FIGURE SUMMARY
Title

Functional drug screening reveals anticonvulsants as enhancers of mTOR-independent autophagic killing of Mycobacterium tuberculosis through inositol depletion

Authors
Schiebler, M., Brown, K., Hegyi, K., Newton, S.M., Renna, M., Hepburn, L., Klapholz, C., Coulter, S., Obregón-Henao, A., Henao Tamayo, M., Basaraba, R., Kampmann, B., Henry, K.M., Burgon, J., Renshaw, S.A., Fleming, A., Kay, R.R., Anderson, K.E., Hawkins, P.T., Ordway, D.J., Rubinsztein, D.C., Floto, R.A.
Source
Full text @ EMBO Mol. Med.

Anticonvulsants stimulate killing of mycobacteria in vitro

A. Screening drugs for effects on survival of intracellular mycobacteria in macrophages. RAW 264.7 cells were infected with a luminescent strain of M. bovis BCG (BCGlux) for 1 h, washed and treated for 24 h with vehicle alone (white), known autophagy enhancers (interferonγ (IFNγ), 200 ng/ml and rapamycin, 200 nM; black), known mTORindependent autophagy inhibitor (forskolin; 24 µM; blue), carbamazepine (CBZ, 50 µM; red), valproic acid (VPA, 3 mM; green) and other examples of hits from a large screen of compounds enhancing intracellular killing of mycobacteria (lithium, 10 mM; rilmenidine, 1 µM; clonidine, 1 µM; calpeptin, 50 µM; grey). Pvalues, unpaired Student′s ttest (e 6) (compared to vehicle alone): IFNγ 0.03; rapamycin 0.003, CBZ 0.001, VPA 0.001, lithium 0.001, rilmenidine 0.001; clonidine 0.02; calpeptin 0.01. Inset: Correlation between measurements of colonyforming units (CFU) and luminescence (RLU) for cultures of M. bovis BCGlux as previously described (Kampmann et al, 2000).

B. Effects on intracellular survival of M. bovis BCGlux of treatment with varying concentrations of CBZ (red) or VPA (green). Pvalues, unpaired Student′s ttest (n = 6) (compared to vehicle alone) for CBZ 30 µM: 9 × 107, 40 µM: 1.9 × 109, 50 µM: 1.3 × 109, 100 µM: 1.6 × 108, 200 µM: 2.7 × 109, VPA 0.5 mM: 6.9 × 108, 1 mM: 1.6 × 109, 3 mM: 4.3 × 1010, 4 mM: 4.2 × 109, 10 mM: 1.1 × 109, 20 mM: 4.2 × 1010. These compounds had no effect on cellfree mycobacterial viability (grey circles).

C. Anticonvulsants enhance intracellular killing of mycobacteria within human alveolar macrophages. Alveolar macrophages, obtained from the bronchoalveolar lavage fluid of three individuals, were infected with M. bovis BCGlux and then treated with carbamazepine (CBZ, 50 µM; red), valproic acid (VPA, 3 mM; green) or vehicle alone (Con; black). Viable intracellular mycobacteria were determined after 24 h of treatment. Unpaired Student′s ttest (n = 3) (compared to vehicle alone): CBZ 0.011; VPA 0.0009.

D. Enhanced killing of intracellular M. tuberculosis (H37Rv) within primary human macrophages by treatment with CBZ (50 µM; red), VPA (3 mM; green) and rapamycin (200 nM; black), compared to control (vehicle alone; white). Unpaired Student′s ttest (n = 6) (compared to vehicle alone): rapamycin 48 h 0.00002, rapamycin 72 h 0.00005, CBZ 48 h 0.00021, CBZ 72 h 0.00022, VPA 48 h 0.0002, VPA 72 h 0.00049.

E. In vivo induction of autophagy by CBZ (50 µM; red) and rapamycin (RAP; 1 µM; blue) compared to vehicle control (white) monitored in zebrafish expressing fluorescent ATG8 cotreated with chloroquine to delay degradation of autophagosomes.

F. Wildtype zebrafish were injected with a red fluorescently tagged M. marinum strain M into the yolk sac circulation valley at 28 hpf. Larvae were imaged at 120 hpf by confocal microscopy and the total mycobacteriaassociated fluorescence quantified using Volocity® software. Data expressed as mean ± SEM (n e 13 fish performed as 3 independent experiments). Pvalues, unpaired Student′s ttest (compared to vehicle alone): 0.0035.

G–I Mice infected via aerosol with a highly virulent clinical strain of multidrugresistant M. tuberculosis CSU 87 were treated from day 20 postinfection with carbamazepine (CBZ, 50 µg/kg i.p. daily), rifampicin/isoniazid (RIF/INH) or vehicle control (= 5 per time point per group). CBZ treatment for 30 days resulted in (G) significantly less viable bacteria detected in lung and spleen, (H) reduced inflammatory pulmonary infiltrates compared to RIF/INHtreated or control animals and (I) decreased lung lesion scores. Unpaired Student′s ttest (n = 5) (compared to vehicle alone): RIF/INH 0.007; CBZ 0.037.

Data information: *P < 0.05; **P < 0.005.

Acknowledgments
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