High-throughput fluorescence-based screening assays for tryptophan-catabolizing enzymes.

Indoleamine 2,3-dioxygenase (IDO1) and tryptophan 2,3-dioxygenase (TDO) are two structurally different enzymes that have a different tissue distribution and physiological roles, but both catalyze the conversion of tryptophan to N-formylkynurenine (NFK). IDO1 has been clinically validated as a small-molecule drug target for cancer, while preclinical studies indicate that TDO may be a target for cancer immunotherapy and neurodegenerative disease. We have developed a high-throughput screening assay for IDO1 and TDO based on a novel chemical probe, NFK Green, that reacts specifically with NFK to form a green fluorescent molecule with an excitation wavelength of 400 nm and an emission wavelength of 510 nm. We provide the first side-by-side comparison of a number of published inhibitors of IDO1 and TDO and reveal that the preclinical IDO1 inhibitor Compound 5l shows significant cross-reactivity with TDO, while the relative selectivity of other published inhibitors was confirmed. The suitability for high-throughput screening of the assays was demonstrated by screening a library of 87,000 chemical substances in 384- or 1536-well format. Finally, we demonstrate that the assay can also be used to measure the capacity of cells to metabolize tryptophan and to measure the cellular potency of IDO1 and TDO inhibitors.

Inhibition of Wnt/ß-catenin signaling by p38 MAP kinase inhibitors is explained by cross-reactivity with casein kinase Iδ/ɛ.

Wnt/ß-catenin signaling plays essential roles in embryonic development, adult stem cell maintenance, and disease. Screening of a small molecule compound library with a ß-galactosidase fragment complementation assay measuring ß-catenin nuclear entry revealed TAK-715 and AMG-548 as inhibitors of Wnt-3a-stimulated ß-catenin signaling. TAK-715 and AMG-548 are inhibitors of p38 mitogen-activated protein kinase, which has been suggested to regulate activation of Wnt/ß-catenin signaling. However, two highly selective and equally potent p38 inhibitors, VX-745 and Scio-469, did not inhibit Wnt-3a-stimulated ß-catenin signaling. Profiling of TAK-715 and AMG-548 against a panel of over 200 kinases revealed cross-reactivity with casein kinase Iδ and ɛ, which are known activators of Wnt/ß-catenin signaling. Our data demonstrate that this cross-reactivity accounts for the inhibition of ß-catenin signaling by TAK-715 and AMG-548 and argue against a role of p38 in Wnt/ß-catenin signaling.

Cryopreserved cells facilitate cell-based drug discovery.

Advances in detection technologies have enabled an increased use of cell-based functional assays in early drug discovery, in particular for G protein-coupled receptors. Screening assays that use live cells are less prone to generate false positives than assays using lysed cell samples. The use of cryopreserved cells instead of cells that are continuously maintained in culture decreases day-to-day variation, removes passage effects and improves the consistency of cell-based assay results. Cryopreservation techniques uncouple cell culturing from drug-screening activities and allow the use of cells as reagents, just like enzymes in biochemical assays.

High-throughput screening using beta-lactamase reporter-gene technology for identification of low-molecular-weight antagonists of the human gonadotropin releasing hormone receptor.

G-protein coupled receptors (GPCRs) signal via G-proteins to intracellular second messengers. Assays that link transcription of a detectable reporter to promoters that are activated by such signaling cascades are highly sensitive and allow screening for compounds that either activate or inactivate a GPCR of interest. This study describes the development and performance of an antagonistic screen on the human gonadotropin releasing hormone receptor (GnRH-R). Compounds (245,000) were tested in a high-throughput screen using a Chinese hamster ovary cell line stably expressing the human GnRH-R and the Ca2+ sensitive reporter nuclear factor activated in T-cells/ activator protein-1-beta-lactamase. In total, 4,160 active compounds were identified. Colored and toxic compounds, as well as dust and compound aggregates, have been depicted as artifacts. To deselect non-target hits, several follow-up assays, including luminescent and fluorescent Ca2+ mobilization assays and radioligand binding, were developed for the GnRH-R. These assays were validated using peptide and low-molecular-weight GnRH-R reference compounds before hits from screening were also profiled in these assays. For several reference compounds the use of different assay technologies resulted in a poor correlation of potency values. In conclusion, beta-lactamase as a primary high-throughput screening assay is a powerful complementation to other screening technologies. The beta-lactamase technology has several advantages, including lack of cell lysis and ratiometric read-out, which augments assay robustness. Based on technology comparison, it is not adequate to assume that the same hits would be found regardless of which assay technology is used.