II. Targeting G4C2 pathogenetic expansions in C9ORF72
The GGGGCC large repeat expansions (from 250 up to >3000 repeats) present in the first intron of the C9ORF72 gene have been identified in patients suffering from ALS or FTD (frontotemporal dementia) (Dejesus-Hernandez et al., 2011; Renton et al., 2011). These hexanucleotide expansions are transcribed bidirectionally and form either RNA foci in the nucleus, that binds and sequesters RNA-binding proteins, or are translated into dipeptide repeat proteins (DPRs) by an abnormal translation process called repeat-associated non-ATG (RAN) translation (Jiang et al., 2019). This results in gain-of-function that leads to neuronal cell toxicity (Jiang et al., 2019). These GGGGCC repeats exhibit somatic instability, thus, it is possible that repeat expansion may not be present in DNA from blood samples although present in CNS. Therefore, a blood test negative for GGGGCC expansion does not necessarily indicate that the patient is not aC9ORF72ExpGGGGCC carrier, and this should be taken into consideration when classifying ALS patients. Evidently, a combination of genetic analysis and determination of poly-(GP) in CSF will be required for a definite clinical diagnosis (Balendra et al., 2017; Lehmer et al., 2017). Approaches to alleviate symptoms or decelerate the course of the disease include targeting the RNA transcribed from GGGGCC-repeats or targeting the DPRs. These RNAs adopt two folded states that are in equilibrium, a hairpin structure and the G-quadruplex structure (Su et al., 2014).
Targeting RNA G-quadruplexes/hairpins. HTS identified the structurally similar chemicals DB1246 (XX ), DB1247 (XXI ), and DB1273 (XXII ), that exhibit high-affinity binding on GGGGCC RNA G-quadruplexes. These chemicals significantly reduced RNA foci in vitro in human iPSC-motor and iPSC-cortical neuron cell lines and decreased DPRs. Decreased levels of DPRs and improved survival of larvae reaching the pupal stage of development was observed in vivo , in GGGGCC repeat-expressing Drosophilastreated with DB1273 (Simone et al., 2018).
The cationic porphyrin (5,10,15,20-tetra(N-methyl-4-pyridyl) porphyrin (TMPyP4)) XXIII binds to GGGGCC RNA G-quadruplexes in a concentration-dependent manner and causes a conformational change in their secondary structures conferring thermal instability of GGGGCC RNA G-quadruplexes. Thus, TMPyP4 blocks the interaction of GGGGCC RNA G-quadruplexes with RNA-binding proteins, such as ASF/SF2 and hnRNPA1 (Zamiri et al., 2014). Another group developed three compounds (XXIV , XXV , XXVI ) targeting the hexanucleotide repeat region of RNAs and tested them for binding to hairpin RNA and reducing RAN in a cell-free model. XXIV andXXV reduced RNA foci and significantly decreased RAN translation in GGGGCC repeat-expressing neurons (Su et al., 2014). Improving the selectivity of XXIV , led to compoundXXVII that binds selectively in the internal loops of the hairpin form of RNA. XXVII blocked polysome assembly and reduced RNA foci and RAN translation in vitro (Wang et al., 2019). The in vivo action of these compounds inC9ORF72 -ALS remains to be validated.
Targeting DPRs. RAN translation of C9ORF72  RNA GGGGCC-repeats in all six-reading frames produces five repeated polypeptides [poly(GA), poly(GP), poly(GR), poly(PR), and poly(PA)] that are toxic to cells through induction of nucleolar stress and defects in mRNA splicing (Mori et al., 2013a; Mori et al., 2013b).
Inhibition of PIKFYVE kinase that converts phosphatidylinositol-3-phosphate (PI3P) to phosphatidylinositol-3,5-biphosphate [PI(3,5)P2] leads to increased PI3P levels that regulate autophagosome formation and engulfment of proteins for degradation. Therefore, it may be important for the destruction of DPRs. Indeed, the PIKFYVE inhibitor apilimod (XXVIII ) reduces DPRs in C9-BAC mice that harbor the humanC9ORF72 with 100-1000 GGGGCC repeats (Staats et al., 2019).
Another way to reduce protein aggregates, including DPRs, is through proteosome activation. Rolipram (XXIX ) is an antidepressant drug that acts via inhibition of phosphodiesterase 4 (PDE4) and promotes proteasome function. Rolipram decreased poly(GA) in primary hippocampal neurons in vitro (Khosravi et al., 2020). DPRs induce integrated stress response (ISR) through ER implicating TMX2(Kramer et al., 2018). ISR involves hyperphosphorylation of elf2a and increased RAN of C9ORF72 RNA GGGGCC-repeats (Cheng et al., 2018).XXX , a selective inhibitor of eIF2α dephosphorylation and tauroursodeoxycholate (TUDCA), a chemical chaperone, showed protection against poly(GA)-induced stress and cell death in vitro (Zhang et al., 2014). In conclusion, there are many compounds that can target the DPRs that need to be evaluated in animal models and then in clinical trials.