Mode of Action of Clofazimine and Combination Therapy with Benzothiazinones against Mycobacterium tuberculosis

Abstract

Clofazimine (CZM) is an antileprosy drug that was recently repurposed for treatment of multidrug-resistant tuberculosis. In Mycobacterium tuberculosis, CZM appears to act as a prodrug, which is reduced by NADH dehydrogenase (NDH-2), to release reactive oxygen species upon reoxidation by O 2 . CZM presumably competes with menaquinone (MK-4), a key cofactor in the mycobacterial electron transfer chain, for its reduction by NDH-2. We studied the effect of MK-4 supplementation on the activity of CZM against M. tuberculosis and found direct competition between CZM and MK-4 for the cidal effect of CZM, against nonreplicating and actively growing bacteria, as MK-4 supplementation blocked the drug's activity against nonreplicating bacteria. We demonstrated that CZM, like bedaquiline, is synergistic in vitro with benzothiazinones such as 2-piperazino-benzothiazinone 169 (PBTZ169), and this synergy also occurs against nonreplicating bacteria. The synergy between CZM and PBTZ169 was lost in an MK-4-rich medium, indicating that MK-4 is the probable link between their activities. The efficacy of the dual combination of CZM and PBTZ169 was tested in vivo, where a great reduction in bacterial load was obtained in a murine model of chronic tuberculosis. Taken together, these data confirm the potential of CZM in association with PBTZ169 as the basis for a new regimen against drug-resistant strains of M. tuberculosis. With approximately 9 million incident cases of tuberculosis (TB) worldwide and around 1.5 million deaths in 2012, Mycobacterium tuberculosis infection is one of the most important causes of death from a single infectious agent (1). The spread of multidrug-resistant TB (MDR-TB), namely, with resistance to isoniazid and rifampin, poses additional challenges to treatment with currently available anti-TB drugs. The situation is exacerbated by the increasing emergence of extensively drug-resistant (XDR) strains of M. tuberculosis, which cause diseases essentially untreatable with existing compounds. It is nowadays widely acknowledged that we need to develop new antibiotic combinations for TB and that these new regimens should be tested together at the preclinical stage, rather than testing a series of single drugs separately, in order to fill the TB drug development pipeline more efficiently (2-4).Some of the compounds in advanced clinical trials for TB are molecules that were originally used to treat other infectious diseases and have been repurposed for TB. Among the repurposed molecules, clofazimine (CZM), a riminophenazine originally developed as a drug to treat TB but overlooked for decades, has been used as a standard component of the treatment of leprosy for 50 years. It was recently repurposed for managing MDR-TB cases, notably following the results of the so-called Bangladesh study, which demonstrated that a CZM-containing regimen can cure such resistant cases in 9 to 12 months (5). Grosset et al. demonstrated the substantial benefit of adding CZM to second-line regimens in mice infected with isoniazid-resistant...

Targeting Mycobacterium tuberculosis Sensitivity to Thiol Stress at Acidic pH Kills the Bacterium and Potentiates Antibiotics

“…3A). Clofazimine, a component of the multidrug treatment regimen for Mycobacterium leprae infection, is a redox cycling compound that can directly compete with menaquinone and spontaneously produce reactive oxygen species (Lechartier and Cole, 2015; Yano, et al, 2011). If AC2P36 functions to deplete thiol pools and diminish resistance to oxidative stress, then AC2P36 is predicted to sensitize Mtb to clofazimine.…”

Antiinfectives targeting enzymes and the proton motive force

“…Clofazimine (17) was originally developed as a TB drug (41) but later was used extensively (42) in treating leprosy, caused by another Mycobacterium, Mycobacterium leprae. There have been several mechanisms of action demonstrated or proposed for clofazimine (17), including a redox cycling reaction involving the generation of reactive oxygen species (43), and clofazimine is currently of interest for use in combination therapies with benzothiazinones (44). We used the sealed, inside-out IMV assay used previously to investigate SQ109 (2) (13) with 9-amino-6-chloro-2-methoxyacridine (ACMA) as a pH-sensitive fluorescence probe of the pH gradient, ΔpH (computed as illustrated in Fig.…”

The M. tuberculosis HAD phosphatase (Rv3042c) interacts with host proteins and is inhibited by Clofazimine

“…Clofazimine (CFZ) itself is a riminophenazine anti-leprosy drug that is being tested for treatment of MDR and XDR TB [9,10]. It has been reported earlier that CFZ acts as a prodrug, which is reduced by NADH dehydrogenase (NDH-2), to release reactive oxygen species upon reoxidation by Oxygen [11]. Excitingly, the inhibition of MtSerB2 by CFZ reverses the various functional effects elicited by the enzyme.…”