In Vitro Combination Studies of Benzothiazinone Lead Compound BTZ043 against Mycobacterium tuberculosis

Abstract

Benzothiazinones (BTZ) are a new class of drug candidates to combat tuberculosis that inhibit decaprenyl-phosphoribose epimerase (DprE1), an essential enzyme involved in arabinan biosynthesis. Using the checkerboard method and cell viability assays, we have studied the interaction profiles of BTZ043, the current lead compound, with several antituberculosis drugs or drug candidates against Mycobacterium tuberculosis strain H37Rv, namely, rifampin, isoniazid, ethambutol, TMC207, PA-824, moxifloxacin, meropenem with or without clavulanate, and SQ-109. No antagonism was found between BTZ043 and the tested compounds, and most of the interactions were purely additive. Data from two different approaches clearly indicate that BTZ043 acts synergistically with TMC207, with a fractional inhibitory concentration index of 0.5. TMC207 at a quarter of the MIC (20 ng/ml) used in combination with BTZ043 (1/4 MIC, 0.375 ng/ml) had a stronger bactericidal effect on M. tuberculosis than TMC207 alone at a concentration of 80 ng/ml. This synergy was not observed when the combination was tested on a BTZ-resistant M. tuberculosis mutant, suggesting that DprE1 inhibition is the basis for the interaction. This finding excludes the possibility of synergy occurring through an off-target mechanism. We therefore hypothesize that sub-MICs of BTZ043 weaken the bacterial cell wall and allow improved penetration of TMC207 to its target. Synergy between two new antimycobacterial compounds, such as TMC207 and BTZ043, with novel targets, offers an attractive foundation for a new tuberculosis regimen. W ith 8.8 million incident cases of tuberculosis (TB) worldwide and around 1.5 million deaths in 2010, Mycobacterium tuberculosis infection is one of the most important causes of death from an infectious disease (14). The spread of multidrug-resistant TB (MDR-TB), namely, resistance to both isoniazid (INH) and rifampin (RIF), 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. Greater effort is required to find more efficacious combinations of molecules in order to meet the desired goals of killing both active and persistent tubercle bacilli while decreasing treatment duration. The TB drug development pipeline now comprises several candidates that are in clinical trials or soon will be (2, 12). The recommendations of the Global TB Alliance for Drug Development are to aim for a completely new TB chemotherapy with innovative molecules in combination in order to decrease the risk of emergence of drug resistance (3). Here, benzothiazinones (BTZ) are an extremely potent class of novel antimycobacterials that act by blocking the synthesis of decaprenyl-phospho-arabinose, the precursor of the arabinans in the mycobacterial cell wall (5). BTZ043, the current lead compound of this class, displays similar activity against all clinical isolates of...

1,4-Azaindole, a Potential Drug Candidate for Treatment of Tuberculosis

“…Similar results were also observed with other compounds from the 1,4-azaindole series described previously (11). Interestingly, BTZ043, which also targets the DprE1 enzyme, exhibited synergy with TMC207 (12,17). The absence of any antagonism with a variety of tested anti-TB drugs supported the potential of 1,4-azaindoles for combination therapy.…”

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

“…Compound activity and effect of MK-4 supplementation were evaluated by the resazurin reduction microplate assay (REMA) as previously described in 7H9 medium (12). When applicable, growth medium was supplemented with MK-4 at the concentrations indicated.…”

“…The lead compound BTZ043 was demonstrated to be fully compatible with all the other approved or experimental TB drugs tested (12). Interestingly, BTZ043 and 2-piperazino-benzothiazinone 169 (PBTZ169), the preclinical drug candidate, were shown to act synergistically in vitro with bedaquiline (BDQ), an ATP synthase inhibitor (13).…”

Inhibition of the First Step in Synthesis of the Mycobacterial Cell Wall Core, Catalyzed by the GlcNAc-1-phosphate Transferase WecA, by the Novel Caprazamycin Derivative CPZEN-45

“…mycolylarabinogalactan is quite different from teichoic acid, the first step in its biosynthesis is identical; both WecA and TagO are polyprenyl-phosphate-GlcNAc-1-phosphate transferases. Because mycolylarabinogalactan is essential and some antituberculous drugs, such as ethambutol, and the new benzothiazinones, such as BTZ043 (33), target enzymes involved in P P P P P P P P P P P P P P P P P P P P P P P mycolylarabinogalactan biosynthesis, WecA may be a promising target for a new generation of antituberculous drugs. In fact, WecA is apparently essential for the growth of M. smegmatis (34); however, WecA was initially reported to be nonessential in M. tuberculosis arising from a comprehensive study of a library of transposon insertion mutants (35,36); more recently, however, WecA was reclassified as essential, through reevaluation of the transposon mutant library by deep sequencing and subsequent statistical analysis (37).…”