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While there is an urgent need to develop new and effective drugs for treatment of tuberculosis (TB) and multi-drug resistant TB (MDR-TB), repurposing FDA (U.S. Food and Drug Administration) -approved drugs for development of anti-TB agents may decrease time and effort from bench to bedside. Here, we employed host cell-based high throughput screening (HTS) assay to screen and characterize FDA-approved, off-patent library drugs for anti- Mycobacterium tuberculosis  (MTB) activities. The cell-based HTS allowed us to identify an anti-cancer drug of bis-biguanide dihydrochloride (BBD) as potent anti-mycobacteria agent. Further characterization showed that BBD could inhibit intracellular and extracellular growth of  M. smegmatis  and slow-growing  M. bovis  BCG. BBD also potently inhibited replication of clinically-isolated MTB and MDR-TB strains. The proof-of-concept study showed that BBD treatment of MTB-infected mice could significantly decrease CFU counts in the lung and spleen. Notably, comparative evaluation showed that MTB CFU counts in BBD-treated mice were lower than those in rifampicin-treated mice. No apparent BBD side effects were found in BBD-treated mice. Thus, our findings support further studies to develop BBD as a new and effective drug against TB and MDR-TB.

Bis-biguanide dihydrochloride inhibits intracellular replication of M. tuberculosis and controls infection in mice