Cysteine biosynthesis in Mycobacterium tuberculosis as potential drug target

Abstract

The bacterium Mycobacterium tuberculosis is the underlying cause of tuberculosis, one of the most devastating infectious diseases, causing 1.4 million deaths in 2015. In addition one third of the global population is infected with latent tuberculosis, during which the bacilli remain viable in a dormant state, characterized by slow growth rates and changes in metabolism. It was shown that the underlying genes of sulfur assimilation and L-cysteine biosynthesis are essential for the survival of Mtb during dormancy. The availability of sulfur and the subsequently produced L-cysteine is directly linked to increased survival of Mtb inside host macrophages, because the first line of defense of Mtb against reactive oxygen and nitrogen species relies on mycothiol, the functional analog to glutathione in mycobacteria and the redox active sulfhydryl group of mycothiol is directly derived from L-cysteine. Hence the enzymes of the sulfur assimilation pathway and L-cysteine biosynthesis have been proposed as potential targets for novel antimycobacterial drugs.

Within the scope of this thesis, mycobacterial CysC, the APS kinase domain of the mycobacterial sulfur activation complex was mechanistically and structurally characterized. Phosphoryl group transfer from ATP to APS was shown to follow a conserved mechanism found in several species. In addition mutation of an L-cysteine residue in the lid region closing off the ATP binding site resulted in impaired ATP binding and prevented catalysis. The structural characterization included a comparison of mycobacterial CysC and the crystal structures to the two human homologs and it was found that the substrate binding sites for APS and ATP in the three enzymes shared a high degree of sequence identity, hence inhibitor development specifically targeting the mycobacterial enzyme was considered to be very challenging.

The three mycobacterial L-cysteine synthases, CysK1, CysK2 and CysM are upregulated during different metabolic states of Mtb. A high-throughput screening campaign of CysM identified a class of urea-based active site binders. Subsequent in vitro validation and organic synthesis of compounds to establish structure activity relationships in combination with structural based methods allowed the identification of seven potent CysM inhibitors. The affinities were found to be in the low micromolar range. The identified compounds did not only decrease CysM activity, but also showed bactericidal potency in a nutrient starvation model. CysK1 and CysK2 were also tested against a library of 71 compounds that were used for in vitro validation of CysM. In total four compounds were found to inhibit CysK1 and CysK2 and were also among the best inhibitors targeting CysM, suggesting that the identified inhibitors might provide a valuable starting point towards the development of a drug targeting all three L-cysteine synthases simultaneously.