Supporting Information

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1 Supporting Information Development of a mitochondriotropic antioxidant based on caffeic acid: proof of concept on cellular and mitochondrial oxidative stress models José Teixeira a,b, Fernando Cagide a, Sofia Benfeito a, Pedro Soares a, Jorge Garrido a,c, Inês Baldeiras d,e José A. Ribeiro a, Carlos M. Pereira a, António F. Silva a, Paula B. Andrade f, Paulo J. Oliveira b*, Fernanda Borges a* a CIQUP/Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, Porto , Portugal b CNC Center for Neuroscience and Cell Biology, UC-Biotech Building, Biocant Park University of Coimbra, Cantanhede , Portugal c Department of Chemical Engineering, School of Engineering (ISEP), Polytechnic Institute of Porto, Porto , Portugal d Faculty of Medicine, University of Coimbra, Coimbra, Portugal e Laboratory of Neurochemistry, Coimbra University Hospital (CHUC), Coimbra, Portugal f REQUIMTE/LAQV-Laboratory of Pharmacognosy, Department of Chemistry, Faculty of Pharmacy, University of Porto, Porto , Portugal These authors contributed equally to the work. Corresponding Authors: Fernanda Borges, CIQ/Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, Porto , Portugal, Porto , Portugal. fborges@fc.up.pt Paulo J. Oliveira, CNC Center for Neuroscience and Cell Biology, University of Coimbra, UC-Biotech, Biocant Park Cantanhede, Portugal. pauloliv@cnc.uc.pt

2 Table of contents of supporting information S1. Data of AntiOxCINs functional mitochondrial toxicity profile S2 S2. References S5 S3 List of figures S6 S4. List of tables S11 S2

3 S1. Data of AntiOxCINs functional mitochondrial toxicity profile Toxicity effects of AntiOxCINs on liver mitochondrial respiration and on mitochondrial transmembrane electric potential ( Ψ) The toxicity profile of the antioxidants (MitoQ 10 and AntiOxCINs) was assessed on isolated RLM fractions by the evaluation of their direct effects on the bioenergetics apparatus. The first studies on mitochondrial respiration were performed with the mitochondrialtargeted antioxidant MitoQ 10 not only as a standard, but also used to set the range of concentration used. At high concentrations, mitochondria-targeted molecules can disrupt mitochondrial respiration by damaging the inner mitochondrial membrane or by inhibiting the respiratory chain, ATP synthesis or export machinery. The highest concentration used was the one at which MitoQ 10 completely disrupted mitochondrial bioenergetics. For all tested concentrations, MitoQ 10 caused a significant decrease of RCR and ADP/O parameters. (Table S1). Moreover, when RLM were incubated with MitoQ 10 concentrations up to 5 µm an increase on state 2, state 4 and oligomycin-inhibited respiration and a decrease on state 3 and FCCP-uncoupled respiration, using glutamate/malate as substrate was observed. When using succinate, RLM were completely uncoupled in the presence of MitoQ 10 at the highest concentration tested (5 µm) (Figure S4). The incubation with increasing concentrations of MitoQ 10 resulted in a progressive decrease of the maximum Ψ obtained upon energization. MitoQ 10 (5 µm) also decreased the ability of Ψ to recover to a value similar to the control. Ψ collapse after ADP addition was observed with 10 µm MitoQ 10, since no repolarization occurred after (Table S1). Compound 1. For all tested concentrations, compound 1 was the antioxidant which showed the less effects on mitochondrial bioenergetics (Figure S4). A decrease in the RCR was verified after glutamate/malate-energization and incubation with 10 µm of compound 1 did not affect Ψ generated by mitochondria, even for the highest concentration, independent of which respiratory substrate was used (Table S2). Compound 22. Significant mitochondrial respiration alterations were observed at 5 µm of this compound (Figure S4). By using complex I substrates, a decrease on state 3 respiration, RCR and ADP/O parameters was observed. However, by using succinate as respiratory substrate, the alterations in mitochondrial respiration were only observed at the highest S3

4 concentration (10 µm). Compound 22 not only stimulated mitochondrial state 2 and state 4 respiration but also oligomycin-inhibition respiration, which resulted into a decrease in RCR and ADP/O (Table S3). RLM incubated with compound 22 generated a similar Ψ compared to the control. After energization with succinate, at the highest concentration tested (10 µm), compound 22 decreased the phosphorylative lag phase, i.e., the time that the Ψm takes to recover after ADP addition (Table S3). Compound 23. An increased state 2, state 4 and oligomycin-inhibition respiration was observed for all tested concentrations after energization with glutamate-malate (Figure S4). As a consequence, a decrease on RCR was also observed. On succinate-energized RLM the increased state 2, state 4 and oligomycin-inhibition respiration was accompanied by an increase on state 3 respiration. Yet, oxidative phosphorylation was still coupled and the mitochondrial phosphorylative efficiency displayed values similar to those of the control. compound 23 did not affect mitochondrial Ψ even for the highest concentration, independent of the respiratory substrate used (Table S4). Compound 24. Increased state 2, state 4 and oligomycin-inhibition respiration was observed for all tested concentrations after energization, independently of the respiratory substrate used (Figure S4). Furthermore, compound 24 caused a stimulation on respiratory state 3 for all concentration tested when glutamate/malate was used as respiratory substrate. The same stimulatory effect was only observed at 10 µm using succinate as substrate. compound 24 caused a decrease in the RCR with glutamate-malate for all tested concentrations but only at 10 µm with succinate (Table S5). The incubation with increasing concentrations of compound 24 resulted in a progressive decrease of the mitochondrial Ψ upon succinate-induced energization. Compound 24 (10 µm) decreased the depolarization amplitude following ADP addition and the ability of RLM to recover Ψ to control values. Incubation with compound 24 (10 µm) decreased the phosphorylative lag phase, i.e., the time that the Ψ takes to recover after ADP addition, upon glutamate/malate-energization (Table S5). Compound 25. Compound 25 (10 µm) decreased state 3 respiration when glutamate/malate was used as substrate (Figure S4). Although no other differences were observed on respiratory states, compound 25 significantly decreased RCR and ADP/O at 5 µm (Table S6). When succinate was used as respiratory substrate, significant alterations of mitochondrial respiration were observed, including stimulated oligomycin-inhibited S4

5 respiration, at concentrations up to 5 µm of compound 25. A significant decrease on RCR and ADP/O was observed in the presence of compound 25 (10 µm). Compound 25 did not affect the Ψ generated by RLM even for the highest concentration tested, when compared to the control, independent of which respiratory substrate was used (Table S6). Compound 26. Increased state 2, state 4 and oligomycin-inhibition respiration was observed for all tested concentrations after energization. The results were independent of the respiratory substrate used (Figure S4). The stimulatory effect on respiration was more evident when using complex I substrates and as a consequence a decrease in the RCR was observed. An increase on FCCP-stimulated uncoupled respiration was verified on succinateenergized RLM incubated with 10 µm compound 26. Mitochondrial phosphorylative efficiency was not affected by compound 26 incubation (Table S7). Compound 26 not only decreased the phosphorylative lag phase, but also succinate-sustained Ψ after ADP addition was significantly lower than control value (Table S7). Compound 27. An increased state 2, state 4 and oligomycin-inhibition respiration was observed for all tested concentrations after energization, independent of which respiratory substrate was used (Figure S4). Furthermore, respiratory state 3 stimulation was also verified for all concentrations, independent of which respiratory substrate was used. Contrarily to the observed pattern, incubating RLM with compound 27 (10 µm) did not affect state 3 respiration but increased FCCP-stimulated respiration. For all tested concentrations, compound 27 decreased the RCR after glutamate/malate-energization (Table S8). Compound 27 (10 µm) also decreased Ψ obtained upon succinate-energization and decreased the ability of mitochondria to recover their Ψ values after (Table S8). S5

6 S2. Supplementary references S3. Supplementary figures 250 na A 100 na B E / V vs Ag/AgCl E / V vs Ag/AgCl Supplementary Figure S1 Representative AntiOxCINs voltammograms: (A) Differential pulse and (B) cyclic voltammograms for a 0.1 mm solution of ( ) compound 23 and ( ) compound 26 in physiological ph 7.4 supporting electrolyte. Scan rate: 8 mv s -1 (DPV) and 50 mv s -1 (CV). S6

7 A NaCl 2 mm BTPPATPBCl x mm AntiOxCIN + Ag AgCl BTPPACl 2 mm 1 mm Tris-HCl 10 mm ph 7.0 AgCl Ag (Water) (DCH) (Water) B Supplementary Figure S2 Evaluation of AntiOxCINs lipophilicity in water/dch system. (A) Schematic representation of the electrochemical cell used in the AntiOxCINs transfer across the aqueous phase/dch micro-interface at ph 7.0. (B) Differential pulse (top) and cyclic (bottom) voltammograms obtained at the water/dch interface in the absence (---) and presence ( ) of 0.32 mm of compound 22 at ph 7.0. The current rise at 0.45 V corresponds to the transfer of the drug from water to DCH whereas the current rise in the reverse scan corresponds to its transfer back to the aqueous phase. Scan rate: 8 mv s -1 (DPV) and 50 mv s -1 (CV). S7

8 Supplementary Figure S3. Effect of (A) non-targeted natural antioxidant caffeic acid, (B) dtpp and MitoQ 10, and AntiOxCINs containing a (C) catechol or (D) pyrogallol core on lipid peroxidation of RLM membranes induced by ADP and Fe 2+ and followed as oxygen consumption. Data are means ± SEM from six independent experiments and are expressed as % of control (control = 100%). S8

9 Supplementary Figure S4 - Effect of MitoQ 10 and AntiOxCINs on RLM respiration supported by (A) 10 mm glutamate + 5 mm malate or (B) 5 mm succinate. The white bars S9

10 refer to the control, while grey bars refer to the experiments where RLM were pre-incubated with AntiOxCINs (2.5µM light grey; 5µM grey; and 10 µm dark grey). The presented results are means ± SEM of seven independent experiments. The statistical significance relative to the different respiratory rates/states was determined using Student s two tailed t- test. Significance was accepted with *P<0.05, **P<0.01, ***P<0.0005, ****P< Supplementary Figure S5 Effect of AntiOxCINs on mitochondrial swelling upon induction of the mitochondrial permeability transition pore (mptp). AntiOxCINs and MitoQ 10 (A) 2.5µM, (B) 5µM and (C) 10µM were pre-incubated with RLM for 5 min before calcium addition. Data are means ± SEM of three independent experiments and are expressed as absorbance at 540 nm. The comparisons were performed using one-way ANOVA between control (Ca 2+ only) vs. assays where AntiOxCIN derivatives were pre-incubated before Ca 2+. The most relevant concentrations are shown in Figure 5. Significance was accepted with *P<0.05, **P<0.01, ***P<0.0005, ****P< S10

11 S4. Supplementary tables Supplementary Table S1. Effect of MitoQ 10 on mitochondrial bioenergetics: mitochondrial respiratory control ratio (RCR), efficiency of the phosphorylative system (ADP/O), and mitochondrial transmembrane potential ( Ψ). Mitochondrial Bioenergetics Control MitoQ µm 5 µm 10 µm Glutamate/Malate Maximum potential ± ± ± 10.6 * ± 10.2 **** ± ± ± ± ± ± 9.4 * Lag Phase (s) 70.7 ± ± ± 7.1 RCR 6.4 ± ± 0.6 * 2.7 ± 0.3 *** 1.3 ± 0.1 **** ADP/O 2.6 ± ± 0.1 * 1.9 ± 0.1 *** 2.0 ± 0.2 ** Succinate Maximum potential ± ± ± 8.1 * ± 3.8 **** ± ± ± ± ± ± 8.7 * Lag Phase (s) ± ± ± 19.4 RCR 4.9 ± ± 0.2 ** 2.4 ± 0.2 ** ADP/O 1.6 ± ± 0.1 * 1.3 ± 0.1 * Effect of MitoQ10 on Ψ, RCR and ADP/O of energized mitochondria (5 mm glutamate / 2.5 malate or 5 mm succinate). Values are means ± SEM of five independent experiments. Statistically significant compared with control using Student s two tailed t-test. Significance was accepted with *P<0.05, **P<0.01, ***P<0.0005, ****P< S11

12 Supplementary Table S2. Effect of compound 1 on mitochondrial bioenergetics: mitochondrial respiratory control ratio (RCR), efficiency of the phosphorylative system (ADP/O), and mitochondrial transmembrane potential ( Ψ). Mitochondrial Bioenergetics Control µm 5 µm 10 µm Glutamate/Malate Maximum potential ± ± ± ± ± ± ± ± ± ± ± ± 9.7 Lag Phase (s) 70.7 ± ± ± ± 5.1 RCR 6.4 ± ± ± ± 0.2 * ADP/O 2.6 ± ± ± ± 0.1 Succinate Maximum potential ± ± ± ± ± ± ± ± ± ± ± ± 8.3 Lag Phase (s) ± ± ± ± 30.4 RCR 4.9 ± ± ± ± 0.2 ADP/O 1.6 ± ± ± ± 0.1 Effect of compound 1 on Ψ, RCR and ADP/O of energized mitochondria (5 mm glutamate / 2.5 malate or 5 mm succinate). Values are means ± SEM of five independent experiments. Statistically significant compared with control using Student s two tailed t-test. Significance was accepted with *P<0.05, **P<0.01, ***P<0.0005, ****P< S12

13 Supplementary Table S3. Effect of compound 22 on mitochondrial bioenergetics: mitochondrial respiratory control ratio (RCR), efficiency of the phosphorylative system (ADP/O), and mitochondrial transmembrane potential ( Ψ). Mitochondrial Bioenergetics Control µm 5 µm 10 µm Glutamate/Malate Maximum potential ± ± ± ± ± ± ± ± ± ± ± ± 8.0 Lag Phase (s) 70.7 ± ± ± ± 25.1 RCR 6.4 ± ± ± 0.6 * 3.4 ± 0.3 ** ADP/O 2.6 ± ± ± 0.1 * 2.2 ± 0.1 * Succinate Maximum potential ( Ψ in - mv) ± ± ± ± ± ± ± ± ± ± ± ± 7.1 Lag Phase (s) ± ± ± ± 12.4 * RCR 4.9 ± ± ± ± 0.2 *** ADP/O 1.6 ± ± ± ± 0.1 ** Effect of compound 22 on Ψ, RCR and ADP/O of energized mitochondria (5 mm glutamate / 2.5 malate or 5 mm succinate). Values are means ± SEM of five independent experiments. Statistically significant compared with control using Student s two tailed t-test. Significance was accepted with *P<0.05, **P<0.01, ***P<0.0005, ****P< S13

14 Supplementary Table S4. Effect of compound 23 on mitochondrial bioenergetics: mitochondrial respiratory control ratio (RCR), efficiency of the phosphorylative system (ADP/O), and mitochondrial transmembrane potential ( Ψ). Mitochondrial Bioenergetics Control µm 5 µm 10 µm Glutamate/Malate Maximum potential ± ± ± ± ± ± ± ± ± ± ± ± 7.2 Lag Phase (s) 70.7 ± ± ± ± 12.1 RCR 6.4 ± ± 0.5 * 3.5 ± 0.5 ** 3.3 ± 0.3 ** ADP/O 2.6 ± ± ± ± 0.1 Succinate Maximum potential ( Ψ in - mv) ± ± ± ± ± ± ± ± ± ± ± ± 7.6 Lag Phase (s) ± ± ± ± 17.6 RCR 4.9 ± ± ± ± 0.6 ADP/O 1.6 ± ± ± ± 0.1 Effect of compound 23 on Ψ, RCR and ADP/O of energized mitochondria (5 mm glutamate / 2.5 malate or 5 mm succinate). Values are means ± SEM of five independent experiments. Statistically significant compared with control using Student s two tailed t-test. Significance was accepted with *P<0.05, **P<0.01, ***P<0.0005, ****P< S14

15 Supplementary Table S5. Effect of compound 24 on mitochondrial bioenergetics: mitochondrial respiratory control ratio (RCR), efficiency of the phosphorylative system (ADP/O), and mitochondrial transmembrane potential ( Ψ). Mitochondrial Bioenergetics Control µm 5 µm 10 µm Glutamate/Malate Maximum potential ± ± ± ± ± ± ± ± ± ± ± ± 20.8 Lag Phase (s) 70.7 ± ± ± ± 8.7 * RCR 6.4 ± ± 0.3 *** 3.3 ± 0.2 ** 2.6 ± 0.1 *** ADP/O 2.6 ± ± ± ± 0.1 Succinate Maximum potential ( Ψ in - mv) ± ± ± 4.7 * ± 9.3 *** ± ± ± ± 11.0 * ± ± ± ± 11.2 *** Lag Phase (s) ± ± ± ± 11.2 RCR 4.9 ± ± ± ± 0.3 * ADP/O 1.6 ± ± ± ± 0.11 Effect of compound 24 on Ψ, RCR and ADP/O of energized mitochondria (5 mm glutamate / 2.5 malate or 5 mm succinate). Values are means ± SEM of five independent experiments. Statistically significant compared with control using Student s two tailed t-test. Significance was accepted with *P<0.05, **P<0.01, ***P<0.0005, ****P< S15

16 Supplementary Table S6. Effect of compound 25 on mitochondrial bioenergetics: mitochondrial respiratory control ratio (RCR), efficiency of the phosphorylative system (ADP/O), and mitochondrial transmembrane potential ( Ψ). Mitochondrial Bioenergetics Control µm 5 µm 10 µm Glutamate/Malate Maximum potential ± ± ± ± ± ± ± ± ± ± ± ± 15.6 Lag Phase (s) 70.7 ± ± ± ± 15.1 RCR 6.4 ± ± ± 0.7 * 3.5 ± 0.5 ** ADP/O 2.6 ± ± ± 0.1 * 2.2 ± 0.1 ** Succinate Maximum potential ( Ψ in - mv) ± ± ± ± ± ± ± ± ± ± ± ± 10.5 Lag Phase (s) ± ± ± ± RCR 4.9 ± ± ± ± 0.4 * ADP/O 1.6 ± ± ± ± 0.02 * Effect of compound 25 on Ψ, RCR and ADP/O of energized mitochondria (5 mm glutamate / 2.5 malate or 5 mm succinate). Values are means ± SEM of five independent experiments. Statistically significant compared with control using Student s two tailed t-test. Significance was accepted with *P<0.05, **P<0.01, ***P<0.0005, ****P< Supplementary Table S7. Effect of compound 26 on mitochondrial bioenergetics: mitochondrial respiratory control ratio (RCR), efficiency of the phosphorylative system (ADP/O), and mitochondrial transmembrane potential ( Ψ). S16

17 Mitochondrial Bioenergetics Control µm 5 µm 10 µm Glutamate/Malate Maximum potential ± ± ± ± ± ± ± ± ± ± ± ± 26.0 Lag Phase (s) 70.7 ± ± ± ± 7.6 RCR 6.4 ± ± 0.4 ** 3.7 ± 0.4 ** 3.2 ± 0.1 ** ADP/O 2.6 ± ± ± ± 0.1 Succinate Maximum potential ( Ψ in - mv) ± ± ± ± ± ± ± ± ± ± ± ± 9.3 * Lag Phase (s) ± ± ± ± 10.2 * RCR 4.9 ± ± ± ± 0.8 ADP/O 1.6 ± ± ± ± 0.1 Effect of compound 26 on Ψ, RCR and ADP/O of energized mitochondria (5 mm glutamate / 2.5 malate or 5 mm succinate). Values are means ± SEM of five independent experiments. Statistically significant compared with control using Student s two tailed t-test. Significance was accepted with *P<0.05, **P<0.01, ***P<0.0005, ****P< S17

18 Supplementary Table S8. Effect of compound 27 on mitochondrial bioenergetics: mitochondrial respiratory control ratio (RCR), efficiency of the phosphorylative system (ADP/O), and mitochondrial transmembrane potential ( Ψ). Mitochondrial Bioenergetics Control µm 5 µm 10 µm Glutamate/Malate Maximum potential ( Ψ in - mv) ± ± ± ± ± ± ± ± ± ± ± ± 18.4 Lag Phase (s) 70.7 ± ± ± ± 5.4 RCR 6.4 ± ± 0.2 ** 3.5 ± 0.3 ** 3.3 ± 0.3 ** ADP/O 2.6 ± ± ± ± 0.1 Succinate Maximum potential ( Ψ in - mv) ± ± ± ± 3.1 * ± ± ± ± ± ± ± ± 5.0 * Lag Phase (s) ± ± ± ± 8.7 RCR 4.9 ± ± ± 0.2 * 3.7 ± 0.3 ADP/O 1.6 ± ± ± ± 0.2 Effect of compound 27 on Ψ, RCR and ADP/O of energized mitochondria (5 mm glutamate / 2.5 malate or 5 mm succinate). Values are means ± SEM of five independent experiments. Statistically significant compared with control using Student s two tailed t-test. Significance was accepted with *P<0.05, **P<0.01, ***P<0.0005, ****P< S18