ABSTRACT
With over 450 genes, solute carriers (SLCs) constitute the largest transporter superfamily responsible for the uptake and efflux of nutrients, metabolites, and xenobiotics in human cells. SLCs are associated with a wide variety of human diseases, including cancer, diabetes, and metabolic and neurological disorders. They represent an important therapeutic target class that remains only partly exploited as therapeutics that target SLCs are scarce. Additionally, many small molecules reported in the literature to target SLCs are poorly characterized. Both features may be due to the difficulty of developing SLC transport assays that fulfill the quality criteria for high-throughput screening. Here, we report one of the main limitations hampering assay development within the RESOLUTE consortium: the lack of a resource providing high-quality information on SLC tool compounds. To address this, we provide a systematic annotation of tool compounds targeting SLCs. We first provide an overview on RESOLUTE assays. Next, we present a list of SLC-targeting compounds collected from the literature and public databases; we found that most data sources lacked specificity data. Finally, we report on experimental tests of 19 selected compounds against a panel of 13 SLCs from seven different families. Except for a few inhibitors, which were active on unrelated SLCs, the tested inhibitors demonstrated high selectivity for their reported targets. To make this knowledge easily accessible to the scientific community, we created an interactive dashboard displaying the collected data in the RESOLUTE web portal (https://re-solute.eu). We anticipate that our open-access resources on assays and compounds will support the development of future drug discovery campaigns for SLCs.
ABSTRACT
The FLIPR (Fluorescent Imaging Plate Reader) system has been extensively used in the early stages of drug discovery for the identification of small molecules as a starting point for drug development, and for the pharmacological characterization of compounds. The main application of the system has been the measurement of intracellular Ca(2+) signals using fluorescent calcium indicators.This chapter describes the application of a protocol for the study and characterization of state-dependent blockers of Voltage-Gated Calcium Channels (VGCC) on the FLIPR(TETRA).The cell line suitable for the application of the protocol, and described hereafter, co-expresses the human CaV1.2 channel and the human inward rectifier K(+) channel Kir2.3. The presence of Kir2.3 allows the modulation of the plasma membrane potential and consequently of the state of the CaV1.2 channel by changing the extracellular K(+) concentration. In this way, CaV1.2 activity can be measured at different membrane voltages, corresponding to either the resting or partial inactivated state, by loading the cells with a calcium probe in extracellular low or high potassium buffer.
