PMC

(a) Structure of the Mtb QcrB protein homology modelled onto the structure of R. sphaeroides QcrB. Close-up of the Q_p-active site containing the inhibitor stigmatellin A (yellow sticks). Leucine-176, mutated in LPZS-resistant mutants, and threonine 313, mutated in imidazopyridine amide (IPA)-resistant mutants, are depicted as cyan sticks. (b) Dose–response curve of the imidazopyridine amide (GSK2111534A) against wild-type Mtb, the L176P mutant and the T313A mutant (mean±s.d. of three individual experiments). (c) Dose–response curve showing that the T313A mutant remains susceptible to LPZS (mean±s.d. of three individual experiments).

(a) Dose response of LPZS against wild-type Mtb, spontaneous-resistant mutants 1–3 and the genetically engineered recombinant L176P strain (rMtb-L176P) (mean±s.d. of triplicates). (b) Mutation in QcrB conferring LPZS resistance. The arrow indicates the L176P mutation that confers resistance to LPZS. The equivalent region of the human QcrB amino-acid sequence is aligned to the Mtb sequence. (c) LPZS depletes ATP levels after 24 h of treatment (mean±s.d. of three individual experiments). The ATPase inhibitor bedaquiline and the cell-wall inhibitor isoniazid were used as controls. Drug concentrations were 5 × the MIC.

The proton-pump inhibitor LPZ is converted to a sulfenic acid intermediate in the acidic environment of the gastric gland lumen outside the parietal cell. Further prodrug activation to a sulfenamide (not shown) allows binding to the gastric H⁺K⁺-ATPase and its inhibition. We were able to show that sulfoxide reduction in the cytoplasm of Mtb-host cells converts LPZ to the potent antituberculous agent LPZS, which is active against MDR-TB. We provide evidence that LPZS targets cytochrome bc₁ (complex III) leading to disruption of the mycobacterial respiratory chain and rapid ATP depletion. Conversion of LPZS to the sulfenic acid intermediate necessary for inactivation of the gastric H⁺K⁺-ATPase is not possible, making LPZS a highly selective lead compound for the tuberculosis drug pipeline.

(a) Protective activity of LPZ and other drugs against Mtb-induced killing of MRC-5 lung fibroblasts. Data are expressed as the mean±s.d. of three individual experiments. Viable fibroblasts were quantified using Prestoblue. (b) Dose response of LPZ in the fibroblast survival assay using Mtb expressing GFP. Grey bars display host cell survival, green bars quantify intracellular Mtb-GFP (mean±s.d. of three independent experiments; right y axes are truncated for better visualization). (c) Dose response of LPZ in Mtb-infected RAW264.7 macrophages. Grey bars display macrophage survival, green bars quantify intracellular Mtb-GFP (mean±s.d. of 3 independent experiments; right y axes are truncated for better visualization). Growth of intracellular bacteria was inhibited with an IC₅₀ of 2.2 μM. (d) Confocal microscopy of Mtb-GFP-infected RAW264.7 macrophages after treatment with LPZ (10 μM) or vehicle (dimethyl sulfoxide (DMSO)). Macrophage nuclei were stained with 4′,6-diamidino-2-phenylindole (scale bar, 20 μm).

(a) Intracellular ratio of LPZ (m/z 370.0834, g mol⁻¹) and its metabolite (m/z 354.0884, g mol⁻¹) determined by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) over a 48-h period in MRC-5 cells. Representative example of three individual experiments; the complete data set can be found in . (b) ESI–MS mass spectra in the range m/z 350–375 measured for experiments performed on the cell lysate of MRC-5 fibroblasts exposed to LPZ (extracted ion chromatograms can be found in ). (c) ESI–MS spectrum at m/z 354.0884 corresponding to the LPZS standard in methanol. (d) Structures of LPZ and LPZS. LPZS is missing the sulfoxide (red), which is essential for LPZ activity on the human proton pump. (e) LPZ/LPZS ratio determined by ESI-Q-TOF-MS over a 48-h period in 7H9 broth. Representative example of three individual experiments; the complete data set can be found in . (f) Dose–response curve of LPZS for Mtb grown in 7H9 broth (mean±s.d. of three individual experiments). (g) Survival of Mtb-infected MRC-5 fibroblasts was quantified at different concentrations of LPZS (mean±s.d. of three individual experiments). (h) Efficacy of LPZS in the mouse model of acute tuberculosis. Bacterial burden (c.f.u.) was determined in the lungs of four mice treated with the vehicle control (TPGS) or four mice treated with LPZS at 300 mg kg⁻¹ b.i.d. given by oral gavage (mean±s.d., Student's t-test was used to compare groups).