Novel Patient Cell-Based HTS Assay for Identification of Small Molecules for a Lysosomal Storage Disease

Abstract

Small molecules have been identified as potential therapeutic agents for lysosomal storage diseases (LSDs), inherited metabolic disorders caused by defects in proteins that result in lysosome dysfunctional. Some small molecules function assisting the folding of mutant misfolded lysosomal enzymes that are otherwise degraded in ER-associated degradation. The ultimate result is the enhancement of the residual enzymatic activity of the deficient enzyme. Most of the high throughput screening (HTS) assays developed to identify these molecules are single-target biochemical assays. Here we describe a cell-based assay using patient cell lines to identify small molecules that enhance the residual arylsulfatase A (ASA) activity found in patients with metachromatic leukodystrophy (MLD), a progressive neurodegenerative LSD. In order to generate sufficient cell lines for a large scale HTS, primary cultured fibroblasts from MLD patients were transformed using SV40 large T antigen. These SV40 transformed (SV40t) cells showed to conserve biochemical characteristics of the primary cells. Using a specific colorimetric substrate para-nitrocatechol sulfate (pNCS), detectable ASA residual activity were observed in primary and SV40t fibroblasts from a MLD patient (ASA-I179S) cultured in multi-well plates. A robust fluorescence ASA assay was developed in high-density 1,536-well plates using the traditional colorimetric pNCS substrate, whose product (pNC) acts as “plate fluorescence quencher” in white solid-bottom plates. The quantitative cell-based HTS assay for ASA generated strong statistical parameters when tested against a diverse small molecule collection. This cell-based assay approach can be used for several other LSDs and genetic disorders, especially those that rely on colorimetric substrates which traditionally present low sensitivity for assay-miniaturization. In addition, the quantitative cell-based HTS assay here developed using patient cells creates an opportunity to identify therapeutic small molecules in a disease-cellular environment where potentially disrupted pathways are exposed and available as targets.

A high-throughput screening assay using Krabbe disease patient cells

“…a dose-response enhancement of GALC residual enzymatic activity [22]. Based on the classification of the CRCs previously described [17; 22] (Table S1 in supplemental data), 19 small molecules (SM) showed active CRC patterns within classes 1 to 3 (Table S1). In this HTS assay, an active CRC represents the percentages of HMU signal increase ( y axis) as a function of the increasing SM concentrations ( x axis) used to treat the SV40T GLD patient fibroblasts in the 1,536-well plates (Fig.5).…”

“…Only small molecules showing dose-response enhancements of residual GALC resulting in active CRCs are identified. Therefore, given small size of chemical compound collection, which is designed for pilot HTS, a low “hit” rate is expected as previously described [17]. …”

“…Measurements of total β-hexosaminidase (Hex), β-galactosidase and glucocerebrosidase enzymatic activity were performed using cultured cell lysates from primary and SV40T fibroblasts as previously described [12; 17]. …”

“…After 48h treatment course with different concentrations of selected small molecules, cell viability assays were performed in 96-well plates utilizing CellQuant-Blue™ reagents as protocol and described earlier [17]. This is a non-radioactive fluorescent assay that is available through BioAssay Systems.…”

“…Recently, we described a patient cell-based assay having as a target a lysosomal enzyme deficient in a neurological LSD [17]. Herein, we report the first GLD patient cell-based assay having the lysosomal GALC as a target.…”