Trojan Horse Strategy: How A Prodrug For Moxifloxacin Kills TB Persisters
When the TB drug moxifloxacin was masked as a prodrug form, the prodrug’s permeability into the bacteria improved significantly. Once inside the bacteria, the prodrug is activated by an enzyme leading to the generation of the active drug moxifloxacin within the bacteria. This strategy significantly enhanced the lethality against TB persisters, thus effectively reducing the population of persisters
We developed a masked form of the frontline antibiotic moxifloxacin that shows significantly enhanced lethality against drug-tolerant, non-replicating Mycobacterium tuberculosis (Mtb). This dormant ‘persister’ subpopulation prolongs tuberculosis treatment and drives relapse. The work demonstrates how altering drug permeability can help address this major problem.
The work was carried out in collaboration with IISc Bangalore, Binghamton University, CSIR-Central Drug Research Institute, and CSIR-National Chemical Laboratory, and published in the Journal of Medicinal Chemistry.
Cracking the challenge of TB persisters
Tuberculosis, caused by the bacterium Mycobacterium tuberculosis (Mtb), remains one of the world’s deadliest infectious diseases. While TB is curable with a six-month course of antimicrobial therapy, global health agencies continue to seek shorter, more effective regimens that improve adherence, reduce relapse, and combat rising drug resistance. A major obstacle to shortening therapy is a small fraction of bacteria that enter a non-replicating, drug-tolerant “persister” state. These persisters behave like hibernating seeds that survive antibiotic exposure and reactivate later.
Even though genetically identical to actively growing bacteria, persisters adopt a slow-growing, stress-adapted state with poor permeability to drugs and elevated drug efflux. The combination of poor permeability and enhanced drug efflux leading to reduced drug accumulation inside the bacteria and target engagement. This results in phenotypic antimicrobial resistance that enables them to withstand prolonged antibiotic exposure. Recent clinical advances have shown that adding moxifloxacin can shorten therapy from six to four months; moxifloxacin now features in updated WHO guidelines for children and for multidrug-resistant TB (MDR-TB).
However, non-replicating TB bacteria continue to remain highly refractory to moxifloxacin and other frontline drugs. This limitation motivated our team to redesign moxifloxacin as a prodrug that penetrates both replicating and non-replicating bacteria,and activates only inside the bacteria. While the prodrug retains lethality in actively growing bacteria, it outperforms moxifloxacin against persisters.
Arming a drug to disarm persisters
We used an ‘arm to disarm’ strategy, where we masked moxifloxacin as a prodrug form. This change improves permeability into the bacteria, and once inside the bacteria, the prodrug is activated by an enzyme nitroreductase (NTR). The active drug is now generated within bacteria. This strategy was found to be effective in reducing the population of persisters, and hence arming a drug to disarm persisters.
To identify the prodrug, we synthesised a focused library of nitro(hetero)aryl-modified moxifloxacin prodrugs and screened them for conversion back to the active drug under conditions associated with mycobacterial persisters, such as a reductive environment. Among these, one prodrug — 2-nitrothiazole ester prodrug1e — was identified as the best candidate, exhibiting rapid and efficient cleavage to release moxifloxacin.
Experimental data with the enzyme was studied by a rigorous computational model that provides the foundation for future prodrug design. The prodrug exhibited efficacy in replicating TB bacteria, multidrug-resistant and extensively drug-resistant (MDR/XDR) patient isolates and macrophage infection models, with phenotypic and genotypic data confirming moxifloxacin-driven lethality. A mathematical model linking permeability, prodrug conversion, and bacterial killing showed that non-replicating TB bacteria require higher intracellular moxifloxacin levels, and helps us rationalise our results.
Together, this study shows that phenotypic antimicrobial tolerance in non-replicating TB bacteria can be addressed by designing compounds that can help enhance permeability. The Trojan horse strategy that we developed for moxifloxacin can be a blueprint for other TB drugs as well. We are currently undertaking these studies at IISER Pune.
