title: Selective intracellular accumulation of the major metabolite issued from the activation of the prodrug ethionamide in mycobacteria.
abstract: BACKGROUND
  Ethionamide is one of the most widely used drugs for the treatment of multidrug-resistant tuberculosis (MDR-TB). Like isoniazid, and pyrazinamide, ethionamide is a prodrug that needs to be activated by a mycobacterial enzyme. Activation pathways of prodrugs are generally problematic to uncover as they produce intermediates potentially difficult to characterize, to purify and that might prove unstable outside of their cellular context.
  
  
  OBJECTIVE
  We have used high resolution magic angle spinning-NMR (HRMAS-NMR) to follow ethionamide activation directly within living mycobacterial cells.
  
  
  RESULTS
  Data indicated that the intracellular metabolization of ethionamide strictly depends on the presence of the monooxygenase EthA and that EthA-dependent activation of ethionamide is coupled to a precise molecular sorting mechanism of the ethionamide metabolites. We found that the previously identified ethionamide metabolite 2-ethyl-4-hydroxymethylpyridine is produced in substantial amounts by the ethionamide-treated mycobacteria and that it is present exclusively outside of the bacteria. In contrast, the still unidentified ethionamide metabolite ETH* is the only ethionamide derivative detected within the bacterial cell. Moreover, ETH* appears to be unable to cross the bacterial envelope and consequently accumulates within the cytoplasm of the ethionamide-treated mycobacteria.
  
  
  CONCLUSIONS
  These results strongly suggest that ETH* is the active antimycobacterial ethionamide derivative and open new perspectives for the understanding of the mode of action of prodrugs.
authors.affiliation: UMR 8525 CNRS-Universíté de Lille 2 Lille, F-59021, France.
authors.forename: Xavier
authors.initials: X
authors.lastname: Hanoulle
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Wieruszeski
authors.forename: Pierre
authors.initials: P
authors.lastname: Rousselot-Pailley
authors.forename: Isabelle
authors.initials: I
authors.lastname: Landrieu
authors.forename: Camille
authors.initials: C
authors.lastname: Locht
authors.forename: Guy
authors.initials: G
authors.lastname: Lippens
authors.forename: Alain R
authors.initials: AR
authors.lastname: Baulard
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D001711:Biotransformation; D003470:Culture Media; D005000:Ethionamide / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000295:instrumentation / Q000379:methods; D020102:Mycobacterium smegmatis / Q000254:growth & development / Q000378:metabolism*; D010105:Oxygenases / Q000378:metabolism; D011355:Prodrugs / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; D011355:Prodrugs; C414178:etaA protein, Mycobacterium tuberculosis; D010105:Oxygenases; D005000:Ethionamide