Sicart, A. | bioRxiv Science

bioRxiv2026

MeFluHyA: a novel fluorescent screening tool for high-throughput identification of HDAC6-selective inhibitors

The cytosolic histone deacetylase 6 (HDAC6) plays a key role not only in cancer but also in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) peripheral neuropathies. Pharmacological inhibition as well as genetic silencing of HDAC6 is able to rescue several defects. Therefore, developing pharmacological compounds targeting this enzyme is of crucial interest. Here, we report the design, synthesis, and characterization of Methyl Fluorescent Hydroxamic Acid (MeFluHyA), a novel fluorescent HDAC6-selective probe designed for cell imaging. By integrating a Cy5 fluorophore into a phenyl hydroxamic acid scaffold, MeFluHyA shows binding to the catalytically active CD2 domain of HDAC6 without significantly inhibiting its deacetylating function in the sub-micromolar range. Biochemical enzymatic activity assays confirmed its selectivity over other HDAC isoforms. Fluorescent imaging studies in HeLa cells demonstrated strong colocalization with HDAC6-eGFP and a commercial HDAC6 antibody. Competitive binding assays revealed that MeFluHyA effectively identifies known HDAC6 inhibitors, with reduced probe-binding serving as a readout for successful target engagement. MeFluHyA is a cell-permeable, fast and easy to use probe that can be added organism-independent, and its red-shifted fluorophore makes it compatible with multiplex, live and fixed imaging. Overall, we provide a novel tool for screening platforms aimed at identifying novel HDAC6-targeting inhibitors.

biochemistry↗

bioRxiv2023

PP2A and GSK3 act as modifiers of FUS-ALS by modulating mitochondrial transport

ALS is a fatal neurodegenerative disease which currently lacks effective treatments. Mutations in the RNA-binding protein FUS are a common cause of familial ALS, accounting for around 4% of fALS cases. Studying the mechanisms by which mutant FUS is toxic to neurons may provide insight into the pathogenesis of both familial and sporadic forms of ALS. Here we identify Protein Phosphatase 2A (PP2A) and Glycogen Synthase Kinase 3 (GSK3) as novel modifiers of FUS-ALS in vivo, looking from fly to human. PP2A-C and GSK3{beta} inhibition rescued FUS-induced toxicity in Drosophila and disease-relevant phenotypes in human iPSC-derived spinal motor neurons (sMNs). In both Drosophila and human iPSC-sMNs, we observed reduced GSK3{beta} inhibitory phosphorylation, suggesting that FUS dysfunction results in GSK3{beta} hyperactivity. We found that PP2A acts upstream of GSK3, affecting its inhibitory phosphorylation, and in synergy they modulate mitochondrial transport through the motor protein kinesin. Our data provide in vivo evidence that PP2A and GSK3 are disease modifiers, and reveal an unexplored mechanistic link between PP2A, GSK3 and kinesin in FUS-associated ALS.

neuroscience↗