Streptomycin-Starved Mycobacterium tuberculosis 18b, a Drug Discovery Tool for Latent Tuberculosis

Mycobacterium tuberculosis 18b, a streptomycin (STR)-dependent mutant that enters a viable but nonreplicating state in the absence of STR, has been developed as a simple model for drug testing against dormant bacilli. Here, we further evaluated the STR-starved 18b (SS18b) model both in vitro and in vivo by comparing the behavior of 22 approved and experimental tuberculosis drugs. Using the resazurin reduction microplate assay (REMA), rifampin (RIF), rifapentine (RPT), TMC207, clofazimine (CFM), and linezolid (LIN) were found to be active against SS18b in vitro, and their bactericidal activity was confirmed by determining the number of CFU. A latent 18b infection was established in mice, and some of the above-mentioned drugs were used for treatment, either alone or in combination with RIF. RIF, RPT, TMC207, CFM, and pyrazinamide (PZA) were all active in vivo, while cell wall inhibitors were not. A comparative kinetic study of rifamycin efficacy was then undertaken, and the results indicated that RPT clears latent 18b infection in mice faster than RIF. Intrigued by the opposing responses of live and dormant 18b cells to cell wall inhibitors, we conducted a systematic analysis of 14 such inhibitors using REMA. This uncovered an SS18b signature (CWPRED) that accurately predicted the activities of cell wall inhibitors and performed well in a blind study. CWPRED will be useful for establishing the mode of action of compounds with unknown targets, while the SS18b system should facilitate the discovery of drugs for treating latent tuberculosis.

More Medicines for Tuberculosis: Fuelling Drug Discovery against Mycobacterium tuberculosis

Shi-Yan Caroline Foo

Tuberculosis (TB), whose etiological agent is Mycobacterium tuberculosis (M. tuberculosis), has plagued humanity since antiquity. Even with chemotherapy available today, TB is the leading cause of death due to an infectious disease. Modern day factors, such as the HIV epidemic and the emergence of drug-resistant TB strains, have redefined the complexities and challenges of tackling the TB pandemic. Current treatments for TB are lengthy, and poor adherence to such prolonged treatment further exacerbates the issue of drug resistance. Moreover, therapies for drug-resistant TB have low cure rates. There is therefore a pressing need for improved therapies that are short and efficacious against all TB strains, which can be achieved through new antimicrobials that are more potent and have novel mechanisms of action, in addition to being affordable, orally bioavailable, and without drug-drug interactions. Such new anti-TB drugs need to be discovered and developed through a long, risky, and costly process, in which attrition rates are high. While there are promising compounds currently being developed, including the benzothiazinones (BTZs), it is necessary to further populate and enhance the Global TB drug pipeline to ensure the availability of new drugs. This thesis aims to address this need through the discovery work of two new, highly promising families, the AX and PB compounds, and of BTZs. The piperazine-based AX analogs are easily synthesised and demonstrate potent activity against M. tuberculosis in vitro and in vivo. Their target, identified in this work, is the QcrB subunit of the cytochrome bc1-aa3 complex, a terminal oxidase of the mycobacterial respiratory chain. Notably, AX compounds are bactericidal in the absence of the alternate terminal oxidase, cytochrome bd. As this family interacts differently in the same binding site of QcrB as Q203, a drug candidate in clinical trials, AX compounds could potentially serve as a backup series for QcrB inhibitors. The PB family, derived from the natural product lapachol, is also easily synthesised and shows substantially improved activity against M. tuberculosis in vitro compared to the parent compound. PB analogs also demonstrated activity against the non-replicating bacillus and in infected macrophages. The mechanism of action of PB compounds relies on the F420 cofactor, although not on the F420-dependent nitroreductase Ddn, therefore this family has a novel mechanism of action which is highly specific to M. tuberculosis. To support the clinical development of PBTZ169, the mechanism of resistance to BTZs was further elucidated in this thesis. Five mutations at cysteine 387 of the target enzyme, DprE1, were identified as mediating resistance to BTZs, which would serve as resistance markers for clinical screening. The impact of these mutations on M. tuberculosis and on DprE1 was further characterised, revealing a fitness cost imposed on the bacillus intracellularly and reduced catalytic efficiency of the enzyme. This thesis additionally contributed to the characterisation of a PBTZ169 backup series and the design of an optimal regimen with other TB drugs. The compounds presented herein merit further optimisation so their full antibiotic potential may be realised for TB, and possibly for other mycobacterial diseases as well. Altogether, this thesis has contributed to the fuelling of drug discovery against M. tuberculosis, and a step towards more medicines for TB.

EPFL2018

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

Stewart Cole, Ruben Hartkoorn, Benoit Lechartier

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.

Amer Soc Microbiology2012

Multitarget Drug Discovery for Tuberculosis and Other Infectious Diseases

Stewart Cole, Benoit Lechartier, Kai Li, Wei Zhu

We report the discovery of a series of new drug leads that have potent activity against Mycobacterium tuberculosis as well as against other bacteria, fungi, and a malaria parasite. The compounds are analogues of the new tuberculosis (TB) drug SQ109 (1), which has been reported to act by inhibiting a transporter called MmpL3, involved in cell wall biosynthesis. We show that 1 and the new compounds also target enzymes involved in menaquinone biosynthesis and electron transport, inhibiting respiration and ATP biosynthesis, and are uncouplers, collapsing the pH gradient and membrane potential used to power transporters. The result of such multitarget inhibition is potent inhibition of TB cell growth, as well as very low rates of spontaneous drug resistance. Several targets are absent in humans but are present in other bacteria, as well as in malaria parasites, whose growth is also inhibited.

Amer Chemical Soc2014

More Medicines for Tuberculosis: Fuelling Drug Discovery against Mycobacterium tuberculosis

Shi-Yan Caroline Foo

EPFL2018