title: RNA editing ligases as novel drug targets in trypanosomatid parasites
Creators: name: Brouck, Laurine
Publisher: name: The University of Edinburgh
Year: 2024
Subjects: subject: RNA editing ligases
subject: trypanosomatid parasites
subject: REL1
subject: REL2
subject: Trypanosoma brucei
subject: Leishmania mexicana promastigotes

description: Trypanosomatids are responsible for devastating human and veterinary diseases, yet current treatments are inadequate. These parasites rely on a unique RNA editing process to express their mitochondrial genome. Two paralogous RNA editing ligases, REL1 and REL2, catalyse the final step of each editing event. While REL1 is an attractive drug target as it is essential for the survival of Trypanosoma brucei, REL2 knockdown has not produced any detectable phenotype. This is unexpected since both ligases are active in vitro and their genes have been under strong purifying selection. While previous studies have suggested a role for REL1 in non-canonical editing, the exact function of REL2 remains elusive. The primary aim of this project was to discern the respective roles of REL1 and REL2 in RNA editing through evolutionary and structure-function analyses. Phylogenetic reconstructions revealed that the REL paralogues started to diverge after the split between prokinetoplastids and metakinetoplastids — the latter encompassing the trypanosomatid family. In silico structural analyses provided evidence of functional specialisation predicted to affect their RNA-binding capabilities. This is consistent with in vitro experiments, wherein REL1 was more tolerant than REL2 towards nucleotide mismatches in RNA oligos mimicking non-canonical substrates. This finding supports the hypothesis that REL1 plays a key role in non-canonical editing. The essentiality of REL1 and non-essential nature of REL2 appear to be conserved among trypanosomatids as they were confirmed in Leishmania mexicana promastigotes using a CRISPR-based knockout strategy. The editing patterns of REL2 null mutants were examined in a preliminary RNA-Seq experiment, which supported a potential proof-reading role for this ligase. However, sequence coverage was very low, warranting additional analyses to corroborate these initial observations. The secondary aim of this project was to advance hit-to-lead development for REL1-targeted drug discovery. Previous high-throughput screens had identified promising compound series that have not yet been validated as bona fide REL1 inhibitors. In this project, target engagement was confirmed for one of these compounds using a truncated REL1 variant in thermal shift assays. A complementary proof-of-concept assay based on fluorescence polarisation was also developed to guide hit prioritisation. In summary, these findings support the functional divergence of REL paralogues and suggest a structural basis for REL1’s versatile substrate specificity, which may underlie its essential role in this highly dynamic process. Moreover, structure-activity relationships are emerging for REL1 inhibitors, paving the way for hit optimisation and in vivo testing against live parasites. By integrating drug discovery with foundational investigations of the REL drug targets, this work contributes to the understanding of RNA editing mechanisms and the development of much-needed drugs against trypanosomatid diseases.