High-Throughput Screening to Identify Small Molecules That Selectively Inhibit APOL1 Protein Level in Podocytes.

High-throughput phenotypic screening is a key driver for the identification of novel chemical matter in drug discovery for challenging targets, especially for those with an unclear mechanism of pathology. For toxic or gain-of-function proteins, small-molecule suppressors are a targeting/therapeutic strategy that has been successfully applied. As with other high-throughput screens, the screening strategy and proper assays are critical for successfully identifying selective suppressors of the target of interest. We executed a small-molecule suppressor screen to identify compounds that specifically reduce apolipoprotein L1 (APOL1) protein levels, a genetically validated target associated with increased risk of chronic kidney disease. To enable this study, we developed homogeneous time-resolved fluorescence (HTRF) assays to measure intracellular APOL1 and apolipoprotein L2 (APOL2) protein levels and miniaturized them to 1536-well format. The APOL1 HTRF assay served as the primary assay, and the APOL2 and a commercially available p53 HTRF assay were applied as counterscreens. Cell viability was also measured with CellTiter-Glo to assess the cytotoxicity of compounds. From a 310,000-compound screening library, we identified 1490 confirmed primary hits with 12 different profiles. One hundred fifty-three hits selectively reduced APOL1 in 786-O, a renal cell adenocarcinoma cell line. Thirty-one of these selective suppressors also reduced APOL1 levels in conditionally immortalized human podocytes. The activity and specificity of seven resynthesized compounds were validated in both 786-O and podocytes.

Development of a colorimetric method for functional chloride channel assay.

Anion channels play significant physiological roles in humans and animals. However, the effort of screening for anion channel modulators was limited by the available assay technologies. This report discusses the development of a cell-based functional chloride channel assay using iodine as the chloride channel functional indicator. Iodine concentrations were measured with modified Sandell-Kolthoff reaction using colorimetric detection. The assay was rapid and quantitative. When WSS-1 cells were activated by gamma-aminobutyric acid (GABA) in the condition that gamma-aminobutyric acid type A receptor (GABAA receptor) conducted outwardly rectifying chloride channel function, the EC50 of GABA was 7.69 microM. IC50s were 0.53 microM for bicuculline and 3.1 microM for picrotoxin, respectively, in the presence of 10 microM GABA. When Capan-1 cells were activated by forskolin, the EC50 was 0.14 microM. The assay can also be applied to inwardly rectifying anion channels as exemplified by GABAA channel with an EC50 of 294 microM. Thus, the assay is universal and reliable and can be used for anion channel high-throughput screening.

Development of a sensitive and HTS-compatible reporter gene assay for functional analysis of human adenosine A2a receptors in CHO-K1 cells.

Adenosine A2a receptor, a member of the G protein-coupled receptor superfamily, has been demonstrated to be an important pharmacological target. It couples to stimulatory G protein and activates adenylate cyclase upon agonist stimulation. Here we attempted to stably transfect Chinese hamster ovary (CHO-K1) cells, which lack any known subtypes of adenosine receptors, with recombinant human adenosine A2a receptors (hA2aR). Rapid down-regulation of hA2aR in a clonal cell line, CHOA2a-2, was observed over a short period of time in culture. This is consistent with other groups' findings of low expression and poor G protein coupling of this receptor in several cell systems. To facilitate pharmacological profiling for hA2aR ligand, we introduced a cyclic AMP response element (CRE)-linked beta-galactosidase reporter gene into CHOA2a-2 cells to generate a stable cell line, CHOA2a-2CREbetagal#26. Robust cyclic AMP signal amplification was obtained using a colorimetric assay measuring beta-galactosidase activity. The EC(50) of 5'-N-ethylcarboxamidoadenosine (NECA), a potent A2a agonist, for inducing beta-galactosidase activity was 23.3 +/- 3.5 nM, similar to 22.7 +/- 3.9 nM, which was the NECA EC(50) in the direct measurement of cyclic AMP of CHOA2a-2 cells in early culture. Subsequently we validated this assay for high throughput screening for hA2aR agonists. The Z' factor for robotic assay performance was 0.79 +/- 0.03, the ratio of signal/noise was 157 +/- 36, and the ratio of signal/background was 10.6 +/- 1.2, demonstrating that this assay is well suitable for quality high throughput screening. High throughput screening of Johnson & Johnson libraries uncovered a couple of distinct series of nonadenosine small molecules, in addition to adenosine analogues, as potential hA2aR agonists with EC(50) values of 2-6 microM. Preliminary characterization of those compounds was presented.

Virus-based expression systems facilitate rapid target in vivo functionality validation and high-throughput screening.

Target validation is one of rate-limiting steps in the modern drug discovery. The authors developed a strategy of combining adenovirus-mediated gene transfer for efficient target functionality validation, both in vivo and in vitro, with baculovirus expression to produce sufficient quantities of protein for high-throughput screening (HTS). The incorporation of green fluorescent protein (GFP) in the adenovirus vectors accelerates recombinant adenovirus plaque purification, whereas the use of epitope and affinity tags facilitates the identification and purification of recombinant protein. In this generalized scheme, the flexible modular design of viral vectors facilitates the transition between target validation and HTS. In the example presented, functional target validation in vivo was achieved by overexpressing the target gene in cell-based models and in the mouse cortex following adenovirus-mediated gene delivery. In this context, target overexpression resulted in the accumulation of a disease-related biomarker both in vitro and in vivo. A baculovirus-based expressional system was then generated to produce enough target protein for HTS. Thus, the use of these viral expression systems represents a generalized method for rapid target functionality validation and HTS assay development, which could be applied to numerous target candidates being elucidated in gene discovery programs.