Journal
JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY
Volume 68, Issue 4, Pages 869-880Publisher
OXFORD UNIV PRESS
DOI: 10.1093/jac/dks483
Keywords
tuberculosis; thioridazine; Ndh; latency; toxicity
Funding
- National Institute of Health Research (NIHR, BRC Liverpool)
- Leverhulme Trust
- Medical Research Council
- Engineering and Physical Sciences Research Council [EP/K039687/1] Funding Source: researchfish
- Medical Research Council [G1002586, MC_PC_12017] Funding Source: researchfish
- EPSRC [EP/K039687/1] Funding Source: UKRI
- MRC [MC_PC_12017, G1002586] Funding Source: UKRI
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Objectives: Phenothiazines have been shown to exhibit in vitro and in vivo activity against Mycobacterium tuberculosis (Mtb) and multidrug-resistant Mtb. They are predicted to target the genetically validated respiratory chain component type II NADH:quinone oxidoreductase (Ndh). Using a set of compounds containing the phenothiazine pharmacophore, we have (i) investigated whether chemical validation data support the molecular target and (ii) evaluated pharmacophore tractability for further drug development. Methods: Recombinant Mtb Ndh was generated and its functionality confirmed by steady-state kinetics. Pharmacodynamic profiling of the phenothiazines, including antitubercular efficacy in aerobic and O-2-limited conditions, time-kill assays and isobole analyses against first-line antituberculars, was performed. Potential mitochondrial toxicity was assessed in a modified HepG2 cell-line assay and against bovine cytochrome bc(1). Results: Steady-state kinetic analyses revealed a substrate preference for coenzyme Q(2) and an inability to utilize NADPH. A positive correlation between recombinant Ndh inhibition and kill of aerobically cultured Mtb was observed, whilst enhanced potency was demonstrated in a hypoxic model. Time-kill studies revealed the phenothiazines to be bactericidal whilst isobolograms exposed antagonism with isoniazid, indicative of intracellular NADH/NAD(+) couple perturbation. At therapeutic levels, phenothiazine-mediated toxicity was appreciable; however, specific mitochondrial targeting was excluded. Conclusions: Data generated support the hypothesis that Ndh is the molecular target of phenothiazines. The favourable pharmacodynamic properties of the phenothiazines are consistent with a target product profile that includes activity against dormant/persistent bacilli, rapid bactericidal activity and activity against drug-resistant Mtb by a previously unexploited mode of action. These properties warrant further medicinal chemistry to improve potency and safety.
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