title: Antitubercular pharmacodynamics of phenothiazines.
abstract: OBJECTIVE
  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 O2-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 bc1.
  
  
  RESULTS
  Steady-state kinetic analyses revealed a substrate preference for coenzyme Q2 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.
authors.affiliation: Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK.
authors.forename: Ashley J
authors.initials: AJ
authors.lastname: Warman
authors.forename: Teresa S
authors.initials: TS
authors.lastname: Rito
authors.forename: Nicholas E
authors.initials: NE
authors.lastname: Fisher
authors.forename: Darren M
authors.initials: DM
authors.lastname: Moss
authors.forename: Neil G
authors.initials: NG
authors.lastname: Berry
authors.forename: Paul M
authors.initials: PM
authors.lastname: O'Neill
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Ward
authors.forename: Giancarlo A
authors.initials: GA
authors.lastname: Biagini
mesh_terms: D000995:Antitubercular Agents / Q000737:chemistry / Q000494:pharmacology*; D042967:Electron Transport Complex I / Q000037:antagonists & inhibitors; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D050296:Microbial Viability / Q000187:drug effects; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000502:physiology; D010640:Phenothiazines / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D000995:Antitubercular Agents; D004791:Enzyme Inhibitors; D010640:Phenothiazines; D042967:Electron Transport Complex I