Tuberculosis (TB) is an infectious disease that is caused by Mycobacterium tuberculosis. Fatty acids and lipids are thought to be important sources of carbon and energy for M. tuberculosis during infection, in particularly when the bacteria are in the latent phase. The enzyme isocitrate lyase (ICL), which catalyses the formation of succinate and glyoxylate from isocitrate, is the first enzyme in mycobacterial glyoxylate cycle. ICL is important for the survival of latent M. tuberculosis as it allows the bacteria to survive with fatty acids as its sole carbon source. Whilst ICL is crucial for the survival of M. tuberculosis, it is not present in humans. As such, ICL is an excellent therapeutic target for the development of new anti-TB treatments. The development of ICL inhibitors for potential anti-TB treatments is hampered by a lack of reliable and efficient in vitro assays for ICL activity. Reported methods included radioactive assay using 14C-labelled isocitrate, which is tedious to set up and is potentially dangerous, or by the use of a chemically-coupled assay with detection by UV/Vis spectroscopy. Herein, we report the development of an NMR-based activity assay that allows the detection of isocitrate consumption and succinate formation in real time. This method was demonstrated by using existing ICL inhibitors, and exemplified by our inhibitor discovery work in tandem with virtual high-throughput screening.

Development and application of an NMR-based activity and inhibition assay for mycobacterial isocitrate lyase

253rd National Meeting (2017)

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