Microplate Reader Detection of a Fluorescent Substrate to Determine Transporter Activity and Identify Novel Inhibitors

In this project, we optimized a high-throughput fluorescent-based assay to assess transporter activity by measuring uptake of the substrate, ASP+  4-(4-(dimethylamino)styryl)-N-methylpyridinium. ASP+ is the fluorescent derivative of the neurotoxin MPP+, both of which have been used to study various transporter properties. The ASP+ microplate method offers an easily teachable platform for pharmacy, graduate, or undergraduate students to perform meaningful, short-term projects. Confocal microscopy and radioligand binding techniques are informative, but are relatively low throughput, require extensive training, expensive equipment, and dedicated laboratory space, or can generate considerable hazardous risks. Our current work focuses on organic cation transporters (OCTs), proteins expressed in the brain and throughout the body that have vital roles in normal physiology and drug responses. OCTs have a low-affinity and high-capacity to transport substrates across the cell membrane, and can quickly take up a broad range of compounds that include pharmacological drugs, endogenous neurotransmitters like dopamine, norepinephrine, and serotonin, and experimental substrates like ASP+. OCT research is limited by the lack of selective ligands for each of the three OCT subtypes. Our initial data, generated by collaborators at RTI International, used an optimized ASP+ uptake for high-throughput screens (measuring 96 samples simultaneously). The goal of this collaborative project is to identify experimental selective inhibitors to propel the study of broadly expressed OCTs. Using human embryonic kidney cells that overexpressed human OCT3, nearly 1500 compounds were screened with the ASP+ uptake model and 105 compounds (used at 10 µM concentrations) were found to inhibit ASP+ uptake similarly to other known OCT3 inhibitors. Ongoing ASP+ experiments are screening the new OCT3 compounds to determine their IC50 values and activity at subtypes OCT1 and OCT2. The ASP+ microplate reader assay offers a rapid, adaptable, economically and environmentally favorable means for studies to identify novel selective ligands, and evaluate the impact of physiological environment, or genetic variations on the function of OCTs and other monoamine transporters.

Learning Objectives:

1. Define the fluorescent molecule, types of transport proteins, and expression system used in the study.

2. Identify 3 differences between radioligand binding and fluorescent substrate microplate detection. 

3. Outline how ASP+ can be used as a preclinical tool to screen transporter inhibitors using high throughput, microplate detection.