Abstract
Screening drug candidates for their affinity and selectivity for a certain binding site is a crucial step in developing targeted therapy. Here, we created a screening assay for receptor binding that can be easily scaled up and automated for the high throughput screening of Kv channel blockers. It is based on the expression of the KcsA-Kv1 hybrid channel tagged with a fluorescent protein in the E. coli membrane. In order to make this channel accessible for the soluble compounds, E. coli were transformed into spheroplasts by disruption of the cellular peptidoglycan envelope. The assay was evaluated using a hybrid KcsA-Kv1.3 potassium channel tagged with a red fluorescent protein (TagRFP). The binding of Kv1.3 channel blockers was measured by flow cytometry either by using their fluorescent conjugates or by determining the ability of unconjugated compounds to displace fluorescently labeled blockers with a known affinity. A fraction of the occupied receptor was calculated with a dedicated pipeline available as a Jupyter notebook. Measured binding constants for agitoxin-2, charybdotoxin and kaliotoxin were in firm agreement with the earlier published data. By using a mid-range flow cytometer with manual sample handling, we measured and analyzed up to ten titration curves (eight data points each) in one day. Finally, we considered possibilities for multiplexing, scaling and automation of the assay.
Highlights
Despite the progress in precision medicine such as genetic, cell-based- and immunotherapies, synthetic and peptide drugs are still indispensable and most effective for various pathologies
We aimed to adapt our spheroplast-based ligand-binding assay for use with flow cytometry
Measurement of 1 μm particles that are near to the lower size limit is challenging since the scattering of such particles is relatively low
Summary
Despite the progress in precision medicine such as genetic-, cell-based- and immunotherapies, synthetic and peptide drugs are still indispensable and most effective for various pathologies. The accelerated appearance of protein structure data and new drug-discovery algorithms gives rise to vast amounts of new drug candidates. This stimulates the demand for fast, affordable and reliable techniques for first-line drug screening [1]. Target proteins used in screening assays can be expressed in E. coli or in eukaryotic cells. The former has a much higher yield and cost efficiency; this is challenging when membrane proteins are needed [2,3]. The expression of transmembrane proteins in the membrane of E. coli might be a superior
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