Schnappinger Lab Research

One of our main goals is to help develop of new medicines for the treatment and prevention of Tuberculosis (TB), an infectious disease that still claims millions of lives each year. We began this work with developing a regulatory system that allows to turn Mtb genes on and off, both in vitro and during infections (1-4). We now use this system to

1. Evaluate Mtb gene products as new targets for TB drug development by documenting the impact of their genetic inactivation on growth and persistence of Mtb in vitro and in mice (5-11);

2. Help elucidate the mechanisms by which small-molecules inhibit the growth of Mtb (12-17);

3. Construct mutants for target-directed whole-cells screens (18); and

4. Measure vulnerability of Mtb to the partial, CRISPRi-mediated inactivation of individual genes (19).

Drug development requires a broad spectrum of expertise and techniques. Our work in this area therefore includes close collaborations with many labs, including those led by Bree Aldridge (Tufts University), Courtney Aldrich (University of Minnesota), Clifton Barry III (NIH), Veronique Dartois (Center for Discovery and Innovation), Deborah Hung (Broad Institute), Sabine Ehrt (Weill Cornell), Valerie Mizrahi (University of Cape Town), Carl Nathan (Weill Cornell), Kyu Rhee (Weill Cornell), Jeremy Rock (Rockefeller University), Eric Rubin (Harvard University), Christopher Sassetti (University of Massachusetts) and several pharmaceutical companies that participate in the TB Drug Accelerator program (20).

Our biology-centric projects are frequently motivated by genes and pathways we initially studied due to their potential for drug development. For example, we first got interested in biotin metabolism due to Mtb’s susceptibility to inhibition of biotin dependent enzymes (21). We then determined the consequences of inhibiting different Mtb enzymes required for protein biotinylation in vitro and during infection (9,22). In ongoing work, we are studying the transcriptomic responses to inhibition of biotin deprivation in Mtb and M. smegmatis, the mechanism of biotin-starvation induced cell death, the accessory factors required to convert dethiobiotin into biotin, biotin import, and the host defence mechanisms that cause killing of biotin auxotrophs in mice.

In more recent and currently unpublished work we began to use our genetic tools to also help with the development and effective vaccines to prevent pulmonary TB in adults. Our specific goals are to

1. Improve safety of the widely used vaccine M. bovis BCG to allow its use for alternative routes of vaccination in humans, and

2. Construct conditionally replicating Mtb strains that are safe enough for human challenge studies.

We pursue these goals in collaboration with Sabine Ehrt (Weill Cornell), JoAnne Flynn (University of Pittsburgh), Sarah Fortune (Harvard University), Eric Rubin (Harvard University) and Robert Seder (NIH)

References

1.         Ehrt, S., et al. Controlling gene expression in mycobacteria with anhydrotetracycline and Tet repressor. Nucleic acids research 33, e21 (2005).

2.         Gandotra, S., Schnappinger, D., Monteleone, M., Hillen, W. & Ehrt, S. In vivo gene silencing identifies the Mycobacterium tuberculosis proteasome as essential for the bacteria to persist in mice. Nature medicine 13, 1515-1520 (2007).

3.         Kim, J.H., et al. A genetic strategy to identify targets for the development of drugs that prevent bacterial persistence. Proceedings of the National Academy of Sciences of the United States of America 110, 19095-19100 (2013).

4.         Klotzsche, M., Ehrt, S. & Schnappinger, D. Improved tetracycline repressors for gene silencing in mycobacteria. Nucleic acids research 37, 1778-1788 (2009).

5.         Botella, L., Vaubourgeix, J., Livny, J. & Schnappinger, D. Depleting Mycobacterium tuberculosis of the transcription termination factor Rho causes pervasive transcription and rapid death. Nat Commun 8, 14731 (2017).

6.         Evans, J.C., et al. Validation of CoaBC as a Bactericidal Target in the Coenzyme A Pathway of Mycobacterium tuberculosis. ACS Infect Dis 2, 958-968 (2016).

7.         Lin, K., et al. Mycobacterium tuberculosis Thioredoxin Reductase Is Essential for Thiol Redox Homeostasis but Plays a Minor Role in Antioxidant Defense. PLoS pathogens 12, e1005675 (2016).

8.         Puckett, S., et al. Glyoxylate detoxification is an essential function of malate synthase required for carbon assimilation in Mycobacterium tuberculosis. Proceedings of the National Academy of Sciences of the United States of America 114, E2225-E2232 (2017).

9.         Tiwari, D., et al. Targeting protein biotinylation enhances tuberculosis chemotherapy. Sci Transl Med 10(2018).

10.      Li, W., et al. Therapeutic Potential of the Mycobacterium tuberculosis Mycolic Acid Transporter, MmpL3. Antimicrob Agents Chemother 60, 5198-5207 (2016).

11.      Beites, T., et al. Plasticity of the Mycobacterium tuberculosis respiratory chain and its impact on tuberculosis drug development. Nat Commun 10, 4970 (2019).

12.      Wang, H., et al. Re-discovery of PF-3845 as a new chemical scaffold inhibiting phenylalanyl-tRNA synthetase in Mycobacterium tuberculosis. J Biol Chem (2021).

13.      Soares de Melo, C., et al. Antitubercular 2-Pyrazolylpyrimidinones: Structure-Activity Relationship and Mode-of-Action Studies. J Med Chem 64, 719-740 (2021).

14.      Ray, P.C., et al. Spirocycle MmpL3 Inhibitors with Improved hERG and Cytotoxicity Profiles as Inhibitors of Mycobacterium tuberculosis Growth. ACS Omega 6, 2284-2311 (2021).

15.      Lee, B.S., et al. Dual inhibition of the terminal oxidases eradicates antibiotic-tolerant Mycobacterium tuberculosis. EMBO Mol Med 13, e13207 (2021).

16.      Grover, S., et al. Two-Way Regulation of MmpL3 Expression Identifies and Validates Inhibitors of MmpL3 Function in Mycobacterium tuberculosis. ACS Infect Dis 7, 141-152 (2021).

17.      Bockman, M.R., et al. Investigation of ( S)-(-)-Acidomycin: A Selective Antimycobacterial Natural Product That Inhibits Biotin Synthase. ACS Infect Dis 5, 598-617 (2019).

18.      Johnson, E.O., et al. Large-scale chemical-genetics yields new M. tuberculosis inhibitor classes. Nature 571, 72-78 (2019).

19.      Bosch, B., et al. Genome-wide gene expression tuning reveals diverse vulnerabilities of M. tuberculosis. Cell (2021).

20.      Aldridge, B.B., et al. The Tuberculosis Drug Accelerator at year 10: what have we learned? Nature medicine (2021).

21.      Duckworth, B.P., et al. Bisubstrate adenylation inhibitors of biotin protein ligase from Mycobacterium tuberculosis. Chemistry & biology 18, 1432-1441 (2011).

22.      Woong Park, S., et al. Evaluating the sensitivity of Mycobacterium tuberculosis to biotin deprivation using regulated gene expression. PLoS pathogens 7, e1002264 (2011).