Molecular tools to unravel the mechanism of action of novel M. tuberculosis phenotypic hits

Aim: The identification of the mode of action of novel hits active against Mycobacterium tuberculosis (Mtb).

Objectives:

1. To determine the mode of action and molecular targets of prioritised hits identified by their activity against Mtb.

2. To validate targets identified by genetic and biochemical approaches.

3. To purify protein targets, develop suitable robust biochemical assays and crystals for structural studies with our phenotypic hits.

The success of whole-cell phenotypic screening in drug discovery rests on there being efficient strategies for elucidating the cellular targets of identified hits. In this regard, the main aim of this iCASE PhD studentship is to determine the mode of action of selected hits identified from a GSK DDW Mtb phenotypic screening campaign, which provided 50 novel compounds for mode of action studies [1]. We will prioritise a selection of these hits for our proposed studies based on their reported in vivo and in vitro Mtb activities, and desriable hit-to-lead properties [1]. This multi-disciplinary project will therefore define new TB targets at UoB and identify hits for further hit/lead optimisation at GSK DDW. Simultaneously, this will also provide new insights into fundamental Mtb biology, as we've shown previously for EchA6 and KasA [2].

Experimental Methods and Research Plan:

1. Target identification: We plan to take three parallel approaches, the first two of which we have successfully applied to identify target(s) of novel hits [2]. The first approach will involve the isolation of spontaneous resistant Mtb mutants generated against the prioritised hit molecule. Whole genome sequencing will be used to identify SNPs or InDels that are potentially responsible for resistance. Through a series of further genetic and biochemcial studies, the target will be identified [2]. The second approach uses our recently published chemical proteomics methodology to identify the respective target [2]. The third approach involves the development of a novel mass spectrometry-based metabolomics platform [3], which would allow one to monitor directly the metabolic flux of specific target inhibition through a whole cell phenotypic hit.

2. Target validation: For this purpose we will use CESTET, a tool for testing gene essentiality and studying loss of gene function through metabolic labeling in mycobacteria [2].

3. Purification of protein targets: For biochemical and structural studies we will express and purify the proteins using a variety of expression vectors and cell lines [2]. The resulting recombinant proteins will be used in in vitro biochemical assays and for structural studies with our phenotypic hits [2].

Expected Outcomes: We will identify novel drug targets and refresh the TB drug pipeline.

References

1. Rebello-Lopez, M (2015) PLoS ONE, 10(12):e0142293
2. Cox, J. (2016) Nat. Microbiology DOI:10.1038/NMICROBIOL.2015.6; Abrahams, K. (2016) Nat. Comm., in press
3. Prosser, S. (2012) PNAS, 109:11354