| dc.description.abstract | Vesicular monoamine transporter 2 (VMAT2) is an essential protein for transport of monoamine neurotransmitters such as dopamine from the cytosol into synaptic vesicles, packing them for subsequent release into the synaptic cleft. Dysfunctional monoamine signalling disturbs neurotransmission and plays a role in several neurological diseases. Thus, VMAT2 holds therapeutic potential for the symptomatic treatment of these diseases. For example, in the case of chorea, a movement disorder associated with Huntington’s disease, and tardive dyskinesia, inhibitors of VMAT2 that hinder the release of dopamine have already been approved. In Parkinson’s disease where there is degeneration of dopaminergic neurons and loss of dopamine signalling, activators of VMAT2 have the potential to increase dopamine release and have been shown to be neuroprotective. We thus set out to establish a functional high-throughput screening assay that can help identify potential VMAT2 activators and inhibitors by using the fluorescent VMAT2 substrate FFN206, enabling the measurement of VMAT2 activity with a microplate reader. The screening assay uses HEK293 cells transiently transfected with human VMAT2 and is based on detection of increase or decrease of VMAT2-specific substrate uptake in the presence of different small molecule compounds. We screened the Prestwick chemical library® containing 1280 compounds, mainly FDA approved drugs, from which the initial screening gave 55 hits showing inhibitory or activating effects for VMAT2. All these initial hits were further evaluated with a concentration dependent substrate uptake assay to remove false positive hits from the initial screening and to investigate potency at lower compound concentrations. Top-ranking hit compounds were selected for further evaluation of EC50 and IC50 for VMAT2 and they were also assayed with human VMAT1, to determine their specificity for VMAT2. The screening and the consequent validation assays resulted in the identification of inhibitors of varying strength. The inhibitors also have a higher IC50 value for VMAT1 than VMAT2, indicating that they have a higher affinity for VMAT2. However, the activators did not show the same clear modulation of VMAT2. By using an inhibition displacement assay to check if the hit compounds compete with the known VMAT2 inhibitor dihydrotetrabenazine, we found that some compounds do compete with this known inhibitor, indicating that it binds to the same site in VMAT2. | |
