High-throughput screening of compounds targeting RNA editing in Trypanosoma brucei: Novel molecular scaffolds with broad trypanocidal effects.

Mitochondrial uridine insertion/deletion RNA editing, catalyzed by a multiprotein complex (editosome), is essential for gene expression in trypanosomes and Leishmania parasites. As this process is absent in the human host, a drug targeting this mechanism promises high selectivity and reduced toxicity. Here, we successfully miniaturized our FRET-based full-round RNA editing assay, which replicates the complete RNA editing process, adapting it into a 1536-well format. Leveraging this assay, we screened over 100,000 compounds against purified editosomes derived from Trypanosoma brucei, identifying seven confirmed primary hits. We sourced and evaluated various analogs to enhance the inhibitory and parasiticidal effects of these primary hits. In combination with secondary assays, our compounds marked inhibition of essential catalytic activities, including the RNA editing ligase and interactions of editosome proteins. Although the primary hits did not exhibit any growth inhibitory effect on parasites, we describe eight analog compounds capable of effectively killing T. brucei and/or Leishmania donovani parasites within a low micromolar concentration. Whether parasite killing is - at least in part - due to inhibition of RNA editing in vivo remains to be assessed. Our findings introduce novel molecular scaffolds with the potential for broad antitrypanosomal effects.

Hammerhead ribozyme-based U-insertion and deletion RNA editing assays for multiplexing in HTS applications.

Untranslatable mitochondrial transcripts in kinetoplastids are decrypted post-transcriptionally through an RNA editing process that entails uridine insertion/deletion. This unique stepwise process is mediated by the editosome, a multiprotein complex that is a validated drug target of considerable interest in addressing the unmet medical needs for kinetoplastid diseases. With that objective, several in vitro RNA editing assays have been developed, albeit with limited success in discovering potent inhibitors. This manuscript describes the development of three hammerhead ribozyme (HHR) FRET reporter-based RNA editing assays for precleaved deletion, insertion, and ligation assays that bypass the rate-limiting endonucleolytic cleavage step, providing information on U-deletion, U-insertion, and ligation activities. These assays exhibit higher editing efficiencies in shorter incubation times while requiring significantly less purified editosome and 10,000-fold less ATP than the previously published full round of in vitro RNA editing assay. Moreover, modifications in the reporter ribozyme sequence enable the feasibility of multiplexing a ribozyme-based insertion/deletion editing (RIDE) assay that simultaneously surveils U-insertion and deletion editing suitable for HTS. These assays can be used to find novel chemical compounds with chemotherapeutic applications or as probes for studying the editosome machinery.