Abstract

Hexanucleotide repeat expansions (HREs) in intron-1 of the C9ORF72 gene are the most common known genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). These microsatellite repeat sequences (GGGGCC, G4C2) contribute to neuronal injury via three non-mutually exclusive mechanisms. A growing body of evidence implicates toxic RNA and protein gain-of-functions due to abnormal nuclear export and repeat-associated non-AUG (RAN) translation of intron-retaining repeat transcripts into neurotoxic dipeptide repeat proteins (DPRs) as one of the main drivers of pathogenicity. Repeat RNA sequestration of SRSF1 triggers the nuclear export of repeat transcripts into the cytoplasm while its depletion inhibits the nuclear export of pathological C9ORF72 transcripts and production of DPRs, thereby preventing neurotoxicity in reporter cells, patient-derived neurons, Drosophila and Zebrafish models of C9ORF72-related disease (Hautbergue et al, 2017).

To identify tool compounds targeting the interaction of SRSF1 with C9orf72, an HTRF assay was established in 1536-well format to detect proximity of tagged SRSF1 to tagged G4C2 RNA. This robust assay (S/B 10.1±0.1, Z’ 0.91±0.01) allowed for high throughput screening (HTS) of 50,000 compounds selected from our compound library for their significant structural diversity and CNS-differentiating physicochemical properties. Following frequent hitter analysis and structural alerts review, 503 compounds were selected based on their ability to inhibit TR-FRET signal. Preliminary clustering analysis showed good SAR.

Our data demonstrates the robustness of our HTRF assay for hit finding and shows how this in collaboration with academia accelerates identification of potential tool compounds. Our current efforts focus on further selection to <100 compounds with selectivity against counter assays. Tool compounds will be validated by assessing their potency in inhibiting the SRSF1-dependent nuclear export of C9orf72-repeat transcripts and the production of DPRs in in vitro (reporter human cells, patient-derived neurons) and in vivo (zebrafish, mouse) models of C9ORF72-ALS/FTD at SITraN, supported by ADME profiling at CRL.