Authors

Z Nare¹; M F Sardis¹; S Zimmermann¹; A Schnaufer¹;  ¹ Institute of Immunology and Infection Research, University of Edinburgh, UK

Discussion

Trypanosomatids utilise a unique method of post transcriptional modification known as uridylyl (U) insertion/deletion RNA editing to produce mature mitochondrial mRNAs. This conversion is catalysed by the RNA editing core complexes (RECCs) of which there are three distinct classes. The RECCs are comprised of a set of twelve common proteins including the enzymes RNA editing ligase 1 (REL1) and RNA editing ligase 2 (REL2). RNA editing involves endonucleolytic cleavage, insertion and/or deletion of a number or uridine nucleotides (Us) and ligation of the edited RNA either by REL1 or REL2. In vivo studies have shown that REL1 is essential for the survival of bloodstream forms of T. brucei while no negative effects have been observed following REL2 downregulation by RNAi-mediated knockout. Although REL1 and REL2 have a high degree of sequence similarity (41% sequence identity and 55% similarity) biochemical studies have shown that there are significant differences in their activities. It remains unclear why TbREL1 is essential for parasite survival in the bloodstream while TbREL2 is not. Previous work in our lab has led to the development of a high throughput screening (HTS) assay compatible with 384-well and 1536-well microplate formats that has been used successfully for the identification of inhibitors of TbREL1. We will report our progress of the following: (1) identification and characterisation of inhibitors of different trypanosomatid REL1 orthologues including T. brucei, T. cruzi, T. vivax and L. donovani, (2) development and optimisation of a surface plasmon resonance based assay as a secondary assay for the validation of small molecule inhibitors of TbREL1, (3) development of an expression and purification protocol for trypanosomatid REL2 orthologues for use in structural and functional studies and as complementary tools for REL1 drug discovery.