Identification and characterization of RANT modulators in the G4C2 expansion

The intronic hexanucleotide repeat expansion, GGGGCC (or G4C2), in the C9ORF72 gene is the most common genetic cause of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). Above 30 and up to hundreds repeats, G4C2 expansion is transcribed in aberrant mRNAs that fold in G-quadruplexes structures and generate RNA foci within motorneurons. Additionally, they also located in the cytoplasm and can be translated via the unconventional repeat- associated non-ATG translation (RANT) into five toxic dipeptide repeat (DPR) proteins that accumulate in cells. Unlike ATG-initiated translation, RANT products accumulate after activation of the integrated stress response (ISR). Both RAN foci and RANT products are considered the causative factors of the disease but, so far, no effective pharmacological approach is currently available. We performed a high-throughput drug-screening to identify modulators of RANT. Effective small molecules were divided into four categories: positive CAP and RANT products modulators, negative CAP and RANT products modulators, positive RANT products and negative CAP products modulators, negative RANT products and positive CAP products modulators. Selective hits were tested to evaluate their toxicity in a dose- response analysis and to assess whether their mechanisms of action affect general transcription and translation. Moreover, we verified whether they interfere with the ISR pathway by modulating the phosphorylation levels of PERK and eIF2D.