Despite the identification of the pathogenic poly-CAG expansion to the huntingtin (Htt) gene as the cause of Huntington’s Disease (HD) over 20 years ago, there remains no effective treatment. Numerous studies have been performed to identify molecules which modulate the toxic effects and/or expression of the mutant HTT protein (mutHTT). These studies have often been based around a single hypothesis such as reducing aggregation or preventing caspase cleavage. Additionally, an exogenous promoter driving expression of a HTT fragment with a polyQ length well beyond a clinically relevant range has been required to generate a sufficient assay signal for reliable screening. These attributes may have contributed to the lack of translatability of hit genes or molecules from one screen to another, and to higher in vivo models of HD.

Here we describe the steps taken to develop a HTS compatible HTRF assay to identify novel small molecules that reduce mutHTT levels using cryopreserved HD patient derived lymphoblasts expressing a clinically relevant mutHTT allele from its endogenous promoter. Parameters such as cell density, compound incubation time, lysis buffer constituents, antibody fluorophore and concentration have been optimized.

To ensure that any hit molecules were not active purely via cytotoxic mechanisms, a parallel cytotoxicity assay was developed. A number of commercial cytotoxicity assays were evaluated for sensitivity and ability to fit into the workflow, and the CellTiter Glo ATP assay was selected as having the best linear range and flexibility to fit into our workflow.

The optimized conditions led to an automated 384-well plate assay that is acceptable for HTS (Z’ >0.3, S/B >2). Cells are plated into 384-well plates and exposed to compounds for 48 h before being lysed, after which mutHTT and total HTT are detected by specific anti-HTT antibodies to defined epitopes on the HTT protein.

A n=2 pilot screen of 8,000 molecules displayed good correlation between replicates as well as between single-point and IC50 screening, validating the ability of the assay to robustly identify reproducible hits. We aim to apply the validated assay to a wider screen of diverse chemical libraries to identify hits that may lead to novel therapeutics regulating the expression of mutHTT to ultimately slow the progression of HD.