The Development of a High-Content Screening Binding Assay for the Smoothened Receptor

Author:

Bee Weilin Tiger1,Xie Wensheng1,Truong Maggie1,Will Matthew2,Turunen Brandon3,Zuercher William J.3,McMillan Lynette2,Li Hu1,Hornberger Keith R.4,Davenport Elizabeth A.1,Ames Robert S.1,Kallal Lorena A.1

Affiliation:

1. Department of Biological Reagents and Assay Development, Molecular Discovery Research, GlaxoSmithKline, Collegeville, PA, USA

2. Department of Biological Reagents and Assay Development, Molecular Discovery Research, GlaxoSmithKline, King of Prussia, PA, USA

3. Discovery Medicinal Chemistry, GlaxoSmithKline, Research Triangle Park, NC, USA

4. Oncology Chemistry, GlaxoSmithKline, Collegeville, PA, USA

Abstract

In this study, the development of an image-based high-content screening (HCS) binding assay for the seven-transmembrane (7TM) receptor Smoothened (Smo) is described. Using BacMam-based gene delivery of Smo, BODIPY-cyclopamine as a fluorescent probe, and a confocal imaging system, a robust 384-well assay that could be used for high-throughput compound profiling activities was developed. The statistically robust HCS binding assay was developed through optimization of multiple parameters, including cell transduction conditions, Smo expression levels, the image analysis algorithm, and staining procedures. Evaluation of structurally diverse compounds, including functional Smo activators, inhibitors, and related analogs, demonstrated good compound potency correlations between high-content imaging binding, membrane fluorescence polarization binding, and gene reporter assays. Statistical analysis of data from a screening test set of compounds at a single 10-µM concentration suggested that the high-content imaging Smo binding assay is amenable for use in hit identification. The 384-well HCS assay was rapidly developed and met statistical assay performance targets, thus demonstrating its utility as a fluorescent whole-cell binding assay suitable for compound screening and profiling.

Publisher

Elsevier BV

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