Discovery of Small Molecule Inhibitors of Huntingtin Exon 1 Aggregation by FRET-Based High-Throughput Screening in Living Cells

Abstract

Huntington’s disease (HD) is the most common inherited neurodegenerative disorder and one of the nine polyglutamine (polyQ) diseases. HD is characterized by the pathological aggregation of the misfolded huntingtin exon 1 protein (Httex1) with abnormally long polyQ expansion due to genetic mutation. While there is currently no effective treatment for HD, inhibition of aggregate formation represents a direct approach in mediating the toxicity associated with Httex1 misfolding. To exploit this therapeutic window, we engineered two fluorescence resonance energy transfer (FRET) based biosensors that monitor the aggregation of Httex1 with different expanded Q-lengths (Q39 and Q72) in living cells. These FRET biosensors, together with a high-precision fluorescence lifetime detection platform, enable high-throughput screening of small molecules that target Httex1 aggregation. We found six small molecules that decreased the FRET of the biosensors and reduced Httex1-Q72-induced neuronal cytotoxicity in N2a cells with nanomolar potency. Using advanced SPR and EPR techniques, we confirmed that the compounds directly bind to Httex1 fibrils and inhibit aggregate formation. This strategy in targeting the Httex1 aggregates can be applicable to other proteins involved in polyQ related diseases.

A new chemoenzymatic semisynthetic approach provides novel insight into the role of phosphorylation beyond exon1 of Huntingtin and reveals N-terminal fragment length-dependent distinct mechanisms of aggregation

“…Although these efforts have led to the identification of several small molecules (such as Niclosamide, AMG 9810, Methylene blue, PGL-135, and arginine ethyl ester) that inhibit the aggregation of polyQ peptides and Httex1, no aggregation inhibitors have advanced to clinical trials. 49,50,51,52 The observed differences in the aggregation mechanism of the longer N-terminal Htt fragments further emphasize the prominent role of cross-talk between the polyQ repeat domain and other flanking C-terminal sequences in determining the Htt aggregation pathway and the ultrastructural properties of the Htt aggregates. Therefore, we propose that future screening efforts to identify Htt aggregation inhibitors should be based on multiple physiologically and pathologically relevant polyQ-containing fragments of different lengths.…”

Molecular Strategies to Target Protein Aggregation in Huntington’s Disease

“…These are small-molecule inhibitors which target either the RNA or the protein and modulate HD levels and aggregation. There are a number of identified inhibitors of mHTT aggregation, in vitro and in vivo ( Pollitt et al, 2003 ; Zhang et al, 2005 ; Chopra et al, 2007 ; Khan et al, 2019 ; Lo et al, 2020 ), however their lack of selectivity for the target protein limits them as potential HD therapeutics.…”

Application of FRET Biosensors in Mechanobiology and Mechanopharmacological Screening

“…Förster or fluorescence resonance energy transfer method combined with subcellular imaging has been successfully applied in versatile HTS assays for different targets and diseases over the past decade. Time resolved (TR)-FRET-based HTS in which FRET ratio change is triggered by protein-protein interactions has been applied to the discovery of small-molecule inhibitors for Httex1 aggregation in Huntington’s disease ( Lo et al, 2020 ), calcium release by endoplasmic reticulum due to familial AD-linked presenilin 1 mutations (FAD-PS1) in Alzheimer’s disease ( Honarnejad et al, 2013 ), binding of anthrax protective antigen and capillary morphogenesis gene 2 (CMG2) protein in angiogenetic cancers and anthrax intoxication ( Rogers et al, 2012 ), I&κB kinase β (IKKβ) and non-canonical ubiquitin-conjugating enzyme UBC13 in inflammatory diseases ( Oh et al, 2010 ; Madiraju et al, 2011 ), and histone methylation activity of lysine demethylase 1 (LSD1) and Jumonji C domain-containing oxygenase D2C (JMJD2C) ( Yu et al, 2011 ). Recent studies employed TR-FRET-based HTS to screen and successfully identify small molecule drugs that affect cardiac sarcoplasmic reticulum Ca-ATPase (SERCA2a) structure ( Gruber et al, 2014 ; Schaaf et al, 2016 ) or increase the affinity between SERCA2a and phospholamban (PLB), presenting a potential therapeutic solution to cardiac contractile dysfunction due to deficient Ca 2+ transport ( Stroik et al, 2018 ).…”