Structural and biochemical characterisation of the de novo cysteine biosynthesis pathway in trypanosomatids.

Neglected tropical diseases (NTD) affect over a billion people throughout the world. Chagas disease, caused by the protozoan parasite T. cruzi and leishmaniasis, a result of over 15 species of Leishmania, are NTDs selected for control and elimination. Under a One Health approach T. theileri infections affecting cattle should also be eradicated. Current treatments for these diseases are inadequate and their use is associated with many severe side effects, often leading to incomplete treatment. These trypanosomatids rely on cysteine biosynthesis for the production of trypanothione, which is essential in allowing the parasite to survive within the host. The de novo pathway of cysteine biosynthesis requires the conversion of L-serine into O-acetyl-L-serine by serine acetyltransferase, and then the transformation of O-acetyl-L-serine into L-cysteine, which is catalysed by cysteine synthase. These enzymes present an attractive potential for drug development against T. cruzi, Leishmania spp. and T. theileri. This work aimed to explore the de novo cysteine biosynthesis pathway furtherthrough exploration of the underlying molecular mechanisms. This was achieved through investigations into the proteins involved. Studies of TcSAT were conducted and it was concluded that further work is required to produce soluble recombinant protein to be used in additional experiments. Biochemical and crystallographic studies of TcCS, LiCS and TthCS were conducted. Crystal structures of the threeenzymes were determined at resolutions of 1.80 Å for TcCS, 1.75 Å for LiCS and 2.75 Å for TthCS. These three homodimeric structures show the same overall fold and support a common reaction mechanism. Interactions between the two proteins, forming a cysteine synthase complex were explored with the characterisation of activity and initial structural studies undertaken. Through use of the cysteine synthase structures, several avenues of de novo drug design were explored. To further this aim, 14 structures of TcCS and LiCS collectively were determined with fragments bound, resulting in the selection of a lead compound to develop further. Overall, understanding of the de novo cysteine biosynthesis pathway in trypanosomatids has been explored and the foundations of drug design have been established.