Nitazoxanide controls virus viability through its impact on membrane bioenergetics

Figures

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  Fig. 1. The three thiazolides: Nitazoxanide, tizoxanide, RM4848 and the uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP).

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  Fig. 2. Effect of TZ, RM or CCCP on the cellular OCR of Phoenix ECO cells. The data in (a, b and c are expressed as the mean of OCR ± SEM of three independent experiments. OCRs are shown for the phosphorylating state (Phos) and non-phosphorylating state (olig) in presence of oligomycin 2 µM final. The relative values of OCRs due to the activity of ATP synthase (OXPHOS-OCR) in d, e and f were calculated as described in the materials and methods.

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  Fig. 3. Effect of TZ, RM or CCCP on cellular viability. Panels (a–c) MTT test with mean values ± SDs of the difference in optical density (OD570nm—OD680nm). Cells were grown in a medium with 1 mM Galactose, low (3 mM) or high (25 mM) glucose. N indicates the number of values considered taking into consideration the number of independent experiments (number in parenthesis) and cell lines. The five experiments with Galactose were made with the parental cell line (Phoenix ECO). In contrast, the five experiments made with glucose (3 or 25 mM) implied also the derivatives of Phoenix ECO prepared for the project: Phoenix ECO GFP-, GFP + , or GFP + + hence the number of values (12) is higher than the number of experiments. The statistical significance of the differences was evaluated with paired t tests on OD570nm—OD680nm values. Panels (d–f) relative units, with 100% as the vehicle (DMSO) and 0% as the control in the presence of oligomycin. The statistical significance (paired t test) of the difference compared with that of DMSO was determined with respect to the original OD values (shown in a–c: * = p < 0.05; ** = p < 0.01; *** = p < 0.001, same code with other symbols (§,#).

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  Fig. 4. Increased impact on OXPHOS and viral RNA release. The number of viral RNA copies in the supernatant of the virus producing cells (Phoenix-ECO GFP + +) was assessed by RTqPCR titration at 24 h posttreatment with TZ, RM or CCCP. The levels of impact on OXPHOS-OCR are indicated below the groups of histograms and are coded from white (0% impact on OXPHOS-OCR) to black (100% impact and full inhibition by oligomycin). The number of data points reveals number of independent experiments and histograms represent mean value ± SD. (a) The reference data points are as follows: hexagons, control (no addition); diamonds, vehicle (DMSO); squares, reference uncoupler (CCCP); and downward triangles, oligomycin. Control, vehicle and oligomycin were references included in each single experiment (N = 6). Thiazolides are indicated by black symbols; circles, tizoxanide and triangles, RM4848. All impact levels with uncouplers (TZ, RM and CCCP) could not be included in each single experiment and N ranged from 2 to 4. (b) To improve comparison between the different experiments, the viral RNA copy number was expressed as the ratio to control in the same experiment and values obtained with the uncouplers…

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  Fig. 5. Increased impact on OXPHOS efficiency and the infectivity of released viruses. Infectivity was evaluated from the percentage of NIH3T3 positive for expression of the green fluorescent protein (see M & M section). Data points from independent experiments are shown and histograms represent mean value ± SD. (a) Measurement of infectivity of virus particles, the reference data points are as follows: hexagons, control (no addition); diamonds, vehicle (DMSO); squares, reference uncoupler (CCCP); downward triangles, oligomycin (100% impact). Thiazolides are indicated by black symbols; circles, TZ and triangles, RM. Experiment with 3 mM shown examined supernatants used previously for viral RNA release (Fig. 4). Statistical significance is shown with regard to vehicle. (b) Expression of infectivity in relative units with uncouplers grouped according to level on OXPHOS-OCR (as in Fig. 4b) more experiments with 3 mM are included (see text). Statistical significance is shown as: * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001, for comparison with vehicle.

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  Fig. 6. Adenine nucleotides. Left: AEC is a dimensionless ratio given by the formula (ATP + 1/2ADP)/(ATP + ADP + AMP), and the Y ordinate starts at 0.5, which is the value of AEC obtained if ADP represents 100% of adenine nucleotides. The level of interference of drugs with OXPHOS in Phoenix-ECO cells is indicated above. The black arrow highlights the oligomycin reference with OXPHOS set to zero. Drugs that interfere with OXPHOS are indicated below the X axis. The data points are shown with their medians (four determinations/independent experiments). Right: X axis: impact on OXPHOS (%); Y axis: ATP/ADP ratios in these same samples. Statistical significance (p value) is shown for comparison with DMSO (One-way ANOVA for repeated measures and Dunnett’s multiple comparison test).

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  Fig. 7. Mitochondrial uncoupling by TZ, RM and CCCP. ATP production (Oxphos). (a–c) Dose response to thiazolides and to CCCP. The values of the OCR (solid line) and mitochondrial membrane potential (milliVolts) are shown starting from oligomycin and the nonphosphorylating rate (X = 0) and with increasing concentrations of TZ (a,c,e), RM (b,d,f) or CCCP (g) at different concentrations, a-d and g mean value ± SD of three or more independent experiments (mitochondrial preparations). Experiments comparing the effects of thiazolides in the presence (gray lines) or absence (black lines) of CAT (c,d) or CSA (e,f one experiment only) are shown without symbols for clarity. (h) Products of the OCR and membrane potential (in mV) for the three uncouplers; the symbols corresponding to (a,b,g) and the TZ and CCCP values are shown up to the maximal value and not for higher concentrations. This product could be converted into power in Watts (see Materials and Methods section), and a Y ordinate of 100 in D indicates 116 µW/mg protein (116 W/kg protein) dissipated by the proton circuit (inset in h).

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  Fig. 8. Mitochondrial ATP production (Oxphos). (a) Relationships between the oxygen consumption rate and the membrane potential in mitochondria at steady state. Individual gray diamonds show individual data points obtained with four independent mitochondrial preparations used for ATP measurement (controls in b–d) “P” indicates the phosphorylating state (ADP present), while L indicates the absence of phosphorylation (oligomycin present). The intermediate data points (with error bars) correspond to the first (left) values shown in Fig. 7a,b,g excluding values when potential dropped or after maximal OCR hence five values with TZ (black circles) four with RM (black triangles) and three with CCCP (empty squares). (b) kinetic of phosphorylation of ADP into ATP in presence/absence of thiazolides, Y represents results for ATP sampling (see M&M section) with control (gray diamonds), TZ (black circles) or RM (empty triangles) it is expressed in relative units: 100% is the ATP produced by the respective control in five minutes, actual values are shown in supplemental 5a. A same dosage of TZ or RM was used in each of the four experiments but varied from one experiment to another to adjust the…

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  Fig. 9. Impact on cellular energy charge viability and virus, experiments with 3 mM Glucose are considered. X axis: impact on OXPHOS-OCR, 0% is vehicle, 25–75% refer to uncouplers (TZ, RM, CCCP) and 100% to full suppression of OXPHOS by oligomycin. Left Y axis: value of the Adenine nucleotide energy charge (AEC); Right Y axis: Relative values: ratio to control (no addition) for cell viability (MTT test) Viral RNA release and infectivity of viruses released. If the reference uncoupler (CCCP) is considered viral RNA release and Cell viability curves are similar (Suppl. 6) the difference observed here is explained by another effect of TZ and RM that increase cell viability in presence of glucose (Fig. 3). The number of independent experiments was 4 (AEC), 8 (MTT), 6 (viral RNA) and 7 (infectivity). The number of data points might be different, it is higher when values obtained with the three uncouplers were pooled (AEC, viral RNA and infectivity) or lower for the 75% level of interference, which was not considered (AEC), or only in one experiment (infectivity, three data points) and two (viral RNA, six data points).