Working Group on New TB Drugs

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The Federal Tuberculosis Taskforce, Diagnostics Workgroup has made increasing access to molecular diagnostic tests a priority. Working with NIH and FDA, CDC has concentrated on collaborative research and development to improve the overall testing for drug resistance. In this meeting, we presented progress since CDC PZA Day, held December 15, 2011, specifically related to tests for pyrazinamide, one of four first line drugs used to treat tuberculosis. Data and analysis from national tuberculosis surveillance system, CDC's Tuberculosis Epidemiologic Studies Consortium, the Preserving Effective Treatment Study (or "PETTS"), and the laboratory branch's applied research team were highlighted.

Critical early discoveries were:

1. The discovery by Konno In McDermott's lab that PZA is a pro-drug.
2. That the requirement of an acid pH for PZA activity is now known to be due to the passive absorption of the active moiety pyrazinoic acid (POA) according to the Henderson-Hasselbach equation.
3. That POA is not active when fed to mice by mouth but we do not know what molecule is actually present in mouse blood after feeding and why it is not active.
4. That BMRC clinical trials in East Africa and Hong Kong established first that PZA was highly effective in reducing the relapse rate after 6 months streptomycin/isoniazid therapy, but that it was ineffective in the continuation phase.

1. Pyrazinamide appears to have no bactericidal activity during the first two days of treatment and very little bactericidal activity during the subsequent 12 days.
2. Of all the single drugs tested, pyrazinamide had the lowest level of bactericidal activity, lower even than of thiacetazone.
3. When given with isoniazid, pyrazinamide demonstrates antagonism by reducing the early bactericidal activity of isoniazid during the first two days of treatment but has no effect on the bactericidal activity of streptomycin.
4. When given with isoniazid and streptomycin together, the3 - drug combination has the greatest early bactericidal activity of all the drugs tested.

• Pyrazinamide (PZA) is a unique frontline sterilizing drug that shortens TB therapy. PZA is an indispensible component in current and likely future drug regimens for the treatment of drug susceptible and drug resistant TB. PZA is a paradoxical and unconventional drug that kills non-replicating persister TB bacteria but has no activity for growing bacteria. PZA acts very differently from common antibiotics (i.e., cell wall, nucleic acid, protein synthesis inhibitors or anti-metabolites) through inhibition of energy production (disrupting membrane potential) and trans-translation pathway (see below).
• Mutations in pncA are a major mechanism of PZA resistance in M. tuberculosis (>85% PZA resistance). Molecular tests by pncA sequencing or based on detection of pncA mutation are urgently needed for rapid detection of PZA resistance.
• We propose to divide MDR-TB based on pncA sequencing into two categories, PZA susceptible MDR-TB (Zs-MDR-TB) and PZA-resistant MDR-TB (ZR-MDR-TB), for improved treatment of MDR-TB, where Zs-MDR-TB treatment may be shortened from 24 months to 9 months.
• We identified a new target of PZA, RpsA (S1 protein) that binds POA. Overexpression of target RpsA conferred PZA resistance in M. tuberculosis, and some PZA-resistant clinical isolates without pncA mutation have been found to harbor RpsA mutations. The active form of PZA, pyrazinoic acid (POA), inhibited the trans-translation through inhibition of tmRNA binding to RpsA. Trans-translation is dispensable for growing bacteria but is required for survival under stress conditions. Inhibition of trans-translation is thought to be important for its unique activity against persisters and its treatment shortening potential of this unique drug.
• Future studies are needed to further understand the mechanisms of action of this unique prototype persister drug by identifying new targets and their mutations in PZA action and resistance. In addition, it would be of interest to develop a new generation of persister antibiotics for more effective treatment of TB based on inhibition of trans-translation. New efforts are required to shorten the treatment of TB by novel PZA combinations.

• Trans-Translation is a mechanism for releasing ribosomes that have translated to the end of an mRNA without terminating protein synthesis.
• Zhang's group (Shi et al., 2011) reported that POA binds ribosomal protein S1 and inhibits trans-translation, so lack of trans-translation activity may be responsible for part of the effects of PZA.
• Genetic data indicates that trans-translation is essential in M.tb.
• High-throughput screening identified small molecule inhibitors of trans-translation in E. coli. These inhibitors kill many species of bacteria in vitro, including M.tb.
• The trans-translation inhibitors should help determine which effects of PZA are due to inhibition of trans-translation, and may help in the development of drugs that have some of the unique properties of PZA.

1. Half of all MDR-TB, but none of the INH/RIF-sensitive, strains from Panama were found to have pncA mutations.
2. The LAM family was highly represented among strains with pncA mutations.
3. pncA mutations were distributed throughout the gene, with 15/36 mutations previously reported to be associated with PZA resistance.
4. The most common mutation (G132 insertion) was found in 8 clustered LAM strains, accounting for a quarter of cases.

The crystal structure of the pyrazinamidase PncA of Mycobacterium tuberculosis has unveiled important structural features of the enzyme, in particular the important role played by the metal ion (likely Fe2+) and the residue His57 for local stabilisation of the protein scaffold. The structure provides an unvaluable tool to investigate the molecular mechanisms of resistance to PZA stemming from aminoacid replacements in PncA. The complementary investigation of the thermal stability of PncA mutants has highlighted the underestimated importance of protein folding and thermal stability in PZase activity of the PncA mutants produced in clinical isolates, a parameter which has to be taken into account to fully understand resistance to PZA.

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PZA is remarkable in that it is far more bactericidal when TB are held at room temperature (18-19oC) than at 37oC, 2. In the proposed test LJ slopes contining graded PZA concentrarions are inoculated with the standard BMRC inoculum and left at room temperature for 2 weeks. They are then incubated at 37oC to allow growth of surviving bacilli. The resulting distribution of sensitive strains is very tight resembling that originally found for streptomycin. There was one strain, found highly resistant in the test, that had a normal pncA gene and requires further study.

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• False resistance to PZA is common on both FDA approved broth susceptibility platforms: the VersaTREK and the BACTEC MGIT 960.
• Confirmation of resistance is expensive and time-consuming and false resistances adds to that burden.
• pncA sequencing is an excellent alternative to broth methods but it is not available in all clinical mycobacteriology laboratories.
• Addition of Tween to the VersaTREK seed bottle provides a more consistent inoculum that reduces false resistance and reduces the number of isolates requiring repeat testing to confirm resistance.

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1. Current PZA susceptibility testing in clinical laboratories is unreliable.
2. We do not know whether a mutation in pncA corresponds to clinical resistance to PZA.
3. The pncA wild type gene correlates with PZA susceptibility.
4. We look to develop high resolution melt curve profiles of lights-on/lights-off technology to screen for wild type pncA.

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• PZA definitely does cause heptotoxicity.
• The evidence regarding the interaction of dosage and hepatotoxicity is conflicting.
• The use of pyrazinamide with companion drugs isoniazid and rifampin appears to contribute to the development of hepatotoxicity.
• In a minority of cases rechallenge with pyrazinamide after an episode of hepatotoxicity will elicit an hepatotoxic response, but in a majority of cases pyrazinamide can be reintroduced without incident.
• The precise mechanism by which pyrazinamide causes hepatotoxicity remains a mystery.

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• (Too) little is known about the mechanism of PZA toxicity, little is known about interactions in terms of toxicity with other first line drugs, more research needs to be done.
• PZA hepatotoxicity seems to be dose related, but the final answer still needs to be determined.
• The best dose to use for PZA in relation to toxicity needs to be better defined, is 20-30 mg/kg too low?
• INH with PZA is hepatotoxic in vitro; it may be hepatoprotective in vivo.

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