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.
Highlights
In the drug discovery process, the use of cell-based assays for high throughput screening (HTS) has become increasingly common as targets are presented in a more relevant biological context than classical in vitro biochemical assays [1]
The increased cell number of cultured SV40 transformed (SV40t) cell lines is explained by their growth being less inhibited by cell contact, which is consistently observed in cultured primary fibroblasts
In SV40t fibroblasts, arylsulfatase A (ASA) enzymatic activity is conserved in both controls with wild type ASA (ASA-WT) and metachromatic leukodystrophy (MLD) patient cells with mutant ASA that retain residual ASA activity
Summary
In the drug discovery process, the use of cell-based assays for high throughput screening (HTS) has become increasingly common as targets are presented in a more relevant biological context than classical in vitro biochemical assays [1]. In most cellular HTS assays, cells utilized are usually not disease relevant. Cellular assays using patient cells provide a unique opportunity to assess a target protein and/or pathway in the context of potentially disrupted biochemical and/or signaling pathways secondary to the disease process. This pathogenic in cellulo setting permits the evaluation of multiple intervention points, which are potentially altered in the disease, as opposed to commonly used cellexpression systems of a specific protein target or a predefined step of a purified or recombinant protein-based assay
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
