Phenylhydrazones are active against non-replicating Mycobacterium tuberculosis

There is an urgent need for the development of shorter, simpler and more tolerable drugs to treat antibiotic tolerant populations of Mycobacterium tuberculosis. We previously identified a series of phenylhydrazones (PHY) active against M. tuberculosis. We selected six representative compounds for further analysis. All compounds were active against non-replicating M. tuberculosis, with two compounds demonstrating greater activity under hypoxic conditions than aerobic culture. Compounds had bactericidal activity with MBC/MIC of <4 and demonstrated an inoculum-dependent effect against aerobically replicating bacteria. Bacterial kill kinetics demonstrated a faster rate of kill against non-replicating bacilli generated by nutrient starvation. Compounds had limited activity against other bacterial species. In conclusion, we have demonstrated that the PHY compounds have some attractive properties in terms of their anti-tubercular activity.


Introduction
Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a global health problem [1].
In 2016, 10.3 million people worldwide became ill with TB and 1.7 million people lost their lives to the disease [1]. While the number of deaths fell ~ 24%, the number of new cases increased slightly to 6.3 million in 2016. Approximately a quarter of the world's population has latent TB in which patients are asymptomatic and non-infectious. Reactivation of latent infection is observed in 10% of cases representing a large reservoir of infection [2, anaerobic conditions followed by 28h outgrowth under aerobic conditions; as a comparator plates were incubated for 6 days under aerobic conditions. Growth was measured by luminescence. Growth inhibition curves were fitted using the Levenberg-Marquardt algorithm. IC 90 was determined as the minimum concentration required to prevent 90% growth.

Minimum Bactericidal Concentration (MBC)
MBCs were determined as described [39]. Briefly, a late log phase culture (OD 590 0.6-1.0) was adjusted to an OD of 0.1 in 7H9-Tw-OADC and 50 µL used to inoculate 5 mL of 7H9-Tw-OADC containing compound. Cultures were incubated standing at 37 o C. Bacterial viability was determined by plating serial dilutions and enumerating CFUs after four weeks of incubation at 37°C. For starvation, M. tuberculosis H37Rv was resuspended in phosphate buffer saline (PBS) plus 0.05% w/v Tyloxapol and incubate standing for 2 weeks. Compound was added after two weeks and CFUs determined.

Spectrum
MICs were determined using the serial dilution agar method. Unless otherwise stated, compounds were prepared as an 8-point 2-fold serial dilution in DMSO starting at 100 µM. MIC 99 was defined as the minimum concentration that prevented 99% of growth.

Results and discussion
The PHY series have good activity against aerobically-cultured, actively growing M. tuberculosis in axenic culture. Our previous work was limited to determining minimum inhibitory concentrations (MICs) under these conditions and demonstrated activity for a range of analogs, with many MICs in the range of 20 µM [14]. However, many compounds that act against rapidly growing mycobacteria are ineffective against non-replicating or intracellular organisms. Since M. tuberculosis can survive under low oxygen tension, we were interested to determine whether our compounds had activity under this setting, which is relevant to the environment encountered during infection. We were also interested in determining if compounds were bactericidal against replicating and nonreplicating bacilli. We selected six representative PHY compounds based on their activity and structure for characterization in other assay systems (Fig 1) [14].

PHY compounds are bactericidal against replicating M. tuberculosis
We first determined whether compounds had bactericidal or bacteriostatic activity under aerobic conditions (Fig 2). We tested four compounds at varying concentrations.  aureus. There was no correlation between the activity against M. tuberculosis and other species, since the most active anti-tubercular compounds were inactive against other species. In conclusion, we have demonstrated that the PHY compounds have some attractive In addition, they demonstrated higher rates of kill against non-replicating than replicating bacilli. Our previous work had demonstrated that they are also more potent against a strain of M. tuberculosis with reduced LepB [14]. Since LepB expression is reduced under both nutrient-starved and hypoxic conditions [6,7], this may account for the increased activity of PHY compounds in these conditions.

Funding
This work was funded by NIAID of the National Institutes of Health under award numbers R01AI095652 and R01AI132634 and by the Bill and Melinda Gates Foundation under grant OPP1024038. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Conflict of interest
Tanya Parish serves on the Editorial Board of PLOS ONE. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.