Authors

L Brouck²; Z Nare²; M Sardis²; J Smith¹; M Wear⁴; A Morrison³; M Speake³; S McElroy³; AG Cook⁴; E Gluenz¹; A Schnaufer²;  ¹ Institute of Infection, Immunity and Inflammation, University of Glasgow, UK;  ² Institute of Immunology & Infection Research, University of Edinburgh, UK;  ³ European Screening Centre Newhouse, UK;  ⁴ Institute of Quantitative Biology, Biochemistry and Biotechnology, University of Edinburgh, UK

Discussion

RNA editing ligase 1 (REL1) plays a crucial role in uridylyl insertion/deletion mRNA editing, a unique and extensive form of post-transcriptional RNA modification in the mitochondria of trypanosomatid parasites. Previous gene knockdown and knockout studies showed that REL1 is essential for the survival of Trypanosoma brucei, the causative agent of sleeping sickness (Schnaufer et al., 2001; Gao & Simpson, 2003). The crystal structure of TbREL1 revealed a well-defined ATP-binding pocket with striking differences to mammalian DNA and RNA ligases but high conservation among trypanosomatid REL1 orthologues (Deng et al., 2004). This offers exciting potential for the development of specific REL1 inhibitors with broad anti-trypanosomatid properties. We developed a high-throughput activity assay for REL1 (Zimmermann et al., 2016) and tested over 600,000 compounds in four independent screening campaigns. Promising REL1 small molecule inhibitors were identified with an average hit rate of ~1%, and interesting structure-activity relationships emerged for some hit series. Several compounds inhibited REL1 from different trypanosomatid species, including Leishmania donovani and Trypanosoma cruzi, but are much less potent against a related bacteriophage RNA ligase, suggesting high specificity in vitro. As part of ongoing hit-to-lead development efforts, we are investigating specificity in vivo, optimising the expression of recombinant REL1 orthologues with the help of differential scanning fluorimetry, and using crystallography and surface plasmon resonance as tools to study REL1-inhibitor interactions. In addition, we will present results from CRISPR-Cas9-based knockout studies which suggest that REL1 is essential in Leishmania mexicana. This confirms the expectation that RNA editing is a promising drug target beyond African trypanosomes. References:Schnaufer,A., Panigrahi, A. K., Panicucci, B., Igo, R. P., Wirtz, E., Salavati, R., Stuart, K. (2001). An RNA ligase essential for RNA editing and survival of the bloodstream form of Trypanosoma brucei, Science, 291:2159-62. Gao, G. and Simpson, L. (2003). Is the Trypanosoma brucei REL1 RNA ligase specific for U-deletion RNA editing, and is the REL2 RNA ligase specific for U-insertion editing?, J Biol Chem, 278:27570-4.Deng, J., Schnaufer, A., Salavati, R., Stuart, K., Hol, W. G. (2004). High resolution crystal structure of a key editosome enzyme from Trypanosoma brucei: RNA editing ligase 1, J Mol Biol, 343:601-13.Trypanosoma brucei editosome enzyme REL1 and other RNA ligases, Nucleic Acids Res, 44:e24.