Arylvinylpiperazine Amides, a New Class of Potent Inhibitors Targeting QcrB of Mycobacterium tuberculosis

New drugs are needed to control the current tuberculosis (TB) pandemic caused by infection with Mycobacterium tuberculosis. We report here on our work with AX-35, an arylvinylpiperazine amide, and four related analogs, which are potent antitubercular agents in vitro. All five compounds showed good activity against M. tuberculosis in vitro and in infected THP-1 macrophages, while displaying only mild cytotoxicity. Isolation and characterization of M. tuberculosis-resistant mutants to the arylvinylpiperazine amide derivative AX-35 revealed mutations in the qcrB gene encoding a subunit of cytochrome bc(1) oxidase, one of two terminal oxidases of the electron transport chain. Cross-resistance studies, allelic exchange, transcriptomic analyses, and bioenergetic flux assays provided conclusive evidence that the cytochrome bc(1)-aa3 is the target of AX-35, although the compound appears to interact differently with the quinol binding pocket compared to previous QcrB inhibitors. The transcriptomic and bioenergetic profiles of M. tuberculosis treated with AX-35 were similar to those generated by other cytochrome bc(1) oxidase inhibitors, including the compensatory role of the alternate terminal oxidase cytochrome bd in respiratory adaptation. In the absence of cytochrome bd oxidase, AX-35 was bactericidal against M. tuberculosis. Finally, AX-35 and its analogs were active in an acute mouse model of TB infection, with two analogs displaying improved activity over the parent compound. Our findings will guide future lead optimization to produce a drug candidate for the treatment of TB and other mycobacterial diseases, including Buruli ulcer and leprosy. IMPORTANCE New drugs against Mycobacterium tuberculosis are urgently needed to deal with the current global TB pandemic. We report here on the discovery of a series of arylvinylpiperazine amides (AX-35 to AX-39) that represent a promising new family of compounds with potent in vitro and in vivo activities against M. tuberculosis. AX compounds target the QcrB subunit of the cytochrome bc(1) terminal oxidase with a different mode of interaction compared to those of known QcrB inhibitors. This study provides the first multifaceted validation of QcrB inhibition by recombineering-mediated allelic exchange, gene expression profiling, and bioenergetic flux studies. It also provides further evidence for the compensatory role of cytochrome bd oxidase upon QcrB inhibition. In the absence of cytochrome bd oxidase, AX compounds are bactericidal, an encouraging property for future antimycobacterial drug development.

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

New 2-Ethylthio-4-methylaminoquinazoline derivatives inhibiting two subunits of cytochrome bc1 in Mycobacterium tuberculosis

Andrej Benjak, Stewart Cole, Shi-Yan Caroline Foo, Andréanne Lupien, Jérémie Piton, Anthony Vocat

The emergence of multi-drug (MDR-TB) and extensively-drug resistant tuberculosis (XDR-TB) is a major threat to the global management of tuberculosis (TB) worldwide. New chemical entities are of need to treat drug-resistant TB. In this study, the mode of action of new, potent quinazoline derivatives was investigated against Mycobacterium tuberculosis (M. tb). Four derivatives 11626141, 11626142, 11626252 and 11726148 showed good activity (MIC ranging from 0.02-0.09 mu g/mL) and low toxicity (TD50 >= 5 mu g/mL) in vitro against M. tb strain H37Rv and HepG2 cells, respectively. 11626252 was the most selective compound from this series. Quinazoline derivatives were found to target cytochrome bc(1) by whole-genome sequencing of mutants selected with 11626142. Two resistant mutants harboured the transversion T943G (Trp312Gly) and the transition G523A (Gly175Ser) in the cytochrome bc(1) complex cytochrome b subunit (QcrB). Interestingly, a third mutant QuinR-M1 contained a mutation in the Rieske iron-sulphur protein (QcrA) leading to resistance to quinazoline and other QcrB inhibitors, the first report of cross-resistance involving QcrA. Modelling of both QcrA and QcrB revealed that all three resistance mutations are located in the stigmatellin pocket, as previously observed for other QcrB inhibitors such as Q203, AX-35, and lansoprazole sulfide (LPZs). Further analysis of the mode of action in vitro revealed that 11626252 exposure leads to ATP depletion, a decrease in the oxygen consumption rate and also overexpression of the cytochrome bd oxidase in M. tb. Our findings suggest that quinazoline-derived compounds are a new and attractive chemical entity for M. tb drug development targeting two separate subunits of the cytochrome bc(1) complex.

2020

High content screening identifies decaprenyl-phosphoribose 2' epimerase as a target for intracellular antimycobacterial inhibitors

Stewart Cole, Ha Pham

A critical feature of Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB), is its ability to survive and multiply within macrophages, making these host cells an ideal niche for persisting microbes. Killing the intracellular tubercle bacilli is a key requirement for efficient tuberculosis treatment, yet identifying potent inhibitors has been hampered by labor-intensive techniques and lack of validated targets. Here, we present the development of a phenotypic cell-based assay that uses automated confocal fluorescence microscopy for high throughput screening of chemicals that interfere with the replication of M. tuberculosis within macrophages. Screening a library of 57,000 small molecules led to the identification of 135 active compounds with potent intracellular anti-mycobacterial efficacy and no host cell toxicity. Among these, the dinitrobenzamide derivatives (DNB) showed high activity against M. tuberculosis, including extensively drug resistant (XDR) strains. More importantly, we demonstrate that incubation of M. tuberculosis with DNB inhibited the formation of both lipoarabinomannan and arabinogalactan, attributable to the inhibition of decaprenyl-phospho-arabinose synthesis catalyzed by the decaprenyl-phosphoribose 2' epimerase DprE1/DprE2. Inhibition of this new target will likely contribute to new therapeutic solutions against emerging XDR-TB. Beyond validating the high throughput/content screening approach, our results open new avenues for finding the next generation of antimicrobials.

2009

More Medicines for Tuberculosis: Fuelling Drug Discovery against Mycobacterium tuberculosis

Shi-Yan Caroline Foo

EPFL2018