Small molecule modulators targeting the interactions of small GTPase ARF1 with C9orf72:SMCR8:WDR41 complexes implicated in ALS/FTD

The hexanucleotide repeat expansion (GGGGCC) in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The C9orf72 protein forms a complex with SMCR8 and WDR41 (CSW), which acts as a GTPase-activating protein (GAP) regulating small GTPases like ARF1 and RABs involved in intracellular trafficking. Although these findings implicated the ARF1 dysregulation in ALS/FTD and the critical need for validation of its inhibition as potential intervention, small molecules that target the interactions between CSW and ARF1 are lacking. In this study, we showed that the tyrosine-phosphorylated form (Tyr-782) of ASAP1, an ARF-GAP that inactivates ARF1, is increased in the motor cortex of both sporadic ALS and ALS with C9orf72 mutations. Overexpression of C9orf72 led to Golgi disorganization, partially mimicking the effects of ARF1 inhibitor brefeldin A on dispersion of Golgi apparatus. To identify a better strategy to enhance C9orf72 and ARF1 interactions, we applied rational design and virtual screening of a 40-million compound library of small molecules targeting the ARF1-CSW interface. Molecular docking, MM-GBSA binding energy, ADME/Tox profiles, and interaction analysis established MCULE-5095997944 as a top candidate for ARF1 modulation. MCULE-5095997944 demonstrated strong binding to ARF1 in the nanomolar range, reduced GTP-bound ARF1 levels upon ARF1 activation, and altered ARF1-dependent Golgi organization. These studies identified the first small molecule targeting ARF1-CSW interaction and further support ARF1 modulation as a potential therapeutic approach for ALS/FTD.