RESEARCHERS at the Scripps Research Institute in La Jolla, California have developed a high-throughput screening (HTS) technique which aims to shed light on the biochemical activities of numerous proteins about which little is currently known.
The researchers say the method combines Activity-Based Protein Profiling (ABPP) with fluorescent activity-based probes.
The developed technique, flupol-ABPP has been designed to make it easier to find molecules which proteins interact with and inhibitor which might block protein ability.
Researchers claim the technique’s initial applications lay in the area of secondary assays to assess the selectivity of lead inhibitors emerging from more conventional HTS assays.
The scientists involved in the development program identified the technique, when combined with fluorescence polarisation; ABPP was able to be used as a primary HTS assay which had the ability to open a fraction of uncharacterised proteome to inhibitor screening.
The fluopol-ABPP technique uses fluorescent probes to tag proteins. A probe attaching to a protein results in a larger molecule with higher fluorescent polarisation values.
When an inhibitor binds efficiently with a protein, it can overshadow a probe, slowing or eliminating a rise in fluorescence polarisation, indicating inhibition.
The researchers claim unlike ABPP, which relies on laborious gel and mass spectrometry techniques, fluopol-ABPP does not require separation or washing steps, so is readily incorporated into automated high-throughput screening systems.
The team has used fluopol-ABPP to study two enzymes from different classes: retinoblastoma-binding protein-9 (RBBP9) and glutathione S-transferase omega 1 (GSTO1).
Both are thought to play a role in some cancers, but researchers had made little progress in learning the focus of their biological activity.
However, the method has an intrinsic time-constraint and fluorescence polarisation is very dependent on ambient temperature. The technology is currently involved in screen runs for researchers to identify and mend flaws.
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