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Working Group on New TB Drugs

Stop TB Partnership

Meetings

"Demystifying Pyrazinamide — Challenges and
Opportunities" Workshop 2012

September 5 - 6, 2012 | Baltimore, Maryland

Meeting Overview

DAY 1 September 5 | 8:00 a.m. - 4:00 p.m.

Some description for Day 1

DAY 2 September 6 | 8:00 a.m. - 4:00 p.m.

Some description for Day 2

Johns Hopkins University School of Medicine | Thomas B. Turner Building
2024 East Monument Street | Baltimore, Maryland 21287

September 5, 2012Day 1 Meeting Introduction
Opening Remarks Richard Hafner

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Welcome Michael J. Klag

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Highlights of 2011 Essentiality of PZA Workshop, Conclusions and Action Items Barbara Laughon

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Highlights of 2011 PZA Day, Conclusions and Action Items Michael Iademarco

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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.
History of PZA and Current Status Denis Mitchison

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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.
PZA Early Bactericidal Activity Amina Jindani

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  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.
Session 1: PZA Mechanisms of Action
Mechanisms of Action and Resistance to PZA: a 20-Year Perspective Ying Zhang

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  • 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.
PZA and Trans-Translation Complex RpsA, SsrA, SmpB, EfTu Ken Keiler

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  • 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.
Genome Sequences of MDR Isolates in the Context of PZA Resistance Petros Karakousis

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  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.
Structure of Pyrazinamidase and Lesson on its Mode of Action Wladimir Sougahoff

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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.

Group Discussion

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Session 2: Drug-Resistance
Towards a More Accurate PZA Susceptibility Test Denis Mitchison

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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.

Improved PZA Broth Susceptibility Testing on the VersaTREK - The Use of Tween Patricia Simner

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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.
Development of a PZA Pipeline to Improve Phenotypic and Molecular Drug Susceptibility Testing for PZA Jamie Posey

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Diagnosing the Wild Type pncA Gene Barry Kreiswirth

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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.
Group Discussion

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September 6, 2012Day 2 Session 3: PZA in Combination Therapy
PZA: A New Look Based on RNASeq, the Hollow Fiber System, and Patient Level Data Tawanda Gumbo

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PZA Activity vs. M. Tuberculosis and M. bovis in Immunocompetent and Nude Mice Nicole Ammerman

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Panel Discussion Nicole Ammerman, Veronique Dartois, Jan Gheuens, Tawanda Gumbo, Eric Nuermberger, Kyu Rhee, Anna Upton, and Courtney Aldrich

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Session 4: PZA Toxicity
Overview of PZA Toxicity: 1950's - Present Peter Donald

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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.
Insights into Mechanisms of PZA Toxicity Martin Boeree

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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.
Panel Discussion / Wrap-up and Next Steps Andreas Diacon, Peter Donald, Susan Dorman, Payam Nahid, and Wing-Wai Yew / Richard Hafner

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Speakers & Panelists

Alland, David

University of Medicine & Dentistry of New Jersey

Ammerman, Nicole

Center for Tuberculosis Research, Dept. of Medicine, Div. of Infectious Diseases, Johns Hopkins School of Medicine

Barry, Clifton

Tuberculosis Clinical Research Team, Division of AIDS, NIAID, NIH, DHHS

Blanchard, John

Department of Biochemistry, Albert Einstein College of Medicine

Boeree, Martin Johan

UMC St Radboud/UCCZ Dekkerswald

Boshoff, Helena

Tuberculosis Research Section, LCID, NIAID, NIH, HHS

Brennan, Patrick

Microbiology, Immunology and Pathology, Colorado State University

Chaisson, Richard

Johns Hopkins University

Dartois, Veronique

Public Health Research Institute

Daum, Luke

Longhorn Vaccines & Diagnostics, LLC

Diacon, Andreas

TASK Applied Sciences

Donald, Peter

Paediatrics and Child Health, Stellenbosch University, Faculty of Health Sciences

Dorman, Susan

Johns Hopkins University School of Medicine

Fourie, Bernard

Faculty of Health Sciences, University of Pretoria

Gheuens, Jan

Bill & Melinda Gates Foundation

Gumbo, Tawanda

Office of Global Health, University of Texas Southwestern Medical Center

Hafner, Richard

Division of AIDS, NIAID, NIH, DHHS

Hoffner, Sven

Swedish Institute for Communicable Disease Control

Iademarco, Michael

Division of Tuberculosis Elimination, National Center for HIV/Aids, Viral Hepatitis, STD and TB Prevention, Centers for Disease Control and Prevention

Jacobs, William

Department of Microbiology & Immunology, Albert Einstein College of Medicine, Michael F. Price Center

Jindani, Amina

Clinical Sciences, St. George's, University of London

Karakousis, Petros

Johns Hopkins University School of Medicine

Keiler, Ken

Biochemistry & Molecular Biology, Penn State University

Kreiswirth, Barry

PHRI TB Center, University of Medicine and Dentistry, New Jersey

Laughon, Barbara

Division of Microbiology and Infectious Diseases, NIAID, NIH

Liang, Li

Clinical Center on Tuberculosis, Chinese Center for Disease Control and Prevention

MacKenzie, William

Tuberculosis Trials Consortium, Centers for Disease Control and Prevention

Mdluli, Khisimuzi

TB Alliance

Mitchison, Denis

St. George's, University of London

Nahid, Payam

Pulmonary & Critical Care, University of California, San Francisco

Nuermberger, Eric

Dept. of Medicine, Div. of Infectious Diseases, Johns Hopkins School of Medicine

Posey, Jamie

Division of Tuberculosis Elimination, Centers for Disease Control and Prevention

Powell, Krista

Division of Tuberculosis Elimination, Centers for Disease Control and Prevention

Rhee, Kyu

Weill Cornell Medical College

Simner, Patricia

Clinical Microbiology, Mayo Clinic

Sougakoff, Wladimir

Laboratory of Bacteriology, Faculty Pitie-Salpetriere, Universite Pierre et Marie Curie

Upton, Anna

TB Alliance

Weinrick, Brian

Albert Einstein College of Medicine

Welch, John

College of Arts and Sciences, University of Albany

Yew, Wing-Wai

Department of Microbiology, Chinese University of Hong Kong

Zhang, Ying

Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health

Zimic, Mirko

Cellular and Molecular Sciences, Universidad Peruana Cayetano Heredia

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