A Cell-Based Ultra-High-Throughput Screening Assay for Identifying Inhibitors of D-Amino Acid Oxidase

Abstract

Enzymes are often considered less "druggable" targets than ligand-regulated proteins such as G-protein-coupled receptors, ion channels, or other hormone receptors. Reasons for this include cellular location (intracellular vs. cell surface), typically lower affinities for the binding of small molecules compared to ligand-specific receptors, and binding (catalytic) sites that are often charged or highly polar. A practical drawback to the discovery of compounds targeting enzymes is that screening of compound libraries is typically carried out in cell-free activity assays using purified protein in an inherently artificial environment. Cell-based assays, although often arduous to design for enzyme targets, are the preferred discovery tool for the screening of large compound libraries. The authors have recently described a novel cell-based approach to screening for inhibitors of a phosphatase enzyme and now report on the development and implementation of a homogeneous 3456-well plate assay for D-amino acid oxidase (DAO). Human DAO was stably expressed in Chinese hamster ovary (CHO) cells, and its activity was measured as the amount of hydrogen peroxide detected in the growth medium following feeding the cells with D-serine. In less than 12 weeks, the authors proved the concept in 96-and then 384-well formats, miniaturized the assay to the 3456-well (nanoplate) scale, and screened a library containing more than 1 million compounds. They have identified several cell-permeable inhibitors of DAO from this cell-based high-throughput screening, which provided the discovery program with a few novel and attractive lead structures. (Journal of Biomolecular Screening 2006:481-487)

The Behavioral and Neurochemical Effects of a Novel d-Amino Acid Oxidase Inhibitor Compound 8 [4H-Thieno [3,2-b]pyrrole-5-carboxylic Acid] and d-Serine

“…Cell-based enzyme activity was evaluated essentially as described by Brandish et al (2006). In brief, enzyme activity was evaluated in Chinese hamster ovary (CHO) cells transiently transfected with human or rat DAAO in pcDNA 3.1ϩ.…”

High Throughput Screening Approach to Lead Discovery

“…More recently, 1536-well format provides throughput of over 100,000 compounds per day (ultra-HTS) [106]. This trend continues, with several reports of biological assays carried out in 3456-well microplates with a total assay volume of 1-2 mL per well, although, there are still significant technological hurdles that need to be addressed before this technology becomes routine [107].…”

Cell-Based Assays for High-Throughput Screening

“…Cell-based HTS in 384-, 1536-, and even 3456-well plate format has been reported (e.g., see refs. [26][27][28]). …”