Development of NMR and thermal shift assays for the evaluation of Mycobacterium tuberculosis isocitrate lyase inhibitors
Ram Prasad Bhusal, Krunal Patel, Brooke XC Kwai, Anne Swartjes, Ghader Bashiri, Johannes Reynisson, Jonathan Sperry, Ivanhoe KH Leung
MEDCHEMCOMM | ROYAL SOC CHEMISTRY | Published : 2017
The enzymes isocitrate lyase (ICL) isoforms 1 and 2 are essential for Mycobacterium tuberculosis survival within macrophages during latent tuberculosis (TB). As such, ICLs are attractive therapeutic targets for the treatment of tuberculosis. However, there are few biophysical assays that are available for accurate kinetic and inhibition studies of ICL in vitro. Herein we report the development of a combined NMR spectroscopy and thermal shift assay to study ICL inhibitors for both screening and inhibition constant (IC50) measurement. Operating this new assay in tandem with virtual high-throughput screening has led to the discovery of several new ICL1 inhibitors.
We thank the University of Auckland for a Doctoral Scholarship (R. P. B) and the Maurice and Phyllis Paykel Trust for funding. G. B. is supported by a Sir Charles Hercus Fellowship awarded through the Health Research Council of New Zealand. We thank Professor Bernard Golding (Newcastle University, UK) for his advice when preparing (2S, 3R)-2-methylisocitrate. We thank Dr M. Schmitz for maintenance of the NMR facility and Ms K. Boxen for the DNA sequencing service.