Principal Investigator (PI)
The Foundation is providing £95,787 in support.
Joanna Bacon
? Expertise on the development of antibiotics for the TB drug pipeline and in HTS, the screening of compound libraries, and staff time in training and supervising the project work
? Providing the compound libraries for us to screen in the project
? Testing the efficacy of the selected candidates in vivo and enable a pathway to development of successful candidates
? Providing all the consumables, supervision, and equipment required for the screening, toxicity, and murine studies to be carried out at GSK
An important aim for improving TB treatment is to shorten the period of antibiotic therapy without increasing relapse rates or encouraging the development of antibiotic-resistant strains. PZA is a key component of front-line chemotherapy against Mycobacterium tuberculosis. It plays an essential role in the shortened 6-month treatment course [1, 2] due to its ability to act upon the non-replicating/slow-growing or antibiotic resistant organisms that emerge following treatment with the other front-line drugs, isoniazid (INH) and rifampicin (RIF). Although new combinations of antibiotics with novel modes of action are being evaluated, optimal dosing and treatment duration can be investigated further for existing antibiotics. Potentiation of frontline drugs is an approach that can extend the life of existing antibiotics, such as PZA. PZA has no detectable early bactericidal activity (EBA) in the first 2 days of treatment [3]. However, despite this lack of efficacy, PZA exerts an effect by shortening treatment only during the first 2 months of treatment and not beyond this. Extending the duration of treatment with PZA has no additional benefit in either humans or murine models [4-6].
Can we be sure that PZA is not active during the early stages of treatment? It has always been assumed that INH clears the fast-growing population during EBA leaving non-replicating/slow growing drug-tolerant bacteria that need to be cleared by other drugs such as PZA. However, in a recent study we showed that both fast and slow growing cells persist through INH exposure and via different growth rate-specific genotypic and phenotypic mechanisms [7]. Therefore, we cannot generalise that fast-growing bacteria are sterilised during EBA and slow growing cells are sterilised later during treatment. The contribution of PZA to the clearance of actively-dividing bacteria during the first phase of treatment has not been fully explored but it may be having hidden and profound effects such as controlling bacterial numbers or preventing antibiotic resistance from arising. Could the activity of PZA be improved during the first phase of treatment by using it in combination with other antibiotics that boost its activity; thereby further shortening treatment times?
Scientific rationale: can the activity of PZA be enhanced?
PZA is a pro-drug that is hydrolysed to its active form of pyrazinoic acid (POA) by M. tuberculosis pyrazinamdidase/nicotinamidase (PncA) in the bacterial cytoplasm, which is excreted from the bacteria by an unidentified POA efflux mechanism. POA diffuses passively back in through the cell wall as the protonated molecule (HPOA) when the pH of the bacterial environment is acidic. The mode of action of PZA is not fully understood; it would appear to have multiple targets that have not fully been elucidated. An assumption has been made, that PZA will only target inactive bacteria. During in vitro studies of PZA activity, the acidity of the growth medium is difficult to control, and acidified medium is necessary for the susceptibility testing of PZA against M. tuberculosis. The optimal pH for the activity of PZA in vitro is pH 5.6 [8]. However, M. tuberculosis will not grow
below pH 5.9 [9] showing that non-dividing cells are always going to be the target organism in every PZA susceptibility assay that uses an acidity level that is below pH 5.9. Assays that that includes acidity levels low enough for PZA activity but also accommodates growth of M. tuberculosis would enable the assessment of PZA activity against actively-dividing bacteria. There are currently no finely-tuned methods that allow for this assessment.
We have shown recently that M. tuberculosis will actively divide at pH 6.0 in the chemostat (where growth rates and pH can be controlled) at a fast growth rate (23.1h MGT). PZA is active against these populations and has an equivalent level of activity against fast-growing and slow-growing M. tuberculosis in the chemostat [10]. This study demonstrates the importance of dissecting out the in vitro conditions for understanding which parameters are directly affecting drug activity. Given that PZA is active against fast-growers, its activity against this phenotype could be boosted further by potentiators