Elucidating the antimycobacterial mechanisms of ciprofloxacin and decoquinate derivative RMB041 on Mycobacterium tuberculosis using metabolomics

Abstract

Tuberculosis (TB) is amongst the leading causes of death brought about by a single infectious agent. Despite all the research done to date since the discovery of the infectious agent, Mycobacterium tuberculosis (Mtb) in 1882, its prevalence is still that of epidemic proportions. This can be ascribed to several factors, including, amongst others: the rapid development of anti-TB drug resistance, poor patient adherence, and inadequate treatment regimens. Subsequently, a better understanding of the mechanisms of action (MoA) of the new anti- TB drugs, as well as the possible mechanisms of resistance (MoR) of Mtb to these, is desperately needed. However, due to the many challenges associated with de novo drug development, including the high costs and the extremely long duration required for the completion of the mandatory clinical trials, the use of existing drugs for which safety and efficiency have already been verified, repurposed as anti-TB drugs, is considered a viable option for faster treatment availability. Drug discovery has also seen promising progress with a computational prediction of growth/replication targets, drug-host interactions, and pharmacokinetic properties. To this end, we investigated and compared the whole metabolome of Mtb treated with ciprofloxacin and decoquinate RMB041, respectively, to those of non-treated Mtb (control samples), by using two-dimensional gas chromatography time-of-flight mass spectrometry (GCxGC-TOFMS) and various uni- and multivariate statistical methods. GCxGC-TOFMS is a powerful analytical technique that separates sample constituents at trace levels while providing accurate molecular structural information of the differentiated compounds. The identified metabolite markers characterizing the Mtb alterations induced by the presence of the drugs were used to elucidate the antimycobacterial MoA against Mtb and possible MoR by Mtb to the drug (in the case of the ciprofloxacin treatment). The treatment-induced metabolomic changes observed indicated that both ciprofloxacin and decoquinate RMB041 cause inhibition/destruction of the cell wall and DNA damage. In the decoquinate RMB041 treated group, inhibition of cell growth/replication and a reaction by Mtb going into a more pronounced state of dormancy, with a lack of DNA repair was additionally observed in response to the drug. Considering that drugs commonly target multiple proteins, we investigated proteins possibly affected by decoquinate RMB041 that would explain the altered metabolic pathways seen in the aforementioned experiments. For this, we used an in silico approach using various software and web servers. Interestingly, all of the identified protein targets identified via the in silico, were annotated to pathways, which also unanimously corresponded with our previously suggested MoA of decoquinate derivative RMB04 determined by the metabolomics experiments. Furthermore, the target list included proteins that were previously suggested as promising targets against dormant Mtb. We additionally investigated the in silico safety profile and oral bioavailability, which also showed promising results. In this pharmacometabolomics study, we identified the biochemical pathways that confirm the previously proposed MoA of ciprofloxacin and decoquinate RMB041 against Mtb, identified previously unknown adaptive response mechanisms by Mtb to these anti-TB agents. We also prove the accuracy of using computational

software for determining/confirming the aforementioned activity of such novel repurposed anti-TB drugs and possible mechanisms of drug resistance by Mtb, and whether or not such medication can be used in combination with existing anti-TB drugs.