Optimization and validation of a DYRK1A TR-FRET assay for high-throughput screening.

Due to its role in brain development, the DYRK1A kinase (dual-specificity tyrosine phosphorylation-regulated kinase 1a) has been proposed as a drug target for Down syndrome, and diseases associated with neurodegeneration including Alzheimer's and Parkinson's. Other diseases in which DYRK1A is implicated include cancer and diabetes. Hence, there is need for potent and selective DYRK1A inhibitors. To screen large diversity compound libraries versus DYRK1A requires the development of a cost-effective high-throughput screen. In this study, we have taken a commercial time-resolved fluorescence energy transfer (TR-FRET)-based assay for DYRK1A and optimized for smaller volumes and homogenous format at room temperature. Tracer and enzyme concentrations were determined. DYRK1A-GST, anti-GST Ab and tracer were pre-combined and total assay volume reduced 2-fold. The assay was validated using whole plate minimum and maximum signal wells with a Z' of 0.7-0.8 determined. Overall, this method:•Results in an optimized low volume, homogenous and validated assay for DYRK1A.•Delivers a cost effective high-throughput assay format for DYRK1A inhibitor screening.

Data supporting a pilot high-throughput screen of a drug library for identification of DYRK1A inhibitors and high-content imaging analysis of identified harmine analogs.

The data presented in this article support the accompanying research article "Identification of harmine and ß-carboline analogs from a high-throughput screen of an approved drug collection; profiling as differential inhibitors of DYRK1A and monoamine oxidase A and for in vitro and in vivo anti-cancer studies" [1]. As DYRK1A (dual-specificity tyrosine phosphorylation-regulated kinase 1a) plays a role in the pathophysiology of a number of diseases including diabetes, cancer and neurodegeneration [2], [3], [4], the identification of DYRK1A inhibitors is of significant interest. This data article details the hits identified from a DYRK1A high-throughput screen of a small molecule compound library containing over 95% approved drugs. Twenty-two compounds were identified with >50% inhibition, including harmine and four of its analogs. Subsequent profiling of these harmine analogs using glioma cancer cell lines and high-content image analysis identified those with effects on growth and cytotoxicity.

Identification of harmine and ß-carboline analogs from a high-throughput screen of an approved drug collection; profiling as differential inhibitors of DYRK1A and monoamine oxidase A and for in vitro and in vivo anti-cancer studies.

DYRK1A (dual-specificity tyrosine phosphorylation-regulated kinase 1a) is highly expressed in glioma, an aggressive brain tumor, and has been proposed as a therapeutic target for cancer. In the current study, we have used an optimized and validated time-resolved fluorescence energy transfer (TR-FRET)-based DYRK1A assay for high-throughput screening (HTS) in 384-well format. A small-scale screen of the FDA-approved Prestwick drug collection identified the ß-carboline, harmine, and four related analogs as DYRK1A inhibitors. Hits were confirmed by dose response and in an orthogonal DYRK1A assay. Harmine's potential therapeutic use has been hampered by its off-target activity for monoamine oxidase A (MAO-A) which impacts multiple nervous system targets. Selectivity profiling of harmine and a broader collection of analogs allowed us to map some divergent SAR (structure-activity relationships) for the DYRK1A and MAO-A activities. The panel of harmine analogs had varying activities in vitro in glioblastoma (GBM) cell lines when tested for anti-proliferative effects using a high content imaging assay. In particular, of the identified analogs, harmol was found to have the best selectivity for DYRK1A over MAO-A and, when tested in a glioma tumor xenograft model, harmol demonstrated a better therapeutic window compared to harmine.

Development of a Cell-Based Fluorescence Polarization Biosensor Using Preproinsulin to Identify Compounds That Alter Insulin Granule Dynamics.

Diabetes currently affects 9.3% of the U.S. population totaling $245 billion annually in U.S. direct and indirect healthcare costs. Current therapies for diabetes are limited in their ability to control blood glucose and/or enhance insulin sensitivity. Therefore, innovative and efficacious therapies for diabetes are urgently needed. Herein we describe a fluorescent insulin reporter system (preproinsulin-mCherry, PPI-mCherry) that tracks live-cell insulin dynamics and secretion in pancreatic ß-cells with utility for high-content assessment of real-time insulin dynamics. Additionally, we report a new modality for sensing insulin granule packaging in conventional high-throughput screening (HTS), using a hybrid cell-based fluorescence polarization (FP)/internal FRET biosensor using the PPI-mCherry reporter system. We observed that bafilomycin, a vacuolar H(+) ATPase inhibitor and inhibitor of insulin granule formation, significantly increased mCherry FP in INS-1 cells with PPI-mCherry. Partial least squares regression analysis demonstrated that an increase of FP by bafilomycin is significantly correlated with a decrease in granularity of PPI-mCherry signal in the cells. The increased FP by bafilomycin is due to inhibition of self-Förster resonant energy transfer (homo-FRET) caused by the increased mCherry intermolecular distance. FP substantially decreases when insulin is tightly packaged in the granules, and the homo-FRET decreases when insulin granule packaging is inhibited, resulting in increased FP. We performed pilot HTS of 1782 FDA-approved small molecules and natural products from Prestwick and Enzo chemical libraries resulting in an overall Z'-factor of 0.52 ± 0.03, indicating the suitability of this biosensor for HTS. This novel biosensor enables live-cell assessment of protein-protein interaction/protein aggregation in live cells and is compatible with conventional FP plate readers.