Development and Validation of a High-throughput Luminescence Assay for Screening Natural Product Extracts as Inhibitors Selective for the Mycobacterium tuberculosis Proteasome.

摘要

Mycobacterium tuberculosis (Mtb) causes disease with a high incidence globally1. Although the disease largely has been controlled in many areas, the mortality rate in healthy and immune-compromised patients still remains enormous1. In the recent past, an increasing number of new targets have been identified for treatment of this disease, one of which is the Mtb proteasome. The Mtb proteasome has been implicated in mediating the survival of Mtb within the host cells during the latent, non-replicating phase2,3,4. The first-line of defense in humans are the macrophages, which mount an attack led by reactive nitrogen intermediates that cause nitrosative and oxidative stress to the Mtb cells. Proteasomes are able to withstand this stress and continue to keep the Mtb viable in a latent state3. Therefore, inhibiting Mtb proteasomes could help in combating Mtb infections that do not respond to drugs used currently in therapy. The proteasome is a cytosolic protein assembly that has different proteolytic functions. The chymotrypsin-like function breaks down bonds at the amino acid tyrosine5,6, . The aim of this project was to develop and validate a biochemical assay that could follow proteasome activity in the presence and absence of plant test extracts. The assay was validated for both mammalian and Mtb proteasomes. A proteasome isolation protocol was validated for isolating crude proteasomes from the H37Ra (avirulent) strain of Mtb4,7. The presence of proteasomes in the crude fraction was established through the assay and SDS-PAGE. We used the Bradford assay to determine the total concentration of protein in the isolated fraction 8. We investigated fluorescence and luminescence assays as potential screening methods to determine their respective robustness and repeatability for use in screening natural product extracts for potential Mtb proteasome inhibitors. We found the fluorescence assay to be subject to interference by the natural fluorescence of compounds in many of the extracts; the luminescence approach is free of this interference. Luminescence is the more suitable assay for screening natural product extracts. As proof of principle, we used the luminescence assay to screen a small set of plant test extracts. The assay has been used for screening a set of 80 test extracts and the percentage of inhibition caused by each test extract was determined. The assay can be used in the future for screening a more extensive library of plant and fungal test extracts. Hits obtained from the screening can be subjected to bioassay-guided fractionation to isolate the single active compound.;1. Global Tuberculosis Report. World Health Organization 2014.;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 2007, 13 (12), 1515--20.;3. Darwin, K. H.; Ehrt, S.; Gutierrez-Ramos, J. C.; Weich, N.; Nathan, C. F., The proteasome of Mycobacterium tuberculosis is required for resistance to nitric oxide. Science 2003, 302 (5652), 1963--6.;4. Lin, G.; Li, D.; de Carvalho, L. P.; Deng, H.; Tao, H.; Vogt, G.; Wu, K.; Schneider, J.; Chidawanyika, T.; Warren, J. D.; Li, H.; Nathan, C., Inhibitors selective for mycobacterial versus human proteasomes. Nature 2009, 461 (7264), 621--6.;5. Lin, G.; Tsu, C.; Dick, L.; Zhou, X. K.; Nathan, C., Distinct specificities of Mycobacterium tuberculosis and mammalian proteasomes for N-acetyl tripeptide substrates. The journal of biological chemistry 2008, 283 (49), 34423--31.;6. Butler, S. M.; Festa, R. A.; Pearce, M. J.; Darwin, K. H., Self-compartmentalized bacterial proteases and pathogenesis. Molecular microbiology 2006, 60 (3), 553--62.;7. Nagy, I.; Tamura, T.; Vanderleyden, J.; Baumeister, W.; De Mot, R., The 20S proteasome of Streptomyces coelicolor. Journal of bacteriology 1998, 180 (20), 5448--53.;8. Bradford, M. M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry 1976, 72, 248--54.