resistant characteristics of cancer cells (Lyakhovich and Lleonart, 2016; Viale et al., 2015). Accordingly, several experimental compounds have been reported to target mitochondria to induce cancer cell death (Neuzil et al., 2013). Indeed, inhibition of oxidative phosphorylation has been identified as a promising anti-cancer target in models of brain cancer and acute myeloid leukemia reliant on OXPHOS, possible due

Journal Pre-proof to a bioenergetics impairment and reduced substrate availability that lead to impaired biosynthetic capability (Molina et al., 2018). The site of ETC inhibited by rapanone seems to be complex III since succinatesupported respiration inhibition was eliminated by TMPD/ascorbate, which fuels electrons by cytochrome c for cytochrome oxidase (complex IV). Complex III, also named cytochrome bc1 complex (cyt bc1), is an essential component of the

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mitochondrial respiratory chain that catalyzes the electron transfer from ubiquinol to cyt

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c, driving protons into the intermembrane space, thus contributing to the

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electrochemical proton gradient building. This proton gradient is largely responsible for

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the mitochondrial membrane potential, fundamental for the organellar ions transport and ATP synthesis (Trumpower, 1990; Xiao et al., 2014). The main function of cyt bc1 in

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energy metabolism makes it an important target for various natural and synthetic

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antibiotics (Gordon et al., 2017; Bald et al., 2017) and also anti-cancer agents (Fiorillo et al., 2016; Song et al., 2016; Shrotriya et al., 2015). As mentioned before, there are two

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binding sites in the cytochrome bc1 complex, a quinone reduction site near the negative

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side of the membrane (Qi site) and a quinol oxidation site close to the positive side of the membrane (Qo site). Due to the structural similarity between rapanone and coenzyme Q, we, respectively, docked rapanone into the Q o and Qi sites and obtained two rapanone-bound complexes, rapanone−Qo, and rapanone−Qi complexes. As shown, the Qo and Qi site binding energies of rapanone (−6.9 and −7.0 kcal/mol, respectively) suggest that this molecule could bind favorably to both sites of complex III, thus blocking coenzyme Q electron transfer.

Journal Pre-proof Our set of experiments in isolated mitochondria revealed that the rapanone inhibitory mechanism may also involve an unspecific interaction with mitochondrial membranes that disrupts membrane structural order, i.e., it means enhanced membrane fluidity (Pottel et al., 1983). It is worth to notice that this molecule is a highly lipophilic compound (theoretical Log P = 6.5) with the potential to incorporate into mitochondrial membranes and perturb their lipid organization. Obviously, due to the

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differences between human and rat’s mitochondria (Benga et al., 1978), this in vitro

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study cannot simply be transposed to in vivo situation and should be interpreted with

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caution.

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In conclusion, the present work demonstrates that rapanone, a natural occurring benzoquinone derivative, is a potent inhibitor of mitochondrial respiration and that this

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property would be involved in its toxicity towards HepG2 cells. Since hepatocellular

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carcinoma is the commonest type of primary liver cancer, increasing in prevalence worldwide with a high mortality rate (Noonan and Pawlik, 2019; Wege et al., 2019),

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these results are of medical importance and greatly contribute to the comprehension of

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the cytotoxic mechanisms of rapanone. Additional studies will be needed to determine whether cancer cells could be specifically targeted by the rapanone cytotoxicity. Conflict of interest statement The authors have no conflicts of interest to declare. Acknowledgments This work was partially supported by CAPES-Brazil/MES-Cuba (064/09) and VLIR project CU2018TEA457A103. References

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Figures Legends

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Figure 1. Rapanone (A) and Coenzyme Q (B) structures. Figure 2. Effects of rapanone on HepG2 (A) and primary rats hepatocytes (B) cell

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viability, mitochondrial membrane potential (C), reactive oxygen species (D), and ATP

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levels (E). HepG2 cells or primary rats hepatocytes (1×105) were treated with rapanone (10, 20, 30 and 40 μM), or 25 μM CCCP (uncoupling control) or 5 µM Antimycin A (AA-

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reactive oxygen species induction control). Control cells only contained rapanone

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vehicle (DMSO 1/1000 v/v). The assay conditions are described in Materials and methods. The bars are mean ± SEM of three different experiments. * Significantly different from control, at P < 0.05. Figure 3. Effects of rapanone on the respiration rates of isolated rat liver mitochondria. Panels show the oxygen consumption rates in the phosphorylating (V 3), resting (V4), and uncoupled respiration (VCCCP) when glutamate (10 mM) plus malate (3 mM) (A), or succinate (5 mM) plus rotenone (2.5 µM) (B) were used as respiratory substrates for complex I and II, respectively. Mitochondria were also exposed to a mixture of

Journal Pre-proof TMPD/ascorbate plus antimycin A (2.5 µM) (C) in the absence or presence of rapanone. Mitochondria (0.5 mg/ml) were incubated in the absence (control) or presence of rapanone ( 2.5, 5, 10, 15, and 20 μM) under conditions detailed in Materials and Methods. For V3 and VCCCP determination, 100 μM ADP and 1 μM CCCP were added, respectively. V4 was determined after ADP exhaustion. The bars represent the mean ± S.E.M. of three independent experiments with at least three replicates. Asterisks in

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panels indicate statistical differences compared to the control group. *p < 0.05,

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according to the ANOVA and post-hoc Newman–Keuls tests.

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Figure 4. Effects of rapanone on mitochondrial swelling (A), membrane potential (B),

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Ca2+ release (C), reactive oxygen species (D), ATP production (E), and cytochrome c release (F) in mitochondria energized with succinate. Mitochondria (RLM, 0.5 mg

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protein/ml) were added in the standard medium, under the conditions described in

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Materials and methods. The additions were: (a) none (control), (b) 5, (c) 10, (d) 15, and (e) 20 µM rapanone. In Panel (A), line (f) represents condition (e) plus 1 µM

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cyclosporine/ 100 µM EGTA. RLM, Ca2+ (20 µM), TMPD (0.25 mM) plus ascorbate (3

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mM), and CCCP (1 μM) were added where indicated. For cytochrome c estimation, RLM (2 mg/ml) were incubated under conditions described for the swelling assay. Results are representative of three experiments with different mitochondrial preparations. Asterisks in panels indicate statistical differences compared to the control group. *p < 0.05, according to the ANOVA and post-hoc Newman–Keuls tests. Figure 5. Complex III activity inhibition by rapanone. Rapanone exhibited an IC 50 value of 12.25 ± 1.81 μM (Inset). Non-inhibited complex III activity of mitochondria (0.25

Journal Pre-proof mg/ml) was 7.65 nmol/min/ml, which was set to 100%. Data represent mean ± S.D. from three independent experiments. Figure 6. Effects of rapanone on mitochondrial membrane fluidity, assessed by fluorescence anisotropy. Mitochondria (0.2 mg/ml) were incubated in standard medium containing 0.5 μM 1,6-diphenyl-1,3,5-hexatriene (DPH) for 30 min, at 37 °C, in the absence (control) or presence of rapanone. Fluorescence anisotropy (R) is expressed

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as the mean ± S.E.M. of three independent mitochondrial preparations with at least

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three replicates. Asterisks indicate statistical differences compared to the control group.

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*p < 0.05 according to the ANOVA and post-hoc Newman–Keuls tests.

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Figure 7. Van der Wall surface representation for each ligand complexes for the best crystallographic-docking pose (RMSD < 2Å) in the Cyt bc1 (Qo binding-site): (A) Cyt bc1

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(Qo-site) showing the unoccupied-active binding-site (red dotted circle); (B) FEB

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(rapanone/Qo binding-site interaction complex) = - 6.9 kcal/mol; (C) FEB (Coenzyme Q10/Qo binding-site interaction complex) = - 6.5 kcal/mol, (D) FEB (stigmatellin/Qo binding-

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site interaction complex) = - 6.5 kcal/mol and. In van der Wall surface representation the

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color-label residue is red (acidic residue and blue for basic residues). Figure 8. Van der Wall surface representation for each ligand complexes for the best crystallographic-docking pose (RMSD < 2Å) in the Cyt bc1 (Qi binding-site) as: (A) Cyt bc1 (Qi-site) showing the unoccupied-binding-site (red dotted circle); (B) FEB (rapanone/Qi binding-site interaction complex) = - 7.0 kcal/mol; (C) FEB (Coenzyme Q10/Qi binding-site interaction complex) = - 8.9 kcal/mol, (D) FEB (Antimycin A/Qi bindingsite interaction complex) = - 9.1 kcal/mol and. In van der Wall surface representation the color-label residue is red (acidic residue and blue for basic residues).

Journal Pre-proof Supplementary Material

Rapanone, a naturally occurring benzoquinone, inhibits mitochondrial respiration and induces HepG2 cell death. Gilberto L Pardo Andreua*, Felipe Zuccolotto Dos Reisb, Michael González-Durruthyc, René Delgado Hernándeza, Richard F.D'Vriesd, Wim Vanden Berghee, Luciane C Alberichi f a

Center for Research and Biological Evaluations, Institute of Pharmaceutical and Foods

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Sciences, University of Havana (UH). Av. 23 # 2317 b/ 214 and 222, La Coronela, La b

Department of Neurosciences, Division of Neurology, Ribeirão Preto Medical School,

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University of São Paulo, São Paulo, SP, Brazil c

Chemistry and Biochemistry, Faculty of Sciences, University of Porto, 4169-007 Porto,

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Portugal.

Facultad de Ciencias Básicas, Universidad Santiago de Cali, Calle 5 # 62-00, Cali,

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d

Valle del Cauca, Colombia e

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Lisa, PO 13600, Havana, Cuba.

Laboratory of Protein Chemistry, Proteomics and Epigenetic Signaling (PPES),

f

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Department of Biomedical Sciences, University of Antwerp (UA), Belgium. Physic and Chemistry Department. Faculty of Pharmaceutical Science of Ribeirao

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Preto, University of Sao Paulo (USP), Brazil

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* Corresponding author at: Center for Research and Biological Evaluations, Institute of Pharmaceutical and Foods Sciences, University of Havana (UH). Av. 23 # 2317 b/ 214 and 222, La Coronela, La Lisa, PO 13600, Havana, Cuba. E-mail address: [email protected] (G.L. Pardo-Andreu).

Journal Pre-proof

To study the rapanone docking mechanisms with Q 0 and Qi sites from human mitochondrial respiratory complex III we use an Autodock Vina scoring function developed by Trott and Olson (2010).1 According to the thermodynamic equation depicted below:

FEBdock  Gbind  GvdW  GH bond  Gelectrost  Gint (1)

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The FEB values or (ΔGbind affinity) for the best docked poses of the formed complexes

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was obtaining by sum the individual molecular mechanics terms of chemical potentials (ΔG) like: van der Waals interactions (ΔGvdW ), hydrogen bond (ΔGH-bond), electrostatic

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interactions (ΔGelectrost), and intramolecular ligands interactions (ΔGinternal) as depicted

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above in equation 1.1

Figure SM1. A) Representation of the full x-ray crystallographic molecular structure model from human mitochondrial respiratory complex III transmembrane highlighting in blue the different region like intermembrane and transmembrane space and mitochondrial matrix. B) Representation of transmembrane region (cytochrome bc1) by domain separation (chains J and V). C) Representation of cytochrome bc1 flexibility properties

Journal Pre-proof highlighting the binding-sites (Q0 and Qi), the color intensity bar indicates the distribution of flexibility through the cytochrome bc1 structure from rigid or low flexibility regions (blue) to high flexibility regions (red). D) DeepSite calculation/prediction of topological-cavities2 from mitochondrial cytochrome bc1 active binding-sites involving the Q0 and Qi site within the volumetric orange regions depicted like Van der Waals surface representation for J and V chains. E) Pymol alpha-helices representation of mitochondrial cytochrome b with the corresponding heme prosthetic groups (heme b l and heme bh) (light green) for both

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chains J (brown) and V (olive green).

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Brief discussion SM1: Flexibility analysis like 3D-colored structure is based on the size of fluctuations from the slowest vibration-modes of the mitochondrial cytochrome b from

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low-flexibility to high-flexibility, which provide information to set a flexible box simulation

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from the active binding site of mitochondrial cytochrome bc1.

Journal Pre-proof Figure SM2. A) Ramachandran diagrams like Phi vs. Psi torsion dihedral angles and spatial distribution of Ramachandran outliers (empty pink circles) in the pdb x-ray structure of human mitochondrial respiratory complex III transmembrane region (cytochrome bc1). All the possible torsion dihedral angles combinations of each aminoacid residues are showed. B) Ramachandran plot quality assessment (cytochrome b) is measured by the percentage (%) of the cytochrome bc1-residues that are in the mostfavored (or purple core) in the regions of the Ramachandran plot vs. crystallographic resolution (Å). To this purpose, it showed a good quality model structure for cytochrome

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bc1 with high resolution of 3.4 Å, with expected % of the favored region over 85 % (red

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dotted line).

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Brief discussion SM2: Ramachandran evaluation is a 2D-projection on the plane from

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3D-crystallographic protein model and all the possible cytochrome bc1 conformations of each residue including the key binding sites residues are defined according to the torsion

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dihedral angles (Psi) and (Phi) around the cytochrome bc1 peptide-bond residues 3. For this instance, allowed torsion values for the Phi vs. Psi dihedral angles are localized within

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the Ramachandran colored purple contour (like conformationally-favored residues). Otherwise, are considered as cytochrome bc1 sterically-disallowed residue related to

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torsion values of dihedral angles Psi vs. Phi appear outside of the Ramachandran colored

SM References

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purple contour (like conformationally non-favored amino acid residues).

1. Trott, O., Olson, A.J., 2010. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem. 31, 455-61. Doi: 10.1002/jcc.21334. 2. Jiménez, J., Doerr, S., Martínez-Rosell, G., Rose, A.S., De Fabrittis, G., 2017. DeepSite: protein-binding site predictor using 3D-convolutional neural networks. Bioinformatics 33(19): 3036-3042. Doi: 10.1093/bioinformatics/btx350. 3. Chen, V.B., Arendall, W.B., Headd, J.J., Keedy, D.A., Immormino, R.M., Kapral, G.J., Murray, L.W., Richardson, J.S., Richardson, D.C., 2010. Acta Cryst. D66, 1221. Doi: 10.1107/S0907444909042073.

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Journal Pre-proof Highlights 

Rapanone induced apoptosis in HepG2 cells.

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Rapanone interfered with ATP synthesis by inhibiting mitochondrial respiration.

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Rapanone inhibited electron transport at Complex III and promotes mitochondrial dysfunction. The results help to unravel the cytotoxic mechanisms of rapanone towards

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cancer cells.

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Journal Pre-proof Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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